Characterization of metabolism of (+)-praeruptorin B and (+)-praeruptorin E in human and rat liver microsomes by liquid chromatography coupled with ion trap mass spectrometry and time-of-flight mass spectrometry

Peucedani Radix is a Chinese medicinal herb noted for its effects on treatments of respiratory and pulmonary disorders. As a part of a systematic pharmacokinetic evaluation of the herb in our laboratory, the present study investigated, for the first time, the metabolic profile of (+)-praeruptorin B (dPB) and (+)-praeruptorin E (dPE), two main bioactive constituents of Peucedani Radix in pooled liver microsomes of rats (RLMs) and humans (HLMs). dPE was eliminated faster than dPB in both species. The incubation of dPB with RLMs and HLMs resulted in eight (B1-B8) and nine (B1-B9) metabolites, respectively, while both RLMs and HLMs converted dPE into 13 metabolites (E1-13). Structures of all the metabolites were proposed through comparing their mass data obtained via tandem mass spectrometry on an MSD ion trap system (IT-MS/MS) coupled with high-resolution mass measurement by time-of-flight mass spectrometry (TOF-MS) with those of the respective parent compound. B1 and E1 were unambiguously identified as (-)-cis-khellactone. The formations of all the metabolites were NADPH-dependent. Oxidation and hydrolysis were demonstrated to be two predominant metabolic pathways of dPB and dPE. Oxidation initiated at either the C-3' or C-4' substituent, while hydrolysis only started from the C-3' substituent. Fragmentation of all metabolites followed similar pathways to those of the parent pyranocoumarins. The information on metabolic properties of dPB and dPE and the mass fragmentation profiles of their metabolites obtained in the present study will aid in characterization of metabolic profiles of other angular-type pyranocoumarins and further investigation of in vivo fates of these pyranocoumarins and the herb.     

Total synthesis of (+)- and (-)-duryne: a potent anticancer agent from the marine sponge Cribrochalina dura. Establishment of the central double bond geometry and the absolute configuration of the chiral centers

Duryne is a C30 polyacetylenic alcohol with C2 symmetry. Despite its potent cytotoxicity, its central double bond geometry and the absolute configuration of the chiral centers were not determined. We report the total syntheses of both enantiomers of the anticancer natural product (+)-duryne and the establishment of its stereochemistry by synthesizing both geometric isomers. The natural (+)-duryne is identified as (15Z) and (3S,28S) as shown in structure 1. The autoxidation/Wittig coupling reaction was employed to synthesize the central (Z)-olefin. The stereochemistry of the (E)-alkene isomer was constructed stereoselectively by using LiAlH4 reduction of the corresponding alkyne. The absolute configurations of the chiral centers are established by using Burgess' enzymatic resolution procedure with Pseudomonas AK lipase.     

Determination of the absolute configuration of the enantiomers of dihydroquinolines, isolated by chiral chromatography, by non empirical analysis of circular dichroism spectra and X-ray analysis

The enantiomers of racemic 3,4-dihydroquinolines with an acetal or thioacetal spiro ring and a quaternary stereogenic carbon have been isolated through semipreparative chiral chromatography using a polysaccharide-derived chiral stationary phase (Chiralpak AD) and n-hexane/ethanol as a mobile phase. The absolute configurations of the enantiomers of four compounds have been determined by a comparison of density functional theory (DFT) calculations of their electronic circular dichroism (ECD) spectra with the experimental ECD data. A detailed conformer population search to achieve a conformational average of these compounds was crucial, due to the flexibility of these molecules. The conformer distribution was evaluated by spartan 02 and the structure of each of the conformers found within 4 kcal/mol energy range was optimized with DFT. The final calculated ECD spectrum obtained after Boltzmann averaging was compared with the ECD spectrum of the less well retained enantiomer and the correlation (R)/(−) was established for all compounds. The monocrystals of both enantiomers of one compound were obtained from the HPLC eluates. Their absolute configurations were determined by X-ray crystallographic analysis and confirmed by ECD analysis. In all cases, the second-eluted enantiomer in chiral HPLC exhibits an (R)-configuration.     

Synthesis, resolution, and absolute configuration of chiral 4,4'-bipyridines

A chiral polyhalogenated 4,4'-bipyridine derivative is described allowing an easy access to a new family of chiral 4,4'-bipyridines by site-selective cross-coupling reactions. The absolute configurations of all the HPLC separated enantiomers were determined by X-ray diffraction and electronic circular dichroism coupled with time-dependent density functional theory calculations.     

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography–tandem mass spectrometry with chiral derivatization

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey’s reagent, N ^α- (5-fluoro-2,4-dinitrophenyl)-d-leucinamide, without extraction. The diastereomers of the N ^α-(2,4-dinitrophenyl)-d-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03?μg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20?μg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.     

Isolation, absolute configuration, and chiral crystallization of optically active seleninic acid

An optically pure seleninic acid was isolated as stable crystals for the first time by chiral crystallization. The relationship between the absolute configurations and the circular dichroism spectra of the enantiomers could be determined by X-ray crystallographic analysis.     

Incarvilleatone, a new cyclohexylethanoid dimer from Incarvillea younghusbandii and its inhibition against nitric oxide (NO) release

Incarvilleatone (1), an unprecedented dimeric cyclohexylethanoid analog with a racemic nature, was isolated from the whole plant of Incarvillea younghusbandii. HPLC chiral separation of 1 gave two enantiomers (-)-incarvilleatone and (+)-incarvilleatone. The structure of 1 was established by spectroscopic methods and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. (-)-Incarvilleatone exhibited a potent inhibitory effect against NO production in LPS-induced RAW264.7 macrophages.     

A new approach to enantiopure inherently chiral calix[4]arenes: determination of their absolute configurations

A new approach to the synthesis of meta-substituted enantiopure inherently chiral calix[4]arenes by introducing L-Boc-proline as dual functions of the chiral auxiliary has been described. Moreover, the absolute configurations of the enantiomers were determined by CD spectra, X-ray crystallographic analysis, and the chemical derivative method.     

三唑农药的手性拆分及对映体的转化

利用Chiralcel OJ-H和Chiralcel OD-H手性柱对烯唑醇、三唑酮和三唑醇的手性拆分进行了研究,进一步测定了烯唑醇和三唑酮对映体的旋光性质,据此确定了两种农药对映体的绝对构型,在此基础上结合三唑酮转化为三唑醇的还原实验确定了三唑醇4个对映异构体的绝对构型。考察了几种三唑农药在有机溶剂和缓冲溶液中的手性稳定性,其中三唑酮在甲醇、乙醇和水中存在明显的对映体转化行为,而烯唑醇和三唑醇则是手性稳定的,升高温度及碱性环境会加快三唑酮的对映体转化。     

Identifying Enantiomers in Mixtures of Chiral Molecules with Broadband Microwave Spectroscopy

Chirality‐sensitive broadband microwave spectroscopy was performed on mixtures of carvone enantiomers and conformers to distinguish enantiomers, measure enantiomeric excesses, and determine the absolute configurations of the enantiomers. This method uses microwave three‐wave mixing and is inherently well‐suited to the analysis of mixtures—a unique advantage over other techniques. In contrast to conventional microwave spectroscopy, the phase of the received signal is also exploited. This phase depends upon the signs of the molecules’ dipole‐moment components and is used to identify the excess enantiomer. The measured signal amplitude determines the size of the excess. The broadband capabilities of the spectrometer were used to simultaneously excite and measure two conformers of carvone, demonstrating the analysis of a sample with multiple chiral species. Employing quantum chemical calculations and the measured phases, the absolute configurations of the enantiomers are determined.     

Discrimination of eight chloramphenicol isomers by liquid chromatography tandem mass spectrometry in order to investigate the natural occurrence of chloramphenicol

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg(-1). It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para-configuration and in the meta-configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta-stereoisomers and four para-stereoisomers. It is known that only RR-p-CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR-p-CAP and SS-p-CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.     

High-throughput synthesis and analysis of acylated cyanohydrins

The yields and optical purities of products obtained from chiral Lewis acid/Lewis base-catalysed additions of alpha-ketonitriles to prochiral aldehydes could be accurately determined by an enzymatic method. The amount of remaining aldehyde was determined after its reduction to an alcohol, whilst the two product enantiomers were analysed after subsequent hydrolysis first by the (S)-selective Candida antarctica lipase B and then by the unselective pig liver esterase. The method could be used for analysis of products obtained from a number of aromatic aldehydes and aliphatic ketonitriles. Microreactor technology was successfully combined with high-throughput analysis for efficient catalyst optimization.     

Chemoenzymatic synthesis of optically active 4-methyl-tetrahydro-5-oxo-2-furancarboxylic acids and esters

Enantiomerically pure 4-methyl-tetrahydro-5-oxo-2-furancarboxylic acids and esters are prepared by enzymatic resolution of the chiral racemic esters. Their stereochemistry as well as their absolute configurations have been established by chemical correlation. The influence of the alkoxycarbonyl group at C-2 and that of the methyl group at C-4 on the sign of the Cotton effect in their CD spectra have been investigated. Formation of enantiomerically pure hydroxydiesters, precursors of the above-mentioned γ-lactones, by baker’s yeast reduction of the corresponding ketodiesters was unsatisfactory.     

Enantiomer separation and indirect chromatographic absolute configuration prediction of chiral pirinixic acid derivatives: Limitations of polysaccharide-type chiral stationary phases in comparison to chiral anion-exchangers

Chiral α-arylthiocarboxylic acids with different substitution patterns, representing new pirinixic acid derivatives with dual PPARα/γ agonistic activities, have been separated into enantiomers on tert-butylcarbamoylquinine and quinidine based chiral anion-exchangers and amylose tris(3,5-dimethylphenylcarbamate) coated silica on analytical and preparative scale. Absolute configurations of individual enantiomers were assigned chromatographically via elution orders on the chiral anion-exchangers and were confirmed by stereoselective syntheses via Ewans auxiliaries that have lead to enantiomeric products with known absolute configurations. The results of both methods were in full agreement. Moreover, the receptor stereoselectivity in PPARα transactivation activities was consistent within the test set of structurally related compounds. Limited correlation (between elution order and substitution) was observed within the set of α-arylthiocarboxylic acids on the amylose tris(3,5-dimethylphenylcarbamate) based chiral stationary phase (CSP), in particular the elution order changed with remote substitution. This clearly demonstrates the risks of chromatographic absolute configuration assignments by prediction from one structural analog to another one, especially with CSPs such as polysaccharide CSPs that are recognized for their broad applicability due to multiple binding and chiral recognition modes. It is therefore of utmost importance that such chromatographic absolute configuration predictions by extrapolation to structural analogs are combined with orthogonal methods for verification of the results.     

Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated beta-CD

CE with multiple isomer sulfated beta-CD as the chiral selector was assessed for the simultaneous analysis of the enantiomers of ketamine and metabolites in extracts of equine plasma and urine. Different lots of the commercial chiral selector provided significant changes in enantiomeric ketamine separability, a fact that can be related to the manufacturing variability. A mixture of two lots was found to provide high-resolution separations and interference-free detection of the enantiomers of ketamine, norketamine, dehydronorketamine, and an incompletely identified hydroxylated metabolite of norketamine in liquid/liquid extracts of the two body fluids. Ketamine, norketamine, and dehydronorketamine could be unambiguously identified via HPLC fractionation of urinary extracts and using LC-MS and LC-MS/MS with 1 mmu mass discrimination. The CE assay was used to characterize the stereoselectivity of the compounds' enantiomers in the samples of five ponies anesthetized with isoflurane in oxygen and treated with intravenous continuous infusion of racemic ketamine. The concentrations of the ketamine enantiomers in plasma are equal, whereas the urinary amount of R-ketamine is larger than that of S-ketamine. Plasma and urine contain higher S- than R-norketamine levels and the mean S-/R-enantiomer ratios of dehydronorketamine in plasma and urine are lower than unity and similar.     

Screening flavonoid metabolites of naringin and narirutin in urine after human consumption of grapefruit juice by LC-MS and LC-MS/MS

The main flavonoids in grapefruit juice, naringin and narirutin, were quantified by LC-MS with structural differentiation by LC-MS/MS. After human consumption of grapefruit juice, urine samples were collected for 24 hours and screened for flavonoid metabolites by LC-MS. The metabolite structures (glucuronides, sulfates, and glucuronide sulfates) were then confirmed via their unique fragmentation patterns by LC-MS/MS. To further verify the identity of the common aglycon (naringenin) shared by the metabolites, enzymatic hydrolysis was performed and the resulting products were analyzed. This work demonstrates that LC-MS and LC-MS/MS techniques can be used for fast metabolite screening without extensive sample preparation.     

Chiral liquid chromatography contribution to the determination of the absolute configuration of enantiomers

The review covers examples in which chiral HPLC, as a source of pure enantiomers, has been combined with classical methods (X-ray, vibrational circular dichroism (VCD), enzymatic resolutions, nuclear magnetic resonance (NMR) techniques, optical rotation, circular dichroism (CD)) for the on- or off-line determination of absolute configuration of enantiomers. Furthermore, it is outlined that chiral HPLC, which associates enantioseparation process and classical purification process, opens new perspectives in the classical determination of absolute configuration by chemical correlation or chemical interconversion methods. The review also contains a discussion about the various approaches to predict the absolute configuration from the retention behavior of the enantiomers on chiral stationary phases (CSPs). Some examples illustrate the advantages and limitations of molecular modeling methods and the use of chiral recognition models. The assumptions underlying some of these methods are critically analyzed and some possible emerging new strategies are outlined.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Total syntheses of (+)- and (−)-1,3,4,5-tetragalloylapiitol and revision of absolute configuration of naturally occurring (−)-1,3,4,5-tetragalloylapiitol

The total syntheses of (+)- and (−)-1,3,4,5-tetragalloylapiitol were achieved in seven steps from d- and l-ribose, respectively. By comparing the optical rotations of both enantiomers with those of the natural product, the absolute configuration at C-3 in the naturally occurring 1,3,4,5-tetragalloylapiitol has been revised to R. The absolute configurations at C-3 in the synthetic (+)- and (−)-1,3,4,5-tetragalloylapiitol were further confirmed by the circular dichroism exciton chirality method.     

The Bip method, based on the induced circular dichroism of a flexible biphenyl probe in terminally protected -Bip-Xaa*- dipeptides, for assignment of the absolute configuration of beta-amino acids

An induced axial chirality of the biphenyl core of the Bip (2',1':1,2;1',2':3,4-dibenzcyclohepta-1,3-diene-6-amino-6-carboxylic acid) residue in the terminally protected dipeptides Boc-Bip-beta-Xaa*-OMe (beta-Xaa* = L-beta(3)-HAla, L-beta(3)-HVal, L-beta(3)-HLeu, L-beta(3)-HPro, trans-(1S,2S)-ACHC, trans-(1R,2R)-ACHC, trans-(1S,2S)-ACPC, trans-(1R,2R)-ACPC) resulted in an induced circular dichroism, revealing the usefulness of the Bip method for a reliable and fast assignment of the absolute configuration of chiral beta-amino acids. Remarkably, the Bip method was also applied to the unique spin-labeled, cyclic, beta-amino acids cis/trans-beta-TOAC and trans-POAC. In particular, this study allowed the assignment of the unknown absolute configurations of the enantiomers of the latter compound.