CN117030878A

CN117030878A - Stable free amino acid underivatization detection method and application thereof

Info

Publication number: CN117030878A
Application number: CN202310972108.6A
Authority: CN
Inventors: 张林林; 冯利兴; 杨兵; 韩继臣
Original assignee: Shanghai Majorbio Bio Pharm Technology Co ltd
Current assignee: Shanghai Majorbio Bio Pharm Technology Co ltd
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2023-11-10

Abstract

The invention provides a stable non-derivatization detection method of free amino acid and application thereof, wherein the detection method comprises the following steps: mixing a sample to be detected with deoxycholate, trichloroacetic acid and an internal standard, centrifuging, taking supernatant, sampling, detecting by liquid chromatography-mass spectrometry, and calculating the content of free amino acid in the sample to be detected according to the detection result and a standard curve. The detection method provided by the invention can be used for simultaneously measuring 21 amino acids in blood plasma under the condition of not using an ion pair reagent, the sample consumption is small, the protein precipitation is complete, the chromatographic column loss caused by the sample can be reduced, the stability is high, the accuracy is high, the detection time is short, and the leucine and the isoleucine can be perfectly separated.

Description

Stable free amino acid underivatization detection method and application thereof

Technical Field

The invention belongs to the field of biochemical detection, and particularly relates to a stable underivatized detection method of free amino acid and application thereof, in particular to a high-accuracy stable underivatized detection method of free amino acid and application thereof.

Background

Amino Acids (AAs) are the core component of metabolism. They are the basic units that make up proteins, and are also regulators of gene expression and precursors to a variety of hormones and neurotransmitters. Of the 20 protein-derived AAs present in humans, most are present in human plasma at a concentration of μm. Of these 9 are essential amino acids (i.e., histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) that must be taken into the body by balanced diet. Changes in amino acid levels have long been used as a biological indicator for disease diagnosis, e.g., metabolic fingerprinting of blood samples from cancer patients shows that AAs levels decrease significantly as disease progresses. AAs fingerprint is also an important parameter for evaluating the nutritional quality of food.

From a chemical point of view, a common feature of amino acids is the simultaneous presence of amine groups and carboxyl groups in their structure. Furthermore, because of the asymmetric α -carbon, optically active L-and D-isomers can be produced, which directly result in that they can be polar, nonpolar, acidic or basic. AAs may be positively/negatively charged or in a zwitterionic state, depending on the pH of the medium. The structure of amino acids determines the complexity of developing analytical methods on a liquid-based basis, but the function and effect of amino acids determine the necessity for accurate detection thereof.

At present, a derivatization method is mostly adopted for amino acid detection, but the reality of the detection result is doubtful due to the complexity of derivatization operation and uncontrollability of derivatization efficiency; the use of commercial derivatization kits, or the provision of accurate, precise, sensitive and reproducible results, is expensive and not suitable for routine amino acid detection assays. The quantitative analysis of amino acids by non-derivatization methods has been reported, and most of the sample pretreatment operations are performed by extracting 50% acetonitrile, which depends on the fact that the sample of the amino acid needs to be protein precipitated, but the amino acid itself is water-soluble, and only 50% acetonitrile can be selected, but the protein precipitation and the extraction efficiency of the amino acid are also compromised. Removing the difficulty of preparing amino acid samples, the amino acid itself is one of the difficulties in chromatographic separation, due to the specificity of the structure, retention separation is also achieved by adding ion pair reagents (alkyl sulfonate or perfluorocarboxylic acid, etc.) in the mobile phase of reversed phase chromatographic separation to achieve effective retention and separation of amino acids in the reversed phase chromatographic column, but the alkyl sulfonate is not volatilized and is not used for MS detection, perfluorocarboxylic acid can be considered for MS detection, but the minimum effective concentration of ion pair reagents must also be selected to limit signal suppression, as all ion pair reagents may cause contamination of the whole LC-MS system, leading to higher than normal background signals and additional noise; there are also those which are directly detected with a reverse phase column without using an ion pair reagent. However, most methods suffer from low retention of amino acids that peak before the column dead time, which is clearly unacceptable for analysis of complex matrices, and desolvation in electrospray ionization (ESI) is very difficult when RPLC analysis is performed using high water mobile phases, resulting in low MS sensitivity. In addition, the HILIC mode can analyze amino acids, can retain all amino acids, has satisfactory peak shape, is completely compatible with MS, has long balancing time, is difficult to find a proper sample diluent, mostly reports that hydrochloric acid is used as the diluent, but does not mention the stability of a method and a sample, obviously is easily volatilized with hydrochloric acid, the prepared mixed standard is more than 24 hours and cannot be stabilized, and the HILIC mode has low resolution ratio between leucine and isoleucine and poorer selectivity than the RPLC mode. Therefore, there is a need for a stable, non-derivatizing, and efficiently retained amino acid detection method that does not use ion pair reagents.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a stable non-derivatization detection method of free amino acid and application thereof, in particular to a high-accuracy and stable non-derivatization detection method of free amino acid and application thereof. The detection method provided by the invention can be used for simultaneously measuring 21 amino acids in blood plasma under the condition of not using an ion pair reagent, the sample consumption is small, the protein precipitation is complete, the chromatographic column loss caused by the sample can be reduced, the stability is high, the accuracy is high, the detection time is short, and the leucine and the isoleucine can be perfectly separated.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in one aspect, the present invention provides a method for the detection of stable free amino acids without derivatization, comprising the steps of:

mixing a sample to be detected with Deoxycholate (DOC), trichloroacetic acid and an internal standard, centrifuging, taking supernatant and sampling, carrying out liquid chromatography-mass spectrometry detection, and calculating the content of free amino acid in the sample to be detected according to a detection result and a standard curve.

According to the method, the sample to be detected is treated by adopting deoxycholate and trichloroacetic acid, the deoxycholate and trichloroacetic acid are synergistic, the protein in the sample can be effectively precipitated, the damage of the protein to a chromatographic column is obviously reduced, the internal standard and a specific liquid phase process are combined, the stability and the accuracy of detection are effectively improved, meanwhile, a derivatization method is not needed, the separation of leucine and isoleucine isomers can be ensured on the premise that an ion pair reagent is not used, and the method has the advantages of wide linear range, high recovery rate, good stability, good repeatability, high sensitivity and the like, and the reliability of qualitative and quantitative results is fully ensured.

Preferably, the sample to be measured is a blood sample.

Preferably, the free amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-cysteine, L-leucine, L-hydroxyproline, L-tryptophan, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, and L-valine.

The method can realize simultaneous detection of the 21 amino acids in a short time, and can ensure separation of leucine and isoleucine isomers without using ion pair reagents.

Preferably, the deoxycholate is used in the form of a solution, and the mass fraction of the deoxycholate solution is 0.13-0.17%.

Preferably, the volume ratio of the sample to be tested, deoxycholate solution and trichloroacetic acid is (8-12): (18-22): 1.

Wherein, the mass fraction of the deoxycholate solution can be 0.13%, 0.14%, 0.15%, 0.16% or 0.17%, etc., the volume ratio of the sample to be measured, the deoxycholate solution and trichloroacetic acid can be 8, 9, 10, 11 or 12, etc., the number of the sample to be measured can be 18, 19, 20, 21 or 22, etc., but not limited to the above-listed values, and other non-listed values in the above-listed value range are equally applicable.

Preferably, the internal standard comprises L-lysine-D4 hydrochloride (Lys-D4), L-glutamine-D5 (Try-D5) and L-tryptophan-D5 (Gln-D4).

Preferably, the chromatographic column of the liquid chromatography is AdvanceBio MS Spent Media.

Preferably, the liquid chromatography is carried out at 35-45 ℃, for example 35 ℃,36 ℃, 37 ℃, 38 ℃, 39 ℃,40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃ or the like, but not limited to the values listed above, and other values not listed in the above-mentioned value ranges are equally applicable.

Preferably, the mobile phase of the liquid chromatograph comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is formic acid-ammonium formate aqueous solution, and the mobile phase B is formic acid-ammonium formate acetonitrile solution.

Preferably, in the mobile phase a and the mobile phase B, the volume fraction of formic acid is independently 0.08-0.12%, and the concentration of ammonium formate is independently 8-12mM, wherein the volume fraction of formic acid may be 0.08%, 0.09%, 0.1%, 0.11%, or 0.12%, and the concentration of ammonium formate may be 8mM, 9mM, 10mM, 11mM, or 12mM, etc., but not limited to the above-listed values, and other non-listed values within the above-listed values are equally applicable.

Preferably, the acetonitrile in the aqueous acetonitrile solution in mobile phase B has a volume fraction of 93-97%, such as 93%, 94%, 95%, 96% or 97%, etc., but is not limited to the values recited above, and other values not recited in the above ranges are equally applicable.

Preferably, the elution procedure of the liquid chromatography is gradient elution, and the flow of the gradient elution is as follows:

0-3min, changing the volume fraction of the mobile phase A from 10% to 40% at uniform speed, changing the volume fraction of the mobile phase B from 90% to 60% at uniform speed, and changing the flow rate of the mobile phase to 0.45-0.55mL/min;

3-4min, the volume fraction of the mobile phase A is 40%, the volume fraction of the mobile phase B is 60%, and the flow rate of the mobile phase is 0.25-0.35mL/min;

4 th to 4.1min, the volume fraction of the mobile phase A is changed from 40% uniform velocity to 10%, the volume fraction of the mobile phase B is changed from 60% uniform velocity to 90%, and the flow rate of the mobile phase is 0.25-0.35mL/min;

4.1-6min, the volume fraction of the mobile phase A is 10%, the volume fraction of the mobile phase B is 90%, and the flow rate of the mobile phase is 0.45-0.55mL/min.

Preferably, the standard curve is prepared by a method comprising the steps of:

mixing an amino acid standard sample with hydrochloric acid to obtain a standard stock solution, diluting the standard stock solution with trichloroacetic acid solution to obtain a series of concentration gradient working solutions, diluting the working solutions with an internal standard solution, detecting according to the liquid chromatography flow, and drawing a standard curve according to the detection result.

The standard curve can effectively improve the stability of the solution by adopting trichloroacetic acid solution for dilution.

Preferably, the supernatant is diluted before injection, and the concentration of trichloroacetic acid in the diluted supernatant is 20-30mM, such as 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM or 30mM, etc., but not limited to the above-listed values, and other non-listed values within the above-listed ranges are equally applicable.

On the other hand, the invention also provides application of the detection method in amino acid content detection.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for detecting the underivatized free amino acid, which is characterized in that deoxycholate and trichloroacetic acid are adopted to jointly treat a sample to be detected, the deoxycholate and trichloroacetic acid are synergistic, the protein in the sample can be effectively precipitated, the damage of the protein to a chromatographic column is obviously reduced, the internal standard and a specific liquid phase process are combined, the stability and the accuracy of detection are effectively improved, meanwhile, the derivatization method is not required, the separation of leucine and isoleucine isomers can be ensured on the premise of not using an ion pair reagent, and the method has the advantages of wide linear range, high recovery rate, good stability, good repeatability, high sensitivity and the like, and the reliability of qualitative and quantitative results is fully ensured.

Drawings

FIG. 1 is a graph of the results of testing plasma samples of example 1;

FIG. 2 is a graph showing the detection results of a blank solution containing an internal standard in example 1;

FIG. 3 is a graph showing the detection results of 21 amino acid mixed labeling solutions in example 1;

FIG. 4 is a superimposed graph of individual chromatographic peaks for leucine, isoleucine and hydroxyproline in example 1;

FIG. 5 is a graph of individual peaks for Ala in example 1;

FIG. 6 is a graph of individual peaks for Asn in example 1;

FIG. 7 is a graph of individual peaks for Asp in example 1;

FIG. 8 is a graph of individual peaks for Gln in example 1;

FIG. 9 is a graph of individual peaks of Glu in example 1;

FIG. 10 is a graph of individual peaks of Gly in example 1;

FIG. 11 is a single peak plot of Lys in example 1;

FIG. 12 is a single peak plot of Ser in example 1;

FIG. 13 is a single peak plot of Thr in example 1;

FIG. 14 is a single peak plot of Arg in example 1;

FIG. 15 is a single peak plot of His in example 1;

FIG. 16 is a single peak plot of Ile in example 1;

FIG. 17 is a peak-to-peak plot of Cys alone in example 1;

FIG. 18 is a single peak plot of Leu in example 1;

FIG. 19 is a single peak plot of Hyd in example 1;

FIG. 20 is a single peak plot of Trp in example 1;

FIG. 21 is a graph of individual peaks for Met in example 1;

FIG. 22 is a single peak plot of Phe in example 1;

FIG. 23 is a single peak plot of Pro in example 1;

FIG. 24 is a single peak plot of Tyr in example 1;

FIG. 25 is a single peak plot of Val in example 1;

FIG. 26 is a graph of the individual peaks of Gln-d5 in example 1;

FIG. 27 is a single peak plot of Lys-d4 in example 1;

FIG. 28 is a graph of individual peaks of Trp-d5 in example 1;

FIG. 29 is a graph of mobile phase optimization results in conditional optimization;

FIG. 30A is a graph of sample size, flow rate, and first portion results of column Wen Youhua for condition optimization;

FIG. 30B is a graph of sample size, flow rate, and second portion results of column Wen Youhua for condition optimization;

FIG. 31 is a graph of the liquid phase results of the conditions of example 1 in condition optimization;

FIG. 32A is a graph showing the results of a first portion of the optimization of a chromatographic column and trichloroacetic acid solution in a condition optimization;

FIG. 32B is a graph showing the results of a second portion of the optimization of the chromatographic column and trichloroacetic acid solution in the condition optimization;

FIG. 33 is a graph of elution gradient optimization process results;

FIG. 34 is a graph of the results of the prior art article and the patent chromatographic condition reproduction test;

fig. 35 is a diagram of the method application detection result.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

All instruments and reagents in the following examples are as follows:

NewClassic MF MS105DU electronic balance (METTLER tolio); SBL-10DT ultrasonic constant temperature washer (Ningbo Xinzhi biotechnology Co., ltd.); centrifuge 5430R high-speed refrigerated Centrifuge (Eppendorf); QTRAP 6500 liquid chromatograph-mass spectrometer (AB SCIEX).

The 21 amino acid standard, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-cysteine, L-leucine (leucine), L-hydroxyproline, L-tryptophan, L-lysine, L-methionine (methionine), L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine are all purchased from Shanghai-derived leaf Biotechnology Co., ltd. And the internal standard L-lysine-D4 hydrochloride, L-glutamine-D5, L-tryptophan-D5 are all purchased from BePure.

Example 1

This example provides a stable non-derivatization assay for free amino acids

Sample solution preparation:

accurately transferring 50 mu L of plasma to be detected, adding 91 mu L of water, mixing 100 mu L of 0.15% DOC, adding 4 mu L of 100 mu g/mL of three internal standard solution (Lys-d 4/Try-d5/Gln-d 4), mixing uniformly, performing ultrasonic treatment for 10min (5 ℃ at 40 KHz), adding 5 mu L of 10M trichloroacetic acid (TCA), mixing uniformly, freezing, standing and precipitating a sample for 10min, centrifuging at 14000rcf at 4 ℃ for 10min, taking 50 mu L of supernatant, adding 350 mu L of water, performing vortex mixing uniformly, and filtering with a biological 0.2 mu M PTFE filter membrane to obtain the plasma.

And (3) sampling the sample solution, performing liquid chromatography-mass spectrometry detection, and calculating by combining a standard curve to obtain a result, wherein the liquid chromatography-mass spectrometry parameters are as follows:

chromatographic column: advanceBio MS Spent Media (2.1X105 mm,2.7 μm), column temperature 40℃and sample loading of 1. Mu.L. Mobile phase a (0.1% formic acid 10mM ammonium formate in water) and mobile phase B (0.1% formic acid 10mM ammonium formate in 95% acetonitrile)

The gradient elution procedure was as follows:

time (min)	Flow rate (mL/min)	A％	B％
				0.0	0.5	10	90
3	0.3	40	60
				4	0.3	40	60
4.1	0.5	10	90
				6	0.5	10	90

Mass spectrometry conditions: AB SCIEX QTRAP 6500+, detected in positive/negative mode, gas curtain gas (CUR) of 35, collision gas (CAD) of medium level, ion spray voltage (IS) of +5500/-4500, ion source Temperature (TEM) of 550, ion source gas 1 (GS 1) of 50, ion source gas 2 (GS 2) of 50.

The mass spectral parameter information for the 21 amino acids and internal standard is as follows, with a residence time of 10ms:

wherein Hyd, ile, leu three amino acids share 132/86 ion channels; gln and Lys share 147/84 and 147/130 ion channels.

The liquid chromatography results are shown in FIG. 1 (time on the abscissa and intensity on the ordinate, and all of FIGS. 2 to 34 below are the same), the blank solution containing the internal standard, the chromatographic results of the 21 amino acid mixed standard are shown in FIGS. 2 to 3, and the superimposed graphs of the individual chromatographic peaks of leucine, isoleucine and hydroxyproline are shown in FIG. 4.

Standard curve preparation:

accurately weighing a proper amount of 21 amino acid standard substances, adding 0.1M HCl water for dissolution and volume fixation to 1 mL, and uniformly vortex mixing to obtain standard substance stock solution (-80 ℃ for storage), wherein the concentration information of the stock solution is as follows:

taking a proper amount of L-Trp-d5, L-Gln-d5 and L-Lys-d4, and preparing an internal standard stock solution (stored at 4 ℃) of 100 mug/mL by using a 5mM TCA aqueous solution;

the stock solution is diluted into 10 working solutions with different concentrations (stored at 4 ℃) by using 5mM TCA solution, and the working solution is diluted by adopting 400ng/mL of internal standard solution in equal volume to obtain a sample-injection standard curve solution, wherein the standard curve concentration information is as follows:

and then carrying out liquid phase mass spectrum detection on the standard curve solution by referring to the method, and drawing a standard curve according to the detection result, wherein the result is as follows:

the individual peak patterns of the 21 amino acids and the internal standard are shown in FIGS. 5-28. The results show that 21 amino acids have good linearity in the linear range, the standard curve range is more than C2-C9, and the correlation coefficient is more than 0.99.

And (3) methodological verification:

stability of

The experimental method comprises the following steps: 3 samples of the sample solution to be tested are respectively prepared for each concentration by adopting low, medium and high concentration quality control, and the stability of the sample is evaluated for 84 hours by using 9 samples and 63 measurement results, wherein the method comprises the following steps: 0,6, 24, 36, 48, 72, 84h, see table 1.

3 samples of the test solution are prepared for each concentration by adopting low, medium and high concentration quality control, and the storage stability is evaluated by measuring the total of 63 samples, wherein the method comprises the following steps: the stability was calculated by analysis according to established analytical methods of LCMS quantification by placing at 20 ℃ for 36h and 48h, at 4 ℃ for 36h and 48h, and at-20 ℃ for 36h, 48h and 72h, as shown in table 2.

Experimental results: the stability accuracy of 21 amino acids at 84h is 87.2-107.4%, and RSD is less than 15%. The stability accuracy of the 21 amino acids under different storage conditions is between 87.9 and 109.7 percent, and the RSD is less than 15 percent.

Table 1 stability test for 84h

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TABLE 2 storage stability experiments

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Intra-batch and inter-batch precision

The experimental method comprises the following steps: the precision adopts 3 different concentrations (low, medium and high quality control concentrations), 5 parts of test sample solutions are respectively prepared for each concentration, 15 measurement results are used for evaluating the precision in the batch, three analysis batches including 3 different concentrations (low, medium and high quality control concentrations) are collected within 3 days, 5 parts of test sample solutions are respectively prepared for each concentration, and 15 measurement results are used for evaluating the precision between batches. The peak areas were recorded by separate injection chromatography-mass spectrometry, and the content and relative standard deviation of 21 amino acids were calculated, see table below.

Experimental results: the daily precision of the amino acids except Arg reaches 124.5%, the daily precision of the rest amino acids is 86.4-113.1%, and the RSD values are all less than 15%.

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Accuracy of

The experimental method comprises the following steps: the accuracy inspection of the method is carried out by adopting a recovery rate method, taking 25 mu L of bovine serum, adding a proper amount of water, uniformly mixing 100 mu L of 0.15% DOC, adding 4 mu L of 100 mu g/mL of three internal standard solution (Lys-d 4/Try-d5/Gln-d 4), adding 10/20/30 mu L (low, medium and high concentration) of standard solution respectively, uniformly mixing, performing ultrasonic treatment for 10min (5 ℃ and 40 KHz), adding 5 mu L of 10M trichloroacetic acid (TCA), uniformly mixing, finally obtaining a sample solution with the volume of 250 mu L, freezing, standing and precipitating the sample for 10min, centrifuging for 10min at the temperature of 14000rcf at the temperature of 4 ℃, taking 50 mu L of supernatant, adding 350 mu L of water, uniformly vortex, and filtering a biological PTFE filter membrane with the thickness of 0.2 mu M. 5 parts of the sample solutions were prepared for each concentration and the recovery rate was evaluated using 15 measurement results.

Precisely sucking 1 μl of each of the control solution and the sample solution, and measuring with injection chromatography-mass spectrometry according to established chromatographic conditions, and calculating recovery rate according to the following formula:

percent recovery = (measured value of measured object in the labeled sample-amount of measured object contained in the sample taken)/amount of reference substance added x 100%. The results are shown in the following table.

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Condition optimization:

the conditions in example 1 were replaced with the same ones including the type and concentration of mobile phase, the amount of sample introduced, the flow rate of mobile phase, the column temperature, the type of chromatographic column, and the concentration and solvent of trichloroacetic acid solution for dilution of the stock solution of standard, respectively, as shown in FIGS. 29 to 32 (changing conditions are shown in the drawing, wherein FIG. 31 is the conditions of example 1)

From FIG. 29 (A chromatographic conditions: mobile phase A (0.1% aqueous formic acid solution) mobile phase B (0.1% aqueous formic acid acetonitrile solution), B chromatographic conditions: mobile phase A (0.1% aqueous formic acid 10mM ammonium formate-95% aqueous solution) mobile phase B (0.1% formic acid 10mM ammonium formate-95% acetonitrile solution), C chromatographic conditions: mobile phase A (0.1% aqueous formic acid 10mM ammonium formate solution) mobile phase B (0.1% formic acid 10mM ammonium formate-95% acetonitrile solution)), it was found that the addition of ammonium formate in the mobile phase significantly improved the separation of leucine from isoleucine and increased the retention of amino acids as a whole; the column temperature is examined at 35, 40 and 45 ℃, the flow rate is examined at 0.45, 0.5 and 0.55mL/min, the sample injection amount is examined at 1 and 2 mu L, the comparison chart in the optimization process of different chromatographic conditions is shown in figure 30A (the sample injection amount of the A chromatographic condition is adjusted to 2 mu L, the default sample injection amount of the B chromatographic condition is adjusted to 1 mu L, the flow rate is adjusted to 0.55mL/min, the C chromatographic condition is adjusted to 1 mu L, the flow rate is adjusted to 0.55 mL/min), the sample injection amount of the B chromatographic condition is 1 mu L, the default flow rate is adjusted to 0.50mL/min, the column temperature is adjusted to 35 ℃, and the influence of the three parameters on amino acid detection is not obvious; for concentration studies of the chromatographic columns and trichloroacetic acid solutions, a comparison of Waters BEH Amide (2.1X100 mm,1.7 μm) with AdvanceBio MS Spent Media (2.1X105 mm,2.7 μm), i.e., amide and AdvanceBio chromatographic columns, was shown in FIG. 32A (both of which were Amide chromatographic columns, A was prepared by diluting 10mM TCA50% acetonitrile aqueous solution, B was prepared by diluting 5mM TCA50% acetonitrile aqueous solution), 32B (A was Amide chromatographic column, B was AdvanceBio chromatographic column, and example 1 conditions were adopted), and it was apparent that AdvanceBio MS Spent Media chromatographic columns were superior to Waters BEH Amide in terms of retention and peak pattern under the same conditions, so AdvanceBio MS Spent Media chromatographic columns were selected as the chromatographic columns used in the present invention.

Elution procedure investigation:

the elution program of the invention is mainly optimized for the separation of leucine and isoleucine and the overall retention degree of amino acid, and is optimized for 23 times in total, and the elution gradient in the three optimization processes are listed below as shown in figure 33 (the elution condition flow rate of A is 0.3mL/min, the elution gradient is 0/3/4/4.1/6min, and the elution gradient corresponds to mobile phase B80/60/60/90/90%; the flow rate of the elution condition of B is 0.35mL/min, the elution gradient is 0/3/4/4.1/6min, corresponding to mobile phase B90/60/60/90/90%, the elution gradient of C is 0/1/3/4/4.1/6min, corresponding to mobile phase B90/90/60/60/90/90%, the corresponding flow rate is 0.5/0.5/0.3/0.5/0.5 mL/min, and the chromatogram reproduced according to the article and the existing patent chromatographic condition is shown in FIG. 34 (A is a chromatographic condition adopting patent application chromatographic column Waters BEH Amide (2.1×100mm,1.7 μm) with reference to publication number CN 114019034A), B is a chromatographic condition detection result mentioned in article about free amino acid detection in article No. Journal of Chromatography A by Bo Yuan et al, no. 2021:3267-high performance liquid chromatography tandem mass spectrometry and interactive machine learning, C is a chromatographic condition detection result of the present patent, and the chromatographic column is a mixture of 21 amino acid standard samples detected by above three detection methods, the reproduction chromatogram of the patent shows that the separation of the amino acid is obviously problematic for 6-9 min, the peak type is extremely bad, the basic line of a large section for 10-14 min is unreasonable to the gradient design, the reproduction chromatogram of the article shows that the overall peak type is good, but the distribution of the overall peak is back, and the leucine and the isoleucine do not reach the separation requirement.

Stability investigation of hydrochloric acid dilution standard curve

The 5mM TCA of example 1 was replaced with 10mM hydrochloric acid 50% acetonitrile and diluted to prepare standard curve whose linearity and range and stability data are as follows:

linearity and range

Because the standard curve range of hydrochloric acid dilution is narrow, the stability of low concentration and medium concentration quality control detection is poor, so only the stability data of high concentration quality control 12, 24 and 48h respectively are shown as follows, wherein Cys, gln, hyd, thr, trp, tyr shows instability at 24 h:

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sample preparation stability investigation

After thawing the plasma sample, sub-packaging a plurality of 50 muL to 1.5mL EP tubes, cryopreserving at-20 ℃, designing and preparing a plurality of samples, simultaneously storing at Room Temperature (RT), 4 ℃ and-20 ℃, respectively measuring 0, 36, 48 and 72h data, calculating stability, wherein the result that the data at-20 ℃ are all abnormally high, and freeze thawing out is possible, or even mixing is needed after freeze thawing, the stability of RT 48h and the stability data at 4 ℃ 48 and 72h are as follows, and in addition, sample preparation detection is carried out on the sub-packaged and cryopreserved samples on the first day, the second day and the twenty-fifth day respectively, wherein the stability data are as follows, and only the stability of Lys data is insufficient.

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Investigation of protein precipitation

The patent designs A/B/C/D/E/F/G/H experiments according to the protein precipitation step of the sample, and proves the advantages of the protein precipitation method in the sample solution preparation method, wherein a/B is a parallel experiment, and the conditions corresponding to the experiments are as follows: a, weighing and recording an EP tube, accurately transferring 50 mu L of plasma to be measured to the EP tube, adding 91 mu L of water, mixing 100 mu L of 0.15% DOC, adding 4 mu L of 100 mu g/mL of a three-internal standard solution (Lys-d 4/Try-d5/Gln-d 4), mixing uniformly, performing ultrasonic treatment for 10min (5 ℃ at 40 KHz), adding 5 mu L of 10M trichloroacetic acid (TCA), mixing uniformly, freezing and standing a sample, precipitating for 10min, centrifuging for 10min at 14000rcf at 4 ℃, completely removing the supernatant, weighing the EP tube, adding the precipitation weight, and calculating to obtain the precipitation weight; b experiment, based on A experiment, adding 91 μL water to 195 μL water, and not adding 0.15% DOC; c, on the basis of the A experiment, adding 91 mu L of water to 96 mu L of water, and not adding 10M trichloroacetic acid; d, based on the A experiment, adding 91 mu L of water to adjust to 150 mu L of acetonitrile, and not adding 0.15% DOC and 10M trichloroacetic acid; e, based on the A experiment, adding 91 mu L of water to adjust to 200 mu L of 50% acetonitrile, and not adding 0.15% DOC and 10M trichloroacetic acid; f, based on the A experiment, adding 91 mu L of water to 250 mu L of methanol, and not adding 0.15% DOC and 10M trichloroacetic acid; g, on the basis of the experiment A, transferring 50 mu L of plasma to be detected to an EP tube, adding 91 mu L of water to adjust to transfer 25 mu L of plasma to be detected to the EP tube, and adding 116 mu L of water; h experiment, based on experiment G, 116 μl of water was adjusted to 216 μl of water, and 0.15% doc was not added; experimental results show that 1) no trichloroacetic acid is added to precipitate protein; 2) The protein precipitation amount of the patent is more than three times of acetonitrile and five times of methanol to precipitate protein; 3) The addition of 0.15% DOC increases the amount of protein precipitated when the protein concentration is not high.

Experimental conditions	Amount of protein precipitate (mg)
		A-a	34.47
A-b	33.28
		B-a	33.74
B-b	33.99
		C-a	0
C-b	0
		D-a	20.13
D-b	19.49
		E-a	22.33
E-b	18.57
		F-a	29.26
F-b	27.35
		G-a	22.24
G-b	22.95
		H-a	17.77
H-b	18.98

Method application

The method of example 1 is used for measuring endogenous free amino acids in human, pig, cow, mouse and rat plasma, and the result is shown in figure 35, and it can be seen that the method of the invention realizes simultaneous measurement of different kinds of free amino acids in plasma by pretreatment of samples and selection of UPLC MS/MS measurement conditions, and the linear range, quantitative limit, stability, precision and accuracy of the method meet analysis requirements, and the method is simple and convenient and has high analysis efficiency.

The applicant states that the present invention is illustrated by the above examples as a method for the detection of the undenaturation of stable free amino acids and its use, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention must be carried out in dependence on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. A method for the detection of stable free amino acids without derivatization, characterized in that it comprises the steps of:

mixing a sample to be detected with deoxycholate, trichloroacetic acid and an internal standard, centrifuging, taking supernatant, sampling, detecting by liquid chromatography-mass spectrometry, and calculating the content of free amino acid in the sample to be detected according to the detection result and a standard curve.

2. The method according to claim 1, wherein the sample to be tested is a blood sample;

3. The detection method according to claim 1 or 2, wherein the deoxycholate is used in the form of a solution, and the mass fraction of the deoxycholate solution is 0.13-0.17%;

4. A detection method according to any one of claims 1-3, wherein the internal standard comprises L-lysine-D4 hydrochloride, L-glutamine-D5 and L-tryptophan-D5.

5. The method according to any one of claims 1 to 4, wherein the column of the liquid chromatograph is AdvanceBio MS Spent Media;

preferably, the liquid chromatography is carried out at a temperature of 35-45 ℃.

6. The method according to any one of claims 1 to 5, wherein the mobile phase of the liquid chromatograph comprises a mobile phase a and a mobile phase B, the mobile phase a being an aqueous formic acid-ammonium formate solution, the mobile phase B being an aqueous formic acid-ammonium formate acetonitrile solution;

preferably, in the mobile phase A and the mobile phase B, the volume fraction of formic acid is independently 0.08-0.12%, and the concentration of ammonium formate is independently 8-12mM;

preferably, the acetonitrile is present in the aqueous acetonitrile solution in mobile phase B in a volume fraction of 93-97%.

7. The method according to claim 6, wherein the elution procedure of the liquid chromatography is gradient elution, and the flow of the gradient elution is as follows:

8. The method of any one of claims 1-7, wherein the standard curve is prepared by a method comprising the steps of:

mixing an amino acid standard sample with hydrochloric acid to obtain a standard stock solution, diluting the standard stock solution with trichloroacetic acid solution to obtain a series of concentration gradient working solutions, diluting the working solutions with an internal standard solution, detecting according to the liquid chromatography flow of any one of claims 1-7, and drawing a standard curve according to the detection result.

9. The method according to claim 8, wherein the supernatant is diluted before sample injection, and the concentration of trichloroacetic acid in the diluted supernatant is 20-30mM.

10. Use of the detection method according to any one of claims 1-9 in the detection of amino acid content.