Simultaneous determination of first‐line anti‐tuberculosis drugs and their major metabolic ratios by liquid chromatography/tandem mass spectrometry

Abstract

Monitoring of anti-tuberculosis drug concentrations and dose adjustment can be helpful in cases that show poor response to treatment. Here, we describe a method that can rapidly and simultaneously measure the blood concentrations of four anti-tuberculosis drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) and two major metabolic ratios (acetylisoniazid/isoniazid and 25-desacetylrifampicin/rifampicin) using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). A C18 reversed-phase column and gradients of methanol in 0.3% formic acid and water were used for HPLC separation. The drug concentrations were determined by multiple reaction monitoring in positive ion mode and the assay performance was evaluated. We determined peak concentration ranges for each drug and acetylisoniazid/isoniazid and 25-desacetylrifampicin/rifampicin ratios by analyzing 2-h post-dose samples in patients treated with standard dosing as a first-line treatment. The preparation of 20 samples including two steps of deproteinization with 50% and 100% methanol was performed within 20 min and chromatographic separation was achieved within 4 min/sample. Interassay calibration variability data obtained over concentrations of 0-8 microg/mL for isoniazid and ethambutol and 0-80 microg/mL for rifampicin and pyrazinamide showed a linear and reproducible curve. Within-run and between-run imprecision (CVs) were 1.9-5.5% and 3.5-10.5% and the lower limits of detection and quantification were 0.01-0.5 microg/mL and 0.05-1.0 microg/mL, respectively. The isoniazid concentration was found to be inversely correlated to the acetylisoniazid/isoniazid ratio (R=-0.739, P<0.001). The devised method allows for the simple, rapid, sensitive and reproducible quantification of isoniazid, rifampicin, pyrazinamide, ethambutol and their two metabolic ratios and should be helpful for therapeutic drug monitoring in tuberculosis patients.

Simultaneous Determination of Isoniazid, Pyrazinamide, Rifampicin and Acetylisoniazid in Human Plasma by High-Performance Liquid Chromatography

“…In our study, the on-column sensitivities (the quantity of drug injected on to the column per injection) of PZA, AcINH, INH and RFP were 0.28, 0.18, 0.46 and 1.08 ng, which were lower than those reported in the literature, 10,18 and almost as sensitive as the LC-MS method (5.0, 0.1 and 1.0 ng of PZA, INH and RFP). 22 The LLOQs of PZA, AcINH, INH and RFP were 0.1, 0.05, 0.1 and 0.2 µg mL -1 , respectively. The LLOQs in our method were also lower than those reported in the literature.…”

“…It then had to be injected two times for each sample to analyze PZA, INH and RFP. LC-MS is selective and sensitive, and several HPLC-MS methods 17,[19][20][21][22][23] have been reported. However, it is very expensive and not always available, particularly in remote areas of developing countries.…”

Quantification of isoniazid, pyrazinamide and ethambutol in serum using liquid chromatography-tandem mass spectrometry

“…Including the preparation of calibration standards and QC samples, it takes approximately 1.5 h to analyse the three drugs in these 12 samples. The maximum capacity is almost 200 samples per 24 h. In the same time, the samples can be analysed for rifampin on a separate LC-MS/MS [7,15] resulting in a processing time similar to the method of Song [6].…”

“…Like for other antimicrobial drugs, it would be ideal to determine all four compounds in the same sample using a single method of analysis [4,5]. Song et al published a method for the simultaneous determination of all firstline anti-TB drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [6]. Unfortunately, the polar compounds isoniazid, pyrazinamide and ethambutol elute near the void volume of the system, co-eluting with endogenous compounds resulting in substantial ion suppression [6,7].…”

“…Song et al published a method for the simultaneous determination of all firstline anti-TB drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [6]. Unfortunately, the polar compounds isoniazid, pyrazinamide and ethambutol elute near the void volume of the system, co-eluting with endogenous compounds resulting in substantial ion suppression [6,7]. As matrix effects may vary within and Quantification of isoniazid, pyrazinamide and ethambutol in serum using liquid chromatography-tandem mass spectrometry , Jan-Willem C. Alffenaar between subjects, ion suppression at the time of elution of the compounds of interest should be avoided or overcome using a stable isotope-labelled internal standard [8][9][10].…”