Validation of a method for the determination of zolpidem in human plasma using LC with fluorescence detection.

A sensitive and selective high-performance liquid chromatography (HPLC) method was developed for the determination of zolpidem in human plasma. Zolpidem and the internal standard (trazodone) were extracted from human plasma that had been made basic. The basic sample was loaded onto a conditioned Bond Elut C18 cartridge, rinsed with water and eluted with methanol. Forty microliters were then injected onto the LC system. Separation was achieved on a C18 column (150 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:50 mM potassium phosphate monobasic at pH 6.0 (4:6, v/v). Detection was by fluorescence, with excitation at 254 nm and emission at 400 nm. The retention times of zolpidem and internal standard were approximately 4.7 and 5.3 min, respectively. The LC run time was 8 min. The assay was linear in concentration range 1-400 ng/ml for zolpidem in human plasma. The analysis of quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated excellent precision with relative standard deviations (RSD) of 3.7, 4.6, and 3.0%, respectively (n = 18). The method was accurate with all intraday (n = 6) and overall (n = 18) mean concentrations within 5.8% from nominal at all quality control sample concentrations. This method was also performed using a Gilson Aspec XL automated sample processor and autoinjector. The samples were manually fortified with internal standard and made basic. The aspec then performed the solid phase extraction and made injections of the samples onto the LC system. Using the automated procedure for analysis, quality control samples for zolpidem (3, 30, and 300 ng/ml) demonstrated acceptable precision with RSD values of 9.0, 4.9, and 5.1%, respectively (n = 12). The method was accurate with all intracurve (n = 4) and overall (n = 12) mean values being less than 10.8% from nominal at all quality control sample concentrations.

Validation of a method for the determination of (R)-warfarin and (S)-warfarin in human plasma using LC with UV detection.

A sensitive and selective chiral high-performance liquid chromatography (HPLC) method was developed for the determination of (R)-warfarin and (S)-warfarin in human plasma. (R)- and (S)-warfarin and the internal standard (oxybenzone) were extracted from human plasma that had been made acidic with 1 N sulfuric acid into ethyl ether. The ethyl ether layer was removed and evaporated, and the residue was reconstituted in 200 microl of acetonitrile. A 50-microl aliquot was injected onto the HPLC system. Separation was achieved on a beta-cyclodextrin column (250 x 4.6 mm, 5 microm) with a mobile phase composed of acetonitrile:glacial acetic acid:triethylamine (1000:3:2.5, v/v/v). Detection was by ultraviolet absorbance at 320 nm. Late-eluting peaks were diverted from the analytical column by using a beta-cyclodextrin precolumn (50 x 4.6 mm, 5 microm) and a column switching device. The retention times of (R)- and (S)-warfarin and the internal standard were approximately 7.7, 6.9 and 4.0 min, respectively. The run time was 15 min. The assay was linear in concentration ranges of 12.5-2500 ng/ml for (R)- and (S)-warfarin in human plasma. The analysis of quality control samples for (R)- and (S)-warfarin (25.0, 400 and 2000 ng/ml) demonstrated excellent precision with relative standard deviations (R.S.D.) for (R)-warfarin of 10.9, 2.8, and 2.8%, respectively (n = 18), and for (S)-warfarin of 7.0, 2.4 and 2.6%, respectively (n = 18). The method was accurate with all overall (n = 18) mean concentrations being less than 6.0% from nominal at all quality control sample concentrations.

Improvements in detection sensitivity for the determination of ivermectin in plasma using chromatographic techniques and laser-induced fluorescence detection with automated derivatization.

Improvement in detection sensitivity for the analysis of ivermectin was observed through utilization of laser-induced fluorescence detection and by manipulation of chromatographic conditions. Gradient elution used in combination with narrow-bore chromatography and conventional fluorescence detection resulted in a limit of quantitation for the major homologue of ivermectin of 0.01 ng/ml in dog plasma. Laser-induced fluorescence detection with isocratic chromatographic conditions also resulted in a limit of quantitation of 0.01 ng/ml in dog plasma, which is a six-fold improvement over previously reported methods. Introduction of an automated procedure for the derivatization and injection of samples reduced the amount of sample handling, eliminated the potential for analyte/internal standard degradation and contributed to the overall ease of analysis.