Determination Of Underivatized Amino Acids Research Articles

Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.

In neonatal screening, amino acids have a significant diagnostic role. Determination of their values may identify abnormal conditions. Early diagnosis and continuous monitoring of amino acid disorders results in a better disease outcome. An easy and simple LC-MS/MS method was developed for the quantitation of underivatized amino acids. Amino acids were separated using a normal-phase HPLC column having a totally porous silica stationary phase and using classical reversed-phase eluents. Mass spectrometry in multiple reaction monitoring mode was used for the analysis, providing high selectivity and sensitivity. A standard addition calibration model was applied for quantitation using only one isotope-labeled internal standard for all amino acids. Five calibration points were used for quantitation, and the method was successfully validated. The slopes of the calibration curves of the individual amino acids in parallel measurements were found to be similar. Since the measured slopes were reproducible, one serum sample could represent every series of serum samples of a given day. The method was tested on human serum samples and adequate results were obtained. This new method can be easily applied in clinical laboratories.

Combining Orbitrap-HRMS acquisition modes and direct injection by a guard column for targeted analysis of underivatized amino acids in urine

Nowadays, there is a great interest in the development of rapid methods for the determination of compounds of clinical interest in biological samples, such as amino acids in urine samples. Mass spectrometry-based methods have been proposed with this objective, although the use of low resolution mass analyzers sometimes cannot provide accurate results. Orbitrap has barely been used with quantitative purposes for the targeted analysis of amino acids in urine although it has been used to a greater extent for non-targeted assays to identify potential biomarkers of different diseases.The use of a guard column for sample introduction into the Orbitrap was evaluated. The coupling of a hydrophilic interaction-guard column significantly improved the MS-signals by producing a matrix cleaning effect with a slight displacement of the target analytes. Possible isobaric interferences were studied and, consequently, different modes of MS data acquisition were tested: Full MS acquisition mode was selected for those analytes for which it was possible their determination by exact mass, and parallel reaction monitoring mode was used for those compounds showing isobaric interferences. Method validation was performed using synthetic urine. Limits of detection between 0.003 and 1.50 mg/L and limits of quantification between 0.10 and 4.95 mg/L were found with recoveries in the 80–130 % range. The runtime was 1.6 min and the total time between injection was less than 3 min. The optimized method was satisfactorily applied to the analysis of urine from healthy volunteers.This work describes for the first time the direct coupling of a hydrophilic interaction-guard column to a Orbitrap high resolution mass spectrometer (HRMS) for the development of a fully validated quantitative method for the determination of underivatized amino acids in urine. The method proposed here represents a high throughput and reliable approach, with an analysis runtime of 1.6 min, much less than the time required when chromatographic techniques were used.

Determination of underivatized amino acids in human plasma using ion pair liquid chromatography/tandem mass spectrometry

Accurate quantification of amino acids (AA) is essential for several applications, including clinical research, food analysis, and pharmaceutical studies. In this study, we developed an analytical method based on liquid chromatography with electrospray ionization coupled to tandem mass spectrometry detection (LC-ESI-MS/MS). This method was devised to accurately quantify a spectrum of amino acids, notably taurine, creatinine, glutathione (GSH), and sulfur-containing amino acids (SAAs) such as methionine, cysteine, and homocysteine, using only 10 μL of human plasma. A stable isotope derivative of each AA is used as an internal standard (IS) for accurate quantification. For retention and separation on a C18 column, heptafluorobutyric acid (HFBA) was employed as an ion pair agent. Multiple reaction monitoring (MRM) in positive mode with the precursor-to-product ion transitions at m/z is used for quantification. The method showed excellent linearity for all AA with a high correlation coefficient (r > 0.9927). The linear fit indicates that the detector response is linear over the tested range of standard concentrations. The accuracy and precision of the method were within the acceptable range of 92–110% and < 15%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were in the range of 0.001–1.80 µM and 0.004–6.0 µM, respectively. No significant ion suppression or carry over was observed. In conclusion, the assay was validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The assay has been successfully applied to the analysis of human plasma.

Determination of seventeen free amino acids in human urine and plasma samples using quadruple isotope dilution mass spectrometry combined with hydrophilic interaction liquid chromatography – Tandem mass spectrometry

Taking into account the growing demand for new analytical procedures that are appropriate for analysis of complex biological samples with increased sensitivity, accuracy and precision, a novel analytical method was described for the determination of underivatized amino acids in human plasma and urine samples. The presented analytical procedure involved the direct analysis of urine samples and the analysis of plasma samples followed by a simple protein precipitation protocol. Samples were analyzed using a simple and fast chromatographic method developed for the determination of 17 different amino acids by liquid chromatography - tandem mass spectrometry. The limit of detection and quantification values for amino acids were ranged between 0.03–2.26 µmol kg−1 and 0.09–7.54 µmol kg−1. Matrix effects of plasma and urine on the quantification of analytes were determined by spiking experiments. The accuracy of method was evaluated by matrix matching and quadruple isotope dilution strategies. Excellent accuracy and precision were obtained with the use isotope labeled amino acids demonstrating the high reliability and reproducibility of the proposed method. The percent recovery values were found to be between 98.70 - 101.68% with%RSD below than 1.62% for human plasma and 99.14 - 101.78% with%RSD below than 2.44% for urine samples.

Optimal Conditions for the Direct RP-HPLC Determination of Underivatized Amino Acids with Online Multiple Detection.

The combined use of a dual-UV detector as well as a fluorimetric and a multielectrode electrochemical detector (equipped with a dual electrode, consisting of a conventional size 3mm diameter glassy carbon electrode (GCE) and of a pair of 30μm thick carbon microfibers) is proposed for the detection of the following 15 underivatized amino acids: L-histidine (His), L-cysteine (Cys), creatine (Crn), S-methyl-L-cysteine (Me-Cys), DL-homocysteine (Hcy), L-methionine (Met), beta-(3,4-dihydroxyphenyl)-L-alanine (DOPA), L-tyrosine (Tyr), DL-m-tyrosine (m-Tyr), L-a-methyl-DOPA (Me-DOPA), L-phenylalanine (Phe), DL-alpha-methyltyrosine (Me-Tyr), 5-hydroxy-tryptophan (5-HTP), 3-nitro-L-tyrosine (NO2Tyr), and L-tryptophan (Trp), as well as of 2 dipeptides L-cystathionine (Cysta) and L-carnosine (Car), and of creatinine (Cre). A multilinear solvent (acetonitrile) gradient elution program, determined by a simple optimization algorithm, is required for the efficient reversed-phase separation of the above mixture of 18 solutes within 27min at a flow rate of 1.0mL/min and at 25°C.

A novel hydrophilic interaction liquid chromatography method for the determination of underivatized amino acids in alimentary supplements

Amino acids playing important roles in metabolic processes are often included in dietary supplements whose use has largely expanded over the last 20 years not only in patients with particular deficiencies, but also in athletes and even common people that want to enrich their regular daily diet. In the present study, a bare silica Kinetex core-shell 2.6μm HILIC column was used for separation of some important hydrophilic amino acids and amino acids-like molecules i.e., aspartic acid, creatine, carnitine, arginine and the tripeptide glutathione (GSH), by optimizing the chromatographic conditions for their determination in complex alimentary supplements. The contribution of partition, adsorption and ion exchange on the retention mechanism was studied by varying parameters such as water content and the counter-ion concentration in the mobile phase. Optimum conditions employed a Phenomenex Kinetex core-shell 2.6μm HILIC (100×4.6mm i.d.) column and a mobile phase of acetonitrile/potassium phosphate buffer (12.5mM; pH=2.8) 85:15, v/v, at the flow rate of 1.4mL/min, using UV detection at 200nm. A reference HPLC method for the selective determination of GSH by using 1,4-naphthoquinone as derivatization reagent was also introduced for comparative purposes.The developed HILIC method was validated and applied to the analysis of the considered compounds in dietary supplements. Interestingly, in some of the real samples, oxidized glutathione which is an inactive impurity of GSH, was found at the level of about 20%. The proposed study confirms the importance of simple analytical methods for a rigorous quality control of dietary supplements containing unstable active ingredients.

Determination of underivatized amino acids in microsamples of a yeast nutritional supplement by LC-MS following microwave assisted acid hydrolysis

In this paper we describe a rapid method for microscale microwave assisted acid hydrolysis followed by quantitative amino acid analysis, using liquid chromatography mass spectrometry UPLC-ESI-MS (QTOF) without derivatization.

Direct determination of underivatized amino acids from Ginkgo biloba leaves by using hydrophilic interaction ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry

Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. In addition to the well-known flavonol glycosides and terpene lactones, G. biloba leaves are also rich in amino acids. To determine the content of free amino acids, a reliable method has been established by using hydrophilic interaction ultra-HPLC coupled with ESI-MS. 20 free amino acids were simultaneously determined without derivatization in 12 min. The proposed method was fully validated in terms of linearity, sensitivity, repeatability, as well as recovery. Furthermore, the principal component analysis was applied to different G. biloba leaves collected in November (after fruit harvest season), which revealed that the samples from different production areas exhibited regional disparity in different clusters in accordance with their various hydrophilic interaction chromatograms coupled with mass profiles. The established approach could be helpful for evaluation of the potential values as dietary supplements and the quality control of G. biloba leaves, which might also be utilized for the investigation of other medicinal herbs containing amino acids.

Capillary electrophoresis coupled to evaporative light scattering detection for direct determination of underivatized amino acids: Application to tea samples using carboxyled single‐walled carbon nanotubes for sample preparation

An improved and efficient method for the determination of underivatized amino acids based on the use of CE coupled to evaporative light scattering detector (ELSD), involving carbon nanotubes, was successfully developed. Carboxyled single-walled carbon nanotubes were used for the first time to perform the clean-up of the analyzed samples, which were afterwards analyzed by CE-ELSD. White tea samples were used to demonstrate the usefulness of the CE-ELSD coupled methodology. A suitable interface, based on a triple tube design sprayer, was developed and successfully used for coupling both instruments. Parameters affecting the separation and determination, including the elimination of interferences, were studied and properly optimized. Under the optimized conditions good resolution was achieved for the separation of seven amino acids. The precision of the method, expressed as RSD, was found within the 3.5-5.3% range. The LOD obtained for the proposed method were in the 1.2-2.1 pg range and the LOQ, were in the 2.0-11.5 pg range, with injection pressure of 5 KPa for 20 s (15.3 nL). This method is simple, rapid, and selective compared with other conventional techniques.

Development of a method for the analysis of underivatized amino acids by liquid chromatography/tandem mass spectrometry: Application on Standard Reference Material 1649a (urban dust)

A liquid chromatography–tandem mass spectrometry analytical procedure has been developed for the detection and quantitative determination of underivatized amino acids at low concentrations in a Standard Reference Material—urban dust. In order to minimize interferences of other compounds, an accelerated solvent extraction followed by a solid phase extraction on two different cartridges was applied prior to LC–MS–MS.Fourteen amino acids were separated by high resolution liquid chromatography, detected and quantified by multiple reaction monitoring on a triple quadrupole. The proposed methodology has been applied for the first time on Standard Reference Material 1649a (urban dust) from the National Institute of Standards and Technology, that does not report certification values for these compounds.This methodology avoids the derivatization step and allows the amino acid quantification in a complex matrix, such as that of atmospheric particulate matter, and represent a good method suitable to analyze this class of compounds in atmospheric aerosol.The selected strategy demonstrated to be fit-for-purpose, by applying it to a real atmospheric sample with the aim to verify the efficacy of the study and to provide information about the organic matter content.

Optimization of dynamic pH junction for the sensitive determination of amino acids in urine by capillary electrophoresis

A capillary electrophoresis method with UV-absorbance detection was studied and optimized for the determination of underivatized amino acids in urine. To improve concentration sensitivity the utility of in-capillary analyte stacking via dynamic pH junction was investigated with phenylalanine (Phe) and tyrosine (Tyr) as model amino acids. Before sample injection, a plug of ammonium hydroxide solution was injected to enable analyte concentration. Samples were 1:1 (v/v) mixed with background electrolyte (1 M formic acid) prior to injection. The effect of the injected sample volume, and the injected ammonium hydroxide volume and concentration on analyte stacking and separation performance was investigated. The optimal volume of ammonium hydroxide depended on the injected sample volume. Using a dynamic pH junction good resolution (1.4) was obtained for a sample injection volume of 10% of the capillary (196 nl) with Phe and Tyr dissolved in water. Limits of detection (LODs) were 0.036 and 0.049 μM for Phe and Tyr, respectively. For urine samples, the optimized procedure comprised a 1.7-nl injection of 12.5% ammonium hydroxide, followed by a 196-nl injection of urine spiked with Phe and Tyr. Satisfactory resolution was obtained and amino acid peak widths at half height were only 1.6 s indicating efficient stacking. Calibration plots for Phe and Tyr in urine showed good linearity (R(2) > 0.96) in the concentration range 10-175 μM, and LODs for Phe and Tyr were 0.054 and 0.019 μM, respectively. RSDs for peak area and migration time for Phe and Tyr were below 7.5% and 0.75%, respectively.

Rapid determination of underivatized amino acids in fertilizers by ultra high performance liquid chromatography coupled to tandem mass spectrometry

The analysis of 19 underivatized protein amino acids by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is studied. Amino acids were separated by reversed phase, adding to the mobile phase pentadecafluorooctanoic acid as ion pairing reagent. The selected amino acids were eluted in less than 8 min. MS/MS parameters were optimized, using electrospray ionization (ESI) in positive mode for the detection of the amino acids. A simple solid-liquid extraction with water and heptafluorobutyric acid was used for the extraction of the compounds from the fertilizer. All amino acids were extracted with recoveries higher than 70% and relative standard deviation (RSD) lower than 22.5% (inter-day precision). Limits of quantification were always lower than 100 mg kg−1 of sample, for all the compounds. The validated method is simple, fast and sensitive and it has been applied for the determination of amino acids in fertilizers.

Direct RP‐HPLC determination of underivatized amino acids with online dual UV absorbance, fluorescence, and multiple electrochemical detection

The combined use of a dual-UV detector, a fluorimetric one and of a multiple electrochemical (EC) detector equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 mum thick carbon microfibers, is proposed for the determination of 15 amino acids, two dipeptides and creatinine. This online coupling of the above detection modes could partially replace amino acid analysis by derivatization methods, since it solves problems concerning the direct detection of selected underivatized amino acids. Additionally, it was proved that the use of multiple-detection allows positive peak identification in a single chromatographic run, yields more information for free amino acids and solves in some cases the problem of chromatographic resolution. In order to optimize the detection conditions of the underivatized amino acids and related compounds by different detectors, their detection characteristics were determined by adequate preliminary experiments. The electro-oxidation characteristics of the underivatized compounds of interest were determined by hydrodynamic voltammetry using a flow cell with a macrodisc GCE and by ex-situ voltammetry using both a GCE of conventional size and a carbon fiber disk microelectrode. Important practical advantages of microfiber and microdisk electrodes with respect to macroelectrodes were demonstrated.

Neutral loss analysis of amino acids by desorption electrospray ionization using an unmodified tandem quadrupole mass spectrometer

A new method to analyze free amino acids using desorption electrospray ionization (DESI) has been implemented. The method is based on the neutral loss mode determination of underivatized amino acids using a tandem quadrupole mass spectrometer equipped with an unmodified atmospheric interface. Qualitative and quantitative optimization of DESI parameters, including ESI voltage, solvent flow rate, angle of collection and incidence, gas flow and temperatures, was performed for amino acids detection. The parameters for DESI analysis were evaluated using a mixture of valine, leucine, methionine, phenylalanine and tyrosine standards. A few microliters of this mixture were deposited on a slide, dried and analyzed at a flow rate of 2 microL/min. The optimal ionization response was obtained using laboratory glass slides and an equivalent solution of water/methanol doped with 2% of formic acid. The method specificity was evaluated by comparing product ion spectra and neutral loss analysis of amino acids obtained either by DESI or by electrospray ionization flow injection analysis (ESI-FIA). To evaluate the quantitative response on amino acids analyzed by DESI, calibration curves were performed on amino acid standard solutions spiked with a fixed amount of labelled amino acids. The method was also employed to analyze free amino acids from blood spots, after a rapid solvent extraction without other sample pretreatment, from positive and negative subjects. The method enables one to analyze biological samples and to discriminate healthy subjects from patients affected by inherited metabolic diseases. The intrinsic high-throughput analysis of DESI represents an opportunity, because of its potential application in clinical chemistry, for the expanded screening of some inborn errors of metabolism.

Application of gradient programs for the determination of underivatized amino acids and small peptides in reversed-phase high-performance liquid chromatography with contactless conductivity detection

The application of gradient methods in HPLC determinations with capacitively coupled contactless conductivity detection was successfully demonstrated for non-UV-absorbing amino acids as well as for small peptides as analytes. The drift in baseline which is caused by the change in the conductivity of the mobile phase during the elution could be minimized by careful optimization of the composition of the mobile phase. For such conditions the remaining baseline drift could be corrected for either by subtraction of a blank chromatogram or by baseline normalization using a software package for chromatographic data processing. Reverse phase separations were carried out using aqueous eluents containing either methanol or acetonitrile and acetic acid or trifluoroacetic acid. Detection limits between 1 and 40 μM were achieved for the amino acids and the di- and tri-peptides investigated. The reproducibilities for the peak areas were typically found to be about 2% and good linearity was obtained for the concentration range tested (up to 1000 μM).

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high‐field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.

Comparison of Capillary Zone Electrophoretic Techniques Combined with Indirect UV, Direct UV, and Mass Spectrometric Detection for the Determination of Underivatized Amino Acids and Vitamin B6 in Infusion Solutions

Different methods based on capillary zone electrophoresis (CZE) for the determination of underivatized amino acids and vitamin B6 in infusion solutions are described and compared. Acidic and basic carrier electrolytes have been investigated with respect to their suitability for the separation of the selected analytes. Focusing on detection, indirect and direct UV detection at different wavelengths, as well as mass spectrometric (MS) detection have been tested, with the main emphasis set on their ability to provide quantitative results for all analytes in a single run. Finally, an acidic carrier electrolyte based on phosphoric acid and direct UV detection at 195 nm, were found to be the best suited solution for the determination of the six amino acids and vitamin B6 in the investigated real samples.

Isocratic ion chromatographic determination of underivatized amino acids by electrochemical detection

An isocratic chromatographic separation with amperometric detection of underivatized amino acids at a copper oxyhydroxide modified glassy carbon electrode is described. A simple and sensitive quantitation procedure of amino acids without the need of tedious and time-consuming derivatization step was achieved by coupling anion-exchange chromatography with electrochemical detection. The effects of hydroxide, nitrate and acetonitrile concentration in the mobile phase on the capacity factors and peak resolution was evaluated. Under the optimized isocratic chromatographic conditions (i.e. 60 mM NaOH) and under constant applied potentials (i.e. 0.55 V versus Ag/AgCl) the detection limit ranged between 4 and 24 pmol injected and the linear dynamic range spanned generally over three or four order of magnitude for all investigated amino acid compounds. Direct determination of several common free amino acids in beer and milk samples were performed.

Determination of underivatized amino acids by high-performance liquid chromatography and electrochemical detection at an amino acid oxidase immobilized CuPtCI6 modified electrode.

An amperometric sensor for amino acids based on the immobilization of amino acid oxidase on the surface of a CuPtCl(6)/GC is described. The amperometric current is due to the oxidation of H2O2 liberated during the enzyme reaction on the surface of the enzyme electrode. The electrode response characteristics as well as kinetic parameters have been evaluated. The enzyme electrode was characterized as an electrochemical biosensor, which was used as detector in high performance liquid chromatography (HPLC) for the determination of a mixture of amino acids with satisfactory results.

Amperometric determination of underivatized amino acids at a nickel-modified gold electrode by anion-exchange chromatography

A nickel-based composite electrode obtained by anodic electrodeposition of nickel (III) oxyhydroxide film on the gold electrode substrate was characterized as an amperometric sensor and successfully applied to the determination of underivatised amino acids in flow-through systems. The electrodeposition of nickel oxyhydroxide films was obtained by cycling a gold electrode between 0.0 V and +1.0 V vs. a saturated calomel electrode in a 80 μ M Ni 2+ solution buffered at pH 10 with NaHCO 3/Na 2CO 3. The resulting Au–Ni composite electrode exhibits good stability in alkaline medium and can be used as an amperometric sensor of underivatised amino acids at a fixed applied potential (+0.55 V vs. Ag/AgCl). The detection limits ( S/N=3) for all investigated compounds ranged between 5 and 30 pmol injected, while the linear ranges spanned over two or three orders of magnitude. The contents of several free amino acids in two sample cheeses from different brands were evaluated by calibration graphs.