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Basic dataStandard ID: SF/T 0063-2020 (SF/T0063-2020)Description (Translated English): (General rule of forensic poison analysis method verification) Sector / Industry: Chinese Industry Standard (Recommended) Classification of Chinese Standard: C06 Word Count Estimation: 14,194 Date of Issue: 2020-05-29 Date of Implementation: 2020-05-29 Regulation (derived from): Announcement of the Ministry of Justice (2020.05.29) Issuing agency(ies): Ministry of Justice of the People's Republic of China SFT0063-2020: (General rule of forensic poison analysis method verification)---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.General rules for method validation in forensic toxicology ICS 07.140 C 06 SF People's Republic of China judicial administration industry standards General rules for verification of forensic toxicology analysis methods 2020-05-29 released 2020-05-29 implementation Issued by the Ministry of Justice of the People's Republic of China Table of contentsForeword...II 1 Scope...1 2 Normative references...1 3 Terms and definitions...1 4 Basic requirements...1 5 Screening analysis...1 6 Qualitative Analysis...2 7 Quantitative analysis...2 8 Verification of Toxicological Analysis Method...2 Appendix A (informative appendix) Method validation example...7ForewordThis standard was drafted in accordance with the rules given in GB/T 1.1-2009. Please note that certain contents of this document may involve patents. The issuing agency of this document is not responsible for identifying these patents. This standard was proposed by the Forensic Science Research Institute. This standard is under the jurisdiction of the Information Center of the Ministry of Justice. Drafting organization of this standard. Forensic Forensic Science Research Institute. The main drafters of this standard. Xiang Ping, Shen Min, Wang Xin, Zhuo Xianyi, Liu Wei, Shen Baohua, Yan Hui, Shi Yan, Chen Hang, Wu Hejian. General rules for verification of forensic toxicology analysis methods1 ScopeThis standard specifies the basic requirements for the verification of analytical methods for biological matrix poisons (drugs) and their metabolites. This standard applies to laboratories for non-standard methods, methods developed by laboratories, standard methods used beyond their intended scope, and expansion, Modified standard methods, etc. for method verification. This standard only involves chromatography and chromatography-mass spectrometry analysis methods and does not include sampling.2 Normative referencesThe following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this document. For undated references, the latest version (including all amendments) applies to this document. GB/T 13966 Terminology for analytical instruments GB/T 27417 Guidelines for Validation and Verification of Chemical Analysis Methods for Conformity Assessment GB/T 32465 Chemical analysis method verification confirmation and internal quality control requirements GB/T 32467 Chemical analysis method verification confirmation and internal quality control terms and definitions GB/T 35655 Chemical analysis method verification confirmation and internal quality control implementation guide chromatographic analysis GB/Z 35959 Liquid chromatography-mass spectrometry analysis method general rule GA/T 122 Terminology for toxicological analysis SF /Z JD0107019 General rules for the qualitative analysis of forensic poisons by organic mass spectrometry3 Terms and definitionsGB/T 13966, GB/T 27417, GB/T 32465, GB/T 32467, GB/T 35655, GB/Z 35959, GA/T 122 and The terms and definitions defined in SF /Z JD0107019 apply to this document.4 Basic requirementsThe laboratory should verify the analytical method based on the same or similar biological matrix as the test sample, and select according to the intended use of the method The method performance index to be verified.5 Screening analysisThe screening analysis method verification performance index requirements are as follows. a) Selectivity; b) Detection limit; c) Dilution reliability (when necessary); d) Stability (when necessary).6 Qualitative analysisThe qualitative analysis method verification performance index requirements are as follows. a) Selectivity; b) Delay effect; c) Matrix effect (applicable to LC-MS analysis); d) Detection limit; e) Dilution reliability (when necessary); f) Stability (when necessary).7 Quantitative analysisThe quantitative analysis method verification performance index requirements are as follows. a) Selectivity; b) Delay effect; c) Matrix effect (applicable to LC-MS analysis); d) Linear range; e) Precision; f) accuracy; g) Detection limit; h) Limit of quantification; i) Extraction recovery rate; j) Reliability of dilution (when necessary); k) Stability (when necessary).8 Verification of toxicological analysis method8.1 Selectivity The analysis method should have a certain degree of selectivity. All screening, qualitative and quantitative analysis methods should be evaluated for interferences such as matrix and internal standards. To ensure the reliability of the test results. Specific requirements include. a) Analyze at least 10 blank samples from different sources, observe the peaks of the chromatogram, and check whether there are any areas where the target substance appears interference; b) Internal standard or potential interference should be added to the blank sample to check whether there is interference; c) If interference exists, measures such as optimizing instrument conditions, optimizing pretreatment methods, and changing chromatographic conditions can be taken to eliminate interference. If indeed The presence of a small amount of interfering substances does not affect the quantitative analysis results, and the accuracy and precision of the quantification limit should be proved to be within acceptable limits Inside. 8.2 Delay effect The delay effect can affect the analysis results of subsequent samples, especially during the batch analysis of test samples. The delay effect may cause qualitative and quantitative analysis. The analysis results are inaccurate. Specific requirements include. a) The blank sample should be analyzed immediately after the sample with high concentration or the highest concentration point of the calibration curve, and the delay should be evaluated by the blank sample. Delay effect. The measurement should be repeated 3 times; b) Optimize analysis methods to eliminate delay effects. However, if the delay effect cannot be eliminated, the measures to control the delay effect should be indicated. Example. Add a blank sample after the high concentration test sample, and the signal response of the subsequent test sample is more than 10 times higher than the signal response of the blank sample, otherwise, The test sample needs to be re-extracted and tested again. See Appendix A for a verification example of the delay effect. 8.3 Linear range The analytical method should determine the linear range of the calibration curve based on the expected concentration or content range of the test sample. Specific requirements include. a) The sample for preparing the calibration curve should contain a blank sample (matrix sample without target and internal standard), a zero-point sample (empty White sample plus internal standard) and a certain gradient of samples with more than 6 concentration points; b) The lowest concentration point of the calibration curve should be far away from the detection limit and near the limit of quantification. The middle point is the average concentration of the analytical target in the daily detection. The highest calibration point concentration is the highest point of the expected concentration range or close to the highest point; c) The concentration points of the calibration curve should be distributed as evenly as possible in the linear range, and each concentration point needs at least 5 parallel samples, and 5 A parallel sample needs to be analyzed in different batches; d) A suitable mathematical model should be selected to express the response value of each concentration point of the calibration curve (usually the peak area of the target substance and the internal standard) The ratio of) and concentration. Usually unary linear regression is suitable for data with equal variation. If the linear regression model has different For variance, the weight coefficient 1/x or 1/x2 can be selected to compensate for heteroscedasticity. If necessary, nonlinear regression can also be used; e) Five parallel samples at different concentration points should be put together for correlation analysis. The calibration curve can be used as a graph method (response value y/ Concentration x) or calculated regression equation (y=ax+b). General control correlation coefficient R≥0.99; f) If a calibration curve cannot meet the relevant requirements in the range of the lowest concentration to the highest concentration, consider making the calibration curve in multiple sections. line. See Appendix A for a verification example of the calibration curve. 8.4 Accuracy Accuracy and precision can be investigated at the same time, and quality control samples (referred to as quality control samples) should be used for evaluation. Prepare the calibration curve and The standard solutions of the quality control samples should be weighed or diluted separately. If possible, use standard materials from different sources. Bias table Indicates that it is the percentage of the difference between the measured average value and the reference value and the reference value. It should be controlled within ±15%, and the limit of quantification should be within ±20%. Specific requirements include. a) The quality control sample includes four concentrations of limit of quantification, low, medium and high. According to the method, the limit of quantification and the low concentration can be the same quality control sample, or The low concentration is 3 times the limit of quantification, the middle concentration is the middle concentration of the calibration curve, and the high concentration is close to the highest concentration of the calibration curve; b) 3 quality control samples should be prepared for each concentration point and analyzed according to the established method to obtain the measured value. The same operation should be performed continuously 5 day; c) When conditions permit, the laboratory can also evaluate accuracy by analyzing certified reference materials and participating in proficiency testing. 8.5 Precision Precision should be expressed in deviation or relative standard deviation (RSD), which is also called coefficient of variation (CV), and can be subdivided into daily Internal precision and daytime precision. The RSD of the quantitative analysis method of biological samples should be controlled within 15%, and the RSD should be less than 20% at the limit of quantification. With Physical requirements include. a) Four quality control samples of quantification limit, low, medium and high concentration are used. At least 3 quality control samples are prepared for each concentration point, according to the established method Method analysis to obtain the measured value. The same operation continues for 5 days; b) The intra-day precision is the precision inspection carried out within one day in the same batch. Calculate the intraday precision according to formula (1). 8.6 Detection limit All methods should determine the detection limit, and the following two methods can be used to calculate if the requirements of qualitative confirmation are met. a) Signal-to-noise ratio method Gradually dilute the low-concentration addition samples, there should be at least three blank matrix addition samples from different sources, and at least three batches should be analyzed. Select A section of the baseline near the peak of the target is used as a reference, and the instrument can automatically calculate the signal-to-noise ratio (S/N) of the selected chromatographic peak. When S/N ≥ 3 and meets Qualitative requirements (such as retention time, peak shape, ion abundance ratio, etc.) are the minimum concentration of the added sample as the detection limit. b) Calibration curve method According to the slope of at least three independent calibration curves and the standard deviation of the intercept on the y-axis, the detection limit is calculated according to formula (3). 8.7 Limit of quantification Quantitative analysis should determine the limit of quantification, which can be calculated by the following two methods, but both should meet the requirements of precision and accuracy. a) Signal-to-noise ratio method Gradually dilute the low-concentration addition samples, there should be at least three blank matrix addition samples from different sources, and at least three batches should be analyzed. Select A segment of the baseline near the peak of the target is used as a reference, the instrument can automatically calculate the signal-to-noise ratio (S/N) of the selected chromatographic peak, and when S/N ≥ 10 and meets The minimum concentration of added sample required for precision and accuracy is the limit of quantification. b) Calibration curve method Use the lowest concentration point of the calibration curve as the limit of quantification. There should be at least three blank matrix addition samples from different sources, and at least three batches should be analyzed Second, to ensure that the requirements of precision and accuracy are met. 8.8 Matrix effect, extraction recovery rate Matrix effect is a very obvious phenomenon in LC-MS analysis, which manifests as ion enhancement or inhibition. Extraction recovery rate and The matrix effect can be investigated simultaneously. Specific requirements include. a) Use low and high concentrations of quality control samples. At least 6 blank samples from different sources should be used for each concentration point. Merge should not be used. The matrix; b) At least 6 blank samples from different sources are used to conduct three sets of experiments (see Table 1), and the reagents and the volume are fixed before the instrument analysis The volume is the same, and then the average value of the peak area of the three groups is obtained, and the matrix effect and extraction recovery rate are calculated according to equations (4) and (5). A--The average value of the peak area of the standard substance solution; B--The average peak area of the standard substance of the corresponding concentration after the blank sample is extracted; C--The average value of the peak area of the standard substance of the corresponding concentration before the blank sample extraction. c) If the matrix effect exceeds ±25% or its relative standard deviation (RSD) exceeds 15%, the investigation base of blank samples from different sources should be added. Whether the qualitative effect has an impact on the detection limit, quantification limit, accuracy and precision. Validation example of extraction recovery rate and matrix effect See Appendix A. 8.9 Stability In order to ensure the accuracy and reproducibility of the analysis results, if necessary, according to the specific situation, if samples, processed samples, etc. exist, The stability can be verified when the unstable factor of the energy. Specific requirements include. a) Freeze-thaw stability 1) Quality control samples with at least two concentration points, low and high, should be used, with 9 replicate quality control samples at each concentration point; 2) Each cycle should be frozen at -20°C for 24h, and then placed at room temperature for 24h to melt; 3) After each freeze-thaw cycle (-20°C to room temperature), 3 quality control samples should be tested. Repeat 3 freeze-thaw cycles in total; 4) The freeze-thaw stability should be repeated at least twice; 5) Determine the signal response of the analyzed target with the processed sample (for example, the peak area of the target or the peak surface of the target and the internal standard) Product ratio) to compare with the signal response of the newly prepared quality control sample target; 6) The bias is within ±15%, which can be considered stable after repeated freezing and thawing. b) Long-term stability 1) Quality control samples at two concentration points, low and high, should be used, with 3 replicate quality control samples at each concentration point; 2) According to daily work requirements, actual samples should be frozen at -20°C, stored for a certain period of time, and then pretreated and analyzed. Determine the signal response of the analytical target (for example. the peak area of the target or the ratio of the peak area of the target to the internal standard), and compare it with Compare the signal response of the newly prepared quality control sample target; 3) The bias is within ±15%, which can be considered as stable for a certain period of time. c) The stability of the sample after treatment 1) Quality control samples at two concentration points, low and high, should be used, with 3 replicate quality control samples at each concentration point; 2) After the extraction process, it should be placed in the autosampler or according to the actual situation for 2h-24h or longer to determine the purpose of analysis. The response of the standard substance (for example. the peak area of the target substance or the ratio of the peak area of the target substance to the internal standard), and compare it with the newly prepared quality control sample Compare; 3) The bias is within ±15%, and the sample can be considered stable after treatment. 8.10 Dilution reliability If necessary, you can choose to verify the reliability of the dilution according to the specific situation, and the accuracy and precision of the sample should not be affected after dilution. Add the target substance to the blank sample to a concentration higher than the highest point of the calibration curve, dilute the sample with the same blank matrix, and then follow Analysis of established methods. At least three batches should be analyzed, the accuracy and precision after dilution should be within acceptable limits, and the bias should be within ±15% The precision RSD should be within 15%. Note. The requirements from 8.1 to 8.10 are the minimum requirements for laboratory verification of toxicant analysis methods, and the serial numbers of performance indicators do not represent the sequence of verification experiments. AAAppendix A(Informative appendix) Method validation example A.1 Overview This appendix introduces the calibration curve, delay effect, matrix effect and extraction method of ketamine in blood by liquid chromatography-tandem mass spectrometry. Take a verification example of the recovery rate. A.2 Calibration curve and delay effect It is expected to establish a calibration curve within the concentration range of 10ng/mL～1000ng/mL with linear regression. The standard substance of ketamine was added to the blank blood to prepare a series of concentrations of 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, 1500ng/mL and.2000ng/mL supplemented samples. The added sample at each concentration point is re Repeat 5 times. A blank sample was analyzed after each concentration point to evaluate the delay effect. The results show that the concentration range of 10ng/mL～1500ng/mL No delay effect was observed. Among the five blank samples analyzed after adding the sample at.2000ng/mL, a small amount of delay effect was observed in two blank samples, but Both are less than 10% of the lowest concentration point of the calibration curve, which is 10ng/mL, so the delay effect is within an acceptable range. The data obtained from the fifth calibration curve is shown in Table A.1.According to the established method analysis, taking the added......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of SFT0063-2020_English be delivered?Answer: Upon your order, we will start to translate SFT0063-2020_English as soon as possible, and keep you informed of the progress. The lead time is typically 1 ~ 3 working days. 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