Separation of amino acids. Possibilities offered by optimized associations of low-voltage electrophoresis and chromatography on finely textured paper and on cellulose thin-layers

Summary Three optimized small-scale methods for amino acid separation are described and compared. Two dimensional separations are based on paper or cellulose thin-layer ionophoresis and chromatography with simple and inexpensive apparatus. The influence of experimental conditions on resolution and amount required for amino acids possessing almost identical mobilities, is studied. The quickest procedure based on the coupling of 200×200×0.25 mm thin-layer, low-voltage electrophoresis (20 V/cm) with chromatography, and the system: cellulose −1.04 mol dm⁻³ formic acid — tertbutanol/methanol/pyridine/formic acid/water (33∶43∶9.6∶0.4∶20), provides very reproducible results with high separating power for small samples (2–10 nmoles).     

New chromogenic reagents for labelling amino groups in peptides. Chromatographic and preparatives aspects

Summary A new type of chromogenic reagent for labelling the amino groups of peptides or amino acids, 5-substituted 2,4-dinitrofluorobenzenes, is presented. These reagents are characterized by a polarity determined by the function included in the substituent in the 5-position and by the nature of this substituent. Preparation and chromatographic behaviour of these reagents, of eventual by-products of reactions and of some N-(2,4-dinitro-5-diethylaminophenyl)-peptides or amino acids are described.Proportions in solvent mixtures are v/v except otherwise indicated. Abbreviations: lit=literature, TLC=thin-layer chromatography, TLE=TL electrophoresis, see also: Figs. 2, 3, 5.     

The two-dimensional characterization of amino acids in actinomycin hydrolysates.

Actinomycins, which belong to a family of chromopeptide antibiotics, consist of a hetero-tricyclic chromophore to which are attached two pentapeptide chains either identical or different in amino acid sequence. The classification of existing actinomycins and the identification of new actinomycins are dependent on the characterization and quantitation of the amino acids present in the peptide chains. A simple, fast and highly reliable two-dimensional separation technique employing electrophoresis in a formic acid/acetic acid buffer (pH 1.9) on thin layers of microcrystalline cellulose followed by thin-layer chromatography with an n-butanol:water:glacial acetic acid (50:40:10) solvent system in a direction perpendicular to the electrophoresis was developed to separate the amino acids contained in hydrolysates of the actinomycins. The separated amino acids were identified by two parameters, the chromatographic Rf value and the electrophoretic mobility calculated relative to some standard migrating compound.     

Lipophilicity determination of some fully protected peptides and amino acids

Summary The lipophilicity of 21 fully protected peptides and amino acids was determined by reversed-phase thin-layer chromatography. The R_M values decreased linearly with growing methanol concentration of the eluent. The sequence and conformation of molecular substructures did not significantly influence the lipophilicity. The presence of salt and ammonia in the eluent had a negligible impact on retention; the effect of the pH value was also low. In the presence of 1M and 2M acetic acid the retention of each compound considerably decreased. Acetic acid also changed markedly the selectivity. Our data suggest that the acetic acid has a preponderant role in changing the retention and selectivity of fully protected peptides and amino acids in reversedphase thin-layer chromatography.     

Associations de chromatographie et d'électrophorèse sur couche mince de celllose amorphe pour la séparation des aminoacides. Influence des équilibires ioniques et des constituants des solvants sur les séparations chromatographiques dans le cas des solvants du type alcool/acide/eau

Résumé Des associations d'électrophorèse et de chromatographie dans des milieux acides sur couche mince de cellulose à fibres courtes sont étudiées: acide formique 1 M.—tertbutanol ou tert-pentanol/éthanol/acide formique/eau (40:38:2:20, v/v) ou propanol/éthianol/acid formique/eau (35:35:5:20). Les relations existant entre le pouvoir séparateur et les constituants (alcools) des solvants ou les équilibres ioniques en chromatographie sont examinés. On discute des problèmes relatifs à l'optimisation des méthodes et des différences de pouvoir séparateur existant entre les nouveau procédés et les procédés antérieurement décrits.

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Coupling of electrophoresis and chromatography on amorphous cellulose thin layers for the separation of amino acids. Influence of ionic equilibria and solvent components on chromatographic separations with alcohols/acid/water solvents

Summary Associations of electrophoresis and chromatography in acidic media on thin layers of cellulose with short fibres, are studied: 1 mol. dm^–3 formic acid-tert. butanol or tert, pentanol/ethiano/formic acid/water (40:38:2:20, v/v) or propanol/ethano/formic acid/water 935:35:5: 20). The relationship between separation power and solvent components (alcohols) or ionic equilibria in chromatography is examined. Optimization of emthods and origins of differences in separation power between niew and previously described procedures are discussed

     

Utility of nonaqueous capillary electrophoresis for the determination of lidocaine and its metabolites in human plasma: a comparison of ultraviolet and mass spectrometric detection

A nonaqueous capillary electrophoresis/electrospray mass spectrometry method for the separation of lidocaine (LID) and two of its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), has been developed. The separation medium was: 70 mM ammonium formate and 2.0 M formic acid in acetonitrile/methanol (60:40 v/v). With a sheath liquid of methanol/water (80:20 v/v) containing 2% formic acid and positive ion detection, reproducible determinations (8-11% relative standard deviation (RSD)) of lidocaine and its metabolites were performed in spiked human plasma. The limits of detection (LODs) were between 69.1 and 337 nM. The influences of sheath liquid composition, nebulizing gas pressure and drying gas temperature on the separation were examined.     

Determination of rosiglitazone and metformin in human serum by CE-ESI-MS

A new method for the determination of anti-diabetic drugs metformin and rosiglitazone based on the use of capillary electrophoresis with electrospray mass spectrometry was developed. The proposed method allowed their separation within 11 min by using 50 mM formic acid at +20 kV. Positive electrospray ionization and selected ion monitoring [M+H](+) of metformin (m/z=130) and rosiglitazone (m/z=358) were performed. Several important experimental parameters influencing electrospray ionization of metformin and rosiglitazone were studied. The final composition of sheath liquid was water/methanol/formic acid (50:49.5:0.5, v/v/v), at a flow rate of 2 μL/min. The developed method was applied for the determination of metformin and rosiglitazone simultaneously in human serum after protein precipitation with acetonitrile. The limits of detection of developed method were 4.42 and 2.14 ng/mL for rosiglitazone and for metformin, respectively, which is sufficient for therapeutic serum concentration levels monitoring for both studied drugs.     

Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection

Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 micromol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.     

Analysis of native amino acid and peptide enantiomers by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry was used for the separation and detection of amino acid and peptide enantiomers. With detection limits as low as 250 pg, 25 amino acids enantiomers were baseline resolved on a Chirobiotic T chiral stationary phase. APCI demonstrated an order of magnitude better sensitivity over electrospray ionization (ESI) for free amino acids and low molecular mass peptides at the high LC flow-rates necessary for rapid analysis. As the peptide chain length increased (peptides with M(r) > or = 300 Da), however, ESI proved to be the more ideal atmospheric pressure ionization source. A mobile phase consisting of 1% (w/w) ammonium trifluoroacetate in methanol and 0.1% (w/w) formic acid in water increased the sensitivity of the APCI method significantly. A step gradient was then used to separate simultaneously all 19 native protein amino acid enantiomers in less than 20 min using extracted ion chromatograms.     

Some effective solvents for rapid and reliable characterization of phenylthiohydantoin-amino acids

Summary One-dimensional chromatography with internal standards permits reliable identification of the phenylthiohydantoins from all the common amino acids with the following TLC systems: silica gel — chloroform/n-butyl acetate (9010), di-isopropylether/ethanol (955), dichloromethane/ethanol/acetic acid (9082) ortrans-dichloroethylene/ethanol/acetic acid (88102); and cellulose with 25% formic acid — heptane/isobutanol/75% fromic acid (40309) or silica gel — chloroform/ ethanol/acetic acid/water (50470.52.5).Abbreviations: PTH=phenylthiohydantoin, TLC=thin-layer chromatography, HPTLC=high-performance TLC; other abbreviations: see end of text. Proportions in solvent mixtures are v/v except where otherwise indicated.     

Characterization of protein hydrolysates of cosmetic use by CE-MS

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid--pH 1.8--in 70:30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75:25-25:75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.     

Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.     

The effects of pH, ionic strength and buffer concentration of mobile phase onR F of acidic compounds in ion-pair TLC

Summary The effects of some factors important in ion-pair, high-performance liquid chromatography were studied in ion-pair, thin-layer chromatography. Tetramethyl and cetyltrimethylammonium salts were used as ion-pairing reagents. As stationary phases, silica gel and chemically bonded reversed-layers (C₁₈) were used. Layers were impregnated with ion-pairing reagent prior to chromatography. In some cases the stationary phase was treated with buffer at different concentrations. The mobile phase contained methanol and water, in one set of experiments buffer, salt for adjustment of ionic strength and ion-pairing reagent were added. The migration behaviour of different benzoic acids was studied. Several problems of ion-pair thin-layer chromatography are discussed. Passed away on 13^th of April, 1998 Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Fast analysis of quaternary ammonium pesticides in food and beverages using cation‐exchange chromatography coupled with isotope‐dilution high‐resolution mass spectrometry

A fast separation based on cation‐exchange liquid chromatography coupled with high‐resolution mass spectrometry is proposed for simultaneous determination of chlormequat, difenzoquat, diquat, mepiquat and paraquat in several food and beverage commodities. Solid samples were extracted using a mixture of water/methanol/formic acid (69.6:30:0.4, v/v/v), while liquid samples were ten times diluted with the same solution. Separation was carried out on an experimental length‐modified IonPac CS17 column (2 × 15 mm²) that allowed the use of formic acid and acetonitrile as mobile phase. Detection limits for food and beverage matrices were established at 1.5 μg/L for chlormequat, difenzoquat and mepiquat, and 3 μg/L for diquat and paraquat, while for drinking water a pre‐analytical sample concentration allowed detection limits of 9 and 20 ng/L, respectively. Precision, as repeatability (RSD%), ranged from 0.2 to 24%, with a median value of 6%, and trueness, as recovery, ranged from 64 to 118%, with a median value of 96%. The method developed was successfully applied to investigate the presence of herbicide residues in commercial commodities (mineral water, orange juice, beer, tea, green coffee bean, toasted coffee powder, cocoa bean, white corn flour, rice and sugar samples).     

Thin-layer chromatographic fingerprinting of the nonvolatile fraction extracted from the medicinal herb Cistus incanus L.

ABSTRACT

The aim of this study was to provide the first from-start-to-end thin-layer chromatographic method of fingerprinting the Cistus incanus L. raw herbal material, with a purpose to further use it for rapid screening, authentication, and quality control of the traded C. incanus L. herbs. To this effect, 12 different C. incanus L. samples purchased as herbal teas from a local market were extracted by means of the accelerated solvent extraction (ASE) with chemometrically optimized solvent extraction mixture and temperature (methanol–water, 27:73, v/v; 130°C), to derive the polar fraction from the plant samples. Then, the extracts were developed in two thin-layer chromatographic systems, both using the commercially precoated silica gel 60 chromatographic plates, yet two different mobile phases (mobile phase 1, ethyl acetate–formic acid–acetic acid–water, 100:11:11:13, v/v/v/v, and mobile phase 2, ethyl acetate–dichloromethane–formic acid–acetic acid–water, 100:10:10:10:11, v/v/v/v/v). The chromatograms were densitometrically scanned in the reflectance mode at the wavelength λ?=?366?nm to obtain fingerprints of the extracts derived from individual C. incanus L. samples. Mobile phase 2 performed slightly better, because with its use, the maximum number of 11 peaks could be seen in the respective fingerprints, whereas with mobile phase 1, the maximum number of 10 peaks only. Then an antioxidant potential of the investigated herbal extracts was assessed, making use of mobile phase 2 and the 0.20% methanol solution of 2,2-diphenyl picrylhydrazyl as a visualizing reagent. The resulting chromatograms were densitometrically scanned in the extinction mode at the wavelength λ?=?550?nm to obtain biological fingerprints of the extracts. Finally, chromatographic and biological fingerprints underwent a semiquantitative evaluation in terms of the contents of the extracted polar fraction and an overall antioxidant potential of the individual plant species.     

Determination of Captopril in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

Abstract

In this study, a fast and sensitive liquid chromatography/tandem mass spectrometry method for the determination of captopril in human plasma was developed and validated. The analyte and enalaprilat, used as the internal standard, were extracted from plasma using methanol directly precipitate protein. Analysis was performed on a Lichrospher CN column with water (containing 0.1% formic acid) and methanol (60∶40, v/v) as the mobile phase. Linearity was assessed from 6.25 to 800 ng/ml in plasma. The analytical method proved to be applicable in a pharmacokinetic study of captopril after oral administration of 20 mg captopril tablet to 20 healthy volunteers.     

Isolation of three glucaric acids from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography

The isomerism of glucaric acids and the complexity of the composition of Leonurus japonicus Houtt. increased the difficulty of the separation of glucaric acids from the herb. In the present study, three glucaric acids were isolated from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography. Cation exchange resin chromatography was applied to remove the alkaloids and enrich the glucaric acid fractions. Preliminary separation of the glucaric acid extract by high-speed countercurrent chromatography was carried out at 45℃ by using an optimized solvent system of ethyl acetate/n-butanol/formic acid/water (1:1:0.01:2, v/v/v/v) with satisfied stationary phase retention and separation factor. The semi-preparative high-performance liquid chromatography was used for further separation and purification of the target fractions, and three monomeric compounds were obtained with purities of 90.0, 91.0, and 95.3%. UV spectroscopy, NMR spectroscopy, and mass spectrometry were employed to identify their structures, which were assigned as 2-syringyl glucaric acid, 2,4-disyringyl glucaric acid, and 3,4-disyringyl glucaric acid, respectively, and 2,4-disyringyl glucaric acid was reported for the first time.     

Amino acid analysis of proteins and peptides by thin-layer electrophoresis in association with chromatography

Summary A rapid and simple method for the qualitative determination of the amino acid composition of proteins and peptides by thin-layer electrophoresis in combination with thin-layer chromatography has been developed which permits a map with the separation of 18–20 amino acids to be obtained in 3–4 hr.Khimiya Prirodnykh Soedinenii, Vol. 2, No. 5, pp. 348–354, 1966     

Determination of 8-hydroxyquinoline sulfate in tuberculin solutions by planar and high performance liquid chromatography

Summary TLC and HPLC methods for the determination of the preservative, 8-hydroxyquinoline sulfate in PPD-T tuberculin solution were developed. The planar chromatography method involved separation of 8-hydroxyquinoline sulfate on a TLC plate using a butyl-acetate: formic acid: 2-propanol mobile phase, detection and quantitation by densitometric scanning. The HPLC method was on a LiChrosorb RP-18 column with acetonitrile-water (65:35 v/v) mobile phase, adjusted to pH 3.05 by phosphoric acid. Linearity, reproducibility and accuracy were found to be satisfactory. Under selected conditions, the limit of detection (LOD) of both methods was similar-about 25 ng. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism

A high performance liquid chromatography (HPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of fifteen glucose, or acetate derived metabolites isolated from tumor cells. Glycolytic and tricarboxylic acid (TCA) cycle metabolites as well as acidic amino acids were separated on a HPLC porous graphitic carbon (PGC) column and simultaneously determined by means of triple quadrupole MS/MS using multiple reaction monitoring (MRM). Target compounds were eluted within 10 min with 8% v/v formic acid as an electronic modifier added to a 4:1 v/v methanol water mobile phase. The calibration is linear in the 1–100 μM concentration range for each analyte. The limit of detection ranges between 0.39 and 2.78 μM for the analytes concerned. To test the PGC–HPLC–MS/MS method in metabolomic studies, ZR-75.1 human mammary adenocarcinoma cells were labeled with U-¹³C glucose or 1-¹³C acetate. Applying the MRM mode, the incorporation of ¹³C into metabolites, isolated from the tumor cells, and derived from glucose or acetate, could be properly identified.