Quantification of β-Sitosterol from Mucuna pruriens by TLC

Mucuna pruriens Linn. one of the popular and important medicinal plants of India is a constituent of more than 200 indigenous drug formulations. β-Sitosterol is one of the most prevalent phytosterols which is ubiquitous throughout the plant kingdom. A sensitive, selective and precise thin-layer chromatographic method has been developed and validated for the analysis of β-sitosterol in Mucuna pruriens roots. Separation and quantification was achieved by TLC using ternary mobile phase of toluene: chloroform; methanol (4:4:1 v/v) (R _F 0.55) on precoated silica gel 60F₂₅₄ aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent in reflection/absorption mode at 527 nm. The calibration curve was linear in the concentration range of 100–600 ng spot⁻¹. The method was validated for precision, repeatability and accuracy. The proposed method was found to be simple, precise, specific, sensitive and accurate for the quantification of β-sitosterol.

     

Stability-Indicating HPTLC Method for Analysis of Moclobemide, and Use of the Method to Study Degradation Kinetics

A sensitive, selective, precise, and stability-indicating HPTLC method for analysis of moclobemide in the bulk drug and in formulations has been established and validated. Aluminium TLC plates precoated with silica gel 60 F₂₅₄ were used with benzene–methanol–40% ammonia 7:3:0.1 (v/v) as mobile phase. Densitometric analysis was performed in absorbance mode at 238 nm. Compact bands were obtained for moclobemide (R _F 0.67 ± 0.02; n = 6). The drug was subjected to acidic and alkaline hydrolysis, oxidation, dry heat treatment, and photodegradation. The drug undergoes degradation under acidic, basic, and oxidising conditions. The degradation products were well resolved from the pure drug with significantly different R _F values, so the method can be regarded as stability-indicating. Response to moclobemide was a linear function of amount in the range 50–600 ng per band, with a correlation coefficient, r ², of 0.9967 ± 0.51. LOD and LOQ, determined experimentally, were 10 and 30 ng per band, respectively. Statistical analysis proves the method is repeatable and specific for analysis of moclobemide. The method was used to investigate the kinetics of alkaline degradation. The Arrhenius plot was constructed and the activation energy calculated.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min^?1 for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.     

Rapid TLC Method for Estimation of Mevalonic Acid in the Leaves of Medicinal Plants

A simple, rapid, specific and sensitive thin layer chromatographic (TLC) method has been developed for the quantitative estimation of mevalonic acid (MVA) in leaves of medicinal plants; Artemisia annua, Psorelia corylifolia, Vinca rosea, Withania somnifera and Barleria proinites. The assay procedure involved conversion of MVA to its lactone, mevalonolactone (MVAL). Mevalonic acid was extracted from the leaf tissues of plants. Separation of MVAL was carried out on silica gel 60 F₂₅₄ TLC plates using benzene:acetone (3:2) as the mobile phase. The densitometric determination of MVAL was performed at 600 nm after derivatization with anisaldehyde reagent in absorption–reflectance mode. The method was validated over the linearity range of 100–500 ng spot^?1 and correlation coefficient for the calibration curve was >0.99. The average recovery of MVAL, used as internal standard, was higher than 98%. The lower limit of detection was found to be 50 ng spot^?1.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

     

TLC Based Method for Standardization of Pongamia pinnata (Karanj) Using Karanjin as Marker

Pongamia pinnata Linn. (Papilionaceae) seeds have gained great commercial and industrial importance owing to their high oil content. Presently, there is no appropriate TLC based method available for standardization of P. pinnata. A simple, robust and reproducible TLC method for the determination of Karanjin is reported in the seeds of P. pinnata. The method involves separation of components by TLC on pre-coated silica gel G 60 F₂₅₄ plates developed on toluene: ethyl acetate (7:3 v/v) and detection at 260 nm in absorbance mode. The sensitivity of the method was found to be 100 ng. The linearity range was 50–300 ng. Four samples of P. pinnata from different geographical locations were tested for their karanjin content using the developed method. The proposed method was found to be robust, precise, and accurate, it therefore holds potential for detection, monitoring and quantification of karanjin in Pongamia pinnata.

     

Stability-Indicating TLC Method for the Determination of Dutasteride in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability-indicating thin-layer chromatographic method for determination of dutasteride both as a bulk drug and as pharmaceutical tablets was developed and validated as per the International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates precoated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile:methanol:dichloromethane in the ratio of 2.0:1.0:2.0, v/v/v. This solvent system was found to give compact spots for dutasteride (R _f value of 0.64 ± 0.02). Densitometric analysis of dutasteride was carried out in the absorbance mode at 244 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9943 with respect to peak area in the concentration range of 100–600 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 7.54 and 22.85 ng per band, respectively. Dutasteride was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug undergoes degradation under acidic, basic conditions, photolytic, oxidative and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of dutasteride as bulk drug and from pharmaceutical tablets is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating.     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.

     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.     

Validation of an LC-MS/MS method for the quantitative determination of mavoglurant (AFQ056) in human plasma

A simple, sensitive, and selective liquid chromatography/tandem mass spectrometry method was validated for the identification and quantification of mavoglurant (AFQ056) in human plasma. The chromatographic separation was performed using a Cosmosil 5 C18 (150?×?4.6 mm, 5 μm) column at 40?±?0.5 °C with a mobile phase consisting of acetic acid in water (0.1 %, v/v)/methanol (10:90, v/v) with a flow rate of 1.0 mL/min followed by quantification with tandem mass spectrometry, operating with electrospray ionization in positive ion mode and applying multiple reaction monitoring. The validated method described in this paper presents high absolute recovery with precision and accuracy meeting the acceptance criteria. The method was precise and accurate for 2- and 10-fold dilution of samples. The method was validated using sodium heparin as specific anticoagulant, and the anticoagulant effect was tested by lithium heparin and K₃EDTA. The method was successfully cross-validated between two bioanalytical sites. The method was specific for mavoglurant within the given criteria for acceptance (apparent peak area at the retention time of mavoglurant in zero samples was less than 20 % compared with the mean peak area at LLOQ) in human plasma. The method was fully validated for the quantitative determination of mavoglurant in human plasma between the range of 2.00 and 2,500 ng/mL.     

Development and validation of a TLC-densitometry method for quantitative analysis of nefopam hydrochloride beside its degradation products

A quantitative densitometric thin-layer chromatographic method for determination of nefopam hydrochloride in pharmaceutical preparations has been established and validated. Nefopam from the formulations was separated and identified on silica gel 60 F₂₅₄ TLC plates with chloroform-methanol-glacial acetic acid (9: 2: 0.1, v/v/v) as mobile phase. Densitometric quantification was performed at absorbance maximum 266 nm. The method was validated for linearity, sensitivity, precision and recovery in accordance with ICH guidelines. The presented method is selective and specific with potential application in pharmaceutical analysis. Nefopam hydrochloride was subjected to acidic and alkaline hydrolysis at different temperatures. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

A Validated and Densitometric HPTLC Method for the Quantification of Withaferin-A and Withanolide-A in Different Plant Parts of Two Morphotypes of Withania somnifera

A simple, sensitive and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the estimation of withaferin-A and withanolide-A in different plant parts such as, leaf, root, stem and fruit of two morphotypes of Withania somnifera. HPTLC of W. somnifera methanolic extract was performed on Si 60 F₂₅₄ (20 cm × 20 cm) plates with toluene:ethyl acetate:formic acid (5:5:1), as mobile phase. Quantitative evaluation of the plate was performed in the absorption-reflection mode at 530 nm. The method was validated for precision, repeatability, and accuracy. The average recovery of withaferin-A and withanolide-A in two levels were 96.0 and 96.7%, showing the excellent reproducibility of the method. The calibration curves were linear for both in the range of 200–3,200 ng. The technique has been applied, for the first time, for the estimation of withaferin-A and withanolide-A in different parts of the two morphotypes of Withania somnifera. The method is simple, precise, specific, sensitive and accurate and can be used for routine analysis as well as for quality control of raw materials and herbal formulations.     

Simultaneous Determination of the Endogenous Free ��-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free ??-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution_(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C₁₈ (150 × 4.6 mm, 5 ??m) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min^?1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r ² = 0.999 in the range of 0.1?C500 and 0.25?C1,000 ng mL^?1 for ??-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for ??-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for ??-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.     

HPTLC Determination of Cefpodoxime Proxetil in Formulations

An HPTLC method has been developed that coelutes both the isomers of cefpodoxime proxetil (CFP). CFP was chromatographed on a silica gel 60 F₂₅₄ TLC plate using toluene:acetonitrile (6:4) as a mobile phase and was quantified at 234 nm. The method was validated with respect to linearity, accuracy, precision and specificity. The limit of detection and limit of quantification for CFP were found to be 0.150 and 0.4 μg spot^?1, respectively. The proposed method was successfully used to determine the amount of CFP present in the marketed tablets and self-nanoemulsifying systems.     

HPTLC Determination of Cefpodoxime Proxetil in Formulations

An HPTLC method has been developed that coelutes both the isomers of cefpodoxime proxetil (CFP). CFP was chromatographed on a silica gel 60 F₂₅₄ TLC plate using toluene:acetonitrile (6:4) as a mobile phase and was quantified at 234 nm. The method was validated with respect to linearity, accuracy, precision and specificity. The limit of detection and limit of quantification for CFP were found to be 0.150 and 0.4 μg spot⁻¹, respectively. The proposed method was successfully used to determine the amount of CFP present in the marketed tablets and self-nanoemulsifying systems.

     

Extraction of Azole Antifungal Drugs from Milk and Biological Fluids Using a New Hollow Fiber Liquid-Phase Microextraction and Analysis by GC-FID

A simple, rapid and sensitive high-performance liquid chromatography LC (HPLC) method of determining the concentration of the novel betulinic acid derivative DRF-4012 (5??-chloro-2,3-didehydroindolo[2??,3??:2,3]betulinic acid) in rat plasma for pharmacokinetic and toxicokinetic purposes has been developed and validated. A simple and fast protein precipitation was performed, and then an extraction using an ethyl acetate:methanol (75:25 v/v) mixture was used to extract DRF-4012 and an internal standard (IS, DRF-4015) from rat plasma. Chromatographic separation was achieved using a Zorbax Eclipse XDB-C8 reversed-phase column with UV detection at 235 nm. The isocratic mobile phase, phosphate buffer (water adjusted to pH 3 with 20% o-phosphoric acid) and acetonitrile (15:85, v/v), was run at a flow rate of 1.2 mL min^?1. The assay was linear (r ² > 0.99) over the concentration range 0.040?C75.0 ??g mL^?1, and presented limits of detection and quantification of 0.020 and 0.040 ??g mL^?1, respectively, in rat plasma. The absolute recovery of both the analyte and the IS was >85% from rat plasma. The intraday accuracy ranged from 99.25 to 102.67% with a precision of 2.62?C4.48%, and the interday accuracy ranged from 98.48 to 104.56% with a precision of 3.87?C5.68%. This developed and validated method was successfully used to determine the DRF-4012 concentration in rat plasma for a pharmacokinetic and toxicokinetic study after the intravenous administration of a 1 mg mL^?1 DRF-4012 nanoparticle formulation at doses of 2?C10 mg kg^?1 in Wistar rats.     

A Validated LC Method for the Quantitation of Cefotaxime in pH-Sensitive Nanoparticles

A sensitive and rapid routine LC method was validated for measuring cefotaxime incorporated in three different pH-sensitive nanoparticles. The drug was chromatographed on a C18 reversed-phase column; the mobile phase used was 0.05 M aqueous ammonium acetate, acetonitrile and tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5 with acetic acid. The flow rate was 1 mL min^?1 and cefotaxime was quantified at 254 nm, with a sensitivity range of 0.005 AUFS. The validated method was specific, linear (R ² ≥ 0.999), precise and accurate in a concentration range of 0.2–50.0 μg mL^?1. The method was rapid, selective and suitable for evaluation of cefotaxime in pH-sensitive Eudragit nanoparticles.     

LC–ESI–MS Determination of Imperatorin in Rat Plasma After Oral Administration and Total Furocoumarins of Radix Angelica dahuricae and its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatography–electrospray ionization mass spectrometry method for the quantification of imperatorin in rat plasma after oral administration and total furocoumarins of Radix Angelica dahuricae has been established. The plasma samples were deproteinized by adding internal standard (IS) osthole solution, which was prepared by acetonitrile. The analysis was performed on a Shim-pack C₁₈ column (150 × 2.0 mm i.d., 5 μm) using acetonitrile and 0.5% formic acid solution (70:30, v/v) as a mobile phase. The detection was performed on a quadrupole mass spectrometer detector with an ESI interface operated in the selected ion monitoring mode. The linear quantification range of the method was 2–4000 ng mL^?1 in rat plasma with a correlation coefficient greater than 0.99, the limit of detection (LOD) was 0.5 ng mL^?1 and the lower limit of quantification (LLOQ) 2 ng mL^?1. The intra- and inter-day relative standard deviations (RSD) were less than 2.5 and 3.5%, respectively. The recoveries were above 90%. The validated method was successfully applied to a pharmacokinetic study of imperatorin in rats after oral administration and total furocoumarins of Radix Angelica dahuricae.     

LC–MS–MS Quantitative Determination of Ursolic Acid in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography–electrospray ionization-tandem mass spectrometry method has been developed and validated for the identification and quantification of ursolic acid in human plasma using glycyrrhetic acid as an internal standard. The method involves extraction with methyl tert-butyl ether. The analyte was separated on a C₁₈ column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M–H]? ions, m/z 455.4 for ursolic acid and m/z 469.5 → m/z 425.5 for glycyrrhetic acid. The method was validated over the concentration range of 0.86–110.0 μg L^?1. The intra- and inter-day precisions were less than 13.53% relative standard deviation (RSD) and the accuracy was within ?4.76% in terms of relative error (RE). The lower limit of quantification was 0.86 μg L^?1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of ursolic acid from spiked drug-free plasma was higher than 68%. The method was used to study the pharmacokinetic profile of ursolic acid in human plasma after oral administration of Jieyu capsules.     

HPLC–MS Analysis of Isoniazid in Dog Plasma

A simple, rapid, and sensitive liquid chromatography–mass spectrometric (LC–MS) method was developed and validated for the determination of isoniazid in dog plasma. Plasma samples were deproteined with methanol and separated on a C₁₈ column interfaced with a single quadrupole mass spectrometer, using 0.1% formic acid–acetonitrile (91:9 v/v) as mobile phase. Detection was performed by positive electrospray ionization with selected ion monitoring at m/z 138 for isoniazid and 152 for entecavir maleate internal standard. Linearity was obtained over the range of 25–5,000 ng mL^?1, with a lower limit of quantification of 25 ng mL^?1. The intra- and inter-day precision was less than 2.7% in terms of relative standard deviation. Accuracy, expressed as relative error, ranged from ?2.0 to 8.0%. Plasma samples were analysed within 5 min. The method was successfully applied to the evaluation of the pharmacokinetics of isoniazid in dog plasma.