1. What are residues and contamination?
Carry-over is a phenomenon caused by a small amount of the analyte in the previous sample remaining in the system and being introduced into the next sample injected, or by the adsorption of the analyte in the injection system.
Residues can be mainly divided into three types:
The first is that the residues in the traditional sense mainly come from the system, mainly due to the dead volume in the system.
· Residues due to adsorption (experimental consumables, pipelines).
• Residues formed by incomplete elution in chromatography.
Contamination is any phenomenon that can interfere with the assay introduced during the experimental operation of sample reception, handling, and analysis. Any form of carryover can be considered contamination, so contamination is more random and multi-source than carryover, which also makes it more difficult to diagnose and correct.
Pollution can be divided into three categories:
• Samples are contaminated with analytes during storage and prior to sample preparation, which is associated with the process of transferring from human/animal samples to sample tubes and storage.
• The sample was contaminated with analytes during preparation, which manifested itself as a process related to the sample preparation from sample preparation to LC-MS analysis.
• Caused by an unknown compound that behaves like the analyte, mainly as a manifestation of ionization inhibition or enhancement due to matrix effects.
Since carryover and contamination both affect the precision and accuracy of the method, they must be carefully monitored and controlled.
2. Evaluation of residues and contamination on experimental results
According to FDA’s bioanalytical guidelines, if the analyte peak area in the residual blank is greater than 20.0% of the LLOQ peak area or the peak area observed for the internal standard should not be greater than that observed in calibration samples, quality control samples, and other samples in the same analytical batch 5.0% of the average peak area of the internal standard, then residues may affect the results of the analysis and need to be evaluated for residues.
Absolute carryover (AC) = residual peak area in blank sample/preceding sample peak area
Unknown sample concentration difference (DC) = preceding sample peak area/subsequent sample peak area
Residual Impact Percentage (ECI%) = ACⅹCDⅹ100%
When the residual influence percentage (ECI%) of the sample is less than 5%, it can be considered that the sample is not affected, on the contrary, the sample needs to be re-analyzed or re-injected.
3. Control and elimination of residues and contamination
Carryover can be assessed by analysis of a blank matrix following a high concentration standard or QC sample. Alternatively, several matrix blank samples can be analyzed at the beginning of the analytical batch to confirm that the instrument system is clean and free of contamination.
Generally speaking, sample analysis experiments with compound residues can be eliminated by replacing residual reagents, chromatographic columns, etc., but it is impossible to eliminate residues in many cases, so it is necessary to arrange the sample sequence reasonably: such as referring to the blood drug metabolism curve Concentration, arrange low-concentration samples in the front, high-concentration samples at the back, or add blank samples after high-concentration samples to reduce carryover.
The presence of contamination can also be judged by monitoring the presence of compounds and internal standards in matrix blanks, reagent blanks and solvent blanks. In addition, in practical experiments, we often use the air injection needle method to judge whether contamination and residues come from the injection plate or the From the instrument pipeline, by selecting an appropriate elimination method to find the cause, the speed of investigation can be effectively accelerated.
If compound peaks are found in the injection air needle, it can be judged that the contamination comes from the instrument pipeline and has nothing to do with the sample. It is necessary to check and repair the pipeline interface, ion source, injection needle, chromatographic column and other components. If compound peaks appear in both the initial matrix blank and the blank after the high-concentration sample, the contamination comes from the matrix or sample preparation, the pipettes, reagent consumables used in the pre-experimental treatment, the personnel operation or the replacement of different batches. the blank matrix for investigation. If the compound peak appears in the reagent or solvent, the contamination may come from the solvent used in the experiment. Replace the experimental reagents of the same brand with different batches or different manufacturers. If contamination occurs only before dosing, in control samples and not in matrix and solvent blanks, it indicates that contamination may have occurred outside of the analytical laboratory, from incorrect dosing or in vitro contamination after sample collection, requiring coordination with other departments Investigate multiple links such as sample collection, drug administration or animal feeding. In severe cases, the entire experiment needs to be repeated.