Enantiomeric separation of metoprolol and α-hydroxymetoprolol by liquid chromatography and fluorescence detection using a chiral stationary phase

A sensitive and stereoselective high-performance liquid chromatographic assay for the determination of the enantiomers of metoprolol (R- and S-) and the diastereoisomers of α-hydroxymetoprolol (IIA, IIB) in plasma is reported. Chromatography involved direct separation of enantiomers using a Chirobiotic T bonded phase column (250×4.6 mm) and a mobile phase consisting of acetonitrile–methanol–methylene chloride–glacial acetic acid–triethylamine (56:30:14:2:2, v/v). Solid-phase extraction using silica bonded with ethyl group (C₂) was used to extract the compounds of interest from plasma and atenolol was used as the internal standard. The column effluent was monitored using fluorescence detection with excitation and emission wavelengths of 225 and 310 nm, respectively. S-Metoprolol,R-metoprolol, IIB and IIA eluted at about 5.9, 6.7, 7.3 and 8.2 min without any interfering peaks. The calibration curve was linear over the range of 0.5 to 100 ng/ml for each isomer of metoprolol and 1 to 100 ng/ml for each isomer of α-hydroxymetoprolol (IIA & IIB). The mean intra-run accuracies were in the range of 96.2 to 114% for R-metoprolol, 94.0 to 111% for S-metoprolol, 90.2 to 110% for IIA, and 94.6 to 106% for IIB. The mean intra-run precisions were all in the range of 2.2 to 12.0% for R-metoprolol, 2.1 to 11.1% for S-metoprolol, 1.9 to 14.5% for IIA, and 3.2 to 11.0% for IIB. The lowest level of quantitation for the enantiomers of metoprolol was 0.5 ng/ml and 1.0 ng/ml for α-hydroxymetoprolol (IIA and IIB). The absolute recoveries for each analyte was ≥95%. The validated method accurately quantitated the enantiomers of parent drug and metabolite after a single dose of an extended release metoprolol formulation.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.     

Enantioselectivity of debrisoquine 4-hydroxylation in Brazilian Caucasian hypertensive patients phenotyped as extensive metabolizers

Debrisoquine (D), an antihypertensive drug metabolized to 4-hydroxydebrisoquine (4-OHD) by CYP2D6, is commonly used as an in vivo probe of CYP2D6 activity and can be used to phenotype individuals as either extensive (EMs) or poor metabolizers (PMs) of such drugs as β-adrenergic blockers, tricyclic antidepressants, and class 1C antiarrhythmics. This report describes reversed-phase HPLC systems by which D and 4-OHD or S-(+) and R-(−)-4-OHD in urine are more selectively quantified without the need for derivatization techniques. We also studied the urinary excretion of R-(−)- and S-(+)-4-hydroxydebrisoquine in EM hypertensive patients in order to determine weather 4-OHD formation exhibits enantioselectivity. Twelve patients with mild to severe essential hypertension were admitted to the study. They received a single tablet of Declinax containing 10 mg debrisoquine sulfate. All the urine excreted during the following 8 h was collected. The debrisoquine metabolic ratio (DMR) was calculated as % of dose excreted as D/% of dose excreted as 4-OHD and the debrisoquine recovery ratio (DRR) was calculated as % of dose excreted as 4-OHD/% of dose excreted as D+4-OHD. Debrisoquine and its metabolite were determined in urine by HPLC using a reversed-phase Select B LiChrospher column, a mobile phase of 0.25 N acetate buffer, pH 5–acetonitrile (9:1, v/v) and a fluorescence detector. The limit of quantitation was determined to be 25.0 ng/ml for D and 18.75 ng/ml for 4-OHD. Intra- and inter-day relative standard deviations (RSDs) were less than 10%. All hypertensive patients studied showed a DMR of less than 12.6 or a DRR higher than 0.12 and were classified as EMs. Direct enantioselective separation on chiral stationary phase involved resolution of S-(+)-4-OHD and R-(−)-4-OHD on a Chiralcel OD-R column with a mobile phase of 0.125 N sodium perchlorate, pH 5–acetonitrile–methanol (85:12:3, v/v/v). The quantitation limit of each enantiomer was 3.75 ng/ml of urine. Intra- and inter-day RSDs were less than 10% for each enantiomer. A high degree of enantioselectivity in the 4-hydroxylation of D favouring the S-(+) enantiomer was observed, resulting in R-(−)-4-OHD not detected in the urine of the EM hypertensive patients studied.     

Stereospecific high-performance liquid chromatographic assay of metoprolol

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)-ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (R_s > 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5–500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Enantioselective high-performance liquid chromatographic method for the determination of methadone and its main metabolite in urine using an AGP and a C8 column coupled serially

A simple and sensitive method for the enantioselective high-performance liquid chromatographic determination of methadone and its main metabolite, EDDP, in human urine is described. (−)-(R)-Methadone, (+)-(S)-methadone, (+)-(R)-EDDP, (−)-(S)-EDDP and imipramine as an internal standard are detected by ultraviolet detection at 200 nm. The enantiomers of methadone and EDDP were extracted from human urine by a simple liquid–liquid extraction procedure. The extracted sample was reconstructed in mobile phase and the enantiomers of methadone and EDDP were quantitatively separated by HPLC on a short analytical LiChrospher RP8 column coupled in series with a chiral AGP column. Determination of all four enantiomers was possible in the range of 0.03 to 2.5 μM. The recoveries of methadone enantiomers and EDDP enantiomers added to human urine were about 90% and 80%, respectively. The method was applicable for determination of methadone enantiomers and the enantiomers of its main metabolite in urine samples from methadone maintenance patients and patients suffering from severe chronic pain.     

Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, (α-keto-β-methylval-erate) in physiological fluids suitable for isotope enrichment analysis is described: after extraction with acid, 2-oxo acids are separated from interfering amino acids by cation-exchange chromatography. Reductive amination of the branched-chain 2-oxo acids by use of - leucine dehydrogenase yields the corresponding -amino acids. -Isoleucine and -alloisoleucine which are formed from S- and R-3-methyl-2-oxopentanoate, respectively, are then quantified by amino acid analysis. The method was used for determination of the R-/S-3-methyl-2-oxopentanoate ratio in plasma of healthy subjects and patients with diabetes mellitus and maple syrup urine disease. Applicability for gas chromatographic-mass spectrometric analysis of ¹³C-label enrichment in plasma S-3-methyl-2-oxopentanoate is demonstrated.     

Enantioselective synthesis of (R)-(+)- and (S)-(-)-higenamine and their analogues with effects on platelet aggregation and experimental animal model of disseminated intravascular coagulation

Optically active tetrahydroisoquinoline alkaloids, (R)-(+)-higenamine (1R) and (S)-(−)-higenamine (1 S), and their optically active 1-naphthylmethyl analogues (2 and 3), were synthesized by enantioselective hydrogenation of the corresponding dihydroisoquinoline intermediates 7 as a key step. The evaluation of the platelet anti-aggregation effect demonstrated clearly that the (S)-(−)-enantiomers, 1S, 2S, and 3S, had higher inhibitory potency than the corresponding (R)-(+)-antipodes, 1R, 2R, and 3R, respectively, to platelet aggregation induced by epinephrine. 1S enantiomer was superior to the corresponding 1R enantiomer in attenuating all of the disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) parameters tested, while the S enantiomers 2S and 3S ameliorated some of the DIC and MOF parameters more effectively than the corresponding antipodes 2R and 3R.     

Assay of moguisteine metabolites in human plasma and urine: conventional and chiral high-performance liquid chromatographic methods

Moguisteine is a novel peripheral non-narcotic antitussive agent. Pharmacokinetic studies in animal and in man showed that no unchanged drug is present in plasma, urine and faeces after oral administration. The main active metabolite, M1, is the free carboxylic acid of moguisteine, which maintains a stereogenic centre and consists of R(+)-M1 and S(−)-M1 enantiomers. M1 is partly metabolized to M2, its sulfoxidation derivative. A conventional HPLC method is described for the simultaneous determination of M1 and M2 in human plasma and urine after administration of therapeutic moguisteine doses. Plasma samples, previously acidified with phosphoric acid, are extracted with dichloromethane; urine samples are analyzed after appropriate dilution with methanol. Chromatography is performed using a Lichrosorb RP2 column and a linear gradient. M1 enantiomers can be determined in plasma extracts and urine samples by a chiral HPLC method using a β-cyclodextrin column. The analytical characteristics of both HPLC procedures proved to be adequate to analyze samples of subjects treated with therapeutic doses of moguisteine during clinical pharmacokinetic studies.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R)-[2(R,S)-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2(R,S)-yl-methyl)-[1,3]dioxolan-4(S)-yl]-5H-furan-2-one and 3-O-octadecyl derivatives

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of α-tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a–d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic α-tocopherol analogue (5) and natural α-tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a–d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 μM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.     

High-performance liquid chromatographic analysis of methocarbamol enantiomers in biological fluids

Methocarbamol enantiomers in rat and human plasma were quantified using a stereospecific high-performance liquid chromatographic method. Racemic methocarbamol and internal standard, (R)-(−)-flecainide, were isolated from plasma by a single-step extraction with ethyl acetate. After derivatization with the enantiomerically pure reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate, methocarbamol diastereomers and the (R)-flecainide derivative were separated on a normal-phase silica column with a mobile phase consisting of hexane—isopropanol (95:5, v/v) at a flow-rate of 1.6 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm. The resolution factor between the diastereomers was 2.1 (α = 1.24). An excellent linearity was observed between the methocarbamol diastereomers/internal standard derivative peak-area ratios and plasma concentrations, and the intra- and inter-day coefficients of variation were always <9.8%. The lowest quantifiable concentration was 0.5 μg/ml for each enantiomer (coefficients of variation of 9.8 and 8.8% for (S)- and (R)-methocarbamol, respectively), while the limit of detection (signal-to-noise ratio 3:1) was approximately 10 ng/ml. The assay was used to study the pharmacokinetics of methocarbamol enantiomers in a rat following intravenous administration of a 120 mg/kg dose of racemic methocarbamol and to evaluate plasma and urine concentrations in a human volunteer after oral administration of a 1000-mg dose of the racemate. The method is suitable for stereoselective pharmacokinetic studies in humans as well as in animal models.     

Capillary electrophoretic separation of nucleotide isomers via complexation with cyclodextrin and borate

The electrophoretic behaviour of monophosphorylated nucleotide isomers can be manipulated using complex-forming reactions with β-cyclodextrin (β-CD) and borate. Resolution of the 2'- and 3'-isomers of nucleotides is possible when the electrophoresis buffer contains 10 mM CD. The effect of β-CD concentration on electrophoretic mobility is used to calculate the formation constant, K, of β-CD—nucleotide complexes. The 3'-isomer of adenosine monophosphate (AMP) forms the strongest complex with β-CD probably as a result of hydrogen bonding between the phosphate group of AMP and hydroxyls of β-CD. In addition, complexation of 5'-nucleotides with borate increases the migration time window and leads to better separation. Complex-forming reactions of guanosine monophosphate and uridine monophosphate are shown to be strongly dependent on buffer pH. A mixture of 12 monophosphorylated nucleotides can be separated in less than 15 min using a buffer of 20 mM borate—10 mM β-CD.     

Theoretical study on chiral recognition mechanism of methyl mandelate enantiomers on permethylated β-cyclodextrin

Host-guest interactions of permethylated β-cyclodextrin (PM-β-CD) with methyl mandelate enantiomers ((R/S)-MMA) were simulated using semiempirical PM3 and ONIOM (B3LYP/6-31G(d):PM3) method. The chiral recognition mechanism of (R/S)-MMA enantiomers on PM-β-CD was investigated. The binding energies for all orientations considered in this research are reported. The most stable geometry structures of the two complexes are different. The benzene ring of (R)-MMA locates horizontally approximately on the wider edge of the PM-β-CD cavity, but the aromatic ring of (S)-MMA is deeply included into the hydrophobic cavity. Furthermore, the results of NBO analysis show that the main driving forces in the inclusion process of PM-β-CD with (R/S)-MMA are hydrogen bonding interaction, dipole-dipole interaction, charge-transfer and hydrophobic interaction. The stabilization energy of the (R)-MMA/PM-β-CD complex is lower than that of the (S)-MMA/PM-β-CD complex. Moreover, the chiral carbon in MMA of (R/S)-MMA/PM-β-CD complexes are close to the C2 and C3 in the glucose unit. The chiral recognition mechanism is thus closely related to the chiral environment provided by C2 and C3 in the glucose unit and the degree of (R/S)-MMA and PM-β-CD inclusion.     

Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine

A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-μm Microbore C₁₈ column in (6R,S)-LV and (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-μm bovine serum albumin column through a Rheodyne valve system and are separated into their distereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4–12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.     

Analysis of some metabolites of organic solvents in urine by high-performance liquid chromatography with β-cyclodextrin

Chromatographic separation of the metabolites derived from toluene, ethylbenzene, styrene and xylene was carried out on untreated urine samples from factory workers. The elution sequence was as follows: phenylglyoxilic acid, 3-hydroxy-2-butanone, hippuric acid, o-methylhippuric acid, p-methylhippuric acid, m-methylhippuric acid, p-cresol, m-cresol and o-cresol. The stability constants (K_G) of cresol and methylhippuric acid derivatives were evaluated. The capacity factor (k′), selectivity factor (α) and resolution (R_s) are described with a variety of mobile phases containing β-cyclodextrin (β-CD). The optimum concentration ratio of ethanol–water–acetic acid–β-CD was determined to be 20:80:0.3:1.4%. Under these conditions, k′ values of the five metabolites were 2<k′<6, and all α values were greater than 1.5. Simultaneous determinations of the metabolites were carried out in real urine samples from factory workers using the standard addition method. Validation of the method and the detection limit are described under the optimum conditions attained in this experiment.     

Synthesis of hydantoin analogues of (2S,3R,4S)-4-hydroxyisoleucine with insulinotropic properties

The first synthesis of an optically pure (2R,3R,4S)-hydantoin 2, analogue of (2S,3R,4S)-4-hydroxyisoleucine, was achieved in two steps in un-optimized 35% overall yield from previously reported aldehyde synthon 1. (2R,3R,4S)-Hydantoin is stable at acidic pH. This solves the major drawback of (2S,3R,4S)-4-hydroxyisoleucine that easily cyclizes into inactive lactone. Furthermore, (2R,3R,4S)-hydantoin stimulates the insulin secretion by 150% at 25 μM compared with 4-hydroxyisoleucine and insulin secretagogue drug repaglinide. In view of its stability and biological activity, (2R,3R,4S)-hydantoin represents a good candidate for type-2 diabetes management and control.     

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with β-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C₁₈ reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 μM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 μM 5-HTOL and a standard solution of 2.0 μM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r² = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic—mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 μM, but after an acute dose of alcohol they increased to 0.5–15 μM.     

Enantioselective gas chromatographic assay with electron-capture detection for amlodipine in biological samples

A sensitive enantioselective gas chromatographic assay has been developed for amlodipine, 2-[(2-aminoethoxy)-methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine, a calcium channel blocking therapeutic agent. The assay involves conversion of the (+)-(R)- and (−)-(S)-enantiomers of amlodipine into their acyl derivatives with the chiral reagent (+)-(S)-α-methoxy-α-trifluoromethylphenylacetyl chloride (Mosher's reagent). Peak separation after chromatography of the diastereomers was larger than 85%, and the lower limit of detection in blood plasma was 0.02 ng/ml for each enantiomer. The method has been used for the measurement of amlodipine enantiomers in human, rat and dog plasma, and in various organs of the rat.     

Rapid chiral high-performance liquid chromatographic assay for salmeterol and α-hydroxysalmeterol: Application to in vitro metabolism studies

A rapid and sensitive chiral high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of salmeterol and its principal human metabolite α-hydroxysalmeterol is described. The two pairs of enantiomers were resolved on a chiral-cellobiohydrolase column and detected by electrochemical detection at +700 mV. Standard curves were linear over the concentration range 0.1 to 4.0 μM for α-hydroxysalmeterol enantiomers and 2.5 to 40.0 μM for salmeterol enantiomers. Intra- and inter-day coefficients of variation were <10%. The method was applied to a study of human hepatic metabolism in vitro which showed that microsomal metabolism of salmeterol to α-hydroxysalmeterol is not stereoselective.