Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization with fluorescein isothiocyanate (FITC) and the conditions for derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

Leaching of zinc from an industrial filter dust withPenicillium,Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

Summary Heterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.     

Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography

Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Analysis of Free Amino Acids in Microbially Colonized Sandstone by Precolumn Phenyl Isothiocyanate Derivatization and High-Performance Liquid Chromatography

A fast, sensitive method for extraction and analysis of soluble free amino acids from microbially colonized sandstone is described. After precolumn phenyl isothiocyanate (PITC) derivatization, the PITC-amino acids were identified and quantified by reversed-phase high-performance liquid chromatography. This kind of analysis could be used to elucidate the role and function of amino acids in the nutrition of epi- and endolithic microorganisms active in biological weathering processes.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Determination of amino acids in human plasma by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination

Amino acids in human plasma were determined by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination. S-Carboxymethyl-l-cysteine, d-phenylglycine and S-aminoethyl-l-cysteine were found to be suitable internal standards. The proposed method is simple, rapid (deproteination time less than 1 min) and reproducible [relative standard deviation below 3% except for low-level aspartic acid (n = 3)]. The average recovery of 25 amino acids was above 90%. The elution time of amino acids in human plasma was approximately 2 h. Protein binding of tryptophan was also determined by the proposed method. The analytical data for amino acids in human plasma deproteinated using the proposed and published methods (5-sulphosalicylic acid and ethanol) were compared.     

Deamidation and succinimide formation by gamma-N-methylasparagine: potential pitfalls of amino acid analysis

The biological function of the post-translationally methylated amino acid gamma-N-methylasparagine (gamma-NMA) in proteins is unknown. We are examining the premise that amide methylation protects against deamidation. The free amino acids Asn, gamma-NMA, Gln, and delta-N-methylglutamine (delta-NMG) were incubated at elevated temperature and a variety of pH conditions to assay for deamidation. Gln disappears 12- to 14-fold more rapidly than delta-NMG, and Asn hydrolyzes to Asp and NH3 as expected. However, the gamma-NMA deamidation rate is severely overestimated by simply measuring the disappearance of starting material because gamma-NMA undergoes a cyclization reaction in preference to deamidation. At pH 1 the predominant gamma-NMA reaction is formation of stable 3-amino-N-methylsuccinimide (NMS) and this occurs greater than 10-fold faster than Asn deamidation. At pH 4.0, 7.4, and 9.0 NMS is readily formed but it is unstable and partitions between the parent compound, gamma-NMA, and a second species, alpha-N-methylasparagine. At pH 7.4 and 9.0 gamma-NMA disappears 4-fold slower than Asn but the methyl amide hydrolysis rate is diminished by as much as 13-fold. The Asn incubations over the pH range 1-9 yield scant evidence of a succinimide intermediate. It is concluded that the amide methylation provides a unique reaction pathway and stabilization for the N-methylsuccinimide species. Amino acid analysis by o-phthalaldehyde postcolumn reaction fails to detect isoasparagine, alpha-N-methylasparagine, and NMS. Amino acid analysis by precolumn derivatization with phenyl isothiocyanate destroys NMS and therefore cannot quantitate this compound. The ninhydrin postcolumn derivatization method is able to detect and quantitate all of these amino acid species.     

13C gas chromatography-combustion isotope ratio mass spectrometry analysis of N-pivaloyl amino acid esters of tissue and plasma samples

We present the analysis of the stable carbon isotope compositions of 14 individual N-pivaloyl-isopropyl (NPP) amino acid esters by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS). The mean reproducibility of derivatization procedure and GC-C-IRMS analysis was 0.45 per thousand (range, 0.12-0.68), whereas the mean analytical error was 0.26 per thousand delta(13)C (range, 0.13-0.42). Furthermore, the delta(13)C values of N-pivaloyl-isopropyl and N-acetyl-n-propyl (NAP) amino acid esters were compared. Due to a reproducible isotopic fractionation introduced by the derivatization process an empirical correction factor for each individual amino acid was derived separately for both derivatives (NPP, -1.13 to -2.52 (lysine, +2.09) per thousand delta(13)C; NAP, -2.36 to -3.97 (lysine, +1.91) per thousand delta(13)C), and the original delta(13)C value of the underivatized amino acid was calculated. Further, we performed an animal study where rats (n = 5) ingested a mixed meal containing uniformly (13)C-labeled casein (indispensable amino acids 1.3 to 1.7 at.%). One hour after the meal delta(13)C values of protein-bound amino acids from small intestinal mucosa and liver and of free amino acids from mucosa and plasma were determined. Significant (13)C enrichments of indispensable amino acids of the free pools of mucosa and plasma (range, 0.0518 to 0.1700 at.% excess) and in mucosa and liver proteins (range, 0.0021 and 0.0161 at.% excess) were observed. The feasibility of various derivatives for the measurement of carbon isotopic composition is discussed.     

反相高效液相色谱法测定血浆游离氨基酸

本文报告了采用高效液相色谱法反相梯度洗脱,邻苯二甲醛和β-巯基乙醇柱前衍生化,荧光检测分血浆游离氨基酸。实验采用线性洗脱,在50分钟内可同时测定18种氨基酸,血浆样品的预处理简单,衍生化反应的时间仅需1分30秒,血浆样品的实际进样量少于1μl。本测定方法的精确度高,各个氨基酸保留时间的变异系数平均为0.89%±0.45%(SD),峰面积的变异系数平均为2.06%±1.76%(SD),各个氨基酸的浓度在15—150μmol/L的范围中,线性关系的相关系数平均为0.985±0.0305(SD)。准确性好,各个氨基酸的回收率平均为97.6%±5.1%(SD)。实验还讨论了氨基酸分离时溶液pH值、柱温、离心速度等因素对分析结果的影响。     

Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS

Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 μL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6-10.1%. Absolute accuracy as determined by comparison of external reference samples was 82-117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 μmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

The influence of extrusion on loss and racemization of amino acids

Summary. The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-β-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 °C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 °C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 °C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L – D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize. Authors’ address: J. Csapó, Faculty of Animal Science, Institute of Chemistry, University of Kaposvár, Guba S. u. 40., H-7400 Kaposvár, Hungary     

Analysis of amino acids in nectar from pitchers of Sarracenia purpurea (Sarraceniaceae)

Sarracenia purpurea L. (northern pitcher plant) is an insectivorous plant with extrafloral nectar that attracts insects to a water-filled pitfall trap. We identified and quantified the amino acids in extrafloral nectar produced by pitchers of S. purpurea. Nectar samples were collected from 32 pitchers using a wick-sampling technique. Samples were analyzed for amino acids with reverse-phase high-performance liquid chromatography with phenylisothiocyanate derivatization. Detectable amounts of amino acids were found in each of the 32 nectar samples tested. Mean number of amino acids in a nectar sample was 9 (SD = 2.2). No amino acid was detected in all 32 samples. Mean amount of amino acids in a nectar sample (i.e., amount per wick) was 351.4 ng (SD = 113.2). Nine amino acids occurred in 20 of the 32 samples (aspartic acid, cysteine, glutamic acid, glycine, histidine, hydroxyproline, methionine, serine, valine) averaging 263.4 ng (SD = 94.9), and accounting for ~75% of the total amino acid content. Nectar production may constitute a significant cost of carnivory since the nectar contains amino acids. However, some insects prefer nectar with amino acids and presence of amino acids may increase visitation and capture of insect prey.     

Free amino acids and protein concentrations in reproductive tract fluids of the mare

Uterine tubal fluids (UTF) were collected daily over a 214-day period (March through August) from three mares. Individual UTF samples identified by day of estrous cycle for five complete cycles within this six-month span were analyzed for free amino acids and total protein. Biochemical comparisons were made to blood plasma by drawing samples daily from each mare. Free amino acids and total protein were determined also on follicular fluids collected from three different mares on days 5 and 6 of standing estrus.The free amino acid level of UTF was significantly greater than was the amino acid concentration in blood plasma or follicular fluid. The highest concentration of amino acids in UTF was on day 13. Cyclic trends were observed for the amino acids, histidine, methionine, half-cystine, serine, proline, glycine, alanine, isolecine, and leucine. Glycine and alanine were found in the highest concentrations in UTF, peaking on day 17 of the estrous cycle. Protein concentration in UTF was highest on day 13 and lowest on days 7 and 19. Protein values for diestrus (33.1 mg/ml) were significantly greater (p<0.05) than for estrus (28.0 mg/ml).     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

Capillary gas chromatographic determination of proteins and biological amino acids as N(O)-tert.-butyldimethylsilyl derivatives

Forty-seven biological amino acids containing all 22 protein amino acids were derivatized to N(O)-tert.-Butyldimethylsilyl (tBDMSi) derivatives by a single-step reaction with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide and successfully separated on an HP-1 capillary column. The relative standard deviations of the relative molar responses of most amino acids were <5%. Cystine seems to be partially converted into cysteine during derivatization. An increase in carrier gas flow-rate towards the end of the analysis by inlet pressure programming with electron pressure control avoided the peak broadening and adsorption of the derivatives with high boiling points on the column and especially increased sensitivity of cystine to 5 pmol. Glutamine was converted almost completely into pyroglutamic acid during prolonged storage of a standard solution prepared in 0.01 M HCl but not during derivatization. These results compared with those for the phenylthiocarbamyl derivatives analysed by HPLC and the analytical results reported in the literature on soybean hydrolysate showed good agreement except for cysteine. The results for the amino acid composition of bovine serum albumin also showed good agreement with results in the literature except for cysteine. In human urine, seventeen free amino acids were detected as tBDMSi derivatives.     

冬虫夏草口服液游离氨基酸和微量元素的分析比较

目的：为了研究冬虫夏草口服液的有效成分和药用价值,测定冬虫夏草口服液中的游离氨基酸和微量元素的不同种类和含量。方法：采用安捷伦1200高效液相色谱仪,C18反相柱梯度分离,邻苯二甲醛一芴甲酯柱前自动衍生化,利用已知浓度氨基酸标准品进行外标法定量,根据已知浓度氨基酸标准品的峰面积与样品峰面积之比计算游离氨基酸的含量。结果：冬虫夏草口服液中含有23种游离氨基酸和18种微量元素,氨基酸总量在口服液中占0．892％,必需氨基酸在游离氨基酸总量中占53．1％;含量最高的游离氨基酸依次是赖氨酸、亮氨酸、精氨酸。结论：游离氨基酸是冬虫夏草口服液中重要的生物有效成分,为冬虫夏草口服液的药效提供了基础数据。