Individual Enantiomers Research Articles

Enantiomeric Fraction Determination of 2‐Arylpropionic Acids in a Package Plant Membrane Bioreactor

Enantiomeric compositions of three 2-arylpropionic acid (2-APA) drugs, ibuprofen, naproxen, and ketoprofen, were monitored in a membrane bioreactor (MBR) treating municipal effluent in a small rural town in Australia. Specific enantiomers were determined as amide diastereomers using the chiral derivatizing reagent, (R)-1-phenylethylamine (PEA), followed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The six individual enantiomers were quantified by isotope dilution and the enantiomeric fractions (EFs) were determined. Over four separate sampling events, ibuprofen EF ranged from 0.88 to 0.94 (median 0.93) in the influent and 0.38 to 0.40 (median 0.39) in the effluent. However, no significant change in ketoprofen EF was observed, with influent EFs of 0.56-0.60 (median 0.58) and effluent EFs 0.54-0.68 (median 0.56). This is the first report of enantiospecific analysis of ketoprofen in municipal wastewater and it is not yet clear why such different behavior was observed compared to ibuprofen. Naproxen EF was consistently measured at 0.99 in the influent and ranged from 0.86 to 0.94 (median 0.91) in the effluent. This study demonstrates that EF is a relatively stable parameter and does not fluctuate according to concentration or other short-term variables introduced by sampling limitations. The enantiospecific analysis of chiral chemicals presents a promising approach to elucidate a more thorough understanding of biological treatment processes and a potential tool for monitoring the performance of key biological pathways.

Chiral high-performance liquid chromatographic separation of evodiamine enantiomers and rutaecarpine, isolated from Evodiae fructus

A rapid, simple and sensitive chiral HPLC method was developed and validated for quantification of biologically important alkaloids namely evodiamine enantiomers and rutaecarpine in Evodiae fructus using diphenhydramine as the internal standard (IS). Chromatographic separations were performed on a Chiralpak AD-H® column (250mm×4.6mm i.d., 5μm) with elution of n-hexane–2-propanol–ethanol (70:20:10, v/v/v) in a flow rate of 0.7ml/min and at λmax 225nm. To identify the order of elution, small quantities of the each evodiamine enantiomer were isolated by semi preparative HPLC method. Extraction samples were prepared by a simple solid phase extraction (SPE) method. All calibration curves showed good linearity (r2≥0.999) within the test ranges. The LOD and LOQ were lower than 0.05 and 0.1μg/ml, respectively. The RSDs of intra- and interday for relative peak areas of three analytes to IS were less than 3.2 and 2.5%, respectively, and the recoveries were 98.0–103.7%. The validated method was successfully applied to the quantitative analysis of three constituents in 13 batches of samples collected from market. The results showed that S-(+)-evodiamine was the main component while R-(−)-evodiamine was present in low concentration. This study provides a qualitative and quantitative method for analysis of evodiamine enantiomers and rutaecarpine, and should be extendable to pharmacological and toxicological studies of the individual evodiamine enantiomers.

Abstract 3357: CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide.

Abstract IMiD-class compounds, including thalidomide, lenalidomide, and pomalidomide, have been developed as racemic mixtures of S- and R-enantiomers. The isolated enantiomers of thalidomide are known to have distinct biological activities. For example, the well-documented sedative effects of thalidomide are correlated with the R-enantiomer (Eriksson et al., 2000), whereas S-thalidomide exhibits enhanced potency for TNF-α inhibition compared to the R-enantiomer (Wnendt et al., 1996; Moreira et al., 1993). We have demonstrated that S-lenalidomide is more potent than racemic or R-lenalidomide in biological activities that are believed to be important for clinical efficacy of lenalidomide. Due to facile in vivo conversion, isolated S-enantiomers of IMiDs have not been developed clinically. Lenalidomide (Revlimid®) is a racemic mixture of S- and R-enantiomers that interconvert through epimerization. Revlimid is labeled for the treatment of 5q-myelodysplastic syndromes (MDS) and multiple myeloma. CTP-221 is a deuterium-modified analog of S-lenalidomide containing deuterium atoms at key positions including lenalidomide's chiral center. Deuterium modification has the potential, albeit unpredictably, to alter the metabolic fate and hence the pharmacokinetic disposition of drugs, especially those that are biotransformed via the cleavage of carbon-hydrogen bonds (Fisher et al, 2006). The effect of deuterium modification on the epimerization rate and pharmacokinetic profile of CTP-221 was investigated in vitro and in vivo in mice, rats and monkeys. The rates of epimerization of CTP-221 and S-lenalidomide were compared in vitro in whole blood from mouse, rat, monkey, and human. It was found that CTP-221 was 2- to 3-fold more stable to epimerization than S-lenalidomide in the four species. To compare the in vivo pharmacokinetic profiles of CTP-221 and racemic lenalidomide, the compounds (10 mg/kg, PO) were administered to mice, rats and monkeys. In all three species, the exposure (AUC) of S- and R-lenalidomide following administration of racemic lenalidomide was about 50-57% and 43-50%, respectively, of the sum of the AUC of the individual enantiomers. However, when CTP-221 was administered, the AUC of CTP-221 and the deuterated R-enantiomers formed in vivo were about 96-99% and 1-4% respectively, of the sum of the AUC of the two enantiomers. Thus, CTP-221 epimerizes to a minimal extent in vivo and its administration provides exposure to very low levels of the R-enantiomer. In conclusion, the stabilization of CTP-221 via deuterium substitution resulted in maximal exposure to the more potent S-enantiomer and minimal exposure to the R-enantiomer. As a result, CTP-221 has the potential for improved potency and therapeutic index in comparison to racemic lenalidomide. Citation Format: Vinita Uttamsingh, Richard Gallegos, Changfu Cheng, Ara Aslanian, Julie Fields Liu, Roger Tung, Lijun Wu. CTP-221, a deuterated S-enantiomer of lenalidomide, is greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3357. doi:10.1158/1538-7445.AM2013-3357

TheCYP2B6*6Allele Significantly Alters theN-Demethylation of Ketamine Enantiomers In Vitro

Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K(m)) for the high-affinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V(max) and K(m) values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N'N'-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 μM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 μM) or the monoclonal antibody against CYP3A4 inhibited ketamine N-demethylation at clinically relevant concentrations. The degree of inhibition was significantly reduced in HLMs with the CYP2B6*6 allele (gene-dose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.

A Single Gas Chromatography/Mass Spectrometry Method for Detection of Individual Enantiomers of a Broad Spectrum of G‐Type Chemical Warfare Nerve Agents

The organophosphorus (OP) nerve agents are among the most toxic chemical compounds known. A subset of OP nerve agents known as G‐series compounds (including GA (tabun), GB (sarin), GD (soman) and GF (cyclosarin)) all share a chiral center at the phosphorus atom, resulting in a pair of stereoisomers designated as P(+) and P(−); the in vitro capacity to inhibit acetylcholinesterase as well as the in vivo toxicity of G‐agents is associated predominantly with the P(−) isomer of each compound. We have developed a gas chromatography/mass spectrometry (GC/MS) method to analyze the stereoisomers of all four G‐series nerve agents using a single chiral GC column. This approach allows successful baseline (or near baseline) resolution of each enantiomer of the G‐series agents under the same chromatographic conditions. Based on a variety of approaches, we have assigned each isomer peak as P(+) or P(−). The utility of the method (and variations thereof) was demonstrated by using this approach to determine the stereospecific rates of hydrolysis of GF as catalyzed by the enzyme OPH (from B. diminuta). The method described here will be generally applicable for screening enzymes to identify candidates that promote rapid hydrolysis of G‐agents, and thus may be effective as Bioscavengers of OP nerve agents.Research support: Defense Threat Reduction Agency (DTRA).

Radiosynthesis and biological evaluation of alpha-[F-18]fluoromethyl phenylalanine for brain tumor imaging

Radiolabeled amino acids have proven utility for imaging brain tumors in humans, particularly those that target system L amino acid transport. We have prepared the novel phenylalanine analogue, (FMePhe, 9), as part of an effort to develop new system L tracers that can be prepared in high radiochemical yield through nucleophilic [(18)F]fluorination. The tumor imaging properties of both enantiomers of this new tracer were evaluated through cell uptake, biodistribution and microPET studies in the mouse DBT model of high grade glioma. The non-radioactive form of 9 and the cyclic sulfamidate labeling precursor were prepared from commercially available racemic α-benzylserine. Racemic [(18)F]9 was prepared from the labeling precursor in two steps using standard[(18)F]fluoride nucleophilic reaction conditions followed by acidic deprotection. The individual enantiomers [(18)F]9a and [(18)F]9b were isolated using preparative chiral HPLC. In vitro uptake inhibition assays were performed with each enantiomer using DBT cells. Biodistribution and microPET/CT studies were performed with each enantiomer in male BALB/c mice at approximately 2 weeks after implantation of DBT tumor cells. Radiolabeling of the cyclic sulfamidate precursor 5 provides racemic [(18)F]9 in high radiochemical yield (60%-70%, n=4) and high radiochemical purity (>96%, n=4). In vitro uptake assays demonstrate that both [(18)F]9a and [(18)F]9b undergo tumor cell uptake through system L transport. The biodistribution studies using the single enantiomers [(18)F]9a and [(18)F]9b demonstrated good tumor uptake with lower uptake in most normal tissues, and [(18)F]9a had higher tumor uptake than [(18)F]9b. MicroPET imaging demonstrated good tumor visualization within 10 min of injection, rapid uptake of radioactivity, and tumor to brain ratios of approximately 6:1 at 60 min postinjection. The novel PET tracer, [(18)F]FMePhe, is readily synthesized in good yield from a cyclic sulfamidate precursor. Biodistribution and microPET studies in the DBT model demonstrate good tumor to tissue ratios and tumor visualization, with enantiomer [(18)F]9a having higher tumor uptake. However, the brain availability of both enantiomers was lower than expected for system L substrates, suggesting the [(18)F]fluorine group in the β-position affects uptake of these compounds by system L transporters.

Self‐Assembly of Racemic Alanine Derivatives: Unexpected Chiral Twist and Enhanced Capacity for the Discrimination of Chiral Species

Twisted ribbons were formed by the self-assembly of racemic alanine derivatives (middle picture), whereas only flat nanostructures were obtained from the individual enantiomers (left). The twist was sensitive to a slight enantiomeric excess and showed remarkable macroscopic chirality. Moreover, it could discriminate various amino acid derivatives (right) and even enabled the determination of the ee value of a mixed system.

Evaluation of Difluoromethyl Ketones as Agonists of the γ-Aminobutyric Acid Type B (GABAB) Receptor

The design, synthesis, biological evaluation, and in vivo studies of difluoromethyl ketones as GABAB agonists that are not structurally analogous to known GABAB agonists, such as baclofen or 3-aminopropyl phosphinic acid, are presented. The difluoromethyl ketones were assembled in three synthetic steps using a trifluoroacetate-release aldol reaction. Following evaluation at clinically relevant GABA receptors, we have identified a difluoromethyl ketone that is a potent GABAB agonist, obtained its X-ray structure, and presented preliminary in vivo data in alcohol-preferring mice. The behavioral studies in mice demonstrated that this compound tended to reduce the acoustic startle response, which is consistent with an anxiolytic profile. Structure-activity investigations determined that replacing the fluorines of the difluoromethyl ketone with hydrogens resulted in an inactive analogue. Resolution of the individual enantiomers of the difluoromethyl ketone provided a compound with full biological activity at concentrations less than an order of magnitude greater than the pharmaceutical, baclofen.

Development of a multi-residue enantiomeric analysis method for 9 pesticides in soil and water by chiral liquid chromatography/tandem mass spectrometry

A novel and sensitive chiral liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous measuring individual enantiomers of 9 pesticides including herbicides, insecticides, and fungicides in soil and water. The separation and determination were performed using reversed-phase chromatography on an amylose chiral stationary phase, a Chiralpak AD-RH column, under gradient elution using a mixture of ACN-2mM ammonium acetate in water as the mobile phase at 0.45mL/min flow rate. The effects of three cellulose-based columns and three amylose-based columns on the separation were also investigated. The QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) method and solid-phase extraction (SPE) were used for the extraction and clean-up of the soil and water samples, respectively. Parameters including the matrix effect, linearity, precision, accuracy and stability were undertaken. Under optimal conditions, the mean recoveries for all enantiomers from the soil and water samples were ranged from 77.8% to 106.2% with the relative standard deviations (RSD) less than 14.2%. Good linearity (at least R2≥0.9986) was obtained for all studied analytes in the soil and water matrix calibration curves over the range from 2.0 to 125μg/L. The limits of detection (LOD) for all enantiomers in the soil and water were less than 1.8μg/kg or μg/L, whereas the limit of quantification (LOQ) did not exceed 5.0μg/kg or μg/L. The results of the method validation confirm that this proposed method is convenient and reliable for the enantioselective determination of the enantiomers of 9 chiral pesticides in soil and water.

Probing the chiral separation mechanism and the absolute configuration of malathion, malaoxon and isomalathion enantiomers by chiral high performance liquid chromatography coupled with chiral detector–binding energy computations

Chiral separation mechanism determination and absolute configuration assignment are fundamental to the development of chiral stationary phases (CSPs) and the evaluation of both the enantioselective bioactivity and fate of chiral compounds. This work investigated the process of chiral separation and the assignment of the absolute configurations of malathion, malaoxon, and isomalathion using chiral high performance liquid chromatography (HPLC) coupled with chiral detector–binding energy computations. Hydrogen bonding was found to be a very important factor in the chiral separation of isomalathion on Chiralpak AD, although it did not exhibit a significant effect on the chiral separation of malathion and malaoxon on Chiralcel OJ. Based on the sign of a chiral detector, the relationships between the cotton effect, optical dispersion and absolute configuration were established for individual enantiomers of malathion, malaoxon, and isomalathion. The elution orders of the enantiomers of malathion and malaoxon on Chiralcel OJ and the stereoisomers of isomalathion on Chiralpak AD predicted by binding energy computations were found to coincide precisely with those observed in the chiral separation experiments. The result suggests that binding energy computations can be used to assign the absolute configuration of the enantiomers of chiral compounds eluted on CSPs.

MethadoneN-Demethylation by the CommonCYP2B6Allelic Variant CYP2B6.6

The long-acting opioid methadone displays considerable unexplained interindividual pharmacokinetic variability. Methadone metabolism clinically occurs primarily by N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), catalyzed predominantly by CYP2B6. Retrospective studies suggest that the common allele variant CYP2B6*6 may influence methadone plasma concentrations. The catalytic activity of CYP2B6.6, encoded by CYP2B6*6, is highly substrate-dependent. This investigation compared methadone N-demethylation by CYP2B6.6 with that by wild-type CYP2B6.1. Methadone enantiomer and racemate N-demethylation by recombinant-expressed CYP2B6.6 and CYP2B6.1 was determined. At substrate concentrations (0.25-2 µM) approximating plasma concentrations occurring clinically, rates of methadone enantiomer N-demethylation by CYP2B6.6, incubated individually or as the racemate, were one-third to one-fourth those by CYP2B6.1. For methadone individual enantiomers and metabolism by CYP2B6.6 compared with CYP2B6.1, Vmax was diminished, Ks was greater and the in vitro intrinsic clearance was diminished 5- to 6-fold. The intrinsic clearance for R- and S-EDDP formation from racemic methadone was diminished approximately 6-fold and 3-fold for R- and S-methadone, respectively. Both CYP2B6.6 and CYP2B6.1 showed similar stereoselectivity (S>R-methadone). Human liver microsomes with diminished CYP2B6 content due to a CYP2B6*6 allele had lower rates of methadone N-demethylation. Results show that methadone N-demethylation catalyzed by CYP2B6.6, the CYP2B6 variant encoded by the CYP2B6*6 polymorphism, is catalytically deficient compared with wild-type CYP2B6.1. Diminished methadone N-demethylation by CYP2B6.6 may provide a mechanistic explanation for clinical observations of altered methadone disposition in individuals carrying the CYP2B6*6 polymorphism.

(±)-Tylopilusins, Diphenolic Metabolites from the Fruiting Bodies of Tylopilus eximius

Racemates of the diphenolic metabolites (±)-tylopilusin A (1) and (±)-tylopilusin B (2) were isolated from the fruiting bodies of Tylopilus eximius. Their structures were elucidated on the basis of spectroscopic analyses (1D and 2D NMR data and ROESY correlations) and X-ray crystallography. Each racemate was separated into its individual enantiomers, and electronic circular dichroism calculations were used to assign the absolute configuration of (+)- and (-)-tylopilusin A (1) and (+)- and (-)-tylopilusin B (2).

Chiral analytical method development and application to pre‐clinical pharmacokinetics of pinocembrin

An analytical method enabling the detection and quantification of the individual enantiomers of racemic (±) pinocembrin is required to fully characterize its pharmacokinetic disposition. Direct resolution of the enantiomers of pinocembrin was achieved using a novel and simple reversed-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry in rat serum. A Chiralcel® AD-RH column was employed to perform baseline separation with electrospray positive-mode ionization with selected ion monitoring detection. The standard curves were linear from 0.5 to 100 µg/mL for each enantiomer. The limit of quantification was 0.5 µg/mL. The assay was applied successfully to stereoselective serum disposition of pinocembrin enantiomers in rats. Pinocembrin enantiomers were detected in serum. Both enantiomers had a serum half-life of ~15 min in rats. Similar values of volume of distribution between the enantiomers were also observed: 1.76 L/kg for S-pinocembrin and 1.79 L/kg for R-pinocembrin. Total clearance was 5.527 L//h/kg for S-pinocembrin and 5.535 L/h/kg for R-pinocembrin, and the area under the curve was 1.821 µg h/mL for S-pinocembrin and 1.876 µg h/mL for R-pinocembrin. The large volume of distribution coupled with the short serum half-life suggests extensive distribution of pinocembrin into the tissues.

Enantiomers of all-cis-5-(4-bromophenyl)-4-tert-butoxycarbonyl-2-methoxycarbonylpyrrolidine: preparative HPLC separation and acylative kinetic resolution of the racemate

The fundamental possibility of acylative kinetic resolution of racemic heterocyclic amines was demonstrated by the example of all-cis-5-(4-bromophenyl)-4-tert-butoxycarbonyl-2-methoxycarbonylpyrrolidine. Individual enantiomers (ee >99%) were obtained in high yields via preparative chiral HPLC.

Enantiomeric resolution of chiral aromatic sulfoxides on non-commercial microcrystalline cellulose triacetate and commercial cellulose acetate plates

This paper reports a number of original thin-layer chromatographic enantioseparations of chiral sulfoxides that are important for their use as drugs and drug metabolites or pesticides, obtained by elution with aqueous-alcoholic mixtures at different ratios. Noncommercial microcrystalline cellulose triacetate and commercial cellulose acetate (CEL 300-10/AC, Macherey-Nagel) plates were compared for their chiral resolution power, evidencing the much better performances of the former. The linearity of the densitometric response as a function of the amount of each enantiomer applied to the plate was investigated for selected compounds. Correlation coefficients higher than or equal to 0.99 were obtained in all cases, and the feasibility of the quantification of the individual enantiomers at tens to hundreds of nanograms spotted was demonstrated.

Abstract CN06-01: Induction of oral cavity cancer in rats by (S)-N'-nitrosonornicotine, a constituent of smokeless tobacco

Abstract Smokeless tobacco products such as moist oral snuff are being promoted by the tobacco industry with a half billion dollars per year in advertising. It is likely that the prevalence of smokeless tobacco use will continue to increase in the U.S. Smokeless tobacco products are widely used in other parts of the world and are considered by the International Agency for Research on Cancer as causes of cancer of the oral cavity, esophagus and pancreas in humans. However, no previous study has reported the presence of a strong oral cavity carcinogen in these products. N'-Nitrosonornicotine (NNN) occurs in significant amounts in smokeless tobacco products, generally being found at levels of 1 - 10 μg/g tobacco. Since NNN has a chiral center, it exists as (S)- and (R)- enantiomers. (S)-NNN is the major enantiomer in smokeless tobacco products. While racemic NNN (a 50:50 mixture of (S)- and (R)-NNN) has been previously shown to cause esophageal tumors in rats, the carcinogenicity of the individual enantiomers has not been reported. Based on our published metabolism and DNA binding studies of (S)-NNN, we hypothesized that it might be a more powerful carcinogen than (R)-NNN and, furthermore, that it might cause oral cancer. Groups of 24 male F-344 rats were treated with either (S)-NNN (15 ppm), (R)-NNN (15 ppm), or racemic NNN (30 ppm) in the drinking water for 15-20 months. Necropsy of 20 rats treated with (S)-NNN demonstrated a 100% incidence of oral cavity tumors. More than 60 oral cavity tumors were observed, including benign and malignant tumors of the buccal mucosa, soft and hard palate, tongue, and pharynx. All rats also had esophageal tumors. (R)-NNN was significantly less carcinogenic than (S)-NNN. Racemic NNN also induced a high incidence of oral cavity and esophageal tumors. The results of this study demonstrate for the first time the presence of a strong oral cavity carcinogen in smokeless tobacco. (S)-NNN is a prime target for elimination from these products as a positive step toward cancer prevention. Citation Format: Stephen S. Hecht, Sandra James-Yi, Gerard O'Sullivan, Irina Stepanov, Mingyao Wang, Fekadu Kassie, Steven Carmella, Pramod Upadhyaya, Silvia Balbo. Induction of oral cavity cancer in rats by (S)-N'-nitrosonornicotine, a constituent of smokeless tobacco. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN06-01.

Stereospecific analytical method development and preliminary in vivo pharmacokinetic characterization of pinostrobin in the rat

The complete pharmacokinetic disposition of the chiral flavonoid (±) pinostrobin remains unknown without the development of an analytical method of detection and quantitation of its individual enantiomers. Resolution of the enantiomers of pinostrobin was achieved using as simple high-performance liquid chromatographic method. A Chiralpak(®) AD-RH column was employed to perform baseline separation with UV detection at 287 nm. The standard curves were linear ranging from 0.5 to 100 µg/mL for each enantiomer. The limit of quantification was 0.5 µg/mL. Precision and accuracy of the assay was < 15% (RSD) and was with a bias <15% for all points on the calibration curve. The assay was applied successfully to stereoselective serum disposition of pinostrobin enantiomers in rats. Both enantiomers had a serum half-life of ~7 h. They also shared similar values of volume of distribution (V(d) S-pinostrobin, 8.2 L/kg; V(d) R-pinostrobin, 8.9 L/kg), total clearance (S-pinostrobin CL(total), 0.959 L//h/kg; R-pinostrobin CL(total), 1.055 L//h/kg), and area under the curve (S-pinostrobin AUC(inf), 23.16 µg h/mL; R-pinostrobin AUC(inf), 21.296 µg h/mL). The large volume of distribution suggests extensive distribution of pinostrobin into tissues.

Enantiomer-specific determination of hexabromocyclododecane in fish by supramolecular solvent-based single-step sample treatment and liquid chromatography–tandem mass spectrometry

A single-step, environmentally friendly sample treatment was developed and used in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitation of hexabromocyclododecane (HBCD) stereoisomers in fish. It was based on the microextraction of the stereoisomers with a supramolecular solvent (SUPRAS) made up of reverse aggregates of decanoic acid (DeA). The procedure involved the stirring of the fish sample (750mg) with 600μL of SUPRAS for five minutes, subsequent centrifugation for extract separation from matrix components and direct analysis of the extract after dilution 1:1 with methanol. Individual enantiomers of α-, β- and γ-HBCD were separated on a chiral stationary phase of β-cyclodextrin and quantified by monitoring of the [M−H]−→Br− transition at m/z 640.9→80.9. Driving forces for the microextraction of HBCD in the SUPRAS involved both dispersion and dipole–dipole interactions. Quantitation limits for the determination of individual HBCD enantiomers in hake, cod, sole, panga, whiting and sea bass were within the intervals 0.5–3.4ngg−1, 0.9–2.5ngg−1, 0.6–1.4ngg−1, 1.0–5.6ngg−1, 0.8–1.3ngg−1 and 0.5–3.5ngg−1, respectively. Recoveries for fish samples fortified at the ngg−1 level ranged between 87 and 114% with relative standard deviations from 1 to 10%. The sample treatment proposed greatly simplifies current procedures for extraction of HBCD stereoisomers and is a useful tool for the development of a large scale database for their presence in fish.

Evidence of stereoselective and segmental-dependant transport of chiral antidiabetic drug nateglinide along the rat small intestine: possible role of efflux transporters

The purpose of the study was to investigate whether efflux and stereoselective mechanisms play any role in the transport of nateglinide (NA) enantiomers in rat. The transport of individual enantiomers and racemate was studied with and without the presence of an inhibitor for P-glycoprotein and MRP2, verapamil using duodenal, jejunal, and ileal intestinal sacs prepared from rat intestine. The intestinal samples were analyzed by a validated chiral HPLC method. Generally, higher concentrations of R-NA and its S-enantiomer were observed when the racemate was administered compared to administration of the individual enantiomers. Transport of NA enantiomers when co-incubated with verapamil was increased in the ileum by twofold to threefold with corresponding decreased secretion by almost threefold, while little change in the duodenum and jejunum. The transport of NA from everted sac segments (basal-to-apical side, secretion) was higher than the transport from the normal sac segments (apical-to-basal side, absorption) indicated an involvement of efflux-mediated transport. It has been found that the transport of NA is stereoselective and also regioselective, i.e., R-NA displayed higher efflux than S-NA and it was higher in ileum than the duodenum and jejunum. This study will really pave the way to understand how efflux transporters influence the absorption of chiral compounds when moving across the gastrointestinal tract.

Capillary electrophoresis to quantitate gossypol enantiomers in cotton flower petals and seed

Gossypol is a toxic compound that occurs as a mixture of enantiomers in cotton plant tissues including seed and flower petals. The (−)-enantiomer is more toxic to non-ruminant animals. Efforts to breed cottonseed with a low percentage of (−)-gossypol requires determination of the (+)- to (−)-gossypol ratio in seed and flower petals. We report a method to quantitatively determine the total gossypol and percent of its enantiomers in cotton tissues using high performance capillary electrophoresis (HPCE). The method utilizes a borate buffer at pH 9.3 using a capillary with internal diameter of 50μm, effective length of 24.5cm, 15kV and cassette temperature of 15°C. This method provides high accuracy and reproducible results with a limit of detection of the individual enantiomers of less than 36ng/mL providing base line separation in less than 6min.