Chiral gas chromatography as a tool for investigations into illicitly manufactured methylamphetamine

The aim of this study was to develop a chiral gas chromatographic method for the separation of compounds likely to be found in the EMDE synthesis of methylamphetamine, a heavily abused stimulant drug. Here we describe the separation of the enantiomers of ephedrine, pseudoephedrine, chlorinated intermediates and methylamphetamine using fluorinated acid anhydrides as chemical derivatization reagents prior to gas chromatographic analysis on a 2,3‐di‐O‐methyl‐6‐t‐butyl silyl‐β‐cyclodextrin stationary phase (CHIRALDEX™ B‐DM). Separation of the enantiomers of pseudoephedrine, methylamphetamine and chloro‐intermediates was achieved using PFPA derivatization, and enantiomers of ephedrine using TFAA derivatization, in run times of less than 40 minutes. The use of HFBA as a derivatization reagent for this set of analytes is also discussed. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Identification of ephedrines as their carbon disulfide derivatives

A method for the separation and identification of the diastereoisomers ephedrine and pseudoephedrine and the diastereoisomers norephedrine and norpseudoephedrine is presented. The compounds were derivatised by reaction with carbon disulfide in the presence of alkali. These derivatives and their trifluoroacetic anhydride derivatives were subjected to gas chromatography with nitrogen-selective detection as well as mass-selective detection and high-performance liquid chromatography with ultraviolet detection. The results showed that ephedrine and pseudoephedrine can easily be differentiated by gas chromatographic analyses of their carbon disulfide derivatives. Norephedrine and norpseudoephedrine can be differentiated by the different chromatographic retention times of their carbon disulfide derivatives and by the fact that norephedrine yielded two products and norpseudoephedrine only one product when reacted with carbon disulfide under the same conditions. Trifluoroacetylation of the latter compounds gave a more pronounced differentiation.     

Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

The hyphenation of enantioselective capillary gas chromatography and mass spectrometry is not always sufficient to distinguish between structural isomers, thus requiring peak identification by NMR spectroscopy. Here the first online coupling of enantioselective capillary gas chromatography with proton nuclear resonance spectroscopy is described for the unfunctionalized chiral alkane 2,4‐dimethylhexane resolved on octakis(6‐O‐methyl‐2,3‐di‐O‐pentyl)‐γ‐cyclodextrin at 60°C. NMR allows constitutional and configurational isomers (diastereomers and enantiomers) to be distinguished. Enantiomers display identical spectra at different retention times, which enable an indirect identification of these unfunctionalized alkanes. The presented method is still at an early development stage, and will require instrumental optimization in the future. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Enantiomer separation of fungicidal triazolyl alcohols by normal phase HPLC on polysaccharide‐based chiral stationary phases

Three fungicidal triazolyl alcohols (triadimenol, hexaconazole, and cis/trans‐1‐4‐chlorophenyl‐2‐1H‐1,2,4‐triazol‐1‐yl‐cycloheptanol) were completely separated into enantiomers by chiral HPLC using polysaccharide‐based chiral stationary phases. A better separation was achieved on cellulose and amylose carbamate phases compared with a cellulose ester phase. Peak shapes were almost symmetrical except for two cases, where tailing of the first eluted enantiomer and unusual symmetric peak broadening were observed. The effect of eluents on enantioseparation was also investigated. Chirality 11:195–200, 1999. © 1999 Wiley‐Liss, Inc.     

Separation of ephedrine and pseudoephedrine enantiomers using a polysaccharide‐based chiral column: A normal phase liquid chromatography–high‐resolution mass spectrometry approach

Chiral considerations are found to be very much relevant in various aspects of forensic toxicology and pharmacology. In forensics, it has become increasingly important to identify the chirality of doping agents to avoid legal arguments and challenges to the analytical findings. The scope of this study was to develop an liquid chromatography–mass spectrometry (LCMS) method for the enantiomeric separation of typical illicit drugs such as ephedrines (ie, 1S,2R(+)‐ephedrine and 1R,2S(?)‐ephedrine) and pseudoephedrine (ie, R,R(?)‐pseudoephedrine and S,S(+)‐pseudoephedrine) by using normal phase chiral liquid chromatography–high‐resolution mass spectrometry technique. Results show that the Lux i‐amylose‐1 stationary phase has very broad and balancing‐enantio‐recognition properties towards ephedrine analogues, and this immobilized chiral stationary phase may offer a powerful tool for enantio‐separation of different types of pharmaceuticals in the normal phase mode. The type of mobile phase and organic modifier used appear to have dramatic influences on separation quality. Since the developed method was able to detect and separate the enantiomers at very low levels (in pico grams), this method opens easy access for the unambiguous identification of these illicit drugs and can be used for the routine screening of the biological samples in the antidoping laboratories.     

Mechanistic information on the reaction of cis- and trans-[RuCl2(DMSO)4] with d(T2GGT2) derived from MALDI-TOF and HPLC studies

Reactions of trans and cis isomers of the Ru(II) complex [RuCl(2)(DMSO)(4)] with single-stranded hexanucleotide d(T(2)GGT(2)) were studied in aqueous solutions in the absence and presence of excess chloride by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Despite the different reactive species formed from the two isomers in aqueous solution, similar reaction products are obtained in their interaction with d(T(2)GGT(2)). Both [RuCl(2)(DMSO)(4)] isomers bind to the oligonucleotide in the bidentate mode to form thermodynamically stable bis-guanosine adducts, Ru(G-N7)(2). Significant differences were observed in the reaction rates, however the reaction with trans- [RuCl(2)(DMSO)(4)] is ca. 5-10 times faster in comparison to that observed for the cis analogue. This difference is interpreted in terms of different rate-limiting steps for the trans and cis complexes, respectively. It is suggested that the rate of the reaction with the trans isomer is controlled by dissociation of a Cl(-) ligand from the initially formed trans,cis,cis-[RuCl(2)(DMSO)(2)(H(2)O)(2)]. In the contrast, release of a dimethyl sulfoxide molecule from the reactive species cis,fac-[RuCl(2)(DMSO)(3)(H(2)O)] is likely to be rate limiting for the cis analogue. Significant influence of electrostatic interactions on the reaction rate was observed for the trans isomer. Mechanistic interpretation of the observed reactivity trends based on data obtained from UV-Vis spectroscopy, HPLC and MALDI-TOF MS studies is presented and discussed within the paper.     

Simultaneous chiral separation of methylamphetamine and common precursors using gas chromatography/mass spectrometry

Methylamphetamine, ephedrine, and pseudoephedrine were derivatized using trifluoroacetic anhydride and enantiomers of each were analyzed using gas chromatography coupled to mass spectrometry (GC/MS) fitted with a γ‐cyclodextrin (Chiraldex™ G‐PN) chiral column. A temperature‐programmed method was developed and optimized and the results compared with those obtained using a previously published isothermal GC method applied to GC/MS analysis. Trifluoroacetylated 3‐(trifluoromethyl)phenethylamine hydrochloride was used as an internal standard, and mass fragmentation patterns are proposed for all derivatives analyzed. Qualitative validation of the optimized chromatographic conditions was completed in accordance with the guidelines published by the United Nations Office on Drugs and Crime (UNODC). Under conditions of repeatability and reproducibility, the method gave relative retention times with a relative standard deviation of less than 0.02% for all six analytes of interest. This surpasses the UNODC's acceptance criteria of 2% for validation of qualitative precision. Ephedrine and pseudoephedrine are common precursors in the clandestine manufacture of methylamphetamine. Seizures of illicit methylamphetamine therefore often contain mixtures of these optically active compounds. The simultaneous enantioseparation of these compounds to produce a profile would provide valuable information to law enforcement agencies regarding the provenance of a methylamphetamine seizure. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Radiosensitizing effect of conjugated linoleic acid on tumor cells MCF-7 and MDA-MB-231

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO-1, the CLA-9cis 11cis at 50 micro mol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.     

Catalytic detection principle for high-performance liquid chromatography: Determination of enantiomeric iodinated thyronines in blood serum

A method for the trace determination of iodinated thyronines with differentiation of the optical isomers by high performance liquid chromatography (HPLC) is described. The detection is effected by means of a catalytic principle based on the iodide-catalysed reaction of chloramine-T and N,N′-tetramethyldiaminodiphenylmethane, producing a coloured complex that can be measured spectrophotometrically at 600 nm. Owing to the selectivity of the catalytic reaction, iodine-containing compounds can be easily determined in a complex matrix such as blood plasma. The sensitivity is sufficient for the detection of plasma levels of iodinated thyronines. The limit of detection for thyroxine is in the sub-nanogram range. The enantiomers of thyronines can be separated on commercial reversed phases after pre-column synthesis of diastereomers. For this derivatization the reagent tert.-butyloxy-carbonyl-l-leucine-N-hydroxysuccinimide ester is used. The coupling of the stereospecific HPLC separation with the catalytic detector offers the possibility of determining both d- and l-thyroxine in human plasma.     

Characterization of secondary amide peptide bond isomerization: thermodynamics and kinetics from 2D NMR spectroscopy

Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH(2) , Ac-I-G-G-NH(2) , Ac-I-G-G-N-NH(2) and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13-0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4-9 × 10(-3) s(-1) . Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints.     

A comparative study of the physical properties and enzymatic reactions of slow reacting substance of anaphylaxis and some leukotriene D4 isomers

Eight synthetic isomers of hydroxy-6-S-cysteinylglycine -7,9,11,14-eicosatetraenoic acid were compared with authentic guinea pig SRS-A using UV spectroscopy, high performance liquid chromatography and soybean lipoxygenase. It was found that only the 5S, 6R 7, 9trans 11,14cis isomer was similar to SRS-A in all respects. The 5S, 6R 7trans, 9,11,14 cis isomer shows similar UV and HPLC characteristics but differs in that it spontaneously undergoes a 1,7 hydride shift reaction and unexpectedly does not react with soybean lipoxygenase.     

The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells

The effects of a conjugated linoleic acid (CLA) mixture of single isomers (50:50, w/w, cis9,trans11:trans10,cis12) and the individual isomers on (a) the production of resting and calcium ionophore stimulated (14)C-eicosanoids and (b) the incorporation of (14)C-arachidonic acid (AA) into membrane phospholipids of human saphenous vein endothelial cells were investigated. The CLA mixture and the individual isomers were found to inhibit resting production of (14)C-prostaglandin F(2a) by 50, 43 and 40%, respectively. A dose dependent inhibition of stimulated (14)C-prostaglandins was observed with the CLA mixture (IC(50) 100 microM). The cis9,trans11 and trans10,cis12 (50 microM) isomers individually inhibited the overall production of stimulated (14)C-prostaglandins (between 35 and 55% and 23 and 42%, respectively). When tested at a high concentration (100 microM), cis9,trans11 was found to inhibit eicosanoid production in contrast to trans10,cis12 that caused stimulation. The overall degree of (14)C-AA incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells and the cis9,trans11 isomer was found to increase the incorporation of (14)C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid and linoleic acid did not alter the overall degree of incorporation of (14)C-AA. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans10,cis12 exhibits pro-inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti-inflammatory action that contributes to its anti-carcinogenic and anti-atherogenic properties.     

Fluorenone 1,4-dihydropyridine derivatives with cardiodepressant activity: Enantiomeric separation by chiral HPLC and conformational aspects

The direct HPLC enantiomeric separation of five fluorenone-1,4-dihydropyridine-3,5-dicarboxylic diesters has been achieved using a Chiralpak AD stationary phase obtaining simultaneously good enantioselectivities, resolution factors, and elution times. CD spectra of the individual enantiomers for two compounds were measured. Thermodynamic parameters associated with the adsorption equilibria of the enantiomers with the chiral stationary phase were obtained from HPLC runs at various temperatures. The conformational preferences of the synperiplanar fluorenone group and of the cis/cis ester groups were obtained by ¹H NMR spectra, including NOE experiments. © 1996 Wiley-Liss, Inc.     

Isomer-specific effects of conjugated linoleic acid on mineralized bone nodule formation from human osteoblast-like cells

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 microM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formation of mineralized bone nodules using bone cells of human origin, and provide evidence for isomer-specific effects of CLA on bone health.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

Comparison of brown adipose tissue thermogenesis induced by congeners and isomers of phenylpropanolamine

The present experiment compared the effects of intraperitoneal injection (.0223 mMol/kg) of several phenethylamine congeners and isomers including amphetamine (AMP), ephedrine (EPH), methoxyphenamine (MET), norpseudoephedrine (NOR), pseudoephedrine (PS) and phenylpropanolamine (PPA) on in vivo interscapular brown adipose tissue temperature in adult male rats. Comparisons of isomer potency revealed that the 1-isomer was more thermogenic than the d-isomer for EPH and PPA but not for AMP, NOR and PS. Congener potency order was: AMP greater than PPA greater than EPH greater than NOR = MET greater than PS. The implications of these data for the weight-reducing activity of these compounds is discussed.     

Construction and application of a mass spectral and retention time index database generated from plant GC/EI-TOF-MS metabolite profiles

The non-supervised construction of a mass spectral and retention time index data base (MS/RI library) from a set of plant metabolic profiles covering major organs of potato (Solanum tuberosum), tobacco (Nicotiana tabaccum), and Arabidopsis thaliana, was demonstrated. Typically 300-500 mass spectral components with a signal to noise ratio > or =75 were obtained from GC/EI-time-of-flight (TOF)-MS metabolite profiles of methoxyaminated and trimethylsilylated extracts. Profiles from non-sample controls contained approximately 100 mass spectral components. A MS/RI library of 6205 mass spectral components was accumulated and applied to automated identification of the model compounds galactonic acid, a primary metabolite, and 3-caffeoylquinic acid, a secondary metabolite. Neither MS nor RI alone were sufficient for unequivocal identification of unknown mass spectral components. However library searches with single bait mass spectra of the respective reference substance allowed clear identification by mass spectral match and RI window. Moreover, the hit lists of mass spectral searches were demonstrated to comprise candidate components of highly similar chemical nature. The search for the model compound galactonic acid allowed identification of gluconic and gulonic acid among the top scoring mass spectral components. Equally successful was the exemplary search for 3-caffeoylquinic acid, which led to the identification of quinic acid and of the positional isomers, 4-caffeoylquinic acid, 5-caffeoylquinic acid among other still non-identified conjugates of caffeic and quinic acid. All identifications were verified by co-analysis of reference substances. Finally we applied hierarchical clustering to a complete set of pair-wise mass spectral comparisons of unknown components and reference substances with known chemical structure. We demonstrated that the resulting clustering tree depicted the chemical nature of the reference substances and that most of the nearest neighbours represented either identical components, as judged by co-elution, or conformational isomers exhibiting differential retention behaviour. Unknown components could be classified automatically by grouping with the respective branches and sub-branches of the clustering tree.     

The retina is more susceptible than the brain and the liver to the incorporation of trans isomers of DHA in rats consuming trans isomers of alpha-linolenic acid

Trans polyunsaturated fatty acids are formed during heat treatments of vegetable oils from polyunsaturated fatty acids containing cis double bonds. After dietary intake, they are distributed in the body and are incorporated into nervous tissues including the retina. Since nervous tissues are known to be rich in n-3 fatty acids such as docosahexaenoic acid (DHA), we studied the ability of the retina and the brain to incorporate trans isomers of DHA formed in vivo from the dietary precursor trans alpha-linolenic acid. Wistar rats were fed with trans isomers of alpha-linolenic acid for 21 months. A linear incorporation of trans DHA and a decrease in cis DHA was observed in the retina, whereas no major changes were observed in the brain. In parallel to the modifications in retinal cis and trans DHA levels, the retinal functionality evaluated by the electroretinogram showed defects in animals that consumed trans alpha-linolenic acid. These results suggest that the mechanisms leading to the incorporation of cis and trans fatty acids are quite different in the retina when compared to the brain and the liver, the retina being more susceptible to changes in the dietary lipid contribution.     

Determination of absolute configuration of ketamine enantiomers by HPLC-CD-UV technique

Recognizing that the stereochemical structure of NMDA receptor antagonist ketamine provides valuable data about the relationship between its conformation and absolute configuration by CD-UV analysis, a method for the identification of ketamine enantiomers is proposed which avoids the need for authentic samples of the enantiomers. The ketamine enantiomers were separated by HPLC using Chiralcel OJ stationary phase. The in situ registration of CD and UV spectra, together with the application of the octant rule for cyclohexanone derivatives, makes possible the direct assignment of the eluted ketamine enantiomers.     

Influence of glycation on cis/trans isomerization and tautomerization in novel morphiceptin-related Amadori compounds

The influence of N-glycation of the N-terminus on amide bond stereochemistry and tautomeric distribution has been explored via the synthesis and NMR analysis of novel N-(1-deoxy-D-fructos-1-yl) derivatives (Amadori compounds) of the exogenous, milk derived, opioid tetrapeptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH2). NMR analysis of the protected Amadori compounds revealed the presence of four configurational isomers in DMSO solution arising from cis/trans isomerization about Tyr1-Pro2 and Phe3-Pro4 peptide bonds. Comparison of the data obtained for protected Amadori compounds with those obtained for morphiceptin showed that equilibrium fraction of all-trans isomers in N-glycated peptide derivatives was smaller than in the parent peptide compound. Spectroscopic investigation of unprotected morphiceptin-related Amadori compound revealed the presence of multiple conformers in solution due to cis/trans isomerization of the peptide backbone and tautomerization of the sugar moiety. The equilibrium composition in DMSO is markedly shifted towards furanose forms, amounting to two-thirds of the mixture. The estimated equilibrium of the tautomeric forms in water solution revealed the -pyranose form as the major tautomer (66%).