Polarimetric detection in high-performance liquid chromatography

Chiroptical detection for HPLC is particularly useful as a selective detection method for chiral molecules, and in enantiomeric purity determination with partial chiral separation or without chiral separation. The recent development of laser-based polarimeters with microdegree sensitivity has increased the applicability of optical rotation detection in HPLC. The detection limit of these instruments is submicrogram on-column for many chiral compounds in analytical HPLC. A variety of applications of the selective detection of optically active molecules are reviewed. The use of polarimetric detection with partial chiral separation is considered, both as an aid to method development and for enantiomeric purity determination. Finally applications to enantiomeric purity determination without chiral separation are reviewed, with the dual use of nonchirally selective and chiroptical detectors to determine the total amount and optical purity of the analyte. Determinations of chiral purity for samples of high enantiomeric excess are described, which with laser-based instrumentation may give accuracies of better than +/- 1% with sample loadings of 50 micrograms on an achiral column. Applications to the study of enantioselective reactions are also considered, with determination of enantiomeric excess in near-racemates to better than +/- 0.1%.     

Sensitivity improvement of circular dichroism detection in HPLC by using a low-pass electronic noise filter: Application to the enantiomeric determination purity of a basic drug

The quality control of chiral drugs requires the determination of their enantiomeric purity. Nowadays, circular dichroism (CD) spectroscopy is gaining increasing importance in pharmaceutical analysis because of the commercially available CD detector in liquid chromatography. The separation of the two enantiomers of a basic drug (efaroxan) was achieved by high performance liquid chromatography using an amylose-derivated column with both UV and CD detections. A baseline-resolved separation (resolution: 5) was obtained after optimization of the mobile phase composition with hexane-ethanol-diethylamine (90:10:0.05; v/v/v). The use of a commercial low-pass electronic noise filter of the CD signal has improved the signal-to-noise ratio by a factor twelve and allowed the quantitation of each enantiomer in the 1.25-300 microg ml(-1) concentration range. The CD linear calibration curve, expressed in terms of stereoisomer height ratio versus concentration ratio, was plotted over the 0.4-6% range. A correlation coefficient greater than 0.999 was obtained by least-squares regression and the limit of detection for the distomer/eutomer ratio was estimated at 0.14%. Although the method validation showed good repeatability on the retention times (RSD < 0.9%), on the peak height ratios (RSD < 8.7%) of each enantiomer only up to 99.2% enantiomeric purity was achieved.     

Synthesis of glycosides of 3-deoxy-4-thiopentopyranosid-2-uloses and their reduction products: 3-deoxy-4-thiopentopyranosides

Michael addition of common thiols to the enone system of (2S)-2-benzyloxy-2H-pyran-3(6H)-one (1) afforded the corresponding 3-deoxy-4-thiopentopyranosid-2-ulose derivatives (2-4). The reaction was highly diastereoselective, and the addition was governed by the quasiaxially disposed 2-benzyloxy substituent of the starting pyranone. As expected from the enantiomeric excess of 1 (ee > 86%) the corresponding thiouloses 2-4 exhibited the same optical purity. However, the enantiomerically pure thioulose 5 was obtained by reaction of 1 with the chiral thiol, N-(tert-butoxycarbonyl)-L-cysteine methyl ester. The thio derivative 7 was also synthesized by reaction of 6 (enantiomer of 1) with the same chiral thiol. Alternatively, 4-thiopent-2-uloses 9-12 were prepared in high optical purity by 1,4-addition of thiols to (2S)-[(S)-2'-octyloxy]dihydropyranone 8. Similarly, reaction of 13 (enantiomer of 8) with benzenemethanethiol afforded 14 (enantiomer of 10). This way, the stereocontrol exerted by the anomeric center on the starting dihydropyranone led to 4-thiopentuloses of the D and L series. Sodium borohydride reduction of the carbonyl function of uloses 10 and 12 gave the corresponding 3-deoxy-4-thiopentopyranosid-2-uloses (16-19). The diastereomers having the beta-D-threo configuration (16, 18) slightly predominated over the beta-D-erythro (17, 19) analogues. However, the reduction of the enantiomeric pyranones 10 and 14 with K-Selectride was highly diastereofacial selective in favor of the beta-D- and beta-L-threo isomers 16 and 20, respectively.     

Preparative enantiomeric separation of new selective CB2 receptor agonists by liquid chromatography on polysaccharide-based chiral stationary phases: determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analysis

The development of high-performance liquid chromatography (HPLC) methods using derivatized amylose chiral stationary phases has permitted preparative enantioseparations of substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with satisfactory yields. These compounds constitute new potent selective agonists of the cannabinoid CB(2) receptor. Analytical enantioseparation methods using UV detection were validated to determine the enantiomeric purity of these compounds. Linear calibration curves in the range from 0.18 to 0.40 mM were obtained; repeatability, limits of detection (LOD), and quantification (LOQ) were determined: LOD varied, for the various solutes, from 0.5 to 1.2 μM. All the separated compounds were prepared with high enantiomeric purities superior to 99.3% Absolute configuration of the enantiomers was unequivocally established by single crystal X-ray diffraction method and correlated to the chiroptical properties of isolated enantiomers.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of tetrahydropalmatine enantiomers in dogs

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mmx4.6 mm, 5 microm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mmx4.6 mm, 5 microm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (-)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (alpha) greater than 1.29. Linear calibration curves were obtained over the range of 0.025-4 microg/ml in plasma for each of (+)-THP and (-)-THP (R2>0.999) with a limit of detection (LOD) of 0.005 microg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.     

Determination of enantiomeric excess of nipecotic acid as 1‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl) derivatives

For the enantiopure synthesis of novel chiral GABA uptake inhibitors, nipecotic acid ( 1 ) is an important key precursor. To characterize accurately the pharmacological activity of these interesting target compounds, the determination of the correct enantiomeric purity of nipecotic acid as the starting material is indispensable. In this report, a sensitive high‐performance liquid chromatography (HPLC) based method for the separation and quantitation of both enantiomers of nipecotic acid as 1‐(7‐nitrobenzo[c ][1,2,5]oxadiazol‐4‐yl) derivatives ( 5 ) on a Chiralpak ID‐3 column (Daicel, Illkirch, France) was established. UV/Vis‐detection at 490 nm was chosen to ensure a selective determination of even highly enantioenriched samples. Reliability was demonstrated by validation of specificity, linearity, lower limit of quantification (LLOQ), accuracy, and precision. By spiking highly enantiopure samples with small amounts of racemic rac ‐ 5 , it was proven that the established HPLC method is able to detect even slight changes in enantiomeric excess (ee) values. Thus, accurate determination of ee values up to 99.87% ee for (R )‐ 5 and 99.86% ee for (S )‐ 5 over a linear concentration range of 1– 1500 μM for (R )‐ 5 and of 1– 1455 μM for (S )‐ 5 could be demonstrated.     

Stereochemistry and enantiomeric purity of a novel anxiolytic agent, deramciclane fumarate.

The synthesis, stereostructure, and enantiomeric separation by chromatography of a new, chiral anxiolytic agent, deramciclane fumarate (2, (-)-[1R,2S,4R]-2-(2-dimethylaminoethoxy)-2-phenyl-1,7, 7-trimethylbicyclo[2.2.1]heptane fumarate, EGIS-3886), is described. The optical antipode and the racemate of compound 2 were also prepared. The structure was determined by single crystal X-ray diffraction analysis. The enantiomeric separation was accomplished by HPLC on Chiralcel OD (250 x 4.6 mm; 10 microm) and hexane-ethanol (99.5:0.5) as mobile phase at room temperature. The enantiomeric purity of the synthesized drug substance proved to be very high (>99. 9%). Some statements published earlier on the stereostructure of deramciclane fumarate are critically discussed.     

Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH.

Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL-1) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL-1) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mg.mL-1), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.     

New methods for determining the enantiomeric purity of erythro -sphingosine

The enantiomeric purity of erythro -sphingosine samples can be determined simply, reliably, and accurately from 1H or 19F nuclear magnetic resonance spectra of the alpha-methoxy-alpha-(trifluoromethyl)phenylacetate (MTPA) derivative. As little as 0.1% of the minor enantiomer could be observed in a 1-mg sample, and detection limits of 1% and 5% were estimated for samples of 100 microg and 10 microg. The two threo -sphingosine enantiomers and four dihydrosphingosine stereoisomers were also differentiated by this technique, which served as an effective method for assessing the purity of sphingosine and dihydrosphingosine samples. Enantiomeric and diastereomeric purities could also be determined by normal-phase high performance liquid chromatographic analysis of the MTPA derivatives.     

Chiral discrimination of multiple profens as diastereomeric (R)-(+)-1-phenylethylamides by achiral dual-column gas chromatography

Profens were converted into diastereomeric (R)-(+)-1-phenylethylamides using ethyl chloroformate and triethylamine in dichloromethane. Gas chromatographic analysis on dual-columns with different polarities provided complete enantioresolution of eight profens, facilitating chiral discrimination based on matching with retention index sets characteristic of each enantiomer. The present method was linear (r >/= 0.9992) with good precision (0.8-6.0%) and accuracy (-9.3 to 0.003%), allowing detection of trace (R)-profens in optical purity test on four (S)-profen mixture in a single run. And the method allowed simultaneous enantiomeric screening for ibuprofen enantiomers and their chiral metabolites excreted in urine following administration of racemic ibuprofen.     

Simultaneous determination of diastereoisomeric and enantiomeric impurities in (1R, 3R)-1-(1,3-benzodioxol-5-yl)-2-(chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester a key intermediate of tadalafil by chiral high-performance liquid chromatography

(1R, 3R)-1-(1, 3-Benzodioxol-5-yl)-2-(chloroacetyl)-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester ((1R, 3R)-Cpe) is a key intermediate used in the synthesis of tadalafil, a highly selective phosphodiesterase type-5 inhibitor. In the present study, a chiral high-performance liquid chromatography method was developed for the simultaneous determination of diastereoisomeric and enantiomeric impurities in (1R, 3R)-Cpe. Separation was performed on an Ultron ES-OVM chiral column (150 mm × 4.6 mm, 5 μm,) with a guard column at a column temperature of 30°C. The gradient elution used was acetonitrile (solvent A) and water (solvent B), and the following elution program was used at a flow rate of 1 ml/min: 0-5 min (80% B), 5-10 min (80-60% B), 10-12 min (60% B). The detection wavelength was 220 nm. The four isomers of Cpe were baseline separated in 12 min. The results of method validation indicated that the method was specific and sensitive and was suitable for the quality control of diastereoisomeric and enantiomeric impurities in (1R, 3R)-Cpe.     

Preparative enantiomeric separation of new aromatase inhibitors by HPLC on polysaccharide-based chiral stationary phases: Determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analysis

The development of high-performance liquid chromatography methods on polysaccharide-based stationary phases (cellulose or amylose derivatives) has permitted preparative enantioseparations of various 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzoxazol-2(3H)-one and 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzothiazol-2(3H)-one, aromatase inhibitors, with satisfactory yields. Analytical enantioseparation methods using both UV and evaporative light-scattering detection (ELSD) were validated to determine the enantiomeric purity of these compounds. Using UV detection, linear calibration curves in the range from 4 x 10(-6) to 4.8 x 10(-4) M range were obtained; repeatability, limits of detection (LD), and quantification (LQ) were determined: LD varied, for the various solutes, from 1 to 80 microg/l and from 2.05 to 10.05 mg/l with UV detection and ELSD, respectively. Single-crystal X-ray analysis was successful in determining the absolute configuration of the individual enantiomers. The relationship between retention order and absolute configuration of the enantiomers was established.     

Bioreduction of aromatic ketones: preparation of chiral benzyl alcohols in both enantiomeric forms

Substituted phenacyl chlorides are reduced with whole-cell biocatalysts to give (R)- or (S)-chlorohydrines in high yields and to make them good for high enantiomeric excess. Yields and enantiomeric purity of the S-enantiomer could be increased by performing bioreduction in the presence of polymeric absorbing resins. With this methodology, 2-chloro-1(S)-(3,4-dichloro-phenyl)-ethanol of 98% e.e. and 2-(R)-(4-nitro-phenyl)-ethanol of 92% e.e. have been prepared and used respectively as precursors in the synthesis of (+)-cis-1(S),4(S)-sertraline and of the β-blocker (R)-nifenalol®.     

Cytotoxic activity of (S)-goniothalamin and analogues against human cancer cells

(R)- and (S)-Goniothalamin (1) and analogues 2-9 were efficiently prepared in high overall yield and enantiomeric purity, and their cytotoxic activities were evaluated against eight human cancer cell lines. A structure-activity relationship study (SAR) allowed us to establish the relevant structural features for the cytotoxic activity of goniothalamin analogues. In addition, we have identified non-natural form of goniothalamin (S)-1 and analogue 5 as the highest and more selective cytotoxic compounds against kidney cancer cell growth (786-0) with IC50 = 4 and 5 nM, respectively, and compound 8 (IC50 = 4 nM) as the more potent against breast cancer cells with resistance phenotype for adryamycin (NCI.ADR).     

Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate

An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.     

Reduction of 2-Substituted Cyclohexanones by Saccharomyces Cerevisiae

An enzymatic reduction of 2-substituted cyclohexanones mediated by Saccharomyces cerevisiae was studied with respect to the stereochemical course and optical purity of the products. Reduction of ketones 1b-1f resulted in separable diastereoisomeric mixtures of cis- and trans-stereoisomers of 2-substituted cyclohexanols (2b-2f and 3b-3f) having the (S) absolute configuration at the chiral center bearing the hydroxyl functionality with high enantiomeric purity. Reduction of ketone 1a yielded mixture of cis-(1S, 2R)- and trans-(1R, 2R)-stereoisomers (2a and 3a) with lower enantiomeric purity. Changes in the nature of the C(2)-substituent affect the stereochemical course of the biotransformation. However, they significantly influenced the enantiomeric purity of the products. The diastereoselectivity of the process was studied as well; high diastereoselectivity was observed with the substrates 1a, 1e and 1f.     

Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography

A rapid reversed-phase type HPLC method for the simultaneous separation and analysis of D- and L-thyroxine (D- and L-T(4)) and triiodothyronine (T(3)) was developed using a quinine-derived chiral stationary phase and applied for a quantitative assay of the enantiomeric impurity of the drugs in pharmaceutical formulations of levothyroxine. The influence of operating parameters has been studied for the optimization of the separation and also in order to gain an insight into the retention mechanism. Validation of the method included linearity, precision and accuracy which revealed R.S.D. values of <3.3% for intra-assay precision and percent error ranging from -6 to +2.1% for various defined validation samples, proving satisfactory accuracy. Quantitation was performed over the range of 0.5-500 microg ml(-1) with limits of detection and quantitation lower than 0.1 and 0.5 microg ml(-1), respectively, for both analytes. Further, the determination of 0.1% impurity, of D-T(4) as well as L- and D-T(3) in levothyroxine sodium tablets proved to be feasible.     

Circular dichroism as a detection method in the screening of enantioselective catalysts

The combination of liquid chromatography (HPLC), UV/Vis-spectroscopy and circular dichroism (CD) can be used to construct a high-throughput screening system to determine the enantioselectivity of enzyme- or metal-catalyzed reduction of acetophenone with formation of (S)- and (R)-1-phenylethanol. Prerequisite for the viability of this system is the experimental finding that the anisotropy factor g is linearly related to the enantiomeric excess (ee) and that it is independent of concentration, thereby excluding possible aggregation effects.     

Simultaneous LC/ESI‐MS Separation Method for the Enantioseparation of Some New Anticonvulsant Drugs

A sensitive and specific method for the simultaneous determination of the enantiomeric purity of 2,6‐dimethylphenoxyacetyl derivatives as trans or cis racemic and enantiomeric forms with 2‐ or 4‐aminocyclohexanol moiety ( 1 , 2 , 3 , 6 ) and their amine analogs ( 8 , 9 ) was developed. The compounds studied are known for their anticonvulsant activity and the most interesting pharmacological results were those for (±)‐trans‐2‐(2,6‐dimethylphenoxy)‐N‐(2‐hydroxycyclohexyl)acetamide ( 1 ) as well as (±)‐trans‐2‐[(2,6‐dimethylphenoxy)ethyl]aminocyclohexanol ( 8 ). The analytical method for determining the enantiomeric purity of the compounds studied is based on direct separation of the analytes using a chiral stationary phase (Chiralpak AS column). The mass spectrometric analysis was done on a coupled liquid chromatograph–mass spectrometer system with an electrospray ionization source (LC/ESI‐MS). For the compounds 1 , 8 , and 9 , the method allows an excellent separation of enantiomers, with a resolution higher than 3.2, and a tailing factor of less than 1.67 with a final enantiomer purity better than 97.5%. Chirality 26:144–149, 2014. © 2014 Wiley Periodicals, Inc.     

Assessment of threonine metabolism in vivo by gas chromatography/mass spectrometry and stable isotope infusion

The fractional contributions (FC) of threonine to glycine and 2-ketobutyrate (KB) fluxes in fed pigs have been assessed by the constant infusion of L-[1-13C]-threonine. The analysis of the enantiomeric purity of labeled threonine by gas chromatography/mass spectrometric (GC/MS) analysis is reported as the N-TFA isopropyl ester derivative. The commercially available [1-13C]threonine comprised 98.7% of the L-enantiomer, enriched at 99 atom percentage excess (APE), and 1.3% of L-allo-threonine contaminant, also enriched at 99 APE. The enantiomeric purity of threonine in plasma of pigs infused for 10 h with [1-13C]threonine showed that the L-allo contaminant did not accumulate. The t-butyl dimethylsilyl derivatives of threonine, glycine, and 2-aminobutyrate (ABA) were used to measure the enrichment of these compounds in plasma and liver samples by GC/MS/selected ion monitoring analysis. Analyses were performed on between 1 and 5 nmol of each amino acid extracted from biological fluids and a 1:10 split injection. GC/MS parameters were assessed with standards at similar quantities and found to be satisfactory; e.g., injection of 1-10 nmol of glycine did not significantly alter the slope and the precision of the standard curve. The coefficient of variation of enrichment determination was less than 10% for standards enriched at 0.4 APE or more and biological samples enriched at 0.6 APE or greater. Within-animal coefficients of variation for four plasma samples obtained at equal intervals between 8 and 10 h of [1-13C]threonine infusion were 4, 21, and 24% for threonine, ABA, and glycine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)