Design and development of chiral reagents for the chromatographic e.e. determination of chiral alcohols

(S)-Trolox methyl ether is known as a powerful chiral reagent for the e.e. determination of chiral alcohols by separation of the corresponding diastereoisomeric esters on achiral GC and SFC columns. In order to further improve his methodology, five possible candidates resultings from variation of structural elements of parent reagent have been tested for derivatization with selected alcohols and subsequent analysis of the diastereoisomeric pairs of esters. The results of this optimization procedure showing the ways to new potent reagents are discussed. © 1996 Wiley-Liss, Inc.     

Derivatization of thiol-containing compounds

The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents contain a functional group, e.g. an N-substituted maleimide, active halogen or aziridine, which react with the thiol group. Derivatization for use in flow injection analysis, thin-layer chromatography or gas chromatography—mass spectrometry is also described.     

Application of cyanuric chloride-based six new chiral derivatizing reagents having amino acids and amino acid amides as chiral auxiliaries for enantioresolution of proteinogenic amino acids by reversed-phase high-performance liquid chromatography

Six dichloro-s-triazine (DCT) reagents having l-Leu, d-Phg, l-Val, l-Met, l-Ala and l-Met-NH₂ as chiral auxiliaries in cyanuric chloride were introduced for enantioseparation of 13 proteinogenic amino acids. Four other DCTs and six monochloro-s-triazine (MCT) reagents having amino acid amides as chiral auxiliaries were also synthesized. These 16 chiral derivatizing reagents (CDRs) were used for synthesis of diastereomers of all the 13 analytes using microwave irradiation, which were resolved by reversed-phase high-performance liquid chromatography (RP-HPLC) using C18 column and gradient eluting mixture of aqueous TFA and acetonitrile with UV detection at 230 nm. It required only 60–90 s for derivatization using microwave irradiation. Better resolution and lower retention times were observed for the diastereomers prepared with CDRs having amino acids as chiral auxiliaries as compared to counterparts prepared with reagents having amino acid amides as chiral auxiliaries. As the best resolution of all the 13 analytes was observed for their diastereomers prepared using the DCT reagent having l-Leu as chiral auxiliary, this CDR was further employed for derivatization of Lys, Tyr, His and Arg followed by RP-HPLC analysis of resulting diastereomers. The results are discussed in light of acid and amide groups of chiral auxiliaries constituting CDRs, electronegativities of the atoms of achiral moieties constituting CDRs and hydrophobicities of side chains of amino acids constituting CDRs and analytes.     

Recent advances in separation and detection methods for thiol compounds in biological samples

Biological aminothiols, such as cysteine, homocysteine, and glutathione, widely occur in animal tissues and fluids. The altered levels of the thiols (reduced forms) and their disulfides (oxidized forms) in physiological liquids have been linked to specific pathological conditions and closely associated with several human diseases. Therefore, it is well recognized that the determination of thiols and disufides is important in order to understand their physiological roles. The derivatization utilizing a suitable labeling reagent followed by chromatographic separation and detection is the most reliable means for sensitive and selective assays. Many reagents have typically been synthesized and successfully used for the determination of thiols and disulfides in biological specimens. The development of new reagents for highly sensitive detection is still continuing. This review describes the approaches for the separation assay of various thiol compounds, obtained through the analytical papers published in 2000–2008. The derivatization reagents are categorized with each type of chromophore and fluorophore and evaluated in terms of their reactivity, stability, detection wavelength, handling, sensitivity and selectivity. Application examples of the reagents for bioanalysis are also described in the text.     

Analysis of enantiomers of chiral phenethylamine drugs by capillary gas chromatography/mass spectrometry/flame-ionization detection and pre-column chiral derivatization

Several important chiral phenethylamine agents such as mexiletine, fenfluramine, amphetamine, methamphetamine and N-n-propylamphetamine show stereoselective disposition in humans and large differences in therapeutic relevance and toxicity. To analyze the enantiomers of chiral amine drugs, stereoselective methods were developed to separate those enantiomers on an achiral capillary gas chromatography by pre-column chiral derivatization with S-(-)-N-(fluoroacyl)-prolyl chloride. The stereoselectivity and sensitivity can be improved by chiral derivatization. The methods established offer enantioselective, simple, flexible and economic approaches for the analysis of chiral amine drug enantiomers in biological fluids. The methods have been used to determine S-(+)-methamphetamine in human forensic samples and to analyze enantiomers of amphetamine and fenfluramine in rat liver microsomes.     

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.     

Molecular and nanoparticle postcolumn reagents for assay of low-molecular-mass biothiols using high-performance liquid chromatography

Determination of low-molecular-mass (LMM) biothiols in biological matrixes is of importance in the studies of their related bio-processes and for the clinical diagnostics of a variety of diseases. Standard method for the assay of the small biothiols is in demand. Postcolumn techniques used in high-performance liquid chromatography (HPLC) allow automation of the derivatization step and, therefore, are suitable for standardization of HPLC analysis. This paper gives an overview of the existing reaction systems useful for the postcolumn assay of the LMM biothiol molecules in conjunction with HPLC. The postcolumn reagents are classified by the types of their reactions with thiol-containing compounds. The chemical reactivity and selectivity as well as the spectroscopic characteristics of the postcolumn reagents have been addressed. The emerging strategies of using nanoparticles as thiol-reactive reagents and their applications in postcolumn detection of the LMM biothiols have also been discussed in detail.     

Resolution of the enantiomers of drugs containing amino alcohol structure after derivatization with bromoacetic acid

The separation of the enantiomers of drugs containing an amino alcohol structure like adrenergic drugs or β-blockers is described. The compounds are resolved on chiral ligand-exchange chromatography phases after derivatization with bromoacetic acid. © 1992 Wiley-Liss, Inc.     

Methods for determining the absolute configurations of marine ladder-shaped polyethers

Marine dinoflagellates produce a unique family of bioactive substances featuring multiple ether rings aligned in a ladder shape. These are large, complex molecules with potent bioactivity. Targeted chiral centers sit on either the skeletal ladders or on the side chains of these compounds. However, the laborious steps of isolation and purification severely diminish the amount of sample available for assigning these chiral centers via structural investigations. Three important methods were used to assign the stereochemistry of the molecules, (a) circular dichroism (CD) spectroscopy, (b) labeling with fluorescent chiral reagents for high-performance liquid chromatography (HPLC) analysis, and (c) derivatization with anisotropic reagents for nuclear magnetic resonance (NMR) analysis. The addition of fluorescent chiral reagents allowed for the use of much less material than typically required. In this review, we present examples of the determination of absolute configurations in ladder-shaped polyethers. The targeted compounds include ciguatoxins (CTXs), gymnocin-B, gambieric acids, prymnesin-2, maitotoxin, yessotoxins, gambierol, brevisamide, and brevisin.     

Resolution of the enantiomers of ibuprofen; comparison study of diastereomeric method and chiral stationary phase method

In this study, an indirect diastereomeric method and a direct method utilizing a chiral stationary phase (CSP) were investigated for the resolution of ibuprofen enantiomers. In the indirect method, ethylchloroformate (ECF) and 2-ethoxy-1-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) were utilized as first-step derivatizing reagents in acetonitrile or toluene. In the direct CSP method, ibuprofen enantiomers were derivatized to p-nitrobenzyl ureides and then resolved on an (R)-(−)-(1-naphthyl)ethylurea CSP column. The derivatization procedure took place in 10 min with an overall inversion efficiency of 90.3%. Racemization was not observed under the derivatization conditions used. The HPLC-CSP method was utilized to study the pharmacokinetics of ibuprofen enantiomers in dog plasma after a single oral administration of 200 mg of ibuprofen racemate.     

Fast chiral separation of drugs using columns packed with sub‐2 μm particles and ultra‐high pressure

The use of columns packed with sub‐2 μm particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and β‐blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4‐tri‐O‐acetyl‐α‐D ‐arabinopyranosyl isothiocyanate (AITC) and N‐α‐(2,4‐dinitro‐5‐fluorophenyl)‐L ‐alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2–5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)‐(S)‐amphetamine and (+)‐(S)‐methamphetamine. Similar results were obtained with β‐blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several β‐blocker enantiomers was performed in 1 min or less. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Derivatization of haemoglobin with periodate-generated reticulation agents: evaluation of oxidative reactivity for potential blood substitutes

Periodate modification of the sugar moiety in sugars, including adenosine triphosphate (ATP), has previously been employed in order to prepare dialdehyde-type reagents, which were then utilized in crosslinking reactions on haemoglobin, yielding polymerized material with useful dioxygen-binding properties and hence proposed as possible artificial oxygen carriers ('blood substitutes'). Here, the periodate protocol is shown to be applicable to a wider range of oxygen-containing compounds, illustrated by starch and polyethylene glycol. Derivatization protocols are described for haemoglobin with such periodate-treated crosslinking agents, and the dioxygen-binding properties and redox reactivities are investigated for the derivatized haemoglobins, with emphasis on pro-oxidative properties. There is a general tendency of the derivatization to result in higher autooxidation rates. The peroxide reactivity of the met (ferric) form is also affected by derivatization, as witnessed, among others, by varying yields of ferryl [Fe (IV)-oxo] and free radical generated. In cell, culture tests (human umbilical vein epithelial cells, HUVEC), the derivatization protocols show no toxic effect.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry

The usual aim in metabolomic studies is to quantify the entire metabolome of each of a series of biological samples. To do this for complex biological matrices, e.g., plant tissues, efficient and reproducible extraction protocols must be developed. However, derivatization protocols must also be developed if GC/MS (one of the mostly widely used analytical methods for metabolomics) is involved. The aim of this study was to investigate how different chemical and physical factors (extraction solvent, derivatization reagents, and temperature) affect the extraction and derivatization of the metabolome from leaves of the plant Arabidopsis thaliana. Using design of experiment procedures, variation was systematically introduced, and the effects of this variation were analyzed using regression models. The results show that this approach allows a reliable protocol for metabolomic analysis of Arabidopsis to be determined with a relatively limited number of experiments. Following two different investigations an extraction and derivatization protocol was chosen. Further, the reproducibility of the analysis of 66 endogenous compounds was investigated, and it was shown that both hydrophilic and lipophilic compounds were detected with high reproducibility.     

Assignment of absolute configuration using chiral reagents and NMR spectroscopy

An overview of chiral reagents that are used to assign the absolute configuration of particular classes of compounds using NMR spectroscopy is presented. The use of chiral derivatizing agents, chiral solvating agents, metal complexes, and liquid crystals is described. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Significance of mobile phase composition in enantioseparation of chiral drugs by HPLC on a cellulose-based chiral stationary phase

Eight randomly selected pharmaceuticals, which included ibuprofen, ketoprofen, albuterol, acebutolol, propafenone, betaxolol, methylphenidate, and homatropine, were directly separated on a cellulose tris(4-methylbenzoate) chiral stationary phase (CSP) without derivatization via normal phase mode HPLC. Enantioresolution was achieved by the optimization of the type and the ratio of mobile phase modifiers and additives. The modifiers included alcohols; the mobile phase additives were trifluoroacetic acid (TFA) and triethylamine (TEA). It was found that methanol and ethanol were superior to isopropanol as mobile phase modifiers for enhancing chiral separation of some of the chiral drugs. The results also demonstrated that TFA has a dominant effect on chiral separations for both acidic and basic chiral drugs, although for some basic drug such as homatropine, TEA was more beneficial at improving enantioseparation. The separation of acebutolol enantiomers was achieved for the first time by adding both TFA and TEA to the mobile phase. The purpose of this paper is to demonstrate that the applicability of cellulose based CSPs can be expanded by controlling the mobile phase compositions through the addition of trace amounts of achiral additives and the selection of the appropriate alcoholic modifier. © 1996 Wiley-Liss, Inc.     

Preliminary evidence for novel fucose-containing lipids in normal and murine sarcoma virus-transformed rat cells.

Two fucose-containing lipids with long chain base as their apolar moiety are described. These compounds were shown to possess a free amino group and a net positive charge by derivatization with specific amino reagents and by cation exchange column chromatography. The presence of long chain base was demonstrated by periodate-NaB[³H]₄ treatment to yield long chain alcohol and by [³H]-dansylation followed by hydrolysis to yield dansyl-long chain base.     

Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6'-OH-O-dma, and cis-4-OH-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds.

The metabolic products of daidzein and genistein, the principal isoflavones of soy, were examined. Six volunteers included soy into their normal diet for a 2-week period and urine samples were analyzed before and after soy consumption. Isolation and characterization of the urinary metabolites were carried out with absorption chromatography on Sephadex LH-20 and gas chromatography-electron ionization mass spectrometry (GC-EIMS). The structures of the isoflavones isolated were confirmed by using authentic reference compounds. Dihydrogenistein, 6'-OH-O-desmethylangolensin, and cis-4-OH-equol were identified, in addition to known isoflavonoids daidzein, genistein, glycitein, and the known metabolites equol, O-desmethylangolensin, and dihydrodaidzein, by comparing the retention times and the spectra of the urinary compounds with those of the synthesized reference standards. The mammalian lignans enterolactone and enterodiol were also identified. Derivatization of the isoflavones for GC-MS was examined by comparing two silylating reagents, N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and pyridine:hexamethyldisilazan:trimethylchlorosilane (QSM), both used for the derivatization of these compounds. The silylation experiments revealed significant differences in the compositions of the derivatization products. Some corrections were made concerning the earlier published data of dihydrogenistein and 6'-OH-O-dma.     

Array synthesis of progesterone receptor antagonists: 3-aryl-1,2-diazepines

New non-steroidal chemotypes are required for the development of drugs targeting the steroid hormone receptors. The parallel array synthesis of 3-aryl-1,2-diazepines employing solid-supported reagents is described. The resulting compounds demonstrated high affinity binding to the progesterone receptor.