Direct determination of enantiomeric purity of FMOC amino acids with high performance liquid chromatography

Summary Analytical methods are described which allow a direct determination of enantiomeric purity of seventeen FMOC amino acids commonly used in peptide synthesis. The corresponding ester derivatives can be separated directly on cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). The methods are suitable for primary as well as secondary FMOC amino acids. The presence of a highly sensitive fluorescence moiety within the molecule, in combination with large separation factors (-values between 1.5–2.2) allowed for a general detection limit below 0.05%. In several cases the antipode has been determined in the ppm-range. An interesting result has been observed with respect to the elution order of the FMOC amino acid esters. The elution order of the Trp enantiomers is opposite to that obtained with the other amino acids. This is contrary to the generally held belief that elution order is identical within a homologous series of racemates when chromatographed under identical conditions on the same chiral stationary phase. In addition, the inversion of elution of the Pro enantiomers depending on the estertype indicates a competition of different separation mechanisms.     

Single-step analysis of low abundance phosphoamino acids via on-line sample preconcentration with chemical derivatization by capillary electrophoresis

New strategies for rapid, sensitive and high-throughput analysis of low abundance metabolites in biological samples are required for future metabolomic research. In this report, a direct method for sub-micromolar analyses of phosphoamino acids was developed using on-line sample preconcentration with 9-fluorenylmethyloxycarbonyl chloride (FMOC) derivatization by capillary electrophoresis (CE) and UV detection. Analyte focusing by dynamic pH junction and FMOC labeling efficiency were influenced by several experimental factors including buffer pH, ionic strength, sample injection length and FMOC concentration. About a 200-fold enhancement in concentration sensitivity was achieved under optimal conditions relative to conventional off-line derivatization, as reflected by a detection limit (S/N approximately 3) of 0.1 microM. In-capillary sample preconcentration with chemical labeling by CE offers a unique single-step analytical platform for high-throughput screening of low abundance metabolites without intrinsic chromophores.     

Liquid chromatographic enantiomer resolution of N-fluorenylmethoxycarbonyl alpha-amino acids and their ester derivatives on polysaccharide-derived chiral stationary phases

The liquid chromatographic enantiomer separation of N-fluorenylmethoxycarbonyl (FMOC) protected alpha-amino acids and their ethyl ester derivatives was performed on polysaccharide-derived chiral stationary phases, Chiralcel OD, Chiralpak AD, and Chiralpak AS. In general, Chiralcel OD and Chiralpak AD showed good performance for resolution of N-FMOC alpha-amino acids and their ethyl esters, respectively. All investigated N-FMOC alpha-amino acid enantiomers were baseline separated on Chiralcel OD or Chiralpak AD, whereas N-FMOC alpha-amino acid ethyl ester enantiomers were baseline resolved (alpha = 1.15-3.03) on Chiralpak AD, except for two analytes. The L-enantiomers of all examined FMOC alpha-amino acid ethyl ester derivatives are preferentially retained on Chiralpak AD, while the elution orders of the other enantiomer separations are not consistent.     

Detection and determination of N-nitrosamino acids by thin-layer chromatography using fluorescamine

A novel procedure is described for the detection and determination of N-nitrosamino acids (NAAs) on activated silica gel thin-layer chromatographic plates. N-Nitrososarcosine, N-nitrosoproline, and N-nitroso-4-hydroxyproline could be detected as fluorophors at the 200-pmole level (20-30 ng) after being irradiated with ultraviolet light and sprayed with fluorescamine reagent. Spectrophotometric determination of the relative fluorescence of 0.4-40 nmoles of NAAs gave rise to similar calibration curves when plotted on a log-log scale. An application of this method to the detection of NAAs in uncooked bacon is described.     

Chiral separation of halogenated amino acids by ligand-exchange capillary electrophoresis

The chiral separation of halogenated amino acids by ligand-exchange CE is described. Halogenated amino acids attracted increasing interest in recent years because of their physiological activities. Different chiral selectors, as there are L-4-hydroxyproline, L-histidine, and N-alkyl derivatives of L-4-hydroxyproline in form of their copper(II) complexes, are compared for their chiral recognition ability for halogenated amino acids. The influence of various parameters, such as selector concentration, pH, organic modifier, and field strength, on the resolution was investigated. All halogenated amino acids investigated were baseline-separated under optimized conditions.     

Quantitation of amino acids in plasma by high performance liquid chromatography: Simultaneous deproteinization and derivatization with 9-fluorenylmethyloxycarbonyl chloride

This paper, as a novelty to this field, presents the deproteinization and derivatization of plasma's free amino acids (PFAAs), simultaneously, in a single step, with the acetonitrile (ACN) containing 9-fluorenylmethyloxycarbonyl chloride (FMOC) reagent. Deproteinization and derivatization, were studied with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection, simultaneously. Model investigations have been carried out as a function of the FMOC concentration, reaction time and reaction conditions: with standard solutions, with human plasma samples in its initial condition and fortified with standard amino acids (excluding tryptophan because it co-elutes with the hydrolyzed FMOC). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 3.0 mM FMOC concentration, at pH 9; derivatization time = 20 min), and characterized with the relative standard deviation percentages of their responses (≤4.4%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 2.5 pmol, except for cystine, ornithine (5 pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10 pmol).     

氨基酸和多肽在碱性硅胶载体上的固相衍生研究

为进行复杂体系中痕量生理活性物质 (如氨基酸和多肽等 )的高灵敏度分析 ,往往需要对其进行柱前或柱后的荧光衍生 -高效液相色谱或毛细管电泳分析 [1] .在一个存在着竞争反应的体系中 ,为保证样品有足够的反应产率 ,往往需使衍生试剂过量很多 ,这就使得衍生后的样品中必然含有高浓度的衍生试剂及其水解后形成的副产物 ,从而大大地干扰了分离与分析 .为解决这一问题 ,通常可采取溶剂萃取[2～ 5] 或加入 1 -金刚烷胺 [6 ,7] 或羟胺 [8,9] 等方法除去过量试剂 .但这些额外的处理使衍生方法更加烦琐 ,有时还导致收率的降低 .也有使用固相化的衍…     

On-column labeling technique and chiral CE of amino acids with mixed chiral selectors and UV detection

A novel method has been developed for the on-column labeling of amino acid enantiomers with 9-fluoroenylmethyl chloroformate (FMOC), followed by chiral CE with a binary chiral selector system and UV detection. Efficient labeling was achieved by sequential injection of amino acids, borate buffer, and FMOC labeling solution at 0.2 psi for 6 s. After injection, the sandwich sections were electrically mixed at 250 V/cm for 6 s and allowed to react (electric field-free) at room temperature for 2 min. With this procedure, successful online-labeling and chiral CE separation of 19 pairs of amino acids (AA) have been conducted, giving 17 pairs fully enantioresolved (R(s) = 1.73-5.79) and two pairs partially resolved (Ala, R(s) = 0.39 and Arg, R(s) = 1.15) using a running buffer of 150 mM borate containing 30 mM beta-CD, 30 mM sodium taurodeoxycholate (STDC), and 15% isopropanol (IPA) at pH 9.0. Chiral CE of some mixed pairs was also demonstrated, much the same as using precolumn labeling. Surprisingly, Met, Asp, Asn, Gln, and His gained even higher enantioresolution (up to 2.5%) compared with the case of precolumn labeling. As validated by both artificially prepared solutions and serum samples, the method was applicable to the quantitative determination of AA, with LODs down to 4.0 microM. The method allowed the determination of D-AA at the ratio of 1:100 (D:L).     

Chiral separation of amino acids and peptides by capillary electrophoresis

Chiral separation of amino acids and peptides by capillary electrophoresis (CE) is reviewed regarding the separation principles of different approaches, advantages and limitations, chiral recognition mechanisms and applications. The direct approach details various chiral selectors with an emphasis on cyclodextrins and their derivatives, antibiotics and chiral surfactants as the chiral selectors. The indirect approach deals with various chiral reagents applied for diastereomer formation and types of separation media such as micelles and polymeric pseudo-stationary phases. Many derivatization reagents used for high sensitivity detection of amino acids and peptides are also discussed and their characteristics are summarized in tables. A large number of relevant examples is presented illustrating the current status of enantiomeric and diastereomeric separation of amino acids and peptides. Strategies to enhance the selectivity and optimize separation parameters by the application of experimental designs are described. The reversal of enantiomeric elution order and the effects of organic modifiers on the selectivity are illustrated in both direct and indirect methods. Some applications of chiral amino acid and peptide analysis, in particular, regarding the determination of trace enantiomeric impurities, are given. This review selects more than 200 articles published between 1988 and 1999.     

分离检测生物活性物质的荧光标记试剂与分子探针及其应用

作者结合自己的研究工作主要评述了用于分离检测氨基化合物（氨基酸、肽、蛋白质和生物胺等）、巯基化合物（谷胱甘肽、半胱氨酸和高半胱氨酸等）及NO等生物活性物质的荧光标记试剂和荧光分子探针的近期进展和应用。除了传统的的OPA、NDA、DNS、FMOC、FITC、NBD-F、AQC、Cy5等在HPLC和CE分离荧光检测应用新进展,还介绍了许多新的荧光分子探针和标记试剂的性能和应用。它们是N－羟基琥珀酰亚胺活性酯荧光标记试剂,6-氧-（N-琥珀酰亚胺乙酸酯）-9-（2'-甲氧羰基）荧光素（SAMF）,1,3,5,7-四甲基-8-苯基-（4'-O-（N-琥珀酰亚胺乙酸酯））-二氟化硼-二吡咯甲烷（TMPAB-Osu,）等和3-碘乙酰胺苯嵌蒽酮荧光探针及MCY5、DSTCY、DCDSTCY和DCTCY等花菁类近红外荧光探针等。     

Chiral separation of amino acids by ligand-exchange capillary electrochromatography using continuous beds

A chiral ligand-exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one-step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica-based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.     

Enantioseparation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids and other zwitterionic compounds on cinchona-based chiral stationary phases

The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit from Waters) is a commercial N-terminal label for proteinogenic amino acids (AAs), designed for reversed-phase separation and quantification of the AA racemates. The applicability of AQC-tagged AAs and AA-type zwitterionic compounds was tested for enantiomer separation on the tert-butyl carbamate modified quinine and quinidine based chiral stationary phases, QN-AX and QD-AX employing polar-organic elution conditions. The investigated test analytes included the enantiomers of the positional isomers of isoleucine (Ile), threonine, homoserine, and 4-hydroxyproline. Furthermore, β-AAs, cyclic, and heterocyclic AAs including trans-2-amino-cyclohexane carboxylic acid and trans-2-aminocyclohexyl sulfonic acid, phenylalanine derivatives substituted with halides with increasing electronegativity and 3,4-dihydroxyphenylalanine, cysteine-related derivatives including homocysteic acid, methionine sulfone, cysteine-S-acetic acid, and cysteine-S-acetamide as well as a small range of aminophosphonic acids were enantioseparated. A mechanistic interaction study of AQC-AAs in comparison with fluoresceine isothiocyanate-labeled AAs was performed. The chiral and chemoselective recognition processes involved in enantiomer separation and retention was systematically discussed. Special emphasis was set on the influential factors exhibited by the chemistry, branching position, and spatial properties of the investigated zwitterionic analytes. The general interest to separate and distinguish between different types of branched-chained AAs and metabolic side products thereof lies in the toxicity of some of these compounds, which makes for instance allo–Ile an attractive candidate in disease-related biomarker research.

Determination of organic acid impurities in lactic acid obtained by fermentation of sugarcane juice

Lactic acid produced by fermentation process mostly contains a number of aliphatic carboxylic acids as impurities. In this work, carboxylic acid impurities in lactic acid samples from a number of sources were determined at ppm levels. A simple HPLC method was developed that utilized a new generation polar embedded reverse phase, 20mM phosphate buffer at pH 2.20 (±0.05) and UV detection at 210 nm. The method enabled quantitative analysis of the above acids in lactic acid matrix. The experimental conditions for column temperature, mobile phase pH and flow rate were optimized. A detailed validation of the method was performed for linearity, precision, accuracy, selectivity, limit of detection (LOD), limit of quantitation (LOQ), ruggedness and repeatability and reproducibility (R&R).     

Determination of organic acids in air by capillary electrophoresis and ion-exclusion chromatography

Summary A capillary electrophoretic (CE) method for the determination of organic acids in the low ppm range is described. The buffer consisted of 5 mM 2,6-pyridinedicarboxylic acid and 0.5 mM cetyltrimethylammonium bromide, pH 5.6. The former served as background electrolyte for indirect UV detection at 200 nm, whereas the latter was used to reverse electroosmotic flow. In <5 min 8 low molecular mass organic acids (oxalic, formic, malonic, glutaric, glycolic, acetic, lactic and propanoic) and two inorganic acids (hydrochloric and sulphuric) were separated. Detection limits for anions tested were 0.04 mg L⁻¹ (lactic acid) to 0.6 mg L⁻¹ (malonic acid). The method was applied to the determination of organic acids in air samples. The CE results when compared with ion-exclusion chromatography (IEC) agreed well. The use of electrokinetic injection in CE proved beneficial for preconcentration of organic acids in real samples. Using electrokinetic injection, preconcentration factors ranging from 14 (hydrochloric acid) to 3 (propanoic acid) were obtained. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

High-performance liquid chromatography of biogenic amines, derivatized with 9-fluorenylmethyl chloroformate

A procedure was elaborated for the analysis of three biogenic amines posing a considerable health hazard. The method takes advantage of the characteristics of the 9-fluorenylmethyl chloroformate (FMOC) derivative, namely specificity, stability and compatibility for either fluorescence or UV-absorbance detection. The FMOC-tyramine derivative was probably adsorbed to labware when acetone served as the solvent for FMOC. Methanol, substituted for acetone, removed this problem. Excellent linearity was obtained with standard solutions of tyramine, tryptamine and phenylethylamine. Meat samples, spiked with the mentioned amines, also showed good linearity. Perchloric acid was chosen for deproteinization, as potassium perchlorate may be eliminated on neutralization. Histamine failed to react with FMOC or was not detected under the test conditions.     

HPLC electrochemical fluorometric detection of amino acids including tryptophan using 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole

It has been shown that by electrochemical oxidation 7-nitrobenzo-2-oxa-1,3-diazole-tryptophan (NBD-T) is converted to fluorophores having the same emission and excitation spectra as those for other NBD-amino acids. NBD-dioxindolylalanine was tentatively assumed to be a main electrochemical oxidation product of NBD-tryptophan. A coulochemical cell placed between an analytical column and a fluorometer showed no detrimental effect on the separation of NBD-amino acids by reversed phase HPLC. Highly sensitive fluorescence detection was achieved for amino and imino acids at 10-100 fmol levels. The detection limit for tryptophan was 50 fmol.     

High-performance liquid chromatographic method for the determination of prolyl peptides in urine

A rapid and accurate method is described for the determination of prolyl peptides in urine, with specific reference to the dipeptide prolylhydroxyproline, and free hydroxyproline and proline. Free amino acids and peptides were isolated from urine on cation-exchange minicolumns, and free imino acids and prolyl-N-terminal peptides were selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of amino acids and N-terminal aminoacyl peptides with o-phthalaldehyde. The highly fluorescent adducts of imino acids and prolyl peptides were separated on a Spherisorb ODS 2 column by isocratic elution for 12 min using as mobile phase 17.5 mM aqueous trifluoracetic acid solution containing 12.5% acetonitrile (eluent A), followed by gradient elution from eluent A to 40% of 17.5 mM aqueous trifluoroacetic acid solution containing 80% acetonitrile in 20 min. Analytes of interest, in particular the dipeptide prolylhydroxyproline, can be easily quantified by fluorimetric detection (epsilon ex = 470 nm, epsilon em = 530 nm) without interference from primary amino-containing compounds.     

Amino acids: aspects of impurity profiling by means of CE

Quality control of active pharmaceutical ingredients (API) is commonly performed by means of HPLC. However, CE offers a suitable alternative, especially for the analysis of easily chargeable substances, i.e., amino acids. The article reviews, on the one hand, CE methods developed for impurity profiling of synthesized amino acid analogs. However, nowadays, production of amino acids/peptides is dominated by fermentation. Therefore, on the other hand, CE methods for the analysis of amino acids and small peptides are reported. The results of CE analysis of glutathione samples according to the monograph in the European Pharmacopoeia (Ph. Eur.) 5.7 and amino acid samples after derivatization with 9-fluorenylmethyl chloroformate (FMOC) and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) may pave the way for impurity profiling of fermentatively produced API by means of CE.     

Chromatographic fingerprints of industrial toluic acids established for their quality control

The chromatographic fingerprints of industrial o-toluic acid, m-toluic acid and p-toluic acid have been established by HPLC-UV detection according to their impurity groups. HPLC separation of all relative substances involved in the groups was developed on a Kromasil C₁₈ column by using methanol-water-NH₄Ac-HAc buffer (100 mM, pH 4.70) 15/65/20 (v/v/v) as the mobile phase at a flow rate of 1.5 mL/min, and detection was operated by UV adsorption at a wavelength of 254 nm. The ultraviolet spectra corresponding to each chromatographic peak were also recorded for further identification of all components. Whether the limits of relative impurities residues in a toluic acid product are qualified or not can be intuitively estimated by analyzing its chromatogram with comparison to the fingerprint. This protocol has successfully provided some Chinese manufacturers with a simple and feasible method for quality control of toluic acids for industrial use.     

Enantioseparation of hydroxy acids on easy-to-prepare continuous beds for capillary electrochromatography

This paper deals with the enantioseparation of hydroxy acids by ligand-exchange capillary electrochromatography. A chiral continuous bed was easily prepared by in situ polymerization of monomers, including an L-4-hydroxyproline derivative. This phase showed chiral recognition for several hydroxy acids, in addition to amino acids.