Chiral Derivatizing Reagents Research Articles

Chiral separation of D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent and a phenylboronic acid complex.

A novel method was developed for D/L-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of D/L isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300nM), and the repeatabilities of the migration times and peak area responses were 0.8% and 7.9% (relative standard deviation; n = 6), respectively. The method was applied to the determination of D/L- galactose in red seaweed.

Synthesis of three strong UV-absorbing Naproxen-based chiral derivatizing agents and their application for enantioseparation of Baclofen by RP-HPLC

A sensitive and stereoselective high-performance liquid chromatography (HPLC) method based on diastereomer formation was developed and fully validated for the simultaneous determination of the two enantiomers of Baclofen. (S)-Naproxen was used to prepare three chiral derivatizing reagents (CDRs) which were used for synthesizing diastereomers of Baclofen. The diastereomers so synthesized were separated on C18 column under reversed phase conditions using a binary mixture of acetonitrile and triethylammonium phosphate, with UV detection at 220 nm. The results obtained for enantioseparation of Baclofen using the three CDRs have been compiled and compared. The conditions for derivatization and chromatographic separation have been optimized. The method was validated for linearity, repeatability, limits of detection, and limit of quantification.

A Rapid, Robust and Ultra-Sensitive HPLC Enantioseparation of β-Amino Alcohols.

Enantioseparation of amino acid analogues racemic β-amino alcohols has hitherto been thought impossible without prior derivatization. Method developers of chromatographic enantioseparation often face detection challenge due to low ultraviolet (UV) absorbance of these molecules. A new chiral derivatizing reagent, benzimidazole-(S)-naproxen amide, was synthesized to provide UV detectable chiral moiety for detection of respective diastereomers of eight β-amino alcohols (dl-alaninol, dl-leucinol, dl-prolinol, dl-phenylalaninol, dl-phenylglycinol, dl-valinol, dl-homophenylalaninol and dl-methioninol). An HPLC method has been developed and validated and both the diastereomers were separated within 31 min runtime. Excellent resolution to a pmol detection limit level was achieved. The developed HPLC method for enantioseparation of β-amino alcohols is the first-ever report of its own kind having such a high detection sensitivity.

Towards the chiral metabolomics: Liquid chromatography–mass spectrometry based dl-amino acid analysis after labeling with a new chiral reagent, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate, and the application to saliva of healthy volunteers

A novel triazine-type chiral derivatization reagent, i.e., (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu), was developed for the highly sensitive and selective detection of chiral amines and amino acids by UPLC–MS/MS analysis. The enantiomers of amino acids were easily labeled with the reagents at room temperature within 40min in an alkaline medium containing triethylamine. The diastereomers derived from proteolytic amino acids, except serine, were well separated under isocratic elution conditions by reversed-phase chromatography using an ODS column (Rs=1.2–9.0). dl-Serine was separated by use of an ADME column which has relatively higher polar surface than the conventional ODS column. The characteristic product ions, i.e., m/z 195.3 and m/z 209.3, were detected from all the diastereomers by the collision-induced dissociation of the protonated molecule. A highly sensitive detection on the amol–fmol level was obtained from the selected reaction monitoring (SRM) chromatogram. The chiral amines (e.g., adrenaline and noradrenaline) labeled with DMT-(S)-Pro-OSu were also well separated and sensitively detected by the present procedure. The method using DMT-(S)-Pro-OSu was used for the determination of dl-amino acids in the human saliva from healthy volunteers. Various l-amino acids were identified in the saliva. Furthermore, d-alanine (d-Ala) and d-proline (d-Pro) were also detected in relatively high concentrations (>5%). The ratio was higher in male saliva than in female saliva. However, the difference in the ratio of d-Ala for one day was not very high and the effect of foods and beverage seemed to be negligible. Based on the results using l-Ala-d3, the d-Ala in saliva seemed to be produced due to the racemization with some enzymes such as racemase. The racemization reaction was reversible, i.e., d-Ala-d3 was also racemized to l-Ala-d3 in saliva. Thus, care should be taken during the analysis of dl-amino acids in saliva. The present method using DMT-(S)-Pro-OSu may be applicable for the determination of chiral amine metabolomics, because the resulting derivatives produce the same product ions without relation to the compounds and show highly sensitive detection in the SRM mode of MS/MS. Consequently, DMT-(S)-Pro-OSu seems to be a useful chiral derivatization reagent for the determination of amines and amino acids in biological samples.

Chirality recognition for assessing the enantiomeric purity of Betaxolol

An efficient and concise approach for recognizing chirality and determining the absolute configuration of the diastereomers of (RS)-Betaxolol, [(RS)-Bet] is described, which in turn is a measure of the enantiomeric purity of Bet since the diastereomers were separated and isolated using preparative TLC. A reverse phase HPLC method was developed and validated for the enantioseparation of (RS)-Betaxolol. Diastereomers were synthesized, using a naproxen based chiral derivatizing reagent, under microwave irradiation. The absolute configuration of both diastereomers was established with the help of 1H NMR spectroscopy and by developing the lowest energy optimized structures of the diastereomers based on density functional theory. The configuration of the diastereomers was established as [(S)-Nap-(R)-Bet] and [(S)-Nap-(S)-Bet]. The determination of the absolute configuration of the two diastereomers lent support to elution order and separation mechanism. The limits of quantitation were found to be 0.80ngmL−1 for the diastereomers.

Preparative enantioseparation of (RS)-baclofen: determination of molecular dissymmetry.

The present work reports preparative enantioseparation of (RS)-baclofen using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Diastereomers were synthesized using a new monochloro-s-triazine-based chiral derivatizing reagent (CDR), namely, N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-phenylalanine, under microwave irradiation. Acetonitrile-0.1% aq. triflouroacetic acid in gradient elution mode and CH3 OH-CH2 Cl2 (4:5; v/v) were successful as mobile phase in HPLC and TLC, respectively. The two diastereomers were isolated by preparative TLC. Molecular dissymmetry was established by developing the lowest energy optimized structures of the diastereomers based on Density Functional Theory and with the help of (1) H NMR showing anisotropic effect associated with aromatic ring of s-triazine (in the CDR). The configuration of diastereomers was established as [L-Phe-(R)-Bac] and [L-Phe-(S)-Bac], where the first notation refers to the configuration of chiral auxiliary (of the CDR) and the second to that of the analyte Bac. Limits of detection were found to be 0.056 and 0.061 ng mL(-1), respectively, for the two diastereomers. Determination of absolute configuration of the two diastereomers lent support to the elution order and separation mechanism.

Evaluation of a Novel Positively-Charged Pyrrolidine-Based Chiral Derivatization Reagent for the Enantioseparation of Carboxylic Acids by LC-ESI-MS/MS

Evaluation of a Novel Positively-Charged Pyrrolidine-Based Chiral Derivatization Reagent for the Enantioseparation of Carboxylic Acids by LC-ESI-MS/MS

Synthesis of variants of Marfey's reagent having d-amino acids as chiral auxiliaries and liquid-chromatographic enantioseparation of (RS)-Mexiletine in spiked plasma: Assessment and comparison with l-amino acid analogs

Five d-amino acids have been used for the first time to synthesize chiral derivatizing reagents (as variants of Marfey's reagent) by nucleophilic displacement of one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene as against the literature reports on application of only l-amino acids or their amides as chiral auxiliaries in dinitrobenzene (DNB) moiety. Five other DNB based reagents were also prepared by nucleophilic substitution of fluorine atom with the set of the same amino acids in l-configuration, as chiral auxiliaries. These reagents were characterized and used for synthesis of diastereomers of (RS)-Mexiletine spiked in human plasma. Diastereomers were synthesized employing microwave irradiation and were separated on reversed-phase C18 column. Performance of the two types of chiral derivatizing reagents was compared. The reagents containing d-amino acids provided enhanced separation of diastereomers than those containing l-amino acids. The best resolution was obtained using mobile phase consisting of acetonitrile and 0.1% trifluoroacetic acid in gradient mode. The detection was carried out at 340nm. The method so developed was validated for linearity, accuracy and precision. The limit of quantitation was found to be approximately 25.2ngmL−1 in human plasma.

High-performance liquid chromatography for analytical and small-scale preparative separation of (R,S)-mexiletine using (S)-(—)-(N)-trifluoroacetyl-prolyl chloride and (1S)-(—)-camphanic chloride and recovery of native enantiomer by detagging

A reversed phase high-performance liquid chromatographic method has been established for enantioseparation of (R, S)-mexiletine. Two volatile and thermally stable acyl chlorides, viz., (S)-(—)-(N)-trifluoro acetyl prolyl chloride and (1S)-(—)-camphanic chloride, were used as chiral derivatizing reagents. Binary composition of aqueous trifluoroacetic acid (0.1%)-acetonitrile as mobile phase was successful with ultraviolet (UV) detection at 210 nm. The method was optimized and validated for accuracy, precision, and limit of detection. The limit of detection was found to be 45 ng mL−1 and 80 ng mL−1 for the two types of diastereomers. Besides, kinetic resolution was achieved, and the experimental conditions optimized for this purpose provided diastereomeric excess up to 74% for (R)-isomer. On achieving a resolution value greater than 2, the optimized method for analytical enantioseparation was scaled-up to smallscale preparative level, and the native (R)-mexiletine was recovered by acid hydrolysis of the diastereomer.

Indirect chiral separation of new recreational drugs by gas chromatography-mass spectrometry using trifluoroacetyl-L-prolyl chloride as chiral derivatization reagent.

New recreational drugs such as amphetamine-, cathinone, and benzofury derivatives gained high popularity on the drug market in recent years. They can be purchased via the Internet from different providers and online portals. Most of these compounds are chiral, which makes the development of chiral separation methods necessary. Besides this, it is useful to find out if the compounds were sold as racemic mixtures. Also, it is important to check whether the new psychoactive compounds contain further ingredients or impurities. The aim of this research was the continuation of the application of a method for indirect chiral separation of 24 new psychoactive compounds recently purchased via the Internet. After derivatization with the chiral derivatization reagent trifluoroacetyl-L-prolyl chloride, chromatographic separation of diastereomers was achieved using a 30 m HP5-MS capillary column. As carrier gas, helium was used with a constant flow of 1.0 ml/min. Three different column temperature programs were tested. Under optimum conditions 13 out of 24 compounds were successfully resolved into their enantiomers obtaining Rs values up to 7.0. The use of a single quadrupole mass spectrometer as the detector allowed the identification of the compounds in multicomponent samples.

Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/MS and the application to saliva of healthy volunteers and diabetic patients.

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Enantioseparation of amino alcohols by reversed-phase high-performance liquid chromatography using cyanuric chloride-based chiral derivatizing reagents having amino acids and their amides as chiral auxiliaries

Summary A high-performance liquid chromatographic (HPLC) method was developed for enantioresolution of amino alcohols using an indirect approach. Two sets of chiral derivatizing reagents (CDRs) having amino acids (CDRs 1–4; set A) and their amides (CDRs 5–8; set B) as chiral auxiliaries in cyanuric chloride (CC) moiety were synthesized and characterized. Diastereomers of eight amino alcohols were synthesized, under microwave irradiation, using each of the eight CDRs. The resulting diastereomers were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using C18 column and eluting mixtures of acetonitrile with aqueous trifluoroacetic acid (aq.TFA) in a linear gradient (45 min) with UV detection at 230 nm. The results (in terms of resolution Rs and retention factor k) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The effects of chiral auxiliaries (in terms of acid and amides variants), constituting CDRs, on separation were also ev...

(S)-1-methyl-4-(5-(3-aminopyrrolidin-1-yl)-2,4-dinitrophenyl)piperazine as a novel chiral derivatization reagent for high-performance liquid chromatographic analysis of carboxylic acid enantiomers

A novel chiral derivatization reagent, (S)-1-methyl-4-(5-(3-aminopyrrolidin-1-yl)-2,4-dinitro-phenyl)piperazine (APy-PPZ) was developed for the enantiospecific determination of chiral carboxylic acids in high-performance liquid chromatography. APy-PPZ reacted with carboxylic acids within 10min at 30°C in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). Epimerization during the derivatization reaction was negligible. Diastereomers derived from non-steroidal anti-inflammatory drugs (NSAIDs) and N-acetylamino acids were completely separated by reversed-phase chromatography using a small particle (3.0μm) ODS column (Rs=1.20–4.12). The calibration curves were linear over the concentration range of 1–100μM for each enantiomer of IBU, FLU, and KET, 10–100μM for each enantiomer of NAc-Trp and NAC-Phe, and 50–200μM for each enantiomer of NAc-Leu and NAc-Met. The detection limits (S/N=3) of NSAIDs (1pmol for each enantiomer of IBU, FLU, KET) were one or two orders magnitude lower than that of N-acetylamino acids (10pmol for each NAc-Trp, NAC-Phe enantiomer, 100pmol for each NAc-Leu, NAc-Met enantiomer). The relative standard deviations (RSD, n=5) were less than 3.4% at 60μM for all carboxylic acid enantiomers. The proposed method was applied to the determination of (S)-IBU enantiomer in a pharmaceutical formulation.

Rapid quantitative chiral amphetamines liquid chromatography–tandem mass spectrometry: Method in plasma and oral fluid with a cost-effective chiral derivatizing reagent

Methamphetamine is a widely abused psychostimulant containing a chiral center. Consumption of over-the-counter and prescription medications may yield positive amphetamines results, but chiral separation of l- and d-methamphetamine and its metabolite amphetamine can help determine whether the source was licit or illicit. We present the first LC–MS/MS method with precolumn derivatization for methamphetamine and amphetamine chiral resolution in plasma and oral fluid collected with the Oral-Eze® and Quantisal™ devices. To 0.5mL plasma, 0.75mL Oral-Eze, or 1mL Quantisal specimen racemic d11-methamphetamine and amphetamine internal standards were added, followed by protein precipitation. Samples were centrifuged and supernatants loaded onto pre-conditioned Phenomenex® Strata™-XC Polymeric Strong Cation solid phase extraction columns. After washing, analytes were eluted with 5% ammonium hydroxide in methanol. The eluate was evaporated to dryness and reconstituted in water. Derivatization was performed with 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide (Marfey's reagent) and heating at 45°C for 1h. Derivatized enantiomer separations were performed under isocratic conditions (methanol:water, 60:40) with a Phenomenex® Kinetex® 2.6μm C18 column. Analytes were identified and quantified by two MRM transitions and their ratio on a 3200 QTrap (AB Sciex) mass spectrometer in ESI negative mode. In all three matrices, the method was linear for all enantiomers from 1 to 500μg/L, with imprecision and accuracy of ≤11.3% and 85.3–108%, respectively. Extraction efficiencies ranged from 67.4 to 117% and matrix effects from −17.0 to 468%, with variation always ≤19.1%. Authentic plasma and OF specimens were collected from an IRB-approved study that included controlled Vicks® VapoInhaler™ administration. The present method is sensitive, selective, economic and rapid (separations accomplished in <10min), and improves methamphetamine result interpretation.

ENANTIORESOLUTION OF (RS)-BUPROPION BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY USING CYANURIC CHLORIDE BASED CHIRAL DERIVATIZING REAGENTS HAVING AMINO ACIDS AS CHIRAL AUXILIARIES

Enantioseparation of (RS)-Bupropion was achieved as its diastereomers which were prepared with chiral derivatizing reagents (CDRs) synthesized from cyanuric chloride. Five new CDRs were prepared by nucleophilic substitution of Cl atom in 6‐butoxy derivative of cyanuric chloride with amino acids (namely, L‐Leu, L‐Val, L‐Phe, D‐Phg and L‐Ala) as chiral auxiliaries to give monochloro-s-triazine reagents and other five dichloro-s-triazine reagents were prepared by monosubstitution with the above-mentioned amino acids. The diastereomers were synthesized under microwave irradiation and by conventional method. Separation efficiencies of the two sets of CDRs, among themselves and among the two groups, have been compared on the basis of resolution (RS) and difference between the retention times of resolved diastereomers. The method was validated for detection limit, linearity, accuracy and precision.

Establishment of reverse-phase high-performance liquid chromatography with chiral reagent derivatization for separation of fexofenadine enantiomers

To establish a precolumn chiral derivatization method for determination of fexofenadine enantiomers, a chiral substrate of OATP1B1, in cellular model. R-(+)-phenylethyl isocyanate was selected as chiral derivatization reagent, which was reacted with fexofenadine to form carbamate derivatives. Enantiomers were identified by LC/MS and separated by RP-HPLC. Under the experimental conditions, the fexofenadine enantiomers were separated completely. The standard curve was linear over the concentration range of 25-100 ng/ml (R(2)=0.9992, 0.9989). Accuracy was 101.1% and 98.3%, intra-precision was 2.4% and 3.1%, inter-precision was 3.1% and 4.0% for D1 and D2, respectively. The method established is sensitive and accurate for determination of fexofenadine enantiomers in cells.

Evaluation of a series of prolylamidepyridines as the chiral derivatization reagents for enantioseparation of carboxylic acids by LC–ESI–MS/MS and the application to human saliva

Mass spectrometry has become a popular analytical tool because of its high sensitivity and specificity. The use of a chiral derivatization reagent for the mass spectrometry (MS) detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-MS/MS analysis is very limited. According to these observations, we are currently in the process of developing novel labeling reagents for chiral molecules in MS/MS analysis. The derivatization reagent that is effective for enhancing not only the electrospray ionization-MS/MS sensitivity but also the reversed-phase LC resolution of carboxylic acid enantiomers should have a highly proton-affinitive moiety and an asymmetric structure near the reactive functional group. Furthermore, the resulting derivative has to provide a characteristic product ion suitable for the selected reaction monitoring. Based upon these considerations, a series of prolylamidepyridines ((S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2), (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-3-yl)amide, and (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-4-yl)amide) was synthesized as ideal labeling reagents for the enantioseparation of chiral carboxylic acids and evaluated in terms of separation efficiency and detection sensitivity by ultra-performance LC (UPLC)-MS/MS. Among the synthesized reagents, PCP2 was the most efficient chiral derivatization reagent for the enantioseparation of carboxylic acid. The Rs values and the detection limits of the derivatives of non-steroidal anti-inflammatory drugs, which were selected as the representative carboxylic acids, were in the range of 2.52-6.07 and 49-260 amol, respectively. The sensitive detection of biological carboxylic acids (detection limits, 32-520 amol) was also carried out by the proposed method using PCP2 and UPLC-MS/MS. The PCP2 was applied to the determination of carboxylic acids in human saliva. Several biological carboxylic acids, such as lactic acid (LA), 3-hydroxybutylic acid, maric acid, succinic acid, α-ketoglutalic acid, and citric acid, were clearly identified in the saliva of healthy persons and diabetic patients. Furthermore, the ratio of D-LA in diabetic patients was higher than that in normal subjects. Judging from these results, PCP2 seems to be a useful chiral derivatization reagent for the determination not only of chiral, but also achiral, carboxylic acids in real samples.

Enantioresolution of dl-selenomethionine by thin silica gel plates impregnated with (−) quinine and reversed-phase TLC and HPLC separation of diastereomers prepared with difluorodinitrobenzene based reagents having l-amino acids as chiral auxiliaries

(−)-Quinine was used as a chiral selector by adopting three approaches in TLC for direct enantioresolution of DL-selenomethionine; these included impregnation of plate by chiral selector and its use as CMPA. Effect of pH, temperature and concentration of chiral selector on enantioresolution is studied. The spots were detected with ninhydrin and iodine vapors. Optically pure amino acids L-phenylalanine, S-benzyl-L-cysteine, and L-methionine were used to synthesize chiral derivatizing reagents (CDRs) based on fluorodinitrobenzene; these CDRs were used to synthesize diastereomers of DL-selenomethionine via nucleophilic substitution of the remaining fluorine atom in these reagents under microwave irradiation for 55 s at 75% (of 800 W) and also by stirring for 50 min at 45 °C. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using aq. trifluoroacetic acid and acetonitrile as mobile phase under gradient elution while aq. triethylamine buffer and acetonitrile was successful as mobile phase for separation of diastereomers by reversed-phase thin layer chromatography. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification.

Enantioseparation of Orciprenaline, Betaxolol, and Propranolol using HPLC and New Chiral Reagents Based on 1,5-Difluoro-2,4-dinitrobenzene

Diastereomers of three β-blockers (orciprenaline, betaxolol, and propranolol) were synthesized using three new chiral derivatizing reagents that were prepared by substituting one fluorine atom in 1,5-difluoro-2,4-dinitrobenzene with two chirally pure amines and one L-amino acid. The reagents were characterized using ultraviolet-visible spectroscopy, infrared spectroscopy, elemental analysis, and proton nuclear magnetic resonance. Diastereomers were synthesized under microwave irradiation at 50 s at 80% of 800 W and also by stirring for 50 min at 45°C. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using acetonitrile and aqueous trifluoroacetic acid as the mobile phase components. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection, and limit of quantification.

LC Enantioseparation of 30-Component Diastereomeric Mixture of Amino Acids and Detection of d-Isomers Using New Reagents with Amines as Chiral Auxiliaries in Cyanuric Chloride

Two enantiomerically pure amines, viz., (R)-(+)-naphthylethyl amine and (S)-(+)-1-benzyl-3-aminopyrrolidine, were used as chiral auxiliaries for nucleophilic substitution of chlorine atoms in cyanuric chloride or its 6-butoxy derivative. The chiral derivatizing reagents so obtained were characterized and their chiral purity was ascertained. Diastereomers of 15 dl-proteinogenic amino acids were synthesized under microwave irradiation using these reagents. Separation of diastereomeric pairs along with separation of a mixture of 30 diastereomers in a single chromatographic run was carried out on a reversed-phase C18 column. Mixtures of acetonitrile with aqueous trifluoroacetic acid were used as mobile phase. The detection was made at 230 nm using photo diode array detector. The separation behavior in terms of retention times and resolutions was compared on the basis of effect of chiral auxiliaries (i.e. amines) and achiral substituents (i.e. chlorine or butoxy group) in the chiral derivatizing reagents and the hydrophobic side chains of amino acids. The separation method was validated in terms of accuracy, precision, linearity, recovery, limit of detection and limit of quantitation. The method was successful for determination of d-amino acids in the absence of pure d-enantiomers and for separation of 19 diastereomers from a mixture of 30.