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. Author manuscript; available in PMC: 2021 Dec 30.
Published in final edited form as: Anal Bioanal Chem. 2020 Feb 4;412(8):1947–1954. doi: 10.1007/s00216-020-02450-w

Fast quantification of free amino acids in food by microfluidic voltage assisted liquid desorption electrospray ionization-tandem mass spectrometry

Hankun Hu 1, Shila Smith 2, Xiangtang Li 2, Zhengming Qian 3, Yaxia Su 1, Manting Lin 1, Jiancheng Tu 1,*, Yi-Ming Liu 2,*
PMCID: PMC8717839  NIHMSID: NIHMS1556606  PMID: 32020315

Abstract

A method based on microfluidic voltage-assisted liquid desorption electrospray ionization tandem mass spectrometry (VAL-DESI-MS/MS) has been developed for fast quantification of free amino acids in food. Food extracts were transferred to the microfluidic platform and analyzed by liquid desorption ESI-MS/MS. Deuterated aspartic acid (i.e. 2,2,3-d3-Asp) was used as internal standard for analysis. The method had linear calibration curves with r2 values > 0.998. Limits of detection were at the level of sub μM for the amino acids tested, i.e. glutamic acid (Glu), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). To validate the proposed method in food analysis, extracts of Cordyceps fungi were analyzed. Amino acid contents were found in the range from 0.63 mg/g (Tyr in Cordyceps sinensis) to 4.44 mg/g (Glu in Cordyceps militaris). Assay repeatability (RSD) was ≤5.2% for all the five amino acids measured in all the samples analyzed. Recovery was found in the range from 95.8% to 105.1% at two spiking concentrations of 0.250 mg/g and 1.00 mg/g. These results prove that the proposed microfluidic VAL-DESI-MS/MS method offers a quick and convenient means of quantifying free amino acids with accuracy and repeatability. Therefore, it may have a potential in food analysis for nutritional and quality assessment purposes.

Keywords: Amino acids, Food, Direct analysis, Desorption electrospray ionization, Mass spectrometry, Microfluidics

Graphical Abstract

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Introduction

Amino acids are starting materials in a living system for synthesis of many bioactive molecules, including proteins, neurotransmitters and hormones. In addition, many amino acids exhibit various types of physiological activity [1–4]. In food amino acids are extensively studied because they may have both nutritional and safety implications. Nowadays bacterially derived food proteins, genetically modified foods, new methods for food processing, and synthetic sources of amino acids for food fortification are widely used in food industry [5–8]. To assess the nutritional safety and compositional adequacy of foods analysis of amino acids is highly desired [9, 10].

Majority of current methods for simultaneous quantification of multiple amino acids involve either chromatographic or electrophoretic separation [11–15]. Challenges include difficult chromatographic separations due to their high polarity and low detection sensitivity because of lacking chromophores in their chemical structures. Pre-column derivatization of amino acids is, therefore, needed in many cases [16–19]. Methods with MS detection offers a unique advantage of providing structural information for analytes detected. Although GC-MS based methods offer high sensitivity and excellent resolution, they generally involve a tedious pre-column derivatization procedure to obtain volatile amino acid derivatives that are separable by GC. CE-MS and LC-MS methods are suitable for quantification of native amino acids. CE–MS methods have advantages such as short analysis times and high separation efficiency. In LC-MS methods, ion-pairing reagents are normally utilized to achieve an efficient separation of native amino acids on reversed phase columns. Hydrophilic LC columns in combination with hydrophobic mobile phases, termed hydrophilic interaction LC (HILIC), provide good separations of amino acids. HILIC-ESI-MS-MS was successfully applied to quantification of amino acids in various sample matrices [20, 21]. Although these methods are sensitive and selective, they are not particularly suitable for high throughput sample screening to obtain amino acid profiles.

Mass spectrometry with electrospray ionization (ESI-MS) is a powerful analytical technique due to its wide applicability, high sensitivity, and specificity. However, signal suppression by sample matrix prohibits direct ESI-MS analysis of samples that contain high levels of salt and macromolecules. To overcome this limitation, desorption electrospray ionization-MS technique (DESI-MS) was developed [22–25]. We recently developed the microfluidic voltage-assisted liquid desorption electrospray ionization-MS/MS (VAL-DESI-MS/MS) technique [26] to enhance the applicability of DESI-MS and the assay repeatability. In this work, microfluidic VAL-DESI-MS/MS was deployed to develop an analytical method for fast profiling of free amino acids in food. Inexpensive and easy-to-prepare PMMA microchips were fabricated and evaluated for the analysis. Analytical figures of merit were assessed. Simultaneous quantification of a group of selected amino acids in Cordyceps fungi by using the present VAL-DESI-MS/MS method was demonstrated.

Materials and methods

Reagents and materials

Amino acids, methanol, sodium hydroxide, and Whatman chromatography paper were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). High purity de-ionized water (18.2 MΩ /cm resistivity) was prepared by a Milli-Q water purification system (Millipore, Bedford, MA) and used throughout the work. Stock solutions of amino acids were prepared in 5mM HCL solution and diluted to appropriate concentrations with water to obtain working standard solutions. All solutions were filtered through a nylon 0.22 μm syringe filter before use unless otherwise described.

Microchip fabrication

The microchip was made of two PMMA plates (24mm long × 40mm wide ×1 mm thick). A laser engraving machine controlled with CAD software was used to engrave microchannels onto the substrate. After being treated in an air plasma cleaner (PDC-32G, Harrick Plasma, Ithaca, NY) for 5 min at 10.5 W under 500mTorr the cover and the substrate were bond together permanently. A piece of capillary was inserted into the microchip for ESI solvent delivery. Prior to use, the microfluidic channels were flushed sequentially with 0.5% hydrogen peroxide, 0.1M NaOH, and then the electrospray solvent for 1min each.

Cordyceps sample preparation

Samples of natural Cordyceps sinensis and cultivated Cordyceps militaris were collected from authority-certified herbal stores and local grocery stores in China. The samples were cryogenically ground and homogenized to obtain a uniform matrix. A portion of the powdered sample (~ 2.0g) was extracted with 10.0mL of 5mM HCl /methanol (1:1) solution at 4 °C for 12 hrs. The mixture was transferred to a centrifuge vial and centrifuged at 5,000rpm for 10 min to obtain the supernatant liquid. The extract was diluted 50 times with a 0.2% (v/v) formic acid solution and spiked with 2,2,3-d3-Asp, the internal standard, before analysis.

VAL-DESI-MS/MS analysis

The MS system consisted of an ion trap mass spectrometer (LCQ Deca, ThermoFinnigan, San Jose, CA), a multi-channel power supply, and a syringe pump. Xcalibur software (ThermoFinnigan) was used to acquire and to process MS data. The MS detection conditions were optimized in positive mode as follows: capillary temperature, 250°C; ion source voltage, 0V; relative collision energy, 20–30%; isolation width, 1.0 u; and activation time, 30ms. VAL-DESI conditions: ESI voltage, +4kV; sampling voltage, +75V; electrospray solvent flow rate, 250 nL/min; MRM detection mode.

A portion of a food extract (25 μL) was transferred into a sample reservoir on the microfluidic VAL-DESI-MS/MS platform. A flow of electrospray solvent (i.e. 80% methanol aqueous solution with 0.2% formic acid) was started by using a digital syringe pump that regulated the flow rate. ESI and sampling voltages were applied at respective Pt electrodes. Tandem mass spectra were obtained by collision-induced dissociation (CID). The most abundant fragment of each compound was selected for quantification. The ion transitions were monitored for the six amino acids selected and internal standard: m/z 137 → 119 (for d3-Asp), m/z 148 → 130 (for Glu), m/z 166 → 120 (for Phe), m/z 175 → 157 (for Arg), m/z 182 → 165 (for Tyr), and m/z 205 → 188 (for Trp) by using MRM detection mode. MS data was recorded for 2 min for each sample. Ratio of the average signal intensity of an amino acid to that of internal standard was used for quantification.

Results and discussion

Microfluidic chip design for VAL-DESI-MS/MS analysis

The analytical platform was built on a microchip integrating all the analytical functions desired and a robust ion trap mass spectrometer. In this study, polymethyl methacrylate (PMMA) microfluidic chips were prepared and evaluated. PMMA is easily available and well known for its ease of fabrication [27–29]. Compared with the microchip design we previously reported for performing VAL-DESI-MS/MS analysis [26], a few modifications were made to improve liquid DESI efficiency. As shown in Figure 1A, in the present design the liquid DESI cavity has a different geometry from the previous version. The two sample channel outlets are no longer flat edges but have a shape of triangle with a surface area greatly diminished. Advantages of the present design include minimized memory effects between sample analyses and easy cleaning of the ionization source. More importantly, the efficiency of VAL-DESI is improved because the sample solution outlet is more precisely positioned against the ESI emitter and the positive charges carried by the sample solution are better focused at the outlet, enhancing the interaction between electrospray droplets and the sample solution. Two sample reservoirs are made in the microchip so that analysis of two samples or a duplicate analysis of one sample can be performed alternatively in one run by simply switching the sampling voltage (+75 V) on-and-off at the respective sample reservoirs. For example, the microchip is particularly suitable for quantification using standard addition method because it has two sample reservoirs. In such an assay, a sample is placed in one sample reservoir and the sample spiked with authentic compounds is placed in the other. They are analyzed in an alternative manner for the best assay accuracy. A picture of the microchip prepared is shown in Fig. 1B.

Fig. 1.

Fig. 1.

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(A) Microchip design for the proposed VAL-DESI-MS/MS analysis, and (B) image of a PMMA microchip prepared and placed in front of a mass spectrometer inlet.

Simultaneous quantification of amino acids by microfluidic VAL-DESI-MS/MS

The liquid DESI-MS/MS analytical technique offers advantages such as direct sampling for high throughput analysis, little sample matrix suppression on MS signal, and MRM detection mode for simultaneous quantification of multiple analytes. However, this technique has never been deployed so far for determination of amino acids in liquid samples likely because desorption of highly hydrophilic amino acids from solution is a challenge. In this work, the proposed VAL-DESI-MS/MS microfluidic platform was evaluated for fast simultaneous quantification of free amino acids in food. Five amino acids were selected as model analytes that included acidic amino acids (i.e. Glu), basic amino acids (i.e. Arg), and aromatic amino acids (i.e. Phe, Tyr, and Trp). To obtain good assay accuracy and repeatability a deuterated amino acid (i.e. 2,3,3-d3-Asp) was used as internal standard. MS spectra of the amino acids and internal standard were acquired (Fig. 2). As shown, a major product ion was produced for each of the compounds tested, ensuring sensitive MRM detection. From these MS2 spectra, the following ion transitions were selected for MRM detection: m/z 137 → 119 (for d3-Asp), m/z 148 → 130 (for Glu), m/z 166 → 120 (for Phe), m/z 175 → 157 (for Arg), m/z 182 → 165 (for Tyr), and m/z 205 → 188 (for Trp). Using the proposed VAL-DESI-MS/MS analytical platform, a mixture of the selected amino acids (5.00μM each) was analyzed. The MRM signal traces obtained are shown in Fig 3. As shown, ion abundance for all targeted compounds reached the maximum within 1.5 min after the run started. The run time was, therefore, set as 2.0 min. Analysis of a sample was completed in less than 2.5 min. It’s worth noting that although all amino acids were at the same concentration in the mixture analyzed their responses to VAL-DESI-MS/MS detection were different. This was likely because of the differences in DESI desorption efficiency for each amino acid that was largely dependent on the volatility of the compound to be desorbed from the sample solution and in CID fragmentation patterns (e.g. in the case of Arg). For quantification, peak height ratio of analyte to internal standard was used for good assay accuracy and repeatability.

Fig. 2.

Fig. 2.

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MS2 spectra of the amino acids involved and internal standard (d3-Asp).

Fig. 3.

Fig. 3.

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MRM signal traces from VAL-DESI-MS/MS analysis of a mixture of five selected amino acids. VAL-DESI-MS/MS conditions: nanoESI voltage, +4 kV; sampling voltage, +75 V; electrospray solvent flow rate, 250 nL/min; MRM detection mode. [compound] = 5.00μM.

To assess the analytical figures of merit for simultaneous quantification of amino acids by VAL-DESI-MS/MS, six-point calibration curves were prepared with authentic mixture standard solutions at concentrations ranging from 2.50 to 30.0 μM in 5mM HCl /methanol (1:1) (the extraction solvent for food samples). Internal standard was added to the extraction solvent at 5.00μM. These solutions were analyzed in triplicate. Ratios of peak height were used for the calculation. Regression analysis of the results yielded linear calibration equations for all five amino acids with R2 values > 0.997 (Table 1). From the calibration curves, limits of detection were estimated to be in the range from 0.46 μM (for Arg) to 0.10 μM (for Tyr) (signal /noise = 3). To assess assay repeatability, a standard mixture containing these amino acids at 2.50 μM each was analyzed five times. For all the five compounds, RSD values were found ≤4.5%. The good repeatability obtained could be attributed in part to the use of a deuterated amino acid (i.e. d3-Asp) as internal standard in the analysis. Most current methods for analysis of amino acids involve precolumn derivatization and /or chromatographic (or electrophoretic) separation. Very few reports are seen in literature on direct sampling mass spectrometric analysis of amino acids [30, 31]. To our knowledge, there has been no report on direct analysis of a real sample to quantify amino acids by using a method based on the liquid desorption ESI-MS/MS technique. Considering its speedy and cost-effective nature, the present microfluidic VAL-DESI-MS/MS method was evaluated for fast profiling of free amino acids in food samples.

Table 1.

Analytical parameters of the proposed VAL-DESI-MS/MS method

Analyte Regression equation* R2 LOD (mol /L)
Glu Y = 16236.1X + 0.348 0.9980 3.0 ×10−7
Arg Y = 12751.7X + 0.923 0.9984 4.6 ×10−7
Tyr Y = 7288.7X + 0.521 0.9972 1.0 ×10−7
Trp Y = 42417.7X + 0.307 0.9970 3.4 ×10−7
Phe Y = 42953.6X + 0.854 0.9975 3.0 ×10−7
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Y is peak height ratio of analyte /internal standard and X is analyte concentration in mol/L.

Simultaneous quantification of amino acids in Cordyceps fungi

Fast profiling amino acids in food is highly significant for food nutritional and quality assessment. Caterpillar fungi such as natural cordyceps sinensis (C. sinensis) and its cultivated substitutes (e.g. C. militaris) are popular tonic foods in Asia. They are known to contain a broad spectrum of biologically active ingredients, including amino acids, nucleosides, polysaccharides, and sterols [32–34]. In this study caterpillar fungi were analyzed to demonstrate the applicability of the proposed VAL-DESI-MS/MS method for fast profiling of amino acids in food. The fungus samples were powdered and extracted by a 5mM HCl /methanol (1:1) solution for 12 hrs. After dilution and spiked with internal standard, a portion (25.0 μL) of the extract was transferred into a sample reservoir on the microfluidic platform and analyzed by VAL-DESI-MS/MS. Figures 4 shows full scan MS spectra obtained from extracts of C. sinensis and C. militaris. As shown, ions whose m/z values were in consistence with molar masses of all the targeted amino acids (i.e. Glu, Arg, Phe, Tyr, and Trp) and the internal standard were detected. It’s worth mentioning that abundance of an ion m/z (i.e. height of a peak in Fig. 4) might be contributed from several chemical species in addition to an amino acid because they had the same molar mass. This potential interference was eliminated by using MRM detection because of their different CID fragmentation pathways. Contents of the targeted amino acids in two caterpillar fungi were determined from triplicate analysis of the extract samples. Results are summarized in Table 2.

Fig. 4.

Fig. 4.

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Full scan MS spectra obtained by the proposed microfluidic liquid desorption ESI-MS method from extracts of C. sinensis (A) and C. militaris (B), showing that the targeted amino acids and internal standard were detected.

Table 2.

Results of quantification of free amino acids in caterpillar fungi and recovery tests

Sample Analyte Found (mg /g) * Added (mg /g) Recovery (%)
C. sinensis Glu 2.62 ± 0.108 0.250 103.5
Arg 3.77 ± 0.079 0.250 101.6
Phe 1.51 ± 0.059 0.250 95.8
Tyr 0.63 ± 0.008 0.250 102.8
Trp 0.83 ± 0.032 0.250 97.7
C. militaris Glu 4.44 ± 0.231 1.000 102.6
Arg 4.19 ± 0.046 1.000 98.3
Phe 0.94 ± 0.034 1.000 99.5
Tyr 1.06 ± 0.023 1.000 104.4
Trp 3.53 ± 0.035 1.000 105.1
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*

mean ± SD, n=3.

All the five amino acids targeted were detected in the cordyceps samples. In both natural and cultivated caterpillar fungi Glu and Arg were found at higher levels than the aromatic amino acids. The contents measured were in the range from 0.63 mg/g (Tyr in C. sinensis) to 4.44 mg/g (Glu in C. militaris), which were in consistent with those reported in literature [33, 34]. The assay repeatability (RSD, n=3) ranged from 1.0 to 5.2%. It’s worth noting that contents of all the amino acids except Phe were significantly higher in cultivated C. militaris than those in natural C. sinensis. This is likely because in the cultivation process the artificial growing conditions are such selected to maximize the contents of certain bioactive components such as cordycepin and amino acids [35–37]. To assess the assay accuracy, recovery of the targeted amino acids at two different concentrations (i.e. 0.250 mg /g and 1.000 mg /g) was investigated. As shown in Table 2, recovery ranged from 95.8% to 105.1%, indicating the analysis was accurate for all amino acids tested. Since no precolumn derivatization nor chromatographic (or electrophoretic) separation were involved in the analysis, multiple amino acids were simultaneously quantified with accuracy and repeatability in a 2.5min run. This method is well suited for fast profiling of free amino acids in food.

Conclusions

A microfluidic voltage assisted liquid DESI-MS/MS (VAL-DESI-MS/MS) method has been developed for quantification of amino acids in aqueous solutions. To our knowledge, this is the first report so far on quantification of highly hydrophilic amino acids in liquid samples by using a method based on the DESI-MS technique. In the proposed method, no precolumn derivatization nor separation are involved. Multiple amino acids can be simultaneously determined in a 2.5 min run with LODs at the sub μM level. Using a deuterated amino acid as internal standard, assay repeatability (RSD) was ≤5.2% in all cases. The method was validated by analyzing cordyceps fungi extracts. Contents of free amino acids in the samples were found in the range from 0.63 to 4.44 mg/g, which were in consistence with the results reported in literature. In addition, recovery tests by using standard additions showed the analysis was accurate with recovery ranging from 95.8% to 105.1%. Considering its facile implement features and speedy nature, the proposed microfluidic VAL-DESI-MS/MS method has the potential to become a powerful tool for fast profiling of free amino acids for various purposes such as food quality and nutritional assessment.

Acknowledgements

This study was funded by Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (znpy2017023 and znpy2018116 to HH), Traditional Chinese Medicine research project of Hubei Provincial Health Commission (ZY2019M033 to HH), and US National Institutes of Health (GM089557 to YML).

Footnotes

Compliance with Ethical Standards There are no conflicts of interest to declare.

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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