Alpibectir: Early Qualitative and Quantitative Metabolic Profiling from a First-Time-in-Human Study by Combining 19F-NMR (Nuclear Magnetic Resonance), 1H-NMR, and High-Resolution Mass Spectrometric Analyses.

Alpibectir (also known as BVL-GSK098 and GSK3729098) is a new chemical entity (NCE) with a novel mechanism for the treatment of tuberculosis. The disposition of alpibectir was determined in subjects from a first-time-in-human trial after a single oral dose of 40 mg and after 7 days repeat dosing at 30 mg. Here we present a combined approach of 19F-NMR (nuclear magnetic resonance), 1H-NMR, and high-resolution mass spectrometry (HRMS) to confidently determine the human metabolic fate of alpibectir. Utilizing multiple sites of fluorination in the molecule, it was possible to fractionate human urine and plasma to confidently detect and quantify the metabolite responses using 19F-NMR. Qualitative detection and structural characterization of F-containing NMR fractions were performed using complementary high-resolution ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analyses to further add confidence to the metabolite responses in these fractions. Subsequent 1H-NMR then provided unequivocal standard-free structural confirmation for key metabolites, which would not be possible with conventional radioactivity detection and LC-MS/MS techniques. Alpibectir was shown to undergo extensive hydrolysis of the central amide moiety, where the resultant N-dealkylated amine and trifluorobutyric acid products were detected initially by unbiased 19F-NMR detection along with major downstream biotransformations to form a carbamoyl glucuronide conjugate and trifluoroacetic acid, respectively. Parallel UHPLC-MS/MS analyses provided confirmatory or additional structural characterization only where relevant. These concerted data allowed for the qualitative metabolic profile and quantitative determination of drug-related material (DRM) in urine and plasma, along with the percentage of dose excreted in urine, to be reported in a comprehensive, efficient, and data-led manner. SIGNIFICANCE STATEMENT: Combining the selectivity of 19F-NMR (nuclear magnetic resonance) for unfractionated samples as first-intent, data-led sample fractionation prior to 19F-NMR and structure-rich 1H-NMR detection, along with the sensitivity of high-resolution ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), a novel alternative for time-efficient detection and quantification of drug-related material (DRM) in human without use of radiolabeled drug is reported. This allowed more complete data rationalization of human metabolism, permitting early risk assessment and progression of the development of antitubercular agent, alpibectir.

A discovery biotransformation strategy: combining in silico tools with high-resolution mass spectrometry and software-assisted data analysis for high-throughput metabolism.

Understanding compound metabolism in early drug discovery aids medicinal chemistry in designing molecules with improved safety and ADME properties. While advancements in metabolite prediction brings increased confidence, structural decisions require experimental data. In vitro metabolism studies using liquid chromatography and high-resolution mass spectrometry (LC-MS) are generally resource intensive and performed on very few compounds, limiting the chemical space that can be examined.Here, we describe a novel metabolism strategy increasing compound throughput using residual in vitro clearance samples conducted at drug concentrations of 0.5 µM. Analysis by robust ultra high-performance liquid chromatography separation and accurate-mass MS detection ensures major metabolites are identified from a single injection. In silico prediction (parent cLogD) tailors chromatographic conditions, with data-dependent tandem mass spectroscopy targeting predicted metabolites. Software-assisted data mining, structure elucidation and automatic reporting are used.Confidence in the globally aligned workflow is demonstrated with 16 marketed drugs. The approach is now implemented routinely across our laboratories. To date, the success rate for identification of at least one major metabolite is 85%. The utility of these data has been demonstrated across multiple projects, allowing earlier medicinal chemistry decisions to increase efficiency and impact of the design-make-test cycle thus improving the translatability of early in vitro metabolism data.

Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays.

Reactive metabolite trapping screens and potential pitfalls: bioactivation of a homomorpholine and formation of an unstable thiazolidine adduct.

Successful early attrition of potential problematic compounds is of great importance in the pharmaceutical industry. The lead compound in a recent project targeting neuropathic pain was susceptible to metabolic bioactivation, which produced reactive metabolites and showed covalent binding to protein. Therefore, as a part of the backup series for this compound several structural modifications were explored to mediate the reactive metabolite and covalent binding risk. A homomorpholine containing series of compounds was identified without compromising potency. However, when these compounds were incubated with human liver microsomes in the presence of GSH, Cys-Gly adducts were identified, instead of intact GSH conjugates. This article examines the formation of the Cys-Gly adduct with AZX ([M+H]+ 486) as a representative compound for this series. The AZX-Cys-Gly-adduct ([M+H]+ 662) showed evidence of ring contraction by formation of a thiazolidine-glycine and was additionally shown to be unstable. During its isolation for structural characterization by 1H NMR spectroscopy, it was found to have decomposed to a product with [M+H]+ 446. The characterization and identification of this labile GSH-derived adduct using LC-MS/MS and 1H NMR are described, along with observations around stability. In addition, various structurally related trapping reagents were employed in an attempt to further investigate the reaction mechanism along with a methoxylamine trapping experiment to confirm the structure of the postulated reactive intermediate.

Direct determination of urinary creatinine by reactive-thermal desorption-extractive electrospray-ion mobility-tandem mass spectrometry.

A direct, ambient ionization method has been developed for the determination of creatinine in urine that combines derivatization and thermal desorption with extractive electrospray ionization and ion mobility-mass spectrometry. The volatility of creatinine was enhanced by a rapid on-probe aqueous acylation reaction, using a custom-made thermal desorption probe, allowing thermal desorption and ionization of the monoacylated derivative. The monoacyl creatinine [M + H](+) ion (m/z 156) was subjected to mass-to-charge selection and collision induced dissociation to remove the acyl group, generating the protonated creatinine [M + H](+) product ion at m/z 114 before an ion mobility separation was applied to reduce chemical noise. Stable isotope dilution using creatinine-d3 as internal standard was used for quantitative measurements. The direct on-probe derivatization allows high sample throughput with a typical cycle time of 1 min per sample. The method shows good linearity (R(2) = 0.986) and repeatability (%RSD 8-10%) in the range of 0.25-2.0 mg/mL. The creatinine concentrations in diluted urine samples from a healthy individual were determined to contain a mean concentration of 1.44 mg/mL creatinine with a precision (%RSD) of 9.9%. The reactive ambient ionization approach demonstrated here has potential for the determination of involatile analytes in urine and other biofluids.

Direct detection of a sulfonate ester genotoxic impurity by atmospheric-pressure thermal desorption-extractive electrospray-mass spectrometry.

A direct, ambient ionization method has been developed using atmospheric pressure thermal desorption-extractive electrospray-mass spectrometry (AP/TD-EESI-MS) for the detection of the genotoxic impurity (GTI) methyl p-toluenesulfonate (MTS) in a surrogate pharmaceutical matrix. A custom-made thermal desorption probe was used to the desorb and vaporize MTS from the solid state, by rapid heating to 200 °C then cooling to ambient temperature, with a cycle time of 6 min. The detection of MTS using EESI with a sodium acetate doped solvent to generate the [MTS+Na](+) adduct ion provided a significant sensitivity enhancement relative to the [M+H](+) ion generated using a 0.1% formic acid solvent modifier. The MTS detection limit is over an order of magnitude below the long-term daily threshold of toxicological concern (TTC) of 1.5 µg/g and the potential for quantitative analysis has been determined using starch as a surrogate active pharmaceutical ingredient (API).

Enhanced performance in the determination of ibuprofen 1-ß-O-acyl glucuronide in urine by combining high field asymmetric waveform ion mobility spectrometry with liquid chromatography-time-of-flight mass spectrometry.

The incorporation of a chip-based high field asymmetric waveform ion mobility spectrometry (FAIMS) separation in the ultra (high)-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) determination of the (R/S) ibuprofen 1-ß-O-acyl glucuronide metabolite in urine is reported. UHPLC-FAIMS-HRMS reduced matrix chemical noise, improved the limit of quantitation approximately two-fold and increased the linear dynamic range compared to the determination of the metabolite without FAIMS separation. A quantitative evaluation of the prototype UHPLC-FAIMS-HRMS system showed better reproducibility for the drug metabolite (%RSD 2.7%) at biologically relevant concentrations in urine. In-source collision induced dissociation of the FAIMS-selected deprotonated metabolite was used to fragment the ion prior to mass analysis, enhancing selectivity by removing co-eluting species and aiding the qualitative identification of the metabolite by increasing the signal-to-noise ratio of the fragment ions.

Direct extraction of urinary analytes from undeveloped reversed-phase thin layer chromatography plates using a solvent gradient combined with on-line electrospray ionisation ion mobility-mass spectrometry.

The direct extraction of urinary analytes deposited on reversed-phase thin-layer chromatography (RP-TLC) plates is demonstrated using a solvent gradient extraction procedure without prior chromatographic development. The surface sample probe TLC-MS interface used for the gradient extraction is compared to direct loop injection into the electrospray ion source for biofluid profiling. The gradient elution is shown to enhance ion intensities, as urinary salts are eluted in aqueous formic acid in the early part of the gradient reducing ion suppression. The retention of urinary components on the C18 RP-TLC plate was confirmed by monitoring analyte responses with, and without, an aqueous wash phase prior to the solvent gradient extraction. The use of gradient elution allows fractionation of the complex biological matrix as a result of differential retention of urine components on the undeveloped RP-TLC plate. The direct gradient analysis of TLC plates has also been combined with ion mobility-mass spectrometry to further resolve the complex urinary profile and identify co-eluting compounds.

Conference Report: High-resolution MS in drug discovery and development: current applications and future perspectives.

The evolving use of high-resolution MS (HRMS) across the pharmaceutical industry has seen its integrated application into both quantitative and qualitative assays. Through an American Association of Pharmaceutical Scientists (AAPS)/ American Society for Mass Spectrometry (ASMS) workshop, held at AAPS, key scientists from world pharma discussed the feasibility of this new paradigm shift through examples and visions for the future, assessing impact on drug discovery and drug development, as well as educating users new to HRMS in the field. This report details selected talks from the 2-day workshop, with particular emphasis on their impact, as well as personal conclusions around certain topics.

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry.

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 µg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).

Evaluation of laser diode thermal desorption (LDTD) coupled with tandem mass spectrometry (MS/MS) for support of in vitro drug discovery assays: increasing scope, robustness and throughput of the LDTD technique for use with chemically diverse compound libraries.

Within the drug discovery environment, the key process in optimising the chemistry of a structural series toward a potential drug candidate is the design, make and test cycle, in which the primary screens consist of a number of in vitro assays, including metabolic stability, cytochrome P450 inhibition, and time-dependent inhibition assays. These assays are often carried out using multiple drug compounds with chemically diverse structural features, often in a 96 well-plate format for maximum time-efficiency, and are supported using rapid liquid chromatographic (LC) sample introduction with a tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) endpoint, taking around 6.5 h per plate. To provide a faster time-to-decision at this critical point, there exists a requirement for higher sample throughput and a robust, well-characterized analytical alternative. This paper presents a detailed evaluation of laser diode thermal desorption (LDTD), a relatively new ambient sample ionization technique, for compound screening assays. By systematic modification of typical LDTD instrumentation and workflow, and providing deeper understanding around overcoming a number of key issues, this work establishes LDTD as a practical, rapid alternative to conventional LC-MS/MS in drug discovery, without need for extensive sample preparation or expensive, scope-limiting internal standards. Analysis of both the five and three cytochrome P450 competitive inhibition assay samples by LDTD gave improved sample throughput (0.75 h per plate) and provided comparable data quality as the IC50 values obtained were within 3 fold of those calculated from the LC-MS/MS data. Additionally when applied generically to a chemically diverse library of over 250 proprietary compounds from the AstraZeneca design, make and test cycle, LDTD demonstrated a success rate of 98%.

Determination of free desmosine and isodesmosine as urinary biomarkers of lung disorder using ultra performance liquid chromatography-ion mobility-mass spectrometry.

The elastin degradation products, desmosine (DES) and isodesmosine (IDES) are highly stable, cross-linking amino-acids that are unique to mature elastin. The excretion of DES/IDES in urine, in the free form and with associated peptide fragments, provides an indicator of lung damage in chronic obstructive pulmonary disease (COPD). A quantitative ion mobility-mass spectrometry (IM-MS) method has been developed for the analysis of free DES/IDES in urine with deuterated IDES as an internal standard. Resolution of DES/IDES isomers was achieved in less than five minutes using ultra performance liquid chromatography (UPLC) combined with ion pairing. The optimized UPLC-IM-MS method provided a linear dynamic range of 10-300 ng/mL and a limit of quantitation of 0.028 ng/mL for IDES and 0.03 ng/mL for DES (0.55 ng and 0.61 ng on column respectively). The method reproducibility (%RSD) was <4% for DES and IDES. The UPLC-IM-MS method was applied to the analysis of urine samples obtained from healthy volunteers and COPD patients. The DES/IDES concentrations in healthy and COPD urine showed an increase in DES (79%) and IDES (74%) in the COPD samples, relative to healthy controls. The incorporation of an IM separation prior to m/z measurement by MS was shown to reduce non-target ion responses from the bio-fluid matrix.

Utility of spatially-resolved atmospheric pressure surface sampling and ionization techniques as alternatives to mass spectrometric imaging (MSI) in drug metabolism.

Tissue distribution studies of drug molecules play an essential role in the pharmaceutical industry and are commonly undertaken using quantitative whole body autoradiography (QWBA) methods. The growing need for complementary methods to address some scientific gaps around radiography methods has led to increased use of mass spectrometric imaging (MSI) technology over the last 5 to 10 years. More recently, the development of novel mass spectrometric techniques for ambient surface sampling has redefined what can be regarded as "fit-for-purpose" for MSI in a drug metabolism and disposition arena. Together with a review of these novel alternatives, this paper details the use of two liquid microjunction (LMJ)-based mass spectrometric surface sampling technologies. These approaches are used to provide qualitative determination of parent drug in rat liver tissue slices using liquid extraction surface analysis (LESA) and to assess the performance of a LMJ surface sampling probe (LMJ-SSP) interface for quantitative assessment of parent drug in brain, liver and muscle tissue slices. An assessment of the utility of these spatially-resolved sampling methods is given, showing interdependence between mass spectrometric and QWBA methods, in particular there emerges a reason to question typical MSI workflows for drug metabolism; suggesting the expedient use of profile or region analysis may be more appropriate, rather than generating time-intensive molecular images of the entire tissue section.

Ambient ionization mass spectrometry: current understanding of mechanistic theory; analytical performance and application areas.

Ambient ionization mass spectrometry allows the rapid analysis of samples or objects in their native state in the open environment with no prior preparation. Over the past six years, the ability of these techniques to provide selective analyte desorption and ionization, in combination with mass spectrometry (MS), has provided a growing number of powerful analytical alternatives across broad application areas, both quantitative and qualitative in nature, including pharmaceutical analysis, process chemistry, biological imaging, in vivo analysis, proteomics, metabolomics, forensics, and explosives detection. With the emergence of new ambient ionization methods, and the complementary nature of existing desorption and/or ionization techniques, additional hyphenated methods have been devised, which pushes the total number of documented methods to almost thirty. To cover all current ambient ionization techniques in detail would be too complex and detract from the main objective of this review. Rather, an overview of the field of ambient ionization MS will be given, followed by broad classification to allow detailed discussion of theory and common mechanistic factors underpinning a number of key techniques. Consideration will be given to experimental design, ease of implementation and analytical performance, detailing subsequent impact on a number of application areas, both established and emerging.

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry.

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).

On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides.

Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 microm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 microm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 microL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies.

Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry.

A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed.

Diagnostic biomarkers differentiating metastatic melanoma patients from healthy controls identified by an integrated MALDI-TOF mass spectrometry/bioinformatic approach.

The prognosis of advanced metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) remains dismal with a 5-year survival rate of 6-18%. In the present study, an integrated MALDI mass spectrometric approach combined with artificial neural networks (ANNs) analysis and modeling has been used for the identification of biomarker ions in serum from stage IV melanoma patients allowing the discrimination of metastatic disease from healthy status with high specificities of 92% for protein ions and 100% for peptide biomarkers. Our ANNs model also correctly classified 98% of a blind validation set of AJCC stage I melanoma samples as nonstage IV samples, emphasizing the power of the newly defined biomarkers to identify patients with late-stage metastatic melanoma. Sequence analysis identified peptides derived from metastasis-associated proteins; alpha 1-acid glycoprotein precursor-1/2 (AAG-1/2) and complement C3 component precursor-1 (CCCP-1). Furthermore, quantitation of serum AAG by an immunoassay showed a significant (p<0.001) increase in AAG serum concentration in stage IV patients in comparison with healthy volunteers; moreover; the quantity of AAG plotted against MALDI-MS peak intensity classified the groups into two distinct clusters. Ongoing studies of other disease stages will provide evidence whether our strategy is sufficiently robust to give rise to stage-specific protein/peptide signatures in melanoma.