Application of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization for enhanced peptide mapping analysis of non-biological complex drug glatiramer acetate using liquid chromatography/electrospray ionization collision-induced dissociation high-resolution mass spectrometry.

RATIONALE: Glatiramer acetate (GA) (Copaxone®) is a non-biological complex drug (NBCD) comprising random-sequence polymer chains of four amino acids (MW ~ 5-9 kDa) with unknown structure. The characterization of NBCDs by reversed-phase liquid chromatography/mass spectrometry (RPLC/MS) peptide mapping is often impeded by insufficient separation and/or low sensitivity. To overcome this issue, pre-column derivatization of GA peptide digest was used to improve RPLC/MS detectability and to generate a comprehensive peptide profile. METHODS: Amino groups of peptides generated by trypsin digestion of GA were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent. The derivatized mixture of random-sequence peptides was analyzed by liquid chromatography/positive-mode electrospray ionization collision-induced dissociation high-resolution mass spectrometry (RPLC/ESI-CID-HRMS/MS). Data-independent LC/MSE mode was used for data acquisition. RESULTS: The derivatization of the GA peptide mixture increased the detectability of RPLC/ESI-CID-HRMS/MS analysis. The efficacy of the procedure was demonstrated by using selected peptides related to different polymeric chain origins. The resultant peptides were derivatized in a predictable manner giving a minimum of side products. The reproducibility of the developed method was demonstrated by comparing peptide elution profiles derived from six Copaxone® lots. CONCLUSIONS: Application of the AQC pre-column derivatization provides a framework that could be used as an attractive approach for monitoring the quality and characterization of NBCD products in the pharmaceutical industry.

In Vivo Targeted Metabolomic Profiling of Prostanit, a Novel Anti-PAD NO-Donating Alprostadil-Based Drug.

Prostanit is a novel drug developed for the treatment of peripheral arterial diseases. It consists of a prostaglandin E1 (PGE1) moiety with two nitric oxide (NO) donor fragments, which provide a combined vasodilation effect on smooth muscles and vascular spastic reaction. Prostanit pharmacokinetics, however, remains poorly investigated. Thus, the object of this study was to investigate the pharmacokinetics of Prostanit-related and -affected metabolites in rabbit plasma using the liquid chromatography-mass spectrometry (LC-MS) approach. Besides, NO generation from Prostanit in isolated rat aorta and human smooth muscle cells was studied using the Griess method. In plasma, Prostanit was rapidly metabolized to 1,3-dinitroglycerol (1,3-DNG), PGE1, and 13,14-dihydro-15-keto-PGE1. Simultaneously, the constant growth of amino acid (proline, 4-hydroxyproline, alanine, phenylalanine, etc.), steroid (androsterone and corticosterone), and purine (adenosine, adenosine-5 monophosphate, and guanosine) levels was observed. Glycine, aspartate, cortisol, and testosterone levels were decreased. Ex vivo Prostanit induced both NO synthase-dependent and -independent NO generation. The observed pharmacokinetic properties suggested some novel beneficial activities (i.e., effect prolongation and anti-inflammation). These properties may provide a basis for future research of the effectiveness and safety of Prostanit, as well as for its characterization from a clinical perspective.

Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.

Rabbit plasma metabolomic analysis of Nitroproston®: a multi target natural prostaglandin based-drug.

INTRODUCTION: Nitroproston® is a novel multi-target drug bearing natural prostaglandin E2 (PGE2) and nitric oxide (NO)-donating fragments for treatment of inflammatory and obstructive diseases (i.e., asthma and obstructive bronchitis). OBJECTIVES: To investigate the effects of Nitroproston® administration on plasma metabolomics in vivo. METHODS: Experimental in vivo study randomly assigning the target drug (treatment group) or a saline solution without the drug (vehicle control group) to 12 rabbits (n = 6 in each group). Untargeted (5880 initial features; 1869 negative-4011 positive ion peaks; UPLC-IT-TOF/MS) and 84 targeted moieties (Nitroproston® related metabolites, prostaglandins, steroids, purines, pyrimidines and amino acids; HPLC-QQQ-MS/MS) were measured from plasma at 0, 2, 4, 6, 8, 12, 18, 24, 32 and 60 min after administration. RESULTS: PGE2, 13,14-dihydro-15-keto-PGE2, PGB2, 1,3-GDN and 15-keto-PGE2 increased in the treatment group. Steroids (i.e., cortisone, progesterone), organic acids, 3-oxododecanoic acid, nicotinate D-ribonucleoside, thymidine, the amino acids serine and aspartate, and derivatives pyridinoline, aminoadipic acid and uric acid increased (p < 0.05 AUCROC curve > 0.75) after treatment. Purines (i.e., xanthine, guanine, guanosine), bile acids, acylcarnitines and the amino acids L-tryptophan and L-phenylalanine were decreased. Nitroproston® impacted steroidogenesis, purine metabolism and ammonia recycling pathways, among others. CONCLUSION: Nitroproston®, a multi action novel drug based on natural prostaglandins, altered metabolites (i.e., guanine, adenine, cortisol, cortisone and aspartate) involved in purine metabolism, urea and ammonia biological cycles, steroidogenesis, among other pathways. Suggested mechanisms of action, metabolic pathway interconnections and useful information to further understand the metabolic effects of prostaglandin administration are presented.

Target Metabolome Profiling-Based Machine Learning as a Diagnostic Approach for Cardiovascular Diseases in Adults.

Metabolomics is a promising technology for the application of translational medicine to cardiovascular risk. Here, we applied a liquid chromatography/tandem mass spectrometry approach to explore the associations between plasma concentrations of amino acids, methylarginines, acylcarnitines, and tryptophan catabolism metabolites and cardiometabolic risk factors in patients diagnosed with arterial hypertension (HTA) (n = 61), coronary artery disease (CAD) (n = 48), and non-cardiovascular disease (CVD) individuals (n = 27). In total, almost all significantly different acylcarnitines, amino acids, methylarginines, and intermediates of the kynurenic and indolic tryptophan conversion pathways presented increased (p < 0.05) in concentration levels during the progression of CVD, indicating an association of inflammation, mitochondrial imbalance, and oxidative stress with early stages of CVD. Additionally, the random forest algorithm was found to have the highest prediction power in multiclass and binary classification patients with CAD, HTA, and non-CVD individuals and globally between CVD and non-CVD individuals (accuracy equal to 0.80 and 0.91, respectively). Thus, the present study provided a complex approach for the risk stratification of patients with CAD, patients with HTA, and non-CVD individuals using targeted metabolomics profiling.

Pharmacokinetic Properties of the Novel Synthetic Cannabinoid 5F-APINAC and Its Influence on Metabolites Associated with Neurotransmission in Rabbit Plasma.

The strong psychoactive effects of synthetic cannabinoids raise the need for the deeper studying of their neurometabolic effects. The pharmacokinetic properties of 5F-APINAC and its influence on metabolomics profiles associated with neurotransmission were investigated in rabbit plasma. Twelve rabbits divided into three groups received 1-mL 5F-APINAC at 0.1, 1 and 2 mg/kg. The intervention groups were compared with the controls. Sampling was performed at nine time points (0-24 h). Ultra-high-performance liquid chromatography-tandem mass spectrometry was used. The pharmacokinetics were dose-dependent (higher curve at a higher dose) with a rapid biotransformation, followed by gradual elimination within 24 h. The tryptophan concentrations abruptly decreased (p < 0.05) in all tested groups, returning to the basal levels after 6 h. 5-hydroxylindole acetic acid increased (p < 0.05) in the controls, but this trend was absent in the treated groups. The aspartic acid concentrations were elevated (p < 0.001) in the treated groups. L-kynurenine was elevated (p < 0.01) in the intervention groups receiving 1 mg/kg to 2 mg/kg. Dose-dependent elevations (p < 0.01) were found for kynurenic acid, xanthurenic acid and quinolinic acid (p < 0.01), whereas the anthranilic acid trends were decreased (p < 0.01). The indole-3-propionic acid and indole-3-carboxaldehyde trends were elevated (p < 0.05), whereas the indole-3-lactic acid trajectories were decreased (p < 0.01) in the intervention groups. 5F-APINAC administration had a rapid biotransformation and gradual elimination. The metabolites related to the kynurenine and serotonergic system/serotonin pathways, aspartic acid innervation system and microbial tryptophan catabolism were altered.

LC-MS/MS Identification and Structural Characterization of Main Biodegradation Products of Nitroproston - A Novel Prostaglandin-based Pharmaceutical Compound.

BACKGROUND: Nitroproston is a novel prostaglandin-based compound modified by NOdonating groups with potential application in obstructive respiratory diseases such as asthma and obstructive bronchitis. Nitroproston has been extensively studied using various pharmacological models. Its biological stability is still uncertain. OBJECTIVE: The aim of the present study was to evaluate Nitroproston stability in vitro, as well as to identify and characterize its major biodegradation products. METHODS: The principal biodegradation products of Nitroproston were identified in vitro using liquid chromatography/ion trap - time-of-flight mass-spectrometry. The postulated structure of metabolites was confirmed using authentic reference standards. Rat, rabbit and human plasma and human whole blood samples were used for comparative in vitro degradation study. Nitroproston and its biodegradation products in biological samples were measured by liquid chromatography/triple -stage quadrupole mass spectrometry. RESULTS: Nitroproston is rapidly hydrolyzed in rat plasma to generate glycerol-1,3-dinitrate and prostaglandin E2. The latter can undergo conversion to cyclopentenone prostaglandins A2 and B2. Thereby less than 5% of the parent compound was observed in rat plasma at the first moment of incubation. A similar pattern was observed for rabbit plasma where half-life (T1/2) of Nitroproston was about 2.0 minutes. Nitroproston biodegradation rate for human plasma was the slowest (T1/2 = 2.1 h) among tested species, occurred more rapidly in whole blood (T1/2 = 14.8 min). CONCLUSION: It was found that Nitroproston is rapidly hydrolyzed in rodent compared to human plasma incubations. Whereas Nitroproston is relatively stable in human plasma an enhanced hydrolytic activity was observed in whole human blood incubations. Extensive metabolism of Nitroproston in human whole blood was mainly associated with red blood cells. The observed interspecies variability highlights the need of suitable animal model selection for Nitroproston follow-up PK/PD studies.

HPLC-MS/MS method for the simultaneous quantification of desmethylmebeverine acid, mebeverine acid and mebeverine alcohol in human plasma along with its application to a pharmacokinetics study.

A new simple, rapid and sensitive high pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for simultaneous analysis of mebeverine metabolites as: mebeverine alcohol (MAL), mebeverine acid (MAC) and desmethylmebeverine acid (DMAC) in human plasma. Sample preparation was performed by protein precipitation following the separation of analytes using an Acquity UPLC BEN C8 column 1.7 mm 2.1×50mm (Waters, USA). 2H5-desmethylmebeverine acid (2H5-DMAC) was used as the internal standard (IS). The proposed method was validated with linear ranges of 0.1-10ng/mL; 1-100ng/mL and 5-1000ng/mL for MAL, MAC and DMAC, respectively. Accuracy for all analytes (%RE), given as deviation between nominal and measured concentration and assay variability (CV) ranged from -4.04% to 4.60% and from 0.31% to 6.43% respectively both for within- and between-run. The overall recoveries for all metabolites were above 85%. The proposed method was used successfully for analysis of real samples from a pharmacokinetics study.

Liquid chromatography electrospray ionization tandem mass spectrometry for the detection of mesocarb abuse in horse doping.

A method is described for the determination of mesocarb abuse in equestrian sport by combining gradient liquid chromatography and electrospray ionization tandem mass spectrometry. Mesocarb was administrated orally to two horses at a dose of 50 µg/kg. Urine samples were collected up to 120 h post administration. Hydrolyzed and conjugated urine fractions were handled using liquid-liquid extraction (LLE). The identity of the parent drug and metabolites was confirmed using liquid chromatography combined with tandem mass spectrometry (MS/MS). Mesocarb and seven metabolites were detected in horse urine. Mono- and two di-hydroxylated metabolites were the main metabolites observed in horse urine samples. Based on the differences in MS/MS spectra it was supposed that these metabolites were been formed by the hydroxylation of the phenylisopropyl moiety of mesocarb whilst the main process of hydroxylation of mesocarb in human occurred in the phenylcarbamoyl moiety. The main metabolites were almost completely glucuroconjugated. Minor metabolites such as p-hydroxymesocarb and three di-hydroxylated metabolites together with parent mesocarb were also presented in the free urine fraction. This study has shown that two mono- and two di-hydroxylated metabolites are useful for controlling the abuse of mesocarb in horses.