Formulation and Stability Testing of Eye Drop Preparations Containing Phenylephrine Hydrochloride

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FORMULATION AND STABILITY TESTING OF EYE DROP PREPARATIONS CONTAINING PHENYLEPHRINE HYDROCHLORIDE

CHINEDUM OLUCHUKWU OKAFOR

FORMULATION AND STABILITY TESTING OF EYE DROP PREPARATIONS CONTAINING PHENYLEPHRINE HYDROCHLORIDE

CHINEDUM OLUCHUKWU OKAFOR

Submitted in fulfillment of the requirements for the degree of MAGISTER

SCIENTIAE in the FACULTY OF HEALTH SCIENCES at the NELSON MANDELA

METROPOLITAN UNIVERSITY

DECEMBER 2012

SUPERVISOR: Mrs. M. Keele

CO-SUPERVISORS: Dr M. Worthington, Prof. G. Kilian

DECLARATION

I, Chinedum Oluchukwu Okafor, 205010351, hereby declare that the dissertation for Magister Scientiae is my own work and that it has not previously been submitted for assessment or completion of any postgraduate qualifcaton to another University or for another qualification.

Chinedum Oluchukwu Okafor

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ACKNOWLEDGEMENTS

I wish to thank the following people and institutions for their assistance during the compiling of this dissertation:

 My exceptional families Okafor and Makamure for their steadfast support and love;  Dr. M Worthington, as without sponsorship and guidance there could be no research;  My supervisors, Mrs. M. Keele, and Prof. G. Kilian for guidance and support;  Prof. Milne, for his unwavering support, no words in the dictionary can describe his help;  Michael (Aspen), Jean, Charne and Arista (NMMU) for her input, support and exceptional skills at sourcing materials for me;  Aspen Pharmacare, for financial assistance and the use of equipment and materials needed to perform my experiments;  All my friends all around the world, every moment with you was a blessing.  The Pharmacy, Biochemistry and Microbiology and Chemistry Departments of the Nelson Mandela Metropolitan University for the use of laboratory facilities and technical assistance;  Above all, God Almighty, only through His Grace can I achieve all things.

SUMMARY

Phenylephrine hydrochloride is a potent adrenergic agent and β-receptor sympathomimetic drug, used in its optically active form (Pandey et al., 2003; Pandey et al., 2006). As an α1-adrenergic receptor agonist, phenylephrine hydrochloride is used ocularly as a decongestant for uveitis and as an agent to dilate the pupil (Lang, 1995). High intraocular doses have been reported to cause tachycardia, hypertension, and headache. These side effects are caused by large amounts of the drop draining into the nasal cavity. Eye drops that contain phenylephrine hydrochloride have proven to have low intra-ocular bioavailability because of a short contact time with the eyes which reduces the amount of drug reaching the site of action. Formulations of phenylephrine hydrochloride eye drops have varying shelf- lives of approximately two to four years. The aim of this study was to formulate and manufacture an eye drop product containing phenylephrine hydrochloride. Important characteristics that were targeted were increased ocular absorption by increasing the viscosity of the product and reduced degradation of phenylephrine hydrochloride.

A variety of phenylephrine hydrochloride formulations were manufactured on a laboratory scale using hydroxypropyl methylcellulose (HPMC), glycerol, and sodium carboxy methylcellulose as viscosity modifying agents (VMA). The concentration of phenylephrine hydrochloride was ten percent. Ten millimeters of each formulation was made in triplicate. The quantity in each was evaluated using a previously validated high performance (pressure) liquid chromatography method. Physicochemical properties including pH and colour were also evaluated. Stability was assessed using real time and accelerated stability conditions in accordance with the International Conference on Harmonization (ICH) guidelines.

Formulations containing hydroxypropyl methylcellulose (HPMC) as the viscosity modifying agents proved to be stable under all storage conditions when compared with formulations containing other viscosity modifying agents (VMA). However, sodium citrate dihydrate; sodium metabisulphite and EDTA also stabilized the formulations to a certain extent.

Changes in the appearance and colour of products containing glycerol under accelerated storage conditions were observed. The sodium carboxy methylcellulose (SCMC) containing formulation was found to be physically and chemically stable in two conditions, namely 30 °C/65%RH and 25 °C/60%RH. The formulations containing hydroxypropyl methylcellulose along with an antioxidant showed to be most stable as it remained aesthetically pleasing did not change colour and did not have a reduction in phenylephrine hydrochloride concentrations. This meant that phenylephrine hydrochloride did not degrade while the viscosity modifying agents remained stable.

Rheological tests showed differences in the viscosities of the formulations as glycerol had increased in viscosity over time while HMPC and SCMC displayed relative similarities. The formulations were compared to a marketed eye drop containing polyvinyl alcohol as a VMA. After rheological analysis the formulation containing HPMC displayed better viscosity than the product with polyvinyl alcohol.

The preservatives in the formulations were active against the microbial organisms use to challenged them.

Key words: Phenylephrine hydrochloride, glycerol, hydroxypropyl methylcellulose, preservatives, storage conditions, viscosity.

TABLE OF CONTENTS DECLARATION ...... iii ACKNOWLEDGEMENTS ...... iv SUMMARY ...... v TABLE OF CONTENTS ...... vii LIST OF ABBREVIATIONS ...... x LIST OF FIGURES ...... xiv LIST OF TABLES ...... xxviii 1. INTRODUCTION ...... 1 1.1 Background and motivation ...... 1 1.2 Aim and objectives ...... 2 1.3 Plan of work ...... 3 2. LITERATURE REVIEW ...... 4 2.1 Anatomy and physiology of the eye ...... 4 2.2 Pathophysiology of the eye ...... 10 2.3 Phenylephrine hydrochloride and its ocular uses ...... 14 2.3.1 Phenylephrine hydrochloride ...... 14 2.3.2 Pharmacological actions and uses ...... 15 2.3.3 Mechanism of action ...... 15 2.3.4 Pharmacokinetics ...... 16 2.3.5 Adverse effects ...... 16 2.3.6 Drug interactions ...... 17 2.3.7 Bioavailability ...... 17 2.3.7.1 Reasons for poor ocular bioavailability ...... 18 2.3.7.2 Strategies for improving drug availability in ocular adminstration ...... 19 2.3.7.2.1 Increasing ocular residence time ...... 19 2.3.7.2.2 Increasing ocular absorption ...... 19 2.3.7.2.3 Altering drug structure ...... 20 2.3.8 Polymorphism and pseudomorphism of phenylephrine hydrochloride ...... 21 2.4 Ophthalmic formulations ...... 21 2.4.1 Eye drops as an ophthalmic dosage form ...... 22 2.4.2 Eye drop formulation characteristics ...... 24 2.4.2.1 Clarity ...... 24 2.4.2.2 Stability, pH and buffer systems ...... 25 2.4.2.3 Tonicity ...... 26

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2.4.2.4 Viscosity ...... 27 2.4.2.5 Additives ...... 30 2.5 Sterilization ...... 34 2.5.1 Steam under pressure as a method of sterilization ...... 35 2.5.2 Filtration as a method of sterilization ...... 35 2.5.3 Laminar-flow principles ...... 35 2.5.4 Preservatives used in eye drop formulations ...... 36 2.5.4.1 Quaternary ammonium compounds ...... 38 2.5.4.2 Parahydroxybenzoic acid esters ...... 40 2.6 Efficacy of antimicrobial preservation ...... 42 2.7 Packaging ...... 43 2.9 Formulation development ...... 44 2.9.1 Validation of HPLC analytical methods ...... 46 2.9.1.1 Stability indicating HPLC analysis ...... 47 2.9.1.2 Choice of analytical column and conditions ...... 48 2.9.1.3 Steps for HPLC method validation ...... 50 2.9.1.4 Linearity ...... 50 2.9.1.5 Accuracy and precision ...... 51 2.9.1.6 Limit of detection and limit of quantification ...... 51 2.9.1.7 Range ...... 51 2.9.1.8 Specificity ...... 51 2.9.2 Active-excipient compatibility studies ...... 53 2.10 Determining formulation stability study ...... 54 3. METHODOLOGY ...... 57 3.1 HPLC method validation ...... 57 3.1.1 Equipment ...... 57 3.1.2 Materials and reagents ...... 57 3.1.3 Mobile phase preparation and standard curve construction ...... 57 3.1.4 Chromatographic conditions ...... 58 3.1.5 Linearity ...... 58 3.1.6 Accuracy and precision ...... 59 3.1.7 Limit of detection and limit of quantification ...... 59 3.1.8 Range and system suitability ...... 60 3.1.9 Specificity ...... 60 3.2 Determination of active–excipient compatibility ...... 62 3.3 Manufacture of products ...... 62

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3.3.1 Materials ...... 63 3.3.2 Product manufacture ...... 63 3.3.2.1 Sterilization for heat sensitive API and exipients ...... 64 3.3.3 Manufacturing methods for products I–V ...... 64 3.4 Stability Tests ...... 69 3.5 Qualitative and quantitative analysis of the formulations ...... 70 3.5.1 Appearance and pH ...... 70 3.5.2 Phenyleprine hydrochloride concentration ...... 70 3.6 Test for preservative efficacy ...... 70 3.6.1 Procedure for standard plate count ...... 71 3.6.2 Procedure for plating the bacteria and fungi ...... 71 3.6.3 Standardization of cultures using turbidimetry method ...... 72 3.6.4 Preservative efficacy ...... 73 3.7 Determination of viscosity ...... 73 3.8 Statistical analysis ...... 74 4. RESULTS AND DISCUSSION ...... 75 4.1 Validation of the stability indicating assay ...... 75 4.1.1 Linearity ...... 75 4.1.2 Accuracy ...... 76 4.1.3 Precision ...... 76 4.1.4 Limit of detection (LOD) and quantification (LOQ) ...... 77 4.1.5 Specificity and system suitability ...... 77 4.2 Active and excipient study ...... 113 4.3 Stability study ...... 123 4.4 Determination of yield point and viscosity of products ...... 137 4.5 Effectiveness of the ophthalmic solution preservatives ...... 144 5. CONCLUSION AND RECOMMENDATIONS ...... 147 REFERENCES ...... 151 APPENDIX A ...... 176 CONCEPT ARTICLE ...... 176 APPENDIX B ...... 192 LIST OF EQUIPMENT ...... 192 APPENDIX C ...... 193 LIST OF SOLUTIONS ...... 193

LIST OF ABBREVIATIONS

API Active Pharmaceutical Ingredient

AUC Area under Curve

Å angstrom

ANOVA Analysis of variance atm atmospheric pressure

BP British Pharmacopeia

CPR Cardio Pulmonary Resuscitation

COMT catechol–O–methyltransferases

R2 Correlation coefficient cAMP cyclic Adenosine Monophosphate

CYP Cytochrome P 450

Da Dalton

EDTA Ethylenediaminetetraacetic acid

ET Eustachian tube

FPLC Fast Protein Liquid Chromatography

FDA Food and Drug Administration

> Greater than

HEPA High Efficiency Particulate Air

HPLC High Performance (pressure) Liquid Chromatography

HIV Human Immunodeficiency Virus

HPMC Hydroxypropyl methylcellulose

ICH International Conference on Harmonization kg kilogram

< Less than log logarithmic m/v Mass per volume

MCC Medicines Control Council

MAO Monoamine oxidases

MIC Minimum Inhibitory Concentration

µg microgram

µL microliter

µm micrometer mg milligram

mm millimeter min minute

M Molar concentration (moles of solute per liter of solution)

N Normal concentration (gram-equivalents of solute per liter of solution)

OTC Over–The–Counter

Pa·s Pascals per second

PAC Perennial Allergic Conjunctivitis

% Percentage psi Pounds per square inch

RH Relative humidity

RSD Relative Standard Deviation

~ roughly similar

SAC Seasonal Allergic Conjunctivitis s seconds

SA South Africa

SD Standard Deviation

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Tf Tailing factor

T Temperature

TPN Total Parenteral Nutrition

UV Ultraviolet

USP United States Pharmacopeia

UK United Kingdom

VMA Viscosity Modifying Agent

λ Wavelength

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LIST OF FIGURES

Figure 1: Anatomy of the eye (Del Amo & Urtti, 2008)...... 4 Figure 2: Structure of Phenylephrine, its base and salts (Trommer et al., 2010). .... 15 Figure 3: Diagram of Ocular Absorption (Nanjawade et al., 2007)...... 17 Figure 4: Diagram of a typical HPLC-UV absorbance peak and plots of noise (or threshold) and purity angles (Krull & Swartz, 2001)...... 52 Figure 5: Laboratory scale 1000 ml manufacturing process of product I ...... 65 Figure 6: Laboratory scale 1000 ml manufacturing process of product II ...... 66 Figure 7: Laboratory scale 1000 ml manufacturing process of product III ...... 67 Figure 8: Laboratory scale 1000 ml manufacturing process of product IV ...... 68 Figure 9: Laboratory scale 1000 ml manufacturing process of product V ...... 69 Figure 10: Graph showing a mean peak area versus concentration of replicate samples of phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1x + 438.55, R2 = 0.9999...... 75 Figure 11: HPLC Chromatogram for mobile phase alone...... 78 Figure 12: HPLC Chromatogram for phenylephrine hydrochloride dissolved in mobile phase with a retention time of 7.80 minutes...... 79 Figure 13: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999...... 79 Figure 14: HPLC Chromatogram for product I dissolved in mobile phase with a retention time of 7.87 minutes...... 79 Figure 15: Peak purity profile calculated using PDA data (from 190–800 nm) for Product I prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999054 ...... 80 Figure 16: HPLC Chromatogram for product II dissolved in mobile phase with a retention time of 7.82 minutes...... 80 Figure 17: Peak purity profile calculated using PDA data (from 190–800 nm) for Product II prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.996482...... 80

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Figure 18: HPLC Chromatogram for product III dissolved in mobile phase with a retention time of 7.83 minutes...... 81 Figure 19: Peak purity profile calculated using PDA data (from 190–800 nm) for Product III prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.998178...... 81 Figure 20: HPLC Chromatogram for product IV dissolved in mobile phase with a retention time of 7.89 minutes...... 81 Figure 21: Peak purity profile calculated using PDA data (from 190–800 nm) for Product IV prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.996947...... 82 Figure 22: HPLC Chromatogram for product V dissolved in mobile phase with a retention time of 7.84 minutes...... 82 Figure 23: Peak purity profile calculated using PDA data (from 190–800 nm) for product V prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999587...... 82 Figure 24: HPLC Chromatogram for phenylephrine hydrochloride stressed under UV light dissolved in mobile phase with a retention time of 7.81 minutes...... 83 Figure 25: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999...... 83 Figure 26: HPLC Chromatogram for product I stressed under UV light dissolved in mobile phase with a retention time of 7.86 minutes...... 84 Figure 27: Peak purity profile calculated using PDA data (from 190–800 nm) for product I prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999054...... 84 Figure 28: HPLC Chromatogram for product II stressed under UV light dissolved in mobile phase with a retention time of 7.82 minutes...... 84 Figure 29: Peak purity profile calculated using PDA data (from 190–800 nm) for product II in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999116...... 85 Figure 30: HPLC Chromatogram for product III stressed under UV light dissolved in mobile phase with a retention time of 7.85 minutes...... 85

Figure 31: Peak purity profile calculated using PDA data (from 190–800 nm) for product III in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116...... 85 Figure 32: HPLC Chromatogram for product IV stressed under UV light dissolved in mobile phase with a retention time of 7.81 minutes...... 86 Figure 33: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.997916...... 86 Figure 34: HPLC chromatogram for product V stressed under UV light dissolved in mobile phase with a retention time of 7.87 minutes...... 86 Figure 35: Peak purity profile calculated using PDA data (from 190–800 nm) for product V in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.997475...... 87 Figure 36: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.86 minutes...... 88 Figure 37: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999574...... 88 Figure 38: HPLC chromatogram of Product I stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.82 minutes...... 88 Figure 39: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116. ... 89 Figure 40: HPLC chromatogram for product II stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.83 minutes...... 89 Figure 41: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.996296. ... 89 Figure 42: HPLC chromatogram for product III stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.91 minutes...... 90 Figure 43: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.995179. .... 90

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Figure 44: HPLC chromatogram for product IV stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.86 minutes...... 90 Figure 45: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.997097. .... 91 Figure 46: HPLC chromatogram of product V stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.80 minutes...... 91 Figure 47: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.999578. .... 91 Figure 48: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.82 minutes ...... 92 Figure 49: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.996847...... 92 Figure 50: HPLC chromatogram of product I stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.89 minutes...... 93 Figure 51: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.995815. .... 93 Figure 52: HPLC chromatogram of product II stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.9 minutes...... 93 Figure 53: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996370. ... 94 Figure 54: HPLC chromatogram for product III stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.79 minutes...... 94 Figure 55: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998771. ... 94 Figure 56: HPLC chromatogram of product IV stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.91 minutes...... 95

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Figure 57: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996930...... 95 Figure 58: HPLC chromatogram for product V stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.85 minutes...... 95 Figure 59: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999587. ... 96 Figure 60: HPLC chromatogram for phenylephrine hydrochloride stressed with 0.2

M H2O2 dissolved in mobile phase with a retention time of 7.82 minutes...... 96 Figure 61: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999987...... 97

Figure 62: HPLC chromatogram for product I stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.81 minutes...... 97 Figure 63: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999522. ... 97

Figure 64: HPLC chromatogram for product II stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.9 minutes...... 98 Figure 65: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999722. ... 98

Figure 66: HPLC chromatogram of product III stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time 7.94 minutes...... 98 Figure 67: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999706. ... 99

Figure 68: HPLC chromatogram for product IV with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.81 minutes...... 99

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Figure 69: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896. ... 99

Figure 70: HPLC chromatogram for product V stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.88 minutes...... 100 Figure 71: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999536. . 100 Figure 72: HPLC chromatogram for phenylephrine hydrochloride stored at 100 °C for 24 hours dissolved in mobile phase with a retention time of 7.8 minutes...... 102 Figure 73: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stored at 100 °C for 24 hours in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999...... 102 Figure 74: HPLC chromatogram for phenylephrine hydrochloride stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.83 minutes...... 103 Figure 75: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999989...... 103 Figure 76: HPLC chromatogram for product I stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.92 minutes...... 103 Figure 77: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.999956. . 104 Figure 78: HPLC chromatogram for product II stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.84 minutes...... 104 Figure 79: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056. . 104 Figure 80: HPLC chromatogram for Product III stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.89 minutes...... 105 Figure 81: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at 65 °C for 1 month in mobile phase. Peak shown in pink and

xix purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999720...... 105 Figure 82: HPLC chromatogram for Product IV stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.87 minutes...... 105 Figure 83: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.999803...... 106 Figure 84: HPLC chromatogram for Product V stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.90 minutes...... 106 Figure 85: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999752...... 106 Figure 86: HPLC chromatogram for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.86 minutes...... 107 Figure 87: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056...... 107 Figure 88: HPLC chromatogram for Product I stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.87 minutes...... 107 Figure 89: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at 40 °C/75% RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.995566...... 108 Figure 90: HPLC chromatogram for Product II stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.84 minutes...... 108 Figure 91: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999579...... 108

Figure 92: HPLC chromatogram for product III stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.81 minutes...... 109 Figure 93: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.994609...... 109 Figure 94: HPLC chromatogram for Product IV stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.85 minutes...... 109 Figure 95: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.994609...... 110 Figure 96: HPLC chromatogram for Product V stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.83 minutes...... 110 Figure 97: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.995332...... 110 Figure 98: HPLC chromatogram for phenylephrine hydrochloride alone dissolved in mobile phase with retention of 7.82 minutes...... 115 Figure 99: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999...... 115 Figure 100: HPLC chromatogram for phenylephrine hydrochloride with sodium citrate dihydrate (1:1) dissolved in mobile phase with a retention time of 7.92 minutes...... 115 Figure 101: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with sodium citrate dihydrate (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896...... 116 Figure 102: HPLC chromatogram for phenylephrine hydrochloride with carboxymethycellulose sodium (1:1) dissolved in mobile phase with a retention time of 7.82 minutes...... 116

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Figure 103: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with carboxymethycellulose sodium (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999...... 116 Figure 104: HPLC chromatogram for phenylephrine hydrochloride with hypromellose (1:1) dissolved in mobile phase with a retention time of 7.81 minutes...... 117 Figure 105: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride with hypromellose (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999...... 117 Figure 106: HPLC chromatogram for phenylephrine hydrochloride with glycerol (1:1) dissolved in mobile phase with a retention time of 7.85 minutes...... 117 Figure 107: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with glycerol (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999989...... 118 Figure 108: HPLC chromatogram for phenylephrine hydrochloride with benzalkonium chloride (1:1) dissolved in mobile phase with a retention time of 7.88 minutes...... 118 Figure 109: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride with benzalkonium chloride (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999918...... 118 Figure 110: HPLC chromatogram for phenylephrine hydrochloride with EDTA (1:1) dissolved in mobile phase with a retention time of 7.86 minutes...... 119 Figure 111: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with EDTA (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999991...... 119 Figure 112: HPLC chromatogram for phenylephrine hydrochloride with boric acid (1:1) dissolved in mobile phase with a retention time of 7.92 minutes...... 119 Figure 113: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with boric acid (1:1) in mobile phase. Peak shown in

xxii pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999...... 120 Figure 114: HPLC chromatogram for phenylephrine hydrochloride with sodium metabisulfite (1:1) dissolved in mobile phase with a retention time of 7.82 minutes...... 120 Figure 115: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with sodium metabisulfite (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999870...... 120 Figure 116: HPLC chromatogram for phenylephrine hydrochloride with disodium edetate (1:1) dissolved in mobile phase with a retention time of 7.82 minutes...... 121 Figure 117: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with disodium edetate (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998891...... 121 Figure 118: HPLC chromatogram for phenylephrine hydrochloride with propyl paraben (1:1) dissolved in mobile phase with a retention time of 7.83 minutes. .... 121 Figure 119: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with propyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999948...... 122 Figure 120: HPLC chromatogram for phenylephrine hydrochloride with methyl paraben (1:1) dissolved in mobile phase with a retention time of 7.82 minutes. .... 122 Figure 121: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with methyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999982...... 122 Figure 122: HPLC chromatogram for product I stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes...... 123 Figure 123: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999950...... 124

xxiii

Figure 124: HPLC chromatogram for product II stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.85 minutes...... 124 Figure 125: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.996473...... 124 Figure 126: HPLC chromatogram for product III stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes...... 125 Figure 127: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996955...... 125 Figure 128: HPLC chromatogram for product IV stored at 30 °C/65% RH for 3 months dissolved in mobile phase with a retention time of 7.88 minutes...... 125 Figure 129: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.998694...... 126 Figure 130: HPLC chromatogram for product V stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes...... 126 Figure 131: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.994533...... 126 Figure 132: HPLC chromatogram for product I stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.91 minutes...... 127 Figure 133: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.995873...... 127 Figure 134: HPLC chromatogram for product II stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.89 minutes...... 128 Figure 135: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink

xxiv and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.999950...... 128 Figure 136: HPLC chromatogram for product III stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.92 minutes...... 128 Figure 137: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998742...... 129 Figure 138: HPLC chromatogram for product IV stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.92 minutes...... 129 Figure 139: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996396...... 129 Figure 140: HPLC chromatogram for product V stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes...... 130 Figure 141: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093...... 130 Figure 142: HPLC chromatogram for product I stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes...... 131 Figure 143: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product I stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093...... 131 Figure 144: HPLC chromatogram for product II stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes...... 131 Figure 145: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product II stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093...... 132 Figure 146: HPLC chromatogram for product III stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes...... 132

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Figure 147: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093...... 132 Figure 148: HPLC chromatogram for product IV stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes...... 133 Figure 149: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093...... 133 Figure 150: HPLC chromatogram for product V stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.91 minutes...... 133 Figure 151: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998093...... 134 Figure 152: A graph showing standard error and phenylephrine hydrochloride left in product I–V after 12 weeks at 30 °C/65%RH, 40 °C/75%RH, 25 °C/60%RH...... 134 Figure 153: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 30 °C/65% RH...... 138 Figure 154: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 30 °C/65% RH...... 138 Figure 155: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 40 °C/75% RH...... 139 Figure 156: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 40 °C/75% RH...... 139 Figure 157: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 25 °C/60%RH...... 140 Figure 158: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 25 °C/60% RH...... 140 Figure 159: Graph showing viscosity of products I–V stored in a stability chamber of

40 °C/75%RH tested at time zero (T0), three months later (T1) and compared to an original marketed product Prefrin®...... 141

xxvi

Figure 160: Graph showing viscosity of products I–V stored in a stability chamber of

25 °C/60%RH tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®...... 141 Figure 161: Graph showing viscosity of products I–V stored in a stability chamber of

30 °C/65%RH tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®...... 142

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LIST OF TABLES

Table 1: Conventional dosage forms and usage (Lang, 1995)...... 24 Table 2: Typical minimum inhibitor concentrations of benzalkonium chloride (Kibbe, 2006)...... 39 Table 3: Minimum inhibitory concentration for propylparaben in aqueous solution (Rieger, 2006b) ...... 40 Table 4: Minimum inhibitory concentrations of methylparaben in aqueous solution (Rieger, 2006a)...... 41 Table 5: Formulation summary of active pharmaceutical ingredient and excipients used in the manufacturing of products I–V ...... 64 Table 6: Criteria for tested microorganisms (USP, 2004) ...... 73 Table 7: Accuracy data for quantification of phenylephrine hydrochloride ...... 76 Table 8: Precision data for quantification of phenylephrine hydrochloride ...... 76 Table 9: Physical appearance of phenylephrine hydrochloride and products I–V before and after storage conditions 40 °C/75%RH for 1 month ...... 101 Table 10: Results showing absence of impurity from a series of stressed and unstressed samples of phenylephrine hydrochloride (API) and products...... 111 Table 11: Results showing phenylephrine hydrochloride left with samples stressed and unstressed (API and Products) ...... 112 Table 12: Assay result showing phenylephrine hydrochloride and excipients in a 1:1 ratio after storage conditions 40 °C/75%RH for 1 month ...... 113 Table 13: Physical appearance of active–excipients samples before and after storage conditions 40 °C/75%RH for 4 weeks ...... 114 Table 14: Results of one-way ANOVA analysis for products I–V stored at 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months...... 135 Table 15: Changes in pH of phenylephrine hydrochloride and products I–V before and after storage conditions 40 °C/75%RH for 1 month...... 136 Table 16: Changes in pH for products I–V before and after varying storage conditions for 3 months ...... 136 Table 17: Results of one-way ANOVA analysis for similarities in pH of products II 25 °C/60%RH, 40 °C/75%RH and IV 25 °C/60%RH after 3 months...... 136

xxviii

Table 18: Physical appearance of products I–V before and after varying storage conditions for 3 months...... 137 Table 19: Results of one-way ANOVA analysis for viscosity of products I–V stored at 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. The values shown indicate differences in p-values and significance in differences of mean was defined as p < 0.05...... 143 Table 20: Antimicrobial preservative efficacy of the eye-drop products I–V challenged with E. coli, S.aureus, P. aeruginosa, C.albicans...... 145

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1. INTRODUCTION

1.1 Background and motivation

Phenylephrine is a sympathomimetic amine drug that undergoes extensive first pass metabolism resulting in a bioavailability of approximately 38% or lower (Trommer et al., 2010). Phenylephrine hydrochloride ((R)-1-(3-hydroxyphenyl)-2-methyl- aminoethanol hydrochloride) is an effective adrenergic agent and β–receptor sympathomimetic drug that is chemically related to epinephrine (Ahmed and Amin, 2007) and used in its optically active form (Pandey et al., 2003; Pandey et al., 2006).

As an α1-adrenergic receptor agonist it is used primarily as a decongestant, for uveitis and as an agent to dilate the pupil (Lang, 1995).

Instilling pupil-dilating agents like phenylephrine hydrochloride allows for maximum dilation of the pupil during ophthalmic examinations as well as during many ocular surgical procedures (Hanyu et al., 2007). For the period of an ophthalmoscopic examination, a perfectly dilated pupil should be large and stable to the intensive light stimulation. A high frequency of drug instillation is needed to produce a satisfactory response due to the rapid clearance of phenylephrine hydrochloride from the ophthalmic surface by the lachrimal system (Zoukhri, 2006). Thus a patient may receive up to 30 drops of phenylephrine hydrochloride during an ophthalmic procedure to maintain an optimal pupil size (Hanyu et al., 2007). Differences in the range of drops received are due to patient’s interdependent variability as not all patients are the same. The doses of phenylephrine hydrochloride administered ocularly could precipitate unwanted side effects, posing a problem for both physician and patient.

The conjunctival sac holds a limited capacity of fluids which poses another problem, as most of the eye drop solution is drained into the nasal cavity thereby reducing the portion of drug that reaches its site of action. Systemic absorption of phenylephrine hydrochloride into the nasal mucosa may produce unwanted side effects such as headache, hypertension and tachycardia (Bartlett & Jaanus, 2008). Moreover, phenylephrine hydrochloride solutions may irritate the eye due to the presence of

1 preservatives such as benzalkonium chloride and methyl paraben amongst others (Giaconi et al., 2009).

The contact time of eye drops is considered as being the most important factor in ophthalmic drug delivery (Agarwal et al., 2002). This research study will focus on formulating an eye drop solution which improves contact time. Various formulations of phenylephrine hydrochloride eye drops have been made in the quest to improve contact time. These include viscous solutions (Saettone et al., 1984), rods (Alani, 1978), gels (Durrani et al., 1996), ointments (Saettone et al., 1980; Gurjar et al., 1998), and a polyvinyl alcohol flag (O’Donnell & Gillibrand, 1995; Maitani et al., 1997).

Phenylephrine hydrochloride have been formulated in varying dosage forms using potassium, sodium and lysine salts or mixed with viscosity modifiers such as HPMC and SCMC to improve its effectiveness. Ocular penetration and retention of phenylephrine hydrochloride demands an ophthalmic solution of acidic pH which could precipitate the drug or increase the ocular irritation potential and viscosity modifiers could solve both problems. Benzalkonium chloride, a cationic preservative, used in eye drops causes eye irritation, caution is needed in its use and concentration. Thus a non irratiting ophthalmic solution of phenylephrine hydrochloride can be formulated by dissolving an eye-friendly water soluble salt, a viscosity modifyier, an antioxidant in purified water and stability in mind with benzalkonium chloride.

However, there is still scope for an eye drop that can be administered easily in less frequent doses which produces consistent, rapid results and minimizes the risk of adverse effects to the patient.

1.2 Aim and objectives

The primary aim of the study was to develop a pharmaceutically stable phenylephrine hydrochloride eye drop. The following objectives were accordingly identified:

 Validate a high performance liquid chromatographic (HPLC) method for the quantitative determination of phenylephrine hydrochloride in the finished product.  Propose formulations of phenylephrine hydrochloride eye drops and manufacture laboratory scale batches of these.  Characterize the physicochemical properties of the formulations by assessing appearance, rheology, pH and degradation.  Conduct real time and accelerated stability studies on the formulations; in accordance with the International Conference for Harmonization (ICH) and Medicines Control Council guidelines.  Determine the efficacy of antimicrobial preservation.

1.3 Plan of work

In order to achieve the above objectives, a well laid out plan was to be followed. Literature reviews, on the theory relating to phenylephrine and its prodrugs were undertaken in an effort to understand the API, eye drops, solutions and product formulation. Active–excipients compatibility studies were conducted using HPLC and various eye drops were formulated and manufactured on a laboratory scale. These underwent stability studies in accordance with International Conference on Harmonization and Medicine Control Council guidelines. Rheological tests and efficacy of antimicrobial preservation were demonstrated.

2. LITERATURE REVIEW

2.1 Anatomy and physiology of the eye

The human eye provides a challenge to formulators who seek to produce dosage forms where the API is administered ocularly. This is due to (a) the permeability of the cornea and (b) the protective operation of the eyelids and lacrimal system. The operation of the eyelids and lacrimal system rapidly removes materials instilled into the eye; however, this clearance does not apply to materials that are small in volume and which are chemically and physiologically compatible with surface tissues (Hughes, 2004).

The eyes are highly specialized organs of photoreception and are protected by eyelids and the orbit in which they are placed (Rathore & Nema, 2009). The eye can be divided into two segments, namely the anterior and posterior segments. The anterior segment comprises of the cornea, iris, the ciliary body, the anterior chamber and the posterior chamber while the posterior segment comprises of retina and the vitreous body as seen in Figure 1 (Ghosh & Jasti, 2005).

Figure 1: Anatomy of the eye (Del Amo & Urtti, 2008).

The unique anatomy, physiology and biochemistry of the eye make it resistant to foreign substances (Del Amo & Urtti, 2008) this protective mechanism poses a

4 challenge to the formulator who is required to bypass the barriers without causing damage to the eye (Meqi & Deshpande, 2002). The corneal barrier poses physiological constraints due to its poor permeability which reduces the absorption of ophthalmic drugs. The cornea is made up of three membranes; the epithelium, the endothelium and inner stroma (Chien et al., 1990). The epithelium has tight junctions that serve as a selective barrier to ion transport, thereby limiting the diffusion of macromolecules via the paracellular route. The stroma is a highly lipophilic layer that lies beneath the epithelium, and the more lipophilic a drug is, the less resistance it will have crossing the stroma (Patel et al., 2010).

The eyelids, conjunctiva, lacrimal systems, cornea–precorneal film and its absorption are discussed below as they play a major role in the absorption metabolism of eye drops.

Eyelids: The eyelids have two functions: mechanical protection of the globe (eye) and creation of an optimum environment for the cornea. The eyelids are lubricated and kept moist by secretions of the lacrimal glands and specialized cells found in the bulbar conjunctiva. The antechamber is shaped in a narrow cleft manner directly over the front of the eyeball, with pocket-like extensions upward and downward. The pockets are called the superior and inferior fornices (vaults), and the entire space is called the cul-de-sac. The oval opening between the eyelids is called the palpebral fissure (Zide, 2006).

Conjunctiva: The conjunctiva is defined as a thin, vascularised mucus membrane that lines the inner surface of the eyelids and covers the anterior part of the sclera up to the cornea (Kaur et al., 2003). Its loose attachment permits free movement of the eyeball. Except for the cornea the conjunctiva is the most exposed portion of the eye (Hughes, 2004). Uptake of drugs applied topically is greater in the conjunctiva than in the cornea because the conjunctiva is porous, has a rich blood flow and a large surface area (Araújo et al., 2009).

Lacrimal system: The conjunctival and lacrimal glands secrete a film of fluid which covers and lubricates the conjunctival and corneal surfaces. The lacrimal glands produce tears which are delivered through a number of fine ducts into the

5 conjunctival fornix. A tear is a clear, watery fluid containing salts, glucose, other organic compounds, approximately 0.7% protein, and the enzyme lysozyme. Small accessory lacrimal glands are situated in the conjunctival fornices. Their secretion provides lubrication and cleansing during ordinary conditions and also maintains a thin fluid film covering the cornea and conjunctiva (the precorneal film). The stability of the film is maintained through the mucin–protein layer. The sebaceous glands of the eyelids secrete an oily fluid that prevents overflowing of tears at the lid margin and reduces evaporation from the exposed surfaces of the eye by spreading over the tear film (Hughes, 2004).

Blinking helps in replenishing the fluid film by pushing a thin layer of fluid ahead of the lid margins as they come together. The excess fluid is directed into the lacrimal lake which is a small, triangular area lying in the angle bound by the innermost portions of the lids. The skin of the eyelids is the thinnest in the body and folds easily which permits rapid opening and closing of the palpebral fissures. The eyelids provide controlled movement such as narrowing of the palpebral fissures in a zipper- like action from the lateral canthus toward the medial canthus. The transport or movement of fluid toward the lacrimal lake is aided by the eyelids (Del Amo & Urtti, 2008).

Tears are drained from the lacrimal lake by two small tubes–the lacrimal canaliculi– which go into the upper part of the nasolacrimal duct, called the lacrimal sac. The drainage of tears into the nose does not depend only on gravity. Fluid moves along the lacrimal canaliculi by capillary attraction supported by aspiration caused by contraction of muscle found in the eyelids. The blinking action causes contraction of the muscles inducing dilation of the upper part of the lacrimal sac and compression of its lower portion (Cohen et al., 2006). Tears are aspirated into the sac, which is collected in its lower part by forcing down the tears through the nasolacrimal duct toward its opening into the nose. As the muscle relaxes, the lids open. Owing to muscle relaxation, the upper part of the sac forces fluid into the lower part where it is simultaneously released from compression. The act of blinking therefore exerts a suction force-pump action in removing tears from the lacrimal lake as well as emptying them into the nasal cavity (Cohen et al., 2006). Lacrimation is induced by reflex action through the stimulation of nerve endings of the cornea or conjunctiva.

The reflex could be abolished by anaesthetization of the surface of the eye and by disorders affecting its nerve components (Cohen et al., 2006).

The cul-de-sac is free of pathogenic organisms and is sterile. The sterility is due to the action of lysozyme, in the tears, which destroys saprophytic organisms but has little action against pathogens. Certain diseases cause the lacrimal gland, to undergo involution, resulting in scanty lacrimal fluid production and a change in the conjunctival glands also leads to changes in the amount of the secretions. This may lead to symptoms of dryness, burning and general discomfort and may interfere with visual acuity (Kaur & Kanwar, 2002).

Precorneal film: The precorneal film is composed of a thin outer lipid layer, a thicker middle aqueous layer, and a thin inner mucoid layer. It is renewed during each blink, and when blinking is suppressed, either by drugs or by mechanical means, it dries in patches. The precorneal film is unaffected by the addition of concentrations of up to 2% sodium chloride into the conjunctiva. The precorneal film, which is part of the tear fluid, maintains the cornea’s moist surfaces and its functionality depends on the condition of the corneal epithelium. A pH below four or above nine causes derangement of the film (Grosvenor, 2007).

Cornea: The cornea has a thickness of 0.5 to 1 mm and consists mainly of the following structures (from the front backward): Corneal epithelium, subtantia propia (stroma), corneal endothelium. The cornea is transparent to visible light due to the special laminar arrangement of the cells and fibres and because of the absence of blood vessels (Chien & Schoenwald, 1990). Cloudiness of the cornea may be due to several factors including excess pressure in the eyeball as in glaucoma, and scar tissue due to injury, infection, deficiency of oxygen or excess hydration such as may occur during the wearing of improperly–fitted contact lenses. Trauma to the cornea usually heals as an opaque patch that can permanently impair vision unless it is located in the periphery of the cornea (Kaur et al., 2003).

The outer surface of the cornea is most responsible for the refraction of light whereby the index of refraction changes from that of air (1.00) to that of precorneal substance (1.38). Alteration in its shape or transparency interferes with the formation

7 of a clear image; therefore any pathological process, however little, may seriously interfere with the resolving power or visual acuity of the eye (Lang et al., 2005).

The normal cornea possesses no blood vessels except at the corneoscleral junction. The cornea derives its nutrition from the aqueous humour by diffusion and has certain permeable characteristics. It also receives nourishment from the fluid circulating through the chambers of the eye and from the air. The corneal epithelium provides an efficient barrier against bacterial invasions due to its poor blood supply. Foreign bodies that either scratch the cornea or lodge or become embedded in the cornea are of serious concern, as the presence of any of these could play a role in permitting pathogenic bacteria to gain access to the cornea. Trauma plays an important part in most of the infectious diseases of the cornea that occurs exogenously (Ghosh & Jasti, 2005).

Corneal absorption: The cornea is a membrane having both hydrophilic and lipophilic layers. It is composed of three general layers: the lipid–rich epithelium, the lipid-poor stroma, and the lipid rich endothelium. The combined lipid content of the corneal epithelium and the corneal endothelium approximately 100 times more than the lipid content of the corneal stroma. Modern ocular drug delivery system designs are based on an understanding of drug deposition pathways within the relative lipid content of these layers and their overall ocular pharmacokinetic/pharmacodynamic profile (Curtin & Cormican, 2003). Only drugs having both lipid and hydrophilic properties have the greatest corneal penetration. Highly water–soluble drugs penetrate less readily. For example, highly water–soluble steroid phosphate esters penetrate the cornea poorly and for better penetration to be attained a poorly soluble but more lipophilic steroid alcohol is used (Holladay, 2006).

Eye drops penetrate the eye primarily through the cornea. Typical drug penetraton occurs as follows:

a) The drug molecule as a free base and the salt will be in an equilibrium depending on the drugs physicochemical characteristics and the pH.

b) If the formulation is slightly acidic at the moment of instillation, the acidity is neutralised by the lacrimal fluid which converts it rapidly to the physiological pH range ( pH 7.4). c) There will be sufficient free base to begin penetration of the corneal epithelium at this pH. An undissociated drug molecule will penetrate the stroma, epithelium, and endothelium because it is water–soluble. d) The dissociated drug leaves the endothelium for the aqueous humour, as it can readily diffuse to the iris, the ciliary body, and the site of the pharmacological actions (Nanjawade et al., 2007).

Martini and colleagues (1997) discussed the implications of the mechanism of precorneal drug loss in the design of ocular drug-delivery systems, including the effect of instilled drug volume on aqueous humour concentrations and the amount of drug available for systemic absorption. Ideally, smaller volumes permit more drugs to be absorbed. For a given instilled concentration the opposite is true; however, a smaller volume instilled remains more efficient (Friedrich et al., 1993).

There are two types of corneal penetrations: transcorneal and non-corneal absorption (Nanjawade et al., 2007). Lang and colleagues (2005) stated that the transcorneal route of absorption of a drug into the eye is the route most effective in bringing a given drug to the anterior portion of the eye. This is enhanced by the water-lipid gradient found in the cornea.

Drugs penetrate the corneal epithelium with the use of transcellular or paracellular pathways which are both types of transcorneal penetration. Transcellular and paracellular pathways are used by both lipophilic and hydrophilic drugs; these pathways involve passive diffusion or modified diffusion through intercellular spaces and most topically–applied drugs diffuse passively along their concentration gradient. However, the stereospecific carrier–mediated transport system is used by certain drugs as their mode of transport such as l-lysine which makes use of the Na+ –K+ – ATPase pump as medium of transport in the cornea (Urtti, 2006). Physicochemical properties of drugs have properties such as lipophilicity, solubility, molecular size and shape, charge and degree of ionization affects the route and rate of permeation in the cornea (Järvinen et al., 1995). The hydrophilic drugs are rate–limited by the

9 lipophilic corneal epithelium, while lipophilic drugs partitioning from the epithelium to the hydrophilic stroma are rate limiting (Järvinen et al., 1995).

Non-corneal absorption pathways involve the penetration of drug across the bulbar conjunctiva and sclera into the uveal tract and vitreous humour. The conjunctiva and sclera provide a route for large hydrophilic molecules for example inulin and p- aminoclonidine (Nanjawade et al., 2007; Lang et al., 2005).

The cornea can be penetrated by small ions through extracellular spaces to a measurable degree. The diameter of the largest particles that can pass across the cellular layers is in the range of 10 to 25 Å. Permeation of instilled drugs can also be reduced by protein binding in the tear fluid and metabolic degradation by enzymes. There are numerous drug metabolizing enzymes present in the cornea like esterases, peptidases, reductases and cytochrome P (CYP) family enzymes among others (Gaynes & Fiscella, 1996; Mashkevisch, 2007). Enzymes help in drug detoxification and metabolism. These enzymes also maintain ocular homeostasis by preventing environmental and systemic injuries (Mashkevisch, 2007). Martini and colleagues (1997) concluded that the following are responsible for precorneal drug loss in a descending order: drainage vasodilation > nonconjunctival loss > induced lacrimation conjunctival absorption > normal tear turnover.

2.2 Pathophysiology of the eye

Ocular manifestation of allergy in which the body produces hypersensitivity to normally harmless substances (allergens) is classified to be an allergic eye disease. Allergy prevalence in Europe is between 15 and 20%; by 2015 the rates are estimated to increase to 50% which is about 20% more (Bilkhu et al., 2011). Improved hygiene practices and increased antibiotic use by modern day lifestyle in addition to environmental factors such as increased air pollution, climate change and increased planting and importation of allergenic plant species have been reported to increase the allergy prevalence, although genetics plays an important role in susceptibility (Bilkhu et al., 2011).

Seasonal allergic conjunctivitis (SAC) is the most common form of allergic eye disease that constitutes of 90% of the allergy cases with perennial allergic conjunctivitis at 5% (Bielory, 2008). SAC is most frequently caused by grass, tree and weed pollens and outdoor moulds which peak at different times of the year (Bielory, 2008; Chigbu, 2009), often as part of seasonal rhinoconjunctivitis (hay fever). However, PAC occurs year round due to house dust mites, animal dander, insects and indoor moulds (Chigbu, 2009). The signs and symptoms of SAC develop gradually but sometimes it can develop suddenly in contact with an offending allergen (Bielory, 2000 & Bielory 2008). Some of the signs and symptoms of SAC includes itching, tearing, eyelid oedema and conjunctival hyperaemia, chemosis and papillary reaction in which the severity often varies with pollen counts (Cox, 2007). PAC and SAC have similar signs and symptoms but differ as PAC is milder and chronic in nature and may have seasonal exacerbations (Bielory, 2008 & Buckley, 1998). The impact of allergic eye disease on the quality of life can be profound even though the signs of symptoms of SAC and PAC are relatively mild (Bielory, 2006). It affects daily activities, productivity at work, school performance and the economy (Pitt et al., 2004; Smith et al., 2005; Palmares et al., 2010). The severities of presentation limits the complications of SAC and are linked to steroid use in cases refractory to conventional treatment (Hingorani and Lightman, 1995; Joss & Craig, 1998; Bielory et al., 2010). The treatments provided for SAC are mainly for preventing and alleviating symptoms rather than cure due to the fact that no cure has been found or produced for SAC or any allergy. However, immunotherapy shows promise (Bielory, 2008).

The pathophysiological mechanism of acute ocular allergy involves acute antibody (immunoglobulin E (IgE))-mediated mast cell degranulation and minimal presence of migratory inflammatory cells. In chronic ocular allergy, the pathophysiology consists of persistent activation of mast cells and eosinoiphil–T–lymphocyte-mediated delayed-type hypersensitivity (DTH) response. Antigen-specific antibodies, such as

IgG and IgE contribute to the pathogenesis of Th2-mediated diseases including allergies. IgE and IgG receptors are expressed on mast cells and as such IgE and IgG may participate in mast cell activation and mediator release. Antibody (IgE or IgG, particularly of the IgG1 isotype–mediated–mast cell activation results in degranulation, with the release of pre-formed and newly formed pro-inflammatory

11 mediators as well as the secretion of chemokines and cytokines (Tkaczyk et al., 2002).

Both SAC and PAC are IgE-mast cell mediated hypersensitivity reactions, divided into two phases with the mast cell playing a central role (Bacon et al., 2000; Choi & Bielory 2008). The reaction involves a very complex series of immunological events coordinated by various mediators initiated by an allergen (Ono & Abelson, 2005 ; Hodges & Kean–Myers, 2007) An allergen such as pollen reacts with specific IgE antibodies bound to a sensitised mast cell, triggering cross linkage of the IgE molecules and an influx of calcium ions into the mast cell. This causes the mast cell to degranulate and release preformed inflammatory mediators such as histamine which cause the signs and symptoms associated with the early phase response in sensitised individuals. The early phase response is immediate and lasts clinically for 20–30 minutes (Leonardi et al., 2008).

With increasing knowledge of the pathophysiology/underlying mechanisms of allergic eye disease, there has been a rapid and large increase in the number of pharmacological anti–allergic medications available for treatment. Several authors have pointed out that ocular allergies have been under-diagnosed and under-treated, in particular seasonal allergic conjunctivitis where the ocular symptoms fall under the umbrella of seasonal hay fever which may underestimate its true prevalence (Cox, 2007; Bielory, 2008; Berdy & Berdy, 2009). A recent study by Wolffsohn and colleagues (2011) highlighted the current poor management of ocular allergies, where patients often self-medicate and rarely undergo an ophthalmic examination. Due to the increasing prevalence of allergy, allergic eye disease is becoming more common and combined with its impact on the quality of life and inadequate management, it is important for practitioners to identify these patients in order to manage them appropriately.

Mast cell degranulaion also initiates a series of cellular and extracellular events which lead to the late phase response, including production of prostaglandins, thromboxanes and leukotrienes derived from arachidonic acid. Mast cells also release cytokines and chemotactic factors which induce the production of IgE from

B-cells, enhance production of Th2-lymphocytes, attract eosinophils and activate

12 vascular endothelial corneal and conjunctival cells to release chemokines and adhesion molecules. The chemokines and adhesion molecules mediate the infiltration of eosinophils, basophils, neutrophils and Th2-lymphocytes to the site of inflammation and coupled with the newly formed mediators and sustained mast cell activation they result in the late phase response (Stahl et al., 2002). This may occur 3–12 hours after the initial reaction and symptoms can continue up to 24 hours. The year round symptoms associated with PAC are the result of chronic mast cell activation and Th2-lymphocyte infiltration (Bonini et al., 1989).

The most important and most effective step in treating allergic eye disease is avoiding the offending allergen to prevent the hypersensitivity reaction from being triggered. This necessitates the identification of the offending allergen and complete avoidance is not always possible (Friedlaender, 2001). In SAC a detailed history is essential as knowledge of the period of time of year symptoms occur can allow identification using a pollen calendar to some extent but peak levels of common causative pollens often overlap. However, effective measures for allergen avoidance in SAC and PAC are based upon control of the environment. Given that pollens are the main cause of SAC, preventative measures include limiting outdoor activity during the symptomatic period, closing windows and using air conditioning when in a car or indoors, avoid touching/rubbing eyes after being outdoors, wash hands after being outdoors and wearing close fitting or wrap around style sunglasses when outdoors (Veys, 2004). As PAC can affect the patient all year round, more thorough avoidance measures are necessary. Dust mite levels in the home can be reduced by using and regularly replacing protective pillow, mattress and duvet covers; washing bedding regularly at least at 60 °C; vacuum and damp dust entire house on weekly basis; reduce humidity to between 35 and 50% and remove or regularly clean carpets, upholstery, curtains and any other areas that gather dust (Gotzche & Johansen, 2008; Scheikh et al., 2010). Animal dander can be reduced by eliminating all pets/animals from the home or keep them outdoors; regular vacuuming; minimising exposure to areas that gather animal dander; avoid touching animals; washing hands and avoid eye touching/rubbing after contact with animals; and washing all clothes that have come into contact with animals (Eggleston & Wood, 1992; Bush, 2008).

Despite implementing these avoidance measures, complete avoidance is not always possible so use of anti–allergic medication may become necessary to prevent and alleviate symptoms. With increased knowledge of the pathophysiology of the hypersensitivity reaction in SAC and PAC over the years, there has been a rapid increase in the number of anti-allergic medications that target the immunological cells and inflammatory mediators involved in the allergic expression. Ophthalmic anti-allergic medications include topical mast cell stabilisers, antihistamines, vasoconstrictors (phenylephrine hydrochloride), antihistamine–vasoconstrictor combinations and dual action agents with mast cell stabilising and antihistaminic properties (Schultz, 2006).

Studies on the ocular use of phenylephrine hydrochloride started in 1933 and were first reported in 1936 (Greaves et al., 1992). The present research or study is based on the insight into phenylephrine hydrochloride and its properties.

2.3 Phenylephrine hydrochloride and its ocular uses

2.3.1 Phenylephrine hydrochloride

Phenylephrine is a sympathomimetic drug, used to relieve pain associated with complicated uveitis, allergic conditions, reduce posterior synechiae formation, and induce mydriasis, and as a topical spray into the ear to reduce the pressure in the Eustachsian tube (Johnson et al., 2008; Ahmed and Amin, 2007). Some of the commonly found salts are: phenylephrine hydrochloride; phenylephrine acid tartrate; phenylephrine bitartrate; phenylephrine tannate and phenylephrine tartrate (Sweetman, 2002). Phenylephrine oxazolidine, which is an ester of phenylephrine, has been found to provide longer retention time with lower plasma levels, thereby reducing chances of side effects like increased blood pressure. There is no salt form for phenylephrine oxazolidine (Rautio et al., 2008).

Phenylephrine hydrochloride was used in this study due to its pharmaceutical stability and availability (Trommer et al., 2010). The following discussion will therefore be limited to phenylephrine hydrochloride.

2.3.2 Pharmacological actions and uses Phenylephrine, or 3-(1–hydroxyl–2–methylamino-ethyl) phenol (Trommer et al., 2010) is a synthetic, imidazole-derivative, completely used in the optically active form (Pandey et al., 2003). Figure 2 shows the structure and available salts of phenylephrine in the pharmaceutical industry.

Figure 2: Structure of Phenylephrine, its base and salts (Trommer et al., 2010).

Ornato and Peberdy (2005) showed that phenylephrine hydrochloride solution could be used in place of epinephrine to increase regional cerebral blood flow following a prolonged cardiac arrest during cardiopulmonary resuscitation.

2.3.3 Mechanism of action

Vasoconstriction of the arterioles of the conjunctiva is due to PE’s action as a potent direct-acting alpha-adrenergic stimulator with weak beta-adrenergic activity (Leikin & Paloucek, 2007). It causes contraction by activating the dilator muscle of the pupil (Leikin & Paloucek, 2007). It also stimulates alpha-adrenergic receptors and intracellular acetylate cyclase are inhibited by phenylephrine, the intracellular adenylate cyclase then inhibits the production of cAMP (Donnelly, 2009). This inhibition of cAMP causes arterial and venous constriction in the conjunctiva thereby decreasing the blood flow and mucosal oedema caused by allergic responses (Donnelly, 2009).

2.3.4 Pharmacokinetics

The protective mechanisms that are present in the eye to shield the visual pathway from foreign chemicals make it hard for drug to be delivered to the eye. These mechanisms have led to the development of optimized ophthalmic drug delivery systems that are based on the understanding of the drug disposition pathways in the eye and the ophthalmic pharmacokinetic profile (Worakul & Robinson, 1997). Phenylephrine hydrochloride is administered topically to avoid extensive first pass metabolism and to reduce systemic side effects (Aschenbrenner & Venable, 2008). Peak ocular concentrations of 1.2% to 5% are achieved after a single dose between 20 to 60 minutes. Maximal dilation of the pupil occurs within 60–90 minutes and the effect lasts 5–7 hours (Rossiter, 2010).

Phenylephrine has a half-life of a few minutes when circulating in the blood. It can be degraded either through methylation by COMT or by deamination by MAO in the blood stream (Flancbaum et al., 1997). The dehydrated form of phenylephrine hydrochloride is a major degradation derivative during pharmaceutical processing and stability determination (Trommer et al., 2010).

2.3.5 Adverse effects

Phenylephrine produces systemic adverse effects such as hypertension, subarachnoid haemorrhage, ventricular arrhythmias and myocardial infarction, trembling, headache and sweating (Dipiro et al., 2002).

The day after administration, rebound miosis has a possibility of occurring particularly for older patients (Aschenbrenner & Venable, 2008).

Eye drops containing 10% of API may have profound effects on the cardiovascular system, and the risk is smaller with 2.5% products, which are said to be equally effective as mydriatics (Rossiter, 2010). The hypertensive effects of phenylephrine may be treated with an alpha–adrenoceptor blocking agent such as phentolamine mesylate, 5 to 10 mg intravenously which should be repeated as necessary (Beyene & Maren, 2004).

2.3.6 Drug interactions

Phenylephrine may produce severe hypertension and cardiac arrhythmias when used concurrently with beta blockers or other antihypertensive (for example reserpine) and, tricyclic antidepressants. Mono amine oxidase inhibitors increase pressor effects of phenylephrine which potentiates the risk of hypertensive crisis (Pray, 2006).

2.3.7 Bioavailability

The average human being has a tear volume of 7 µL while the cul-de-sac has a maximum capacity of about 30 µL. Many ophthalmic formulations range from 50 to 75 µL in volume; however, volumes in excess of 50 µL are probably unable to enter the cul-de-sac (Greaves et al., 1992). Systemic absorption occurs through solution drainage into the nose, which causes the loss of an instilled drug as shown in Figure 3 (Jarvinen et al., 1995).

Drug in tear fluid

Ocular absorption Systemic absorption (5% of the dose) (50-100% of the dose)

-Conjunctiva of the eyes -Nose Corneal route Conjunctival and Minor routes: - 1 route Scleral route -Larcrimal drainage -Large, system - Small, lipophilic drugs hydrophilic drugs -pharynx GI-tract -Skin at the cheek and Aqueous humour lids -Aqueous humour -Inner ocular tissues

Ocular tissues

Figure 3: Diagram of Ocular Absorption (Nanjawade et al., 2007).

The equation describing the above processes in terms of drug concentration is:

Where F is the fraction of dose absorbed, D is the dose, k and K are absorption and elimination rate constants, respectively, and V, is the apparent volume of distribution (Lee & Robinson, 1979; Worakul &Robinson, 1997; Lee & Robinson, 1986).

An ideal eye drop preparation possesses a high concentration of drug in a minimum drop volume. Martini and partners (1997) reported that equal tear-film concentrations were obtained whether either 5 µL of 1.61 x 1010 M or 25 µL of 1.0 x 1010 M pilocarpine nitrate solution were administered. The higher bioavailability of the 5 µL solution was due to decreased drainage loss. However, there is a practical limitation to the minimum dosage volume used. Dropper configuration that delivers small volumes poses difficulties in its design and patients often cannot sense or detect the administration of volumes in the 5.0 to 7.5 µL dose volume ranges (Martini et al., 1997).

2.3.7.1 Reasons for poor ocular bioavailability

The most common method for the administration of therapeutic treatment for ocular diseases is the topical delivery of eye drops. The rapid elimination of drugs from the precorneal lachrymal fluid by solution drainage, lachrymation, and poor absorption by the conjunctiva creates problems for topical delivery (Bartlett & Jaanus, 2008).

The eye has the ability to permanently maintain its residence volume at 7–10 µL due to high drainage rate. The volumes instilled with eye drops range from 20–50 µL, therefore the residence time of solutions is limited to a few minutes, causing the overall absorption of a topically applied drug to be limited to 1–10% (Felt et al., 1999).

2.3.7.2 Strategies for improving drug availability in ocular adminstration

There are three main ways to maximise the systemic bioavailability of drugs administered ocularly, these are:

 Increase ocular residence time.  Increase ocular absorption.  Alter the drug structure to change physicochemical properties (Lang et al., 2005).

2.3.7.2.1 Increasing ocular residence time

Lacrimal secretions and drainage systems act to remove foreign bodies and substances from the corneal epithelium as quickly as possible. In order to increase residence time, or delay clearance, drops should be instilled in the anterior segment of the ocular cavity. Products can also be formulated with polymers such as methylcellulose, hydroxypropyl methylcellulose or polyacrylic acid (Carbopol), which increases the viscosity of the formulation and act as bio–adhesives with the ocular tissue (Patel et al., 2010).

2.3.7.2.2 Increasing ocular absorption

Enhancers act by increasing the rate at which drugs penetrate the epithelium. They alter the structure of the epithelial cells to increase absorption; this is achieved without destruction of the cells. Some examples are dimethyl sulfoxide, decamethonium, EDTA and glycocholate. Ideal absorption enhancers should possess the following qualities:

 They should provide an effective increase in the absorption of the drug.  Should not cause permanent damage.  Must be non–irritant.  Must be effective in small quantities.  Effect should be fully reversible and temporary.  Should not have a lag effect when absorption is required.

 Should be stable (Saettone et al., 1996; Patel et al., 2010).

2.3.7.2.3 Altering drug structure

Modifications to a drug molecule are usually done to achieve certain physicochemical properties in order to, amongst others, increase aqueous solubility and improve partitioning. Caution should be applied in drug modification as it could pose the risk of changing a drug’s therapeutic and toxicological profile. Subsequent changes in salts or molecules could result in increased costs and lengthy investigations due to regulatory requirements. Some factors that have been found to influence drug absorption are its weight and pH (Krishnamoorthy & Mitra 1998).

Molecular size and weight: Ocular absorption of small compounds, approximately 100 Daltons, is higher, (around 80%), when compared to oral absorption. This however reduces markedly as the molecular weight increases. There is a restriction to the molecular weights that can pass through pathways and channels (molecular weight cut–off) as only molecules that are smaller than the channels can diffuse through them (Akiba et al., 1993).

The effect of pH and the partition coefficient: The partition coefficients of drugs are dependent upon environmental pH which affects the ionization of drugs (local pH can be modified by ocular formulation). The rate of absorption is increased when the drug or substance is un–ionized. The movement of ionizable drugs through membranes depends on the chemical equilibrium between ionized and unionized drug in the eye drop and the lacrimal fluid (Geroski & Edelhauser, 2000). The ionized form does not easily penetrate the lipid membrane compared to the unionized molecule. The partitioning of ionized molecule does not only depend on the degree of ionization but also on the charge of the molecules (Liaw et al., 1992). The corneal epithelium is negatively charged (isoelectric point is pI 3.2), and as a result, hydrophilic–charged cationic substances permeate easily through the cornea than anionic compounds (Liaw et al., 1992). Compounds below pI 3.2 are negatively- charged and pass through the cornea making the pH too acidic, and irritating for clinical use. Consequently, in practice, charge–discriminating effects of the corneal

20 epithelium decrease the absorption of negatively charged compounds (Rojanasakul & Robinson, 1989; Conroy & Maren, 1995; Taylor, 2002; Lee & Robinson, 2009; Hecht, 2000).

2.3.8 Polymorphism and pseudomorphism of phenylephrine hydrochloride

A polymorph is a solid substance with the ability to crystallize in at least two different crystal structures that produce distinct crystal species. Polymorphism is important in the drug development process as it can affect drug dissolution, chemical stability, as well as drug bioavailability (Yoshihashi et al., 2002). Polymorphs are the collection of different crystal structures that can exist for a single chemical entity and its hydrates. The difference between a solvate and polymorph is that a solvate contains different quantities of solvent trapped within the crystal structure (as is the case for

MgSO4.7H2O and MgSO4.10H2O) and polymorphism is when there is no solvent trapped in the crystal structure and the same chemical species are found in various crystal structures. Solvates or false polymorphs are called pseudopolymorphs. Pseudopolymorphs can be obtained by changing the recrystallizing solvent. Common solvents used to induce polymorphic change are water, methanol, ethanol, and acetone and chloroform (Lee et al., 2006). Phenylephrine hydrochloride does not exhibit polymorph or solvate forms, its chemical stability will therefore not be hampered when dissolved in solvents like water and glycerol (Yoshihashi et al., 2002).

2.4 Ophthalmic formulations

Eye drops are sterile products, free from foreign particles, compounded and packaged for ocular drug delivery. Eye drops can be prepared as single or multidose products (Van Santvliet & Ludwig, 2004).

Compared to the buccal, nasal, rectal, vaginal or dermal routes, eye drops are easier to use, non-invasive, accurate and less expensive for systemic delivery of drugs (Pillion et al., 1994). The ocular route helps eliminate painful parenteral injections or gastro-intestinal degradation (Chiou et al., 1991 and Pillion et al., 1994).

Absorption of the eye drops across the mucosa in the nasal cavity that is near to the conjunctival sac helps eye drops with active pharmaceutical ingredients reach the bloodstream (Winfield et al., 2009). Physicochemical drug properties, such as lipophilicity, solubility, molecular size and shape (Huang et al., 1989; Liaw & Robinson, 1992), charge (Rojanasakul et al., 1992; Liaw et al., 1992) and degree of ionization (Brechu & Maren, 1993) affect the route and rate of permeation in the cornea.

Drugs can be absorbed through the naso-lacrymal system and reach systemic circulation without passing metabolism by the liver only if there is (a) drainage loss of topically-applied solutions,(b) increase in bioavailability of the instilled drug (Saettone et al., 1984), and (c) an increase in viscosity (Flach, 2002). Mild to severe side effects may therefore be observed. The phenomenon has led to the reduction in the size of eye drops (Saettone et al., 1984; Ludwig & Van Ooteghem, 1989; Salminen, 1990; Flach, 2002).

2.4.1 Eye drops as an ophthalmic dosage form

Liquid formulations (eye drops and eye lotions), semisolids (creams, ointment and gels), solids (biodegradable implants) are various ophthalmic dosage forms used to deliver therapeutic agents topically (Kaur & Kanwar, 2002). Eye drops are conventional dosage forms that account for 90% of the currently accessible ophthalmic formulations. Eye drop solutions are common dosage form as they offer advantages like administrations, easy preparation and low costs. Eye drops also have some disadvantages like short contact time with ophthalmic surface and nasolacrimal drainage which causes poor bioavailability of the drug (Ali & Lehmussaari, 2006). Ophthalmic delivery systems are being investigated in order to increase the corneal permeability and prolong the contact time with the ocular surface through the addition of viscosity modifying agents such as hydroxypropyl methylcellulose, carboxy methylcellulose sodium and other cellulose derivatives (Gad, 2008). Suspensions are defined as the dispersion of finely divided, relatively insoluble drug substances in aqueous vehicle containing suitable suspending and dispersing agents (Hecht, 2000). Due to the tendency of particles being retained in the cul-de-

22 sac, the contact time and duration of action of a suspension could theoretically exceed that of a solution. There are difficulties associated with suspension dosage forms such as dosage uniformity, crystal growth and polymorphism. Dosage uniformity requires a brisk “shake well” approach to distribute the suspended drug. This can contribute towards poor patient compliance as significant numbers of patients do not adhere to instructions (shake well before use). Polymorphism can occur during storage resulting in an increased or decreased solubility whereby changes are reflected in increased or decreased bioavailability (Hecht, 2000). Ointments offer longer contact time, greater total drug bioavailability albeit with slower onset and time to peak absorption. Ointments interfere with vision and are usually restricted to bedtime use. They remain a popular paediatric dosage form and for postoperative use (Gad, 2008).

Solutions are the preferred ophthalmic dosage forms for treatment of conditions such as uveitis (Ali & Lehmussaari, 2006). These forms are preferred because:

a) All the ingredients are completely in solution with uniformity. b) There is reduced systemic toxicity. c) Rapid onset of action is achieved. d) The required dose over time is decreased. e) There is little physical interference with vision. f) It offers a convenient mode of administration (Kaur et al., 2003: Ghosh & Jasti, 2005).

Limited bioavailability due to short contact time between the eye and the active pharmaceutical ingredients is one shortcoming of solutions. The short contact time between the active pharmaceutical ingredient and the eye surface can be increased by the inclusion of a viscosity-modifying agent such as methylcellulose. The optimum level of drug retention and visual comfort is within the viscosity range of 0.15 to 0.25 Pa·s (Lang et al., 2005).

There are factors to be considered in the preparation of eye drop solutions. These include sterility, clarity, buffer, buffer capacity and pH, tonicity, viscosity, stability, comfort, additives, particle size, packaging and preservatives. These factors are

23 interrelated and assessed collectively in the preparation of an eye drop product. The buffer system is considered along with tonicity and comfort. Stability of the eye drop product depends on pH, buffer system, and packaging. Viscosity-modifying agents which may or may not be present should not affect the therapeutic effectiveness of the active ingredient (Lang et al., 2005).

The pH and buffer capacity is a compromise between stability of the drug and comfort in the eye. Optimum patient comfort is found at the pH of the tear fluid, or at 7.4 pH, while optimum stability for many drugs is lower than 5 pH. Buffers are therefore needed to maintain the desired pH for drug stability and allow it to be altered to 7.4 immediately after instillation in the eye (Aulton, 2002).

Table 1: Conventional dosage forms and usage (Lang, 1995).

Formulation Number % Gels 2 0.7 Injectables 11 3.8 Inserts 11 3.8 Ointments 50 17.4 Orals 9 3.1 Solutions 179 62.4 Suspensions 25 8.7

2.4.2 Eye drop formulation characteristics

2.4.2.1 Clarity

Eye drop solutions should be clear which is achieved by filtration. Filtration equipment, which has a pore size of 0.45 µm, must be sterile particle free so that particulate matter is not found in the solution by equipment designed to remove it. Sterile techniques must be performed in clean surroundings: and using laminar-flow hoods and proper garments will aid in preparing solutions free from foreign particles. With sophisticated machines clarity and sterility can be achieved in the same filtration step. It is important to note that solution clarity is equally dependent on the cleanliness of the intended container and closure. Both container and its closure must be sterile. This means that the container or closure must not contribute

24 particles to the solution during prolonged contact such as shelf-life storage (Michael & Richards, 2009).

2.4.2.2 Stability, pH and buffer systems

Stability is not only the chemical stability of a single product component but the total product. A well-planned stability programme will consider and evaluate the chemical stability of the active ingredient’s preservative efficacy against selected test organisms, and test the adequacy of the package over time (McDonnell, 2007).

The stability of a drug in an eye drop product depends on the physicochemical nature of the drug substance, pH, method of preparation (temperature exposure), additives, and type of packaging. Initially eye drop solutions had a short shelf life however; with recent advancement in technology the stability of eye drop products is expressed in terms of years usually between two-three years (McDonnell, 2007).

Ideally, the prepared eye drop should be formulated at a pH equivalent to the tear fluid value of pH 7.4. The majorities of active ingredients used in eye drops are salts of weak bases and are stable at acidic pH. The acidic pH selected should therefore be maintained throughout the product’s shelf life, however, if the buffer capacity is sufficient to resist adjustment by tear fluid, and the overall eye pH remains acidic for a considerable period, stinging and discomfort may result (Florence & Attwood, 2006). The buffer capacity should thus be adequate for stability but minimized, as far as possible, to allow the overall pH of the tear fluid to be disrupted only momentarily (Florence & Attwood, 2006). Common buffering agents found in eye drop formulations are sodium citrate dihydrate and boric acid (Florence & Attwood, 2006).

Sodium citrate dihydrate is used primarily to adjust the pH of solutions. It is used at concentrations of 0.1–2.0% in ophthalmic products. It is a stable material and can be easily sterilized by autoclaving (Amidon, 2006). The bulk material should be stored in a well–closed container protected from light, in a cool, dry, place (Harwood, 2006).

Boric acid is odourless, colourless and greasy to the touch. It has weak bacteriostatic and fungistatic properties. It is commonly used as a buffer and antimicrobial in eye drops at concentrations of 1.22%. Boric acid volatilizes in steam (Stewart, 2004).

Oxygen sensitivity is an important factor to be considered as certain active ingredients may require the inclusion of an antioxidant (Ansel et al., 2011; Sutton et al., 1998 and Mitra, 2009).

Phenylephrine hydrochloride is stable at a pH range of 3.5–8. It shows signs of degradation by changing its clear colour to yellow or brown, when dissolved in aqueous solutions (Lang et al., 2005).

2.4.2.3 Tonicity

Tonicity refers to the osmotic pressure exerted by salts in aqueous solution. An eye drop solution is isotonic with another solution when the magnitudes of the colligative properties of the solutions are equal. An eye drop solution is considered isotonic when its tonicity is equal to that of a 0.9% sodium chloride solution. The calculation of tonicity at one time was stressed rather heavily to the detriment of other factors such as sterility and stability. In actuality the eye is much more tolerant of tonicity variations than it was formerly thought. The eye tolerates solutions equivalent to a range of 0.5 to 1.8% sodium chloride (Ansel et al., 2011).

Glycerol is used in ophthalmic pharmaceutical formulations as a tonicity modifying agent with an antimicrobial and viscosity-modifying functions when used at concentrations of >20% and 0.5–3% respectively. Glycerol is hygroscopic and not prone to oxidation by the atmosphere under ordinary storage conditions, but decomposes on heating, with the evolution of toxic acrolein. Mixtures of glycerol with water, ethanol, and propylene glycol are chemically stable. Black discolouration of glycerol occurs in the presence of light on contact with zinc oxide or basic bismuth nitrate (Price, 2006).

2.4.2.4 Viscosity

Medicated eye drops usually have poor bioavailability due to the barrier created by the precorneal area. The site of absorption is cleared rapidly by protective mechanisms such as blinking and nasolacrimal drainage (Ali & Lehmussaari, 2006). This necessitates frequent instillation, increasing side effects associated with the active pharmaceutical ingredient (Di Colo et al., 2009). There is need therefore to increase API residence in the eye by increasing or adding the polymer that prolongs drug contact time with the ocular surface (Wilson, 2004).

Substances such as methylcellulose, polyvinyl alcohol, and hydroxypropyl methylcellulose can be added to increase viscosity and prolong contact time in the eye which enhances drug absorption and activity. Viscosity ranging from 0.15 to 0.25 Pa·s significantly improves contact time in the eye. Results tend to plateau beyond the 0.25 Pa·s as higher viscosity values offer no significant advantage (Winfield & Richards, 2004).

Hydroxypropyl methylcellulose is a widely used thickening agent in topical pharmaceutical formulation especially for ophthalmic formulations (Romanelli et al., 1994) Compared with methylcellulose; hydroxypropyl methylcellulose produces solutions of greater clarity, with fewer undispersed fibres present, thus making it preferable for formulations for ophthalmic use. Its concentration is between 0.45- 1.0%w/w (Harwood, 2006).

For an aqueous solution to be prepared, hydroxypropyl methylcellulose should be dispersed and thoroughly hydrated in about 20–30% of the required amount of water (Harwood, 2006).The water used should be vigorously stirred and heated to between 80–90 °C and the remaining hydroxypropyl methylcellulose added. Cold water should be added to produce the required volume (Harwood, 2006).

Aqueous solutions of HPMC are enzyme-resistant, and provide viscosity stability during long-term storage. However, aqueous solutions are liable to microbial spoilage and should be preserved with an antimicrobial preservative. When used as a viscosity-increasing agent in ophthalmic solutions, benzalkonium chloride is

27 commonly used for antimicrobial protection. Aqueous solutions may be sterilized by autoclaving; the coagulated polymer must be redispersed on cooling by shaking (Harwood, 2006).

Carboxymethylcellulose sodium is widely used as a topical pharmaceutical formulation primarily for its viscosity-modifying properties and its concentrations range between 3–6%. Viscous aqueous solutions are used to suspend powders intended for topical administration (Parsons, 2006).

Carboxymethylcellulose sodium is a stable hygroscopic material. Under high humidity conditions carboxymethylcellulose sodium can absorb a large quantity of water (> 50%). Aqueous solutions are stable between pH 2–10, precipitation can occur below pH 2 solution and viscosity rapidly increase above pH 10 (Gopferich, 1997). Generally, solutions exhibit maximum viscosity and stability at pH 7–9. Aqueous solutions may be sterilized by heating although this may results in some reduction in viscosity. After autoclaving, viscosity is reduced by about 25% although this reduction is less marked than for solutions prepared from material sterilized in the dry state. Aqueous solutions stored for prolonged periods should contain an antimicrobial preservative. The bulk material should be stored in a well-closed container in a cool, dry, place (Parsons, 2006).

Viscosity-increasing agents are extensively used in the formulation of many pharmaceuticals. In solution formulations, cellulose derivatives are used most frequently. As increased viscosity can offer both advantages and disadvantages with respect to use of the preparation, therefore, knowledge of viscosity and flow properties is important in selecting appropriate ingredients. If sterility of the preparation is required, the alteration in viscosity properties due to the sterilization process and various additives, namely electrolytes, should be investigated (Šklubalová and Zatloukal, 2011).

The viscosity modifying agents used in ophthalmology are water-soluble naturally occurring, semi-synthetic or synthetic polymers (Shell, 1982; Lee and Robinson, 1986). The natural polymers include botanical polysaccharides (guar gum, locust bean gum) semi-synthetic cellulose derivatives, such as cellulose ethers

(hydroxypropyl methylcellulose, hydroxyl propylcellulose, hydroxyl ethylcellulose, methylcellulose and carboxy methylcellulose). Cellulose esters are celluloseacetophtalate, microbial polysaccharides (dextran, xanthan gum, gellan, gum and scleroglucan), algal polysaccharides (sodium alginate and carrageenan) and animal polysaccharides (sodium hyaluronate, chondroitin sulphate and chitosan).

Polyvinyl alcohol, polyvinylpyrrolidon and polyacrylic acid (Carbopol®) are commonly used synthetic polymers (Sintzel et al., 1996). In general, viscous ophthalmic solutions exhibit a pseudoplastic rheological behaviour. Pseudoplastic solutions offer less resistance to movement of the eyelids over the globe and, therefore, are expected to be more comfortable in the eye than Newtonian solutions (Dudinski et al., 1983 and Van Ooteghem, 1987). The ideal viscosity of an ophthalmic solution is estimated at 15–30 mPa·s, except in the case of viscoelastic polymers where a higher viscosity is well tolerated by the patient (Ludwig & Van Ooteghem, 1988).

Ideally, small volumes of an ophthalmic solution should be instilled to diminish the drainage rate and to reduce systemic absorption (Urtti & Salminen, 1993). To produce eye drops with the ideal volume of 5–25 μl, dropper tips with small- dimension capillaries are necessary. The inner aperture and outer orifice diameter determine the flowing of the liquid through the capillary and, consequently, could influence the viscosity and surface tension of the solution to be dispensed (Saettone et al., 1984; Chang et al., 1988; Podder et al., 1992; Urtti and Salminen, 1993; Meseguer et al., 1996). In 1984, Jho and Carreras stated that the formation of a drop under flowing conditions at the orifice of a calibrated capillary depended primarily on the surface tension of the solution as according to Tate's law, but was also influenced by hydrodynamic effects including drop formation rate, gravitational and viscous forces.

In this study the following excipients were used as the viscosity modifying agent’s carboxy methylcellulose sodium, glycerol and hydroxylpropyl methylcellulose (Rowe et al., 2003). They were used to thicken and stabilize the formulation (Swarbrick et al., 2000). They also lack reactivity with other excipients and PE and do not irritate the eye (Ranucci & Silverstein, 1998).

2.4.2.5 Additives

Very few additives in eye drop solutions are permissible because they either reduce the efficacy of the active pharmaceutical ingredient or are toxic for use in the ocular region. Antioxidants like sodium bisulfite or sodium metabisulfite are permitted in concentrations of up to 0.3%, particularly in solutions containing phenylephrine and epinephrine salts. The antioxidant acts as a stabilizer to minimize oxidation of PE and epinephrine. Other types of antioxidants are ascorbic acid and acetylcysteine (Järvinen et al., 1995).

Non-ionic surfactants which are least toxic to the ophthalmic tissues are permitted at concentrations of approximately 0.005%–0.2%. Surfactants are used as co-solvents to increase solubility and to improve topical bioavailability of the API and to improve the therapeutic response of an ophthalmic drug. Some surfactants can prolong ocular residence time of the drug enhancing ocular permeation of the drug molecules (Urtti & Salminen, 1993; Järvinen et al., 1995).

Benzalkonium chloride is a cationic surfactant and a preservative used frequently in eye drop solutions. Concentrations that are used are in the range of 0.005–0.02%, with toxicity the limiting factor on the concentration used. The benzalkonium cation has a large molecular weight and is inactivated by macromolecules possessing an opposite charge or by sorption. Benzalkonium chloride is the preservative of choice and is used in many commercial eye drops, as it has low irritation potential to the eye, has a broad-spectrum bactericidal activity at very low concentrations and is compatible with many API (Winfield et al., 2009).

A common penetration enhancer is EDTA which loosens the tight junctions between the superficial epithelial cells, facilitating paracellular transport (Saettone et al., 1996; Hochman & Artursson, 1994). It is used in concentration of 0.1%.

The sorption characteristics of surfactants have to be identified before being included in a formulation. The reason being is that surfactants can absorb preservatives and thus render the formulation unpreserved (Baudouin et al., 2010).

Sodium metabisulfite has antioxidant, antimicrobial (in acidic preparations), and antibrowning agent properties. In acidic preparations, sodium metabisulfite is used at concentrations of 0.01–1% and less than 0.5% in alkaline preparations is slowly oxidized to sodium sulfate on exposure to air and moisture. Sodium metabisulfite is + + immediately converted to sodium ions (Na ) and bisulfite ions (HSO3 ) when mixed with water. The crystals also disintegrate when exposed to air and moisture. Decomposition of aqueous sodium metabisulfite occurs as it exposed to air. The bulk material should be stored in a well-closed container protected from light, in a cool, dry, place (Stewart, 2006).

Edetic acid and edetate salts are used in ophthalmic pharmaceutical formulations as chelating agents. Edetic acid and edetates are primarily used as antioxidant synergists by sequestering trace amounts of metal ions, particularly copper, iron, and manganese, which might otherwise catalyze autoxidation reactions. Edetic acid and edetates may be used alone or in combination with true antioxidants, the usual concentration employed being in the range 0.005–0.1% w/v. Edetic acid possesses antimicrobial activity against Gram-negative microorganisms, Pseudonomas aeruginosa, some yeasts, and fungi, although this activity is insufficient for edetic acid to be used effectively as an antimicrobial preservative on its own. Many solutions used for the cleaning, storage and wetting of contact lenses contain disodium edentate. Edetic acid occurs as a white crystalline powder (Weller, 2006). Aqueous solutions of edetic acid or edetate salt may be sterilized by autoclaving, and should be stored in an alkali-free container. Edetic acid and edentates should be stored in well-closed containers in a cool, dry, place (Weller, 2006).

The most widely used vehicle for pharmaceutical products which is also a solvent is water. This is due to its physiological compatibility with many compounds and absence of toxicity. It has a high dielectric constant which is necessary for ensuring the dissolution of a wide range of ionisable materials (Mido et al., 2003).

There are two types of water used in the production of pharmaceutical products: purified water and water for injections. Purified water must be free of ionic and organic chemicals and microbial increase. It is produced by mains water going through a unit that deionizes and distills the water, has an ion-exchange, provides for

31 reverse osmosis and finally filters. All purified water systems must be validated. Purified water should be supplied constantly at ≥ 80 °C (Potdar, 2007).

Water for injection is used in the production of injections and other pharmaceutical applications like cleaning of critical equipment and preparation of pharmaceutical products. The water for injection gets its feed from purified water, which is subjected to further distillation and reverse osmosis. The requirements for bacterial endotoxin tests are much higher values as it must be free of microbial contamination and endotoxins. Water for injection must show no reaction with the limulus amebocyte lysate (LAL) reagent which is used to test for microbes (Potdar, 2007).

An important factor for eye drops is that they should be sterile when dispensed in a multiple-application container (Reddy & Ganesan, 1996). Aseptic techniques during the manufacture of injections have to be adopted when manufacturing sterile eye drops (Turco & King, 2005).

Essentially two strategies are adopted in the manufacture of microbiologically acceptable pharmaceutical formulations. The first and most important is to minimize the introduction of microorganisms from sources such as air, water, raw materials and personnel; and the second is to formulate the final product so as to be hostile to microorganisms, by the addition of preservatives. Common waterborne organisms found are the Pseudomonas-Achromobacter-Alcaligenes types, including occasionally P. aeruginosa (McDonnell, 2007).

Purified water is a typical source of microorganisms as during use, the ion-exchange column may become contaminated from the water passing through and the entrapped organisms rapidly and multiplies to produce high counts in the outflowing water. Water produced by reverse osmosis might present a problem if the osmosis membrane is not disinfected at regular intervals. Distilled water may also be a significant source of contamination, because the chlorine that protects tap water is lost on distillation, and Gram-negative bacteria may grow to concentrations as high as 105 to 106 ml-1 within a few days of storage at ambient temperature. With the above in mind the correct approaches to preservation are based on two important principles. The first is that a preservative must not be added to a product to mask

32 any deficiencies in the manufacturing procedures, and the second is that the preservative should be an integral part of the formulation, chosen to afford protection in that particular environment (Parker, 2002).

Sterility is one of the important characteristics for ophthalmic solutions. It is defined as the absence of all living organism (McDonnell, 2007). This especially includes microorganisms, such as bacteria, yeasts, molds, and viruses. The presence of even one bacterium in an eye drop container renders its non-sterile. Sterility could be determined or proved by inoculating the eye drop with microbiological cultural media. If sterile, no microbial growth will be observed; if not, the culture medium will become turbid as a result of microbial proliferation (Hanlon, 2002).

Serious ocular infection can result from the use of contaminated eye drop solutions. Such solutions are the cause of corneal ulcers and loss of eyesight. Contaminated solutions have been found in use and dispensed on prescription in community and hospital pharmacies (Hodges, 2002).

Microbes commonly found as a contaminants are the Staphylococcus group. Pseudomonas aeruginosa is a less frequent contaminant; however it is often found as a contaminant in sodium flourescein. P aeruginosa is an opportunistic and very dangerous organism that grows well on most culture media and produces both toxins and antibacterial products. The latter tend to kill off other contaminants and allow the P aeruginosa to grow in pure culture. This gram-negative bacillus also grows readily in eye drop solutions, making it a source of extremely serious infections of the cornea. Its rapid infection rate can cause complete loss of sight in 24 to 48 hours. In concentrations tolerated by tissues of the eye, only certain antimicrobial agents are effective against some strains of this organism (Hanlon, 2002).

Sterile eye drop solutions in multiple-dose containers can be contaminated easily. For example, if a dropper bottle is used, the tip of the dropper while out of the bottle can touch the surface of a table or shelf if laid down or it can touch the eyelid or eyelash of the patient during administration. The solution may be an effective antimicrobial agent, but the next use of the contaminated solution may occur before enough time has elapsed for all of the organisms to be killed. In this manner living

33 organisms penetrate through an abrasion into the corneal stroma, within the corneal stroma, traces of antimicrobial agents are neutralized by tissue components and the organisms finds an excellent culture medium for rapid growth and dissemination through the cornea and the anterior segment of the eye (Fassihi, 2001).

When the vitreous humour is infected by Bacillus subtils, it produces an abscess which can cause blindness. Aspergillus fumigates is a pathogenic fungus found in eye solutions and is responsible for accelerating deterioration of the active drugs. In the late 1930s there was an epidemic of keratoconjunctivitis, it was caused by one bottle of virus contaminated tetracaine solution. Virus contamination is difficult to control because none of the preservatives available is virucidal. In addition, viruses are not removable by filtration they are however destroyed by autoclaving. Recent studies show that the adenoviruses (Type III and VIII), are causative agents of viral conjunctivitis such as the epidemic keratoconjunctivitis (McDonnell, 2007).

2.5 Sterilization

Sterilization is defined as a process used to render a surface or product free from viable organisms, including bacterial spores. Common methods of sterilization include moist heat under pressure, dry heat, filtration, gas sterilization, and ionizing radiation (McDonnell, 2007). A process, whereby a product is sterilized in its final container and, which permits the measurement and evaluation of quantifiable microbial lethality is called terminal sterilization. This process differs from aseptic manufacture, which requires a series of sterile techniques and provides a passive process of protection of the dosage form from contamination. If a finished product is suitable for terminal sterilization, then this is the method of choice (Hanlon, 2002).

Sterilization is a requirement during the manufacture of eye products. The methods used depend on the active ingredient, product resistance to heat and on the packaging used. The sterile solution will usually contain an antimicrobial preservative to protect against unintended contamination during use. Preservatives are not used to produce a sterile but rather to maintain sterility (Prabhasawat et al., 2005).

2.5.1 Steam under pressure as a method of sterilization

Terminal sterilization by autoclaving is an effective method of sterilization but the solution components have to be heat-resistant in order to avoid degradation. Effective sterilization is achieved when steam penetrates the autoclave load uniformly. Products in their primary containers are autoclaved under pressure (1–2 atm) at 121 °C for 15 minutes. A technique has been developed which is called air- over-steam autoclave. This allows pressure adjustments to be made during the autoclave cycle to permit the autoclave sterilization of materials such as, polypropylene containers that deform when exposed to heat (Lang et al., 2005).

2.5.2 Filtration as a method of sterilization

Filtration is defined as the process of removing solids or suspended matter in a liquid or gas by passing matter through a porous medium where solids are retained or entrapped (Twitchell, 2002). The USP (2004) states that sterile membrane filters must have a pore size of 0.2 µm. Filtration is a method of sterilization as it offers the substantial advantage of room temperature operation with none of the damaging effects of exposure to heat or sterilizing gas. Filtration sterilization involves the transfer of the finished sterile product into sterilized containers, through a filter paper using aseptic techniques. The membrane filter is either hydrophobic or hydrophilic; the right choice should be made based its compatibility with the API and excipients, so as to reduce sorption. Sorption could significantly reduce the efficacy of the manufactured product. The membrane filtration equipment itself usually is sterilised by autoclaving. Filtration permits extemporaneous preparation of eye drop solutions that have a high probability of being sterile under aseptic conditions (Gould, 2004).

2.5.3 Laminar-flow principles

A laminar-flow work area is used to prepare sterile, particulate free solutions. Laminar-flow is defined as airflow in which the total body of air moves with uniform velocity along parallel lines with a minimum of eddies. Laminar-flow with the HEPA filters reduces the possibility of airborne microbial contamination by providing air free of viable particles and free of practically all inert particulates. Laminar-flow units can

35 be found in different shapes and sizes and in two categories, horizontal and vertical laminar-flow (Turco & King, 2005). Laminar-flow is not a guarantee of sterility and correct procedures and sterile techniques are necessary (McHugh, 2010).

Laminar flow areas are supplied with air passed through high effiecincy particulate filter (HEPA) filters which are high density mats composed of randomly arranged fibres and are used as air filters to remove much smaller particles such as dust pollutants and microbes. The fibres, made of fibreglass, have a diameter-range of 0.5 and 2.0 µm.

Factors that affect function of HEPA filters are fibre diameter, filter thickness, and face velocity (Abdel-Magid & Caron, 2006).

The HEPA filters remove finer particles in air passed through them, and virtually free it from foreign matter. Ultraviolet light (UV) is installed in the air ducts on the downstream side of the filter to kill microbes that may have escaped through or around the filter (Brooks et al., 2007). The hood air flow should have an air velocity of 100 ft. / minute (Potdar, 2007).

2.5.4 Preservatives used in eye drop formulations

Preservative substances must be evaluated as part of the total eye drop preparation in the proposed package. The growth or presence of microorganisms in the eye drop can lead to destruction of the product or transmission of disease to the consumer. Destruction can be in the form of chemical degradation of drugs or excipients by the enzymes produced by the microorganism or a breakdown of the physical attributes such as tactile change, visible change in colour, or smell (Amin et al., 2010).

The USP (2004) states that eye drop solutions may be packaged in multiple-dose containers and must contain a substance or mixture of substances to prevent the growth of, or to destroy, microorganisms introduced accidentally when the container is opened during use. Eye drop solutions prepared and packaged for a single application, need not contain a preservative because they are not intended for reuse.

Preservatives are not to be used in solutions intended for intraocular use because of the risk of irritation.

The selection of an eye drop preservative can be cumbersome because of the relatively small number of suitable agents. As there is no ideal preservative, however the following criteria are useful in preservative selection:  The agent should have a broad spectrum and be active against gram-positive and gram-negative organisms as well as against fungi.  The agent should exert a rapid bactericidal activity, particularly against known virulent organisms such as P aeruginosa strains.  The agent should be stable over a wide range of conditions for example autoclaving temperatures and a wide pH range.  Compatibility should be established with other components in the product and with packaging systems.  Lack of toxicity and irritation should be established with a margin of safety (Lang et al., 2005).

Sterile eye drop solutions in multiple-dose containers can be contaminated easily. For example, if a dropper bottle is used, the tip of the dropper, while out of the bottle, can touch the surface of a table or shelf if laid down or it can touch the eyelid or eyelash of the patient during administration (Amin et al., 2010). The solution may have an effective antimicrobial, but the next use of the contaminated solution may occur before enough time has elapsed for all of the organisms to be killed (Hodges, 2002). In this manner living organisms penetrate through an abrasion into the corneal stroma, within the corneal, traces of antimicrobial agents are neutralized by tissue components and the organism finds an excellent culture medium for rapid growth and dissemination through the cornea and the anterior segment of the eye (Fassihi, 2001).

The USP (2004) provides a test for preservative effectiveness; preservative effectiveness as an immediate measure, its adequacy or stability as a function of time must also be ascertained. This is achieved by measuring both chemical stability and preservative effectiveness over a given period of time and under varying

37 storage conditions. Common compounds used as preservatives are discussed below.

2.5.4.1 Quaternary ammonium compounds

Benzalkonium chloride is a quaternary ammonium compound and the most common preservative used in eye drop formulations. Reviews have indicated that it is well suited for use as an eye drop preservative. It has antimicrobial, antiseptic and disinfectant properties (Kibbe, 2006). Benzalkonium chloride is found in 65% of commercial eye drop products as a preservative (Rojanasakul et al., 1992). As a cationic surface-active agent of high molecular weight it is not compatible with anionic compounds, this defines its limitations, despite its broad use. It is incompatible with salicylates and nitrates and inactivated by high-molecular-weight non-ionic compounds (Le Bourlais et al., 1998).

Benzalkonium chloride has excellent chemical stability and desirable antimicrobial characteristics. It is a mixture of alkyl benzyldimethylammonium chlorides ranging from n-C8H17 through n-C16H33. The n-C12H25 homolog content is not less than 40% on an anhydrous basis (Ali et al., 2009). It is one of the most widely used preservatives, at a concentration of 0.01–0.02% w/v. It is used in combination with other preservatives or excipients, particularly, 0.1% w/v disodium edetate, to enhance its antimicrobial activity against strains of Pseudomonas (Rozet et al., 2011).

Benzalkonium chloride occurs as a white or yellowish-white amorphous powder, a thick gel, or gelatinous flakes. It is hygroscopic, soapy to the touch, and has a mild aromatic odour and has a very bitter taste (Kibbe, 2006).

Benzalkonium chloride solutions are active against a wide range of bacteria, yeasts, and fungi. Activity is more marked against Gram-positive than Gram-negative bacteria and minimal against bacterial endospores and acid-fast bacteria. The antimicrobial activity of benzalkonium chloride is significantly dependent upon the alkyl composition of the homolog mixture. Benzalkonium chloride is ineffective against some Pseudomonas aeruginosa strains, Mycobacterium tuberculosis,

Trichophyton interdigitale, and Trichophyton rubrum. However, combined with disodium edetate (0.01–0.1%w/v), benzyl alcohol, phenylethanol, or phenylpropanol, the activity against Pseudonomas aeruginosa is increased. Antimicrobial activity may also be enhanced by the addition of phenylmercuric acetate, phenylmercuric borate, chlorhexidine, cetrimide, or m-cresol. In the presence of citrate and phosphate buffers (but not borate), activity against Pseudonomas can be reduced. Benzalkonium chloride is relatively inactive against spores and moulds, but is active against some viruses including human immunodeficiency virus (HIV). Inhibitory activity increases with pH although antimicrobial activity occurs between pH 4–10 and organisms inhibited at specific concentrations can be seen below in Table 2 (Kibbe, 2006).

Table 2: Typical minimum inhibitor concentrations of benzalkonium chloride (Kibbe, 2006).

Microorganism MIC (µg/mL) Aerobacter aerogenes 64 Clostridium histolyticum 5 Escherichia coli 16 Pseudomonas aeruginosa 30 Salmonella typhosa 4 Staphylococcus aureus 1.25 Staphylococcus pyrogenes 1.25 Vibrio cholera 2

Benzalkonium chloride is hygroscopic and may be affected by light, air, and metals. Solutions are stable over a wide pH and temperature range and may be sterilized by autoclaving without loss of effectiveness. Solutions may be stored for prolonged periods at room temperature. It is incompatible with aluminium, anionic surfactants, citrates, flourescein, hydrogen peroxide, iodides, kaolin, lanolin, nitrates, and non- ionic surfactants in high concentration (Kibbe, 2006).

Other types of quaternary ammonium compounds occasionally used include benzethonium chloride and cetyl pyridinuim chloride. Few quaternary ammonium compounds have been formulated, by attaching soluble high molecular weight polymers (Ali et al., 2009).

2.5.4.2 Parahydroxybenzoic acid esters

Mixtures of methylparaben and propylparaben are sometimes used as ophthalmic antimicrobial preservatives. The concentration of methylparaben used is in the range of 0.1 to 0.2%, while that of propylparaben approaches its solubility in water ( 0.04%). They are not considered efficient bacteriostatic agents and are slow in their antimicrobial action. Ocular irritation and stinging are attributed to their use in eye drop preparation (Furrer et al., 2002).

Propylparaben is widely used as an antimicrobial preservative in pharmaceutical formulations. It may be used alone, in combination with other paraben esters, or with other antimicrobial agents. It is effective over a wide pH range and has a broad spectrum of antimicrobial activity although it is most effective against yeasts and moulds. It is known for its poor solubility; particularly the sodium salt. This may cause the pH of poorly buffered formulations to become more alkaline. Use in ophthalmic formulations is in a concentration range of 0.005–0.01%, Table 3 shows organisms inhibited at various concentrations. Propylparaben is found as a white, crystalline, odourless, and tasteless powder (Rieger, 2006b).

Table 3: Minimum inhibitory concentration for propylparaben in aqueous solution (Rieger, 2006b)

Microorganism MIC (µg/mL) Aspergillus niger ATCC 9642 500 Bacillus subtilis ATCC 6633 500 Candida albicans ATCC 10231 250 Escherichia coli ATCC 8739 500 Klebsiella pneumonia ATCC 8308 500 Pseudomonas aeruginosa ATCC 9027 >1000 Salmonella typhosa ATCC 6539 500 Staphylococcus aureus ATCC 6538P 500 Staphylococcus epidermidis ATCC 12228 500 Trichophyton mentagrophytes 65

Aqueous propylparaben solutions at pH 3–6 can be sterilized by autoclaving, without decomposition. At pH 3–6 aqueous solutions are stable for up to about 4 years at

40 room temperature while solutions at pH 8 or above are subject to rapid hydrolysis (Rieger, 2006b).

Methylparaben may be used either alone, in combination with other parabens, or with other antimicrobial agents. Preservative efficacy is also improved by the addition of 2–5% propylene glycol, or by using parabens in combination with other antimicrobial agents such as imidurea (Rieger, 2006a). In ophthalmic formulations it is used between 0.015–0.2 percent. Methylparaben occurs as colourless crystals or a white crystalline powder. It is odourless or almost odourless and has a slight burning taste (Rieger, 2006a).

Methylparaben exhibits antimicrobial activity between pH 4–8. Preservative efficacy decreases with increasing pH due to the formation of the phenolate anion. Parabens are more active against yeasts and moulds than against bacteria, Tables 3 and 4 show ophthalmic concentrations and organisms inhibited respectively. They are also more active against Gram-positive bacteria than against Gram-negative bacteria. Aqueous solutions of methylparaben, at pH 3–6, may be sterilized by autoclaving at 120 ˚C for 20 minutes, without decomposition. Aqueous solutions at pH 3–6 are stable for up to about 4 years at room temperature, while aqueous solutions at pH 8 or above are subject to rapid hydrolysis (Rieger, 2006a).

Table 4: Minimum inhibitory concentrations of methylparaben in aqueous solution (Rieger, 2006a).

Microorganism MIC (µg/mL) Aspergillus niger ATCC 10254 1000 Bacillus subtilis ATCC 6633 2000 Candida albicans ATCC 10231 2000 Escherichia coli ATCC 8739 1000 Klebsiella pneumonia ATCC 8308 1000 Pseudomonas aeruginosa ATCC 9027 4000 Salmonella typhosa ATCC 6539 1000 Staphylococcus aureus ATCC 6538P 2000 Staphylococcus epidermidis ATCC 12228 2000 Trichophyton mentagrophytes 250

2.6 Efficacy of antimicrobial preservation

Pharmaceutical formulations that do not have adequate antimicrobial activity are protected with antimicrobial preservatives particularly in aqueous formulations. This is to prevent the proliferation or to limit microbial contamination which, during normal conditions of storage and use, particularly for multidose containers, could occur and present a hazard to the patient. The efficacy of an antimicrobial preservative may be enhanced or diminished by the active constituent of the preparation or by the formulation in which it is incorporated or by the container and closure used. The antimicrobial activity of the formulation in its final container is investigated to ensure that its activity has not been impaired by storage. Such investigation may be carried out on samples removed from the final container immediately prior to testing (B.P, 2011).

During development of a pharmaceutical formulation, it should be demonstrated that the antimicrobial activity in the formulation including preservatives provides adequate protection from adverse effects that may arise from microbial contamination or proliferation during storage and use of the preparation. The efficacy of the preservatives on microbial activity can be achieved by tests which consist of challenging the preparation in its final container, with prescribed inoculums of suitable micro-organism, storing the inoculated formulation at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples removed (B.P, 2011).

The preservative properties of the formulation are adequate if, in the conditions of test, there is a significant fall or no increase, as appropriate, in the number of microorganisms in the inoculated product after the times and at the temperatures prescribed (B.P, 2011).

Laboratories can isolate different strains of desired micro-organisms by swabbing infected patients, isolating strains from contaminated food, cosmetic or pharmaceutical products and many other sources. Therefore strains obtained in various manners vary in resistance to antimicrobial chemicals. Strains obtained from human and animals are more resistant to antimicrobial chemicals than those from

42 other sources. Strains derived from contaminated medicines will be more resistant to preservatives. This is because the characteristics of the organism (including its resistance to antimicrobial chemicals) over a period of time changes as a result of mutation and natural selection (Curtin and Cormican, 2003). To get results that are reproducible by a variety of laboratories it is necessary to specify the strain of the organism used for the experiment. The common strains used are cultures of Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus. Methods used to assess the activity of antimicrobial preservatives involve an inoculum of the test organisms which are added to a solution of the product in question. Samples are then removed over a period of time, the preservative is inactivated and the surviving cells calculated (Brooks et al., 2007).

In all cases of measurement of antimicrobial activity it is necessary to standardise and control factors such as the concentration of the test organism, its origin (species and strain employed), the culture medium in which the cells are placed, temperature and the of incubation time of cells after exposure to the chemical (Brooks et al., 2007).

2.7 Packaging

According to the American Society for Testing and Materials, a plastic is a material that contains, as an essential ingredient, one or more polymeric organic substances of large molecular weight, is solid in its finished state and at some stage of manufacture or processing into finished articles it can be shaped by flow (Rabinow & Roseman, 2005). Important mechanical properties required in plastic packaging materials are: tensile strength, impact strength, stiffness, flex resistance, tear strength, coefficient of friction, blocking, fatigue resistance and creep failure (Massey, 2004).

Optical properties are important qualities needed in the plastics as a method of packaging. Some needed characteristics are light transmission, clarity, haze and gloss (Marriot, 2006).

Ophthalmic glass containers with glass droppers have largely been replaced by plastic bottles, only in rare cases are glass containers still in use (Van Santvliet & Ludwig, 2004). Plastic containers are used more often due to their increased resistance to shock, they are lightweight, and they present more options with regard to design opportunities (Jenkins & Osborn, 1993).

Plastic containers used to store ophthalmic products should have the following characteristics:

 Flexibility (ability to deform).  Elasticity (ability to return to its original form after deformation).  Stiffness (resistance to deformation) (Smith and Hui, 2004).

Most plastic containers are shaped with round or oval bases containing substances of 3 to 15 ml (Santvliet & Ludwig, 2004). There are disadvantages in plastic containers, these are:

 Minimal transparency (poor visuals of solutions inside the containers).  Low-density polyethylene allows permeability to vapours and light.  Adsorption of molecules from its contents and cracking due to intensive use (McDonald and Parkin, 1995).

Plastic packaging is not interchangeable with glass (Shah et al., 2008). Plastic packages may contain a variety of extraneous substances such as mould-release agents, antioxidants, and reaction quenchers amongst others which can readily leach out of the plastic and into the contained solution (Tokiwa & Calabia, 2004) and label glues, inks and dyes also have been reported to penetrate (Van Santvliet & Ludwig, 2004). Commercially-produced plastic eye drop bottles are made of polyethylene or polypropylene. They are common containers for various drugs including phenylephrine hydrochloride solutions (Winfield et al., 2009).

2.9 Formulation development

Each formulation and API is unique. Formulation experiments begin with a well- structured formulation plan. The formulation strategy is a result of thorough analysis

44 of the preformulation data report and manufacturing process. The following criteria are to be met in order to begin formulation development:

 Relevant patents have been accessed and investigated.  The appropriate literature search has been sourced.  The regulatory and formulation strategies have been well-known.  The desired APIs have been ordered and received (Al-Shaalan, 2010).

A beneficial approach to formulation development of a generic product is to critically evaluate and where possible, to characterize, the innovator product with respect to composition and other qualitative and or quantitative analyses which may be feasible. A simple approach to achieve the above analysis, is to determine the pH of the innovator drug product dispersed in a small volume of pH adjusted purified water, and then to compare the result with that yielded by a similar dispersion of the trail formulation. The approach is based on the principle that if two dispersions provide comparable pHs’, the excipients compositions of the innovator and generic formulations are probably similar. However caution is needed since this test may not be sufficient to provide all results needed. Initial trials and selection of batches should be done using similar excipients. Possible instability or incompatibility can be overlooked, as long as the same excipients provided in text are used. Small changes in the concentration of key excipients can alter the appearance and physical attributes, while impact on the drug product stability and dissolution can be significant (Miller & Ermer, 2005).

The assessment of the final formulation can be achieved by scaling to require pilot batches, and packaged into all possible configurations intended for future commercialization. The batch is placed into accelerated (40 °C/75%RH) storage conditions for a period of three months. The batches are tested using validated analytical methods, should the batch prove to be stable over the 3 months period of exposure, it would have a high degree of probability that the product is stable and formulae succeeded. It is pivotal to ensure that all desirable characteristics are achieved. The generic drug product must demonstrate the minimum 3 months satisfactory stability, before the following are achieved:

 Development of specifications for both API and the dosage form.  Ordering of the API and excipients for batch manufacturing.  Ordering of all relevant tooling, change parts.  Completion of development report (Ansel et al., 2011).

2.9.1 Validation of HPLC analytical methods

Guidelines provided by the international conference on harmonization are deemed paramount for the registration of pharmaceuticals for human use as they represent the regulatory agencies of the three largest pharmaceutical markets (U.S. Food and drug administration, European Commission for the European Union, and Japanese ministry for Health and Welfare (MHW)). The three regulatory agencies came together to address issues of efficacy, safety, quality and harmonized guidelines. Validation is a basic requirement to ensure quality and reliability of the results for all analytical applications (Miller & Ermer, 2005). It is a requirement by registration bodies like the MCC and FDA that the method be validated. A typical validation technique would be the use of HPLC for its pharmaceutical analysis (Karcher et al., 2005). Physical separation technique employed by HPLC is conducted in the liquid phase in which a sample is separated into its constituent components (or analytes) by distributing between the mobile phase (a flowing liquid) and a stationary phase (sorbents packed inside a column). An online detector used by the HPLC monitors the concentration of each separated component in the column effluent and generates a chromatogram (Cecil & Sheinin, 2005). These detectors are then able to quantify the major components in a purified sample, differentiate among components of a reaction mixture and show trace impurities in a complex sample matrix (Wells, 2002).

Reversed-phase HPLC is the method of choice for stability-indicating assays because the samples are generated in aqueous solutions. The advantages of reversed-phase chromatography are due to convenience, a broad range of samples that can be chromatographed conveniently and the speed of column equilibration is shorter when compared to normal–phase chromatography (Nakashima et al., 2002; Marin et al., 2005; Wong et al., 2006; Palabiyik & Onur, 2007; Heydari, 2008; Das

Gupta & Parasrampuria, 1987; Bastos & de Oliveira, 2009). Most reversed phase HPLC separations are done in the isocratic mode, where the composition of the mobile phase is held constant during the analysis. The external standard method is the most general method for determination of the concentration of a compound in an unknown sample. It involves the construction of a calibration plot using external standards of the analyte. A fixed volume of each standard solution of known concentration is injected onto the column and the peak response of each injection is plotted versus the concentration of the standard solution. The standards are called ‘external standards’ because they are prepared and analyzed separately from the unknown samples. After constructing the calibration plot, the unknown sample is prepared, injected and analyzed in exactly the same manner. The concentration of the analyte in the unknown sample is then determined from the calibration plot (Wong, 2006).

Other types of standards are the internal standard, mass balance and area normalization. The internal standard procedure allows the analyst to identify a region of the chromatogram that is devoid of peaks (quiet region). The analyst then attempts to identify a compound of known purity, that is structurally related to the analyte, and which elutes in the quiet region of the chromatogram. The internal standard should have a relative response factor that is about the same as the analyte (Cecil & Sheinin, 2005).

Reversed phase HPLC has been extensively used for the pharmaceutical analysis of phenylephrine hydrochloride in pharmaceutical dosage forms (Sprieck, 1974; Muhammad and Bodnar, 1980; Chien & Schoenwald, 1985; Zeise et al., 1996; Muszalska et al., 2000; Goicoechea & Olivieri, 2001; Savic et al., 2008; Samadi– Maybodi & Nejad–Darzi, 2009; & Al-Shaalan, 2010).

2.9.1.1 Stability indicating HPLC analysis

Analytical procedures used for the assay of the API alone or in the final product during stability studies should be stability indicating. A stability indicating assay is one that accurately quantifies the API without interference from degradation products, process impurities, excipients, or other potential impurities. Samples are

47 obtained by placing the pure API under stress intentionally, for example, by subjecting the API to acid, base, heat, light or oxidation. This process is often called forceful degradation or purposeful degradation. Usually, the goal is to degrade the parent API by 10–20%. Degradation much greater than 10–20% could result in secondary degradants that will complicate the development process (ICH Harmonised Tripartite Guideline Q2A, 1994).

In the mass balance approach, all impurities are quantified and subtracted from the absolute API value of 100%. This approach will result in a purity value that, if all impurities are accounted for, is more accurate than the external or internal standard methods. However, the ability to identify all impurities in a given drug substance may require the use of hyphenated detection techniques and could be extremely costly to complete on a regular basis. A related approach is called area normalization, and is often used where the majority of the impurities can be identified and quantified in a single chromatogram. All of the impurities would be assumed to have the same relative response factor as the parent drug. The quantification of the individual impurities would be reported as a percentage of the parent drug rather than an absolute value in milligrams (Cecil & Sheinin, 2005).

2.9.1.2 Choice of analytical column and conditions

The C18 (2)-bonded phase [-CH2-(CH2)16-CH3] is the separation material contained within columns used in the early development of HPLC. It is available in a wide variety of forms and differs based on for example, silica type, pore size, surface area. As most laboratories with HPLC equipment will have a C18 column available, it is the first choice for initial experiments. In addition, with the broad range of applications accessible in the literature or from commercial sources, it is often easy to find a separation that is similar, allowing for selection of mobile phase conditions that are likely to be suitable for solving a particular analytical problem. C18 columns were utilised most prevalently in the analytical HPLC methods sourced in the literature for the quantitative determination of phenylephrine hydrochloride solutions.

The advantages noted for the C18 (2)-bonded are flexibility in pH, ligand stability at acidic pH and hydrolytic stability of the bonded phase based on a bi silane at low pH

48 which is five times higher than that of a monofunctional bonded phase (Saettone et al., 1980; O’Donnell & Gillibrand, 1995; Palabiyik & Onur, 2007).

An ion-pairing agent is commonly used with reversed–phase chromatography as it is a better alternative to ion–exchange chromatography for the separation of ionic species. The most common ion–pairing agents are the tetrabutylammonium salts and alkylsulphonic salts (octane-1-sulfonic acid sodium salt). Properties of the two are as follows:

Tetrabutylammonium salts are:

 Buffered to pH 7.5.  Form ion pairs with strong and weak acids.  Suppresses weak base ions.

While alkylsulphonic salts are:

 Buffered to pH 3.5.  Form ion–pairs with strong and weak bases.  Buffering suppresses weak acid ions.  The longer the alkyl chain, the greater is the retention time (Florence and Attwood, 2006).

Ion–pairing agents and pH increase the retention time with increasing concentration of ion–pairing agent. The true origins of the ions which pair with drug ions is not clear, but there is evidence that ion–pair formation aids absorption. Ion–pairing provides the interaction between a drug ion and an organic ion of opposite charges to form an absorbable neutral species. The formation of ion pairs will depend on solvent–ion interactions: hydrophobic ions might be encouraged to form ion pairs by the mechanism of water-structure enforced ion–pairing in which water attempts to minimize the disturbance on its structuring, and achieves this end by reducing the polarity of the species in solution by ion–pair formation (Florence and Attwood, 2006).

2.9.1.3 Steps for HPLC method validation

Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of pharmaceutical substances and products. The process of validating a method cannot be separated from the actual development of the method conditions, because the developer will not know whether the method conditions are acceptable until validation studies are performed. Results of validation studies may indicate that a change in the procedure is necessary, which may then require revalidation. During each validation study, key method parameters are determined and then used for all subsequent validation steps. The method developed for this study was validated according to the International Conference on Harmonization guidelines and the following were determined for the HPLC method:

 Specificity.  System suitability.  Linearity.  Accuracy.  Precision.  Limit of detection.  Limit of quantification.  Range (ICH Harmonized Tripartite Guideline Q2A, 1994).

2.9.1.4 Linearity

Linearity of an analytical procedure is its ability, within a given range, to obtain test results that are directly proportional to the concentration of analyte in the sample (ICH Harmonized Tripartite Guideline Q2A, 1994). The requirements for linearity are that the correlation coefficient of the regression line must be greater than or equal to 0.9999 and that the y-intercept must not be significantly different from zero (ICH Harmonized Tripartite Guideline Q2A, 1994).

2.9.1.5 Accuracy and precision

Accuracy of an analytical procedure expresses the closeness of agreement between the value that is found and the value that is accepted (either as a conventional true value or an accepted reference value). Precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions (ICH Harmonized Tripartite Guideline Q2A, 1994). The requirement for accuracy is that the percentage recovery of API for each solution prepared must be within the 98.00 to 102.00% limit. The requirement for precision is that the relative standard deviations at any one concentration must be less than or equal to 2.00%.

2.9.1.6 Limit of detection and limit of quantification

The limit of detection (LOD) is the lowest analyte concentration that produces a response detectable above the noise level of the system and the limit of quantification (LOQ) is the lowest level of analyte that can be accurately and precisely measured (Kupiec et al., 2004). The LOD is thus the lowest concentration for which the relative standard deviation of multiple injections is less than 5.0%. By convention, the LOD value is taken as 0.3 times the LOQ (Thomsen et al., 2003).

2.9.1.7 Range

Range of an analytical procedure is the interval between the upper and lower concentrations of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of linearity, accuracy and precision (ICH Harmonized Tripartite Guideline Q2A, 1994).

2.9.1.8 Specificity

Specificity of an analytical procedure is its ability to assess, unequivocally, the analyte in the presence of components that may be expected to be present (ICH Harmonized Tripartite Guideline Q2A, 1994). Specificity suggests that a given

51 analyte peak is indeed the desired analyte structure and is 100% pure (Krull & Swartz, 2001). The peak purity results from the photodiode-array analysis must show that the phenylephrine hydrochloride peak is pure. In other words the purity angle must be less than the threshold angle. Depending on the wavelength, a tungsten lamp and a deuterium lamp are used as light sources. The polychromatic light beam is focused on a flow-cell and subsequently dispersed by a holographic grating or quartz prism. The spectral light then reaches a chip that contains 100 to 1000 light- sensitive diodes arranged side by side. Each diode only registers a well-defined fraction of the information and in this way all wavelengths are measured at the same time. At the end of the run, a three-dimensional spectrochromatogram (absorbance as a function of wavelength and time) is stored on the computer and can be evaluated qualitatively and quantitatively. Peak identity and peak homogeneity (peak purity) can be investigated by analyzing spectrum collected during chromatographic seperations and detection, a pure compound will produce a peak with spectra that have the same shape across the peak. In contrast any interefence from coeluting analytes will produce composite spectra with varying degrees of spectra dissimilarities across the peak. This is the basis of peak purity index. Diode array detection offers the advantage that knowledge of the spectra of compounds of interest enables interfering peaks to be eliminated such that an accurate quantification of peaks of interest can be achieved despite less than optimal resolution. Peak purity is of the utmost importance in the quality control of pharmaceutical products (Kupiec et al., 2004).

Figure 4: Diagram of a typical HPLC-UV absorbance peak and plots of noise (or threshold) and purity angles (Krull & Swartz, 2001). Figure 4 above shows a typical HPLC-UV absorbance peak with plots of noise and purity angles. Stress testing is undertaken to demonstrate specificity when

52 developing stability indicating methods (Reynolds et al., 2002). The API and finished products will be subjected to stress studies in order to force phenylephrine hydrochloride degradation and thereby verify or exclude the presence of co-eluting impurities or degradation products in the mobile phase, solvent or unstressed / stressed solutions.

Impurities should be between 10 – 20 % of the API. This should include samples stored under relevant stress conditions such as: light, heat, humidity, acid/base hydrolysis and oxidation. Peak purity tests are used to show that the analyte’s chromatographic peak is not attributable to more than one component (ICH Harmonized Tripartite Guideline Q2 (R1), 2005).

2.9.2 Active-excipient compatibility studies

Impurities can be formed in pharmaceutical products as a result of an interaction between the active ingredient and excipient introduced by formulation. In an ideal product, the excipient used in the formulations should not interact with the drug substance or introduce unwanted substances capable of accelerating the formation of new impurities. Incompatibility found between drugs and excipients in pharmaceutical products can alter the stability and bioavailability of drugs, thereby affecting its safety and efficacy. Stress studies of the active-excipients mixture generate high amounts of degradation products which can be identified using various analytical procedures even though the potential degradation products are in low concentrations (Douša et al., 2011).

Binary mixture compatibility testing is a commonly used method. In this approach, binary (1:1 or customized) mixtures of the drug and excipient, with or without, added water and sometimes compacted or prepared as slurries, are stored under stressed conditions (also known as isothermal stress testing (IST)) and analyzed using a stability-indicating method, e.g. HPLC. The water slurry approach allows the pH of the drug-excipient blend and the role of moisture to be investigated. Alternatively, binary mixtures can be screened using other thermal methods, such as differential scanning calorimetry (DSC) (Clas et al., 1999).

DSC is a technique that is used extensively in the field of pharmaceutics. The main benefit of DSC, compared to stress storage methods, is its ability to quickly screen potential excipients for incompatibilities derived from the appearance, shifts or disappearances of peaks and/or variations in the corresponding peak. DSC is mostly used to show instability resulting from solid-solid interactions. Another feature of DSC possesses is low sample consumption making it an attractive method. Although DSC is unquestionably a valuable technique, interpretation of the data may not be straightforward. In this method, the sample is exposed to high temperatures (up to 300 °C or more), which in reality is not experienced by the dosage form. Thus, DSC results should be interpreted carefully, as the conclusions based on DSC results alone can be often misleading and inconclusive. The results obtained with DSC should therefore always be confirmed with isothermal stress testing, IST (Giron, 1998).

Isothermal stress testing involves storage of drug-excipient blends with, or without moisture, at elevated temperatures for a specific period of time, typically 3–4 weeks to accelerate drug degradation and interaction with excipients. The samples are then visually observed for any changes in color or physical characteristics, and the drug content, along with any degradants, is determined quantitatively. Although more useful, the disadvantage with this method is that it is time consuming and requires quantitative analysis using e.g. HPLC (Verma & Garg, 2004).

Ideally, both techniques, DSC and IST should be used in combination for the selection of excipients. That was not the case in this research study and only IST was used as it could satisfactorily indicate the results.

2.10 Determining formulation stability study

Stability, with respect to drug dosage form, refers to the chemical and physical integrity of the dosage unit and the ability of the dosage unit to maintain protection against microbiological contamination. The shelf life of the dosage form is the time lapse from initial preparation to the specified expiration date. The product within its shelf life must fulfil the monograph specification for identity, strength, quality, and

54 purity. Stability parameters of a drug dosage form can be influenced by environmental conditions of storage (temperature, light, air, and humidity) as well as packaging (USP, 2004).

Stability studies on dosage forms should be conducted by means of specific temperatures and relative humidities representing storage conditions experienced in the distribution chain of climatic zones of South Africa (climatic zones I and II). Stability studies performed on eye drops in the pharmaceutical industry follow an integral part of a formulation development programme. The standardized approach applied to the formulation or design of the eye drop is similar to those applied to other dosage forms, which are:

 Selection of batches.  Test procedure and criteria.  Specification storage test conditions.  Testing frequency.  Packaging material.  Evaluation statements and labelling (Miller–Meeks et al., 1991).

Stability study is one of the most important areas, in relation to the registration of pharmaceutical products, as it predicts shelf life and storage instructions for batches. It also determines degradation of products, mechanism of breakdown and conditions under which the breakdown occurs. With the help of stability studies, any parameter subject to change within the eye drop during storage can be measured, such as appearance, pH, viscosity and density, (where relevant), solubility time (reconstitution and appearance thereof) sterility, preserving ability and preservative content (where relevant). Tests are also performed to ensure compatibility between the container-closure system and the product. Stability testing is the cornerstone of drug development or formulation (Jeffs, 2009).

During storage, one or more of the following changes may take place:

a) Chemical interaction involving drug, excipients or container, or many combinations of these. b) Alteration of physical form.

c) Mould or bacterial growth commonly referred to as biological change.

Their effect may be to make the preparation unusable on account of:

i. Loss of active agent. ii. Development of a toxic decomposition product. iii. Poor aesthetics to the patient.

Chemical change involves the drug itself because this is normally the most reactive component of the system. Occasionally, it doesn’t involve the drug but is limited to the excipient and/or the container for example the rusting of cans or flaking of glass, this thus gives plastics added advantage. A hazardous occurrence within plastic is the extraction of substances from the walls of plastic containers. Other chemical changes, such as discoloration, may be harmless and quantitatively negligible but could have serious effect on the acceptability of the product. Chemical change occurs when stimulated by heat light, moisture and aeration. Thus antioxidants, buffers and chelating agents are commonly added to offer protection against light and moisture. Physical change may make a product inconvenient or impossible to use and occasionally can lead to danger to patient. The growth of micro-organisms can cause spoilage either by their appearance or because they have induced significant chemical change (Huynh-Ba, 2009).

3. METHODOLOGY

3.1 HPLC method validation

3.1.1 Equipment

The HPLC system consisted of a complete FPLC Shimadzu® HPLC system which has a SPD-M20A Prominence diode array detector, SIL-20A Prominence auto- sampler, DGU-20A5 Prominence degasser, LC-20AB Prominence liquid chromatography and CTO-10AS vp Prominence column oven ( Shimadzu, Tokyo, Japan). The stationary phase consisted of a reverse phase Phenomenex® Luna C18 (2) column 250 mm × 4.60 mm, 5 μm particle size (Separations, Johannesburg, SA).

3.1.2 Materials and reagents

Phenylephrine hydrochloride, sodium citrate dihydrate, boric acid, disodium ededate, sodium metabisulphite, and benzalkonium chloride were kindly donated by Aspen Pharmacare (Port Elizabeth, SA). Water for chromatography was produced by an Ultra Clear TWF/El-Ion® system which was pre-treated and made ultrapure (reverse osmosis) (Separations, Johannesburg, SA). HPLC grade methanol and octane-1- sulfonic acid sodium salt were obtained from Sigma-Aldrich (Pty) Ltd (Kempton Park, SA). Analytical/technical grades of sodium hydroxide pellets, carboxy methylcellulose sodium, hydroxylpropyl methylcellulose, glycerol, hydrochloride acid solution 33% and pyrophosphoric acid (phosphoric acid) were obtained from Merck Laboratory Supplies (Pty) Ltd (Midrand, SA) and hydrogen peroxide 30% was sourced from Saarchem (Pty) Ltd (Johannesburg, SA).

3.1.3 Mobile phase preparation and standard curve construction

A mass of 1.1 g of octane-1-sulfonic acid sodium salt was dissolved in a Schott bottle that contained one litre of a mixture of methanol and water (1:1). The pH was adjusted to 3.0 with pyrophosphoric acid. The resulting solution was mixed, degassed by ultrasonication (Ultrasonic LC 130, Labotec, Germany) and vacuum filtered through a 0.45 μm Millipore filter (Millipore Corporation, Bedford,

Massachusetts, USA) prior to use. The dilution solvent was prepared by mixing HPLC grade methanol and water in a 1:1 ratio and adjusted to a pH of 3 with pyrophosphoric acid.

The standard curve: Phenylephrine hydrochloride stock solution was prepared by accurately weighing 2 mg of phenylephrine hydrochloride material into a 100 ml volumetric flask, dissolving it in dilution solvent and making up to volume with dilution solvent. Calibration standards containing 0.0125, 0.025, 0.05, 0.075, 0.1 and 0.15 mg/ml were prepared by making appropriate solvent dilutions of the working stock solution. Five millimetre of calibration standard was filtered through a 0.45 μm Millex® Syringe Driven Filter Unit prior to injection

3.1.4 Chromatographic conditions

The flow rate was set at one millimetre per minute. The column temperature was set at its temperature of 40 °C and column back-pressure varied between 2800 to 3000 psi. A PDA was used, therefore all runs were analysed at wave length, 280 nm. The injection volume was 20 μl (USP, 2004). The assay was performed utilizing a reverse phase Phenomenex® Luna C18 (2) column (250 mm × 4.60 mm, 5 μm particle size).

To identify the phenylephrine hydrochloride, the retention times of the peaks were noted and to quantitate the amount of the API, values found of AUC (peak area) which is computer-stored and generated was used. A graph of the concentrations vs. peak area was plotted. Equation of the line was calculated using Microsoft Excel® program. With the above, the following were determined: specificity, system suitability, linearity, accuracy, precision, limit of detection/limit of quantification and range.

3.1.5 Linearity

A 2 mg/ml stock solution was prepared and diluted to concentrations of 0.0125, 0.025, 0.05, 0.075, 0.1 and 0.15 mg/ml using the dilution solvent. The concentration of 0.1 mg/ml was defined as 100% while 0.15 mg/ml was taken to be 150%. Each of the concentrations was assayed in triplicate. The calibration curve was constructed

58 by plotting the peak areas of phenylephrine hydrochloride versus the respective phenylephrine hydrochloride concentrations and a linear regression trend line was fitted to the plot on Microsoft Excel® 2007, (Microsoft Corporation).

3.1.6 Accuracy and precision

Accuracy and precision were determined by replicate injection (n=3 and 6, respectively) of three phenylephrine hydrochloride solutions, at the upper, middle, and lower limits of the concentration range studied. The accuracy of a measurement system is the degree of closeness of measurements of a quantity to that quantity's actual (true) value (Green, 1996). The precision of a measurement system, also called reproducibility or repeatability, is the degree to which repeated measurements under unchanged conditions show the same results (ICH Harmonized Tripartite Guideline Q2A, 1994). The concentration ranges were 0.0095 mg/ml (lower limit), 0.054 mg/ml (middle limit) and 0.138 mg/ml (upper limit). The theoretical concentrations were calculated from the linear regression curve, and compared to the actual concentrations tested. The actual mean concentration and standard deviation were calculated at each theoretical concentration. The mean concentrations and percentage recovery of phenylephrine hydrochloride obtained for the replicate injections were a measure of the accuracy of the method, whilst the relative standard deviations at any one concentration provided a measure of precision. The requirement for accuracy is that the percentage recovery of phenylephrine hydrochloride for each solution prepared must be within the 98.00 to 102.00% limit. The requirement for precision is that the relative standard deviations at any one concentration must be less than or equal to 2.00%.

3.1.7 Limit of detection and limit of quantification

Sensitivity of the method was determined by means of the detection limit (LOD) and quantification limit (LOQ). Calculations for LOD were based on the standard deviation of the calibration curve (σ) and the slope of curve (S), using the equation LOD = 3.3 × σ divided by S. LOQ was calculated using the equation LOQ = 3.33 multiplied by LOD (ICH Harmonized Tripartite Guideline Q2 (R1), 1994). Standard solutions of decreasing concentration were produced by successive dilution of the

59 lowest calibration standard and the resulting solutions were injected in triplicate. A volume or concentration of 0.01 ml, the least calibration standard concentration was diluted 10 times.

3.1.8 Range and system suitability

The range was determined by observing the interval between the upper and lower concentration of phenylephrine hydrochloride for which the HPLC analytical assay has suitable level of linearity, accuracy and precision (ICH Harmonized Tripartite Guideline Q2 (R1), 1994).

System suitability is a measure of the performance and chromatographic quality of the total analytical system, namely instrumentation and procedure (ICH Harmonized Tripartite Guideline Q2B, 1994). Six replicate injections of the standard solution of phenylephrine hydrochloride were performed. The requirements for system suitability are that the percentage relative standard deviation of the peak responses due to for six injections must be less than or equal to 1.0 %. The tailing factor of the peak must not be more than 2.0. A peak is labeled as tailing or asymmetrical when it deviates from the ideal, symmetrical shape of a Gaussian peak. The later-eluted half of the peak is wider than the front half and the broadening appears to be emphasized near the baseline. Peak tailing is measured using the USP tailing factor (Tf) (Dolan,

2003). The tailing factor is expressed by: Tf = a + b 2a where Tf = USP tailing factor a = front half-width measured at 5 % of peak height b = back half-width measured at 5 % of peak height (USP, 2004).

3.1.9 Specificity

Stress testing of API and finished product is undertaken to demonstrate specificity when developing stability indicating methods (Reynolds et al., 2002). The API and finished products were stressed in order to force phenylephrine hydrochloride degradation. This would verify or exclude the presence of co-eluting impurities or degradation products arising from the mobile phase, solvent, unstressed and

60 stressed products. The products are referred to as product I, II, III, IV, V and were formulated as indicated in Table 5 (section 3.3.2). The nature of the specificity samples and the relevant stress conditions are listed below:

All five products and the phenylephrine hydrochloride were subjected to the following stress conditions after which they were analysed:

 0.2 M NaOH for 30 minutes (reflux system).  0.2 M HCl for 30 minutes (reflux system).

 0.2 M H2O2 for 30 minutes (reflux system).  UV lights (17 hours inside a stability chamber).  100 °C (24 hours inside a stability chamber).  65 °C (1 month inside a stability chamber).  40 °C / 75% RH (1 month inside a stability chamber).  Unstressed batch of phenylephrine hydrochloride and products I–V.

A quantity of 10 mg of unstressed phenylephrine hydrochloride was dissolved in 100 ml of dilution solvent and analysed using the HPLC method being validated. A volume of 0.3 ml of unstressed products I–V was dissolved in 10 ml dilution solvent and analysed using the HPLC method being validated.

A mass of 10 mg of phenylephrine hydrochloride was diluted to 100 ml with 0.2 M HCl, 5 ml of this mixture was diluted to 25 ml with 0.2 M HCl and then refluxed for 30 minutes; the same manipulation was applied when using NaOH and H2O2. A measured volume of 0.5 ml from product I–V was diluted to 100 ml with 0.2 M HCl and refluxed for 30 minutes, 10 ml of this was diluted to 100 ml using the dilution solvent, filtered and analysed using the HPLC method being validated.

Phenylephrine hydrochloride and products I–V were stressed in a stability chamber (Binder, SA) which emitted both UV and visible light through a window glass filter (type 2) and conformed to the requirements of the ICH guidelines. The stability chamber had an irradiance level of 318 watts/m2 in order to expose the samples to an overall illumination of not less than 1.2 million lux hours and an integrated near

UV energy of not less than 200 watt hours/m2 all according to ICH Harmonized Tripartite Guideline Q1B, (2005). All impurities and degradation products should be between 10 – 20 % of the API. This includes samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation (ICH Harmonized Tripartite Guideline Q2 (R1), 2005). Degradants and impurities will be labeled as unknown (Ω) and if more than one is present will be denoted as Ωi, Ωii, Ωiii and so on.

3.2 Determination of active–excipient compatibility

Active-excipients mixtures were achieved by mixing in a Schott beaker accurately weighed 10 mg of phenylephrine hydrochloride and 10 mg of excipient, both were dissolved in 10 ml ultra-pure water and transferred into plastic eye drop bottles. They were stored at accelerated conditions for one month. A quantity of 5 ml was filtered and analysed in triplicate using the validated HPLC method. The samples were analysed before and after storage. The potency of the phenylephrine hydrochloride was then calculated as a percentage of the initial potency. Significant change would be defined as a 5% potency loss from the initial assay value of the API (Thakur et al., 1999).

The assayed results of the active-excipient mixture were calculated on the percentage of phenylephrine hydrochloride remaining in each sample from the linear regression curve, y = 8541.1x + 438.55, taking the initial assay of phenylephrine hydrochloride in each vial to be 100%. Physical stability was analyzed by visually assessing the appearance and colour of the sample contents.

3.3 Manufacture of products

An important factor for eye drops is that they should be sterile when dispensed in a multiple-application container. The preservative should be effective against accidental introduction of micro-organisms and contaminants. The formulations were manufactured in a clean environment in a laminar-flow hood using aseptic technique.

3.3.1 Materials

The hotplate magnetic stirrer utilized during the manufacturing process was a Heidolph® MR 3002 (Labotec®, Midrand, SA), the storage chamber (BINDER® KMF series, BINDER Inc, New York, USA) and the autoclave (Hirayama, New York, USA), Natural dropper bottle 15 ml (LDPE) polyethylene container with closure component (plugs with cap).Eeye drop bottles 13 MM CTRL dropper tip 0.031 needle natural (Amcor pharmaceutical packaging, New Jersey, USA), beakers and graduated glass bottles (SCHOTT® North America, Inc. New York, USA), membrane filter paper 0.45 µm (Millipore Corporation, Bedford, Massachusetts, USA), alcohol preparation swabs WEBCOLTM, plastipak syringes (10 and 5 mL), HiCare int. latex gloves large, avacare hypodermic needles (green) 21 g, alcohol 70% (particle free) were all supplied by Alpha Pharm. Pty (Ltd), Port Elizabeth, South Africa.

3.3.2 Product manufacture

1 M Sodium Hydroxide: A mass of 40 g of sodium hydroxide was mixed in a round bottom flask with one liter of purified water. Hydroxypropyl methylcellulose (0.3%): A mass of 3 g of HPMC grade E5 was dispersed in a beaker with 150 ml of purified water at 90 °C, when thoroughly hydrated; 850 ml of ice water was added and stirred until a clear homogenous liquid was formed. Sodium carboxy methylcellulose (0.2%): A mass of 2 g of SCMC was dispersed in a beaker with 150 ml of purified water at 90 °C, when thoroughly hydrated; 850 ml of ice water was added and stirred until a clear homogenous liquid was formed. The summary of the formulation excipients, API and concentration can be seen below in Table 5.

Table 5: Formulation summary of active pharmaceutical ingredient and excipients used in the manufacturing of products I–V

Lot Size: 1L Material Product Product Product Product Product I II III IV V Phenylephrine Hydrochloride 10% w/v 10% w/v 10% w/v 10% w/v 10% w/v Sodium citrate 0.1% w/v 0.1% w/v - - - Dihydrate Sodium - 1% w/v - - - Metabisulfite Boric Acid - - - 1.9% w/v - Benzalkonium 0.1% v/v 0.1% v/v - - - Chloride Hydroxypropyl methylcellulose 0.3% w/v 0.3% w/v - - 0.3% w/v QS to Methyl hydroxybenzoate - - 0.18% w/v - 0.18% w/v Propyl hydroxybenzoate - - 0.02% w/v - 0.02% w/v Sodium carboxy Methylcellulose - - - 0.2 % w/v - to Water Purified (RO) 10% v/v 10% v/v 10% v/v 10% v/v 10% v/v Ethylenediaminetetraacetic - - - 0.1% w/v - Acid Glycerol to - - 100% v/v -

1 M Phosphoric acida a a

1 M Sodium Hydroxidei i i i i and a – For pH adjustment only, ( - ) means it was absent from the formulation

3.3.2.1 Sterilization for heat sensitive API and exipients

 The API and excipients were sterilized as follows: The heat sensitive PE was steamed in a water bath at 100 °C for 30 minutes. Autoclave is not recommended as phenylephrine hydrochloride has tendency to decompose on heating.  All other equipments and excipients were autoclaved as follows: 121 °C for 15 minutes at 1 atm (200 kPa or 15 psi).  After sterilization the bottles containing products were shaken every 15 minutes until cool, to make sure HPMC and SCMC stayed fully mixed and hydrated.

3.3.3 Manufacturing methods for products I–V

The manufacturing processes followed the same general method, with slight variations in the processes based on certain excipeints. The few exceptions are seen with the preservatives methyl and propyl hydroxybenzoate. Ingredients were added

64 sequentially to the main portion of solution while mixing with the glass rod. The formulation was mixed until visually homogenous before addition of the next ingredient. The methyl and propyl hydroxybenzoate were dissolved in ultra-pure water with the aid of gentle heat and mixing with a glass rod. Addition of the dissolved preservatives to the bulk solution was done to ensure complete quantitative transfer of all preservatives to the bulk formulation. The solution was then transferred to a calibrated plastic bottle, the dropper inserted and the bottle capped. The stability of the formulations was determined in the primary plastic packaging. The laboratory scale manufacturing processes for the five products (products I–V) can be seen in figures 5 to 9.

1) Ultra pure water 10% v/v

2) Disperse and mix with a glass i) Phenylephrine hydrochloride 10% w/v rod until all dissolve

ii) Sodium citrate dihydrate 0.1% w/v

3) Filtered and pasteurized at Benzalkonium chloride 0.1% v/v 100 °C for 30 minutes in HPMC 0.3% w/v to 100% water bath

4) Cooled to room iii) Disperse and mix with a glass temperature of 25 °C rod until all dissolve

5) Aseptically and iv) Autoclave mixture at 121 °C for quantitatively mixed 15 minutes

6) A volume of 15 ml was aseptically and quantitatively transferred to the calibrated plastic eye-drop bottle which

was capped.

Figure 5: Laboratory scale 1000 ml manufacturing process of product I

1) Ultra pure water 10%v/v

i) Phenylephrine hydrochloride 10% w/v 2) Disperse and mix with a glass rod until all dissolve

ii) Sodium citrate dihydrate 0.1% w/v

Sodium metabisulfite 0.1% w/v 3) Filtered and pasteurized at

100 °C for 30 minutes in HPMC 0.3 % w/v to 100% water bath

4) Cooled to room iii) Disperse and mix with a glass temperature of 25 °C rod until all dissolve

5) Aseptically and quantitatively mixed iv) Autoclave mixture at 121 °C for 15 minutes

6) A volume of 15 ml was aseptically and quantitatively transferred to the calibrated plastic eye-drop bottle which was capped

Figure 6: Laboratory scale 1000 ml manufacturing process of product II

1) Ultra pure water 10% v/v

2) Disperse and mix with a i)Phenylephrine hydrochloride 10% glass rod until all dissolve w/v

ii)Methyl hydroxy benzoate 0.18% w/v 3) Filtered and pasteurized at Propyl hydroxy benzoate 0.02% w/v 100 °C for 30 minutes in water bath Glycerol to 100% v/v

iii) Disperse and mix with a glass 4) Cooled to room temperature of 25 °C rod until all dissolve

5) Aseptically and iv) Autoclave mixture at 121 °C for quantitatively mixed 15 minutes

6) A volume of 15 ml was aseptically and quantitatively transferred to the calibrated plastic eye-drop bottle which

was capped.

Figure 7: Laboratory scale 1000 ml manufacturing process of product III

1) Ultra pure water 10% v/v

2) Disperse and mix with a glass i) Phenylephrine hydrochloride 10% rod until all dissolve w/v

ii) Boric acid 1.9% w/v 3) Filtered and pasteurized at EDTA 0.1% w/v 100 °C for 30 minutes in water

bath SCMC 0.2% w/v to 100%

4) Cooled to room iii) Disperse and mix with a glass rod temperature of 25 °C

until all dissolve

5) Aseptically and iv) Autoclave mixture at 121 °C for 15 quantitatively mixed minutes

6) A volume of 15 ml was aseptically and quantitatively

transferred to the calibrated plastic eye-drop bottle which was capped.

Figure 8: Laboratory scale 1000 ml manufacturing process of product IV

1)Ultra pure water 10% v/v

i)Phenylephrine hydrochloride 10% w/v 2) Disperse and mix with a glass rod until all dissolve

ii) Methyl hydroxy benzoate 0.18% w/v 3) Filtered and pasteurized at Propyl hydroxy benzoate 0.02% w/v 100 °C for 30 minutes in water bath HPMC 0.3% w/v to 100%

iii) Disperse and mix with a glass rod 4) Cooled to room until all dissolve temperature of 25 °C

5) Aseptically and iv) Autoclave mixture at 121 °C for quantitatively mixed 15 minutes

6) A volume of 15 ml was aseptically and quantitatively transferred to the calibrated

plastic eye-drop bottle which was capped.

Figure 9: Laboratory scale 1000 ml manufacturing process of product V

3.4 Stability Tests

Testing for stability is crucial in product development. It usually involves at least two stages: firstly, accelerated tests on a prototype and secondly, storage under probable conditions of the manufactured product. In SA, the submission of stability data is compulsory before sale is permitted, result from accelerated stability studies are usually acceptable for this purpose (Zahn, 2008).

Stability studies on the dosage forms were conducted at specific temperatures and relative humidities representing storage conditions experienced in the climatic zones

69 of South Africa (climatic zones I and II). The formulations were stored at 40 °C/75%RH, 25 °C/40%RH and 30 °C/65%RH. The five formulations were stored at all three of the above mentioned conditions and tested in triplicate at 0, 3, and 6 months. The most appropriate and stable formulation was chosen based on the level of degradation of the phenylephrine hydrochloride, pH change and appearance.

3.5 Qualitative and quantitative analysis of the formulations

3.5.1 Appearance and pH

The bulk appearance of the prepared products was visually examined for colour, homogeneity, and for the appearance of any precipitate. The pH of the solutions was measured using a pH/mV/ °C meter (744 pH meter, metrohm). Each solution was stirred with a magnetic stirrer (Hanna instruments supplied by Tecnilab, SA) for 30 seconds before pH measurement.

3.5.2 Phenyleprine hydrochloride concentration

The unknown peaks have to be less than or equal to 1% while the phenylephrine hydrochloride left must be between 95%–105% to be accepted as a successful formulation. The mean peak area was divided by the theoretical amount of drug found in the formulation multiplied as a percentage to give the final result of phenylephrine hydrochloride left.

3.6 Test for preservative efficacy

The test challenged the formulation in its final primary container, with a prescribed inoculum of suitable micro-organisms, storing the inoculated product at a prescribed temperature, withdrawing the samples from the container at specified intervals of time and counting the organisms in the samples removed. The two methods used for determining bacterial numbers were the standard (viable) plate count method and spectrophotometric (turbidimetric) analysis. The standard plate method reveals information related to live bacteria while indirectly measuring cell density. The

70 spectrophotometric method is based on turbidity and indirectly measures all bacteria (cell biomass), dead or alive (Harley and Prescott, 1998).

3.6.1 Procedure for standard plate count

Materials: Cultures of Escherichia coli (ATCC No. 38218), Candida albicans (ATCC No. 66027), Pseudomonas aeruginosa (ATCC No. 27853), Staphylococcus aureus (ATCC No. 43300) were obtained from the Department of Biomedical Technology at the Nelson Mandela Metropolitan University. The bacteria were chosen because of their classification as well as pathogenicity. Pipettes, petri plates, curvettes (Hellma®, precision cells, Lasec, Johannesburg, SA) were obtained from Lasec (Johannesburg, SA). Bunsen burner, platinum loop wire dispensers (1/200 mL) (Lasec, Johannesburg, SA), water was produced by Ultra Clear TWF/El-Ion® system which was pre-treated and made ultrapure (reverse osmosis) (Separations, Johannesburg, SA). Tryptone soya broth and agar, Sabouraud broth and agar were supplied by Sigma-Aldrich (Pty) Ltd (Kempton Park, SA). Sodium chloride was obtained from Merck Laboratory Supplies (Pty) Ltd (Midrand, SA). Hockey stick was obtained from Lasec (Johannesburg, SA). Tecam® SB-16 Shaking Water bath was obtained from Spellbound Laboratory solutions (Port Elizabeth, SA). Vacutec (Johannesburg, SA) supplied the P Spectra colony counter, Shimadzu® UV spectrophotometer (UV-1800) (Shimadzu, Tokyo, Japan).

Media preparations: All broths and agars poured into 1000 ml Schott bottles with a screw cap and autoclaved at 121 °C for 15 minutes. Tryptone soya broth was made by weighing 25 g and dissolving 1000 ml of distilled water. Tryptone soya agar was made by weighing 40 g which was dissolved in 1000ml of distilled water. Sabouraud broth was made by weighing 30 g which was dissolved in 1000 ml of distilled water. Sabouraud agar was made by weighing 45 g which was dissolved in 1000 ml of distilled water.

3.6.2 Procedure for plating the bacteria and fungi

All Petri dishes and saline bottles were labelled accordingly. Using aseptic technique, 0.1 ml was added to a 9.9 ml sterile saline and was shaken vigorously to

71 distribute the bacteria. A volume of 0.1 ml of this was aseptically transferred to a second 9.9 ml sterile saline, it was then capped and the second dilution was shaken vigorously. The process was repeated until 10-8 dilution was attained. Volumes of 0.1 ml and 0.01 ml from each dilution of microbes were aseptically transferred to petri plates. The tryptone soya agar at 48 °C was aseptically poured into a petri dish. The agar and sample were immediately mixed by gently moving the plate in a figure-eight motion. The process was repeated for the other plates which were then left to cool; they were inverted and incubated at 35 °C for 24 hours. The plates were counted using the P Spectra® colony counter. Plates with more than 250 colonies were not counted and were labelled too numerous to count (TNTC). Plates with fewer than 25 colonies were designated too few to count (TFTC). The colony forming unit per millilitre was calculated by dividing the number of colonies by the dilution factor (Harley and Prescott, 1998).

3.6.3 Standardization of cultures using turbidimetry method

One empty tube and five tubes containing 3 ml of sterile tryptone soya broth were placed in a test-tube rack and labelled A–E, with the exception of the empty tube. The five tubes of broth were used to make five serial dilutions of the culture. With the aid of a spectrophotometer (Shimadzu® UV spectrophotometer (UV-1800) and curvettes (Hellma®, precision cells) the different cultures were standardized at a wavelength of 600 nm; the blank was a sterile broth without the culture. From the culture broth, different absorbance concentrations of 0.200 to 1.0 were achieved with sterile saline using the serial dilution method. Each absorbance concentration was plated and the colony counted in triplicate. A straight line graph was constructed by plotting the absorbance of the culture broth at 600 nm versus the number of colony forming units, and a linear regression trendline was fitted to the plot (Microsoft Excel® 2007, Microsoft Corporation). A linear relationship was found with all the organisms, as indicated by the linear correlation coefficient (R2), which was greater than 0.99, even though the y-intercepts were significantly different to zero (Harley and Prescott, 1998). This method was applied to the different microorganism.

3.6.4 Preservative efficacy

The preservative properties of the formulation are adequate if, in the conditions of the test, there is a significant fall or no increase in the number of micro-organisms in the inoculated product after the times and at the temperatures prescribed. Negative controlled samples were prepared by excluding preservatives, and inoculums of 100 µl of 106 CFU/ml were added to both control and products I–V. The inoculated samples were stored away from light at 25 °C for 6 hours, 24 hours, 7 days, 14 days and 28 days. Aliquots of 1 ml were removed from each sample at zero, six and 24 hours and after 7, 14 and 28 days intervals. The sampled were then enumerated.

One milliliter aliquots were transferred to a sterile 10 ml nutrient broth and plated in duplicate on tryptone soya agar (for bacteria) or Sabouraud dextrose agar (for fungi). Plates were incubated at 35 °C for 48 hours for bacteria and 25 °C for 72 hours for fungi. Raw data counts were converted to log10 values and the reduction from inoculum values was calculated for evaluation against compendial requirements found in Table 6 below. Table 6: Criteria for tested microorganisms (USP, 2004)

Category 1 products such as eye drops Bacteria Not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days. Yeast and No increase from the initial calculated count at 7, 14, and 28 days. molds

The samples were diluted in a 1:10 ratio at the time of testing, 10 CFU (or 1.0 log reduction) is the lowest sensitivity allowed by the test.

3.7 Determination of viscosity

3.7.1 Equipments and method

All measurements were performed with an Anton Paar RheolabQC rheometer with cylinder measuring CC27 according to ISO 3219. ISO 3219 describes the dimensions of the cylinder geometry and defines the ratio of measuring cup radius to measuring bob radius as 1.0847. This guarantees an industrial standard for shearing

73 the sample in the measuring gap, independent of the measuring system size and manufacturer. The temperature of the measuring system was controlled using a thermostat (Anton Paar ViscoTherm VT2) directly connected to the Antor Paar rheometer (Šklubalova & Zatloukal, 2011). For all measurements temperature was 23 °C this was kept constant as determination of the viscosity η and yield point is very dependent on the temperature.

The testing conditions for the determination of the yield point were as follows:

1. Preshearing with = 5 s-1 over t = 1 minute to homogenise and control the temperature of the product. 2. Rest phase with = 0 s-1 over t = 30 seconds. Intervals 1 and 2 increase the reproducibility of the measurements. 3. Shear stress ramp of = 1 Pa to 30 Pa with 200 measuring points per 1s (duration t = 200 seconds).

3.8 Statistical analysis

Descriptive and inferential statistics were used. Standard deviation (+/-) and mean were calculated from measured replicates using the software package GraphPad Prism version 6.01 (GraphPad Software Inc., San Diego, USA). Comparisons of means were achieved using one-way ANOVA. Statistically significant differences were tested using Student’s t-test with significance in difference defined as p<0.05. The experimental design was based on a non-matching or pairing of mean, with the mean of each observation being compared with the mean of every other observation. The Bonferroni test was chosen as it corrected for multiple comparisons and indicated confidence intervals and significance.

4. RESULTS AND DISCUSSION

4.1 Validation of the stability indicating assay

The method used to analyse the formulations was obtained from the USP. Though it was a pharmacopoeial method its suitability for use on the present system had to be validated. The validation process followed ICH guidelines and the results are indicated below.

4.1.1 Linearity

Linearity is determined by a calibration curve which was constructed by plotting the area of the phenylephrine hydrochloride peak versus phenylephrine hydrochloride concentration. Figure 10 below shows linearity over the concentration range of 0.0125 to 0.15 mg/ml. The linear regression equation was y = 8541.1x + 438.55, with a correlation coefficient, R2, equal to 0.9999. The requirements for linearity were attained, as the correlation coefficient of the regression line was greater than 0.999 and the percentage relative standard deviations for the phenylephrine hydrochloride peak areas of multiple injections 0.0125, 0.025, 0.05, 0.075, 0.1 and 0.15 mg/ml were all less than 1.5%. This means that the results achieved are directly proportional to the concentration of phenylephrine hydrochloride within a given range. 8541.1 defines the slope, y, is 438.55.

linearity graph of phenylephrine hydrochloride standards 1400000 1200000 1000000

800000 600000 400000

peak area peak 200000 0 0 0.05 0.1 0.15 0.2 concentration (mg/ml) Figure 10: Graph showing a mean peak area versus concentration of replicate samples of phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1x + 438.55, R2 = 0.9999.

4.1.2 Accuracy

Accuracy of the method was within acceptable range as indicated in Table 7 below. The percentage relative standard deviations calculated for the samples at the lower, middle and upper limits of the concentration range were all below 0.5%. This means by applying the analytical method of a known purity concentration (linearity) and comparing the results of accuracy the differences were not greater than 0.5%.

Table 7: Accuracy data for quantification of phenylephrine hydrochloride

Theoretical Actual Concentration, Relative Standard Percentage Concentration Mean (n = 3) Deviation Recovery (%) (mg/ml) (mg/ml) (% RSD)

9.5 9.46 0.36 99.5 54 54.1 0.02 100.1 138 136.89 0.05 99.1

The recovery percentage for the samples was between the limits of 99.00 to 100.10%.

4.1.3 Precision

Precision was within acceptable range as seen below in Table 8. The percentage relative standard deviations calculated for the samples at the lower, middle and upper limits of the concentration range were all below 0.5%. The recovery percentage for the samples was between the limits of 99.00 to 100.00%. The method being validated showed precision was achieved as the degree of agreement among individual test results was demonstrated by the relative standard deviation.

Table 8: Precision data for quantification of phenylephrine hydrochloride

Theoretical Actual Relative Standard Percentage Concentration Concentration, Recovery (%) (mg/ml) Mean (n=3) Deviation (mg/ml) (% RSD) 9.5 9.48 0.42 99.8 54 53.86 0.06 99.7 138 137.29 0.16 99.4

4.1.4 Limit of detection (LOD) and quantification (LOQ)

The LOD was determined by serial dilutions of the lowest concentration (0.0125 mg/ml) of the attained phenylephrine hydrochloride standard and found to be 12.3 μg/ml. The amount stated is the lowest amount of phenylephrine hydrochloride in a sample that can be detected. The LOD is the lowest concentration for which the relative standard deviation of multiple injections is less than 5.0%. The LOQ was found to be 41 μg/ml. The amount shows the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy. By convention, the LOD value is taken as 0.3 times the LOQ (Armbruster & Pry, 2008) while LOQ = 3.33 LOD (Thomsen et al., 2003). The results of the two did fit this mathematical assumption. These methods are HPLC specific and are dependent on the type of HPLC conditions; therefore re-determination in each laboratory is necessary. The range was 0.0125 to 0.15 mg/ml.

4.1.5 Specificity and system suitability

Specificity of a chromatographic method is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. Specificity is useful to show that an analyte response cannot be attributed to more than one component (Rozet et al., 2011). The chromatograms were examined for the presence of compounds, metabolites, impurities, degradants and matrix components that may interfere or partly co-elute with the phenylephrine hydrochloride peak.

The mean peak area for the 6 replicate injections of phenylephrine hydrochloride had a percentage relative standard deviation of 0.14 %. The tailing factor was 2.0. The analytical system thus complied with the requirements specified by the system suitability as the percentage relative standard deviation of the peak responses due to phenylephrine hydrochloride for six injections was less than 1.0 %. The column was thus deemed suitable for analysis. There is a peak tailing as potrayed in the chromatograms, some reasons could be due to more than one retention mechanism is present in a separation, and one of those mechanisms is overloaded. At the surface, the polymer terminates in silanol groups. There are different possible silanol configurations which are abundant at the surface of the columns as free silanols, but

77 silicon atoms with two hydroxyl groups in a geminal configuration also are present. If the silanol groups are positioned next to each other, an associated silanol group can share a proton with an adjacent group. The free silanol groups are more acidic than the geminal and associated groups, and they interact more strongly with basic solutes. The result is peak tailing commonly associated with the separation of bases. To further complicate matters, trace metals (e.g., iron and aluminium) can be present in the silica matrix. These trace metals can act as ion-exchange sites or, when adjacent to free silanols, can withdraw electrons, which makes the silanols even more acidic. To reduce the incidence of tailing or fronting, concentrations should be reduced proportionally by appropriate dilutions (Dolan, 2003).

Mobile phase chromatogram showed no interference as it produced a chromatogram which had a steady baseline and no ghost peaks as seen in Figure 11.

Figure 11: HPLC Chromatogram for mobile phase alone.

The peak produced by phenylephrine hydrochloride shows no interference from contaminants or impurities. Figure 12 below shows it eluted after 7.8 minutes and had an average retention time of 7 minutes. This means the conditions and concentrations were suitable for the phenylephrine hydrochloride standard. The purity peak shows the phenylephrine hydrochloride standard was pure as the value was 1.0, the closer the value is to one the purer a compound is. The start of the mobile phase for the gradient elution was different from the dilution solvent; as a result a constant peak is noted between 2.5 and 3.5 minutes as seen in the figures below. They are known as ghost or false peaks. It takes that amount of time for the injected solvent to reach the detector and the polarity differences caused by the changes in pH and solvent phase causes it to be identified as a peak. The peaks were not symmetrical and fronting or tailing was found to be 2.0.

mAU 280nm,4nm (1.00) 45

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 12: HPLC Chromatogram for phenylephrine hydrochloride dissolved in mobile phase with a retention time of 7.80 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

Figure 13: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.

Products I–V without any stress are indicated below in figures 14–23. They show purity indexes of 1.0 and chromatograms showing no co-elution or impurities. The average retention time for PE was 7.85 minutes.

mAU 60 280nm,4nm (1.00)

-5 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 14: HPLC Chromatogram for product I dissolved in mobile phase with a retention time of 7.87 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

Figure 15: Peak purity profile calculated using PDA data (from 190–800 nm) for Product I prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999054

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 16: HPLC Chromatogram for product II dissolved in mobile phase with a retention time of 7.82 minutes.

Figure 17: Peak purity profile calculated using PDA data (from 190–800 nm) for Product II prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.996482.

mAU 280nm,4nm (1.00) 50

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 18: HPLC Chromatogram for product III dissolved in mobile phase with a retention time of 7.83 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min Figure 19: Peak purity profile calculated using PDA data (from 190–800 nm) for Product III prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.998178.

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 20: HPLC Chromatogram for product IV dissolved in mobile phase with a retention time of 7.89 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min Figure 21: Peak purity profile calculated using PDA data (from 190–800 nm) for Product IV prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.996947.

mAU 280nm,4nm (1.00) 45

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 22: HPLC Chromatogram for product V dissolved in mobile phase with a retention time of 7.84 minutes.

Figure 23: Peak purity profile calculated using PDA data (from 190–800 nm) for product V prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999587.

Stress under UV light. Products I–V were stressed using UV light, their chromatograms did not show interference or co-elution with phenylephrine hydrochloride. Figures 24–35 below indicate that the excipients did not react with the phenylephrine hydrochloride and no degradants co-eluted with the phenylephrine hydrochloride. The plastic bottle did not change in colour as the solutions were still clear and homogenous. The average retention time for the products was 7.85 minutes. Peak purity index was 1.0 for products I, II, IV and V. Product III had a peak purity index of 0.999999.

mAU 280nm,4nm (1.00) 45

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 24: HPLC Chromatogram for phenylephrine hydrochloride stressed under UV light dissolved in mobile phase with a retention time of 7.81 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

Figure 25: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.

mAU 60 280nm,4nm (1.00)

-5 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 26: HPLC Chromatogram for product I stressed under UV light dissolved in mobile phase with a retention time of 7.86 minutes.

mAU Purity Curve Peak Zero Line 60 0.75 50

40 0.50

0.25 20

10 0.00 0 7.75 8.00 8.25 min Figure 27: Peak purity profile calculated using PDA data (from 190–800 nm) for product I prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999054.

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 28: HPLC Chromatogram for product II stressed under UV light dissolved in mobile phase with a retention time of 7.82 minutes.

mAU Purity Curve Peak Zero Line 30.0 0.75 25.0

20.0 0.50

15.0

0.25 10.0

5.0

0.00 0.0 7.75 8.00 8.25 min

Figure 29: Peak purity profile calculated using PDA data (from 190–800 nm) for product II in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999116.

mAU 280nm,4nm (1.00) 50

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 30: HPLC Chromatogram for product III stressed under UV light dissolved in mobile phase with a retention time of 7.85 minutes. mAU Purity Curve Peak Zero Line 35.0 0.5 30.0

0.4 25.0

0.3 20.0

15.0 0.2

10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 32: HPLC Chromatogram for product IV stressed under UV light dissolved in mobile phase with a retention time of 7.81 minutes.

mAU Purity Curve Peak Zero Line 35.0 0.75 30.0

25.0 0.50 20.0

15.0 0.25 10.0

5.0 0.00 0.0 7.75 8.00 8.25 min

Figure 33: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.997916.

mAU 280nm,4nm (1.00) 45

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 34: HPLC chromatogram for product V stressed under UV light dissolved in mobile phase with a retention time of 7.87 minutes.

mAU 0.5 Purity Curve Peak Zero Line 35.0 0.4 30.0

0.3 25.0

20.0 0.2 15.0

0.1 10.0

5.0 0.0 0.0 7.75 8.00 8.25 min

Figure 35: Peak purity profile calculated using PDA data (from 190–800 nm) for product V in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.997475.

No colour change was observed with products I–V and phenylephrine hydrochloride remained stable under the UV light stress. If it was unstable a colour chang would be observed from clear to dark brown. The average concentrations of the products remained at 99.01%. Using the above mentioned statistical analysis (one-way ANOVA), there were no significant difference in the API concentration among the products (I–V) manufactured at time zero (T0). They had a P-value of p > 0.9999, this meant that the products during formulation and testing maintained their API at T0.

Physical and chemical degradation are known to occur in phenylephrine hydrochloride and this is sometimes accompanied by a change in color, e.g. changing from a white or almost white colour into a darker, brownish color. Discoloration is accelerated by light, but it occurs eventually even in light-protected solutions. Degradation of phenylephrine hydrochloride may be caused by a variety of factors including the presence of oxygen, moisture, reducing sugars, bases and high temperature (Douša et al., 2011)

Stress with HCl: Products I–V were stressed with HCl, their chromatograms did not show interference or co-elution with phenylephrine hydrochloride. Figures 36–47 below indicates that the excipients did not react with the phenylephrine hydrochloride and no degradants co-eluted with the phenylephrine hydrochloride. Discolorations were observed during the reflux of products I–V with HCl, the colours ranged from

87 very light yellow to yellow. The greatest change was observed in product III, as it turned bright yellow. These could mean glycerol, HCl and phenylephrine hydrochloride were undergoing a reaction of oxidation or reduction. The average retention time for the products was 7.85 minutes. Peak purity index was 1.0 for products I–V however; the average API concentration had reduced to 79.64 %.

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 36: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.86 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

Figure 37: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999574.

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 38: HPLC chromatogram of Product I stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.82 minutes.

mAU Purity Curve Peak Zero Line 35.0 0.5 30.0

0.4 25.0

0.3 20.0

15.0 0.2

10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

Figure 39: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116.

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 40: HPLC chromatogram for product II stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.83 minutes. mAU Purity Curve Peak Zero Line 30.0 0.75 25.0

0.50 20.0

15.0

0.25 10.0

5.0 0.00 0.0 7.75 8.00 8.25 min

Figure 41: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.996296.

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 42: HPLC chromatogram for product III stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.91 minutes.

mAU Purity Curve Peak 0.75 Zero Line 25.0

20.0 0.50 15.0

0.25 10.0

5.0

0.00 0.0 7.75 8.00 8.25 min

Figure 43: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.995179.

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 44: HPLC chromatogram for product IV stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.86 minutes.

mAU Purity Curve Peak Zero Line 35.0 0.75 30.0

25.0 0.50 20.0

15.0 0.25 10.0

5.0 0.00 0.0 7.75 8.00 8.25 min

Figure 45: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.997097.

Figure 46: HPLC chromatogram of product V stressed with 0.2 M HCl dissolved in mobile phase with a retention time of 7.80 minutes.

mAU Purity Curve Peak Zero Line 60 0.75 50

40 0.50

0.25 20

10 0.00 0 7.75 8.00 8.25 min

Figure 47: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.999578.

Stress with NaOH: Products I–V were stressed with NaOH, their chromatograms did not show interference or co-elution with phenylephrine hydrochloride. Figures

48–59 below indicate that the excipients did not react with the phenylephrine hydrochloride nor did degradation products of products I–V co-elute with the phenylephrine hydrochloride. Discolorations were observed during the reflux of products I–V with NaOH, the colours changed from clear to brown. The average retention time for the products was 7.84 minutes. Due to the caustic nature of NaOH the products had a reduced average API concentration of 42.36%.

mAU 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 48: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.82 minutes

mAU Purity Curve Peak 30.0 Zero Line 0.6 25.0 0.5 20.0 0.4

0.3 15.0

0.2 10.0

0.1 5.0

0.0 0.0 7.75 8.00 8.25 min

Figure 49: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.996847.

mAU 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 50: HPLC chromatogram of product I stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.89 minutes.

mAU Purity Curve Peak 30.0 Zero Line 0.6 25.0 0.5 20.0 0.4

0.3 15.0

0.2 10.0

0.1 5.0

0.0 0.0 7.75 8.00 8.25 min

Figure 51: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =1.000000; Single point threshold = 0.995815.

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 52: HPLC chromatogram of product II stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.9 minutes.

mAU Purity Curve Peak Zero Line 17.5 0.75 15.0

12.5 0.50 10.0

7.5 0.25 5.0

2.5 0.00 0.0 7.75 8.00 8.25 min

Figure 53: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996370.

mAU 37.5 280nm,4nm (1.00)

35.0

32.5

30.0

27.5

25.0

22.5

20.0

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 54: HPLC chromatogram for product III stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.79 minutes.

mAU Purity Curve Peak Zero Line 0.75 15.0

12.5

0.50 10.0

7.5

0.25 5.0

2.5 0.00 0.0 7.75 8.00 8.25 min

Figure 55: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998771.

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 56: HPLC chromatogram of product IV stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.91 minutes.

mAU Purity Curve Peak 35.0 0.6 Zero Line 30.0 0.5 25.0 0.4 20.0 0.3 15.0 0.2 10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

mAU 45 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 58: HPLC chromatogram for product V stressed with 0.2 M NaOH dissolved in mobile phase with a retention time of 7.85 minutes.

mAU Purity Curve Peak 0.75 Zero Line 25.0

20.0 0.50 15.0

0.25 10.0

5.0

0.00 0.0 7.75 8.00 8.25 min

Figure 59: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999587.

Stress with H2O2: Products I–V were stressed with H2O2, their chromatograms did not show interference or co-elution with phenylephrine hydrochloride. Figures 60–71 below indicates that the excipients did not react with the phenylephrine hydrochloride or produce degradants found in products I–V which co-eluted with the phenylephrine hydrochloride. Discolorations were observed during the reflux of products I–V with

H2O2, the colours changed from clear to pale yellow (products I, II, IV and V) and from clear to lime green (product III). The average retention time for the products was 7.82 minutes. Due to the oxidizing nature of H2O2 the products had a reduced average API concentration of 26.43%.

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 60: HPLC chromatogram for phenylephrine hydrochloride stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.82 minutes.

mAU Purity Curve Peak Zero Line 0.75 15.0

12.5

0.50 10.0

7.5

0.25 5.0

2.5 0.00 0.0 7.75 8.00 8.25 min Figure 61: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999987.

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 62: HPLC chromatogram for product I stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.81 minutes. mAU 0.6 Purity Curve Peak 15.0 Zero Line

0.5 12.5

0.4 10.0

0.3 7.5

0.2 5.0

0.1 2.5

0.0 0.0 7.75 8.00 8.25 min

Figure 63: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999522.

mAU 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 64: HPLC chromatogram for product II stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.9 minutes.

mAU Purity Curve Peak Zero Line 17.5 0.75 15.0

12.5 0.50 10.0

7.5 0.25 5.0

2.5 0.00 0.0 7.75 8.00 8.25 min

Figure 65: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999722.

mAU 60 280nm,4nm (1.00)

-5 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 66: HPLC chromatogram of product III stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time 7.94 minutes.

mAU Purity Curve Peak Zero Line 17.5

0.75 15.0

12.5

0.50 10.0

7.5 0.25 5.0

2.5 0.00 0.0 7.75 8.00 8.25 min

Figure 67: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999706.

mAU 280nm,4nm (1.00) 55

0 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 68: HPLC chromatogram for product IV with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.81 minutes. mAU 0.7 Purity Curve Peak Zero Line 12.5 0.6

0.5 10.0

0.4 7.5 0.3 5.0 0.2

0.1 2.5

0.0 0.0 7.75 8.00 8.25 min

mAU 50 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 70: HPLC chromatogram for product V stressed with 0.2 M H2O2 dissolved in mobile phase with a retention time of 7.88 minutes.

mAU Purity Curve Peak 35.0 0.6 Zero Line 30.0 0.5 25.0 0.4 20.0 0.3 15.0 0.2 10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

Figure 71: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999536.

Stress with heat and humidity: Products I–V were stressed with heat and humidity; their chromatograms did not show interference or co-elution of impurities with phenylephrine hydrochloride. Figures 72–98 below indicates that the excipients did not react with the phenylephrine hydrochloride. In Figure 72, phenylephrine hydrochloride stressed at 100 °C for 24 hours in a stability chamber showed a reduction of its API to 95% while showing no physical discolorations. Heat therefore has an effect in reducing the potency of phenylephrine hydrochloride.

Chromatograms of products I–V stressed at the 65 °C for one month are shown in figures 74–85 below. The API concentration was reduced and products I and III

100 showed degardants or impurities eluting at 2, 5 and 10 minutes. Tailing was observed in the all products stressed under this condition, which could mean the API had reduced however the peak purity profile indicated that it was still pure. Degradants did not interfere or co-elute with phenylephrine hydrochloride. Changes in physical appearance were recorded in Table 9 below.

Table 9: Physical appearance of phenylephrine hydrochloride and products I–V before and after storage conditions 40 °C/75%RH for 1 month

Item Physical appearance Initial After 4 weeks of storage Physical appearance 40 °C/75%RH 65 °C

Phenylephrine Clear solution, no No change in initial Yellow, no precipitate formed Hydrochloride precipitate/residue, no appearance colour change Product I Clear solution, no No change in initial Light yellow, no precipitate precipitate/residue, no appearance formed colour change Product II Clear solution, no No change in initial Tinge brown, precipitate/residue, no appearance No precipitate formed colour change Product III Clear solution, no No change in initial Slight yellow, No precipitate precipitate/residue, no appearance formed colour change Product IV Clear solution, no No change in initial Tinge brown, No precipitate precipitate/residue, no appearance formed colour change Product V Clear solution, no No change in initial Tinge brown, precipitate/residue, no appearance No precipitate formed colour change Bold print indicates physical mixtures that showed a change in appearance after 4 weeks of storage

Table 9 above shows changes in appearance at 65 °C for all products. The same did not occur at the 45 °C/75%RH storage condition where none of the samples underwent any physical change.

Chromatograms of products I–V stressed at the 40 °C/75%RH for one month are indicated in figures 86–97 below. The API concentration was reduced, however all concentrations were above the 95%, except for product V which was at 94.97%. Products I–V were able to withstand the heat and humidity without showing any change in physical appearance. Tailing was observed in the all products stressed under this condition, which could mean the API had slightly reduced however the peak purity profile indicated that it was still pure. Degradants did not interfere or co- elute with phenylephrine hydrochloride.

101

mAU 280nm,4nm (1.00) 45

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 72: HPLC chromatogram for phenylephrine hydrochloride stored at 100 °C for 24 hours dissolved in mobile phase with a retention time of 7.8 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

Figure 73: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stored at 100 °C for 24 hours in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

102

mAU 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 74: HPLC chromatogram for phenylephrine hydrochloride stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.83 minutes.

mAU Purity Curve Peak Zero Line 0.5 30.0

25.0 0.4

20.0 0.3

15.0 0.2 10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

Figure 75: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999989.

Figure 76: HPLC chromatogram for product I stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.92 minutes.

103

mAU Purity Curve Peak Zero Line 35.0 0.5 30.0 0.4 25.0

0.3 20.0

0.2 15.0

10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

Figure 77: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.999956.

mAU 280nm,4nm (1.00) 60

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 78: HPLC chromatogram for product II stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.84 minutes. mAU Purity Curve Peak Zero Line 35.0 0.5 30.0 0.4 25.0

0.3 20.0

0.2 15.0

10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 min

Figure 79: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056.

104

mAU 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 80: HPLC chromatogram for Product III stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.89 minutes.

mAU Purity Curve Peak 0.5 Zero Line 35.0

30.0 0.4 25.0 0.3 20.0

0.2 15.0

10.0 0.1 5.0 0.0 0.0 7.75 8.00 min

Figure 81: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999720.

mAU 280nm,4nm (1.00) 40.0

37.5

35.0

32.5

30.0

27.5

25.0

22.5

20.0

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 82: HPLC chromatogram for Product IV stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.87 minutes.

105

mAU Purity Curve Peak Zero Line 0.6 25.0

0.5 20.0

0.4 15.0 0.3

10.0 0.2

0.1 5.0

0.0 0.0 7.75 8.00 min Figure 83: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =0.999999; Single point threshold = 0.999803.

mAU 50 280nm,4nm (1.00)

-5 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 84: HPLC chromatogram for Product V stored at 65 °C for 1 month dissolved in mobile phase with a retention time of 7.90 minutes. mAU Purity Curve Peak 70 0.7 Zero Line 60 0.6 50 0.5

0.4 40

0.3 30

0.2 20

0.1 10 0.0 0 7.5 8.0 8.5 min

Figure 85: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999752.

106

mAU 280nm,4nm (1.00) 35.0

32.5

30.0

27.5

25.0

22.5

20.0

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

-2.5 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 86: HPLC chromatogram for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.86 minutes.

mAU Purity Curve Peak 0.6 Zero Line 20.0 0.5

0.4 15.0

0.3 10.0 0.2

0.1 5.0

0.0 0.0 7.50 7.75 8.00 8.25 min

Figure 87: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056.

mAU 280nm,4nm (1.00) 35.0

32.5

30.0

27.5

25.0

22.5

20.0

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

-2.5 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 88: HPLC chromatogram for Product I stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.87 minutes.

107

mAU Purity Curve Peak Zero Line 0.6 25.0 0.5 20.0 0.4

15.0 0.3

0.2 10.0

0.1 5.0

0.0 0.0 7.50 7.75 8.00 8.25 min

Figure 89: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at 40 °C/75% RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.995566.

mAU 280nm,4nm (1.00) 100

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 90: HPLC chromatogram for Product II stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.84 minutes. mAU 0.8 Purity Curve Peak Zero Line 0.7 25.0 0.6 20.0 0.5

0.4 15.0

0.3 10.0 0.2

0.1 5.0

0.0 0.0 7.50 7.75 8.00 8.25 min

Figure 91: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999579.

108

mAU 50 280nm,4nm (1.00)

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 92: HPLC chromatogram for product III stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.81 minutes.

mAU Purity Curve Peak Zero Line 25.0 0.75

20.0

0.50 15.0

10.0 0.25

5.0

0.00 0.0 7.50 7.75 8.00 8.25 min

Figure 93: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.994609.

mAU 280nm,4nm (1.00) 50

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 94: HPLC chromatogram for Product IV stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.85 minutes.

109

mAU Purity Curve Peak Zero Line 25.0 0.75

20.0

0.50 15.0

10.0 0.25

5.0

0.00 0.0 7.75 8.00 8.25 min

Figure 95: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.994609.

mAU 280nm,4nm (1.00) 60

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 96: HPLC chromatogram for Product V stored at 40 °C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.83 minutes. mAU Purity Curve Peak Zero Line 0.75 25.0

20.0 0.50 15.0

0.25 10.0

5.0

0.00 0.0 7.50 7.75 8.00 8.25 min

Figure 97: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.995332.

110

Peak purities of all samples of stressed and unstressed phenylephrine hydrochloride and finished product solutions are indicated in Table 10 below. Phenylephrine hydrochloride alone showed loss to the combination of heat and humidity.

Table 10: Results showing absence of impurity from a series of stressed and unstressed samples of phenylephrine hydrochloride (API) and products. Number Sample name Co-eluting Image reference impurities found 1 Mobile phase only None Figure 11 2 Active: unstressed None Figure 12 3 Product I, unstressed None Figure 14 4 Product II, unstressed None Figure 16 5 Product III, unstressed None Figure 18 6 Product IV, unstressed None Figure 20 7 Product V, unstressed None Figure 22 8 Active, stressed under UV for 17 None Figure 24 hours 9 Product I, stressed under UV for None Figure 26 17 hours 10 Product II, stressed under UV for None Figure 28 17 hours 11 Product III, stressed under UV for None Figure 30 17 hours 12 Product IV, stressed under UV for None Figure 32 17 hours 13 Product V, stressed under UV for None Figure 34 17 hours 14 Active, stressed with HCl None Figure 36 15 Product I, stressed with HCl None Figure 38 16 Product II, stressed with HCl None Figure 40 17 Product III, stressed with HCl None Figure 42 18 Product IV, stressed with HCl None Figure 44 19 Product V, stressed with HCl None Figure 46 20 Active, stressed with NaOH None Figure 48 21 Product I, stressed with NaOH None Figure 50 22 Product II, stressed with NaOH None Figure 52 23 Product III, stressed with NaOH None Figure 54 24 Product IV, stressed with NaOH None Figure 56 25 Product V, stressed with NaOH None Figure 58 26 Active, stressed with H2O2 None Figure 60 27 Product I, stressed with H2O2 None Figure 62 28 Product II, stressed with H2O2 None Figure 64 29 Product III, stressed with H2O2 None Figure 66 30 Product IV, stressed with H2O2 None Figure 68 31 Product V, stressed with H2O2 None Figure 70 32 Active, stressed at 100 °C None Figure 72 33 Active, stressed at 65 °C None Figure 74 34 Product I, stressed at 65 °C None Figure 76 35 Product II, stressed at 65 °C None Figure 78 36 Product III, stressed at 65 °C None Figure 80 37 Product IV, stressed at 65 °C None Figure 82 38 Product V, stressed at 65 °C None Figure 84 39 Active, stressed at 40 °C/75%RH None Figure 86

111

40 Product I, stressed at 40 stressed None Figure 88 at 40 °C/75%RH 41 Product II, stressed at 40 None Figure 90 °C/75%RH

42 Product III, stressed at 40 None Figure 92 °C/75%RH

43 Product IV, stressed at 40 None Figure 94 °C/75%RH 44 Product V, stressed at 40 None Figure 96 °C/75%RH

The products were decomposed (reduction in API concentration) by the combination of heat and humidity, fairly decomposed by heat, and significantly broken (p<0.05) down by hydrogen peroxide and base as seen in Table 11.

Table 11: Results showing phenylephrine hydrochloride left with samples stressed and unstressed (API and Products)

Sample number Sample condition Phenylephrine hydrochloride left 2 Active, unstressed 99.57% 3 Product I, unstressed 100.20% 4 Product II, unstressed 97.09% 5 Product III, unstressed 97.20% 6 Product IV, unstressed 98.68% 7 Product V, unstressed 97.31% 8 Active, stressed under UV light 99.10% 9 Product I, stressed under UV light 99.10% 10 Product II, stressed under UV light 99.61% 11 Product III, stressed under UV light 99.66% 12 Product IV, stressed under UV light 98.33% 13 Product V, stressed under UV light 98.63% 14 Active, stressed with HCl 75.26% 15 Product I, stressed with HCl 77.67% 16 Product II, stressed with HCl 81.11% 17 Product III, stressed with HCl 78.43% 18 Product IV, stressed with HCl 82.18% 19 Product V, stressed with HCl 83.21% 20 Active, stressed with NaOH 33.86% 21 Product I, stressed with NaOH 43.11% 22 Product II, stressed with NaOH 42.56% 23 Product III, stressed with NaOH 30.29% 24 Product IV, stressed with NaOH 50.77% 25 Product V, stressed with NaOH 53.57% 26 Active, stressed with H2O2 23.73% 27 Product I, stressed with H2O2 26.79% 28 Product II, stressed with H2O2 26.29% 29 Product III, stressed with H2O2 32.42% 30 Product IV, stressed with H2O2 23.55% 31 Product V, stressed with H2O2 25.80% 32 Active, stressed at 100 °C 95.42%

112

33 Active, stressed at 65 °C 92.42% 34 Product I, stressed at 65 °C 86.92% 35 Product II, stressed at 65 °C 90.74% 36 Product III, stressed at 65 °C 91.47% 37 Product IV, stressed at 65 °C 80.52% 38 Product V, stressed at 65 °C 88.21% 39 Active, stressed at 40 °C/75%RH 90.20% 40 Product I, stressed at 40 °C/75%RH 96.57% 41 Product II, stressed at 40 °C/75%RH 98.48% 42 Product III, stressed at 40 °C/75%RH 95.99% 43 Product IV, stressed at 40 °C/75%RH 95.37% 44 Product V, stressed at 40 °C/75%RH 94.97%

The HPLC method used for the phenylephrine hydrochloride and finished products was found to be suitable because the degradants showed no interference. The range retention time for phenylephrine was 7.78–7.92 minutes. The impurities did not interfere or co-elute with phenylephrine hydrochloride, as its peak was distinct and clear.

4.2 Active and excipient study

Three replicates of active and excipient mixtures at a ratio of 1:1 were stored at 40 ºC/75%RH in plastic containers for three months. They were then assayed using the HPLC method. The percentage of phenylephrine hydrochloride remaining in each sample was calculated from the linear regression curve, y = 8541.1x + 438.1 taking the initial quantity of phenylephrine hydrochloride in each container to be 100%. The potency of samples stored at 40 ºC/75%RH and the unstressed samples were within 95–105%. Figures 98–121 show the chromatograms of phenylephrine hydrochloride alone and phenylephrine hydrochloride:combined with excipients; there was no interference observed between the phenylephrine hydrochloride and excipients. Table 12 below shows that all the samples were within the required potency range of 95–105%. Table 12: Assay result showing phenylephrine hydrochloride and excipients in a 1:1 ratio after storage conditions 40 °C/75%RH for 1 month Phenylephrine hydrochloride and excipients Percentage of phenylephrine hydrochloride (API (left) % ± % RSD) 40 °C / 75 % RH Phenylephrine hydrochloride alone 98.15 ± 0.41 Boric acid 99.48 ± 1.53 Benzalkonium Chloride 98.63 ± 0.25 EDTA 97.73 ± 0.48

113

Glycerol 95.79 ± 0.22 Methyl Paraben 96.33 ± 1.86 Propyl Paraben 96.73 ± 0.36 Sodium metabisulfite 97.58 ± 0.53 Sodium citrate dehydrate 95.10 ± 0.39 Carboxymethylcellulose Sodium 96.24 ± 1.44 Hydroxypropyl methylcellulose 98.59 ± 1.33

Table 13: Physical appearance of active–excipients samples before and after storage conditions 40 °C/75%RH for 4 weeks Phenylephrine hydrochloride and Initial Physical appearance excipients physical appearance after 4 weeks of storage 40 °C/75%RH Phenylephrine hydrochloride alone Clear solution, no precipitate or No change residue, no colour change Benzalkonium chloride and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Boric acid and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Sodium carboxy methylcellulose and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change EDTA and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Glycerol and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change HPMC and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Methyl paraben and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Propyl paraben and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Sodium citrate dihydrate and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change Sodium metabisulfite and Clear solution, no precipitate or No change phenylephrine hydrochloride residue, no colour change The physical appearance of the phenylephrine hydrochloride and excipient blends can be found in Table 13 above. No colour change and precipitate formation were observed in any of the 1:1 mixtures after four weeks of storage. There were no reductions or increase in liquid level after storage, meaning that the plastic container was not permeable to moisture. The data also proved that the active and excipients did not interfere or interact with each other.

114

mAU 280nm,4nm (1.00)

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 98: HPLC chromatogram for phenylephrine hydrochloride alone dissolved in mobile phase with retention of 7.82 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min Figure 99: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

Figure 100: HPLC chromatogram for phenylephrine hydrochloride with sodium citrate dihydrate (1:1) dissolved in mobile phase with a retention time of 7.92 minutes.

115

mAU Purity Curve Peak 0.6 Zero Line 200 0.5

0.4 150

0.3 100 0.2

0.1 50

0.0 0 8.0 8.5 min

Figure 101: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with sodium citrate dihydrate (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896.

mAU 280nm4nm (1.00) 90

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 102: HPLC chromatogram for phenylephrine hydrochloride with carboxymethycellulose sodium (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

116

mAU 280nm4nm (1.00) 90

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 104: HPLC chromatogram for phenylephrine hydrochloride with hypromellose (1:1) dissolved in mobile phase with a retention time of 7.81 minutes.

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min Figure 105: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride with hypromellose (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

mAU 280nm4nm (1.00) 90

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 106: HPLC chromatogram for phenylephrine hydrochloride with glycerol (1:1) dissolved in mobile phase with a retention time of 7.85 minutes.

117

mAU 0.8 Purity Curve Peak Zero Line 0.7 60

0.6 50

0.5 40 0.4 30 0.3

0.2 20

0.1 10 0.0 0 7.5 8.0 min

Figure 107: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with glycerol (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999989.

mAU 280nm4nm (1.00) 90

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 108: HPLC chromatogram for phenylephrine hydrochloride with benzalkonium chloride (1:1) dissolved in mobile phase with a retention time of 7.88 minutes.

Figure 109: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine hydrochloride with benzalkonium chloride (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999918.

118

Figure 110: HPLC chromatogram for phenylephrine hydrochloride with EDTA (1:1) dissolved in mobile phase with a retention time of 7.86 minutes.

mAU Purity Curve Peak Zero Line 0.7 250

0.6 200 0.5

0.4 150

0.3 100 0.2

0.1 50

0.0 0 8.0 8.5 min

Figure 111: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with EDTA (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999991.

Figure 112: HPLC chromatogram for phenylephrine hydrochloride with boric acid (1:1) dissolved in mobile phase with a retention time of 7.92 minutes.

119

mAU Purity Curve Peak 0.6 Zero Line 20.0 0.5

0.4 15.0

0.3 10.0 0.2

0.1 5.0

0.0 0.0 7.50 7.75 8.00 8.25 min

Figure 113: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with boric acid (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.

mAU 280nm,4nm (1.00) 80

-10

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 114: HPLC chromatogram for phenylephrine hydrochloride with sodium metabisulfite (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.

mAU 0.5 Purity Curve Peak Zero Line 175.0 0.4 150.0

0.3 125.0

100.0 0.2 75.0

0.1 50.0

25.0 0.0 0.0 8.0 8.5 min

Figure 115: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with sodium metabisulfite (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999870.

120

Figure 116: HPLC chromatogram for phenylephrine hydrochloride with disodium edetate (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.

mAU 0.8 Purity Curve Peak Zero Line 50 0.7

0.6 40 0.5 30 0.4

0.3 20 0.2

0.1 10

0.0 0 8.00 8.25 8.50 8.75 min

Figure 117: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with disodium edetate (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998891.

Figure 118: HPLC chromatogram for phenylephrine hydrochloride with propyl paraben (1:1) dissolved in mobile phase with a retention time of 7.83 minutes.

121

mAU Purity Curve Peak 175.0 0.6 Zero Line 150.0 0.5 125.0 0.4 100.0 0.3 75.0 0.2 50.0 0.1 25.0 0.0 0.0 7.5 8.0 8.5 min

Figure 119: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with propyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999948.

Figure 120: HPLC chromatogram for phenylephrine hydrochloride with methyl paraben (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.

mAU 1.00 Purity Curve Peak 500 Zero Line

0.75 400

300 0.50

200 0.25 100

0.00 0 9.0 9.5 10.0 min

Figure 121: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine hydrochloride with methyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999982.

122

4.3 Stability study

Stability studies were performed on products I–V at climatic zone II storage conditions these were 40 °C/75%RH, 25 °C/40%RH and 30 °C/65%RH. The five products were stored at all three of the above mentioned conditions in triplicate and tested at zero, one and three months. The most aesthetically pleasing and stable formulation was chosen based on the phenylephrine hydrochloride left in the product, pH change and appearance.

The results for storage time zero and one month 40 °C/75%RH (accelerated condition) were stated in specificity above.

Products I–V stored for 3 months at 30 °C/65%RH: The chromatograms of the products showed that there was no co-elution and no interference with the API as shown in Figures 122–131 below. The API left in from products I–V was 72.86%, 96.79%, 75.14%, 98.35% and 61.73% respectively. The difference in mean were statistically different as p<0.01. The products I and V failed aesthetically as they turned slightly yellow with no residue, precipitate and no solution loss. The remaining products remained clear and aesthetically pleasing.

mAU 280nm,4nm (1.00) 7

-1

-2

-3

-4

-5

-6

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 122: HPLC chromatogram for product I stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes.

123

mAU 0.65 Purity Curve Peak 27.5 Zero Line 0.60 25.0 0.55 22.5 0.50

0.45 20.0

0.40 17.5

0.35 15.0 0.30 12.5 0.25

0.20 10.0

0.15 7.5

0.10 5.0 0.05 2.5 0.00 0.0 7.75 8.00 8.25 8.50 min

Figure 123: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.999950.

mAU 280nm,4nm (1.00) 7.5

5.0

2.5

0.0

-2.5

-5.0

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 124: HPLC chromatogram for product II stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.85 minutes.

mAU Purity Curve Peak 0.60 Zero Line 27.5 0.55 25.0 0.50 22.5 0.45 20.0 0.40

0.35 17.5

0.30 15.0

0.25 12.5

0.20 10.0 0.15 7.5 0.10 5.0 0.05 2.5 0.00 0.0 7.75 8.00 8.25 8.50 min

Figure 125: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.996473.

124

mAU 280nm,4nm (1.00)

7.5

5.0

2.5

0.0

-2.5

-5.0

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 126: HPLC chromatogram for product III stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes.

mAU Purity Curve Peak Zero Line 0.8 32.5 30.0 0.7 27.5

25.0 0.6 22.5 0.5 20.0

17.5 0.4 15.0 0.3 12.5

10.0 0.2 7.5

0.1 5.0

2.5 0.0 0.0 7.75 8.00 8.25 8.50 min

Figure 127: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996955.

mAU 15.0 280nm,4nm (1.00)

12.5

10.0

7.5

5.0

2.5

0.0

-2.5

-5.0

-7.5 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 128: HPLC chromatogram for product IV stored at 30 °C/65% RH for 3 months dissolved in mobile phase with a retention time of 7.88 minutes.

125

mAU 0.7 Purity Curve Peak Zero Line 55.0

50.0 0.6

45.0

0.5 40.0

35.0 0.4 30.0

0.3 25.0

20.0 0.2 15.0

0.1 10.0

5.0 0.0 0.0 7.75 8.00 8.25 8.50 min

Figure 129: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.998694.

mAU 280nm,4nm (1.00) 7

-1

-2

-3

-4

-5

-6 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 130: HPLC chromatogram for product V stored at 30 °C/65%RH for 3 months dissolved in mobile phase with a retention time of 7.87 minutes.

mAU 0.65 Purity Curve Peak Zero Line 0.60 25.0

0.55 22.5 0.50 20.0 0.45

0.40 17.5

0.35 15.0

0.30 12.5 0.25 10.0 0.20

0.15 7.5

0.10 5.0 0.05 2.5 0.00 0.0 7.75 8.00 8.25 8.50 min

Figure 131: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.994533.

Products I–V stored for 3 months at 25 °C/60%RH: The products showed no co- elution, no interference, and the API left in products I–V was 82.77%, 96.84%,

126

86.78%, 97.06%, and 70.88% respectively. There was a statistic difference of p<0.05. The products III and V failed aesthetically as they turned slightly yellow and brown respectively, with no residue, precipitate and loss of solution. The remaining products remained clear and aesthetically pleasing. The figures 132–141 below show the chromatograms of the products.

mAU 17.5 280nm,4nm (1.00)

15.0

12.5

10.0

7.5

5.0

2.5

0.0

-2.5

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 132: HPLC chromatogram for product I stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.91 minutes.

mAU Purity Curve Peak Zero Line 0.9 45.0 0.8 40.0 0.7 35.0 0.6 30.0 0.5 25.0 0.4 20.0 0.3 15.0 0.2 10.0 0.1 5.0 0.0 0.0 7.75 8.00 8.25 8.50 min Figure 133: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.995873.

127

mAU 280nm,4nm (1.00) 100

-10

-20 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 134: HPLC chromatogram for product II stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.89 minutes.

mAU 0.45 Purity Curve Peak Zero Line 400 0.40 350 0.35

300 0.30

250 0.25

0.20 200

0.15 150

0.10 100

0.05 50

0.00 0 7.50 7.75 8.00 8.25 8.50 min

Figure 135: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999998; Single point threshold = 0.999950.

mAU 15.0 280nm,4nm (1.00)

12.5

10.0

7.5

5.0

2.5

0.0

-2.5

-5.0

-7.5 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 136: HPLC chromatogram for product III stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.92 minutes.

128

mAU Purity Curve Peak Zero Line 55.0 0.6 50.0

45.0 0.5 40.0

0.4 35.0

30.0 0.3 25.0

0.2 20.0

15.0

0.1 10.0

5.0 0.0 0.0 7.75 8.00 8.25 8.50 min

Figure 137: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998742.

mAU 280nm,4nm (1.00)

7.5

5.0

2.5

0.0

-2.5

-5.0

0.0 2.5 5.0 7.5 10.0 12.5 min Figure 138: HPLC chromatogram for product IV stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.92 minutes.

mAU 0.8 Purity Curve Peak Zero Line 32.5

30.0 0.7 27.5

0.6 25.0

22.5 0.5 20.0

0.4 17.5

15.0 0.3 12.5

0.2 10.0 7.5 0.1 5.0

2.5 0.0 0.0 7.75 8.00 8.25 8.50 min

Figure 139: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.996396.

129

mAU 12.5 280nm,4nm (1.00)

10.0

7.5

5.0

2.5

0.0

-2.5

-5.0

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 140: HPLC chromatogram for product V stored at 25 °C/60%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

mAU Purity Curve Peak 0.60 Zero Line 45.0 0.55 40.0 0.50

0.45 35.0

0.40 30.0 0.35 25.0 0.30

0.25 20.0

0.20 15.0 0.15

0.10 10.0

0.05 5.0 0.00 0.0 7.75 8.00 8.25 8.50 min

Figure 141: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093.

Products I–V stored for 3 months at 40 °C/75%RH: The products showed no co- elution and no interference. The API left from product I–V were 61.63%, 96.50%, 53.10%, 77.16%, and 54.79% respectively. There was a statistical difference as p<0.0001. The products I, III and V failed aesthetically as they turned slightly yellow, dark yellow and brown in colour respectively, with no residue, precipitate and loss of solution. The remaining products II and IV remained clear and aesthetically pleasing. The figures 143–152 below show the chromatograms of the products.

130

mAU 280nm,4nm (1.00)

17.5

15.0

12.5

10.0

7.5

5.0

2.5

0.0

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 142: HPLC chromatogram for product I stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

mAU 0.65 Purity Curve Peak Zero Line 0.60 45.0

0.55 40.0 0.50 35.0 0.45

0.40 30.0

0.35 25.0 0.30

0.25 20.0

0.20 15.0 0.15

0.10 10.0

0.05 5.0 0.00 0.0 7.75 8.00 8.25 8.50 min Figure 143: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product I stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093.

mAU 280nm,4nm (1.00) 7.5

5.0

2.5

0.0

-2.5

-5.0

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 144: HPLC chromatogram for product II stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

131

mAU Purity Curve Peak 0.60 Zero Line 27.5 0.55 25.0 0.50 22.5 0.45 20.0 0.40

0.35 17.5

0.30 15.0

0.25 12.5

0.20 10.0 0.15 7.5 0.10 5.0 0.05 2.5 0.00 0.0 7.75 8.00 8.25 8.50 min

Figure 145: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product II stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093.

mAU 280nm,4nm (1.00)

7.5

5.0

2.5

0.0

-2.5

-5.0

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 146: HPLC chromatogram for product III stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

mAU Purity Curve Peak 35.0 0.8 Zero Line

0.7 30.0

0.6 25.0

0.5 20.0 0.4

15.0 0.3

0.2 10.0

0.1 5.0

0.0 0.0 7.75 8.00 8.25 8.50 min

132

mAU 280nm,4nm (1.00) 6

-1

-2

-3

-4

-5

-6

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 148: HPLC chromatogram for product IV stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.84 minutes.

mAU Purity Curve Peak 0.9 Zero Line 22.5

0.8 20.0

0.7 17.5

0.6 15.0

0.5 12.5

0.4 10.0

0.3 7.5

0.2 5.0 0.1 2.5 0.0 0.0 7.75 8.00 8.25 8.50 min Figure 149: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.998093.

mAU 280nm,4nm (1.00) 6

-1

-2

-3

-4

-5

-6

-7 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 150: HPLC chromatogram for product V stored at 40 °C/75%RH for 3 months dissolved in mobile phase with a retention time of 7.91 minutes.

133

mAU Purity Curve Peak 1.0 Zero Line 22.5 0.9

20.0 0.8

0.7 17.5

0.6 15.0

0.5 12.5

0.4 10.0

0.3 7.5

0.2 5.0 0.1 2.5 0.0 0.0 7.75 8.00 8.25 8.50 min

Figure 151: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point threshold = 0.998093.

Figure 152 below summarises the API remaining in the given storage conditions. Product II was stable and within the potency range for all storage conditions while product IV was stable and potent within two storage conditions. The remaining Products (I, III and V) failed to retain their potency at various conditions. According to the results obtained, product II and IV were stable and with the most API remaining in the product. Products II and IV were also the most aesthetically pleasant. This could be due to the presence of protectors and anti-oxidants such as sodium citrate dihydrate, EDTA, sodium metabisulfite, boric acid.

Phenylephrine hydrochloride left 120

100 Product V Product IV 80 Product III 60

Product II product 40 Product I

Percentageof s API in left the 0 1 2 3 1 - 30 °C / 65 % RH 2 - 40 °C / 75 % RH 3 - 25 °C / 60 % RH Figure 152: A graph showing standard error and phenylephrine hydrochloride left in product I–V after 12 weeks at 30 °C/65%RH, 40 °C/75%RH, 25 °C/60%RH.

134

Table 14: Results of one-way ANOVA analysis for products I–V stored at 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. Products I II III IV V I II III IV V I II III IV V °C/%RH 25/ 25/ 25/ 25/ 25/ 30/ 30/ 30/ 30/ 30/ 40/ 40/ 40/ 40/ 40/ 60 60 60 60 60 65 65 65 65 65 75 75 75 75 75 I 25/60 **** **** **** **** **** **** **** **** **** **** **** **** **** **** II 25/60 **** **** ns **** **** ns **** ** **** **** ns **** **** **** III 25/60 **** **** **** **** **** **** **** **** **** **** **** **** **** **** IV 25/60 **** ns **** **** **** ns **** * **** **** ns **** **** **** V 25/60 **** **** **** **** *** **** **** **** **** **** **** **** **** **** I 30/65 **** **** **** **** *** **** **** **** **** **** **** **** **** **** II 30/65 **** ns **** **** **** **** **** ** **** **** ns **** **** **** III 30/65 **** **** **** **** **** **** **** **** **** **** **** **** **** **** IV 30/65 **** ** **** **** **** **** ** **** **** **** *** **** **** **** V 30/65 **** **** **** **** **** **** **** **** **** ns **** **** **** **** I 40/75 **** **** **** **** **** **** **** **** **** ns **** **** **** **** II 40/75 **** ns **** **** **** **** **** **** *** **** **** **** **** **** III 40/75 **** **** **** **** **** **** **** **** **** **** **** **** **** ** IV 40/75 **** **** **** **** **** **** **** **** **** **** **** **** **** **** V 40/75 **** **** **** **** **** **** **** **** **** **** **** **** ** **** ns - Not Significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

Above is Table 14 which shows results of statistical comparisons of all the products. The quantity of phenylephrine hydrochloride in product II did not change significantly when the remaining amounts of API for the various storage conditions were compared. The quantity of API present for the other products changed significantly, this means that product II was stable at all three storage conditions.

Summarised below in tables 15 and 16 are the changes in pH for products I–V. Table 15 below shows pH change in products I–V stored at 40 °C/75%RH for one month, Table 16 below shows pH changes in products I–V stored at various storage conditions after three months of storage. Changes were observed for some products, as they deviated from the pH of four they were initially formulated at. This could be due to oxidation or reduction reactions of the API or excipients; it should be noted that phenylephrine hydrochloride is stable in the pH range of 3.5–8 and none of the products exceeded this limits even though changes were noted (Giahi et al., 2009). Over time products I, III and V became more basic, product II and IV were stable and remained close to their original pH for all storage conditions. According to the pH results obtained, product II and product IV showed favourable stability.

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Table 15: Changes in pH of phenylephrine hydrochloride and products I–V before and after storage conditions 40 °C/75%RH for 1 month.

Item Temperature reading pH before Temperature pH after 1 month (°C) reading (°C) Phenylephrine 20 4.0 20 3.59 ± 0.89 Hydrochloride Product I 20 4.0 20 6.25 ± 0.10 Product II 20 4.0 20 3.95 ± 0.87 Product III 20 4.0 20 5.82 ± 0.45 Product IV 20 4.0 20 3.99 ± 0.38 Product V 20 4.0 20 6.10 ± 0.25 Bold print indicates a change in pH after 4 weeks of storage

Table 16: Changes in pH for products I–V before and after varying storage conditions for 3 months

Item Temperature pH Temperature 40 30 25 reading (°C) before reading (°C) °C/75%RH °C/65%RH °C/60%RH before storage storage after storage Product I 20 4.0 20 5.9 ± 0.16 6.12 ± 6.18 ± 0.16 0.16 Product II 20 4.0 20 3.63 ± 0.57 3.88 ± 3.90 ± 0.82 0.39 Product III 20 4.0 20 4.67 ± 0.65 4.87 ± 6.03 ± 0.41 0.31 Product IV 20 4.0 20 3.63 ± 0.27 3.63 ± 3.96 ± 0.25 0.68 Product V 20 4.0 20 3.23 ± 0.47 4.2 ± 1.4 5.68 ± 0.17 Bold print indicates a change in pH after 3 months of storage

Statistical differences were determined using the one-way ANOVA analysis. All pH results were significantly different from each other as p<0.0001; similarities were summarized in Table 17 below.

Table 17: Results of one-way ANOVA analysis for similarities in pH of products II 25 °C/60%RH, 40 °C/75%RH and IV 25 °C/60%RH after 3 months.

°C/% I II III IV V I II III IV V I II III IV V RH 25/ 25/ 25/ 25/ 25/ 30/ 30/ 30/ 30/ 30/ 40/ 40/ 40/ 40/ 40/ 60 60 60 60 60 65 65 65 65 65 75 75 75 75 75 IV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 25/60 II **** **** **** **** **** **** **** **** ns **** **** **** **** **** **** 40/75 II **** **** **** ns **** **** ns **** **** **** **** **** **** **** **** 25/60 NS - Not Significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

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Table 18: Physical appearance of products I–V before and after varying storage conditions for 3 months.

Item Physical appearance Initial After 3 months of storage Physical appearance 40 °C/75%RH 30 °C/65%RH 25 °C/60%RH

Product I Clear solution, no Clear solution, Slightly yellow, No change in initial precipitate or residue, yellow in colour, clear, no appearance, no no colour change no residue, no residue, no loss of solution precipitate, no precipitate, no loss of solution loss of solution Product II Clear solution, no No change in Clear, no residue, No change in initial precipitate or residue, initial appearance, no precipitate, no appearance, no no colour change no loss of solution loss of solution loss of solution Product III Clear solution, no Clear solution, Clear, no residue, Slight yellow, no precipitate or residue, tinge yellow, no no precipitate, no precipitate formed no colour change residue, no loss of solution precipitate, no loss of solution Product IV Clear solution, no No change in Clear, no residue, No change in initial precipitate or residue, initial appearance, no precipitate, no appearance, no no colour change no loss of solution loss of solution loss of solution Product V Clear solution, no Dark brown, no Brown, no Tinge brown, precipitate or residue, precipitate residue, no no precipitate no colour change formed, presence precipitate, no formed, no loss of of residue, no loss of solution solution, no loss of solution residue Bold print indicates a change after 3 months of storage

Changes in physical appearance were judged visually. Aesthetically pleasing products were noted and above in Table 18 is a summary of what they looked like after three months, products II and IV were the only ones that did not have physical changes.

4.4 Determination of yield point and viscosity of products The viscosity of a solution is a particluarly important parameter, especially during production as continous quality control is essential in order to achive consitently high quality despite the immense production volume. The graphs seen in figures 153–158 below show the flow curves of products I–V and a marketed product Prefrin® at different storage conditions. With the aid of Herschel/Bulkley formula, yield points were calculated and measured by Anton Paar rheometer (RheoPlusTM).

Good viscosity properties of the VMA is important in this experiment, as viscosity shows the flow movement, while yield points corresponds directly to the elastic properties of the formulation. The product must flow out of the plastic eye drop bottle but should be viscous enough for accurate drop dosing.

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TAU

0 30 10 hpmc product 5 30/65 3 t0 2 Pa CC27-SN25781; d=0 mm Shear Stress

26 Viscosity Pa·s glycerol product 3 30/65 3 t0 2 24 CC27-SN25781; d=0 mm 22 Shear Stress Viscosity -1 20 10 hpmc product 1 30/65 3 t0 2

18 CC27-SN25781; d=0 mm Shear Stress

16 Viscosity

hpmc product 2 30/65 3 t0 1 14 CC27-SN25781; d=0 mm

12 Shear Stress Viscosity -2 10 10 scmc product 4 30/65 3 t0 1 CC27-SN25781; d=0 mm 8 Shear Stress 6 Viscosity Prefrin 3 1

4 CC27-SN25781; d=0 mm

Shear Stress 2 Viscosity -3 0 10 0 100 200 300 400 500 600 1/s 700 . Shear Rate

Anton Paar GmbH Figure 153: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 30 °C/65% RH.

Figures 153–158 show variance in viscosity between the different viscosity modifiers (glycerol, hydroxy propyl methylcellulose and sodium carboxy methyl cellulose). At time zero, Prefrin® and product I–V display slightly similar flow. After three months glycerol had a marked increase in its viscosity, this is seen in the figures 154, 156 and 158 below. Other viscosity modifiers show slight changes.

TAU

0 30 10 glycerol product 330/65 3 1 Pa CC27-SN25781; d=0 mm Shear Stress

26 Viscosity Pa·s hpmc product 2 30/65 3 1 24 CC27-SN25781; d=0 mm 22 Shear Stress Viscosity -1 20 10 hpmc product 1 30/65 3 1

18 CC27-SN25781; d=0 mm Shear Stress

16 Viscosity

hpmc product 5 30/65 3 1 14 CC27-SN25781; d=0 mm

12 Shear Stress Viscosity -2 10 10 scmc product 4 30/65 3 1 CC27-SN25781; d=0 mm 8 Shear Stress 6 Viscosity Prefrin 3 1

4 CC27-SN25781; d=0 mm

Shear Stress 2 Viscosity -3 0 10 0 100 200 300 400 500 600 1/s 700 . Shear Rate

Anton Paar GmbH Figure 154: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 30 °C/65% RH.

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TAU

0 30 10 hpmc product 1 40/75 3 t0 1 Pa CC27-SN25781; d=0 mm Shear Stress

26 Viscosity Pa·s hpmc product 2 40/75 3 t0 1 24 CC27-SN25781; d=0 mm 22 Shear Stress Viscosity -1 20 10 hpmc product 5 40/75 3 t0 1

18 CC27-SN25781; d=0 mm Shear Stress

16 Viscosity

glycerol product 3 40/75 3 t0 1 14 CC27-SN25781; d=0 mm

12 Shear Stress Viscosity -2 10 10 scmc product 4 40/75 3 t0 1 CC27-SN25781; d=0 mm 8 Shear Stress 6 Viscosity Prefrin 3 1

4 CC27-SN25781; d=0 mm

Shear Stress 2 Viscosity -3 0 10 0 100 200 300 400 500 600 1/s 700 . Shear Rate Anton Paar GmbH Figure 155: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 40 °C/75% RH.

TAU

0 30 10 scmc product 4 40/75 3 1 Pa CC27-SN25781; d=0 mm Shear Stress

26 Viscosity Pa·s hpmc product 2 40/75 3 1 24 CC27-SN25781; d=0 mm 22 Shear Stress Viscosity -1 20 10 hpmc product 1 40/75 3 1

18 CC27-SN25781; d=0 mm Shear Stress

16 Viscosity

hpmc product 5 40/75 3 1 14 CC27-SN25781; d=0 mm

12 Shear Stress Viscosity -2 10 10 glycerol product 3 40/75 3 1 CC27-SN25781; d=0 mm 8 Shear Stress 6 Viscosity Prefrin 3 1

4 CC27-SN25781; d=0 mm

Shear Stress 2 Viscosity -3 0 10 0 100 200 300 400 500 600 1/s 700 . Shear Rate Anton Paar GmbH Figure 156: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 40 °C/75% RH.

139

TAU

0 30 10 hpmc product 1 25/60 3 t0 1 Pa CC27-SN25781; d=0 mm Shear Stress

26 Viscosity Pa·s hpmc product 2 25/60 3 t0 1 24 CC27-SN25781; d=0 mm 22 Shear Stress Viscosity -1 20 10 hpmc product 5 25/60 3 t0 1

18 CC27-SN25781; d=0 mm Shear Stress

16 Viscosity

glycerol product 3 25/60 3 t0 1 14 CC27-SN25781; d=0 mm

12 Shear Stress Viscosity -2 10 10 scmcproduct 4 25/60 3 t0 1 CC27-SN25781; d=0 mm 8 Shear Stress 6 Viscosity Prefrin 3 1

4 CC27-SN25781; d=0 mm

Shear Stress 2 Viscosity -3 0 10 0 100 200 300 400 500 600 1/s 700 . Shear Rate

Anton Paar GmbH Figure 157: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition 25 °C/60%RH.

TAU

0 30 10 glycerol product 3 25/60 3 1 Pa CC27-SN25781; d=0 mm Shear Stress

26 Viscosity Pa·s hpmc product 1 25/60 3 1 24 CC27-SN25781; d=0 mm 22 Shear Stress Viscosity -1 20 10 hpmc product 5 25/60 3 1

18 CC27-SN25781; d=0 mm Shear Stress

16 Viscosity

scmc product 4 25/60 3 1 14 CC27-SN25781; d=0 mm

12 Shear Stress Viscosity -2 10 10 hpmc product 2 25/60 3 1 CC27-SN25781; d=0 mm 8 Shear Stress 6 Viscosity Prefrin 3 1

4 CC27-SN25781; d=0 mm

Shear Stress 2 Viscosity -3 0 10 0 100 200 300 400 500 600 1/s 700 . Shear Rate Anton Paar GmbH Figure 158: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage condition 25 °C/60% RH.

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Prefrin® was choosen as the product against which others would be measured because polvinyl alcohol was used as its VMA, while phenylephrine hydrochloride as its API. This made it a good chioce for comparison as it had a VMA and a similar API. It proved to be not significantly as different to the rest as seen in figures 154– 159 as p>0.05. Products I–V did not lose water from the containers during the stability study, thus the change in viscosity cannot be attributed to loss of volume.

1 0.9 0.8

0.7 0.6

0.5 T1 40/75 0.4 T0 40/75

Viscosity (mPa.s)Viscosity 0.3 0.2 0.1 0 Product I Product II Product III Product IV Product V Prefrin®

Figure 159: Graph showing viscosity of products I–V stored in a stability chamber of 40 °C/75%RH tested at time zero (T0), three months later (T1) and compared to an original marketed product Prefrin®.

0.9 0.8

0.7

0.6 0.5 t1 25/60 0.4 T0 25/60

0.3 Viscosity (mPa.s) Viscosity 0.2 0.1 0 Product I Product II Product III Product IV Product V Prefrin®

Figure 160: Graph showing viscosity of products I–V stored in a stability chamber of 25 °C/60%RH tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®.

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0.9

0.8

0.7

0.6

0.5 T1 30/65 T0 30/65

Viscosity (mPa.s) Viscosity 0.4

0.3

0.2

0.1

0 Product I Product II Product III Product IV Product V Prefrin®

Figure 161: Graph showing viscosity of products I–V stored in a stability chamber of 30 °C/65%RH tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®. Using the above graphs in figures 159–161 product II showed stability and good rheology properties. The higher the value is to 1 mPa.s, the more viscous a product was.

Statistically no difference was found among most products manufactured and the marketed product. However, product III had significant differences (p<0.05) in viscosity at various storage conditions and time frames as seen in Table 19 below. Product III had a significantly higher viscosity than the other products after the 3 months stability period. The products with “ns” showed that the formulations were the same in mean flow and yield points.

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Table 19: Results of one-way ANOVA analysis for viscosity of products I–V stored at 25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. The values shown indicate differences in p-values and significance in differences of mean was defined as p < 0.05. mPa PI PII PIII PI PV PI PII PIII PI V I II III IV V Prefr .s 25/ 25/ 25/ V 25/ 30/ 30/ 30/ V 30/ 40/ 40/ 40/ 40/ 40/ in® 60 60 60 25/ 60 65 65 65 30/ 65 75 75 75 75 75 60 65 PI ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 25/6 0 PII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 25/6 0 PIII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ** 25/6 0 PIV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 25/6 0 PV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 25/6 0 PI ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 30/6 5 PII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 30/6 5 PIII ns ns ns ns ** ns ns * ns *** * ns ** * **** 30/6 5 PIV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 30/6 5 PV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 30/6 5 PI ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 40/7 5 PII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 40/7 5 PIII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ** 40/7 5 PIV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 40/7 5 PV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 40/7 5 Prefr ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns in® ns - Not Significant, *= p < 0.05, ** = p < 0.01, ***=p < 0.001, **** = p < 0.0001

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4.5 Effectiveness of the ophthalmic solution preservatives

Ophthalmic drops are sterile formulations which are usually packed in multi-dose containers. Microbial contamination from multiple uses may lead to product degradation or result in ocular infection (Sutton et al., 2002). Protection of these multiple dose products against microbial contamination is usually achieved by addition of a suitable preservative system. The antimicrobial effectiveness test is designed to provide a laboratory test that gauges the level of antimicrobial activity by a pharmaceutical product and to evaluate how well a product withstands microbial contamination while being used (Nostro et al., 2004 and Sutton et al., 2002).

The antimicrobial preservative efficacy of the eye-drops challenged with E. coli, S. aureus, P. aeruginosa, and C. albicans showed varied results as seen in Table 20 below. The control eye-drop formulation failed in its preservative efficacy as it lacked any form of preservative. After six hours all formulations containing preservatives showed reduction in the initial microbial count. Four of the eye-drops eradicated the inoculated microorganisms by more than 3 logs in 24 hours, except product IV, which remained as log 2.

The number of P. aeruginosa, C. albicans, E. coli and S. aureus in product IV decreased gradually while the remaining products decreased by over two to three logs. This means that the EDTA, boric acid, sodium metabisulfite and sodium citrate dihydrate acted synergistically to prevent increase in microbial count of other products. For the stated excipients, EDTA had antioxidant and antimicrobial properties; boric acid had antiseptic properties while sodium metabisulfite and sodium citrate had small preservative powers. Products III and V, which contained methylparaben and propylparaben showed the most decrease in microbial load.

After 14 days, all the eye-drops had varied log reduction against all the challenging organisms which were all within acceptable limits. In all cases the number of fungi after 7, 14 and 28 days were acceptable as they were reduced by 3 and 4 logs which surpassed the requisite of fungi count.

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Table 20: Antimicrobial preservative efficacy of the eye-drop products I–V challenged with E. coli, S.aureus, P. aeruginosa, C.albicans. Microorganism Eye-drop Sampling time/Log reduction 0 6 hours 24 7 days 14 days 28 days hour hours Product I 2.9 x 3 2 - - NR 106 E. coli Product II 2.7 x 3 3 - - NR 106 ATCC 38218 Product III 2.7 x 4 1 - - NR 106 Product IV 2.9 x 2 2 - - NR 106 Product V 2.9 x 4 2 - - NR 106

Product I 1.1 x 3 2 - - NR 106

Product II 1.1 x 3 3 - - NR S.aureus 6 10

Product III 2.1 x 3 2 - - NR ATCC 43300 6 10 Product IV 2.0 x 2 2 - - NR 106 Product V 1.1 x 4 2 - - NR 106

P. aeruginosa Product I 3.8 x 3 2 - - NR 106 ATCC 27853 Product II 3.8 x 3 3 - - NR 106 Product III 3.5 x 4 1 - - NR 106 Product IV 3.5 x 2 2 - - NR 106 Product V 3.5 x 4 2 - - NR 106

C. albicans Product I 2.1 x - - 3 2 NI 106 ATCC 66027 Product II 2.0 x - - 3 - NI 106 Product III 2.0 x - - 4 2 NI 106 Product IV 2.1 x - - 3 2 NI 106 Product V 2.0 x - - 4 - NI 106 NR – no recovery of microbial load, NI – no increase of fungi growth.

Eye drops are classified under category 1 in the USP (2007). The criteria for category one under the USP (2007) goes as thus, “bacteria, not less than 1.0 log reduction from the initial calculated count at seven days, not less than 3.0 log

145 reduction from the initial count at 14 days, and no increase from 14 days’ count at 28 days”. The fungus that was specified showed no increase from initial calculated count at 7, 14 and 28 days. No increase was defined as not more than 0.5 log10 unit higher than the previous value measured. The requirements for antimicrobial effectiveness of all the products were met.

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5. CONCLUSION AND RECOMMENDATIONS

The HPLC method used in the present study permitted rapid and precise determination of phenylephrine hydrochloride and complied with the requirements for specificity, linearity, accuracy, precision and range. The method was accepted as valid, and was therefore deemed suitable for quantitative assay of phenylephrine hydrochloride in the finished products during initial formulation analysis and for the ensuing stability studies.

Active-excipient compatibility studies were carried out in order to determine which excipients would have a potential for reacting unfavourably with phenylephrine hydrochloride. From the above results, HPLC showed it could be used as a method of analysis for active-excipient mixtures of phenylephrine hydrochloride products. Excipients analyzed in the active-excipient compatibility study, demonstrated compatibility with phenylephrine hydrochloride as no interfering peaks or co-eluting peaks were found. If such occurred it would be regarded as an impurity to the API. A 1:1 ratio of active-excipient mixture was used; while, in the actual formulation, the ratio of excipients to phenylephrine hydrochloride was small, minimizing the potential for interaction. The chromatograms showed no interference from impurities or degradation of product. The results showed compatibility between phenylephrine hydrochloride and its excipients.

The products were analyzed in order to determine which formulations produced a physically and chemically stable product after stability testing. The API remaining after the three storage conditions differed among the products I–V; product II and IV stayed within accepted concentrations of greater than 90% among the three different storage conditions, however, product II was the only one that showed no statistical difference during storage. The remaining products failed to achieve the same concentrations. Product III and V fared better in the 25 °C/60%RH but the concentrations were still below the accepted range. The better stability could be due to the fact that products II and IV had excipients with antioxidant and buffering properties, sodium metabisulfite and sodium citrate dihydrate for product II, EDTA and boric acid for product IV. Product II was stable and within potency range in all

147 storage condition whereas product IV was stable and potent in two storage conditions namely 25 °C/60%RH and 30 °C/65%RH. The remaining Products (I, III, V) failed to retain their potency at most storage conditions.

Colour and pH changes were observed mostly with no loss or increase of solution. Observations with regard to no loss of solution meant that the container was suitable for the formulations and it was impervious to moisture. Further observations were that products III and V showed degradation of phenylephrine hydrochloride. This was clearly depicted as phenylephrine hydrochloride within the product formulation of III and V was brown or reddish in colour. This meant the phenylephrine hydrochloride had oxidized fully over the period of three months in which the products were stressed in variable storage conditions. It shows that phenylephrine hydrochloride formulations which are in solutions require antioxidants or protective agents and buffering agents to reduce its quick breakdown, product II and IV had both.

The pH of the products varied from 3.5 to 8, all within the stable range of phenylephrine hydrochloride. From the assay results of HPLC, phenylephrine hydrochloride loses its potency when reacted with hydrogen peroxide and sodium hydroxide. As the pH of the products increases there is a decrease in the concentration of phenylephrine hydrochloride. Product II an IV stayed within the pH of 3 meaning acidity kept PE from degrading. Any future research could focus on optimizing buffers and chemicals that protect phenylephrine from degrading.

Rheological tests conducted displayed notable findings especially with glycerol included in product III. Prefrin® (polyvinyl alcohol) was used as standard to which the products were compared. In all, three viscosity-modifying agents were used for products I–V and they were HPMC, SCMC and glycerol. Figures 154–158 indicate that Prefrin® did not display superior viscosity when compared to products I–V. Glycerol increased in its viscosity over time, the reason is still unknown as it is said to be a stable excipient. A study should be conducted on the glycerol containing product to determine why there is an increase in viscosity over time. Viscosity modifying agents, HPMC and SCMC are similar in function but physicochemically dissimilar (Kibbe, 2006). The viscosity of SCMC loses 25% of its original strength when autoclaved while HPMC only gets denatured at extremely high temperatures

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(Parsons, 2006). Generally, increased temperature (autoclaving and storage conditions) decreases the viscosity of the stated three viscosity modifying agents, the important function needed is the ability to return back to their original state. With this being noted, product IV recovered its viscosity during rheological tests of time zero and three months later while product II also recovered but not completely. Product I failed to recover at the storage condition 40 °C/75%RH. This could mean that SCMC and HPMC could have lost some of their viscosity due to cellulose gum depolymerization. This in turn adds to the reason why it in many cases needs additives and preservatives. Another fact that was observed from literature written by Junyan and colleagues (2009) was that EDTA protects cellulose from loss of viscosity; this could have enabled SCMC and HPMC in achieving their return to initial viscosity. Product II, III and IV showed best performances with regard to their flow.

According to the results obtained, product II and IV were favourable. Any future research could focus on optimizing favourable viscosity-modifying range for hydroxylpropyl methylcellulose and carboxy methylcellulose sodium. The optimal value for phenylephrine hydrochloride protectors such as sodium citrate dihydrate, EDTA, sodium metabisulfite, boric acid should be studied.

Another aspect from the objectives was to find whether the products were provided with preservatives which were effective to inhibit microbial growth. The result showed a positive answer. All products reduced the microbial load that they were inoculated with. Also they passed the compendia requisite for category 1 products (eye drops).

Product I, II, III and V were successful in fulfilling the compendial requirement of preservative efficacy.

Overall, product II was selected as the best among the five products. It would be beneficial to approach its development on a more intensive and rigorous scale as to determine if it would be passed as a generic product for the market at large.

Some limitations of the study were due to reduced sample size. It could have been increased to ensure a representative distribution in differences and possibly similarities among products. An increase in formulations samples could lead to

149 increased chances of a more favourable and stable solution. This allows for wider research to find the best fit ingredients to be mixed together to provide the desired solution. Reliable reports on phenylephrine hydrochloride in solutions would enhance quicker decisions in stabilization, as those available are patented and require substantial amount of money to acquire and use. Extra methods could have been applied in testing the available samples. An example is the full factorial experimental design, this design makes it possible to examine all possible combinations of ingredient variant are served with equal probability.

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APPENDIX A

CONCEPT ARTICLE

The following manuscript is intended to be submitted for publication.

Use and validation of high performance liquid chromatography for phenylephrine hydrochloride estimation

Chinedum Okafor a, Mbali Keele a*, Gareth Kilianb, Matthew Worthingtonc a Department of Pharmacy, PO Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth, 6031 b University of Western Cape c Aspen Pharmacare, 7 Fairclough Road, Korsten, Port Elizabeth, 6014

* E-mail: [email protected]

Abstract: Simple, accurate and reproducible high performance liquid chromatography (HPLC) method was validated for the estimation of phenylephrine hydrochloride in pharmaceutical eye drop formulations. Phenylephrine hydrochloride (PE) was assayed using HPLC at concentration range of 0.0125 to 0.15 mg/ml. Linearity, y = 8541.1 x + 438.55 was achieved as the range were directly proportional to the concentration of phenylephrine hydrochloride within a given range (r2 = 0.9999). The method was tested and validated for various parameters according to the ICH (International Conference on Harmonization) guidelines. The detection and quantification limits were found to be 12.3 and 41 μg/ml respectively. The proposed method was successfully applied for the determination of phenylephrine hydrochloride in pharmaceutical eye drop formulations. The results demonstrated that the procedure is accurate, precise and reproducible, while being simple, cheap

176 and less time consuming, and hence can be suitably applied for the estimation of phenylephrine hydrochloride in eye drops.

Key words: phenylephrine hydrochloride; HPLC; formulations; eye drops

Introduction

Phenylephrine hydrochloride (PE) is a potent adrenergic agent and β–receptor sympathomimetic drug, used in its optically active form (Pandey et al., 2003; Pandey et al., 2006). As an α1-adrenergic receptor agonist it is used primarily as a decongestant, for uveitis and as an agent to dilate the pupil (Lang, 1995).

The drainage of phenylephrine hydrochloride into the nasal mucosa could result in systemic absorption of this agent and produce many unwanted systemic side effects including tachycardia, hypertension, and headache (Bartlett and Jaanus, 2008). Also, the eye drop solutions were either blinked out or only a small portion of the drug reached its site of action. The use of viscosity modifying agents is included in solutions with the aim of obtaining thickening effects. However, these components may have other effects, whether independently or as a consequence of interactions with other components, these effects being mostly due to electrostatic, steric, electrosteric, or depletion mechanisms (Duro et al., 1999).

Various methods have been reported in the literature for the analysis of phenylephrine hydrochloride including spectrophotometer (Collado et al., 2000; Erk, 2000; Solich et al., 2000; Shama, 2002; Knochen & Giglio 2004), spectrophotometry with chromogenic reagent (Ahmed and Amin, 2007), fluorometry (Martin et al., 1993), High-performance liquid chromatography (Marin et al., 2002; Olmo et al., 2005; Galmier et al., 2000) spectro-fluorimetric and derivative spectrophotometric methods (Sabry et al., 2000), have also been reported for the determination of phenylephrine hydrochloride.

The aim of the study was is to use and validate a HPLC analytical method for the estimation of phenylephrine hydrochloride in pure form and in pharmaceutical eye

177 drop formulations. The method was validated as per ICH (International Conference on Harmonization) guidelines and MCC requirements.

Materials and methods

Materials

Phenylephrine hydrochloride, sodium citrate dihydrate, boric acid, disodium edentate, octane-1-sulfonic acid sodium salt, sodium metabisulphite, and benzalkonium chloride were kindly donated by Aspen Pharmacare (Port Elizabeth, SA). Water for chromatography was produced by Ultra Clear TWF/El-Ion® system which has been pre-treated and made ultrapure (reverse osmosis) (Separations, Johannesburg, SA). HPLC grade methanol and octane-1-sulfonic acid sodium salt was obtained from Sigma-Aldrich (Pty) Ltd (Kempton Park, SA). Analytical / technical grades of sodium hydroxide pellets, carboxymethylcellulose sodium, hydroxymethylcellulose, glycerol, methanol hydrochloride acid solution 33%, pyrophosphoric acid (phosphoric acid) was obtained from Merck Laboratory Supplies (Pty) Ltd (Midrand, SA) and hydrogen peroxide 30% were sourced from Saarchem (Pty) Ltd (Johannesburg, SA).

Instruments

The HPLC system consisted of a complete FPLC Shimadzu® HPLC system which has a SPD-M20A Prominence diode array detector, SIL-20A Prominence auto- sampler, DGU-20A5 Prominence degasser, LC-20AB Prominence liquid chromatography and CTO-10AS vp Prominence column oven ( Shimadzu, Tokyo, Japan). Column was a reverse phase Phenomenex® Luna C18 (2) column 250 mm × 4.60 mm, 5 μm particle size (Separations, Johannesburg, SA).

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Calibrations

1.1g of octane-1-sulfonic acid sodium salt was dissolved in one litre mixture of methanol and water (1:1) and the pH was adjusted to 3.0 with pyrophosphoric acid. The resulting solution was mixed, degassed by ultrasonication (Ultrasonic LC 130, Labotec, Germany) and vacuum filtered through a 0.45 μm Millipore filter (Millipore Corporation, Bedford, Massachusetts, USA) prior to use. Dilution solvent was prepared as a mixture of HPLC grade methanol and water (1:1) and adjusted to a pH of 3. The stock solution was prepared by accurately weighing 200 mg of phenylephrine hydrochloride material into a 100 ml volumetric flask, dissolving it and making up to volume with dilution solvent. Calibration standards containing 0.0125, 0.025, 0.05, 0.075, 0.1 and 0.15 mg / ml were prepared by making appropriate solvent dilutions of the working stock solution. Each calibration standard was filtered through a 0.45 μm Millex® syringe driven filter unit prior to injection.

Sample preparation

Eye drop formulations had PE concentration of 100 mg / ml. The eye drop formulations were filtered and 0.3 ml was dissolved into 10ml of dilution solvent to get a final concentration of 0.03 mg / ml. The samples were analyzed using the following analytical method.

Methods The samples were analyzed using the following analytical method:

Linearity

Linearity of an analytical procedure is its ability, within a given range, to obtain test results that are directly proportional to the concentration of analyte in the sample (ICH Harmonized Tripartite Guideline Q2A, 2005). A calibration curve was prepared and linearity demonstrated over a phenylephrine hydrochloride concentration range. A stock solution was prepared having a known concentration of 2 mg / ml (defined as 100%) and dilution of the stock solution to final concentrations of 0.0125 to 0.15 mg / ml were prepared with reverse osmosis ( RO) water and filtered through 23 mm 0.45

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µm PVDF syringe filters (Millex-HV, Millipore, Billerica, USA). Each of the standards was assayed in triplicate. The calibration curve was constructed by plotting the peak areas of phenylephrine hydrochloride versus the respective phenylephrine hydrochloride concentrations and a linear regression trend line was fitted to the plot on Microsoft Excel® 2007, Microsoft Corporation.

Accuracy and precision

Accuracy and precision were determined by replicate injection (n=6) of three phenylephrine hydrochloride solutions, at the upper, middle, and lower limits of the concentration range studied. The concentration ranges were 0.0095 lower limit, 0.054 middle limit and 0.138 upper limit (mg/ml). The theoretical concentrations were calculated from the linear regression curve, and compared to the actual concentrations obtained. The actual mean concentration and standard deviation were calculated at each theoretical concentration. The mean concentrations and percentage recovery of phenylephrine hydrochloride obtained for the replicate injections were a measure of the accuracy of the method, whilst the relative standard deviations at any one concentration provided a measure of precision. The requirement for accuracy is that the percentage recovery of phenylephrine hydrochloride for each solution prepared must be within the 98.00 to 102.00% limit. The requirement for precision is that the relative standard deviations at any one concentration must be less than or equal to 2.00%.

Limit of detection and limit of quantification

Standard solutions of decreasing concentration were produced by successive dilution of the lowest calibration standard and the resulting solutions were injected in triplet. 0.01 ml of the least calibration standard concentration was diluted 10 times.

Specificity

All five products and the phenylephrine hydrochloride were subjected to the following stress conditions after which they were manipulated and analysed:

 0.2 M NaOH for 30 minutes (reflux system);  0.2 M HCl for 30 minutes (reflux system);

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 0.2 M H2O2 for 30 minutes (reflux system);  UV lights (17 hours inside a stability chamber);  100 °C (24 hours inside a stability chamber);  65 °C (1 month inside a stability chamber);  40 °C/75%RH (1 month inside a stability chamber);  Unstressed batch of phenylephrine hydrochloride and Products I–V

A mass of 10 mg of unstressed phenylephrine hydrochloride was dissolved in 100 ml of dilution solvent and analysed using the HPLC method being validated. A volume of 0.3 ml of unstressed Product I–V was dissolved in 10 ml dilution solvent and analysed using the HPLC method being validated. The same method was applied for the samples (phenylephrine hydrochloride and products I–V) stressed within the stability chamber (UV lights, 65 °C, 100 °C, 40 °C/75%RH). Phenylephrine hydrochloride (10 mg) was diluted to 100ml 0.2 M HCl, 5 ml was diluted to 25 ml 0.2 HCl and refluxed for 30 minutes; the same manipulation was applied when using

NaOH and H2O2. 0.5ml of product I–V was diluted to 100 ml 0.2 M HCl and refluxed for 30 minutes, 10 ml was diluted to 100ml dilution solvent, and analysed using the HPLC method being validated. The same manipulation was applied when using

NaOH and H2O2. Samples (phenylephrine hydrochloride and Products I–V) were stressed in a stability chamber (Binder, SA) which emitted both UV and visible light through a window glass filter (type 2) and did conform to the requirements of the ICH guidelines. The stability chamber had an irradiance level of 318 watts / m2 in order to expose the samples to an overall illumination of not less than 1.2 million lux hours and an integrated near UV energy of not less than 200 watt hours / m2 all according to ICH Harmonized Tripartite Guideline Q1B, 2005.

Results and discussion

Linearity

Linearity indicates that the method a calibration curve was constructed by plotting the area of the phenylephrine hydrochloride peak versus phenylephrine hydrochloride concentration. The figure below shows linearity over the concentration

181 range. The linear regression equation for the concentration range of 0.0125 to 0.15 mg/ml was y = 8541.1x + 438.55, with a correlation coefficient, R2, equal to 0.9999. The requirements for linearity were attained, as the correlation coefficient of the regression line was greater than 0.999 and the percentage relative standard deviations for the phenylephrine hydrochloride peak areas of multiple injections were all less than 1.5 %. The y-intercept was found to be 2 > z > -2. This means that the results achieved are directly proportional to the concentration of phenylephrine hydrochloride within a given range.

1400000

1200000

1000000

800000

600000 peak area peak 400000

200000

0 0 50 100 150 200 concentration (mg/ml)

Figure 1: Graph showing a mean peak area versus concentration of replicate samples of phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1 x + 438.55, R2 = 0.9999.

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Accuracy

Accuracy was within acceptable range as noted in Table 1. The percentage relative standard deviations calculated for the samples at the lower, middle and upper limits of the concentration range were all below 0.5%. The recovery percentage for the samples was between the limits of 99.00 to 100.10%. This means by applying the analytical method of a known purity concentration (linearity) and comparing the results of a second, well characterised method, the difference should not be greater than 0.5%.

Precision

Precision was within acceptable range as seen above in Table 2. The percentage relative standard deviations calculated for the samples at the lower, middle and upper limits of the concentration range were all below 0.5%. The recovery percentage for the samples was between the limits of 99.00 to 100.00%. The precision method had the degree of agreement among individual test results when the method was applied repeatedly to the three concentrations chosen of phenylephrine hydrochloride. As above it is expressed in RSD, showing that it can be reproduced or repeated under normal operating conditions.

Limit of detection (LOD)

The LOD was found to be 12.3 μg/ml. The amount stated is the lowest amount of phenylephrine hydrochloride in a sample that can be detected. The LOD is the lowest concentration for which the relative standard deviation of multiple injections is less than 5.0%. By convention, the LOD value is taken as 0.3 times the LOQ (Armbruster & Pry, 2008).

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Limit of quantification (LOQ)

The LOQ was found to be 41 μg/ml. The amount shows the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy. LOQ = 3.33 LOD (Thomsen et al., 2003).

Specificity

Specificity of a chromatographic method is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. Specificity is useful to show that an analyte response cannot be attributed to more than one component (Rozet et al., 2011). The chromatograms were examined for the presence of compounds, metabolites, impurities, degradants that may interfere or partly co-elute with the phenylephrine hydrochloride peak. The results are shown below:

1. Mobile phase chromatogram showed no interference. The mobile phase produced a chromatogram which had a steady baseline and no ghost peaks as seen in Figure 2. 2. Phenylephrine hydrochloride peak observed, it shows no interference from contaminants or impurities. It eluted (had a retention time) of 7.8 minutes. The peak was symmetrical not fronting or tailing. 3. All products that were stressed using UV light, heat, humidity, HCl and NaOH, did not produce compounds which interfered or co-eluted with phenylephrine hydrochloride. This means the excipients did not react with the phenylephrine hydrochloride or any degradants co-eluting with the phenylephrine hydrochloride. The products changed from clear solution to a brownish colour during base stress refluxing, while a pale yellow–lime green colour was observed during peroxide stress refluxing. No change in colour was observed during UV stress testing. 4. The mobile phase was different from the extraction as a result a constant peak is noted between 2.5 and 3.5 minutes as observed in figures 2–6. It takes that amount of time for the injected sample to reach the column. This peak is constituent for all chromatograms. Peak purities were observed in all samples of stressed and unstressed phenylephrine hydrochloride and finished product solutions. Phenylephrine

184 hydrochloride showed loss to the combination of heat and humidity. It was, however, potent against heat alone. There was decomposition by acid, peroxide and base. Phenylephrine hydrochloride was stable to UV light.

Conclusion

High performance liquid chromatography method was used successfully for phenylephrine hydrochloride determinations in eye drop formulations. The analytical method was simple, sensitive, rapid and specific and it can be conveniently used for analysis and quality control of phenylephrine hydrochloride in eye drop formulations. The method was suitable to determine concentrations in the range 0.0125–0.15 mg/ml precisely and accurately. The limits of detection and quantification were detected as 12.5 and 41 μg/ml respectively.

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Table 1: Accuracy data for quantification of phenylephrine hydrochloride

Theoretical Actual Relative Standard Percentage

Concentration Concentration, Recovery (%) Deviation (mg/mL) Mean (n = 6) (%RSD) (mg/mL) 9.5 9.46 0.36 99.5 54 54.1 0.02 100.1 138 136.89 0.05 99.1

Table 2: Precision data for quantification of phenylephrine hydrochloride

Theoretical Actual Relative Standard Percentage

Concentration Concentration, Recovery (%) Deviation (mg/mL) Mean (n = 6) (%RSD) (mg/mL) 9.5 9.48 0.42 99.8 54 53.86 0.06 99.7 138 137.29 0.16 99.4

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Figure 2: Chromatogram of mobile phase alone

mAU 280nm,4nm (1.00) 45

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 3: Chromatogram of phenylephrine hydrochloride only

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mAU 60 280nm,4nm (1.00)

-5 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 4: Chromatogram of Product I

mAU 280nm,4nm (1.00) 45

0 0.0 2.5 5.0 7.5 10.0 12.5 min Figure 5: Chromatogram of Product II

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mAU 280nm,4nm (1.00) 50

0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 6: Chromatogram of Product III

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APPENDIX B

LIST OF EQUIPMENT Autoclave Hirayama Manufacturing Corp, Japan

Spectrophotometer Lasec Cecil LE 2021, Wehingen, Germany

HPLC system LC2020 system, Kyoto, Japan

HPLC column Phenomenex Luna C8

PDA detector SPD M20A PDA detector, Kyoto, Japan

Incubator Labcon Incubator, Labex, Orange/grove

Edelstahl Rostfrei, Memmert, NT Laboratory

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APPENDIX C

LIST OF SOLUTIONS Nutrient agar Nutrient agar 16 grams RO water to 1000 ml

0.9% Sodium chloride solution NaCl 9.0 grams Reverse osmosis water to 1000 ml

1 M sodium hydroxide Sodium hydroxide 40 grams

Reverse osmosis water to 1000 ml

Hydroxypropyl methylcellulose 0.3%

Hydroxy propyl methylcellulose 3 grams

Reverse osmosis water to 1000 ml

Sodium carboxy methylcellulose 0.2%

Sodium carboxy methylcellulose 2 grams

Reverse osmosis water to 1000 ml

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