Validated liquid chromatography-tandem mass spectrometry method for simultaneous determination of clopamide, reserpine and dihydroergotoxine: Application to pharmacokinetics in human plasma.

A simple, sensitive and rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous quantitation of clopamide, reserpine and dihydroergotoxine (ergoloid mesylates) in human plasma. Under basic conditions, liquid-liquid extraction using ethyl acetate was efficiently used for extraction of the analytes from plasma samples in presence of indapamide as internal standard (IS). The analytes were separated with isocratic elution on Phenomenex(®) Synergi Fusion-RP 80A column (50×4.6mm, 4µm). With positive ion electrospray ionization (ESI), the analytes were quantified and monitored on a triple quadrupole mass spectrometer using Multiple Reaction Monitoring (MRM) scanning mode. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. The method was linear in the concentration range of 0.04-30.00ng/mL for reserpine, 1-96.00ng/mL for clopamide, and 0.05-40.00ng/mL for dihydroergotoxine alkaloids, respectively. For all analytes, the high sensitivity of HPLC-MS/MS method revealed sufficient lower limit of quantification (LLOQ) ranged from 0.04-1ng/mL using 1mL of plasma. The recoveries from spiked control samples were ≥86.16% for all analytes and IS. The intra- and inter-day precision variations were lower than 13.03% while the accuracy values ranged from 91.76% to 111.50%. The developed method was successfully applied to pharmacokinetic study of fixed dose combination of clopamide, reserpine and dihydroergotoxine in healthy male volunteers.

Sensitive high-performance liquid chromatographic determination of famotidine in plasma. Application to pharmacokinetic study.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 microns ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5-500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.

Gas-liquid chromatography-mass spectroscopy determination of clopamide in plasma.

A gas-liquid chromatography-mass spectroscopy (GLC-MS) method for the determination of clopamide (1) in human plasma was developed to evaluate the pharmacokinetics and bioavailability of 1 in humans. The method is specific, sensitive, and rapid and allows routine analysis as required for extensive pharmacokinetic studies. The assay procedure involves addition of furosemide (2) as an internal standard to plasma, separation on Sep Pack-C18 cartridges, elution by ether: methanol (1:1), evaporation, and subsequent derivatization with trimethylanilinium hydroxide in methanol (Methelute). Then, 5 microL of the reaction mixture is injected into a GLC-MS system which consists of a 1% SE-30 on Gas Chrom Q (100-120 mesh) glass column. The MS information was obtained under the following conditions: ionization beam energy 70 eV, ion source 200 degrees; and m/e 372 for single ion monitoring. The retention times for 1 and 2 were 0.6 and 1.0 min, respectively. The limit of detection is 10 ng/mL of 1 in plasma and the calibration curve was shown to be linear between 10 and 500 ng/mL of 1. After repeated analysis of spiked plasma samples, the coefficient of variation ranged from 5.1 to 8.3%. The recovery from the extraction procedure was 92 +/- 2.2%. Spiked samples frozen at -20 degrees C were stable for at least 8 weeks. The method has been successfully used in a pharmacokinetic study with po dosing of 5 mg of 1 to eight healthy volunteers. Peak plasma concentrations of 197 +/- 56 ng/mL were observed after 1.1 +/- 0.34 h. No measurable concentration of 1 beyond 12 h after dosing was observed.

Clopamide: plasma concentrations and diuretic effect in humans.

Clopamide pharmacokinetics were determined after oral doses of 5, 10, and 20 mg in normal volunteers. Maximum plasma concentrations occurred within 2 hours and were followed by a monoexponential decline with an elimination half-life of approximately 10 hours. There was an approximately linear relationship between dose and the AUC. Urinary sodium, chloride, and potassium excretion rates indicated that the peak diuretic activity corresponded with peak plasma drug concentrations and probably continued for 12 to 24 hours. There was little difference between the total sodium and chloride output after each dose of clopamide, suggesting that 5 mg may have been close to the top of the dose-response curve. Chlorothiazide, 500 mg, caused less sodium and chloride output with similar potassium loss. During chronic administration to patients with hypertension, hypokalemia was more marked with clopamide, 10 mg daily, than with clopamide, 5 mg, or chlorothiazide, 500 mg daily.