Kalpana G. Patel

Central Composite Design for Validation of HPTLC Method for Simultaneous Estimation of Olmesartan Medoxomil, Amlodipine Besylate and Hydrochlorothiazide in Tablets.

High performance thin layer chromatographic method for simultaneous estimation of olmesartan medoxomil, amlodipine besylate and hydrochlorothiazide was developed and validated as per ICH guidelines. Moreover, robustness testing was performed applying a central composite design with k factor having 2k factorial runs, 2k axial experiments and two center points. High performance thin layer chromatographic separation was performed on aluminium plates precoated with silica gel 60F254 and toluene:chloroform:methanol:acetonitrile:formic acid (2:7:1.8:0.8:0.2% v/v) as optimized mobile phase. The detection wavelength for simultaneous estimation of three drugs was 232nm. The Rf values for olmesartan medoxomil, amlodipine besylate and hydrochlorthiazide were 0.78, 0.20 and 0.45, respectively. Percent recoveries in terms of accuracy for the marketed formulation was found to be 101.3-104.4, 100.7-104 and 101.5-103.9 for, olmesartan medoxomil, amlodipine besylate and hydrochlorthiazide, respectively. The pooled %relative standard deviation values for repeatability studies and intermediate precision studies was found to be less than 2% for olmesartan medoxomil, amlodipine besylate and hydrochlorthiazide, respectively. All the three factors evaluated in the robustness testing by central composite design were found to have an insignificant effect on the retention factor. However, methanol content in total mobile phase as a factor appeared to have significant effect on robustness, compared to band size and developing distance and hence it is important to be carefully controlled. In summary, a novel, simple, accurate and reproducible high performance thin layer chromatographic method was developed, which would be of use in quality control of these tablets.

Development and validation of an HPTLC method for the simultaneous estimation of Clonazepam and Paroxetine hydrochloride using a DOE approach

Abstract The present study examines simultaneous multiple response optimization using Derringers desirability function for the development of an HPTLC method to detect Clonazepam and Paroxetine hydrochloride in pharmaceutical dosage form. Central composite design (CCD) was used to optimize the chromatographic conditions for HPTLC. The independent variables used for the optimization were the n-butanol content in the mobile phase, the chamber saturation time and the distance travelled. HPTLC separation was performed on aluminium plates pre-coated with silica gel 60 F254 as the stationary phase using n-butanol:glacial acetic acid:water (9:2:0.5% v/v/v) as the mobile phase. Quantification was achieved based on a densitometric analysis of Clonazepam and Paroxetine hydrochloride over the concentration range of 40–240 ng/band and 300–1800 ng/band, respectively, at 288 nm. The method yielded compact and well-resolved bands at R f of 0.77 ± 0.02 and 0.34 ± 0.02 for Clonazepam and Paroxetine hydrochloride, respectively. The linear regression analysis for the calibration plots produced r 2 = 0.9958 and r 2 = 0.9989 for Clonazepam and Paroxetine hydrochloride, respectively. The precision, accuracy, robustness, specificity, limit of detection and limit of quantitation of the method were validated according to the ICH guidelines. The factors evaluated in the robustness test were determined to have an insignificant effect on the selected responses. The results indicate that the method is suitable for the routine quality control testing of marketed tablet formulations.

Stability Indicating HPTLC Method for Analysis of Rifaximin in Pharmaceutical Formulations and an Application to Acidic Degradation Kinetic Study

A specific stability indicating high-performance thin-layer chromatographic method for analysis of rifaximin both as a bulk drug and in formulations was developed and validated. The method employed HPTLC aluminium plates precoated with silica gel 60 F254 as the stationary phase. The optimized mobile phase system consisted of n-hexane : 2-propanol : acetone : ammonia (5 : 4.1 : 1, v/v/v/v), which gave compact spots for rifaximin at of 0.59 ± 0.03. Rifaximin was subjected to forced degradation studies in order to check the specificity of the method. Densitometric analysis of rifaximin was carried out in the absorbance reflectance mode at 443 nm. The calibration plots showed linear relationship in the concentration range of 400–3200 ng per band. Moreover, linearity was also confirmed by verification of homoscedasticity of variance. According to validation studies, the developed method was repeatable and specific as revealed by % RSD less than 2 and hence can be used for routine analysis of pharmaceutical formulation. Moreover, the method could effectively separate the drug from its degradation products; hence it can be employed as a stability indicating one. The kinetics of acid degradation process at various temperatures was also investigated and first-order rate constant, half-life, shelf life, and activation energy were computed.

Stability-indicating high-performance thin-layer chromatographic method for the estimation of ambroxol hydrochloride and doxofylline in a pharmaceutical formulation using experimental design in robustness study

A sensitive, specific, and accurate stability-indicating high-performance thin-layer chromatographic method for the analysis of ambroxol hydrochloride and doxofylline, for both bulk drug and formulation, was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The densitometric analysis of both drugs was carried out in the absorbance mode at 276 nm using diethyl ether—n-butanol—ammonia (9:0.9:0.1, v/v) as the solvent system. This system was found to give compact spots for ambroxol hydrochloride at RF of 0.74 ± 0.01 and doxofylline at 0.41 ± 0.01. Moreover, both drugs were subjected to acid and alkali hydrolysis, oxidation, and photodegradation. Also, the degraded products were well resolved from the pure drug with different RF values. Linearity was found in the range of 20–100 and 100–500 ng band−1 for ambroxol hydrochloride and doxofylline. The limit of detection (LOD) and limit of quantitation (LOQ) for ambroxol hydrochloride and doxofylline were 1.17 and 3.57 ng band−1 and 30.68 and 92.97 ng band−1, respectively. “Bartlett’s test” and “Lack of fit” applied on peak area for linearity additionally proved validity of the linearity of the developed method. Good accuracy and precision were obtained as revealed from percent relative standard deviation (% RSD) less than 2. Moreover, robustness testing was performed applying fractional factorial design, 24-1. All the four factors: volume of diethyl ether, volume of n-butanol, solvent front, and chamber saturation time, evaluated in the robustness testing, were found to have an insignificant effect on the retention factor. Similarly, no interference was observed from common excipients in tablet formulation as well as degradation product, indicating specificity of the method. As the method could effectively separate both drugs from their degradation products, it can be used as a stability-indicating method.

Cleaning Validation: Quantitative Estimation of Atorvastatin in Production Area

Carefully designed cleaning validation and its evaluation can ensure that residues of active pharmaceutical ingredient will not carry over and cross-contaminate the subsequent product. UV spectrophotometric and total organic carbon–solid sample module (TOC-SSM) method was developed and validated for the verification and determination of atorvastatin residues in the production area and to confirm the efficiency of the cleaning procedure as per ICH guideline. Atorvastatin was selected on the basis of a worst-case rating approach. It exhibited good linearity in the range of 5 to 25 μg/mL for UV spectrophotometric and 7300 to 83800 μg for the TOC-SSM method. The limit of detection was 0.419 μg/mL and 4.19 μg in the UV spectrophotometric and TOC-SSM methods, respectively. The limit of quantitation was 1.267 μg/mL and 12.69 μg in UV spectrophotometric and TOC-SSM methods, respectively. Percentage recovery from spiked stainless steel plates was found to be 95.37% and 92.82% in UV spectrophotometric and TOC-SSM methods, respectively. The calculated limit of acceptance per swab for atorvastatin (35.65 μg/swab) was not exceeded during three consecutive batches of production after cleaning procedure. Both proposed methods are suitable for quantitative determination of atorvastatin on manufacturing equipment surfaces well below the limit of contamination. The ease of sample preparation permits fast and efficient application of the proposed methods in quantitation of atorvastatin residue with precision and accuracy. Above all, the methodology is of low cost, and is a simple and less time-consuming alternative to confirm the efficiency of the cleaning procedure in pharmaceutical industries. LAY ABSTRACT: Carefully designed cleaning validation and its evaluation can ensure that residues of active pharmaceutical ingredient will not carry over and cross-contaminate the subsequent product. Atorvastatin was identified as a potential candidate among existing drug substances in production areas based on a worst-case rating approach. Atorvastatin residues were detected and quantified below acceptance limits after cleaning of production equipment using two proposed methods, namely, the UV spectrophotometric and total organic carbon–solid sample module (TOC-SSM) methods. The ease of sample preparation permits fast and efficient application of the proposed methods in quantitation of atorvastatin residue in production equipment area to confirm the efficiency of the cleaning procedure in pharmaceutical industries. Above all, the methodology is of low cost, and is a simple and less time-consuming alternative for cleaning validation.

Validated HPTLC method for quantification of myricetin in the stem bark of Myrica esculenta Buch. Ham. Ex D. Don, myricaceae

A simple, rapid, and precise high-performance thin-layer chromatographic method has been established for quantitative analysis of myricetin in the stem bark of Myrica esculenta, family Myricaceae. The marker myricetin was separated from other components of stem bark extract on silica gel 60 F254 HPTLC plates with toluene-ethyl acetate-formic acid-methanol 3:3:0.6:0.4 (v/v) as mobile phase. Densitometry in absorbance-reflection mode at 268 nm was used for quantitative analysis of myricetin. The stem bark of M. esculenta was found to contain 0.225% (w/w) myricetin. The method was validated for linearity, accuracy, precision, and specificity in accordance with ICH guidelines. The calibration plot was linear between 0.4 and 2.0 µg per band for myricetin. The limits of detection and quantitation were 0.0939 and 0.2845 µg per band, respectively. The method can be used as a quality control-method for fingerprint profiling and quantitative analysis of the stem bark of Myrica esculenta.

Box–Behnken design-assisted optimization for simultaneous estimation of quercetin, kaempferol, and keto-β-boswellic acid by high-performance thin-layer chromatography method

A simple, specific, and quantitative high-performance thin-layer chromatography (HPTLC) method has been developed for the simultaneous determination of quercetin, kaempferol, and keto-β-boswellic acid in the herbal extracts of Spinacia oleracea and Boswellia serrata. Chromatographic development was performed using n-hexane—ethyl acetate—glacial acetic acid (6.4:3.6:0.2, % v/v) as optimized mobile phase on TLC aluminum plate precoated with silica gel 60 F254 and optimized resolution between quercetin and kaempferol (R12) and between kaempferol and KBBA (R23) were 2.11 and 2, respectively. The detection of quercetin, kaempferol, and keto-β-boswellic acid was carried out at 272 nm. Box—Behnken design was applied for optimization of the chromatographic conditions and combination of factors such as the volume of n-hexane (A), solvent front (B), and chamber saturation time (C) which were likely to affect RF, and resolution of quercetin, kaempferol, and keto-β-boswellic acid were identified from preliminary trials and further optimized using response surface design. The drugs were satisfactorily resolved with RF values of 0.22 ± 0.02, 0.40 ± 0.02, and 0.54 ± 0.02 for quercetin, kaempferol, and keto-β-boswellic acid, respectively. The linear ranges were found to be 1500–3500, 200–600, and 1040–3120 ng band−1 for quercetin, kaempferol, and keto-β-boswellic acid. The accuracy and precision measured were less than 2% relative standard deviation for all markers. The sensitivity of the method in terms of limit of detection (LOD) and limit of quantitation (LOQ) were measured. Among three factors, the significant factor found was the volume of hexane that resulted in a change in the RF of all three markers. The proposed method was found to be accurate, precise, reproducible, robust, and specific and can be applicable for the simultaneous determination of quercetin, kaempferol, and keto-β-boswellic acid for the quality control testing of extracts and medicinal plants.

Optimizing derivatization conditions using an experimental design and simultaneous estimation of artemether and lumefantrine by ratio first order derivative spectrophotometric method

Abstract Artemether–lumefantrine is the most widely used artemisinin-based combination therapy for malaria. The present work aims to develop and validate a simple, accurate, precise and rapid ratio first order derivative spectrophotometric method for the simultaneous estimation of artemether and lumefantrine in a fixed dose combination tablet. The first step in development of the method was to derivatize artemether. As artemether does not show absorption in the UV region, it was derivatized using hydrochloric acid as the derivatizing agent. The derivatizing conditions were further optimized by full factorial multivariate approach, where the independent variables were volume of concentrated hydrochloric acid and time taken for artemether derivatization at room temperature. Furthermore, based on the statistical analysis, derivatizing conditions were optimized i.e. 1.3 ml of conc. HCl at room temperature for 30 min. At this condition, the artemether was found to absorb in the UV region satisfactorily, and the absorbance of lumefantrine was found to remain unaffected. The developed method showed good calibration data in the range of 5–30 μg/ml for artemether and 2–12 μg/ml for lumefantrine. The mean % recovery values were found to be 99.96–100.49% and 99.48–100.31% for artemether and lumefantrine, respectively. Additionally, the developed method was effectively applied in the estimation of artemether and lumefantrine in a commercial tablet (ARH-L DS tablets), suggesting that it can be practically applied for quality control of routinely examined drugs in combined dosage forms with the reduced expenditure of time.

Simultaneous Estimation of Stigmasterol and Withaferin A in Union Total Herbal Formulation Using Validated HPTLC Method.

Union Total is herbal formulation made in the form of capsule which contains two standardized plant extracts Cissus quadrangularis (CQ) and Withania somnifera (WS). The present work describes development and validation of High Performance Thin Layer Chromatographic method for simultaneous analysis of Stigmasterol (STG) in Cissus quadrangularis (CQ) and Withaferin A (WFA) in Withania somnifera (WS). Stigmasterol and Withaferin A were identified on silica G60 F254 HPTLC plates by post derivatization technique and robustness study was performed by applying a central composite design (CCD) with k factor having 2k factorial runs, 2k axial experiments and five center points. In HPTLC good separation was obtained with chloroform: methanol: toluene: formic acid (6.5: 0.5: 3: 0.25 v/v/v/v) as mobile phase and anisaldehyde sulphuric acid as a derivatizing reagent at detection wavelength 530 nm. Linearity was obtained in the concentration range of 100-200 ng/band for WFA and 200-700 ng/band for STG and the % recoveries were found in the range of 100.06 % to 100.46 % for WFA and 99.97 % to 100.94 % for STG respectively. HPTLC method was found to be sensitive, precise, accurate and reproducible, which would be of use in quality control of these tablets.

Validated HPTLC Method for Quantification of Luteolin and Apigenin in Premna mucronata Roxb., Verbenaceae.

A simple, rapid, and precise high-performance thin-layer chromatographic method was developed for quantitative estimation of luteolin and apigenin in Premna mucronata Roxb., family Verbenaceae. Separation was performed on silica gel 60 F254 HPTLC plates using toluene : ethyl acetate : formic acid (6 : 4 : 0.3) as mobile phase for elution of markers from extract. The determination was carried out in fluorescence mode using densitometric absorbance-reflection mode at 366 nm for both luteolin and apigenin. The methanolic extract of Premna mucronata was found to contain 10.2 mg/g % luteolin and 0.165 mg/g % of apigenin. The method was validated in terms of linearity, LOD and LOQ, accuracy, precision, and specificity. The calibration curve was found to be linear between 200 and 1000 ng/band for luteolin and 50 and 250 ng/band for apigenin. For luteolin and apigenin, the limit of detection was found to be 42.6 ng/band and 7.97 ng/band while the limit of quantitation was found to be 129.08 ng/band and 24.155 ng/band, respectively. This developed validated method is capable of quantifying and resolving luteolin and apigenin and can be applicable for routine analysis of extract and plant as a whole.