Executive Summary for Fusion System

Multi-drug and Cannabinoids Screen in Blood and Urine by LC/Q-TOF Using SLE+ Extraction Plates

Overview

Idaho State Police Forensic Services (ISPFS) has established methods for the screening and confirmation of over 100 drugs utilizing an SLE+ extraction and running on an LC/MS/MS.   The screening extraction is performed and run on the instrument and after the data is analyzed, the confirmation extraction and run are performed.  These methods work well, and even though we are using different acquisition methods, mobile phases, and columns, best practice is to use two different types of technology for screening and confirmations.  The goal of the Multi-drug and Cannabinoids Screen validations were to develop and assess methods utilizing the LC/Q-TOF that could be used in place of the established screening methods that utilize the LC/MS/MS.

Methods

ISPFS already utilizes proven extraction methods (AM 25, 26, 27, and 28), so these methods were used for the validation with the following modifications:  (1) during the transfer to the SLE plate, 450µl of blood + base mixture or urine + base mixture was transferred and (2) 20% methanol in water (LC/MS grade) was used as the reconstitution solvent.

The following evaluation criteria was used for the methods: (1) the retention time for a peak must be within +/- 0.1minutes of the calibrator and (2) the sample must have a mass accuracy of +/- 10 and/or (3) the sample must have a mass abundance of 40 or greater.  Samples may also be evaluated as positive (typically based on peak presence and shape) or negative (typically based on peak absence or clear indicators that the “peak” is actually background noise) at the analyst’s discretion.  A sample may be moved forward for confirmation with a retention time that is outside the allowable window if it is due to the sample being at a high concentration (which is causing peak widening/shifting).

Results/Conclusions

Limits of detection were established for all compounds that met criteria.  Twenty samples that were previously analyzed were analyzed with the new methods and the results were consistent for seventeen of the twenty samples.  Inconsistencies in the three samples were due to the compound(s) not being included in the scope of the prior testing or not typically being reported (as they were metabolites).

Accuracy, precision and reproducibility, reportable range and sensitivity, specificity and selectivity, ion suppression and enhancement, carryover, interference and robustness, mass identifiers and TOF parameters, and stability were all investigated during the validations and were found to be acceptable for all but three of the compounds in the overall validation.  The three compounds that did not meet the criteria (specifically mass accuracy and/or mass abundance) were not were not included in the final approved method.  There were thirteen compounds that worked in the runs done at the Pocatello laboratory but not in the runs done in the Coeur d’Alene laboratory and as such, the Coeur d’Alene instrument was not approved for use for casework until troubleshooting was done and a performance verification was completed to demonstrate that it was working properly. 

The methods were approved for implementation by the Idaho State Police Forensic Services toxicology section and the analysts that participated in the evaluation were approved to perform the methods.

Multi-drug Screen in Blood and Urine by LC/MS/MS Using SLE+ Extraction Plates

Overview

Idaho State Police Forensic Services (ISPFS) has established methods for the screening and confirmation of over 100 drugs utilizing an SLE+ extraction and running on an LC/MS/MS or LC/Q-TOF.   This validation was performed to determine the suitability of adding new compounds and making modifications to the methods to increase efficiency.

Methods

Analytical methods #25 and #29 are approved, published methods for the testing and detection of multiple drugs by LC/MS/MS and LC/Q-TOF, respectively.  These methods use a sample volume of 250µl blood or urine, which is added to an analytical plate, extracted, and injected on the instrument.  One modification tested within the validation was a reduction of the sample volume from the required 250µl to 50µl of blood or urine.  This reduction in sample volume affected the method performance as there was a reduction in abundances of the compounds of interest and the internal standards.   To compensate for the reduction in sample volume, modifications were made to the acquisition parameters of the LC/MS/MS instrument that reduced the number of transitions being monitored for each compound from two to one to potentially increase the response.

The second modification tested was to eliminate the 15-minute plate shaking/incubation step that takes place after adding sample to the analytical plate and prior to adding ammonium hydroxide.  The purpose of removing this step was to reduce the extraction time.  Comparison runs were performed where the extraction was run as currently written versus run with eliminating the 15-minute shaking/incubation step.  The data showed that this change did not cause any issues with sample recovery or chromatography and as such, the analytical methods will be modified to remove this unnecessary step.  Since the extraction for AM #29 is identical to AM #25 and AM #28, this modification will also be implemented for it as well.   

The other portion of the validation was to determine if additional compounds could be added to the method without affecting the already existing compounds.  While some of the compounds worked well, there were a few that did not.  Overall, there appeared to be a loss in sensitivity with all the compounds, including those that were already validated and part of the existing methods.  This decrease in sensitivity is likely due to lowering the sample volume rather than adding in the new compounds.  As such, the addition of these compounds will be evaluated by adding the compounds to the analytical plate and evaluating them over the course of several runs (the addition of compounds to an existing run is detailed in AM #24).

Results/Conclusions

The validation was completed in the Idaho State Police Pocatello and Coeur d’Alene forensic laboratories using the following instruments: Agilent 1290 HPLC with Agilent 6470 MS/MS (Pocatello property #: 069910, Coeur d’Alene property #: 69679).  A total of eight validation runs were completed. 

Due to the reduction in sensitivity with the lower volume and only having one transition to evaluate for each compound rather than two, we have concluded those two updates to the method will not be employed. 

Screening Suspected Overdose Blood Samples with Randox MultiSTAT Analyzer

Overview
Idaho State Police Forensic Services (ISPFS) established and validated a method for the screening of suspected overdose samples using the Randox MultiSTAT Analyzer. This method is a preliminary screen, the screen results are an indication of drugs that may be present in a sample. The weight or confidence in screening results cannot be given the same as a confirmatory test. This is not a quantitative method and quantitative results cannot be reported from this method. If these results are reported it must be clear on the report that it is an indicator the drug is present in the sample, not a confirmation of the drug in the sample.

Results/Conclusions
Ten blood samples that were previously analyzed using the current ISPFS LC/MS/MS screening and confirmation methods were analyzed with the Randox MultiSTAT Analyzer. In addition to the case comparison samples, positive controls, negative controls, and negative blood samples were also analyzed.

The results of all the controls were consistent with the scope of the screening method, based on cross reactivities and the cutoffs.

There were some differences noted with the case comparison samples. One sample that had previously been test had methamphetamine confirmed at ~128ng/mL but did not screen positive for methamphetamine using the Randox analyzer. Most of the samples we receive that are positive for methamphetamine are believed to be the illicit (+/-) form of the drug. The assay is calibrated again the S(+)-methamphetamine and has 100% cross-reactivity for it. The cross-reactivity for the (+/-) form of the drug is 41.7%, and the expected concentration that would produce a positive result is 120ng/mL. Considering the uncertainty associated with the concentration of methamphetamine in our confirmation method (+/- 27%), I would expect that any sample that has methamphetamine confirmed with our methods over 160ng/mL to screen positive using the Randox MultiSTAT Analyzer, if it is strictly in the +/- form. For DUI cases, this number would not be sufficient, however, as this instrument is being used to screen suspected overdose samples, and the concentrations associated with methamphetamine overdoses are much larger than this, this increased cutoff for methamphetamine is appropriate. No case comparison samples were run that had amphetamine at a concentration that would be expected to screen positive on the Randox instrument. However, since that assay is also calibrated against the S(+) form and we would expect to see the (+/-) form for most cases, it is safe to assume that a concentration higher than the listed 50ng/mL cutoff would be required for the sample to screen positive on the Randox instrument.

There was a case comparison sample that was analyzed and screened positive for cannabinoids.

The sample showed a response of ~3.1ng/mL for carboxy-THC with the LC/MS/MS screen method.

The cutoff for the Randox method is 10ng/mL so it was not apparent why the sample screened positive at that level but since we had detected the carboxy-THC in our testing, and since the assay does react with other cannabinoids, we did not consider this a false positive.