Enantioseparation of hydrochloride salts using carbon dioxide-based mobile phases with on-line polarimetric detection

Most HPLC enantioseparations of amine analytes are performed using normal-phase systems containing mobile phases of heptane with ethanol (or 2-propanol) and an amine additive. Since salt-forms of amine analytes are usually insoluble in normal-phase eluents, free-base forms are synthesized for preparative chromatography. It would be highly desirable to directly chromatograph salt forms of amine analytes using mobile phases of carbon dioxide (CO(2)) and methanol (MeOH). Such separations would be readily suitable for preparative chromatography, since most amine salts are highly soluble in MeOH. In this article, advantages are shown for the use of supercritical fluid chromatography (SFC) instrumentation with tandem UV and polarimetric detection for confirming enantioseparation as well as for determining optimum preparative column injections. Examples are shown for racemic mixtures of propranolol HCl (I), thioridazine HCl (II), tramadol HCl (III), and flurbiprofen (IV), all of which resolved on Chiralpak AD-H chiral stationary phase using mobile-phase systems of CO(2) and MeOH without the use of basic or acidic additives.     

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.     

High performance liquid chromatographic enantioseparation of chiral bridged polycyclic compounds on chiralcel OD-H and chiralpak OT(+)

The HPLC enantiomeric separation of 29 racemic bridged polycyclic compounds was examined on commercially available Chiralcel OD-H and Chiralpak OT(+) columns. The separations were evaluated under normal-phase mode (hexane containing mobile phase) for Chiralcel OD-H and under normal-phase as well as under reversed-phase mode (pure MeOH, temperature 5 degrees C) for Chiralpak OT(+). Almost all compounds were resolved either on Chiralcel OD-H or on Chiralpak OT(+), in some cases on both. The use of trifluoroacetic acid (TFA), as modifier of the hexanic mobile phase, had a beneficial effect on the enantioseparation of some polar and acidic compounds on Chiralcel OD-H. The influence of small chemical structural modifications of the analytes on the enantioseparation behavior is discussed. A structure-retention relationship has been observed on both stationary phases. This chromatographic evaluation may provide some information about the chiral recognition mechanism: in the case of Chiralcel OD-H, hydrogen bonding, pi-pi and distereoselective repulsive are supposed to be the major analyte-CSP interactions. In the case of Chiralpak OT(+), a reversed-phase enantioseparation could take place through hydrophobic interactions between the aromatic moiety of the analytes and the chiral propeller structure of the CSP. The synthesis of some unknown racemic bromobenzobicyclo[2.2.1] analytes is also described.     

The chiral pyrethroid cycloprothrin: stereoisomer synthesis and separation and stereoselective insecticidal activity

The synthesis and separation of the isomers of the pesticide cycloprothrin have been realized for the first time. Complete separation was achieved on a DAICEL CHIRALCEL OJ-H column (25 x 0.46 cm) for (1R, alpha*)-cycloprothrin isomers and on a DAICEL CHIRALCEL OD-H column (25 x 0.46 cm()) for (1S, alpha*)-cycloprothrin isomers. The insecticidal activity of (1R, alphaR)-cycloprothrin for the larvae of Mythimaseparata and Aphismedicagini was found to be about six times and four times higher, respectively, than that of racemic cycloprothrin.     

Liquid chromatographic enantiomer resolution of N-hydrazide derivatives of 2-aryloxypropionic acids on a crown ether derived chiral stationary phase

The liquid chromatographic separation of the enantiomers of several N-hydrazide derivatives of 2-aryloxypropionic acids was performed on a crown ether type chiral stationary phase derived from (18-crown-6)-2,3,11,12-tetracarboxylic acid. The behavior of chromatographic parameters by the change of mobile phases and additives for the resolution of these analytes was investigated. The enantiomers of all analytes were base-line resolved with a mobile phase of 100% methanol containing 20 mM H2SO4. These results are the first reported for enantiomer resolution of chiral acids of 2-aryloxypropionic acids as their N-hydrazide derivatives.     

Bioanalytical liquid chromatography tandem mass spectrometry methods on underivatized silica columns with aqueous/organic mobile phases

This review article summarizes the recent progress on bioanalytical LC-MS/MS methods using underivatized silica columns and aqueous/organic mobile phases. Various types of polar analytes were extracted by using protein precipitation (PP), liquid/liquid extraction (LLE) or solid-phase extraction (SPE) and were then analyzed using LC-MS/MS on the silica columns. Use of silica columns and aqueous/organic mobile phases could significantly enhance LC-MS/MS method sensitivity, due to the high organic content in the mobile phase. Thanks to the very low backpressure generated from the silica column with low aqueous/high organic mobile phases, LC-MS/MS methods at high flow rates are feasible, resulting in significant timesaving. Because organic solvents have weaker eluting strength than water, direct injection of the organic solvent extracts from the reversed-phase solid-phase extraction onto the silica column was possible. Gradient elution on the silica columns using aqueous/organic mobile phases was also demonstrated. Contrary to what is commonly perceived, the silica column demonstrated superior column stability. This technology can be a valuable supplement to the reversed-phase LC-MS/MS.     

Enantiomeric separation of substituted 2-arylindoles on derivatised polysaccharide chiral stationary phases

The enantiomeric separation of a series of 2-arylindoles, developed as 5HT(2A) receptor antagonists for the treatment of schizophrenia, was investigated. Evaluation of a number of chiral stationary phases (CSPs) suggested that Chiralcel OD-H and Chiralpak AD were the most versatile for these compounds, and were employed for more detailed studies. A degree of complementarity between the CSPs was observed, such that Chiralcel OD-H was more effective for piperidine-containing molecules and Chiralpak AD for piperazine- and morpholine-containing molecules. The presence of a basic secondary amine was detrimental chromatographically, but resolution was improved substantially by employing diethylamine (DEA) in the mobile phase. All separations were either enthalpy-controlled or showed no temperature dependence. Differential temperature effects between series highlighted the possibility of multiple binding modes on these CSPs. Based on this study, it is possible to make a more rational selection of chromatographic conditions for future novel analogues.     

Effect of acidic mobile phase additives on chiral selectivity for phenylalanine analogs using subcritical fluid chromatography

A variety of acidic mobile phase additives were investigated as to their effects on retention, selectivity, efficiency, and overall chiral resolution for a number of chiral N‐substituted phenylalanine analogs under subcritical conditions. These mobile phase additives showed significant effects for all of the chromatographic parameters evaluated in this study. All of the phenylalanine analogs showed decreasing retention as the pK_a of the additive decreased. Plots of selectivity, efficiency, and chiral resolution showed pronounced improvement using acidic additives with pK_a values near −1. These results demonstrated that the choice of acidic mobile phase additives had a significant effect on the resulting chromatography for these chiral analytes under subcritical conditions. Chirality 11:91–97, 1999. © 1999 Wiley‐Liss, Inc.     

The covalently bonded cellulose tris(3,5-dimethylphenylcarbamate) on a silica monolithic capillary column for enantioseparation in capillary electrochromatography

A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-microm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.     

The effect of acidic and basic additives on the enantioseparation of basic drugs using polysaccharide-based chiral stationary phases

The enantioseparation of nine commercially available basic drugs was achieved on polysaccharide-based chiral stationary phases with the acidic additive ethanesulfonic acid and the basic additive butylamine. Seven different commercially available CSPs were used for the study (AD, AS, OD, OJ, OG, OB, and OC). Mobile phase additives have been proven to be essential in obtaining satisfactory enantio-resolution in terms of both efficiency and selectivity. Significantly improved selectivities were obtained for the basic probe drugs with the acidic additive, ethanesulfonic acid, rather than the basic additive, butylamine. This is best seen with Chiralpak AS CSP. No enantioseparation for the nine drugs was observed when butylamine was used as an additive; however, satisfactory enantioseparation for the nine drugs was achieved using ethanesulfonic acid. Higher column efficiencies were observed with the acidic additive, especially when isopropanol was used as a modifier. Higher sensitivity was also achieved with ethanesulfonic acid because of the significantly lower background at the UV detection wavelength. The acidic additive was demonstrated to be superior to the basic additive for the enantioseparation of basic drugs using seven different polysaccharide-based CSPs. These results are counterintuitive to the common "rule of thumb" in enantioseparation that states acidic additives work best for acidic analytes and basic additives work best for basic analytes. The beneficial effects of acidic additive in enantioseparations observed in this study could significantly improve the applicability of polysaccharide-based CSPs for the enantioseparation of basic analytes.     

Enantiomeric separations of amino alcohols by packed-column SFC on Hypercarb with L-(+)-tartaric acid as chiral selector

The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min.     

Enantioselective analysis of praziquantel in plasma samples

A direct enantioselective high-performance liquid chromatography method is described for the quantitative determination of praziquantel enantiomers in plasma samples. The method involves two-step extraction of plasma with toluene, evaporation of the solvent and chromatography on a Chiralcel OD-H column using hexane-ethanol (85:15, v/v) as the mobile phase and detection at 220 nm. The assay satisfies all of the criteria required for use in clinical pharmacokinetic studies.     

Liquid chromatography-mass spectrometry using the hydrogen/deuterium exchange reaction as a tool for impurity identification in pharmaceutical process development

HPLC-MS employing deuterium oxide and common MS-compatible deuterated additives in the mobile phase with electrospray ionization is shown to be a viable approach for the structural elucidation of impurities in pharmaceutically active agents following initial studies with protic mobile phases. This approach incorporates the hydrogen/deuterium (H/D) exchange reaction where deuterium is substituted for hydrogen at labile sites. Some developmental compounds studied include an amide, amine, lipopeptide, indole and methyl sulfone. H/D exchange is rapid and the chromatographic performance using deuterated mobile phases is comparable to protic counterparts.     

Enantiomeric separations of remoxipride,propranolol, and trimipramine on CHIRAL-AGP using micellar chromatography and anionic additives

The retentions and enantiomeric resolutions of remoxipride, propranolol, and trimipramine were studied using a CHIRAL-AGP column with micellar mobile phases and aliphatic, anionic additives. The retentions of the compounds, which in neat buffer solution were very high (k′ > 50), could be decreased to k′ < 10 by adding a mixture of Tween® 20 and heptanoic acid to the mobile phase. The presence of the aliphatic acid was essential in order to increase the enantiomeric selectivity. An efficiency enhancement was obtained by increasing the temperature. With a mobile phase composition optimized for the separation of remoxipride, the possibility of detecting levels of the enantiomeric impurity (R-remoxipride) down to 0.025% in the drug was demonstrated. © 1993 Wiley-Liss, Inc.     

High-performance liquid chromatographic enantioseparation of 1-(aminoalkyl)-2-naphthol analogs on polysaccharide-based chiral stationary phases

The enantiomers of various 1-(alpha-aminobenzyl)-2-naphthol and 1-(aminoalkyl)-2-naphthol analogs were separated on cellulose-tris-3,5-dimethylphenyl carbamate-based chiral stationary phases (Chiralcel OD-H and Chiralcel OD-RH), using n-hexane/2-propanol/diethylamine or phosphate buffer/organic modifier mobile phases. The 3,5-dimethylphenyl carbamoylated cellulose columns were effective in both normal and rev ersed-phase modes. The effects of the mobile phase composition, the pH, the buffer concentration, and the structures of the substituents on the 2-naphthol on the enantioseparations were studied. The absolute configuration and elution sequence were determined for 1-(1-amino-2-methylpropyl)-2-naphthol: the elution sequence was S < R.     

Study on the HPLC‐based separation of some ezetimibe stereoisomers and the underlying stereorecognition process

The enantioseparation of ezetimibe stereoisomers by high‐performance liquid chromatography on different chiral stationary phases, ie, 3 polysaccharide‐based chiral columns, was studied. It was observed that cellulose‐based Chiralpak IC column exhibited the best resolving ability. After the optimization of mobile phase compositions in both normal and reversed phase modes, satisfactory separation could be obtained on Chiralpak IC column, especially in normal phase mode. The use of prohibited solvents as nonstandard mobile phase gave rise to better resolution than that of standard mobile phases (n‐hexane/alcohol system). In addition, the presence of ethanol in nonstandard mobile phase has played an important role in enhancing chromatographic efficiency and resolution between ezetimibe stereoisomers. Various attempts were made to comprehensively compare the chiral recognition capabilities of immobilized versus coated polysaccharide‐based chiral columns, amylose‐based versus cellulose‐based chiral stationary phases, reversed versus normal phase modes, and standard versus nonstandard mobile phases. Moreover, possible solute‐mobile phase‐stationary phase interactions were derived to explain how stationary and mobile phases affected the separation. Then the method validation with respect to selectivity, linearity, precision, accuracy, and robustness was carried out, which was demonstrated to be suitable and accurate for the quantitative determination of (RRS)‐ezetimibe impurity in ezetimibe bulk drug.     

Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS

It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase μLC-ICP-MS or nanoLC-ESI-MS. Using μLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga³⁺-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.     

Direct high-performance liquid chromatographic enantioseparation of beta-lactam stereoisomers

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of 12 beta-lactams. Direct separations were performed on chiral stationary phases (CSPs) containing cellulose-tris-3,5-dimethylphenyl carbamate (Chiralcel OD-RH and OD-H columns), the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column), or teicoplanin aglycone (Chirobiotic TAG column) as the chiral selector. It was clearly established that, with teicoplanin-based columns, the teicoplanin aglycone was most often responsible for the enantioseparation of the beta-lactams. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was in the range between 0.02 and 0.97 kJ mol(-1) for these beta-lactam stereoisomer separations. The separations were carried out with high selectivity and resolution, and the method was therefore suitable for monitoring of the enantiomeric excess after chiral synthesis. The Chirobiotic and Chiralcel columns appear to be highly complementary to one another. The best separation of this class of beta-lactam compound could be obtained using the Chirobiotic TAG in the polar-organic mode plus the Chiralcel OD-H in the normal-phase mode. The elution sequence was also determined.     

Additive concentration effects on enantioselective separations in supercritical fluid chromatography

Polar additive concentration effects in supercritical fluid chromatography were studied on chiral stationary phases having either a macrocyclic glycopeptide or a derivatized polysaccharide as the chiral selector. Two basic additives, isopropylamine and triethylamine, were incorporated into the methanol modifier at various concentrations and the effects on retention, selectivity, and resolution were monitored. Many of the analytes failed to elute from the macrocyclic glycopeptide stationary phase in the absence of an additive and the most noticeable effect of increasing additive concentration was a significant decrease in retention. On the derivatized polysaccharide stationary phase the additives had little effect on retention, but they did foster significant improvements in peak shape and resolution.     

Separation of nicotine and nornicotine enantiomers via normal phase HPLC on derivatized cellulose chiral stationary phases

This paper describes the enantiorecognition of (±)nicotine and (±)nornicotine by high-performance liquid chromatography using two derivatized cellulose chiral stationary phases (CSPs) operated in the normal phase mode. It was found that different substituents linked to the cellulose backbone significantly influence the chiral selectivity of the derivatized CSP. The results showed that, in general, the tris(4-methylbenzoyl) cellulose CSP (Chiralcel OJ) surpasses tris(3,5-dimethylphenyl carbamoyl) cellulose CSP (Chiralcel OD). On the former column, the resolution (±)nicotine and (±)nornicotine enantiomers depended largely on mobile phase compositions. For the separation of the nicotine enantiomers, the addition of trifluoroacetic acid to a 95:5 hexane/alcohol mobile phase greatly improved the enantioresolution, probably due to enhanced hydrogen bonding interactions between the protonated analytes and the CSP. For (±)nornicotine separation, a reduction in the concentration of alcohol in the mobile phase was more effective than the addition of trifluoroacetic acid. Possible solute-mobile phase-stationary phase interactions are discussed to explain how different additives in the mobile phase and different substituents on the cellulose glucose units of the CSPs affect the separation of both pairs of enantiomers. Chirality 10:364–369, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.