Simultaneous Analysis of Phenylephrine Hydrochloride, Guaiphenesin, Ambroxol Hydrochloride, and Salbutamol (As Salbutamol Sulphate) by Use of a Validated High-Performance Liquid

Acta Chromatographica 23(2011)1, 109–119 DOI: 10.1556/AChrom.23.2011.1.7

Simultaneous Analysis of Phenylephrine Hydrochloride, Guaiphenesin, Ambroxol Hydrochloride, and Salbutamol (as Salbutamol Sulphate) by Use of a Validated High- Performance Liquid Chromatographic Method

S. JOSHI1,*, C. BHATIA1, C.S. BAL2, AND M.S.M. RAWAT3

1Department of Chemistry, K.L.D.A.V. (P.G.) College, Roorkee, Uttarakhand, India-247667 2Talwar Pharma, Jhabrera Road, Manglore, Roorkee, Uttarakhand, India-247656 3Department of Chemistry, H.N.B. Garhwal University, Srinagar, Uttarakhand (India) E-mail: [email protected]

Summary. A simple, reversed-phase HPLC method has been developed for rapid, simultaneous quantification of phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol (as salbutamol sulphate) in a commercial cough–cold liquid formulation. The compounds were separated on a 250 mm × 4.6 mm C8 column with a gradient prepared from pH 3.0 phosphate buffer and 1:1 methanol–acetonitrile as mobile phase at a flow rate of 1.0 mL min−1. Elution of the analytes was achieved in less than 15 min. Detection was by UV absorbance at 273 nm for phenylephrine hydrochloride and guaiphenesin and 225 nm for ambroxol hydrochloride and salbutamol. Percentage recovery and RSD were, respectively, 100.09% and 0.22% for phenylephrine hydrochloride, 100.43% and 0.50% for guaiphenesin, 100.91% and 0.70% for ambroxol hydrochloride, and 100.54% and 0.55% for salbutamol. The components of the syrup formulation were quantified on the basis of the peak areas obtained from freshly prepared standard solutions. The method was validated in accordance with ICH guidelines.

Key Words: simultaneous quantification, validation, cough–cold syrup, RP-HPLC

Intro-duction

Complete prevention of infections induced by the cold virus is not yet pos- sible, which has led to increasing demand for formulations for relief of cough and allergy symptoms. Cough and cold formulations are available commercially as over-the-counter (OTC) products. These multicomponent formulations include dextromethorphan hydrobromide, ambroxol hydrochloride, guaiphenesin, phenylephrine hydrochloride, levocetirizine hydrochloride, salbutamol, and pseudoephedrine hydrochloride, among others. The presence of different classes of active chemicals and the disparity in

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concentration and formulation matrix poses an analytical challenge which is attracting the attention of analytical chemists. Our earlier work on formulations of non-steroidal anti-inflammatory drugs (NSAIDs) [1] and anti-hypertensive drugs [2] has directed us toward developing rapid and reliable HPLC methods for analysis of a multicomponent cough formulation containing phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol (as salbutamol sulphate) as active ingredients. Guaiphenesin and ambroxol hydrochloride are expectorants, phenylephrine hydrochloride is an alpha-adrenergic receptor agonist, and salbutamol sulphate is a beta-adrenoceptor agonist [3]. The chemical structures of these compounds are shown in Fig. 1.

H CH 2 HC CH2OH O H OH OH OCH HO N .HCl 3 CH3

Phenylephrine Hydrochloride Guaiphenesin

OH H CH Br 3 N HO CCH3NC CH3 .H2SO4 H . HCl OH CH HOH C 3 NH2 2 Br 2 Ambroxol hydrochloride Salbutamol Sulphate

Fig. 1. The chemical structures of phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol sulphate

Analysis of multi-component anti-cough formulations with two or three active ingredients by HPLC [4–9] is more common than analysis of those with four or more ingredients [10, 11]. Other techniques, including micellar electrokinetic chromatography [12], non-aqueous capillary electro- phoresis [13], gas chromatography [14], and derivative spectrophotome- try [15], have been used to determine a few of these compounds. To the best of our knowledge, however, no analytical method has been reported for simultaneous determination of these four compounds in the presence of pre- servative and excipients which might interfere with analysis of the active ingredients.

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In this investigation a simple HPLC method with gradient elution was used for analysis of a cough–cold formulation. The method was validated in accordance with ICH guidelines [16].

Experimental

Chemicals, Reagents, and Solutions

Reference standards were gifts from Roorkee Research and Analytical Laboratories, Roorkee, India. Solvents were of HPLC-grade and chemicals were of analytical grade (Qualigens India, Mumbai). The commercial formulation Viscodyne-S syrup containing phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride and salbutamol (as salbutamol sulphate), manufactured in India by Wockhardt, was purchased locally. Approximately 100 mg phenylephrine hydrochloride, 500 mg guaiphenesin, 150 mg ambroxol hydrochloride, and 10 mg salbutamol sulphate (reference standards) were accurately weighed, transferred to a 100-mL volumetric flask, dissolved in diluent prepared by mixing methanol and water in the ratio 20:80, sonicated for 10 min, and made up to volume with diluent to produce a stock solution. Aliquots of the stock solution were suitably diluted with diluent to produce sample solutions of suitable concentration. Sample solutions were filtered through a 0.45-μm pore size membrane filter, injected (20 μL) in triplicate on to the column, and the peak areas and retention times were recorded. Average peak areas were used for calculations after ensuring that the RSD was <2%.

Sample Preparation

Syrup equivalent to approximately 10 mg phenylephrine, 50 mg guaiphenesin, 15 mg ambroxol hydrochloride, and 1 mg of salbutamol was accurately weighed and transferred to a 100-mL volumetric flask. The weighed sample was dissolved in 70 mL diluent, sonicated for 10 min, and diluted to volume with diluent. The solution was filtered through a 0.45 μm pore size Nylon filter discarding the first 5–10 mL of filtrate. The dilution step prevents problems because of the presence of hard syrup matrix in the sample.

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Chromatography

Analysis was performed with a Shimadzu gradient high-performance liquid chromatograph (Prominence model), binary pump (model LC-20AT), man- ual injector (Rheodyne model 7725i) of capacity 20 μL, and a UV–visible de- tector (model SPD-20A). Compounds were separated on a 250 mm × 4.6 mm i.d., 5-μm particle, Luna C8 column (Phenomenex, USA). The mobile phase was a gradient prepared from aqueous buffer solution (component A) and a 1:1 (v/v) mixture of methanol and acetonitrile (component B). To prepare solution A (phosphate buffer, 10 mM, pH 3.0), potassium dihydro- gen phosphate (2 g) and hexane sulphonic acid sodium salt (750 mg) were accurately weighed and dissolved in approximately 800 mL water. Tri- ethylamine (2 mL) was added and the pH was adjusted to 3.0 with ortho- phosphoric acid. The resulting solution was diluted to 1000 mL with water. Before use the mobile phase components were filtered through a 0.45-μm membrane filter, degassed by sparging, and pumped from the respective solvent reservoirs to the column at a flow rate of 1 mL min−1. The run time was set at 15 min, the column temperature was ambient, and the injection volume was always 20 μL. Before analysis, the column was equilibrated for at least 15–20 min with the mobile phase. Data were acquired, stored, and analyzed with LC Solutions software (Shimadzu).

Results and Discussion

To develop and validate a simple HPLC method for simultaneous analysis of the active ingredients phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol in a pharmaceutical dosage form, detection wavelength and mobile phase composition were investigated. Be- cause buffered low pH mobile phases keep basic compounds in their ion- ized form, it reduces their retention and hence makes the method selec- tive [17, 18]. Thus phosphate buffer (pH 3.0) was used with acetonitrile which also supports easy sample elution, thereby maintaining column life. It was further modified by addition of ion-pairing reagent hexane sulphonic acid sodium salt to affect retention of the ion-paired solutes expected at this pH. To further improve resolution between peaks, methanol was added to the organic component of mobile phase (methanol–acetonitrile 1:1). System- atic variation of their ratio and the detection wavelength resulted in good peak separation and short analysis time. The detection wavelengths were 273 nm for guaiphenesin and phenylephrine and 225 nm for salbutamol and ambroxol. The mobile phase gradient is given in Table I. A gradient was pre-

Unauthenticated | Downloaded 09/26/21 02:40 AM UTC HPLC Analysis of a Cough–Cold Formulation 113 ferred for good resolution of all the compounds because salbutamol and ambroxol required a low buffer to organic solvent ratio whereas guaiphenesin and phenylephrine required a high ratio. System suitability tests were performed (Table II). All four components of the formulation were clearly resolved with retention times (RT) between 0 and 11 min. Before implemen- tation for quantitative analysis of the active ingredients in the pharmaceutical preparation, this method was thoroughly validated in accordance with ICH guidelines [16].

Table I. The optimum multi-linear gradient

Mobile phase Time (min) Wavelength (nm) Conc. A (%) Conc. B (%) 0 0 100 225 3 5 95 225 6 95 5 273 10 95 5 273 15 0 100 273

Table II. System suitability test

Property Salbutamol Ambroxol Guaiphenesin Phenylephrine

Retention time (min) 1.54 4.54 6.77 10.11

Resolution (RS) – 3.72 2.76 3.42 Selectivity (α) – 2.94 1.49 1.49 Tailing factor 1.23 1.09 0.98 0.84

Method Validation

Assay performance was evaluated by determination of specificity, linear range, accuracy, precision, and robustness. Specificity was investigated by assessment of chromatograms obtained from a standard solution of the active ingredients (Fig. 2) and Viscodyne-S Syrup (Fig. 3); peaks of the active components were well resolved in the presence of endogenous compounds.

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Fig. 2. Representative chromatogram obtained from a standard solution of salbutamol (RT 1.54 min), ambroxol (RT 4.54 min), guaiphenesin (RT 6.77 min), and phenylephrine (RT 10.11 min)

Fig. 3. Chromatogram obtained from Viscodyne-S syrup showing the peaks of the active ingredients salbutamol (RT 1.53 min), ambroxol (RT 4.51 min), guaiphenesin (RT 6.79 min), and phenylephrine (RT 10.12 min) (indicated by arrows) and excipients

Calibration plots were generated after chromatography of solutions at five concentrations: 50–250 μg mL−1 for phenylephrine hydrochloride, 250– 1250 μg mL−1 for guaiphenesin, 75–375 μg mL−1 for ambroxol hydrochloride, and 5–25 μg mL−1 for salbutamol and measurement of the correspond- ing peak areas. The linearity of the method was excellent in these ranges and calibration plots could be described by the equation y = ax + b. The cor- relation coefficients (R2 > 0.99) obtained for the compounds (Table III) sug- gest the linearity of the method is good. The range of a method is defined as lowest and highest concentrations for which accuracy, precision, and linearity are adequate. To demonstrate the range of the method, six sample solutions each of low concentration (50% of target level) and high concentration (150% of target level) were prepared and analysed in duplicate. Recovery and RSD were 101.48–99.87% and <1.04%, respectively (Table IV).

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Table III. Regression analysis

Phenylephrine Salbutamol Guaiphenesin Ambroxol HCl HCl sulphate R 0.99968 0.99984 0.99638 0.99976 R2 0.99936 0.99969 0.99278 0.99952 Standard 51628.48 661147.59 219196.60 10755.72 error Slope 22484.8 82743.6 18782.8 53950.3 Intercept 142532.4 493945.1 56604.1 −2210.9

Table IV. Range data

Lower range Higher range (50% target level) (150% target level) Compound Mean recovery RSD Mean recovery RSD (%) (%) (%) (%) Phenylephrine HCl 100.46 0.43 99.98 0.54 Guaiphenesin 100.48 0.86 99.88 0.39 Ambroxol hydrochloride 101.47 1.03 99.99 0.89 Salbutamol sulphate 101.40 0.97 99.87 1.23

The accuracy of method was evaluated by measurement of the recovery of the compounds, by comparing the response obtained from sample solutions with that from identical standard solutions prepared in the diluent (Table V). Recovery and RSD, respectively, were 100.09% and 0.22% for phenylephrine hydrochloride, 100.43% and 0.50% for guaiphenesin, 100.91% and 0.70% for ambroxol hydrochloride, and 100.54% and 0.55% for salbutamol. To determine inter-day precision seven different sample solutions containing the drugs were analyzed in triplicate under same conditions and mean peak area and the relative standard deviations of peak-area response were calculated. Relative standard deviations were 1.12% for phenylephrine hydrochloride, 1.54% for guaiphenesin, 0.67% for ambroxol hydrochloride, and 1.62% for salbutamol. Intra-day precision was established by perform- ing the same analysis in triplicate on four different days. Relative standard deviations of peak-area responses were 0.43% for phenylephrine hydrochloride, 0.75% for guaiphenesin, 0.84% for ambroxol hydrochloride, and 1.45% for salbutamol. All these values are therefore within ICH guidelines (< 2.0%).

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Table V. Recovery of analytes

Theoretical Theoretical Amount Recovery Bias Number amount (% of target determined (%) (%) (mg per 5 mL) level) (mg per 5 mL) Phenylephrine HCl 1 5.00 50.00 5.023 100.46 0.46 2 7.50 75.00 7.509 100.12 0.12 3 10.00 100.00 10.002 100.02 0.02 4 12.50 125.00 12.486 99.89 –0.11 5 15.00 150.00 14.997 99.98 –0.02 Overall mean (n = 5) 100.09 Overall % RSD 0.22 Guaiphenesin 1 25.00 50.00 25.12 100.48 0.48 2 37.50 75.00 37.84 100.91 0.91 3 50.00 100.00 50.46 100.92 0.92 4 62.50 125.00 62.47 99.95 –0.05 5 75.00 150.00 74.91 99.88 –0.12 Overall mean (n = 5) 100.43 Overall % RSD 0.50 Ambroxol hydrochloride 1 7.50 50.00 7.61 101.47 1.47 2 11.25 75.00 11.39 101.24 1.24 3 15.00 100.00 15.23 101.53 1.53 4 18.75 125.00 18.81 100.32 0.32 5 22.50 150.00 22.498 99.99 –0.01 Overall mean (n = 5) 100.91 Overall % RSD 0.70 Salbutamol sulphate 1 0.50 50.00 0.507 101.40 1.40 2 0.75 75.00 0.754 100.53 0.53 3 1.00 100.00 1.006 100.60 0.60 4 1.25 125.00 1.254 100.32 0.32 5 1.50 150.00 1.498 99.87 –0.13 Overall mean (n = 5) 100.54 Overall % RSD 0.55

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Table VI. Robustness

Mean RT ± SD Condition Modification Mean area ± SD (min) Salbutamol 0.9 285899.6 ± 01869.158 1.63 ± 0.013 Mobile phase flow rate (mL min−1) 1 263680.6 ± 00381.269 1.53 ± 0.015 1.1 276297.0 ± 02446.926 1.46 ± 0.014 35 284462.2 ± 01824.916 1.71 ± 0.015 Oven temperature (°C) 40 263680.6 ± 00381.269 1.53 ± 0.010 45 247903.8 ±04292.716 1.43 ± 0.011 2.9 293458.2 ± 01087.197 1.53 ± 0.015 Mobile phase pH 3 263680.6 ± 00381.269 1.53 ± 0.015 3.1 264672.2 ± 05708.802 1.53 ± 0.015 Ambroxol 0.9 32139245.2 ± 323825.17 4.52 ± 0.008 Mobile phase flow rate (mL min−1) 1 30273907.6 ± 386241.79 4.36 ± 0.057 1.1 28543387.2 ± 226963.33 4.33 ± 0.056 35 33333407.6 ± 338030.08 4.68 ± 0.082 Oven temperature (°C) 40 30273907.6 ± 386241.79 4.36 ± 0.057 45 28619766.8 ± 203609.37 4.25 ± 0.035 2.9 31537010.2 ± 264861.03 4.58 ± 0.025 Mobile phase pH 3 30273907.6 ± 386241.79 4.36 ± 0.057 3.1 32101148.8 ± 502261.89 4.58 ± 0.020 Guaiphenesin 0.9 42162232.2 ± 405831.96 6.82 ± 0.037 Mobile phase flow rate (mL min−1) 1 39683074.4 ± 470162.06 6.77 ± 0.025 1.1 39289066.8 ± 581966.58 7.32 ± 0.112 35 40523645.2 ± 313548.10 7.60 ± 0.125 Oven temperature (°C) 40 39683074.4 ± 470162.06 6.77 ± 0.025 45 38317155.0 ± 586571.28 6.32 ± 0.019 2.9 39901964.6 ± 318973.18 6.48 ± 0.032 Mobile phase pH 3 39683074.4 ± 470162.06 6.77 ± 0.025 3.1 42640261.8 ± 762914.63 6.23 ± 0.054 Phenylephrine 0.9 2359079 ± 017131.98 10.17 ± 0.135 Mobile phase flow rate (mL min−1) 1 2350555 ± 005382.06 10.11 ± 0.119 1.1 2190746 ± 011140.07 09.54 ± 0.123 35 2213121 ± 008431.43 11.04 ± 0.142 Oven temperature (°C) 40 2350555 ± 005382.06 10.11 ± 0.119 45 2245947 ± 119613.80 09.26 ± 0.019 2.9 2334228 ± 032082.83 10.54 ± 0.058 Mobile phase pH 3 2350555 ± 05382.064 10.11 ± 0.119 3.1 2257503 ± 027468.79 09.98 ± 0.172

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To determine the robustness of the method, the experimental conditions were deliberately changed and the results obtained were examined (Table VI). The relative standard deviations of peak-area responses and retention times were less than 2% for all four compounds. These results show that separation and quantification of the analytes was not much affected, thus confirming the ruggedness of the method. To further ascertain the accuracy and validity of the method, recovery experiments were performed. Pre-analyzed syrup solution was spiked with pure drug at three different levels and the total was found by use of the method, each determination being repeated three times. Recovery of phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol was quantitative and revealed that common additives and excipients did not interfere with the determination.

Conclusion

This HPLC method for quantitative analysis of phenylephrine hydrochloride, guaiphenesin, ambroxol hydrochloride, and salbutamol in a pharmaceutical dosage form is simple and economical. Gradient elution efficiently solved the problems associated with simultaneous determination of four compounds. Validation results show that it is suitable for routine analysis and quality control of pharmaceutical preparations of these drugs.

Acknowledgment

The authors acknowledge the help and support of Roorkee Research and Analytical Labs Pvt. Ltd., Roorkee, Uttarakhand (India).

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Accepted by DA

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