Validation and application of an online extraction and liquid chromatography tandem mass spectrometry assay for the analysis of delamanid and its DM-6705 metabolite in human breast milk.

We developed and validated a bioanalytical assay to quantify delamanid and its key metabolite (DM-6705) in breast milk and aimed to quantify the secretion of these compounds in breast milk. Due to the hydrophobic nature of the analytes, special care was taken during sample preparation to prevent the formation of fatty deposits during protein precipitation. This was followed by online solid phase extraction and liquid chromatography with tandem mass spectrometry for detection. A Restek Viva BiPh C18 column (1.0 mm×50 mm, 5 µm) was used for extraction while chromatographic separation was performed using a Waters Xterra MS C18 (2.1 mm×100 mm, 5 µm) analytical column with an isocratic mobile phase consisting of acetonitrile, methanol, and 5 mM ammonium carbonate. The mass spectrometric detection of the analytes was performed using an AB Sciex 3200 mass spectrometer employing electrospray ionisation in the positive mode with multiple reaction motoring of the relevant precursor and product ions. Delamanid-d4 and OPC-14714 were used as internal standards. A quadratic (weighted 1/x concentration) regression was used to fit calibration curves for delamanid and DM-6705 over the concentration range of 10.0 - 1000 ng/mL. The intra- and inter-day validation accuracies of the quality control samples were between 92.1% and 98.3% for delamanid, and 97.0% and 102.8% for DM-6705. The percentage coefficient of variation (precision) was less than 7.8%. To our knowledge, this is the first report describing the concentrations of delamanid and DM-6705 in the breast milk of patients treated for rifampicin-resistant tuberculosis.

Liquid chromatography-tandem mass spectrometry analysis of delamanid and its metabolite in human cerebrospinal fluid using protein precipitation and on-line solid-phase extraction.

The penetration of the antituberculosis drug delamanid into the central nervous system is not established. The distribution of delamanid and its major metabolite, DM-6705, into the cerebrospinal fluid requires investigation. A liquid chromatography-tandem mass spectrometry method for the quantification of delamanid and DM-6705 in human cerebrospinal fluid was developed and validated. The calibration range for both analytes was 0.300 - 30.0 ng/mL. The deuterium-labelled analogue of delamanid (delamanid-d4) and OPC-14714 were used as internal standards for delamanid and DM-6705, respectively. Samples were processed by protein precipitation followed by on-line solid-phase extraction and high-performance liquid chromatography on an Agilent 1260 HPLC system. A Phenomenex Gemini-NX C18 (5.0 µm, 50 mm × 2.0 mm) analytical column was used for on-line solid-phase extraction, and a Waters Xterra MS C18 (5.0 µm, 100 mm × 2.1 mm) analytical column for chromatographic separation using gradient elution, at a flow rate of 300 µL/min. The total run time was 7.5 min. Analytes were detected by multiple reaction monitoring on an AB Sciex 5500 triple quadrupole mass spectrometer at unit mass resolution, with electrospray ionization in the positive mode. Accuracy and precision were assessed over three independent validation batches. Extraction recoveries were more than 98% and were consistent across the analytical range. Both analytes in CSF exhibited non-specific adsorption to polypropylene tubes. The method was used to analyse cerebrospinal fluid samples from patients with pulmonary tuberculosis in an exploratory pharmacokinetic study.

Development and validation of a liquid chromatography-tandem mass spectrometry method for quantifying delamanid and its metabolite in small hair samples.

New all-oral regimens for rifampin-resistant tuberculosis (RR-TB) are being scaled up globally. Measurement of drug concentrations in hair assesses long-term drug exposure. Delamanid (DLM) is likely to be a key component of future RR-TB treatment regimens, but a method to describe its quantification in hair via liquid chromatography-tandem mass spectrometry (LC-MS/MS) has not previously been described. We developed and validated a simple, fast, sensitive, and accurate LC-MS/MS method for quantifying DLM and its metabolite DM-6705 in small hair samples. We pulverized and extracted two milligrams of hair in methanol at 37 °C for two hours, and diluted 1:1 with water. A gradient elution method eluted DLM, DM-6705, and the internal standard OPC 14714 within 3 min, bringing overall analysis time to 5.5 min. The method has limits of detection (LOD) of 0.0003 ng/mg for DLM and 0.003 ng/mg for DM-6705. The established linear dynamic ranges are 0.003-2.1 ng/mg and 0.03-21 ng/mg for DLM and DM-6705, respectively. Eleven of 12 participant hair samples had concentrations within DLM's linear dynamic range, while all 12 samples had concentrations within the quantifiable range for DM-6705. The ranges of concentrations observed in these clinical samples for DLM and DM-6705 were 0.004-0.264 ng/mg hair and 0.412-12.041 ng/mg hair respectively. We demonstrate that while DLM was detected in hair at very low levels, its primary metabolite DM-6705 had levels approximately 100 times higher. Measuring DM-6705 in hair may accurately reflect long-term adherence to DLM-containing regimens for drug-resistant TB.