Journal of Chromatographic Science 2015;53:903– 908 doi:10.1093/chromsci/bmu142 Advance Access publication November 3, 2014
Article
Development and Validation of a Stability-Indicating RP-UPLC Method for Determination of Cloxacillin Sodium in Its Bulk Form and Formulation Nikita Patel1*, Pooja Contractor1,2, Rajesh Keshrala1, Parag R. Patel1 and Bhimagoni Shridhar1 1
Parul Institute of Pharmacy, Waghodia, Limda, Gujarat, India and 2Piramal Health Care, Ahmedabad, Gujarat, India
*Author to whom correspondence should be addressed. Email: [email protected] Received 10 May 2014; revised 14 September 2014
A simple, linear gradient, rapid, precise and stability-indicating RPUPLC method was developed for the determination of Cloxacillin Sodium in its bulk form and formulation. Ultra performance liquid chromatography, a most promising advancement in a world of chromatography, reduces analysis time, increases reliability through higher resolution, sensitivity and selectivity as well as used as an economic method due to reducing solvent consumption. A chromatographic separation of a drug as well as its degradants was achieved using Waters acquity BEH, 2.1 3 100 mm, 1.7 mm C18 column with gradient of mobile phase A: phosphate buffer, pH 6.8 and mobile phase B: methanol:acetonitrile (75:25). The drug and degradants were monitored at a detection wavelength of 225 nm with a flow rate of 0.35 mL/min and an injection volume of 10 mL. The temperature of the column and auto sampler compartments was at 3088 C and 2588 C + 188 C, respectively. The retention time of the drug was ∼6.9 min. The resolution of the drug and degradant peak was >1.5 in all cases. Force degradation of CLOX SOD was carried under alkaline, acidic, oxidative, thermal, photo degradation conditions and it was analyzed by the proposed method. The drug degrades under alkaline, acidic and oxidative conditions but was stable in temperature and light. A developed method was validated as per ICH guidelines using validation parameters such as precision, linearity and range, limit of quantification, specificity, assay and robustness.
Introduction Cloxacillin, chemically known as monosodium(2S,5R,6R)6-[o-(2-chlorophenyl)-5-methyl-4-isoxazole carboxamido]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]-heptane-2-carboxylate monohydrate, is a semi-synthetic antibiotic in the same class as penicillin. It used against staphylococci that produce b-lactamase (1). The chemical structure of Cloxacillin Sodium is depicted in Figure 1. The ACQUITY ultra performance liquid chromatography (UPLC) system eliminates significant time and cost per sample from an analytical process while improving the quality of the results. What differentiates the system’s holistic design is Waters’ patented sub-2-mm hybrid particle chemistry, which offers significant benefits over today’s high performance liquid chromatography (HPLC) systems equipment. These particles operate at elevated mobile phase linear velocities to affect which cause increase in resolution, sensitivity and speed of analysis. Because of its speed and sensitivity, this technique has gained considerable attention in recent years for pharmaceutical and biomedical analysis (2–6). A literature survey reveals that few spectrophotometric and HPLC methods were available for the estimation of Cloxacillin Sodium individually as well as in combination with other drugs. No analytical method was reported for estimation of Cloxacillin Sodium by UPLC
in bulk and pharmaceutical dosage form (7 – 16). Hence, it was thought worthwhile to develop and validate the stability-indicating RP-UPLC method for determination of Cloxacillin Sodium in its bulk form and formulation. Experimental Chemicals API (CLOX SOD-99.8% Purity) was supplied by Centuarian Laboratories, GIDC, Vadodara, India. Capsules (CLOXI-500 mg) were obtained from Savorite Pharmaceuticals, Alkapuri, Vadodara, India. Methanol (HPLC grade) and sodium hydroxide pellets (AR Grade) were obtained from Finar Reagent, Ahmedabad, India. Furthermore, acetonitrile (HPLC grade) was obtained from Sigma-Aldrich, and H2O2 (30% v/v), HCl and NaOH were obtained from Merck Specialties Pvt Ltd, Ahmedabad, India. Potassium dihydrogen orthophosphate (AR Grade) was obtained from Fluka Reagent, Vadodara, India. Chromatographic conditions and equipment LC was carried out on a Waters Acquity UPLC with a photodiode array detector. For optimization of chromatographic conditions, the effects of various method parameters such as mobile phase, column, flow rate and solvent ratio were studied and the chromatographic parameters such as asymmetric factor, resolution and column efficiency were calculated. The separation was achieved on an Acquity H-UPLC C18 100 mm 2.1 mm, BEH 1.7 mm column using mobile phase A: 20 mM phosphate buffer, pH 6.8, and mobile phase B: methanol:acetonitrile (75:25) using a gradient program. The drug and degradation products were detected (see Table I). By keeping the column temperature of 308C, flow rate of 0.35 mL/min and injection volume of 10 mL, all peaks were separated, and the gradient pattern did not interfere with the CLOX SOD peak which was detected by a PDA detector at 225 nm. Preparation of solutions An accurately weighed 50 mg of CLOX SOD API was transferred into a 100 mL-volumetric flask. First, it was dissolved in 50 mL buffer and sonicated, then diluted up to the mark with buffer to get the concentration of CLOX SOD (500 mg/mL). Concentrations ranging from 0.025 to 75 mg/mL for CLOX SOD were prepared from stock solution and different validation parameters were performed. Validation procedure Validation of the newly developed method was studied in terms of linearity and range, precision, accuracy, robustness, limit of
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Table II System Suitability Data S no.
Parameter
Drug
1 2 3 4 5
Peak area Number of theoretical plates Retention time (min) Asymmetry/USP tailing % RSD
3,079,906 48,559 6.956 1.2 0.8
RSD, relative standard deviation.
Figure 1. Chemical structure of CLOX SOD.
Table I Gradient Programme for Method Development Time (min)
Initial 2 8 10 10.01 13
Gradient program A%
B%
75 70 35 35 75 75
25 30 65 65 25 25
volumetric flask and to this solution, 2 mL of 0.03% hydrogen peroxide was added. This solution was kept for 1 h at room temperature. The volume was made up by buffer to make a final concentration of 50 mg/mL. For thermal degradation, 50 mg/mL solution of CLOX SOD (working standard) was exposed to 608C for 3 days. As well as API of CLOX SOD was also exposed to the same condition for 3 days and the photo degradation was done by prepared 50 mg/mL solution of CLOX SOD (working standard) was exposed to 200 lux h for 3 days. Results and Discussion
Mobile phase A: 20 mM, phosphate buffer, pH 6.8. Mobile phase B: methanol:acetonitrile (75:25 v/v).
detection (LOD) and limit of quantification (LOQ) as per the ICH guidelines (17). For linearity, the solutions were prepared from the stock solution (500 mg/mL) ranging from 0.025 to 75 mg/mL, the calibration curve was generated, and regression parameters were obtained. The LOQ was estimated from the set of five calibration curves of CLOX SOD. The precision of the method was studied by checking the repeatability, interday and intraday precision for three concentrations thrice. Accuracy of the method was determined by calculating percentage recoveries of CLOX SOD by the standard addition method. A known amount of standard solutions of CLOX SOD (0.025, 50 and 75 mg/mL) was added to a preanalyzed sample solution of CLOX SOD (50 mg/mL). Robustness of the method was studied by changing the temperature of column compartment by +5 units and flow rate by +5 mL/min. The changes in the response of CLOX SOD were noted and compared with the original one. System suitability parameters are mentioned in Table II. Specificity-forced degradation studies To prove the stability-indicating property of the method, forced degradation studies were performed on CLOX SOD. For acid degradation, 2 mL of standard stock solution (500 mg/mL) was added to a 20-mL volumetric flask and to this 2 mL of 0.1 N HCl solution was added and kept at room temperature for 30 min. Then, 2 mL of 0.1 N NaOH was added to neutralize it and the volume was made up by buffer solution to make a final concentration of 50 mg/mL. For base degradation, 2 mL of standard stock solution (500 mg/mL) was added to a 20-mL volumetric flask. To this solution, 2 mL of 0.05 N NaOH solution was added and the solution was kept at room temperature for 5 min. Then, 2 mL of 0.05 N HCl was added to neutralize it and the volume was made up by buffer solution to make a final concentration of 50 mg/mL. For oxidation degradation, 2 mL of standard stock solution (500 mg/mL) was added to 20 mL of the 904 Patel et al.
UPLC method development The main aim of the chromatographic method was to achieve separation of degradation products in the presence of drug substance. CLOX SOD was soluble in water. Practically, the solubility of CLOX SOD was observed in alcohol and water. The diluent selected in the finalized method was phosphate buffer, pH 6.8, as it was found compatible with the mobile phase. The pKa of CLOX SOD was reported as 2.79. Therefore, the pH of the mobile phase was selected on the basis of pKa. Initially, isocratic HPLC conditions were optimized for CLOX SOD but degradation products were not separated properly in HPLC and CLOX SOD was eluted late. Therefore, the gradient system is used to reduce run time and due to less particle size in UPLC, it gives more separation of degradation products. It is also observed that the elution time of CLOX SOD in UPLC was reduced 10-fold to that of HPLC. Therefore, using the UPLC method instead of the HPLC method gives good resolution and also reduces tailing of drug substance. The objective of the study to develop and validate the stability-indicating RP-UPLC method for determination of Cloxacillin Sodium in its bulk form and formulation was accomplished using the Acquity H-UPLC C18 100 mm 2.1 mm, BEH 1.7 mm column with PDA detection at 225 nm. Initially, various mobile phase compositions were tried to elute complex but mobile phase comprising of mobile phase A: 20 mM phosphate buffer, pH 6.8, and mobile phase B: methanol:acetonitrile (75:25 v/v) was found to be giving better resolution with a run time of 13 min and a flow rate 0.35 mL/min. The retention time was found to be 6.9 min. Specificity The forced degradation studies were performed to check the possible degradation of the active pharmaceutical ingredient when exposed to various conditions such as acidity, alkalinity and thermal conditions. The analytical method was used to measure the analyte response in the presence of its degradation products. The degradation results for all the stress conditions are discussed (see Table III). From the chromatograms of
degradation under different conditions, we concluded that CLOX SOD was stable in temperature and light (see Figures 2–6). The purity of the principle and other chromatographic peaks was found to be satisfactory which confirmed the stability-indicating power of the UPLC method. Linearity and range Linearity was determined by preparing solutions over the range of 0.05–150% of the assay concentration. Linear correlation was Table III Forced Degradation Study Data Stress condition/duration/state
% Assay
% Degradation
Mass balance (%)
Acidic/0.1 N HCl/30 min/solution Alkaline/0.05 N NaOH/05 min/solution Oxidative/0.03% H2O2/1 h/solution Thermal/608C/3 days/solid Solution Photo/UV light/3 days/solid Solution
90.28 87.63 86.43 99.04 71.59 99.04 71.67
8.76 11.41 12.61 ND 27.45 ND 27.53
99.04 99.04 99.04 99.04 99.04 99.04 99.20
Figure 4. Chromatogram of CLOX SOD oxidative degradation.
Figure 5. Chromatogram of CLOX SOD thermal degradation. Figure 2. Chromatogram of CLOX SOD acid degradation.
Figure 3. Chromatogram of CLOX SOD base degradation.
Figure 6. Chromatogram of CLOX SOD photo degradation.
Determination of Cloxacillin Sodium in Its Bulk Form and Formulation 905
Table IV Summary of Validation Parameters with Acceptance Criteria Characteristics
Acceptance criteria
Results
Specificity LOQ
Peak purity factor 1.000 RSD ,2% s/n ratio 10 Correlation coefficient r 2 . 0.995 Recovery 98–102% (individual and mean) RSD ,2% 1. % RSD of six replicate sample preparations should not be .2.0%. 2. Individual impurities 0.1%, %RSD must be 30%. Record the % RSD for other individual impurities as well as total impurities. RSD ,2% Overall RSD ,2% Difference of the mean assay ,2% Theoretical plates: .2,500 % RSD of replicates: ,2% Asymmetry (tailing factor): ,2%
0.999 1.4 10 0.997
Linearity Accuracy Method precision
Intermediate precision
Robustness
100.86 0.73 1. % RSD of six replicate sample preparations is 0.08%. 2. % RSD for individual impurities is below 30%. 0.9 1.04 0.2 Meets
RSD, relative standard deviation. Table V Accuracy (Recovery) Data for Method Level
Area
Mean area
Added amount (mg/mL)
Recovered amount (mg/mL)
% Recovery
50%
1,556,734 1,590,341 1,511,263 1,566,843 1,508,829 1,553,927 3,042,147 3,044,578 3,060,125 3,051,691 3,061,691 3,061,691 4,644,276 4,651,427 4,624,452 4,689,452 4,605,876 4,672,876
1,573,538
25
25.9318
101.9
1,539,053
25
25.4313
100.1
1,531,378
25
25.1279
101.7
3,043,363
50
50.5147
99.2
3,055,908
50
50.6328
100.1
3,061,691
50
50.7578
100.6
4,647,852
75
75.3589
101.4
4,656,952
75
75.824
102
4,639,376
75
75.4478
101.5
100%
150%
Mean
Mean % recovery + SD 101.2 + 0.986577
% RSD 1
100 + 0.70946
0.7
101.4 + 0.321455
0.5
100.86
0.73
RSD, relative standard deviation.
obtained between the peak area versus concentration of CLOX SOD. The linearity was observed in the range of 0.025–75 mg/mL. The R 2 obtained was 0.998. The regression results suggested an excellent correlation between the peak area and concentration values (see Table IV). LOQ The LOQ was estimated from the set of five calibration curves (see Table IV). The LOQ was calculated by using the formula LOQ ¼
10SD S
where SD, is the standard deviation of the response and S is the slope of calibration curve of the analyte. Precision The precision of the proposed method was determined by carrying out repeatability and intermediate studies. The repeatability 906 Patel et al.
Table VI Assay Data of CLOX SOD Capsule Parameters
Capsule formulation (n ¼ 5)
Standard area
30,21,702 30,16,359 30,19,287 30,14,862 30,03,692 30515180 30,39,802 30,39,887 30,39,845 99.1 + 0.021213
Mean area Sample area Mean area % Assay
was checked by repeatedly (n ¼ 6) injecting sample solutions and computing the relative standard deviation (% RSD) of the assay results. The % RSD observed was 0.08, which was well within the acceptance criteria and the study concludes repeatability of the method. The method precision was repeated on a different day, by a different analyst, using a different UPLC system. The % RSD was calculated and found to be 0.9%, which was within the
Figure 7. Chromatogram for assay of Cloxacillin SOD.
Conclusion
Table VII Summary of Robustness Data Factor
Level
Retention time
Theoretical plate
Asymmetry
Flow rate (mL/min)
0.3 0.35 0.4 25 30 35
7.36 7.056 6.776 7.583 7.056 6.488 0.392714 5.567908
36,255 36,965 36,322 37,764 36,965 37,920 698.74 1.88685804
1.4 1.2 1.3 1.3 1.2 1.4 0.08994427 6.91879
Temperature (8C)
CV % RSD
The new stability-indicating method for determination of Cloxacillin Sodium in bulk and marketed formulations was developed and validated. In this method, the use of RP-UPLC helped in giving faster retention time and better resolution of peaks than that of HPLC. Hence this method exhibited excellent performance in terms of sensitivity and speed. The method was fully validated, showing satisfactory data for all the parameters tested. There were no common degradation products for Cloxacillin Sodium in all degradation conditions.
CV, coefficient of variation; RSD, relative standard deviation.
Supplementary material limit of 2%. Hence, the method is precise and rugged (see Table V).
Supplementary materials are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org).
Accuracy The accuracy of the method was determined for the CLOX SOD by spiking its stock solution in a blank matrix in triplicate at levels 50, 100 and 150% of the specified limit. The mean % recovery at the 50, 100 and 150% level was found to be 101.2, 100.0 and 101.4, respectively, and % RSD was found to be 1.0, 0.7 and 0.5, respectively, which meets the established acceptance criteria. Thus, the study proves that the method is accurate in the considered range (Table V).
Acknowledgments
Assay Analysis of samples of marketed antibiotic capsules containing 500 mg of CLOX SOD was carried out, and the amount recovered was found to be 99.1 (Table VI and Figure 7). % Assay CLOX SOD was within the acceptance range of 98 –102%. Robustness To prove the reliability of the analytical method during the normal usage, some small but deliberate changes were made in the analytical method. The robustness of the method was studied by changing the flow rate (0.30, 0.35 and 0.40 mL/min), and the column oven temperature (25, 30 and 358C), the retention time, theoretical plates, asymmetry factor were observed. Theoretical plates and asymmetry values were obtained from the first injection of the system suitability set and were found well within the acceptance criteria. Thus, the study proves the reliability of the test method for minor changes under chromatographic conditions. Hence, the method can be termed as robust (Table VII).
The authors gratefully acknowledge industrial guide Mr Sandeep Rana and Mr Anirban Roy, Group Leader of Piramal Healthcare, Ahmedabad, for allowing to complete this work in the industry. They also thankful to Centuarian Laboratories, GIDC, Vadodara, and Savorite Pharmaceuticals, Alkapuri, Vadodara, for providing samples of API and capsules, respectively. References 1. Drug profile of Cloxacillin Sodium. (2009). www.drugbank.ca/drugs/ DB01147.Drug profile of Cloxacillin Sodium. (accessed January 24, 2013). 2. The United State Pharmacopoeia XXIV, National Formulary XX. The US Pharmaceutical Convention, Inc., Rockville, MD, (2002). 3. Sharma, Y.R.; Elementary organic spectroscopy. 4th ed. S. Chand & Company Ltd, New Delhi, (2004), pp. 9 –60. 4. Kasture, A.V., Wadodkar, S.G., Mahadik, K.R., More, H.M.; Introduction to instrumental techniques. Vol. II, 12th ed. Nirali Prakashan, Pune, (2002), pp. 1–3. 5. Jeffery, G.H., Bassett, J., Mendham, J., Denny, R.C.; Vogel’s textbook of quantitative chemical analysis. 5th ed. Longmann Scientific & Technical, England, (1989), pp. 3 –14. 6. Ahuja, S., Scypinski, S.; Handbook of modern pharmaceutical analysis. Vol. 3, 2nd ed. Elseveir, London, (2001), pp. 96– 99. 7. Indian Pharmacopoeia. The Indian Pharmacopoeia Commission, Ghaziabad, Govt. of India Ministry of Health and Family Welfare (2007), Volume I, Appendix 2.5.3, pp. 1123. 8. United State Pharmacopoiea, vol II (2008), pp. 1831. 9. British Pharmacopoeia. Department of health, published by the stationery office on behalf of the Medicines and Healthcare products
Determination of Cloxacillin Sodium in Its Bulk Form and Formulation 907
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Regulatory Agency (MHRA), vol. 4. HMSO Publication Volume IV, London, (2008), pp. 532. Rathinavel, G., Mukherjee, P., Valarmathy, J., Samueljoshua, L., Ganesh, M., Sivakumar, T., et al.; A validated RP-HPLC method for simultaneous estimation of cefixime and cloxacillin; Journal of Chemistry, (2008); 5(3): 648–651. Biswas, A., Rajendiran, A., Rawat, I.S., Dey, A.K.; Validated estimation procedure of amoxycillin trihydrate, cloxacillin sodium in pharmaceutical formulation by RP-HPLC method; Research Journal of Pharmaceutical Biological and Chemical Sciences, (2010); 1(4): 344. Sarode, M.R., Chaple, D.R., Nikhade, P.A., Yeole, M.P.; Simultaneous spectrophotometric estimation of cefixime and cloxacillin in tablet dosage form; International Journal of Uni. Pharmacy and Life Sciences, (2012); 2(4): 548. Kumar, V., Bhutani, H., Singh, S.; Validated stability-indicating HPLC method for simultaneous determination of ampicillin and cloxacillin
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in combination drug products; Journal of Pharmaceutical and Biomedical Analysis, (2007); 43(2): 769– 773. Evaggelopoulou, E.N., Samanidou, V.F.; Development and validation of an HPLC method for the determination of six penicillin and three amphenicol antibiotics in gilthead seabream (Sparus aurata) tissue; Food Chemistry, (2013); 3(4): 346. Qureshi, S.Z., Qayoom, T., Helalet, M.I.; Simultaneous spectrophotometric and volumetric determinations of amoxycillin, ampicillin and cloxacillin in drug formulations: reaction mechanism in the base catalysed hydrolysis followed by oxidation with iodate in dilute acid solution; Journal of Pharmaceutical and Biomedical Analysis. (1999); 21(3): 473–482. Zeng, L.-g.; HPLC determination of cloxacillin sodium for injection and its related substance; Chinese Journal of Pharmaceutical Analysis, (2004); 22: 154–159. ICH Q1A (R2). Stability testing of new drug substances and products (2003).
Article
Development and Validation of a Stability-Indicating RP-UPLC Method for Determination of Cloxacillin Sodium in Its Bulk Form and Formulation Nikita Patel1*, Pooja Contractor1,2, Rajesh Keshrala1, Parag R. Patel1 and Bhimagoni Shridhar1 1
Parul Institute of Pharmacy, Waghodia, Limda, Gujarat, India and 2Piramal Health Care, Ahmedabad, Gujarat, India
*Author to whom correspondence should be addressed. Email: [email protected] Received 10 May 2014; revised 14 September 2014
A simple, linear gradient, rapid, precise and stability-indicating RPUPLC method was developed for the determination of Cloxacillin Sodium in its bulk form and formulation. Ultra performance liquid chromatography, a most promising advancement in a world of chromatography, reduces analysis time, increases reliability through higher resolution, sensitivity and selectivity as well as used as an economic method due to reducing solvent consumption. A chromatographic separation of a drug as well as its degradants was achieved using Waters acquity BEH, 2.1 3 100 mm, 1.7 mm C18 column with gradient of mobile phase A: phosphate buffer, pH 6.8 and mobile phase B: methanol:acetonitrile (75:25). The drug and degradants were monitored at a detection wavelength of 225 nm with a flow rate of 0.35 mL/min and an injection volume of 10 mL. The temperature of the column and auto sampler compartments was at 3088 C and 2588 C + 188 C, respectively. The retention time of the drug was ∼6.9 min. The resolution of the drug and degradant peak was >1.5 in all cases. Force degradation of CLOX SOD was carried under alkaline, acidic, oxidative, thermal, photo degradation conditions and it was analyzed by the proposed method. The drug degrades under alkaline, acidic and oxidative conditions but was stable in temperature and light. A developed method was validated as per ICH guidelines using validation parameters such as precision, linearity and range, limit of quantification, specificity, assay and robustness.
Introduction Cloxacillin, chemically known as monosodium(2S,5R,6R)6-[o-(2-chlorophenyl)-5-methyl-4-isoxazole carboxamido]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]-heptane-2-carboxylate monohydrate, is a semi-synthetic antibiotic in the same class as penicillin. It used against staphylococci that produce b-lactamase (1). The chemical structure of Cloxacillin Sodium is depicted in Figure 1. The ACQUITY ultra performance liquid chromatography (UPLC) system eliminates significant time and cost per sample from an analytical process while improving the quality of the results. What differentiates the system’s holistic design is Waters’ patented sub-2-mm hybrid particle chemistry, which offers significant benefits over today’s high performance liquid chromatography (HPLC) systems equipment. These particles operate at elevated mobile phase linear velocities to affect which cause increase in resolution, sensitivity and speed of analysis. Because of its speed and sensitivity, this technique has gained considerable attention in recent years for pharmaceutical and biomedical analysis (2–6). A literature survey reveals that few spectrophotometric and HPLC methods were available for the estimation of Cloxacillin Sodium individually as well as in combination with other drugs. No analytical method was reported for estimation of Cloxacillin Sodium by UPLC
in bulk and pharmaceutical dosage form (7 – 16). Hence, it was thought worthwhile to develop and validate the stability-indicating RP-UPLC method for determination of Cloxacillin Sodium in its bulk form and formulation. Experimental Chemicals API (CLOX SOD-99.8% Purity) was supplied by Centuarian Laboratories, GIDC, Vadodara, India. Capsules (CLOXI-500 mg) were obtained from Savorite Pharmaceuticals, Alkapuri, Vadodara, India. Methanol (HPLC grade) and sodium hydroxide pellets (AR Grade) were obtained from Finar Reagent, Ahmedabad, India. Furthermore, acetonitrile (HPLC grade) was obtained from Sigma-Aldrich, and H2O2 (30% v/v), HCl and NaOH were obtained from Merck Specialties Pvt Ltd, Ahmedabad, India. Potassium dihydrogen orthophosphate (AR Grade) was obtained from Fluka Reagent, Vadodara, India. Chromatographic conditions and equipment LC was carried out on a Waters Acquity UPLC with a photodiode array detector. For optimization of chromatographic conditions, the effects of various method parameters such as mobile phase, column, flow rate and solvent ratio were studied and the chromatographic parameters such as asymmetric factor, resolution and column efficiency were calculated. The separation was achieved on an Acquity H-UPLC C18 100 mm 2.1 mm, BEH 1.7 mm column using mobile phase A: 20 mM phosphate buffer, pH 6.8, and mobile phase B: methanol:acetonitrile (75:25) using a gradient program. The drug and degradation products were detected (see Table I). By keeping the column temperature of 308C, flow rate of 0.35 mL/min and injection volume of 10 mL, all peaks were separated, and the gradient pattern did not interfere with the CLOX SOD peak which was detected by a PDA detector at 225 nm. Preparation of solutions An accurately weighed 50 mg of CLOX SOD API was transferred into a 100 mL-volumetric flask. First, it was dissolved in 50 mL buffer and sonicated, then diluted up to the mark with buffer to get the concentration of CLOX SOD (500 mg/mL). Concentrations ranging from 0.025 to 75 mg/mL for CLOX SOD were prepared from stock solution and different validation parameters were performed. Validation procedure Validation of the newly developed method was studied in terms of linearity and range, precision, accuracy, robustness, limit of
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Table II System Suitability Data S no.
Parameter
Drug
1 2 3 4 5
Peak area Number of theoretical plates Retention time (min) Asymmetry/USP tailing % RSD
3,079,906 48,559 6.956 1.2 0.8
RSD, relative standard deviation.
Figure 1. Chemical structure of CLOX SOD.
Table I Gradient Programme for Method Development Time (min)
Initial 2 8 10 10.01 13
Gradient program A%
B%
75 70 35 35 75 75
25 30 65 65 25 25
volumetric flask and to this solution, 2 mL of 0.03% hydrogen peroxide was added. This solution was kept for 1 h at room temperature. The volume was made up by buffer to make a final concentration of 50 mg/mL. For thermal degradation, 50 mg/mL solution of CLOX SOD (working standard) was exposed to 608C for 3 days. As well as API of CLOX SOD was also exposed to the same condition for 3 days and the photo degradation was done by prepared 50 mg/mL solution of CLOX SOD (working standard) was exposed to 200 lux h for 3 days. Results and Discussion
Mobile phase A: 20 mM, phosphate buffer, pH 6.8. Mobile phase B: methanol:acetonitrile (75:25 v/v).
detection (LOD) and limit of quantification (LOQ) as per the ICH guidelines (17). For linearity, the solutions were prepared from the stock solution (500 mg/mL) ranging from 0.025 to 75 mg/mL, the calibration curve was generated, and regression parameters were obtained. The LOQ was estimated from the set of five calibration curves of CLOX SOD. The precision of the method was studied by checking the repeatability, interday and intraday precision for three concentrations thrice. Accuracy of the method was determined by calculating percentage recoveries of CLOX SOD by the standard addition method. A known amount of standard solutions of CLOX SOD (0.025, 50 and 75 mg/mL) was added to a preanalyzed sample solution of CLOX SOD (50 mg/mL). Robustness of the method was studied by changing the temperature of column compartment by +5 units and flow rate by +5 mL/min. The changes in the response of CLOX SOD were noted and compared with the original one. System suitability parameters are mentioned in Table II. Specificity-forced degradation studies To prove the stability-indicating property of the method, forced degradation studies were performed on CLOX SOD. For acid degradation, 2 mL of standard stock solution (500 mg/mL) was added to a 20-mL volumetric flask and to this 2 mL of 0.1 N HCl solution was added and kept at room temperature for 30 min. Then, 2 mL of 0.1 N NaOH was added to neutralize it and the volume was made up by buffer solution to make a final concentration of 50 mg/mL. For base degradation, 2 mL of standard stock solution (500 mg/mL) was added to a 20-mL volumetric flask. To this solution, 2 mL of 0.05 N NaOH solution was added and the solution was kept at room temperature for 5 min. Then, 2 mL of 0.05 N HCl was added to neutralize it and the volume was made up by buffer solution to make a final concentration of 50 mg/mL. For oxidation degradation, 2 mL of standard stock solution (500 mg/mL) was added to 20 mL of the 904 Patel et al.
UPLC method development The main aim of the chromatographic method was to achieve separation of degradation products in the presence of drug substance. CLOX SOD was soluble in water. Practically, the solubility of CLOX SOD was observed in alcohol and water. The diluent selected in the finalized method was phosphate buffer, pH 6.8, as it was found compatible with the mobile phase. The pKa of CLOX SOD was reported as 2.79. Therefore, the pH of the mobile phase was selected on the basis of pKa. Initially, isocratic HPLC conditions were optimized for CLOX SOD but degradation products were not separated properly in HPLC and CLOX SOD was eluted late. Therefore, the gradient system is used to reduce run time and due to less particle size in UPLC, it gives more separation of degradation products. It is also observed that the elution time of CLOX SOD in UPLC was reduced 10-fold to that of HPLC. Therefore, using the UPLC method instead of the HPLC method gives good resolution and also reduces tailing of drug substance. The objective of the study to develop and validate the stability-indicating RP-UPLC method for determination of Cloxacillin Sodium in its bulk form and formulation was accomplished using the Acquity H-UPLC C18 100 mm 2.1 mm, BEH 1.7 mm column with PDA detection at 225 nm. Initially, various mobile phase compositions were tried to elute complex but mobile phase comprising of mobile phase A: 20 mM phosphate buffer, pH 6.8, and mobile phase B: methanol:acetonitrile (75:25 v/v) was found to be giving better resolution with a run time of 13 min and a flow rate 0.35 mL/min. The retention time was found to be 6.9 min. Specificity The forced degradation studies were performed to check the possible degradation of the active pharmaceutical ingredient when exposed to various conditions such as acidity, alkalinity and thermal conditions. The analytical method was used to measure the analyte response in the presence of its degradation products. The degradation results for all the stress conditions are discussed (see Table III). From the chromatograms of
degradation under different conditions, we concluded that CLOX SOD was stable in temperature and light (see Figures 2–6). The purity of the principle and other chromatographic peaks was found to be satisfactory which confirmed the stability-indicating power of the UPLC method. Linearity and range Linearity was determined by preparing solutions over the range of 0.05–150% of the assay concentration. Linear correlation was Table III Forced Degradation Study Data Stress condition/duration/state
% Assay
% Degradation
Mass balance (%)
Acidic/0.1 N HCl/30 min/solution Alkaline/0.05 N NaOH/05 min/solution Oxidative/0.03% H2O2/1 h/solution Thermal/608C/3 days/solid Solution Photo/UV light/3 days/solid Solution
90.28 87.63 86.43 99.04 71.59 99.04 71.67
8.76 11.41 12.61 ND 27.45 ND 27.53
99.04 99.04 99.04 99.04 99.04 99.04 99.20
Figure 4. Chromatogram of CLOX SOD oxidative degradation.
Figure 5. Chromatogram of CLOX SOD thermal degradation. Figure 2. Chromatogram of CLOX SOD acid degradation.
Figure 3. Chromatogram of CLOX SOD base degradation.
Figure 6. Chromatogram of CLOX SOD photo degradation.
Determination of Cloxacillin Sodium in Its Bulk Form and Formulation 905
Table IV Summary of Validation Parameters with Acceptance Criteria Characteristics
Acceptance criteria
Results
Specificity LOQ
Peak purity factor 1.000 RSD ,2% s/n ratio 10 Correlation coefficient r 2 . 0.995 Recovery 98–102% (individual and mean) RSD ,2% 1. % RSD of six replicate sample preparations should not be .2.0%. 2. Individual impurities 0.1%, %RSD must be 30%. Record the % RSD for other individual impurities as well as total impurities. RSD ,2% Overall RSD ,2% Difference of the mean assay ,2% Theoretical plates: .2,500 % RSD of replicates: ,2% Asymmetry (tailing factor): ,2%
0.999 1.4 10 0.997
Linearity Accuracy Method precision
Intermediate precision
Robustness
100.86 0.73 1. % RSD of six replicate sample preparations is 0.08%. 2. % RSD for individual impurities is below 30%. 0.9 1.04 0.2 Meets
RSD, relative standard deviation. Table V Accuracy (Recovery) Data for Method Level
Area
Mean area
Added amount (mg/mL)
Recovered amount (mg/mL)
% Recovery
50%
1,556,734 1,590,341 1,511,263 1,566,843 1,508,829 1,553,927 3,042,147 3,044,578 3,060,125 3,051,691 3,061,691 3,061,691 4,644,276 4,651,427 4,624,452 4,689,452 4,605,876 4,672,876
1,573,538
25
25.9318
101.9
1,539,053
25
25.4313
100.1
1,531,378
25
25.1279
101.7
3,043,363
50
50.5147
99.2
3,055,908
50
50.6328
100.1
3,061,691
50
50.7578
100.6
4,647,852
75
75.3589
101.4
4,656,952
75
75.824
102
4,639,376
75
75.4478
101.5
100%
150%
Mean
Mean % recovery + SD 101.2 + 0.986577
% RSD 1
100 + 0.70946
0.7
101.4 + 0.321455
0.5
100.86
0.73
RSD, relative standard deviation.
obtained between the peak area versus concentration of CLOX SOD. The linearity was observed in the range of 0.025–75 mg/mL. The R 2 obtained was 0.998. The regression results suggested an excellent correlation between the peak area and concentration values (see Table IV). LOQ The LOQ was estimated from the set of five calibration curves (see Table IV). The LOQ was calculated by using the formula LOQ ¼
10SD S
where SD, is the standard deviation of the response and S is the slope of calibration curve of the analyte. Precision The precision of the proposed method was determined by carrying out repeatability and intermediate studies. The repeatability 906 Patel et al.
Table VI Assay Data of CLOX SOD Capsule Parameters
Capsule formulation (n ¼ 5)
Standard area
30,21,702 30,16,359 30,19,287 30,14,862 30,03,692 30515180 30,39,802 30,39,887 30,39,845 99.1 + 0.021213
Mean area Sample area Mean area % Assay
was checked by repeatedly (n ¼ 6) injecting sample solutions and computing the relative standard deviation (% RSD) of the assay results. The % RSD observed was 0.08, which was well within the acceptance criteria and the study concludes repeatability of the method. The method precision was repeated on a different day, by a different analyst, using a different UPLC system. The % RSD was calculated and found to be 0.9%, which was within the
Figure 7. Chromatogram for assay of Cloxacillin SOD.
Conclusion
Table VII Summary of Robustness Data Factor
Level
Retention time
Theoretical plate
Asymmetry
Flow rate (mL/min)
0.3 0.35 0.4 25 30 35
7.36 7.056 6.776 7.583 7.056 6.488 0.392714 5.567908
36,255 36,965 36,322 37,764 36,965 37,920 698.74 1.88685804
1.4 1.2 1.3 1.3 1.2 1.4 0.08994427 6.91879
Temperature (8C)
CV % RSD
The new stability-indicating method for determination of Cloxacillin Sodium in bulk and marketed formulations was developed and validated. In this method, the use of RP-UPLC helped in giving faster retention time and better resolution of peaks than that of HPLC. Hence this method exhibited excellent performance in terms of sensitivity and speed. The method was fully validated, showing satisfactory data for all the parameters tested. There were no common degradation products for Cloxacillin Sodium in all degradation conditions.
CV, coefficient of variation; RSD, relative standard deviation.
Supplementary material limit of 2%. Hence, the method is precise and rugged (see Table V).
Supplementary materials are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org).
Accuracy The accuracy of the method was determined for the CLOX SOD by spiking its stock solution in a blank matrix in triplicate at levels 50, 100 and 150% of the specified limit. The mean % recovery at the 50, 100 and 150% level was found to be 101.2, 100.0 and 101.4, respectively, and % RSD was found to be 1.0, 0.7 and 0.5, respectively, which meets the established acceptance criteria. Thus, the study proves that the method is accurate in the considered range (Table V).
Acknowledgments
Assay Analysis of samples of marketed antibiotic capsules containing 500 mg of CLOX SOD was carried out, and the amount recovered was found to be 99.1 (Table VI and Figure 7). % Assay CLOX SOD was within the acceptance range of 98 –102%. Robustness To prove the reliability of the analytical method during the normal usage, some small but deliberate changes were made in the analytical method. The robustness of the method was studied by changing the flow rate (0.30, 0.35 and 0.40 mL/min), and the column oven temperature (25, 30 and 358C), the retention time, theoretical plates, asymmetry factor were observed. Theoretical plates and asymmetry values were obtained from the first injection of the system suitability set and were found well within the acceptance criteria. Thus, the study proves the reliability of the test method for minor changes under chromatographic conditions. Hence, the method can be termed as robust (Table VII).
The authors gratefully acknowledge industrial guide Mr Sandeep Rana and Mr Anirban Roy, Group Leader of Piramal Healthcare, Ahmedabad, for allowing to complete this work in the industry. They also thankful to Centuarian Laboratories, GIDC, Vadodara, and Savorite Pharmaceuticals, Alkapuri, Vadodara, for providing samples of API and capsules, respectively. References 1. Drug profile of Cloxacillin Sodium. (2009). www.drugbank.ca/drugs/ DB01147.Drug profile of Cloxacillin Sodium. (accessed January 24, 2013). 2. The United State Pharmacopoeia XXIV, National Formulary XX. The US Pharmaceutical Convention, Inc., Rockville, MD, (2002). 3. Sharma, Y.R.; Elementary organic spectroscopy. 4th ed. S. Chand & Company Ltd, New Delhi, (2004), pp. 9 –60. 4. Kasture, A.V., Wadodkar, S.G., Mahadik, K.R., More, H.M.; Introduction to instrumental techniques. Vol. II, 12th ed. Nirali Prakashan, Pune, (2002), pp. 1–3. 5. Jeffery, G.H., Bassett, J., Mendham, J., Denny, R.C.; Vogel’s textbook of quantitative chemical analysis. 5th ed. Longmann Scientific & Technical, England, (1989), pp. 3 –14. 6. Ahuja, S., Scypinski, S.; Handbook of modern pharmaceutical analysis. Vol. 3, 2nd ed. Elseveir, London, (2001), pp. 96– 99. 7. Indian Pharmacopoeia. The Indian Pharmacopoeia Commission, Ghaziabad, Govt. of India Ministry of Health and Family Welfare (2007), Volume I, Appendix 2.5.3, pp. 1123. 8. United State Pharmacopoiea, vol II (2008), pp. 1831. 9. British Pharmacopoeia. Department of health, published by the stationery office on behalf of the Medicines and Healthcare products
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