CN115184517B - Online derivatization detection method for blood plasma amino acid - Google Patents

Abstract

The invention relates to an online derivatization detection method of blood plasma amino acid, which comprises the following steps: pretreating a plasma sample to be tested, to which an amino acid internal standard solution is added, to obtain a pretreatment test solution; and detecting the amino acid in the pretreatment test solution by adopting a pre-column online derivatization high performance liquid chromatography. The invention avoids cross contamination among reagents caused by residue of derivatization reagent, and reduces the risk of unstable and easy decomposition of derivatization product; meanwhile, the needle is washed by the solvent after the pretreatment test solution is sucked, so that the carrying effect is avoided.

Description

An on-line derivatization detection method for plasma amino acids

Technical Field

The invention relates to the technical field of biological detection, in particular to an online derivatization detection method for plasma amino acids.

Background Art

Among the various components of the human body, the most important function of amino acids is to synthesize proteins, peptides, etc. They exist in two main forms: one is in the form of free state in the body's blood; the other is in the form of bound state in the blood vessels' proteins. The dynamic balance of plasma free amino acids can help understand the metabolism of amino acids in the body. The lack of related enzymes and other protein factors in the body, or serious lesions in organs related to amino acid metabolism such as the liver and kidneys, can lead to amino acid metabolism disorders. Diseases related to amino acid metabolism disorders can occur at any age, and most of them are particularly obvious in infancy or early childhood. If not diagnosed and treated in time, it will lead to mental retardation or even death.

The content of free amino acids and their metabolites is the most sensitive indicator of various amino acid metabolic diseases. Therefore, the clinical diagnosis of amino acid metabolic disorders can be carried out by testing the content of various amino acids in the blood of children (such as screening of neonatal inherited amino acid metabolic diseases including phenylketonuria, tyrosinemia, maple syrup urine disease and homocystinuria), so that children can receive pre-symptomatic diagnosis and treatment to prevent physical and intellectual development disorders. In addition, by regularly analyzing the content of plasma free amino acids, it is possible to monitor whether the diet is reasonable, evaluate the nutritional health status of children in the growth and development period, and provide a reference for the treatment and recovery of patients with endocrine diseases, liver diseases, renal failure and large-area burns.

Among the current methods for detecting free amino acids in plasma: sample pretreatment can be divided into two types: derivatization and non-derivatization, of which the non-derivatization method is the most common; detection instruments include high performance liquid chromatography (HPLC), capillary electrophoresis (CE), liquid chromatography mass spectrometry (LC-MS/MS), gas chromatography mass spectrometry (GC-MS), etc., of which HPLC and LC-MS/MS are the most common. As for the need to simultaneously detect multiple plasma free amino acids, due to the low content of some amino acids in the human body, if non-derivatization methods are used for pretreatment, it is necessary to use highly sensitive detection instruments such as LC-MS/MS to obtain better detection results. The related instruments are expensive, the detection cost is relatively high, and it is difficult to achieve widespread popularization. The use of derivatization methods can greatly improve the sensitivity of the detection method and improve the detection effect. It can achieve the detection of low-concentration amino acids with conventional HPLC. The method is simple, economical and easy to popularize.

The reversed-phase high performance liquid chromatography method of pre-column derivatization of o-phthalaldehyde (OPA) and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) is widely used due to its advantages such as simple operation, fast analysis speed, high sensitivity and easy automation. The reaction principle is to first use OPA to fully derivatize the primary amino acid, and then use FMOC-Cl to derivatize the secondary amino acid. However, the derivatives of OPA and amino acids are unstable and need to be injected and analyzed immediately after derivatization; FMOC-Cl is easily hydrolyzed, and the reagent itself and its hydrolysis product (FMOC-OH) are fluorescent, which will cause certain interference to chromatographic separation. In order to minimize the degradation of OPA derivatives, the hydrolysis of FMOC-Cl derivatives and itself, and at the same time maximize the yield of amino acid derivatives, it is necessary to accurately control the amount of derivatization reagents, derivatization time, pH of the derivatization system and other conditions of the online derivatization procedure, and the introduction of water into the FMOC derivatization reagent should be avoided.

Summary of the invention

Based on the technical problems existing in the traditional technology, the purpose of the present invention includes providing an online derivatization detection method for the plasma amino acid content. The detection method is used to detect plasma amino acids, which can avoid the decomposition of derivatives during the detection process.

The purpose of the present invention can be achieved through the following technical solutions:

An online derivatization detection method for plasma amino acids, the detection method comprising the following steps:

Pre-treating the plasma sample to be tested to which the amino acid internal standard solution is added to prepare a pre-treatment test solution;

The amino acids in the pre-treatment test solution are detected by using a pre-column online derivatization high performance liquid chromatography method;

Wherein, in the pre-column online derivatization high performance liquid chromatography method, the procedure of pre-column online derivatization treatment includes:

Draw a borate buffer solution, draw the pretreatment test solution, and mix them;

Wash the needle with solvent A, wash the needle with solvent B, aspirate the o-phthalaldehyde, and mix;

Wash the needle with the solvent B, wash the needle with the solvent C, wait, aspirate 9-fluorenylmethoxycarbonyl chloride, and mix;

Wash the needle with the solvent C, aspirate the diluent, wash the needle with the solvent A, and mix;

The solvent B and the solvent C are organic solvents, and the solvent A is a mixed solution of methanol, acetonitrile and water.

In some embodiments of the present invention, the solvent B is methanol, and the solvent C is acetonitrile.

In some embodiments of the present invention, the volume ratio of the borate buffer, the o-phthalaldehyde, the 9-fluorenylmethoxycarbonyl chloride, the diluent and the pretreatment solution is (0.4-0.7): (0.15-0.35): (0.15-0.3): (7-9): (0.3-0.5).

In some embodiments of the present invention, the chromatographic conditions in the pre-column online derivatization high performance liquid chromatography method include:

The stationary phase was a C18 column;

Mobile phase A comprises an aqueous solution of 1.5 mM to 2.5 mM potassium dihydrogen phosphate and 20 mM to 30 mM potassium dihydrogen phosphate;

The mobile phase B comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52):(47-52):(8-12);

The elution method adopted gradient elution;

The gradient elution procedure includes:

0min～0.35min, the volume percentage of the mobile phase A is 95%～99%;

0.35min-15min, the volume percentage of the mobile phase A decreases from 95%-99% to 40%-45%;

15min-15.1min, the volume percentage of the mobile phase A decreases from 40%-45% to 0%;

15.1min-17.6min, the volume percentage of the mobile phase A is maintained at 0%;

17.6min-17.7min, the volume percentage of the mobile phase A increases from 0% to 90%-98%;

From 17.7 min to 25 min, the volume percentage of the mobile phase A is maintained at 90% to 98%.

In some embodiments of the present invention, the chromatographic conditions further include: a column temperature of 25°C to 35°C.

In some embodiments of the present invention, the chromatographic conditions further include: an injection volume of 0.35 μL to 0.45 μL.

In some embodiments of the present invention, the chromatographic conditions further include: an injection temperature of 3.5°C to 4.5°C.

In some embodiments of the present invention, the chromatographic conditions further include: a flow rate of 0.3 mL/min to 0.5 mL/min.

In some embodiments of the present invention, the chromatographic conditions further include: excitation wavelength: 250-260 nm; emission wavelengths include: 445nm-455nm for primary amino acids and 310nm-320nm for secondary amino acids.

In some embodiments of the invention, the amino acids include primary amino acids and secondary amino acids;

The secondary amino acids include one or two of the following amino acid types: hydroxyproline and proline;

The primary amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the following amino acid types: aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, citrulline, arginine, alanine, tyrosine, taurine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine and lysine.

In some embodiments of the present invention, the amino acid internal standard in the amino acid internal standard solution comprises norvaline and sarcosine.

In some embodiments of the present invention, the concentration of norvaline is 750 μmol/L to 850 μmol/L.

In some embodiments of the present invention, the concentration of sarcosine is 150 μmol/L to 250 μmol/L.

In some embodiments of the present invention, the solvent A comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52):(47-52):(8-12).

In some embodiments of the present invention, the diluent is an acidic buffered saline solution comprising 1.5 mM to 2.5 mM potassium dihydrogen phosphate and 20 mM to 30 mM dipotassium hydrogen phosphate.

In some embodiments of the present invention, the concentration of the borate buffer is 0.35M to 0.45M, and the pH is 10 to 10.5

In some embodiments of the present invention, in the procedure of pre-column online derivatization treatment: the mixing method adopts air mixing, and the number of air mixing is 5 to 10 times.

In some embodiments of the present invention, the waiting time is 0.1 min to 0.3 min.

In some embodiments of the present invention, the needle washing time is 2 to 5 seconds.

In some embodiments of the present invention, the pre-processing step includes:

The pretreatment test solution is prepared by adding a mixed solution of formic acid and methanol to the plasma to be tested with the amino acid internal standard solution, freezing, centrifuging, collecting the supernatant, removing the solvent, re-dissolving with hydrochloric acid, centrifuging, collecting the supernatant.

In some embodiments of the present invention, in the mixed solution of formic acid and methanol, the volume percentage of formic acid is 0.04% to 0.08%.

In some embodiments of the present invention, the freezing conditions include: a temperature of -25°C to -15°C and a time of 20 min to 40 min.

In some embodiments of the present invention, the solvent is removed by drying with nitrogen.

In some embodiments of the present invention, the concentration of hydrochloric acid used for re-dissolution is 0.035 mol/L to 0.045 mol/L.

Compared with the traditional technology, the present invention has the following beneficial effects:

The inventors of the present invention have found in their research that in order to achieve rapid, efficient and low-cost simultaneous detection of multiple amino acids in plasma, a pre-column online derivatization combined with a reversed-phase high-performance liquid chromatography detection scheme can be used to achieve detection. Further, in terms of online derivatization, the present invention uses a derivatization reagent system of OPA and FMOC, and reduces the instability of OPA derivative products by implementing specific derivatization steps; at the same time, the derivatization process uses multiple solvents and step-by-step needle washing steps, which greatly reduces the carryover contamination between reagents caused by the residues of samples and reagents, and controls and delays the risk of hydrolysis of FMOC and its derivatives. The detection method of the present invention is convenient, rapid, low in detection cost, and highly sensitive, and can accurately detect multiple free amino acids in human plasma.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

FIG1 is a graph showing the results of the standard curve determination of 22 free amino acid standards in Example 1 (primary amino acids);

FIG2 is a graph showing the results of the standard curve determination of 22 free amino acid standards in Example 1 (secondary amino acids);

FIG3 is a blank matrix chromatogram of primary amino acids and internal standard extraction chromatograms thereof in Example 1;

FIG4 is a blank matrix chromatogram of secondary amino acids and internal standard extraction chromatograms thereof in Example 1;

FIG5 is a secondary amino acid S0 chromatogram of a needle washed with 50% methanol in Example 2;

FIG6 is a chromatogram of the primary amino acid S0 in the organic solvent-free needle washing step in Example 2.

DETAILED DESCRIPTION

The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

the term

Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

The terms "and/or", "or/and", and "and/or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", and "and/or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and/or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

In the present invention, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

In the present invention, % (w/w) and wt% both represent weight percentage, % (v/v) refers to volume percentage, and % (w/v) refers to mass volume percentage.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and/or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

Traditionally, the contents of various amino acid components in plasma vary greatly. To achieve simultaneous detection of multiple plasma amino acids, the detection method must ensure the effective separation of amino acids with similar polarity in the derivative products while covering the accurate determination of amino acids at different concentrations. It must also overcome the problem of very limited chromatographic column life and high project operating costs when a highly alkaline buffer salt system is used as the mobile phase.

The present invention provides an online derivatization detection method for plasma amino acid content, the detection method comprising the following steps:

Pre-treating the plasma sample to be tested to which the amino acid internal standard solution is added to prepare a pre-treatment test solution;

The amino acids in the pre-treated test solution are detected by pre-column online derivatization high performance liquid chromatography; wherein, in the pre-column online derivatization high performance liquid chromatography, the pre-column online derivatization treatment procedure includes:

Draw a borate buffer solution, draw the pretreatment test solution, and mix them;

Wash the needle with solvent A, wash the needle with solvent B, aspirate the o-phthalaldehyde, and mix;

Wash the needle with the solvent B, wash the needle with the solvent C, wait, aspirate 9-fluorenylmethoxycarbonyl chloride, and mix;

Wash the needle with the solvent C, aspirate the diluent, wash the needle with the solvent A, and mix;

The solvent B and the solvent C are organic solvents, and the solvent A is a mixed solution of methanol, acetonitrile and water.

In some embodiments of the present invention, the solvent B is methanol, and the solvent C is acetonitrile.

In some embodiments of the present invention, the needle can be cleaned by carrying the solvent in the injection bottle and moving the injection needle to enter, or by carrying the relevant solvent through the equipment cleaning port, or by other cleaning methods. The present invention does not limit the cleaning method.

In some embodiments of the present invention, the volume ratio of the borate buffer, the o-phthalaldehyde, the 9-fluorenylmethoxycarbonyl chloride, the diluent and the pre-treatment test solution is (0.4-0.7): (0.15-0.35): (0.15-0.3): (7-9): (0.3-0.5). The volume ratio of the borate buffer, the o-phthalaldehyde, the 9-fluorenylmethoxycarbonyl chloride, the diluent and the pre-treatment test solution is, for example, 0.4: 0.15: 0.15: 9: 0.5, 0.7: 0.35: 0.3: 7: 0.3, 0.6: 0.2: 0.2: 8: 0.4. In the detection method provided by the present invention, the derivatization reagent mixing ratio system used can ensure that the pH value of the derivatization environment is in an alkaline state, producing a better derivatization effect; and the loss of the chromatographic column is low, which has the advantages of increasing the service life of the chromatographic column. Since the buffer salt of the amino acid project is alkaline, the stability of general silica gel columns is poor under alkaline conditions. The present invention optimizes the method to increase the service life from 6300 mL of the volume of the mobile phase flowing through the chromatographic column to 10080 mL.

In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography include:

The stationary phase was a C18 column;

Mobile phase A comprises an aqueous solution of 1.5 mM to 2.5 mM potassium dihydrogen phosphate and 20 mM to 30 mM potassium dihydrogen phosphate;

The mobile phase B comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52):(47-52):(8-12);

The elution method adopted gradient elution;

The gradient elution procedure includes:

0min～0.35min, the volume percentage of the mobile phase A is 95%～99%;

0.35min-15min, the volume percentage of the mobile phase A decreases from 95%-99% to 40%-45%;

15min-15.1min, the volume percentage of the mobile phase A decreases from 40%-45% to 0%;

15.1min-17.6min, the volume percentage of the mobile phase A is maintained at 0%;

17.6min-17.7min, the volume percentage of the mobile phase A increases from 0% to 90%-98%;

From 17.7 min to 25 min, the volume percentage of the mobile phase A is maintained at 90% to 98%.

In the detection method provided by the present invention, the mobile phase A is, for example, an aqueous solution containing 2mM potassium dihydrogen phosphate and 25mM dipotassium hydrogen phosphate, an aqueous solution containing 1.5mM potassium dihydrogen phosphate and 30mM dipotassium hydrogen phosphate, and an aqueous solution containing 2.5mM potassium dihydrogen phosphate and 20mM dipotassium hydrogen phosphate.

In the detection method provided by the present invention, in the mobile phase B, the volume ratio of methanol, acetonitrile and water is, for example, 40:50:10, 38:47:12, or 42:52:8.

In the detection method provided by the present invention, the selected mobile phase reagent system and gradient elution procedure can effectively separate multiple amino acids in the plasma sample, especially amino acids with similar polarity of derivative products.

Further, the chromatographic conditions include:

The stationary phase was a C18 column;

Mobile phase A comprises an aqueous solution of 1.5 mM to 2.5 mM potassium dihydrogen phosphate and 20 mM to 30 mM potassium dihydrogen phosphate;

The mobile phase B comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52):(47-52):(8-12);

The elution method adopted gradient elution;

The gradient elution procedure includes:

0min～0.35min, the volume percentage of the mobile phase A is 96%;

0.35min～15min, the volume percentage of the mobile phase A decreases from 96% to 43%;

15min-15.1min, the volume percentage of the mobile phase A decreases from 43% to 0%;

15.1min-17.6min, the volume percentage of the mobile phase A is maintained at 0%;

17.6min-17.7min, the volume percentage of the mobile phase A increases from 0% to 96%;

From 17.7 min to 25 min, the volume percentage of the mobile phase A was maintained at 96%.

In some embodiments of the present invention, the chromatographic conditions further include: a column temperature of 25°C to 35°C. The column temperature is, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

In some embodiments of the present invention, the chromatographic conditions further include: an injection volume of 0.35 μL to 0.45 μL, for example, an injection volume of 0.35 μL, 0.36 μL, 0.37 μL, 0.38 μL, 0.39 μL, 0.4 μL, 0.41 μL, 0.42 μL, 0.43 μL, 0.44 μL, 0.45 μL.

In some embodiments of the present invention, the chromatographic conditions further include: an injection temperature of 3.5°C to 4.5°C, for example, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C, 4.5°C.

In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography further include: a flow rate of 0.3 mL/min to 0.5 mL/min. The flow rate is, for example, 0.3 mL/min, 0.32 mL/min, 0.34 mL/min, 0.36 mL/min, 0.38 mL/min, 0.40 mL/min, 0.42 mL/min, 0.44 mL/min, 0.46 mL/min, 0.48 mL/min, 0.5 mL/min.

In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography also include: excitation wavelength: 250-260nm (for example: 250nm, 251nm, 252nm, 253nm, 254nm, 255nm, 256nm, 257nm, 258nm, 259nm, 260nm); emission wavelengths include: primary amino acids are 445nm-455nm (for example: 445nm, 446nm, 447nm, 448nm, 449nm, 450nm, 451nm, 452nm, 453nm, 454nm, 455nm), secondary amino acids are 310nm-320nm (for example: 310nm, 311nm, 312nm, 313nm, 314nm, 315nm, 316nm, 317nm, 318nm, 319nm, 320nm).

Furthermore, the chromatographic conditions of the high performance liquid chromatography include:

The stationary phase was a C18 column;

Mobile phase A comprises an aqueous solution of 1.5 mM to 2.5 mM potassium dihydrogen phosphate and 20 mM to 30 mM potassium dihydrogen phosphate;

The mobile phase B comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52):(47-52):(8-12);

The elution method adopted was gradient elution;

The gradient elution procedure includes:

0min～0.35min, the volume percentage of the mobile phase A is 96%;

0.35min～15min, the volume percentage of the mobile phase A decreases from 96% to 43%;

15min-15.1min, the volume percentage of the mobile phase A decreases from 43% to 0%;

15.1min-17.6min, the volume percentage of the mobile phase A is maintained at 0%;

17.6min-17.7min, the volume percentage of the mobile phase A increases from 0% to 96%;

17.7min～25min, the volume percentage of the mobile phase A is maintained at 96%;

Column temperature is 25℃～35℃;

The injection volume is 0.35 μL to 0.45 μL;

The injection temperature is 3.5℃～4.5℃.

The flow rate is 0.3mL/min～0.5mL/min.

Excitation wavelength: 250-260nm (for example: 250nm, 251nm, 252nm, 253nm, 254nm, 255nm, 256nm, 257nm, 258nm, 259nm, 260nm); emission wavelengths include: 445nm-455nm for primary amino acids (for example: 445nm, 446nm, 447nm, 448nm, 449nm, 450nm, 451nm, 452nm, 453nm, 454nm, 455nm) and 310nm-320nm for secondary amino acids (for example: 310nm, 311nm, 312nm, 313nm, 314nm, 315nm, 316nm, 317nm, 318nm, 319nm, 320nm).

In some embodiments of the invention, the amino acids include primary amino acids and secondary amino acids;

The secondary amino acids include one or two of the following amino acid types: hydroxyproline and proline;

The primary amino acid is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the following amino acid types: aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, citrulline, arginine, alanine, tyrosine, taurine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine and lysine.

In some embodiments of the present invention, the amino acid internal standard in the amino acid internal standard solution comprises norvaline and sarcosine.

In some embodiments of the present invention, the concentration of norvaline is 750 μmol/L to 850 μmol/L, for example, 750 μmol/L, 760 μmol/L, 770 μmol/L, 780 μmol/L, 790 μmol/L, 800 μmol/L, 810 μmol/L, 820 μmol/L, 830 μmol/L, 840 μmol/L, and 850 μmol/L.

In some embodiments of the present invention, the concentration of sarcosine is 150 μmol/L to 250 μmol/L, for example, 150 μmol/L, 160 μmol/L, 170 μmol/L, 180 μmol/L, 190 μmol/L, 200 μmol/L, 210 μmol/L, 220 μmol/L, 230 μmol/L, 240 μmol/L, and 250 μmol/L.

Furthermore, the solvent in the amino acid internal standard solution comprises hydrochloric acid with a concentration of 0.05 mol/L to 0.15 mol/L, for example, 0.05 mol/L, 0.07 mol/L, 0.09 mol/L, 0.11 mol/L, 0.13 mol/L, or 0.15 mol/L.

In some embodiments of the present invention, the solvent A comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52):(47-52):(8-12), for example, 40:50:10, 38:47:12, 42:52:8.

In some embodiments of the present invention, the diluent is an acidic buffered saline solution comprising 1.5 mM to 2.5 mM potassium dihydrogen phosphate and 20 mM to 30 mM dipotassium hydrogen phosphate.

In some embodiments of the present invention, the concentration of the borate buffer is 0.35M to 0.45M (e.g., 0.35M, 0.37M, 0.39M, 0.41M, 0.43M, 0.45M), and the pH is 10 to 10.5 (e.g., 10, 10.1, 10.2, 10.3, 10.4, 10.5).

In some embodiments of the present invention, in the procedure of the online derivatization treatment: the mixing method adopts air mixing, and the number of air mixing is 5 to 10 times, for example, 5 times, 6 times, 7 times, 8 times, 9 times, and 10 times.

In some embodiments of the present invention, the waiting time is 0.1 min to 0.3 min, for example, 0.1 min, 0.15 min, 0.2 min, 0.25 min, or 0.3 min.

In some embodiments of the present invention, the needle washing time is 2 to 5 seconds, for example, 2 seconds, 3 seconds, 4 seconds, or 5 seconds.

In some embodiments of the present invention, the pre-treatment step includes:

The pretreatment test solution is prepared by adding a mixed solution of formic acid and methanol to the plasma to be tested with the amino acid internal standard solution, freezing, centrifuging, collecting the supernatant, removing the solvent, re-dissolving with hydrochloric acid, centrifuging, collecting the supernatant.

In some embodiments of the present invention, in the mixed solution of formic acid and methanol, the volume percentage of formic acid is 0.04% to 0.08%, for example, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%.

In some embodiments of the present invention, the freezing conditions include: a temperature of -25°C to -15°C (for example, -25°C, -23°C to 21°C, -20°C, -19°C, -17°C, -15°C), and a time of 20min to 40min (for example, 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, 40min).

In some embodiments of the present invention, the solvent is removed by drying with nitrogen; the concentration of hydrochloric acid used for re-dissolution is 0.035mol/L to 0.045mol/L, for example, 0.035mol/L, 0.037mol/L, 0.039mol/L, 0.041mol/L, 0.043mol/L, 0.045mol/L.

Specific embodiments

The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

Example 1

1. Reagent preparation

(1) Preparation of standard curve solution

6 kinds of supplementary amino acid solutions: weigh 10 mg of asparagine, glutamine, citrulline, taurine, tryptophan, and tryptophan respectively into 6 10 mL volumetric flasks, add ultrapure water to dissolve and make up to volume, and then dilute step by step with pure water to a series of concentrations.

16 Mixed amino acid solution: Take Sigma-Aldrich 79248-5X2mL mixed solution and dilute it step by step with 0.1mol/L HCl to a series of concentrations.

The above 6 kinds of supplementary amino acid solutions and 16 mixed amino acid series solutions were mixed in equal volumes to obtain the standard working curve working solution.

(2) Preparation of internal standard solution

Weigh 23.44 mg of norvaline and 8.92 mg of sarcosine respectively and place them in the same 10 mL volumetric flask, dissolve and make up to volume with 0.1 mol/L HCl, and dilute to the required concentration with 0.1 mol/L HCl.

2. Sample pretreatment

The sample pretreatment steps are as follows:

(1) Pipette 100 μL of plasma, add 25 μL of internal standard solution, and vortex mix for 30 seconds;

(2) adding 600 μL of a 0.05% formic acid/methanol mixed solution, vortexing for 5 min, and freezing at -20°C for 30 min;

(3) Centrifuge for 5 min (12000 rpm, 4°C) and transfer the supernatant;

(4) Nitrogen blowing for 15 min (temperature 60°C);

(5) Add 125 μL of 0.04 M HCl aqueous solution to re-dissolve and vortex mix for 2 min;

(6) Centrifuge for 5 min (12,000 rpm, 4°C), transfer the supernatant to a 96-well filter plate for filtration, and seal the plate.

3. Online derivatization

The borate buffer, pretreatment solution, OPA and FMOC reagent were derivatized online according to the set program in Table 1.

Table 1. Derivatization procedures of the present invention

step parameter Reagents 1 0.5μL Borate buffer, 0.4 M, pH = 10.2 2 0.3μL Pretreatment test solution 3 Mix the mixture 5 times / 4 Cleaning needle, 3s Mixed solution (solvent A) 5 Cleaning needle, 3s Methanol (Solvent B) 6 0.23μL OPA 7 Mix the mixture 10 times / 8 Cleaning needle, 3s Methanol (Solvent B) 9 Cleaning needle, 3s Acetonitrile (Solvent C) 10 Wait 0.2min / 11 0.18μL FMOC 12 Mix the mixture 10 times / 13 Cleaning needle, 3s Acetonitrile (Solvent C) 14 Draw 8μL Diluent, 25 mM K ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH = 2.5 15 Cleaning needle, 3s Mixed solution (solvent A = methanol: acetonitrile: water = 40:50:10, v:v:v) 16 Injection /

4. High performance liquid chromatography detection

Liquid chromatography conditions: chromatographic column Agilent ZOEBAX Eclipse Plus-C18 (100×2.1mm, 1.8μm), mobile phase A = 25mM K ₂ HPO ₄ (containing 2mM KH ₂ PO ₄ ), mobile phase B = methanol: acetonitrile: water (v:v:v = 40:50:10); column temperature: 35°C; injector temperature: 4°C; injection volume: 0.4μL. Excitation wavelength: 256; emission wavelength: primary free amino acid, 450nm; secondary free amino acid 315nm. Gradient elution was used, and the elution program was as follows:

0-0.35min, the volume percentage of mobile phase A is 96%;

From 0.35 to 15 min, the volume percentage of mobile phase A decreased from 96% to 43%;

From 15 to 15.1 min, the volume percentage of mobile phase A decreased from 43% to 0%;

15.1-17.6 min, the volume percentage of mobile phase A is 0%;

From 17.6 to 17.7 min, the volume percentage of mobile phase A increased from 0% to 96%;

17.7～25min, the volume percentage of mobile phase A is 96%.

5. Experimental results

According to the above liquid chromatography conditions, the standard curves of the 22 free amino acids were determined, and the results are shown in Figure 1 (primary amino acids) and Figure 2 (secondary amino acids). It can be seen that the 22 free amino acids can be completely separated. The curve range and linear correlation of the 22 plasma free amino acids are shown in Table 2.

Table 2. Detection limits, linear ranges and linear correlations of 22 plasma free amino acids

Serial number Compound Detection limit μmol/L Linear range μmol/L Regression curve R ² 1 Asp 0.75 1.92～750.0 y＝2.1915x+0.0045 0.9995 2 Glu 0.75 1.92～750.0 y＝1.3372x+0.0014 0.9992 3 Asn 0.32 0.96～320.0 y＝1.4926x+0.0006 0.9998 4 Ser 0.75 1.92～750.0 y＝1.6134x+0.0009 0.9993 5 Gln 0.50 1.54～1200. y＝1.6516x-0.0021 0.9983 6 His 0.35 0.96～750.0 y＝1.4203x-0.0004 0.9995 7 Gly 0.75 1.92～750.0 y＝1.8078x-0.0010 0.9998 8 Thr 0.75 1.92～750.0 y＝1.4549x+0.0016 0.9989 9 Cit 0.32 0.82～320.0 y＝1.9102x-0.0001 0.9995 10 Arg 0.75 1.92～750.0 y＝1.9832x-0.0017 0.9994 11 Ala 0.75 1.92～750.0 y＝1.5987x+0.0006 0.9998 12 Tau 0.40 1.02～400.0 y＝2.0070x-0.0011 0.9991 13 Tyr 0.35 0.96～750.0 y＝2.3223x+0.0006 0.9993 14 Val 0.75 1.92～750.0 y＝1.7847x+0.0014 0.9991 15 Met 0.35 0.96～750.0 y＝2.0658x-0.0019 0.9997 16 Trp 0.24 0.72～240.0 y＝3.7180x-0.0012 0.9992 17 Phe 0.35 0.96～750.0 y＝2.2534x+0.0024 0.9997 18 Ile 0.35 0.96～750.0 y＝2.0323x+0.0004 0.9995 19 Leu 0.35 0.96～750.0 y＝2.1487x+0.0001 0.9998 20 Hyp 0.75 1.92～320.0 y＝1.6242x+0.0002 0.9991 twenty one Lys 0.75 1.92～750.0 y＝1.8637x-0.0007 0.9994 twenty two Pro 0.75 1.92～750.0 y＝1.6557x-0.0016 0.9999

Precision verification:

The mixed patient samples at low and high concentration levels were prepared in advance and the intermediate precision experiment was carried out: 2 groups of data were tested for each batch of samples at each level, and the test was carried out continuously for 10 days; the mean X, standard deviation SD and coefficient of variation CV of the test results at each level were calculated. The calculation results showed that the coefficient of variation of the intermediate precision of the 22 free amino acids in the samples was in the range of 1.1% to 8.5%, and the intermediate precision of the method was good.

Accuracy Verification:

Patient matrix samples and spiked samples of different concentrations were processed in parallel for 3 samples at each concentration, and the average value (basic concentration) of the patient matrix samples and the recovery rate of the matrix samples corresponding to the actual measured concentration of each spiked sample were calculated. The results showed that the recovery rates of 22 free amino acids in the samples were in the range of 85.7% to 102.9%, and the method had good accuracy.

Matrix specificity validation:

Take the blank matrix used to prepare the standard curve for detection to observe whether there are interfering peaks near the target peak.

The verification results are shown in Figures 3 and 4. No peaks were detected in the blank matrix at the peaks corresponding to the 22 amino acids. The method has strong specificity and the matrix specificity meets the requirements.

Example 2

In conventional automated derivatization procedures, the injection needle draws up a number of different reagents multiple times. If the injection needle is not fully cleaned, carryover contamination of the injection needle or the system may occur. In addition, since the derivatization reagent (FMOC) and its derivatized products are extremely susceptible to hydrolysis, it is necessary to avoid the introduction of moisture during use and storage to ensure the effect of the derivatization reaction. The present invention adopts a multi-solvent and step-by-step organic solvent needle washing step, which can not only avoid carryover contamination of the injection needle, but also remove moisture carried by the injection needle, thereby avoiding hydrolysis of the derivatization reagent (FMOC) and its derivatized products.

This example uses different organic solvents to wash the needle to verify their effects on the detection effect of this detection scheme. The detection scheme of Example 1 was used, and the needle washing steps were respectively adopted with non-pure organic solvents (relative to Example 1, only acetonitrile aqueous solution with a volume ratio of 50% acetonitrile was used to replace solvent C) and pure organic solvents (relative to Example 1, only solvent A was replaced by acetonitrile) to observe their effects on the detection, and the results are shown in Figures 5 and 6, respectively.

As shown in Figure 5, after washing the needle with a non-pure organic solvent, the FMOC hydrolysis product (A) responds strongly at the set fluorescence wavelength, raising the baseline of the chromatogram. At the same time, the derivative by-product (B) interferes with the quantification of the secondary amino acid internal standard. If only the organic solvent is used to wash the needle, the primary amino acid is prone to carryover contamination. As shown in Figure 6, when a high-concentration sample is injected into the instrument system and then a reagent blank is injected, it will be found that multiple components to be tested have carryover effects.

The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims (10)

1. An on-line derivatization detection method of plasma amino acids, which is characterized by comprising the following steps:

Pretreating a plasma sample to be tested, to which an amino acid internal standard solution is added, to prepare a pretreatment test solution;

detecting amino acid in the pretreatment test solution by adopting a pre-column online derivatization high performance liquid chromatography;

In the pre-column online derivatization high performance liquid chromatography, the pre-column online derivatization treatment procedure comprises the following steps:

extracting borate buffer solution, extracting the pretreatment test solution, and mixing;

Washing the needle with a solvent A, washing the needle with a solvent B, sucking phthalic dicarboxaldehyde, and mixing;

washing the needle with the solvent B, washing the needle with the solvent C, waiting, sucking 9-fluorenylmethoxycarbonyl chloride, and mixing;

Washing the needle with the solvent C, sucking the diluent, washing the needle with the solvent A, and mixing;

the solvent B and the solvent C are organic solvents, and the solvent A is a mixed solution of methanol, acetonitrile and water;

the solvent B is methanol; the solvent C is acetonitrile;

The volume ratio of the borate buffer solution, the phthalic dicarboxaldehyde, the 9-fluorenylmethoxycarbonyl acyl chloride, the diluent and the pretreatment test solution is (0.4-0.7): (0.15-0.35): (0.15-0.3): (7-9): (0.3 to 0.5);

Chromatographic conditions in the pre-column on-line derivatization high performance liquid chromatography include:

the stationary phase is a C18 chromatographic column;

mobile phase a comprises an aqueous solution of 1.5mM to 2.5mM monopotassium phosphate and 20mM to 30mM dipotassium phosphate;

the mobile phase B comprises a mixed solution of methanol, acetonitrile and water with the volume ratio of (38-52) to (47-52) to (8-12);

The elution mode adopts gradient elution;

the gradient elution procedure included:

0 to 0.35min, wherein the volume percentage of the mobile phase A is 95 to 99 percent;

0.35-15 min, wherein the volume percentage of the mobile phase A is reduced from 95-99% to 40-45%;

15-15.1 min, wherein the volume percentage of the mobile phase A is reduced to 0% from 40-45%;

15.1 to 17.6 minutes, wherein the volume percentage of the mobile phase A is kept at 0 percent;

17.6 to 17.7 minutes, wherein the volume percentage of the mobile phase A is increased from 0 percent to 90 to 98 percent;

17.7-25 min, wherein the volume percentage of the mobile phase A is kept between 90 and 98 percent.

2. The method for on-line derivatization detection of plasma amino acids according to claim 1, wherein the chromatographic conditions further comprise: the column temperature is 25-35 ℃; or/and the sample injection amount is 0.35 mu L-0.45 mu L; or/and the sampling temperature is 3.5-4.5 ℃; or/and the flow rate is 0.3 mL/min-0.5 mL/min; or/and, excitation wavelength: 250-260 nm; the emission wavelength includes: the primary amino acid is 445 nm-455 nm, and the secondary amino acid is 310 nm-320 nm.

3. The method for on-line derivatization detection of plasma amino acids according to any one of claims 1 to 2, wherein the amino acids comprise primary and secondary amino acids;

The secondary amino acid comprises 1 or 2 of the following amino acid classes: hydroxyproline and proline;

The primary amino acids comprise 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the following amino acid species: aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, citrulline, arginine, alanine, tyrosine, taurine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine and lysine.

4. The method for on-line derivatization detection of plasma amino acids according to any one of claims 1 to 2, wherein the amino acid internal standard in the amino acid internal standard solution comprises norvaline and sarcosine.

5. The method for on-line derivatization detection of plasma amino acids according to claim 4, wherein the concentration of norvaline is 750. Mu. Mol/L to 850. Mu. Mol/L; or/and the concentration of the sarcosine is 150 mu mol/L-250 mu mol/L.

6. The method for on-line derivatization detection of plasma amino acids according to any one of claims 1 to 2 and 5, wherein the solvent a comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38 to 52): 47 to 52): 8 to 12.

7. The method for on-line derivatization assay of plasma amino acids according to any one of claims 1 to 2 and 5, wherein the diluent is an acidic buffer salt solution comprising 1.5mM to 2.5mM potassium dihydrogen phosphate and 20mM to 30mM dipotassium hydrogen phosphate, pH2 to 3; or/and the concentration of the borate buffer solution is 0.35M-0.45M, and the pH value is 10-10.5.

8. The method for on-line derivatization assay of plasma amino acids according to any one of claims 1 to 2 and 5, wherein in the pre-column on-line derivatization procedure: the mixing mode adopts air mixing, and the frequency of air mixing is 5-10 times; and/or waiting for 0.1 min-0.3 min; and/or the time for washing the needle is 2-5 seconds.

9. The method for on-line derivatization detection of plasma amino acids according to any one of claims 1 to 2 and 5, wherein the step of pretreatment comprises: adding a mixed solution of formic acid and methanol into the plasma sample to be tested, which is added with the amino acid internal standard solution, freezing, centrifuging, collecting supernatant, removing solvent, redissolving hydrochloric acid, centrifuging, collecting supernatant, and preparing the pretreatment test solution.

10. The method for on-line derivatization detection of plasma amino acids according to claim 9, wherein the volume percentage of formic acid in the mixed solution of formic acid and methanol is 0.04% -0.08%; or/and, freezing conditions include: the temperature is between 25 ℃ below zero and 15 ℃ below zero for 20 to 40 minutes; or/and, the solvent is removed by adopting nitrogen to blow-dry; or/and, the concentration of hydrochloric acid adopted by the redissolution is 0.035mol/L to 0.045mol/L.