LC-MS/MS in Drugs of Abuse Testing

Drugs of abuse testing is performed to identify drug abuse, to monitor someone with a substance abuse problem, or to detect drug intoxication and overdose. The identification of drug abuse in biological samples can be used in court as scientific evidence, and it can help to improve the quality of clinical management during emergencies. One of the most common screening methods used for the detection of drugs in urine and other matrices are immunoassays. They are convenient, but they have their limitations: results are often class-specific and cannot be attributed to a specific drug or drug metabolite. Antibodies are also susceptible to cross-reactivity with structurally related and unrelated compounds, which increases the risk of false-positive results. This is why data gathered by immunoassays are considered as presumptive.

GC-MS vs LC-MS/MS

An immunoassay requires a second analytical procedure to confirm the quantitative determination, and this is usually performed by either GC-MS or LC-MS. The mass spectra obtained from the GC-MS can be compared with large databases, enabling the unknown abusive drugs to be identified – this is one of the reasons why GC-MS has been the gold standard in drugs of abuse testing for many years. However, most compounds of interest need to undergo a chemical derivatisation to make them more volatile and compatible with GC analysis – without derivatisation, GC-MS generally offers poor peak shapes, lower resolutions and reduced sensitivities. However, undertaking more sample preparation steps also increases the risk of errors, and acidic derivatisation can be prone to uncertainties, such as the reagent quality, the presence of interferences, and variable lab conditions.

In contrast, LC-MS is ideal for polar and non-volatile molecules such as those analysed in drugs of abuse testing. An efficient separation and ion generation can be achieved without derivatisation and LC-MS generally requires less sample preparation than GC-MS. Among the different mass spectrometry platforms, triple quadrupole mass spectrometry with multiple reaction monitoring (MRM) is the most commonly adapted technique.

 

LC-MS/MS – From Theory to Practice

A laboratory tested a commercial LC-MS/MS assay (MassTox® Drugs of Abuse Testing, Chromsystems) and compared it with GC-MS, with a focus on routine analysis [1]. The sample prep for the amount they routinely deal with usually takes 6 hours (excluding hydrolysis), but by using the commercial assay, the lab was able to reduce the time down to 2 hours. The switch from GC-MS to LC-MS reduced the resources required for the sample prep, and the sample volume required for the sample preparation was also significantly lower (see Table 1).

The lab also conducted comparative analysis between the commercial assay and an in-house LC-MS assay used by an external accredited laboratory. The values obtained correlate very well with each other across a range of concentrations demonstrating a high accuracy, as showcased by the linear results. Therefore, the commercial assay (Chromsystems) is suitable for replacing LC-MS/MS in-house methods and allows for the target screening and/or quantitative confirmation of 106 drugs in a single run (Fig. 1). Proficiency testing schemes from GTFCh and RfB, in which the assay has been used, also confirmed its accuracy [1].

 

  GC-MS in-house methods LC-MS/MS Assay
Sample Prep Time (excluding hydrolysis) 6 h per day 2 h per day
Sample Volume 1000-5000 µl 50 µl
Run Time 10-15 min 15 min

Table 1: Comparison of GC-MS with LC-MS/MS

Fig. 1: Comparison of a commercial assay with an in-house method. Both assays provide similar data

The lab also conducted comparative analysis between the commercial assay and an in-house LC-MS assay used by an external accredited laboratory. The values obtained correlate very well with each other across a range of concentrations demonstrating a high accuracy, as showcased by the linear results. Therefore, the commercial assay (Chromsystems) is suitable for replacing LC-MS/MS in-house methods and allows for the target screening and/or quantitative confirmation of 106 drugs in a single run (Fig. 1). Proficiency testing schemes from GTFCh and RfB, in which the assay has been used, also confirmed its accuracy [1].

Fig. 3: Chromatogram of the commercial assay – more than 100 drugs can be tested within a single run

Target Screening and Confirmation in One Run

Immunoassays often require an alternative method to confirm the results, and this is how many organisations have laid out their drug abuse testing schemes. However, LC-MS/MS has an accuracy and selectivity that is a sufficient to do both in one step. This is why commercial assays, such as those from Chromsystems, enable the target screening and quantitative confirmation of more than 100 drugs in a single run (Fig. 3), including benzodiazepines, opioids, booster, and Z-drugs. In the case of a positive result, the quantification can be evaluated straight away from the same peak. Labs might find this option in drug of abuse testing appealing, as it reduces the resources required without compromising on the accuracy.


Reference/Original Study:

[1] Geffert et al., Validation of a New LC-MS/MS Assay for the Analysis of Drugs in Urine and Comparison with Established Analytical Methods (GC-MS and LC-MS/MS): Advantages for daily Laboratory Routine. GTFCh Symposium 2019.

[2] Wang P et al., Incomplete Recovery of Prescription Opioids in Urine using Enzymatic Hydrolysis of Glucuronide Metabolites. J. Analytical Toxicology, (2019), 571-575.

[3] Hackett LP et al., Optimizing the hydrolysis of codeine and morphine glucuronides in urine. Ther Drug Monit., (2002), 652-657.

[4] Opiate & Benzodiazepine Confirmations: To Hydrolyze or Not to Hydrolyze is the Question. J. of Appl. Lab Med., (2018), 1-9.

Article originally published in Clinical Laboratory International, September 2019, p. 24-25


LC-MS/MS in Amino Acid Analysis

The quantitative amino acid analysis (AAA) is crucial for the diagnosis of inherited diseases that affect amino acid metabolism. The most frequent are classical phenylketonuria (PKU) and Maple syrup urine disease (MSUD), but there are also many other rare diseases. Amino acid analysis requires high sensitivity and specificity. Over the years, the gold standard technique has been Ion Exchange Chromatography (IEC). As with most techniques, IEC does have some drawbacks, and a recent study by Carling et al suggests that it might be time to rethink how clinical laboratories approach quantitative amino acid analyses.

The Problems with Ion Exchange Chromatography (IEC)

Even though IEC is considered the go-to method in clinical diagnostic settings, its operation has been unchanged for over four decades. Nevertheless, there are enough drawbacks to warrant looking into other techniques: some of the practical disadvantages of IEC are a poor retention of acidic compounds, single point calibrations, and the need for post-experimental analysis programmes to negate some of these limitations. However, one major roadblock for clinical laboratories is a long analysis time of more than 120 minutes, which limits the speed of reporting results for clinically urgent samples and inhibits the efficiency in the routine laboratory workflow.  

LC-MS/MS: A New Approach to AAA

A new study looked at LC-MS/MS as an alternative method. The researchers compared IEC with different commercial LC-MS/MS assays for the determination in human plasma samples, including the Chromsystems MassChrom® Amino Acid Analysis test. Generally, not all LC-MS/MS methods showed the performance required for all analytes. However, the analysis carried out with the MassChrom® assay showed a performance comparable to IEC, but with a higher degree of specificity and a much quicker analysis time of less than 19 minutes for 48 amino acids. “Sample preparation is quick and simple, taking approximately 20 minutes and utilising sensible volumes for a manual assay”, the authors said. The specificity was achieved via a combination of chromatographic resolution and selected reaction monitoring (SRM) approaches. “The underivatised approach has an impact on the sensitivity of the analytes with a small m/z.”, the authors also concluded. The Chromsystems assay was the only one in this study to also separate the isobaric compounds Leucine, Iso-Leucine and Allo-Leucine, eliminating the need for second line testing for these analytes.

Fig.1: The Chromsystems-assay provides an efficient separation of all isobars such as Leu, Ile and Allo-Ile.

Conclusion

LC-MS/MS in general, and the MassChrom® assay in particular, demonstrated an analytical performance that is comparable to that of an IEC analysis. However, the amino acid analysis is performed much faster and with a higher degree of specificity. The availability of a commercial CE-IVD compliant reagent kit from Chromsystems can simplify reagent management procedures required for accreditation to UKAS standards. “The benefit of this in the routine clinical laboratory should not be underestimated”, the authors wrote.
The study ultimately concluded that because the same - or better - results can be obtain faster, then there is no longer a need to use IEC and it should no longer be classified as the gold standard method for plasma amino acid analysis. Results in the study were also presented in a webinar that you can watch on-demand here.

Watch Webinar Rachel Carling

 

Dr. Rachel Carling, Consultant Scientist and Director of Biochemical Sciences, Viapath, UK

Rachel is working at Guy's and St Thomas’ Hospital where she is a Consultant Scientist, Director of Newborn Screening and Clinical Lead, Biochemical Sciences. She is also the Scientific Director for Viapath at the GSTT site. Her area of interest is inherited metabolic disease with a particular interest in tandem mass spectrometry.


Reference/Original Study:

Carling R. S. et al, Challenging the status quo: A comparison of ion exchange chromatography with liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods for the measurement of amino acids in human plasma, Annals of Clinical Biochemistry¸ 2020, DOI: 10.1177/0004563220933303