Solvent-Casted Films to Assist Polymer Selection for Amorphous Solid Dispersions During Preclinical Studies: In-vitro and In-vivo Exploration.

PURPOSE: The use of two solvent-casted film methods to select optimal polymer compositions for amorphous solid dispersions prepared to support preclinical pharmacokinetic and toxicology studies is described. METHODS: Evaporation of solvent from cover slips by using nitrogen flow, and solvent removal from vials by using rotary evaporation were employed. The films prepared on cover slips were evaluated under the microscope to determine crystallinity. The methods were validated by scaling up corresponding SDDs, evaluating SDD's dissolution, and comparing those results to the dissolution of drug-polymer films. Subsequently, SDD suspensions were prepared and dosed orally to rats to determine pharmacokinetic parameters. This was done by using three compounds from our pipeline and evaluating multiple polymers. RESULTS: The dissolution of generated films showed good agreement with the dissolution of spray dried dispersions when the films were fully amorphous (Compound A and B). In contrast, there was disagreement between film and SDD dissolution when the films had crystallized (Compound C). The in vivo exposure results indicated that the polymer choice based on the film screening methods would have been accurate for drug-polymer films that were amorphous (Compound A and B). Two additional case studies (Compound D and E) are presented, showing good agreement between in vivo and in vitro results. CONCLUSION: This study established the ability of two film casting screening methods to predict the in vitro and in vivo performance of corresponding SDDs, provided that the films are fully amorphous.

An integrated Qual/Quan strategy for ganglioside lipidomics using high-resolution mass spectrometry and Skyline software.

RATIONALE: Gangliosides (GS) are attractive targets in biomarker discovery because of their physiological significance in numerous human diseases including certain cancers and developmental and metabolic disorders. The robust strategy described here enables the profiling of numerous GS while obtaining quantitative data of exploratory biomarkers present in human plasma and whole blood. METHOD: The GS from human blood, human plasma, and several cell lines were extracted using a mixture of methanol and isopropanol/0.1% formic acid followed by direct analysis of the supernatant. The simultaneous Qualitative and Quantitative (Qual/Quan) approach involves micro flow (20 µL/min) high pressure liquid chromatography (HPLC)/high-resolution mass spectrometry (HRMS) and post-acquisition data processing with Skyline software for profiling numerous GS in biological matrices. The quantitative assay involves reverse-phase liquid chromatography/HRMS and calibration curves using commercially available GS. RESULTS: Protein precipitation resulted in ~60%-80% GS recovery from biological matrices. Direct injection of the extract allowed for quantification of targeted GS in human blood, plasma, and cancer cell lines. The lower limit of detection for the target analytes, GM1, GT1, GD1, spiked into 1% BSA/PBS, ranged from 1 to 10 ng/mL. Human lung cancer cell lines contained variable amounts (1-130 ng/mL) of soluble Fuc-GM1 analogs, potential biomarkers of lung cancer. CONCLUSIONS: A combination of simple extraction and micro-HPLC/HRMS allowed for quantification of GS in human serum and whole blood. Integration of HRMS with Skyline allowed for GS profiling in the same samples using post-acquisition HRMS data without the need for reanalysis. The strategy presented here is expected to play an important role in profiling exploratory GS biomarkers in discovery bioanalytical research.

Development of BET inhibitors as potential treatments for cancer: A search for structural diversity.

We describe our efforts to identify structurally diverse leads in the triazole-containing N1-carboline series of bromodomain and extra-terminal inhibitors. Replacement of the N5 "cap" phenyl moiety with various heteroaryls, coupled with additional modifications to the carboline core, provided analogs with similar potency, improved pharmacokinetic properties, and increased solubility compared to our backup lead, BMS-986225 (2). Rapid SAR exploration was enabled by a convergent, synthetic route. These efforts provided a potent BET inhibitor, 3-fluoropyridyl 12, that demonstrated robust efficacy in a multiple myeloma mouse tumor model at 1 mg/kg.

Fucosyl monosialoganglioside: Quantitative analysis of specific potential biomarkers of lung cancer in biological matrices using immunocapture extraction/tandem mass spectrometry.

RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.

BMS-871: a novel orally active pan-Notch inhibitor as an anticancer agent.

This Letter describes synthesis, SAR, and biological activity of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhibitors of γ-secretase mediated signaling of Notch receptors. Optimization of this series led to the identification of BMS-871 (compound 30) which displayed robust in vivo efficacy in Notch-dependent leukemia and solid tumor xenograft models.

Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides.

Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.

Quantification of Linrodostat and its metabolites: Overcoming bioanalytical challenges in support of a discovery Indoleamine 2,3 dioxygenase program.

BMS-986205 (Linrodostat) is a small molecule inhibitor of Indoleamine 2, 3 dioxygenase (IDO) that is currently being evaluated in clinical trials for the oral treatment of advanced cancer. Initially, there were concerns regarding possible toxicity following administration, since BMS-986205 undergoes metabolism to form 4-chloroaniline. However, it was later determined that the downstream metabolites of 4-chloroaniline might be a greater concern. To evaluate the potential toxicity of these metabolites, a sensitive LC-MS/MS analytical method was needed to quantify both the parent compound and multiple metabolites. This presented a challenge since the method required the analysis of multiple analytes while still retaining the analytical sensitivity required to support studies. By utilizing a multi-function analytical method, we were able to quantify the necessary analytes using a complex LC-MS/MS-based method including the application of both negative and positive electrospray ionization.

Discovery and Preclinical Evaluation of BMS-986242, a Potent, Selective Inhibitor of Indoleamine-2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing dioxygenase enzyme implicated in cancer immune response. This account details the discovery of BMS-986242, a novel IDO1 inhibitor designed for the treatment of a variety of cancers including metastatic melanoma and renal cell carcinoma. Given the substantial interest around this target for cancer immunotherapy, we sought to identify a structurally differentiated clinical candidate that performs comparably to linrodostat (BMS-986205) in terms of both in vitro potency and in vivo pharmacodynamic effect in a mouse xenograft model. On the basis of its preclinical profile, BMS-986242 was selected as a candidate for clinical development.

Conformational-Analysis-Guided Discovery of 2,3-Disubstituted Pyridine IDO1 Inhibitors.

IDO1 inhibitors have shown promise as immunotherapies for the treatment of a variety of cancers, including metastatic melanoma and renal cell carcinoma. We recently reported the identification of several novel heme-displacing IDO1 inhibitors, including the clinical molecules linrodostat (BMS-986205) and BMS-986242. Both molecules contain quinolines that, while being present in successful medicines, are known to be potentially susceptible to oxidative metabolism. Efforts to swap this quinoline with an alternative aromatic system led to the discovery of 2,3-disubstituted pyridines as suitable replacements. Further optimization, which included lowering ClogP in combination with strategic fluorine incorporation, led to the discovery of compound 29, a potent, selective IDO1 inhibitor with robust pharmacodynamic activity in a mouse xenograft model.

Discovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug Design.

Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an additional lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematologic and solid tumor models, BMS-986158 was selected as a candidate for clinical evaluation.

Differential induction of N(2),3-ethenoguanine in rat brain and liver after exposure to vinyl chloride.

Although vinyl chloride (VC) clearly induces hepatic angiosarcoma in humans and rodents, a causal association with brain tumors has not been definitively established with the available epidemiological and experimental evidence. Because VC acts by genotoxic mechanisms, DNA adduct formation is thought to be a sensitive biomarker of early events in carcinogenesis. Adult male Sprague Dawley rats were exposed to 0 or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week) by inhalation. Male weanlings were similarly exposed for 5 days. Another group of male adults was exposed to 1100 ppm [(13)C(2)]VC in a nose-only inhalation apparatus for 5 days (6 h/day). A sensitive gas chromatography high-resolution mass spectrometry assay was used to measure the major promutagenic DNA adduct, N(2),3-ethenoguanine (N(2),3-epsilonG), in rat brain and hepatocyte (HEP) DNA. The respective concentrations of N(2),3-epsilonG in control rat brain DNA at 1 and 4 weeks were 5.0 +/- 0.9 and 5.6 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. There was no change in N(2),3-epsilonG in adult rat brain after exposure to 1100 ppm VC for 1 or 4 weeks. In HEPs from the same animals, these adduct concentrations increased from 5.5 +/- 1.4 to 55 +/- 2.0 N(2),3-epsilonG/10(8) unmodified guanine after a 1-week exposure and from 3.0 +/- 0.3 to 110 +/- 20 N(2),3-epsilonG/10(8) unmodified guanine after a 4-week exposure. When weanlings were exposed to 1100 ppm VC for 5 days, there was a statistically significant (P = 0.04) increase in N(2),3-epsilonG in brain from 1.5 +/- 0.2 to 4.4 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. Weanlings exposed to 1100 ppm VC had an even greater increase in N(2),3-epsilonG in HEPs from 1.6 +/- 0.1 to 97 +/- 5.0 N(2),3-epsilonG/10(8) unmodified guanine. [(13)C(2)]N(2),3-epsilonG was not detected in brain DNA from adult rats exposed to 1100 ppm [(13)C(2)]VC for 5 days but was present in HEP DNA at 55 +/- 4.0 [(13)C(2)]N(2),3-epsilonG/10(8) unmodified guanine. The concentrations of the endogenous adduct in both organs were unchanged after this exposure. 7-(Oxoethyl)guanine (OEG), the major DNA adduct formed by VC, was reduced to 7-(2-hydroxyethyl)guanine and measured by liquid chromatography-electrospray ionization-tandom mass spectrometry in brain and HEP DNA from rats exposed to 1100 ppm VC for 1 week. Whereas 4.0 +/- 0.8 OEG/10(6) unmodified guanine were present in HEP DNA from VC-exposed rats, no adducts were detectable in brain DNA (detection limit, 0.3 OEG/10(6) unmodified guanine). These findings indicate that the genotoxic metabolite of VC is not formed in or transported to adult rat brain. Thus, it is unlikely that N(2),3-epsilonG or other VC-induced promutagenic DNA adducts play a significant role in initiating carcinogenesis in adult rat brain after exposure to VC. The data for weanling rats are less clear. Whereas a small increase in N(2),3-epsilonG in the brains of weanlings was found after exposure to 1100 ppm VC, the resulting adduct concentration was similar to that measured in unexposed adults. Future exposures of weanling rats to the stable isotopically labeled compound will be necessary to conclusively determine whether this increase was due to VC.

Molecular dosimetry and repair of N(2),3-ethenoguanine in rats exposed to vinyl chloride.

Although the DNA adducts of vinyl chloride (VC) have been well characterized, previous studies have used single concentrations of VC that are well above contemporary human exposures. This study examined the exposure response to VC in male Sprague Dawley rats with respect to the molecular dose of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonG). Adult rats were exposed by inhalation to 0, 10, 100, or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week). Weanling rats were similarly exposed for 5 days. The amount of N(2),3-epsilonG in hepatocyte (HEP) and nonparenchymal cell (NPC) fractions obtained from the liver was measured with a sensitive immunoaffinity/gas chromatography/high-resolution mass spectrometry assay. Endogenous N(2),3-epsilonG was present in HEPs and NPCs from all unexposed rats. The exposure response to VC in each group and cell population was supralinear, with a linear increase from 0 to 100 ppm, and a plateau between 100 and 1100 ppm. There was no statistically significant difference in N(2),3-epsilonG concentrations between HEPs and NPCs in any adult exposure group, which suggests that factors other than adduct concentrations contribute to the particular susceptibility of NPCs to VC-induced carcinogenesis. The accumulation of N(2),3-epsilonG with respect to time was nearly linear in rats exposed to 600 ppm VC for 1, 2, 4, or 8 weeks (4 h/day, 5 days/week), and no repair of N(2),3-epsilonG was detected in rats exposed to VC for 4 weeks and allowed to recover for 1 week. N(2),3-epsilonG concentrations in HEPs from weanling rats were 2-3-fold greater than those in adult rats exposed for the same time. Higher adduct concentrations in young rats may contribute to their greater susceptibility to VC-induced hepatic angiosarcoma as well as their particular susceptibility to hepatocellular carcinoma. The molecular dosimetry of N(2),3-epsilonG in liver appears to be a sensitive and informative biomarker of genotoxic effect after exposure to VC. N(2),3-epsilonG was the predominant etheno adduct measured in vivo after exposure to VC, and the saturable nature of VC metabolism was reflected in its molecular dose. The relationships between endogenous N(2),3-epsilonG and that formed by low exposures to VC were demonstrated. Conclusions drawn from these exposures may be more relevant for risk assessment purposes than those drawn from high exposures where activation, detoxication, and repair pathways may be saturated or otherwise perturbed. These data are well suited for consideration in future risk assessments of VC that incorporate nontumor mode of action data.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors.

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route.

The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N-HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N-HPVal measurements in humans, similar binding was found.

Biomarkers in Czech workers exposed to 1,3-butadiene: a transitional epidemiologic study.

A multiinstitutional, transitional epidemiologic study was conducted with a worker population in the Czech Republic to evaluate the utility of a continuum of non-disease biological responses as biomarkers of exposure to 1,3-butadiene (BD)* in an industrial setting. The study site included two BD facilities in the Czech Republic. Institutions that collaborated in the study were the University of Vermont (Burlington, Vermont, USA); the Laboratory of Genetic Ecotoxicology (Prague, the Czech Republic); Shell International Chemicals, BV (Amsterdam, The Netherlands); the University of North Carolina at Chapel Hill (Chapel Hill, North Carolina, USA); University of Texas Medical Branch at Galveston (Galveston, Texas, USA); Leiden University (Leiden, The Netherlands); and the Health and Safety Laboratory (Sheffield, United Kingdom). Male volunteer workers (83) participated in the study: 24 were engaged in BD monomer production, 34 in polymerization activities, and 25 plant administrative workers served as unexposed control subjects. The BD concentrations experienced by each exposed worker were measured by personal monitor on approximately ten separate occasions for 8-hour workshifts over a 60-day exposure assessment period before biological samples were collected. Coexposures to styrene, benzene, and toluene were also measured. The administrative control workers were considered to be a homogeneous, unexposed group for whom a series of 28 random BD measurements were taken during the exposure assessment period. Questionnaires were administered in Czech to all participants. At the end of the exposure assessment period, blood and urine samples were collected at the plant; samples were. fractionated, cryopreserved, and kept frozen in Prague until they were shipped to the appropriate laboratories for specific biomarker analysis. The following biomarkers were analyzed: * polymorphisms in genes involved in BD metabolism (Prague and Burlington); * urinary concentrations of 1-hydroxy-2-(N-acetylcysteinyl)-3-butene and 2-hydroxy-1-(N-acetylcysteinyl)-3-butene (M2 [refers to an isomeric mixture of both forms]) (Amsterdam); * urinary concentrations of 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (M1) (Amsterdam); * concentrations of the hemoglobin (Hb) adducts N-(1-[hydroxymethyl]-2-propenyl)valine and N-(2-hydroxy-3-butenyl)valine (HBVal [refers to an isomeric mixture of both forms]) (Amsterdam); * concentrations of the Hb adduct N-(2,3,4-trihydroxybutyl)valine (THBVal) (Chapel Hill); * T cell mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene (autoradiographic assay in Galveston with slide review in Burlington; cloning assay in Leiden with mutational spectra determined in Burlington); and * chromosomal aberrations by the conventional method and by fluorescence in situ hybridization [FISH]), and cytogenetic changes (sister chromatid exchanges [SCEs] (Prague). All assay analysts were blinded to worker and sample identity and remained so until all work in that laboratory had been completed and reported. Assay results were sent to the Biometry Facility in Burlington for statistical analyses. Analysis of questionnaire data revealed that the three exposure groups were balanced with respect to age and years of residence in the district, but the control group had significantly more education than the other two groups and included fewer smokers. Group average BD exposures were 0.023 mg/m3 (0.010 ppm) for the control group, 0.642 mg/m3 (0.290 ppm) for the monomer group, and 1.794 mg/m3 (0.812 ppm) for the polymer group; exposure levels showed considerable variability between and within individuals. Styrene exposures were significantly higher in the polymer group than in the other two groups. We found no statistically significant differences in the distributions of metabolic genotypes over the three exposure groups; genotype frequencies were consistent with those previously reported for this ethnic and national population. Although some specific genotypes were associated with quantitative differences in urinary metabolite concentrations or Hb adduct dose-response characteristics, none indicated a heightened susceptibility to BD. Concentrations of both the M2 and M1 urinary metabolites and both the HBVal and THBVal Hb adducts were significantly correlated with group and individual mean BD exposure levels; the Hb adducts were more strongly correlated than the urinary metabolites. By contrast, no significant relations were observed between BD exposures and HPRT gene mutations (whether determined by the auto-radiographic or the cloning method) or any of the cytogenetic biomarkers (whether determined by the conventional method or FISH analysis). Neither the mutational nor the cytogenetic responses showed any association with genotypes. The molecular spectrum of HPRT mutations in BD-exposed workers showed a high frequency of deletions; but the same result was found in the unexposed control subjects, which suggests that these were not due to BD exposure. This lack of association between BD exposures and genetic effects persisted even when control subjects were excluded from the analyses or when we conducted regression analyses of individual workers exposed to different levels of BD.

Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties.

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

Integrated quantitative and qualitative workflow for in vivo bioanalytical support in drug discovery using hybrid Q-TOF-MS.

BACKGROUND: UHPLC coupled with orthogonal acceleration hybrid quadrupole-TOF (Q-TOF)-MS is an emerging technique offering new strategies for the efficient screening of new chemical entities and related molecules at the early discovery stage within the pharmaceutical industry. In the first part of this article, we examine the main instrumental parameters that are critical for the integration of UHPLC-Q-TOF technology to existing bioanalytical workflows, in order to provide simultaneous quantitative and qualitative bioanalysis of samples generated following in vivo studies. MATERIAL & METHODS: Three modern Q-TOF mass spectrometers, including Bruker maXis™, Agilent 6540 and Sciex TripleTOF™ 5600, all interfaced with UHPLC systems, are evaluated in the second part of the article. The scope of this work is to demonstrate the potential of Q-TOF for the analysis of typical small molecules, therapeutic peptides (molecular weight <6000 Da), and enzymatically (i.e., trypsin, chymotrypsin and pepsin) cleaved peptides from larger proteins. RESULTS & DISCUSSION: This work focuses mainly on full-scan TOF data obtained under ESI conditions, the major mode of TOF operation in discovery bioanalytical research, where the compounds are selected based on their pharmacokinetic/pharmacodynamic behaviors using animal models prior to selecting a few desirable candidates for further development. Finally, important emerging TOF technologies that could potentially benefit bioanalytical research in the semi-quantification of metabolites without synthesized standards are discussed. Particularly, the utility of captive spray ionization coupled with TripleTOF 5600 was evaluated for improving sensitivity and providing normalized MS response for drugs and their metabolites. The workflow proposed compromises neither the efficiency, nor the quality of pharmacokinetic data in support of early drug discovery programs.

Development of an on-line automated sample clean-up method and liquid chromatography-tandem mass spectrometry analysis: application in an in vitro proteolytic assay.

Fluorescence detection has been a method of choice in industry for screening assays, including identification of enzyme inhibitors, owing to its high-throughput capabilities, excellent reproducibility, and sensitivity. Occasionally, inhibitors are identified that challenge the fluorescence assay limit, necessitating the development of more sensitive detection methods to assess these compounds. For data mining purposes, however, original assay conditions may be required. A direct method transfer to highly sensitive and specific LC-MS-based methods has not always been possible due to the presence of MS-incompatible neutral detergents and non-volatile salts in the assay matrix. Utilizing an in vitro proteolytic screening assay for the serine protease hepatitis C virus (HCV) nonstructural (NS) 3 protease as a test case, we report the development of an automated sample clean-up procedure implemented on-line with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to complement fluorescence detection. Ion exchange and peptide microtraps were employed to remove MS-incompatible assay matrix components. Three protease inhibitors were used to validate the MS/MS method. Comparable potencies were achieved for these compounds when assessed by fluorescence and MS/MS detection. Furthermore, four-fold less enzyme could be utilized when employing the MS/MS method compared to fluorescence detection. The longer analysis time, however, resulted in reduced sample capacity. The potency of our designed HCV NS3 protease inhibitors are thus routinely evaluated using a continuous fluorescence-based assay. Only pertinent inhibitors approaching the fluorescence assay sensitivity limit are subsequently analyzed further by LC-MS/MS. This methodology allows us to maintain a database and to compare results independent of the detection method. Despite the relatively slow sample turnaround time of this LC-MS approach, the versatility of the automated on-line clean-up procedure and sample analysis can be applied to assays containing reagents which were historically considered to be MS incompatible.

Analysis of M1G-dR in DNA by aldehyde reactive probe labeling and liquid chromatography tandem mass spectrometry.

A novel method for the measurement of pyrimido[1,2-a]purin-10(3H)one (M1G) has been developed. Previous methods for analysis of M1G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form. Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins. We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M1G-deoxyribose (ARP-M1G-dR) conjugate to minimize adduct loss. This conjugate has increased the hydrophobicity that enhances separation of ARP-M1G-dR from unmodified DNA nucleosides by using solid phase extraction. In addition, measuring ARP-M1G-dR by selective reaction monitoring (SRM) of the [ARP-M1G-dR + H]+ (635) --> [M1G + H]+ (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M1G-dR by SRM. For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used. High purity 15N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing (15NH4)2SO4. The 15N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M1G adducts to prepare the IS and AS DNA, respectively. A consistent calibration curve was established from the analysis of 200 microg of blank DNA, 23 ng of IS DNA (400 fmol of 15N5-M1G-dR), and AS DNA containing 0-810 fmol of M1G-dR. With the use of this novel IS DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures. By this assay, two M1G adducts/10(8) guanines can readily be detected. Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.

Quantitation of choline and its metabolites in tissues and foods by liquid chromatography/electrospray ionization-isotope dilution mass spectrometry.

Choline is important for normal membrane function, acetylcholine synthesis, lipid transport, and methyl metabolism. The U.S. National Academy of Sciences recently set requirements for choline in the human diet. In tissues and foods, there are multiple choline compounds that contribute to choline content. Betaine, a derivative of choline, is also important because of its role in donation of methyl groups to homocysteine to form methionine. Radioisotopic, high-pressure liquid chromatography, and gas chromatography/isotope dilution mass spectrometry (GC/IDMS) methods are available for measurement of choline. However, these existing methods are cumbersome and time-consuming, and none measures all of the compounds of interest. In this study, we describe a new method for quantitation of choline, betaine, acetylcholine, glycerophosphocholine, cytidine diphosphocholine, phosphocholine, phosphatidylcholine, and sphingomyelin in liver, plasma, various foods, and brain using liquid chromatography/electrospray ionization-isotope dilution mass spectrometry (LC/ESI-IDMS). Choline compounds were extracted by and partitioned into organic and aqueous phases using methanol and chloroform and analyzed directly by LC/ESI-IDMS without the need for isolation and derivatization of each compound separately as was required by the GC/IDMS method. The new LC/ESI-IDMS method was validated using the existing published GC/IDMS method.