Researchers and forensic agencies that test samples or screen post mortem patient specimens for drugs of abuse currently employ immuno- or colourimetric assays. These assays suffer from a number of limitations:
They lack specificity.
They are not as commonly available for novel sample matrices such as saliva as they are for urine or serum samples.
They can be relatively costly due to the high volume of reagents used.
They are not commercially available for many significant drugs and their metabolites.
In addition, with conventional approaches, testing for drugs of abuse is done separately for each of several classes of compounds (e.g. opiates, benzodiazepines, etc.), which requires time-consuming multiple assays. To screen for a portfolio of drugs from different compound classes, it is often necessary to perform repeat analysis on indvidual samples.
This application note describes a novel LC/MS/MS research method for the simultaneous screening of 23 drugs of abuse from extracted oral fluid samples. This method provides equal or better sensitivity than traditional immunoassays, while also being more specific, more cost effective, and less time consuming.
Sensitive and selective Multiple Reaction Monitoring (MRM) scan function provides linear quantitative analysis with wide dynamic range.
The patented LINAC collision cell within the Applied Biosystems/MDS SCIEX API 3000TM Mass Spectrometer increases the number of compounds that can be analyzed simultaneously, without loss of sensitivity or increased LC separation.
The quantitative facility of the Applied Biosystems/MDS SCIEX Analyst Software organizes and displays results for quick interpretation.
Results and Discussion
Calibration samples at or below current established cut-off levels were provided by Leeds Teaching Hospitals NHS Trust. The following data were generated from these calibration samples as well as patient samples kindly provided by Leeds Teaching Hospitals NHS Trust
Comparison with Current Methods
The following figures summarize the ability of the method to detect concentrations of the drugs of abuse described earlier at levels at or significantly below current established cut-off levels.
Fig. 1 shows a selection of opiates at 1 ng/ml in an oral fluid matrix. This is equivalent to approximately 10 times less than the current cut-off level.*
Figure 2 shows a range of benzodiazepines at 1 ng/ml in oral fluid matrix. This is equivalent to the current established cut off level.*
Multiple Compound Screening
As the method presented in this research study allows the screening of multiple compounds from various classes, it is possible to detect the presence of abused drugs in the presence of prescribed drugs. Figure 4 shows a patient sample with a large signal for methadone. However, smaller signals for morphine, codeine and 6-monoacetyl morphine can also be seen at levels above the cut-off.* The detection of 6-monoacetyl morphine is of particular significance, as it is a primary product from the metabolism of heroin.
Multi-compound screening also makes it possible to look at various drug metabolites. This gives further credence to suspected positive results by monitoring for the major metabolite. Figure 5 shows an actual patient sample, once again with a high methadone result. In this case, however, the sample also shows positive results for cocaine and for benzoylecgonine, a cocaine metabolite.
Another potential application for multi-compound screening is for determining whether or not a sample has been adulterated. For example, screening for methadone metabolites could establish if methadone had actually been taken or merely added to the sample prior to analysis.
Interpretation of Results Using the Analyst software, numerical results can be generated and results tables can be customized for easy interpretation and use. A typical results table, generated using a single point calibrator at the cut-off level, is shown in Figure 6. This table provides the analyst with immediate information as to whether the levels detected in the samples are above or below the accepted cut-off levels.*
* Refers to cut-off levels at time of going to press.
The LC/MS/MS method described here provides a rapid, simple and reliable test to detect 23 drugs of abuse in oral fluid. Based on the results of this study:
This method has potential as a fully quantitative testing method in research and forensic applications.
The method can be used to ascertain whether the detected level is above or below an established cut-off value, giving a simple yes/no test in the style of commercially available immunoassay systems.
Due its high sensitivity, the method has the potential to minimize sample preparation, in some cases, even to the point of direct sample injection.
Application of this method is further simplified by using the Analyst software to customize the presentation of results, allowing only relevant information to be displayed for rapid interpretation and use.
Applied Biosystems/MDS SCIEX acknowledges Stephen Lock, Daniel Blake and Michael Baynham, and our collaborator Keith Allen from Leeds Teaching Hospitals NHS Trust, UK for providing the method, samples and data for this application note .