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 invention relates to the technical field of metrological chemical analysis and detection, in particular to a human urine glucose detection standard substance and its preparation and determination. Background Art

[0002] Diabetes is a global chronic disease, and the prevention and control of its complications have become a major challenge in the medical field. Early diabetic kidney damage, a common microvascular complication of diabetes, requires early detection and timely intervention to slow disease progression and improve patient outcomes. Diabetic nephropathy is a common complication of diabetes. Its pathological basis is primarily hyperglycemia and the accumulation of advanced glycation end products, which lead to glomerular and tubular damage. Early diabetic nephropathy is typically asymptomatic. As the disease progresses, patients may develop signs of renal impairment, such as hypertension, edema, and anemia. In the late stages of diabetic nephropathy, renal function gradually deteriorates, and symptoms of uremia develop. Early diagnosis and intervention are crucial for delaying the progression of diabetic nephropathy. Currently, numerous clinical methods are available for detecting diabetic kidney damage. Among them, urinalysis is simple, reproducible, non-invasive, and inexpensive, making it easily accepted by patients and widely used in clinical practice. Impaired renal function can impair the renal absorption of glucose in urine, leading to the excretion of some glucose in the urine and elevated urine glucose levels. Positive urine sugar indicates that the patient has abnormal hyperglycemia metabolism, which can intuitively reflect the blood sugar control situation and facilitate clinical adjustment of treatment plans.

[0003] Therefore, urine glucose testing, as a non-invasive screening method, has important clinical value in early diagnosis, disease monitoring and efficacy evaluation. Changes in urine sugar levels can indirectly reflect the glomerular filtration function and determine the degree of renal damage in diabetic patients. However, the urine sample matrix is ​​complex and is easily affected by interference factors during the detection process, 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, establishing a highly accurate and traceable urine glucose standard substance is crucial for unifying detection standards, improving method consistency and ensuring the quality of clinical testing.

[0004] Currently, research on reference materials, both domestically and internationally, focuses on the serum matrix, while reference materials for urine glucose are still lacking. While existing serum reference materials are partially used for the calibration of urine testing systems, matrix differences can lead to biased measurement transfer. Furthermore, the stability, homogeneity, and storage conditions of urine samples differ fundamentally from those of serum, necessitating the development of reference materials specifically for the urine matrix to meet the specific needs of clinical testing. Summary of the Invention

[0005] This invention addresses the need for traceability and transmission of urine glucose measurement values ​​and the calibration of fully automated biochemical analyzers. It provides a method for preparing and determining the value of a standard substance for glucose detection in human urine. This method aims to provide technical support for clinical laboratory quality evaluation, test system performance verification, and laboratory capability comparison, further promoting the standardization and precision of laboratory medicine.

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

[0007] The first step is to process and prepare urine glucose standard materials. The raw materials come from urine collected from healthy people. Then use a high-speed refrigerated centrifuge at 12000rpm and 4℃ for 15min, take the supernatant, filter it with a 0.22μm filter membrane, and collect the filtrate; use a pipette to add ProClin300 as a preservative at a ratio of 0.1% (v / v), and mix it thoroughly after adding to ensure the stability of the urine. Use a fully automatic biochemical analyzer or mass spectrometer to perform a preliminary determination of the glucose concentration in the urine. When the preliminary determination results meet the design requirements of the standard material, the packaging process can be entered; when the preliminary determination results are lower than the design requirements of the standard material, the glucose purity standard material solution is added to increase the glucose content in the urine. When adding, ensure that the volume of the glucose purity standard material solution added does not exceed 10% of the urine volume. After addition, mix at room temperature for (6 to 12) hours.

[0008] Urine glucose standard materials should be dispensed in colorless or brown screw-capped sample bottles, preferably low-temperature-resistant, low-adsorption brown sample bottles. The urine raw material should be dispensed into sample bottles or cryotubes under shaking conditions and sealed or sealed. The standard materials for each urine glucose concentration level should be numbered consecutively according to the order of dispensing and stored frozen at -20°C, -40°C, or -80°C.

[0009] The uniformity test of urine glucose requires random sampling of samples according to the number of units in the packaging. The number of units in the packaging is counted as N. When 100<N≤200, the number of units to be sampled shall not be less than 11; when 200 <N≤500时,抽取单元数不少于15个;当500<N≤1000时,抽取单元数不少于25个;当总体单元数N> 1000, the number of samples taken shall not be less than 30. At each urine glucose concentration level, units that meet the above quantity requirements shall be randomly drawn for uniformity testing. The experimental method for uniformity testing is biochemical analyzer method or isotope dilution mass spectrometry, preferably isotope dilution mass spectrometry. Each standard substance unit drawn shall be measured 3 times using the above method. During analysis, in order to eliminate the error caused by instrument drift during the measurement process, the samples shall be analyzed three times in the order of 1→15, 15→1, and odd and even interspersed. The analyzed samples shall be tested for uniformity and the results shall be statistically analyzed according to the following formula:

[0010] Assume there are a units of reference material, and unit i is measured ni times (i = 1, ..., a). For a simple balanced design, the number of repeated measurements ni is the same for all units, that is, n1 = n2 = ... = na = n0. One-way 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 value of the i-th unit; μ is the total number of all possible results (observation result x ij (true) mean value of the value assumed to be derived from it; i is the effect of unit i on the result, that is, the (true) deviation between unit i and μ; ε ij is the random error of the jth observation value of the i-th unit, also known as the residual term. According to formulas 2-2 and 2-3, the mean square between units M can be obtained between and residual mean square M within ;

[0013] Degree of freedom v1=a-1 Formula (2)

[0014] degrees of freedom

[0015] in;

[0016]

[0017] Make statistic F:

[0018]

[0019] According to the degrees of freedom (v1, v2) and the given significance level α, the critical F α (v1, v2) value, and then compare it with the F value calculated by the formula. <Fα则认为组内与组间无明显差异,样品是均匀的。否则,需进行标准物质的重新制备和均匀化处理。

[0020] 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. During the long-term stability test period, at least 5 time points k are selected according to the principle of close analysis at the beginning and sparse analysis at the end. At each time point, at least 2 units of standard substances are extracted. Each unit of standard substance is analyzed 3 times using a biochemical analyzer or isotope dilution mass spectrometry method. The arithmetic mean of these measurement results is calculated to obtain the measurement result X at each time point. i , fit the measurement results and measurement time according to the following linear model:

[0021] Y=b0+bX formula (7)

[0022] Where:

[0023] b0—intercept; b—regression coefficient; X—time; Y—measurement result of glucose content of standard substance.

[0024] Calculate the regression coefficient and intercept according to Equation 8 and Equation 9:

[0025]

[0026] Where:

[0027] X i —the i-th time point;

[0028] Y i —The result of urine glucose standard substance determination at time point i;

[0029] —mean value of all time points;

[0030] —Determination results of urine glucose standard substance at all time points;

[0031] k—number of time points.

[0032] Calculate the s value according to formula 10:

[0033]

[0034] The meanings of the symbols in the formula are the same as those in Formula 8 and Formula 9.

[0035] Calculate the value of s(b) according to Equation 11:

[0036]

[0037] Look up the t distribution table and get t 0.95.k-2 value.

[0038] If |b|<t 0.95，k-2 ·s(b), the slope is not significant and no instability is observed, indicating that the urine glucose standard substance is stable.

[0039] The long-term stability of urine glucose standard substances was investigated at -20°C, -40°C or -80°C, preferably -80°C. The short-term stability of urine glucose standard substances was investigated and calculated at -20°C, 4°C, 25°C and 60°C.

[0040] The method for determining the value of urine glucose standard substance is to use high performance liquid chromatography-isotope dilution mass spectrometry to determine the standard value of urine glucose, including the steps of adding isotope labeled internal standard and measuring on the machine. When using high performance liquid chromatography-isotope dilution mass spectrometry to determine urine glucose, the isotope label selected 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 pre-treating the sample, 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, preferably an aqueous solution, with a preferred concentration of 0.5 mg / g. The same amount of [ 13 C6] labeled glucose, adding [ 13The volume ratio of C6] labeled glucose to the volume ratio of the sample is between (1:100 and 10:1), preferably 1:1. 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. The chromatographic column used is a C4, C8, or C18 column, preferably an Agilent Eclipse Plus C18 (4.6×150 mm, 5 μm) column. The mobile phase A used is water containing (0.1% to 1%) formic acid, or water containing (0.1% to 1%) trifluoroacetic acid, or (5 to 100) mmol / L ammonium formate, or (5 to 100) mmol / L ammonium acetate solution, containing (0% to 50%) methanol or acetonitrile. The mobile phase B used is acetonitrile or methanol containing (0.1% to 1%) formic acid, or acetonitrile or acetonitrile or methanol containing (0.1% to 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 a mobile phase A:B ratio of 100:0 to 50:50 over a period of 5 to 60 minutes. The preferred mobile phase examples are shown in the table below:

[0041] TotalTime(min) A(％) B(％) 0.00 95.0 5.0 10.00 95.0 5.0

[0042] The flow rate used is (100-2000) mL / min, preferably 300 mL / min. The injection volume used is (1-100) mL, preferably 5 mL. Mass spectrometry detection is performed in MRM mode or SIM mode. When the MRM mode is used, glucose in the sample (m / z = 512.4 of the glucose parent ion after derivatization, m / z = 175.0 of the glucose daughter ion) and glucose isotope markers in the standard are detected respectively. 13 C6](glucose isotope label after derivatization[ 13 C6] parent ion m / z = 518.4, glucose isotope label [ 13 C6] daughter ion m / z = 175.0); when the SIM mode was used for detection, glucose in the sample (glucose ion m / z = 512.44) and glucose isotope markers in the standard [ 13 C6](glucose isotope label[ 13 C6] ion m / z = 518.4) signal. MRM mode is preferred for detection. The quantitative standard used is glucose purity standard material and [ 13 C6] labeled glucose prepared standard solution, glucose concentration in the standard solution, [ 13 C6] marks the glucose concentration and glucose and [ 13 C6] glucose concentrations in the standard substance to be tested, 13C6] glucose concentration and ratio are close, and the ratio range is (0.90~1.10). 13 The peak areas of the C6-labeled glucose extraction chromatograms were integrated. Quantification was performed using the single-point method, bracket method, or standard curve method based on the ratio of the sample peak area to the standard peak area. The preferred ratio range was (0.95-1.05), and the bracket method was the preferred quantification method.

[0043] The method for determining the value of urine glucose standard substances, in which the identification value of the standard substances is determined by high performance liquid chromatography-isotope dilution mass spectrometry in conjunction with multiple laboratories. According to the number requirements of independent data groups in JJF 1343-2022, each set of data obtained should be based on the measurement of 2 units, each unit should be measured at least twice, and no less than 4 to 6 independent repeated measurement data should be provided. All data are regarded as a new set of measurement data. According to the KS single sample normality test, and the Grubbs method and / or Dixon method are used for outlier test, and finally the Cochran rule is used to test the homogeneity of variance of the measurement data; after passing the above statistical tests, the arithmetic mean of all results is taken as the standard value of the measurement result of the high performance liquid chromatography-isotope dilution mass spectrometry method. The uncertainty u of the determination result of the glucose standard substance in urine comes from the uncertainty u introduced in the determination process. char , uncertainty u introduced by the homogeneity of the standard material bb and the uncertainty u introduced by long-term stability lts . According to formula 12, the synthesis

[0044]

[0045] Where k is the inclusion factor, usually k=2.

[0046] where u char Includes the uncertainty u from HPLC-MS char,HPLC-IDMS .u char,HPLC-IDMS It includes the uncertainty component u introduced from the repeatability of HPLC-ISODMS measurement results. char,HPLC-IDMS,A (according to Equation 13), and the uncertainty component u introduced by multiple balance weighings char,HPLC-IDMS,wi (According to Equation 16) and the uncertainty component u introduced by the glucose purity standard substance char,HPLC-IDMS,P (According to Equation 15) The various uncertainty components are synthesized according to Equation 16.

[0047]

[0048] In Equation 13, s is the standard deviation and n is the number of measurements;

[0049]

[0050] In Equation 14, U(E) is the uncertainty in the balance calibration certificate, and k is the coverage factor

[0051]

[0052] In Equation 15, U is the uncertainty in the glucose purity standard material certificate, and k is the coverage factor;

[0053]

[0054] Where q is the number of times the balance is weighed, c is i is the sensitivity coefficient of the i-th weighing, c p is the sensitivity coefficient of the purity of the standard substance.

[0055] When the between-group mean square is greater than the within-group mean square, the uncertainty introduced by the uniformity is calculated according to Formula 17.

[0056]

[0057] When the between-group mean square is smaller than the within-group mean square, calculate according to Formula 18.

[0058]

[0059] The long-term stability of the standard substance is calculated according to formula 19:

[0060] u lts =s k,lts ·t lts Formula (19)

[0061] Where,

[0062] s k,lts is s(b),t during long-term stability test lts The duration of long-term stability testing.

[0063] 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:

[0064] x±U

[0065] Where x is the certified value and U is the expanded uncertainty of the certified value.

[0066] The present invention addresses the problem that the lack of human urine glucose standard substances causes inaccurate and non-comparable urine glucose test results. It provides a method for preparing and determining the value of a urine glucose standard substance, which can be used for the development of urine glucose detection reagents and method performance verification, as well as for the traceability of the value of urine glucose test results, ensuring the accuracy and comparability of clinical urine glucose test results, and facilitating clinical testing of diseases such as chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is a chromatographic ion flow chart of a standard substance for detecting glucose in human urine according to the present invention;

[0068] Figure 2 It is a packaged standard substance for human urine glucose testing;

[0069] Figure 3 It is the glucose derivatization process of ID LC-MS / MS method;

[0070] Figure 4 It is a glucose derivative cleavage product by ID LC-MS / MS method. DETAILED DESCRIPTION

[0071] The present invention is further described below with reference to the following examples, but is not intended to limit the present invention in any way. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be encompassed by the protection of the present invention.

[0072] Example 1: Processing and preparation of urine glucose standard material;

[0073] The raw material for the urine glucose standard material was urine collected from healthy individuals. The collected urine was centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant was removed and filtered through a 0.22 μm filter membrane. 0.1% Proclin 300 was added to the collected urine as a preservative to ensure the stability of the standard material, and the solution was thoroughly mixed after addition. The glucose concentration in urine was initially determined using an automated biochemical analyzer. The glucose solution required for the target glucose concentration was prepared using a sample of GBW10062 glucose purity standard material. The amount to be added was calculated based on the designed target concentration range. The glucose solution was pipetted directly into the urine and mixed at room temperature for 8 hours to ensure the homogeneity of the standard material. The human urine glucose standard material was aliquoted into cryogenically resistant, low-adsorption brown vials under shaking conditions, totaling 500 units (1500 μL per unit). The cryovials and boxes were labeled with the designed concentration, sample number, and aliquot date and stored at -80°C. Urine glucose standard substances such as Figure 2 shown.

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

[0075] The homogeneity of the standard material was tested using a CS-1200 fully automatic biochemical analyzer and an enzymatic glucose assay kit. According to JJF 1343-2022, when the total number of units is ≤500, ≥15 units should be sampled. Therefore, 15 units were randomly selected from each candidate level and each candidate unit was measured three times using the fully automatic biochemical analyzer. To eliminate errors caused by instrument drift during the measurement process, the samples were analyzed three times in the order of 1 → 15, 15 → 1, alternating odd and even numbers.

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

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

[0078] Level 1 times 1 times 2 times 3 Number of observations Sum average 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 urine glucose standard substance was subjected to one-way ANOVA using Excel software. The results are shown in Table 2. <Fα,因此认为组内与组间无明显差异,样品是均匀的。

[0080] Table 2 Results of one-way ANOVA of the homogeneity data of urine glucose standard material

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

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

[0083] The stability of the standard substance was investigated over a 10-month period using a CS-1200 fully automatic biochemical analyzer and a glucose enzymatic glucose assay kit. The average results of the stability investigation were obtained by taking three standard substance samples each time. The stability investigation results are shown in Table 3.

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

[0085] Time / month 1 2 3 average 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] The above data were subjected to linear regression analysis using Excel software, the regression parameters were calculated, and the slope β was subjected to t-test. The results are listed in Table 4.

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

[0088] Stability time / month β s(β) t s(β)·t in conclusion 10 0.0010 0.0005 2.571 0.0014 Stablize

[0089] Long-term stability testing showed that the glucose standard in human urine was stable for 10 months at -80°C. Short-term stability testing was conducted using a CS-1200 fully automatic biochemical analyzer and an enzymatic glucose assay kit at -20°C, 4°C, 25°C, and 60°C. The testing period at each temperature was seven days. Tables 5 to 8 list the average stability test results for three samples taken at different intervals.

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

[0091] Time / day 1 2 3 average 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 Results of short-term stability study of urine glucose standard substance at 4°C (mmol / L)

[0093] Time / day 1 2 3 average 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 Results of short-term stability study of urine glucose standard substance at 25°C (mmol / L)

[0095] Time / day 1 2 3 average 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 substance at 60℃ (mmol / L)

[0097] Time / day 1 2 3 average 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 substance (mmol / L)

[0099]

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

[0101] Open-bottle stability testing of standard substance samples was performed using a fully automated biochemical analyzer. Three glucose standard substance samples were taken at each test and the average results were measured at 4°C and 25°C for 1, 2, 3, 4, 5, and 6 hours to assess open-bottle stability. The results are shown below.

[0102] Table 10 Open bottle stability test results of glucose standard substance in human urine at 4°C (mmol / L)

[0103] Time / h 1 2 3 average 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 Open bottle stability test results of glucose standard substance in human urine at 25°C (mmol / L)

[0105] Time / h 1 2 3 average 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 Opening stability test results of glucose standard substance in human urine (mmol / L)

[0107]

[0108] After opening the bottle, the human urine glucose standard substance sample is stable for 4 hours at 4°C (judged by the uncertainty, the values ​​within 4 hours are within the uncertainty range of subsequent calculations), and is stable for 4 hours at 25°C (judged by the standard deviation of β1, using a t-test, β1 = 0.0133, which is less than t(0.95, n-2)·s(β1) = 0.0176). Therefore, it is recommended that the standard substance be used within 4 hours after opening the bottle.

[0109] The freeze-thaw stability of the standard substance was tested using a CS-1200 fully automatic biochemical analyzer and an enzymatic glucose assay kit. Three glucose standard samples were taken each time and repeatedly frozen and thawed. The average results after 1, 2, 3, 4, 5, 6, and 7 freeze-thaw cycles were measured to investigate the freeze-thaw stability of the standard substance. The results are shown below.

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

[0111] Freeze-thaw cycles 1 2 3 average 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 freeze-thaw stability test of glucose standard substance in human urine (mmol / L)

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

[0114] After repeated freezing and thawing, the human urine glucose standard material sample can remain stable within 7 times, so the standard material can be repeatedly frozen and thawed for at least 7 times after opening the bottle.

[0115] According to the stability test results of the above-mentioned standard substances, the glucose standard substance in human urine can be stably stored for 10 months under long-term storage conditions of -80°C; it can be stably stored for 7 days at -20°C, 4°C, and 25°C, and no obvious turbidity is observed. The sample is unstable at 60°C; it can remain stable within 6 hours after opening the bottle at 4°C, and within 4 hours after opening the bottle at 25°C; it can be repeatedly frozen and thawed 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;

[0117] ① Preparation of derivatization reagents

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

[0119] 400mmol / L ammonia water is prepared with 25% ammonia water and ultrapure water. To prepare 30mL, 0.9mL ammonia water and 29.1mL ultrapure water are required.

[0120] Prepare 10 mL of derivatization reagent: weigh 0.4355 g of PMP and dissolve it in 10 mL of 400 mmol / L ammonia water. If the pH is higher than 9.0, adjust it with acetic acid.

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

[0122] ② Preparation of standard solution

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

[0124] Glucose purity standard material was used and prepared by weight method to approximately 0.5 mg / g.

[0125] Labeled glucose was used and the concentration was adjusted to approximately 0.5 mg / g using a gravimetric method.

[0126] ③ Mixing of standard solution and internal standard

[0127] According to the concentration of glucose in the standard sample, pipette [ 13 Weigh the glucose standard solution and record the amount. Add the glucose standard solution according to the concentration of the standard solution. Gently shake to mix and balance thoroughly to prepare a calibration ratio mixed solution. High standard: 78 μg standard solution + 100 μg internal standard solution + 22 μg ultrapure water; low standard: 52 μg standard solution + 100 μg internal standard solution + 48 μg ultrapure water. Simultaneously weigh the standard solution and 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, that is, 70 μg of the diluted sample to be tested + 100 μg of the internal standard solution + 30 μg of ultrapure water. The masses of the urine sample and the internal standard solution are weighed at the same time.

[0130] ⑤Deproteinization

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

[0132] ⑥ Derivatization

[0133] Take 25 μL of supernatant, add 10 μL of PMP-ammonia derivatization reagent, and react at 70°C for 90 min. Vortex and shake for 30 s every 30 min to ensure full reaction.

[0134] The derivatization process involves reactions such as Figure 3 As shown;

[0135] The final derivative product has the molecular formula: C 26 H 30 N4O7, whose molecular mass is 510.41. If isotope-labeled glucose is used for derivatization, the molecular formula of the derivatized product is 13 C612 C 20 H 30 N4O7, its molecular mass is 516.41. The derivatized product mainly undergoes fragmentation in the mass spectrum, such as Figure 4 shown.

[0136] Whether it is an isotope-labeled glucose-derived product or a natural glucose-derived product, the molecular formula of the main fragment ion peak is: C 10 H 10 N2O has a molecular mass of 174.08. Therefore, the electrospray ionization source (ESI) positive ion mode and multiple reaction monitoring (MRM) mode can be used for analysis, and the ion pairs detected are m / z = 512.4 → 175.0 (native glucose) and m / z = 518.4 → 175.0 ([ 13 C6] glucose).

[0137] ⑦Remove excess derivatization reagent

[0138] After the reaction, let it cool to room temperature. Then, add 50 μL of acetic acid (1 mol / L) and mix thoroughly to neutralize the reaction system. Add 1 mL of deionized water and 1 mL of chloroform. Vortex and extract for 5 minutes, then centrifuge at 8000 rpm for 5 minutes. The aqueous phase is collected and analyzed.

[0139] Glucose was determined by high performance liquid chromatography tandem with triple quadrupole mass spectrometry.

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

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

[0142] Table 15 Mobile phase gradient

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

[0144] Mass spectrometry conditions: Multiple reaction monitoring (MRM) mode was used, ion pairs m / z = 512.4 → 175.0 (native glucose) m / z = 518.4 → 175.0 ([ 13 The mass spectrometry parameters for [C6] glucose were set as follows.

[0145] Table 16 Mass spectrometry parameter settings

[0146]

[0147] The glucose concentration in the sample was calculated according to formula 1:

[0148]

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

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

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

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

[0153] W low is the mass ratio of glucose in the low standard to the internal standard;

[0154] W Hi is the mass ratio of glucose in the high standard to the internal standard;

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

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

[0157] D S is the density of urine;

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

[0159] A typical chromatogram is Figure 1 shown.

[0160] Example 5: Statistical calculation of the certified value of urine glucose standard substance and uncertainty assessment of the determined value results.

[0161] This proficiency test adopted a joint determination method. Since a reference measurement procedure for glucose in urine has not yet been published in the JCTLM, the development of this human urine glucose reference material represents a new method development. Therefore, research institutions with strong analytical method development capabilities, such as universities, research institutes, and analytical testing centers, were primarily selected. Their analytical proficiency was confirmed through blind sample testing. Randomly distributed, deidentified GBW(E)091148 human serum glucose reference material was used as a proficiency testing sample. The proficiency testing data for each institution are shown in Table 17. The measured values ​​of the certified reference material were all within the uncertainty range of the certified values, confirming that the proficiency testing passed and allowing for joint determination. The raw data for the joint determination are shown in Tables 18 and 19. All data were treated as a new set of measurements, and the grand mean and standard deviation of the raw data were calculated. SPSS software was used to test the normality of the combined measurement data for each level of reference material. All results met the normal distribution assumption. The analysis results are shown in Table 20. The raw data at each level were tested for outliers using the Dixon and Grubbs outlier tests, respectively. No outliers were found. The analysis results are shown in Table 21. The average value was finally calculated as the determination result of the glucose standard substance in urine.

[0162] Table 17 Proficiency testing of glucose combined value units

[0163]

[0164] Table 18 Raw data of glucose combined determination (mmol / L)

[0165]

[0166] Table 19 Original data of fixed values ​​of this unit (mmol / L)

[0167] Sample name 1 2 3 average value 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 average 2.534

[0168] All data were regarded as a new set of measurement data, and the normality test of the measurement data of the standard substances was performed using IBM SPSS software. All measurement results were in line with the normal distribution assumption. The analysis results are shown in Table 20.

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

[0170]

[0171] The original data in Tables 18 and 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 Outlier test of ID LC-MS / MS method fixed value data

[0173]

[0174] Cochran's rule was used to test the homogeneity of variance of the measurement data. Analysis showed that for the human urine glucose standard substance, the C value between the combined determination methods was less than the critical C value, indicating that the combined determination results had homogeneity of variance and the same precision.

[0175] Table 22 Test for homogeneity of variance of joint fixed values ​​(Cochran method)

[0176]

[0177] The final calculated average value is taken as the determination result of the glucose standard substance in human urine, as shown in Table 23.

[0178] Table 23 Determination results of glucose standard substances in human urine (mg / g)

[0179] sample Fixed value results Human urine glucose standard material 0.45

[0180] The density of this standard substance at 20°C is (1.003±0.009) g / mL, i.e. 1 mg / g = 5.97 mmol / L. Therefore, the volumetric concentration of glucose standard substance in human urine is as follows:

[0181] Table 24 Results of glucose standard substances in urine (mmol / L)

[0182] sample Fixed value results Human urine glucose standard material 2.53

[0183] The uncertainty of the determination results of glucose standards in urine comes from the uncertainty introduced in the determination process, the uncertainty introduced by the homogeneity of the standard material and the uncertainty introduced by long-term stability.

[0184]

[0185] (1) Uncertainty assessment of fixed value results

[0186] Glucose standard substance in urine is quantified using isotope dilution liquid phase mass spectrometry. The calculation model for the quantification is:

[0187]

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

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

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

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

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

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

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

[0195] M Ser is the mass of 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 balance weighing, glucose purity standard material, density measurement and analytical method repeatability, among which:

[0199] ①Weighing on a balance

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

[0201]

[0202] During the experiment, the solution part was weighed a total of 3 times, namely the weighing of the high-standard medium standard solution, the weighing of the low-standard medium standard solution and the weighing of the urine sample, and each time was repeated twice (empty pan and gross weight).

[0203] When weighing a standard solution in a high-standard medium, the relative standard uncertainty introduced by the balance weighing is:

[0204]

[0205] When weighing a low-standard medium standard solution, the relative standard uncertainty introduced by the balance weighing is:

[0206]

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

[0208]

[0209] ② Preparation of standard solution

[0210] During the experiment, GBW10062 glucose purity standard material was used to prepare 100g standard solution by weight method, and [ 13 C6] Prepare 50g of glucose standard solution.

[0211] The preparation of the unlabeled glucose standard solution involves two weighing processes. The calibration certificate gives the uncertainty of the balance measurement, including the two weighing processes (glucose and deionized water). Each weighing should be repeated twice (empty pan and gross weight). The uncertainty introduced by the preparation process of the glucose standard solution is

[0212]

[0213] ③Uncertainty introduced by purity standard substances

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

[0215]

[0216] ④Uncertainty introduced by density measurement

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

[0218]

[0219] Similarly, the density determination process involves weighing and volume determination, in which the weighing part: the standard weight should be repeated 20 times (5 parallel measurements, each time including the empty pan and gross weight), then Volume determination: According to JJG 646-2006 “Pipette Verification Procedure”, the tolerance of the volume of a 200μL pipette is ±3μL. Assuming that the ambient temperature differs from the measurement temperature by ±3°C, the water volume expansion coefficient In summary, the volume uncertainty caused by the difference between the calibration temperature and the operating temperature of the volumetric flask is: Synthesized The relative standard uncertainty of density determination obtained by combining the two is 0.870%.

[0220] ⑤Uncertainty introduced by method repeatability

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

[0222]

[0223] The uncertainties introduced by the two isotope dilution mass spectrometry methods are the synthesis of the above uncertainties:

[0224]

[0225] (2) Uncertainty assessment of uniformity and stability

[0226] ①Uncertainty introduced by sample uniformity

[0227] According to JJF1343-2022, the uncertainty introduced by the homogeneity of the reference material is equal to the standard deviation of the homogeneity between bottles. When the mean square between groups is greater than the mean square within groups, the calculation is performed according to formula (9).

[0228]

[0229] When the between-group mean square is smaller than the within-group mean square, the calculation is performed according to formula (10).

[0230]

[0231] ②Uncertainty introduced by sample stability

[0232] The uncertainty introduced by the long-term stability of the reference material is calculated according to formula 11, where t is the result of 10 months of long-term stability monitoring plus an additional 6 months of expected stability time, i.e., t = 10 + 6 = 16:

[0233] u lts =s k,lts ·t formula 11

[0234] (3) Uncertainty assessment results

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

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

[0237]

[0238]

[0239] Table 26 Determination of values ​​and uncertainty assessment results of glucose reference materials in urine (mmol / L)

[0240] sample Fixed value results Glucose standard substance in human urine 2.53±0.06

[0241] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A human urine glucose detection standard substance, characterized in that: The typical chromatogram of the reference substance 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 substance 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 substance to be prepared, immediately subpackage the above urine to obtain the urine glucose reference substance; When the preliminarily determined glucose concentration of the above urine is lower than the glucose concentration of the urine 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 glucose concentration of the urine glucose reference substance 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 substance.

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 substance 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 steps are as follows: Test the homogeneity of uric acid in the subpackaged and stored human urine glucose reference substance. Under the condition of magnetic stirring, subpackage the above human urine glucose reference substance into 2 mL low-temperature resistant and low-adsorption brown sample bottles respectively, and seal or close them. According to the subpackaging order, 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 substance 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, alternating odd and even numbers until the nth test of the uric acid in the urine glucose reference substance 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 substance in the subpackaging unit; Assume that there are a units of reference material, and unit i is measured ni times (i = 1, ..., a). For a simple balanced design, the number of repeated measurements ni is the same for all units, that is, n1 = n2 = ... = na = n0. One-way analysis of variance is used for analysis; the statistical model is as follows: Formula 1 in: is the jth observation value of the i-th unit; μ is the total number of all possible results (observation results the (true) mean value assumed to arise from it; is the effect of unit i on the result, that is, the (true) deviation of unit i from μ; is the random error of the jth observation value of the i-th unit, also known as the residual term; According to formulas 2 and 3, the mean square M between units can be obtained between and residual mean square M within ; , degrees of freedom Formula 2 , degrees of freedom Formula 3 in: Formula 4 Formula 5 Make statistic F: Formula 6 According to the degree of freedom ( , ), and the given significance level α, the critical ( , ) 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 obvious 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 test steps are: divided into long-term stability test and short-term stability test; 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 the long-term stability test as an example, within the time period of the long-term stability test, select no less than 5 time points k. At each time point, extract no less than 2 subpackaging units of human urine glucose standard substances. Use a biochemical analyzer or isotope dilution mass spectrometry method to perform 3 repeated tests on the urine glucose of the human urine glucose standard substances in each subpackaging unit. Then calculate the arithmetic mean of these measurement results to obtain the arithmetic mean Y of the measurement results at each time point. i , fit the arithmetic mean of the above measurement results and the corresponding measurement time according to the following linear model: Formula 7 In Formula 7: b0—intercept, b—regression coefficient; X—test time, unit (month); Y—arithmetic mean of the results of the determination of uric acid content in urine glucose standard substance; The regression coefficient and intercept are calculated according to Formula 8 and Formula 9: Formula 8 Formula 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 packaging unit at the i-th time point; —The average of the detection time at all time points, in months; —The arithmetic mean of the urine glucose determination results of the human urine glucose standard substance in the above packaging units at all time points; k—the number of time points; Calculate the s value according to formula 10: Formula 10 The meanings of the symbols in Formula 10 are the same as those in Formula 7, Formula 8, and Formula 9. Calculate the value of s(b) according to formula 11: Formula 11 Look up the t distribution table and get The value of like , it indicates that the slope is not significant and no instability is observed, indicating that the human urine glucose standard substance is stable in the long term; the long-term stability of the urine glucose standard substance is investigated at -20°C, -40°C or -80°C, preferably -80°C; the short-term stability of the urine glucose standard substance is investigated and calculated at -20°C, 4°C, 25°C and 60°C.

7. The standard substance according to claim 4, characterized in that The steps of determining the standard value by high performance liquid chromatography-isotope dilution mass spectrometry are: adding isotope labeled internal standard and measuring on the machine; when urine glucose is determined by high performance liquid chromatography-isotope dilution mass spectrometry, the isotope label selected 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, 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 [ 13 C6] labeled glucose, adding [ 13 The volume ratio of C6] labeled glucose to sample 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. The chromatographic column used is a C4, C8, or C18 column. The mobile phase A used is water containing 0.1% to 1% formic acid, or water containing 0.1% to 1% trifluoroacetic acid, or 5 to 100 mmol / L ammonium formate, or 5 to 100 mmol / L ammonium acetate solution, which contains 0% to 50% methanol or acetonitrile. The mobile phase B used is acetonitrile or methanol containing 0.1% to 1% formic acid, or acetonitrile or acetonitrile or methanol containing 0.1% to 1% trifluoroacetic acid. The mobile phase used is a mobile phase A:B ratio of 100:0 to 50:50 within 5 to 60 minutes. The flow rate used is 100 to 2000 mL / min, and the injection volume used is 1 to 100 mL. Mass spectrometry detection was performed in MRM mode or SIM mode. When the MRM mode was used, glucose in the sample (m / z = 512.4 of the glucose parent ion after derivatization, m / z = 175.0 of the glucose daughter ion) and glucose isotope markers in the standard were detected respectively. 13 C6] (glucose isotope label after derivatization [ 13 C6] parent ion m / z = 518.4, glucose isotope label [ 13 C6] product ion m / z = 175.0) signal; when the SIM mode was used for detection, glucose in the sample (glucose ion m / z = 512.44) and glucose isotope markers in the standard [ 13 C6] (glucose isotope label [ 13 C6] ion m / z = 518.4) signal; the quantitative standards used were glucose purity standard substances and [ 13 C6] labeled glucose prepared standard solution, glucose concentration in the standard solution, [ 13 C6] marks the glucose concentration and glucose and [ 13 C6] glucose concentrations in the standard substance to be tested, 13 C6] glucose concentration and ratio are close, and the ratio range is 0.90~1.10; the obtained glucose extraction chromatogram and [ 13 The peak areas of the C6] labeled glucose extraction chromatograms were integrated separately, and quantification was performed using the single-point method, bracket method, or standard curve method based on the ratio of the sample peak area to the standard peak area.

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 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 from 0.00 to 10.00 min was 95.0:5.0, 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 adopts MRM mode detection, and the quantitative method is the bracket method.

10. The standard substance according to any one of claims 4 to 9, characterized in that: The fixed value result is: 0.45 mg / g or 2.53 mmol / L.

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