Simultaneous determination of pioglitazone and candesartan in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

A simple and rapid liquid chromatography-tandem mass spectrometric(LC-MS/MS) assay method has been developed and fully validated for simultaneous quantification of pioglitazone and candesartan in human plasma.Irbesartan was used as an internal standard.The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 mm polymeric sorbent.The reconstituted samples were chromatographed on a C18 column by using a 80:20(v/v) mixture of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of 0.8 mL/min.The calibration curves obtained were linear(rZ0.99) over the concentration range of 15-3000 ng/mL for pioglitazone and 5-608 ng/mL for candesartan.The results of the intra-and inter-day precision and accuracy studies were well within the acceptable limits.A run time of 2.7 min for each sample made it possible to analyze more than 300 plasma samples per day.The proposed method was found to be applicable to clinical studies.

High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma:Pharmacokinetic application

A rapid and sensitive liquid chromatography-tandem mass spectrometric（LC-MS/MS） assay method has been developed and fully validated for the simultaneous quantification of pravastatin and aspirin in human plasma.Furosemide was used as an internal standard.Analytes and the internal standard were extracted from human plasma by liquid-liquid extraction technique using methyl tertiary butyl ether.The reconstituted samples were chromatographed on a Zorbax SB-C;8 column by using a mixture of 5 mM ammonium acetate buffer and acetonitrile（20:80,v/v） as the mobile phase at a flow rate of 0.8 mL/min.The calibration curve obtained was linear（r≥0.99） over the concentration range of 0.50-600.29 ng/mL for pravastatin and 20.07-2012.00 ng/mL for aspirin.Method validation was performed as per FDA guidelines and the results met the acceptance criteria.A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day.The proposed method was found to be applicable to clinical studies.

HPLC Method Development and Validation of S(-)-Carvedilol from API and Formulations

A simple chiral HPLC method was developed and validated for quantification of S(-)-Carvedilol in Active Pharmaceutical Ingredient (API) and marketed tablet formulation of racemic Carvedilol. Chiral resolution of enantiomers of Carvedilol was achieved on Phenomenex Lux-cellulose–4 (250 mm × 4.6 mm;5 μ particle size) chiral column by using a mobile phase, Isopropanol and n-Heptane (60:40 v/v), at a flow rate of 1.0 ml/min and by employing UV detection at 254 nm wavelength. The method was validated according to the ICH guidelines and was proved to be specific, linear, precise and accurate for the analysis of S(-)-Carvedilol.

Synthesis and biological evaluation of some novel-3-(5-substituted benzimidazol-2-yl)-5-arylisoxazolines

The synthesis of a new series of 3-（5-substituted benzimidazol-2-yl）-5-arylisoxazolines （6a-h） was achieved in excellent yields by the condensation of 1-（1H-benzimidazol-2-yl）-3-（substituted phenyl）prop-2-en-l-ones （5a-h） with hydroxylamine at room temperature. These 1-（1H-benzimidazol-2-yl）-3-（subsfituted phenyl）prop-2-en-l-ones （Sa-h） were obtained by the condensation of 2-acetyl benzimidazoles （4a-d） with different aromatic aldehydes, which in turn were obtained by the oxidation of 2-（α- hydroxy）ethyl benzimidazoles （3a-d） prepared by the reaction of o-phenylenediamines （lad） with ot-hydroxy propiohic acid 2. The synthesized compounds were characterized by their IR, ^1H NMR and MS analyses. These compounds were screened for their antibacterial and antifungal activity by standard methods and found some of them active.

Analytical Method Development and Validation of Filgrastim by UV and RP-UFLC Methods

The research work was carried out for establishing a new Ultra Violet (UV)— Visible spectroscopy and Reverse phase-Ultra Fast Liquid Chromatography (RP-UFLC) method for the analysis and quantification of a biosimilar drug, Filgrastim. Filgrastim or recombinant methionyl granulocyte colony stimulating factor (rGCSF) is a glycoprotein. It has a biological action essential for proliferation and differentiation of hematopoetic and progenitor cells. The UV and RP-UFLC work was carried on a Shimadzu UV1800 Spectrophotometer and Shimadzu Prominence LC-20AD UFLC systems, respectively. The λmax of filgrastim was found to be 215 nm. The correlation coefficient by UV spectroscopy was found to be 0.9994 for the concentration range of 1 to 3 μg/ml in double distilled water. The Reverse phase UFLC was done by using Phenomenex C4 (25 cm × 0.46 cm internal diameter) 15 μ, 300 A° analytical column. The optimized mobile phase for binary elution was Acetonitrile and double distilled water (80:20) with a flow rate of 1 ml/min. The retention time of drug was at 3.2 min. It was observed that the response of the detector was linear in the range of 5 - 15 μg/ml with correlation coefficient value of 0.999. After developing the methods, it was assured for the intended use by validation of the analytical parameters like linearity, accuracy, precision, limit of detection, limit of quantitation, ruggedness and robustness. The results of all the parameters for both the methods were found to be within the acceptance criteria as per the International Council for Harmonisation (ICH) guidelines.

Development of Ultra Fast Liquid Chromatography (UFLC) Method for Fluorescence Detection of Domperidone in Human Serum and Application to Pharmacokinetic Study

A simple and sensitive fluorescence detection of domperidone by ultra fast liquid chromatographic method was developed and validated in human serum. For the evaluation of new drug delivery systems, conducting of pharmacokinetic studies in human volunteers is essential for approval to marketing after preclinical evaluation in animal models. The present method consists of protein precipitation, extraction of analytes from human serum into dichloromethane and separation using reversed-phase C18 column. Propranolol hydrochloride was used as an internal standard and the eluent was monitored by fluorescence detector at excitation 282 and emission 328 nm. The mobile phase used was 62:38 ratio of 10 mM phosphate buffer pH adjusted to 3.1 with OPA and methanol at a flow rate of 1 mL·min-1. The method was evaluated for assay, LLOD, LLOQ, recovery and stability studies. The retention times for domperidone and propranolol hydrochloride were found to be 6.36 and 7.94 minutes respectively. The intraday and inter-day coefficient of variation and percent error values of assay method were less than 5%;mean recovery was more than 96% for each analyte and the method was found to be precise, accurate and specific during study. The method was successfully applied for pharmacokinetic study of immediate and controlled release bioadhesive hot melt extruded buccal patches of domperidone after buccal administration to healthy human volunteers. The Cmax, Tmax, and AUC0–24 of domperidone from immediate and controlled release buccal patches were found to be 129.7 ng·mL-1, 1.5 h, 455.1 ng·h·mL-1 and 145.7 ng·mL-1, 5.25 h, 911.0 ng·h·mL-1 respectively. A simple, sensitive and reliable method for the fluorescence determination of domperidone in human serum by UFLC method was developed and validated.

Establishment of inherent stability of pramipexole and development of validated stability indicating LC-UV and LC-MS method

Pramipexole belongs to a class of nonergot dopamine agonist recently approved for the treatment of early and advanced Parkinson＇s disease.A validated specific stability indicating reversed-phase liquid chromatographic method has been developed for the quantitative determination of pramipexole in bulk as well as in pharmaceutical dosage forms in the presence of degradation products.Forced degradation studies were performed by exposition of drug to hydrolytic（acidic and basic）,oxidative and photolytic stress conditions,as defined under ICH guideline Q1A（R2）.Significant degradation was observed under hydrolytic,oxidative and photolytic conditions and the degradation products formed were identified by LC-MS.

Reversible antispermatogenic and antisteroidogenic activities of Feronia limonia fruit pulp in adult male rats

Objective:To explore the antispermatogenic and testicular antisleroidogenic activities of Feronia limonia fruit pulp southern India.Methods:Fourty Wistar male albino rats(Ratios norvegicus)were equally divided into four groups.Experimental groups were administered with the ethanolic extract of Feronia lirnonia(F.limoni)fruit pulp at doses of 250 and 500 nig/kg body weight once daily for 55 days.All treated rats had corresponding recovery groups.At the end of each treatment periods,various spermatological indices,tissue biochemicals and testicular enzymes levels were analysed.Blood profiles were also estimated.Results:Compared with the control,the F.lirnonia.fruit pulp at both dose leveb did not decrease body weight,which were associated with decline in epididymal sperm count,motility,viability and increased percent of abnormal sperm.Further,F.limonia.fruit pulp at 500 mg/kg body weight markedly reduced the epididymal and testicular protein content by 24.58%and 29.86%,respectively,as well as the glucose-6-phosphate dehydrogenase and△~5-3β-hydroxy steroid dehydrogenase)levels by 42.82%and 38.08%,respectively,while a significant elevation was observed in testicular cholesterol and ascorbic acid content.A gradual recovery of all parameters was observed after 55 days of treatment withdrawal.No significant alterations in haematological indices were observed.Conclusions:The present findings indicate that F.lirnonia fruit pulp may have reversible antispermatogenic and antisteroidogenic properties,and could partially support the traditional use as mate contraceptive.

Preliminary study on antifertility activity of Enicostemma axillare leaves and Urena lobata root used in Indian traditional folk medicine

Objective:To evaluate the possible antifertility activity of Enicostemma axillare(E.axillare) leaves and Urena lobata(U.lobata) root in adult male Wistar albino rats.Methods:Six groups of rats were treated with ethanolic(70%v/v) extracts of E.axillare(375 and 750 mg/kg body weight) and U.lobata root(300 and 600 mg/kg body weight) once daily for 55 days.Control groups received the distilled water and vehicle.All the treated rats had corresponding recovery groups.At the end of each treatment periods,animals were killed and organ weights,sperm characteristics,testicular and epididymal biochemicals as well as testicular enzymes were assessed.Results:The E. axillare and U.lobata at tested doses did not decrease body weight,whereas the weight of testes, epididymides and seminal vesicles were significantly(P【0.01) reduced.Significantly(P【0.01) more reductions in the sperm motility,viability and counts,epididymal and testicular protein contents were noted in the rats treated with higher dose of both the plants.Both the plants at the higher dose caused a marked increase(P【0.01) in sperm morphological abnormalities,testicular cholesterol and ascorbic acid contents were remarkably increased(P【0.01),while,the activities of testicular glucose-6-phosphate dehydrogenase(G-6-PDH) andΔ~5-3β-hydroxy steroid dehydrogenase(Δ~5-3β-HSD) were significantly reduced(P【0.01).However,reversal of these changes occurred after 55 days of treatment withdrawal.Conclusions:This study suggests that the ? axillare leaves and U.lobata root reversibly inhibited spermatogenesis and steroidogenesis indicating reversible antifertility activity which could partially support the traditional of these plants as male contraceptives.

Enantioselective Resolution of(R,S)-Carvedilol to(S)-(2)-Carvedilol by Biocatalysts

Among the microorganisms employed in the study,Aspergillus niger(GUFCC5443),Escherichia coli(ATCC9637),Streptomyces halstedii(CKM-2),Pseudomonas putida(NCIB9494),Cunninghamella elegans(NCIM689)and Sphingomonas paucimobilis(NCTC11030)were capable for the enantioselective conversion of racemic Carvedilol.Immobilization technique enhanced the enantioselectivity of microorganisms and thus increased the enantiomeric purity of the drug.Excellent enantiomeric ratios(E)were found in reactions catalyzed by immobilized A.niger and E.coli with values 174.44 and 104.26,respectively.Triacylglycerol lipase from Aspergillus niger was also employed in this study as a biocatalyst which resulted in the product with 83.35%enantiomeric excess(ee)and E of 11.34 while the enzyme on immobilization has yielded 99.08%ee and 216.39 E.The conversion yield(C%)of the drug by free-enzyme was 57.42%,which was enhanced by immobilization to 90.51%.Hence,our results suggest that immobilized triacylglycerol lipase from A.niger(Lipase AP6)could be an efficient biocatalyst for the enantioselective resolution of racemic Carvedilol to(S)-(-)-Carvedilol with high enantiomeric purity followed by immobilized cultures of A.niger and E.coli.

Development and Validation of a High Performance Thin Layer Chromatographic Method for Quantitative Analysis of Saxagliptin

A simple, specific, accurate and precise high performance thin layer chromatographic method (HPTLC) was developed for the quantitative analysis of Saxagliptin in active pharmaceutical ingredients (APIs) and pharmaceutical dosage forms. The method was achieved using silica gel aluminum plate 60 F254 (10 × 10 cm) as stationary phase and Methanol:Chloroform (6:4 v/v) as mobile phase. The developed plate was scanned densitometrically using UV 222 nm wavelength. The Rf value of Saxagliptin was found to be 0.50 ± 0.02. The developed method was validated according to ICH guidelines. Limit of detection (LOD) and limit of quantification (LOQ) of Saxagliptin by this method were found as 7.96 ng/spot and 26.54 ng/spot, respectively. The method was found to be sensitive, specific, linear, accurate, precise and robust for the quantitative analysis of Saxagliptin in both APIs and marketed tablet formulation.

<i>In Vivo</i>and <i>in Vitro</i>Evaluation of Anti Diabetic and Insulin Secretagogue Activities of <i>Capparis zeylanica</i>

Since ancient times, traditional medicines have been in the usage for the treatment of Diabetes mellitus. An edible fruit from traditional medicinal plant Capparis zeylanica (CZ) was studied for its anti diabetic, insulin secretagogue activities and mechanisms involved in it. In Streptozotocin induced diabetes rats, oral administration of Capparis zeylanica methanolic extract (CZME) (200 mg/kg body weight) for 28 days showed a significant reduction in blood glucose levels by 35.53% and enhanced circulating insulin levels by 81.82% than the diabetic control rats. The insulin secretagogue activity mechanisms of the extract were evaluated by using mouse insulinoma beta cell line (MIN6-β). The extract stimulated insulin release in dependent manner of glucose concentration (3 - 16.7 mM) and extract dose (5 - 500 μg/mL). The insulin releasing effect of the extract was significantly enhanced by 3-isobutyl-1-methyl xanthine, glibenclamide, elevated extracellular calcium and K+ depolarized media. This insulin release was significantly reduced in calcium blocking conditions (by nifedipine and EGTA), in the presence of potassium channel opener (diazoxide). Hence, anti diabetic activity of CZME might be a result of its stimulatory effect on insulin release from pancreatic beta cells via KATP channel dependent and independent ways. These results indicate that CZ fruits have the potential to use in diabetes therapy.

Determination and Validation of HPTLC Method for Cinacalcet Hydrochloride

Cinacalcet Hydrochloride (CIN) is a new calcimimetic agent indicated for the use in hypercalcemia. The present work is aimed at development and validation of a novel and simple high-performance thin-layer chromatographic (HPTLC) method for the analysis of Cinacalcet Hydrochloride (active pharmaceutical ingredient, API. In the method, Aluminum-backed silica gel 60 F254 plates (10 × 10 cm) were used as stationary phase and chloroform: acetonitrile (6:4, v/v) as the mobile phase, which showed compact bands of Cinacalcet HCl (RF 0.30 ± 0.02). Quantitative analysis was carried out by densitometry at a wavelength of 282 nm. Linear regression analysis for the calibration spots showed good correlation ship with regression co-efficient r2 = 0.9994 in the range of 40 - 160 ng/band. The developed method suitability for quantification of CIN was learned by validating it as per the ICH guidelines. CIN detection limit was 0.48 ng/band and the quantification limit was 1.59 ng/band. The proposed method was found to be linear (r2 = 0.999), precise (%RSD < 2% for intraday and intermediate precision), accurate, specific, and robust. Further, the developed method was validated and found suitable for stress induced studies, since presence of degradants has no effect on CIN estimation. The proposed method was found to be simple, sensitive, precise, accurate and reproducible for the estimation of CIN.

Quantitative Determination of Nitidine from Roots and Plant Tissue Culture Extracts of <i>Toddalia asiatica</i>(Linn.) Using HPTLC

Toddalia asiatica Linn. is an important medicinal plant belonging to the family Rutaceae. The plant is well known for its antimalarial activity, which has been attributed to the presence of benzophenanthridine alkaloid nitidine in the roots of plants. A simple, rapid, sensitive, accurate, repeatable and robust HPTLC method has been developed and validated for the quantitative determination of nitidine in the dried roots and plant tissue culture extracts of T. asiatica. Nitidine was estimated at 332 nm by densitometry using Silica gel 60 F254 as stationary phase and chloroform:methanol (7:1, v/v), and as mobile phase. Linearity was observed in the concentration range of 25 -200 ng/spot for nitidine. The limit of detection and limit of quantitation were found to be 0.026 and 0.086 ng/spot respectively for nitidine. Developed method was validated according to the ICH guidelines with respect to precision, accuracy, specificity and robustness. The technique has been applied for the first time for the estimation of nitidine in roots and plant tissue culture extracts of T. asiatica. Statistical analysis data indicate the accuracy and reliability of the method.

Analytical Method Development and Validation of Some Biosimilar Drugs by High Performance Thin Layer Chromatography

A simple and rapid HPTLC analytical method has been developed and validated for the determination of Etanercept and Filgrastim in pure form and in marketed formulation. Both the drugs were chromatographed on silica gel 60 F254s HPTLC plates, as stationary phase. The mobile phase optimized for Filgrastim and Etanercept was Water: n-butanol (7.5:2.5 v/v) and Isopropyl alcohol: water (6.5:4.5 v/v), respectively. The chromatogram obtained was scanned at 225 nm and 222 nm for filgrastim and etanercept which resulted in a retention factor of 0.45 ± 0.07 and 0.32 ± 0.03, respectively. The method was validated for parameters like linearity, accuracy, precision, specificity and robustness. Recovery studies were performed at three concentration levels, to demonstrate suitability, accuracy and precision of proposed method. Statistical analysis proved that the proposed method is accurate and reproducible with linearity in the range of 500 to 3000 ng/band for filgrastim and 200 to 1200 ng/band for etanercept. The limit of detection and limit of quantification for filgrastim was found to be 63.418 ng/band and 192.177 ng/band. For etanercept, LOD and LOQ were found to be 33.381 ng/band and 101.153 ng/band, respectively. The proposed method can be employed for the routine analysis of selected biosimilars.

Simultaneous Quantitative Determination of Nitidine, Chelerythrine and Sanguinarine Using HPTLC from Callus Extract of <i>Zanthoxylum rhetsa</i>

Nitidine, Chelerythrine and Sanguinarine, all these three alkaloids are benzophenanthridine alkaloids. Nitidine was used as an anti-HIV, anti-malarial and anti-cancer. Chelerythrine had anti-cancer and anti-inflammatory activities. Sanguinarine was widely used as an anti-plaquestic and anti-cancer. High performance thin layer chromatography (HPTLC) method was used for simultaneous quantification of Nitidine, Chelerythrine and Sanguinarine in callus extract of Zanthoxylum rhetsa by using Silica gel 60 F254 as stationary phase and ethyl acetate:methanol:water:diethylamine (30:5:2:0.5 v/v) as mobile phase at 280 nm. The linearity concentration range was 5 - 160 μg/band of each alkaloid. The Rf values of Nitidine, Chelerythrine and Sanguinarine were found to be 0.28, 0.49 and 0.73. The limit of detection and limit of quantification were found to be 0.026, 0.088 μg/spot and 0.010 and 0.033 μg/spot, 0.0104 and 0.035 μg/spot respectively for Nitidine, Chelerythrine and Sanguinarine. HPTLC method was developed and validated according to ICH guidelines for simultaneous estimation of Nitidine, Chelerythrine and Sanguinarine and proved to be simple, specific, accurate, robust and rapid.

Analytical Method Development and Validation of Etanercept by UV and RP-UFLC Methods

The research work was carried out using Ultraviolet (UV)—visible spectroscopy and Reverse Phase-Ultra Fast Liquid Chromatography (RP-UFLC) for establishing novel methods for the analysis and quantification of Biosimilar drug, Etanercept. The maximum absorbance of Etanercept was found to be 215 nm and it obeyed Beer-Lamberts law in the range of 5 to 200 μg/ml and 1 to 32 μg/ml for UV and RP-UFLC, respectively. The correlation coefficient (r2) value was found to be between 0.999 and 0.9995. All the validation parameters like linearity, accuracy, and precision, Limit of Detection (LOD), Limit of Quantitation (LOQ) and Robustness were found to be within acceptance criteria as per ICH guidelines. The results of accuracy studies (99.0% to 100.38%) indicated the methods to be accurate. The RSD % for interday and intraday precision studies was found to be less than 2%. Robustness and ruggedness were expressed in terms of RSD % which were also in the specified limits. LOD and LOQ of proposed method was calculated and found to be 1.257 and 3.809 μg/ml by UV, and 0.1073 μg/ml and 0.3251 μg/ml by RP-UFLC method, respectively. The developed methods were observed to be simple, rapid and cost-efficient. It can be easily applied for the estimation of Etanercept in the marketed formulations and for routine analysis of the Biosimilar drug.

Enantioselective Conversion of Racemic Felodipine to S(-)-Felodipine by Aspergillus niger and Lipase AP6 Enzyme

The present study involves the enantioselective resolution of racemic Felodipine by using free and immobilized forms of microbial cultures as well as an enzyme (Lipase AP6). Among the microbial cultures employed in the present study, Aspergillus niger, Sphingomonas paucimobilis, Cunninghamella elegans, Escherichia coli, Pseudomonas putida and Cunninghamella blakesleeana were found to possess capability of enantioselective resolution of racemic Felodipine. The enantiomeric excess (ee%) of Felodipine after reaction catalyzed by whole-cell A. niger and S. paucimobilis was found as 81.59 and 71.67%, respectively. Immobilization enhanced the enantioselectivity (enantiomeric ratio (E)) of the biocatalysts and hence this led to enhanced enantiomeric purity of the drug. The ee% values were found to be enhanced in reactions catalyzed by A. niger and S. paucimobilis cultures after immobilization as 98.27 and 93.56%, respectively. Enantiomeric ratio (E) of the reactions catalyzed by all the biocatalysts has been improved after immobilization. E value of the reaction catalyzed by immobilized A. niger was found to be excellent (E > 100) and hence the drug showed high enantiomeric purity. In lipase AP6 catalyzed study, the enantioselectivity was enhanced after immobilization with excellent E value, which led to enhanced enantiomeric purity of the drug (99.21% ee%).

Validated Chiral Ultra Fast Liquid Chromatographic Method for Quantitative Analysis of Enantiomeric Vildagliptin

A rapid, accurate, and precise chiral Ultra fast liquid chromatography (UFLC) method was developed and validated for enantiomeric separation of racemic vildagliptin and S-vildagliptin according to the guidelines of the International Conference on Harmonization (ICH). The chiral chromatographic separation was achieved with a mobile phase consisting of 20 mM borax buffer (pH 9.0 ± 0.05), ACN, and 0.1% Triethylamine (50:50:0.1, v/v/v) at a flow rate of 1 ml/min using a chiralcel OD-RH column, tris(3,5-dimethyl phenyl carbamate) (250 mm × 4.6 mm, 5 μm) column. The UFLC analysis was monitored at 210 nm. The method showed good linearity with a regression coefficient (r2) of 0.999 in the range of 1 - 12 μg/ml for S-vilda. The detection limit (LOD), quantitation limit (LOQ), and the average percentage recovery for S-vilda were found to be 0.024, 0.075 μg/mL, and 99.19% to 100.4%, respectively. The percentages of relative standard deviation (% RSD) for intra- and inter-day precision were found to be 0.346% and 0.364%, respectively. The developed method proved to be reproducible as % RSD was <2% and it had robustness within the acceptable limit. The percentage purity of pharmaceutical preparations of S-vilda was found to be 99.19 w/w. The proposed chiral method can be put in application for the enantiomeric purity determination of S-vilda formulations.

A note on the occurrence of lichens on Vainateya Godavari mangroves in East Godavari district of Andhra Pradesh India

During the collection of manglicolous lichens for the project work on pharmacological evaluation,three lichen species collected from a remote“Lanka”in less known Vainateya Godavari river basin in the year 2015.There is no record of any of these lichen species on the mangroves of Andhra Pradesh like Dirinaria consimilis(Stirton)D.D.Awasthi,Parmotrema tinctorum(Despr.ex Nyl.)Hale and Roccella montagnei Bel.em.D.D.Awasthi on the host is Excoecaria agallocha.Interestingly the authors find no such lichen flora even in the Coringa wild life sanctuary the second largest stretch of mangrove forests of India due to its close proximity to the port city of Kakinada.