Human urine glucose detection standard substance and preparation and valuing thereof

Abstract

The invention relates to a human urine glucose detection standard substance as well as preparation and valuing thereof. The method comprises the following steps: processing and preparing the standard substance; performing uniformity test on the urine glucose standard substance; testing the stability of the urine glucose standard substance; determining a standard value of the urine glucose standard substance by a high performance liquid chromatography-isotope dilution mass spectrometry method; and carrying out statistical calculation on the determination value of the urine glucose standard substance and carrying out uncertainty evaluation on the determination result. The invention provides a method for preparing and valuing a urine glucose standard substance, aiming at the problem that the urine glucose detection result is inaccurate and incomparable because no human urine glucose standard substance exists at present, so that the method is used for developing a urine glucose detection reagent and verifying the performance of the method. And the kit can be used for measuring value traceability of urine glucose detection results, so that accuracy and comparability of clinical urine glucose detection results are guaranteed, and clinical examination of diseases such as chronic kidney diseases is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of metrological chemical analysis detection, in particular to a human urine glucose detection standard substance and preparation and value determination thereof. BACKGROUND

[0002] Diabetes mellitus is a global chronic disease, and the prevention and control of its complications have become an important challenge in the medical field. Early diabetic kidney damage, as one of the common microvascular complications of diabetes, its early detection and timely intervention are of great significance to delay the progression of the disease and improve the prognosis of patients. Diabetic nephropathy is a common complication of diabetes, and its pathological basis is mainly the damage of glomerulus and renal tubule caused by high blood sugar and accumulation of glycosylation end products. Early diabetic nephropathy usually has no obvious symptoms, and as the disease progresses, patients may have kidney function impairment, such as hypertension, edema, anemia, etc. After the development to the late stage of diabetic nephropathy, the kidney function gradually deteriorates, and the symptoms of uremia appear. Early diagnosis and intervention are of great significance to delay the development of diabetic nephropathy. At present, there are many detection methods for diabetic kidney damage in clinic. Among them, urine routine operation is simple, repeatable, non-invasive, low cost, easy to be accepted by patients, and widely used in clinical application. Kidney damage can affect the absorption of glucose in urine by the kidney, causing some urine glucose to be excreted with urine, resulting in an increase in urine glucose level. Urine glucose positive indicates that the patient has abnormal glucose metabolism, which can directly reflect the blood glucose control and facilitate the adjustment of clinical treatment plan.

[0003] Therefore, urine glucose detection as a non-invasive screening method has important clinical value in early diagnosis, disease monitoring and efficacy evaluation. Changes in urine glucose level can indirectly reflect the glomerular filtration function and judge the degree of kidney damage in diabetic patients. However, urine sample matrix is complex and is easily affected by interference factors during detection, resulting in differences in results between different detection systems, which directly affects the accuracy of clinical judgment and the comparability of data between laboratories. Therefore, the establishment of high-accuracy, traceable urine glucose standard substance is of great importance to unify the detection standard, improve the method consistency and ensure the quality of clinical examination.

[0004] At present, the research of standard substance at home and abroad focuses on serum matrix, and the standard substance for urine glucose is still blank. Although the existing serum standard substance is partially applied to the calibration of urine detection system, the difference in matrix may cause deviation in value transmission. In addition, there are essential differences in stability, uniformity and storage conditions between urine samples and serum, and it is urgent to develop standard substance specially used for urine matrix to meet the specific needs of clinical examination. SUMMARY

[0005] The present application aims at the demand of urine glucose detection value traceability transmission and the need of automatic biochemical analyzer detection calibration, and provides a preparation and value determination method of human urine glucose detection standard substance.

[0006] The human urine glucose detection standard substance provided by the present application comprises: processing and preparation of urine glucose standard substance; uniformity test of urine glucose standard substance; stability test of urine glucose standard substance; determination of standard value of urine glucose standard substance by high performance liquid chromatography-isotope dilution mass spectrometry; statistical calculation of urine glucose standard substance certified value and uncertainty evaluation of value determination result.

[0007] Firstly, the urine glucose standard substance is processed and prepared, and the raw material is collected from the urine of healthy ordinary people. Then, a high-speed refrigerated centrifuge is used to centrifuge at 12000 rpm and 4 DEG C for 15 min, the supernatant is taken, filtered by a 0.22 mu m filter membrane, and the filtrate is collected; a pipette is used to add ProClin300 as a preservative at a proportion of 0.1% (v / v), and the urine is mixed thoroughly after the addition to ensure the stability of the urine. A fully automatic biochemical analyzer or a mass spectrometer is used to preliminarily determine the glucose concentration in the urine, when the preliminary determination result meets the design requirements of the standard substance, the sub-packaging process can be entered; when the preliminary determination result is lower than the design requirements of the standard substance, the glucose purity standard substance solution is added to increase the glucose content in the urine. When adding, the added volume of the glucose purity standard substance solution is ensured to be not more than 10% of the volume of the urine. After adding, the mixture is mixed at room temperature for 6-12 hours.

[0008] The container for sub-packaging the urine glucose standard substance is a colorless or brown screw sample bottle, preferably a low-temperature resistant, low-adsorption brown sample bottle. The urine raw material is sub-packaged into sample bottles or cryogenic tubes under oscillation, and is sealed or sealed, each urine glucose concentration level standard substance is sequentially numbered according to the sub-packaging order, and is stored in a refrigerator at-20 DEG C or-40 DEG C or-80 DEG C.

[0009] The homogeneity of urine glucose is tested by randomly sampling from the sub-packaged units. The number of sub-packaged units is N, when 100 < N ≤ 200, the number of units sampled is not less than 11; when 200 < N ≤ 500, the number of units sampled is not less than 15; when 500 < N ≤ 1000, the number of units sampled is not less than 25; when the total number of units N > 1000, the number of samples is not less than 30. The homogeneity of each urine glucose concentration level is tested by randomly sampling units according to the above requirements. The experimental method for homogeneity testing is biochemical analyzer method or isotope dilution mass spectrometry, preferably isotope dilution mass spectrometry. Each unit of the standard material sampled is measured 3 times by the above method. During analysis, in order to eliminate the error caused by instrument drift during measurement, the samples are analyzed in the order of 1→15, 15→1, and odd-even interlacing. The samples are tested for homogeneity according to the following formula and the results are counted:

[0010] Assuming there are a standard material units, unit i is measured ni times (i = 1, …, a), for a simple balanced design, the number of repeated measurements ni of all units is the same, i.e. n1 = n2 = … = na = n0. Single factor analysis of variance is used for analysis. The statistical model is as follows:

[0011] x ij = μ + δ i + ε ij Formula (1)

[0012] Where: x ij is the jth observation of the ith unit; μ is the (true) mean of the population of all possible results (observation x ij is generated from); δ i is the effect of unit i on the result, i.e. the (true) deviation of unit i from μ; ε ij is the random error of the jth observation of the ith unit, also known as the residual term. According to formulas 2-2 and 2-3, the inter-unit mean square M between and the residual mean square M within can be obtained;

[0013]

[0014] Where:

[0015]

[0016] The statistic F is calculated:

[0017]

[0018] According to the degrees of freedom (v1, v2) and the given significance level α, the critical F is obtained from the tableα (v1, v2) values, and the F value calculated by the formula. If F < Fα, it is considered that there is no significant difference between groups and within groups, and the sample is uniform. Otherwise, the preparation and homogenization of the standard substance need to be reprocessed.

[0019] The stability test of urine glucose is divided into long-term stability test and short-term stability test. The duration of long-term stability test is not less than 6 months. In the period of long-term stability test, not less than 5 time points k are selected according to the principle of more dense in the front and less dense in the back, not less than 2 units of standard substance are extracted at each time point, each unit of standard substance is repeatedly analyzed 3 times by biochemical analyzer or isotope dilution mass spectrometry method, the arithmetic mean of these measurement results is calculated, and the measurement result X i The measurement results and measurement time are fitted according to the following linear model:

[0020] Y = b0 + bX Formula (7)

[0021] In the formula:

[0022] b0—intercept; b—regression coefficient; X—time; Y—determination result of glucose content of standard substance.

[0023] The regression coefficient and intercept are calculated according to formula 8 and formula 9:

[0024]

[0025] In the formula:

[0026] X i —the i-th time point;

[0027] Y i —determination result of urine glucose standard substance at the i-th time point;

[0028] —average value of all time points;

[0029] —determination result of urine glucose standard substance at all time points;

[0030] k—number of time points.

[0031] The s value is calculated according to formula 10:

[0032]

[0033] The meanings of various symbols in the formula are the same as those in formula 8 and formula 9.

[0034] The value of s(b) is calculated according to formula 11:

[0035]

[0036] Referring to the t-distribution table, the value of t 0.95.k-2 is obtained.

[0037] If |b| < t 0.95，k-2 · s(b), it indicates that the slope is not significant, no instability is observed, and the urine glucose reference material is stable.

[0038] The long-term stability of the urine glucose reference material is investigated at -20℃, -40℃ or -80℃, preferably -80℃. The short-term stability of the urine glucose reference material is investigated at -20℃, 4℃, 25℃ and 60℃.

[0039] The method for determining the value of the urine glucose reference material uses high-performance liquid chromatography-isotope dilution mass spectrometry to determine the standard value of urine glucose, including the steps of adding an isotope-labeled internal standard and determining on the machine. When determining urine glucose by high-performance liquid chromatography-isotope dilution mass spectrometry, the selected isotope-labeled substance is 13 C6] labeled glucose, the number of isotope-labeled atoms is not less than 3, and the isotope abundance is not less than 99%. When performing sample pretreatment, first, use a solvent such as water or methanol or acetonitrile to prepare a solution of 13 C6] labeled glucose with a concentration of (0-100) mg / g, preferably an aqueous solution, and preferably a concentration of 0.5 mg / g. Add the same amount of 13 C6] labeled glucose as the initial determination of the glucose content in the urine glucose reference material sample, and the volume ratio of the added 13 C6] labeled glucose to the sample is between (1:100-10:1), and the preferred ratio is 1:1. After mixing well, filter with a 0.22 mm filter membrane, and then perform high-performance liquid chromatography-isotope dilution mass spectrometry analysis. The used chromatographic column is a C4 or C8 or C18 chromatographic column, and the preferred model is Agilent Eclipse Plus C18 (4.6x150mm, 5μm) chromatographic column. The mobile phase A used is water containing (0.1%-1%) formic acid or (0.1%-1%) trifluoroacetic acid or (5-100) mmol / L ammonium formate or (5-100) mmol / L ammonium acetate solution, which contains (0%-50%) methanol or acetonitrile. The mobile phase B used is acetonitrile or methanol containing (0.1%-1%) formic acid or acetonitrile or methanol containing (0.1%-1%) trifluoroacetic acid. The preferred mobile phase A is ultrapure water, and the preferred mobile phase B is acetonitrile containing 0.1% formic acid. The mobile phase used is mobile phase A:B (100:0-50:50) within (5-60) min. The preferred mobile phase ratio is shown in the following table:

[0040] Total Time (min) A(％) B(％) 0.00 95.0 5.0 10.00 95.0 5.0

[0041] The flow rate used is (100–2000) mL / min, with a preferred flow rate of 300 mL / min. The injection volume used is (1–100) mL, with a preferred injection volume of 5 mL. Mass spectrometry detection is performed using either MRM or SIM mode. When using MRM mode, the glucose in the sample (m / z = 512.4 for the glucose precursor ion after derivatization and m / z = 175.0 for the glucose daughter ion) and the glucose isotope label in the standard are detected separately. 13 C6](Derivatized glucose isotope markers[ 13 The C6] precursor ion has an m / z of 518.4, and the glucose isotope label [ 13 The signal of the C6] daughter ion (m / z = 175.0) was detected; when using SIM mode for detection, the glucose in the sample (glucose ion m / z = 512.44) and the glucose isotope label in the standard were detected respectively. 13 C6](glucose isotope markers) 13 The signal of C6 ions (m / z = 518.4) was used. MRM mode was preferred for detection. The quantitative standards used were glucose purity standards and […]. 13 C6] A standard solution of glucose was prepared, wherein the glucose concentration in the standard solution, [ 13 C6] indicates glucose concentration and glucose and [ 13 The proportion of C6] glucose is related to the glucose concentration in the standard substance to be tested, [ 13 The concentration and ratio of glucose in C6 were close, ranging from 0.90 to 1.10. The obtained glucose extraction chromatogram and [ 13 The peak areas of the glucose extraction chromatograms labeled with C6 were obtained by integration. Quantification was performed using the single-point method, bracket method, or standard curve method, based on the ratio of sample peak area to standard peak area. The preferred ratio range was (0.95–1.05), and the preferred quantitative method was the bracket method.

[0042] The method for determining the value of urine glucose standard material, wherein the determination of the certified value of the standard material is carried out by high performance liquid chromatography-isotope dilution mass spectrometry combined with multiple laboratories, according to the number of independent data sets in JJF 1343-2022, each set of data obtained by measurement should be based on 2 units, each unit is measured at least 2 times, not less than 4-6 times of independent repeated measurement data is provided, all data is regarded as a new set of measurement data, according to K-S single sample normality test, and abnormal value test is carried out by using Grubbs method and / or Dixon method, finally, the determination data is carried out by variance homogeneity test by using Cochran rule; after the above statistical test, the arithmetic mean value of all results is taken as the standard value of the determination result of high performance liquid chromatography-isotope dilution mass spectrometry. The uncertainty u of the standard value of glucose in urine is derived from the uncertainty u introduced in the determination process char , the uncertainty u introduced by the uniformity of the standard material bb and the uncertainty u introduced by the long-term stability lts . According to formula 12 for synthesis

[0043]

[0044] In the formula, k is a containing factor, usually k=2.

[0045] Where u char includes the uncertainty u char,HPLC-IDMS from high performance liquid chromatography-isotope dilution mass spectrometry. u char,HPLC-IDMS includes the uncertainty component u char,HPLC-IDMS,A from high performance liquid chromatography-isotope dilution mass spectrometry measurement results repeatability (according to formula 13), and the uncertainty component u char,HPLC-IDMS,wi from multiple balance weighing (according to formula 16) and the uncertainty component u char,HPLC-IDMS,P from glucose purity standard material (according to formula 15). Each uncertainty component is synthesized according to formula 16.

[0046]

[0047] In formula 13, s is the standard deviation, and n is the number of measurements;

[0048]

[0049] In formula 14, U(E) is the uncertainty in the balance calibration certificate, and k is the containing factor

[0050]

[0051] In formula 15, U is the uncertainty in the glucose purity standard material certificate, and k is the containing factor;

[0052]

[0053] In the formula, q is the number of times of balance weighing, c i is the sensitivity coefficient at the i-th weighing, c p is the sensitivity coefficient of the purity of the standard substance.

[0054] When the inter-group mean square is greater than the intra-group mean square, the uncertainty introduced by homogeneity is calculated according to formula 17.

[0055]

[0056] When the inter-group mean square is less than the intra-group mean square, the calculation is performed according to formula 18.

[0057]

[0058] The long-term stability of the standard substance is introduced and calculated according to formula 19:

[0059] u lts = s k,lts · t lts Formula (19)

[0060] In the formula,

[0061] s k,lts is s(b) at the long-term stability test, t lts is the time length of the long-term stability test.

[0062] The above results are synthesized according to formula 12 to obtain the expanded uncertainty U of the certified value of the standard substance. Therefore, the certified value result of the urine glucose standard substance can be expressed as:

[0063] x ± U

[0064] In the formula, x is the certified value, and U is the expanded uncertainty of the certified value.

[0065] The present application aims at the problem that there is no human urine glucose standard substance at present, which causes the urine glucose detection result to be inaccurate and incomparable, and provides a preparation and certification method of urine glucose standard substance, so as to be used for the development of urine glucose detection reagent and the performance verification of method, and be used for the quantity traceability of urine glucose detection result, ensures the accuracy and comparability of clinical urine glucose detection result, and is helpful for the clinical examination of chronic kidney disease and other diseases. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 is the chromatographic ion flow diagram of the human urine glucose detection standard substance of the present application;

[0067] Figure 2 is the sub-packaged human urine glucose detection standard substance;

[0068] Figure 3 is the glucose derivative cleavage product of the ID LC-MS / MS method.

[0069] Figure 4 is the glucose derivative cleavage product of the ID LC-MS / MS method. DETAILED DESCRIPTION

[0070] The application will be further described below in connection with the examples, but the application is not limited in any way by the examples, and the application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection of the application.

[0071] Example 1: Processing and preparation of urine glucose standard material

[0072] The raw material of the urine glucose standard material is collected from the urine of healthy ordinary people. The collected urine is centrifuged at 12000 rpm and 4°C for 15 min by using a high-speed refrigerated centrifuge, the supernatant is filtered by using a 0.22 μm filter membrane, the filtrate is collected, 0.1% Proclin 300 is added as a preservative to ensure the stability of the standard material, and the mixture is mixed thoroughly after the addition. The glucose concentration in the urine is preliminarily determined by using a full-automatic biochemical analyzer. The glucose concentration in the urine is determined by using a glucose oxidase-peroxidase (GOD-POD) method.

[0073] The glucose solution required for preparing the target glucose concentration of the GBW10062 glucose purity standard material sample is added directly into the urine by using a pipette, mixed at room temperature for 8 h to ensure the uniformity of the standard material. The above-mentioned human urine glucose standard material is divided into 500 units under oscillation conditions, each unit is 1500 μL, and is divided into low-temperature-resistant, low-adsorption brown sample bottles. The designed concentration, sample number and division date are marked on the freeze tube and freeze box respectively, and are placed in a-80°C refrigerator for storage. The divided urine glucose standard material is as shown in Table 1. Figure 2

[0074] Example 2: Uniformity test of urine glucose standard material

[0075] ​The homogeneity of the standard substance was tested by CS-1200 full-automatic biochemical analyzer and glucose determination kit. According to the requirements of JJF 1343-2022, when the total unit number is ≤500, the number of units extracted is ≥15. Therefore, 15 units of each level of candidate were randomly taken out, and each unit of candidate was repeatedly determined for 3 times by full-automatic biochemical analyzer. In order to eliminate the error caused by instrument drift in the determination process, the samples were analyzed in the order of 1→15, 15→1, and odd-even interlacing.

[0076] The homogeneity test data of urine glucose standard substance is shown in Table 1, and the precision (CV) of the test method is less than 2.0%.

[0077] Table 1 Homogeneity test data of urine glucose standard substance (mmol / L)

[0078] Level 1 Time 1 Time 2 Time 3 Observations Sum Mean Variance 1 2.67 2.70 2.74 3 8.11 2.70 0.0012 2 2.67 2.63 2.74 3 8.04 2.68 0.0031 3 2.66 2.68 2.74 3 8.08 2.69 0.0017 4 2.79 2.69 2.68 3 8.16 2.72 0.0037 5 2.69 2.73 2.77 3 8.19 2.73 0.0016 6 2.75 2.65 2.72 3 8.12 2.71 0.0026 7 2.70 2.66 2.73 3 8.09 2.70 0.0012 8 2.70 2.73 2.77 3 8.20 2.73 0.0012 9 2.67 2.63 2.68 3 7.98 2.66 0.0007 10 2.69 2.75 2.67 3 8.11 2.70 0.0017 11 2.69 2.74 2.73 3 8.16 2.72 0.0007 12 2.75 2.68 2.79 3 8.22 2.74 0.0031 13 2.68 2.72 2.62 3 8.02 2.67 0.0025 14 2.70 2.75 2.70 3 8.15 2.72 0.0008 15 2.67 2.68 2.66 3 8.01 2.67 0.0001

[0079] The single factor variance analysis of urine glucose standard substance was carried out by Excel software, and the results are shown in Table 2. In the table, the F of urine glucose standard substance is less than Fα, so it is considered that there is no significant difference between groups and within groups, and the sample is uniform.

[0080] Table 2 Single factor variance analysis results of homogeneity data of urine glucose standard substance

[0081] Source of Variation SS df MS F P-value Fcrit Between Groups 0.0248 14 0.0018 1.0167 0.4635 2.0374 Within Groups 0.0523 30 0.0017

[0082] Example 3: Stability test of urine glucose standard substance;

[0083] The stability of the standard substance within 10 months was investigated by CS-1200 full-automatic biochemical analyzer and glucose determination kit, and the average results of 3 standard substance samples taken out each time for stability investigation are shown in Table 3.

[0084] Table 3 Average results of long-term stability investigation of urine glucose standard substance (mmol / L)

[0085] Time / Month 1 2 3 Mean 0 2024 / 8 / 2 2.74 2.76 2.75 1 2024 / 9 / 5 2.74 2.75 2.74 2 2024 / 10 / 5 2.73 2.74 2.75 3 2024 / 11 / 4 2.75 2.74 2.76 7 2025 / 3 / 19 2.76 2.77 2.74 9 2025 / 5 / 5 2.77 2.76 2.74 10 2025 / 6 / 3 2.75 2.73 2.77

[0086] Linear regression analysis was carried out on the above data by Excel software, the regression parameters were calculated, and t-test was carried out on the slope β, and the results are shown in Table 4.

[0087] Table 4 Statistical analysis of stability of urine glucose standard substance

[0088] Stabilization Time / Month β s(β) t s(β)·t Conclusion 10 0.0010 0.0005 2.571 0.0014 Stable

[0089] According to the long-term stability test results, the human urine glucose standard material can be stored stably for 10 months at -80℃. The short-term stability of the standard material was tested by using a CS-1200 automatic biochemical analyzer and a glucose enzyme method glucose determination kit. The short-term stability test temperatures were -20℃, 4℃, 25℃ and 60℃, respectively. The observation time at each temperature was 7 days. Tables 5-8 list the average results of the stability test of 3 samples taken at different intervals.

[0090] Table 5 Short-term stability test results of urine glucose standard material at -20℃ (mmol / L)

[0091] Time / Day 1 2 3 Mean 0 2.71 2.76 2.66 2.71 1 2.81 2.79 2.81 2.80 2 2.37 2.70 2.76 2.61 3 2.42 2.69 2.74 2.62 4 2.82 2.75 2.63 2.73 5 2.73 2.75 2.72 2.73 6 2.75 2.74 2.49 2.66 7 2.73 2.68 2.75 2.72

[0092] Table 6 Short-term stability test results of urine glucose standard material at 4℃ (mmol / L)

[0093] Time / Day 1 2 3 Mean 0 2.68 2.74 2.71 2.71 1 2.68 2.77 2.77 2.74 2 2.77 2.76 2.75 2.76 3 2.73 2.76 2.72 2.74 4 2.73 2.78 2.68 2.73 5 2.76 2.73 2.78 2.76 6 2.77 2.76 2.75 2.76 7 2.70 2.71 2.75 2.72

[0094] Table 7 Short-term stability test results of urine glucose standard material at 25℃ (mmol / L)

[0095] Time / Day 1 2 3 Mean 0 2.76 2.69 2.79 2.75 1 2.74 2.78 2.75 2.76 2 2.77 2.87 2.74 2.79 3 2.72 2.77 2.76 2.75 4 2.72 2.75 2.70 2.72 5 2.74 2.73 2.79 2.75 6 2.72 2.80 2.77 2.76 7 2.76 2.74 2.80 2.77

[0096] Table 8 Short-term stability test results of urine glucose standard material at 60℃ (mmol / L)

[0097] Time / Day 1 2 3 Mean 0 2.70 2.69 2.73 2.71 1 2.74 2.81 2.77 2.77 2 2.76 2.68 2.56 2.67 3 2.71 2.76 2.73 2.73 4 2.65 2.54 2.69 2.63 5 2.63 2.65 2.67 2.65 6 2.57 2.61 2.64 2.61 7 2.56 2.61 2.58 2.58

[0098] Table 9 Short-term stability test results of urine glucose standard material (mmol / L)

[0099]

[0100] According to the short-term stability test results of the samples in Table 9, the human urine glucose standard material can be stored stably for 7 days at -20℃, 4℃ and 25℃, and for 3 days at 60℃ (judged by uncertainty, the values within 3 days are within the uncertainty range calculated subsequently), so it is determined that the standard material should be transported at 4℃ within 7 days.

[0101] The standard material samples were tested for bottle opening stability by using an automatic biochemical analyzer. 3 glucose standard material samples were taken each time, and the average results of the standard material at different intervals of 1h, 2h, 3h, 4h, 5h and 6h were detected at 4℃ and 25℃ to investigate the bottle opening stability. The results are as follows.

[0102] Table 10 Results of the glucose standard substance in human urine at 4°C (mmol / L)

[0103] Time / h 1 2 3 Mean 0 2.72 2.71 2.75 2.73 1 2.75 2.77 2.81 2.78 2 2.79 2.77 2.85 2.80 3 2.80 2.78 2.83 2.80 4 2.81 2.84 2.83 2.83 5 2.84 2.88 2.90 2.87 6 2.83 2.87 2.88 2.86

[0104] Table 11 Results of the glucose standard substance in human urine at 25°C (mmol / L)

[0105] Time / h 1 2 3 Mean 0 2.73 2.71 2.73 2.72 1 2.70 2.72 2.71 2.71 2 2.69 2.74 2.74 2.72 3 2.73 2.77 2.73 2.74 4 2.77 2.79 2.76 2.77 5 2.76 2.80 2.78 2.78 6 2.78 2.84 2.86 2.83

[0106] Table 12 Results of the glucose standard substance in human urine (mmol / L)

[0107]

[0108] After opening, the human urine glucose standard substance sample can be stable for 4h at 4°C (judged by uncertainty, the values within 4h are all within the uncertainty range calculated subsequently), and can be stable for 4h at 25°C (judged by t-test with the standard deviation of β1, β1=0.0133, less than t(0.95, n-2)·s(β1)=0.0176), therefore it is recommended that the standard substance should be used up within 4h after opening.

[0109] The repeated freeze-thaw stability of the standard substance was tested by using CS-1200 automatic biochemical analyzer and glucose enzyme method glucose determination kit, 3 glucose standard substance samples were taken out each time, repeated freeze-thaw, the average results of the standard substance after 1, 2, 3, 4, 5, 6, 7 freeze-thaw were detected, and the repeated freeze-thaw stability was investigated. The results are shown below.

[0110] Table 13 Results of the repeated freeze-thaw stability of the glucose standard substance in human urine (mmol / L)

[0111] Number of Freeze-Thaw Cycles 1 2 3 Mean 1 2.69 2.72 2.70 2.70 2 2.77 2.74 2.79 2.77 3 2.76 2.79 2.76 2.77 4 2.68 2.73 2.76 2.72 5 2.85 2.82 2.79 2.82 6 2.77 2.78 2.84 2.80 7 2.70 2.73 2.77 2.73

[0112] Table 14 Results of the repeated freeze-thaw stability test of the glucose standard substance in human urine (mmol / L)

[0113] 1 3 4 5 6 7 β s(β) t s(β)·t Conclusion 2.70 2.77 2.72 2.82 2.80 2.73 0.0071 0.0085 2.571 0.0218 Stable

[0114] After repeated freeze-thaw, the human urine glucose standard substance sample can remain stable within 7 times, therefore the standard substance after opening can be used for at least 7 times of repeated freeze-thaw.

[0115] According to the above standard substance stability test results, the human urine glucose standard substance can be stored stably for 10 months under long-term storage conditions at-80℃; can be stored stably for 7 days at-20℃, 4℃ and 25℃, and no obvious turbidity is observed, and the sample is unstable at 60℃; can be kept stable within 6h after opening at 4℃, and can be kept stable within 4h after opening at 25℃; can be repeatedly frozen and thawed for at least 7 times.

[0116] Example 4: Determination of standard value of urine glucose standard substance by high performance liquid chromatography-isotope dilution mass spectrometry method;

[0117] 1. Preparation of derivatization reagent

[0118] The derivatization reagent is PMP-ammonia solution, wherein the PMP concentration is 250 mmol / L, the ammonia concentration is 400 mmol / L, and the pH is about 9.0. Among them:

[0119] 400 mmol / L of ammonia is prepared by using 25% ammonia and ultrapure water, and when preparing 30 mL, 0.9 mL of ammonia and 29.1 mL of ultrapure water are needed.

[0120] Prepare 10 mL of derivatization reagent: weigh 0.4355 g of PMP and dissolve it in 10 mL of 400 mmol / L ammonia, and if the pH is higher than 9.0, acetic acid should be used for adjustment.

[0121] 1 mol / L of acetic acid needs to be prepared by using glacial acetic acid and ultrapure water, and the ratio is glacial acetic acid: water = 0.5:8.25.

[0122] 2. Preparation of standard solution

[0123] In order to meet the determination of all samples to be tested, a standard solution of about 0.5 mg / g is prepared.

[0124] Use glucose purity standard substance, and prepare about 0.5 mg / g by weight method.

[0125] Use labeled glucose, and prepare about 0.5 mg / g by weight method.

[0126] 3. Mixing of standard solution and internal standard

[0127] According to the concentration of glucose in the standard substance sample, use a pipette to take 13C6] glucose standard solution, weigh, record the count, according to the mass concentration of the standard solution, add glucose standard solution to it, shake gently, mix well, and fully equilibrate to prepare a calibration ratio mixed solution. High mark: standard solution 78 μg + internal standard solution 100 μg + ultrapure water 22 μg; low mark: standard solution 52 μg + internal standard solution 100 μg + ultrapure water 48 μg, while weighing the mass of the standard solution and the internal standard solution.

[0128] ④ Mixing of urine sample and internal standard

[0129] Glucose in urine and 13 The ratio of C6] glucose is approximately 1:1, i.e. after dilution, the sample to be tested 70 μg + internal standard solution 100 μg + ultrapure water 30 μg, while weighing the mass of the urine sample and the internal standard solution.

[0130] ⑤ Deproteinization

[0131] After mixing, 1.5 ml of anhydrous ethanol is added to the sample, which is vortexed for 5 min and then centrifuged at 8000 rpm for 5 min.

[0132] ⑥ Derivatization

[0133] Take 25 μL of the supernatant, add 10 μL of PMP-ammonia derivatization reagent, and react at 70°C for 90 min, vortexing for 30 s every 30 min to mix and fully react.

[0134] The reaction involved in this derivatization process is shown in Figure 3 .

[0135] The final derivatization product has the molecular formula C 26 H 30 N4O7, and a molecular mass of 510.41. If isotopically labeled glucose is used for derivatization, the final derivatization product has the molecular formula 13 C6 12 C 20 H 30 N4O7, and a molecular mass of 516.41. The main fragmentation process of the derivatization product in mass spectrometry is shown in Figure 4 .

[0136] Whether it is an isotopically labeled glucose derivatization product or a natural glucose derivatization product, the molecular formula of the main fragment ion peak is: C 10 H 10 N2O, with a molecular mass of 174.08. Therefore, analysis can be performed using an electrospray ionization source (ESI) in positive ion mode and multiple reaction monitoring (MRM) mode, with the ion pair m / z = 512.4→175.0 (natural glucose) and m / z = 518.4→175.0 ([13 C6]glucose).

[0137] ⑦removing excess derivatization reagent

[0138] After reaction, cool to room temperature, add 50 μL acetic acid solution (1 moL / L) and mix, neutralize the reaction system. Add 1 mL deionized water and 1 mL chloroform, vortex extraction for 5 min, then centrifuge at 8000 rpm for 5 min, take the water phase to the machine for analysis.

[0139] Determination of glucose by high performance liquid chromatography tandem triple quadrupole mass spectrometry.

[0140] Liquid chromatography and mass spectrometry conditions are as follows:

[0141] Liquid chromatography conditions: injection volume 5 μL; chromatographic column Agilent Eclipse Plus C18 (4.6 x 150 mm, 5 μm); flow rate 300 μL / min, time 10 min. Mobile phase A is ultrapure water, mobile phase B is acetonitrile, isocratic elution as follows.

[0142] Table 15 Gradient of mobile phase

[0143] Total Time (min) A(％) B(％) 0.00 60.0 40.0 12.00 60.0 40.0

[0144] Mass spectrometry conditions: using multiple reaction monitoring (MRM) mode, ion pair m / z = 512.4→175.0 (natural glucose) m / z = 518.4→175.0 (13C6 glucose) mass spectrometry parameter settings as follows. 13 C6]glucose) mass spectrometry parameter settings as follows.

[0145] Table 16 Mass spectrometry parameter settings

[0146]

[0147] According to formula 1, the glucose concentration in the sample is calculated:

[0148]

[0149] In the formula: C is the concentration of glucose in urine (unit: mmol / L);

[0150] I sam The peak area ratio of glucose to internal standard in the sample (determined value);

[0151] I low The peak area ratio of glucose to internal standard in the low standard (determined value);

[0152] I Hi The peak area ratio of glucose to internal standard in the high standard (determined value);

[0153] Wlow mass ratio of low-mark glucose to internal standard;

[0154] W Hi mass ratio of high-mark glucose to internal standard;

[0155] M Is mass of internal standard in urine sample (unit: mg);

[0156] M Ser mass of urine sample (unit: g);

[0157] D S density of urine;

[0158] 180.16 is the relative molecular mass of glucose.

[0159] A typical chromatogram is shown in Figure 1 .

[0160] Example 5: Statistical calculation of the certified value of urine glucose reference material and uncertainty evaluation of the certified value.

[0161] The joint certification method was adopted in this certification. Since there is no reference measurement procedure for glucose in urine in JCTLM at present, the development of the human urine glucose reference material belongs to the development of a new method. Therefore, research institutions with strong analytical method development capabilities, such as colleges, research institutes, and analysis and testing centers, were mainly selected to confirm their analytical capabilities through blind sample testing. The glucose reference material in GBW(E)091148 human serum without identification was randomly distributed as a proficiency testing sample. The proficiency testing data of each unit is shown in Table 17. The measured certified reference material was within the uncertainty range of the certificate value, indicating that the proficiency testing was passed and joint certification could be performed. The joint certification raw data is shown in Tables 18 and 19. All data were considered as a new set of measurement data, and the total average value and standard deviation of all raw data were calculated. The combined measurement data of each level of reference material were tested for normality using SPSS software. All measurement results met the normal distribution assumption, and the analysis results are shown in Table 20. No abnormal values were found in the raw data of each level using the Dixon and Grubbs outlier test methods, and the analysis results are shown in Table 21. The final average value was calculated as the certified value of the urine glucose reference material.

[0162] Table 17 Proficiency testing of glucose joint certification units

[0163]

[0164] Table 18 Raw data of glucose joint certification (mmol / L)

[0165]

[0166] Table 19 The original data of the unit value (mmol / L)

[0167] Sample Name 1 2 3 Mean Sample 1 2.530 2.546 2.506 2.527 Sample 2 2.517 2.542 2.544 2.534 Sample 3 2.526 2.477 2.489 2.497 Sample 4 2.546 2.564 2.549 2.553 Sample 5 2.573 2.563 2.546 2.561 Sample 6 2.513 2.554 2.529 2.532 Overall Mean 2.534

[0168] All data were considered as a new set of measurement data. The determination data of the reference material were tested for normality using IBM SPSS software. All determination results were in accordance with the normal distribution assumption. The analysis results are shown in Table 20.

[0169] Table 20 The value data of ID LC-MS / MS method Kolmogorov-Smirnov test

[0170]

[0171] The original data in Table 18 and Table 19 were tested for outliers using the Dixon and Grubbs outlier test methods respectively. No outliers were found. The results are shown in Table 21.

[0172] Table 21 The outlier test of the value data of ID LC-MS / MS method

[0173]

[0174] The determination data were tested for homogeneity of variance using the Cochran rule. According to the analysis, for human urine glucose reference material, the C value between the joint value methods was less than the critical value C, i.e. the joint value result had homogeneity of variance and the same precision.

[0175] Table 22 Homogeneity of variance test (Cochran method) of joint value

[0176]

[0177] The final calculated average value was taken as the value result of human urine glucose reference material, as shown in Table 23.

[0178] Table 23 The value result of human urine glucose reference material (mg / g)

[0179] Sample Certified Value Human Urine Glucose Reference Material 0.45

[0180] The density of the reference material at 20℃ is (1.003±0.009) g / mL, i.e. 1 mg / g = 5.97 mmol / L. Therefore, the concentration of human urine glucose reference material is as follows:

[0181] Table 24 The value result of urine glucose reference material (mmol / L)

[0182] Sample Certified Value Human Urine Glucose Reference Material 2.53

[0183] The uncertainty of the glucose standard material in urine is derived from the uncertainty introduced in the valuation process, the uncertainty introduced by the homogeneity of the standard material, and the uncertainty introduced by the long-term stability.

[0184]

[0185] (1) Uncertainty evaluation of the valuation results

[0186] The glucose standard material in urine is valued by isotope dilution liquid chromatography-mass spectrometry. The valuation calculation model is:

[0187]

[0188] C is the concentration of glucose in urine (unit: mmol / L);

[0189] I sam is the peak area ratio of glucose to internal standard in the sample (determined value);

[0190] I low is the peak area ratio of glucose to internal standard in the low standard (determined value);

[0191] I Hi is the peak area ratio of glucose to internal standard in the high standard (determined value);

[0192] W low is the mass ratio of glucose to internal standard in the low standard;

[0193] W Hi is the mass ratio of glucose to internal standard in the high standard;

[0194] M Is is the mass of the internal standard in the urine sample (unit: mg);

[0195] M Ser is the mass of the urine sample (unit: g);

[0196] D S is the density of urine;

[0197] 180.16 is the relative molecular mass of glucose.

[0198] The uncertainty of isotope dilution mass spectrometry mainly comes from the balance weighing, glucose purity standard material, density determination, and analytical method repeatability, etc., among which:

[0199] ① Balance weighing

[0200] The uncertainty introduced by the balance weighing is calculated according to the maximum allowable error in the balance verification certificate according to formula (4):

[0201]

[0202] During the experiment, the weighing of the solution part was counted for a total of 3 times, which were the weighing of the high standard solution, the weighing of the low standard solution, and the weighing of the urine sample, each repeated twice (empty plate and gross weight).

[0203] The relative standard uncertainty introduced by the balance weighing of the high standard solution was:

[0204]

[0205] The relative standard uncertainty introduced by the balance weighing of the low standard solution was:

[0206]

[0207] The relative standard uncertainty introduced by the balance weighing of the urine sample was:

[0208]

[0209] ②Preparation of standard solution

[0210] During the experiment, 100 g of standard solution was prepared using the weight method with GBW10062 glucose purity standard substance, and 50 g of standard solution was prepared with C6] glucose. 13 C6] glucose.

[0211] During the preparation of the non-labeled glucose standard solution, the standard solution preparation process involved two weighing processes, and the calibration certificate provided the uncertainty of the balance measurement, including two weighing processes (glucose and deionized water), each weighing should be repeated twice (empty plate and gross weight), then the uncertainty introduced by the preparation of the glucose standard solution was

[0212]

[0213] ③Uncertainty introduced by purity standard substance

[0214] The uncertainty introduced by the glucose purity standard substance was calculated according to the uncertainty in the standard substance certificate and the corresponding inclusion factor according to formula (5):

[0215]

[0216] ④Uncertainty introduced by density measurement

[0217] Density measurement was determined using a calibrated balance and a pipette, and the density measurement was calculated according to formula ρ = m / V, so the uncertainty introduced by the density measurement was calculated according to formula (6):

[0218]

[0219] Similarly, the weighing and constant volume processes are involved in the density determination process. The weighing part: the standard component should be calculated repeatedly for 20 times (5 times of parallel determination, each containing empty tray and gross weight), then The constant volume part: according to JJG 646-2006 "Pipette Verification Regulation", the capacity tolerance of 200 μL pipette is ± 3 μL, then Assuming that the ambient temperature and the measurement temperature differ by ± 3℃, the volume expansion coefficient of water is The volume uncertainty caused by the difference between the calibration temperature and the use temperature of the volumetric flask is: The synthesis is obtained The relative standard uncertainty of density determination by the two is 0.870%.

[0220] ⑤Uncertainty introduced by method repeatability

[0221] The uncertainty introduced by repeatability is calculated according to the standard deviation and the number of measurements n:

[0222]

[0223] The uncertainty introduced by the two kinds of isotope dilution mass spectrometry is the synthesis of the above uncertainties:

[0224]

[0225] (2) Uncertainty evaluation of homogeneity and stability

[0226] ① Uncertainty introduced by sample homogeneity

[0227] According to JJF1343-2022, the uncertainty introduced by the homogeneity of the standard material is equal to the standard deviation of the inter-bottle homogeneity. When the inter-group mean square is greater than the intra-group mean square, calculate according to formula (9).

[0228]

[0229] When the inter-group mean square is less than the intra-group mean square, calculate according to formula (10).

[0230]

[0231] ② Uncertainty introduced by sample stability

[0232] The uncertainty introduced by the long-term stability of the standard material is calculated according to formula 11, t is the long-term stability monitoring result of 10 months, plus the predicted stable time of 6 months, that is, t = 10 + 6 = 16:

[0233] u lts = sk,lts • t Formula 11

[0234] (3) Uncertainty evaluation results

[0235] The final calculation results are shown in Table 25:

[0236] Table 25 Uncertainty evaluation results of glucose standard material in urine

[0237]

[0238]

[0239] Table 26 Glucose standard material in urine and uncertainty evaluation results (mmol / L)

[0240] Sample Certified Value Human Urine Glucose Reference Material 2.53±0.06

[0241] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the claims of the present application.

Claims

1. A standard substance for detecting glucose in human urine, characterized in that, The typical chromatogram of the reference material is shown in Figure 1 of the specification.

2. The standard substance according to claim 1, characterized in that, The preparation method of the reference material is as follows: Collect about 1200 - 1500 mL of normal human urine, centrifuge at 12000 rpm and 4°C for 15 min using a high-speed refrigerated centrifuge, take the supernatant, filter it by suction filtration through a 0.22 μm filter membrane, and collect the filtrate; Add a preservative using a pipette at a ratio of 0.1% by volume, and mix well after adding to ensure the stability of the urine; Then, preliminarily determine the glucose concentration in the urine using a biochemical analyzer or isotope dilution mass spectrometry. When the preliminarily determined glucose concentration in the above urine is not lower than the glucose concentration of the urine glucose reference material to be prepared, immediately subpackage the above urine to obtain the urine glucose reference material; When the preliminarily determined glucose concentration of the above urine is lower than the urine glucose concentration to be prepared, add glucose with a purity of more than 95% to the above urine so that the glucose concentration in the above urine is not lower than the urine glucose concentration to be prepared, and the volume of the added pure glucose shall not exceed 10% of the total volume of the above urine. After adding, mix the above urine at room temperature for 6 - 12 h to obtain the human urine glucose reference material.

3. The standard substance according to claim 2, characterized in that, The preservative is Proclin 300, and its concentration is 0.1%.

4. The standard substance according to claim 1, characterized in that, The value determination method of the reference material includes: homogeneity test, stability test, and determination of the reference value by high performance liquid chromatography - isotope dilution mass spectrometry.

5. The standard substance according to claim 4, characterized in that, The homogeneity test step is: Test the homogeneity of uric acid in the subpackaged and stored human urine glucose reference material. Under the condition of magnetic stirring, subpackage the above human urine glucose reference material into 2 mL low-temperature resistant and low-adsorption brown sample bottles respectively, and seal or close. According to the subpackaging sequence, continuously number each subpackaging unit, and the number of subpackaging units is P; Randomly select and test the continuously numbered subpackaging units. When 100 < P ≤ 200, the number of subpackaging units m selected is not less than 11; When 200 < P ≤ 500, the number of subpackaging units m selected is not less than 15; When 500 < P ≤ 1000, the number of subpackaging units m selected is not less than 25; When the number of subpackaging units P > 1000, the number of subpackaging units m selected is not less than 30. According to the order of selection, re-number and arrange the m selected subpackaging units in sequence, and use the biochemical analyzer method or isotope dilution mass spectrometry to test the homogeneity of the urine glucose in the human urine glucose reference material in the above selected subpackaging units. During the test, in order to eliminate the error caused by instrument drift during the determination process, analyze the samples three times in the order of 1→15, 15→1, odd - even interspersed until the nth test of the uric acid in the urine glucose reference material in each selected subpackaging unit, where n is an integer greater than or equal to 3, so as to obtain the n test results, calculate according to the following formula and statistically obtain the homogeneity of uric acid in the human urine glucose reference material in the subpackaging unit; Assuming there are *a* standard material units, and unit *i* is measured *ni* times (i = 1, ..., a), for a simple equilibrium design, the number of repeated measurements *ni* is the same for all units, i.e., n1 = n2 = ... = na = n0. A one-way ANOVA is used for analysis; the statistical model is as follows: x ij = μ + δ i + ε ij Formula 1 Where: x ij Let x be the j-th observation of the i-th unit; μ is the total number of possible outcomes (observation x). ij Assuming the (true) mean is derived from it; δ i ε represents the effect of element i on the result, i.e., the (true) deviation of element i from μ; ij The random error of the j-th observation in the i-th unit is also known as the residual term; according to formulas 2 and 3, the mean square between units M can be obtained. between and residual mean square M within ; in: Statistic F: According to the degrees of freedom (v1, v2) and the given significance level α, the critical F α (v1, v2) value is obtained from the table and then compared with the F value calculated by the formula; if F < Fα, it is considered that there is no significant difference between within-group and between-group, and the sample is homogeneous.

6. The standard substance according to claim 4, characterized in that, The stability testing steps are divided into long-term stability testing and short-term stability testing. The duration of long-term stability testing shall be no less than 6 months, and the duration of short-term stability testing shall be no less than 7 days. Taking long-term stability testing as an example, within the long-term stability testing period, at least 5 time points k are selected. At each time point, human urine glucose standard material is extracted from at least 2 dispensing units. The urine glucose of the human urine glucose standard material in each dispensing unit is tested three times using a biochemical analyzer or isotope dilution mass spectrometry. Then, the arithmetic mean of these measurement results is calculated to obtain the arithmetic mean Y of the measurement results at each time point. i The arithmetic mean of the above measurement results is fitted to the corresponding measurement time using the following linear model: Y=b0+bX Formula 7 In Formula 7: b0—intercept; b—regression coefficient; X—test time, in months; Y—arithmetic mean of the results of uric acid content determination in urine glucose standard substance; The regression coefficients and intercepts were calculated using formulas 8 and 9: In Formula 8: X i —The detection time at the i-th time point, in months; Y i —The arithmetic mean of the urine glucose determination results of the human urine glucose standard substance in the above-mentioned dispensing unit at the i-th time point; —The average detection time across all time points, in months; —The arithmetic mean of the urine glucose determination results of the human urine glucose standard substance in the above-mentioned dispensing unit at all time points; k — the number of time points; Calculate the value of s according to formula 10: The meanings of the symbols in Formula 10 are the same as those in Formulas 7, 8, and 9. Calculate the value of s(b) according to Formula 11: Look up the t-distribution table to get t 0.95,k-2 The value; If |b| < t 0.95,k-2 If s(b) is not significant, it indicates that no instability was observed, suggesting that the human urine glucose standard is stable in the long term. The long-term stability of the urine glucose standard was examined at -20℃, -40℃, or -80℃, with -80℃ being preferred. The short-term stability of the urine glucose standard was examined and calculated at -20℃, 4℃, 25℃, and 60℃.

7. The standard substance according to claim 4, characterized in that, The procedure for determining the standard value using high-performance liquid chromatography-isotope dilution mass spectrometry includes: adding an isotope-labeled internal standard and performing the determination on the instrument; when using high-performance liquid chromatography-isotope dilution mass spectrometry to determine urinary glucose, the selected isotope label is [ 13 C6]-labeled glucose, with at least 3 isotopic labeled atoms and an isotopic abundance of at least 99%; during sample pretreatment, first use a solvent such as water, methanol, or acetonitrile to […]. 13 C6] labeled glucose was prepared into a solution with a concentration of 0-100 mg / g, and the same amount of glucose as initially determined was added to the urine glucose standard sample. 13 C6]-labeled glucose, added [ 13 The volume ratio of C6-labeled glucose to sample volume is between 1:100 and 10:

1. After addition, the mixture is thoroughly mixed and filtered through a 0.22 mm filter membrane before high-performance liquid chromatography-isotope dilution mass spectrometry analysis. A C4, C8, or C18 column is used. Mobile phase A is water containing 0.1%–1% formic acid, water containing 0.1%–1% trifluoroacetic acid, or a 5–100 mmol / L ammonium formate or ammonium acetate solution containing 0%–50% methanol or acetonitrile. Mobile phase B is acetonitrile or methanol containing 0.1%–1% formic acid, or acetonitrile or methanol containing 0.1%–1% trifluoroacetic acid. The mobile phase ratio of A:B is 100:0 to 50:50 over a period of 5–60 min. The flow rate is 100–2000 mL / min, and the injection volume is 1–100 mL. Mass spectrometry detection was performed in either MRM or SIM mode. In MRM mode, glucose in the sample (m / z = 512.4 for the glucose precursor ion after derivatization, m / z = 175.0 for the glucose daughter ion) and glucose isotope labeling in the standard were detected separately. 13 C6](Derivatized glucose isotope markers[ 13 The C6] precursor ion has an m / z of 518.4, and the glucose isotope label [ 13 The signal of the C6] daughter ion (m / z = 175.0) was detected; when using SIM mode for detection, the glucose in the sample (glucose ion m / z = 512.44) and the glucose isotope label in the standard were detected respectively. 13 C6](glucose isotope markers) 13 The signal of C6 ions (m / z = 518.4) was used; the quantitative standard used was a glucose purity standard and [ 13 C6] A standard solution of glucose was prepared, wherein the glucose concentration in the standard solution, [ 13 C6] indicates glucose concentration and glucose and [ 13 The proportion of C6] glucose is related to the glucose concentration in the standard substance to be tested, [ 13 The concentration and ratio of glucose in C6 were close, with a ratio range of 0.90–1.10; the extracted glucose chromatogram and [ 13 The peak areas of the C6-labeled glucose extraction chromatograms were obtained by integration. Based on the ratio of sample peak area to standard peak area, quantification was performed using the single-point method, bracket method, or standard curve method.

8. The standard substance according to claim 7, characterized in that, The solvent is prepared as an aqueous solution with a concentration of 0.5 mg / g; the addition of [ 13 The volume ratio of C6-labeled glucose to the sample volume was 1:1; the chromatographic column used was an Agilent Eclipse Plus C18 (4.6 × 150 mm, 5 μm) column; the mobile phase A was ultrapure water, and the mobile phase B was acetonitrile containing 0.1% formic acid; the mobile phase ratio was 95.0:5.0 for 0.00–10.00 min, the flow rate was 300 mL / min, and the injection volume was 5 mL.

9. The standard substance according to claim 7, characterized in that, The mass spectrometry detection was performed in MRM mode, and the quantification method was the bracket method.

10. The standard substance according to any one of claims 4-9, characterized in that, The determined values ​​are: 0.45 mg / g or 2.53 mmol / L.

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