Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma

Abstract

A new, sensitive and fast method for the simultaneous determination of pyrazinamide, isoniazid, streptomycin, ethambutol, and rifampicin in human plasma was developed and validated. The method required only 100 μL of plasma and one step for sample preparation by protein precipitation. The drugs were separated by using a hydrophilic interaction liquid chromatography (HILIC) column. The mobile phase was methanol and water (0.1% formic acid and 5 mM ammonium acetate, pH 3.0 ± 0.1) in a ratio of 65:35 (v/v), which was eluted at an isocratic flow rate of 0.5 mL/min. Tandem mass spectrometry was performed with a triple-quadrupole tandem mass spectrometer. By use of the HILIC column, the detection was free of ion-pair reagents in the mobile phase, with no significant matrix effects. The total run time was less than 2 min for each sample. The method was validated by evaluating its selectivity, sensitivity, linearity, accuracy, and precision according to US Food and Drug Administration guidelines. The lower limit of quantification was 4.0 ng/mL for pyrazinamide, isoniazid, and rifampicin, 0.5 ng/mL for ethambutol, and 10.0 ng/mL for streptomycin. The intraday precision and interday precision were less than 9%, with the accuracy ranging between -9.3 and 7.3%. The method was successfully applied to therapeutic drug monitoring of 33 patients with tuberculosis after administration of standard antituberculosis drugs. The method has been proved to meet the high-throughput requirements in therapeutic drug monitoring.

Evaluation of the Effects of Sample Dilution and Volume in Hydrophilic Interaction Liquid Chromatography

“…In contrast to reversed-phase liquid chromatography (RPLC), HILIC has several advantageous features such as its increased retention of polar compounds with complete avoidance of ion-pair reagents [2,3], its good peak shape for basic compounds [4], its high sensitivity in mass spectrometry because of the highly organic mobile phase [5,6], and the possibility of applying higher flow rates, which can shorten the analysis time significantly [7]. A large number of HILIC applications have been reported recently, most of which include the determination of highly polar compounds such amino acids [8], organic acids [9], and polar drugs [10].…”

Simultaneous quantification of isoniazid, rifampicin, ethambutol and pyrazinamide by liquid chromatography/tandem mass spectrometry

“…Quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measurement of anti-TB drug levels in plasma have been reported (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). However, most LC-MS/MS methods only allow quantification of a limited number of the first-line drugs making plasma concentration determination of all four drugs time consuming.…”

LC–QToF–MS method for quantification of ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma and its application

“…In this context, therapeutic drug monitoring of these drugs is an important tool to control dosages in patients under treatment. Some methods are available in the literature for simultaneous analysis of anti‐tuberculosis drugs, but use HILIC mode, ion pairs or monolithic columns owing to the polar characteristics of some analytes (Gao et al, ; Prahl et al, ; Zhou et al, ). Therefore, the aim of this study was to develop and validate a rapid and sensitive LC–QToF–MS method using a C 18 column for simultaneous quantitation of PZA, INH, EMB and RIF in human plasma for application to therapeutic drug monitoring and pharmacokinetic studies.…”