Robust and Comprehensive Targeted Metabolomics Method for Quantification of 50 Different Primary, Secondary, and Sulfated Bile Acids in Multiple Biological Species (Human, Monkey, Rabbit, Dog, and Rat) and Matrices (Plasma and Urine) Using Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) Analysis.

Bile acids (BAs) are biomolecules synthesized in the liver from cholesterol and are constituents of bile. The in-vivo BA pool includes more than 50 known diverse BAs which are unconjugated, amino acid conjugated, sulfated, and glucuronidated metabolites. Hemostasis of bile acids is known to be highly regulated and an interplay between liver metabolism, gut microbiome function, intestinal absorption, and enterohepatic recirculation. Interruption of BA homeostasis has been attributed to several metabolic diseases and drug induced liver injury (DILI), and their use as potential biomarkers is increasingly becoming important. Speciated quantitative and comprehensive profiling of BAs in various biomatrices from humans and preclinical animal species are important to understand their significance and biological function. Consequently, a versatile one single bioanalytical method for BAs is required to accommodate quantitation in a broad range of biomatrices from human and preclinical animal species. Here we report a versatile, comprehensive, and high throughput liquid chromatography-high resolution mass spectrometry (LC-HRMS) targeted metabolomics method for quantitative analysis of 50 different BAs in multiple matrices including human serum, plasma, and urine and plasma and urine of preclinical animal species (rat, rabbit, dog, and monkey). The method has been sufficiently qualified for accuracy, precision, robustness, and ruggedness and addresses the issue of nonspecific binding of bile acids to plastic for urine samples. Application of this method includes comparison for BA analysis between matched plasma and serum samples, human and animal species differences in BA pools, data analysis, and visualization of complex BA data using BA indices or ratios to understand BA biology, metabolism, and transport.

Determination of a novel antifibrotic small molecule GDC-3280 in human plasma and urine by liquid chromatography tandem mass spectrometry to support its first-in-human clinical trial.

A specific and robust LC-MS/MS method was developed and validated for the quantitative determination of GDC-3280 in human plasma and urine. The nonspecific binding associated with urine samples was overcome by the addition of CHAPS. The sample volume was 25 µL for either matrix, and supported liquid extraction was employed for analyte extraction. d6-GDC-3280 was used as the internal standard. Linear standard curves (R2 > 0.9956) were established from 5.00 to 5000 ng/mL in both matrices with quantitation extended to 50,000 ng/mL through dilution. In plasma matrix, the precision (RSD) ranged from 1.5 to 9.9% (intra-run) and from 2.4 to 7.2% (inter-run); the accuracy (RE) ranged from 96.1 to 107% (intra-run) and from 96.7 to 104% (inter-run). Similarly, in urine the precision was 1.5-6.2% (intra-run) and 1.9-6.1% (inter-run); the accuracy was 83.1-99.3% (intra-run) and 87.1-98.3% (inter-run). Good recovery (>94%) and negligible matrix effect were achieved in both matrices. Long-term matrix stability was established for at least 703 days in plasma and 477 days in urine. Bench-top stability of 25 h and five freeze-thaw cycles were also confirmed in both matrices. The method was successfully implemented in GDC-3280's first-in-human trial for assessing its pharmacokinetic profiles.

A validated surrogate analyte LC-MS/MS assay for quantitation of endogenous kynurenine and tryptophan in human plasma.

AIM: Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) catalyze the initial and rate-controlling step of tryptophan metabolism through the kynurenine pathway, which plays an important role in mediating immune response. Accurate measurement of tryptophan and kynurenine is critical for monitoring the activity of IDO/TDO. Experimental: Surrogate analytes ([15N2]-Tryptophan and [13C6]-Kynurenine) were used for preparation of calibration standard and quality control. A fit-for-purpose validation using an approach of surrogate analyte and authentic matrix was carried out. RESULTS: Acid precipitation was used in sample preparation, which yielded good recovery without significant matrix effect. Precision and accuracy results were well within the acceptance criteria. The assay demonstrated successful application to a clinical study to confirm a transient depletion of kynurenine upon IDO inhibition. CONCLUSION: A robust, specific and simple LC-MS/MS method was developed and validated with a fit-for-purpose style for measuring tryptophan and kynurenine in human plasma samples.

Definitive profiling of plasma bile acids as potential biomarkers for human liver diseases using UPLC-HRMS.

AIM: Investigation of bile acids (BAs) as biomarkers for liver and kidney diseases has gained momentum recently to fulfill the needs in drug development and clinical practice, but a thorough and rapid profiling of BAs in human plasma has been hindered by the large interindividual variability and lack of selective methods. RESULTS: A selective and efficient UPLC-high resolution mass spectrometry method was developed and fully validated for the definitive profiling of 26 BAs in human plasma with a curve rage of 1-1000 ng/ml and a runtime of 7.2 min. CONCLUSION: Four BA combinations with good sensitivity and specificity show potential biomarker applications for liver injury and diseases.

Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Development of a multi-matrix LC-MS/MS method for urea quantitation and its application in human respiratory disease studies.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.

Simultaneous determination of itraconazole, hydroxy itraconazole, keto itraconazole and N-desalkyl itraconazole concentration in human plasma using liquid chromatography with tandem mass spectrometry.

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ) and N-desalkyl itraconazole (ND-ITZ) concentration in human plasma. One hundred and fifty microliters of human plasma were extracted using a solid-supported liquid extraction (SLE) method and the final extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. The standard curve range is 5-2500 ng/mL for ITZ and OH-ITZ and 0.4-200 ng/mL for keto-ITZ and ND-ITZ. The curve was fitted to a 1/x(2) weighted linear regression model for all analytes. The precision and accuracy of the LC-MS/MS assay based on the five analytical quality control (QC) levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery was 97.4% for ITZ, 112.9% for OH-ITZ, 103.4% for keto-ITZ, and 102.3% for ND-ITZ across their respective curve range. Matrix factor was close to 1.0 at both high and low QC levels of all 4 analytes, which indicates minimal ion suppression or enhancement in our validated assay. Itraconazole and all three metabolites are stable in human plasma for 145 days stored at -70 °C freezers. The validated assay was successfully applied to a clinical study, which has a drug-drug interaction (DDI) arm using ITZ as a cytochrome P450, family 3, subfamily A (CYP3A) inhibitor.

Design and synthesis of 4-heteroaryl 1,2,3,4-tetrahydroisoquinolines as triple reuptake inhibitors.

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

Isolation and identification of five impurities of ALB 109564(a) drug substance.

Four low-level impurities were detected during the development and scale up of the synthesis of ALB 109564(a). These impurities were isolated and characterized in order to determine how they originated in the drug substance. The information allowed the elimination of one impurity and a significant reduction in the relative abundance of the other three. A fifth impurity was detected in an accelerated stability study sample of the drug substance. The degradant was found to be the free acid resulting from the hydrolysis of the methyl ester within the indoline moiety of ALB 109564(a). The characterization of this impurity allowed for changes in the handling of the drug substance which minimized the formation of the impurity.

Two minor diterpene glycosides from the leaves of Stevia rebaudiana.

Two new new diterpene glycosides, 13-[(2-O-(6-O-beta-D-glucopyranosyl)-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy] kaur-16-en-18-oic acid beta-D-glucopyranosyl ester (1) and 13-[(2-O-beta-D-glucopyranosyl-3-O-beta-D-fructofuranosyl-beta-D-glucopyranosyl)oxy] kaur-16-en-18-oic acid beta-D-glucopyranosyl ester (2) were isolated from the leaves of Stevia rebaudiana, along with the known steviol glycosides stevioside, rebaudiosides A-F and dulcoside A. The structures of the two new compounds were established on the basis of extensive 2D NMR (COSY, HSQC, and HMBC), MS and chemical studies.

Gas-phase intramolecular elimination reaction studies of steviol glycosides in positive electrospray and tandem mass spectrometry.

This paper reports the first study of the gas-phase intramolecular elimination reaction of steviol glycosides in positive electrospray mass spectrometry. The observed glycosylated product ions are proposed to be formed via an intramolecular elimination of sugar units from the parent molecule ion. It was further proven by MS/MS studies and deuterium labeling experiments with one of the steviol glycosides, rebaudioside A. These mass spectrometric results confirmed that the new glycosylated product ions observed are most likely formed by the combination of glucose moieties (Glu) II-IV and Glu I via a gas-phase intramolecular elimination reaction.

Citreamicins with potent gram-positive activity.

Two new xanthone antibiotics, citreamicin delta (1) and epsilon (2), with potent activity against Gram-positive pathogens including multidrug-resistant Staphylococcus aureus (MDRSA) were discovered. Compounds 1 and 2 exhibited MIC values < 1 microg/mL versus a number of resistant strains. The compounds were obtained from EtOAc extracts of Streptomyces vinaceus and were purified by countercurrent chromatography and reversed-phase HPLC. Their structures were elucidated using primarily NMR and mass spectroscopy.

Mutactimycin E, a new anthracycline antibiotic with gram-positive activity.

Resistance to currently available antibiotics has become a widely recognized crisis in the medical community. To address this, many companies and researchers are refocusing their attention towards natural products, which have an excellent track record of producing effective antibacterial drugs. The AMRI natural product library was screened for activity against multi-drug resistant Staphylococcus aureus (MDRSA). The active samples were counter screened for cytotoxicity against the human hepatocellular carcinoma HepG2 cell line to determine an in vitro therapeutic index (in vitro TI). Those samples with a high in vitro TI were selected for fractionation and dereplication. This led to the discovery of a new anthracycline structure. This metabolite, named mutactimycin E (1), exhibited moderate activity against several gram positive organisms. Here we report the isolation, structure elucidation and biological activities of this new compound.

Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography-electrospray ionization mass spectrometry.

Unusual amino acids such as beta-methoxytyrosine (beta-MeOTyr), allo-threonine (allo-Thr) and allo-isoleucine (allo-Ile) were derivatized with N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among beta-MeOTyr isomers was poor. The best separation of beta-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.

Gymnangiamide, a cytotoxic pentapeptide from the marine hydroid Gymnangium regae.

A cytotoxic aqueous extract from the marine hydroid Gymnangium regae provided a novel linear pentapeptide, designated gymnangiamide (1). The planar structure of 1 was elucidated by interpretation of spectral data as well as chemical degradation and derivatization studies. In addition to the amino acids isoleucine and phenylserine, this peptide contained N-desmethyldolaisoleuine, O-desmethyldolaproine, and alpha-guanidino serine, three residues that have not previously been reported in a natural product. The absolute configurations of the constituent amino/guanidino acids were determined by chemical degradation and derivatization, followed by HPLC and LC-MS comparison with authentic standards. Gymnangiamide (1) was moderately cytotoxic against a number of human tumor cell lines in vitro.

Cyclonellin, a new cyclic octapeptide from the marine sponge Axinella carteri.

Cyclonellin (1), a new cyclic octapeptide, was isolated from an aqueous extract of the marine sponge Axinella carteri. Its structure was elucidated by interpretation of NMR spectral data of the intact compound and N-terminal Edman sequencing of linear peptide fragments obtained by partial hydrolysis of 1. The absolute configurations of the constituent amino acids were determined by acid hydrolysis, derivitization with FDAA, and LC-MS analyses.

Caulibugulones A-F, novel cytotoxic isoquinoline quinones and iminoquinones from the marine bryozoan Caulibugula intermis.

An extract of the marine bryozoan Caulibugula intermis, collected in the Indo-Pacific off Palau, produced a distinct pattern of differential cytotoxicity in the National Cancer Institute's 60 cell line antitumor screen. Bioactivity-directed fractionation of the extract provided six new compounds, caulibugulones A-F (1-6). The structures of these novel metabolites were determined by spectrochemical analyses including LC-MS, HRFABMS, 1-D and 2-D NMR experiments, and by comparison with related compounds. The structures of compounds 2 and 3 were confirmed by chemical interconversion. The isolated compounds exhibited IC(50)'s of 0.03-1.67 microg/mL against murine tumor cells in an in vitro cytotoxicity assay.

Geographic distribution of three alkaloid chemotypes of Croton lechleri.

Three known alkaloids, isoboldine (2), norisoboldine (1), and magnoflorine (8), have been isolated for the first time from Croton lechleri, a source of the wound healing latex "sangre de grado". An HPLC system was developed, and a large number of latex and leaf samples of C. lechleri from 22 sites in northern Peru and Ecuador were analyzed to gain an understanding of the natural variation in alkaloid content for the species. Up to six alkaloids were found to occur in the leaves including, in addition to those listed above, thaliporphine (3), glaucine (4), and taspine (9), whereas the latex contained only 9. Taspine (9) is the component that has been previously found to be responsible for the wound healing activity of C. lechleri latex, and its mean concentration throughout the range examined was found to be 9% of the latex by dry weight. In addition, three chemotypes are defined based on the alkaloid content of the leaves, and the geographic distribution of these chemotypes is discussed along with a quantitative analysis of the alkaloid content as a function of chemotype.