Establishment and validation of analytical methods for biological samples


biological samples analytical

1. Specificity
It must be demonstrated that the substance measured is the expected analyte and that endogenous substances and other metabolites must not interfere with the determination of the sample. For the chromatographic method, at least 6 different individual blank biological sample chromatograms, blank biological samples plus reference substance chromatograms (indicate the concentration) and biological sample chromatograms after medication should be examined. For detection methods based on soft ionization mass spectrometry (LC-MS, LC-MS/MS, etc.), attention should be paid to the investigation of medium effects in the analysis process, such as ion suppression.

2. Standard curve and quantitative range
– A standard curve must be built with at least 6 concentrations, more concentration points may be required for non-linear correlations. The quantitative range should cover the concentration range of all biological samples to be tested, and the concentration of unknown samples should not be calculated by extrapolating the quantitative range;
– Blank biological samples should be accompanied when establishing the standard curve, but this point is not included in the calculation and is only used for evaluating interference;
– When the deviation between the measured value of each concentration point of the standard curve and the marked value is within the acceptable range, the standard curve can be judged to be qualified. The acceptable range is generally defined as the deviation of the lowest concentration point within ±20%, and the deviation of the remaining concentration points within ±15%. Only a qualified standard curve can quantitatively calculate the clinical samples to be tested.

3. Precision and Accuracy
– It is required to select 3 concentrations of quality control samples to conduct the precision and accuracy inspection of the method at the same time;
– The low concentration is selected near the lower limit of quantification, and its concentration is within 3 times of the lower limit of quantification; the high concentration is close to the upper limit of the standard curve; one concentration is selected in the middle;
– At least 5 samples are determined per batch for each concentration. To obtain inter-assay precision, at least 3 consecutive analysis batch samples shall be determined;
-Precision is expressed by the relative standard deviation (RSD) between batches and between batches of quality control samples, the relative standard deviation should generally be less than 15%, and the relative standard deviation near the lower limit of quantification should be less than 20%;
-Accuracy should generally be in the range of 85% to 115%, and should be in the range of 80% to 120% near the lower limit of quantification.

4. Lower limit of quantification
The lower limit of quantification is the lowest concentration point on the standard curve, and it is required to meet at least the drug concentration in the sample when measuring 3 to 5 half-lives, or the drug concentration when the Cmax is 1/10 to 1/20, and its accuracy should be within the true concentration. Within the range of 80% to 120%, the RSD should be less than 20%. It shall be evidenced by the test results of at least 5 standard samples.

5. Sample stability
According to the specific situation, the stability of the drug-containing biological samples at room temperature, under freezing or freeze-thaw conditions, and different storage times is investigated to determine the storage conditions and time of the biological samples. Attention should also be paid to the stability of the stock solution and the stability of the analyte in solution after sample processing.

– Determination after placing at room temperature for 8 hours;
-Place at -20°C for 15 days, freeze-thaw twice in the middle, and measure after the third freeze-thaw;
– After the third freeze-thaw, place it at 4°C for 8 hours and measure;
– The stability of the freeze-thaw process should be considered in three concentrations: high, medium and low, n=5.

6. Extraction recovery rate
Recovery is an important indicator to be examined when establishing a method, and it is divided into relative recovery and absolute recovery. Extraction recoveries at high, medium and low concentrations should be examined, with n=5 or 6, and the results should be precise and reproducible.

-Relative recovery rate: also known as the accuracy, the calculation method is to substitute the response value measured after the treatment of the drug-added sample into the standard curve of the sample, calculate the corresponding concentration value (A), and then divide it by the amount of the drug in the sample. Add the concentration value. The relative recovery mainly examines the standard curve and the accuracy of the established method;
-Absolute recovery: Also known as extraction recovery, it is calculated by dividing the measured response value of the dosing sample after treatment by the measured response value of the same concentration of standard dissolved in the mobile phase.

7. Problems that should be paid attention to when measuring
– Testing of unknown samples should begin after the validation of the biological sample analytical method has been completed;
– Each unknown sample is generally measured once, and re-measurement can be carried out if necessary. In pharmacokinetic comparison tests, biological samples from the same individual are preferably tested in the same analytical batch;
– A standard curve should be established for each analysis batch (in the case of tissue distribution test, it may be determined according to the specific situation), and three quality control samples of high, medium and low concentrations should be measured along with each other. At least two samples of each concentration should be uniformly distributed in Unknown samples are in the test sequence;
– When the number of unknown samples in an analysis batch is large, the number of quality control samples of each concentration should be increased, so that the number of quality control samples is greater than 5% of the total number of unknown samples;
– The deviation of the measurement results of the quality control samples should generally be less than 15%, and the deviation of the low concentration point should generally be less than 20%. At most, the results of 1/3 of the quality control samples are allowed to exceed the limit, but they cannot appear in the same concentration. If the test results of the quality control samples do not meet the above requirements, the test results of the analysis batch of samples will be invalid;
– Samples whose concentration is higher than the upper limit of quantification should be diluted with the corresponding blank medium and re-measured. For samples whose concentration is lower than the lower limit of quantification, in the pharmacokinetic analysis, the sample taken before reaching Cmax should be calculated as zero value, and the sample taken after reaching Cmax should be calculated as Not detectable (ND), To reduce the effect of zero value on the AUC calculation.