Mass spectrometry enantiomer separation

Mass spectrometry (MS) is also being used to add another dimension of analysis to achiral-chiral analysis. Recently, an achiral-chiral column-switching LC/LC-MS/MS method was reported for the pindolol enantiomers in human serum (Motoyama et al., 2002) and phenprocoumon metabolites (Kammerer et al., 1998). For analytes that have very poor chromophores or cannot naturally fluoresce, MS detection can be more sensitive for the underivatized form of the analyte. Also, MS detection can be particularly useful when very similar analytes that differ in mass (such as some amino acids and metabolites) cannot be satisfactorily separated chromatographically,... 

Pihlainen, K. and Kostiainen, R., Effect of the eluent on enantiomer separation of controlled drugs by liquid chromatography-ultraviolet absorbance detection-electrospray ionisation tandem mass spectrometry using vancomycin and native fi-cyclodextrin chiral stationary phases, J. Chromatogr. A, 1033, 91, 2004. 

When using PFT with a neutral selector, it is quite difficult to avoid any entrance of the chiral selector into the ionization source, particularly at a high pH, where EOF is important. The use of BGE at low pH and/or coated capillary to minimize EOF is therefore mandatory. However, the coaxial sheath gas, which generally assists the ionization process, leads to an aspirating phenomenon of the chiral selector in the MS direction. Javerfalk et al. were the first to apply PFT with a neutral methyl-/i-CD for the separation of racemic bupivacaine and ropivacaine with a polyacrylamide-coated capillary and an acidic pH buffer (pH 3). Cherkaoui et al. employed another neutral CD (HP-/1-CD) with a PVA-coated capillary for the analysis of amphetamines and their derivatives. To prevent a detrimental aspiration effect, analyses were carried out without nebulization pressure. Numerous other studies presented excellent results such as the enantioselective separation of adrenoreceptor antagonist drugs using tandem mass spectrometry (MS/MS) the separation of clenbuterol enantiomers after solid-phase extraction (SPE) of plasma samples or the use of CD dual system for the simultaneous chiral determination of amphetamine, methamphetamine, dimethamphetamine, and p-hydroxymethamphetamine in urine. 

Otsuka, K., Smith, C. J., Grainger, J., Barr, J. R., Patterson, J., Tanaka, N., and Terabe, S. (1998). Stereoselective separation and detection of phenoxy acid herbicide enantiomers by cyclodextrin-modified capillary zone electrophoresis-electrospray ionization mass spectrometry. /. Chromatogr. A 817, 75-81. 

Toussaint, B., Palmer, M., Chiap, P., Hubert, P., and Crommen, J. (2001). On-line coupling of partial filling-capillary zone electrophoresis with mass spectrometry for the separation of clenbuterol enantiomers. Electrophoresis 22, 1363 — 1372. 

Von Brocke, A., Wistuba, D., Gfrorer, P., Stahl, M., Schurig, V, and Bayer, E. (2002). On-line coupling of packed capillary electrochromatography with coordination ion spray-mass spectrometry for the separation of enantiomers. Electrophoresis 23, 2963 — 2972. 

Y Tanaka, Y Kishimoto, S Terabe. Separation of acidic enantiomers by capillary electrophoresis-mass spectrometry employing a partial filling technique. J Chromatogr A 802 83-88, 1998. 

Reetz and coworkers developed a highly efficient method for screening of enantioselectivity of asymmetrically catalyzed reactions of chiral or prochiral substrates using ESI-MS [60]. This method is based on the use of isotopically labeled substrates in the form of pseudo-enantiomers or pseudo-prochiral compounds. Pseudo-enantiomers are chiral compounds which are characterized by different absolute configurations and one of them is isotopically labeled. With these labeled compounds two different stereochemical processes are possible. The first is a kinetic separation of a racemic mixture, the second the asymmetric conversion of prochiral substrates with enantiotopic groups. The conversion can be monitored by measuring the relative amounts of substrates or products by electrospray mass spectrometry. Since only small amounts of sample are required for this method, reactions are easily carried out in microtiter plates. The combination of MS and the use of pseudo-enantiomers can be used for the investigation of different kinds of asymmetric conversion as shown in Fig. 3 [60]. 

In addition to sensory and physical properties, the content of certain typical components is determined. Problems concerning the natural, botanical, and geographical origins of these products are also solved by using modern chromatographic methods such as enantiomer separations [843-843c], and spectroscopic analytical techniques such as isotope ratio mass spectrometry (IRMS) [844-844b],... 

Capillary gas chromatography (GC) using modified cyclodextrins as chiral stationary phases is the preferred method for the separation of volatile enantiomers. Fused-silica capillary columns coated with several alkyl or aryl a-cyclo-dextrin, -cyclodextrin and y-cyclodextrin derivatives are suitable to separate most of the volatile chiral compounds. Multidimensional GC (MDGC)-mass spectrometry (MS) allows the separation of essential oil components on an achiral normal phase column and through heart-cutting techniques, the separated components are led to a chiral column for enantiomeric separation. The mass detector ensures the correct identification of the separated components [73]. Preparative chiral GC is suitable for the isolation of enantiomers [5, 73]. 

Figure 25-8 Baseline separation of enantiomers of the drug Ritalin by HPLC with a chiral stationary phase. One enantiomer is pharmacologically active for treating attention deficit disorder and narcolepsy. The other enantiomer has little activity but could contribute to undesired side effects. Pharmaceutical companies are moving toward providing enantiomerically pure drugs, which could be safer than mixtures of optical isomers. [From R. Bakhtiar, L Ramos, and F. L. S. Tse, "Quantification of Methylphenidate in Plasma Using Chiral Uquid-Chromatography/Tandem Mass Spectrometry Application to Taxicokinetic Studies," Anal. Chim. Acta 2002, 469.261.]...

Chromatography—mass spectrometry. Figure 25-8 shows the separation of enantiomers of the drug Ritalin. 

HPLC with column switching and mass spectrometry was applied to the online determination and resolution of the enantiomers of donepezil HC1 in plasma [38]. This system employs two avidin columns and fast atom bombardment-mass spectrometry (FAB-MS). A plasma sample was injected directly into an avidin trapping column (10 mm x 4.0 mm i.d.). The plasma protein was washed out from the trapping column immediately while donepezil HC1 was retained. After the column-switching procedure, donepezil HC1 was separated enantioselectivity in an avidin analytical column. The separated donepezil HC1 enantiomers were specifically detected by FAB-MS without interference from metabolites of donepezil HC1 and plasma constituents. The limit of quantification for each enantiomer of donepezil HC1 in plasma was 1.0 ng/ml and the intra-and inter-assay RSDs for the method were less than 5.2%. The assay was validated for enantioselective pharmacokinetic studies in the dog. 

Stenhoff et al. [117] determined enantiomers of omeprazole in blood plasma by normal-phase liquid chromatography and detection by atmospheric-pressure ionization tandem mass spectrometry. The enantioselec-tive assay of omeprazole is using normal-phase liquid chromatography on a Chiralpak AD column and detection by mass spectrometry. Omeprazole is extracted by a mixture of dichloromethane and hexane and, after evaporation, redissolution and injection, separated into its enantiomers on the chiral stationary phase. Detection is made by a triple quadrupole mass spectrometer, using deuterated analogs and internal standards. The method enables determination in plasma down to 10 nmol/1 and shows excellent consistency suited for pharmacokinetic studies in man. 

It also was reported by Lagerstroem et al that pantoprazole and its enantiomers can be separated by using normal-phase liquid chromatography, and detection by atmospheric pressure ionization tandem mass spectrometry [13]. 

Other more conventional detectors that might ostensibly outperform CD in selectivity are nmr and mass spectrometry, and in fact they do for the analysis of diastereomers, although quantitation is a much more difficult task. They cannot compete with chiroptical methods for the distinction between enantiomers. In nmr detection, derivatization to diastereomers is a prerequisite to enantiomer analysis, and chiral forms of lanthanide reagents can been used with good effect [16,17]. For the analysis of mixtures by either nmr or mass spectrometry, total chromatographic separation is a necessity, so the completeness of the baseline separation is the limiting step not the detector. In contrast CD can be applied to the analysis of enantiomers in mixtures in methods that require no prior separation. 

Recently, the enantiomeric excess of a-amino acid ester hydrochlorides has been determined directly by using FAB (fast atom bombardment) mass spectrometry without chromatographic separation of the enantiomers. ... 

The chromatographic separation of enantiomers as diastereomers was first developed using GLC (1). Subsequently, many separations using GLC were reported, but modern LC has dominated the field of enantiospedfic drug analysis in recent years. Nevertheless, the arrival of high-resolution capUlary GLC has revived interest in the use of indirect enantiomer separation via this type of chromatography, and today GLC remains important in the analytical separation of enantiomers after derivatization with CDAs. The availability of sensitive detection methods, for example, mass spectrometry, electron capture, etc., enhances the applicability of GLC in indirect enantiospedfic drug analysis. 

Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry" (72). The direct separation and determination of the enantiomeric composition of terbutaline in human plasma at the nmol/L level... 

Blaschke, G. Separation of brompheniramine enantiomers by capillary electrophoresis and study of chiral recognition mechanisms of cyclodextrins using NMR spectroscopy, UV spectrometry, electrospray ionization mass spectrometry and X-ray crystallography. J. Chromatogr., A 2000, 875, 471-484. 

Although TLC-MS (mass spectrometry) has been shown to be technically feasible and applicable to a variety of problems, thin-layer chromatography is generally coupled with spectrophotometric methods for quantitative analysis of enantiomers. Optical quantitation can be achieved by in situ densitometry by measurement of UV-vis absorption, fluorescence or fluorescence quenching, or after exctraction of solutes from the scraped layer. The evaluation of detection limits for separated enantiomers is essential because precise determinations of trace levels of a d- or L-en-antiomer in an excess of the other become more and more important. Detection limits as low as 0.1% of an enantiomer in the other have been obtained. 

Instead by solvent extraction [207], aroma compounds from aqueous media, e.g. fruit juices, can even be separated and enriched by techniques of solid phase micro extraction (SPME), preferably from the headspace [208] , corresponding devices can often be directly connected to GC systems. These techniques provide the complete spec-tmm of the individual compounds of an aroma. As it will normally not be possible and even not necessary to analyse all components of the complex mixture, the separation of its main compounds may demand a multi-dimensional (MD) gas chromatographic system [209[ as displayed in Fig. 6.14 [210[. Examples for the multi-ele-ment/multi-compound isotope analysis by such systems will be given later (6.2.2.4.4, [211[) they can even integrate the identification of the compounds by molecular mass spectrometry and a simultaneous determination of the enantiomer ratios of isomers [210, 211 [. The importance of enantiomer analysis as a tool for authenticity assessment is extensively treated in chapter 6.2.3. 

Capillary approaches have been shown to be useful for many chiral separations as well as achiral separations. For chiral separations, separation buffer additives containing chirogenic centers (tecoplainin, erythromycin, vancomycin, or cyclodextrans) have facilitated the resolution of enantiomers [26,30,31]. Chiral capillary separations could readily be combined with mass spectrometry because the volume of effluent moving from the separation capillary to the ion source is small and makeup solvent is commonly added by means of an union to stabilize the ion beam. Chiral capillary separations provide an attractive alternative to analytical-scale normal-phase separations when using atmospheric pressure ion-ization mass spectrometry. 

Scheme 18 illustrates the proposed stages in 6-MSA biosynthesis in which the first and second condensation steps proceed with inversion to give the triketide (63). Ketoreduction gives the alcohol (64) and then elimination followed by a final malonyl condensation generates the tetraketide (65) which cyclises via an intramolecular condensation and enolises to give the aromatic nucleus of (66). In the first set of experiments (J )- and (S)-[l- C, H]nialonales were incubated separately with 6-MSA synthase purified from Penicillium patulum [56]. Isotope incorporations were determined by mass spectrometry. All the possible isotope patterns for retention or loss of the pro-J or pro-S hydrogens from C-3 and C-5 were permutated. Comparison with the actual spectra obtained demonstrated that opposite prochiral hydrogens were eliminated. The absolute stereochemistry was established in an analogous experiment [57] where the chiral malonates were incubated with acetoacetyl CoA rather than acetyl CoA. Subsequent mass spectral analysis showed that it is the Hr proton that is retained at C-3 of 6-MSA and so it can be deduced that the hydrogen at C-5 must be derived from the opposite prochiral hydrogen, Hg. The overall result is summarised in Scheme 18. In a recent collaborative study we have synthesised the triketide alcohol (64) as its NAC thioester and shown that it is indeed a precursor as, on incubation with 6-MSA synthase and malonyl CoA, 6-MSA production is observed [unpublished results]. Current work is aimed at synthesis of both enantiomers of (64) to study the overall stereochemistry of the ketoreduction and elimination reactions.

A new n.m.r. shift reagent, tris-[3-(t-butylhydroxymethylene)-d-camphorato]-europium(m), should prove to be useful for the determination of enantiomeric purity of chiral / -phen ethyl amines.2 For example, it was found that the CHNH2 resonances of (R)- and (S)-amphetamines were separated by 0.7 p.p.m. in a carbon tetrachloride solution of the europium reagent ( 0.15 mol 1 l. In comparison to the use of optically active solvents for the same purpose, this technique has the advantage of showing very large shifts between resonances of enantiomers. Mass spectrometry has been used in the detection of mescaline and tetrahydro-isoquinoline precursors as biochemical intermediates.3 Spectral differences of 4-chloro-2-nitrobenzenesulphonyl derivatives of ephedrine and related compounds have been used for identification purposes.4... 

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