Whole exome sequencing reveals a novel de novo FOXC1 mutation in a patient with unrecognized Axenfeld-Rieger syndrome and glaucoma.

We report the identification of a novel mutation in the fork-head box C1 (FOXC1) gene which occurred de novo in an Italian patient with unrecognized Axenfeld-Rieger syndrome. He was previously diagnosed as having late recognized primary congenital glaucoma at the age of 14 years and was subsequently subjected to multiple surgical interventions due to uncontrolled intraocular pressure and progressive visual field loss. After exclusion of mutations in CYP1B1 and MYOC, trio-whole-exome sequencing revealed de novo in frame deletion in the coding region of the FOXC1 gene (c.407_409delGTC, p.V137del) leading to a deletion of the evolutionary conserved amino acid Valine at position 137 of the protein. Molecular modeling predicted that Val137 deletion impairs FOXC1 DNA-binding capacity and transcriptional activation. Since loss-of-function mutations in FOXC1 are associated with Axenfeld-Rieger syndrome, the genetic findings in combination with re-evaluation of the patient's clinical data resulted in a corrected diagnosis of Axenfeld-Rieger syndrome with developmental glaucoma. We therefore suggest that in addition to CYP1B1 and MYOC, FOXC1 should be included in the genetic analysis of cases with unclear glaucomatous phenotypes to ensure proper diagnosis, adequate treatment and appropriate genetic counseling.

A puzzle over several decades: eye anomalies with FRAS1 and STRA6 mutations in the same family.

Fraser syndrome (FS) and microphthalmia syndromic 9 (MCOPS9) are autosomal recessive conditions with distinct, and some overlapping features affecting the ocular, respiratory and cardiac systems. Mutations in FRAS1 and FREM2 occur in FS, and mutations in STRA6 occur in MCOPS9. We report two sibships, in the same family, where four deceased offspring had ocular, respiratory and cardiac abnormalities. Two sibs with microphthalmia had syndactyly and laryngeal stenosis, suggesting a clinical diagnosis of FS. Our results indicate that they were compound heterozygotes for novel FRAS1 mutations, p.Cys729Phe and p.Leu3813Pro. The other two sibs, first cousins to the first sib pair, had anophthalmia, lung hypoplasia and cardiac anomalies, suggesting a retrospective diagnosis of MCOPS9. Our results indicate compound heterozygous STRA6 mutations, a novel frameshift leading to p.Tyr18* and a p.Thr644Met mutation. The one surviving individual from these sibships is heterozygous for the p.Tyr18*STRA6 mutation and has bilateral ocular colobomata and microphthalmia. This work emphasises the need for careful phenotypic characterisation to determine genes for assessment in ocular syndromic conditions. It also indicates that heterozygous STRA6 mutations may rarely contribute to microphthalmia and coloboma.

Insulin-sparing effect of hydroxychloroquine in diabetic rats is concentration dependent.

To study the effect of hydroxychloroquine (HCQ) on glucose and insulin homeostasis, healthy rats were dosed with 160 mg x kg (-1) x day(-1) of HCQ orally, and streptozocin-induced diabetic rats received 80, 120, and 160 mg x kg(-1) x day(-1) of HCQ, while controls received normal saline. Ten days after treatment with HCQ, healthy animals were challenged intravenously with insulin or glucose, while diabetic rats were given only an i.v. injection of insulin. In healthy rats, the areas within and under the glucose concentration - time curve following insulin and glucose challenge were estimated. In diabetic animals, the areas under the curve for both the percent change in serum glucose from baseline (AUG) and the percent change in serum insulin from baseline (AUI) were used as pharmacodynamic end points. In healthy rats, HCQ did not influence fasting serum glucose concentrations or glycemic profiles following i.v. administration of glucose or insulin. In diabetic rats, AUG and AUI were increased dependent on blood HCQ concentrations. The normal homeostatic mechanisms responsible for insulin-glucose regulation may compensate for possible HCQ-induced reduction of insulin metabolism in healthy rats. The HCQ dose- or concentration-effect relationships for glucose and insulin were linear over the range of HCQ concentrations tested. It is concluded that HCQ significantly elevated insulin blood concentration resulting in reduced glucose levels in a concentration-dependent fashion in diabetic rats. HCQ may have therapeutic potential in the treatment of type I and type II diabetes.

Inhibition of insulin metabolism by hydroxychloroquine and its enantiomers in cytosolic fraction of liver homogenates from healthy and diabetic rats.

To elucidate the mechanism by which hydroxychloroquine (HCQ) affects glucose metabolism, the effect of this drug and its enantiomers on insulin metabolism was studied using the cytosolic fraction of liver homogenates from healthy and diabetic rats. Eadie-Hofstee plots were monophasic suggesting that only a one-component enzyme system is involved in insulin degradation in the fraction used. Reaction velocity (V) vs substrate concentration plots were consistent with a Vmax model. HCQ caused a significant reduction in Vmax and Vmax/Km values in both healthy (Vmax, 3.63 +/- 0.46 vs 1.97 +/- 0.13, ng/min/mg; protein P < 0.001; and Vmax/Km 0.265 +/- 0.015 vs 0.112 +/- 0.004, ml/min/g protein) and diabetic rats (Vmax, 0.718 +/- 0.06 vs 0.360 +/- 0.024, ng/min/mg protein; and Vmax/Km, 0.05 +/- 0.002 vs 0.023 +/- 0.001, ml/min/g protein). Significant reduction in the V was observed in the presence of racemic (rac)-, R-, or S-HCQ. Ranking of the inhibitory potency was HCQ > S = R except at highest examined concentration (20 mg/mL) which was HCQ > S > R. In conclusion, the effect of HCQ on insulin degradation appears to be, in part, through inhibition of cytosolic insulin metabolizing enzyme. The effect is not stereoselective except at high concentrations. The R- and S-HCQ may have synergistic effects on inhibition of insulin degradation.

Effect of experimental diabetes mellitus and arthritis on the pharmacokinetics of hydroxychloroquine enantiomers in rats.

PURPOSE: To study the effect of experimental diabetes and arthritis on the pharmacokinetics of hydroxychloroquine (HCQ) enantiomers in rats. METHODS: The pharmacokinetic studies were carried out following administration of 40 mg/kg of racemic HCQ to diabetic, insulin-treated diabetic, adjuvant arthritic and control rats. RESULTS: Renal (70% and 62% for R- and S-HCQ, respectively) and non-renal clearance (100% and 145% for R- and S-HCQ, respectively) of HCQ enantiomers were significantly increased in diabetic rats. Diabetes-induced alterations in the disposition of HCQ were reversed by insulin treatment. In arthritic rats, systemic clearance (CL) of HCQ enantiomers was significantly reduced (1.05 +/- 0.15 and 1.3 +/- 0.19 l/h/kg for R- and S-HCQ, respectively) compared to controls (1.69 +/- 0.32 and 1.93 +/- 0.34 l/h/kg for R- and S-HCQ, respectively). The fraction unbound of the R- and S-HCQ were 49.4% and 50.5% lower in platelet rich plasma of arthritic rats compared to healthy rats. Increased blood concentrations of HCQ enantiomers in arthritic rats were significantly related to the degree of inflammation. CONCLUSIONS: Diabetes significantly increased the CL of both R- and S-HCQ by increasing renal and non-renal clearance. Arthritis caused a significant decrease in CL of HCQ enantiomers through increased binding and a decreased intrinsic clearance. The effect of the diseases on the pharmacokinetics of HCQ, however, was not stereoselective.

Stereoselective metabolism of rac-mexiletine by the fungus Cunninghamella echinulata yields the major human metabolites hydroxymethylmexiletine and p-hydroxymexiletine.

rac-Mexiletine is an orally effective class 1b antiarrhythmic agent used to treat ventricular arrhythmias. In vivo experiments have demonstrated. It is predominantly metabolized by the liver with < 10% excreted as unchanged drug. The major mammalian metabolites have been identified as p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM). The purpose of our study was to determine whether the fungus Cunninghamella echinulata, which possesses a cytochrome P450 system analogous to that found in humans, could be used as a suitable in vitro model for studying the oxidative metabolism of rac-mexiletine. To accomplish this, a high performance liquid chromatographic assay was used that was capable of simultaneously quantifying the enantiomers of mexiletine, HMM, and PHM. Utilizing this procedure, it was demonstrated that C. echinulata stereoselectively converted rac-mexiletine into HMM (4% of added drug) and PHM (32% of added drug) after an incubation period of 50 hr. In addition, metabolite biosynthesis could be optimized by altering fermentation media components. Seven media values and seven pH values were evaluated. It was determined that a medium at pH 7.0 containing yeast extract and sucrose yielded optimal amounts of metabolites.

Renal, biliary and intestinal clearance of sotalol enantiomers in rat model: evidence of intestinal exsorption.

Biliary clearance (Clb) of sotalol (STL) enantiomers was assessed in anaesthetized Sprague-Dawley rats (419 +/- 9 g, mean +/- SEM, n = 4) following administration of a 10 mg kg-1 i.v. dose of the racemate. Clb for S- and R-STL (0.0675 +/- 0.0090 and 0.0662 +/- 0.0089 mL min-1 kg-1, respectively) represented approximately 0.3% of systemic clearance (Cls) values for S- and R-STL (20.4 +/- 2.2 and 20.7 +/- 2.0 mL min-1 kg-1, respectively). Bile:plasma concentration ratios at 1, 2, and 3 h post-dose were approximately 1.4, 1.3, and 1.2 for both STL enantiomers. Renal clearance (Clr) and intestinal clearance (Cli) of STL enantiomers were assessed in conscious Sprague-Dawley rats (325 g, n = 4) following administration of a 10 mg kg-1 i.v. dose of the racemate. STL enantiomers were predominantly eliminated intact in the urine: Clr for S- and R-STL (26.3 +/- 3.2 and 28.7 +/- 4.2 mL min-1 kg-1 respectively) accounted for approximately 96% of Cls for S- and R-STL (27.5 +/- 3.3 and 29.9 +/- 4.2 mL min-1 kg-1, respectively). Approximately 4% of the dose was recovered in the faeces, corresponding to Cli values of 1.16 +/- 0.17 and 1.26 +/- 0.19 mL min-1 kg-1 for S- and R-STL, respectively. Total recovery of the administered dose in urine and faeces was 99.7 +/- 0.2 and 99.8 +/- 0.5% for S- and R-STL, respectively. It is concluded from these results in the rat model that (i) STL enantiomers are predominantly eliminated intact in urine; (ii) STL enantiomers are excreted intact in bile, and to a much larger extent in the faeces, thus suggesting the presence of intestinal exsorption of STL; (iii) STL does not appear to be metabolized; and (iv) Cls, Clr, Clb, and Cli are negligibly stereoselective.

Influence of cimetidine coadministration on the pharmacokinetics of sotalol enantiomers in an anaesthetized rat model: evidence supporting active renal excretion of sotalol.

Sotalol (STL) is an amphoteric, chiral beta-adrenergic blocking drug useful in the treatment of both hypertension and ventricular arrhythmias. In the human and rat, STL enantiomers are predominantly cleared from the body by the kidney as intact drug. The renal clearance (Clr) of STL enantiomers substantially exceeds the glomerular filtration rate (GFR) in the human and rat. In this report, the hypothesis that STL enantiomers are excreted by an active renal transport system was investigated in the rat by coadministering racemic STL (10 mg kg-1) with cimetidine, an inhibitor of renal tubular secretion of organic cations. To compare the effects of short-term and sustained cimetidine exposure on STL enantiomer disposition, cimetidine was administered either as a single bolus (30 mg kg-1, n = 7) immediately prior to the STL dose, or as a 30 mg kg-1 bolus plus a 50 mg kg-1 infusion over the 6 h study period (n = 7). Blood and urine samples were collected over 6 h, during which time anaesthesia was maintained via intraperitoneal administration of pentobarbital. Cimetidine bolus and cimetidine infusion reduced STL enantiomer Clr by 43 and 59%, respectively, compared with respective saline controls. Significant stereoselectivity was observed in the cimetidine infusion group: systemic clearance, Clr (R > S), and AUC (S > R), although the magnitude of stereoselectivity was less than 5%. This study supports the hypothesis that STL enantiomers are predominantly cleared from the rat via a renal cationic transport mechanism and that this system can be competitively inhibited by the presence of cimetidine.

Pharmacokinetics of verapamil and norverapamil enantiomers after administration of immediate and controlled-release formulations to humans:evidence suggesting input-rate determined stereoselectivity.

Verapamil is a racemic calcium channel-blocking drug that undergoes extensive hepatic first-pass metabolism to an active metabolite, norverapamil. The enantiomers of verapamil and norverapamil have differing negative inotropic, chronotropic, and dromotropic activities and differing effects on vascular smooth muscles; the S-enantiomers having greater activity. It is hypothesized that the R/S concentration ratio of verapamil enantiomers may be input-rate dependent. The pharmacokinetics of verapamil and norverapamil enantiomers were studied in 11 young, healthy male and female volunteers after oral administration of 80 mg immediate-release (IR) verapamil every 8 hours, and a 240 mg dose once daily of controlled-release (CR) formulation on two separate occasions. Both dosage regimens were continued for 1 week with a minimum 1-week period between the two drug treatments. After the last dose of each regimen, plasma samples were collected over the period corresponding to the dosing interval. Enantiomer concentrations were determined using a microwave-facilitated precolumn derivatization with high performance liquid chromatographic quantification. Stereospecific assay revealed that: (1) stereoselective R- and S-enantiomer disposition occurred regardless of formulation administered; (2) a trend of R:S concentration ratios of verapamil differed between the two formulations; and (3) fluctuations between Cmax and Cmin values of the two formulations were statistically different over respective dosing intervals (greater fluctuation after CR administration). Using nonstereospecific data analyses, however, the pharmacokinetic parameters for verapamil and norverapamil were similar for both formulations over a 24-hour period. We suggest that kinetic differences can be attributed to differences in release rates of drug from the tablet matrices. The relative bioavailabilities of verapamil and norverapamil from the two products may, therefore, be subject to input rate-dependent processes.

Protein binding of sotalol enantiomers in young and elderly human and rat serum using ultrafiltration.

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32 +/- 2 years, n = 5) and elderly (73 +/- 6 years, n = 5) male and female humans, and young (8 weeks, n = 4) and elderly (60 weeks, n = 3) male Sprague-Dawley rats. Serum samples were collected and immediately frozen at -20 degrees C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL-1 and 500 ng mL-1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37 degrees C, and centrifuged at 1850g for 1.5 h at 37 degrees C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.

Stereospecific high-performance liquid chromatographic assay of lomefloxacin in human plasma.

This report describes an HPLC assay developed for the quantification of the enantiomers of lomefloxacin (LFLX), a quinolone antibiotic, in plasma. Following addition of racemic acebutolol (internal standard, IS), plasma samples were extracted at pH 7 with a mixture of chloroform-isopentyl alcohol-diethyl ether (71.25:3.75:25, v/v/v). The organic layer was evaporated, and LFLX and IS enantiomers in the resulting residue were derivatized with chloroform solutions of 1% triethylamine and 1% (S)-(+)-(1-naphthyl)ethyl isocyanate, followed by 2% ethyl chloroformate (ECF) 1 min later. Ethanolamine was added 30 s after the addition of ECF. The enantiomers were separated as diastereomers on an 8 x 100 mm Radial Pak normal phase column using a mobile phase of hexane-chloroform-methanol (64.5:33:2.5, v/v/v) pumped at 2.0 ml min-1. The IS was detected by fluorescence at 245 and 420 nm (excitation and emission, respectively) during the first 12 min, after which time the wavelengths were 280 and 470 nm for detection of LFLX. The method: (1) was sensitive and showed excellent linearity (10-1000 ng ml-1, r2 > 0.99) between added enantiomer concentrations and peak-area-ratio (LFLX/IS); and (2) separated LFLX and IS enantiomers within 25 min. The assay is suitable for the quantification of LFLX enantiomers in plasma samples.

Inclusion complexation of heptakis(2,6-di-O-ethyl)-beta-cyclodextrin with tiaprofenic acid: pharmacokinetic consequences of a pH-dependent release and stereoselective dissolution.

beta-Cyclodextrin was ethylated at the 2- and 6-hydroxyl positions. Diethyl sulfate was employed as an alkylating reagent. NMR spectra data indicate that heptakis(2,6-di-O-ethyl)-beta-cyclodextrin (DCD) is the principal component of the product obtained. In addition, the FAB mass spectra obtained in nitrobenzyl alcohol and glycerin matrices gave pseudo-molecular ions with m/z ratios of 1630.75 and 1711.90 corresponding to C70H126O35[2Na.NaCl] and C70H126O35[2C3H8O3], respectively. The dissolution of tiaprofenic acid (TA) enantiomers, from TA powder (10 mg) and inclusion complex and/or coprecipitate (IC) (TA:DCD 1:1 molar ratio, equivalent to 10 mg of free TA), were examined using the dispersion method at pH values of 1.5, 3.0, and 7.4. Complex formation with the hydrophobic DCD resulted in a significant reduction in the release rate of both R- and S-TA, as compared to that observed with the powder. At pH 1.5, tiaprofenic acid enantiomers were not released from IC, compared to 20.52 +/- 1.47% of R-TA and 20.47 +/- 1.64% of S-TA dissolved from the powder. The greatest stereoselectivity in release profiles was found at pH 3.0 from IC [S:R 24 h cumulative percent release (sigma R24) ratio of 0.88 +/- 0.04]. Elevation of the pH to 7.4, which resulted in a faster dissolution and greater sigma R24 of enantiomers from both powder and IC, was accompanied by a parallel reduction in the stereoselectivity. Following single 20 mg/kg oral doses of racemic TA as both powder or IC to Sprague-Dawley rats, significant stereoselectivity was observed in the plasma concentration profiles of the enantiomers (S:R AUC(0-infinity) = 1.5). Despite significant reduction in the rate and extent of absorption, there was not a significant difference in the observed in vivo stereoselectivity between the two formulations. Therefore the in vivo importance of the observed stereoselectivity in release at pH 3.0 is ruled out. Nevertheless, consideration must be given to the possibility of stereoselective release when chiral excipients are used in the formulation of racemic drugs.

Stereospecific high-performance liquid chromatographic assay of zopiclone in human plasma.

A high-performance liquid chromatographic (HPLC) assay for the analysis of the enantiomers of zopiclone (ZPC), a cyclopyrrolone hypnotic, in plasma was developed. Following the addition of chlordiazepoxide as internal standard (I.S.), plasma containing the ZPC enantiomers and I.S. was extracted by liquid-liquid extraction at an alkaline pH. After evaporation of the organic layer, the drug and I.S. were reconstituted in ethanol-hexane (80:20, v/v) and injected onto the HPLC column. The enantiomers were separated at ambient temperature on a 25-cm Chiralcel OD-H column with ethanol-hexane (60:40, v/v) as the mobile phase pumped at a flow-rate of 0.6 ml/min. The enantiomers of ZPC were quantified by fluorescence detection with excitation and emission wavelengths of 300 and 470 nm, respectively. The assay described allows for the direct quantitation of ZPC without pre-column derivatization, and is suitable for clinical studies of ZPC in humans after administration of therapeutic doses.

Stereospecific evaluation of sotalol pharmacokinetics in a rat model: evidence suggesting an enantiomeric interaction.

Sotalol (STL) is a chiral beta-adrenergic blocking drug, which is useful clinically as the racemate in treating hypertension, and is also useful as a class III antiarrhythmic when administered as the pure S-enantiomer. Utilizing a stereospecific high-performance liquid chromatographic (HPLC) assay, the enantiomeric disposition of STL is reported after administration of racemate and both pure enantiomers to a rat model. After administration of the racemate, enantiomers of STL had similar plasma concentration-time profiles. Following administration of the pure S-enantiomer of STL, however, systemic clearance was significantly reduced; R-STL disposition after pure enantiomer administration was not significantly altered. Changes in systemic clearance of S-STL after either racemate or enantiomer dosing were explained by corresponding changes in renal clearance. Renal clearance values of S-STL were significantly reduced from 33.7 +/- 6.0 to 28.9 +/- 5.6 ml min-1 kg-1 for administration as racemate and pure enantiomer, respectively. As clearance of STL approximates reported values of renal blood flow, renal perfusion changes caused by the beta-blocking effects of R-STL may explain changes in S-STL disposition. It is suggested that dosing of STL as either racemate or pure enantiomer, depending on the clinical indication for use, may result in significantly altered enantiomer disposition.

High-performance liquid chromatographic method for resolving the enantiomers of mexiletine and two major metabolites isolated from microbial fermentation media.

(+/-)-Mexiletine is a class Ib antiarrhythmic drug useful in the treatment of premature ventricular contractions. It is predominantly metabolized by the liver with less than 15% being excreted in urine as unchanged drug. p-Hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM) are the two major mammalian metabolites. The purpose of our study was to develop a stereospecific high-performance liquid chromatographic (HPLC) method to determine whether the fungus, Cunninghamella echinulata (UAMH 4145), was able to biosynthesize these same two metabolites from the substrate (+/-)-mexiletine. Furthermore, it was desirable to ascertain whether metabolism of mexiletine was stereoselective. The method requires pre-column derivatization of the drug and metabolites with S-(+)-1-(1-naphthyl)ethyl isocyanate (NEIC) followed by normal-phase HPLC. Mexiletine, PHM, HMM and (+/-)-1-(4-hydroxyphenoxy)-3-isopropylaminopropan-2-ol (internal standard) were extracted from microbial broth using two volumes of diethyl ether after basifying with sodium carbonate. The combined ether extracts were evaporated to dryness, using a gentle stream of nitrogen, and reconstituted in 0.3 ml of chloroform to which was added 0.075 ml of NEIC (0.1%, v/v, in chloroform). This solution was immediately evaporated to dryness under a nitrogen stream. The residue was reconstituted with 0.220 ml of chloroform and 0.030 ml of n-butylamine (0.33%, v/v, in chloroform) and injected into the HPLC system.

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 (alpha = 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 microgram/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.

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.

Mirror images: the analysis of pharmaceutical enantiomers.

Pharmaceutical enantiomers often exhibit different pharmacodynamic and pharmacokinetic properties. Stereospecific chromatographic assays are available to separate these stereoisomers. Therapeutic agents often contain chemical functional groups (e.g. amino, hydroxyl, carbonyl, and carboxylic acid). These can be reacted with enantiomerically pure reagents to give diastereoisomers suitable for analysis on achiral gas chromatographic (GC) and high performance liquid chromatographic (HPLC) columns. Alternatively, derivatized or underivatized drugs may be resolved on chiral chromatographic phases. A wide variety of GC (e.g. amino acid, cyclodextrin, and metal-complex) and HPLC (mobile phase additive, crown ether, pi-pi interaction and related phases, protein, cyclodextrin, polysaccharide, methacrylate and amide polymer, and ligand exchange) columns are commercially available. This article reviews the chromatographic separation of enantiomers.

Stereospecific high-performance liquid chromatographic determination of tocainide.

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.