Simultaneous determination of atorvastatin, metformin and glimepiride in human plasma by LCâMS/MS and its application to a human pharmacokinetic study

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LCâMS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin, metformin and glimepiride in human plasma. Carbamazepine was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via protein precipitation using acetonitrile. The reconstituted samples were chromatographed on a Alltima HP C18 column by using a 60:40 (v/v) mixture of acetonitrile and 10 mM ammonium acetate (pH 3.0) as the mobile phase at a flow rate of 1.1 mL/min. The calibration curves obtained were linear (r2â¥0.99) over the concentration range of 0.50â150.03 ng/mL for atorvastatin, 12.14â1207.50 ng/mL for metformin and 4.98â494.29 ng/mL for glimepiride. The API-4000 LCâMS/MS in multiple reaction monitoring (MRM) mode was used for detection. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. All the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers. Keywords: Atorvastatin, Metformin, Glimepiride, LCâMS/MS, Human plasma, Pharmacokinetic

A rapid and sensitive liquid chromatographyâtandem mass spectrometric assay for duloxetine in human plasma: Its pharmacokinetic application

This paper describes a simple, rapid and sensitive liquid chromatographyâtandem mass spectrometry assay for the determination of duloxetine in human plasma. A duloxetine stable labeled isotope (duloxetine d5) was used as an internal standard. Analyte and the internal standard were extracted from 100 μL of human plasma via solid phase extraction technique using Oasis HLB cartridges. The chromatographic separation was achieved on a C18 column by using a mixture of acetonitrileâ5 mM ammonium acetate buffer (83:17, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r2â¥0.99) over the concentration range of 0.05â101 ng/mL. Multiple-reaction monitoring mode (MRM) was used for quantification of ion transitions at m/z 298.3/154.1 and 303.3/159.1 for the drug and the internal standard, respectively. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 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. Keywords: Duloxetine in human plasma, Solid-phase extraction (SPE), Liquid chromatographyâtandem mass spectrometry, Method validation, Pharmacokinetic studie

Simultaneous Determination of Atorvastatin and Aspirin in Human Plasma by LC–MS/MS: Its Pharmacokinetic Application

A simple, rapid, and sensitive liquid chromatography tandem mass spectro-metric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin and aspirin in human plasma using a polarity switch. Proguanil and furosemide were used as the internal standards for the quantification of atorvastatin and aspirin, respectively. The analytes were extracted from human plasma by the liquid–liquid extraction technique using methyl tert-butyl ether. The reconstituted samples were chromatographed on a Zorbax XDB Phenyl column by using a mixture of 0.2% acetic acid buffer, methanol, and acetonitrile (20:16:64, v/v) as the mobile phase at a flow rate of 0.8 mL/min. Prior to detection, atorvastatin and aspirin were ionized using an ESI source in the multiple reaction monitoring (MRM) mode. The ions were monitored at the positive m/z 559.2→440.0 transition for atorvastatin and the negative m/z 179.0→136.6 transition for aspirin. The calibration curve obtained was linear (r2 ≥ 0.99) over the concentration range of 0.20–151 ng/mL for atorvastatin and 15.0–3000 ng/mL for aspirin. The method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 3.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The proposed method was found to be applicable to clinical studies

Development of quinazolinone and vanillin acrylamide hybrids as multi-target directed ligands against Alzheimer’s disease and mechanistic insights into their binding with acetylcholinesterase

In view of Multi-Target Directed Ligand (MTDL) approach in treating Alzheimer’s Disease (AD), a series of novel quinazolinone and vanillin cyanoacetamide based acrylamide derivatives (9a-z) were designed, synthesized, and assessed for their activity against a panel of selected AD targets including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), amyloid β protein (Aβ), and also 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and neuroprotective activities. Five of the target analogs 9e, 9h, 9 l, 9t and 9z showed elevated AChE inhibitory activity with IC50 values of 1.058 ± 0.06, 1.362 ± 0.09, 1.434 ± 0.10, 1.015 ± 0.10, 1.035 ± 0.02 µM respectively, high inhibition selectivity against AChE over BChE and good DPPH radical scavenging activity. Enzyme kinetic studies of the potent hybrids in the series disclosed their mixed inhibition approach. Active analogs were found to be non-toxic on SK-N-SH cell lines and have excellent neuroprotective effects against H2O2-induced cell death. Strong modulating affinities on Aβ aggregation process were observed for most active compounds since; they irretrievably interrupted the morphology of Aβ42 fibrils, increased the aggregates and declined the Aβ-induced toxicity in neurons. From the fluorescence emission studies, the binding constants (K) were determined as 2.5 ± 0.021x103, 2.7 ± 0.015x103, 3.7 ± 0.020x103, 2.4 ± 0.013x104, and 5.0 ± 0.033x103 M−1 and binding free energies as −5.82 ± 0.033, −6.07 ± 0.042, −6.26 ± 0.015, −7.71 ± 0.024, and −6.29 ± 0.026 kcal M−1 for complexes of AChE–9e, 9h, 9 l, 9t and 9z, respectively. Moreover, the CD analysis inferred the limited modifications in the AChE secondary structure when it binds to 9e, 9h, 9 l, 9t and 9z. On the basis of docking studies against AChE, the most active congeners were well oriented in the enzyme’s active site by interacting with both catalytic active site (CAS) and peripheral anionic site (PAS). In summary, these quinazolinone and vanillin acrylamide hybrid analogs can be used as promising molecular template to further explore their in vivo efficiency in the development of lead compound to treat AD. Communicated by Ramaswamy H. Sarma</p