A Thesis

entitled

Physicochemical compatibility and stability of 4% sodium citrate and ethanol when

stored in silicone coated and non-silicone coated glass containers

by

Megha Krishnakumar

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Pharmaceutical Sciences

Industrial Pharmacy

______Dr. Jerry Nesamony, Committee Chair

______Dr. Gabriella Baki, Committee Member

______Dr. Jeffrey G. Sarver, Committee Member

______Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

May 2020 Copyright 2020 Megha Krishnakumar

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Physicochemical compatibility and stability of 4% sodium citrate and ethanol when stored in silicone coated and non-silicone coated glass containers

by

Megha Krishnakumar

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Pharmaceutical Sciences Industrial Pharmacy

The University of Toledo May 2020

Background: Hemodialysis patients are at high risk of infection because the process of hemodialysis requires frequent use of catheters or insertion of needles to access the bloodstream. Indwelling catheters deliver lifesaving therapy for chronically ill patients but frequently cause infections. Ethanol 20 to 74% concentration has proven efficacy in eradicating various planktonic pathogens as well as microbial organisms embedded in biofilms of indwelling central venous catheters (CVCs). Centre for disease control (CDC) report of emerging infectious diseases suggests 2 million people are infected with bacteria resistant to antibiotics.

Objective: Even though 70% sodium citrate and 30% ethanol solution is used routinely as catheter lock, there is a paucity of guidance and methodological approaches to ensure safe and effective admixture storage when compounded in hospitals. The aim of this research project was to investigate the compatibility of 4% sodium citrate and ethanol mixtures (v/v) containing greater than 30% v/v of ethanol for potential use as a catheter lock solution. The influence of light, temperature and type of storage container

iii on the physico-chemical compatibility and stability of admixtures was studied over

48hours.

Method: Increasing % (v/v) concentrations of 4% sodium citrate was admixed

with ethanol. Samples were studied under four conditions: (1) at 25°C with artificial

indoor white LED tube light, (2) at 25°C without light wrapped using aluminum foil, (3)

at 37°C with artificial indoor white LED tube light, and (4) at 37°C without light

wrapped using aluminum foil. Two types of containers were used: (1) silicone-coated and

(2) non-coated glass test tubes. Physical compatibility, chemical compatibility and

stability were assessed at 0, 8, 24 and 48 hours.

Results: Physical compatibility tests indicated that regardless of the storage condition and nature of container 70% volume of ethanol with 4% sodium citrate formed a crystalline precipitate. A statistically significant difference p≤0.05 in chemical compatibility as indicated by the UV/Vis absorbance at 546 nm was observed between sample admixtures incubated in silicone coated and non-coated tubes and storage conditions such as light and temperature influenced chemical compatibility. The stability study data followed a pattern similar to, albeit statistically insignificant, chemical compatibility studies. Samples stored at 37°C without light wrapped using aluminum foil showed better sodium citrate recovery when compared to samples stored at 25°C with artificial indoor white LED tube light.

iv Physical and chemical compatibility tests of admixtures prepared with a 50% or higher volume % of 4% sodium citrate and ethanol demonstrated compatibility regardless of the type of container and storage conditions. Chemical stability tests indicated a sodium citrate recovery of 90 to100%. There was no statistically significant difference observed in physico-chemical compatibility and stability between different types of containers, light conditions, and temperature of storage.

Conclusions: 70% parts by volume (v/v) ethanol should not be mixed with 4% sodium citrate since this would lead to drug loss and adverse consequences. The admixtures containing 50% or 30% (v/v) ethanol and 4% sodium citrate and were physically and chemically compatible and chemically stable for 48 hours regardless of light and temperature conditions. Pattern plots of physico-chemical compatibility and stability parameters indicated reversible reaction kinetics between sodium citrate and ethanol.

Better safety and compatibility outcomes may be expected when ethanol and sodium citrate admixtures are stored at 37°C without light wrapped using aluminum foil

v Dedicated to my parents Bindu and Krishnakumar and my younger sister Neha.

vi Acknowledgements

I would like to acknowledge the faculty at the University of Toledo for sharing knowledge, providing feedback, motivation and support throughout my period as a graduate student. I would like to thank Dr. Mariann D.Churchwell for providing resources, material, advise, and subject matter that was the foundation for this project. I would like to thank Dr. Jerry Nesamony, Dr. Jeffrey G. Sarver and Dr. Gabriella Baki for imparting knowledge and providing subject expertise, which made this work possible. I would also like to thank the College of Graduate Studies for the continued financial support throughout my graduate studies.

vii Table of Contents

Abstract...... iii

Acknowledgments...... vii

Table of Contents...... viii

List of Tables ...... xii

List of Figures...... xiii

List of Abbreviations ...... xv

List of Symbols...... xvii

1 Introduction ...... 1

1.1 Central Venous Catheter...... 1

1.2 Pathogenesis of CRBSI...... 2

1.3 IDSA Guidelines on CRBSI ...... 3

1.4 Catheter Lock Solution ...... 4

1.4.1 Flushing, Flushing Regimen & Locking of ALT...... 5

1.4.1.1 Flushing...... 5

1.4.1.2 Flushing Regimen ...... 5

1.4.1.3 Locking Volume & Locking Regimen ...... 6

1.5 Physicochemical Compatibility & Stability of CLS...... 6

1.5.1 Physical Stability ...... 8

viii 1.5.2 Chemical Stability...... 8

1.5.3 Microbiological Stability ...... 9

1.6 Factors affecting Drug Stability...... 9

1.6.1 pH...... 10

1.6.2 Temperature ...... 10

1.6.3 Light...... 10

1.6.4 Concentration...... 11

1.6.5 Containers ...... 11

2 Sodium Citrate and ethanol Catheter Lock Solution ...... 12

2.1 Pharmacology of Sodium Citrate in CLS ...... 12

2.2 Pharmacology of Ethanol in CLS ...... 13

2.3 Physicochemical properties of Sodium Citrate (API)...... 13

2.4 Sodium Citrate and Ethanol Lock...... 16

2.4.1 Heparin VS Sodium Citrate ...... 14

2.4.2 Ethanol VS Antimicrobials ...... 14

2.4.3 Dosage Form...... 15

3 Aim of Research ...... 17

4 Materials and Methods...... 18

4.1 Materials ...... 18

4.1.1 Active Ingredients...... 18

4.1.2 Analytical Reagents ...... 18

ix 4.1.2 Storage Containers...... 19

4.2 Methods ...... 19

4.2.1 Preparation of Sample Admixtures ...... 19

4.3 Evaluation of Physical Compatibility ...... 20

4.3.1 Visual Clarity...... 20

4.3.2 pH Evaluation ...... 20

4.4 Evaluation of Chemical Compatibility ...... 21

4.4.1 UV/Vis Spectrophotometry ...... 21

4.4.1.1 Turbidity ...... 21

4.4.1.2 Absorbance Ratio at 240nm...... 21

4.5 Stability Study of Admixtures ...... 22

4.5.1 HPLC Method ...... 22

4.5.2 Preparation of Mobile Phase ...... 23

4.5.3 Preparation of Stock and Standard Solution ...... 23

4.5.4 Stability Study of Sample admixtures...... 24

4.6 Statistical Analysis...... 24

5 Results and Discussion ...... 25

5.1 Visual Clarity...... 25

5.2 Determination of pH ...... 27

5.2.1 Admixture 1 ...... 27

x 5.2.2 Admixture 2 ...... 30

5.2.3 Admixture 3 ...... 32

5.3 Determination of Turbidity...... 34

5.3.1 Admixture 1 ...... 34

5.3.2 Admixture 2 ...... 36

5.3.3 Admixture 3 ...... 38

5.4 Determination of Absorbance Ratio at 240nm ...... 40

5.4.1 Admixture 1 ...... 41

5.4.2 Admixture 2 ...... 42

5.4.3 Admixture 3 ...... 44

5.5 Chemical Stability of Sodium Citrate in Admixtures...... 46

5.5.1 Validation of HPLC method ...... 46

5.5.2 Stability study of sodium citrate in admixtures ...... 48

5.5.2.1 Admixture 1 ...... 48

5.5.2.2 Admixture 2 ...... 53

5.5.2.3 Admixture 3 ...... 56

5.5.2 Admixture 1 ...... 52

5.5.3 Admixture 3 ...... 54

6 Conclusions ...... 59

7 Future Studies ...... 63

8 References ...... 64

xi List of Tables

1.1 USP Chapter 1191: Types of Stability...... 7

4.2 Composition of Admixtures...... 20

4.5 Values of method validation parameters...... 26

5.1 Visual compatibility of admixtures...... 28

5.2.1 Average pH of ADX 1 ...... 29

5.2.2 Average pH of ADX 2 ...... 31

5.2.3 Average pH of ADX 3 ...... 33

5.3.1.1 Average UV/Vis absorbance (OD) at 546nm of ADX 1 ...... 36

5.3.1.2 Average UV/Vis absorbance (OD) at 546nm of ADX 2 ...... 37

5.3.1.3 Average UV/Vis absorbance (OD) at 546nm of ADX 3 ...... 39

5.4.1 Average UV absorbance ratio at 240nm of ADX 1...... 41

5.4.2 Average UV absorbance ratio at 240nm of ADX 2...... 42

5.4.3 Average UV absorbance ratio at 240nm of ADX 3...... 45

5.5.2.1 Average Recovery (%) of ADX 1...... 49

5.5.2 Average Recovery (%) of ADX 2...... 55

5.5.3 Average Recovery (%) of ADX 3...... 56

xii List of Figures

1 – 1 Central venous catheter ...... 2

1 – 2 . Biofilm formation on catheter ...... 3

2 – 2 Structure of sodium citrate...... 13

4 – 1 Spectral line processing of sodium citrate ...... 22

5 – 1 Prepared ADX 1 at 0hr ...... 26

5 – 2 ADX 1 at 8,24 and 48hr...... 26

5 – 3 White crystalline precipitate at the bottom of tube...... 27

5 – 4 Change in pH profiles of ADX 1 ...... 29

5 – 5 pH profiles of ADX 1 ...... 30

5 – 6 Change in pH profiles of ADX 2 ...... 32

5 – 7 pH profiles of ADX 2 ...... 34

5 – 8 Change in pH profiles of ADX 3 ...... 34

5 – 9 pH profiles of ADX 3 ...... 34

5 – 10 UV/Vis Absorbance at 546nm profiles of ADX 1...... 36

5 – 11 UV/Vis Absorbance at 546nm profiles of ADX 2...... 38

5 – 12 UV/Vis Absorbance at 546nm profiles of ADX 3...... 40

5 – 13 UV absorbance at 240nm profiles of ADX 1...... 41

xiii 5 – 14 UV absorbance at 240nm profiles of ADX 2...... 44

5 – 15 UV absorbance at 240nm profiles of ADX 3...... 46

5 – 16 Calibration curve of sodium citrate...... 47

5 – 17 A. Silicone coated container B. Non-coated container average recovery (%) ADX

1 plot ...... 50

5 – 18 A. Silicone coated container B. Non-coated container Time VS Concentration

ADX 1 plot...... 50

5– 19 Average recovery (%) of ADX 2 plot...... 52

5– 20 A. Silicone coated container B. Non-coated container Time Vs Concentration

ADX 2 plot...... 55

5 – 21 Average recovery (%) of ADX 3 plot...... 56

5 – 22 A. Silicone coated container and B. Non-coated container Time Vs Concentration

ADX 3 plot ...... 57

xiv List of Abbreviations

AI ...... Active Ingredient ADX...... Admixture ALT...... Antimicrobial Lock Technique API ...... Active Pharmaceutical Ingredient AMR ...... Antimicrobial Resistance ANOVA ...... Analysis of Variance

BTL...... Body temperature exposed to light BTNL ...... Body temperature not exposed to light

CVC ...... Central Venous Catheter CLS ...... Catheter Lock Solution CRB...... Catheter-Related Bacteremia CRBSI...... Catheter-Related Blood Stream Infections

DI ...... Deionized

FDA...... Food and Drug Administration

HCL...... Hydrochloric acid HPLC ...... High-Pressure Liquid Chromatography

IDSA ...... Infectious Diseases Society of America INR...... International Normalized Ratio IV ...... Intravenous

LED...... Light-emitting diode LOD ...... Limit of detection LOQ ...... Limit of Quantitation

NF ...... National Formulary NS ...... Normal Saline

OD...... Optical density

PDA...... Photodiode Array Detector PT...... Prothrombin Time PICC...... Peripheral Inserted Central Line Catheter

xv RP-HPLC ...... Reverse Phase High Pressure Liquid Chromatography RTL...... Room temperature exposed to light RTNL ...... Room temperature not exposed to light

SAEs ...... Serious Adverse Events

TPN...... Total Parenteral Nutrition

USP ...... United States Pharmacopeia UV/Vis ...... Ultraviolet Visible

xvi List of Symbols

% ...... Percent °C ...... Degree Celsius ® ...... Registered TM...... Trademark

µg ...... Microgram µg/ml...... Microgram/milliliter µL...... Microlitre µm ...... Micrometer g...... Gram(s) hr ...... Hour(s) mg ...... Milligram min ...... Minute(s) mL...... Milliliter mm ...... Millimeter mM...... Millimolar Mohm-cm...... Resistance of pure water n...... Number of replicates N...... Normality nm ...... Nanometer OD...... Optical density pH...... Negative logarithm of hydrogen ion concentration pKa...... Negative logarithm of ionization constant v/v ...... volume/volume w/v...... weight/volume

xvii Chapter 1

Introduction

1.1 Central venous catheters (CVC)

Central venous catheters (CVCs) are the most common form of vascular access for hemodynamic monitoring, fluid and drug administration, chemotherapy, total parental nutrition (TPN), in hemodialysis (1)(2) as shown in Fig.1.1.A. Critically ill hospitalized patients need multiple intravenous therapies, and a frequent problem is the lack of sufficient number of vascular access sites to deliver each therapy separately(3) . These critically ill patients in whom vascular access is difficult for long term (>14 days) and need multiple intravenous therapies, nutrition and fluids utilize indwelling CVCs

(3)(4)(5). Long term catheter uses such as those used in hemodialysis (Fig.1.1. A.) causes catheter-related bloodstream infection (CRBSI) when catheter replacement is not a feasible option(6). Hemodialysis CVCs have arterial and venous ports that remain outside the body of the patient. A catheter lock solution (CLS) is injected to dwell in such catheter ports when not in use(5).

1 Fig 1.1.A. Central venous catheter(Image by BruceBlaus,CC-BY-3.0 https://creativecommons.org/licenses/by-sa/3.0/, no changes made)(7)

1.2 Pathogenesis of CRBSI

CRBSI is defined as the presence of bacteremia originating from an intravascular catheter. Long-term implantation of a CVC on critically ill patients can cause a high rate of infection, thrombus-related dysfunction, or occlusion(2) .Skin and catheter hubs are the frequent sources of microbial colonization of catheters. The catheter hubs are colonized through contaminated hands of healthcare personnel handling, while skin borne organisms such as Coagulase-negative staphylococci and Staphylococcus aureus are pathogens causing CRBSI through the extraluminal route(8). Fig.1.2 Within 24hrs of catheter insertion, the catheter gets colonized by microorganisms producing what is called a biofilm, formed from inoculation of skin microorganisms

2 Once adhered to the surface, microorganisms proliferate to form multilayered clusters or

biofilm(9)(10). Eventually, these microbes detach from biofilm and seed the bloodstream

causing CRBSI(10).

Fig. 1.2 Biofilm formation on catheter (Image by 24Adrianus. Cropped for clarity, see original at https://commons.wikimedia.org/wiki/File:Biofilm_id.JPG. CC-BY-2.5 https:// creativecommons.org/licenses/by/2.5/deed.en)(7)

1.3 IDSA guidelines on CRBSI

Clinical practice guidelines recommend the use of the antimicrobial lock

technique (ALT) for prophylactical and therapeutic management of CRBSI. The

Infectious Diseases Society of America (IDSA) guideline for diagnosis and management

of CRBSI recommends the use of antibiotic locks for catheter salvage in cases when a

catheter cannot be removed from patients(11). CVC removal remains the first-line of

therapy for the management of CRBSI, especially in cases of Staphylococcus aureus and the resistant gram-negative pathogen, Pseudomonas aeruginosa(11), and presents significant challenges when catheter removal is not always a feasible option in patients(6).

3 The prophylactic catheter lock solution should not be used routinely for preventing

CRBSI but used only under special circumstances (e.g., in treating a patient with a long- term cuffed or tunneled catheter or port who has a history of multiple CRBSIs despite optimal maximal adherence to aseptic technique due to potential concerns on the development of antimicrobial resistance) (12).

1.4 Catheter lock solutions (CLS)

The technique of using CLS or ALT in catheters involves instilling highly concentrated antimicrobial-anticoagulant solutions in volumes sufficient to fill the catheter lumen and allowing them to dwell for a specified period to sterilize the catheter lumen(13). Catheter lock solutions are placed or “locked” in the catheter lumen at the end of each dialysis session or between treatments and aspirated before the next treatment to maintain catheter patency and to prevent thrombosis(14). IDSA guideline states that the dwelling period of lock solution should not exceed 48 hour before the re-installation of

CLS, but in hemodialysis patients, lock solution should be renewed after every dialysis session(15)(6).

For CRBSI, antibiotic lock therapy should not be used as monotherapy but should be used in conjunction or as an adjunct with systemic antimicrobial therapy(16).

Systemic antibiotics fail to treat biofilm-associated infections since sessile bacteria within the biofilms are less susceptible to antimicrobial agents when compared to rapidly growing planktonic cells. Filling catheter lumen with CLS containing high concentrations of antibiotics for extended periods achieves supraphysiologic antibiotic concentrations

4 that overcome AMR of sessile bacteria. However, with this approach, there is a potential for development of resistant organisms and risk of systemic toxicity due to leak of antibiotics from the catheter lumen to systemic circulation(17)(10).

1.4.1 Flushing, flushing regimen and locking of ALT

1.4.1.1 Flushing

Flushing is a process intending to clear the catheter chamber which is in active use. An adequate flush volume is required to remove debris and fibrin deposits in the catheter reservoir and port reservoir. Recommendations state to “use at least twice the volume of the catheter and add-on device” usually 5-10 ml(18). When viscous material such as blood products and TPN are passed through a catheter due to difficulty in removal, a flush volume of 20 ml is to be used to prevent early chances of infection.

Adherence of lipid, fibrin, and other drug deposits to the reservoir wall from such material, results in colonization of microorganisms and CRBSI(18).

1.4.1.2 Flushing regimen

The flushing regimen recommendation to flush before and after administration of medication or any procedure involving use of a catheter is also known as SAS/SBS acronym. The order of IV injection is as follows: a normal saline flush (S), followed by the administration of drugs or fluids (A)/blood sampling(B), followed by saline flush (S).

The SAS/SBS procedure should end with locking the catheter with CLS. This regimen is called ALT(18).

5 1.4.1.3 Locking volume and locking regimen

The locking volume of the catheter lock solution should be enough to fill the volume of the entire catheter. Considering the risk of leakage of the “lock” over time, which has been proved, the catheter should be overfilled by approximately 15-20%, provided the catheter lock solution does not cause systemic adverse effects when injected systemically. The total lock volume of CVC lumen is 1 ml, an extra volume of 0.2 ml should be theoretically used in practice. However, in current guidelines and clinical practice usage of twice the internal volume of catheters of 5-10 ml is advised (18).

The locking regimen is based on the concentration of the active ingredient in the lock solution. For low concentration lock solutions, when a lock is renewed the new lock solution can be instilled without aspirating or flushing with normal saline. Whereas, for high concentration locks, where there is a risk of adverse effects when injected into circulation, the lock solution must be aspirated before the renewal of the lock(18).

1.5 Physicochemical compatibility & stability

Incompatibility is defined as “a physicochemical phenomenon which occurs when a drug is mixed with others and produces an unsuitable product”(19). The new product is unsuitable for administration because the “active ingredient” has been modified (e.g., increase in toxicity) or because of physical changes (e.g., solubility change) has occurred.

Incompatibility is different from instability but must be considered in the overall stability evaluation of the preparation(20).

6 The United States Pharmacopeia 36/National Formulary 31 (USP 36/NF 31), Chapter

<1191> “Stability considerations in dispensing practice” and USP general chapter <795>

“Pharmaceutical Compounding-Nonsterile Preparations” defines stability as the extent to which a product retains within specified limits and throughout its period of storage and use (i.e., its shelf life), the same properties and characteristics that it possesses at the time of compounding(21)(22). The purpose of stability testing is to provide evidence on how the quality of the compounded admixture varies with time under the influence of a variety of environmental factors such as temperature, humidity and light and to establish shelf- life of product and recommend storage conditions. USP, recognizes 5 types of stability as shown below in Table 1.1(22):

Table 1.1 USP Chapter <1191>: Types of stability(22)

Types of Stability Condition maintained throughout the shelf life of drug product

Chemical Each active ingredient retains its chemical integrity and

labeled potency within specified limit

Physical The original physical properties including appearance,

palatability, uniformity, dissolution and suspendability are

retained

Microbiological Sterility or resistance to microbial growth is retained

according to specified requirement.

Therapeutic Therapeutic effects remain unchanged

Toxicological No significant increase in toxicity occur

7 1.5.1 Physical stability

When a physical change occurs, the same drug or chemicals would be present in admixture, but its physical state would be altered resulting in the drug precipitating out of the solution or drug adsorbing on to the walls of the container which may be lethal and unacceptable when used in patients(22).

According to USP <790>, all products intended for parenteral admixture must be visually inspected and evaluated for cloudiness, color change, evolution of gas and observable precipitation such as crystals, haziness, or turbidity when reconstituted(23)(24). Fig. 1.4 describes USP Chapter <1191>, criteria for acceptable physical stability.

1.5.2 Chemical stability

Chemical instability is the loss of potency of the original drug molecule and forms distinctly different chemical entities (degradation products) that can be both therapeutically inactive and possibly exhibit greater toxicity(20)(22). According to USP

Chapter <1191>, loss of potency may be the result of chemical reactions such as hydrolysis, epimerization, dehydration, decarboxylation, oxidation-reduction, and photolysis. A chemical change occurs through the interaction between ingredients with an admixture or between products and container without an obvious visual or olfactory evidence of the occurrence (22). An apparent loss of potency of active ingredient (AI) may result when drug diffuses into the surface of the container-closure system.

8 An apparent gain in potency usually is caused by solvent evaporation or leaching of materials from the container-closure system (Hunt,2020),(25). Fig.1.4 USP Chapter

<1191> lists the criteria for acceptable levels of chemical stability. Pharmacists should understand and avoid ingredients and conditions that result in excessive physical deterioration or chemical decomposition of drug preparations during compounding of

CLS(25).

1.5.3 Microbiological stability

Chapter <1191> of the USP provides the following criteria for acceptable levels of microbiologic stability: “Sterility or resistance to microbial growth is retained according to the specified requirements. Antimicrobial agents that are present retain effectiveness within the specified limits”. Areas used for compounding sterile preparations should be well-lighted, clean, separated and distinct(22)(21). Fig.1.4 USP

Chapter <1191>lists the criteria for acceptable levels of chemical stability.

1.6 Factors affecting drug stability

The primary environmental factors that can reduce stability include exposure to adverse temperature, light, humidity, oxygen, and carbon dioxide. The major dosage form factors that influence drug stability include particle size, pH, solvent system composition, the concentration of the active ingredient, and primary container(25).

9 1.6.1 pH

The degradation of drugs in solution accelerates or decelerates exponentially as the pH is

decreased or increased over a specific range of pH values. Improper pH with exposure to

elevated temperature is a factor most likely to cause significant loss of drug due to

hydrolysis and oxidation reactions. Drug solutions may be stable for days, weeks or years

in their original formulation, but when admixed with another liquid that changes the pH,

they can degrade rapidly in minutes or days. A pH change of only 1 unit decreases drug

stability by a factor of 10 or greater(25) (26). Weakly acidic and basic drugs show good solubility when ionized but they also decompose faster when ionized(26).

1.6.2 Temperature

The rate of chemical reaction increases exponentially for each 10-degree increase

in temperature. The actual factor of rate increase depends on the activation energy of the

reaction. The activation energy is a function of the specific reactive bond and the drug

formulation (e.g., solvent, pH, and additives)(25).

1.6.3 Light

Drug stability is affected through energy or thermal effects which leads to

oxidation called photochemical decomposition(26). Possible strategies to handle drugs

subjected to oxidation is to wrap the drug containers and IV bags or store in light-

sensitive containers (22).

10 1.6.4 Concentration

If a product is diluted or when two products are admixed, the pharmacist should observe good professional and scientific procedures to guard against incompatibility and instability. For example, when products containing high concentrations of alcohol are mixed with aqueous systems, they can develop a precipitate. Combining parenteral products demands the utmost care, aseptic technique, judgment, and diligence. Because of potential problems of unobservable sterility and chemical stability, compounded parenteral preparations are to be used within 24 hours(25).

1.6.5 Containers

All containers used for compounded parenteral preparations should maintain the integrity of the preparation sterile, pyrogen-free, and pure until used. Glass is the material of choice for parenteral products and may either be sealed or closed with rubber stoppers.

Type-1 glass also known as “neutral” is a borosilicate glass with good chemical resistance(27).Containers of Type-1 glass are best for aqueous preparations.

Siliconization, i.e., the application of a thin film of silicone to coat the inside surface of containers has helped prevent interaction of the product with the glass surface(28). The application of a coating on the glass surface reduces the number of sites for hydrophilic bonding, it replaces the -OH groups on the glass surface to Si-O-Si (Silyl Ether groups)(22).

11 Chapter 2

Sodium citrate and ethanol CLS

2.1 Pharmacology of sodium citrate in CLS

The citrate anion in the sodium citrate 4% solution acts as an anticoagulant by chelating

ionized calcium(Ca2+)in the blood and tissue preventing the activation of calcium-

dependent procoagulants (29) .This mechanism of action of sodium citrate is by lysing

blood clots formed inside catheters during blood sampling procedures or dialysis and

thereby preventing thrombus occlusion. Sodium citrate chelates magnesium ions (Mg²+) as well and interferes with cellular integrity by degrading the bacterial cell wall membrane. The chelation of Ca2+ and Mg²+ and the hyperosmolality of the solution

prevents biofilm formation, which holds bacteria in firm glycocalyx. Sodium citrate can

be used in various concentrations as an antimicrobial and CLS(30)(29). 0.5% Sodium

citrate concentration inhibits biofilm formation and growth of Staphylococcus aureus and

Staphylococcus epidermis. A concentration of 30% is effective in killing Pseudomonas

aeruginosa and Escherichia choli(31).

12 2.2 Pharmacology of ethanol in CLS

Ethanol acts as an antiseptic, demonstrates bactericidal activities against Gram- positive and Gram-negative bacteria and has fungicidal activity. It works by penetrating the bacterial cell and denaturing cell proteins required for metabolic functions. It eliminates bacterial cells by targeting the cell membrane causing membrane leakage(32)(33). Ethanol act as a sterilizing agent for hemodialysis catheter (32). No known resistance for ethanol has been reported to date(33)(10). Ethanol also has an innate and proven ability to reduce CRBSI in hemodialysis patients(34).

2.3 Physicochemical properties of sodium citrate (API)

Sodium citrate is a white hygroscopic crystalline drug(35)(36). It is acidic with a pKa of

3.5(35)(36).It is freely soluble in water and practically insoluble in alcohol(36).Quantification of sodium citrate is most popularly performed using reverse- phase liquid chromatography (RP-HPLC)(37).

Fig 2.2 Structure of sodium citrate (Image by Rifleman82, self-published work https://creativecommons.org/licenses/by-sa/3.0/deed.en, no changes made)(38)

13 2.4 Sodium citrate and ethanol lock

2.4.1 Heparin Vs sodium citrate

In the past heparin was widely used as anticoagulants in catheter locks(39).

Spillage of lock solution into the blood circulation (intentional/unintentional) can cause heparin-induced bleeding complications and interference with prothrombin time/International Normalized Ratio (PT/INR ratio) leading to heparin-induced thrombocytopenia in patients. Sodium citrate at 30% concentration is known to reduce catheter-related bacteremia (CRB) and act as an antimicrobial and anticoagulant.

Inadvertent flushing of sodium citrate when used as catheter lock leads to calcium complexation and hypocalcemia(40). There is an 85% cost reduction when using sodium citrate instead of heparin as the intradialytic anticoagulant in hemodialysis catheters(41).

Additionally, heparin should not be concomitantly used with ethanol due to the rapid formation of a precipitate(41)(42). Sodium citrate is commonly used clinically in 4% w/v strength due to its reported efficacy and to avoid adverse effects of myocardial infarction and death associated with concentrated 46.7% sodium citrate(42).

2.4.2 Ethanol Vs antibiotics

Due to the spillage of lock solution into blood circulation, antibiotics such as gentamicin, telavancin, vancomycin, which were combined with sodium citrate developed antimicrobial resistance in patients(43). Ethanol is inexpensive, easily

14 available, and effective for use in prophylactic lock regimens(43). Ethanol in varying concentrations (20-74%) is used to eradicate planktonic pathogens as well as microbial organisms embedded in the intraluminal biofilm of indwelling CVCs(2). An important consideration while selecting ethanol as a component in lock solutions is the characteristics of adherent pathogens and the impact of materials of CVCs, including silicone, polyurethane, and polycarbonate on the antimicrobial activity of ethanol and water. Recommendations suggest aspirating ethanol from catheter to avoid inadvertent spillage into systemic circulation especially in vulnerable population(43).

A 30% ethanol concentration produces a rapid kill of planktonic bacteria and should prevent biofilm formation between dialysis sessions(1).The combination of 30% ethanol and 4% sodium citrate catheter locking solution prevented biofilm formation in CRBSI and is very successful at eliminating planktonic MRSA,MSSA, P. aeruginosa and E.coli in vitro(33)(1).There is a possibility of seepage of blood into CVC due to density difference especially with greater than 30% ethanol which could be avoided by adjusting catheter position in patients to jugular(subclavian)-vertical position(2),and highly emphasized to remove the lock solution before each dialysis sessions(2)(1).

2.4.3 Dosage Form

Sodium citrate formulations have not been approved to be commercially available by the

FDA and most formulations are prepared in hospital pharmacies as salvage therapy for patients in United States(39). A prefilled catheter lock syringe containing a solution of

30% ethyl alcohol and 4% sodium citrate is commercially available in Canada(44)(43).

15 Ethanol/ Sodium citrate locking solution is reported to have a positive effect on catheter survival compared to heparin(34). This combination prevents CRBSIs and catheter dysfunction issues without demonstrating toxicity. The solution is compatible with carbothane and silicone catheters with no serious adverse events (SAEs) (34)(45).

16 Chapter 3

Aim of the Research

Clinically 70% sodium citrate and 30% ethanol are used routinely as catheter lock.

However, there is a paucity of guidance and methodological approaches to ensure safety and effective admixture storage when it is compounded in hospitals. The aim of this research project was to investigate the compatibility of 4% sodium citrate and ethanol mixtures (v/v) containing greater than 30% v/v of ethanol for potential use as catheter lock solution. In our study, 4% w/v sodium citrate was a premixed solution of 4g of sodium citrate in 100ml water and ethanol were both solutions, a (v/v) admixture composition was used for expressing concentration. The influence of light, temperature and type of storage container on the physico-chemical compatibility and stability of admixtures was studied over 48hrs

17 Chapter 4

Materials and Methods

4.1Materials

4.1.1 Active ingredients

Sodium citrate 4% w/v 250 ml Intravenous (IV) bag (Lot no.: Y295238) was received from Fenwal Inc./Baxter Healthcare Corporation (Deerfield, IL, USA). Ethanol

200 proof anhydrous USP (1 Gallon) (Lot no.:067914) was received from Decon

Laboratories Inc (King of Prussia, PA, USA).

4.1.2 Analytical reagents

Hydrochloric acid, potassium phosphate monobasic, triethylamine and orthophosphoric acid were procured from Fischer Scientific (Hampton, NH). All reagents used were of analytical grade.

18 4.1.3 Storage containers

Admixtures were stored in 16×100 mm glass containers with silicone coating (BD vacutainer, no additive, no interior coating, glycerin lubricated stopper, lot.no.: 5249852) received from BD (Franklin Lakes, NJ), non-coated glass containers (BD vacutainer, no additive, silicone interior coating, silicone lubricated stopper, lot.no.: 9004549) received from Beckton Dickinson and company (Franklin, NJ).

4.2 Methods

4.2.1 Preparation of sample admixtures

The antimicrobial sodium citrate was compounded with 100% ethanol under a horizontal laminar airflow hood to obtain the concentrations described in Table 1-1. Reconstitution was performed in a sodium citrate intravenous (IV) bag by adjusting the volumes of sodium citrate, followed by instilling ethanol in enough volume required for each admixture through the IV port. Aliquots of 5 ml each were drawn out of the bags and placed in glass containers with silicone interior coating and non-silicone coated tubes.

Samples were stored under four different conditions: (1) at 25°C with artificial indoor white LED tube light (RTL), (2) at 25°C without light wrapped using aluminum foil

(RTNL), (3) at 37°C with artificial indoor white LED tube light (BTL), and (4) at 37°C without light wrapped using aluminum foil (BTNL) stored in two different containers: (1) silicone-coated and (2) non-coated glass test tubes. Samples were analyzed in triplicate at

0, 8, 24, and 48hour time points

19 Table 4.2 Composition of Admixtures

Components Composition(v/v) ADX 1 ADX 2 ADX 3 Sodium citrate 4% 30% 50% 70%

Ethanol 70% 50% 30%

4.3 Evaluation of physical compatibility

4.3.1 Visual Clarity

Visual clarity was evaluated by visual inspection of the samples, i.e., moving the samples against the black and white background. A positive control was prepared using

2.5 ml of anhydrous calcium chloride and 2.5 ml of sodium phosphate tribasic heptahydrate. The negative control comprised of deionized (DI) water. The samples were graded on an ordinal scale, where 0 meant no evidence of precipitation, 1 meant trace evidence of precipitation, 2 meant slight haze, 3 meant medium haze, and 4 meant dense haze(6)(46)(47).

4.3.2 pH Evaluation

The pH of admixtures was determined using Mettler Toledo Seven Multi pH meter, Schwarzenbach, Switzerland with a Mettler Toledo LE409 electrode, Griefensee,

Switzerland pre-calibrated with standard buffer solutions of pH 4, 7, and 10 before each run. The electrode was directly dipped in the admixtures. A change in pH of > 0.1 pH unit, compared with the 0-hour reading was considered as a demonstrative change

20 indicating incompatibility(47). pH evaluations were done in triplicate samples of each admixture.

4.4 Evaluation of chemical compatibility

4.4.1 UV/Vis spectrophotometry

A UV Spectrophotometer, UV-Spectramax 2000, Sunnyvale, CA with quartz microcuvette of path length 1 cm was used. Blank samples consisting of ethanol: deionized water in a similar v/v composition as that of the sample were used during measurements.

4.4.2 Turbidity

The presence of visible and sub-visible precipitates in the samples was estimated by measuring optical density (OD) of the samples at 546 nm(46). At 546 nm, neither the blank nor the sample itself absorbs and only suspended precipitates scatter incident light causing a pseudo-absorbance(48)(47)(46).

4.4.3 Absorbance ratio at 240nm

The optical densities before incubation, at the time of mixing (t = 0 h) and after incubation (time points: 8, 24, and 48 h) were measured using the same device and blank used in turbidity tests. Figure 4-1 shows the spectral line processing of sodium citrate at

240 nm where the absorbance of sodium citrate was 1 OD. At the wavelength maxima of sodium citrate at 240 nm, both the precipitate, which is a derivative of sodium citrate formed as a result of incompatibility during incubation, and the non-precipitated sodium

21 citrate in the solution absorb UV/Vis. The supernatant would be utilized for analysis in the event of precipitation. In the absence of incompatibility, the ratio between absorbance

(OD) at 240 nm before and after incubation is theoretically 1. Precipitation during storage was estimated from the ratio of absorbance at baseline (0 hour) to various time points of analysis after incubation or storage. The change in absorbance at 240 nm indicated citrate loss from the solution due to precipitation, which in turn indicated turbidity (48).

nm 240=ג Figure 4.1: Spectral line processing of sodium citrate at

4.5 Stability study of admixtures

4.5.1 HPLC Method

An isocratic elution was carried out on a Waters® HPLC, Milford, MA with e2695

22 separation module equipped with 2998 Photodiode array (PDA) detector system using

C18 reverse phase Water’s column with a pore size of 3.5µm: 4.6 × 75mm (Part

#WAT066224). The mobile phase was 100% 50 mM phosphate buffer at pH 2.80.

The HPLC system operated at a flow rate of 0.8 ml/min, the injection volume was 10 µl, and triplicate injections were performed for every sample. The detection wavelength was

215 nm using PDA detector. Analysis time was 3 min with wash/equilibration time of 15 min. The output signal was monitored and processed using Empower-3 software.

4.5.2 Preparation of mobile phase

The 50 mM phosphate buffer solution used as the mobile phase was prepared by dissolving 6.82 g of monobasic potassium phosphate in deionized water and filtered using a 0.22 µm nylon membrane by vacuum filtration to obtain HPLC grade mobile phase(37). 1 ml triethylamine was added to the buffer to reduce peak tailing followed by adding 1.5 ml concentrated phosphoric acid adjusting the pH to 2.80 while monitoring with a calibrated pH meter. The solution was then diluted using deionized water to make up to 1000 ml. The buffer was sonicated further for 30 seconds.

4.5.3 Preparation of stock and standard Solution

A stock solution of 500 µl (200 µg) sodium citrate in 100 ml 0.1 N hydrochloric acid (HCl) was prepared. Working standards of 5 to 25 µg/ml were prepared by diluting the stock solution with 0.1 N HCl to required concentrations before use. Calibration

23 standards were run (n = 6) and the average peak area was obtained A calibration curve was made by plotting the average peak area against the concentration of sodium citrate

(µg/ml). Analytical method validation parameters including linearity, limit of detection

(LOD), limit of quantitation (LOQ), and accuracy were determined.

4.5.4 Stability study of sample admixtures

Samples of ADX1 from coated and non-coated silicone tubes exposed to (room temperature exposed to artificial indoor white LED light) RTL, (room temperature not exposed to light wrapped using aluminum foil) RTNL, (body temperature exposed to artificial indoor white LED light) BTL, and (body temperature not exposed to light wrapped using aluminum foil) BTNL of 5 ml volume were prepared. A dilution factor of

1:500 was used to bring the nominal concentration of sodium citrate in the admixture to

24 µg. Care was taken not to agitate admixtures upon sampling(4)

Samples of ADX 2 and 3 from non-coated and silicone-coated tubes were diluted

1:500 using 0.1 N HCl to obtain concentrations within the linearity range of 5 to -25 µg/ml of the calibration curve, adequate for final measurement. At each time point of the study period, samples were taken in triplicate for analysis.

4.6 Statistical analysis

Statistical analyses were performed with IBM® SPSS® (Version 25). All statistical analyses were performed at the 95% significance level and confidence interval.

In each variable of analysis, the dependent variable was the variable itself and the

24 independent variables were containers, conditions, and time period. Two-Way ANOVA was used to analyze the effect of independent variables on dependent variable. Results were statistically significant at p value ≤ 0.05.

25 Chapter 5

Results and Discussion

5.1 Visual clarity

Admixture 1 exhibited slight haze (ordinal scale=2) at the time of mixing as shown in

Figure 5-1, and progressed to have dense haze (ordinal scale=4) at 8, 24, and 48 hours,

Figure 5-2, irrespective of whether they were stored at 25 ± 0.5°C and 37 ± 0.5 °C or exposed to artificial indoor white LED tube light and not exposed to light wrapped using aluminum foil exposure. Admixture 1 appeared clear and colorless at the top layer of the solution with a white crystalline precipitate settling down at the bottom of the tube at 8,

24, and 48 hours as shown in Figure 5-2(42).

All the admixtures of 2 & 3, stored at 25 ± 0.5°C and 37 ± 0.5°C exposed to artificial indoor white LED tube light and not exposed to light wrapped using aluminum foil were clear and colorless from the time of mixing until 48 hours after mixing irrespective of whether they were stored in silicone-coated and non-coated glass containers.

26 Table 5.1 Visual compatibility of admixtures

Admixture Appearance of admixture Compatibility

ADX 1 Precipitates No

ADX 2 Clear Yes

ADX 3 Clear Yes

Fig. 5-1 Prepared ADX 1 at 0hr Fig. 5-2 ADX 1 at 8,24,48hr

27 Fig.5-3 White crystalline precipitate at the bottom of the tube

5.2 Determination of pH

5.2.1 Admixture 1

Changes in pH is important since any change in the acid-base environment of the drug is a factor of instability(49). The pKa of sodium citrate is 6.40 (35)(36), and the admixture 1 pH range is 6.98 to 7.80 (Table 5.2.1). The percent of unionized sodium citrate ranges from 21 to 4% as calculated using the Henderson-Hasselbalch equation for weak acids:

( −) Where (A-) and (HA) represent the concentration = + log of ionized and unionized species. The () optimal pH of the admixture has been shown to be one unit below the pKa of weakly acidic drugs. pH determination of admixtures points to its impact on drug solubility.

Sodium citrate is an acidic drug whose solubility is pH dependent. As pH increases, the

28 percentage of ionized species increases over unionized species. The greater the concentration of ionized species in the admixture, greater the tendency to form a salt or precipitate with a co-component in the admixture with ethanol raising admixture pH closer to 8. The pH profile of ADX1 is suggestive of a pH-dependent solubility of ADX1 as demonstrated in Fig 5-5 (50)(3). The non-coated tubes exposed to RTL, RTNL, BTL, and BTNL (8 and 24 h), and silicone-coated tubes exposed to RTL, BTNL, and BTL (8 and 24 h) did not exhibit a pH change greater than 0.1, thus considered compatible

(Fig.5-4). Hence ADX1 exposed to all conditions was further tested for confirming incompatibility and stability.

Influence of Conditions and Time with Containers on pH: A two-way ANOVA statistical analysis was performed to analyze the effect of fixed factor (containers: silicone-coated and non-coated), and random factor (conditions and time) on pH values.

There was no statistically significant interaction between container and time on pH (F =

1.306, p = 0.331), or between container and conditions on pH (F = 1.013, p = 0.431).

Therefore, it can be concluded that there is no difference between pH values of silicone coated and non-coated containers when various conditions used in the study, and different time points of analysis were taken into consideration.

29 Table 5.2.1 Average pH of ADX1

ADX Container Time pH (Mean ± SD)

(h) RTL RTNL BTL BTNL

0 7.72±0.04 6.98±0.05 7.68±0.01 7.66±0.02

Silicone- 8 7.66±0.02 7.69±0.04 7.71±0.00 7.69±0.01

Coated 24 7.71±0.04 7.74±0.05 7.61±0.04 7.67±0.03

ADX 1 48 7.60±0.01 7.67±0.03 7.67±0.03 7.80±0.04

0 7.76±0.05 7.69±0.05 7.76±0.02 7.54±0.05

Non- 8 7.75±0.05 7.69±0.02 7.75±0.01 7.58±0.07

Silicone 24 7.63±0.02 7.67±0.02 7.46±0.08 7.63±0.04

Coated 48 7.71±0.05 7.65±0.03 7.66±0.05 7.78±0.03

ADX 1-Absolute pH change 120% 100% 80% 60% 40% 20% 0% RTL RTNL BTNL BTL RTL RTNL BTNL BTL Silicone coated Non-coated

Compatible Incompatible

Fig 5-4: Change in pH profiles of ADX1

30 A. Silicone-coated container B. Non-coated container

Fig 5-5: pH profiles of ADX1

5.2.2. Admixture 2

All admixtures exposed to RTL, RTNL, BTL, and BTNL stored in silicone and non- silicone coated tubes over 48 h dwell time did not exhibit a pH change greater than 0.1,

Fig 5-6. Table 5.2.2 and Fig 5-7 summarizes the mean pH obtained from triplicate samples of ADX 2. ADX2 was observed to be compatible(6).

Influence of Containers, Conditions and Time on pH: A two-way ANOVA statistical analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), random factor (conditions and time) on pH values. There was no statistical significance of interaction between container and time on pH (F = 0.124, p = 0.944), or between container and conditions on pH (F = 4.283, p = 0.039). Therefore, it can be concluded that there is no difference between pH values of silicone coated and non- coated containers when the influence of various conditions used in study, and different time points of analysis are taken into consideration.

31 Table 5.2.2 Average pH of ADX 2

Admixture Container Time pH (Mean ± SD)

(h) RTL RTNL BTL BTNL

0 7.24±0.01 7.25±0.01 7.58±0.05 7.29±0.02

Silicone- 8 7.32±0.01 7.37±0.01 7.46±0.03 7.39±0.02

Coated 24 7.33±0.01 7.31±0.01 7.55±0.01 7.37±0.02

ADX 2 48 7.22±0.01 7.19±0.01 7.62±0.01 7.41±0.04

0 7.27±0.01 7.27±0.01 7.53±0.02 7.23±0.08

Non- 8 7.31±0.01 7.34±0.01 7.53±0.05 7.29±0.06

Silicone 24 7.36±0.03 7.31±0.01 7.57±0.03 7.33±0.01

Coated 48 7.29±0.02 7.28±0.02 7.62±0.02 7.24±0.08

ADX 2-Absolute pH Change 100% 80% 60% 40% 20% 0% RTL RTNL BTNL BTL RTL RTNL BTNL BTL Silicone coated Non-coated

Compatible Incompatible

Fig 5-6: Change in pH profiles of ADX 2

32 A. Silicone coated container B. Non-Coated container

B. Fig 5-7: pH profiles of ADX 2

5.2.3. Admixture 3

Table 5.2.3 and Fig. 5-9 summarizes the average pH values obtained from triplicate samples of ADX 3. All admixtures exposed to RTL, RTNL, BTL, and BTNL stored in silicone coated and non-silicone coated tubes exhibited a pH change within 0.1 and remained compatible over the 48 hours (Fig.5-8)(46).

Influence of containers, conditions and time on pH: A two-way ANOVA statistical analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), random factor (conditions and time) on pH values. There was no statistical significance of interaction between container and time on pH (F = 0.189, p = 0.901), between container and conditions on pH (F = 2.268, p = 0.150).

33 Therefore, there is no difference between pH values of silicone coated and non-coated containers when the influence of various conditions used in study, and different time points of analysis are taken into consideration.

Table 5.2.3 Average pH of ADX 3

Admixture Container Time pH (Mean ± SD)

(h) RTL RTNL BTL BTNL

0 7.04±0.02 7.06±0.02 7.07±0.04 7.2±0.01

Silicone- 8 7.05±0.01 7.11±0.05 7.12±0.03 7.1±0.04

Coated 24 7.00±0.03 7.09±0.02 7.13 ±0.02 7.1±0.01

ADX 3 48 7.01±0.01 7.00±0.01 7.13±0.02 7.03±0.03

0 7.05±0.01 7.05±0.02 7.10±0.02 7.06±0.01

Non- 8 7.09±0.03 7.1±0.04 6.96±0.01 7.12±0.04

Silicone 24 7.05±0.04 7.09±0.02 7.033±0.04 7.13±0.02

Coated 48 7.07±0.05 7.05±0.02 6.95±0.02 7.13±0.02

34 ADX 3-Absolute pH Change 100% 80% 60% 40% 20% 0% RTL RTNL BTNL BTL RTL RTNL BTNL BTL Silicone coated Non-coated

Compatible Incompatible

Fig. 5-8: Change in pH profiles of ADX 3

A. Silicone coated container B. Non-coated container

Fig. 5-9: pH profiles of ADX 3

5.3 Determination of turbidity

5.3.1 Admixture 1

Admixture 1 that contained 70%v/v of ethanol and 30%v/v sodium citrate, whether stored in silicone-coated or non-silicone coated glass containers exposed to RTL,

35 RTNL, BTL, and BTNL conditions demonstrated an absorbance greater than 0.015 OD at

all time points for 48 hours in the study period (Table 5.3.1.1). Admixture 1 was thus

considered incompatible(6).

Influence of light & temperature: The average UV/Vis Absorbance at 546 nm of

samples is plotted in Fig.5-10. Admixtures kept in silicone-coated tubes showed the

highest turbidity at 0 h under all exposure conditions and then decreased to more or less

similar turbidity at 8, 24, and 48 h. In samples kept in noncoated tubes the rank-ordered

severity of turbidity was RTL>BTNL>BTL>RTNL at all time points. Admixtures

exposed to light stored in silicone coated and non-coated tubes were turbid at all storage

temperatures.

Influence of containers on different conditions and time: A two-way ANOVA was performed to analyze the effect of fixed factor (containers: silicone and non-coated), random factor (conditions and time) on UV/Vis absorbance at 546 nm. There was a statistically significant difference in interaction between container and time on UV/Vis absorbance (F = 10.138, p = 0.003), between container and conditions on UV/Vis absorbance (F = 11.228, p = 0.002). There is a statistically significant difference between

UV/Vis absorbance values of silicone coated and non-coated containers when the influence of various conditions used in the study, and different time points of analysis are taken into consideration.

36 Table 5.3.1 Average UV/Vis Absorbance (OD) at 546nm of ADX 1

Container Storage Absorbance (Mean ± S.D) (OD) ADX 1 0hr 8hr 24hr 48hr RTL 0.302±0.271 0.013±0.013 0.072±0.017 0.013±0.014 30% Silicone RTNL 1.032±0.407 0.017±0.001 0.036±0.019 0.029±0.015 Sodium Coated BTL 0.973±0.422 0.084±0.060 0.086±0.039 0.099±0.091 citrate BTNL 1.673±0.036 0.015±0.002 0.019±0.012 0.028±0.035 4% RTL 0.852±0.433 0.595±0.382 0.811±0.799 0.622±0.640 & Non- RTNL 0.018±0.010 0.015±0.004 0.053±0.022 0.025±0.025 70% Coated BTL 0.052±0.037 0.024±0.022 0.050±0.087 0.089±0.097 Ethanol BTNL 0.489±0.658 0.062±0.036 0.209±0.145 0.075±0.049

A. Silicone coated container B. Non-coated container

Fig. 5-10: UV/Vis Absorbance at 546 nm profiles of ADX 1

5.3.2 Admixture 2

Table 5.3.2 summarizes the average UV/Vis absorbance values of ADX 2 samples exposed to various conditions. Admixture 2 stored in silicone-coated and non- coated glass containers exposed to RTL, RTNL, BTL, BTNL conditions exhibited a

UV/Vis absorbance less than 0.015 and hence considered to be compatible over 48 hours.

The average of UV/Vis absorbance at 546 nm is plotted in Fig.5-11.

37 Table 5.3.2 Average UV/Vis Absorbance (OD) at 546nm of ADX 2

ADX 2 Container Storage Absorbance (Mean ± S.D) (OD) 0 h 8 h 24 h 48 h RTL 0.002±0.001 -0.001±0.001 0.007±0.006 0.015±0.003 50% Silicone RTNL 0.003±0.001 0.001±0.007 0.002±0.001 0.013±0.003 Sodium Coated BTL 0.000±0.002 0.005±0.003 0.004±0.004 0.008±0.005 citrate BTNL 0.013±0.022 0.003±0.004 0.002±0.002 0.014±0.004 4% RTL -0.006±0.003 -0.002±0.006 0.015±0.024 0.014±0.004 & Non- RTNL -0.002±0.005 0.006±0.004 -0.002±0.006 0.011±0.001 50% Coated BTL 0.000±0.003 0.006±0.004 0.003±0.002 0.005±0.004 EtOH BTNL -0.001±0.009 -0.005±0.004 0.006±0.005 0.006±0.005

Influence of containers on different conditions and time: A two-way ANOVA analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), random factor (conditions and time) on UV/Vis absorbance at 546 nm. There were no statistically significant differences between container and time on UV/Vis absorbance (F = 2.025, p = 0.181), and between container and conditions on UV/Vis absorbance (F = 0.976, p = 0.446). There is no statistically significant difference between

UV/Vis absorbance values of ADX 2 in silicone coated and non-coated containers under various conditions used in the study, and different time points of analysis.

38 A. Silicone coated container B. Non-coated container

Fig. 5-11: UV/Vis Absorbance at 546 nm profiles of ADX 2

5.3.3 Admixture 3

Table 5.3.3 summarizes the average UV/Vis absorbance values of ADX 3 exposed to various conditions. Admixture 3 stored in silicone-coated and non-coated glass containers exposed to RTL, RTNL, BTL, and BTNL conditions exhibited a UV/Vis absorbance < 0.015 and hence considered to be compatible over 48 hours. The Average of UV/Vis absorbance at 546 nm is represented in Fig.5-12.

39 Table 5.3.3 Average UV/Vis Absorbance (OD) at 546nm of ADX 3

ADX 3 Container Storage Absorbance (Mean ± S.D) (OD) 0 h 8 h 24 h 48 h RTL 0.011±0.006 0.010±0.001 0.009±0.003 0.002±0.009 70% Silicone RTNL 0.005±6E-04 0.009±0.002 0.008±0.005 -0.006±0.001 Sodium Coated BTL 0.002± 6E-04 0.005±0.001 0.007±0.005 0.002±0.001 citrate BTNL 0.003±0.001 0.015±0.005 0.012±0.004 0.009±0.002 4% RTL 0.009±0.005 0.006±0.005 0.008±0.009 0.004±0.001 & Non- RTNL 0.011±0.006 0.010±0.001 0.009±0.003 0.002±0.009 30% Coated BTL -0.008±0.010 0.005±0.003 0.007±0.001 0.004±0.006 EtOH BTNL 0.002±6E-04 0.005±0.001 0.007±0.005 0.002±0.001

Influence of containers on different conditions & time: A two-way ANOVA statistical analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), random factor (conditions and time) on UV/Vis absorbance at 546 nm. There was no statistically significant difference between container and time on UV/Vis absorbance (F = 0.770, p = 0.539), and between container and conditions on UV/Vis absorbance (F = 3.203, p = 0.076). There was no statistically significant difference between UV/Vis absorbance values of ADX 3 stored in silicone coated and non-coated containers under various conditions used in the study, and different time points of analysis.

40 A. Silicone coated container B. Non coated container

Fig. 5-12: UV/Vis Absorbance at 546 nm profiles of ADX 3

5.4 Determination of absorbance ratio at 240 nm

5.4.1 Admixture 1

Aggregation is inherently nucleation and growth phenomenon where aggregates accumulate, eventually exceeding solubility and precipitate out of solution forming visible particles. Table 5.4.1 demonstrates an initial lag phase during which sodium citrate remains in the supernatant solution utilized for analysis at 0 h. As dwelling time progress, the lag phase is followed by rapid, often complete, sodium citrate loss from the supernatant solution resulting in the formation of an insoluble precipitate(51). This type of nucleation-dependent aggregation is seen in immunoglobulins or therapeutic protein aggregation(52). This pattern is dynamic as is evident in Fig.5-13 & 5-14 is due to sodium citrate successively dissolving and precipitating continuously (53).

41 Table 5.4.1. Average UV Absorbance Ratio at 240nm of ADX 1

ADX 1 Container Storage Absorbance Ratio (Mean ± S.D) (OD) 0:8 h 0:24 h 0:48 h RTL 0.42±0.46 0.50±0.47 0.01±0.92 30% Silicone RTNL 0.75±0.12 0.70±0.11 1.689±1.76 Sodium Coated BTL 0.80±1.43 -0.38±1.10 -0.69±1.41 citrate BTNL 1.09±0.18 14.72±15.01 3.23±2.65 4% RTL 1.04±0.04 0.965±0.12 1.14±0.4 & Non-Coated RTNL -0.05±1.01 -0.03±0.91 -0.64±0.37 70% EtOH BTL 0.72±0.11 0.81±0.05 2.31±2.39 BTNL 0.81±0.14 2.80±6.63 -8.31±18.81

Influence of containers on different conditions and time: A two-way ANOVA analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), random factor (conditions and time) on UV absorbance at 240 nm. There was no statistically significant difference between container and time on UV absorbance (F =

0.628, p = 0.566), or between container and conditions on UV absorbance (F = 4.2, p =

0.065).

A. Silicone-coated container B.Non-coated container

Fig. 5-14: UV Absorbance ratio profiles at 240 nm of ADX 1

42 5.4.2 Admixture 2

Table 5.4.2 summarizes the mean ratio of UV absorbance values of ADX 2 samples at 240 nm. There was no detectable sub-visible precipitation during storage at all container, temperature, and light conditions. Precipitation during storage was estimated from the ratio of absorbance ADX 2 before to that after storage. ADX 2, did not demonstrate a significant difference in absorbance values during the study period. Fig 5-

15 demonstrates absorbance ratio plots of ADX 2, indicating ratio closer 1 at different conditions and time period(48)

Table 5.4.2. Average UV Absorbance Ratio at 240 nm of ADX 2

ADX 2 Container Storage Absorbance Ratio (Mean ± S.D) (OD) 0:8 h 0:24 h 0:48 h RTL 1.07±0.01 1.09±0.05 1.26±0.08 50% Silicone RTNL 1.04±0.02 1.05±0.01 1.25±0.02 Sodium Coated BTL 0.96±0.03 0.98±0.02 1.41±0.06 citrate BTNL 0.98±0.09 1.02±0.06 1.09±0.08 4% RTL 0.81±0.26 0.81±0.12 0.89±0.09 & Non-Coated RTNL 1.06±0.04 1.08±0.03 0.98±0.16 50% BTL 0.98±0.11 1.01±0.09 0.98±0.05 EtOH BTNL 1.05±0.02 1.10±0.00 1.21±0.06

43 Influence of containers on different conditions and time: A two-way ANOVA analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), and random factor (conditions and time) on UV absorbance at 240 nm. There were no statistically significant differences between container and time on UV absorbance (F = 3.286, p = 0.109). There was statistically significant difference on UV absorbance between container and conditions (F = 5.010, p = 0.045).

From Fig 5-15, the UV absorbance ratio plot of silicone-coated container indicates there was a clear distinction in the order between conditions, at 0:8 and 0:24 h

RTL>RTNL>BTNL>BTL. But at 0:48hr, BTL exceeds other conditions in the same order. In non-coated containers at all time point ratios BTNL>RTNL>BTL>RTL, almost reversing the pattern which was seen with silicone-coated containers. This pattern possibly indicates the role of body temperature and not exposed to light conditions in sample reaction kinetics. When comparing y axis scales (mean absorbance ratio) between types of containers depicts RTL shows a significant difference. In silicone-coated containers, as time progress RTL moves from less than 1.1 to 1.27 whereas in non-coated containers 0.8 to 0.9. Thus, it can be concluded from the nature of containers that BTL,

BTNL, and RTL are the conditions that caused a significant difference in ADX 2.

44 A. Silicone-coated container B. Non-coated container

Fig. 5-15: UV Absorbance ratio profiles of ADX 2 at 240 nm

5.4.3 Admixture 3

Table 5.4.3 summarizes the mean ratio of UV absorbance values of ADX 3 samples at 240 nm. There was no sub-visible precipitation during storage at all temperatures used in this study. ADX 3, did not demonstrate a significant difference in absorbance values during the study period(48). Fig 5-16 mean absorbance plot of ADX 3 indicates a reversed pattern in BTL condition between silicone-coated and non-coated containers while all other conditions showed similar pattern indicating BTL enhancing reaction kinetics in ADX 3.

45 Table 5.4.3. Average UV absorbance ratio at 240nm of ADX 3

ADX 3 Container Storage Absorbance Ratio (Mean ± S.D) (OD) 0:8 h 0:24 h 0:48 h RTL 1.05±0.01 1.06±0.01 1.16±0.05 30% Silicone RTNL 1.01±0.01 1.02±0.01 1.13±0.00 Sodium Coated BTL 1.31±0.07 0.77±0.08 0.87±0.12 citrate BTNL 0.97±0.02 1.01±0.01 1.08±0.01 4% RTL 1.02±0.05 0.98±0.06 1.05±0.04 & Non-Coated RTNL 1.03±0.04 1.00±0.02 1.07±0.04 70% EtOH BTL 0.74±0.07 1.21±0.05 1.26±0.04 BTNL 1.06±0.02 1.13±0.10 1.13±0.02

Influence of containers on different conditions and time: A two-way ANOVA analysis was performed to analyze the effect of fixed factor (containers: silicone and non- coated), and random factor (conditions and time) on UV absorbance at 240 nm. There was no statistically significant difference between container and time on UV absorbance

(F = 0.708, p = 0.530), and between container and conditions on UV absorbance (F =

0.217, p = 0.881). There was no significant difference between UV absorbance values of

ADX 3 in silicone coated and non-coated containers under various conditions used in the study.

46 A. Silicone coated container B. Non-coated container

Fig. 5-16: UV Absorbance ratio profiles of ADX 3 at 240 nm

5.5 Chemical stability of sodium citrate in admixtures

5.5.1 Validation of HPLC method

The HPLC method was successfully developed and validated for the analysis of sodium citrate in admixtures ADX1, ADX2, ADX3. From the optimized parameters, the retention time of sodium citrate was found to be 2.1 min. From the calibration curve

Fig.4.5, a straight line with the following equation was obtained with an R-squared value of 0.9991:

y=272.48x+30.417 Eq. 5.5.1

47 8000

7000

6000

5000 y = 272.48x + 30.417 4000 R² = 0.9991

3000

2000

1000

0 0 5 10 15 20 25 30 Concentration of sodium citrate (µg/ml)

Figure 5-17: Calibration curve of sodium citrate

Table 5.5.1 Values of the method validation parameters

PARAMETER VALUE LOD 1.09µg/ml LOQ 3.30µg/ml Linearity 5-25µg/ml, R²=0.9991, y-intercept=272.48 Accuracy 100.37± 2.06%

48 From the method validation parameters, the method was found to be linear over

5 to 25 µg/ml sodium citrate concentration range. The accuracy of the method, as determined by the percent recovery of sodium citrate, was found to be well within the suggested limits of 95 to 105%(54). The inter-assay RSD of the measurements was found to be 12.4%(39).

5.5.2 Chemical stability study of sodium citrate in admixtures

HPLC sodium citrate recovery (%) data were expressed as mean ± Standard

Deviation (SD). At time zero, the initial concentration of the drugs was designated as

100%; all subsequent concentrations were expressed as a percentage of the initial concentration. The admixture was considered chemically stable if they retained 90% or more of the initial concentration.

5.5.2.1 Admixture 1

Table 5.5.2.1. shows that the HPLC recovery of sodium citrate from ADX 1 was

< 90% at all the time points of analysis indicating instability of 30% sodium citrate in

70% ethanol. Incompatibility decreased the sodium citrate concentration in the admixture and subsequent decreased recovery (%)(42). Fig.5-18 demonstrates the average recovery

(%) of ADX 1 samples exposed to different conditions.

49 Table 5.5.2.1. Average Recovery (%) of ADX 1

ADX 1 Container Initial Storage Initial Drug Conc. Remaining drug (%)(Mean ± S.D) con. 0 h 8 h 24 h 48 h (µg/ml) RTL 3.09±1.07 2.15±0.68 1.66±0.99 0.53±0.06 Silicone RTNL 1.81±1.08 7.93±5.16 2.06±1.26 3.71±0.84 70% Coated BTL 0.49±0.30 6.55±0.78 10.03±6.27 7.17±4.27 Sodium BTNL 1.46±0.55 2.74±1.64 3.99±8.51 8.04±3.16 citrate 12 RTL 1.49±0.35 0.28±0.65 3.50±1.87 1.79±0.80 4% Non- RTNL 1.02±0.44 0.25±0.09 2.64±0.16 6.21±5.78 & Coated BTL 1.89±0.93 9.63±1.36 7.43±2.37 17.4±5.77 30% BTNL 0.83±0.27 -0.47±7E-17 13.84±5.17 12.55±3.36 Ethanol

Influence of containers on different conditions & time: A two-way ANOVA analysis was performed to analyze the effect of fixed factor (containers: silicone and non-coated), and random factor (conditions and time) on recovery (%). There was no statistically significant difference between container and time on sodium citrate concentration (F =

2.593, p = 0.117), and between container and conditions on sodium citrate concentration

(F = 1.204, p = 0.363). There was no significant difference between recovered concentration values of ADX 1 in silicone coated and non-coated containers under various conditions used in the study and different time points of analysis.

50 A. Silicone coated container B. Non-coated container

Fig.5-18Average Recovery (%) ADX 1 Plot

Time VS Concentration Plot

ADX1 Silicone-Coated ADX 1-Noncoated MAX 2 6 MAX 1 4 2 0 0 8 24 48 0 0 8 24 48 Time (hr) -2 Time(hr)

RTL RTNL RTL RTNL BTL BTNL

Fig. 5-19. A Silicone coated container Fig.5-19.B Non-coated container

Time VS Concentration ADX 1 Plot

Fig.5-19. A and Fig.5-19. B depict an increase in the concentration of sodium citrate recovered as dwell time of admixture progressed to 48th hour in silicone coated and non-

coated containers.

51 1. Fig.5-18. A and Fig.5-18.B demonstrates all samples regardless of the nature of

tubes, temperature, and lighting conditions exhibited recovery of less than 90%.

2. Among the 4 time points of ADX 1, the maximum concentration of sodium citrate

recovered Fig.5-19.A from silicone-coated containers were at 8th hour from

RTNL (2 µg/ml). Similarly, Fig.5-19. B in non-coated containers at 24th hour

from BTNL (4.2 µg/ml). Other conditions such as RTL, BTL recovered lesser

quantitatively.

3. In Fig.5.9. A and 5.9.B concentration of sodium citrate recovered from RTNL

exceeded RTL in both silicone-coated containers and non-coated containers.

Table 5.5.1 shows that in silicone-coated containers, recovery of sodium citrate

(%) from RTL consistently decreased (3-0.5%) as time progressed. In non-coated

containers, recovery (%) of sodium citrate from RTL decreased as time

progressed, but the trends were fluctuating at alternate time points. In silicone-

coated containers, maximum recovery (%) of sodium citrate in RTNL was 8% at

8th hour reducing to half, i.e., 4% at 48th hour whereas in non-coated containers

recovery from RTNL increased 1-6.2% as time progressed.

52 4. Comparing BTL and BTNL using Table 5.5.1 concludes in general terms that, in

both BTL and BTNL condition non-coated tubes have greater recovery (%) of

sodium citrate compared to silicone-coated tubes. In non-coated tubes, maximum

recovery of BTL was 17.4% (4.2 µg/ml) at 48th hour and BTNL recovered a

maximum of 13-14% (2 µg/ml) at 48th and 24th hour, respectively. In silicone-

coated tubes the maximum BTL recovered was 7-10% (1.4 µg/ml) and BTNL

recovered almost similar 8%. Comparing trends of BTL and BTNL in Fig.5.9. A

and 5.9.B indicate in silicone-coated tubes BTNL had increasing sodium citrate

concentration as time progressed up to 48 hours. Similarly, in non-coated tubes,

BTNL showed an increase in concentration as time progressed up to 24 hours

followed by a fall in recovered concentration at 48th hour. In the case of BTL in

silicone-coated containers, concentration increased with time with fluctuating

trends whereas in non-coated containers concentration decreased with time.

5. Influence of Light and Temperature: From above 4&5 points, it can be

concluded samples exposed to light have lower recovery when compared to

samples not exposed to light. This pattern possibly indicates that the admixtures

not exposed to light provided greater recovery compared to samples exposed to

light(55). This suggests that exposure to light, facilitates chemical reaction

kinetics, aggregation, and increased precipitation. Samples stored at elevated

temperature showed greater recovery at 48 hours possibly indicating the

redissolution of the precipitate into the solution at 37 °C (56)(57).

53 6. Influence of containers on different conditions and time: A two-way ANOVA

was performed to analyze the effect of fixed factor (containers: silicone and non-

coated), and random factor (conditions and time) on recovered concentration

(µg/ml). There were no statistically significant differences between container and

time on concentration (F = 9.693, p = 1.920), or between container and conditions

on concentration (F = 1.366, p = 0.271). There was no significant difference

between recovered concentration values of ADX 1 in silicone coated and non-

coated containers under various conditions used in the study.

5.5.2.2 Admixture 2

Table 5.5.2.2 shows that the HPLC recovery (%) of sodium citrate from ADX 2 samples was 90-110% at all condition combinations. Though recovery of 90-110% was retained at all conditions in ADX 2, Fig.5-20 shows the fluctuations in the recovery (%) in each condition combinations when plotted against time (hour) in both silicone-coated and non-coated containers. Some of the samples when assayed showed recovery that exceeded 100%, as the assays had a coefficient of variation of 12.4%(39).

54 Table 5.5.2.2. Average Recovery (%) of ADX 2

ADX 2 Container Initial Storage Initial Drug Conc. Remaining drug (%)(Mean ± S.D) con. 0 h 8 h 24 h 48 h (µg/ml) RTL 102.0 96.0±1.5 98.0±1.1 100.0±8.1 Silicone RTNL ±19.095.0±11.0 98.0± 12.0 98.0 ±3.0 100.0±7.4 50% Coated BTL 94.0± 2.0 94.0±2.3 99.0±3.0 100.0±2.3 Sodium Container BTNL 91.0± 12.4 100.0±22.2 95.0±5.0 97.0±4.0 citrate 4% 10 RTL 94.0±4.0 92.0±2.0 102.0±17.4 95.4±2.0 & Non- RTNL 95.0±10.3 101.0±11.0 100.0±7.0 105.0±14.0 50% Coated BTL 103.0±3.0 99.0±11.1 98.0±10.3 95.0±4.0 Ethanol Container BTNL 92.2±5.0 90.0±19.0 100.0±8.0 99.0±6.0

Influence of containers on different conditions and time: A two-way ANOVA analysis was performed to analyze the effect of fixed factor (containers: silicone coated and non-coated), and random factor (conditions and time) on recovery (%). There were no statistically significant differences between container and time on sodium citrate concentration (F = 0.360, p = 0.783), or between container and conditions on sodium citrate concentration (F = 0.811, p = 0.519). There were no significant differences between recovered concentration of ADX 2 in silicone coated and non-coated containers under various conditions used in study.

55 Fig.5-20. A. Silicone coated container Fig.5-20.B. Non-coated container

Fig. 5-20: Average Recovery (%) of ADX 2 Plot

ADX 2- Silicone coated ADX 2-Non coated 10.5 11 10 10 9.5 9 9 8.5 8 0hr 8hr 24hr 48hr 0hr 8hr 24hr 48hr Time(hr) Time(hr)

RTL RTNL BTL BTNL RTL RTNL BTL BTNL

Fig 5.21.A Silicone-coated container Fig.5.21.B Non-coated container

Time Vs Concentration plot ADX 2

56 From Fig.5.21. A and Fig.5.21.B it can be seen that in silicone-coated and non-coated containers all the condition combinations retained 90% of the initial concentration of

ADX 2. In silicone-coated containers, RTL showed flattening of curve at 9 µg/ml at 0,

48th hour while retaining 10 µg/ml at 8th, 24th hour. RTNL retained the same (10 µg/ml) throughout while dipping (9 µg/ml) at the 48th hour. BTL and BTNL increased to 9-10

µg/ml as time progress. In non-coated containers, all condition combinations retained 9-

10 µg/ml throughout the admixture dwell period.

5.5.2.3 Admixture 3

Table 5.5.5.3 shows that the HPLC recovery (%) of sodium citrate from ADX 3 was 90-110% of the initial concentration of ADX 3 at all condition combinations during the entire dwell time of admixture. Fig.5.5.3.3.

Table 5.5.2.3. Average Recovery (%) of ADX 3

ADX 3 Container Initial Storage Initial Drug Conc. Remaining (%) drug (Mean ± S.D) con. 0 h 8 h 24 h 48 h (µg/ml) RTL 101.4 ±9.1 93.0±3.4 103.4±5.0 94.0±23.0 Silicone RTNL 93.0±13.4 97.0±20.2 103.0 ± 3.0 106.0±6.1 70% Coated BTL 100.0± 8.0 104.2±1.3 102.2±6.0 109.2±9.0 Sodium Container BTNL 102.2± 5.2 102.3±2.1 101.0±5.0 108.0±5.2 citrate 4% 11.2 RTL 95.0±12.0 96.0±4.2 102.0±3.0 99.4±5.0 & Non- RTNL 101.0±5.0 97.4±3.0 98.0±11.0 96.3±13.0 30% Coated BTL 94.0±14.2 101.0±9.4 100.0±1.0 97.1±3.0 Ethanol Container BTNL 103.0±3.3 97.0±6.0 97.4±2.0 101.0±8.1

57 Influence of containers on different conditions and time: A two-way ANOVA

analysis was performed to analyze the effect of fixed factor (containers: silicone coated and non-coated), and random factor (conditions and time) on recovery (%). There was no statistically significant difference between container and time on concentration (F =

0.641, p = 0.607), or between container and conditions on concentration (F = 0.864, p =

0.494). There were no significant differences between recovered concentration values of

ADX 3 in silicone coated and non-coated containers under various conditions used in study.

A. Silicone coated container B. Non coated container

Fig. 5-21: Average Recovery (%) of ADX 3 Plot

58 ADX 3-Silicone coated ADX 3-Non-coated 13 12 12 11.5 11 11 10 10.5 9 10 0hr 8hr 24hr 48hr 0hr 8hr 24hr 48hr Time(hr) Time(hr)

RTL RTNL BTL BTNL RTL RTNL BTL BTNL

Fig.5-22.A Silicone-coated container-ADX3 Fig.5-22.B Non-coated container-ADX3 Time VS Concentration

From Fig.5-22.A and Fig.5-22.B it can be seen that in both silicone-coated and non-

coated containers ADX 3 retained 90% of the initial drug concentration from which the

analysis started. In silicone-coated containers, RTL, BTNL showed fluctuating

concentration at alternate time points. RTNL and BTL showed an increase in

concentration (11-12 µg/ml) as time progressed. In non-coated containers, RTL showed an increase in concentration (11-12 µg/ml) as time progressed.

59 Chapter 6

Conclusions

In the present study, various admixtures containing different % (v/v) concentrations of

4% sodium citrate were mixed with ethanol. Samples were studied under four conditions:

(1) at 25°C with artificial indoor white LED light, (2) at 25°C without light wrapped using aluminum foil, (3) at37°C with artificial indoor white LED light, and (4) at 37°C without light wrapped using aluminum foil stored in two different types of containers: (1) silicone-coated and (2) non-coated glass test tubes. The physical compatibility, chemical compatibility, and stability were assessed at 0, 8, 24, and 48 hours after the admixtures were prepared.

Physicochemical compatibility and stability of ADX 1:

ÿ Physical compatibility: ADX 1 (70%v/v ethanol and 4% sodium citrate)

immediately developed haze at the time of mixing (0 h). The haze eventually

progressed to form a white crystalline precipitate that settled to the bottom of both

types of containers at all time points of analysis. The pH of the ADX 1 was found

to be 6.98-7.80. ADX 1 exhibited a pH change greater than 0.1 and hence was

physically incompatible.

60 ÿ Chemical Compatibility: At 546 nm, the absorbance of ADX 1 was greater than

0.015 OD in two types of containers and under all conditions of exposure. At 240

nm, the absorbance ratio of ADX1 was less than1, indicating subvisible

precipitation in different types of containers and under all conditions of exposure.

ADX 1 was chemically incompatible.

ÿ Stability Study: ADX 1 recovery data indicated sodium citrate was nearly

completely precipitated with a maximum HPLC recovery of 14%. The recovery

was less than 90% in two different containers at all exposure conditions and

analysis timepoints. ADX 1 comprising of 30% v/v 4% sodium citrate solution in

70% ethanol was determined to be unstable.

ÿ Influence of conditions (light, temperature and time) on types of container:

Two-way ANOVA analysis indicated that there was no significant difference in

pH, absorbance ratio, and stability between different types of containers.

However, there was a significant difference in UV/Vis absorbance between

containers at 546 nm. Analyzing UV/Vis absorbance plots of ADX 1 between the

two types of containers showed that samples exposed to light were turbid

compared to samples not exposed to light. This correlates well with ADX 1

stability plots between types of containers. Though there was no statistical

significance as dwell time progressed, samples not exposed to light, and stored at

body temperature showed greater recovery compared to samples exposed to light

and room temperature.

61 Physicochemical compatibility and stability of ADX 2 and 3:

ÿ Physical compatibility: ADX 2 (50%v/v ethanol and 50% v/v 4% sodium citrate)

and ADX 3(30%v/v ethanol and 70% v/v 4% sodium citrate) remained clear in

both types of containers at all time points of analysis under all storage conditions.

The pH range of ADX 2 was 7.19 to 7.62 and that of ADX 3 was 6.95 to 7.20. As

the pH change was less than 0.1 from baseline measurement ADX 2 and 3 were

physically compatible.

ÿ Chemical compatibility: ADX 2 and 3 absorbance values were less than 0.015

OD and the absorbance ratio were equal to 1. The admixtures were not turbid and

did not indicate subvisible precipitation. Hence, ADX 2 and 3 were chemically

compatible in both types of containers, at all-time points, and under all exposure

conditions.

ÿ Stability study: ADX 2 and 3 indicated sodium citrate recovery between 90 to

110% in two different containers at all conditions and time points.

Influence of conditions (light, temperature, and time) on types of container:

Two-way ANOVA analysis indicated that there was no significant difference in

pH, UV/Vis absorbance at 546 nm and stability in ADX 2 under all conditions of

this study. There was a significant difference observed in the UV absorbance ratio

at 240 nm in ADX 2. The absorbance ratio values of ADX 2 at 240 nm between

the two types of containers indicated a possible role of body temperature and

storage away from light in chemical reaction kinetics. There was no statistical

significance in pH, UV/Vis absorbance at 546 nm, absorbance ratio and stability

62 study in ADX 3

Chemical reaction kinetics of 4% sodium citrate and ethanol: The pH, UV/Vis absorbance at 546 nm, and HPLC recovery at various times for ADX 1, ADX 2 and ADX

3 fluctuated in a decreasing and then increasing pattern potentially indicating reversible reaction kinetics and pH-dependent solubilization of 4% sodium citrate in ethanol(50).

63 Chapter 7

Future Studies

∑ Evaluating antimicrobial activity of 50% v/v ethanol and 50% v/v 4% sodium

citrate will be useful in understanding admixture’s spectrum of activity as this

admixture was found to be physically and chemically compatible and stable.

Further studies to evaluate efficacy and safety in hemodialysis patients may be

needed.

∑ A controlled solubility study of 4% sodium citrate with ethanol would help to get

insight on the chemical reaction kinetics between the components of the

admixture.

∑ Performing long-term stability studies will help to investigate the shelf-life of

compatible admixtures offering information about the beyond use date of the

compounded admixtures.

∑ The influence of light must be further investigated by titrating sodium citrate in

ethanol.

∑ To identify the chemical nature of the precipitated compound. inclusion of parameters such

as particle sizing, zeta potential analysis, and characterization of crystalline precipitate

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