5.11.4 If the result of the microbial analyses is outside the specified acceptance criteria in Appendix A, Table 2, the cannabis testing entity shall determine the cause and take steps to remedy the problem until the result is within the specified acceptance criteria. Microbiology
Culture Methods - Qualitative and Quantitative
The quality control (QC) samples that are required for culturing of cannabis and cannabis products using qualitative and quantitative methods are included in Appendix A, Table 2.
Molecular Assays/Methods
The QC samples that are required for molecular (i.e., polymerase chain reaction (PCR), gel electrophoresis and probe-based qPCR with or without melting curve analyses) analysis of cannabis and cannabis products are listed in Appendix A, Table 3.
PCR positive DNA controls are used to verify that the PCR master mix and reagents were prepared correctly to produce amplification of the target nucleic acid. This type of positive control is analyzed with each PCR run.
A PCR run is defined as a group of samples that are analyzed at the same time under the same amplification conditions, using the same PCR master mix, and in the same thermocycler. A PCR run may contain more than one extracted sample batches.
A PCR run with multiple assays must have a DNA positive control for each assay.
Inhibition controls are used to verify that interfering constituents from a cannabis form, which may be carried over during isolation of nucleic acids or organisms during sample processing, do not inhibit the PCR. Because cannabis forms are constantly changing, inhibition positive controls must be performed in every extracted sample.
PCR DNA negative controls are used to verify that the PCR master mix and reagents were prepared correctly to produce amplification of the target nucleic acid. This type of negative control is analyzed with each PCR run. A PCR run is defined as a group of samples that are analyzed at the same time under the same amplification conditions, using the same PCR master mix, and in the same thermocycler. A PCR run may contain more than one extracted sample batches.
A PCR run with multiple assays must have a DNA negative control for each assay to verify that the amplification conditions are working properly.
No template controls are used to verify no contaminating nucleic acid has been introduced into the master mix. These controls are prepared when template is added to the master mix. They are prepared as separate PCR reactions to which aliquots of molecular-grade water or buffer are added to the master mix in place of target nucleic acid or sample. A negative result with this control indicates that the master mix and final processing reagents are not contaminated. This type of negative control is analyzed with each PCR run. A PCR run is defined as a group of samples that are analyzed at the same time under the same amplification conditions, using the same PCR master mix, and in the same thermocycler. A PCR run may contain more than one extracted sample batch. A PCR run with multiple assays must have not template controls for each assay to verify that the sterility of the assays.
One duplicate sample is required per run. A duplicate sample is subjected to all of the same steps as the original sample. For qualitative analyses, if the duplicate sample does not equal the sample result, the sample and its duplicate must be reanalyzed. Consideration should also be given to possibility of re-preparing and reanalyzing all associated samples. For quantitative analyses, if the RPD of the sample and duplicate is greater than 100, the parent sample and duplicate sample must be reanalyzed. Consideration should also be given to possibility of re-preparing and reanalyzing all associated samples. When data are accepted, the result for the sample portion designated as the "original sample" is reported.
5.11.5 Chemistry - Analytical, Organic and Inorganic (Metals). Quality control must be performed for each analytical, organic and metal chemistry method.
Each cannabis testing entity shall maintain sufficient raw data records to ensure the QC was performed at the frequency specified.
'Bracketing' of QC samples, rotating from across the calibration curve range, is required.
QC samples must follow the first twenty (20) samples after an initial calibration, every twenty (20) samples thereafter, and at the end of testing samples. This would also apply to a continuing calibration.
Initial Calibration
A. Samples results must be associated with an acceptable initial calibration. If the initial calibration is not acceptable, corrective actions must be performed and all associated samples re-analyzed.B. No sample results are to be reported nor data qualified for a failed initial calibration.C. Samples must be analyzed under an initial calibration that was performed no more than one month prior.D. The following items are required elements of an initial calibration: 1. The details of the initial calibration procedures including calculations, integrations, acceptance criteria, and associated statistics must be included or referenced in the method SOP. When initial calibration procedures are referenced in the method SOP, then the referenced material must be retained by the cannabis testing entity and be available for review;2. Sufficient raw data records must be retained to permit reconstruction of the initial calibration (e.g., calibration date, method, instrument, analysis date, each analyte name, and analyst or technician's initials or signature; concentration and response, calibration curve or response factor; or unique equation or coefficient used to reduce instrument responses to concentration);3. The cannabis testing entity must use the most recent initial calibration analyzed prior to the analytical batch;4. Standards used for calibration must be traceable to an international or national standard, when commercially available; and5. The cannabis testing entity must have a written procedure addressing removal and replacement of calibration standards.E. The lowest calibration standard must be at or below the lowest concentration for which quantitative data are to be reported without qualification.F. The highest calibration standard shall be at or above the highest concentration for quantitative data are to be reported without qualification.G. Sample results must be quantitated from the initial calibration and may not be quantitated from any continuing calibration verification.H. Criteria for the acceptance of an initial calibration must be established including any calculations (e.g., relative error, relative standard deviation). 2. Curve recovery of ±20% (and ±30% for the lowest point) for all points must be maintained.I. The cannabis testing entity must use and document a measure of relative error in the calibration as specified in the method SOP.Initial Calibration Verification
A. All initial calibrations must be verified with a standard obtained from a second manufacturer or a separate lot prepared independently by the same manufacturer.B. Initial calibration verification is performed by analyzing a test solution of known analyte concentration(s) after initial calibration and prior to sample analysis.C. In general, the check must be ± 20% (± 30% for the lowest point) of the known value. Some individual methods may require tighter tolerances (±10% of the known value).Continuing Calibration Verification
A. The validity of the initial calibration must be verified prior to sample analyses by a continuing calibration verification with each analytical batch.B. A CCV is performed by analyzing a test solution of known analyte concentration(s) prior to sample testing on each testing day and continued periodically during the analytical batch run, no less frequently than once after each set of 20 samples, and at the end of each run.C. The CCV must be a standard that is from the same vendor/lot that is used for the calibration curve.D. In general, the check must be ± 20% (and ± 30% for the lowest point) of the known value.E. Calibration must be verified for each compound, element, or other discrete chemical analyte, except for multi-component analytes where a representative chemical, related substance or mixture can be used.F. Instrument continuing calibration verification must be performed at the beginning and end of each analytical batch, and at the frequency defined in the method.G. Sufficient raw data records must be retained to permit reconstruction of the continuing instrument calibration verification (e.g., method, instrument, analysis date, each analyte name, concentration and response, calibration curve or response factor, or unique equations or coefficients used to convert instrument responses into concentrations).H. Continuing calibration verification records must explicitly connect the continuing calibration verification data to the initial calibration.I. If the continuing instrument calibration verification results obtained are outside the established acceptance criteria, the following steps must be taken:1. If a cause for the calibration verification failure is identified that impacts only the calibration verification sample (e.g., a missed autosampler injection), then analysis may proceed if a second calibration verification sample is analyzed immediately and the result is within acceptance criteria. Samples analyzed previously must be considered valid if bracketed by a passing calibration verification sample. The cause for the failure of the first calibration verification result must be documented; and2. If the cause for the calibration verification failure is not identifiable or has impacted other samples, then corrective action must be performed and documented. Prior to analyzing samples, the cannabis testing entity must demonstrate acceptable performance after corrective action with calibration verification or a new initial calibration must be performed. Samples analyzed prior to the calibration verification failure must be reanalyzed.J. Data associated with an unacceptable calibration verification must not be reported with a qualifier. Qualifying the data is not an acceptable approach.Low Level Continuing Calibration Verification
A. A LLCCV will be run at the end of each analytical batch.B. The measured value must be within ± 30 % of the prepared value.C. The cannabis testing facility entity shall prepare and analyze at least one of each of the following QC samples for each analytical batch: 1. Negative Control, Method Blank; and2. Positive Control, Laboratory Control Sample (LCS);4. Duplicate matrix spike sample; andD. The required QC is summarized in Appendix 2, Table 4.Negative Control - Method Blank (MB)
A. A method blank must be analyzed at a minimum of one (1) per preparation batch.B. The MB must be processed along with and under the same conditions as the associated samples to include all steps of the preparation and analytical procedure.C. The MB is used to assess the samples in the preparation batch for possible contamination during the preparation and processing steps.D. The measured concentration of each analyte in the MB or LRB must be < LOQ or MRL.E. Procedures must be in place to determine if a MB or LRB is contaminated. While the goal is to have no detectable contaminants, each method blank must be critically evaluated as to the nature of the interference and the effect on the analysis of each sample within the batch.F. The source of contamination must be investigated and measures taken to minimize or eliminate the problem and affected samples reprocessed if the concentration of a targeted analyte in the blank is at or above the LOQ, if the blank contamination otherwise affects the sample results as per the method requirements or the individual project data quality objectives, and a blank is determined to be contaminated. Samples associated with a contaminated blank must be evaluated as to the best corrective action for the samples (e.g., reprocessing or data qualifying codes). In all cases, the corrective action must be documented.G. Any affected samples associated with a contaminated MB or LRB must be reprocessed for analysis.Positive Control - Laboratory Control Sample (LCS)
A. The LCS is used to evaluate the performance of the total analytical system, including all preparation and analysis steps.B. The LCS must be carried through the entire sample preparation process and analyzed.C. The LCS must be spiked with all target analytes at a mid-level concentration in the curve.D. The LCS must be analyzed at a minimum of one (1) per preparation batch.E. The LCS is a quality system matrix, known to be free of analytes of interest, spiked with known concentrations of analytes that are within the calibration range.1. A laboratory control sample (LCS) may be used in place of a continuing calibration verification (CCV) (but not as a replacement for a failing CCV) for methods where the calibration goes through the same process as the LCS. Note that the more stringent acceptance criteria must be met.2. The matrix spike may be used in place of this control as long as the acceptance criteria are as stringent as for the LCS.3. The lab may use commercially available or pre-prepared standards (separate from calibrators) for QC.F. All analyte concentrations must be within the calibration range of the methods.G. The individual LCS must be compared to the acceptance criteria stated in the standard operating procedure. The results of the individual batch LCS are calculated in percent recovery or other appropriate statistical technique that allows comparison to established acceptance criteria. The cannabis testing entity must document the calculation.H. When the acceptance criteria for the positive control are exceeded, those sample results must be investigated, and a corrective action implemented.Matrix Spikes and Matrix Spike Duplicates
A. Analyze an actual sample with a known amount of standard added (matrix spike. MS). A second portion of the actual sample used to prepare the MS that is spiked and processed in the same manner as the MS (matrix spike duplicate, MSD). 1. For potency testing, a "representative matrix" may be used to prepare the MS/MSD.2. MS/ MSD shall be spiked at a midlevel concentration with the target analytes.B. Calculate the relative percent difference (RPD) between first sample and replicate. The calculations must be documented, and the target value must be close to the first value and have a RPD of less than 20%.C. Matrix-specific QC samples indicate the effect of the sample matrix on the precision and accuracy of the results generated using the selected method. The information from these controls is sample/matrix specific and would not normally be used to determine the validity of the entire batch.D. For methods that include one (1) to twenty (20) targets, spike all components.E. For methods with more than twenty (20) targets, randomly spike at least sixteen (16) components.Sample Duplicate
A. Analyze the same sample twice, using two separate preparations. The sample should be chosen at random and run together on the same analytical run.B. Calculate the relative percent difference (RPD) between first sample and replicate. Calculations must be documented, and the target value must be close to the first value and have a RPD of less than twenty percent (20%). Variability may be introduced during sample preparation. To account for this, if more than one staff member is prepping samples, each staff must also prepare and analyze a sample matrix duplicate for each set of prepared samples.