The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine

Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme. The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed. Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom. These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate. The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period. The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.     

Stereoselective degradation of fungicide triadimenol in cucumber plants

The stereoselective degradation of triadimenol in different cucumber plant tissues (root, stem, leaf, and fruit) has been investigated. Rac‐triadimenol was applied to cucumber plants by root irrigation mode under field conditions. The degradation kinetics and the enantiomer fraction were determined by normal‐phase high‐performance liquid chromatography with diode array detector and on‐line optical rotatory dispersion detector on Chiralpak® AS‐H column. It has been shown that the degradation of triadimenol in cucumber plants was stereoselective under field conditions. The results indicated that RS enantiomer was degraded faster than SR enantiomer, and SS enantiomer was degraded faster than RR enantiomer, which resulted in plants enriched with SR and RR enantiomers. Furthermore, it was found that leaf was the dominating location for triadimenol enantiomer accumulation and stereoselective degradation, comparing with the root, stem, and fruit tissue. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Enantiomers of a nonylphenol isomer: absolute configurations and estrogenic potencies

Enantiomers of 4-(1,1,2-trimethylhexyl)phenol, a chiral isomer of the endocrine disrupting chemical nonylphenol, have been resolved and isolated by preparative chiral HPLC. The absolute configurations of the enantiomers were then determined by an X-ray crystallographic study of the (-)-camphanoyl derivative of the first eluted enantiomer NP(35)E1. The first enantiomer (NP(35)E1) and the second enantiomer (NP(35)E2) eluted were found to have the S and R absolute configurations, respectively. The estrogenic potencies of the S and R enantiomers were tested by the E-screen assay. A slight difference was observed in the relative proliferative effect between the S enantiomer and R enantiomer in the E-screen assay.     

Stereoselective pharmacokinetics of tetrahydropalmatine after oral administration of (-)-enantiomer and the racemate

Tetrahydropalmatine (THP) is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). THP is a racemic mixture which contains 50% of the (+) and 50% of (-) enantiomer. The (-) enantiomer accounts for most of the analgesic effects. Plasma concentrations of THP enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OJ column with quantification by UV at 230 nm. The method was used to determine the pharmacokinetics of THP enantiomers in rats and dogs after oral administration of rac-THP or (-)-THP. The pharmacokinetic profiles of the two enantiomers after dosing with rac-THP were significantly different both in rats and dogs. The mean C(max) and AUC(0-infinity) values in rats were 1.93 +/- 0.36 microg/ml and 6.65 +/- 2.34 microg x h/ml for the (-) enantiomer, and 1.11 +/- 0.25 microg/ml and 2.03 +/- 0.45 microg x h/ml for the (+) enantiomer. The mean C(max) and AUC(0-infinity) in dogs were 1.60 +/- 0.81 microg/ml and 9.88 +/- 2.58 microg x h/ml for the (-) enantiomer, while 0.36 +/- 0.21 microg/ml and 1.22 +/- 0.40 microg x h/ml for the (+) enantiomer. rac-THP at 40 mg/kg and (-)-THP at 20 mg/kg had very similar plasma concentration-time profiles, and C(max), AUC(0-infinity), and t(1/2) of the (-) enantiomer in both rats and dogs, indicating that the two treatments were equivalent with respect to the pharmacokinetic properties of the (-) enantiomer.     

Distribution of enantiomers of volatile organic compounds in selected fruit distillates

The enantiomer ratios of chiral volatile organic compounds in fruit distillates were determined by multidimensional gas chromatography using solid‐phase microextraction (SPME) as a sample treatment procedure. Linalool and its oxides, limonene, α‐terpineol, and nerolidol, were present at the highest concentration levels, while significantly lower amounts of β‐citronellol and lactones were found in the studied samples. However, almost all terpenoids mainly occur as a racemic or near‐racemic mixture; enantiomer distribution of some chiral organic compounds in fruit distillates correlated to a botanical origin. In particular, a significant enantiomeric excess of (R)‐linalool and (S)‐α‐terpineol was found only for pear brandy, and likewise the dominance (R)‐limonene and the second eluted enantiomer of nerolidol for Sorbus domestica and strawberry, respectively. The distribution of γ‐lactones stereoisomers was more nonspecific, with a general excess of the R‐enantiomer.     

Pharmacokinetics of nateglinide enantiomers and their metabolites in Goto‐Kakizaki rats,a model for type 2 diabetes mellitus

The pharmacokinetics of (?)‐N‐(trans‐4‐isopropylcyclohexanecarbonyl)‐D ‐phenylalanine (nateglinide) and its enantiomer (L‐enantiomer) was studied in Goto‐Kakizaki (GK) rats after intravenous administration of nateglinide or L‐enantiomer at a dose of 40 μmol/kg body weight. Nateglinide, its L‐enantiomer and their metabolites in serum, bile and urine were determined. The total clearance (CL_tot) and the volume of distribution (V_d) was slightly higher for nateglinide than those for L‐enantiomer in control rats, although the differences were not statistically significant. The cumulative excretions of L‐M1 (major metabolite of L‐enantiomer) and L‐M2 (major metabolite of L‐enantiomer) into bile were almost the same as that of M1 (major metabolite of nateglinide)and M2 (major metabolite of nateglinide). In GK rats, CL_tot and V_d were higher for nateglinide than those for L‐enantiomer. The cumulative excretion of L‐M1 and L‐M2 were not different from those of M1 and M2, respectively, into bile or urine. CL_tot and V_d for nateglinide or L‐enantiomer in GK rats were not different from those in control rats. The total excretion of M1, M2, L‐M1, and L‐M2 into bile or urine in GK rats was not substantially different from that of control rats. These results suggest that the L‐enantiomer of nateglinide shows higher CL_tot and V_d compared with nateglinide, especially in the diabetic state. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).     

Effects of pure enantiomers of flurbiprofen in comparison to racemic flurbiprofen on eicosanoid release from various rat organs ex vivo.

The effects of oral treatment of rats with pure enantiomers of flurbiprofen in comparison to racemic flurbiprofen on ex vivo release of eicosanoids from gastric mucosa, jejunum, lung, brain and clotting whole blood were investigated. With the S(+) enantiomer and the racemate dose-dependent inhibition of release of cyclooxygenase products of arachidonate metabolism in all tissues tested was observed, while release of leukotriene (LT) C4 was inhibited in gastric mucosa, but not in jejunum and lung. On the other hand, the R(-) enantiomer inhibited cyclooxygenase in the various tissues less potently and to a variable degree with no significant effect in the jejunum. The R(-) enantiomer had no effect on LTC4 release from any of the tissues investigated. Furthermore, the effect of a high dose of 25 mg/kg of the S(+) enantiomer on release of cyclooxygenase products from the various tissues was much longer lasting than that of an identical dose of the R(-) enantiomer. Stereoselective pharmacokinetics of the flurbiprofen enantiomers and/or organ specific cyclooxygenase activities could underly these results. The more potent cyclooxygenase inhibition by the S(+) enantiomer correlates with its higher anti-inflammatory activity and gastrointestinal toxicity. On the other hand, both enantiomers have been shown previously to be almost equally effective analgesics. Inhibition of brain cyclooxygenase might contribute to this effect.     

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4- and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.     

Enantiomer selection in the addition reaction of optically active 3-methyl-5-substituted hydantoins to N-[(S)-α-methylbenzyl]glycine N-Carboxyanhydride: Model Reaction for the Stereoselective Polymerization of α-amino acid N-carboxyanhydride

In order to investigate the contribution from the chiral penultimate unit to the enantiomer selection in the activated N-carboxyanhydride (NCA) polymerizations, the addition reaction to N-[(S)-methylbenzyl]glycine NCA of various α-amino acid hydantoins activated by the tertiary amines was investigated in different solvents. The reactions of activated Ala, Val, and Phe hydantoins were stereoselective and suggested the participation of the penultimate unit in the enantiomer selection of the activated NCA type of polymerization. The degree of enantiomer selection was not well correlated with the structure of hydantoins. Taking into account the dipole repulsion and the orbital overlapping between the reaction species, the transition-state model was proposed, which gave a good explanation of the selectivity for (R)-hydantoin in PhNO₂ and CH₃CN and the selectivity for (S)-hydantoin in AcNMe₂ and HCONMe₂. In these two types of solvents the orientation of the methylbenzyl group with respect to the NCA ring is so different that the direction of the approach of the activated hydantoin to the NCA is different. This difference leads to the inversion of enantiomer selection in amide solvents and in others. Cationic species derived from tertiary amines and the chiral amide compound were found to affect the enantiomer selection in the model reaction. The implications of these findings with regard to enantiomer selection in the activated NCA type of polymerization are discussed.     

Parallel SFC/MS-MUX screening to assess enantiomeric purity

Enantiomeric excess (ee) was evaluated for two internally synthesized compound libraries using a high-throughput automated, intelligent four-channel parallel supercritical fluid chromatography/mass spectrometry system equipped with a multiplexed ion source interface (SFC/MS-MUX). The two libraries contained compounds spanning a wide range of enantiomeric ratios with structurally diverse chemical scaffolds and stereogenic centers. The system analyzed each sample simultaneously against four chiral columns using up to six organic modifiers. Enhancements to our previously published parallel supercritical fluid chromatography/mass spectrometry system were implemented to address the challenges associated with automated trace enantiomer identification and quantitation. A reversal of enantiomer elution order was observed for several samples across multiple CSPs and modifiers. The relationship between elution order and % ee accuracy is presented for compounds exhibiting high, middle and low % ee values. Despite incidences in which the minor enantiomer eluted prior to the major enantiomer with less than baseline resolution, the overall % ee was in agreement with separations in which full baseline resolution was achieved. The methods presented here demonstrate the value and utility of high-throughput ee determinations to support drug discovery and development programs.     

Complete microbial degradation of both enantiomers of the chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propionic acid] in an enantioselective manner by Sphingomonas herbicidovorans sp. nov.

Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp. strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source. When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release. However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. These results suggest the involvement of specific enzymes for the degradation of each enantiomer. This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop. Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer. Nevertheless, such cells could transform (S)-mecoprop at low rates. Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate. Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.     

Pharmacokinetics of the active antifungal enantiomer, SCH 42427 (RR), and evaluation of its chiral inversion in animals following its oral administration and the oral administration of its racemate genaconazole (RR/SS)

Genaconazole (SCH 39304) is a potent triazole antifungal agent that is active both orally and topically. Genaconazole is a racemic mixture which contains 50% of the RR (SCH 42427) and 50% of the SS (SCH 42426) enantiomers. The RR isomer accounts for most of the antifungal activity of genaconazole. Serum concentrations of the RR and SS enantiomers were analyzed by a chiral HPLC method which involved extraction of serum with organic solvent followed by separation on a Cyclobond I column and quantification by UV absorbance at 205 nm. The bioavailability and pharmacokinetic profiles of the two enantiomers after oral administration of the racemate (genaconazole) were very similar in cynomolgus monkeys. In rats following dosing with genaconazole, the RR enantiomer had a lower C(max) and a longer t(1/2) than the SS enantiomer, while the AUC(I) values of the two enantiomers were similar. Based on chiral HPLC analysis, there was no evidence for the inversion of the RR to the SR isomer, or of the SS to the SR isomer, indicating that there was no chiral inversion of the RR or SS enantiomers in either species. Genaconazole at 20 mg/kg and the RR (SCH 42427) enantiomer at 10 mg/kg had very similar serum concentration-time profiles and C(max), AUC(I), and t(1/2) values for the RR enantiomer in both rats and monkeys, indicating that the two treatments were equivalent with respect to the bioavailability of the RR enantiomer.     

Spectral study on inclusion interaction and enantiorecognition of 2-aryl carboxylic acids with hydroxypropyl-β-cyclodextrin

The inclusion interaction between hydroxypropyl-β-cyclodextrin (HP-β-CD) and 21 2-aryl carboxylic acids was investigated by UV (ultraviolet) spectrophotometer. The inclusion constant of each 2-aryl carboxylic acids with HP-β-CD was determined by Benesi–Hildebrand's equation. According to our previous work, it was found that a high inclusion constant for inclusion complex formed by a racemate and cyclodextrin was always observed with the fact that a high enantioseparation factor was achieved for the racemate in enantioseparation by liquid–liquid chromatography, which suggested that high binding combination between racemate and cyclodextrin is very important for a successful enantioseparation in enantioselective liquid–liquid extraction. Among all the studied subjects, mandelic acid enantiomer, 2,3-diphenylpropionic acid enantiomer, and naproxen enantiomer were selected for the further study. The inclusion constants of enantiomers of these three subjects were determined by UV spectra, which indicated that a necessary difference in inclusion constants between enantiomer and cyclodextrin was also essential. It was found in UV spectra that the absorbance of the analytes with the addition of cyclodextrin would increase or decrease, which was determined by the type of electron excitation. The conformation changes of small molecules can lead to the changes of chromophore valence electron clouds distribution, causing the HOMO-LUMO energy difference decreased. Thus, a red shift of the wavelength of the maximum absorption was produced indicating that the possibility of the molecular interaction of enantiomers with HP-β-CD exists.     

Stereoselective metabolism of propranolol glucuronidation by human UDP‐glucuronosyltransferases 2B7 and 1A9

Stereoselective metabolism of propranolol side‐chain glucuronidation was studied for two recombinant human uridine diphosphate glucuronosyltransferases (UGTs), UGT1A9 and UGT2B7. The S‐ and R‐propranolol side‐chain glucuronides produced in the incubation mixtures were assayed simultaneously by RP‐HPLC with fluorescent detector. The excitation and emission wavelengths were set at 310 nm and 339 nm, respectively. UGT1A9 prefers catalyzing S‐enantiomer to R‐enantiomer and the intrinsic clearance (CL_int) ratios of S‐enantiomer to R‐enantiomer are 3.8 times and 6.5times for racemic propranolol and individual enantiomers, respectively. UGT2B7, however, catalyzes slightly less S‐enantiomer than R‐enantiomer and the CL_int ratio of S‐enantiomer to R‐enantiomer is 0.8 times. The high concentration of racemic propranolol (>0.57 mmol/l) and individual enantiomers (>0.69 mmol/l) exhibited substrate inhibition of glucuronidation for UGT2B7, but only the S‐enantiomer (>0.44 mmol/l) in racemic propranolol exhibited substrate inhibition for UGT1A9. The substrate inhibition constants (K_si) were all similar (P > 0.05). Drug–drug interactions were also found between S‐ and R‐enantiomer glucuronidation metabolisms by UGT1A9 and UGT2B7. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

cis-chlorobenzene dihydrodiol dehydrogenase (TcbB) from Pseudomonas sp. strain P51, expressed in Escherichia coli DH5alpha(pTCB149), catalyzes enantioselective dehydrogenase reactions

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5alpha(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3, 4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1, 2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.     

Contribution from the chirality of the growing chain to the enantiomer selectivity in activated-NCA polymerization of α-amino acid N-carboxyanhydride

As a model compound for the growing chain in the activated-NCA type of polymerization of α-amino acid N-carboxyanhydride (NCA), 3-[ω-acetylglycyl-poly(α-amino acid) acyl]-α-amino acid NCA (called the prepolymer) having various degrees of polymerization (DPs) was synthesized by the polymerization of Phe, Val, Glu(OEt), and Asp(OBzl) NCA in the presence of AcGly NCA by the tertiary amine. Activated (S)-Phe, Val, Glu(OEt), and Asp(OBzl) NCA were added to the terminal cyclic group of the corresponding (S)- or (R)- prepolymer, and the enantiomer selectivity in the reaction was investigated. With prepolymers having DPs ranging from 1 to 15, the addition reaction always took place preferentially between species having the same configuration, and the degree of the enantiomer selection increased with increasing DP of the prepolymer. With prepolymers having DP = 1 and 2, we found contributions from the chiral terminal unit and the chiral penultimate unit to the enantiomer selection, respectively. Prepolymer having DP = 5 was shown to take a β-type conformation, which led to higher enantiomer selection; and prepolymers having DP = 10 and 15 were shown to take an α-helix conformation, which led to much higher enantiomer selection than did the β-type conformation. In the present investigation the mechanisms of terminal-unit control, penultimate-unit control and conformational control of the enantiomer selection in the activated-NCA type of polymerization were clearly observed.     

Impact of microorganisms,humidity, and temperature on the enantioselective degradation of imazethapyr in two soils

Imazethapyr (IM) is a chiral herbicide composed of an (−)‐R‐enantiomer and an (+)‐S‐enantiomer with differential herbicidal activity. In this study, the effects of microbial organisms, humidity, and temperature on the selective degradation of the (−)‐R‐ and (+)‐S‐enantiomers of IM were determined in silty loam (SL) and clay loam (CL) soil with different pH values. The (−)‐R‐enantiomer of IM was preferentially degraded in two soils under different microorganism, humidity, and temperature conditions. The average half‐lives of R‐IM ranged from 43 to 66.1 days and were significantly shorter (P <  0.05) than those of S‐IM, which ranged from 51.4 to 79.8 days. The enantiomer fraction (EF = (+)‐S‐enantiomer/((−)‐R‐enantiomer + (+)‐S‐enantiomer)) values were used to describe the enantioselectivity of degradation of IM were >0.5 (P <  0.05) in two unsterilized soils under different humidity and temperature conditions. The highest EF values were observed at unsterilized CL soil samples under 50% maximum water‐holding capacity (MWHC) and 25 °C environmental conditions. The EF values of the IM enantiomers were significantly higher (P <  0.05) in CL soils (higher pH = 5.81) and were 0.581 (unsterilized) and 0.575 (50% MWHC; 25 °C) compared with those recorded in SL soil (lower pH = 4.85). In addition, this study revealed that microbial organisms preferentially utilized the more herbicidal active IM enantiomer.     

Flavonoids enantiomer distribution in different parts of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.) and phacelia (Phacelia tanacetifolia Benth.)

Plant material is a rich source of valuable compounds such as flavanones. Their different forms influence bioavailability and biological activity, causing problems with the selection of plant material for specific purposes. The purpose of this research was to determine selected flavanone (eriodictyol, naringenin, liquiritigenin, and hesperetin) enantiomer contents in free form and bonded to glycosides by an RP‐UHPLC‐ESI‐MS/MS method. Different parts (stems, leaves, and flowers) of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.), and phacelia (Phacelia tanacetifolia Benth.) were used. The highest content of eriodictyol was found in goldenrod flowers (13.1 μg/g), where it occurred mainly as the (S)‐enantiomer, and the greatest proportion of the total amount was bonded to glycosides. The richest source of naringenin was found to be lucerne leaves (4.7 μg/g), where it was mainly bonded to glycosides and with the (S)‐enantiomer as the dominant form. Liquiritigenin was determined only in lucerne, where the flowers contained the highest amount (1.2 μg/g), with the (R)‐enantiomer as dominant aglycone form and the (S)‐enantiomer as the dominant glycosylated form. The highest hesperetin content was determined in phacelia leaves (0.38 μg/g), where it was present in the form of a glycoside and only as the (S)‐enantiomer. A comparison of the different analyte forms occurring in different plant parts was performed for the first time.     

Nonlinear stereoselective pharmacokinetics of ketoconazole in rat after administration of racemate

The stereoselective pharmacokinetics of ketoconazole (KTZ) enantiomers were studied in rat after i.v. and oral administration of (+/-)-KTZ. Sprague-Dawley rats were administered racemic KTZ as 10 mg/kg i.v. or orally over the range 10-80 mg/kg as single doses. Serial blood samples were collected over a 24-h period via surgically placed jugular vein cannulae. Plasma was assayed for KTZ enantiomer concentrations using stereospecific HPLC. Enantiomeric plasma protein binding was determined using an erythrocyte partitioning method at racemic concentrations of 10 and 40 mg/L. Stereoselective metabolism was tested by incubating the racemate (0.5-250 microM) with rat liver microsomes. In all rats, (+)-KTZ plasma concentrations were higher (up to 2.5-fold) than (-)-KTZ. The clearance and volume of distribution of the (-) enantiomer were approximately twofold higher than antipode. Half-life did not differ between the enantiomers. After oral doses the t(max) was not stereoselective. For both enantiomers with higher doses the respective half-life were found to increase. The mean unbound fraction of the (-) enantiomer was found to be up to threefold higher than that of the (+) enantiomer. At higher concentrations nonlinearity in plasma protein binding was observed for both enantiomers. There was no evidence of stereoselective metabolism by liver microsomes. Stereoselectivity in KTZ pharmacokinetics is attributable to plasma protein binding, although other processes such as transport or intestinal metabolism may also contribute.