Walter A. Korfmacher

It is time for a paradigm shift in drug discovery bioanalysis: from SRM to HRMS

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.

Utility of solution electrochemistry mass spectrometry for investigation the formation and detection of biologically important conjugates of acetaminophen

On-line formation and detection of glutathione and cysteine conjugates of acetaminophen were accomplished by the interfacing of a coulometric electrochemical cell with a thermospray mass spectrometer in a flow-injection experiment using a liquid chromatographic pump. Formation of the conjugates occurred only after acetaminophen was oxidized electrochemically by a two-electron transfer to N-acetyl-p-benzoquinoneimine and reacted in a mixing tee with either glutathione or cysteine. The newly formed conjugate was detected by thermospray mass spectrometry by observing the [M + H]+ ion for the acetaminophen-glutathione conjugate at m/z 457, or the [M + H]+ ion for the acetaminophen cysteine conjugate at m/z 271. Both the glutathione and cysteine conjugates produced a common fragment ion at m/z 184. The on-line reaction of glutathione and electrochemically generated N-acetyl-p-benzoquinoneimine was monitored at varying pH. At pH 8.5 the ion intensity for the acetaminophen-glutathione conjugate was greater than at lower pH, indicating that lower proton concentration enhanced the reaction of glutathione with N-acetyl-p-benzoquinoneimine. This on-line electrochemical-thermospray mass spectrometric method demonstrated that acetaminophen conjugates may be formed and detected in the time frame of 1 s.

High-performance liquid chromatography—thermospray mass spectrometry of ten sulfonamide antibiotics: Analysis in milk at the ppb level

Ten sulfonamide antibiotics including sulfanilamide (SNL), sulfamethazine (SMZ), sulfamethizole (SMTZ), sulfachloropyridazine and sulfaquinoxaline (SQX), were analyzed by thermospray (TSP) mass spectrometry on-line with a high-performance liquid chromatography-UV detection system. Except for the pairs SMZ-SMTZ and sulfadimethoxine-SQX, the standards were resolved in both the UV and TSP profiles. Co-eluting compounds could be differentiated in TSP by their different relative molecular masses. The [M+H]+ ion was the base peak for all the standards except SNL, which showed an [M+NH4]+ ion. Collision-induced dissociation of the [M+H]+ ions afforded daughter ion spectra characterized by common ions at m/z 92, 108 and 156, and ions derived from the amine substituent ([MH-155]+). TSP detection limits [signal-to-noise ratio (S/N) > 3] were below 20 ng (scan mode), 2 ng (selected reaction monitoring, daughter ions from [M+H]+) and 400 pg (selected ion monitoring). UV detection limits were ca. 2 ng (S/N > 5). Results obtained from the multi-residue analysis of spiked cow milk samples at the low ng/ml level are presented.

Rapid Determination of Pharmacokinetic Properties of New Chemical Entities: In vivo Approaches

There is a continuing need for increased throughput in the evaluation of new chemical entities in terms of their pharmacokinetic (PK) parameters as part of new drug discovery. This review summarizes various approaches that have been used to increase throughput in this area. The article divides the approaches into two areas: assay enhancement and sample reduction.

The emergence of high-resolution MS as the premier analytical tool in the pharmaceutical bioanalysis arena

Consultant for MS Imaging, Discovery BA & Discovery Drug Metabolism and PK, Westfield, NJ 07090, USA “Despite the recent widespread adoption and the benefits of using UHPLC–HRMS for drug metabolism, pharmacokinetics and metabonomic studies, challenges around automated user-friendly software tools, data file comparison techniques and ultimate quantitative sensitivity similar to triple quadrupole MS remain unconquered.”

Fast mass spectrometry-based methodologies for pharmaceutical analyses.

Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.

Fungal transformations of antihistamines: metabolism of methapyrilene, thenyldiamine and tripelennamine to N-oxide and N-demethylated derivatives

1. Strains of the fungus Cunninghamella elegans ATCC 9245 and 36112 were tested for their ability to transform the antihistamines methapyrilene (I), thenyldiamine (II) and tripelennamine (III). 2. Antihistamine metabolites were isolated by h.p.l.c., and identified by their 1H-n.m.r. and mass spectral properties. 3. All three drugs were transformed by both C. elegans strains to N-oxidized and N-demethylated derivatives. Metabolism during 96 h of incubation amounted to 85% for (I), 64% for (II), and 83% for (III). Metabolites soluble in organic solvents amounted to 62% to 86% of the total metabolism; approximately 88% to 95% of the organic-soluble metabolites were N-oxide derivatives of each antihistamine.

Fungal transformations of antihistamines : metabolism of cyproheptadine hydrochloride by Cunninghamella elegans

1. Metabolites formed during incubation of the antihistamine cyproheptadine hydrochloride with the zygomycete fungus Cunninghamella elegans in liquid culture were determined. The metabolites were isolated by hple and identified by mass spectrometric and proton nmr spectroscopic analysis. Two C elegans strains, ATCC 9245 and ATCC 36112, were screened and both produced essentially identical metabolites. 2. Within 72 h cyproheptadine was extensively biotransformed to at least eight oxidative phase-I metabolites primarily via aromatic hydroxylation metabolic pathways. Cyproheptadine was biotransformed predominantly to 2-hydroxycyproheptadine. Other metabolites identified were 1- and 3-hydroxycyproheptadine, cyproheptadine 10,11-epoxide, N-desmethylcyproheptadine, N-desmethyl-2-hydroxycyproheptadine, cyproheptadine N-oxide, and 2-hydroxycyproheptadine N-oxide. Although a minor fungal metabolite, cyproheptadine 10,11-epoxide represents the first stable epoxide isolated from the microbial biotransformation of drugs. 3. The enzymatic mechanism for the formation of the major fungal metabolite, 2-hydroxycyproheptadine, was investigated. The oxygen atom was derived from molecular oxygen as determined from 18O-labelling experiments. The formation of 2-hydroxycyproheptadine was inhibited 35, 70 and 97% by cytochrome P450 inhibitors metyrapone, proadifen and 1-aminobenzotriazole respectively. Cytochrome P450 was detected in the microsomal fractions of C. elegans. In addition, 2-hydroxylase activity was found in cell-free extracts of C. elegans. This activity was inhibited by proadifen and CO, and was inducible by naphthalene. These results are consistent with the fungal epoxidation and hydroxylation reactions being catalysed by cytochrome P450 monooxygenases. 4. The effects of types of media on the biotransformation of cyproheptadine were investigated. It appears that the glucose level significantly affects the biotransformation rates of cyproheptadine; however it did not change the relative ratios between metabolites produced.

Analysis of 2,3,7,8-tetrachlorodibenzofuran by fused silica GC combined with atmospheric pressure ionization MS

Abstract A detection limit of 0.5 pg was obtained for 2,3,7,8-tetrachlorodibenzofuran using GC-atmospheric pressure ionization MS with nitrogen as the makeup gas. All of the tetrachlorodibenzofuran isomers studied showed the same mass spectrum. The possible interference of hexachlorodiphenyl ethers was also examined.

The Role of Hyphenated Chromatography-Mass Spectrometry Techniques in Exploratory Drug Metabolism and Pharmacokinetics

The advances in high-speed synthesis technologies have produced a large number of biologically active new chemical entities (NCEs) for developability assessment. Current drug discovery efforts have been focused on identifying drug metabolism and pharmacokinetic (DMPK) issues at the earliest possible stage in order to reduce the attrition rate of drug candidates during the development phase. Mass spectrometry (MS) has proven a powerful tool in providing rapid qualitative and quantitative measurements of drug molecules for DMPK studies in both drug discovery and development. Although mass spectrometers can serve as separation devices, for most pharmaceutical applications, some form of chromatography is combined with MS. For most MS-based methods, tandem mass spectrometry (MS/MS) utilizes atmospheric pressure ionization (API) and chromatographic techniques. This review describes the major hyphenated chromatography-mass spectrometry techniques and their applications in supporting exploratory DMPK studies including various in vitro and in vivo PK and metabolite identification profiles.