Amino Acid Analysis Procedure Research Articles

A-317 Development of a patient-focused volumetric absorptive microsampling strategy to assess urinary amino acid excretion

Abstract Background Biochemical determinations of urinary amino acid excretion patterns can diagnose specific inborn errors of metabolism (IEM). Clinical management of these genetic metabolic patients may require frequent urine amino acid tests. A simplified process for transporting home collected urine specimens would benefit the IEM community. Volumetric absorptive microsampling (VAMS) is an emergent alternative sampling technique for the collection of dried biological specimens. We describe the development and preliminary validation results of a VAMS strategy for the analysis of creatinine and amino acids (N=22) from microsampled dried urine. Methods Urine creatinine and amino acids were respectively measured in a clinical laboratory setting using established assays on a CobasPro (Roche) and a MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Two 30 µL VAMS tips from a Mitra device (Neoteryx) were used to sample each random urine specimen. An ultrasonication-based solvent extraction procedure was optimized to extract the amino acids and creatinine from the VAMS tips. Solvent extracts were subsequently dried by centrifugal evaporation. Extract from one tip was re-dissolved in water and tested for creatinine using an enzymatic method. The other extract was re-dissolved in 0.02 N sodium hydroxide and tested according to the MassTrak AAA procedure, which deploys pre-column AccQTag derivatization with reversed-phase UPLC on a C18 column (2.1 × 150 mm; 1.7 µm) and UV detection at 260 nm. Accuracy, within-batch precision and short-term stability of the optimized VAMS protocol were determined. Results Extraction solvent with a 60:40 (v/v) water:methanol mixture provided the highest average amino acid recovery. This extraction solvent produced higher average amino acid recoveries at pH 11 (89±13%), than pH 3 (82±10%) or pH 7 (72±11%). Creatinine measured from the VAMS tips (N=44) had a consistent negative bias of 20% relative to neat urine (R=0.9905, slope=0.802, intercept=0.02). The creatinine corrected urine amino acid results from the Mitra device were corrected for this observed concentration bias. The accuracy of the amino acid results derived from the VAMS tip vs neat urine was 104 ± 14%. The average within-batch precision (CV%) of the amino acids was 9.8% (range=6.7% (cystine) to 17.6% (arginine)) and 6.2% for creatinine. The average 1 week storage stability at ambient temperature was 91% (range=73% (ornithine) to 107% (glycine)) for the amino acids and 107% for creatinine. The devised VAMS strategy was applied to the analysis of microsampled dried urine from a patient with cystinuria (OMIM no. 220100). Marked elevations in arginine, cystine, lysine and ornithine were respectively observed and are consistent with the biochemical diagnosis of the patient. Conclusions Creatinine corrected amino acid results from the devised VAMS strategy exhibit good precision and accuracy when compared to tradition analyses of neat urine. The potential use of microsampled dried urine for the diagnosis and monitoring of selected IEM has been demonstrated. The analytical and clinical performance of this VAMS protocol must be further validated, including the use of microsampled dried urine from home specimen collections.

Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC–MS/MS) and gas chromatography with mass spectrometry (GC)–MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as “in-house” standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC–MS and LC–MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.Graphical abstract

A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases. Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds. Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time = 9min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode. The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified. We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.

A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC.

Octreotide as a synthetic cyclic octapeptide is a somatostatin analog with longer half-life and more selectivity for inhibition of the growth hormone. The acetate salt of octreotide is currently used for medical treatment of somatostatin-related disorders such as endocrine and carcinoid tumors, acromegaly, and gigantism. Octreotide contains both cysteine and tryptophan residues which make the hydrolysis part of its amino acid analysis procedure very challenging. The current paper introduces a fast and additive-free method which preserves tryptophan and cysteine residues during the hydrolysis. Using only 6 M HCl, this hydrolysis process is completed in 30 min at 150 °C. This fast hydrolysis method followed by pre-column derivatization of the released amino acids with 4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride (DABS-Cl) which takes only 20 min, makes it possible to do the complete amino acid analysis of an octreotide sample in a few hours. The highly stable-colored DABS-Cl derivatives can be detected in 436 nm in a reversed phase chromatographic system, which eliminates spectral interferences to a great extent. The amino acid analysis of octreotide acetate including hydrolysis, derivatization, and reversed phase HPLC determination was validated according to International Conference of Harmonization (ICH) guidelines.

Available versus digestible dietary amino acids

Available amino acids are those absorbed from the gastrointestinal tract in a form suitable for body protein synthesis. True ileal digestible amino acids are determined based on the difference between dietary amino acid intake and unabsorbed dietary amino acids at the terminal ileum. The accuracy of ileal digestible amino acid estimates for predicting available amino acid content depends on several factors, including the accuracy of the amino acid analysis procedure. In heat processed foods, lysine can react with compounds to form nutritionally unavailable derivatives that are unstable during the hydrochloric acid hydrolysis step of amino acid analysis and can revert back to lysine causing an overestimate of available lysine. Recently, the true ileal digestible reactive (available) lysine assay based on guanidination has provided a means of accurately determining available lysine in processed foods. Methionine can be oxidised during processing to form methionine sulphoxide and methionine sulphone and cysteine oxidised to cysteic acid. Methionine sulphoxide, but not methionine sulphone or cysteic acid, is partially nutritionally available in some species of animal. Currently, methionine and cysteine are determined as methionine sulphone and cysteic acid respectively after quantitative oxidation prior to acid hydrolysis. Consequently, methionine and cysteine are overestimated if methionine sulphone or cysteic acid are present in the original material. Overall, given the problems associated with the analysis of some amino acids in processed foodstuffs, the available amino acid content may not always be accurately predicted by true ileal amino acid digestibility estimates. For such amino acids specific analytical strategies may be required.

Biosensors for Protein Detection: A Review

There is considerable demand for the rapid low‐cost determination of proteins, particularly in the food and beverage industry. The most widely used tests are based on colorimetric procedures in which proteins react to produce colored complexes. These methods (Lowry et al. 1951; Bradford 1976; Gornall et al. 1949; Smith et al. 1985) are dependent upon a number of factors other than absolute protein quantity, including amino acid composition, protein purity, and association state. The successful application of these methods relies on using representative calibration standards. Time‐consuming and complex methodologies such as the Kjeldahl technique and quantitative amino acid analysis procedures have been also reported. Apart from these methods, biosensors are interesting tools offering certain operational advantages over standard photometric methods, notably with respect to rapidity, ease‐of‐use, cost, simplicity, portability, and ease of mass manufacture. By appropriate recognition element selection, it is possible to detect either a particular target protein or a broad range of proteins. This review presents an overview of the different biological recognition elements and the various transduction systems that have been reported in the literature to design biosensors for protein detection. Examples are given to illustrate the different possibilities. It must be noticed that this review reports detection of proteins in solution and not proteins immobilized on membranes. Briefly, immobilized proteins can be detected by antibodies (Western blotting) or oligonucleotides such as aptamers (“Eastern blotting”) bearing reporter molecules, fluorescently labeled or radiolabeled.

AMINO ACID ANALYSIS OF PEPTIDES USING HPLC WITH EVAPORATIVE LIGHT SCATTERING DETECTION

A new procedure for the amino acid analysis of peptides has been devised utilizing high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). This procedure eliminates the need for complex derivatization schemes inherent of previous amino acid analysis procedures since the ELSD detects the amino acids directly. This quantitative method detects and separates 18 of the common amino acids in a one hour run time using cation exchange chromatography coupled with ELSD. The procedure was tested by analyzing the hydrolysate of human parathyroid hormone 1–34 (PTH), a synthetic polypeptide. A standard digestion consisting of 24 hour hydrolysis at 110°C in 6 N hydrochloric acid with 3% phenol was used. Validation data reveal this is an accurate and precise procedure for the amino acid analysis of peptides. The sensitivity of this technique is low compared to other state-of-the-art analytical techniques. Detection limits varied depending on the amino acid being analyzed and were as low as 200 picomoles for this new procedure. This approach yields a simple, universal method that is well suited for the amino acid analysis of peptides, of which sufficient quantity is available. Only basic HPLC instrumentation with an ELSD is necessary for this procedure.

Comparison of Amino Acid Analyses by Phenylisothiocyanate and 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate Precolumn Derivatization

An extensive retrospective comparison was conducted of the long-term repeatability and consistency of amino acid analyses using phenylisothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) precolumn derivatization. Amino acid standards were analyzed more than 130 times on more than 60 independent occasions by each of these two precolumn derivatization methodologies, during routine amino acid analysis procedures. Similar coefficients of variation were seen only when very freshly prepared derivatives were analyzed. When realistic aging for <20 h was taken into account, the extreme stability of the AQC derivatives stood out. Chromatography of AQC derivatives using HPLC solvents prepared on a large scale provided the lowest coefficients of variation. The superiority of AQC over PTC methodology was clearly apparent.

Flow-injection spectrophotometric determination of lysine in feed samples

A flow-injection spectrophotometric method is described for the determination of lysine, based on the product of the reaction between lysine and 1,2-naphthoquinone-4-sulphonate in basic medium. The influence of different physical, chemical and hydrodynamic parameters on absorbance were studied. At 480 nm, the response is linear up to 1.5 × 10 −4 M, the detection limit is 1.9 × 10 −6 M and the relative standard deviation is 0.8%. Sample throughtput is 80 h −1. The method was applied to the determination of free lysine in commercial feed samples, by means of multivariate calibration techniques, without the need of chromatographic separation. Good concordance is shown between this method and the standard procedure for amino acid analysis.

Development of an HPLC method to estimate hexosamines and its application to determine mucin content in rat and human gastric mucosa

1. 1. An HPLC analysis of hexosamines and related compounds modified from the Waters Pico-Tag amino acid analysis procedure is described. 2. 2. This method is applicable to determine glucosamine and galactosamine not only in the hydrolyzate of purified glycoconjugate such as gastric mucin and ovalbumin but also in crude materials such as rat gastric mucosal extract and powdered bovine nasal cartilage. 3. 3. Compared with a conventional colorimetric hexosamine determination, sensitivity of this method is almost 100 times higher. 4. 4. By applying this method, a simple but highly sensitive procedure to determine mucin content in a crude stomach specimen of rat and that in a biopsied human gastric mucosa was also developed.

Determination of m-nitrophenol and nipecotic acid in mouse tissues by high-performance liquid chromatography after administration of the anticonvulsant m-nitrophenyl-3-piperidinecarboxylate hydrochloride

AbstractThe nipecotic acid ester m-nitrophenyl-3-piperidinecarboxylate (MNPC) possesses anticonvulsant activity. In the present study, the metabolites m-nitrophenol and nipecotic acid were determined in mouse blood and brain tissue after administration of MNPC. This determination was used as an indication of the distribution of the parent compound MNPC and to provide information regarding the differences in distribution between the enantiomers of MNPC, the times of onset, and effectiveness when (±)MNPC was administered by subcutaneous (sc) and intraperitoneal (ip) routes. m-Nitrophenol was determined by a previously reported high-performance liquid chromatography (HPLC) method. There was no significant difference in m-nitrophenol distribution after sc administration of (+)MNPC and (−)MNPC (400 mg/kg each). This similar pattern of distribution is in agreement with the earlier reported equi-effectiveness of the enantiomers of anticonvulsants. Peak m-nitrophenol levels in blood, which occurred at 15min, were three times greater when (±)MNPC was administered by ip injection as compared with sc injection. This significant difference is most likely due to enhanced absorption and the peripheral metabolism of MNPC by the liver when the ip route is employed. A novel HPLC assay for the determination of nipecotic acid in mouse brain was developed, based on a modification of a reported amino acid analysis procedure. The results of the brain distribution studies showed that nipecotic acid brain levels peaked at 30min after sc administration of (−)MNPC. These findings correlate with m-nitrophenol distribution in the blood and the previously reported onset of the anticonvulsant effect of MNPC. The HPLC assays used in this study are simple, rapid, and sensitive. Furthermore, the HPLC brain assay for nipecotic acid can be used to examine the distribution of other esters of nipecotic acid reported to possess anticonvulsant activity.

HPLC analysis of hexosamine phosphates in biological samples

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.

ESTIMATION OF FUNGAL CONTAMINATION OF CEREAL GRAINS AS DETERMINED BY MEASURING GLUCOSAMINE CONCENTRATION

The degree of fungal contamination of a grain or feed sample can be estimated from the determination of its glucosamine (GlcN) content, using a modified amino acid analysis procedure. A sample (100 mg) was hydrolyzed in 6 N HCl for 7 h at 121 °C, evaporated to dryness, reconstituted in citrate buffer and applied to an ion exchange column of an amino acid analyzer. Hydrolysis conditions were optimized for the recovery of GlcN, released by the breakdown of chitin, a major fungal cell wall component. The GlcN and galactosamine (GalN) concentrations of several species of fungi indigenous to western Canada, grown on liquid culture, were determined to provide reference values for estimating fungal contamination. A fungal contamination index (FCI) is proposed, based on a comparison of the GlcN concentration of a sample to the specific reference fungus (if the fungal species has been characterized) or an average from several species. Based on the fungal species tested, a suggested average for use with most naturally contaminated grain samples is 76 mg GlcN g−1 of fungal dry matter. This approximation would be valid for most cases of mixed or unknown fungal species. Knowing the fungal content of a grain sample will aid in feed formulation and thereby reduce costly losses to the livestock producer. Key words: Fungal contamination, glucosamine, chitin, grain, amino acid analysis

Amino acid analysis using phenylisothiocyanate prederivatization: elimination of the drying steps.

Results of experiments on the procedure for amino acid analysis via analysis of the phenylthiocarbamyl amino acids are reported. It was found that yields of some amino acids varied in the presence of salt and with changes in the vacuum drying steps. An improved procedure is described which includes a standard addition of salt to the hydrolysate before drying it; the redrying step is omitted and the post derivatization drying is replaced by a simple addition of heptane to the reaction mixture.

High-performance liquid chromatographic analysis of galactosamine, glucosamine, glucosaminitol, and galactosaminitol

Both N-acetylgalactosamine and N-acetylglucosamine covalently link oligosaccharides to peptide in glycoproteins. In order to identify the N-acetylhexosamine involved in this linkage, the corresponding hexosaminitol generated by alkaline borohydride treatment must be determined. An HPLC method modified from the Waters PICO-TAG amino acid analysis procedure is described. Phenylisothiocarbamyl derivatives of galactosamine, glucosamine, glucosaminitol, galactosaminitol, and the internal standard, p-aminophenyl-β- d-galactoside, are eluted from the Waters PICO-TAG column at 3.9, 4.3, 6.9, 8.1, and 10.1 min, respectively. The standard curves for the hexosamines and hexosaminitols are linear between 1 and 75 nmol. In addition to p-aminophenyl-β- d-galactoside, several synthetic hexosamines and hexosaminitols can be employed as internal standard. These include 3-allosamine, 3-glucosamine, allosamine, 3-allosaminitol, mannosaminitol, and allosaminitol, which are eluted at 3.1, 3.4, 4.8, 6.4, 7.4, and 7.8 min, respectively. The analysis time is 15 min but can be shortened to 10 min if only hexosamines are to be analyzed and either 3-glucosamine or 3-allosamine is used as the internal standard. This rapid method is superior to previous methods for the analysis of hexosamines in glycoconjugates and hexosaminitols generated from glycoconjugates following alkaline borohydride treatment.

The use of reverse-phase high-performance liquid chromatography and precolumn derivatization with dansyl chloride for quantitation of specific amino acids in collagen and elastin

A rapid and accurate reverse-phase high-performance liquid chromatography procedure for amino acid analysis of connective tissue proteins has been optimized. The method is based on quantitative dansyl chloride precolumn derivatization of protein hydrolyzates and chromatographic separation of the dansyl dervatives on Ultrasphere ODS C18 column. High molar uv absorption of Pro and Hyp derivatives, quantitation of low Des and Ide amounts (60 pmol), and good separation of all the amino acid derivatives (CV mostly <0.1%) including Hyl overcome the difficulties of other methods in producing reliable single run amino acid analysis of collagen and elastin. The use of fluorescence detector and the possibility of concentrating derivatized samples would give even higher sensitivity to the system. The procedure appeared to be suitable for single run analysis of other proteins, particularly those having an unbalanced amino acid composition.

Rat plasma levels of amino acids and related compounds during stress

Forty-one amino acids and related compounds were measured (using an HPLC physiological amino acid analysis procedure fully validated for plasma studies) in rat plasma obtained through an indwelling jugular catheter before, during and following a 30 min period of immobilization. Taurine, phosphoethanolamine, aspartic acid, glutamic acid, alanine, cystine, tyrosine, β-alanine and ethonolamine were increased during the period of stress; whereas, valine, tryptophan and arginine were decreased. Most of these alterations were restored toward normal during the 30 min of rest following the stress period. However, cystine, ethanolamine and β-alanine remained significantly elevated, and valine, tryptophan and arginine remained significantly reduced. Serine, isoleucine, leucine and glutamine were not significantly altered during the stress period, but became significantly reduced during the 30 min following the stress period. While the patterns of amino acid alterations were generally consistent from animal to animal, the magnitude of the responses were variable with some rats demonstrating much larger responses than others. These results may implicate amino acids as important markers for stress related pathologies. The individual differences noticed may explain why some individuals show more stress effects than others.

Comparison of amino acid levels in rat blood obtained by catheterization and decapitation

A novel, sensitive and highly resolving amino acid analysis procedure was developed and used to compare two methods of obtaining blood from experimental animals. The procedure, utilizing a meter long microbore HPLC column containing spherical cation-exchange resin and fluorescence detection following postcolumn reaction with o-phthaldialdehyde, was shown to reliably measure forty-one primary amine components in rat plasma. Comparison of values from blood obtained by decapitation and by catheterization documented the significant artifactual influence of the decapitation procedure on approximately half of the measured constituents.

A sensitive and accurate method for amino acid analysis using isotopic double labeling with fluorodinitrobenzene

A double-labeling procedure for amino acid analysis using 3H-labeled 1-fluoro-2,4-dinitrobenzene and 14C-labeled amino acids as internal standards is described. The procedure was tested by analyzing lysozyme and insulin B chain, and the results obtained were in good agreement with their accepted amino acid compositions. Analysis of samples containing from 100 pmol of each amino acid can be achieved at an accuracy comparable to that obtained by conventional automated amino acid analysis methods, many of which require considerably more material. An important advantage is that amino acids present in low molar proportions can be separated and measured more readily than by column chromatography.

Three-directional thin-layer chromatographic method for detection of amino acids in urine

In this paper, the standard two-directional chromatographic procedure for amino acid analysis is modified such that it is less liable to error, and is suitable for routine laboratory screening for metabolic abnormalities. The cellulose layer is divided into two equal halves, and the sample is spotted onto the left side and the standard is spotted onto the right side. The plate is developed in the first direction with one solvent system. Then for the second and third directions, the left and right sides of the plate are developed in opposite directions with another solvent system. After color development, it is demonstrated that the two sides are almost mirror images, and therefore, errors in qualitative analysis are unlikely.