Simultaneous Quantification of Betulinic Acid, Lupeol, and ß-Sitosterol in Madhuca longifolia Methanolic Extract of Bark by Liquid Chromatography-Tandem Mass Spectrometric Method.

BACKGROUND: Liquid chromatography with tandem mass spectrometry is used widely used for the quantitative analysis of phytoconstituents present in medicinal plants to assess the quality of extract used for different investigations. OBJECTIVE: A sensitive, precise, and accurate liquid chromatographic method with tandem mass spectrometric detection was developed for simultaneous quantification of lupeol, betulinic acid, and ß-sitosterol in the methanolic extract of Madhuca longifolia bark. METHOD: The three compounds were eluted with a stationary phase Gemini C18 column (50 × 2.0 mm, 3 µm id) and the temperature of the column was maintained by a column oven at 40 ± 0.3°C; mobile phase A (water and 0.1% formic acid) and mobile phase B [acetonitrile-methanol (50+50, v/v) and 0.1% formic acid] were used in a gradient mode and the flow rate was 0.4 mL/min. RESULTS: With these conditions, the retention time for betulinic acid, lupeol, and ß-sitosterol was found to be 1.25, 3.08, and 3.53 minutes, respectively. The total run time was 5.0 min. Detection and quantitation of all three phytoconstituents were carried out by the mass spectrometer, a triple quadrupole equipped with atmospheric pressure chemical ionization, and multiple reaction monitoring using the predominantly positive ion mode and obtained much higher and more stable response nebulizer gas flow at 3.0 L/min. Linear responses were exhibited for all three phytoconstituents with a dynamic linear range of 10-100 µg/mL with the values of the regression coefficient more than 0.995 for betulinic acid, lupeol, and ß-sitosterol. The values of percentage RSD for intraday and interday precision were found to be within the accepted limits for analytical methods (<15%). Selectivity, linearity, LOD, LOQ, accuracy, and precision were evaluated for all three phytoconstituents as per International Conference on Harmonization guidelines. CONCLUSIONS: The proposed method is accurate and sensitive and can be used for the routine quantification of betulinic acid, lupeol, and ß-sitosterol from the herbal extract and its poly-herbal formulations.

Stability Indicating TLC Method for Quantification of Brexpiprazole in Bulk and Its Pharmaceutical Dosage Form and Determination of Content Uniformity.

A sensitive, selective and precise high performance thin layer chromatographic method has been developed and validated for the quantification of Brexpiprazole in bulk drug and in pharmaceutical dosage form. The method employed HPTLC aluminum plates (pre-coated with silica gel 60 F254) as stationary phase while n-butanol was used as mobile phase. The Rf value of Brexpiprazole was observed to be 0.38. The densitometric analysis was carried out in absorbance mode at 215 nm. The linear regression analysis data for the calibration plots showed a good linear relationship for Brexpiprazole over a concentration range of 200-1,600 ng band-1. The limit of detection and limit of quantification for Brexpiprazole was found to be 66 and 200 ng band-1. To find out the possible degradation pathway, forced degradation studies were performed. The stock solutions of Brexpiprazole (1,000 µg mL-1) were subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photo degradation. The drug was found to be susceptible to acid and alkali hydrolysis, chemical oxidation, photo degradation and dry heat. The degraded product peaks were well resolved from the pure drug peak with significant difference in their Rf values. Stressed samples were analyzed using developed HPTLC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of Brexpiprazole in marketed formulation and determination of content uniformity of tablet formulation. Statistical analysis showed that the method is repeatable, selective, and precise.

A Synchronous Fluorescence Spectrofluorometric Method for the Simultaneous Determination of Clonazepam and Paroxetine Hydrochloride in Combined Pharmaceutical Dose Form.

OBJECTIVES: First derivative synchronous spectrofluorimetry has been found to be superior because of its highly specific spectral discrimination and readily available solvent, it is economical, eco-friendly, and lacks an extraction procedure. MATERIALS AND METHODS: In the present study, a new simple, sensitive, and time-saving first derivative synchronous spectrofluorimetry method has been developed for simultaneous estimation of clonazepam (CLO) and paroxetine hydrochloride (PH) in pharmaceutical dose forms. RESULTS: CLO was determined at the emission wavelength of 512.79 nm (zero-crossing wavelength point of PH). Similarly, PH was measured at 336.00 nm (zero-crossing wavelength point of CLO). The first derivative amplitude-concentration plots were rectilinear over the range of 1-5 µg/ mL for CLO and 5-25 µg/mL for PH. The method was validated statistically as per the ICH guidelines. The limits of detection were 0.055 and 0.033 µg/mL and quantification limits were 0.169 and 0.102 µg/mL for CLO and PH, respectively. The percentage recovery in commercial formulation was found to be in the range 100.45% and 99.38% for CLO and PH, respectively, by the proposed method, and percent relative standard deviation values for precision and accuracy studies were found to be less than 2. CONCLUSION: This spectrofluorimetry method has been found to have several advantages such as simple spectra, high selectivity, and low interference. By virtue of its high sensitivity, this method could be applied to the analysis of both CLO and PH in their co-formulated dose forms.

Development and validation of a liquid chromatographic method for estimation of dicyclomine hydrochloride, mefenamic Acid and paracetamol in tablets.

Liquid chromatographic method was developed for simultaneous quantitative determination of dicyclomine hydrochloride, mefenamic acid and paracetamol in their combined dosage form. The separation was achieved using a C18 column (250×4.6 mm id, 5 µm) using acetonitrile:20 mM potassium dihydrogen phosphate 70:30 (v/v) adjusted to pH 4 using orthophosphoric acid as mobile phase at a flow rate of 1 ml/min and detection at 220 nm. Separation was completed within 12 min. The retention times of dicyclomine hydrochloride, mefenamic acid and paracetamol were 3.8, 9.3 and 2.5 minutes respectively. The proposed method was found to have linearity in concentration range of 10-100 µg/ml for dicyclomine hydrochloride, 0.05-10 µg/ml for mefenamic acid and 0.1-20 µg/ml for paracetamol. The developed method has been statistically validated and was found to be simple, precise, reproducible and accurate. The developed and validated method was successfully used for the quantitative analysis of commercially available dosage form.

Comparative pharmacokinetic studies of fast dissolving film and oral solution of ondansetron in rats.

Ondansetron, selective serotonin (5-HT3) receptor blocker, is used in treating chemotherapy induced nausea and vomiting in cancer patients. Mouth dissolving films containing ondansetron were developed to have better onset and patient compliances. The drug content of prepared films was within 85%-115%. The films were found to be stable for 4 months when stored at 40 %°C and 75% RH. In-vitro dissolution studies suggested a rapid disintegration, in which most of ondansetron was released (91.5±3.4%) within 90 sec. Subsequently, Sprague-Dawley rats were used to compare pharmacokinetic parameters of the formulated films with oral administration of pure drug solution. Pharmacokinetic parameters were similar between the two groups in which AUC0-t (ng h/ml), AUC0-∞ (ng h/ml), Cmax (ng/ml), Tmax (min), Kel (h(-1)) and t1/2 (h) of reference was 109.091±15.73, 130.32±18.56, 28.5±4.053, 60, 0.1860±0.0226, and 3.771±0.498 respectively; and for formulated film 113.663±16.64, 151.79±16.54, 30±3.51, 60, 0.1521±0.0310 and 4.755±0.653 respectively. These results suggest that the fast dissolving film containing ondansetron is likely to become one of the choices to treat chemotherapy induced nausea and vomiting.

Stability-indicating liquid chromatographic method for the quantification of the new antipsychotic agent asenapine in bulk and in pharmaceutical formulation.

A simple, specific and stability-indicating reversed phase high performance liquid chromatographic method was developed for the quantitative determination of asenapine in tablet dosage form. A SunFire C(18), 5 µm column having 250×4.6 mm i.d. in isocratic mode, with mobile phase containing 0.02 M potassium dihydrogen phosphate: acetonitrile (95:05, v/v, pH 3.5 adjusted with 1% o-phosphoric acid) was used. The flow rate was 1.0 mL min(-1) and effluents were monitored at 232 nm. The retention time of asenapine was 5.51 min. The linearity for asenapine was in the range of 0.1-20 µg/ml. The recoveries obtained for asenapine were 98.31-101.51%. Asenapine stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, sunlight and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. Stressed samples were assayed using developed LC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of asenapine in tablet dosage form.

Development and characterization of pharmacokinetic parameters of fast-dissolving films containing levocetirizine.

A fast-dissolving film containing levocetirizine, a non-sedative antihistamine drug, was developed using pullulan, xanthan gum, propylene glycol, and tween 80 as the base materials. The drug content of the prepared films was within an acceptable limit as prescribed by the USP. The film exhibited excellent stability for four months when stored at 40 °C and 75% humidity. In vitro dissolution studies suggested a rapid disintegration, in which most of levocetirizine (93.54 ± 3.9%) dissolved within 90 seconds after insertion into the medium. Subsequently, Sprague-Dawley rats were used to compare the pharmacokinetic properties of the film preparation administered to the oral cavity, to those with oral administration of the pure drug solution. The pharmacokinetic parameters were similar between the two groups in which AUC(0-t) (ng h/ml), AUC(0-∞) (ng h/ml) C(max) (ng/ml), T(max) (min), K(el) (h(-1)), and t(1/2) (h) of the reference were 452.033 ± 43.68, 465.78 ± 48.16, 237.16 ± 19.87, 30, 0.453 ± 0.051, and 1.536 ± 0.118, respectively, for the film formulation 447.233 ± 46.24, 458.22 ± 46.74, 233.32 ± 17.19, 30, 0.464 ± 0.060, and 1.496 ± 0.293, respectively. These results suggest that the present levocetirizine containing fast-dissolving film is likely to become one of the choices to treat different allergic conditions.

Development and validation of a stability-indicating RP-HPLC method for duloxetine hydrochloride in its bulk and tablet dosage form.

The objective of the present work was to develop a stability-indicating RP-HPLC method for duloxetine hydrochloride (DUL) in the presence of its degradation products generated from forced decomposition studies. The drug substance was found to be susceptible to stress conditions of acid hydrolysis. The drug was found to be stable to dry heat, photodegradation, oxidation and basic condition attempted. Successful separation of the drug from the degradation products formed under acidic stress conditions was achieved on a Hypersil C-18 column (250 mm à 4.6 mm id, 5Îm particle size) using acetonitrile: 0.01 M potassium dihydrogen phosphate buffer (pH 5.4 adjusted with orthophosphoric acid) (50:50, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Quantification was achieved with photodiode array detection at 229 nm over the concentration range 1â25 Îg/ml with range of recovery 99.8â101.3 % for DUL by the RP-HPLC method. Statistical analysis proved the method to be repeatable, specific, and accurate for estimation of DUL. It can be used as a stability-indicating method due to its effective separation of the drug from its degradation products.