Human urine creatinine detection standard substance and preparation and fixed value thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of metrological chemical analysis and detection technology, and in particular to a standard substance for detecting human urine creatinine, its preparation, and its determination. Background Technology

[0002] Chronic kidney disease (CKD) has become a significant challenge in global public health, with its high incidence, high disability rate, and high medical burden having a profound impact on patient health and socioeconomic status. Urinary creatinine measurement, as an important laboratory test in clinical medicine, primarily assesses kidney function and assists in the diagnosis of various kidney diseases by quantitatively analyzing the concentration of creatinine in urine. Creatinine is the end product of muscle metabolism; its production is closely related to muscle mass, and its daily excretion is relatively constant. Therefore, urinary creatinine concentration reflects the glomerular filtration capacity and is one of the core parameters for estimating the glomerular filtration rate (eGFR). In the staging management of CKD, the ratio of urinary creatinine to serum creatinine, combined with factors such as age, sex, and weight, is used to calculate eGFR using formulas (such as the CKD-EPI or MDRD formula). This accurately determines the degree of kidney function impairment and guides the timing of clinical intervention. For example, in CKD stage 3 (eGFR 30-59 mL / min / 1.73 m... 2 Patients require close monitoring and medication dosage adjustments to slow progression. Furthermore, urinary creatinine measurement is of significant value in the early diagnosis of acute kidney injury (AKI). Dynamic monitoring of urinary creatinine changes can rapidly identify prerenal, renal, or postrenal causes. A sharp drop in urinary creatinine accompanied by an increase in serum creatinine indicates severe glomerular filtration function impairment, requiring emergency treatment to reverse the damage. In the management of diabetic nephropathy, urinary creatinine is often used as a correction factor in calculating the urinary albumin / creatinine ratio (UACR) to eliminate interference from urine concentration or dilution on proteinuria quantification. For example, an UACR ≥ 30 mg / g indicates early diabetic kidney injury, requiring initiation of RAAS inhibitor therapy (such as valsartan) to reduce protein leakage and protect renal function. For hypertensive nephropathy, urinary creatinine combined with microalbumin testing can assess tubulointerstitial damage and guide the selection of antihypertensive drugs.

[0003] Furthermore, UACR is an important measurement for identifying kidney damage in patients with kidney-related diseases. Urinalysis is often the first step in CKD diagnosis and a crucial part of follow-up management. As a non-invasive testing method, urine testing not only provides diagnostic and staging data for CKD but also offers insights into disease prognosis and progression. However, the accuracy and comparability of testing systems are highly dependent on the traceability of metrological standards, which is essential for the reliability of clinical test results.

[0004] Existing research on reference materials largely focuses on serum matrices, with fewer developments targeting urinary creatinine. Urine matrices are complex, and their composition differs significantly from serum; directly using serum reference materials for calibration or quality control may lead to systematic bias. Therefore, developing a human urinary creatinine reference material with good interoperability and traceability to the International System of Units (SI) is crucial for achieving comparability of test results and promoting standardization in clinical testing. Summary of the Invention

[0005] This invention addresses the need for traceability and transmission of urinary creatinine measurement values ​​and the calibration requirements of fully automated biochemical analyzers by providing a standard material for human urinary creatinine detection, along with its preparation and determination method. The aim is to provide technical support for clinical laboratory quality evaluation, testing system performance verification, and laboratory proficiency testing, further promoting the standardization and precision of laboratory medicine.

[0006] The present invention provides a standard reference for the detection of human urinary creatinine, comprising: processing and preparation of the urinary creatinine standard reference; homogeneity testing of the urinary creatinine standard reference; stability testing of the urinary creatinine standard reference; determination of the standard value of the urinary creatinine standard reference by high performance liquid chromatography-isotope dilution mass spectrometry; statistical calculation of the determination value of the urinary creatinine standard reference and uncertainty assessment of the determination result.

[0007] The first step involves the processing and preparation of the urinary creatinine standard. The raw material for the urinary creatinine standard comes from urine collected from healthy individuals. The sample is then centrifuged at 12000 rpm for 15 minutes at 4°C using a high-speed refrigerated centrifuge. The supernatant is collected and filtered through a 0.22 μm filter membrane. The filtrate is then added as a preservative at a ratio of 0.1% (v / v) using a pipette, ensuring thorough mixing to guarantee urine stability. The concentration of creatinine in the urine is initially determined using an automated biochemical analyzer or mass spectrometer. If the initial result meets the design requirements of the standard, the sample can proceed to the dispensing process. If the initial result is lower than the design requirements, a creatinine purity standard solution is added to increase the creatinine content in the urine. During addition, the volume of the added creatinine purity standard solution should not exceed 10% of the urine volume. After addition, the sample is mixed at room temperature for 6–12 hours.

[0008] The containers used for dispensing the urinary creatinine standard are colorless or brown screw-top sample bottles, preferably low-temperature resistant and low-adsorption brown sample bottles. The urine sample is dispensed into sample bottles or cryovials under shaking conditions and then sealed. According to the dispensing order, the standard for each urinary creatinine concentration level is sequentially numbered and stored frozen at -20°C, -40°C, or -80°C.

[0009] For the homogeneity test of the urinary creatinine reference material, samples need to be randomly selected according to the number of packaging units for the homogeneity test. The number of packaging units is denoted as N. When 100 < N ≤ 200, the number of selected units is not less than 11; when 200 < N ≤ 500, the number of selected units is not less than 15; when 500 < N ≤ 1000, the number of selected units is not less than 25; when the total number of units N > 1000, the number of selected samples is not less than 30. At each urinary creatinine concentration level, units meeting the above quantity requirements are randomly selected for the homogeneity test. The experimental methods for the homogeneity test are the biochemical analyzer method or the isotope dilution mass spectrometry method, with the isotope dilution mass spectrometry method being preferred. Each reference material unit selected is measured 3 times respectively using the above method. During analysis, in order to eliminate the error caused by instrument drift during the measurement process, the samples are analyzed in the order of 1 → 15, 15 → 1, alternating odd and even numbers. The analyzed samples are subjected to the homogeneity test and the results are statistically analyzed according to the following formula:

[0010] Suppose there are a reference material units, and the unit i is measured ni times (i = 1,..., a). For a simple balanced design, the repeated measurement times ni of all units are the same, that is, n1 = n2 =... = na = n0. The one-way analysis of variance method 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 ith unit; μ is the (true) mean of the population of all possible results (assuming it is generated from the observed results x ij ); δ 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 ith unit, also known as the residual term. According to Formulas 2-2 and 2-3, the mean square between units M between and the residual mean square M within can be obtained;

[0013]

[0014] where;

[0015]

[0016] Take the statistic F:

[0017]

[0018] According to the degrees of freedom (v1, v2) and the given significance level α, the critical F value is obtained from the tableα (v1, v2) values are 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. Otherwise, the reference material needs to be re-prepared and homogenized.

[0019] The stability test of the urinary creatinine reference material is divided into long-term stability test and short-term stability test. The duration of the long-term stability test is not less than 6 months. During the period of the long-term stability test, not less than 5 time points are selected according to the principle of denser in the front and sparser in the back. For each time point, not less than 2 units of the reference material are sampled, and each unit of the reference material is repeatedly analyzed 3 times by a biochemical analyzer or isotope dilution mass spectrometry method. Calculate the arithmetic mean of these measurement results to obtain the measurement result X at each time point i The measurement results and measurement times 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 - the measurement result of the creatinine content of the reference material.

[0023] Calculate the regression coefficient and intercept according to Formula 8 and Formula 9:

[0024]

[0025] In the formula:

[0026] X i — the i-th time point;

[0027] Y i — the measurement result of the urinary creatinine reference material at the i-th time point;

[0028] — the average value of all time points;

[0029] — the measurement results of the urinary creatinine reference material at all time points;

[0030] k — the number of time points.

[0031] Calculate the s value according to Formula 10:

[0032]

[0033] The meanings of each symbol in the formula are the same as those in Formula 8 and Formula 9.

[0034] Calculate the value of s(b) according to Formula 11:

[0035]

[0036] Look up the t-distribution table to get t 0.95.k-2 The value of .

[0037] If |b| <t 0.95,k-2 If ·s(b), it indicates that the slope is not significant and no instability was observed, suggesting that the urinary creatinine standard is stable.

[0038] The long-term stability of the urinary creatinine standard was investigated at -20℃, -40℃, or -80℃, with -80℃ being preferred. The short-term stability of the urinary creatinine standard was investigated and calculated at -20℃, 4℃, 25℃, and 60℃.

[0039] The method for determining the standard value of urinary creatinine using high-performance liquid chromatography-isotope dilution mass spectrometry (HPLC-IOSD-MS) involves several steps, including adding an isotope-labeled internal standard and performing the determination. When determining urinary creatinine using HPLC-IOSD-MS, the selected isotope label is [D3]-labeled creatinine, with at least three isotope-labeled atoms and an isotope abundance of at least 99%. For sample pretreatment, [D3]-labeled creatinine is first prepared into a solution with a concentration of (0–100) mg / g using a solvent such as water, methanol, or acetonitrile, preferably with water, and preferably at a concentration of 10 mg / g. [D3]-labeled creatinine with the same concentration as initially determined is added to the urinary creatinine standard sample. The volume ratio of added [D3]-labeled creatinine to the sample volume is between (1:100–10:1), preferably 1:1. After addition, mix thoroughly and filter through a 0.22 mm filter membrane. Perform high-performance liquid chromatography-isotope dilution mass spectrometry (HPLC-IOSD-MS) analysis using a C4, C8, or C18 column, preferably an Agilent Eclipse Plus C18 (4.6 × 150 mm, 5 μm) column. Mobile phase A is a solution of water containing (0.1%–1%) formic acid, water containing (0.1%–1%) trifluoroacetic acid, or a solution of (5–100) mmol / L ammonium formate or (5–100) mmol / L ammonium acetate, containing (0%–50%) methanol or acetonitrile. Mobile phase B is a solution of acetonitrile or methanol containing (0.1%–1%) formic acid, or a solution of acetonitrile or methanol containing (0.1%–1%) trifluoroacetic acid. Preferably, mobile phase A is water containing 10 mmol / L ammonium acetate, and preferably mobile phase B is acetonitrile containing 0.1% formic acid. The mobile phase used is isocratic elution, with a displacement of mobile phase A:B (100:0 to 50:50) over a time period of (5–60) min. Preferred mobile phase ratios are shown in the table below:

[0040] TotalTime(min) A(％) B(％) 0.00 96.0 4.0 10.00 96.0 4.0

[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 signals of creatinine in the sample (m / z = 114.1 for the precursor ion and m / z = 44 for the daughter ion) and the creatinine isotope label [D3] in the standard (m / z = 117.1 for the precursor ion and m / z = 47 for the daughter ion) are detected separately. When using SIM mode, the signals of creatinine in the sample (m / z = 114.1 for the creatinine ion) and the creatinine isotope label [D3] in the standard (m / z = 117.1 for the creatinine isotope label [D3] ion) are detected separately. MRM mode is preferred for detection. The quantitative standard used is a standard solution prepared with creatinine purity standard and [D3]-labeled creatinine. The concentrations of creatinine, [D3]-labeled creatinine, and the ratio of creatinine to [D3]-creatinine in the standard solution are close to those in the analyte, with a ratio range of (0.90–1.10). Peak areas are obtained by integrating the creatinine extraction chromatogram and the [D3]-labeled creatinine extraction chromatogram. Quantification is performed using the single-point method, bracket method, or standard curve method based on the peak area ratio of the sample and the standard. The preferred ratio range is (0.95–1.05), and the preferred quantitative method is the bracket method.

[0042] In the above method, the determination of the standard substance's identification value is carried out by high-performance liquid chromatography-isotope dilution mass spectrometry in conjunction with multiple laboratories. According to the requirement for the number of independent data sets in JJF 1343-2022, each set of data should be based on measurements of two units, with each unit measured at least twice, providing no less than 4 to 6 independent repeated measurements. All data are considered as a new set of measurement data. The KS single-sample normality test is performed, and outlier tests are conducted using the Grubbs method and / or Dixon method. Finally, the Cochrane rule is used to test the homogeneity of variance of the measured data. After the above statistical tests, the arithmetic mean of all results is taken as the standard value of the result determined by the high-performance liquid chromatography-isotope dilution mass spectrometry method. The uncertainty u of the determination result of the creatinine standard in urine originates from the uncertainty u introduced during the determination process. char Uncertainty u introduced by the homogeneity of standard materials bb Uncertainty u introduced by long-term stability lts Synthesize according to Equation 12.

[0043]

[0044] In the formula, k is the coverage factor, which is usually taken as k=2.

[0045] Where u char This includes the uncertainty u from high performance liquid chromatography-isotope dilution mass spectrometry. char,HPLC-IDMS u char,HPLC-IDMS This includes the uncertainty component u introduced by the repeatability of high performance liquid chromatography-isotope dilution mass spectrometry measurement results. char,HPLC-IDMS,A (Based on Equation 13), and the uncertainty component u introduced by multiple balance weighings. char,HPLC-IDMS,wi (Based on Equation 16) and the uncertainty component u introduced by the creatinine purity standard. char,HPLC-IDMS,P (Based on Equation 15). The various uncertainty components are combined according to Equation 16.

[0046]

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

[0048]

[0049] In Equation 14, U(E) represents the uncertainty in the balance calibration certificate, and k is the coverage factor.

[0050]

[0051] In Equation 15, U is the uncertainty in the creatinine purity standard material certificate, and k is the coverage factor;

[0052]

[0053] In the formula, q represents the number of weighings performed by the balance, and c i Let c be the sensitivity coefficient for the i-th weighing. p The sensitivity coefficient for the purity of the standard substance.

[0054] When the mean square between groups is greater than the mean square within groups, the uncertainty introduced by homogeneity is calculated according to Equation 17.

[0055]

[0056] When the mean square between groups is less than the mean square within groups, it is calculated according to Equation 18.

[0057]

[0058] The long-term stability of the standard material is calculated according to Equation 19:

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

[0060] In the formula,

[0061] s k,lts For the long-term stability test, s(b),t lts This refers to the duration of the long-term stability test.

[0062] The above results are synthesized according to Equation 12 to obtain the expanded uncertainty U of the standard reference's determined value. Therefore, the determined value of the urinary creatinine standard reference can be expressed as:

[0063] x±U

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

[0065] This invention addresses the problem of inaccurate and incomparable urine creatinine test results due to the lack of urine creatinine standard materials. It provides a method for preparing and determining the value of urine creatinine standard materials, which can be used for the development of urine creatinine test reagents and the performance verification of methods, as well as for the traceability of urine creatinine test results. This ensures the accuracy and comparability of clinical urine creatinine test results and is helpful for clinical testing of diseases such as chronic kidney disease. Attached Figure Description

[0066] Figure 1 This is the chromatographic ion chromatogram of the human urine creatinine detection standard material of the present invention;

[0067] Figure 2 It is a repackaged standard substance for detecting creatinine in human urine. Detailed Implementation

[0068] The present invention will be further described below with reference to embodiments, but this is not intended to limit the invention in any way. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection of the present invention.

[0069] Example 1: Processing and preparation of urinary creatinine standard material;

[0070] The raw material of the urinary creatinine reference material is derived from the urine collected from healthy ordinary people. The collected urine is centrifuged at 12,000 rpm and 4°C for 15 minutes using a high-speed refrigerated centrifuge. The supernatant is taken, filtered by suction filtration through a 0.22-μm filter membrane, and the filtrate is collected; 0.1% Proclin 300 is added to the collected urine as a preservative to ensure the stability of the reference material, and it is thoroughly mixed after addition. The creatinine concentration in the urine is preliminarily determined using an automatic biochemical analyzer. The creatinine solution required to be added for preparing the target creatinine concentration is prepared using the NIST914b creatinine reference material sample in the United States. According to the designed target concentration range, the addition amount is calculated, and the creatinine solution is directly added to the urine using a pipette and mixed at room temperature for 8 hours to ensure the homogeneity of the reference material. The above human urine creatinine reference material is sub-packed into low-temperature-resistant and low-adsorption brown sample bottles under oscillating conditions, totaling 500 units, with 1500 μL for each unit. The designed concentration, sample number, and sub-packing date are marked on the cryotubes and cryoboxes respectively, and they are stored in a -80°C refrigerator. The sub-packed urinary creatinine reference material is as Figure 2 shown.

[0071] Example 2: Homogeneity test of urinary creatinine reference material;

[0072] The homogeneity of the reference material is tested using a CS-1200 automatic biochemical analyzer and a creatinine assay kit based on the sarcosine oxidase method. According to the requirements of JJF 1343-2022, when the total number of units ≤ 500, the number of extracted units ≥ 15. Therefore, 15 units of the candidate substance at each level are randomly taken out, and the candidate substance of each unit is repeatedly measured 3 times using an automatic biochemical analyzer. In order to eliminate the error caused by instrument drift during the measurement process, the samples are analyzed in the order of 1→15, 15→1, odd and even interspersed three times.

[0073] The homogeneity test data of the urinary creatinine reference material are shown in Table 1, and the precision (CV) of the test method is less than 2.0%.

[0074] Table 1 Homogeneity test data of urinary creatinine reference material (mmol / L)

[0075] Level 1 1 time 2 times 3 times Observations Summation average variance 1 15.899 15.939 16.098 3 47.935 15.978 0.011 2 15.540 15.979 15.939 3 47.457 15.819 0.059 3 15.899 15.500 16.218 3 47.617 15.872 0.129 4 16.178 16.456 15.580 3 48.215 16.072 0.200 5 15.540 15.779 16.337 3 47.657 15.886 0.167 6 16.377 15.819 15.779 3 47.976 15.992 0.112 7 16.098 15.540 15.979 3 47.617 15.872 0.086 8 16.098 16.178 15.899 3 48.174 16.058 0.021 9 16.257 16.138 16.058 3 48.453 16.151 0.010 10 15.540 15.859 15.779 3 47.178 15.726 0.027 11 16.178 15.859 15.899 3 47.935 15.978 0.030 12 16.058 16.297 16.297 3 48.652 16.217 0.019 13 15.899 16.537 16.178 3 48.613 16.204 0.102 14 15.859 16.019 16.297 3 48.174 16.058 0.049 15 15.859 16.257 16.297 3 48.413 16.138 0.059

[0076] One-way analysis of variance of the urinary creatinine reference material is performed using Excel software, and the results are shown in Table 2. In the table, F of the urinary creatinine reference material < Fα, so it is considered that there is no significant difference between within-group and between-group, and the samples are homogeneous.

[0077] Table 2 Results of one-way analysis of variance of homogeneity data of urinary creatinine reference material

[0078] Source of difference SS df MS F P-value Fcrit Between groups 0.891 14 0.064 0.882 0.584 2.037 within the group 2.166 30 0.072

[0079] Example 3: Stability test of urinary creatinine standard material;

[0080] The stability of the standard substances was investigated over a period of 10 months using a CS-1200 fully automated biochemical analyzer and a creatinine oxidase assay kit. Three standard substance samples were taken each time for stability testing, and the average results are shown in Table 3.

[0081] Table 3. Average results of long-term stability study of urinary creatinine standard reference (mmol / L)

[0082] Time / Month 1 2 3 average 0 15.979 15.819 15.872 15.890 1 16.317 15.779 15.999 16.032 2 16.058 16.297 16.337 16.231 3 16.357 16.018 15.799 16.058 7 16.031 16.191 16.165 16.129 9 16.058 15.779 15.859 15.899 10 15.859 15.899 15.819 15.859

[0083] The data above were analyzed using Excel software to calculate the regression parameters and perform a t-test on the slope β. The results are shown in Table 4.

[0084] Table 4. Statistical analysis of the stability of urinary creatinine standard references

[0085] Stable time / month β s(β) t s(β)·t in conclusion 10 -0.0122 0.0178 2.571 0.0458 Stablize

[0086] Based on the long-term stability results of the samples, the standard substance of creatinine in human urine can be stably stored for 10 months at -80℃.

[0087] The short-term stability of the standard substance was tested using a CS-1200 fully automated biochemical analyzer and a creatine oxidase method creatinine assay kit. The short-term stability test temperatures were -20℃, 4℃, 25℃ and 60℃. The test time was 7 days at each temperature. Tables 5 to 8 show the average results of stability testing with 3 samples taken at different intervals.

[0088] Table 5. Results of short-term stability study of urinary creatinine standard at -20℃ (mmol / L)

[0089] Time / day 1 2 3 average 0 15.540 15.779 16.019 15.779 1 14.704 15.739 14.982 15.142 2 14.704 15.381 15.819 15.301 3 15.381 15.381 16.079 15.613 4 15.500 15.859 15.540 15.633 5 15.660 15.739 15.819 15.740 6 15.660 16.416 14.982 15.686 7 15.321 15.819 15.461 15.534

[0090] Table 6. Results of short-term stability study of urinary creatinine standard material at 4℃ (mmol / L)

[0091]

[0092]

[0093] Table 7. Results of short-term stability study of urinary creatinine standard at 25℃ (mmol / L)

[0094] Time / day 1 2 3 average 0 16.178 16.178 15.939 16.098 1 15.939 15.919 15.461 15.773 2 16.377 15.222 15.062 15.554 3 15.899 16.138 15.262 15.766 4 15.421 16.377 15.899 15.899 5 15.381 15.660 15.262 15.434 6 15.102 15.819 15.341 15.421 7 15.341 15.381 15.102 15.275

[0095] Table 8. Results of short-term stability study of urinary creatinine standard at 60℃ (mmol / L)

[0096] Time / day 1 2 3 average 0 14.863 16.058 16.218 15.713 1 14.066 14.424 14.026 14.172 2 12.990 12.711 12.711 12.804 3 12.552 12.193 12.352 12.366 4 11.516 11.595 11.317 11.476 5 10.759 10.480 10.958 10.732 6 10.560 9.843 10.360 10.254 7 9.643 9.563 9.523 9.577

[0097] Table 9. Short-term stability test results of urinary creatinine standard reference (mmol / L)

[0098]

[0099] According to the short-term stability test results of the samples in Table 9, the human urine creatinine standard can be stably stored for 7 days at -20℃ and 4℃ without obvious turbidity. It can be stably maintained for 4 days at 25℃ (judged by the standard deviation of β1 using the t-test, |β1|=0.0405, which is less than t(0.95,n-2)·s(β1)=0.2199). The sample was found to be unstable in the statistical test at 60℃. Therefore, it is determined that the standard should be transported within 7 days at 4℃.

[0100] The open-bottle stability of the standard substances was tested using a CS-1200 fully automated biochemical analyzer and a creatinine oxidase method creatinine assay kit. Three creatinine standard substance samples were taken each time, and the average results were measured at different time intervals of 1h, 2h, 3h, 4h, 5h, and 6h at 4℃ and 25℃ to examine their open-bottle stability. The results are shown in Tables 10 and 11.

[0101] Table 10. Results of the stability test (mmol / L) of creatinine standard in human urine after opening at 4℃.

[0102] Time / h 1 2 3 average 0 15.543 15.454 15.477 15.491 1 15.873 15.732 16.082 15.896 2 16.080 15.315 15.857 15.751 3 15.830 16.136 16.525 16.163 4 16.024 15.996 16.372 16.131 5 15.913 15.927 16.205 16.015 6 15.732 15.996 16.427 16.052

[0103] Table 11 Results of the stability test (mmol / L) of creatinine standard substance in human urine after opening at 25℃

[0104] Time / h 1 2 3 average 0 15.737 15.776 15.776 15.763 1 16.291 16.053 16.073 16.139 2 16.172 16.172 16.113 16.152 3 15.678 15.401 16.014 15.697 4 16.211 16.073 16.765 16.350 5 16.666 15.677 16.409 16.251 6 16.350 15.697 16.132 16.060

[0105] Table 12 Results of Stability Tests for Creatinine Standard Material in Urine After Opening (mmol / L)

[0106]

[0107] According to the results of the sample opening stability test in Table 12, the human urine creatinine standard substance sample can be stable for 6 hours at 4℃ and 25℃ after opening. Therefore, it is recommended that the standard substance should be used up within 6 hours after opening.

[0108] The repeated freeze-thaw stability of the standard substances was tested using a CS-1200 fully automated biochemical analyzer and a creatinine oxidase method creatinine assay kit. Three creatinine standard substance samples were taken each time and repeatedly frozen and thawed at 25℃ and -80℃. The average result after 1, 2, 3, 4, 5, 6, and 7 freeze-thaw cycles was measured to examine its repeated freeze-thaw stability. The results are shown in Table 13.

[0109] Table 13 Results of repeated freeze-thaw stability study of creatinine standard in human urine (mmol / L)

[0110] Number of freeze-thaw cycles 1 2 3 average 1 16.038 16.058 16.357 16.151 2 15.640 15.440 16.397 15.826 3 16.218 16.098 16.357 16.224 4 15.879 15.919 16.118 15.972 5 15.759 15.899 16.377 16.012 6 16.098 15.819 16.258 16.058 7 16.616 16.357 16.676 16.550

[0111] Table 14 Results of Repeated Freeze-Thaw Stability Tests for Creatinine Standard Substance in Human Urine (mmol / L)

[0112]

[0113] According to the results of the repeated freeze-thaw stability test of the samples in Table 14, the human urine creatinine standard substance sample can remain stable within 7 freeze-thaw cycles. Therefore, the standard substance can be used at least 7 times after being opened.

[0114] 4. Determination of standard values ​​of urinary creatinine standard material by high performance liquid chromatography-isotope dilution mass spectrometry;

[0115] ① Preparation of standard solutions

[0116] Using a creatinine purity standard, the concentration was prepared to approximately 1 mg / g by gravimetric method, and then diluted with water to approximately 10 μg / g.

[0117] Using labeled creatinine, the solution was prepared to approximately 20 μg / g by gravimetric method.

[0118] ② Mixing of standard solution and internal standard

[0119] Based on the concentration of creatinine in the standard substance sample, pipette [D3] creatinine standard solution, weigh it, and record the count. Add creatinine standard solution to the standard solution according to its mass concentration, gently shake to mix thoroughly, and prepare a calibration ratio mixed solution. High standard: 310 μg standard solution + 60 μg internal standard solution + 630 μg ultrapure water; Low standard: 210 μg standard solution + 60 μg internal standard solution + 730 μg ultrapure water. Weigh the added standard solution and internal standard solution simultaneously.

[0120] ③ Dilution of the sample to be tested

[0121] Take 40 μg of urinary creatinine standard substance, dilute the urinary creatinine standard substance 150 times to prepare a urine diluent, and weigh the urinary creatinine standard substance and ultrapure water at the same time.

[0122] ④ Mixing urine samples with internal standards

[0123] The ratio of creatinine to [D3]creatinine in urine is approximately 1:1, i.e., 260 μg of urine diluent + 60 μg of internal standard solution + 680 μg of ultrapure water. The mass of the added urine diluent and internal standard solution is weighed simultaneously.

[0124] Creatinine was determined by high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

[0125] The liquid chromatography and mass spectrometry conditions are as follows:

[0126] Liquid chromatography conditions: injection volume 5 μL; column Agilent Eclipse Plus C18 (4.6 × 150 mm, 5 μm); flow rate 300 μL / min; time 10 min. Mobile phase A was 10 mmol / L ammonium acetate in water, and mobile phase B was acetonitrile. Isocratic elution is shown in Table 12.

[0127] Table 12 Gradient of the mobile phase

[0128] TotalTime(min) A(％) B(％) 0.00 96.0 4.0 10.00 96.0 4.0

[0129] Mass spectrometry conditions: Multiple reaction monitoring (MRM) mode was used; ion-to-mass ratio: 114.1 → 44 (natural creatinine)

[0130] The mass spectrometry parameters for 117.1→47 ([D3]creatinine) are shown in Table 13.

[0131] Table 13 Mass Spectrometry Parameter Settings

[0132]

[0133] The creatinine concentration in the sample was calculated according to Equation 1:

[0134]

[0135] In the formula: C is the concentration of creatinine in urine (unit: mmol / L);

[0136] I sam This is the peak area ratio (measured value) of creatinine to internal standard in the sample;

[0137] I low The peak area ratio (measured value) of creatinine to internal standard in the low standard;

[0138] I Hi The peak area ratio (measured value) of creatinine in the high standard to that of the internal standard;

[0139] W low This represents the mass ratio of creatinine to internal standard in the low standard;

[0140] W Hi This represents the mass ratio of creatinine to internal standard in the high-standard formulation;

[0141] M Is The mass of the internal standard in the urine sample (unit: mg);

[0142] M Ser Mass of urine sample (unit: g);

[0143] D S The density of urine;

[0144] 113.12 is the relative molecular mass of creatinine.

[0145] Typical chromatograms are as follows Figure 1 As shown.

[0146] Example 5: Statistical calculation of the determination value of urinary creatinine standard material and evaluation of the uncertainty of the determination result.

[0147] This determination adopted a joint determination method. Since the JCTLM currently lacks a reference measurement procedure for creatinine in urine, the development of this human urinary creatinine standard material constitutes the development of a new method. Therefore, research institutions with strong analytical method development capabilities, such as universities, research institutes, and analytical testing centers, were primarily selected. Their analytical capabilities were confirmed through blind sample testing. This determination involved eight institutions collaborating on the human urinary creatinine standard material. First, proficiency testing was conducted on each institution. GBW09170 and GBW09171 frozen human serum creatinine component analysis standard materials (without labels) were randomly distributed as proficiency testing samples. The proficiency testing data for each institution are shown in Table 14. All certified standard materials were within the uncertainty range of their certificate values, indicating successful proficiency testing and allowing for joint determination. The raw data for joint determination are shown in Tables 15 and 16. All data were considered as a new set of measurements, and the overall mean and standard deviation of all raw data were calculated. The normality of the combined measurement data for each level of standard material was tested using SPSS software. All measurement results conformed to the normal distribution assumption, and the analytical results are shown in Table 17. Outlier tests were performed on the raw data at each level using both Dixon and Grubbs outlier tests, and no outliers were found. The results are shown in Table 18. The final average value was calculated as the determination result for the creatinine standard in urine.

[0148] Table 14 Proficiency Testing of Combined Units for Creatinine Values

[0149]

[0150] Table 15 Raw data for combined creatinine determination (mmol / L)

[0151]

[0152] Table 16. Original data for this unit's set values ​​(mmol / L)

[0153]

[0154]

[0155] All data were treated as a new set of measurement data. The normality of the measurement data of the standard substances was tested using IBM SPSS software. All measurement results met the normal distribution assumption. The analysis results are shown in Table 17.

[0156] Table 17 ID LC-MS / MS method setpoint data Kolmogorov-Smirnov test

[0157]

[0158] Outlier tests were performed on the original data in Tables 15 and 16 using the Dixon and Grubbs outlier tests respectively, and no outliers were found. The results are shown in Table 18.

[0159] Table 18. Outlier Detection of LC-MS / MS Method Setpoint Data

[0160]

[0161] The homogeneity of variance of the measurement data was tested using Cochrane's rule. The results are shown in Table 19. Analysis showed that for the human urine creatinine standard, the C-value among the joint determination methods was less than the C-critical value, indicating that the joint determination results had homogeneity of variance and the same accuracy.

[0162] Table 19. Joint test for homogeneity of variance of constant values ​​(Cochran's method)

[0163] S1 S2 S3 S4 S5 S6 S7 S8 S9 smax ∑s C C(0.05,9,6) 0.194 0.088 0.129 0.182 0.050 0.201 0.118 0.098 0.252 0.252 1.312 0.283 0.329

[0164] The final calculated average value was taken as the determination result of the standard substance of creatinine in human urine, as shown in Table 20.

[0165] Table 20 Results of Creatinine Standard Reference Values ​​in Urine (mg / g)

[0166] sample Fixed value result Urine creatinine standard material 1.80

[0167] The density of this standard reference material at 20℃ is (1.002±0.009) g / mL, which is 1 mg / g = 8.86 mmol / L. Therefore, the volumetric concentration of the creatinine standard reference material in human urine is as follows:

[0168] Table 21 Results of Standard Reference Values ​​for Creatinine in Human Urine (mmol / L)

[0169] sample Fixed value result Urine creatinine standard material 15.94

[0170] The uncertainty in the determination of creatinine standard values ​​in urine comes from the uncertainty introduced during the determination process, the uncertainty introduced by the homogeneity of the standard substance, and the uncertainty introduced by long-term stability.

[0171]

[0172] (1) Uncertainty assessment of the set value result

[0173] The creatinine standard in urine was determined using isotope dilution liquid chromatography-mass spectrometry. The calculation model for the determined value is as follows:

[0174]

[0175] C represents the concentration of creatinine in urine (unit: mmol / L);

[0176] I sam This is the peak area ratio (measured value) of creatinine to internal standard in the sample;

[0177] I low The peak area ratio (measured value) of creatinine to internal standard in the low standard;

[0178] I Hi The peak area ratio (measured value) of creatinine in the high standard to that of the internal standard;

[0179] W low This represents the mass ratio of creatinine to internal standard in the low standard;

[0180] W Hi This represents the mass ratio of creatinine to internal standard in the high-standard formulation;

[0181] M Is The mass of the internal standard in the urine sample (unit: mg);

[0182] M Ser Mass of urine sample (unit: g);

[0183] D S The density of urine;

[0184] 113.12 is the relative molecular mass of creatinine.

[0185] The uncertainty of isotope dilution mass spectrometry mainly stems from factors such as balance weighing, creatinine purity standards, density determination, and the repeatability of the analytical method.

[0186] ① Weighing with a balance

[0187] The uncertainty introduced by the balance weighing is calculated according to formula (4) based on the maximum permissible error in the balance calibration certificate:

[0188]

[0189] During the experiment, the solutions were weighed a total of three times: the standard solution in the high-concentration standard, the standard solution in the low-concentration standard, and the urine sample. Each weighing was repeated twice (empty plate and gross weight). In addition, the dilution of the high-concentration urine sample to the low-concentration urine sample also involved two weighing processes for urine and deionized water.

[0190] The relative standard uncertainty introduced by the balance weighing of the high-standard solution is:

[0191]

[0192] The relative standard uncertainty introduced by the weighing of the standard solution in the low standard is:

[0193]

[0194] The relative standard uncertainty introduced by weighing a urine sample using a balance is:

[0195]

[0196] The relative standard uncertainty introduced by the weighing of diluted urine samples using a balance is:

[0197]

[0198] ② Preparation of standard solutions

[0199] During the experiment, 50 mL standard solutions were prepared using NIST 914b creatinine purity standard material by gravimetric method, and 1 mL standard solutions were prepared using [D3] creatinine.

[0200] The preparation of the unlabeled creatinine standard solution involves five weighing processes, including the preparation of the creatinine stock solution and the dilution of the creatinine working solution. The calibration certificate specifies the uncertainty of the balance measurement, including two weighing processes (creatinine and deionized water). Each weighing should be calculated twice (empty pan and gross weight). Therefore, the uncertainty introduced by the preparation process of the creatinine standard solution is:

[0201]

[0202] ③ Uncertainty introduced by purity standard substances

[0203] The uncertainty introduced by the creatinine purity standard material is calculated according to formula (5) based on the uncertainty in the standard material certificate and the corresponding coverage factor:

[0204]

[0205] ④ Uncertainty introduced by density measurement

[0206] Density was determined using a calibrated balance and pipette, and the uncertainty introduced by density measurement was calculated according to the formula ρ=m / V. Therefore, the uncertainty introduced by density measurement was calculated according to formula (6):

[0207]

[0208] Similarly, density determination involves weighing and volume adjustment. For the weighing portion: the standard weight should be calculated 20 times (5 parallel measurements, each including both empty and gross weights). Volume adjustment section: According to JJG 646-2006 "Verification Procedure for Pipettes", the permissible volume tolerance for a 200μL pipette is ±3μL. Assuming the ambient temperature differs from the measurement temperature by ±3℃, what is the effect on 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 as follows: Synthesized The combined results yielded a relative standard uncertainty of 0.870% for the density determination.

[0209] ⑤ Uncertainty introduced by method repeatability

[0210] The uncertainty introduced by repeatability is calculated based on the standard deviation and the number of measurements n:

[0211]

[0212] The uncertainties introduced by isotope dilution mass spectrometry are all a combination of the above uncertainties:

[0213]

[0214] (2) Uncertainty assessment of homogeneity and stability

[0215] ① Uncertainty introduced by sample homogeneity

[0216] According to JJF1343-2022, the uncertainty introduced by the homogeneity of the standard substance 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, it is calculated according to formula (9).

[0217]

[0218] When the mean square between groups is less than the mean square within groups, it is calculated according to formula (10).

[0219]

[0220] ② Uncertainty introduced by sample stability

[0221] The short-term and long-term stability of the standard material are calculated according to formulas (12) and (13), where t is taken as the result of 10 months of long-term stability monitoring plus the expected stabilization time of 6 months, i.e., t = 10 + 6 = 16:

[0222] u lts =s k,lts ·t Formula 11 (3) Uncertainty assessment results

[0223] The final calculation results are shown in Table 22:

[0224] Table 22 Uncertainty Assessment Results of Standard Reference Material for Creatinine in Human Urine

[0225]

[0226] Table 23. Results of Standard Value and Uncertainty Assessment of Creatinine in Urine (mmol / L)

[0227] sample Fixed value result Human urine creatinine standard material 15.94±0.58

[0228] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

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

2. The standard substance according to claim 1, characterized in that, The preparation method of the reference material is as follows: Collect the urine of healthy ordinary people, then use a high-speed refrigerated centrifuge at 12,000 rpm and 4°C for 15 minutes, take the supernatant, filter it by suction filtration with a 0.22 μm filter membrane, and collect the filtrate; Add preservatives using a pipette at a ratio of 0.1% by volume, and mix well after adding to ensure the stability of the urine; Use an automatic biochemical analyzer or isotope dilution mass spectrometry to preliminarily measure the creatinine concentration in the urine. When the preliminary measurement result meets the design requirements of the reference material, it can enter the sub-packaging process; When the preliminary measurement result is lower than the design requirements of the reference material, add a creatinine purity reference material solution to increase the creatinine content in the urine; When adding, ensure that the added volume of the creatinine purity reference material solution does not exceed 10% of the urine volume. After adding, mix well at room temperature for 6 - 12 hours to obtain a human urine creatinine 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 steps are as follows: Test the homogeneity of urinary creatinine in the sub-packaged and stored human urine creatinine reference material. Under the condition of magnetic stirring, sub-pack the above human urine creatinine reference material into 2 mL low-temperature resistant and low-adsorption brown sample bottles respectively, and seal or close them. According to the sub-packaging order, continuously number each sub-packaging unit, and the number of sub-packaging units is P; Randomly select and test the continuously numbered sub-packaging units. When 100 < P ≤ 200, the number of sub-packaging units m selected is not less than 11; When 200 < P ≤ 500, the number of sub-packaging units m selected is not less than 15; When 500 < P ≤ 1000, the number of sub-packaging units m selected is not less than 25; When the number of sub-packaging units P > 1000, the number of sub-packaging units m selected is not less than 30. According to the order of selection, re-number and arrange the m selected sub-packaging units in sequence, and use the biochemical analyzer method or isotope dilution mass spectrometry to test the homogeneity of urinary creatinine in the human urine creatinine reference material in the above selected sub-packaging units. During the test, in order to eliminate the error caused by instrument drift during the measurement process, analyze the samples three times in the order of 1 → 15, 15 → 1, odd - even interspersed until the nth test of urinary creatinine in the human urine creatinine reference material in each selected sub-packaging 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 urinary creatinine in the human urine creatinine reference material in the sub-packaging unit; Assume there are a reference material units, and the unit i is measured ni times (i = 1,..., a). For a simple balanced design, the number of repeated measurements ni for all units is the same, that is, n1 = n2 =... = na = n0; Use the one-way analysis of variance method for analysis, and 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 ; Where: Make the 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 test steps are as follows: Divide 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 long-term stability testing as an example, within the long-term stability testing period, at least 5 time points k are selected according to the principle of denser analysis at the beginning and sparser analysis at the end. At least 2 units of standard material are extracted at each time point. The standard material of each unit is analyzed 3 times using a biochemical analyzer or isotope dilution mass spectrometry. The arithmetic mean of these measurement results is calculated to obtain the measurement result X at each time point. i The measurement results and measurement time were fitted using the following linear model: Y=b0+bX Formula 7 In the formula: b0—intercept; b—regression coefficient; X—time; Y—determination result of creatinine content in standard substance; Calculate the regression coefficients and intercepts according to Equations 8 and 9: In the formula: X i —The i-th time point; Y i —Results of urine creatinine standard substance measurement at the i-th time point; —The average value across all time points; —Results of urine creatinine standard reference measurements at all time points; k — the number of time points; Calculate the value of s according to Equation 10: The meanings of the symbols in the formula are the same as those in formulas 8 and 9; Calculate the value of s(b) according to Equation 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), it indicates that the slope is not significant and no instability was observed, indicating that the urine creatinine standard is stable. The long-term stability of the urine creatinine standard was examined at -20℃, -40℃ or -80℃, with -80℃ being preferred. The short-term stability of the urine creatinine 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 (HPLC-IOSD-MS / MS) includes several steps: adding an isotope-labeled internal standard and performing the determination. When determining urinary creatinine using HPLC-IOSD-MS / MS, the selected isotope label is [D3]-labeled creatinine, with at least three isotope-labeled atoms and an isotope abundance of at least 99%. For sample pretreatment, firstly, a solution of [D3]-labeled creatinine with a concentration of 0–100 mg / g is prepared using a solvent such as water, methanol, or acetonitrile. Then, [D3]-labeled creatinine with the same initial creatinine content is added to the urinary creatinine standard sample. The volume ratio of the added [D3]-labeled creatinine to the sample volume is between 1:100 and 10:

1. After addition, the mixture is thoroughly mixed, and the solution is obtained using a 0.22 mm... After filtration through a filter membrane, high-performance liquid chromatography-isotope dilution mass spectrometry (HPLC-IOSD-MS) was performed. The chromatographic column used was a C4, C8, or C18 column. Mobile phase A consisted of 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 consisted of acetonitrile or methanol containing 0.1%–1% formic acid, or acetonitrile or methanol containing 0.1%–1% trifluoroacetic acid. The mobile phase was isocratic elution, with a ratio of mobile phase A:B of 100:0 to 50:50 over 5–60 min. The flow rate was 100–2000 mL / min, and the injection volume was 1–100 mL. Mass spectrometry detection was performed in either MRM or SIM mode. In MRM mode, the signals of creatinine in the sample (m / z = 114.1 for the precursor ion, m / z = 44 for the daughter ion) and the creatinine isotope label [D3] in the standard (m / z = 117.1 for the precursor ion, m / z = 47 for the daughter ion) were detected separately. In SIM mode, the signals of creatinine in the sample (m / z = 114.1 for the precursor ion, m / z = 44 for the daughter ion) and the creatinine isotope label [D3] in the standard were detected separately. The signal of the [D3] ion (m / z = 117.1) was used; the quantitative standard was a standard solution prepared using creatinine purity standard material and [D3]-labeled creatinine. The concentrations of creatinine, [D3]-labeled creatinine, and the ratio of creatinine to [D3]-creatinine in the standard solution were close to those in the standard material to be tested, with a ratio range of 0.90 to 1.

10. The peak areas of the obtained creatinine extraction chromatogram and [D3]-labeled creatinine extraction chromatogram were obtained by integration. Quantification was performed using the single-point method, bracket method, or standard curve method based on the peak area ratio of the sample and the peak area ratio of the standard.

8. The standard substance according to claim 7, characterized in that, The solvent was prepared with water at a concentration of 10 mg / g; the volume ratio of the added [D3]-labeled creatinine to the sample volume was 1:1; the chromatographic column used was an Agilent EclipsePlus C18 (4.6 × 150 mm, 5 μm) column; the mobile phase A was water containing 10 mmol / L ammonium acetate, and the mobile phase B was acetonitrile containing 0.1% formic acid; the mobile phase ratio was 96.0:4.0 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: 1.80 mg / g or 15.94 mmol / L.

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