Vol. 8(42), pp. 1086-1092, 15 November, 2014 DOI: 10.5897/AJPP2014.4092 Article Number: 065080948819 ISSN 1996-0816 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJPP
African Journal of Pharmacy and Pharmacology
Full Length Research Paper
Development and validation for the determination of residual solvents of pharmaceutical formulations of flucloxacillin sodium using head space gas chromatography Sk Manirul Haque Department of Chemical Engineering and Process Technology, Jubail Industrial College, Royal Commission of Jubail, Al –Jubail Industrial City, P. B. No – 10099, Zip Code: 31961, KSA. Received 8 April, 2014; Accepted 8 September, 2014
An accurate and novel method was developed for the determination of residual solvents of flucloxacillin sodium by using head space gas chromatography (HSGC). The determination was done on Clarus 400 (PerkinElmer) with Total Chrome Navigator software. The residual solvents were separated on Elite - 5 fused silica capillary column 30 m long and 0.32 mm in internal diameter. Beer’s law is obeyed in the concentration ranges of methanol, ethyl acetate and toluene 50 to 2000, 50 to 1000, 100 to 500 ppm for flucloxacillin sodium. The method was validated for specificity, linearity, precision, accuracy, limit of detection, limit of quantitation, robustness and solution stability. The degrees of linearity of the calibration curves, the percent recoveries, relative standard deviation for the method were determined. The correlation coefficient for methanol, ethyl acetate and toluene are found as 0.9956, 0.9941 and 0.9863 for flucloxacillin sodium. Key words: Flucloxacillin, residual solvents, methanol, ethyl acetate, toluene, head space gas chromatography (HSGC), validation.
INTRODUCTION Flucloxacillin (INN) or floxacillin (USAN) is a narrowspectrum beta-lactam antibiotic of the penicillin class. It is used to treat infections caused by susceptible grampositive bacteria. Unlike other penicillins, flucloxacillin has activity against beta-lactamase-producing organisms
such as Staphylococcus aureus as it is beta-lactamase stable. However, it is ineffective against methicillin – resistant Staphylococcus aureus (MRSA). It is most commonly used to treat infections such as: chest, ear, nose and throat infections (for example, tonsillitis,
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sinusitis, pneumonia); skin and soft tissue infections (for example, boils, burns, wounds, abscesses, infected eczema, infected acne); other infections including those of the heart (endocarditis), bones and joints (osteomyelitis), membranes of the brain (meningitis), guts (enteritis), blood (septicaemia) and the kidney, bladder or urethra. Flucloxacillin can also be used to prevent infections during major surgical procedures, particularly in heart or orthopedic surgery. Flucloxacillin is insensitive to beta-lactamase (also known as penicillinase) enzymes secreted by many penicillin-resistant bacteria. The presence of the isoxazolyl group on the side-chain of the penicillin nucleus facilitates the β-lactamase resistance, since they are relatively intolerant of side-chain steric hindrance. Thus, it is able to bind to penicillin-binding proteins (PBPs) and inhibit peptidoglycan crosslinking, but is not bound by or inactivated by β-lactamases. The therapeutic importance of flucloxacillin was behind the development of numerous methods for its determination. The methods adapted to the analysis of FCS include high–performance liquid chromatography (HPLC) (Zhou et al., 2007; Pullen et al., 2007; Dhiraj et al., 2009) and spectrophotometry(Dey et al., 2010; Reddy and Reddy, 2012; Singh et al., 2009; Prakash et al., 2012; Fiorentino and Salgado, 2012; Flavia and Herida, 2012). There is no official method for the determination of residual solvent of flucloxacillin sodium. Therefore, it is very imperative to develop a simple and suitable analytical method for the determination of residual solvent in bulk and pharmaceutical formulations. Head space gas chromatography (HSGC) is the technique of choice in research laboratories and pharmaceutical industries.
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processing system were run with Total Chrome Navigator software which was connected with PC. In the present method, the chromatograph is equipped with flame ionization detector. The solvents are separated on elite - 5 fused silica capillary column 30 m long and 0.32 mm in internal diameter. The column temperature is maintained at 50°C for 2 min, then raised at a rate of 20°C per min at 220°C and maintained at 220°C for 2 min. The injection port and detector temperature was maintained at 250°C. The carrier gas nitrogen was passed with a velocity of 37.3 cm per second with pressure 10 Kpa and split ratio of 1:1. The injections, pressurized, withdraw and thermostat times are 0.1, 2.0, 0.2 and 10 min. The gas chromatography cycle time is only 35 min.
Validation Specificity Diluent blank: 2 ml of dimethyl sulphoxide (DMSO) was transfered into one headspace sample (HSS) vial and sealed with vial septa followed by aluminum cap. Preparation of solvents standard solution for specificity: 0.25 g of methanol was weighed in 100 ml volumetric flask diluted with DMSO up to mark and 2 ml solution taken in headspace vial. 0.25 g of ethyl acetate was weighed in 100 ml volumetric flask diluted with DMSO up to mark 2 ml solution taken in headspace vial. 0.25 g of toluene was weighed in 100 ml volumetric flask diluted with DMSO up to mark and 2 ml solution taken in headspace vial. Procedure: Diluent blank and standard solutions of methanol was injected, with ethyl acetate and toluene in singlet. The retention time of each solvent was identified and the retention time of methanol, ethyl acetate and toluene recorded.
Precision Instrument precision
MATERIALS 1. Flucloxacillin Sodium (Sigma Aldrich, USA), 2. Dolopen (Techno drugs, Gujarat, India), 3. Enoclox (Modern Pharmaceuticals Ltd, Kerala, India), 4. Floxason (Hudson Pharmaceuticals Ltd, Dhaka, Bangladesh), 5. Methanol (Spectrochem, Mumbai, India), 6. Ethyl Acetate (Spectrochem, Mumbai, India), 7. Toluene (Spectrochem, Mumbai, India), 8. Dimethyl Sulphoxide (Spectrochem, Mumbai, India).
Diluent blank: 2 ml of DMSO was transfered into one headspace sample (HSS) vial and sealed with vial septa, followed by aluminum cap. Preparation of standard solution: 3.0 g of methanol and 5.0 g ethyl acetate and 0.89 g toluene was weighed in 100 ml volumetric flask and diluted with DMSO up to mark. 5 ml of this was take in 50 ml volumetric flask and diluted up to 50 ml with DMSO and 2 ml solution taken in headspace vial. Procedure: 2 ml of standard solution was injected six times and the area response recorded to calculate % RSD.
METHODOLOGY HSGC instrument and conditions
Method precision
HSGC used was model Clarus 400, PerkinElmer (Shelton, USA) (Serial No: 646N8111901). The Clarus 400 Gas chromatograph (GC) is a microprocessor controled gas chromatograph with an optional built – in auto sampling system. The GC system is supported with additional equipment TurboMatrix 40 Sampler. All gas conduits that deliver carrier gas or any detector gas to the Clarus 400 GC must be formed from copper or stainless steel tubing that is free of grease, oil, or other organic material. The data
Diluent blank: 2 ml of DMSO was transfered into one headspace sample (HSS) vial, sealed with vial septa, followed by aluminum cap. Preparation of sample solution: 0.2 g of sample was weighed in 100 ml DMSO. 2 ml of this was take in headspace vial. Procedure: 2 ml of sample solution was injected three times and
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the area response recorded. The % RSD for area response of sample was calculated.
Linearity and range Linearity solutions Preparation of standard stock solution (5000 ppm): 0.5 g of methanol, 0.5 g m ethyl acetate and 0.5 g m toluene was weighed in 100 ml volumetric flask and diluted with DMSO up to mark.
Procedure: 2 ml of each linearity solution was injected. A plot of concentration (ppm) of methanol, ethyl acetate, toluene in the linearity solution (X-axis) versus peak area responses of the respective component peak (Y-axis) was drawn. The plot for its linearity was also observed. Regression line was calculated by least square method and correlation coefficient (r2), y-intercept and slope of regression line sorted out.
Accuracy Preparation of stock standard solution
50 ppm standard solution: Take 0.5 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 100 ppm standard solution: Take 1.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 200 ppm standard solution: Take 2.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 300 ppm standard solution: Take 3.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 500 ppm standard solution: Take 5.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 800 ppm standard solution: Take 8.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO.
0.5 g of methanol, 0.5 g toluene and 0.5 g ethyl acetate was weighed in 100 ml volumetric flask and diluted with DMSO up to mark.
Accuracy solution preparation 200 ppm standard solution: Take 2.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 300 ppm standard solution: Take 3.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 500 ppm standard solution: Take 5.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO.
1000 ppm standard solution: Take 10.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO.
Procedure: 2 ml of 200, 300 and 500 ppm was injected in different vials.
2000 ppm standard solution: Take 20.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO.
The % recovery for all the solvents was calculated by using following formula:
Concentration by correlation graph (linearity equation) % Recovery of solvent = × 100 Actual concentration
Limit of detection and limit of quantitation Preparation of stock standard solution 0.5 g of methanol, 0.5 g ethyl acetate and 0.5 g toluene was weighed in 100 ml volumetric flask and diluted with DMSO upto mark.
Procedure: 2 ml of 30 ppm LOD/LOQ solutions was injected in triplicate. % RSD for methanol, ethyl acetate, toluene was calculated for lowest detection concentration. Relative standard deviation (% RSD) of the area responses of methanol, ethyl acetate, toluene peaks obtained with triplicate injections of each LOQ and LOD solution preparation was calculated.
Robustness LOQ and LOD solution preparation 30 ppm standard solution: Take 0.3 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO. 50 ppm standard solution: Take 0.5 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO 100 ppm standard solution: Take 1.0 ml of standard stock solution in 50 ml volumetric flask and dilute to 50 ml with DMSO.
Robustness is the measure of variation in end results by changing routine analytical parameters. In order to establish the robustness, experiments were performed with the following changes. Experiment a: Initial oven temperature decrease 5° from normal oven temperature. Experiment b: Initial oven temperature increase 5° from normal oven temperature. The experiment was performed as explained. The relative standard
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Table 1. Instrument precision of the proposed method
Injection Standard - 1 Standard - 2 Standard - 3 Standard - 4 Standard - 5 Standard - 6 Mean % RSD
Peak area of methanol 2915226 2893734 2874768 2885792 2888466 2902449 2893406 0.486
Peak area of ethyl acetate 5074778 5055477 5064266 5040532 5062370 5055758 5058864 0.226
Peak area of toluene 2728156 2729454 2716695 2721325 2728798 2724377 2724801 0.185
RSD = Relative standard deviation
Table 2. Method precision of the proposed method
Sample injection Sample injection - 1 Sample injection - 2 Sample injection - 3 Mean % RSD
Peak area of methanol Not detected Not detected Not detected -
deviation (% RSD) of the results obtained was calculated with the experiments (a - b). Procedure: 2 ml of three standards was injected at normal analytical condition and at varied oven temperature in triplicate. Relative standard deviation for methanol, ethyl acetate, toluene peak response from three injections of standard solutions was calculated.
Peak area of ethyl acetate 1034538 1133875 1062966 1077126 4.75
Peak area of toluene Not Detected Not Detected Not Detected -
No. 42 filter paper (Whatman International Limited, Kent, UK) and washed with DMSO. The residue was washed well with DMSO for complete recovery of product and was further diluted to give a final concentration of 2 mg/ml. An aliquot of the diluted solution was analysed for residual solvent content following the recommended procedure.
RESULTS AND DISCUSSIONS Solution stability Preparation of standard solution: 1.5 mg of methanol, 5.0 mg ethyl acetate and 0.89 mg toluene was weighed in 100 ml volumetric flask and diluted with DMSO up to mark. Take 5 ml of this in 50 ml volumetric flask and dilute up to 50 ml with DMSO. 2 ml solution was taken in headspace vial. Procedure: 2 ml of standard solution was injected three times after 24 h of solution preparation and the area response recorded to calculate % RSD.
Procedure for determination of residual solvents flucloxacillin sodium in commercial dosage forms
of
To determine the content of residual solvents in commercial dosage forms, the contents of 500 mg capsules were weighed and finely powdered. A portion of the powder equivalent to 200 mg of active ingredient was weighed accurately, stired well with 20 ml DMSO and let standing for 10 min. The residue was filtered on Whatmann’s
Specificity Under experimental conditions, the retention times of DMSO, methanol, ethyl acetate and toluene were found to be 8.0, 2.8, 4.4 and 6.5, respectively.
Precision For instrument precision the relative standard deviation of methanol, ethyl acetate and toluene were found to be 0.49, 0.23 and 0.19 for flucloxacillin sodium (Table 1). For method precision, the RSD value of ethyl acetate was found to be 4.75% (Table 2) whereas methanol and toluene solvents were not detected. These values are acceptable within limit of RSD value 15%.
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Table 3. Analytical characteristics of the proposed method.
Concentration (ppm) Area (m) Co-relation factor Value of slope Value of intercept Equation
50 100 200 33329 234890 373169 0.9956 1214.3 89144 Y = 1214.3X + 89144
Concentration (ppm) Area Co-relation factor Value of slope Value of intercept Equation
50 100 200 18307 372307 674055 0.9941 2960.1 26918 Y = 2960.1X + 26918
Concentration (ppm) Area Co-relation factor Value of slope Value of intercept Equation
50 100 200 489899 965476 0.9963 4067.6 125413 Y = 4067.6X + 125413
Linearity and range Under experimental conditions, the peak area and concentration plot for methanol, ethyl acetate and toluene were found to be rectilinear over the range of 50 to 2000, 50 to 1000 and 100 to 500. The correlation factors of methanol, ethyl acetate and toluene were found to be 0.9956, 0.9941 and 0.9963. The results are summarized in Table 3.
Methanol 300 500 487377 732671
800 1041793
1000 1320646
2000 2500043
Ethyl acetate 300 500 942144 1583067
800 2410947
1000 2919978
2000 -
Toluene 300 500 1390458 2130222
800 -
1000 -
2000 -
ppm (Table 5).
Robustness The RSD values of methanol, ethyl acetate and toluene were found to be 1.30, 0.58 and 0.67 when oven temperature decreases by 5°. But when increased by 5°, the RSD values of methanol, ethyl acetate and toluene were found to be 0.73, 0.34, 0.76 and 0.97 (Table 6).
Accuracy Solution stability As can be seen from Table 4 that the recovery values of methanol, ethyl acetate and toluene were found to be 105.99 to 116.95, 103.06 to 109.31 and 98.58 to 103.67 % at 200, 300 and 500 ppm.
The relative standard deviation of methanol, ethyl acetate and toluene were found to be 1.83, 1.10 and 1.84 after injecting for 24 h of sample preparation (Table 7). The RSD values are acceptable within limit of 15 %.
Limit of detection and limit of quantitation The relative standard deviation of methanol, ethyl acetate and toluene were found to be 3.81, 7.68 and 4.77 at 30
Commercial formulation products The concentration of solvents present in the pharmaceutical
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Table 4. Accuracy and recovery
Solvent 200 ppm Methanol Ethyl Acetate Toluene
Peak area
Concentration by graph
% recovery
373169 674055 965476
233.9002 218.6200 206.5255
116.95 109.31 103.26
300 ppm Methanol Ethyl Acetate Toluene
487377 942144 1390458
327.9527 309.1875 311.0053
109.32 103.06 103.67
500 ppm Methanol Ethyl Acetate Toluene
732671 1583067 2130222
529.9572 525.7083 492.8727
105.99 105.14 98.58
Table 5. LOD and LOQ of the proposed method
Injection of lowest detection (30 ppm) Injection - 1 Injection - 2 Injection - 3 Mean % RSD
Peak area of methanol
Peak area of ethyl acetate
Peak area of toluene
200306 215189 212715 209403 3.81
128995 149099 146176 141423 7.68
120357 131056 130685 127366 4.77
Table 6. Robustness of the proposed method
Condition Oven temperature decrease 5°C
Oven temperature increase 5°C
Peak area of methanol 3031322 2964905 2963900 RSD = 1.29
Peak area of ethyl acetate 5282146 5232449 5226522 RSD = 0.58
Peak area of toluene 2635013 2608901 2601683 RSD = 0.67
2874076 2895831 2916078 RSD = 0.73
4867619 4868647 4896894 RSD = 0.34
2587351 2601302 2636236 RSD = 0.97
product was determined by using the calibration graph. The concentration of ethyl acetate were found to be 0.46, 38 and 0.14 ppm whereas methanol and toluene were
not present in the formulation products dolopen (500 mg capsule), enoclox (500 mg capsule) and floxason (500 mg capsule), respectively.
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Table 7. Solution stability of the proposed method
Standard after 24 h injection -1 injection -2 injection-3 Mean % RSD
Peak area for methanol 2911973 2939494 2837332 2896266 1.83
Conclusions After review of results it is concluded that the analytical method used for the analysis of residual solvents flucloxacillin is valid. This method is highly sensitive and accurate for the determination of methanol, ethyl acetate and toluene for FCS. The present method is not time consuming whereas there is no official method for its determination. The present method has wider linear dynamic range with good accuracy and precision. The relative standard deviation value was obtained, which is accurate and within the limit. Thus the present method is satisfactorily a better method for the determination of residual solvents for flucloxacillin sodium in bulk and pharmaceutical formulations.
Conflict of interest Authors declare that they have no competing interests. REFERENCES Zhou Q, Ruan Z, Yuan H, Jiang B, Xu D (2007). RP – HPLC analysis of flucloxacillin in human plasma: validation and application to a bioequivalence study. Pharmazie 62:101– 4.
Peak area for ethyl acetate 4970259 5021198 4912392 4967950 1.10
Peak area for toluene 2593181 2598880 2514520 2568860 1.84
Pullen J, Stolk LM, Neef C, Zimmermann LJ (2007). Microanalysis of amoxicillin, flucloxacillin, and rifampicin in neonatal plasma. Biomed Chromatogr. 21, 1259–65. Dhiraj SN, Chandrakant GB, Surana SJ, Venkateshwarlu G, Dekate PG (2009). Development and Validation of RP – HPLC Method for Simultaneous Estimation of Amoxicillin trihydrate and Flucloxacillin sodium in capsule dosage form. Int. J. Pharm. Tech. Res. 1:935–939. Dey S, Ratnakar C, Vaithiyanathan S, Samal HB, Vikramreddy Y, Balakrishna Y, Reddy A, Navinkumar G, Mohapatra, S (2010). Spectrophotometric method developed for the estimation of Flucloxacillin in bulk and dosage form using UV-VIS spectrophotometric method. Int. J. Pharma Bio. Sci. 1:35–43. Reddy CMB, Reddy GV (2012). Spectrophotometric estimation of flucloaxacillin in pure drug and pharmaceutical dosage formulation. IOSR J. Pharm. Bio. Sci. 2:46–48. Singh RS, Haque SM, Shanker PA (2009). Sensitive validated Spectrophotometric Method for the Determination of Flucloxacillin Sodium. E – J. Chem. 6:S397–S405. Prakash V, Konari SN, Prakash GS (2012). Development and validation of spectrophotometric method for the estimation of Flucloxacilin. Int. J. Pharm. Sci. Res. 3:1806–1808. Fiorentino FAM, Salgado HRN (2012). Development and validation of a stability indicative agar diffusion assay to determine the potency flucloxacillin sodium in capsules. Int. J. Microbiol. Res. 4:217–222. Flavia AMF, Herida RNS (2012). Development and Validation of a UVSpectrophotometric Method for Determination of Flucloxacillin Sodium in Capsules. Curr. Pharm. Anal. 8:101–106.
