A battery of tandem mass spectrometry assays with stable isotope-dilution for the quantification of 15 anti-tuberculosis drugs and two metabolites in patients with susceptible-, multidrug-resistant- and extensively drug-resistant tuberculosis.

OBJECTIVE: Anti-tuberculosis (antiTB) drugs are characterized by an important inter-interindividual pharmacokinetic variability poorly predictable from individual patients' characteristics. Therapeutic drug monitoring (TDM) may therefore be beneficial for patients with Mycobacterium tuberculosis infection, especially for the management of multidrug/extensively drug resistant- (MDR/XDR)-TB. Our objective was to develop robust HPLC-MS/MS methods for plasma quantification of 15 antiTB drugs and 2 metabolites, namely rifampicin, isoniazid plus N-acetyl-isoniazid, pyrazinamide, ethambutol (the conventional quadritherapy for susceptible TB) as well as combination of agents against MDR/XDR-TB: bedaquiline, clofazimine, delamanid and its metabolite M1, levofloxacin, linezolid, moxifloxacin, pretomanid, rifabutin, rifapentine, sutezolid, and cycloserine. METHODS: Plasma protein precipitation was used for all analytes except cycloserine, which was analyzed separately after derivatization with benzoyl chloride. AntiTB quadritherapy drugs (Pool1) were separated by Hydrophilic Interaction Liquid Chromatography (column Xbridge BEH Amide, 2.1 × 150 mm, 2.5 µm, Waters®) while MDR/XDR-TB agents (Pool 2) and cycloserine (as benzoyl derivative) were analyzed by reverse phase chromatography on a column XSelect HSS T3, 2.1 × 75 mm, 3.5 µm (Waters®). All runs last <7 min. Quantification was performed by selected reaction monitoring electrospray tandem mass spectrometry, using stable isotopically labelled internal standards. RESULTS: The method covers the clinically relevant plasma levels and was extensively validated based on FDA recommendations, with intra- and inter-assay precision (CV) < 15% over the validated ranges. Application of the method is illustrated by examples of TDM for two patients treated for drug-susceptible- and MDR-TB. CONCLUSION: Such convenient extraction methods and the use of stable isotope-labelled drugs as internal standards provide an accurate and precise quantification of plasma concentrations of all major clinically-used antiTB drugs regimens and is optimally suited for clinically efficient TDM against tuberculosis.

Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4.

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.