Kyunghoon Lee (Presenter)
Seoul National University Hospital
Authorship: Kyunghoon Lee (1), Sun-Hee Jun (2), Sang Hoon Song (1), Jong Sun Park (3), Jae Ho Lee (3), Kyoung Un Park (1,2), Junghan Song (1,2)
(1) Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul (2) Department of Laboratory Medicine, SNUBH, Seongnam (3) Department of Internal Medicine, SNUBH, Seongnam
| Short Abstract We developed an UPLC-MS/MS method for simultaneously measuring blood concentrations of ethambutol, pyrazinamide, rifampicin, isoniazid in Dried Blood Spots (DBSs). Each DBS sample was analyzed on the UPLC system and the assay performance was evaluated. All drugs were clearly separated within 3 min. Within-run and Between-run precisions were 6.1%–11.3% and 12.2%-28.8%. Linearity was acceptable for each drug. Inter-assay calibration variability data on five consecutive days showed a linear and reproducible curve in the observed analytical ranges. Favorable correlations between drug concentrations in DBSs and sera were observed except for rifampicin and isoniazid. |
Long Abstract
Introduction
Therapeutic drug monitoring (TDM) of anti-tuberculosis (TB) drugs is increasingly important to treat patients and to control TB. We already applied for the assays for first-line anti-TB drugs: ethambutol, pyrazinamide, rifampicin, isoniazid in human sera using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). However, a method in dried blood spots (DBSs) was not yet developed. We developed an UPLC-MS/MS method for simultaneously measuring blood concentrations of these anti-TB drugs in DBSs.
Methods
DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to each well of a 96-well microplate. Each sample was analyzed on an UPLC system. Total running time was 3 min. All drug concentrations were determined by multiple reaction monitoring in positive ion mode as the same conditions as the preceding our study, and assay performance was evaluated.
Results
All drugs were clearly separated in UPLC-MS/MS system. Within-run precisions were 6.3%–11.3% at the low concentration and 6.1%–11.1% at the high concentration. Between-run precisions were 15.1%–28.8% at the low concentration and 12.2%–27.5% at the high concentration. Linearity was acceptable at five concentrations for each drug (R2 > 0.9961). Inter-assay calibration variability data obtained over concentrations 0.5–8 ¥ìg/mL for ethambutol and isoniazid, 5–80 ¥ìg/mL for pyrazinamide and rifampicin on five consecutive days shows a linear and reproducible curve in the observed analytical ranges. For ethambutol and pyrazinamide, Passing Bablok regression analysis revealed favorable correlations between drug concentrations in DBSs and sera. However, for rifampicin and isoniazid, these correlations were not good for clinical application.
Conclusions
We developed the method to measure first-line anti-TB drugs in DBSs using UPLC-MS/MS. The performance of our detection technique in DBSs, comparable to those of preceding method in sera, was generally acceptable to apply TDM of ethambutol and pyrazinamide. Further study for the other drugs should be carried out to optimize our multiplex assay.
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