Underivatized Amino Acids Research Articles

Proteinaceous binders identification in the works of art using ion-pairing free reversed-phase liquid chromatography coupled with tandem mass spectrometry

A simple, fast and reliable procedure for the proteinaceous binders identification in the works of art samples is presented. The procedure consisted of ammonia extraction in order to suppress pigment interferences, acidic hydrolysis and quantification of underivatized amino acids using reversed phase liquid chromatography coupled with electrospray tandem mass spectrometry (RP-LC–ESI-MS/MS). Fourteen underivatized amino acids were quantified without the addition of ion-pairing agents (IPA) using the multiple reaction monitoring (MRM) mode. The chromatographic separation was optimized by testing three C18 columns and three different eluent compositions. The optimal chromatographic resolution and the ionization efficiency were achieved with Symmetry C18 column and the eluent consisting of water with methanol. The amino acids composition of the proteins commonly found in the paint binding media, eggs, casein and animal glues, was determined. The procedure was tested using a set of naturally aged samples. Calculated detection and quantification limits indicated that the method is suitable for the analysis of protein binders in the paint micro-samples. Animal glues, casein and eggs were identified in the samples from 18th and 19th century paintings by Jacek Malczewski, while eggs and casein were detected in the mural painting samples from the 13th century UNESCO-listed church of Yemrehanna Krestos. The LC/MS/MS based method of the protein binders identification described here can be used as an alternative for the approach based on the gas chromatography coupled with mass spectrometry (GC/MS).

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.

Electromembrane extraction of amino acids from body fluids followed by capillary electrophoresis with capacitively coupled contactless conductivity detection

Electromembrane extraction (EME) proved to be a simple and rapid pretreatment method for analysis of amino acids and related compounds in body fluid samples. Body fluids were acidified to the final concentration of 2.5 M acetic acid and served as donor solutions. Amino acids, present as cations in the donor solutions, migrated through a supported liquid membrane (SLM) composed of 1-ethyl-2-nitrobenzene/bis-(2-ethylhexyl)phosphonic acid (85:15 (v/v)) into the lumen of a porous polypropylene hollow fiber (HF) on application of electric field. The HF was filled with 2.5 M acetic acid serving as the acceptor solution. Matrix components in body fluids were efficiently retained on the SLM and did not interfere with subsequent analysis. Capillary electrophoresis with capacitively coupled contactless conductivity detection was used for determination of 17 underivatized amino acids in background electrolyte solution consisting of 2.5 M acetic acid. Parameters of EME, such as composition of SLM, pH and composition of donor and acceptor solution, agitation speed, extraction voltage, and extraction time were studied in detail. At optimized conditions, repeatability of migration times and peak areas of 17 amino acids was better than 0.3% and 13%, respectively, calibration curves were linear in a range of two orders of magnitude ( r 2 = 0.9968–0.9993) and limits of detection ranged from 0.15 to 10 μM. Endogenous concentrations of 12 amino acids were determined in EME treated human serum, plasma, and whole blood. The method was also suitable for simple and rapid pretreatment and determination of elevated concentrations of selected amino acids, which are markers of severe inborn metabolic disorders.

Screening for Human Milk Amino Acids by HPLC-ESI-MS/MS

Amino acids have a determining effect on the nutritional status of the newborn, and their appropriate levels in breast milk are vital for this first stage of life. The amino acids tryptophan, arginine, glutamate, and taurine, for example, are suggested to have a positive effect on immune functions. The purpose of the present study was to develop a new method for the assay of amino acids in human milk by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after hydrolysis. Breast milk samples were collected from 77 healthy mothers living in the community of Extremadura (Spain) with less than 2 months of lactation. The samples were then stored at −80 °C. The HPLC-ESI-MS/MS technique proved to be a sensitive and efficient tool for the assay of amino acids in breast milk. The method could be used for the qualitative screening of 40 underivatized amino acids, and for the assay of 20. The resulting data will serve to improve commercial infant formula milks and bring them closer to the reference standard represented by human milk.

A simple method for the analysis by MS/MS of underivatized amino acids on dry blood spots from newborn screening

The analysis by electrospray-ionization tandem mass spectrometry of amino acids with butyl esterification and isotopically labeled internal standard is routine in newborn screening laboratories worldwide. In the present study, we established a direct analysis method of higher accuracy that uses a non-deuterated internal standard. The automatic sampler and the pump of an LC apparatus were used to inject sample and mobile phase to MS, but no LC column was needed. The dry blood spot (DBS) material was prepared at levels of low, medium and high concentration; the running time was 1min. In parallel to the new procedure, we applied the established method to analyze nine amino acids on DBS of healthy newborns and phenylketonuria newborns. The newly proposed method of product ion confirmation scan along with multiple reaction monitoring resulted in a very accurate identification of each amino acid. Our innovative protocol had high sensitivity and specificity in the analysis of cases of suspected metabolic diseases.

Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C‐abundance determination by isotope ratio mass spectrometry

Amino acids are the building blocks of proteins and the analysis of their (13)C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ(13)C determination. Mixtures of underivatised amino acids (0.1-0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO 3-, 5-25 mM). The total run time was 70 min. The average δ(13)C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

Advances in Liquid Chromatographic and Voltammetric Analysis of Underivatized Amino Acids

The problems associated with liquid chromatographic analysis of underivatized amino acids are identified and, through reviewing the recent ten years literature, possible solutions are described. Emphasis has been placed on amperometric as well as on multi mode detection. The advantages offered by preliminary voltammetric studies at microelectrodes (under mass transfer conditions similar to those of a flow cell detector) in optimising the operation of amperometric detectors are discussed and the use of environmentally friendly Bi electrode materials for the stripping voltammetric determination of amino acids is also proposed. This review is also aimed at giving a broad outline of the potential for improvement of the separation of underivatized amino acids offered by multimode gradient elution and/or ion-chromatography provided that the relevant separation theory and optimization algorithms are available. Keywords: Underivatized amino acids, liquid chromatographic separation, ultraviolet, conductivity, evaporative light scattering, mass spectrometry, electrochemical, voltammetric analysis, Conductometric detection, capacitively coupled contactless conduc-tivity detection, C4D, RI, refractive index detector, ELS, ELS detector, Chemiluminescent nitrogen, CLN, CLN detector, Fluorescence (FL) Detection, nuclear magnetic resonance, NMR, Electrochemical (EC) or Amperometric Detection, homocystinuria, sinusoidal voltammetry, SV, Linear Sweep, Differential Pulse Voltammetry, LSV, DPV, Adsorptive Stripping Voltammetry, CSV, AdSV, cathodic stripping voltammetry, square wave cathodic stripping voltammetry, SWCSV, Carbon Paste Electrodes, CPEs, Reversed-phase liquid chromatography, RP-HPLC, reversed phase ion-pair chromatography, IPC, drophilic interaction chromatography, HILIC

The Use of N‐Type Ligands in the Enantioselective Liquid–Liquid Extraction of Underivatized Amino Acids

AbstractThe first palladium based extraction system using chiral N‐based ligands in the enantioselective liquid–liquid extraction (ELLE) of underivatized amino acids, is presented. The system shows the highest selectivity for the ELLE of methionine with metal complexes as hosts reported to date. Furthermore, the host can be prepared in situ from commercially available compounds. The dependency of the system on parameters such as pH, organic solvent, and temperature has been established. The intrinsic selectivity was deduced by determination of the association constants of the palladium complex with the tryptophan enantiomers.

Quantitative determination of un-derivatised amino acids in artistic mural paintings using high-performance liquid chromatography/electrospray ionization triple quadrupole mass spectrometry

The tempera painting technique is one of the most common methods used throughout art history. Tempera is defined by the type of binders used and in this work we study protein-based temperas. Proteinaceous binders can be characterized by the chromatographic determination of the amino acids present where techniques are either based on gas chromatography or high-performance liquid chromatography (HPLC) coupled to mass spectrometry. The objective of this work was to develop a derivatisation-free HPLC method with triple quadrupole tandem mass spectrometric detection (HPLC/ESI-MS/MS) of 21 amino acids contained in the protein-based binders of tempera paints. The analytical method identifies the painting techniques of two contemporary artists: Sironi and DeLuigi. The sample data are compared to painting material standards. The results show that the samples from works by DeLuigi contain mainly animal glue binders, while the samples from Sironi paintings contain binders that are an amino acid mixture with an overall composition between that of eggs and casein.

Hydrophilic interaction LC combined with electrospray MS for highly sensitive analysis of underivatized amino acids in rhizosphere research

Analysis of underivatized amino acids is challenging regarding the separation as well as the detection of these small polar analytes. Hydrophilic interaction LC using a 2.1x150 mm ZIC (ZIC, zwitterionic)-hydrophilic interaction LC from SeQuant as stationary phase with 1% v/v formic acid in water and ACN as eluents was combined with MS/MS in multiple reaction monitoring mode for the separation and the detection of 16 underivatized amino acids. Regression coefficients of eight or seven point calibrations varied from 0.9454 to 0.9993. Absolute LODs and LOQ (on column) were in the fmol range (0.1-12 and 0.4-41 fmol, respectively). A fast screening method of 19 min total runtime has been developed offering applicability to samples from rhizosphere studies--characterized by low analyte concentrations and complex matrices. A successful application to the analysis of tyrosine in samples from soil adsorption experiments is presented as well as an evaluated enrichment procedure for amino acids derived from plant culture in nutrient solution.

Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids

The combined effect of the ion-pairing reagent concentration, C(ipr), and organic modifier content, phi, on the retention under phi-gradient conditions at different constant C(ipr) was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D phi-gradient retention data obtained under the same phi-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C(ipr) values in the eluent. The second approach is based on a retention model describing the combined effect of C(ipr) and phi on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C(ipr) values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C(ipr) values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by phi-variation.

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.

Combined Contactless Conductometric, Photometric, and Fluorimetric Single Point Detector for Capillary Separation Methods

This work for the first time combines three on-capillary detection methods, namely, capacitively coupled contactless conductometric (C(4)D), photometric (PD), and fluorimetric (FD), in a single (identical) point of detection cell, allowing concurrent measurements at a single point of detection for use in capillary electrophoresis, capillary electrochromatography, and capillary/nanoliquid chromatography. The novel design is based on a standard 6.3 mm i.d. fiber-optic SMA adapter with a drilled opening for the separation capillary to go through, to which two concentrically positioned C(4)D detection electrodes with a detection gap of 7 mm were added on each side acting simultaneously as capillary guides. The optical fibers in the SMA adapter were used for the photometric signal (absorbance), and another optical fiber at a 45 degrees angle to the capillary was applied to collect the emitted light for FD. Light emitting diodes (255 and 470 nm) were used as light sources for the PD and FD detection modes. LOD values were determined under flow-injection conditions to exclude any stacking effects: For the 470 nm LED limits of detection (LODs) for FD and PD were for fluorescein (1 x 10(-8) mol/L) and tartrazine (6 x 10(-6) mol/L), respectively, and the LOD for the C(4)D was for magnesium chloride (5 x 10(-7) mol/L). The advantage of the three different detection signals in a single point is demonstrated in capillary electrophoresis using model mixtures and samples including a mixture of fluorescent and nonfluorescent dyes and common ions, underivatized amino acids, and a fluorescently labeled digest of bovine serum albumin.

Amino Acid Ionic Liquids as Chiral Ligands in Ligand‐Exchange Chiral Separations

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1-alkyl-3-methylimidazolium L-proline (L-Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers-dl-phenylalanine (dl-Phe), dl-histidine (dl-His), dl-tryptophane (dl-Trp), and dl-tyrosine (dl-Tyr)-were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R(s))=3.26-10.81 for HPLC; R(s)=1.34-4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L-Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L-Pro are consequently attached to the support surface, thus inducing an ion-exchange type of retention for the dl-enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand-exchange-based chiral separation. It also reveals the tremendous application potential of this new type of task-specific ILs.

Enantioseparation of amino acids, α‐hydroxy acids, and dipeptides by ligand‐exchange CEC using silica‐based chiral stationary phases

This work deals with the application of silica-based ligand-exchange chiral stationary phases (CSPs) for the enantioseparation of underivatized amino acids, alpha-hydroxy acids, and dipeptides with packed CEC. Two different possibilities of preparing silica-based CSPs are presented. One phase contains L-4-hydroxyproline chemically bonded via a spacer to 3 mum silica material. The other approach makes use of N-decyl-L-4-hydroxyproline dynamically coated on a reversed-phase packed capillary. Dynamical coating of reversed-phase material represents a simple alternative to prepare CSP. A comparison of the chemically bonded phase with the dynamically coated CSP by means of resolution of complex-forming analytes is presented. The chemically bonded phase was found to be superior to the dynamically coated phase in terms of resolution of amino acids and dipeptides. However, the dynamically coated CSP was found to be especially suitable for the separation of alpha-hydroxy acids. Both techniques are applicable for enantiomer purity tests.

Chiral Separation of Underivatized Amino Acids by Reactive Extraction with Palladium−BINAP Complexes

In answer to the need for a more economic technology for the separation of racemates, a novel system for reactive enantioselective liquid-liquid extraction (ELLE) is introduced. Palladium (S)-BINAP complexes are employed as hosts in the separation of underivatized amino acids. The system shows the highest selectivity for the ELLE of tryptophan with metal complexes as hosts reported to date and shows a good selectivity toward a range of natural and unnatural amino acids. Furthermore, the host can be prepared in situ from commerically available compounds. Bulk-membrane transport in the form of U-tube experiments demonstrates the enantioselective and catalytic nature of the transport. The dependency of the system on parameters such as pH, organic solvent, and host-substrate ratio has been established. (31)P NMR spectroscopy has been used to confirm the preferred enantiomer in the extraction experiments. The intrinsic selectivity was deduced by determination of the association constants of the palladium complex with the tryptophan enantiomers.

Application of amino acid analysis using hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for peptide and protein quantification

Amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography (HILIC) coupled with isotope dilution mass spectrometry (IDMS) has been developed for the accurate quantification of underivatized amino acids from hydrolyzed protein/peptide. Sufficient separation of amino acids on a zwitterion chromatography (ZIC)-HILIC column was achieved after removal of chloride ions in the hydrolyzate. The detection limits and quantification limits as concentration of the four amino acids ranged from 0.003 to 0.04 pmol μL −1 and from 0.01 to 0.1 pmol μL −1, respectively. The analytical results for the certified reference materials, angiotensin I and bovine serum albumin (BSA), were satisfactory. Furthermore, the quantitative results by this method were compared with those by the commercially available precolumn method, derivatizd with aminoquinolylhydroxysuccinimidyl carbamate (AQC method), and better recovery and more precise data were obtained with this method.

Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism

Background An electro-spray ionisation ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) application for the quantitative analysis of amino acids was developed. The suitability for the detection and follow-up of patients suffering from inborn errors of metabolism (IEM) was assessed by extensive cross-validation with ion-exchange liquid chromatography (IEX-LC) with post-column ninhydrin derivatisation, participation in external quality control (ERNDIM) and analysis of samples of patients with confirmed IEM. Methods Prior to analysis plasma and urine samples were merely diluted 150-fold in mobile phase. Amino acids were detected in the multiple reaction monitoring mode (MRM) in the ESI-positive mode. The analytical results were compared with IEX-LC. External quality control scheme performance is presented. Results Comprehensive analysis of amino acids in plasma and urine was achieved with a run-to-run time of 30 min. Validation results were satisfactory and there was a very good correlation between UPLC-MS/MS and IEX-LC. Analytical results obtained in the external quality control scheme were essentially the same as those of the other participants. Patients suffering from IEM were readily identified. Conclusion UPLC-MS/MS analysis of amino acids in body fluids is rapid, reliable and suitable for the diagnosis and follow-up patients with IEM.

Direct UV detection of underivatized amino acids using capillary electrophoresis with online sweeping enrichment

This is an original report proposed a CE method for direct analysis of the underivatized amino acids using UV detection with relatively higher sensitivity, which was based on coordination interactions between amino acids and Cu (II) ions. In addition, an online sweeping preconcentration technique was easily combined to improve the detection sensitivity. Satisfying separations of the amino acids were obtained under optimized conditions: 50 mmol/L CuSO4-0.05% HAc-H2O (pH 4.5), and the separation voltage of 15 kV. The LODs for the analytes ranged from 0.1 to 0.5 micromol/L. The linearity of detection for all analytes was two orders of magnitude with the correlation coefficients greater than 0.99. The repeatability was displayed with an RSD less than 3% for migration time and peak height (n = 5). Moreover, some amino acids in real samples of human saliva and green tea were analyzed by this direct UV detection CE method with acceptable sensitivity.

A three-phase liquid chromatographic method for δ 13C analysis of amino acids from biological protein hydrolysates using liquid chromatography–isotope ratio mass spectrometry

We report a three-phase chromatographic method for the separation and analysis of δ 13C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Both precision and accuracy of δ 13C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ 13C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ 13C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology.