Analysis Of Underivatized Amino Acids Research Articles

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.

Fast, Direct Analysis of Underivatized Amino Acids in Infant Formula

Fast, Direct Analysis of Underivatized Amino Acids in Infant Formula

High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS.

Amino acids and acylcarnitines are important biomarkers of the body's energy state and can be used as diagnostic markers of certain inborn errors of metabolism. Few multianalyte methods for high-throughput analysis in serum exist for these compounds, but micromethods suitable for use in young children and infants are lacking. Therefore, we developed a quantitative high-throughput multianalyte hydrophilic interaction liquid chromatography-tandem mass spectrometry method preceded by a derivatization-free sample preparation using minimum amounts of serum (25 μL). Isotopically labeled standards were utilized for quantification. Forty amino acids and amino acid derivatives and 22 acylcarnitines were detected by applying a multiple reaction monitoring mode within a 20 min run. The method was comprehensively validated, comprising linearity, accuracy, (intraday/interday) precision, and quantitation limits, of which the latter ranged from 0.25 to 50 nM for acylcarnitines and from 0.005 to 1 μM for amino acids and their derivatives. Application of the method to 145 serum samples of three- to four-month-old healthy infants showed excellent reproducibility for multiday analyses and enabled simultaneous amino acid and acylcarnitine profiling in this age group.

Combining Orbitrap-Hrms Acquisition Modes and Direct Injection by a Guard Column for Targeted Analysis of Underivatized Amino Acids

Combining Orbitrap-Hrms Acquisition Modes and Direct Injection by a Guard Column for Targeted Analysis of Underivatized Amino Acids

Capillary electrophoresis-mass spectrometry as a tool for the noninvasive target metabolomic analysis of underivatized amino acids for evaluating embryo viability in assisted reproduction.

Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 μL)ofspent culture media collected after the cultivation of in vitro fertilized embryos. Special attention was focused on the sample preparation procedure. The sample was processed with acetonitrile, which facilitates online sample preconcentration via field-amplified sample stacking, and undesired sample evaporation was significantly reduced by the simultaneous addition of dimethyl sulfoxide. Key parameters that affected electrophoretic separation and mass spectrometric detection were investigated, including the type of buffers and organic solvent, optimization of their concentrations, and finally the settings for their ionization. The separation and quantification of 19 amino acids were achieved using 15% acetic acid as the background electrolyte with a sheath liquid consisting of an equimolar mixture of methanol and water. The applicability of the optimized system was demonstrated by determining the amino acid profile in 40 samples of spent cultivation medium in this pilot study. This developed method also has great potential for amino acid analyses in minute sample volumes of other biological matrices.

Rapid and reliable analysis of underivatized amino acids in urine using tandem mass spectrometry

Nowadays there is a great interest in the development of fast and reliable methods for the determination of amino acids (AAs) in biological samples due to their biological importance. In the present work, a method based on the use of a guard column (gC) prior to tandem mass spectrometry (gC-MS/MS) is proposed for the determination of proteinogenic AAs in urine. Heptafluorobutyric acid (HFBA) is used as ion pairing reagent. Comparison of the gC-MS/MS method versus tandem mass spectrometry (MS/MS) in stand-alone mode showed improved sensitivity and peak shape, and solved some problems related to interfering compounds, with a total analysis time of 2.8 min. All the proteinogenic AAs were adequately determined using the gC-MS/MS method, except glutamic acid (Glu).To confirm quantitative results obtained with gC-MS/MS for individual AAs, an ion-pair liquid chromatography tandem mass spectrometry method (LC-MS/MS) has also been developed. Both methods (gC-MS/MS and LC-MS/MS) were validated using synthetic urine. For the gC-MS/MS method, LODs and LOQs values were found to be between 0.004 and 0.425 mg/L and 0.01 and 1.40 mg/L, respectively. Aspartic acid (Asp) showed the highest LOD and LOQ values (3 mg/L and 9 mg/L, respectively). A one-point standard addition method and internal standard normalization were used for the quantification because matrix effects were observed. L-alanine-1-13C (Ala-13C) and L-leucine-1-13C (Leu-13C) were used as isotopically labeled internal standards.To demonstrate the applicability of the gC-MS/MS method in the reliable determination of AAs in real samples, urine from eighteen healthy volunteers were analyzed using both gC-MS/MS and LC-MS/MS methods. Similar quantitative results were obtained for individual AAs with both of them. In addition, possible differences in AAs concentrations related to sex were checked, but the results did not show significant differences for the evaluated compounds.

Performance of ion pairing chromatography and hydrophilic interaction liquid chromatography coupled to charged aerosol detection for the analysis of underivatized amino acids

The charged aerosol detector (CAD) is frequently employed in liquid chromatography for the analysis of small polar and ionizable compounds such as amino acids and amino sugars, which provide a weak chromophore only. Separation of these compounds is achieved by means of ion pair chromatography (IPC), and, more recently, hydrophilic interaction chromatography (HILIC) techniques. However, as the CAD's response is highly dependent on the mobile phase composition, the substantial differences in the mobile phase composition of IPC and HILIC have a distinct impact on the detector's performance. This study was aimed at systematically comparing the performance of IPC and HILIC when coupled to the CAD. Therefore, the separation techniques characterized by their specific mobile phase compositions were evaluated for their influence on the CAD response and the signal-to-noise ratio (S/N) of the amino acids L-alanine, L-leucine, and L-phenylalanine applying the response surface methodology (RSM). The RSM results derived from flow injection analysis (FIA) indicated that the CAD response and thus the obtainable S/N are significantly higher in HILIC compared to IPC where the S/N decreased with the chain length of the applied ion-pairing reagent. In addition, an IPC and a HILIC method, respectively, were developed for the impurity profiling of the branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. The beneficial effects of the HILIC conditions on the S/N observed under FIA conditions were partly offset by moderate column bleed effects when using an amide functionalized column, which facilitates the separation in the HILIC method. Satisfactory LOQs (3-10 ng on column) were obtained with both methods; however, the HILIC method was found to be slightly superior in terms of sensitivity and separation efficiency.

Rapid quantification of underivatized alloisoleucine and argininosuccinate using mixed-mode chromatography with tandem mass spectrometry

Plasma elevations of the amino acids alloisoleucine and argininosuccinic acid (ASA) are pathognomonic for maple syrup urine disease and argininosuccinate lyase deficiency, respectively. Reliable detection of these biomarkers is typically achieved using methods with tedious sample preparations or long chromatographic separations, and many published amino acid assays report poor specificity and/or sensitivity for one or both of these compounds. This report describes a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method that provides rapid quantification of alloisoleucine and ASA in human plasma. The basis of this method is a mixed-mode solid phase separation that achieves baseline resolution of alloisoleucine from isobaric interferents without the use of derivatization or ion pairing agents. The inject-to-inject time is 6 min including elution, column washing and re-equilibration. Validation studies demonstrate excellent limits of quantification (1 μmol/L), linearity (r = 0.999 from 1 to 250 μmol/L), accuracy (bias = −3.8% and −10.1%), and inter-assay imprecision (CV < 8.06%) for plasma analyses. Data from long-term clinical application confirms chromatographic consistency equivalent to more traditional reversed-phase or HILIC based columns. Additional matrix studies indicate low suppression (<10%) for a wide range of amino acids and compatibility with other matrixes such as blood spot analyses. Finally, analysis of our first 257 clinical specimens demonstrates high analytic specificity and sensitivity, allowing the detection of subtle but clinically relevant elevations of alloisoleucine and ASA that may be missed by other less sensitive methods. In conclusion, the novel LC-MS/MS method reported here overcomes a number of the challenges associated with alloisoleucine and ASA quantification. Combining this approach with published incomplete amino acid quantification methods allows, for the first time, a rapid and comprehensive LC-MS/MS analysis of underivatized amino acids without the use of ion pairing agents.

A rapid, sensitive, and widely applicable method for quantitative analysis of underivatized amino acids in different biological matrices by UHPLC-MS/MS.

A rapid, sensitive, and widely applicable method for the simultaneous quantitative analysis of 20 underivatized amino acids in different biological matrices, including serum, plasma, and tissue homogenates, using ultra high performance liquid chromatography with tandem mass spectrometry was developed and validated. Only 4µL of serum, plasma, or tissue homogenate was extracted with 996 µL of solution (1.7mM ammonium formate in 85% acetonitrile containing 0.1% formic acid) containing 100ng/mL phenylalanine-d5 as an internal standard without any further derivatization step. In addition, the matrix effects were small because a large volume of extraction solution was used. The total run time including reequilibration was 13min. The results of linearity, accuracy, repeatability, precision, limits of detection, limits of quantification, and sample stability were sufficient to allow the measurement of the amino acids in different biological matrices. We conclude that our method is rapid, sensitive, and widely applicable and represents an improvement over other currently available technologies.

Direct Analysis of Underivatized Amino Acids in Plant Extracts by LC-MS/MS (Improved Method).

In this chapter we describe a method for quantification of 20 proteinogenic amino acids by liquid chromatography-mass spectrometry which affords neither derivatization nor the use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5μ SCX 100Å) with 5% acetic acid in water (A) and 75mM ammonium acetate in water (B). Quantification is accomplished by use of d2-phenylalanine as internal standard achieving limits of detection of 5-50nM. The method was successfully applied for the determination of proteinogenic amino acids in plant extracts.

Analysis of Underivatized Amino Acids: Zwitterionic Hydrophilic Interaction Chromatography Combined with Triple Quadrupole Tandem Mass Spectrometry.

Analysis of underivatized amino acids is challenging regarding both separation and detection of this small, polar, and largely UV-inactive compounds. Additives for reversed phase chromatography such as ion pairing reagents can hamper mass spectrometric detection. Zwitterionic hydrophilic interaction chromatography using MS compatible eluents together with tandem mass spectrometry in multiple reaction monitoring mode for selective detection is an attractive approach to overcome the abovementioned issues.

Quantitative Analysis of Underivatized Amino Acids by Liquid Chromatography-Tandem Mass Spectrometry.

Quantitative amino acid analysis has diverse applications in clinical diagnostics, biomedical research, and agriculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and specific detection of amino acids in comparison to traditional, gold-standard ninhydrin-based methods. However, triple quadrupole mass spectrometers are unable to definitively differentiate isomers and are susceptible to ion suppression, both of which prevent accurate quantitation. Therefore, appropriate chromatography must be applied before ionization.We have shown that two-dimensional LC enables rapid and specific amino acid quantitation without derivatization by resolving isomers, such as alloisoleucine, isoleucine, and leucine, and reducing matrix effects (Le et al., J Chromatogr B Analyt Technol Biomed Life Sci 944:166-174, 2014). In this clinically validated protocol, we provide an updated description of the chromatographic setup and selected reaction monitoring (SRM) transitions. Then, we describe sample processing for serum, plasma, urine, cerebral spinal fluid, and dried blood spots. Most importantly, we outline a singular quantitative design for efficient data analysis of the listed sample types and quality assurance strategies to ensure test fidelity. Lastly, we share extensive knowledge critical to the success of this method. A liquid sample can be processed and be ready for injection within 5min, and each sample is analyzed by the MS in 14.5min.

Quantitative analysis of underivatized amino acids in the sub- to several-nanomolar range by ion-pair HPLC using a corona-charged aerosol detector (HPLC–CAD)

The separation and quantification of underivatized naturally occurring amino acids (AAs) by high-performance liquid chromatography (HPLC) is advantageous for reducing preparation time, running costs, and analytical errors, and is compatible with the isolation of intact AAs. This study establishes and validates an analytical method for the separation and quantification of underivatized AAs and taurine (Tau) in the sub- to several-nanomolar range (ca. 0.1–1.6 nmol) by optimizing ion-pair HPLC coupled to a corona charged aerosol detector (corona CAD). Chromatographic separation of 19 AAs and Tau was achieved using a porous graphite carbon (PGC) column and nonafluoropentanoic acid (NFPA) as a volatile ion-pair reagent. The response of the corona CAD to the AAs was highly dependent on the eluate composition, whereas these response factors were similar for AAs in eluate with similar compositions. Regression curves and coefficients (r2) >0.998 were obtained for plots of injection amount versus peak area, except for Arg which co-eluted with a background peak. On the other hand, all plots of injection amount versus peak height regressed to curves with r2 > 0.997. Repeat quantification based on peak area showed lower relative standard deviations (RSDs) (typically better than 5%) than those based on peak height. The present method is useful for quantifying AAs from proteins and Tau in the sub- to several-nanomolar range without derivatization, and constant repeatability can be maintained by quantification using peak areas.

A rapid and precise method for the analysis of underivatized amino acids in natural samples using volatile-ion-pairing reverse-phase liquid chromatography–electrospray ionization tandem mass spectrometry

Amino acids are effective tracers of organic carbon and nitrogen cycling in natural substrates. However, total analytical time can be long due to extraction, derivatization, and chromatography. Here, we present a liquid chromatographic separation of 19 naturally occurring amino acids requiring no derivatization using triple-quadrupole mass spectrometry. This method builds on recent advances in volatile ion pairing and was applied to natural soil samples collected from two distinct systems with highly variable organic C contents. Separation and quantification of amino acids were achieved with an 18 min sample-to-sample run time employing a one-point standard addition to account for variable matrix effects during ionization. Detection limits ranged from 5.9 to 187.5 fmol (mean 30.0 fmol) while instrumental precision averaged 5.8% and 12.0% for intra- and inter-day error, respectively. The highest yields (mean > 100 µmol/g C) in our natural substrates were observed for glycine, aspartic acid, alanine, and glutamic acid while low yields (mean < 20 µmol/g C) were observed for all non-proteinogenic amino acids as well as histidine and tyrosine. A typical hot acid hydrolysis in 6N HCl was used and hydrolyzates were diluted and filtered rather than being subjected to solid-phase extraction or similar techniques, which require significant investment of time during sample preparation. This method enables high throughput and reliable analysis of hydrolyzable amino acids, while also reducing user workload and instrument time compared to previous techniques. The Alaskan tundra system had significantly higher absolute yields of amino acids (mean 88.4 vs 25.6 µmol/g dry sediment), in part driven by higher organic C concentrations, while the Louisiana delta system had significantly higher organic C normalized yields of amino acids (mean 1492.5 vs 541.6 µmol/g C). Despite these differences, both systems exhibited broadly similar mole-percent amino acid compositions.

イオンペアクロマトグラフィー／電子スプレーイオン化質量分析法（LC/ESI-MS）によるアミノ酸のマススペクトル解析

イオンペアクロマトグラフィー／電子スプレーイオン化質量分析法（LC/ESI-MS）によるアミノ酸のマススペクトル解析

Amino acid analysis using chromatography–mass spectrometry: An inter platform comparison study

The analysis of amino acids has become a central task in many aspects. While amino acid analysis has traditionally mainly been carried out using either gas chromatography (GC) in combination with flame ionization detection or liquid chromatography (LC) with either post-column derivatization using ninhydrin or pre-column derivatization using o-phthalaldehyde, many of today’s analysis platforms are based on chromatography in combination with mass spectrometry (MS). While derivatization is mandatory for the GC-based analysis of amino acids, several LC platforms have emerged, particularly in the dawn of targeted metabolite profiling using hydrophilic interaction liquid chromatography (HILIC) coupled to MS, allowing the analysis of underivatized amino acids. Among the numerous analytical platforms available for amino acid analysis today, we here compare three prominent approaches, being GC–MS and LC–MS after amino acid derivatization using chloroformate and HILIC–MS of underivatized amino acids. We compare and discuss practical issues as well as performance characteristics, e.g., the use of 13C-labeled internal standards, of the different platforms and present data on their practical implementation in our laboratory. Finally, we compare the real-life applicability of all three platforms for a complex biological sample. While all three platforms are very-well suited for the analysis of complex biological samples they all show advantages and disadvantages for some analytes as discussed in detail in this manuscript.

A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.

Amino acid analysis: current topics and trends

The history of amino acids begins with the discovery of aspartic acid in asparagus sprouts in 1806. Thereafter, various naturally occurring amino acids of more than 360 species were detected from living organisms. Amino acid analysis was first performed by a postcolumn derivatization method using ninhydrin, which was originally developed by Moore and Stein [1]. They analyzed 124 amino acids and the ribonuclease structure using an automatic and systematic analyzer, and were awarded the Nobel Prize in Chemistry in 1972. Amino acid analysis is an integral part of analytical biochemistry. Amino acid analysis generally includes derivatization, i.e., precolumn, on-column, and postcolumn, coupled with chromatographic separation. A wide variety of separation techniques, including ion-exchange chromatography, reversed-phase chromatography, hydrophilic interaction chromatography, gas chromatography, capillary electrophoresis, and microchip electrophoresis, have been used. Various detection techniques, such as ultraviolet–visible, fluorescence, chemiluminescence, and electrochemical detection, have also been developed for amino acid analysis. On the basis of recent advances in the software and hardware used in mass spectrometry (MS), the variety of amino acid analysis methods has dramatically evolved and use has shifted to various types of MS detection techniques. In the meantime, a number of relatively old techniques, based on derivatization and conventional separation, have acquired a new look. On the basis of these observations, The Japan Society of Amino Acid Analysis (JSA3) was established in the autumn of 2011 to promote and follow the bioanalytical and/or technological advances relating to analysis of amino acids and related molecules. Various labeling techniques using ultraviolet–visible and fluorescent reagents are still used for the trace analysis of amino acids by the combination of chromatographic separation and MS detection. Of course, electrospray ionization MS allows the analysis of underivatized amino acids. Another new aspect is high-throughput analysis, such as the use of a high-pressure liquid-chromatographic system such as ultraperformance liquid chromatography using a sub-2-μm particle column, and miniaturization analysis such as the use of a microfluidic system. However, the most important issue in amino acid analysis today seems to be in the context of metabolomics studies as part of systems biology. Besides, naturally occurring D-amino acids, such as D-serine and D-aspartic acid, were found in awide variety of living organisms, both in their free form and as isomeric residues in many peptides. Their detection is due to the tremendous advances in the analytical techniques in recent years. Stereoselective metabolism of amino acids has also been gradually unveiled, and D-amino acids are now considered as novel physiologically active substances and biomarkers even in mammals. In view of the growing importance of the enantioselective analysis of amino acids, articles related to D-amino acid analysis are provided in this issue. Several articles regarding the enantioseparation of DL-amino acids by direct methods using a chiral stationary phase column are also provided. This topical collection covers a broad range of amino acid analysis, with contributions from the most prominent researchers in the field. The review articles are focused on the analytical methods applied to amino acids and related molecules using liquid chromatography, capillary electrophoresis, and microchip electrophoresis. The topics of the original research articles are expanded to the research field of peptides and proteins. Clarification of both the quantity and the distribution of amino acids in biological samples, including physiological fluids and foods, is also presented. Several research articles describe new methods of amino acid analysis, such as the use of a pillar column and a novel immunometric assay. This topical collection is focused on analysis of amino acids, including peptides, and provides a glimpse of the current research activity in the field. As a guest editor, it is my pleasure to present this collection of research articles and critical reviews which address the many challenging areas of amino acid analysis, including chiral recognition. Thanks are due to all of the authors for their excellent and timely contributions as well as the editorial staff of Published in the topical collection Amino Acid Analysis with guest editor Toshimasa Toyo’oka.

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.

Hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry for highly rapid and sensitive analysis of underivatized amino acids in functional foods

This work presented a new analytical methodology based on hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode for analysis of 24 underivatized free amino acids (FAAs) in functional foods. The proposed method was first reported and validated by assessing the matrix effects, linearity, limit of detections and limit of quantifications, precision, repeatability, stability and recovery of all target compounds, and it was used to determine the nutritional substances of FAAs in ginkgo seeds and further elucidate the nutritional value of this functional food. The result showed that ginkgo seed turned out to be a good source of FAAs with high levels of several essential FAAs and to have a good nutritional value. Furthermore, the principal component analysis was performed to classify the ginkgo seed samples on the basis of 24 FAAs. As a result, the samples could be mainly clustered into three groups, which were similar to areas classification. Overall, the presented method would be useful for the investigation of amino acids in edible plants and agricultural products.