CN115184517A - An online derivatization detection method for plasma amino acids - Google Patents

Abstract

The invention relates to an online derivatization detection method of plasma amino acid, which comprises the following steps: pretreating the plasma sample to be detected, which is added with the amino acid internal standard solution, 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 method avoids cross contamination among reagents caused by the residue of the derivatization reagent, and reduces the risks of instability and easy decomposition of the derivatization product; meanwhile, the carrying effect is avoided by adopting the solvent to wash the needle after the pretreatment of the test solution is absorbed.

Description

An online derivatization detection method for plasma amino acids

technical field

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

Background technique

Among the various components of the human body, the most important function of amino acids is to synthesize proteins and peptides. There are two main forms of their existence: one is in the form of free state in the body's blood; the other is in the form of binding in the form of peptide blood vessels in the blood. The dynamic balance of plasma free amino acids can help to understand the metabolism of amino acids in the body. Lack of related enzymes and other protein factors in the body, or severe lesions in organs related to amino acid metabolism, such as liver and kidney, will lead to amino acid metabolism disorders. Diseases related to amino acid metabolism disorders can appear at any age, and most of them manifest in infancy or early childhood. If not diagnosed and treated in time, they can lead to mental retardation and even death.

The content of free amino acids and their metabolites is the most sensitive indicator to reflect various amino acid metabolism diseases, so the clinical diagnosis of amino acid metabolism disorders can be carried out by detecting the content of various amino acids in the blood of children (such as neonatal genetic amino acid metabolism). Diseases include phenylketonuria, tyrosinemia, maple syrup urine disease and homocystinuria screening), so that children can get pre-diagnosis and treatment, and prevent physical and intellectual developmental disabilities. In addition, regular analysis of plasma free amino acid content can monitor whether the diet is reasonable, evaluate the nutritional health status of children during growth and development, and provide reference for the treatment and recovery of patients with endocrine diseases, liver diseases, renal failure, and extensive burns.

Among the current plasma free amino acid detection methods: 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., among which HPLC and LC-MS/MS are the most common. In terms of the need to detect multiple free amino acids in plasma at the same time, due to the low content of some amino acids in the human body, if the non-derivatized method is used for pretreatment, it is necessary to use a detection instrument with high sensitivity, such as LC-MS/MS, etc. To obtain a better detection effect, the relevant instruments are expensive, and the detection cost is relatively high, making it difficult to achieve widespread popularization. The derivatization method can greatly improve the sensitivity of the detection method and improve the detection effect. With conventional HPLC, the detection of low-concentration amino acids can be realized. The method is simple, economical, and easy to widely popularize.

Pre-column derivatization of ortho-phthalaldehyde (OPA) combined with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) by reversed-phase high performance liquid chromatography is widely used due to its simple operation, fast analysis speed, high sensitivity and easy automation. use. The reaction principle is to use OPA to fully derivatize the primary amino acid, and then use FMOC-Cl to derivatize the secondary amino acid. However, OPA and amino acid derivatives are unstable and need to be injected and analyzed immediately after derivatization; while FMOC-Cl is easily hydrolyzed, and the reagent itself and its hydrolysis product (FMOC-OH) have fluorescence, which will interfere with chromatographic separation. In order to minimize the degradation of OPA-derived products, FMOC-Cl-derived products and their own hydrolysis, and at the same time maximize the yield of amino acid-derived products, it is necessary to precisely control the amount of derivatization reagents, derivatization time, and pH of the derivatization system in the online derivatization program. And FMOC derivatization reagent to avoid the introduction of moisture.

SUMMARY OF THE INVENTION

Based on the technical problems existing in the traditional technology, the purpose of the present invention includes providing an on-line derivatization detection method for plasma amino acid content. Using the detection method to detect plasma amino acids can avoid the decomposition of derivatives in the detection process.

The object of the present invention can be realized through the following technical solutions:

A kind of on-line derivatization detection method of plasma amino acid, described detection method comprises the steps:

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

Detect amino acids in the pretreatment test solution by using pre-column online derivatization high performance liquid chromatography;

Wherein, in the pre-column online derivatization high-performance liquid chromatography, the pre-column online derivatization processing program includes:

Extract the borate buffer, extract the pretreatment test solution, and mix;

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

Wash the needle with the solvent B, wash the needle with the solvent C, wait, suction 9-fluorenemethoxycarbonyl 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-fluorenemethoxycarbonyl chloride, the diluent and the pretreatment test solution is ( 0.4 to 0.7): (0.15 to 0.35): (0.15 to 0.3): (7 to 9): (0.3 to 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 is a C18 chromatographic column;

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

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 adopts 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%;

From 15.1min to 17.6min, the volume percentage of the mobile phase A remains 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 remains at 90% to 98%.

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

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

In some embodiments of the present invention, the chromatographic conditions further include: the injection temperature is 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: primary amino acids are 445 nm-455 nm, and secondary amino acids are 310 nm-320 nm.

In some embodiments of the invention, the amino acid comprises a primary amino acid and a secondary amino acid;

The secondary amino acids comprise one or two of the following amino acids: 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: 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˜850 μmol/L.

In some embodiments of the present invention, the concentration of sarcosine is 150 μmol/L˜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-0.45M, and the pH is 10-10.5

In some embodiments of the present invention, in the procedure of pre-column online derivatization treatment: air mixing is used as the mixing method, and the number of times 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 time for washing the needle is 2-5 seconds

In some embodiments of the present invention, the steps of pre-processing include:

Add a mixture of formic acid and methanol to the plasma to be tested with the amino acid internal standard solution added, freeze, centrifuge, collect the supernatant, remove the solvent, redissolve in hydrochloric acid, centrifuge, collect the supernatant, and prepare the pretreatment test solution.

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

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

In some embodiments of the present invention, the method of removing the solvent is nitrogen blow-drying.

In some embodiments of the present invention, the concentration of hydrochloric acid used for reconstitution 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 research that to achieve rapid, efficient and low-cost simultaneous detection of various amino acids in plasma, the detection scheme can be achieved by using pre-column online derivatization combined with reversed-phase high performance liquid chromatography. In terms of online derivatization, the present invention adopts the derivatization reagent system of OPA and FMOC, and reduces the instability of OPA derivative products by implementing specific derivatization steps; meanwhile, the derivatization process adopts multi-solvent and step-by-step needle washing steps, which not only greatly It reduces the carryover contamination between reagents caused by the residues of samples and reagents, and controls and delays the hydrolysis risk of FMOC and its derivative products. The detection method of the invention has convenient and rapid detection, low detection cost and high sensitivity, and can accurately detect various free amino acids in human plasma.

Description of 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 briefly introduces the accompanying drawings required in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the determination result (primary amino acid) of carrying out the standard curve to the standard substance of 22 kinds of free amino acids in embodiment 1;

Fig. 2 is the measurement result (secondary amino acid) of carrying out the standard curve to the standard substance of 22 kinds of free amino acids in embodiment 1;

Fig. 3 is a blank matrix chromatography primary amino acid and its internal standard extraction chromatogram in Example 1;

Fig. 4 is a blank matrix chromatographic secondary amino acid and its internal standard extraction chromatogram in Example 1;

Fig. 5 is the secondary amino acid SO chromatogram of 50% methanol washing needle in embodiment 2;

Fig. 6 is the first-level amino acid SO chromatogram without organic solvent needle washing step in Example 2.

Detailed ways

The present invention will be described in further detail below with reference to the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and not to limit the scope of the present invention, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and complete. 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 departing from the connotation of the present invention to obtain The equivalent forms of , also fall within the protection scope of the present application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention, it being understood that the present invention may be practiced without one or more of these details.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are only for the purpose of describing the embodiments and examples, and are not intended to limit the present invention.

the term

Unless otherwise stated or otherwise contradicted, terms or phrases used herein have the following meanings:

As used herein, the terms "and/or," "or/and," and "and/or" are inclusive of any one of two or more of the associated listed items, as well as any of the associated listed items. and all combinations, including any combination of any two of the related listed items, any more of the related listed items, or all of the 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, it includes technical solutions that are connected by "logical AND", and also includes technical solutions that are connected by "logical OR". For example, "A and/or B" includes three parallel schemes of A, B and A+B. For another 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, all connected by "logical OR" technical solution), also includes any and all combinations of A, B, C, D, that is, includes the combination of any two or any three of A, B, C, D, and also includes A, B, C The four combinations of , D (that is, the technical solutions that are connected by "logical AND").

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

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

Herein, "suitable" is described in "appropriate combination", "suitable manner", "any suitable manner", etc., so as to be able to implement the technical solution of the present invention, solve the technical problem of the present invention, and realize the expectation of the present invention The technical effect shall prevail.

Herein, "preferred", "better", "better" and "suitable" are only to describe embodiments or examples with better effects, and it should be understood that they do not limit the protection scope of the present invention.

In the present invention, "further", "further", "specially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as limiting the protection scope of the present invention.

In the present invention, "optionally", "optional" and "optional" refer to optional, that is to say, any one of the two side-by-side schemes of "with" or "without". If there are multiple "optionals" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "optional" is independent of each other.

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

In the present invention, the technical features described in an open style include a closed technical solution composed of the listed features, and an open technical solution including the listed features.

In the present invention, a numerical interval (that is, a numerical range) is involved. Unless otherwise specified, the optional numerical distribution is considered to be continuous within the above-mentioned numerical interval, and includes the two numerical endpoints of the numerical range (that is, the minimum value and the maximum value). value), and every value between those two numerical endpoints. Unless otherwise specified, when a numerical range only refers to an integer within the numerical range, including the two endpoint integers of the numerical range, and each integer between the two endpoints, in this paper, it is equivalent to directly enumerating each integer An integer, for example, t is an integer selected from 1 to 10, which means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise indicated, ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameters in the present invention, if not particularly limited, are allowed to be constant temperature treatment, and are also allowed to vary within a certain temperature range. It will be appreciated that the described constant temperature treatment allows the temperature to fluctuate within the precision of the instrument control. It is allowed to fluctuate within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C.

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

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. Unless it conflicts with the purpose of the invention and/or the technical solution of the present application, the citations involved in the present invention are cited in their entirety and purpose. When the cited documents are involved in the present invention, the definitions of related technical features, terms, nouns, phrases, etc. in the cited documents are also cited together. When cited documents are involved in the present invention, examples and preferred modes of the cited related technical features may also be incorporated into the present application by reference, but are limited to being able to implement the present invention. It should be understood that, when the cited content conflicts with the description in the present application, the present application shall prevail or be adapted according to the description of the present application.

Traditionally, the content of various amino acid components in plasma varies greatly. To achieve simultaneous detection of multiple plasma amino acids, the detection method needs to ensure the effective separation of amino acids with similar polarities in derivative products, and at the same time cover the accurate determination of amino acids at different concentrations. , and can also overcome the problem of very limited service life of the chromatographic column and high project operation cost when the buffer salt system with strong alkalinity is used as the mobile phase.

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

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

The amino acid in the pretreatment test solution is detected by using pre-column online derivatization high-performance liquid chromatography; wherein, in the pre-column online derivatization high-performance liquid chromatography, the pre-column online derivatization process includes:

Extract the borate buffer, extract the pretreatment test solution, and mix;

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

Wash the needle with the solvent B, wash the needle with the solvent C, wait, suction 9-fluorenemethoxycarbonyl 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 sample bottle and entering by moving the sample needle, or by carrying the relevant solvent through the cleaning port of the equipment, or cleaning by other cleaning methods. Limit the cleaning method.

In some embodiments of the present invention, the volume ratio of the borate buffer, the o-phthalaldehyde, the 9-fluorenemethoxycarbonyl chloride, the diluent and the pretreatment test solution is ( 0.4 to 0.7): (0.15 to 0.35): (0.15 to 0.3): (7 to 9): (0.3 to 0.5). The volume ratio of the borate buffer, the o-phthalaldehyde, the 9-fluorene methoxycarbonyl chloride, the diluent and the pretreatment 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 adopted derivatization reagent mixing ratio system can ensure that the pH value of the derivatization environment is in an alkaline state, resulting in a better derivatization effect; and the loss of the chromatographic column is low, and the chromatographic column is improved. advantages of longevity. Since the buffer salt of the amino acid item is alkaline, the general silica gel column has poor stability under alkaline conditions.

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

The stationary phase is a C18 chromatographic column;

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

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 adopts 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%;

From 15.1min to 17.6min, the volume percentage of the mobile phase A remains 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 remains at 90% to 98%.

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

In the detection method provided by the present invention, in the mobile phase B, the volume ratios of methanol, acetonitrile and water are, for example, 40:50:10, 38:47:12, 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 the amino acids whose derivative products are relatively similar in polarity.

Further, the chromatographic conditions include:

The stationary phase is a C18 chromatographic column;

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

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 adopts 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 decreased from 96% to 43%;

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

From 15.1min to 17.6min, the volume percentage of the mobile phase A remains 0%;

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

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

In some embodiments of the present invention, the chromatographic conditions further include: the column temperature is 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, and 35°C.

In some embodiments of the present invention, the chromatographic conditions further include: the injection volume is 0.35 μL to 0.45 μL. The injection volume is, for example, 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, and 0.45 μL.

In some embodiments of the present invention, the chromatographic conditions further include: the injection temperature is 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. Flow rates are, for example, 0.3mL/min, 0.32mL/min, 0.34mL/min, 0.36mL/min, 0.38mL/min, 0.40mL/min, 0.42mL/min, 0.44mL/min, 0.46mL/min, 0.48mL /min, 0.5mL/min.

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

Further, the chromatographic condition of described high performance liquid chromatography comprises:

The stationary phase is a C18 chromatographic column;

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

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 adopts 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 decreased from 96% to 43%;

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

From 15.1min to 17.6min, the volume percentage of the mobile phase A remains 0%;

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

From 17.7min to 25min, the volume percentage of the mobile phase A remains at 96%;

The column temperature is 25℃～35℃;

The injection volume is 0.35μL～0.45μL;

The injection temperature was 3.5°C to 4.5°C.

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: 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).

In some embodiments of the invention, the amino acid comprises a primary amino acid and a secondary amino acid;

The secondary amino acids comprise one or two of the following amino acids: hydroxyproline and proline;

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

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 ranges from 750 μmol/L to 850 μmol/L, such as 750 μmol/L, 760 μmol/L, 770 μmol/L, 780 μmol/L, 790 μmol/L, 800 μmol/L L, 810 μmol/L, 820 μmol/L, 830 μmol/L, 840 μmol/L, 850 μmol/L.

In some embodiments of the present invention, the concentration of sarcosine is 150 μmol/L˜250 μmol/L, such as 150 μmol/L, 160 μmol/L, 170 μmol/L, 180 μmol/L, 190 μmol/L, 200 μmol/L , 210μmol/L, 220μmol/L, 230μmol/L, 240μmol/L, 250μmol/L.

Further, the solvent in the amino acid internal standard solution contains hydrochloric acid with a concentration of 0.05mol/L～0.15mol/L, such as 0.05mol/L, 0.07mol/L, 0.09mol/L, 0.11mol/L, 0.13 mol/L, 0.15mol/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 borate buffer has a concentration of 0.35M to 0.45M (eg, 0.35M, 0.37M, 0.39M, 0.41M, 0.43M, 0.45M), and a pH of 10 to 0.45M. 10.5 (eg 10, 10.1, 10.2, 10.3, 10.4, 10.5).

In some embodiments of the present invention, in the procedure of the on-line derivatization treatment: the mixing method adopts air mixing, and the number of times of air mixing is 5 to 10 times, such as 5 times, 6 times, 7 times, and 8 times. , 9 times, 10 times.

In some embodiments of the present invention, the waiting time ranges from 0.1 min to 0.3 min, for example, 0.1 min, 0.15 min, 0.2 min, 0.25 min, 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, and 5 seconds.

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

Add a mixture of formic acid and methanol to the plasma to be tested with the amino acid internal standard solution added, freeze, centrifuge, collect the supernatant, remove the solvent, redissolve in hydrochloric acid, centrifuge, collect the supernatant, and prepare the pretreatment test solution.

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

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

In some embodiments of the present invention, the method of removing the solvent adopts nitrogen to dry; the concentration of hydrochloric acid used for reconstitution is 0.035mol/L～0.045mol/L, such as 0.035mol/L, 0.037mol/L , 0.039mol/L, 0.041mol/L, 0.043mol/L, 0.045mol/L.

specific embodiment

The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods that do not indicate specific conditions, preferably refer to the guidelines given in the present invention, and can also follow the experimental manuals or conventional conditions in the field, and can also follow the conditions suggested by the manufacturer, or refer to the existing conditions in the art. known experimental methods.

In the following specific examples, the measurement parameters related to raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. Involves temperature and time parameters, allowing acceptable deviations from instrument test accuracy or operational accuracy.

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 in 6 10 mL volumetric flasks, add ultrapure water to dissolve and make up to volume, and use Pure water is diluted stepwise to a range of concentrations.

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

The standard working curve working solution was obtained by mixing the above 6 kinds of supplementary amino acid solutions and 16 mixed amino acid series solutions in equal volumes respectively.

(2) Internal standard solution preparation

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

2. Sample pretreatment

The sample preprocessing steps are as follows:

(1) Aspirate 100 μL of plasma, add 25 μL of internal standard solution, and vortex to mix for 30s;

(2) Add 600 μL of formic acid methanol mixed solution with a volume percentage of 0.05% formic acid, vortex to mix for 5 minutes, and freeze at -20°C for 30 minutes;

(3) Transfer the supernatant after centrifugation for 5min (12000rpm, 4°C);

(4) nitrogen blowing for 15min (temperature 60℃);

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

(6) Centrifuge for 5 min (12000 rpm, 4° C.), transfer the supernatant to a 96-well filter plate for filtration, and seal the membrane.

3. Online derivatization

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

Table 1. The derivatization procedure of the scheme of the present invention

step parameter reagent 1 0.5μL Borate buffer, 0.4M, 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 Withdraw 8 μL Diluent, 25mM K₂HPO₄, 2mM KH₂PO₄, pH=2.5 15 Cleaning needle, 3s Mixed solution (solvent A=methanol:acetonitrile:water=40:50:10, v:v:v) 16 inject /

4. Detection by high performance liquid chromatography

Liquid chromatography conditions: Column Agilent ZOEBAX Eclipse Plus-C18 (100 x 2.1 mm, 1.8 μm), mobile phase A = 25 mM K ₂ HPO ₄ (containing 2 mM 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: 0.4 μL. Excitation wavelength: 256; Emission wavelength: primary free amino acid, 450 nm; secondary free amino acid, 315 nm. Gradient elution was used, and the elution procedure was as follows:

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

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

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

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

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

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

5. Experimental results

According to the above liquid chromatography conditions, the standard curve of 22 kinds of free amino acids was determined. The results are shown in Figure 1 (primary amino acid) and Figure 2 (secondary amino acid), it can be seen that the 22 kinds of free amino acids can be completely separated. The curve ranges and linear correlations of the 22 plasma free amino acids are shown in Table 2.

Table 2. Detection limit, linear range and linear correlation of 22 plasma free amino acids

serial number compound Detection limit μmol/L Linear range μmol/L regression curve R² 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:

Take pre-prepared mixed patient samples of low and high concentration levels, and conduct intermediate precision experiments: each level of samples is tested for 2 groups of data in each batch for 10 consecutive days; the mean value X of the test results for each level is calculated , standard deviation SD and coefficient of variation CV. The calculation results showed that the coefficients of variation of the intermediate precision of the 22 free amino acids in the samples were all in the range of 1.1% to 8.5%, and the intermediate precision of the method was good.

Accuracy Verification:

The patient matrix samples and the spiked samples with different concentrations were processed in parallel for 3 samples at each concentration, and the average value (basal concentration) of the patient matrix samples was calculated, and the actual measured concentration of each sample after adding the standard corresponds to the matrix sample. recovery rate. The results showed that the recoveries of 22 free amino acids in the samples were all in the range of 85.7% to 102.9%, and the accuracy of the method was good.

Matrix-specific validation:

Take the blank matrix prepared with the standard curve for detection, and observe whether there are interference peaks near the target peak.

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

Example 2

In a conventional automated derivatization procedure, the syringe draws several different reagents multiple times, which may lead to carryover contamination of the syringe or the system if the syringe is not sufficiently cleaned. In addition, since the derivatization reagent (FMOC) and its derivatized product are easily hydrolyzed, it is necessary to avoid introducing moisture during use and storage to ensure the effect of the derivatization reaction. The invention adopts the multi-solvent and step-by-step organic solvent needle washing steps, which can not only avoid the pollution carried by the injection needle, but also remove the water carried by the injection needle, and avoid the hydrolysis of the derivatization reagent (FMOC) and its derivatized product.

In this example, the needle washing scheme of different organic solvents is used to verify its influence on the detection effect of this detection scheme. The detection scheme of Example 1 was adopted, respectively using non-pure organic solvents (relative to Example 1, only an aqueous solution of acetonitrile with a volume ratio of 50% of acetonitrile was used to replace solvent C) and pure organic solvents (relative to Example 1, only The solvent A was replaced by acetonitrile) the needle washing step, and its influence on the detection was observed. The results are shown in Figure 5 and Figure 6, respectively.

As shown in Figure 5, after needle washing with impure organic solvent, the FMOC hydrolyzate (A) responds strongly at the set fluorescence wavelength, which raises the baseline of the chromatogram, while the derivative by-product (B) interferes with secondary amino acids. If only the solution of washing the needle with organic solvent is adopted, the first-level amino acid is likely to bring carry-over pollution. As shown in Figure 6, if a high-concentration sample is entered into the instrument system, then the reagent blank is entered, and multiple components to be tested will be found. There is a carryover effect.

The technical features of the above-mentioned embodiments and embodiments may be combined in any suitable manner. In order to simplify the description, all possible combinations of the technical features in the above-mentioned embodiments and embodiments are not described. However, as long as these There is no contradiction in the combination of technical features, and all should be considered to be within the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, which are convenient for a specific and detailed understanding of the technical solutions of the present invention, but should not be construed as a limitation on the protection scope of the invention patent. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several modifications 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 teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the obtained equivalent forms also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent of the present invention should be based on the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims (13)

1. An online derivatization detection method for plasma amino acid is characterized by comprising the following steps:

pretreating a to-be-detected plasma sample added with an amino acid internal standard solution 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 method, the procedure of the pre-column online derivatization treatment in the pre-column online derivatization high performance liquid chromatography comprises the following steps:

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

washing the needle with the solvent A, washing the needle with the solvent B, sucking the o-phthalaldehyde, and mixing;

washing the needle with the solvent B, washing the needle with the solvent C, waiting, sucking 9-fluorenylmethoxycarbonyl acyl 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.

2. The method for detecting online derivatization of plasma amino acid according to claim 1, wherein the solvent B is methanol; or/and the solvent C is acetonitrile.

3. The method for detecting online derivatization of plasma amino acid according to claim 1, wherein the volume ratio of the borate buffer solution, the o-phthalaldehyde, 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): (0.3-0.5).

4. The method for detecting the online derivatization of plasma amino acid according to any one of claims 1 to 3, wherein the chromatographic conditions in the pre-column online 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 potassium dihydrogen phosphate and 20mM to 30mM dipotassium hydrogen phosphate;

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

gradient elution is adopted as an elution mode;

the procedure for gradient elution included:

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

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

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

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

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

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

5. The method for detecting the online derivatization of plasma amino acids according to claim 4, wherein the chromatographic conditions further comprise: the column temperature is 25-35 ℃; or/and the sample injection amount is 0.35-0.45 mu L; or/and the sample introduction 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 wavelengths include: the primary amino acid is 445 nm-455 nm, and the secondary amino acid is 310 nm-320 nm.

6. The method for detecting the online derivatization of plasma amino acids according to any one of claims 1 to 3 and 5, wherein the amino acids comprise primary amino acids and secondary amino acids;

the secondary amino acids comprise 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.

7. The method for detecting the online derivatization of plasma amino acids according to any one of claims 1 to 3 and 5, wherein the amino acid internal standards in the amino acid internal standard solution comprise norvaline and sarcosine.

8. The method for detecting online derivatization of plasma amino acids according to claim 7, wherein the norvaline concentration is 750-850 μmol/L; or/and the concentration of the sarcosine is 150-250 mu mol/L.

9. The method for detecting the online derivatization of plasma amino acid according to any one of claims 1 to 3, 5 and 8, wherein the solvent A comprises a mixed solution of methanol, acetonitrile and water in a volume ratio of (38-52) to (47-52) to (8-12).

10. The method for detecting the online derivatization of plasma amino acids according to any one of claims 1 to 3, 5 and 8, wherein the diluent is an acidic buffered salt solution containing 1.5mM to 2.5mM potassium dihydrogen phosphate and 20mM to 30mM dipotassium hydrogen phosphate, and the pH is 2 to 3; or/and the concentration of the borate buffer solution is 0.35-0.45M, and the pH value is 10-10.5.

11. The method for detecting plasma amino acid derivatization online according to any one of claims 1 to 3, 5 and 8, wherein in the procedure of the pre-column online derivatization treatment: the mixing mode adopts air mixing, and the air mixing frequency is 5-10 times; and/or the waiting time is 0.1 min-0.3 min; and/or the time for washing the needle is 2-5 seconds.

12. The method for detecting the online derivatization of plasma amino acids according to any one of claims 1 to 3, 5 and 8, wherein the pretreatment step comprises: and adding a mixed solution of formic acid and methanol into the to-be-detected plasma sample added with the amino acid internal standard solution, freezing, centrifuging, collecting supernate, removing a solvent, redissolving hydrochloric acid, centrifuging, collecting supernate, and preparing the pretreatment test solution.

13. The method for detecting online derivatization of plasma amino acids according to claim 12, wherein the volume percentage of formic acid in the mixed solution of formic acid and methanol is 0.04-0.08%; or/and, the conditions of freezing include: the temperature is-25 ℃ to-15 ℃, and the time is 20min to 40min; or/and the solvent is removed by blowing with nitrogen; or/and the concentration of hydrochloric acid adopted by redissolution is 0.035-0.045 mol/L.

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