A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids.

The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate determination of amino acids in biological samples and in metal-assisted peptide hydrolysis reactions. It is especially critical for the relative fluorescence intensities (RFI) of equal amounts of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as organic solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although determination of tryptophan is problematic, this difficulty is overcome by the addition of beta-cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring amino acids.     

Practical gas—liquid chromatographic method for the determination of amino acids in human serum

Application of the gas—liquid chromatographic method previously reported by us was made to the analysis of the 22 amino acids including asparagine and glutamine in serum. The method permitted that aqueous serum samples obtained after deproteinization with perchloric acid were directly subjected to derivatization without any further clean-up procedure such as ion-exchange chromatography. The N-ethyloxycarbonyl methyl esters, which were prepared in the same manner as the N-isobutyloxycarbonyl methyl esters, were introduced for the determination of leucine, isoleucine, arginine and tyrosine. Both derivatives were prepared by two-step procedures involving alkyloxycarbonylation in aqueous media and esterification with diazomethane, and simultaneously analyzed by using the dual set of columns with the same thermal conditions. The advantages of this method are that the sample pretreatment and derivatization are very simple and rapid, and that both asparagine and glutamine along with other amino acids in serum can be determined.     

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.     

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible.     

Simple plasma work-up for a fast chromatographic analysis of homocysteine,cysteine, methionine and aromatic amino acids

Simplified sample workup obviating protein precipitation and eluent evaporation commonly employed in earlier reports using chloroformate-mediated derivatization of aminothiols prior to mass spectrometric (MS) detection is presented. The reduction of disulfides in plasma is accomplished with dithiothreitol within minutes. A simultaneous derivatization with ethyl chloroformate (ECF) and extraction of derivatives into organic phase takes place within seconds. Along with S-amino acids, also aromatic amino acids can be determined during a 5-min run. Gas chromatography with flame ionization detection (GC-FID) proved to be sensitive enough to reach plasma homocysteine levels. A prerequisite for a reliable quantitation was fulfilled under the given conditions. Intra-assay precision was <5%, recoveries from spiked plasma complete (101.2%), detection and quantitation limits for homocysteine came to <1 and 3 micro mol/l. Our results were in full agreement with those obtained by liquid chromatography (r=0.999 for homocysteine and 0.987 for cysteine), and were close to two homocysteine immunoassays (r=0.991 and 0.939, respectively).     

Advancement in the derivatizations of the amino groups with the o-phthaldehyde-thiol and with the 9-fluorenylmethyloxycarbonyl chloride reagents

An overview is presented on the advancement of the two most frequently used derivatization protocols applying the o-phthalaldehyde (OPA)-thiol and the fluorenylmethyloxycarbonyl (FMOC) chloride reagents, prior to the high performance liquid chromatographic analysis of amino acids. This review pays special attention (i) to the blank value, to the composition, to the stability and to the life time of the reagents, (ii) to the optimum pH conditions for the interactions and derivatives' elutions, (iii) to the characteristics and behavior of those amino acids which might provide more than one derivative correlating with the molar ratios of the reagent to the reactants, and (iv) on the practical applicability of the optimized protocols.     

Development of an LC-MS-MS method for the quantification of taurine derivatives in marine invertebrates

Sulfur amino acids, such as taurine, hypotaurine, and thiotaurine, were found in high quantities in tissues of marine symbiotic organisms (e.g., bivalves, tubeworms) living close to hydrothermal vent sites. Therefore, they are assumed to play a key role in the S-oxidizing base metabolism or sulfide detoxification. We propose here a specific, rapid, and original analytical procedure for the direct determination of sulfur amino acids at the level of a few parts per billion in biological samples, avoiding the classical low specific post-column ortho-phthaldialdehyde derivatization step required by non-ultraviolet-absorbing molecules. Indeed, by coupling liquid chromatography on a porous graphitic stationary phase under isocratic conditions (10 mM ammonium acetate buffer adjusted to pH 9.3) to tandem mass spectrometry (ionization process by pneumatically assisted electrospray in negative ion mode), it is possible to perform specific quantification of these metabolites in less than 10 min directly in biological matrices without any derivatization step or other tedious sample treatments. Thus, taurine, hypotaurine, and thiotaurine have been identified and assayed in several deep sea organisms, showing that the developed method is well suited for this kind of application.     

Engineering surfaces for bioconjugation: developing strategies and quantifying the extent of the reactions

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene) (PTFE) surfaces for subsequent conjugation of biologically relevant molecules. First, PTFE films were treated by a radiofrequency glow discharge (RFGD) ammonia plasma to introduce amino groups on the fluoropolymer surface. This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups, as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy (XPS). In a second step, these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides, carboxylic acids, acetals, aldehydes, and thiols, that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins, DNA, drugs, etc. In the present study, glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface. Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface, providing a diethylacetal function, which is a latent form of aldehyde functionality. Reactions with cross-linkers such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB) were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules. Traut reagent (2-iminothiolane) was also conjugated to introduce a thiol group onto the fluoropolymer surface. PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer. Finally, surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.     

High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave

A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.     

两种柱前衍生反相高效液相色谱法测定血液和尿液中游离氨基酸

建立以PITC法和AQC法为柱前衍生试剂测定血液和尿液中游离氨基酸含量的测定方法。采用Waters-e2695操作系统,色谱柱为Shim-vp,ODS(250mm×4.6mm,5μm)(日本,岛津公司),以甲醇/乙腈/水和醋酸钠溶液(pH 6.5)为流动相,梯度洗脱。分别采用紫外和荧光检测器对血液和尿液中游离氨基酸进行含量测定。结果显示,两种衍生化方法灵敏度好、分离度高,具有良好的线性范围(r>0.990 0),准确度高(平均回收率为75.1%～127.0%),进样精密度好(RSD为0.12%～3.42%)。PITC法在尿液中游离氨基酸含量测定中显示了良好的测试准确性;而AQC法测定尿液中组氨酸、苏氨酸、脯氨酸超出线性范围,需要对尿样的前处理进行深入研究。     

Synthesis of 1,3-diacylaminopropan-2-ols and corresponding 2-acyl derivatives as amide isosteres of natural lipids.

N,N'-diacyl-1,3-diaminopropan-2-ols as amide isosteres of 1,3-diacylglycerols are obtained in 50-79% yields by treatment of 1,3-diaminopropan-2-ol with acid chlorides either in ether or THF in the presence of triethylamine and catalytic amounts of 4-dimethyl-aminopyridine. Subsequent acylation of the secondary alcohol function with a variety of carboxylic acids (e.g. fatty acids, N-protected amino acids, drug compounds) can be effected by various techniques (i.e. acid chlorides, symmetrical anhydrides, isopropenyl chloroformate) with yields ranging from 56% to 76%.     

Polymer-bound alkyltriazenes for mild racemization-free esterification of amino acid and peptide derivatives.

A novel tool for polymer-assisted solution phase (PASP) esterification of amino acid and peptide derivatives has been developed. When treated with carboxylic acids, polymer-bound alkyltriazenes react with a loss of nitrogen and transfer of the alkyl moiety to the carboxylate anion to form the corresponding alkyl esters. There are no limitations with regard to either the protecting groups or the nature of the amino acid. Furthermore no racemization occurs at the chiral centers of the amino acids as demonstrated by chiral GC-MS analyses. Alkyltriazene-resins were also applied successfully to the esterification of peptide acids and other peptidic structures, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). The triazene-mediated esterification reaction is exceptionally mild, and there is no need for prior activation of the carboxy groups. This method is therefore particularly suitable for the alkylation of complex peptidomimetic structures prone to racemization and for acid-sensitive structures.     

Enzyme Catalyzed Degradation and Formation of Peroxycarboxylic Acids

The ability of hydrolases to catalyze perhydrolysis, i.e. lysis of acyl substrates with hydrogen peroxide to form peroxycarboxylic acids, has been investigated. Lipases, esterases and cholinesterases were found to catalyze perhydrolysis but the preference of the enzymes for hydrogen peroxide relative to water as nucleophile was only 10-100 fold, even in the best cases. Hence, perhydrolysis proceeds with a very low efficiency in aqueous systems. Furthermore, all lipases, esterases and cholinesterases tested degrade peroxycarboxylic acids to the corresponding carboxylic acid and hydrogen peroxide. This reaction is most pronounced in the case of lipases while less so for cholinesterases. Consequently, cholinesterases are superior to the other hydrolases studied in catalyzing net formation of peracids in aqueous systems. In organic solvents, immobilized lipases efficiently catalyze formation of peracids from either triglycerides or the parent carboxylic acid. Proteases and phospholipase A-2 were found to neither degrade peracids nor catalyze perhydrolysis of carboxylic esters or phospholipids, respectively.     

Chromium mobilization and plant availability — the impact of organic complexing ligands

Pot experiments were conducted to investigate the effect of various organic acids (carboxylic and amino acids) on the uptake and translocation of root-absorbed trivalent chromium by tomato ( Lycopersicum esculentum) plants grown in sand and soil culture. Statistically significant increases in chromium accumulation from Cr(III) treated plants in the presence of increasing concentrations of organic acid suggest the existence of Cr(III) — organic acid interactions in the soil-plant system. However, the amino acids have been less effective in the mobilization of chromium compared to carboxylic acids. The results are discussed on the basis of the potential of organic acids to form complexes with Cr(III). This revised version was published online in June 2006 with corrections to the Cover Date.     

Polymeric 6-aminoquinoline, an activated carbamate reagent for derivatization of amines and amino acids by high-performance liquid chromatography

A new polymeric reagent containing the 6-aminoquinoline (6-AQ) tag was developed and applied for the off-line derivatization of amines and amino acids in high-performance liquid chromatography (HPLC). The synthesis and characterization of this polymeric reagent are described. An authentic external standard of a typical amine was synthesized and characterized for the determination of the derivatization efficiency. All amines had a derivatization efficiency higher than 50%; the derivatization of amino acids was performed under optimized phase-transfer catalysis reaction conditions. Derivatized amines and amino acids were separated under conventional reversed-phase conditions and determined by UV and FL detectors. To investigate the practical applications, this polymeric reagent was also used to derivatize protein hydrolysates.     

Rapid and selective derivatizatin method for the nitrogen-sensitive detection of carboxylic acids in biological fluids prior to gas chromatographic analysis

A rapid and selective derivatization procedure is described for the pre-column labelling of carboxylic acids with a nitrogen-containing label. The carboxylic acid function is activated with 2-bromo-1-methylpyridinium iodide and the activated carboxylic acid function reacts with a primary or a secondary amine to yield an amide. With flurbiprofen as the test compound and dipropylamine as a label the acid was completely converted to the corresponding amide. The method was tested for several aliphatic, aromatic and for phenylacetic or phenylpropionic carboxylic acid dervatives, and was found to result in the complete derivatization of these compounds with a few exceptions only. The derivatization procedure is potentially useful for drug monitoring purposes, as is shown with the analysis of valproic acid and flurbiprofen in plasma.     

Determination of isokinetic ratios necessary for equimolar incorporation of carboxylic acids in the solid-phase synthesis of mixture-based combinatorial libraries

The methods used to study the relative reaction rates of 45 different aliphatic and aromatic carboxylic acids when coupled to resin-bound amino acid amides is described. Competition experiments involving the coupling of incoming carboxylic acids to resin-bound amino acid amides were performed. The relative composition of each N-acylated amino acid amide in the resulting mixtures was compared to controls prepared by physically mixing equal aliquots of individual compounds in order to study the relative reaction rates of the incoming carboxylic acids. The ratios of the incoming carboxylic acids were then iteratively adjusted to yield as close to equimolar products as possible. As expected, the steric and electronic nature of the incoming carboxylic acids was found to influence their relative reaction rates. The steric hindrance of the resin-bound amino acid appears to have a proportional effect on the reaction rates of the incoming carboxylic acids. N-acylated amino acid amides in the final mixtures, prepared using the final isokinetic ratios, were found to be approximately equimolar.     

Femtomole detection of amino acids and dipeptides by gas chromatography—negative-ion chemical ionization mass spectrometry following alkylation with pentafluorobenzyl bromide

Amino acids and di- and tripeptides were derivatized by extractive alkylation using pentafluorobenzyl bromide (PFBBr) followed by reaction with heptafluorobutyric anhydride (HFBA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Good chromatographic separation and the formation of intense diagnostic ions were observed for the derivative when examined using gas chromatography—negative-ion chemical ionization mass spectrometry (GC—NICI-MS). Of the 20 amino acids investigated, only Arg and Glu could not be detected by this method. Also, dipeptides which included neutral amino acid residues were derivatized with more success than those containing either acidic or basic residues. Each of the amino acids or dipeptides formed one major derivative with the exception of Asn which formed two derivatives with either one or two HFB groups. This derivatization method was optimized with respect to the reaction temperature, reaction time, and choice of derivatizing reagents. Recoveries of derivatized [³H]-labeled Phe, Lys, and Thr were 76, 55, and 34%, respectively. Linearity was observed from 10 to 2000 pg of Ala per vial; selected-ion monitoring provided a detection limit of less than 150 fg with a signal-to-noise (S/N) ratio of 80 to 1. This method has proven to work well with urine samples and shows great promise for the detection of small peptides at low levels.     

N-(9-acridinyl)-bromoacetamide--a powerful reagent for phase-transfer-catalyzed fluorescence labeling of carboxylic acids for liquid chromatography

Phase-transfer-catalyzed esterification of various carboxylic acids has been carried out on a microanalytical scale with a new reagent, N-(9-acridinyl)-bromoacetamide, in aqueous/organic two-phase systems. The procedure gives esters showing very high fluorescence intensity in acid solution. The large Stokes' shift (greater than 120 nm) means that a low background can be achieved even when using a large emission bandwidth. These derivatives show very good chromatographic behavior on reversed-phase liquid chromatographic columns with the use of aqueous acetonitrile, containing 0.2% of phosphoric acid, as the mobile phase. Detection limits (S/N = 2) as low as 10 fmol can be obtained under optimal conditions.     

A modified spray reagent for the detection of amino acids on thin layer chromatography plates

Summary. Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced. Received March 14, 2000 Accepted August 31, 2000