Pre-column Chiral Derivatization Research Articles

Determination of d- and l-Amino Acids in Garlic Foodstuffs by Liquid Chromatography–Tandem Mass Spectrometry

Black garlic is currently attracting interest as a health food and constituent of commercial supplements; however, no data regarding the d-amino acids within black garlic have been reported. Therefore, the amino acid compositions of methanol extracts from fresh and black garlic were compared herein. We investigated the contents of the d- and l-forms of amino acids in commercial fresh, black, and freeze-dried garlic foodstuffs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pre-column chiral derivatization reagent, succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl-5-oxoimidazolidin-4-yl) acetate. Several d-amino acids, namely, the d-forms of Asn, Ala, Ser, Thr, Glu, Asp, Pro, Arg, Phe, Orn, Lys, and Tyr, were observed in the methanol extract of black garlic, whereas only d-Ala was detected in that of fresh garlic foodstuffs. These data suggest that several d-amino acids can be produced during fermentation for preparing black garlic.

Determination of γ-D-glutamyl-meso-diaminopimelic acid in rumen fluid of dairy cows by pre-column chiral derivatization-HPLC

High concentrate (HC) diet feeding leads to the lysis of rumen microbes and the release of hazardous metabolites, which can trigger inflammatory responses, thereby impairing dairy cow health and production. γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which constitutes the peptidoglycan (PGN) layer of bacteria, is the minimum PGN structure capable of activating inflammatory signaling pathways. This research paper aimed to determine the iE-DAP concentration and investigate the effects of an HC diet on the concentration of iE-DAP in the rumen fluid of dairy cows. However, there are limited studies on the determination of iE-DAP concentration. Hence, we established a high-performance liquid chromatography (HPLC) method combined with pre-column chiral derivatization to detect the concentration of iE-DAP in rumen fluid. Moreover, we conducted an animal experiment that included 12 lactating Holstein cows, which were randomly divided into a low-concentrate (LC) group and an HC group. The results showed that the linear range of iE-DAP was 5–500 µg/mL and that the intra- and inter-day RSDs were lower than 7%. Meanwhile, this method was successfully applied to the analysis of iE-DAP in rumen fluid, and the results revealed that long-term feeding with an HC diet elevated the concentration of iE-DAP in rumen fluid of dairy cows.

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization.

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization.

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization.

Ammuxetine (AMT), a novel chiral antidepressant candidate compound, exhibits better antidepression effects than duloxetine in different animal models. In this article, a chiral derivatization method, combined with online solid phase extraction (online SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed for the chiral separation of AMT enantiomers after administration of racemic AMT to dogs. The derivatization reaction employed 2,3,4,6-tetra-O-acetyl-b-glucopyr-anosyl isothiocyanate (GITC) as a precolumn chiral derivatization reagent. A SPE column Retain PEP Javelin (10×2.1mm) was used to remove proteins and other impurities in plasma samples. The enantiomeric derivatives were separated on a ZORBAX SB-C18 column (50×2.1mm×3.5μm) with an isocratic elution procedure. The selected multiple reaction monitoring mode of the positive ion was performed and the parent to the product transitions m/z 681.0/543.1 and m/z 687.4/543.1 were used to measure the derivatives of AMT and duloxetine (internal standard) with electrospray ionization. The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The method was applied to a pharmacokinetics study of AMT racemate in dogs. The results suggested that the pharmacokinetic of AMT enantiomers might be stereoselective in dogs.

Resolution of fluorinated aminophosphonic acid enantiomers by precolumn derivatization with following reverse phase liquid chromatography

GRAPHICAL ABSTRACT

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.

Analysis of flurbiprofen, ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug–protein binding in human plasma

A stereoselective reversed-phase high-performance liquid chromatography (HPLC) assay to determine the enantiomers of flurbiprofen, ketoprofen and etodolac in human plasma was developed. Chiral drug enantiomers were extracted from human plasma with liquid–liquid extraction. Then flurbiprofen and ketoprofen enantiomers reacted with the acylation reagent thionyl chloride and pre-column chiral derivatization reagent ( S)-(−)-α-(1-naphthyl)ethylamine ( S-NEA), and etodolac enantiomers reacted with S-NEA using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C 18 (4.6 mm × 250 mm, 5 μm) column with a mixture of acetonitrile–0.01 mol·L −1 phosphate buffer (pH 4.5) (70:30, v/v) for flurbiprofen enantiomers, acetonitrile–0.01 mol·L −1 phosphate buffer (pH 4.5) (60:40, v/v) for ketoprofen enantiomers and methonal–0.01 mol·L −1 potassium dihydrogen phosphate buffer (pH 4.5) (88:12, v/v) for etodolac enantiomers as mobile phase. The flow of mobile phase was set at 0.8 mL·min −1 and the detection wavelength of UV detector was set at 250 nm for flurbiprofen and ketoprofen enantiomers and 278 nm for etodolac enantiomers. The assay was linear from 0.5 to 50 μg·mL −1 for each enantiomer. The inter- and intra-day precision (R.S.D.) was less than 10% and the average extraction recovery was more than 87% for each enantiomer. The limit of quantification for the method was 0.5 μg·mL −1 (R.S.D. < 10%, n = 5). The method developed was used to study the drug–protein binding of flurbiprofen, ketoprofen and etodolac enantiomers in human plasma. The results showed that the stereoselective binding of etodolac enantiomer was observed and flurbiprofen and ketoprofen enantiomers were not.

Determination of Optical Impurity of Pregabalin by HPLC with Pre-Column Chiral Derivatization

A new method for determining the optical impurity of Pregabalin is developed. The method is based on Pregabalin, and its isomers can be derivatized with Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide. These derivated compounds can be separated by an ordinary chromatography column (Inertsil ODS-2.5 microm, 250 mmx4.6 mm i.d.). Phosphoric acid buffer and acetonitrile (55:45, v/v) are used as mobile phase and 1.0 mL/min flow rate at room temperature. The detective wavelength is fixed at 340 nm. The results indicate that the limit of detection of R model optical impurity 1.1x10(-8) g/mL (signal-to-noise=3), accuracy, and repeatability is satisfied. Therefore, the method can be used for the quality control of Pregabalin.

Thiosugars – Derivatization agents for chiral resolution of homoleucines

o-Phthaldialdehyde was used in combination with a series of six thiosugars for the pre-column chiral derivatization of selected primary amino acids. The diastereomers were resolved on a conventional reversed-phase column and with fluorescence detection. A surprisingly effective resolution of 1-isoindolyl-(1-thioglycosides) derived from 1-thio-beta-L-fucose was observed.

Stereoselective determination of demethyl‐ and didemethyl‐citalopram in rat plasma and brain tissue by liquid chromatography with fluorescence detection using precolumn derivatization

A rapid and sensitive HPLC enantioselective method with fluorescence detection was developed to determine (-)-(R) and (+)-(S) enantiomers of the metabolites of citalopram, demethyl- and didemethyl-citalopram in plasma and brain tissue. This assay involves pre-column chiral derivatization with (-)-(R)-1-(1-naphthyl)ethyl isocyanate followed by separation on a normal-phase silica column. The developed liquid-liquid extraction procedure permits quantitative determination of analytes with recoveries ranged between 81 and 88% with intra- and inter-day relative standard deviations less than 10.5%. Linearity was obtained over the concentration range 5-1000 ng/mL and 100-10,000 ng/g for spiked drug-free plasma and brain tissue, respectively, with detection limits lower than 2.1 ng/mL and 42.8 ng/g.

Stereoselective behavior of a novel biodegradable racemic ketoprofen injectable implant in rats

The stereoselectivity of release of ketoprofen (KET) enantiomers from a biodegradable injectable implant containing racemic KET (rac-KET) was investigated in vivo. A pre-column chiral derivatization RP-HPLC method was employed to assay diastereoisomeric derivatives of R- and S-KET. The rac-KET injectable implant, once injected subcutaneously in rats, produced long-lasting plasma levels of S-KET, which were always greater than those of R-KET. The difference in enantiomer concentration was to be related to stereoselective release, due to stereoselective interaction between D,L-PLG in the implant and KET enantiomers, as well as the chiral inversion of KET in vivo. The rac-KET injectable implant provided the sustained release of S-KET with effective plasma levels maintained for about 8 wk after a single injection.

Determination of fluoxetine, norfluoxetine and their enantiomers in rat plasma and brain samples by liquid chromatography with fluorescence detection

Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection ( λ exc = 227 nm, λ em = 305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5–1000 ng ml −1 and 100–40,000 ng g −1 for doped samples, with detection limits of 3.2 and 2.1 ng ml −1 in plasma and 31.5 and 26.1 ng g −1 in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD ( λ exc = 224 nm, λ em = 336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2–1000 ng ml −1 and 20–7000 ng g −1 with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml −1 in plasma and between 3.0 and 8.2 ng g −1 in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV < 9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection ( λ=224 nm) after chiral derivatization. 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 μg/ml for each enantiomer of esmolol and 0.07–8.0 μg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.

Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(−) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid–liquid extraction of esmolol from human plasma, using S-(−)-propranolol as the internal standard, and employed 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-μm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at λ=224 nm with UV detector. The assay was linear from 0.035 to 12 μg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(−)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 μg/ml and the limit of quantification for the method was 0.035 μg/ml (RSD<14%). The reproducibility of the assay was satisfactory.

Enantioselective assay of S(+)- and R(-)-propafenone in human urine by using RP-HPLC with pre-column chiral derivatization

Enantioselective assay of S(+)- and R(-)-propafenone in human urine by using RP-HPLC with pre-column chiral derivatization

Enantioselective assay of S+ - and R- -propafenone in human urine by using RP-HPLC with pre-column chiral derivatization.

The enantioselective assay for S(+)- and R(-)-propafenone (PPF) in human urine that developed in this work involves extraction of propafenone from human urine and using S(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-beta-D-glucopranosyl isothiocyanate, and quantitation by an RP-HPLC system with UV detection (lambda=220 nm). A baseline separation of propafenone enantiomers was achieved on a 5-microm reverse phase ODS column, with a mixture of acetic acid (25:12:0.02,v/v) as mobile phase. There was good linear relationship from 24.9 ng/ml to 1875.0 ng/ml for both of enantiomers. The regression equations of the standard curves based on C(S-PPF) (or C(R-PPF)) versus ratio of A(S-PPF)/A(S) (or A(R-PPF)/A(S)) were y=0.0032x-0.081, (r=0.999) for S-PPF and y=0.0033x+0.0039, (r=0.998) for R-PPF, respectively. The method's limit of detection was 12.5 ng/ml for both enantiomers, and the method's limit of quantitation was 28.2+/-0.52 ng/ml for S-PPF, 30.4+/-methanol:water:glacial 0.53 ng/ml for R-PPF (RSD<8%, n=5). The analytical method yielded average recovery of 98.9% and 100.4% for S-PPF and R-PPF, respectively. The relative standard deviation was no more than 6.11% and 6.22% for S-PPF and R-PPF, respectively. The method enabled study of metabolism of S(+)- and R(-)-propafenone in human urine. The results from 7 volunteers administered 150 mg racemic propafenone indicated that propafenone enantiomers undergo stereoselective metabolism and that in the human body, S(+)-propafenone is metabolized more extensively than R(-)-propafenone.

Enantioselective assay of S(+)- and R(-)-propafenone in human urine by using RP-HPLC with pre-column chiral derivatization

Enantioselective assay of S(+)- and R(-)-propafenone in human urine by using RP-HPLC with pre-column chiral derivatization

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.

Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.

HPLC enantioseparation of amino acids and their protected derivatives used in peptide synthesis with pre-column chiral derivatization

Indirect chiral separation methods based on enantiomeric derivatizations were developed in order to monitor optical purity of uncoded amino acids and a new series of amino acid derivatives. Marfey's reagent was used for derivatization of amino groups: whilst boxyl groups were derivatised with (1R, 2R)-or (1S, 2S)-2-amino-1(4-nitrophenyl)-1,3-propanediol reagents were used, respectively. The separations of diastereomeric derivatives formed via derivatization were optimized in RP-HPLC and NP-HPLC systems.