A clinical study was undertaken in 12 healthy volunteers. At first, subjects received metronidazole (CAS 443-48-1; a substrate for cytochrome CYP3A4 and CYP2C9) alone at a dose of 400 mg every 8 h for 3 days. On day 4, blood and urine were collected at different time points and metronidazole levels were measured. After a washout period (> 10 half-lives) of one week silymarin (CAS 22888-70-6) was given at a daily dose of 140 mg for 9 days. From day 7 both silymarin (140 mg/day) and metronidazole (3 x 400 mg/day) were given till the 9th day. On day 10, blood and urine were collected as above and the levels of metronidazole and its metabolite were measured by HPLC. Administration of silymarin increased the clearence of metronidazole and its major metabolite, hydroxy-metronidazole (HM) by 29.51% and 31.90%, respectively, with a concomitant decrease in half-life, Cmax and AUC(0-48). Urinary excretions of acid-metronidazole (AM), HM as well as metronidazole in 48 h were decreased. The results indicate that silymarin might induce both intestinal P-glycoprotein and CYP3A4 upon multiple dose administration.

Diclofenac sodium is a non-steroidal anti-inflammatory drug (NSAID). It undergoes extensive Phase I and Phase II metabolism and in vitro it is a specific CYP2C9 substrate. The first part of the study consisted of oral administration of 100 mg of diclofenac sodium (Voveran100) to 12 healthy male volunteers. Blood samples were collected from the antecubital vein at intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours. The second part of the study was conducted after a washout period of 7 days. Treatment with 500 mg p.o. of diosmin (Venex 500) was given daily for 9 days. On day 10, 100 mg of diclofenac sodium (Voveran 100) was administered. Blood samples were obtained as mentioned earlier and pharmacokinetic parameters of diclofenac before and after pretreatment with diosmin analyzed by HPLC. Diosmin pretreatment significantly enhanced AUC, C(max) and t1/2 with a concomitant reduction in CL/f. Diosmin might have inhibited the microsomal CYP2C9 mediated oxidation of diclofenac sodium.

An analytical method based on liquid chromatographic-tandem mass spectrometry (LC-MS/MS) was developed for the determination of the non-nucleoside reverse transcriptase inhibitor rilpivirine in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from human plasma by liquid-liquid extraction. The reconstituted samples were chromatographed on a C(18) column using a mixture of acetonitrile and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The linearity was confirmed in the concentration range 0.51-200 ng/mL in human plasma. Multiple reaction monitoring mode was used for quantification of ion transitions at m/z 367.2/195.1 and 267.1/226.1 for the drug and the internal standard, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. Extraction recoveries of drug from plasma were >69.5%. A run time of 2.50 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed method is simple, rapid and sensitive for the determination of rilpivirine concentrations in real-time plasma samples obtained from pharmacokinetic studies.

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and validated for simultaneous quantification of sitagliptin and simvastatin in human plasma. Carbamazepine was used as an internal standard (IS). The analytes and IS were extracted from the human plasma by liquid-liquid extraction technique. The reconstituted samples were chromatographed on an Alltima HP C(18) column using an isocratic solvent mixture [acetonitrile-5 mm ammonium acetate (pH 4.5), 85:15 (v/v)] at a flow rate of 1.0 mL/min. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curves obtained were linear (r(2)  ≥ 0.99) over the concentration range of 0.10-501 and 0.05-105 ng/mL for sitagliptin and simvastatin, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. Both 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 3.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay was successfully applied to a pharmacokinetic study in human volunteers.

A high performance liquid chromatographic (HPLC) method for the simultaneous determination of phenytoin (CAS 57-41-0), phenobarbital (CAS 50-06-6, phenobarbitone) and carbamazepine (CAS 298-46-4) is described. The serum was extracted with ethyl acetate, the dried extract was reconstituted in mobile phase and the aliquot was injected. The eluent drugs were detected at 230 nm. The mobile phase consisting of methanol: water: glacial acetic acid mixture (67:33:1, v/v/v) was used at a flow rate of 1 ml/min on C-18 column. The absolute recovery was above 96% of all the three drugs over a concentration range of 0.5-50.0 micrograms/ml. The inter-day and intra-day precision relative standard deviation (RSD) ranged from 0.79-5.13% and 0.11-6.81%, respectively. The method is simple, rapid, accurate and sensitive and is presently used for therapeutic drug monitoring in epileptic patients. The results obtained with this method showed very good clinical correlation.