OBJECTIVE To develop HPLC-MS/MS method to quantify rifampicin levels in human plasma, with clinical sampling and delivery protocols designed according to whole blood stability findings.

METHODS The plasma samples underwent precipitation with acetonitrile, followed by dilution of the supernatant with water in a volumetric ratio of 1∶1. The mobile phase utilized in the chromatographic separation consisted of 0.1% formic acid-acetonitrile in a volumetric ratio of 55∶45. The separation was achieved using an Ultimate AQ-C₁₈ column (3.0 mm×100 mm, 3.0 μm) coupled with a pre-column SecurityGuard^TM Standard C₁₈ (2.0 mm×4.0 mm, 2.0 μm). The flow rate was set at 0.4 mL∙min⁻¹, with a column temperature of 45 ℃. Mass spectrometry analysis was conducted using an electrospray ion source, employing a multi-reaction monitoring mode for quantitative assessment. The study examined the stability of rifampicin in whole blood and plasma samples under various conditions. The findings from the whole blood stability analysis informed the development of clinical sampling and examination protocols, as well as the implementation of therapeutic drug monitoring in clinical practice.

RESULTS Rifampicin exhibited linear correlation within the concentration range of 0.16−45.00 μg∙mL⁻¹ (r=0.9998), with a lower limit of quantification of 0.16 μg∙mL⁻¹. The recovery rates for low, medium, and high concentrations ranged from 86.68% to 114.26%, and the precision RSD for intra-day and inter-day analyses was below 4.04%. Stability studies revealed that whole blood samples containing heparin sodium remained stable at room temperature(18−23 ℃)for 24 hours and in the refrigerator at 4 ℃ for 48 hours under dark conditions, surpassing the stability of EDTA-K2 samples under the same conditions (stable for 4 hours and 12 hours, respectively). The plasma quality control samples demonstrated stability when stored at an automatic injector at 4 ℃ for 16 hours following pretreatment. Additionally, the plasma samples remained stable at room temperature (18−23 ℃) for 4 hours, were frozen at −40 ℃ for a period of 30 days, and underwent three freeze-thaw cycles at −40 ℃.

CONCLUSION The validated rifampicin therapeutic drug monitoring method and clinical sampling protocol contribute to the maintenance of rifampicin stability and the precision of analytical outcomes, thereby offering insights for the establishment of standardized practices in rifampicin therapeutic drug monitoring.