A rapid,sensitive, and greener stability-indicating normal-phase HPTLC method with univariate calibration for the estimation of chlorhexidine acetate in its commercial formulations

Till date, there is no report on high-performance thin-layer chromatography (HPTLC) analysis of chlorhexidine (CHD) or its salts like CHD acetate (CHD-A), CHD gluconate, and CHD hydrochloride. Therefore, a rapid, sensitive, and greener normal-phase HPTLC method has been reported for the analysis of CHD-A in its four different commercial dosage forms. The quaternary combination of ethyl acetate: ethanol: water: formic acid (75:10:10:5, v v v v^?1) was optimized as the green solvent system/mobile phase for CHD-A analysis. The CHD-A detection was carried out at 264 nm. The greener normal-phase HPTLC assay was linear in the range of 10–2000 ng band^?1. In addition, the proposed method was accurate, precise, sensitive, and selective for CHD-A analysis. The greener normal-phase HPTLC method was able to detect CHD-A in the presence of its degradation products, suggesting the stability-indicating property of this method. The content of CHD-A in commercial products A, B, C, and D was detected as 0.18, 2.02, 1.46, and 0.19% w w^?1, respectively. The greenness scale for the greener normal-phase HPTLC assay was estimated as 0.88 utilizing “analytical GREEnness (AGREE)” calculator, suggested the greener nature of the normal-phase HPTLC assay. Overall, these data suggested that the greener normal-phase HPTLC method can be successfully used for the determination of CHD-A in its commercial products.     

An HPTLC method for the determination and the purity control of ciprofloxacin HCl in coated tablets

A high-performance thin-layer chromatographic method (HPTLC) has been developed for the determination and the purity control of a synthetic fluoroquinolone antibiotic ciprofloxacin hydrochloride in coated tablets when desfluoro compound, ethylene diamine compound, by-compound A and fluoroquinolonic acid are considered as impurities. Silica gel F₂₅₄ was used as a stationary phase and a mixture of acetonitrile, ammonia 25%, methanol and methylene chloride (1:2:4:4, v/v/v/v) as a mobile phase. The method was validated in terms of linearity (range), selectivity (placebo and related compounds), precision, accuracy (Recovery), system suitability (repeatability, peak symmetry, resolution) and impurities limit of detection (LOD). The analysis of variance (ANOVA) and t-test were applied to correlate the results of ciprofloxacin hydrochloride determination in coated tablets by means of the HPTLC method and the official British Pharmacopoeia (BP 1999) high-performance liquid chromatographic (HPLC) method.     

Development and validation of a high-performance thin-layer chromatographic method for the quantitative analysis of vitexin in Passiflora foetida herbal formulations

The aim of this study is the development of validated HPTLC method for the quantification of vitexin from Passiflora foetida commercial herbal formulations. The developed method was validated, in accordance with ICH guidelines for precision, accuracy, specificity and robustness. The plate was developed using ethyl acetate:methanol:water:formic acid 30:4:2:1(%, v/v/v/v) on 20 × 10 cm glass coated silica gel 60 F₂₅₄ plates and the developed plate was scanned and quantified densitometrically at λ = 340 nm. Linear regression analysis revealed a good linear relationship between peak area and amount of vitexin in the range of 100–700 ng/spot. The amount of vitexin in nine commercial herbal formulations was successfully quantified by the developed HPTLC method. The developed and validated high performance thin layer chromatographic method offers a new sensitive and reliable tool for quantification of vitexinin in various herbal formulations containing Passiflora foetida.     

Central composite design expert-supported development and validation of HPTLC method: Relevance in quantitative evaluation of protopine in Fumaria indica

The current study was executed for method development, validation and to estimate the concentration of protopine in methanolic extract of Fumaria indica by high-performance thin-layer chromatography (HPTLC). Isolation of bioactive compounds was carried out using the mobile phase, toluene:ethyl acetate:diethyl amine (8:2.5:0.5 v/v/v), and detected at wavelength 290 nm. This method was validated for precision, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), etc. The calibration range was found to be 500–5000 ng/spot for the bioactive compound. Protopine was separated with an R_f value of 0.22 ± 0.03. The method was validated for linearity (r2 ≥ 0.996 ± 0.082), accuracy 98.75–102.12%), and RSD of precision (0.49–2.07) with a calibration curve range of 500.00–5000.00 ng/spot. The LOD and LOQ were found to be 83.92 ng/spot and 254.30 ng/spot., respectively. The Central Composite design expert was applied for the validation of robustness. Three independent variables such as the volume of toluene in solvent system, chamber saturation time and wavelength were investigated. The results indicated that a slight change in these variables had no significant effect on the peak response. This developed HPTLC method is simple, precise, robust, specific, rapid, and cost effective. It could be used for quality control study and quantification of protopine in the plant extract and different herbal formulations containing the plant species.     

HPTLC method for simultaneous determination of ascorbic acid and gallic acid biomarker from freeze dry pomegranate juice and herbal formulation

A new rapid, simple, sensitive and high performance thin layer chromatography (HPTLC) has been established for the simultaneous determination of ascorbic acid and gallic acid in the freeze-dried pomegranate fruit juice and herbal formulation. HPTLC method was carried out using ethyl acetate: acetone: water: formic acid, 10:6:2:2 (%, v/v/v/v)) on 20 × 10 cm glass coated silica gel 60 F₂₅₄ plates and scanned at 254 nm for ascorbic acid and gallic acid. Ascorbic acid and gallic acid in the freeze-dried pomegranate fruit juice were identified by comparing their single spot at R_f = 0.54 ± 0.02 and R_f = 0.83 ± 0.01 respectively. The value of regression equation (r² ≥ 0.9992) revealed a good linear relationship between peak area and amount of ascorbic acid and gallic acid in the range of 100–800 ng/band. The method was validated for precision, accuracy, robustness LOD and LOQ. The method proposed can be useful for routine determination of ascorbic acid and gallic acid in various crude as well as herbal formulations as a quality control tool.     

Validated high-performance thin-layer chromatographic (HPTLC) method for simultaneous determination of nadifloxacin,mometasone furoate,and miconazole nitrate cream using fractional factorial design

A high-performance thin-layer chromatographic method for simultaneous determination of nadifloxacin, mometasone furoate, and miconazole nitrate was developed and validated as per International Conference on Harmonization guidelines. High-performance thin-layer chromatographic separation was performed on aluminum plates precoated with silica gel 60F₂₅₄ and methanol:ethyl acetate:toluene: acetonitrile:3M ammonium formate in water (1:2.5:6.0:0.3:0.2, % v/v) as optimized mobile phase at detection wavelength of 224 nm. The retardation factor (R_f) values for nadifloxacin, mometasone furoate, and miconazole nitrate were 0.23, 0.70, and 0.59, respectively. Percent recoveries in terms of accuracy for the marketed formulation were found to be 98.35–99.76%, 99.36–99.65%, and 99.16–100.25% for nadifloxacin, mometasone furoate, and miconazole nitrate, respectively. The pooled percent relative standard deviation for repeatability and intermediate precision studies was found to be < 2% for three target analytes. The effect of four independent variables, methanol content in total mobile phase, wavelength, chamber saturation time, and solvent front, was evaluated by fractional factorial design for robustness testing. Amongst all four factors, volume of methanol in mobile phase appeared to have a possibly significant effect on retention factor of miconazole nitrate compared with the other two drugs nadifloxacin and mometasone furoate, and therefore it was important to be carefully controlled. In summary, a novel, simple, accurate, reproducible, and robust high-performance thin-layer chromatographic method was developed, which would be of use in quality control of these cream formulations.     

Quantitative analysis of rutin,quercetin, naringenin,and gallic acid by validated RP- and NP-HPTLC methods for quality control of anti-HBV active extract of Guiera senegalensis

Context: Guiera senegalensis J.F. Gmel (Combretaceae) is a folk medicinal plant used in various metabolic and infectious diseases. In addition to its antiviral activities against herpes and fowlpox, the anti-HBV efficacy is very recently reported.

Objective: To develop and validate simple, sensitive RP-/NP-HPTLC methods for quantitative determination of biomarkers rutin, quercetin, naringenin, and gallic acid in the anti-HBV active G. senegalensis leaves ethanol-extract.

Materials and methods: RP-HPTLC (rutin &; quercetin; phase- acetonitrile:water, 4:6) and NP-HPTLC (naringenin &; gallic acid; phase- toluene:ethyl acetate:formic acid, 6:4:0.8) were performed on glass-backed silica gel plates 60F₂₅₄-RP18 and 60F₂₅₄, respectively. The methods were validated according to the ICH guidelines.

Results: Well-separated and compact spots (R_f) of rutin (0.52?±?0.006), quercetin (0.23?±?0.005), naringenin (0.56?±?0.009) and gallic acid (0.28?±?0.006) were detected. The regression equations (Y) were 12.434x?+?443.49, 10.08x?+?216.85, 11.253x?+?973.52 and 11.082x?+?446.41 whereas the coefficient correlations (r²) were 0.997?±?0.0004, 0.9982?±?0.0001, 0.9974?±?0.0004 and 0.9981?±?0.0001, respectively. The linearity ranges (ng/spot) were 200–1400 (RP-HPTLC) and 100–1200 (NP-HPTLC). The LOD/LOQ (ng/band) were 33.03/100.1 (rutin), 9.67/29.31 (quercetin), 35.574/107.8 (naringenin), and 12.32/37.35 (gallic acid). Gallic acid (7.01?μg/mg) was the most abundant biomarker compared to rutin (2.42?μg/mg), quercetin (1.53?μg/mg) and naringenin (0.14?μg/mg) in the extract.

Conclusion: The validated NP-/RP-HPTLC methods were simple, accurate, and sensitive for separating and quantifying antiviral biomarkers in G. senegalensis, and endorsed its anti-HBV activity. The developed methods could be further employed in the standardization and quality-control of herbal formulations.     

Box-Behnken supported validation of stability-indicating high performance thin-layer chromatography (HPTLC) method: An application in degradation kinetic profiling of ropinirole

Stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of ropinirole HCl was developed and validated as per the ICH guidelines. The method employed the mobile phase and toluene-ethyl acetate-6 M ammonia solution (5:6:0.5, v/v/v) was optimized with the help of a design expert. Densitometric analysis of ropinirole HCl was carried out in the absorbance mode at 250 and 254 nm. Compact spots for ropinirole HCl were found at R_f value of 0.58 ± 0.02. The linear regression analysis data for the calibration plots showed R² = 0.9989 ± 0.0053 with a concentration range of 100–3000 ng spot⁻¹. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were 12.95 and 39.25 ng spot⁻¹ respectively. Drug was subjected to acidic, alkaline, oxidative, dry heat, wet heat and photo degradation stress. All the peaks of degradation products were well resolved from the standard drug peak with significant difference of R_f. The acidic and alkaline stress degradation kinetics of ropinirole, were found to be in first order, showing high stability (t_1/2, 146.37 h⁻¹; t_0.9, 39.11 h⁻¹) in the acidic medium and low stability (t_1/2, 97.67 h⁻¹; t_0.9, 14.87 h⁻¹) in the alkaline environment.     

Isolation and quantitative analysis of B<Subscript>1</Subscript>, B<Subscript>2</Subscript>, B<Subscript>6</Subscript> AND B<Subscript>12</Subscript> vitamins using high-performance thin-layer chromatography

A new, simple and accurate high-performance thin-layer chromatography (HPTLC) method has been developed for the separation and simultaneous analysis of B1, B2, B6 and B12 vitamins. Standard and sample solutions of vitamins were applied onto precoated aluminum-backed silica gel G 60 F254 HPTLC plate. The plates were developed with about thirty different solvent systems, and the ethanol – chloroform – acetonitrile – toluene – ammonia – water (7 : 4 : 4.5 : 0.5 : 1 : 1, v/v) mixture was chosen as the optimum mobile phase. UV detection was performed densitometrically at 254 nm. The retention factors of B1, B2, B6 and B12 vitamins were 0.36, 0.60, 0.85 and 0.46, respectively. The linearity range for indicated compounds was 10 – 2500 ng per spot and the correlation coefficients were R ² = 0.97 – 0.98. The limits of quantification (LOQ) were 141.72, 42.41, 100.31 and 11.50 ng and the limits of detection (LOD) were 42.52, 12.72, 30.09 and 3.45 ng for B1, B2, B6 and B12 vitamins, respectively. Analyses of standard solutions showed good precision and repeatability.     

Development and comprehensive greenness assessment for MEKC and HPTLC methods for simultaneous estimation of sertaconazole with two co-formulated preservatives in pharmaceutical dosage forms

Four greenness assessment tools: National Environmental Methods Index (NEMI), Ecoscale Assessment (ESA), Green Analytical Procedure Index (GAPI) and the most recent Analytical GREEness metric (AGREE) were tested to evaluate two proposed MEKC and HPTLC methods for the simultaneous determination of the antifungal drug sertaconazole (SER) and two co-formulated preservatives methyl paraben (MEP) and sorbic acid (SOR). Method I was micellar electrokinetic capillary chromatography (MEKC). Electrophoretic conditions were optimized to improve separation, sensitivity, and rapidity. The proposed method used a fused silica capillary (50 cm × 50 μm id), and the background electrolyte was 50 mM acetate buffer pH 4.6 containing 15 mM SDS with 17 s injection time. The applied voltage was 30 kV. The diode array detector (DAD) was set at 210 nm for measurement of SER and 254 nm for both MEP and SOR. The Three compounds were resolved in less than 6 min with migration times 3.07, 4.29, and 5.97 min for SER, MEP, and SOR respectively. The described method was linear over the range of 5–100 μg/mL for SER, MEP and SOR with correlation coefficients >0.9995. For method II: high-performance thin-layer chromatography (HPTLC) analysis was carried out on aluminum-backed sheet of silica gel using chloroform-ethyl acetate (3:7) as mobile phase. Retardation factors were 0.20, 0.71, and 0.88 for SER, MEP, and SOR respectively. Quantification was achieved with UV densitometry at 210 nm for SER and 254 nm for MEP and SOR. The linearity ranges were 0.1–1 μg/spot for the three drugs with correlation coefficients >0.9998. The analytical performance of both methods was thoroughly validated according to International Conference on Harmonization (ICH) guidelines with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The proposed MEKC and HPTLC methods were successfully applied for estimation of the studied drugs in laboratory prepared mixtures and in the cream and spray dosage forms.     

Development of validated high-performance thin layer chromatography for quantification of aristolochic acid in different species of the Aristolochiaceae family

This study was undertaken to isolate and quantify aristolochic acid in Aristolochia indica stem and Apama siliquosa root. Aristolochic acid is an important biomarker component present in the Aristolochiaceae family. The isolation method involved simple solvent extraction, precipitation and further purification, using recrystallization. The structure of the compound was confirmed using infrared spectroscopy, mass spectrometry and nuclear magnetic resonance. A specific and rapid high-performance thin layer chromatography (HPTLC) method was developed for analysis of aristolochic acid. The method involved separation on the silica gel 60 F₂₅₄ plates using the single solvent system of n-hexane: chloroform: methanol. The method showed good linear relationship in the range 0.4–2.0 μg/spot with r² = 0.998. The limit of detection and limit of quantification were 62.841 ng/spot and 209.47 ng/spot, respectively. The proposed validated HPTLC method was found to be an easy to use, accurate and convenient method that could be successfully used for standardization and quality assessment of herbal material as well as formulations containing different species of the Aristolochiaceae family.     

Effective separation and analysis of E- and Z-guggulsterones in Commiphora mukul resin, guggulipid and their pharmaceutical product by high performance thin-layer chromatography-densitometric method

A high performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of the hypolipidemic agents, E- and Z-isomers of guggulsterone in Commiphora mukul resin, guggulipid (ethyl acetate extract of resin), and its pharmaceutical formulation, was developed. The developed system was efficient enough to separate both isomers from their conger, 17,20-dihydroguggulsterone (3). HPTLC glass plates, pre-coated with silica gel 60F-254, were used as a stationary phase. The mobile phase consisted of toluene:acetone (9.3:0.7, v/v) which gave well resolved spots for E- and Z-guggulsterones (R_f: 0.52 ± 0.01, and 0.67 ± 0.01, respectively) following double development of chromatoplate with the same mobile phase under unsaturated conditions. The analyte stability towards the developed chromatographic procedure was also investigated by two-dimensional (2D) HPTLC analysis. 17,20-Dihydroguggulsterone (3) was identified by the electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) analysis.     

Development of HPTLC method for the estimation of ondansetron hydrochloride in bulk drug and sublingual tablets

A new, simple, rapid, accurate and precise high performance thin layer chromatography (HPTLC) method has been developed for the estimation of ondansetron hydrochloride in bulk and sublingual tablets. The mobile phase composition was chloroform : ethyl acetate : methanol : ammonia (9:5:4:0.1 v/v). Spectrodensitometric analysis of ondansetron was carried out at 254 nm and a symmetrical, well‐resolved, well‐defined peak was obtained at mean retardation factor (R_f) 0.52 ± 0.02. The calibration plot was linear in the range 200‐1200 ng/spot and showed good linear relationship with coefficient of regression, R² = 0.9952 with respect to peak area. The method was validated according to the guidelines of the International Conference on Harmonization (ICH Q2(R1). The limit of detection and quantitation were 14.83 and 44.92 ng per spot, respectively. The recovery study was carried out by standard addition method and the percentage recovery was found to be 99.34 ± 1.08. Therefore it was concluded that the proposed developed HPTLC method can be applied for identification and quantitative determination of ondansetron in bulk drug and dosage forms. Copyright © 2011 John Wiley & Sons, Ltd.     

Uzara – A quality control perspective of Xysmalobium undulatum

Context: Xysmalobium undulatum (L.) Aiton f var. (Asclepiadaceae), commonly known as uzara, is an ethnomedicinally important plant from southern Africa used to treat a variety of ailments. In addition to local use in African Traditional Medicine (ATM), formulations containing uzara have been successfully marketed by a number of pharmaceutical companies. Despite its commercialization, published adequate quality control (QC) protocols are lacking.

Objective: The study was conducted to develop QC protocols for uzara based on chromatographic and spectroscopic techniques.

Materials and methods: High performance thin layer chromatography (HPTLC) and liquid chromatography coupled to mass spectrometry (LC-MS) were used to develop phytochemical fingerprints of ethanolic root extracts of 47 uzara samples collected from eight distinct localities in South Africa. Mid-infrared (MIR) spectroscopy was also explored as a suitable alternative technique for rapid and economic quantification of uzarin.

Results: Adequate chromatographic profiles were obtained using both HPTLC and LC-MS analyses. The chromatographic patterns showed qualitative similarities among plants collected from different locations. The levels of uzarin, the major constituent of uzara, were highly variable between locations, ranging from 17.8 to 139.9?mg/g (dry weight). A good coefficient of determination (R^2?=?0.939) and low root mean square error of prediction (RMSEP?=?7.9?mg/g) confirmed the accuracy of using MIR-PLS calibration models for the quantification of uzarin.

Discussion and conclusion: Both HPTLC and LC-MS can be used as tools in developing quality control procedures for uzara. MIR in combination with chemometrics provides a fast alternative method for the quantification of uzarin.     

High-Performance Liquid Chromatographic and High-Performance Thin-Layer Chromatographic Method for the Quantitative Estimation of Dolutegravir Sodium in Bulk Drug and Pharmaceutical Dosage Form

Simple, sensitive, precise, and specific high-performance liquid chromategraphic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for the determination of dolutegravir sodium in bulk drug and pharmaceutical dosage form were developed and validated. In the HPLC method, analysis of the drug was carried out on the ODS C₁₈ column (150 × 4.6 mm, 5 μm particle size) using a mixture of acetonitrile: water (pH 7.5) in the ratio of 80:20 v/v as the mobile phase at the flow rate 1 mL/min at 260 nm. This method was found to be linear in the concentration range of 5–35 μg/mL. The peak for dolutegravir sodium was observed at 3.0 ± 0.1 minutes. In the HPTLC method, analysis was performed on aluminum-backed plates pre-coated with silica gel G60 F₂₅₄ using methanol: chloroform: formic acid in the proportion of 8:2:0.5 v/v/v as the mobile phase. This solvent system was found to give compact spots for dolutegravir sodium with the Rf value 0.77 ± 0.01. Densitometric analysis of dolutegravir sodium was carried out in the absorbance mode at 265 nm. Linear regression analysis showed good linearity with respect to peak area in the concentration range of 200–900 ng/spot. The methods were validated for precision, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and specificity. Statistical analysis showed that both of the methods are repeatable and specific for the estimation of the said drug. The methods can be used for routine quality control analysis of dolutegravir sodium.     

Matrix Solid-Phase Dispersion Extraction and Quantification of Alpinetin in Amomum Seed using Validated HPLC and HPTLC Methods

Alpinetin is a flavonoidal constituent of seeds of Amomum subulatum Roxb., recently reported to possess vasorelaxant and antiHIV activities. Simple, accurate and precise HPLC and HPTLC methods were developed for the analysis of alpinetin in A. subulatum seed extracts and extraction technique was optimized to get maximum yield using conventional, ultrasonic and matrix solid phase dispersion extraction. HPLC was performed on a C18 column with methanol and water (70:30, v/v) as mobile phase at a flow rate of 1.0 ml/min whereas HPTLC on silica aluminum sheet (60F₂₅₄) using toluene, dichloromethane and ethyl acetate as solvent system. A sharp peak was obtained for alpinetin at a retention time (R_t) of 5.7 min by HPLC and retardation factor (R_f) of 0.48 by HPTLC. Both methods were validated as per the ICH guidelines and the content of alpinetin was estimated in different extracts. Matrix solid phase dispersion technique was found most suitable for extracting alpinetin as compared to other techniques. Validation data are indicative of good precision and accuracy and proved the reliability of the methods.     

巴戟天不同炮制品的高效薄层色谱指纹图谱研究

目的以巴戟天药材生品为基础,建立巴戟天不同炮制品的高效薄层色谱(HPTLC)指纹图谱,并比较分析生品的指纹图谱。方法硅胶GF254高效预制薄层板,乙酸乙酯部位以石油醚-乙酸乙酯(5∶5.5)为展开剂,上行展开8 cm,10%硫酸乙醇溶液显色;正丁醇部位以正丁醇-冰醋酸-水(4∶1∶5)上层溶液为展开剂,上行展开8 cm,α-萘酚浓硫酸试剂显色。显色后置紫外光灯365 nm或自然光下检视,得薄层色谱指纹图谱,并进行相关分析。结果巴戟天生品的薄层荧光色谱指纹图谱与巴戟肉(蒸制)、盐巴戟天、制巴戟天(甘草制)图谱比较分析,彼此具有明显的区别,可用于生品与巴戟天炮制品的鉴别。结论通过HPTLC指纹图谱考察,生品巴戟天与巴戟肉、盐巴戟天、制巴戟天的区别较大,应分别制定质量评价的标准。     

Validated HPTLC Method for Simultaneous Determination of Quinapril Hydrochloride and Hydrochlorothiazide in a Tablet Dosage Form

Quinapril hydrochloride and hydrochlorothiazide were simultaneously determined by HPTLC in pharmaceutical formulations. The drugs were separated on silica gel 60 F₂₅₄ plates using suitable combination of solvents as mobile phase. The validation parameters, tested in accordance with the requirements of ICH guidelines, prove the suitability of methods.     

Concurrent analysis of bioactive triterpenes oleanolic acid and β-amyrin in antioxidant active fractions of Hibiscus calyphyllus,Hibiscus deflersii and Hibiscus micranthus grown in Saudi Arabia by applying validated HPTLC method

In this study, we developed a validated HPTLC method for concurrent analysis of two natural antioxidant triterpenes, oleanolic acid (OA) and β-amyrin (BA) in the biologically active fractions (petroleum ether, toluene, chloroform, ethyl acetate and n-butanol) of aerial parts of three Hibiscus species (H. calyphyllus, H. deflersii and H. micranthus). The chromatography was conducted on normal HPTLC (ready to use glass-plate coated with silica gel 60 F254) plate with chloroform and methanol (97:3, V/V) used as mobile phase. The derivatization of the developed plate was done with p-anisaldehyde and scanned at λ_max?=?575?nm. Well resolved and intense peaks of OA and BA were obtained at Rf?=?0.36 and 0.57, respectively. The linear regression equation/correlation coefficient (r²) for OA and BA were Y?=?6.65x?+?553.35/0.994 and Y?=?9.177x?+?637.23/0.998, respectively in the linearity range of 100–1200?ng/spot indicated good linear relationship. The low values of %RSD for intra-day/inter-day precision of OA (1.45–1.61/1.38–1.59) and BA (1.52–1.57/1.50–1.53) suggested that the method was precise. The recovery/RSD (%) values for OA and BA were found to be 99.21–99.62/1.39–1.95 and 98.75–99.70/1.56–1.80, respectively assures the reasonably good accuracy of the proposed method. Fifteen samples were analyzed to check the content of OA and BA by using the developed HPTLC methods. The content of OA in different samples were found to be 3.87 (HmP)?>?1.212 (HcP)?>?0.673 (HdC)?>?0.493 (HdP)?>?0.168 (HdE)?>?0.059 (HcC)?>?0.015 (HcE)?>?0.008 (HmT) µg/mg of the dried weight of extract. However the content of BA was found as: 2.293 (HmP)?>?1.852 (HdT)?>?0.345 (HdC)?>?0.172 (HmT)?>?0.041 (HdE)?>?0.008 (HcC) µg/mg of the dried weight of extract. Some Hibiscus species fractions exhibited good antioxidant potential like: HcE (IC50?=?17.6?±?1.8)?>?HdB (IC50?=?32.16?±?0.9)?>?HmP (IC50?=?80.4?±?4.5)?>?HmT (IC50?=?99.7?±?8.2) when compared with ascorbic acid (IC50?=?14.2?±?0.5), while other fractions exhibited only mild antioxidant capability. The developed HPTLC method can be further exploited for analysis of these markers in the quality assessment of raw material as well as herbal formulations available in the market.     

High performance thin-layer chromatographic method for the determination of sparfloxacin in human plasma and its use in pharmacokinetic studies

A rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method has been developed for the measurement of sparfloxacin in human plasma and its use for pharmacokinetic study has been evaluated. Detection and quantitation were performed without using an internal standard. A single stage extraction procedure was followed for extracting sparfloxacin from plasma and a known amount of the extract was spotted on precoated silica gel 60 F254 plates using a Camag Linomat IV autosampler. Sparfloxacin was quantified using a Camag TLC Scanner 3. The recovery study of authentic analytes added to plasma at 0.1 to 0.8 μg ml⁻¹ was 94.9±0.98% and the lowest amount of sparfloxacin that could be detected was 50 ng ml⁻¹ plasma. The method provides a direct estimate of the amount of sparfloxacin present in plasma. The method was used for the determination of plasma levels as well as pharmacokinetic parameters of sparfloxacin after oral administration of two marketed preparations to healthy volunteers.