CN110849830A - Serum copper standard substance and preparation method and application thereof - Google Patents
Serum copper standard substance and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a serum copper standard substance and a preparation method and application thereof. The concentration fixed value of the serum copper standard substance is 0.50-1.40 mg/L; the factor k is 2, and the uncertainty of the concentration fixed value is 0.10-0.12 mg/L. The preparation method of the standard substance of the fixed value serum copper is to determine by flame atomic absorption spectrophotometry and inductively coupled plasma mass spectrometry and to fix the value by two methods. The serum copper standard substance fills the blank of the lack of single element standard substances of the serum copper at present, provides economic and practical single element standard substances for serum copper detection, and provides quality control products for quality control of clinical detection.
Description
Technical Field
The invention relates to a serum copper standard substance and a preparation method and application thereof, belonging to the technical field of standard value transmission in chemometry.
Background
Copper is a necessary metal trace element for normal physiological activities of human body, and is an important component of protein and enzyme in the body. Copper is involved in various metabolism in vivo, and plays an important role in development and function of blood, central system and immune system, hair, skin and skeletal tissue, brain tissue and liver, heart and other internal organs. Therefore, the disorder of copper metabolism will cause the abnormality of the normal physiological function of the body. Compared with a control group, the serum copper has obvious difference in the diagnosis and treatment process of various diseases, the correlation between trace elements in a human body and the diseases is always a focus of attention of Chinese and foreign students, and the serum copper is used as a biomarker of the diseases caused by abnormal copper metabolism. Hepatolenticular degeneration (HLD), also known as Wilson's Disease (WD), is an autosomal recessive genetic disease with primary copper metabolism disorders. Serum copper content is one of the important diagnostic indicators of the disease and is also used for monitoring the curative effect. The determination of the concentration of copper element in human body has important significance for early stage diagnosis and later stage curative effect observation of diseases, and currently, a single element standard substance for serum copper detection is lacked in China. Most laboratories still adopt the mode of adding standard and recycling to carry out quality control, and the method can only eliminate accidental errors and cannot eliminate system errors. And thus lack an assessment of the accuracy of the test data.
Suitable standard substances are the basis and key for performing accurate elemental analyses. The reference substance is a material or substance and is an important component of the reference measurement system. The ISO guidelines state that standard substances are used primarily for calibrating measuring devices, evaluating measuring methods, assigning materials and quality control. The preparation method of the standard substance mainly comprises the following steps: artificially synthesizing, purifying natural substances, purifying human or animal tissue sources or recombining standard substances by genetic engineering. In consideration of interoperability, the ion standard should use the same substance as the actual sample as the raw material, preferably human serum specimen.
At present, the National Institute of Standards and Technology (NIST) of the United states possesses a multi-element standard substance in serum but does not contain copper elements, and a primary standard substance GBW09152 sold by the national center for standards of China in the frozen human serum contains 9 elements, so that the cost is high, and the state of goods failure is present. Therefore, a blank lacking of a serum copper single element standard substance is needed to meet the actual requirement of clinical laboratory detection.
Disclosure of Invention
The invention aims to provide a serum copper standard substance and a preparation method and application thereof.
The concentration fixed value of the serum copper standard substance is 0.50-1.40 mg/L; and when the factor k is 2, the uncertainty of the concentration fixed value is 0.10-0.12 mg/L.
In the serum copper standard substance, the concentration fixed value of the serum copper standard substance is 0.749-1.025 mg/L; when the factor k is 2, the uncertainty of the concentration fixed value is 0.095-0.113 mg/L.
In the serum copper standard substance, the concentration fixed value of the serum copper standard substance is 0.749mg/L or 1.025 mg/L; when the factor k is 2, the uncertainty of the concentration fixed value is 0.095mg/L or 0.113mg/L respectively.
The invention also provides a preparation method of the standard substance of the fixed value serum copper, which comprises the following steps:
(1) sampling a copper single element solution standard substance with a matrix of 1% nitric acid, preparing at least 3 copper ion working solutions with known concentrations on the detection day, respectively determining corresponding absorbance values and copper element response values by adopting a flame atomic absorption spectrophotometry and an inductively coupled plasma mass spectrometry, establishing a standard curve 1 by using the copper ion concentration of the copper ion working solution as a horizontal coordinate and the corresponding absorbance value to obtain a flame atomic absorption spectrophotometry regression equation 1, and establishing a standard curve 2 by using the copper ion concentration of the copper ion working solution as the horizontal coordinate and the corresponding copper element response value to obtain an inductively coupled plasma mass spectrometry regression equation 2;
(2) sampling a serum standard substance, respectively adopting the conditions of the flame atomic absorption spectrophotometry and the inductively coupled plasma mass spectrometry in the step (1) to determine an absorbance value and a copper element response value, then respectively substituting the absorbance value and the copper element response value into the regression equation 1 and the regression equation 2, calculating to obtain the concentration 1 and the concentration 2 of the serum standard substance, and solving the average value determined by the two methods to obtain the concentration of the serum copper standard substance with a fixed value.
In the invention, the copper single element solution is a national first-class standard substance of a standard substance, and the product number is GBW 08615; the copper ion working solution takes a national first-grade standard substance copper single element solution standard substance GBW08615 as a standard curve sample, and the detection result of the copper element is 90-110% of the nominal value.
In the preparation method, the serum and the standard substance are weighed by weight;
the preparation of the serum standard substance comprises the following steps: 1) screening serum: removing hemolytic, lipemic and jaundice samples and positive samples of hepatitis B, hepatitis C, AIDS and syphilis from the frozen normal human serum sample after redissolution to obtain a screened serum sample;
2) the screened normal human serum sample is 0.6-1.0L per bottle, and the low-copper serum is 0.4-0.5L per bottle. Measuring the absorbance value by adopting a flame atomic absorption spectrophotometry, and calculating the concentration of copper in each mixed serum according to the regression equation 1 in the step (1); then independently mixing the screened serum samples into two samples;
3) centrifuging and filtering in a grading way: centrifuging two serum samples obtained after mixing in the step 2), taking centrifugal liquid, and then filtering by adopting a filter membrane to obtain the serum standard substance.
In the preparation method, the number of the serum samples for preparing the serum standard substance is 200-2000, specifically 500-1000;
the mixing temperature in the step 2) for preparing the serum standard substance is 2-8 ℃; when the screened serum samples are independently mixed into two samples with the same volume: wherein the concentration of one sample is 0.7-1.4 mg/L and is marked as a high concentration group; the other sample is obtained by mixing serum with the concentration of 0.7-1.4 mg/L and serum with the concentration of 0.3-0.5 mg/L to obtain serum with the concentration of 0.5-0.8 mg/L, and the serum is marked as a low concentration group; specifically, the screened serum samples can be independently mixed into 1L of two samples;
the conditions of centrifugation in step 3) were as follows: centrifuging at 4000-7500 rpm/min at 2-8 ℃ for 20-45 minutes, specifically 6000rpm/min, and centrifuging at 4 ℃ for 30 minutes, wherein the aperture of the filter membrane used in the fractional filtration can be 0.45 μm and 0.22 μm in sequence;
the step 3) also comprises the step of subpackaging and freezing the serum standard substance.
In the invention, in order to keep the consistency of the matrix, the serum standard substance is properly adjusted by using relative low-concentration serum copper in the step 2) of preparing the serum standard substance, so that the concentration is divided into a high level and a low level, the concentration of the copper in the serum of a normal person is 0.7-1.4 mg/L, and the concentration of the two mixed serum copper is about 1.0mg/L, thus obtaining a high-concentration group; in order to obtain the serum standard substance with low concentration, a certain amount of serum of a person with low concentration of copper is specially added into the serum of a normal person, so that the serum copper standard substance with the copper concentration of about 0.7mg/L is obtained, namely the serum copper standard substance is a low concentration group.
In the invention, hemolytic, lipemic and jaundice samples are removed, and light yellow clear liquid serum is selected as a raw material. (ii) a Detecting pathogenic microorganisms in serum by an immunological method, wherein the detection items comprise HBsAg, HCV antibody, HIV-1/HIV-2 antibody and Tp antibody, removing a serum sample with a positive result, and reserving samples with negative HBsAg, HCV antibody, HIV-1/HIV-2 antibody and Tp antibody detection results;
and (3) subpackaging and freezing: and (4) filling the mixture into a screw cap freezing tube according to 1.0mL per branch, and capping the sample within 10 minutes after filling. The sub-packaging is completed within 3 hours; subpackaging the samples for storage in a refrigerator at-80 ℃.
In the preparation method, when the flame atomic absorption spectrophotometry is adopted to measure the absorbance value, the absorbance value is measured jointly by a plurality of laboratories;
the conditions of the flame atomic absorption spectrophotometry are as follows:
acetylene-air flame atomic absorption photometer, which is provided with a copper hollow cathode lamp; copper detection wavelength 324.8 nm; the width of the slit is 1 mm; lamp current 7 mA; the gas flow is 1.7L/min;
the inductively coupled plasma mass spectrometry conditions are as follows:
the flow rate of the atomizer is 1.02L/min; the flow rate of the plasma torch auxiliary gas is 1.2L/min; the plasma torch cooling gas flow is 15L/min;
ion lens voltage 5.5V; inductively coupled plasma emitter power 1350W; the analog step voltage of the detector is-1850V;
detector pulse step voltage 950V; the scanning time is 20 ms; an auto lens On; the gas flow of the first reaction tank is 0.6ml/min, and ammonia gas;
the alternating voltage of the quadrupole rods of the reaction tank is 0.6V.
In the invention, the absorbance value is measured by adopting a flame atomic absorption spectrophotometry and the copper element analysis peak response value is measured by adopting an inductively coupled plasma mass spectrometry, and the data are processed as follows according to the common knowledge in the field: after abnormal value result test, normal distribution test and equal precision test, determining that the abnormal value is not abnormal, the normal distribution is normal and the equal precision is achieved.
In the above preparation method, step (2) further comprises the step of sampling the serum standard for uniformity test and stability test;
the homogeneity test is specifically carried out by adopting a variance analysis method according to the requirements of JJG1006 and 1994 'first-class standard substance technical specification';
the stability test specifically monitors the stability according to the second edition of Standard materials and techniques for their use.
In the preparation method, the step (2) further comprises the step of carrying out statistical analysis on the quantitative serum copper standard substance concentration data according to the general principle and statistical principle of quantitative JJF1343-2012 standard substance on the quantitative serum copper standard substance cooperative quantitative measurement data.
In the invention, the concentration data of the standard substance of the serum copper with fixed value is statistically analyzed according to the general principle and statistical principle of fixed value of JJF1343-2012 standard substance: removing suspicious values in the array according to a Koclen criterion; the measurements measured in each laboratory are listed: raw data, average value, standard deviation and measurement times; verifying the normal distribution of the data according to a Charulor-Wilkelk method; in the case that the data obey normal distribution, regarding the average value measured by each laboratory as a single measurement value constitutes a new set of measurement data; statistically rejecting the abnormality again according to the dickson and Grubbs method; and checking whether the precision among the groups of data is equal or not by using a method of comparing the calculated value of the Kokern method with the check table value. When the data are of equal precision, the overall mean and standard deviation are calculated.
In the above preparation method, the step (2) further comprises the step of performing uncertainty analysis on the fixed value of the concentration of the standard substance of serum copper.
In the present invention, the source of uncertainty consists of three components: the first part is the uncertainty introduced by the fixed value (uchar), the uncertainty introduced by the linearity of the standard curve (us), the second part is the uncertainty introduced by the homogeneity of the standard substance (ubb), and the third part is the uncertainty introduced by the variability of the standard substance over the useful life, i.e. the long-term stability of the standard substance (ults) and the short-term stability (usts).
The standard substance of serum copper is applied to at least one of the following items 1) to 5):
1) the method is used for quality control of serum copper detection in health monitoring and medical research;
2) calibrating the analytical instrument as a standard substance;
3) tracing the material;
4) verifying the capability;
5) evaluation of the Chamber.
The serum copper standard substance is used for quality control of serum copper detection in health monitoring and medical research, and is specifically realized by measuring the concentration of copper ions in human blood. Specifically, the method can be used for measuring the copper concentration in the human blood caused by the hepatolenticular degeneration and the abnormal copper metabolism.
The invention has the following advantages:
the hepatolenticular degeneration is one of a few of currently-treatable nervous system genetic diseases, is very easy to misdiagnose, the determination of the copper concentration of a human body has important significance for early stage definite diagnosis and later stage copper discharge treatment, and the domestic unit for carrying out serum copper detection is suffered from the lack of economic and practical copper standard substances. At present, no single copper element standard substance of serum matrix is seen at home and abroad. Can provide accurate and reliable standard reference substances for diagnosis and treatment of diseases related to hepatolenticular degeneration and abnormal copper metabolism. The research and development of the serum copper standard substance has good application prospect, and fills the gap that the single element standard substance of the serum copper is lacked at present.
Drawings
FIG. 1 is a standard curve 1 of flame atomic absorption spectrophotometry according to the present invention.
FIG. 2 is a standard curve 2 of inductively coupled plasma mass spectrometry of the present invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples 1,
Preparation of serum standard substance
According to the relevant national standards, the serum is collected and the human serum copper standard substance is prepared by referring to the ISO guidelines and WHO related technical data. The expected purpose is achieved through process control and inspection.
1. Serum standard preparation:
two concentration levels of frozen human serum copper standards were prepared, 1000 each.
(1) Raw material screening
And (3) appearance inspection: eliminating hemolytic, lipemic and icteric samples, and selecting light yellow clarified liquid serum as a raw material.
Serum immunological test and screening: and (3) detecting pathogenic microorganisms in serum by an immunological method, wherein the detection items comprise HBsAg, HCV antibodies, HIV-1/HIV-2 antibodies and Tp antibodies, removing a serum sample with a positive result, and reserving samples with negative HBsAg, HCV antibodies, HIV-1/HIV-2 antibodies and Tp antibody detection results.
Mixing the serum: independently mixing the screened serum samples into two parts, and preliminarily measuring the concentration of copper in each part of mixed serum; the concentration of copper element was divided into two levels by appropriate adjustment with low-concentration serum. Stirring in a biological safety cabinet, and mixing uniformly.
Centrifuging and filtering in a grading way: transferring the uniformly mixed serum into a treated centrifuge bottle in a biological safety cabinet, centrifuging for 30 minutes at 4 ℃ at 6000rpm/min in a high-speed refrigerated centrifuge to remove solid substances such as fibrin, collecting the centrifugate, primarily filtering by using analysis filter paper, and further removing the solid substances generated by centrifugation. In a biosafety cabinet, serum is subjected to negative pressure suction filtration sequentially through 0.45 μm and 0.22 μm pore size filters (Millipore), and the filtrate is transferred to a sterilized bottle. And (3) placing the bottle filled with the filtered serum on a stirrer for stirring to prevent the blood serum from being layered up and down to cause unevenness.
Subpackaging and freezing: mixing the above serum in biological safety cabinet, adding into screw cap freezing tube with stirring at a volume of 1.0 mL/piece, and covering the sample within 10 min. The dispensing was completed within 3 hours. Subpackaging the samples for storage in a refrigerator at-80 ℃.
And (3) biological safety inspection: randomly selecting 1 from each level candidate standard substance, and detecting that the HBsAg, HCV antibody, HIV-1/HIV-2 antibody and Tp antibody are negative by an immunological method.
2. Uniformity test
A subset of 20 cells was selected from a batch of cells (1000), and uniformity verification was performed by repeating 3 measurements for 20 cells. The method for selecting the subset is to perform random sampling according to the principle of covering the whole batch.
Inhomogeneities are generally of two types:
the first type of non-uniformity is inter-vial non-uniformity, which is reflected by inter-vial uniformity studies. The results of the inter-vial uniformity study can be used as a component of uncertainty in the evaluation of the valuation model, which can vary widely in magnitude, depending primarily on the nature of the candidate standard substance.
The second type of non-uniformity is in-vial non-uniformity, where the standard substance is reconstituted to a clear solution, where the concentration of the standard solution should be non-uniform over a few microliters, while in-vial non-uniformity can be significantly reduced by providing proper instructions for use. The instructions may include details of the time of thawing the sample and the manner of mixing.
(1) Homogeneity testing method
The uniformity of the standard substance is an evaluation of the overall spatial distribution of the standard substance sample, and is one of the important contents in the development of the standard substance. In general, standard material uniformity refers to the degree to which a characteristic quantity value is uniform in spatial distribution. Analysis of variance using the F test and calculation of s according to equation (1)bb,
When the value of F is > 1, ubb=sbb(ii) a When the F value is less than 1, calculating u according to the formula (2)bb:
In formulas (1) and (2):
-variance between bottles, MSBetween groupsInter-group mean square, MSIn groupMean square in group, n0Effective degree of freedom, n-number of measurements,-degree of freedom in group,sr-standard deviation within the group.
3. Results and analysis of results
(1) The results of the homogeneity test are shown in table 1:
TABLE 1 serum copper uniformity test results (unit mg/L)
The results of the homogeneity analysis of variance are shown in Table 2.
TABLE 2 homogeneity test results of analysis of variance (in mg/L) for serum copper
The data results in tables 1 and 2 show that the measured values of the uniformity of the standard substances at the low and high concentrations are significantly higher than 0.05, indicating that no significant difference is observed between the concentrations in the bottle and the bottle when the precision of the measurement results is good, and that the standard substances at the two concentrations are uniform.
(2) And (3) stability test:
the stability of the standard substance is studied, and the following two main substances are: long term stability (shelf life stability) and short term stability (shipping stability, investigating the effect on the sample of possible extremes; decap stability, investigating the effect on stability of the sample of external factors after opening the sealed package).
1) And (3) testing the long-term stability: the standard substances were stored at-80 ℃ under the prescribed storage conditions, 1 sample was taken out at each concentration level after 0, 1, 2, 3, 4, 5,6, 17 and 24 months of storage, and the long-term stability was measured by flame atomic absorption spectrometry (FF-AAS method), and the protocol for long-term stability is shown in Table 3.
TABLE 3 Long term stability protocol
Long-term stability on storage at 80 ℃: the standard substance was stored at-80 ℃ for 24 months, and the measurement results are shown in Table 4.
TABLE 4 Long-term stability measurement of copper at 80 ℃ (unit: mg/L)
And (4) conclusion: the serum copper standard substance has no significant change in measurement results at a confidence level of 95% within 24 months of storage at-80 ℃, and the concentration value stability is good within 24 months.
Long-term stability on storage at 20 ℃: the standard substance was stored at-20 ℃ for 24 months, and the measurement results are shown in Table 5.
TABLE 5 Long-term stability measurement of copper at 20 ℃ (unit: mg/L)
And (4) conclusion: the serum copper standard substance has no significant trend in the measurement result within 24 months of storage at-20 ℃ at a confidence level of 95 percent, and the concentration value stability within 24 months is good.
2) Short term stability
The stability of the candidate standards was tested for storage at 2-8 ℃ for 10 days and at 25 ℃ for 5 days, respectively, according to the relevant requirements, which are extreme conditions that may be encountered under shipping conditions or prior to use by the customer. Short-term stability studies were performed under repetitive conditions using a synchronous method, and the results are shown in table 6.
TABLE 6 short term stability protocol
Short term stability on storage at 2-8 ℃ for 10 days: the standard substance was stored at 4 ℃ for 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 3 days, 7 days, 8 days, 9 days, and 10 days, and the measurement results are shown in Table 7.
TABLE 7 short-term stability of copper (2-8 ℃ C. for 10 days) test results (unit: mg/L)
Date of measurement (sky) | Level 1(mg/L) | Level 2(mg/L) |
0 | 0.922 | 0.662 |
1 | 0.967 | 0.681 |
2 | 0.974 | 0.652 |
3 | 0.957 | 0.672 |
4 | 0.939 | 0.688 |
5 | 0.942 | 0.687 |
6 | 0.960 | 0.679 |
7 | 0.963 | 0.640 |
8 | 0.970 | 0.686 |
9 | 0.944 | 0.678 |
10 | 0.979 | 0.667 |
Slope (β 1) | 0.001945455 | 0.000381818 |
Intercept (β 0) | 0.946363636 | 0.670090909 |
Standard deviation (S) | 0.017011701 | 0.016247952 |
Slope standard deviation [ S (β 1)] | 0.001622002 | 0.001549181 |
t statistic | 1.199415573 | 0.246464463 |
t critical value | 2.262157158 | 2.262157158 |
Conclusion | No significant change in | No significant change in |
From the above results, it can be seen that the measurement results of the serum copper standard substance stored at 2-8 ℃ for 10 days did not significantly change at a confidence level of 95%.
Short-term stability after 5 days of storage at room temperature (18-25 ℃): the standard substance was stored at room temperature for 0 day, 0.3 day, 1 day, 2 days, 3 days, 4 days, and 5 days, and the measurement results are shown in Table 8.
TABLE 8 short-term stability of copper (5 days at room temperature) results (unit: mg/L)
Date of measurement (sky) | Level 1(mg/L) | Level 2(mg/L) |
0 | 0.931 | 0.633 |
0.3 | 0.970 | 0.645 |
1 | 0.956 | 0.694 |
2 | 0.982 | 0.689 |
3 | 0.959 | 0.665 |
4 | 0.959 | 0.695 |
5 | 0.937 | 0.682 |
Slope (β 1) | -0.001024152 | 0.007902864 |
Intercept (β 0) | 0.958524218 | 0.65458374 |
Standard deviation (S) | 0.019249528 | 0.021673901 |
Slope standard deviation [ S (β 1)] | 0.00413719 | 0.004658247 |
t statistic | -0.247547742 | 1.696531905 |
t critical value | 2.570581835 | 2.570581835 |
Conclusion | No significant change in | No significant change in |
From the above results, it can be seen that the measurement results of the serum copper standard substance stored at room temperature of 18-25 ℃ for 5 days did not significantly change at the 95% confidence level; the stability of the concentration values is good within 5 days.
Two, constant value
According to the relevant regulations of JJG1006-1994 first-class standard substance technical specification, the characteristic quantity values of the two developed standard substances are determined by adopting a method of determining the value of data generated by a plurality of determination methods by adopting the cooperation of a plurality of laboratories. The fixed value test is carried out by referring to a flame atomic absorption spectrometry method of copper in WS/T93-1996 serum and standard operating procedures for ICP-MS (direct injection dynamic reaction cell) measurement of 21 elements in the serum. In order to ensure the accuracy of the fixed value data, the experiment carries out strict quality control:
using SeronrmTMThe Trace element human Serum standard substance Trace Elements Serum L-1 is used as the accompanying quality control, and the measurement results are required to be within a 95% confidence interval.
The national first-grade standard substance copper single element solution standard substance (GBW08615) is used as a standard curve sample, and the detection result of the copper element is required to be within 90-110% of the nominal value.
The range of the results of 6 independent constant values of each unit is controlled within 10 percent of the average value of the detection results.
1. Participate in the laboratory of the fixed value.
1) The university of capital medical science attaches a poison chemical analysis laboratory of Beijing Chaoyang Hospital and a laboratory dedicated to chemical poison detection and analysis, and can detect substances such as metal elements, non-metal inorganic poisons, organic poisons and the like.
2) Beijing Huiyizitakang pharmaceutical technology Limited company, located in 14 street, No. 18 building 2 unit 3 of Kao, 99 Kechuang district, Beijing City, the qualification aspect of the laboratory has passed CMA (qualification certification of inspection and detection institution) and CNAS (China qualification national committee) certification, the detection parameters cover all elements in the standard substance;
3) the Beijing analysis center of Shimadzu corporation (China) can detect metal elements, non-metal inorganic poisons, organic poisons and other substances.
2. Method of valuing
Quantitative testing of serum copper standards was performed in several independent laboratories using classical atomic absorption spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS).
Wherein, the conditions of the flame atomic absorption spectrophotometry are as follows:
acetylene-air flame atomic absorption photometer, which is provided with a copper hollow cathode lamp; copper detection wavelength 324.8 nm; the width of the slit is 1 mm; lamp current 7 mA; the gas flow is 1.7L/min.
The conditions for inductively coupled plasma mass spectrometry are as follows:
the flow rate of the atomizer is 1.02L/min; the flow rate of the plasma torch auxiliary gas is 1.2L/min; the plasma torch cooling gas flow is 15L/min;
ion lens voltage 5.5V; inductively coupled plasma emitter power 1350W; the analog step voltage of the detector is-1850V;
detector pulse step voltage 950V; the scanning time is 20 ms; an auto lens On; the gas flow of the first reaction tank is 0.6ml/min, and ammonia gas;
reaction cell quadrupole alternating voltage (0.6V)
3. Constant value data statistics
(1) According to the general principle and the statistical principle of the JJF1343-2012 standard substance definite value, the measured data is processed by the following steps when a plurality of laboratories cooperate to definite values:
(2) after technically accounting for the outliers generated and eliminated for each individual set of measurements by the operator, the outliers are statistically eliminated again using the dickson and grubbs method. The results of each operator measurement are listed: raw data, mean, standard deviation, number of measurements.
(3) All the measured data are collected, and the normal classification of the data is verified according to a Charulo-Wilck method.
(4) In the case where the data obeys a normal distribution, considering the average value measured by each laboratory as a single measurement value constitutes a new set of measurement data. The anomalies are statistically rejected again according to dickson and grubbs.
(5) And checking whether the precision among the groups of data is equal or not by using a method of comparing the calculated value of the Kokern method with the check table value. When the data are of equal precision, the overall mean and standard deviation are calculated.
(6) The atomic absorption spectrophotometry and ICP-MS methods are respectively adopted in a plurality of fixed value laboratories for determination, and the fixed value data are summarized in the following table.
By the dickson methodAnd (4) abnormal value judgment: arranging the n times of measurement data in a sequence from small to big: x is the number of1≤x2......xn-1≤xnThe measured data is calculated as follows, and the critical value Q is found(α,n),
Q1=(x2-x1)/(xn-x1) Formula (3)
Qn=(xn-xn-1)/(xn-x1) Formula (4)
If Q1>QWatch (0.05,6)Then x is determined1Abnormal values should be eliminated. If Qn>QWatch (0.05,6)Then x is judgednAbnormal values should be eliminated.
And (3) judging an abnormal value by adopting a Grublas method: in a set of measurements, e.g. a measurement xiHaving residual error s is the standard deviation of a set of numbers. When | vi|>λ(α,n)Then xiShould be rejected. Lambda [ alpha ](α,n)Is a number associated with the number of measurements and a given significance level α, and may be obtained by looking up a table.
TABLE 9 abnormal value test of fixed value result of Cu element in low concentration serum standard substance
TABLE 10 evaluation of abnormal values of Cu element in high-concentration serum standards
Measurement of sequence number | No.01 | No.02 | No.03 | No.04 | No.05 | No.06 |
1 | 0.979 | 1.045 | 1.047 | 1.060 | 1.023 | 1.016 |
2 | 1.009 | 1.016 | 1.023 | 1.040 | 1.015 | 0.997 |
3 | 0.990 | 1.025 | 1.005 | 1.060 | 1.061 | 1.011 |
4 | 0.990 | 1.047 | 1.025 | 1.050 | 1.034 | 1.057 |
5 | 1.006 | 1.036 | 1.008 | 1.050 | 1.038 | 1.013 |
6 | 0.992 | 1.053 | 1.022 | 1.060 | 1.030 | 0.980 |
Mean value of | 0.994 | 1.037 | 1.022 | 1.053 | 1.034 | 1.012 |
Q1=(X2-X1)/(Xn-X1) | 0.36 | 0.24 | 0.07 | 0.50 | 0.17 | 0.22 |
Qn=(Xn-Xn-1)/(Xn-X1) | 0.11 | 0.16 | 0.52 | 0.00 | 0.50 | 0.53 |
QWatch (0.05,6)=0.628 | No suspect value | No suspect value | No suspect value | No suspect value | No suspect value | No suspect value |
Q1=(Xmin-X)/s | 1.36 | 1.42 | 1.11 | 1.63 | 1.17 | 1.26 |
Qn=(Xmax-X)/s | 1.32 | 1.23 | 1.69 | 0.82 | 1.75 | 1.74 |
QWatch (0.05,6)=1.887 | No suspect value | No suspect value | No suspect value | No suspect value | No suspect value | No suspect value |
The results of examination of 3 families of 6 groups of data by the dickson and grubbs method show that all the data are in accordance with the requirements and have no abnormal values.
Arranging a group of measurement data in sequence from small to large by adopting a Chariro-Wilk method for the data after the suspicious value test, and carrying out normal distribution test according to the following formula when W is more than W(n,p)In this case, the measured data is normally distributed.
TABLE 11 results of normal distribution test of fixed value data of serum copper standard substance
The result of normal distribution investigation shows that the w values of the copper elements in the serum standard substances with two concentrations are both larger than the table-checking value, so that all the fixed value result data conform to normal distribution.
And then, according to accuracy testing criteria such as the Koclen method testing, whether the accuracy among the groups of data is equal is tested. The test results are shown in tables 12-13 below:
TABLE 12 results of constant value results and other precision tests of low concentration serum copper standard substance
TABLE 13 results of high-concentration serum copper standard substance quantitative results and other precision tests
The equal-precision test result shows that the result values of six groups of data of Cokronen test are all smaller than the table look-up value, which shows that the six groups of data measured by multiple laboratories have equal precision for the measurement results of two copper elements with concentration. The average value of 6 groups of data which meet the equal precision is regarded as a single measurement value to form a group of new measurement data, and abnormal value detection also meets the requirements without abnormal values.
4. Investigation of accuracy
Using fixed value method to pair SeronrmTMThe Trace element human serum standard substance Trace Elements serum L-1 was subjected to accuracy verification and three independent analyses, and the measurement results are shown in Table 14.
TABLE 14 method accuracy test results (mg/L)
The measured values are all in the uncertainty range of the standard values, which shows that the method has better accuracy.
5. Result of constant value
The concentration of the candidate serum copper standard substance in the subject is jointly set by 6 sets of independent detection systems adopted by Beijing Kogyang Hospital affiliated to the university of capital medical science, Beijing Virginiangtaikang pharmaceutical technology Co., Ltd, Shimadzu corporation management (China) Co., Ltd. When the AAS and ICP-MS are adopted to measure the concentration of copper in serum, 36 bottles of serum standard substances developed in the research are randomly extracted, the total number of the 36 bottles is 72, each concentration level of each detection system is measured for 6 bottles, each bottle of serum is measured for 3 times, and the average value is taken. The average of the measurements of 36 sera was used as the standard for the standard substance, and the results are shown in Table 15.
TABLE 15 serum copper standard quantitation results (mg/L)
6. Measurement of candidate standard substance density
The density of the horizontal 1 serum was measured 10 times at 21 ℃ using a 10mL volumetric flask, XS105 dual range balance (d 0.1mg, max weight 140g, Mettler) to give a density value of 1.024 g/mL; the density of the horizontal 2 serum was measured 10 times to obtain a density value of 1.025 g/mL.
TABLE 16 Density measurement results of the standard substances
Low concentration (mg/L) | High concentration (mg/L) | |
1 st time | 1.0246 | 1.0242 |
2 nd time | 1.0243 | 1.0241 |
3 rd time | 1.0251 | 1.0241 |
4 th time | 1.0245 | 1.0243 |
5 th time | 1.0246 | 1.0246 |
6 th time | 1.0248 | 1.0248 |
7 th time | 1.0255 | 1.0241 |
8 th time | 1.0241 | 1.0251 |
9 th time | 1.0245 | 1.0236 |
10 th time | 1.0253 | 1.0241 |
Mean value | 1.0247 | 1.0243 |
Standard deviation of | 0.0004 | 0.0004 |
7. And (3) uncertainty evaluation:
the sources of uncertainty for the standard substance are mainly composed of three parts: the first part is the uncertainty (u) introduced by the constant valuechar) Uncertainty introduced by the linearity of the standard curve (u)s) The second part is the uncertainty (u) introduced by the homogeneity of the standardbb) The third part is the variability of the standard substance over the life cycle, i.e.the uncertainty caused by the long-term stability of the standard substance (u)lts) And short term stability-induced uncertainty (u)sts)。
(1) Uncertainty (u) introduced by constant valuechar)
The human serum copper standard substance developed by the project adopts the atomic absorption spectrophotometer method and the inductively coupled plasma mass spectrometry method to determine the value, the Kokronen test shows that the value data obtained by different methods have equal precision, and the methods have no significant difference, so the uncertainty introduced by the determined value is obtained by the uncertainty of each method. N times from a plurality (m) of laboratories (or methods), each laboratory (or method) being measured, calculated according to the following formula:
the uncertainty of the measured result of each fixed value method comprises A-type uncertainty and B-type uncertainty, wherein the A-type uncertainty is the relative standard deviation of the mean value of the measured values,
while the class B uncertainty is mainly measured by the sample (u)1) Standard curve used (u)2) And a single element solution standard substance (u)3) The introduced uncertainty is composed of three parts,
1) uncertainty introduced by sample weighing (u)1)
u1Weighing by adopting a weight loss method, and the uncertainty brought by the sensitivity of the balance can be ignored. According to the metering certificate, all the balances are one-hundred-thousandth electronic balance e is 0.03mg, and according to uniform consideration, all the balance nonlinear errors are respectively u (delta, nonlinear), the balance is automatically zeroed as 1-time skin buckling, a0The error caused by the automatic scale Zeroing is u (Δ, Zeroing) and the uncertainty of the scale regardless of the repetitive error is:
the relative uncertainties of the weighing of the test object and of the diluent are respectively
2) uncertainty introduced by the standard curve (u)2) Calculated according to the following formula:
if the standard curve is denoted by y ═ ax + b, then the uncertainty (u) introduced by the standard curve4):
(wherein: P-number of tests C, n-number of tests of standard solution, b-slope of standard curve,-testing the average value of C,the mean value of the test standard solution, Sy-standard error,-the sum of the squares of X. )
The results are shown in Table 17.
TABLE 17 results of uncertainty analysis
(2) Uncertainty of standard substance introduction (u)3)
Copper element, U3.rel=0.001
The results of the valuing are combined for uncertainty as shown in table 18.
TABLE 18 results of uncertainty analysis of fixed value results
Uniformity-induced uncertainty (u)bb)
Using variance analysis method to calculate and analyze uniformity detection data, when the repeatability of the measurement method is good, then u isbbCan be calculated using equation (13):
in the formula:is the variance within the group of the measured data,is the variance between groups and n is the number of measurements within a group.
When the repeatability of the measurement method of the uniformity evaluation data is poor, the probability is thatWhen, or when, the standard deviation s between the bottles is estimatedbbLess than the standard deviation s of repeatabilityrTo sbbEquation (13) cannot be used for the influence of (c). At this time, it can be calculated by equation 14:
the results of the sample uniformity-induced uncertainty calculations are listed in table 19.
TABLE 19 human serum copper standard uniformity determination uncertainty
(3) Uncertainty due to stability
Seven times of short-term stability (standing for 5 days at room temperature) and ten times of long-term stability (24 months) of standard substances with two concentrations of human serum copper are respectively examined, the precision of a detection method, a value-fixing method and the like is calculated, the uncertainty values of the short-term stability and the long-term stability are calculated by adopting a variance method, and a formula is calculated: u. ofs=s(β1) X, the calculation results are shown in tables 20 and 21.
TABLE 20 human serum copper standards short term stability determination uncertainty
TABLE 21 Long term stability determination uncertainty for human serum copper standards
(4) Synthesis of uncertainty
The three types of uncertainty components were synthesized, and the total uncertainty of each element is shown in table 22, where k is 2.
Uc.rel=k·uc.relFormula (16)
In the formula:
uc.rel-synthetic relative standard uncertainty
Uc.rel-synthetic relative expansion uncertainty
Table 22 total uncertainty of human serum copper standards (k ═ 2)
And (3) comparison and verification:
the results of comparative detection of the subject prepared standards by flame atomic absorption spectrometry using a conventional measurement method are shown in table 23.
TABLE 23 human serum copper standards comparison
Expression of human serum copper standard substance characteristic quantity value result
Table 24 human serum copper standard substance characteristic values and uncertainties (mg/L, k ═ 2)
The density of the standard substance at 20 ℃ is (1.024 +/-0.001) g/mL.
Uncertainty evaluation includes uncertainty components such as fixed value procedures, fixed value measurements, uniformity, stability, etc.
Measuring method of characteristic quantity value
A plurality of laboratories are organized, and a quantitative test is carried out by using an atomic absorption method and an inductively coupled plasma mass spectrometry (ICP-MS) to obtain 6 groups of quantitative data of characteristic quantity values. And comparing with foreign standard substances of the same grade. The result shows that the fixed value result of the standard substance is accurate and reliable.
Fourth, traceability description
The standard substance is traced to the national first-class standard substance.
Fifth, description of correct use
The standard substance is frozen human serum, and needs to be re-melted and stabilized at room temperature for at least half an hour before use. After re-melting of the sample, immediate use is recommended. Before use, the sample is ensured to be fully mixed. The samples were protected from exposure to intense ultraviolet light or direct sunlight.
Sixthly, transportation and storage
The standard substance was packed in 1.0mL of 1.5mL screw-packed vials.
The standard substance is normally transported at room temperature in long-distance transportation, dry ice transportation is needed in high-temperature weather, and no special measures are needed when the temperature is not higher than 25 ℃. The standard substance is stored at a temperature of-20 ℃ or lower.
Claims (10)
1. A standard substance of serum copper, which is characterized in that: the concentration fixed value of the serum copper standard substance is 0.5-1.40 mg/L; and when the factor k is 2, the uncertainty of the concentration fixed value is 0.10-0.12 mg/L.
2. The standard substance of serum copper according to claim 1, characterized in that: the concentration fixed value of the serum copper standard substance is 0.749-1.025 mg/L; when the factor k is 2, the uncertainty of the concentration fixed value is 0.095-0.113 mg/L;
the concentration fixed values of the serum copper standard substance are specifically 0.749mg/L and 1.025 mg/L; the uncertainty of the concentration fixed value is 0.095mg/L and 0.113mg/L respectively when the factor k is 2.
3. The method for preparing a serum copper standard substance according to claim 1 or 2, comprising the steps of:
(1) sampling a copper single element solution standard substance with a matrix of 1% nitric acid, preparing at least 3 copper ion working solutions with known concentrations, respectively determining corresponding absorbance values and copper element response values by adopting a flame atomic absorption spectrophotometry and an inductively coupled plasma mass spectrometry, establishing a standard curve 1 by using the copper ion concentration of the copper ion working solution as a horizontal coordinate and the corresponding absorbance value to obtain a flame atomic absorption spectrophotometry regression equation 1, and establishing a standard curve 2 by using the copper ion concentration of the copper ion working solution as a horizontal coordinate and the corresponding copper element response value to obtain an inductively coupled plasma mass spectrometry regression equation 2;
(2) sampling a serum standard substance, respectively adopting the conditions of the flame atomic absorption spectrophotometry and the inductively coupled plasma mass spectrometry in the step (1) to determine an absorbance value and a copper element response value, then respectively substituting the absorbance value and the copper element response value into the regression equation 1 and the regression equation 2, calculating to obtain the concentration 1 and the concentration 2 of the serum standard substance, and solving the average value determined by the two methods to obtain the concentration of the serum copper standard substance with a fixed value.
4. The production method according to claim 3, characterized in that: weighing the serum standard substance by a gravimetric method;
the preparation of the serum standard substance comprises the following steps: 1) screening serum: removing hemolytic, lipemic and jaundice samples and positive samples of hepatitis B, hepatitis C, AIDS and syphilis from the frozen normal human serum sample after redissolution to obtain a screened serum sample;
2) measuring the absorbance value of the screened serum sample by adopting a flame atomic absorption spectrophotometry, and calculating according to a regression equation 1 in the step (1) to obtain the concentration of copper in each mixed serum; then independently mixing the screened serum samples into two samples;
3) centrifuging and filtering in a grading way: centrifuging two serum samples obtained after mixing in the step 2), taking centrifugal liquid, and then filtering by adopting a filter membrane to obtain the serum standard substance.
5. The method of claim 4, wherein: the number of the serum samples for preparing the serum standard substance is 200-2000;
the mixing temperature in the step 2) for preparing the serum standard substance is 2-8 ℃; when the screened serum samples are independently mixed into two samples with the same volume: one sample is 0.7-1.4 mg/L in concentration and is marked as a high-concentration group; the other sample is obtained by mixing serum with the concentration of 0.7-1.4 mg/L and serum with the concentration of 0.3-0.5 mg/L to obtain serum with the concentration of 0.5-0.8 mg/L, and the serum is marked as a low concentration group;
the conditions of centrifugation in step 3) were as follows: centrifuging at 4000-7500 rpm/min at 2-8 ℃ for 20-45 minutes, wherein the aperture of a filter membrane adopted during fractional filtration is 0.45 mu m and 0.22 mu m in sequence;
the step 3) also comprises the step of subpackaging and freezing the serum standard substance.
6. The production method according to any one of claims 2 to 5, characterized in that: when the flame atomic absorption spectrophotometry is adopted to measure the absorbance value, the measurement is carried out in a plurality of laboratories in a combined way;
the conditions of the flame atomic absorption spectrophotometry are as follows: acetylene-air flame atomic absorption photometer, which is provided with a copper hollow cathode lamp; copper detection wavelength 324.8 nm; the width of the slit is 1 mm; lamp current 7 mA; the gas flow is 1.7L/min;
the inductively coupled plasma mass spectrometry conditions are as follows:
the flow rate of the atomizer is 1.02L/min;
the flow rate of the plasma torch auxiliary gas is 1.2L/min;
the plasma torch cooling gas flow is 15L/min;
ion lens voltage 5.5V;
inductively coupled plasma emitter power 1350W;
the analog step voltage of the detector is-1850V;
detector pulse step voltage 950V;
the scanning time is 20 ms;
an auto lens On;
the gas flow of the first reaction tank is 0.6ml/min, and ammonia gas;
the alternating voltage of the quadrupole rods of the reaction tank is 0.6V.
7. The production method according to any one of claims 2 to 6, characterized in that: the step (2) further comprises the step of carrying out a homogeneity test and/or a stability test on the serum standard sample;
the homogeneity test is specifically carried out by adopting a variance analysis method according to the requirements of JJG1006 and 1994 'first-class standard substance technical specification';
the stability test specifically monitors the stability according to the second edition of Standard materials and techniques for their use.
8. The production method according to any one of claims 3 to 7, characterized in that: and (2) processing and correcting the measured data of the serum copper standard substance with the fixed value in the cooperative fixed value process of each laboratory according to the general principle and the statistical principle of the fixed value of the JJF1343-2012 standard substance.
9. The production method according to any one of claims 3 to 8, characterized in that: the step (2) also comprises a step of carrying out uncertainty analysis on the fixed value of the concentration of the standard substance of the serum copper.
10. Use of the standard substance for serum copper according to claim 1 or 2 in at least one of the following 1) to 5):
1) the method is used for quality control of serum copper detection in health monitoring and medical research;
2) calibrating the analytical instrument as a standard substance;
3) tracing the material;
4) verifying the capability;
5) evaluation of the Chamber.
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