Stereoselective degradation of tebuconazole in rat liver microsomes

The aim of this study was to assess the stereoselectivity of two tebuconazole [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol] enantiomers in in vitro system (rat liver microsomes). The analytes were extracted with acetic ether and concentrations were determined by high performance liquid chromatography (HPLC) with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase. The degradation of rac-tebuconazole (15 μM) followed first-order kinetics, and the degradation of the S-tebuconazole (t(1/2) = 22.31 min) was faster than that of the R-tebuconazole (t(1/2) = 48.76 min), but no significant difference between the enantiomers was found in the respective incubation (7.5 μM for each). Kinetic assays showed that the K(m) was different between the two enantiomers (K(mR) = 14.83 ± 2.19, K(mS) = 12.23 ± 2.72). The interaction results revealed that there was competitive inhibition between S- and R-form, and there was a significant difference between the IC(50) of R- to S-tebuconazole and S- to R-tebuconazole (IC(50R/S)/IC(50S/R) = 4.98).     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography–mass spectrometry (LC–MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC–tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.     

LC–MS/MS and chiroptical spectroscopic analyses of multidimensional metabolic systems of chiral thalidomide and its derivatives

Enantiomeric thalidomide undergoes various kinds of biotransformations including chiral inversion, hydrolysis, and enzymatic oxidation, which results in several metabolites, thereby adding to the complexity in the understanding of the nature of thalidomide. To decipher this complexity, we analyzed the multidimensional metabolic reaction networks of thalidomide and related molecules in vitro . Characteristic patterns in the amount of various metabolites of thalidomide and related molecules generated during a combination of chiral inversion, hydrolysis, and hydroxylation were observed using liquid chromatography–tandem mass spectrometry and chiroptical spectroscopy. We found that monosubstituted thalidomide derivatives exhibited different time‐dependent metabolic patterns compared with thalidomide. We also revealed that monohydrolyzed and monohydroxylated metabolites of thalidomide were likely to generate mainly by a C‐5 oxidation of thalidomide and subsequent ring opening of the hydroxylated metabolite. Since chirality was conserved in most of these metabolites during metabolism, they had the same chirality as that of nonmetabolized thalidomide. Our findings will contribute toward understanding the significant pharmacological effects of the multiple metabolites of thalidomide and its derivatives.     

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

In this study the analysis and confirmation of flumequine enantiomers in rat plasma by ultra‐fast liquid chromatography coupled with electron spray ionization mass spectrometry (using propranolol as an internal standard [IS]) was developed and validated. Plasma samples were prepared by liquid–liquid extraction using methyl tert‐butyl ether as the extraction solvent. Direct resolution of the R‐ and S‐isomers was performed on a CHIRALCEL OJ‐RH column (4.6 × 150 mm, 5 μm) using acetonitrile / 0.1% formic acid / 1 mM ammonium acetate as the mobile phase. Detection was operated by electron spray ionization in the selected ion monitoring and positive ion mode. The target ions at m/z 262.1 and m/z 260.1 were selected for the quantification of the enantiomers and IS, respectively. The linear range was 0.5–500 ng/mL. The precisions (coefficient of variation, CV%) and recoveries were 1.43–8.68 and 94.24–106.76%, respectively. The lowest quantitation limit for both enantiomers is 0.5 ng/mL, which is sensitive enough to be applied to sample analysis in other related studies.     

Stereoselective metabolism of benalaxyl in liver microsomes from rat and rabbit

Benalaxyl (BX), methyl‐N‐phenylacetyl‐N‐2,6‐xylyl alaninate, is a potent acylanilide fungicide and consist of a pair of enantiomers. The stereoselective metabolism of BX was investigated in rat and rabbit microsomes in vitro. The degradation kinetics and the enantiomer fraction (EF) were determined using normal high‐performance liquid chromatography with diode array detection and a cellulose‐tris‐(3,5‐dimethylphenylcarbamate)‐based chiral stationary phase (CDMPC‐CSP). The t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rat liver microsomes were 22.35 and 10.66 min of rac‐BX and 5.42 and 4.03 of BX enantiomers. However, the t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rabbit liver microsomes were 11.75 and 15.26 min of rac‐BX and 5.66 and 9.63 of BX enantiomers. The consequence was consistent with the stereoselective toxicokinetics of BX in vitro. There was no chiral inversion from the (?)‐R‐BX to (+)‐S‐BX or inversion from (+)‐S‐BX to (?)‐R‐BX in both rabbit and rat microsomes. These results suggested metabolism of BX enantiomers was stereoselective in rat and rabbit liver microsomes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.     

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization

Ammuxetine (AMT), a novel chiral antidepressant candidate compound, exhibits better antidepression effects than duloxetine in different animal models. In this article, a chiral derivatization method, combined with online solid phase extraction (online SPE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), was developed for the chiral separation of AMT enantiomers after administration of racemic AMT to dogs. The derivatization reaction employed 2,3,4,6‐tetra‐O‐acetyl‐b‐glucopyr‐anosyl isothiocyanate (GITC) as a precolumn chiral derivatization reagent. A SPE column Retain PEP Javelin (10 × 2.1 mm) was used to remove proteins and other impurities in plasma samples. The enantiomeric derivatives were separated on a ZORBAX SB‐C₁₈ column (50 × 2.1 mm × 3.5 μm) with an isocratic elution procedure. The selected multiple reaction monitoring mode of the positive ion was performed and the parent to the product transitions m/z 681.0/543.1 and m/z 687.4/543.1 were used to measure the derivatives of AMT and duloxetine (internal standard) with electrospray ionization. The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The method was applied to a pharmacokinetics study of AMT racemate in dogs. The results suggested that the pharmacokinetic of AMT enantiomers might be stereoselective in dogs.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Enantioseparation and determination of flumequine enantiomers in multiple food matrices with chiral liquid chromatography coupled with tandem mass spectrometry

The present work firstly described the enantioseparation and determination of flumequine enantiomers in milk, yogurt, chicken, beef, egg, and honey samples by chiral liquid chromatography‐tandem mass spectrometry. The enantioseparation was performed under reversed‐phase conditions on a Chiralpak IC column at 20°C. The effects of chiral stationary phase, mobile phase components, and column temperature on the separation of flumequine enantiomers have been studied in detail. Target compounds were extracted from six different matrices with individual extraction procedure followed by cleanup using Cleanert C18 solid phase extraction cartridge. Good linearity (R²>0.9913) was obtained over the concentration range of 0.125 to 12.5 ng g^‐1 for each enantiomer in matrix‐matched standard calibration curves. The limits of detection and limits of quantification of two flumequine enantiomers were 0.015‐0.024 and 0.045‐0.063 ng g^‐1, respectively. The average recoveries of the targeted compounds varied from 82.3 to 110.5%, with relative standard deviation less than 11.7%. The method was successfully applied to the determination of flumequine enantiomers in multiple food matrices, providing a reliable method for evaluating the potential risk in animal productions.     

Simultaneous determination of dimethylamphetamine and its metabolites in rat hair by gas chromatography–mass spectrometry

In order to study the disposition of dimethylamphetamine (DMAP) and its metabolites, DMAP N-oxide, methamphetamine (MA) and amphetamine (AP), from plasma to hair in rats, a simultaneous determination method for these compounds in biological samples using gas chromatography–mass spectrometry with selected ion monitoring (GC–MS-SIM) was developed. As DMAP N-oxide partially degrades to DMAP and MA during GC–MS analysis, it was necessary to avoid conditions which co-extract the N-oxide in the sample preparation so as to assure no contribution of artifactual products from DMAP N-oxide in the detection of the other compounds. For confirmation of the satisfactory separation of DMAP N-oxide from the others, the internal standards used for quantification were labeled with different numbers of deuterium atoms. Determination of unchanged DMAP was performed without any derivatization, that of DMAP N-oxide was carried out after conversion into trifluoroacetyl-MA by reaction with trifluoroacetic anhydride, and MA and AP were quantified after trifluoroacetyl-derivatization.After intraperitoneal administration of DMAP HCl to pigmented hairy rats (5 mg kg⁻¹ day⁻¹, 10 days, n=3), concentrations of DMAP and its metabolites in urine, plasma and hair were measured by GC–MS-SIM. The area under the concentration versus time curves (AUCs) of DMAP, DMAP N-oxide, MA and AP in the plasma were 397.2±97.5, 279.7±68.3, 18.4±1.2 and 15.9±2.2 μg min ml⁻¹, while their concentrations in the hair newly grown for 4 weeks after administration were 4.82±0.67. 0.45±0.09, 3.25±0.36 and 0.89±0.05 ng mg⁻¹, respectively. This fact suggested that the incorporation tendency of DMAP N-oxide from plasma into hair was distinctly low in comparison with the other compounds.     

Measurement of dexfenfluramine metabolism in rat liver microsomes by gas chromatography–mass spectrometry

A specific and useful method was developed for the determination of dexfenfluramine metabolism by microsomal systems utilising GC–MS. The synthesis of two metabolites 1-(3-trifluoromethylphenyl)propan-2-ol (‘alcohol') and 1-(3-trifluoromethylphenyl)-1,2-propanediol (‘diol') via straightforward routes, were confirmed by MS and NMR spectra. The conditions for extraction from alkalinised microsomal mixtures of the metabolites nordexfenfluramine, 1-(3-trifluoromethylphenyl)propan-2-one (‘ketone'), alcohol and diol, their conversion to trifluoroacetate derivatives and analysis by GC–MS–SIM are described. Calibration curves were constructed between 48 and 9662 nM and fitted to quadratic equations (r²>0.999). The method precision was good over low (121 nM) medium (2415 nM) and above medium (9662 nM) concentrations for all metabolites; the within- and day-to-day coefficients of variation ranged between 2.5–12.4% and 6.7–17.5%, respectively. The accuracy, measured as bias, was very good both within- and day-to-day (range: −0.4–12.6%, 0.8–18.9%). For most metabolites, the C.V. for the assay and bias increased at 121 nM. Dexfenfluramine metabolism by rat liver microsomes was investigated using the assay method and showed a concentration dependent increase in nordexfenfluramine and ketone metabolites over the substrate range of 5–200 μM.     

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry (HILIC–ESI–MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC–pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.     

Validation of salivary cortisol and testosterone assays in chimpanzees by liquid chromatography‐tandem mass spectrometry

Owing to its high temporal sensitivity, saliva has distinct advantages for measuring steroids, compared with other noninvasive samples such as urine and feces. Here, we report the validity of assaying salivary cortisol (C) and testosterone (T) using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) in captive male chimpanzees, Pan troglodytes. For both the C and T concentrations, we found positive relationships between saliva and plasma. The concentrations of C and T in saliva showed clear patterns of diurnal fluctuation, whereas those in urine and feces did not. These results suggest that the salivary steroid concentrations can be regarded as good indicators of circulating steroid levels. We also developed and validated an efficient method for collecting saliva samples from cotton rope. Although rope includes inherent steroid‐like compounds and may affect the accuracy of steroid measurements, our rope‐washing procedures effectively removed intrinsic steroidal materials. There was a significant association between the C and T concentrations measured from saliva collected from rope licked by the chimpanzees and those measured from saliva collected directly from the mouth. Salivary T values estimated by LC/MS‐MS were similar to those measured by radioimmunoassay. The results indicate the usefulness of saliva as a noninvasive steroid measure and that steroids in the saliva of chimpanzees can be accurately measured by LC‐MS/MS. Am. J. Primatol. 71:696–706, 2009. © 2009 Wiley‐Liss, Inc.     

Amino acid composition of rat and human liver microsomes in normal and pathological conditions

The amino acid composition of proteins from liver microsomes has been studied in rats and in human subjects with normal liver, with obstructive jaundice or liver cirrhosis. The pattern of the amino acid composition of microsomes appeared to be species-specific. Phenylalanine, threonine, serine, proline, histidine and [aspartic acid plus asparagine] were increased, while alanine, tyrosine, glycine and arginine were decreased in the human compared to the rat microsomes. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in leucine and phenylalanine could be noticed, while in the case of liver cirrhosis amino acid composition was markedly changed.     

Ethyl-glucuronide and ethyl-sulfate in placental and fetal tissues by liquid chromatography coupled with tandem mass spectrometry

The aim of this study was to develop a method for the determination of ethyl-glucuronide (EtG) and ethyl-sulfate (EtS), two direct ethanol metabolites, in early placental and fetal human tissues, as potential biomarkers of transplacental ethanol transfer from the mother to the fetus. Placental and fetal tissue samples were obtained from women undergoing voluntary termination of pregnancy at 12 weeks of gestation. Samples were deproteinized and directly injected into a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) system. Limits of detection of 13.0 and 23.0 pmol/g and lower limits of quantification of 22.0 and 40.0 pmol/g were reached for EtG and EtS, respectively. Inter- and intraday imprecision and accuracy were always lower than 15%. The method was applied to 70 samples (35 placentas and 35 fetal tissues). Of 35 samples, 4 samples collected from 4 women tested positive for EtG and EtS, always showing higher concentrations for EtG. The placenta/fetal tissue ratio for EtG was 2.9 ± 0.9, whereas EtS showed a ratio of 1.7 ± 0.7. Preliminary results suggest that these metabolites are present in both tissues. Further studies should now corroborate the hypothesis, not yet confirmed, that transplacental transfer of ethanol takes place not only for the parent compound but also for EtG and EtS.     

Quantitative determination of dihydroetorphine in rat plasma and brain by liquid chromatography–tandem mass spectrometry

The extraordinarily strong analgesic dihydroetorphine (DHE) was registered as one of the most strictly controlled narcotic drugs by the United Nations in 1999. However, an effective detection method for DHE in biological samples has not yet been established. We developed a quantitative method for assay of DHE in rat plasma and brain by liquid chromatography–tandem mass spectrometry equipped with an ionspray interface. A 0.5-ml volume of plasma and brain homogenate spiked with buprenorphine (internal standard) was purified by the solid-phase extraction column Bond Elute Certify. DHE produced numerous weak fragment ions by collision induced dissociation. Therefore, collision energy was utilized to decompose the interferences, and the protonated molecular ion was used for both precursor and product ion monitoring. As a result of the method validation, the dynamic concentration range was determined as 0.05–10 ng/ml. DHE in these samples was stable for 2 months at −4°C and for 24 h at ambient temperatures. Using the present method, DHE was detected in rat plasma and brain tissue after intravenous injection (0.5 μg/kg).     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry

The profile of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was determined from a Chilean strain of the marine dinoflagellate Alexandrium catenella. The toxin composition was compared with that of toxic shellfish, presumably contaminated by natural blooms of A. catenella from the same region in southern Chile. Ion pair-liquid chromatography with post-column derivatization and fluorescence detection (LC-FD) was employed for relative quantitative analysis of the toxin components, whereas unambiguous identification of the toxins was confirmed by tandem mass spectrometry (LC–MS/MS). In the dinoflagellate strain from Chile, the N-sulfocarbamoyl derivatives (C1/C2, B1) and the carbamoyl gonyautoxins GTX1/GTX4 comprise >90% of the total PSP toxin content on a molar basis. This toxin composition is consistent with that determined for A. catenella populations from the Pacific coast in the northern hemisphere. The characteristic toxin profile is also reflected in the shellfish, but with evidence of epimerization and metabolic transformations of C1 and C2 to GTX2 and GTX3, respectively. This work represents the first unequivocal identification and confirmation of such PSP toxin components from the Chilean coast.