Liquid chromatographic-electrospray tandem mass spectrometric determination of clarithromycin in human plasma

Abstract

A rapid, sensitive and specific LC-MS-MS method has been developed for the determination of clarithromycin (CLA) in human plasma using roxithromycin (ROX) as the internal standard. Samples were prepared via liquid-liquid extraction with methyl tert-butyl ether (MTBE) and chromatographed on a Supelco RP 18 (4.6 × 50 mm, 3 µm particle size) column with a mobile phase consisting of acetonitrile:methanol:60 mM (pH 3.5) ammonium acetate buffer (32.5:32.5:35) at a constant flow rate of 0.8 mL/min. The run time was 3 min with retention times of approximately 1.65 and 1.70 min for CLA and ROX, respectively. Detection was performed on a PE Sciex API 365 mass spectrometer equipped with a turboionspray ionization source in multiple reaction monitoring (MRM) mode. The MRM pairs were m/z 748.5 → m/z 158.2 for CLA and m/z 837.7 → m/z 679.3 for ROX, respectively, with dwell times of 200 ms for each transition. The validated calibration curve range was 5.00-5000 ng/mL, based on 0.100 mL plasma sample volume with signal-to-noise ratio (S/N) greater than 60 for CLA at the lower limit of quantification level (5.00 ng/mL). The correlation coefficients (r 2 ) of the calibration curves were better than or equal to 0.996. The inter-day (n = 18) precision and accuracy of the quality control (QC) samples were less than 3.58% RSD (relative standard deviation) and −10.8% bias, respectively. The intra-day (n = 6) precision and accuracy of the quality control samples were less than 5.0 and 12.6%, respectively. There was no significant deviation from the nominal values after a 10-fold dilution of high concentration QC samples using blank matrix. The QC samples were stable when left on the bench for 24 h or after three freeze-thaw cycles. The processed samples were also stable in HPLC autosampler at 10°C for over 72 h. No matrix ionization suppression was observed when extracted blank matrix or reconstitution solvent was injected onto the system with post-column infusion of clarithromycin and roxithromycin. No carryover was observed when an extracted blank plasma sample was injected immediately after a 5000 ng/mL ULOQ (the upper limit of quantification) standard. The mean recovery was 81.5 and 78.3%, respectively, for clarithromycin and internal standard.

Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry

“…Stability experiments have therefore to be performed in the local setting during method development. Samples should be protected from light and be analyzed directly or frozen at −20 • C. Although, ranges of the calibration curves are sometimes wider [17,23], in our study these were based on the expected plasma concentrations of the components (depending on the dose and individual variation) and on the MIC of Mycobacterium ulcerans. Much lower LLOQs do not contribute significantly to the area under the concentration time curve and require liquid-liquid extraction (LLE) [17].…”

“…Samples should be protected from light and be analyzed directly or frozen at −20 • C. Although, ranges of the calibration curves are sometimes wider [17,23], in our study these were based on the expected plasma concentrations of the components (depending on the dose and individual variation) and on the MIC of Mycobacterium ulcerans. Much lower LLOQs do not contribute significantly to the area under the concentration time curve and require liquid-liquid extraction (LLE) [17]. Due to the observed carryover of rifampicin the calibration range had to be narrowed compared to the method of analysis of Hartkoorn et al [23].…”

“…Chromatographic methods with fluorescent, ultraviolet, electrochemical or amperometric detection of the individual drugs [7][8][9][10][11][12][13][14][15] are time consuming, mostly require large sample volumes and are less sensitive than methods with mass spectrometric detection. The reported chromatographic methods for clarithromycin with tandem mass spectrometric detection (LC/MS/MS) [16][17][18][19] achieve sufficient sensitivities in short run times, but suffered from lacking the active 14-hydroxymetabolite. In one LC/MS/MS method the 14-hydroxyclarithromycin concentration was only estimated [20].…”

Development and Validation of RP-HPLC Method for Quantitation of Clarithromycin in Matrix Tablet Dosage Form

“…Literature survey revealed that various methods have been developed for the estimation of clarithromycin from laboratory prepared mixtures, pharmaceutical preparations and biological matrices (such as human plasma) through automated solid phase extraction and electrochemical detection, liquid chromatographic electrospray tandem mass spectrometry and high performance liquid chromatography. [4][5][6][7] But most of these reported methods were cumbersome due to poor resolution, troublesome mobile phase (buffers) preparation and lengthy run time, hence difficult for routine analysis. Therefore, the aim of the present work was to establish a rapid, simple, precise, specific, accurate, and cost-effective RP-HPLC method according to ICH guidelines Q2 (R1) for routine analysis of clarithromycin in pharmaceutical formulations.…”

Determination of clarithromycin in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

“…Various analytical methods have been developed to determine clarithromycin in formulations and biological samples, such as high-performance liquid chromatography (HPLC) with fluorescent [3], ultraviolet [4,5], electrochemical [6][7][8][9][10][11][12][13][14][15][16][17], amperometric [18] and mass spectrometric detection [19][20][21][22][23][24]. The fluorescence detection method [3] needs derivatization before the analysis.…”