Chiral Derivatizing Reagents Research Articles

Enantiomeric Fraction Determination of 2‐Arylpropionic Acids in a Package Plant Membrane Bioreactor

Enantiomeric compositions of three 2-arylpropionic acid (2-APA) drugs, ibuprofen, naproxen, and ketoprofen, were monitored in a membrane bioreactor (MBR) treating municipal effluent in a small rural town in Australia. Specific enantiomers were determined as amide diastereomers using the chiral derivatizing reagent, (R)-1-phenylethylamine (PEA), followed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The six individual enantiomers were quantified by isotope dilution and the enantiomeric fractions (EFs) were determined. Over four separate sampling events, ibuprofen EF ranged from 0.88 to 0.94 (median 0.93) in the influent and 0.38 to 0.40 (median 0.39) in the effluent. However, no significant change in ketoprofen EF was observed, with influent EFs of 0.56-0.60 (median 0.58) and effluent EFs 0.54-0.68 (median 0.56). This is the first report of enantiospecific analysis of ketoprofen in municipal wastewater and it is not yet clear why such different behavior was observed compared to ibuprofen. Naproxen EF was consistently measured at 0.99 in the influent and ranged from 0.86 to 0.94 (median 0.91) in the effluent. This study demonstrates that EF is a relatively stable parameter and does not fluctuate according to concentration or other short-term variables introduced by sampling limitations. The enantiospecific analysis of chiral chemicals presents a promising approach to elucidate a more thorough understanding of biological treatment processes and a potential tool for monitoring the performance of key biological pathways.

High‐performance liquid chromatographic enantioseparation of (RS)‐bupropion using isothiocyanate‐based chiral derivatizing reagents

A high-performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate-based chiral derivatizing reagents, (S)-1-(1-naphthyl) ethyl isothiocyanate, (S)-NEIT, and (R)-α-methyl benzyl isothiocyanate, (R)-MBIT. The diastereomers synthesized with (S)-NEIT were enantioseparated by reversed-phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)-MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040-0.043 µg/mL for each of the diastereomers prepared with (S)-NEIT.

Application of optically pure amines as chiral auxiliaries to develop trichloro‐s‐triazine‐based new chiral derivatizing reagents for reversed‐phase high‐performance liquid chromatographic enantioseparation of dl‐selenomethionine

(R)-(+)-naphthylethyl amine and (S)-(+)-1-benzyl-3-aminopyrrolidine were incorporated as chiral auxiliaries, by nucleophilic substitution of chlorine atoms, in cyanuric chloride (CC) or its 6-butoxy derivative. There were obtained four new chiral derivatizing reagents (CDRs) as two dichloro and two monochloro triazine reagents. The CDRs so obtained were characterized and their optical purity was ascertained. Diastereomers of dl-selenomethionine were synthesized under microwave irradiation for 60 or 90 s (at 80% power of 800 W). Reversed-phase high-performance liquid chromatographic separation of diastereomers was carried out on a C18 column using mixtures of acetonitrile with aqueous trifluoroacetic acid as mobile phase. The detection was made at 230 nm using a photodiode array detector. The separation behaviors in terms of retention times and resolutions were compared. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.

Novel chiral derivatization reagents possessing a pyridylthiourea structure for enantiospecific determination of amines and carboxylic acids in high-throughput liquid chromatography and electrospray-ionization mass spectrometry for chiral metabolomics identification

This paper reports the synthesis and the application of novel derivatization reagents possessing a pyridylthiourea structure for the enantiospecific determination of chiral amines and carboxylic acids in high-throughput LC–ESI-MS/MS. The novel reagents, i.e., (R)-N-(3-pyridylthiocarbamoyl)pyrrolidine-2-carboxylic acid (PyT-C) and (S)-3-amino-1-(3-pyridylthiocarbamoyl)pyrrolidine (PyT-N), were evaluated as chiral derivatization reagents for the enantiomeric determination of chiral amines and carboxylic acids, respectively, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The chiral amines and carboxylic acids were easily labeled with PyT-C and PyT-N, respectively, at 60°C in 60min in the presence of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) as the activation reagents. The resulting diastereomers were completely separated by reversed-phase chromatography using a small particle (1.7μm) ODS column (Rs=3.54–6.00 for carboxylic acids and Rs=3.07–4.75 for amines). A highly sensitive detection at the sub-fmol level was also obtained from the SRM chromatograms at a single monitoring ion, m/z 137.0 (0.72–1.46fmol for carboxylic acids and 0.55–1.89fmol for amines). The proposed procedure using PyT-C and PyT-N was applied to the determination of chiral amines and carboxylic acids spiked into human saliva, as a model study of chiral metabonomics identification. dl-Amino acid methyl esters and N-acetyl dl-amino acids, which are the representatives as the chiral amines and carboxylic acids, in the saliva were clearly identified by the present method. Because the same product ion at m/z 137.0 was obtained from collision-induced dissociation (CID) of protonated molecular ions of all the derivatives, the proposed procedure using both reagents (i.e., PyT-C and PyT-N) seems to be useful for chiral metabolomics identification having selected functional groups (i.e., amines and carboxylic acids).

Indirect enantioseparation of selenomethionine by reversed‐phase high‐performance liquid chromatography using a newly synthesized chiral derivatizing reagent based on (S)‐naproxen moiety

(S)-Naproxen was reacted with N-hydroxyphthalimide in the presence of coupling reagent dicyclohexylcarbodiimide, and a new chiral derivatizing reagent, phthalimidyl-(S)-naproxen ester, was synthesized. It was characterized and was used for synthesis of diastereomers of selenomethionine via microwave irradiation or vortexing. The reaction conditions were optimized. Diastereomeric pairs synthesized by two approaches were successfully separated by reversed-phase high-performance liquid chromatography using binary mixtures of aqueous triethylammonium phosphate and acetonitrile. Detection was carried out at 231 nm. The limit of detection was found to be 0.11 and 0.10 pmol/mL for diastereomers of d- and l-SeMet, respectively. The method was validated for accuracy, precision and limit of detection. The new chiral derivatizing reagent was capable of enantioseparation of dl-SeMet in the form of diastereomers having higher stability, enhanced resolution and lower limits of detection in comparison to the diastereomers prepared with other chiral derivatizing reagents reported in the literature. Optimized structures of the two diastereomers were drawn using the Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G basis set to explain the separation mechanism.

Study on the Regioselectivie of Grignard Reagent Addition Reaction of Maleopimaric Acid Trimethyl Ester

Two regioselectivite adducts (compounds 3 and 4) were firstly synthesized from the Grignard reagent and maleopimaric acid trimethyl ester (2). The structures of all compounds were characterized by elemental analyses, NMR and MS, and stero-structures of 3 were further determined by COSY, HMQC and HMBC. The spatial structure of 3 was further confirmed by X-ray single crystal diffraction analysis method. Owing to the addition reaction selectivity of 2 was dependent on the steric hindrance of three methyl groups, the TADDOL (tetraaryl-1,3-dioxolane-4,5-dimethanols) product was prevented because of the formation of lactone 3, but the stereo-structure of the rosin-ring did not changed. Products of 3 and 4 are expected to apply as chiral derivatization reagents. Keywords maleopimaric acid trimethyl ester; grignard reagent; 2D NMR; regioselectivity

Indirect enantioseparation of proteinogenic amino acids using naproxen‐based chiral derivatizing reagent and HPLC

Diastereomers of 18 proteinogenic amino acids were synthesized under microwave irradiation and by vortexing using (S)-naproxen-benzotriazole as chiral derivatizing reagent. The diastereomers synthesized by two approaches were found to be identical in terms of their characterization and chromatographic data. A linear gradient of triethylammonium phosphate (pH 3.5)-acetonitrile (30-65%, within 35 min) was found to be successful using reversed-phase high-performance liquid chromatography for their separation. Detection was carried out at 231 nm and sharp peaks were obtained. The method was validated for accuracy, precision and limit of detection.

Chiral separation of new cathinone- and amphetamine-related designer drugs by gas chromatography–mass spectrometry using trifluoroacetyl-l-prolyl chloride as chiral derivatization reagent

Since cathinone derivatives gained high popularity on the recreational drugs market within the past 5years the development of analytical methods for the achiral and chiral determination of this substance class is of great interest. Not at least because it is obvious that the pharmacological potency differs between both enantiomers. Cathinones are structurally closely related to amphetamines, which have similar stimulating effects and are somehow better investigated. The goal of this research was to perform indirect enantioseparation of novel psychoactive cathinone and amphetamine derivatives. Trifluoroacetyl-l-prolyl chloride was served as chiral derivatization agent (CDA). Chromatographic separation was performed using a commercially available HP5-MS capillary column with a length of 30m. Helium was used as carrier gas with a constant flow of 1.0ml/min. Under optimum conditions 14 amphetamine derivatives were successfully resolved into their enantiomers and detected with the single quadrupol detector. Racemic methcathinone derivatives analyzed with the same method showed different peak areas for each of the produced diastereomeric isomers, even if they are structurally closely related to the amphetamines. Derivatization experiments with the single isomers of methcathinone led to both diastereomers whereas the S(−) enantiomer seemed to racemize more likely. Based on comparative experiments with R-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA) as CDA, racemization due to the keto-enol-tautomerism of the cathinone derivatives seemed to be responsible for this phenomenon. Nevertheless, 18 cathinone derivatives were successfully enantioseparated and an approach of quantitative evaluation is demonstrated.

Chiral amines as reagents for HPLC–MS enantioseparation of chiral carboxylic acids

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non-steroidal anti-inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed-phase chromatography, and detection sensitivity by electrospray ionization (ESI)-MS/MS. Among the tested eight chiral amines, i.e. (R)-(+)-4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-APy), (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine (PMP), L-prolinamide, (3R)-(-)-1-benzyl-3-aminopyrrolidine, (S)-(+)-1-cyclohexyl-ethylamine, (3R)-(+)-3-(trifluoroacetamido)-pyrrolidine (TFAP), (R)-(-)-1-aminoindan (AI), and (S)-(+)-tetrahydrofurfuryl-amine, DBD-APy, PMP, AI, and TFAP could be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29-3.85 and 0.57-0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma.

(S)‐Naproxen as a platform to develop chiral derivatizing reagent for reversed‐phase high‐performance liquid chromatographic enantioseparation of analytes having a carbonyl functional group

(S)-Naproxen was used to synthesize a chiral reagent, (S)-2-(6-methoxynaphthalen-2-yl)propanehydrazide, by itsreaction with hydrazine hydrate in the presence of dicyclohexylcarbodiimide as coupling agent. The reagent was characterized and its chiral purity was established. It was used as a chiral derivatizing reagent for the synthesis of hydrazone diastereomers, under microwave irradiation, of certain chiral aldehydes and ketones. The respective diastereomers were separated by reversed-phase high-performance liquid chromatography using a binary solvent combination containing trifluoroacetic acid. The diastereomers were detected at 231 nm. The method was validated for accuracy, precision, and limit of detection (LOD). For a series of hydrazones the LOD was found to be in the range 1.62-1.65 pmol/mL.

Rapid enantiomeric separation and simultaneous determination of phenethylamines by ultra high performance liquid chromatography with fluorescence and mass spectrometric detection: application to the analysis of illicit drugs distributed in the Japanese market and biological samples

A rapid enantiomeric separation and simultaneous determination method based on ultra high performance liquid chromatography (UHPLC) was developed for phenethylamine-type abused drugs using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((R)-(-)-DBD-Py-NCS) as the chiral fluorescent derivatization reagent. The derivatives were rapidly enantiomerically separated by reversed-phase UHPLC using a column of 2.3-µm octadecylsilica (ODS) particles by isocratic elution with water-methanol or water-acetonitrile systems as the mobile phase. The proposed method was applied to the analysis of products containing illicit drugs distributed in the Japanese market. Among the products, 1-(3,4-methylenedioxyphenyl)butan-2-amine (BDB) and 1-(2-methoxy4,5-methylenedioxyphenyl)propan-2-amine (MMDA-2) were detected in racemic form. Furthermore, the method was successfully applied to the analysis of hair specimens from rats that were continuously dosed with diphenyl(pyrrolidin-2-yl)methanol (D2PM). Using UHPLC-fluorescence (FL) detection, (R)- and (S)-D2PM from hair specimens were enantiomerically separated and detected with high sensitivity. The detection limits of (R)- and (S)-D2PM were 0.12 and 0.21 ng/mg hair, respectively (signal-to-noise ratio (S/N) = 3).

P.4.006 Ketamine infusion in bipolar disorder resistant to treatment with antidepressants

Racemate separation can be achieved according to two modes. The first mode is direct separation method, and the second is indirect method. The first mode of separation includes the application of a chiral stationary phase (cellulose and its derivatives, CLEC-system, or silica), a stationary phase impregnated with a chiral selector (enantiomers of various chiral compounds, for example acids, alkaloids, amino acids, and their derivatives) or chiral mobile phase additives (e.g., various cyclodextrins and their derivatives, alkaloids). The second mode of separation principle consists of a reaction between a racemate and a chiral derivatizing reagent, which yields separate diastereomers in an achiral chromatographic system. The examples of both types of chiral resolution of a racemate using thin-layer chromatography will be presented.

HPLC enantioresolution of (R,S)‐baclofen using three newly synthesized dichloro‐s‐triazine reagents having amines and five others having amino acids as chiral auxiliaries

Enantioresolution of (R,S)-baclofen was accomplished using a newly synthesized set of three chiral derivatizing reagents (CDRs) having amines [(S)-(-)-α,4-dimethylbenzylamine, (-)-cis-myrtanylamine and (R)-(-)-1-cyclohexylethylamine] as chiral auxiliaries in cyanuric chloride and another set of five CDRs having amino acids (L-Leu, D-Phg, L-Val, L-Met and L-Ala) as chiral auxiliaries. These eight CDRs were used for synthesis of diastereomers of (R,S)-baclofen under microwave irradiation. The diastereomers were separated on a reversed-phase C(18) column using mixtures of methanol with aqueous trifluoroacetic acid with UV detection at 230 nm. Chromatographic data obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for limit of detection, linearity, accuracy and precision.

Enantiomeric Purity of Chiral Derivatizing Reagents for Enantioresolution

BioanalysisVol. 3, No. 18 EditorialEnantiomeric purity of chiral derivatizing reagents for enantioresolutionRavi BhushanRavi BhushanDepartment of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India. Search for more papers by this authorEmail the corresponding author at rbushfcy@iitr.ernet.inPublished Online:26 Sep 2011https://doi.org/10.4155/bio.11.211AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: chiral derivatizing reagentsenantiomeric purityenantioresolutionHPLCracemic drugsBibliography1 Bhushan R, Martens J. Amino Acids: Chromatographic Separation and Enantioresolution. HNB Publishing, NY, USA, 32–35 (2010).Google Scholar2 Bhushan R, Brückner H. Use of Marfey’s reagent and analogs for chiral amino acid analysis: assessment and applications to natural products and biological systems. J. Chromatogr. B DOI: 10.1016/j.jchromb.2011.05.058 (2011) (Epub ahead of print).Google Scholar3 Bhushan R, Kumar R. Analysis of multicomponent mixture and enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger’s reagent. Anal. Bioanal. Chem.394,1697–1705 (2009).Crossref, Medline, CAS, Google Scholar4 Bhushan R, Tanwar S. Synthesis of dinitrophenyl-L-Pro-N-hydroxysuccinimide ester and four new variants of Sanger’s reagent having chiral amines and their application for enantioresolution of mexiletine using reversed-phase high-performance liquid chromatography. J. Chromatogr. A1216,5769–5773 (2009).Crossref, Medline, CAS, Google Scholar5 Bhushan R, Kumar V. Indirect enantioseparation of α-amino acids by reversed-phase liquid chromatography using newly synthesized chiral derivatizing reagents prepared from s-triazine chloride. J. Chromatogr. A1201,35–42 (2008).Crossref, Medline, CAS, Google Scholar6 Bhushan R, Kumar V, Tanwar S. Liquid chromatographic enantioseparation of non-protein α-amino acids after derivatization with Marfey’s reagent and its structural variants. Amino Acids36,571–579 (2009).Crossref, Medline, CAS, Google Scholar7 Bhushan R, Tanwar S. RP-LC resolution of (R,S)-atenolol via diastereomerization with Marfey’s reagent and its structural variants under conventional and microwave heating. Chromatographia68,849–853 (2008).Crossref, CAS, Google Scholar8 Bhushan R, Dixit S. Application of hydrazino dinitrophenyl-amino acids as chiral derivatizing reagents for liquid chromatographic enantioresolution of carbonyl compounds. Chromatographia DOI: 10.1007/s10337–011–2063-x (2011) (Epub ahead of print).Google Scholar9 Bhushan R, Dixit S. Enantioresolution of five β-blockers by reversed-phase high-performance liquid chromatography using fifteen chiral derivatizing reagents having amino acids or their amides as chiral auxiliaries on cyanuric chloride platform. Biomed. Chromatogr. DOI: 10.1002/bmc.1653 (2011) (Epub ahead of print).Google ScholarFiguresReferencesRelatedDetailsCited BySynthesis and Chiral Separation of Some 4-thioflavones9 February 2021 | Journal of Chromatographic Science, Vol. 59, No. 9Derivatization procedures and their analytical performances for HPLC determination in bioanalysis24 November 2020 | Biomedical Chromatography, Vol. 35, No. 1Bioanalysis and enantioseparation of dl-carnitine in human plasma by the derivatization approachVinod Kumar Vashistha & Ravi Bhushan12 October 2015 | Bioanalysis, Vol. 7, No. 19Quantitative analysis of drugs in biological matrices by HPLC hyphenated to fluorescence detectionEmmanuelle Lipka & Claude Vaccher14 April 2015 | Bioanalysis, Vol. 7, No. 6Preparative Enantioseparation of ( RS )-Baclofen: Determination of Molecular Dissymmetry14 February 2015 | Chirality, Vol. 27, No. 4Quantitative liquid chromatography–electrospray ionization-mass spectrometry analysis of amine-containing metabolites derivatized with cyanuric chloride and methylamine isotopologuesJournal of Chromatography A, Vol. 1388Amino acids as chiral auxiliaries in cyanuric chloride-based chiral derivatizing agents for enantioseparation by liquid chromatography18 August 2014 | Biomedical Chromatography, Vol. 28, No. 11Enantioseparation 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 auxiliariesActa Chromatographica, Vol. 26, No. 3Liquid chromatographic enantioseparation of ( RS )-mexiletine and ( RS )-fluoxetine using chiral derivatizing reagents synthesized with ( S )-naproxen moiety25 May 2014 | Biomedical Chromatography, Vol. 28, No. 6Indirect enantioseparation of selenomethionine by reversed-phase high-performance liquid chromatography using a newly synthesized chiral derivatizing reagent based on ( S )-naproxen moiety21 March 2013 | Biomedical Chromatography, Vol. 28, No. 1LC Enantioseparation of 30-Component Diastereomeric Mixture of Amino Acids and Detection of d-Isomers Using New Reagents with Amines as Chiral Auxiliaries in Cyanuric Chloride18 July 2013 | Chromatographia, Vol. 76, No. 17-18LC-MS Bioanalysis of Chiral Compounds30 August 2013Indirect enantioseparation of proteinogenic amino acids using naproxen-based chiral derivatizing reagent and HPLC7 December 2012 | Biomedical Chromatography, Vol. 27, No. 6Application of optically pure amines as chiral auxiliaries to develop trichloro- s -triazine-based new chiral derivatizing reagents for reversed-phase high-performance liquid chromatographic enantioseparation of dl -selenomethionine26 March 2013 | Biomedical Chromatography, Vol. 15 Vol. 3, No. 18 Follow us on social media for the latest updates Metrics Downloaded 175 times History Published online 26 September 2011 Published in print September 2011 Information© Future Science LtdKeywordschiral derivatizing reagentsenantiomeric purityenantioresolutionHPLCracemic drugsFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. 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Validated high‐performance liquid chromatographic enantioseparation of selenomethionine using isothiocyanate based chiral derivatizing reagents

A high-performance liquid chromatographic (HPLC) method for enantioseparation of selenomethionine (SeMet) was developed using two isothiocyanate-based chiral derivatizing reagents [(R)-methyl benzyl isothiocyanate (MBIC) and (S)-1-(1-naphthyl) ethyl isothiocyanate (NEIC)] and UV detection. Diastereomers of selenomethionine were synthesized either via stirring (using MBIC) or by microwave irradiation (using NEIC). Derivatization conditions were optimized and the synthesized diastereomers were successfully resolved using triethyl ammonium phosphate buffer and acetonitrile on a reversed-phase column. The method was validated for accuracy, precision and limit of detection. The mechanism of separation is also discussed.

Analysis of chiral non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac binding with HSA

The protein binding of non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac with human serum albumin (HSA) was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. S-(–)-1-(1-naphthyl)-ethylamine (S-NEA) was utilized as chiral derivatization reagent and pre-column derivatization RP-HPLC method was established for the separation and assay of the three pairs of enantiomer. The method had good linear relationship over the investigated concentration range without interference. The average extraction efficiency was higher than 85% in different systems, and the intra-day and inter-day precisions were less than 15%. In serum albumin, the protein binding of etodolac enantiomers showed significant stereoselectivity that the affinity of S-enantiomer was stronger than R-enantiomer, and the stereoselectivity ratio reached 6.06; Flurbiprofen had only weak stereoselectivity in HSA, and ketoprofen had no stereoselectivity at all. Scatchard curves showed that all the three chiral drugs had two types of binding sites in HSA.

Enantioresolution of five β‐blockers by reversed‐phase high‐performance liquid chromatography using fifteen chiral derivatizing reagents having amino acids or their amides as chiral auxiliaries on a cyanuric chloride platform

Enantioseparation of five β-blockers, namely, (R,S)-atenolol, (R,S)-propranolol, (R,S)-bisoprolol, (R,S)-metoprolol and (R,S)-carvedilol, was achieved as their diastereomers prepared with chiral derivatizing reagents (CDRs) synthesized on a cyanuric chloride platform. Fifteen CDRs were synthesized by nucleophilic substitution of the Cl atom in cyanuric chloride or its 6-methoxy derivative with amino acids (namely, L-Leu, L-Val, D-Phg, L-Met and L-Ala) or their amides as chiral auxiliaries. The diastereomers were synthesized under microwave irradiation for 70 or 100 s at 85% power. Separation of diastereomers was carried out on a C(18) column and gradient eluting mixtures of methanol with aqueous trifluoroacetic acid with UV detection at 230 nm. Separation efficiencies of the reagents were compared on the basis of effect of chiral auxiliaries (i.e. amino acids or amino acid amides) and achiral substituents (i.e. chlorine or methoxy group) in the CDRs. The method was validated for detection limit, linearity, accuracy and precision.

Application of Hydrazino Dinitrophenyl-Amino Acids as Chiral Derivatizing Reagents for Liquid Chromatographic Enantioresolution of Carbonyl Compounds

A new series of chiral derivatizing reagents (CDRs) consisting of five hydrazino dinitrophenyl (HDNP)-amino acids (CDR 1–5) was prepared by a two-step synthesis procedure starting from 1,5-difluoro-2,4-dinitrobenzene (DFDNB). In the first step, five fluoro-dinitrophenyl (FDNP)-reagents, namely FDNP-l-Leu, FDNP-l-Val, FDNP-l-Phe, FDNP-l-Ala and FDNP-d-Phg were synthesized by substituting one of the fluorine atoms in DFDNB moiety with amino acids l-Leu, l-Val, l-Phe, l-Ala and d-Phg, respectively. In the following step, the remaining fluorine atom of the FDNP reagents was substituted with hydrazine hydrate to obtain five HDNP reagents (i.e. CDR 1–5; HDNP-l-Leu, HDNP-l-Val, HDNP-l-Phe, HDNP-l-Ala and HDNP-d-Phg). These five CDRs were used for synthesis of diastereomers of six racemic carbonyl compounds which were resolved by high-performance liquid chromatography using C18 column and gradient eluting mixture of acetonitrile or methanol with triethylammonium phosphate buffer with UV detection at 348 nm. Microwave irradiation was used for synthesis of both the CDRs and the diastereomers. The newly synthesized CDRs were observed to be superior in comparison to their counterparts having amino acid amides as chiral auxiliaries in terms of cost effectiveness and providing better resolution of diastereomers. The method was validated for limit of detection, linearity, accuracy and precision.

Synthesis of ( S)-naproxen-benzotriazole and its application as chiral derivatizing reagent for microwave-assisted synthesis and indirect high performance liquid chromatographic separation of diastereomers of penicillamine, cysteine and homocysteine

( S)-Naproxen-benzotriazole was synthesized by the reaction of ( S)-naproxen with 1 H-benzotriazole using coupling reagent dicyclohexyl carbodiimide and 4-dimethylamino pyridine (DCC/DMAP). It was used as chiral derivatizing reagent for microwave irradiated synthesis of diastereomers of penicillamine, cysteine and homocysteine. The diastereomers were separated by reversed phase high performance liquid chromatography using gradient elution of triethylammonium phosphate (pH 3.5)–acetonitrile (30–65% within 30 min). The method was validated for accuracy, precision, and limit of detection.

Indirect enantioresolution of (R,S)‐mexiletine by reversed‐phase high‐performance liquid chromatography via diastereomerization with [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride], (S)‐naproxen and nine chiral reagents synthesized as variants of Marfey's reagent

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)-mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)-O,O'-di-p-toluoyl tartaric acid anhydride] was synthesized and (S)-naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed-phase high-performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10-30 pmol.