Determination and stability assessment of clarithromycin in human plasma using RP-LC with electrochemical detection.

A selective, sensitive and stability-indicating reversed-phase high-performance liquid chromatography method was developed and validated for the determination of clarithromycin antibiotic in human plasma. Liquid chromatography was performed on a 5-µm (100 × 4.6 mm) C8 column at 40°C. The mobile phase consisted of acetonitrile with 0.045M H(3)PO(4) (37:63, v/v) adjusted to pH 6.7 and pumped at a flow rate of 1.2 mL/min. Detections were monitored on an electrochemical detector operated at a potential of 0.85 V with glassy carbon electrode against Ag/AgCl reference electrode. Each analysis required 13 min and quantification over the range of 0.05-5.0 µg/mL of plasma was linear, as indicated by a correlation coefficient (R(2)), 0.9999. The method was validated according to international guidelines. Data with respect to accuracy, within-run and between run, were close to 100% with 4% precision. Absolute recovery was 95%. The limit of quantification was 0.05 µg/mL. Neither endogenous substances nor commonly used drugs were found to interfere with the retention times of analytes. Stock solutions and calibration standards of the drug and quality control preparations were demonstrated to be stable at room temperature and -20°C for long and short periods of time. Eventually, the proposed method was successfully applied to quantify clarithromycin in spiked human plasma and real samples from healthy volunteers, indicating the utility and throughput of this method for clinical and bioavailability studies.

LC-MS and LC-PDA vs. phytochemical analysis of Colchicum brachyphyllum.

In this paper, reliability and validity of a recently in-house developed dereplication strategy based on LC-MS and LC-UV/Vis PDA techniques were verified. The dereplication strategy was applied to investigate the alkaloid rich fraction of Colchicum brachyphyllum Boiss. & Haussk. ex Boiss. (Colchicaceae) which has been previously analyzed phytochemically. Both studies results, LC-MS and LC-PDA and phytochemical analysis, were matched in seven compounds namely: (-)-colchicine (5), 3-demethyl-(-)-colchicine (4), (-)-cornigerine (7), beta-lumi-(-)-colchicine (6), (-)-demecolcine (3), 3-demethyl-(-)-demecolcine (2), and 2,3-didemethyl-(-)-demecolcine (1). LC-MS and LC-PDA based dereplication strategy was not able to detect two of the compounds that were reported in the phytochemical study namely: (+)-demecolcinone and (-)-androbiphenyline. This finding could raise a question about the "naturality" of (+)-demecolcinone. On the other hand apigenin (8) was identified only using this dereplication strategy. Four compounds which had molecular ions at m/z 372 and 400 in the stems, and at m/z at 372 and 358 in flowers, could not be dereplicated, and were thus considered unknown. Their spectral data were not matched with neither the compounds that have been isolated previously from this species nor with any other colchicinoid; hence they should be pursued as potential new compounds.

Liquid chromatographic-electrospray mass spectrometric determination of cyclosporin A in human plasma.

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) assay has been developed for determination of cyclosporin A (CyA) in human plasma; cyclosporin B (CyB) was used as internal standard (IS). The method utilized a combination of a column-switching valve and a reversed-phase symmetry column. The mobile phase was a 25:75 (v/v) mixture of 10% aqueous glacial acetic acid and acetonitrile. Running time per single run was less than 10 min. Sample preparation included C8 SPE of human plasma spiked with the analyte and internal standard, evaporation of the eluate to dryness at 50 degrees C under N2 gas, and finally reconstitution in the mobile phase. Detection of cyclosporin A and the IS was performed in selected ion-monitoring mode at m/z 601.3 and 594.4 Da for CyA and IS, respectively. Quantitation was achieved by use of the regression equation of relative peak area of cyclosporin to IS against concentration of cyclosporin. The method was validated according to FDA guideline requirements. The linearity of the assay in the range 5.0-400.0 ng mL(-1) was verified as characterized by the least-squares regression line Y = (0.00268+/-1.9 x 10(-4))X+(0.00078+/-1.8 x 10(-3)), correlation coefficient, r = 0.9986+/-1.1 x 10(-3) (n = 48). Intra and inter-day quality-control measurements in the range 5.0-350.0 ng mL(-1) revealed almost 100% accuracy and < or = 9% CV for precision. The mean absolute recovery of CyA was found to be 84.01+/-9.9% and the respective relative recovery was 100.3+/-9.19. The limit of quantitation (LOQ) achieved was 5 ng mL(-1). Eventually, stability testing of the analyte and IS in plasma or stock solution revealed that both chemicals were very stable when stored for long or short periods of time at room temperature or -20 degrees C.

RP-LC method for the determination of cetirizine in serum.

The development and evaluation of HPLC method for quantifying cetirizine in human serum is described. The method involves liquid phase extraction of cetirizine in methylene chloride, adding diazepam as an internal standard, followed by separation on a reversed phase C18 Novapak column (150 x 3.9 nm; 4 microm), and employing a UV-detection set at 230 nm at ambient temperature. The mobile phase consists of a 13 mM phosphoric acid solution and acetonitrile (61:39 v/v) adjusted to pH 2.8 with 5 M NaOH. The assay is linear from 10 to 500 ng ml(-1) with a detection limit of 5 ng ml(-1) and a mean recovery of 96.5%. The applicability of this method in pharmacokinetic studies is evaluated.