Investigation of low‐abundant in vitro metabolites of stable isotope‐labelled BAL4815 by accurate mass capillary‐LC‐ESI‐qTof‐MS and MS/MS

Abstract

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

“…Another issue in biological analysis is the limited sample amount, which directs attention to columns with reduced inner diameter, such as microcolumns or capillary HPLC columns [ 76 , 77 ]. The most recent developments deal with the use of separation on a chip, which may be easily coupled with nanoelectrospray ionization [ 78 ] with extremely low sample and solvent consumption and enormous sensitivity.…”

“…The most widespread tandem mass analyzer providing high mass accuracy used in the metabolite identification is the hybrid double-quadrupole TOF instrument [ 19 , 64 , 77 , 101 , 112 ]. Other analyzers based on Fourier transform instruments exhibit even better mass accuracies, but at significantly higher instrument costs, such as ion cyclotron resonance (ICR) [ 104 , 113 ] and Orbitrap as the newest types of mass analyzer [ 102 , 103 ].…”

“…The oxidation is, in general, the introduction of an oxygen atom by various reactions, such as hydroxylation (one or multiple times) [ 23 , 24 , 40 , 45 , 47 , 51 , 52 , 76 , 83 , 102 , 108 , 112 , 124 , 130 , 131 , 135 – 139 ] and aliphatic and aromatic epoxidation [ 77 , 124 , 140 , 141 ], which is often followed by hydrolysis leading to dihydrodiol formation [ 141 ]. Epoxides have the same elemental composition as hydroxylated metabolites and similar retention behavior, so their differentiation depends on the interpretation of their mass spectra and UV spectra for aromatic epoxides.…”

Elucidation of the in vitro metabolic profile of stable isotope labeled BAL19403 by accurate mass capillary liquid chromatography/quadrupole time‐of‐flight mass spectrometry and isotope exchange

“…A further reduction of the i.d. of the column to 300 µm was also tested (data not shown)9 but resulted in only a slight gain in sensitivity for BAL19403 and its metabolites and showed significant limitations. The LC separation was more time‐consuming and the necessity was given to use a less robust and less reproducible nanoESI source instead of the standard interface.…”

“…An elegant alternative is the use of stable isotope labeled drug analogues. Mixtures of labeled and unlabeled drug yield a characteristic isotopic pattern during MS analysis, which can be used to help identify metabolites 9–12. This approach, which is particularly advantageous in the case that unexpected or atypical biotransformations occur, was chosen for the elucidation of the metabolic pattern of BAL19403 in conjunction with structural elucidation of the observed metabolites.…”