To address the most appropriate endogenous biomarker for drug–drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate‐3‐glucuronide, glycochenodeoxycholate‐3‐sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose‐dependent change in area under the plasma concentration‐time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC0‐24h between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR−1 vs. rifampicin plasma Cmax (maximum total concentration in plasma)) to yield an estimate of the inhibition constant of rifampicin. These endogenous substrates can complement existing OATP1B‐mediated drug–drug interaction risk assessment approaches based on agency guidelines in early clinical trials.

This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long-acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N-oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides.

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D 2 O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H 2 O or D 2 O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site uring the process of drug discovery, it is highly desirable to increase the number of successful drug candidates for preclinical, clinical and commercial development. Therefore, the drug discovery process is constantly scrutinized and improved [1]. Adding to this pressure is the generation of vast numbers of new chemical entities resulting from combinatorial chemistry technology [2]. Drug metabolism plays an important role in the drug discovery process [3]. Specifically, the identification of metabolites during the early stage of development can be helpful to medicinal chemists trying to block some of the metabolic hot spots and produce an appropriate drug that is less susceptible to metabolism and increase the half-life of the drug. Therefore, rapid identification of drug metabolites is imperative for drug development [4,5].Hydrogen/deuterium exchange is a well-established technique for studying structure, stability, folding dynamics, and intermolecular interactions in proteins in solution [6]. During solution phase H/D exchange, labile protons in the side chains and amide hydrogens, which are not protected from solution generally exchange rapidly. Exchanges of these unprotected hydrogens occur on the order of a few to a few tenths per second under the experimental conditions described in the aforementioned studies. If however, amide or side chain hydrogens are protected from solution (e.g., when they are hydrogen-bonded in structurally stable secondary-structure elements), the exchange rates can be considerably reduced. Methods in which H/D exchange experiments are combined with either nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry are also well-established [7,8]. NMR methods, when coupled with H/D exchange are the ideal choice for monitoring individual residues or each amide hydrogen; however, these methods are limited to highly purified proteins or metabolites that are soluble at high concentrations, thus eliminating the possibility of determining structural features of drugs and metabolites that are i...

It can be argued that the last true paradigm shift in the bioanalytical (BA) arena was the shift from high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection to HPLC with tandem mass spectrometry (MS/MS) detection after the commercialization of the triple quadrupole mass spectrometer in the 1990s. HPLC-MS/MS analysis based on selected reaction monitoring (SRM) has become the gold standard for BA assays and is used by all the major pharmaceutical companies for the quantitative analysis of new drug entities (NCEs) as part of the new drug discovery and development process. While LC-MS/MS continues to be the best tool for drug discovery bioanalysis, a new paradigm involving high-resolution mass spectrometry (HRMS) and ultrahigh-pressure liquid chromatography (uHPLC) is starting to make inroads into the pharmaceutical industry. The ability to collect full scan spectra, with excellent mass accuracy, mass resolution, 10-250 ms scan speeds and no NCE-related MS parameter optimization, makes the uHPLC-HRMS techniques suitable for quantitative analysis of NCEs while preserving maximum qualitative information about other drug-related and endogenous components such as metabolites, degradants, biomarkers and formulation materials. In this perspective article, we provide some insight into the evolution of the hybrid quadrupole-time-of-flight (Qq-TOF) mass spectrometer and propose some of the desirable specifications that such HRMS systems should have to be integrated into the drug discovery bioanalytical workflow for performing integrated qualitative and quantitative bioanalysis of drugs and related components.

The aim of this study was to investigate the sensitivity and specificity of endogenous glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) as substrates for organic anion transporting polypeptide 1B1 (OATP1B1) in humans. We measured fasting levels of plasma GCDCA-3G and GDCA-3G using liquid chromatography-tandem mass spectrometry in 356 healthy volunteers. The mean plasma levels of both compounds were ~ 50% lower in women than in men (P = 2.25 × 10 −18 and P = 4.73 × 10 −9). In a microarray-based genome-wide association study, the SLCO1B1 rs4149056 (c.521T>C, p.Val174Ala) variation showed the strongest association with the plasma GCDCA-3G (P = 3.09 × 10 −30) and GDCA-3G (P = 1.60 × 10 −17) concentrations. The mean plasma concentration of GCDCA-3G was 9.2-fold (P = 8.77 × 10 −31) and that of GDCA-3G was 6.4fold (P = 2.45x10 −13) higher in individuals with the SLCO1B1 c.521C/C genotype than in those with the c.521T/T genotype. No other variants showed independent genome-wide significant associations with GCDCA-3G or GDCA-3G. GCDCA-3G was highly efficacious in detecting the SLCO1B1 c.521C/C genotype with an area under the receiver operating characteristic curve of 0.996 (P < 0.0001). The sensitivity (98-99%) and specificity (100%) peaked at a cutoff value of 180 ng/mL for men and 90 ng/mL for women. In a haplotype-based analysis, SLCO1B1*5 and *15 were associated with reduced, and SLCO1B1*1B, *14, and *35 with increased OATP1B1 function. In vitro, both GCDCA-3G and GDCA-3G showed at least 6 times higher uptake by OATP1B1 than OATP1B3 or OATP2B1. These data indicate that the hepatic uptake of GCDCA-3G and GDCA-3G is predominantly mediated by OATP1B1. GCDCA-3G, in particular, is a highly sensitive and specific OATP1B1 biomarker in humans.