Polysaccharide-type Chiral Stationary Phases Research Articles

Development and implementation of a stereoselective normal-phase liquid chromatography–tandem mass spectrometry method for the determination of intrinsic metabolic clearance in human liver microsomes

The stereoselective determination of stereoisomers in biological samples provides vital information on stereospecific metabolism and pharmacokinetic profiles of the drugs. Despite the unique advantage and the great success of normal-phase (NP) HPLC for the separations of drug stereoisomers using polysaccharide-type chiral stationary phases (CSPs), the technique is rarely applied to quantitative HPLC–MS–MS bioanalysis. This is, at least in part, due to the incompatibility between the usual mobile phase ( n-hexane or n-heptane) in normal-phase HPLC and the MS ionization sources which poses a potential detonation hazard. An environmentally friendly and nonflammable alternative solvent, ethoxynonafluorobutane (ENFB), was reported previously to potentially provide an ideal solution for combining the powers of stereoselective NP chromatographic separation and MS–MS detection. In this study, a stereoselective NP-HPLC–MS–MS method was developed using ENFB to quantify a pair of Bristol Myers Squibb (BMS) proprietary drug stereoisomers and their ketone metabolite for an in vitro study, which demonstrated, for the first time, the practical applicability and utility of ENFB for bioanalysis in pharmaceutical industry. The effects of different organic modifiers and temperature, as well as the comparison between ENFB and the usual solvent, heptane, for the separation, are discussed. The resolution of the stereoisomers was achieved using 63% of 3:1 mixture of ethanol and methanol with 37% ENFB on a Chiralpak AD-H column at 50 °C. High sensitivity was obtained using the MS–MS detection in the positive ion atmospheric pressure chemical ionization (APCI) mode. The lower limit of quantitation (LLOQ) for the first stereoisomer and the ketone metabolite was 5 ng/mL, and was 10 ng/mL for the second isomer in the human liver microsome–potassium phosphate buffer matrix. The linear dynamic range of 5–1000 ng/mL for both isomers and 10–1000 ng/mL for the metabolite were demonstrated with R 2 ≥ 0.997. The precision of the analysis was <5% R.S.D. at or above the nominal concentration of 80 ng/mL, and <20% R.S.D. at 8 ng/mL. The mean bias was less than 15%. Extraction recovery and acceptable matrix interference were demonstrated using one isomer and the ketone, and better than 75% recovery and less than 25% ion suppression or interference were found. The method was successfully implemented for an in vitro intrinsic metabolic clearance study.

Enantioseparation of 2‐aryl‐1,3‐dicarbonyl analogues by high performance liquid chromatography using polysaccharide type chiral stationary phase

The HPLC chiral separation of 21 kinds of 2-aryl-1,3-dicarbonyl analogues was investigated in normal phase mode with amylose tris(3,5-dimethylphenylcarbamate), amylose tris((S)-1-phenylethylcarbamate), cellulose tris(3,5-dimethylphenylcarbamate), and cellulose tris(4-methylbenzoate) chiral stationary phases, respectively. The whole set of 2-aryl-1,3-dicarbonyl analogues shows better enantioselectivity and enantioseparation on amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak AD-H). The temperature dependence of enantioselectivity was studied to improve the enantioseparation. In addition, efforts are made to relate analyte structure with the quality of the achieved chiral separation.

Chiral separation of selected proline derivatives using a polysaccharide-type stationary phase by supercritical fluid chromatography and comparison with high-performance liquid chromatography

Chiral separation of selected proline derivatives was conducted on a polysaccharide-type chiral stationary phase (CSP) using supercritical fluid chromatography (SFC). Effect of both modifier percentage and column temperature was studied. As modifier content decreased, the retention and selectivity increased. However, retention was decreased as temperature increased from 30 °C to 40 °C. In comparison to high-performance liquid chromatography (HPLC), better resolution was achieved for Boc-2-methylproline using SFC along with a shorter run time. Selectivity is impacted less by change of ethanol content in the mobile phase under SFC conditions than with HPLC. Chiral recognition mechanism under SFC and HPLC conditions was also examined. With SFC, the contribution from hydrogen bonding is most likely a dominant interaction for retention rather than chiral recognition. This is supported by the limited thermodynamic data and the elution order change for Boc-proline between SFC and HPLC. Linearity, injection repeatability and limit of quantitation were determined for Boc-2-methylproline.

Chiral separation of lipoxygenase metabolites utilizing high-performance liquid chromatography

An isocratic, reversed-phase HPLC assay has been developed for the separation of the enantiomers of four lipoxygenase metabolites, without the need for a derivatization step. Separation of the enantiomers was studied on a polysaccharide type chiral stationary phase column. Upon determination of suitable mobile phase composition, the assay was evaluated at various temperatures. In all cases the R enantiomer eluted before the S enantiomer. The best separations were observed at 0 °C.

Comparative Liquid Chromatographic Enantiomer Resolution on Two Chiral Stationary Phases Derived from Amylose Tris(3,5-dimethylphenylcarbamate)

Polysaccharide-derived chiral stationary phases (CSPs) are known to show high chiral recognition ability in HPLC and have been extensively used to separate a broad range of racemic compounds. These type CSPs are usually prepared coating or adsorbing the polysaccharide derivatives on silica gel. Therefore, the solvents such as chloroform, methylene chloride, and tetrahydrofuran which dissolve or swell the chiral selectors of the polysaccharide derivatives cannot be used as mobile phases. For example, in case of Chiralpak AD prepared by coating amylose tris(3,5dimethylphenylcarbamate) derivative, one of the widely used polysaccharide type CSPs, suitable mobile phases like hexane, 2-propanol and ethanol should be used for the column safety. It may be damaged in case of even the use of only a little amount of inappropriate solvents used as mobile phases and/or sample solvents. These limitations represent a disadvantage for new applications using these CSPs and, especially, preparative separation due to solubilization of analytes. To overcome these problems, therefore, the development of polysaccharide-derived covalently bonded CSPs has been of great interest and various results of different attempts have been reported. Recently, Chiralpak IA prepared by chemically bonding amylose tris(3,5-dimethylphenylcarbamate) derivative on silica gel, which is used as the same chiral selector of coated type Chiralpak AD has been introduced. In this study, we present the comparative liquid chromatographic enantiomer resolution of N-fluorenylmethoxycarbonyl (FMOC) protected αamino acids ethyl ester derivatives on two polysaccharidederived CSPs, covalently bonded type Chiralpak IA and coated type Chiralpak AD. This is the first report concerning the comparison of the chiral separations on Chiralpak IA and Chiralpak AD using normal mobile phases. For enantiomer separation of N-FMOC α-amino acids ester derivatives, very a few results on CSPs have been reported. Rizzi has separated three FMOC α-amino acids methyl esters enantiomers on cellulose triacetate type column. Miyazawa et al. have reported on the resolution of only one analyte of FMOC 2-aminobutanoic acid methyl ester enantiomers among many different N-protected amino acid derivatives. Kusters et al. have reported on the resolution of fifteen N-FMOC α-amino acids methyl and isopropyl esters enantiomers on a polysaccharide-derived CSP. To our knowledge, our results are the first reported for the enantiomer resolution of N-FMOC α-amino acids ethyl ester derivatives. Table 1 shows the effect of mobile phase on the enantiomer separation of some N-FMOC α-amino acids ethyl esters on Chiralpak IA. The enantioselectivities and retention times are greatly influenced by the nature of mobile phase. As shown in Table 1, in general, 5% 2propanol in hexane as a mobile phase afforded the greatest enantioselectivity with the highest resolution factor, whereas 20% chloroform in hexane afforded the lowest enantioselectivity. It is notable that the elution orders of three analytes using 10% tetrahydrofuran or 10% ethyl acetate or 20% chloroform in hexane are different from those using 5% 2-propanol in hexane. Table 2 shows the comparative data of enantiomer separation of N-FMOC α-amino acids ethyl esters on Chiralpak IA and Chiralpak AD using 2-propanol in hexane as a mobile phase. In general, Chiralpak IA showed slightly lower enantioseparation than Chiralpak AD for enantioresolution of N-FMOC α-amino acids ethyl esters. Several results have reported that a certain decrease in the enantio-

Enantioseparation in capillary chromatography and capillary electrochromatography using polysaccharide-type chiral stationary phases.

AbstractPolysaccharide esters and phenylcarbamates represent powerful chiral stationary phases (CSPs) for enantioseparations in high-performance liquid chromatography (HPLC) (1). Within the last few years, these materials have been increasingly used for enantioseparations in capillary liquid chromatography (CLC) and capillary electrochromatograpy (CEC) (2,3).KeywordsChiral SelectorCapillary Liquid ChromatographyOrganic Mobile PhasisPolysaccharide DerivativeChiral PolymerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

HPLC and mass spectrometry analysis of the enzymatic hydrolysis of anti-HIV pronucleotide diastereomers

In one current strategy to develop membrane-soluble pronucleotides, the phosphoramidate derivatives of the approved anti-HIV nucleosides 2 ′,3 ′-didehydro-3 ′-deoxythymidine (d4T), 3 ′-azido-3 ′-deoxythymidine (AZT), (−)-β- l-2 ′,3 ′-dideoxy-3 ′- thiacytidine (3TC), and 2 ′,3 ′-dideoxyadenosine (ddA) exhibit promising antiviral activity. However, the non-stereoselective synthetic route results in a mixture of diastereoisomers, which differ in the configuration of the phosphorus chiral center. Since it is believed that enzymatic ester hydrolysis is the first step in the intracellular activation of these prodrugs and that this process could be dependent on the stereochemistry at the phosphorus center, analytical methods must be developed. In the present work, in vitro evaluation of the selectivity of pig liver esterase (PLE) towards each diastereomer of d4T, AZT, 3TC, and ddA prodrugs has been investigated, applying our recently published HPLC-MS procedure using a polysaccharide-type chiral stationary phase [1]. This method has been used to analyze the products of the PLE-catalyzed hydrolysis of the pronucleotides. It was found that both diastereomers of the four prodrugs were substrates for PLE.

Liquid chromatographic separation of phosphoramidate diastereomers on a polysaccharide-type chiral stationary phase

To improve the therapeutic potential of anti-HIV nucleoside analogues (d4T, AZT, 3TC and ddI), the delivery of the corresponding monophosphate from neutral, membrane-permeable prodrugs has been realised by the synthesis of lipophilic phosphoramidate triester prodrugs, such as the simple phenyl- l-alaninephosphate derivatives. However, the present non-stereoselective synthesis results in a mixture of 1:1 diastereomers, which differ from the configuration of the phosphorus atom asymmetric center. Since each diastereomer may have different biological activity and pharmacokinetic profile, analytical methods have to be developed for their separation. This work aims at showing the ability of a polysaccharide-type chiral stationary phase to resolve such diastereomers in reversed-phase high-performance liquid chromatography. The influence of operating parameters has been studied to optimise the separation; a thermodynamic approach has also been investigated to gain an insight in the retention mechanism of the prodrugs. Preliminary validation study (linearity, accuracy, repeatability) has yielded good results; in addition, the feasibility of HPLC–electrospray-mass spectrometry (HPLC–ESI-MS) coupling has been demonstrated and it is expected that this will lead to lower detection limits.

Enantioseparation of tetramisole by capillary electrophoresis and high performance liquid chromatography and application of these techniques to enantiomeric purity determination of a veterinary drug formulation of L-levamisole

The enantioseparation of the chiral anthelmintic drug tetramisole was studied by capillary electrophoresis using various cyclodextrins as chiral selectors. In particular, the following aspects have been addressed: (1) the enantiomer affinity pattern of tetramisole towards various cyclodextrins; (2) development of a capillary electrophoretic method for quantitative determination of the enantiomeric impurity, i.e. of the R-(+) enantiomer, in S-(–)-tetramisole (levamisole); (3) application of the capillary electrophoretic method for the determination of the enantiomeric impurity of R-(+)-tetramisole in commercial samples and in a veterinary drug formulation of S-(–)-tetramisole; and (4) comparison of capillary electrophoretic and traditional pharmacopoeial polarimetric methods for the enantiomeric impurity determination in S-(–)-tetramisole. Opposite affinity order of tetramisole enantiomers was observed towards different cyclodextrins, depending on the nature of the substituents and the substitution pattern. In addition, an enantioseparation of tetramisole was developed by high performance liquid chromatography on polysaccharide type chiral stationary phase. This method was compared to the aforementioned capillary electrophoretic method. Preliminary validation results indicated that capillary electrophoresis represents a flexible, fast, and inexpensive technique for determination of the enantiomeric impurity of R-(+)-tetramisole in the commercial samples and drug formulations of S-(–)-tetramisole (levamisole). Both of the commercial samples as well as the veterinary drug formulation of levamisole contained R-(+)-tetramisole as enantiomeric impurity. Capillary electrophoresis represents a superior method for enantiomeric impurity determinations compared to the traditional polarimetric method still used in the major pharmacopoeias. CE offers the advantages of miniaturised techniques, as well as higher peak efficiency, flexibility, and shorter method development time compared to HPLC.

Comparative enantioseparation of selected chiral drugs on four different polysaccharide-type chiral stationary phases using polar organic mobile phases

The enantiomers of randomly selected chiral drugs and drug analogs of various structural and pharmacological groups were resolved on four different polysaccharide-type chiral stationary phases (CSP) using pure methanol and acetonitrile as mobile phases. Polysaccharide phenylester type CSP, Chiralcel-OJ although resolving the enantiomers of some chiral drugs was less universal in the combination with methanol and acetonitrile as mobile phases. Among polysaccharide phenylcarabamates amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak-AD) was superior over the corresponding cellulose derivative, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). However, another derivative of cellulose, namely, cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) exhibited higher chiral recognition ability compared to Chiralpak-AD material. This study confirms previous findings about the applicability of polysaccharide type CSPs in so called polar organic mode as well as shows high potential of CDCPC as a practically useful CSP for High performance liquid chromatography (HPLC) enantioseparations.

Comparative HPLC enantioseparation of new chiral hydantoin derivatives on three different polysaccharide type chiral stationary phases

The enantioseparation of eighteen new chiral hydantoin derivatives was studied on three different polysaccharide type chiral stationary phases (CSP) Chiralpak AD, Chiralcel OD and Chiralcel OJ in the normal-phase HPLC mode. Chiralpak AD material exhibited the most universal chiral resolving ability and allowed the enantioseparation of 17 out of 18 compounds followed by Chiralcel OD (10 enantioseparations of 12 tested compounds) and Chiralcel OJ (eight enantioseparation from 13 tested analytes). Some complementary separations were observed and all of 18 compounds could be resolved at least with one of the three chiral CSP under the conditions of this study. With regard to the structure of the analytes, bulky electron rich substituents at C5 of the hydantoin nucleus appear to favor stereoselective interactions.

Diastereomeric resolution of nucleoside analogues, new potential antiviral agents, using high-performance liquid chromatography on polysaccharide-type chiral stationary phases

This paper describes the separation of the four sets of stereoisomers of nucleoside analogs, new potential antiviral agents by direct analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases. The resolution was made using normal-phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol or 2-propanol) in various percentages, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ) and a silica-based amylose tris-3,5-dimethylphenylcarbamate (Chiralpak AD) or tris-( S)-1-phenylethylcarbamate (Chiralpak AS). The effects of structural features on the extent of discrimination between the stereoisomers were examined through the retention, the selectivity and the resolution factors as well as the elution order. Baseline separation ( R s>1.5) was easily obtained in many cases. The resolution results were complementary between the different columns.

Enantioseparation of selected chiral sulfoxides using polysaccharide-type chiral stationary phases and polar organic, polar aqueous–organic and normal-phase eluents

HPLC enantioseparation of selected chiral sulfoxides was studied using cellulose and amylose phenylcarbamate derivatives as chiral stationary phases (CSPs). The contributions of various functional groups of a chiral analyte as well as the polysaccharide derivatives in the analyte retention and chiral recognition were evaluated. A very high enantioseparation factor exceeding 110 was observed in the enantioseparation of 2-(benzylsulfinyl)benzamide (BSBA) on cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) CSP by using 2-propanol as a mobile phase. The enantiomer elution order was opposite on cellulose and amylose phenylcarbamates. For the polysaccharide-type CSPs, pure alcohols such as methanol, ethanol and 2-propanol represent a valuable alternative to more common alcohol–hydrocarbon and reversed-phase eluents.

Simultaneous separation and enantioseparation of thalidomide and its hydroxylated metabolites using high-performance liquid chromatography in common-size columns, capillary liquid chromatography and nonaqueous capillary electrochromatography

The separation of thalidomide (TD) and its hydroxylated metabolites including their simultaneous enantioseparation was studied using three different polysaccharide-type chiral stationary phases (CSPs) in combination with polar organic mobile phases. Three different techniques, high-performance liquid chromatography in common-size columns, capillary LC and nonaqueous capillary electrochromatography were compared in terms of separation. As this study illustrates, polar organic mobile phases represent a valuable extension for less polar and polar aqueous–organic mobile phases in combination with polysaccharide CSPs. Chiralpak AD consisting of 25% of amylose-tris(3,5-dimethylphenylcarbamate) coated on wide-pore aminopropylsilanized silica gel exhibited higher resolving ability compared to the similar cellulose derivative (Chiralcel OD) as well as to cellulose-tris(4-methylbenzoate) (Chiralcel OJ) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of all three compounds could be achieved under optimized separation conditions.

Simultaneous determination of enantioselective plasma protein binding of aminohydantoins by ultrafiltration and chiral high-performance liquid chromatography

Chiral HPLC methods were developed and utilized for the simultaneous determination of plasma protein binding of enantiomers of two racemic aminohydantoin compounds. Reversed-phase HPLC with the use of a polysaccharide-type chiral stationary phase column was employed for the separation and quantitation of the enantiomers of the two compounds with detection limits in the range 5–10 ng/ml in the plasma matrix. The chiral HPLC methods were selective, sensitive and reproducible. The R and S enantiomers of both compounds were baseline-resolved under the chromatographic conditions employed. Ultrafiltration techniques were applied to determining the plasma protein binding for each enantiomer in rat, dog and human plasma. The results clearly show stereoselective binding of the two enantiomers of each compound with higher protein binding of the R enantiomer than the S enantiomer in rat, dog and human plasma. Binding association constants were also determined to be in the range 1.01–14.0·10 4 M −1 at 37°C. Both the protein binding percentage and binding association constant were enantioselective and species-dependent. Such information is important for a clear understanding of the differences in biological activity as well as in pharmacokinetic and pharmacodynamic properties between the two enantiomers of each compound in the drug discovery and development process.

Recent applications of polysaccharide-type chiral stationary phases for the enantioselective analysis of chiral drugs with one and two stereogenic centers.

Recent applications of polysaccharide-type chiral stationary phases for the enantioselective analysis of chiral drugs with one and two stereogenic centers.