Simultaneous determination of binary diffusion coefficients from multiple response curves by chromatographic measurements

A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic D-amino acids in the presence of large amounts of their L-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the D-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of D-Leu were found in all tissues and physiological fluids tested (trace-1.3 nmol/g tissue), and in the urine, the presence of high amounts of D-allo-Ile (D-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. D-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual D-amino acid and the potential need to clarify its unique metabolism in mammals.     

A sensitive and simple ultra-high-performance-liquid chromatography-tandem mass spectrometry based method for the quantification of D-amino acids in body fluids

D-Amino acids are increasingly being recognized as important signaling molecules in mammals, including humans. D-Serine and D-aspartate are believed to act as signaling molecules in the central nervous system. Interestingly, several other D-amino acids also occur in human plasma, but very little is currently known regarding their function and origin. Abnormal levels of D-amino acids have been implicated in the pathogenesis of different diseases, including schizophrenia and amyotrophic lateral sclerosis (ALS), indicating that D-amino acid levels hold potential as diagnostic markers. Research into the biological functions of D-amino acids is hindered, however, by the lack of sufficiently sensitive, high-throughput analytical methods. In particular, the interference of large amounts of L-amino acids in biological samples and the low concentrations of D-amino acids are challenging. In this paper, we compared 7 different chiral derivatization agents for the analysis of D-amino acids and show that the chiral reagent (S)-NIFE offers outstanding performance in terms of sensitivity and enantioselectivity. An UPLC-MS/MS based method for the quantification of D-amino acids human biological fluids was then developed using (S)-NIFE. Baseline separation (R(s)>2.45) was achieved for the isomers of all 19 chiral proteinogenic amino acids. The limit of detection was <1 nM for all amino acids except d-alanine (1.98 nM), d-methionine (1.18 nM) and d-asparagine (5.15 nM). For measurements in human plasma, cerebrospinal fluid and urine, the accuracy ranged between 85% and 107%. The intra-assay and inter-assay were both <16% RSD for these three different matrices. Importantly, the method does not suffer from spontaneous racemization during sample preparation and derivatization. Using the described method, D-amino acid levels in human cerebrospinal fluid, plasma and urine were measured.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citratesodium chloride buffer. Enantioseparation is by subsequent injection of 3 l heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Enantioselective micro-2D-HPLC determination of aspartic acid in the pineal glands of rodents with various melatonin contents

Enantioselective determination of aspartic acid (Asp) in the pineal gland of rodents with various melatonin contents was performed using a highly sensitive and selective two-dimensional HPLC system. After derivatization of the amino group with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), NBD-Asp was separated using a capillary monolithic ODS column in the first dimension. The fraction of NBD-Asp was automatically collected and transferred to the second dimension, and the D- and L-Asp were separated and determined using a narrowbore enantioselective column. Large amounts of D-Asp were observed in the pineal gland of the rats and specific strains of mice (C3H and CBA) possessing a high concentration of melatonin in their pineal gland. On the other hand, the amounts of D-Asp were small in the pineal gland of mice possessing a trace or no melatonin in their pineal gland (ddY, ICR, C57BL and BALB/c). In other tissues and physiological fluids, no significant strain-dependent changes of the D-Asp amounts were observed. These results indicate that large amounts of D-Asp are present only in the pineal gland containing large amounts of melatonin, and special care should be taken when selecting mouse strains for the investigation of D-Asp.     

Determination of slight contamination by D-amino acids in natural amino acids

Conclusions The problem of using gas chromatography for the estimation of slight contamination by D-amino acids in natural L-amino acids has been examined. The threshold amount of D-isomer detectable by the gas chromatographic method, using a drift conpensating integrator, amounts to 0.5%.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 12, pp. 2771–2772, December, 1967.     

Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept

A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios.     

Fluorescence high-performance liquid chromatographic determination of free and conjugated bile acids in serum and bile using 1-bromoacetylpyrene as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile-methanol-water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile.     

Gas chromatographic quantification of free D-amino acids in higher vertebrates

D-amino acids were determined in brain, body fluids (urine, blood coagulate, serum, plasma) and faeces of animals belonging to nine out of 11 taxonomic orders of vertebrates (Artiodactyla, Aves, Carnivora, Lagomorpha, Marsupalia, Osteichthyes, Primates, Rodentia, Tubilidentata). Free amino acids were isolated by means of cation exchangers and converted into volatile N(O)-perfluoroacylamino acid propyl esters. Derivatives of amino acids were separated into D- and L-enantiomers using Chirasil-L-Val capillary columns and detected by selected ion monitoring mass spectrometry. Quantification of amino acids was achieved by comparison of analytes with amino acid standards using L-norleucine as internal standard. Large relative amounts of D-serine were determined in brains of mammals but not of birds. In body fluids the D-enantiomers of most proteinogenic L-amino acids were detected, largest absolute and relative amounts were found in urine. Therein quantities of D-Ala and D-Ser exceeded 50% relative to the L-enantiomers in many instances. Feeding animals with diet fortified with DL-Met resulted in excretion of almost racemic Met in urine. D-Amino acids were also abundant in faeces of rodents. The data confirm that d-amino acids are common in body fluids and certain tissues of vertebrates.     

Chromatographic determination of amino acid enantiomers in beers and raw materials used for their manufacture

Using gas chromatography (GC) on a chiral stationary phase, accompanied by high-performance liquid chromatography, beers and raw materials used for manufacturing (hops, barley grains, malts) were investigated for the pattern and quantities of amino acid enantiomers. Although L-amino acids were most abundant, certain D-amino acids were detected in all beers and most of the raw materials. Highest amounts of D-amino acids were detected in special beers such as Berliner Weisse that underwent bottle-conditioning with lactic cultures, and Belgian fruit beers produced by spontaneous fermentation. It is demonstrated that GC on chiral stationary phases is highly suitable for the quantitative determination of amino acid enantiomers in beers and raw materials used for their manufacture. Quantities, relative amounts and pattern of amino acid enantiomers can serve in particular as chiral markers for the authenticity of special beers.     

Separation of alpha-amino acid enantiomers by reversed-phase high-performance liquid chromatography after derivatization with o-phthaldialdehyde and a sodium salt of 1-thio-beta-D-glucose

A sensitive system for D,L-amino acid analysis has been developed, using fluorescence derivatization with o-phthaldialdehyde in the presence of sodium salt of 1-thio-beta-D-glucose. The reagents rapidly form fluorescent diastereoisomeric derivatives with primary amino acids. These derivatives are efficiently separated on a conventional reversed-phase column with an analysis time of 60 min. Simultaneous determination of enantiomers of various amino acids was achieved by a simple binary gradient elution with methanol in 0.05 M aqueous sodium acetate.     

Enantioselective screen-printed amperometric biosensor for the determination of D-amino acids

D-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of D-amino acids has been constructed by the immobilization of D-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for D-alanine in the concentration range from 5 to 200 microM. Excellent enantioselectivity towards D-amino acids was discovered. During the experiment, D-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements.     

Occurrence and functions of free D-aspartate and its metabolizing enzymes

D-Aspartate is one of a few D-amino acids that attracted attention at an early date, since it was detected in various tissues of mammals as a protein component. The occurrence of free D-aspartate in nature was recognized a little later, and raised questions about its physiological functions and metabolism. This amino acid has been gradually accepted, based on various experimental observations, to be a physiological substrate of D-aspartate oxidase, whose role had been considered enigmatic since its early discovery in the 1940s. Mammalian enzymes that serve to liberate D-aspartyl residue in proteins have been identified. One enzyme hydrolyzes peptide bond at the amino side of D-aspartyl residue in a dipeptide and another enzyme hydrolyzes that at the carbonyl side of the residue in proteins. The first pyridoxal 5'-phosphate-dependent aspartate racemase has been purified and cloned from a bivalve species. The enzyme supports the high contents of D-aspartate comparable to those of L-aspartate in the bivalve, and the enantiomers are consumed when hypoxia is imposed on the bivalve. In some yeast species, assimilation of D-aspartate has been found to depend on inducible D-aspartate oxidase, which also serves to detoxify acidic D-amino acids.     

Simultaneous determination of dl-lactic acid and dl-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC–ESI-MS/MS

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7?min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12?fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.     

Novel enantioselective receptors for N-protected glutamate and aspartate

A series of chiral bisthiourea macrocycles 1-4 have been prepared and their binding properties with various dicarboxylate salts have been examined by using NMR titration and isothermal calorimetry experiments. Macrocycle 1, in particular, favours the 1:1 binding of N-protected L-glutamate and aspartate, but favours 1:2 binding of the corresponding D-amino acids in polar solvents (dimethyl sulfoxide and acetonitrile). The macrocycles, however, do not bind carboxylates at all in the less competitive solvent chloroform. The binding properties of these macrocyles are sensitive to small structural changes as demonstrated by the altered binding properties of macrocycles 2-4 compared with 1.     

Determination of urinary beta-phenylethylamine as its N-benzenesulphonamide derivative by gas chromatography with flame photometric detection.

A selective and sensitive method for the determination of urinary beta-phenylethylamine (PEA) by gas chromatography (GC) has been developed. After extraction of the urine sample with n-pentane, PEA was converted into its N-benzenesulphonamide derivative and then determined by GC with flame photometric detection using a DB-1301 capillary column. By using this method, nanogram amounts of PEA in urine could be accurately and precisely determined without any influence from coexisting substances. Analytical results for the determination of PEA in urine samples from normal subjects are presented.     

Production of α-keto acids with alginate-entrapped whole cells of the yeastTrigonopsis variabilis

The yeast,Trigonopsis variabilis, was immobilized by entrapment in alginate. The immobilized cells containing high amounts of D-amino acid oxidase were used to convert D-amino acids to their corresponding α-keto acids.     

High-performance liquid chromatographic detection of L- and D-amino acids by use of immobilized enzyme electrodes as detectors

Two enzyme electrodes based on immobilized L- and D-amino acid oxidases give specific responses to L- and D-amino acids, respectively. They are used as amperometric detectors for high-performance liquid chromatography, by splitting the flow after elution from the column and detecting D-isomers in one line, L-isomers in the other. The detection limit is about 2 pmol for some amino acids (methionine, tyrosine, leucine, and phenylalanine). The procedure is useful for the specific detection of L- and D-amino acids without complicated pretreatment. The electrodes retain most of their original activities after repetitive use for one month.     

Quantification of D-amino acids in the central nervous system of Aplysia californica by liquid chromatography/tandem mass spectrometry

A sensitive, specific and reliable liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for simultaneous determination of D-amino acids in the central nervous system (CNS) of Aplysia californica. In order to correct for any potential matrix effects on measured signals, deuterium-labeled L-Asp-d3 was used as an internal standard. Pre-column derivatization of the sample with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) allowed both effective in-line pre-concentration and sensitive MS/MS detection of the analytes. An extraction column (50x0.25 mm, 5 microm C18 silica particles) was used to pre-concentrate/stack samples. Enantiomeric separation of amino acid enantiomers was achieved on a chiral column packed with teicoplanin aglycone bonded silica particles (170x0.25 mm, 5 microm) with an MS-friendly mobile phase. The characteristic precursor to product ion transitions, m/z 297-->279 (for NBD-Asp), m/z 269-->223 (For NBD-Ser), m/z 311-->293 (for NBD-Glu) and m/z 300-->282 (for NBD-L-Asp-d3) were monitored for the quantification. Samples from the CNS of A. californica and heart tissues were analyzed. D-Asp was detected at high levels in all the ganglia and nerve tissues, but not in the heart tissue. Further, neither D-Ser nor D-Glu was detected in Aplysia, a widely used neuronal model.     

高效液相色谱法测定银杏果中有机酸的含量

建立了反相高效液相色谱测定银杏果中有机酸含量的方法.方法采用ultimate-C18色谱柱,以pH=2.0的磷酸盐缓冲溶液为流动相,流速0.5 mL/min,检测波长210 nm,在22 min内实现了7种有机酸的基线分离.有机酸的检出限在0.001～0.067μg/mL之间.所建立的方法具有分析速度快、线性范围宽、灵...     

Homochirality and life

Before the emergence of life, left-handed amino acids (L-enantiomers) were selected and right-handed amino acids (D-enantiomers) were eliminated on the primal earth. Nevertheless, with the progress of analytical methods, D-amino acids have recently been found in higher order living organisms in the form of free amino acids, peptides, and proteins. Free D-amino acids have numerous physiological functions. D-amino acids containing animal peptides are well known as opioid peptides. D-amino acids in protein are related to aging. In this review, we describe the D-amino acids that are present and function as D-amino acid biosystems in our bodies.