When declaring a new drug, the analytical method of the drug quality standard must be verified; when the drug production process is changed, the composition of the preparation is changed and the original analysis method is revised, the quality standard analysis method must be verified;
The methodological verification of GMP drug analysis in drug analysis and test is the prerequisite and basis for ensuring the accuracy of drug analysis results and also the necessary factor for the realization of GMP in drug analysis.
Constitute the drug analysis of the detection method validation, which involves the following aspects:
1, the analysis method to verify the prerequisite for success:
(1) The instrument has been confirmed, corrected and expired
(2) Trained personnel
(3) reliable and stable reference substance
(4) reliable and stable experimental reagents
(5) confirm the stability of the test solution, no degradation within the specified time.
2, analytical methodological verification required verification content:
(1) Determination of content
(2) Determination of impurity content
(3) qualitative identification of drugs
(4) drug content uniformity determination
(5) Microbial detection of drugs
(6) detection of bacterial endotoxin of drugs
Verification content:
Accuracy:
Accuracy is measured by the method and the actual value of the results or reference value close to the degree, expressed as a percentage recovery.
The specific method of measuring the recovery rate R (recovery) can be measured by the sample recovery test method. Add sample recovery test has been accurately measured the actual sample of drug content P + known amount of A reference substance (or standard) determination, the measured value of M. Data requirements: within the scope of the provisions of at least 9 times the evaluation of the evaluation, such as the preparation of high, medium and low three different concentrations of each measured three times
Precision:
(Repeatability, intermediate precision and reproducibility Precision refers to the degree of closeness between the results of multiple samples taken from the same uniform sample under the specified conditions, and the deviation (d), standard deviation (SD) Relative standard deviation (RSD) (coefficient of variation, CV).
(1) Repeatability: The same analyst shall determine the precision of the results obtained; under the same conditions, at least 9 times the results of the evaluation, such as the preparation of three different concentrations of each test three times Or the measured concentration as 100%, at least 6 times to evaluate
(2) intermediate precision The same laboratory, at different times by different analysts with different equipment resulting precision
(3) reproducibility Different laboratories, different analysts to determine the precision of the results
(4) Data Requirements: Report SD, RSD and Trusted limits.
Specialties:
Refers to other methods (impurities, degradants, excipients, etc.) may exist in the case of the method used to accurately measure the characteristics of the measured object can reflect the method of coexistence when the test material for accurate and exclusive determination of ability, Refers to the law is used for complex sample analysis whether the degree of mutual interference measure
• The difference between the two test results, ie the specificity, is usually obtained by comparing the results obtained with those of the samples without the aforementioned ingredients by analyzing the samples containing impurities, degradation products, chemicals or placebo components
• Discrimination reactions should be distinguishable from substances or structurally similar compounds that may coexist, samples that are not analyzed, and compounds of similar structure or composition, should be negatively responsive
• Determination of content and determination of impurities --- Chromatography and other methods should be accompanied by a representative map, also shows the specificity. The figure should indicate the location of each component, the chromatographic separation should meet the requirements
• In the case of impurities, impurities or excipients may be added to the sample to determine whether the test result is interfered with or not; for impurities, a certain amount of impurities may also be added to the sample to see whether the impurities are separated
Detection limit:
Detection limit refers to the sample under certain experimental conditions, the measured substance can be detected by the lowest concentration or content. It is a limit test performance index, without quantitative determination, as long as the above or below the specified concentration can be.
• Non-Instrumental Analysis Visual Method: Using a test substance of known concentration, the lowest concentration or amount that can be reliably detected
• Signal-to-noise ratio method: An analytical method (instrument analysis method) for displaying a baseline noise is to compare a signal of a known low-concentration sample with a signal of a blank sample to calculate a signal that can be reliably detected The lowest concentration or amount. The detection limit is generally determined at the signal to noise ratio of 3: 1 or 2: 1 at the appropriate concentration or amount injected into the instrument
• Standard deviation can also be used:
Blank value = 0;
① The background is measured 10 times or more to find the standard deviation σ
② multiply σ by three times;
③ in the working curve to find the corresponding concentration of 3σ X, is the method of detection value;
Blank value is not equal to 0;
① measuring the background more than 10 times, find the standard deviation σ;
② multiply σ by three times;
③ in the working curve to find the corresponding concentration of 3σ X;
④ The corresponding concentration value obtained by adding the blank value of the detection limit of the method
Limit ofquantitation LOQ
• refers to the sample can be measured in the quantitative determination of the lowest amount, the result should have a certain accuracy and precision requirements
• Commonly used signal-to-noise method to determine the limit of quantification, usually at a signal to noise ratio (S / N) of 10: 1 corresponding to the concentration or the amount of injected equipment to determine, you can also use the instrument to measure the background of the blank response standard deviation (SD) 10 times the estimated value, and then test the actual determination of the lower limit of the method
The limit of quantitation:
Refers to the sample can be measured in the quantitative determination of the minimum amount, the result should have a certain accuracy and precision requirements
• Commonly used signal-to-noise method to determine the limit of quantification, usually at a signal to noise ratio (S / N) of 10: 1 corresponding to the concentration or the amount of injected equipment to determine, you can also use the instrument to measure the background of the blank response standard deviation (SD) 10 times the estimated value, and then test the actual determination of the lower limit of the method
Linear:
Within the scope of the design, the test result is directly proportional to the analyte concentration in the sample. Linear shape is usually least square method to process the data to obtain the slope of the regression curve (Slope) to represent. Data Requirement: At least five concentrations are required for investigation of linearity. The correlation coefficient, y intercept (which is the possible bias of the assay), regression slope and variance should be provided. The regression equation and the linear graph
Note: When doing linear, it should be noted that as far as possible to do more, the linear range of a wide range of concentrations, the benefits of doing so is: When you examine the accuracy, you do the concentration of the recovery rate is larger, the preparation of the solution It is easy to operate, otherwise, when you do a linear study, you do linear range of concentration is not wide, then when you examine the recovery rate, when you use the access method to investigate the concentration range is not wide, the operating concentration is difficult to control , Resulting in practical difficulties. I came across a person who was doing a methodological study. Since she did not have a linear concentration when examining the linearity, the amount of added linearity caused by the amount of addition after the recovery was made exceeded the concentration of the linear range of investigation, and I said that Is not acceptable. Because when doing the analysis and calculation, it is precisely calculated by using the linear relationship. The result is certainly doubtful and the result can not be affirmed! (This is the experience summary)
range:
Refers to a certain precision, accuracy and linearity of the test method for the application of high and low concentration or amount of range. The range should be based on the specific application of analytical methods and linearity, accuracy, precision results and requirements identified.
• The range of APIs and preparations is 80% -120%; the content uniformity of preparations is 70% -130%; the determination of impurities should be 120% of the reported impurities; the dissolution should be within ± 20%. If the limits are set, -20% of the lower limit to + 20% of the upper limit, such as 1h70% of the extended release tablets, the validation range is 0-90%.
Durability:
Refers to a slight change in the measurement conditions, the results are not affected by the degree of acceptance, to provide the basis for routine testing. Is a measure of the reproducibility of laboratory results between laboratories and staff under normal conditions; it should be noted if the method is susceptible to the conditions of the analysis or to the requirements. Typical changes include: analysis of solution stability, extraction time and so on.
Methodological Verification of Drug Analysis (2)
Acceptance criteria for content determination methods
* Capacity analysis method: the precision of the raw material drug product (content> 99.5%) or reference substance inspection method, the relative standard deviation should not be more than 0.2%; conduct the recovery test. The recovery rate is generally between 99.7 ~ 100.3%
* UV method: Determination of the appropriate concentration of refined products, the RSD is generally not more than 1%. Formulation determination, the recovery rate should generally be between 98% ~ 102%; Linear: Absorbance A is generally 0.2 ~ 0.7, concentration point n = 5. A linear regression of concentration c to A yields a straight line equation, where r should be 0.9999 (n = 5) and the intercept of the equation should be close to the acceptance criterion validated by the zero content determination method
* HPLC method:
* Requires RSD%, recovery rate between 98% ~ 102%.
* Linear range: Preparation of a series of standard solution with refined products, the concentration point n should be 5 to 7, with the concentration of c or peak height h or the response of the measured value of the regression processing, the establishment of regression equation, r should be greater than 0.999 , Intercept should tend to zero
Analysis Method Verification Steps:
* Verification scheme development
* Verification purposes, methods to improve the background,
* The APIs and products, equipment outline, reagents and reference materials provided
* Method to be verified, project, eligibility criteria
* Implement staff training
*references
* Verify the implementation
* Collect complete verification process records and original maps
* Review
* Verification Report
* Evaluate whether the method is validated
Method validation and method transfer
* Method validation of new products is usually completed in the product development phase; post-mass production verification usually includes method validation and method transfer
* Confirmation of analytical method refers to the confirmatory experiment in our laboratory that is applicable to the products involved when the national or pharmacopoeia standards are intended to change or have changed
* Verify that the old and new methods should be checked before identifying all the changes
* Test sample information (batch number, size), change the project's acceptance criteria
* Confirmed test samples usually include normal products, stability products
* Method transfer refers to the test performed when the transfer method is transferred from the transferor to the receiver.
* PH (± 0.5)
* Mobile phase composition (organic phase ± 5%)
* Column (three batches of the same manufacturer, or two different manufacturers (with the stationary phase and filler, size)
* Temperature (20-25C) and flow rate (± 10%)
* Detection wavelength (± 5nm)
Inspection items and verification summary
* Discrimination test in addition to specificity, durability, the other is not required.
* Extent of impurity inspection In addition to specificity, detection limit, durability, the other is not required
* Quantitative determination of impurities in addition to the detection limit, the other requirements.
* Determination of content and dissolution in addition to detection limits, quantitative limits, the other are required
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