阅读说明：本技术 一种测定细胞基质中尿囊素浓度的衍生化结合lc-ms方法 (derivatization combined LC-MS method for determining allantoin concentration in cell matrix ) 是由 吴凌云 於江华 王晓霞 彭帅 于 2019-10-26 设计创作，主要内容包括：本发明提供一种测定细胞基质中尿囊素浓度的衍生化结合LC-MS方法,该方法利用尿囊素碱水解和酸水解后生成的乙醛酸与2,4-二硝基苯肼发生缩合反应,形成稳定衍生物乙醛酸-2,4-二硝基苯腙,该衍生物具有较高的质谱电喷雾离子化效率,利用5-硝基吲哚-2-甲酸作为内标物,能够对细胞样品中的尿囊素进行定量化的分析。(The invention provides a derivatization combined LC-MS method for determining the concentration of allantoin in a cell matrix, which utilizes glyoxylic acid generated after the alkali hydrolysis and acid hydrolysis of the allantoin to perform condensation reaction with 2, 4-dinitrophenylhydrazine to form a stable derivative glyoxylic acid-2, 4-dinitrophenylhydrazone, wherein the derivative has higher mass spectrum electrospray ionization efficiency, and 5-nitroindole-2-formic acid is used as an internal standard substance to quantitatively analyze the allantoin in a cell sample.)

1. A derivatization coupled LC-MS method for determining the concentration of allantoin in a cell matrix, the method comprising the steps of:

Step 1, preparation of a detection solution of a sample to be detected:

Taking 10 mu L of transfer sample liquid, adding 90 mu L of blank cell matrix, reacting 20 mu L of 0.3mol/L NaOH in a water bath kettle at 85 ℃ for 60min, and then adding 40 mu L of 0.25mmol/L DNPH in the water bath kettle at 85 ℃ for reacting for 20min to obtain a reaction product GLX-DNPH;

Step 2, preparing a standard solution:

Taking 100 mu L allantoin liquid, adding 20 mu L0.3mol/L NaOH to react in a water bath kettle at 85 ℃ for 60min, adding 40 mu L2.5 mmol/L DNPH to react in the water bath kettle at 85 ℃ for 20min to obtain a reaction product GLX-DNPH; taking 16 mu L of reaction product, adding 84 mu L of blank cell matrix on an ice box, and then adding 20 mu L of 0.6mol/L NaOH and 40 mu L of 2mol/L HCl;

step 3, preparation of internal standard solution:

Weighing 10.31mg of 5-nitroindole-2-formic acid in a 50ml measuring flask, using methanol to fix the volume to a scale mark to obtain a mother solution of 1000 mu mol/L, and using ultrapure water to dilute the mother solution to 1.5 mu mol/L for later use;

And 4, detection:

Respectively adding 40 mu L of 1.5 mu mol/L internal standard solution and 200 mu L of acetonitrile precipitated protein into 160 mu L of sample detection solution and standard solution to be detected, filtering to obtain cell transfer experiment sample solution of the sample to be detected and standard curve preparation sample solution, preparing the standard curve preparation sample solution into samples with the concentration of 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 3.75 and 5 mu mol/L, respectively mixing the samples uniformly, centrifuging at 12000rpm and 4 ℃ for 10min, taking 130 mu L of supernatant, injecting the sample into LC-MS to obtain a chromatogram, preparing a sample chromatogram according to the standard curve to draw the standard curve, and calculating the allantoin concentration of the sample to be detected according to the sample chromatogram and the standard curve;

Wherein: the chromatographic conditions were as follows:

Chromatographic column VP-ODS column (2.0mM i.d.. times.150 mM,4.6 μm, Shimadzu, Kyoto, Japan), 5mM ammonium formate as phase A, 90% acetonitrile as phase B, mobile phase flow rate of 0.3ml/min, sample amount of 5 μ L, gradient elution program of 0.01-4.50min: 22% B, 4.50-10.00: 22% B-35% B, 10.00-12.00: 35% -90% B, 12.00-15.00: 90% B, 15.00-17.00min: 90% -22% B, 17.00-22.00: 22% B; the mass spectrum acquisition time is 22 min; the interface voltage is-3.5 kv; the atomization airflow speed is 1.5L/min; the drying airflow rate was 15L/min.

Technical Field

The invention belongs to the field of analytical chemistry methods, and particularly relates to a method for determining the concentration of allantoin in cells by using derivatization and LC-MS (liquid chromatography-mass spectrometry) technology.

Technical Field

Allantoin aluminum, also known as dihydroxyallantoin aluminum; aluminum hydroxide in urine; weichangning (gastrointestinal disease remedy); the medicine is prepared into tablets for preparing acid and protecting gastric mucosa, and the dosage form is as follows: 100mg, 55mg of allantoin and 45mg of aluminum hydroxide. Pharmacological action of allantoin aluminum: can accelerate the growth of normal granulation tissues of the injured part and promote the regeneration of mucosal epithelial cells. Indications for aluminum allantoin: protecting medicine for digestive tract mucous membrane. Can be used for treating gastric ulcer, duodenal bulbar ulcer, and chronic gastritis. In the allantoin aluminum, the structural formula of the allantoin is as follows: allantoin is a derivative of urea and is,Agricultural and light chemical industries. Allantoin is widely found in nature, such as in the urine of fetuses and in all but human and simian allantoic fluids of mammals. Allantoin is also found in some plants, such as tobacco seeds, wheat seeds, sugar beets, medical cynoglossum, wisteria flowers and roots of comfrey in the family Boraginaceae.

the existing allantoin detection method also comprises a liquid chromatography-tandem triple quadrupole mass spectrometry method, wherein 13C1,15N 1-allantoin is often used as an isotope internal standard, and a normal phase chromatographic column is used for increasing the retention of the allantoin on the chromatographic column. Another method is that derivatization is combined with an HPLC method, and quantitative detection can be carried out at 360nm, but the method has low sensitivity and cannot detect trace endogenous substances. For example, the method for measuring the content of allantoin in a diabolo capsule by a derivatization method is disclosed in the 'Zhongnan pharmacy' 2008 th stage 02, 435-phase 438 in the 5 th stage of 2004, and the method for measuring the concentration of the allantoin in the human plasma by a pre-column derivatization high performance liquid chromatography is disclosed.

the invention provides a derivatization and LC-MS (liquid chromatography-mass spectrometry) method, which can accurately measure glyoxylic acid generated by hydrolysis of exogenous allantoin in a cell sample and endogenous glyoxylic acid in a cell matrix so as to calculate the true content of the allantoin. Glyoxylic acid is a basic organic chemical raw material and is widely applied to the fields of medicines and daily chemicals. Endogenous glyoxylic acid is an intermediate product of light respiration in plants, and it has also been reported in the literature that endogenous glyoxylic acid is also contained in plasma and urine of animals. The glyoxylic acid has the following structural formula:

the method provided by the invention realizes accurate quantitative analysis of polar small-molecule allantoin in the cell matrix, avoids strong matrix effect generated by more endogenous polar small molecules in a biological sample and damage of salt-containing samples to chromatographic columns and instruments through positive-phase column analysis, and breaks through the combination of liquid chromatography and triple quadrupole mass spectrometry (LC-MS/MS)¹³C₁,¹⁵N₁The method using allantoin as the isotope internal standard has higher requirement on instruments and internal standards, and makes up for the defects that the derivatization is combined with the HPLC method, so that the sensitivity is poor and the trace allantoin cannot be accurately quantified.

disclosure of Invention

The invention aims to determine the allantoin concentration in cells by adopting a derivatization combined liquid chromatography-mass spectrometry (HPLC-MS, LC-MS) technology, namely a liquid chromatography-mass spectrometry technology, so as to avoid the problem that the matrix effect is too strong due to the fact that the polar small molecules are easily interfered by other endogenous polar small molecule substances in the matrix when being directly determined.

in order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

a derivatization combined LC-MS method for measuring the allantoin concentration in cell matrix is used to accurately measure the concentration of internal/external glyoxylic acid in cells, and the method utilizes the hydrolysis of allantoin to generate glyoxylic acid, and the glyoxylic acid reacts with 2, 4-dinitrophenylhydrazine to generate a stable derivatization product glyoxylic acid-2, 4-dinitrophenylhydrazone. The derivative has higher mass spectrum electrospray ionization efficiency, and simultaneously, the internal standard substance 5-nitroindole-2-formic acid with similar structure and molecular weight to the derivative product is utilized to carry out quantitative analysis on allantoin in cells by combining the liquid chromatography-mass spectrometry technology.

To this end, the invention provides a derivatization coupled with LC-MS method for determining the allantoin concentration in a cell matrix, said method comprising the steps of:

Step 1, preparation of a detection solution of a sample to be detected:

Taking 10 mu L of transfer sample liquid, adding 90 mu L of blank cell matrix, reacting 20 mu L of 0.3mol/L NaOH in a water bath kettle at 85 ℃ for 60min, adding 40 mu L of 0.25mmol/L DNPH, and reacting in the water bath kettle at 85 ℃ for 20min to obtain a reaction product GLX-DNPH (liquid state);

Step 2, preparing a standard solution:

Taking 100 mu L of allantoin liquid (see the specification [ 0012 ]), adding 20 mu L of 0.3mol/L NaOH to react in a water bath kettle at 85 ℃ for 60min, then adding 40 mu L of 2.5mmol/L DNPH to react in the water bath kettle at 85 ℃ for 20min to obtain a reaction product GLX-DNPH (liquid state); taking 16 mu L of reaction product, adding 84 mu L of blank cell matrix on an ice box, and then adding 20 mu L of 0.6mol/L NaOH and 40 mu L of 2mol/L HCl;

Step 3, preparation of internal standard solution:

Weighing 10.31mg of 5-nitroindole-2-formic acid in a 50ml measuring flask, using methanol to fix the volume to a scale mark to obtain a mother solution of 1000 mu mol/L, and using ultrapure water to dilute the mother solution to 1.5 mu mol/L for later use;

And 4, detection:

Respectively adding 40 mu L of 1.5 mu mol/L internal standard solution and 200 mu L of acetonitrile precipitated protein into 160 mu L of sample detection solution and standard solution to be detected, filtering to obtain cell transfer experiment sample solution of the sample to be detected and standard curve preparation sample solution, preparing the standard curve preparation sample solution into samples with the concentration of 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 3.75 and 5 mu mol/L, respectively mixing the samples uniformly, centrifuging at 12000rpm and 4 ℃ for 10min, taking 130 mu L of supernatant, injecting the sample into LC-MS to obtain a chromatogram, preparing a sample chromatogram according to the standard curve to draw the standard curve, and calculating the allantoin concentration of the sample to be detected according to the sample chromatogram and the standard curve;

Wherein: the chromatographic conditions were as follows:

Chromatographic column VP-ODS column (2.0mM i.d.. times.150 mM,4.6 μm, Shimadzu, Kyoto, Japan), 5mM ammonium formate as phase A, 90% acetonitrile as phase B, mobile phase flow rate of 0.3ml/min, sample amount of 5 μ L, gradient elution program of 0.01-4.50min: 22% B, 4.50-10.00: 22% B-35% B, 10.00-12.00: 35% -90% B, 12.00-15.00: 90% B, 15.00-17.00min: 90% -22% B, 17.00-22.00: 22% B; the mass spectrum acquisition time is 22 min; the interface voltage is-3.5 kv; the atomization airflow speed is 1.5L/min; the drying airflow rate is 15L/min; .

Wherein the content of the first and second substances,

Transferring the sample liquid: cell samples obtained from Caco-2 cell transport experiments; called transport sample solution or quality control sample working solution, the Caco-2 cell transport experiment is shown in example 2.

sources of the standards: allantoin standard (Chinese food and drug testing institute)

The source of the internal standard: 5-nitroindole-2-carboxylic acid (purity is more than or equal to 98 percent, and the purity is of a source leafy organism);

Blank cell matrix: the cells were incubated for 2h in HBSS buffer.

intracellular/exogenous glyoxylate: cell endogenous glyoxylate-glyoxylate that Caco-2 cells contain themselves;

cell-exogenous glyoxylate, namely the glyoxylate generated by the hydrolysis of the added allantoin in a Caco-2 cell transport experiment.

The process of hydrolyzing allantoin to glyoxylic acid under alkaline (such as sodium hydroxide) conditions:

the reaction of glyoxylic acid and 2, 4-dinitrophenylhydrazine to generate a stable derivative product glyoxylic acid-2, 4-dinitrophenylhydrazone:

the lower limit of the quantification of the derivative glyoxylic acid-2, 4-dinitrophenylhydrazone can reach 0.08 mu mol/L, which is far higher than that of the derivative glyoxylic acid-2, 4-dinitrophenylhydrazone which is directly used for detecting allantoin in a cell matrix.

according to the derivatization and LC-MS method for determining the concentration of allantoin in the cell matrix, the method utilizes derivatization reaction to realize the analysis of polar molecules by reversed phase chromatography, improves ionization efficiency of mass spectrum, reduces matrix effect, and reduces the cost of instruments and reagents used in experiments.

the experimental method is obtained by screening, and the screening process is as follows.

using the following reagents: allantoin bulk drug (provided by Wuxi Jimin credible Shanhe pharmaceutical Co., Ltd., purity > 99%); 2, 4-dinitrophenylhydrazine (the purity is more than or equal to 99 percent, Adamas-beta); 5-nitroindole-2-carboxylic acid (purity is more than or equal to 98 percent, and the purity is of a source leafy organism); acetonitrile, methanol (chromatographically pure, Tedia, usa).

allantoin, internal standard substance and derivative mother liquor are prepared respectively. Accurately weighing 15.81mg of allantoin in a 100ml measuring flask, and metering the volume to a scale mark by using ultrapure water, namely mother liquor of 1000 mu mol/L; diluting with ultrapure water to obtain a standard curve working solution: 0.8, 1.6, 3.1, 6.3, 12.5, 25, 37.5, 5 μmol/L; quality control sample working solution: 0.8, 2.35, 12.5, 37.5 mu mol/L. Accurately weighing 10.31mg of 5-nitroindole-2-formic acid in a 50ml measuring flask, using methanol to fix the volume to a scale mark to obtain 1000 mu mol/L mother liquor, and using ultrapure water to dilute the mother liquor to 1.5 mu mol/L for later use. Precisely weighing 24.77mg of 2, 4-dinitrophenylhydrazine in a 25ml measuring flask, using 2mol/L HCl to fix the volume to a scale mark, obtaining 5mmol/L mother liquor, and using 2mol/L HCl to dilute the mother liquor to 0.5mmol/L for later use.

And (3) performing derivatization reaction, namely performing alkaline hydrolysis on allantoin at 85 ℃ for 60min, then continuously reacting the allantoin with a derivatization reagent 2, 4-dinitrophenylhydrazine at 85 ℃ for 20min, finally adding internal standard 5-nitroindole-2-formic acid and acetonitrile (used for precipitating protein), centrifuging at a high speed at a low temperature, and then putting the mixture into a liquid phase small bottle for liquid mass analysis.

The conditions of the derivatization reaction are as follows: performing alkaline hydrolysis of allantoin at 85 deg.C for 60min, reacting with derivatization reagent at 85 deg.C for 20min, adding internal standard and acetonitrile, centrifuging, and performing liquid quality analysis.

In the invention, a standard curve without endogenous glyoxylate interference is prepared, and the endogenous glyoxylate concentration in a blank matrix and the real allantoin concentration in a quality control sample are measured, and the specific sample preparation method is shown in example 1.

The method for determining the concentration of allantoin in a cell sample according to one of the described derivatization for determining the concentration of allantoin in a cell matrix in combination with the LC-MS method is as described above for calibration standards.

the sample to be detected is taken from a Caco-2 cell transport experiment. See example 2 for a specific sample preparation method.

Compared with the prior art, the method has the advantages of high sensitivity, high specificity to allantoin, small matrix effect and the like:

(1) the method can accurately measure the glyoxylate generated by the hydrolysis of the exogenous allantoin in a cell sample and the endogenous glyoxylate in a cell matrix, thereby calculating the true content of the exogenous allantoin.

(2) The reverse phase chromatography can be used for accurately and quantitatively analyzing the polar small molecule allantoin in the cell matrix.

(3) can avoid the stronger matrix effect generated by more endogenous polar small molecules in the biological sample and the damage to a chromatographic column and an instrument caused by the normal phase column analysis of the salt-containing sample.

(4) Tandem triple quadrupole mass spectrometry (LC-MS/MS) binding compared to liquid chromatography¹³C₁,¹⁵N₁the method of taking allantoin as an isotope internal standard greatly reduces the higher requirements on instruments and internal standards, and simultaneously makes up the defects that the derivatization is combined with the HPLC method, the sensitivity is poor, and trace allantoin cannot be accurately quantified.

the method of the invention utilizes derivatization reaction to realize the analysis of polar molecules by reversed phase chromatography, improves ionization efficiency of mass spectrum, reduces matrix effect and reduces cost of instruments and reagents used in experiments.

compared with the prior art, such as 435-438 in 5 th stage 2004 of China New drug journal, the method for determining the allantoin concentration in human plasma by the pre-column derivatization high performance liquid chromatography has the difference that:

the method is different from the method used in 435-438 of 5 th phase 2004 of China New drug journal, the method uses a mass spectrum detector, and the previous method uses a PDA detector. Has the advantages that: the sensitivity is greatly improved by using a mass spectrum detector, and the method can be used for measuring the concentration of trace allantoin and even detecting the content of endogenous glyoxylic acid in a cell matrix.

drawings

FIG. 1 is a schematic diagram of allantoin derivatization reaction

FIG. 2LC-MS methodological specificity chromatogram (A. true blank matrix without endogenous glyoxylic acid B.LLOQ C. blank matrix D. Permeability Experimental sample)

FIG. 3 apparent permeability coefficients (P) of AP → BL side and BL → AP side of different concentrations of allantoin aluminum_app) Drawing (A)

Detailed Description

The following description will further explain the substance of the present invention by using the embodiments of the present invention with reference to the accompanying drawings, but the present invention is not limited thereto.

The data acquisition and processing of the embodiment of the invention are carried out according to the following method:

All LC-MS experiments related to the present invention were performed on Shimadzu LC-MS 202. Gradient elution was performed using a SHIMADZU column (2.0mM i.d.. times.150 mM,4.6 μm) and a SHIMADZU guard column ((2.0mM i.d.. times.5 mM,4.6 μm) for separation of allantoin derivatives, using 5mM ammonium formate (a) and 90% acetonitrile (B) as mobile phases.

The mass spectrum ion source is an electrospray ion source (ESI), data are collected in a negative ion SIM mode, the product glyoxylic acid-2, 4 dinitrophenylhydrazone has strong response in a negative ion mode (m/z 253), and the internal standard 5-nitroindole-2-formic acid has strong response in a negative ion mode (m/z 205).