阅读说明：本技术 检测血液中甲基丙二酸含量的方法 (Method for detecting content of methylmalonic acid in blood ) 是由 邱天祎 贾永娟 许丽 倪君君 于 2021-01-05 设计创作，主要内容包括：本发明提供了检测血液中甲基丙二酸含量的方法,该方法包括：在一定检测条件下,利用高效液相串联质谱仪分别检测至少三个标准溶液,得到各个标准溶液的色谱图,各标准溶液中均含有浓度已知的甲基丙二酸标准品及内标物；根据各个标准溶液的色谱图拟合得到标准曲线方程；将内标工作液和血液样本混匀,加入乙腈并混匀,离心取上清液,通过加热氮吹的方式吹干上清液,吹干后加入含0-2％甲酸和0-20％乙腈的水溶液并混匀,离心取上清液得到待测样本；再同样检测待测样本得到其色谱图；根据待测样本的色谱图和标准曲线方程,计算血液样本中甲基丙二酸的含量。本方案提供了检测血液中甲基丙二酸含量的方法,样本前处理操作较简单,且成本较低。(The invention provides a method for detecting the content of methylmalonic acid in blood, which comprises the following steps: under certain detection conditions, respectively detecting at least three standard solutions by using a high performance liquid tandem mass spectrometer to obtain a chromatogram of each standard solution, wherein each standard solution contains a methylmalonic acid standard product with known concentration and an internal standard substance; fitting according to the chromatogram of each standard solution to obtain a standard curve equation; uniformly mixing the internal standard working solution and the blood sample, adding acetonitrile, uniformly mixing, centrifuging to obtain a supernatant, blow-drying the supernatant in a heating nitrogen blowing mode, adding an aqueous solution containing 0-2% of formic acid and 0-20% of acetonitrile, uniformly mixing, centrifuging to obtain a supernatant to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the content of the methylmalonic acid in the blood sample according to the chromatogram and the standard curve equation of the sample to be detected. The scheme provides a method for detecting the content of methylmalonic acid in blood, and the sample pretreatment operation is simple and the cost is low.)

1. The method for detecting the content of methylmalonic acid in blood is characterized by comprising the following steps:

respectively detecting at least three standard solutions by using a high performance liquid chromatography tandem mass spectrometer under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a methylmalonic acid standard substance with known concentration and an internal standard substance, and the concentration of the methylmalonic acid standard substance in different standard solutions is different;

fitting to obtain a standard curve equation of the methylmalonic acid according to the chromatogram of each standard solution;

uniformly mixing a certain amount of internal standard working solution and a certain amount of blood sample, adding a certain amount of acetonitrile, uniformly mixing, centrifuging to obtain supernatant, blow-drying the supernatant in a heating nitrogen blowing mode, adding a certain amount of complex solution after blow-drying, uniformly mixing, centrifuging to obtain the supernatant to be detected, wherein the internal standard working solution contains an internal standard substance with known concentration, and the complex solution is an aqueous solution containing 0-2% of formic acid and 0-20% of acetonitrile;

detecting a sample to be detected by using a high performance liquid chromatography tandem mass spectrometer under the same detection condition to obtain a chromatogram of the sample to be detected;

and calculating the content of the methyl malonic acid in the blood sample according to the chromatogram of the sample to be detected and the standard curve equation of the methyl malonic acid.

2. The method of claim 1,

mix a certain amount of interior label working solution and a certain amount of blood sample mixing, add a certain amount of acetonitrile and mixing again, the supernatant is got in the centrifugation, weathers the supernatant through the mode that the heating nitrogen blows, adds a certain amount of compound solution and mixing after weathering, and the supernatant is got in the centrifugation obtains the sample that awaits measuring, includes:

transferring 50-200 mu L of blood sample into a 1.5mL centrifuge tube by using a liquid transfer gun, wherein the blood sample is serum or plasma obtained by processing at least 1mL of blood to be detected, adding a certain amount of internal standard working solution, and mixing for 20s-2min by vortex oscillation at the rotating speed of 1000-2500 rpm;

adding 200-;

blowing the supernatant with nitrogen at 35-70 ℃, adding 50-200 mu L of complex solution, mixing for 1-3min by vortex oscillation at the rotation speed of 1000-2500rpm, then centrifuging for 5-12min at the temperature of 4-10 ℃ and the rotation speed of 10000-14000rpm, and taking the supernatant to obtain a sample to be detected.

3. The method of claim 2,

the internal standard working solution contains 0.5-20nmol/mL of methylmalonic acid-d 3, and the addition amount is 10 muL.

4. The method of claim 1,

the liquid phase conditions in the detection conditions include: a hexyl-phenyl chromatographic column, wherein the mobile phase A is an aqueous solution containing 0.4 percent of formic acid and 2mM ammonium formate, the mobile phase B is acetonitrile, a gradient elution mode is adopted in the elution process, the analysis time is 3-7min, the column temperature is 20-45 ℃, the sample injection amount is 3-20 mu L, and the flow rate is 0.20-0.70 mL/min.

5. The method of claim 4,

the hexyl-phenyl chromatography column comprises: phenomenex Gemini C6-Phenyl chromatographic column, the length of chromatographic column is 150mm, the internal diameter is 2.0mm, and the particle size of filler is 3 μm.

6. The method of claim 4,

the flow rate is 0.40 mL/min;

the elution process comprises:

time (min) Mobile phase A (%) Mobile phase B (%) 0.00 97 3 1.00 97 3 1.50 5 95 2.50 5 95 2.51 97 3 5.00 97 3

。

7. The method of claim 1,

tandem mass spectrometry conditions among the detection conditions include: ESI (-) detection mode, MRM acquisition mode, 20-55psi of air curtain pressure, 1-12psi of collision air pressure, 1500-4500V of ion spray voltage value, 750 ℃ of ionization temperature, 10-90psi of atomization air pressure, and 10-90psi of auxiliary air pressure.

8. The method of claim 7,

the air curtain pressure was 20psi, the collision air pressure was 5psi, the ion spray voltage was-2000V, the ionization temperature was 550 deg.C, the atomization air pressure was 80psi, and the auxiliary air pressure was 60 psi.

9. The method of claim 1,

before the separately detecting at least three standard solutions, further comprising:

preparing at least three standard working solutions, wherein the standard working solutions contain methyl malonic acid standard substances with known concentrations, the concentration of the methyl malonic acid standard substances in the standard working solutions is within the range of 0.4-51.2nmol/mL, and the concentrations of the methyl malonic acid standard substances in different standard working solutions are different;

transferring 10 mu L of standard working solution and 10 mu L of internal standard working solution into a centrifuge tube by using a pipette, and then transferring and adding 80 mu L of the complex solution;

and (3) uniformly mixing the centrifugal tube in a vortex manner at the rotation speed of 1000-2500rpm for 0.5-1min to obtain a standard solution.

10. The method of claim 9,

the concentration of the methylmalonic acid standard in each standard working solution was 0.4nmol/mL, 0.8nmol/mL, 1.6nmol/mL, 3.2nmol/mL, 6.4nmol/mL, 12.8nmol/mL, 25.6nmol/mL, and 51.2nmol/mL, respectively.

Technical Field

The invention relates to the technical field of clinical chemistry, in particular to a method for detecting the content of methylmalonic acid in blood.

Background

Popularity ofPathologically survey data show vitamin B12 deficiency rates of about 20% in the us and canadian investigational population, up to 40% in latin america and even up to 80% in indian children. Women of childbearing age, infants and the elderly are at high risk for vitamin B12 deficiency. Vitamin B12 deficiency can cause pathologies in the blood system, metabolism and nervous system, especially increase the risk of fertility in pregnant women and affect the cognitive development of infants and young children. Some elderly people with mild reduction in serum vitamin B12 have been shown to have Methylmalonic acid (MMA, molecular formula C) in their serum₄H₆O₄) The significant increase suggested that the measurement of the metabolite MMA was more sensitive than that of serum vitamin B12, and that the measurement of serum MMA was also used as a criterion for determining whether the vitamin B12 supplementation was sufficient.

At present, the content of methylmalonic acid in blood can be detected by adopting a liquid chromatography-mass spectrometry combined method, wherein a pretreatment method of a blood sample can adopt derivatization treatment or ultrafiltration centrifugal pretreatment. However, the derivatization treatment is complicated, and the ultrafiltration centrifugal pretreatment is a little simple in operation, but the material cost of the ultrafiltration centrifugal tube is high, which results in high detection cost.

Disclosure of Invention

The invention provides a method for detecting the content of methylmalonic acid in blood, and the sample pretreatment operation is simple and the cost is low.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides a method for detecting the content of methylmalonic acid in blood, which comprises the following steps:

respectively detecting at least three standard solutions by using a high performance liquid chromatography tandem mass spectrometer under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a methylmalonic acid standard substance with known concentration and an internal standard substance, and the concentration of the methylmalonic acid standard substance in different standard solutions is different;

fitting to obtain a standard curve equation of the methylmalonic acid according to the chromatogram of each standard solution;

uniformly mixing a certain amount of internal standard working solution and a certain amount of blood sample, adding a certain amount of acetonitrile, uniformly mixing, centrifuging to obtain supernatant, blow-drying the supernatant in a heating nitrogen blowing mode, adding a certain amount of complex solution after blow-drying, uniformly mixing, centrifuging to obtain the supernatant to be detected, wherein the internal standard working solution contains an internal standard substance with known concentration, and the complex solution is an aqueous solution containing 0-2% of formic acid and 0-20% of acetonitrile;

detecting a sample to be detected by using a high performance liquid chromatography tandem mass spectrometer under the same detection condition to obtain a chromatogram of the sample to be detected;

and calculating the content of the methyl malonic acid in the blood sample according to the chromatogram of the sample to be detected and the standard curve equation of the methyl malonic acid.

Preferably, mixing a certain amount of internal standard working solution and a certain amount of blood sample, adding a certain amount of acetonitrile and mixing, centrifuging to obtain supernatant, blow-drying the supernatant by heating nitrogen blowing, adding a certain amount of complex solution after blow-drying and mixing, centrifuging to obtain the supernatant to be tested, including:

transferring 50-200 mu L of blood sample into a 1.5mL centrifuge tube by using a liquid transfer gun, wherein the blood sample is serum or plasma obtained by processing at least 1mL of blood to be detected, adding a certain amount of internal standard working solution, and mixing for 20s-2min by vortex oscillation at the rotating speed of 1000-2500 rpm;

adding 200-;

blowing the supernatant with nitrogen at 35-70 ℃, adding 50-200 mu L of complex solution, mixing for 1-3min by vortex oscillation at the rotation speed of 1000-2500rpm, then centrifuging for 5-12min at the temperature of 4-10 ℃ and the rotation speed of 10000-14000rpm, and taking the supernatant to obtain a sample to be detected.

Preferably, the internal standard working solution contains 0.5-20nmol/mL of methylmalonic acid-d 3, and the addition amount is 10 muL.

Preferably, the liquid phase condition of the detection conditions includes: a hexyl-phenyl chromatographic column, wherein the mobile phase A is an aqueous solution containing 0.4 percent of formic acid and 2mM ammonium formate, the mobile phase B is acetonitrile, a gradient elution mode is adopted in the elution process, the analysis time is 3-7min, the column temperature is 20-45 ℃, the sample injection amount is 3-20 mu L, and the flow rate is 0.20-0.70 mL/min.

Preferably, the hexyl-phenyl chromatography column comprises: phenomenex Gemini C6-Phenyl chromatographic column, the length of chromatographic column is 150mm, the internal diameter is 2.0mm, and the particle size of filler is 3 μm.

Preferably, the flow rate is 0.40 mL/min;

the elution process comprises:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0.00	97	3
1.00	97	3
			1.50	5	95
2.50	5	95
			2.51	97	3
5.00	97	3

。

Preferably, tandem mass spectrometry conditions among the detection conditions include: ESI (-) detection mode, MRM acquisition mode, 20-55psi of air curtain pressure, 1-12psi of collision air pressure, 1500-4500V of ion spray voltage value, 750 ℃ of ionization temperature, 10-90psi of atomization air pressure, and 10-90psi of auxiliary air pressure.

Preferably, the air curtain pressure is 20psi, the collision air pressure is 5psi, the ion spray voltage is-2000V, the ionization temperature is 550 ℃, the atomization air pressure is 80psi, and the auxiliary air pressure is 60 psi.

Preferably, before the separately detecting at least three standard solutions, further comprising:

preparing at least three standard working solutions, wherein the standard working solutions contain methyl malonic acid standard substances with known concentrations, the concentration of the methyl malonic acid standard substances in the standard working solutions is within the range of 0.4-51.2nmol/mL, and the concentrations of the methyl malonic acid standard substances in different standard working solutions are different;

transferring 10 mu L of standard working solution and 10 mu L of internal standard working solution into a centrifuge tube by using a pipette, and then transferring and adding 80 mu L of the complex solution;

and (3) uniformly mixing the centrifugal tube in a vortex manner at the rotation speed of 1000-2500rpm for 0.5-1min to obtain a standard solution.

Preferably, the concentration of the methylmalonic acid standard in each standard working solution is 0.4nmol/mL, 0.8nmol/mL, 1.6nmol/mL, 3.2nmol/mL, 6.4nmol/mL, 12.8nmol/mL, 25.6nmol/mL, 51.2nmol/mL, respectively.

The invention provides a method for detecting the content of methylmalonic acid in blood, which comprises the following steps: under certain detection conditions, respectively detecting at least three standard solutions by using a high performance liquid tandem mass spectrometer to obtain a chromatogram of each standard solution, wherein each standard solution contains a methylmalonic acid standard product with known concentration and an internal standard substance; fitting according to the chromatogram of each standard solution to obtain a standard curve equation; uniformly mixing the internal standard working solution and the blood sample, adding acetonitrile, uniformly mixing, centrifuging to obtain a supernatant, blow-drying the supernatant in a heating nitrogen blowing mode, adding an aqueous solution containing 0-2% of formic acid and 0-20% of acetonitrile, uniformly mixing, centrifuging to obtain a supernatant to obtain a sample to be detected; detecting the sample to be detected to obtain a chromatogram map; and calculating the content of the methylmalonic acid in the blood sample according to the chromatogram and the standard curve equation of the sample to be detected. The invention provides a method for detecting the content of methylmalonic acid in blood, and the sample pretreatment operation is simple and the cost is low.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a flowchart of a method for detecting the level of methylmalonic acid in blood according to an embodiment of the present invention;

FIG. 2 is a chemical structural formula of methylmalonic acid provided according to an embodiment of the present invention;

FIG. 3 is a chromatogram of a methylmalonic acid standard in a standard solution provided by an embodiment of the present invention;

FIG. 4 is a chromatogram of methylmalonic acid-d 3 in a standard solution provided by an embodiment of the present invention;

FIG. 5 is a mass spectrum of a methylmalonic acid standard and methylmalonic acid-d 3 in a standard solution according to an embodiment of the present invention;

FIG. 6 is a chromatogram of methylmalonic acid in a sample to be tested according to an embodiment of the present invention;

FIG. 7 is a chromatogram of methylmalonic acid-d 3 in a test sample according to an embodiment of the present invention;

FIG. 8 is a mass spectrum of a methylmalonic acid standard and methylmalonic acid-d 3 in a sample to be tested according to an embodiment of the present invention;

FIG. 9 is a graph of the linear relationship of methylmalonic acid provided in accordance with one embodiment of the present invention.

In the drawings, 1: methylmalonic acid, 2: methylmalonic acid-d 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

As shown in figure 1, the embodiment of the invention relates to detection of endogenous substances of organic acids, and particularly provides a method for detecting the content of methylmalonic acid in blood, wherein the detection method is not used for diagnosis and treatment of diseases and can comprise the following steps:

step 101: and respectively detecting at least three standard solutions by using a high performance liquid chromatography tandem mass spectrometer under certain detection conditions to obtain the chromatogram of each standard solution, wherein any standard solution contains a methylmalonic acid standard substance with known concentration and an internal standard substance, and the concentration of the methylmalonic acid standard substance in different standard solutions is different.

Referring to fig. 2, fig. 2 shows the chemical structural formula of methylmalonic acid.

In an embodiment of the present invention, preferably, the hplc-tandem mass spectrometer can be Sciex 6500 plus. When the instrument is used for detecting the content of the methylmalonic acid in blood, the problems of insufficient sensitivity and high baseline noise easily caused by using other detection instruments can be solved.

Step 102: and fitting to obtain a standard curve equation of the methylmalonic acid according to the chromatogram of each standard solution.

Step 103: uniformly mixing a certain amount of internal standard working solution and a certain amount of blood sample, adding a certain amount of acetonitrile, uniformly mixing, centrifuging to obtain supernatant, blow-drying the supernatant in a heating nitrogen blowing mode, adding a certain amount of complex solution after blow-drying, uniformly mixing, centrifuging to obtain the supernatant to be detected, wherein the internal standard working solution contains an internal standard substance with known concentration, and the complex solution is an aqueous solution containing 0-2% of formic acid and 0-20% of acetonitrile.

In detail, acetonitrile is a precipitant, and by adding acetonitrile, protein impurities in a sample to be detected can be removed, and methylmalonic acid is reserved.

In detail, considering that methylmalonic acid is a water-soluble substance, the reconstituted solution in the embodiment of the present invention may be pure water. When pure water is used as the compound solution, the preparation of the compound solution can be simplified, and the use of organic reagents can be reduced.

In the embodiment of the invention, the internal standard and the blood sample are mixed uniformly, acetonitrile is added and mixed uniformly to precipitate protein, the supernatant is centrifugally taken and dried by heating and nitrogen blowing, the complex solution is added for redissolution after drying, and finally the supernatant is centrifugally taken to obtain the sample to be detected. The sample pretreatment not only can remove the interferents while retaining the methylmalonic acid to the maximum extent, but also is simple and easy to operate, consumes short time, and can obtain a sample to be detected without adopting complicated derivatization treatment, thereby detecting the content of the methylmalonic acid in blood. In addition, the cost of the sample pretreatment is obviously lower than that of the existing ultrafiltration centrifugal pretreatment, so that the sample pretreatment method is beneficial to ensuring lower sample detection cost.

Step 104: and detecting the sample to be detected by using a high performance liquid chromatography tandem mass spectrometer under the same detection condition to obtain a chromatogram of the sample to be detected.

Step 105: and calculating the content of the methyl malonic acid in the blood sample according to the chromatogram of the sample to be detected and the standard curve equation of the methyl malonic acid.

In the embodiment of the invention, based on the blood sample pretreatment process, the sample pretreatment can be simplified while the detection effect is ensured, the operation is simple, and the error caused by the operation can be reduced or avoided, so that the more accurate quantification can be realized. In addition, the pretreatment method is simple to operate and low in detection cost, so that the application and popularization of actual detection are facilitated.

In an embodiment of the present invention, preferably, in the step 103, mixing a certain amount of internal standard working solution and a certain amount of blood sample uniformly, adding a certain amount of acetonitrile and mixing uniformly, centrifuging to obtain a supernatant, blow-drying the supernatant by heating nitrogen, adding a certain amount of complex solution and mixing uniformly after blow-drying, and centrifuging to obtain a supernatant to obtain the sample to be tested, includes:

transferring 50-200 μ L (such as 50, 100, 150 or 200 μ L) of blood sample obtained by processing at least 1mL of blood to be detected into a 1.5mL centrifuge tube by using a pipette, adding a certain amount (such as 10 μ L) of internal standard working solution, and mixing for 20s-2min (such as 20, 40, 60 or 120s) by vortex at the rotation speed of 1000-2500rpm (such as 1000, 1500, 2000 or 2500 rpm);

adding 200-;

blowing the supernatant with nitrogen at 35-70 deg.C (such as 35, 40, 50, 60 or 70 deg.C), adding 50-200 μ L (such as 50, 100, 150 or 200 μ L) of complex solution, vortex-shaking at 2500rpm (such as 1000, 1500, 2000 or 2500rpm) for 1-3min, centrifuging at 14000rpm (such as 10000, 11000, 12000, 13000 or 14000rpm) at 4-10 deg.C (such as 4, 6, 8 or 10 deg.C) for 5-12min (such as 5, 7, 9, 10 or 12min), and collecting the supernatant to obtain the sample to be tested.

In the embodiment of the invention, the compound solution can be an aqueous solution containing 0-2% of formic acid and 0-20% of acetonitrile, and the dosage of the compound solution can be 50-200 mu L. In addition, the sample injection volume of the complex solution is small, and the influence of the content of formic acid in the complex solution on the peak shape is small.

In detail, the blood sample can be obtained by processing at least 1mL of blood to be tested. The dosage of blood to be detected is not high and can be as low as 1mL, and the blood sampling amount is less, so that the blood sampling experience of a detected person is good. Typically, blood samples are stored frozen at-20 ℃ until ready for analysis. After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.

Based on the above, in one embodiment of the present invention, the internal standard working solution preferably contains 0.5-20nmol/mL (e.g., 0.5, 1, 5, 10 or 20nmol/mL) of methylmalonic acid-d 3, and the addition amount is 10. mu.L.

In detail, isotopes of methyl malonic acid are preferably used as internal standards in the examples of the present invention. Wherein d3 represents 3H atoms substituted by deuterium atoms. The isotope label of the methylmalonic acid-d 3 is used as an internal standard substance, so that the detection of the blood sample is not easily influenced by matrix and the like, the identification of the methylmalonic acid is more accurate, the analysis time is short, the interference is small, the internal standard quantification is proper, the specificity is strong, and the accuracy and the sensitivity are high.

Preferably, the diluent used for preparing the standard stock solution, the standard working solution, the internal standard stock solution and the internal standard working solution can be methanol solution with the water content of 5-60%. For example, the moisture proportion in the dilution may be 5, 10, 20, 30, 40 or 60%.

In one embodiment of the present invention, the liquid phase condition in the detection condition includes: a hexyl-phenyl chromatographic column, wherein the mobile phase A is an aqueous solution containing 0.4 percent of formic acid and 2mM ammonium formate, the mobile phase B is acetonitrile, a gradient elution mode is adopted in the elution process, the analysis time is 3-7min, the column temperature is 20-45 ℃, the sample injection amount is 3-20 mu L, and the flow rate is 0.20-0.70 mL/min.

For example, the analysis time may be 3, 4, 5, 6 or 7min, the column temperature may be 20, 25, 30, 35, 40 or 45 ℃, the sample size may be 3, 5, 10, 15 or 20 μ L, and the flow rate may be 0.20, 0.30, 0.40, 0.50, 0.60 or 0.70 mL/min.

In detail, when the liquid phase condition is used for detecting the content of the methyl malonic acid in blood, the methyl malonic acid and the isomer impurity (succinic acid) thereof can be separated quickly and better, and the interference of the high-concentration isomer impurity on the detection of the methyl malonic acid is avoided, so that the quick and accurate detection of the content of the methyl malonic acid in the blood sample is realized. In addition, when the liquid phase condition is used, the analysis time can be about 5.0min, the analysis time is short, and the method has the characteristics of stable retention time among batches, good target peak-appearing effect, good repeatability of separation effect and the like.

Based on the above, in one embodiment of the present invention, preferably, the hexyl-phenyl chromatographic column comprises: phenomenex Gemini C6-Phenyl chromatographic column, the length of chromatographic column is 150mm, the internal diameter is 2.0mm, and the particle size of filler is 3 μm.

In the embodiment of the invention, the chromatographic column is used for detecting the content of the methyl malonic acid in blood, the filler is special, the inner diameter is small, the column length is long, and the good separation of the methyl malonic acid and the impurity succinic acid can be realized. In addition, compared with a small-particle-size chromatographic column, the chromatographic column has medium particle size, so that the chromatographic column is not easy to block and has long service life.

Based on the above, in one embodiment of the present invention, the flow rate is preferably 0.40 mL/min; the elution process can be as shown in table 1 below.

In table 1, mobile phase a is an aqueous solution containing 0.4% formic acid and 2mM ammonium formate and mobile phase B is acetonitrile.

TABLE 1

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0.00	97	3
1.00	97	3
			1.50	5	95
2.50	5	95
			2.51	97	3
5.00	97	3

In one embodiment of the present invention, the tandem mass spectrometry condition among the detection conditions includes: ESI (-) detection mode, MRM acquisition mode, 20-55psi of air curtain pressure, 1-12psi of collision air pressure, 1500-4500V of ion spray voltage value, 750 ℃ of ionization temperature, 10-90psi of atomization air pressure, and 10-90psi of auxiliary air pressure.

For example, the air curtain pressure may be 20, 30, 40, 50, or 55psi, the collision air pressure may be 1, 3, 5, 7, 10, or 12psi, the ion spray voltage may be-1500, -2500, -3500, or-4500V, the ionization temperature may be 200, 350, 500, 650, or 750 ℃, the atomizing air pressure may be 10, 30, 50, 70, or 90psi, and the auxiliary air pressure may be 10, 30, 50, 70, or 90 psi.

Based on the above, in one embodiment of the present invention, it is preferable that the air curtain pressure is 20psi, the collision air pressure is 5psi, the ion spray voltage is-2000V, the ionization temperature is 550 ℃, the atomization air pressure is 80psi, and the auxiliary air pressure is 60 psi.

In general, at least three coordinate points are required for establishing the standard curve equation to ensure the accuracy of the established equation, so at least three standard solutions need to be prepared in advance, and the standard curve equation of the methylmalonic acid can be fitted according to the chromatogram obtained by detecting each standard solution.

In detail, a linear range can be set by combining the detected population, the dosage of blood to be detected, the dilution degree of a blood sample, the dilution degree of a standard working solution, the approximate content range of methylmalonic acid in a human body and the like, so that most of the detection results of clinical samples are within a reportable range.

In one embodiment of the present invention, before the separately detecting at least three standard solutions, further comprising: preparing at least three standard working solutions, wherein the standard working solutions contain methyl malonic acid standard substances with known concentrations, the concentration of the methyl malonic acid standard substances in the standard working solutions is within the range of 0.4-51.2nmol/mL, and the concentrations of the methyl malonic acid standard substances in different standard working solutions are different; transferring 10 mu L of standard working solution and 10 mu L of internal standard working solution into a centrifuge tube by using a pipette, and then transferring and adding 80 mu L of the complex solution; the centrifuge tube is vortexed and mixed at the rotation speed of 1000-.

As mentioned above, the double solution may be pure water, i.e., pure water may be used to dissolve the standard to prepare a standard solution.

In one embodiment of the present invention, preferably, the concentration of the methylmalonic acid standard in each standard working solution is 0.4nmol/mL, 0.8nmol/mL, 1.6nmol/mL, 3.2nmol/mL, 6.4nmol/mL, 12.8nmol/mL, 25.6nmol/mL, 51.2nmol/mL, respectively.

In detail, the standard curve equation fitted to methylmalonic acid may be generally y ═ k × x + b. The two variables x and y can be the peak area ratio of the methylmalonic acid standard substance to the corresponding internal standard substance in the chromatogram of each standard solution, and the concentration ratio of the methylmalonic acid standard substance to the corresponding internal standard substance in each standard solution. Therefore, according to the peak area ratio of the methylmalonic acid to the internal standard substance in the chromatogram of the sample to be detected and the concentration of the internal standard substance in the sample to be detected, the concentration of the methylmalonic acid in the sample to be detected can be calculated by substituting the standard curve equation.

In general, the standard curve equation needs to be re-determined before each test, and the standard curve equation obtained each time is usually only applied to one time of the current test, such as one time of the on-off period of the high performance liquid tandem mass spectrometer.

In conclusion, the embodiment of the invention provides a method for detecting the content of methylmalonic acid in blood, and an internal standard method is combined with a high performance liquid chromatography-tandem mass spectrometry method, so that the accuracy of a quantitative result is improved, and the system error is eliminated; the sample pretreatment is simple, and the sample pretreatment time is greatly saved; the pretreatment of the sample can be realized only by using common reagents and consumables, so that the detection cost is reduced; the sample detection is carried out by combining specific detection conditions, so that the detection process is simple, convenient and quick, the sample analysis time is shortened, the monitoring of the content of the methylmalonic acid in a patient body in clinical treatment is facilitated, whether the supply amount of the vitamin B12 is sufficient can be quickly and accurately judged, an experimental basis can be provided for personalized administration of related medicines, and the occurrence of vitamin B12 deficiency symptoms is reduced or avoided, and the medication of the patient is facilitated to be guided.

The present invention will be described in detail below by way of examples, but the present invention is not limited to the following examples.

Example 1

The embodiment of the invention is used for obtaining the standard curve equation.

1.1 preparation of Standard stock solutions

Purchasing a standard solution of methylmalonic acid, namely a standard solution of methylmalonic acid dissolved in acetonitrile to obtain a standard stock solution, wherein the concentration of the standard solution of methylmalonic acid is 1mg/mL, and the standard solution of methylmalonic acid is stored at-80 ℃ for 1 year.

1.2 preparation of stock solutions for internal standards

Precisely weighing 2mg of a methylmalonic acid-d 3 standard substance in a 2mL volumetric flask, adding an aqueous solution containing 50% methanol for dissolving, and fixing the volume to 2mL to obtain an internal standard stock solution, wherein the concentration of the methylmalonic acid-d 3 standard substance is 1000 mug/mL, the internal standard stock solution is stored at-80 ℃, and the effective period is 1 year.

1.3 Instrument for detection

High performance liquid tandem mass spectrometer: sciex 6500 plus.

1.4 Mass Spectrometry conditions

ESI (-) detection mode, MRM acquisition mode, 20psi air curtain pressure, 5psi collision gas, -2000V ion spray voltage value, 550 ℃ ionization temperature, 80psi atomization gas pressure; the assist gas pressure was 60 psi.

For other parameters of the mass spectrometry conditions, see table 2 below.

TABLE 2

Categories	Name of substance	Retention time	Parent ion	Daughter ions	DP	EP	CE	CXP
									Characterization of nature	MMA	2.35min	117.1	117.1	0	-3	0	-9
Quantification of	MMA	2.35min	117.1	73.0	0	-3	-13	-9
									Qualitative and quantitative	IS/MMA-d3	2.35min	120.0	76.0	0	-3	-13	-9

In Table 2, MMA was a characterization of methylmalonic acid, IS/MMA-d3 was a characterization of methylmalonic acid-d 3, DP was the optimized declustering voltage for positive ion mode, EP was the focusing voltage, CE was the collision voltage, and CXP was the collision cell ejection voltage.

1.5 liquid phase conditions

1.5.1 chromatography columns

Phenomenex Gemini C6-Phenyl column (2X 150mm,3 μm).

1.5.2 Mobile phase

Mobile phase A: an aqueous solution containing 0.4% formic acid and 2mM ammonium formate;

mobile phase B: and (3) acetonitrile.

1.5.3 elution mode

Gradient elution was used, with the mobile phase ratios and flow rates as shown in table 1 above.

1.5.4 others

The online filter is SSI COL PRE-FILTER WATER 1/160.5M; the analysis time is 5.0 min; the column temperature was 25 ℃; the sample injection amount is 5 mu L; the flow rate was 0.4 mL/min.

1.6 preparation of Standard working solution

Taking appropriate amount of standard stock solution, diluting with diluent (50% methanol-containing aqueous solution, the same below) to obtain eight standard working solutions with concentration of 0.4-51.2nmol/mL for methyl malonic acid standard, and storing at-80 deg.C.

Among the eight standard working solutions, the concentrations of the methylmalonic acid standard were 0.4nmol/mL, 0.8nmol/mL, 1.6nmol/mL, 3.2nmol/mL, 6.4nmol/mL, 12.8nmol/mL, 25.6nmol/mL, and 51.2nmol/mL, respectively.

1.7 preparation of internal standard working solution

Sucking 400 mu L of internal standard stock solution into a 10mL volumetric flask, fixing the volume by using diluent, and uniformly mixing for 3min in a vortex manner at the rotating speed of 2500r/min to obtain internal standard intermediate solution; sucking 94.5 mu L of internal standard intermediate solution into a 5mL volumetric flask, adding diluent to constant volume to 5mL, oscillating and mixing uniformly to obtain internal standard working solution with the concentration of 8nmol/mL of methylmalonic acid-d 3 standard, and storing at-80 ℃.

1.8 preparation of Standard solution

For each standard working solution, 10 μ L of the internal standard working solution and 80 μ L of the complex solution (aqueous solution containing 1% formic acid and 5% acetonitrile, the same below) are respectively transferred by a liquid transfer machine and placed in a 1.5mL centrifuge tube, and then mixed uniformly by vortexing at the rotation speed of 2500rpm for 1min to obtain a mixed solution, and the mixed solution is transferred as the standard solution to be detected.

Thus, eight standard solutions were obtained for eight standard working solutions.

1.9 detecting the standard solution to generate a standard curve equation

After each standard solution is obtained, the eight standard solutions can be respectively detected by using a high performance liquid tandem mass spectrometer, and a chromatogram and a mass spectrogram of each standard solution are correspondingly obtained.

Referring to fig. 3-5, fig. 3 shows chromatograms of a methylmalonic acid standard in a standard solution, fig. 4 shows a chromatogram of methylmalonic acid-d 3 in a standard solution, and fig. 5 shows mass spectrograms of a methylmalonic acid standard and methylmalonic acid-d 3 in a standard solution.

As can be seen from the chromatograms shown in FIGS. 3-5, the detection method provided by the embodiment of the invention has the advantages of accurate identification of the target compound, short analysis time, small interference and strong specificity.

From the chromatogram of the standard solution, the chromatographic peak area of the methyl malonic acid standard and the chromatographic peak area of the methyl malonic acid-d 3 can be obtained, and then the standard curve equation of the methyl malonic acid can be obtained by combining the known concentrations of the methyl malonic acid standard and the methyl malonic acid-d 3 in each standard solution.

Referring to fig. 9, fig. 9 shows a linear relationship diagram of the obtained methylmalonic acid. According to the linear relation chart, a standard curve equation of the methylmalonic acid can be obtained: Y2.22X +0.00127, R0.9991, correlation coefficient R²When the concentration is more than 0.999, Y is the peak area ratio of the methyl malonic acid to the methyl malonic acid-d 3, and X is the concentration ratio of the methyl malonic acid to the methyl malonic acid-d 3.

It can be seen that the correlation coefficient R²> 0.9900, indicating good linearity. When the content of the methylmalonic acid in the blood is calculated based on the standard curve equation, the accuracy is high, and the error is small.

And after a standard curve equation is obtained, pretreating the blood sample to obtain a sample to be detected, detecting the sample to be detected under the same detection condition, and combining the obtained standard curve equation to obtain the content of the methylmalonic acid in the blood sample.

Example 2

The embodiment of the invention is used for detecting the content of the methylmalonic acid in the blood.

2.1 obtaining blood samples

Taking at least 1mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, taking supernatant to obtain serum, and storing the serum as a blood sample at 4 ℃ under refrigeration until the serum is reserved before analysis.

After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded.

2.2 blood sample pretreatment

Transferring 100 mu L of serum into a 1.5mL centrifuge tube by using a pipette gun, then adding 10 mu L of internal standard working solution, and mixing for 1min by vortex oscillation at the rotating speed of 2500 rpm; then adding 300 mu L of acetonitrile, mixing for 3min by vortex oscillation at the rotating speed of 2500rpm, then centrifuging for 5min at the rotating speed of 12000rpm, and transferring 350 mu L of supernate to another 1.5mL centrifuge tube; blowing the transferred supernatant with nitrogen at 55 ℃, redissolving with 100 mu L of redissolution, mixing for 1min at the rotation speed of 2500rpm by vortex oscillation, then centrifuging at the high speed of 14000rpm for 5min at 10 ℃, and transferring 90 mu L of supernatant obtained by high-speed centrifugation to obtain the sample to be detected.

2.3 detection of samples to be tested

Under the detection conditions of the embodiment 1, the same high performance liquid tandem mass spectrometer is used for detecting the sample to be detected, and the chromatogram of the sample to be detected is obtained.

Referring to fig. 6-8, fig. 6 shows a chromatogram of methylmalonic acid in a test sample, fig. 7 shows a chromatogram of methylmalonic acid-d 3 in a test sample, and fig. 8 shows mass spectra of methylmalonic acid and methylmalonic acid-d 3 in a test sample.

Referring to fig. 3 to 8, it can be seen that the retention time of the methylmalonic acid in the sample to be detected is consistent with the retention time of the methylmalonic acid standard in the standard solution, and the retention time of the methylmalonic acid-d 3 in the sample to be detected is consistent with the retention time of the methylmalonic acid-d 3 in the standard solution, and the detection method provided by the embodiment of the present invention has the advantages of more accurate identification of the target compound, short analysis time, less interference, proper internal standard quantification, strong specificity, high accuracy and sensitivity, and the like.

Referring to fig. 6, the retention time of the methylmalonic acid in the sample to be detected is 2.35min, the retention time of the succinic acid is about 2.00min, and the difference between the two retention times is about 0.35min, which indicates that the separation condition of the methylmalonic acid and the succinic acid is good, and the effective chromatographic separation is realized, and the chromatographic peaks of both the methylmalonic acid and the succinic acid have no tailing condition, which indicates that the detection effect of the methylmalonic acid and the succinic acid is good and the detection accuracy is high. Based on this, although succinic acid is an isomer impurity of methyl malonic acid, in the embodiment of the present invention, succinic acid does not affect the accurate detection of methyl malonic acid.

In addition, because the retention time of both methylmalonic acid and methylmalonic acid-d 3 is 2.35min, the analysis time can be as low as 5min, or even lower.

2.4 calculation of Methylmalonic acid content in the sample to be tested

And correspondingly substituting the chromatographic peak areas of the methyl malonic acid and the methyl malonic acid-d 3 in the chromatogram of the sample to be detected and the known concentration of the methyl malonic acid-d 3 in the sample to be detected into the standard curve equation, so as to calculate the content of the methyl malonic acid in the sample to be detected.

The concentration range of the methylmalonic acid standard substance in the standard working solution prepared in the above step 1.6 was 0.4 to 51.2nmol/mL, and it was found that there was a 10-fold conversion relationship between the standard dilution and the sample treatment by combining the standard solution preparation process described in the above step 1.8 and the blood sample pretreatment process described in the above step 2.2, and therefore, it was considered that the linear range (clinically reportable range) of methylmalonic acid was 0.04 to 5.12nmol/mL, and the linearity of methylmalonic acid was good in the range of 0.04 to 5.12 nmol/mL.

Example 3

The embodiment of the invention is used for detecting the content of the methylmalonic acid in the blood plasma.

In step 2.1, blood to be tested is treated to obtain serum. In contrast, the present embodiment treats blood to be tested to obtain plasma, and the obtained plasma is used as a blood sample, while the other treatments are consistent with the above embodiment 2.

By measuring the content of methylmalonic acid in the plasma, it was found that the obtained measurement result was consistent with the measurement result obtained in example 2 within an allowable error range.

Example 4

The embodiment of the invention is used for determining the quantitative limit and the detection limit.

The control samples with low concentration were selected and diluted with physiological saline to different degrees to prepare blood sample dilutions with different concentrations, and these blood sample dilutions were measured in the blood sample pretreatment method and measurement conditions in example 2. The detection results show that the detection limit and the quantification limit of the methylmalonic acid are as follows:

(1) limit of detection (LOD): 0.01126 nmol/mL.

(2) Limit of quantitation (LOQ): 0.0379 nmol/mL.

In this example, the detection limit of methylmalonic acid can be as low as 0.01126nmol/mL, and the quantitation limit can be as low as 0.0379 nmol/mL. Therefore, the detection method provided by the embodiment of the invention has high sensitivity, can improve the parallelism of low-concentration samples, enhances the accuracy and can meet clinical requirements.

Due to the high sensitivity, the requirement of the sample volume of the blood to be detected in the embodiment of the invention can be wider, so that the overall accuracy of sample detection is improved. In addition, the embodiment of the invention can accurately quantify the biological sample with very low content of the methylmalonic acid, thereby ensuring the high accuracy and the wide applicability of the detection method.

Example 5

The embodiment of the invention is used for measuring the recovery rate and the precision.

The standard working solution containing the methylmalonic acid standard substance is respectively taken to prepare high, medium and low concentrations of 3, sample loading recovery rate experiments and precision experiments are carried out, the detection method in the embodiment 2 is used for detection, 3 batches of analysis and determination are repeated, and the recovery rate and precision of the methylmalonic acid are shown in Table 3.

TABLE 3

Adding quantity of scalar	0.08nmol/mL	0.64nmol/mL	2.56nmol/mL
				Average recovery rate	98％	99％	104％
Precision (RSD)	1.56％	2.02％	1.47％

It can be seen that the methyl malonic acid has an average recovery rate of 98-104% in the range of 3 addition levels of low, medium and high, has good reproducibility and good sample addition recovery rate, has a relative standard deviation of 1.47-2.02%, has high accuracy of detection results, and can eliminate system errors.

Based on the above, the detection method described in the above embodiment 2 has the advantages that the technical indexes such as detection limit, quantitative limit, recovery rate and precision meet the requirements, the reproducibility is good, the sample recovery rate is high, and the accuracy of the detection result is improved.

In summary, the method for detecting the blood concentration content of the methylmalonic acid in the blood provided by the embodiment of the invention combines an internal standard method with a high performance liquid chromatography-mass spectrometry combined method, so that interference factors are greatly reduced, sample pretreatment is simple, pretreatment of a labeled yeast is not required, the sample pretreatment time is greatly saved, the quantification is accurate, the reproducibility is good, the specificity is strong, the sensitivity is high, the detection result is more accurate, the cost is low, the analysis time is short, the clinical requirements are met, the method is suitable for detecting a large batch of clinical blood samples, and an experimental basis can be provided for personalized administration of related medicines and reduction or avoidance of the occurrence of vitamin B12 deficiency.

Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.