Development and validation of a multiplex UHPLC-MS/MS method for the determination of the investigational antibiotic against multi-resistant tuberculosis macozinone (PBTZ169) and five active metabolites in human plasma

Abstract

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 μm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169.

Fig 1. Chemical structures of PBTZ169, in vitro known metabolites and internal standards.

In brackets, the names reported in the Investigator’s Brochure.

Fig 2

(A) UHPLC-MS/MS separation of PBTZ169, six in vitro known metabolites and internal standards in spiked human plasma (calibration sample at 500 ng/mL). Each targeted compound is reported as a black chromatographic peak at the selected m/z transition (i.e. to distinguish them from position isomers signals). (B) Selectivity for matrix interferences: overlaid UHPLC-MS/MS profiles (dotted lines) of methanolic extracts from ten blank human plasma. (C) Selectivity for analytical interferences: UHPLC-MS/MS profiles for checking mutual interferences between analytes and ISTD. The background profiles (dotted lines) reported in the analytes LC-MS/MS traces are obtained after precipitation of a human blank plasma with processed with only ISTD solution at 40 ng/mL, whereas background profiles depicted in the ISTD LC-MS/MS traces are obtained after precipitation of the highest calibrator level (2000 ng/mL) with pure MeOH. For (B) and (C), retention times and chromatographic LC-MS/MS profiles of PBTZ169, active metabolites, Met-amino and ISTD obtained during experiments were superimposed for interpretation. (D) Carryover: overlaid UHPLC-MS/MS profiles (n = 3) obtained for the first injection of blank solvent (MeOH) after the highest calibration sample (2000 ng/mL).

Fig 3. Accuracy profiles over the considered validation domain in plasma of PBTZ169 and its in vitro known active metabolites.

Bias (red solid line), upper and lower β-expectation tolerance intervals (β = 90%) (blue dotted lines) and acceptance limits (λ = ± 30%), green dotted lines) are shown.

Fig 4

(A) Chromatographic profiles of PBTZ169 and metabolites in plasma collected in an healthy volunteer 30 minutes after the oral intake of 320 mg PBTZ169.HCl. (B) PK profile of PBTZ169 in plasma from the same volunteer, followed for 2 days after the administration of a single dose of 320 mg PBTZ169.HCl.