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 derivatization to make them more volatile and compatible with GC analysis – without derivatization, 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 derivatization 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 derivatization 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].
100% hydrolysis, 0% doubt
In the human body, many drugs undergo glucuronidation, which requires an enzymatic or acidic hydrolysis prior to the analysis – a challenge for many assays. Enzymatic hydrolysis varies in its effectiveness depending on the drug and the enzyme [2]. Erratic quality assurance results for codeine – one of the more difficult to hydrolyse compounds – are considered to be based on an incomplete hydrolysis [3]. Consequently, some papers recommend the use of an acidic hydrolysis, however, this can degrade both opioids and other substances [4]. Furthermore, this approach can convert oxycodone to oxymorphone, and codeine to morphine by demethylation, which increases the risk of false-negative or false-positive results. To overcome this drawback, our lab has developed an enzymatic hydrolysis process that is effective for hydrolysing all glucuronides within 2 hours. This has been achieved by using a carefully selected enzyme that ensures a complete and selective hydrolysis of all 106 drugs that are covered in the assay, including codeine. The effectiveness has been demonstrated by measuring the hydrolysis of several substances over time: Easy-to-hydrolyse glucuronides become fully hydrolysed quickly, while others, such as codeine, require longer. After 2 hours, the hydrolysis is complete for all the compounds (Fig. 3).

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.2), 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.
[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.

The author
Marc Egelhofer, PhD, Head of Marketing Communications, egelhofer@chromsystems.de

Offering the most advanced screening technology on the market, Randox has transformed the landscape of emergency point-of-care testing with innovative drug of abuse (DoA) and stroke diagnosis and detection.

The Evidence MultiSTAT – Utilizing Randox’s Biochip Technology
Designed to work across a wide variety of matrices, this revolutionary multi-analyte testing platform allows clinicians to achieve a complete immunoassay profile in the initial screening phase. Offering the most advanced screening technology on the market, Randox has transformed the landscape of emergency point of care testing. The Randox drugs of abuse Biochip Arrays form part of an unrivalled toxicology test menu capable of detecting over 500 drugs and drug metabolites along with the new addition of the Randox Stroke Biochip. Moving away from traditional single analyte assays, Biochip Technology boasts cutting-edge multiplex testing capabilities providing rapid and accurate detection and diagnosis from a single sample.

Based on ELISA principles, the biochip is a solid-state device with discrete test sites onto which antibodies specific to different compounds are immobilized and stabilized, offering a highly sensitive screen.

Using chemiluminescence as a measurement principle, the Evidence MultiSTAT consistently delivers accurate results and offers a highly sensitive method of detecting and diagnosing. Biochip Technology has a proven high standard of accurate test results with CVs typically <10% with a 98% agreement with confirmatory methods.

Each biochip can have up to 49 Discrete Test Regions (DTR). This means that up to 44 tests can be carried out simultaneously. The additional DTRs are reserved for internal quality control and visual reference, a unique Biochip Array Technology feature.

Rapid, accurate stroke diagnosis and differentiation
A ground-breaking new test which improves the accuracy of stroke diagnosis has been developed by Randox scientists. The Randox Stroke Biochip is a rapid and highly sensitive blood test that will complement and enhance existing CT scanning technology to facilitate accurate classification of stroke patients and improve patient care pathways.
With a unique ability to differentiate between ischemic and hemorrhagic stroke, the Randox Stroke Biochip takes less than 30 minutes to complete – making sure patients get the right diagnosis as fast as possible.

Dr Peter FitzGerald, Managing Director of Randox Laboratories, whose team developed the test, commented:
“There is great tragedy in the fact that the majority of stroke damage can be minimized if intervention is delivered on time, yet too often the window closes before a diagnosis is made. For doctors, nothing is more frustrating.
“Excellent work has been undertaken to assist the public in recognizing the signs of a stroke so people can get to hospital as quickly as possible. Our stroke test is the vital next step – assisting clinicians in making a rapid diagnosis and
differentiation between hemorrhagic and ischemic stroke, so their patients get the right treatment at the right time.”

Using Randox revolutionary patented biochips, the Randox Stroke Biochip provides a unique solution for simultaneous detection of multiple stroke biomarkers from a single sample, facili-
tating fast and accurate classification of stroke patients in an emergency setting.

Benefits of the Randox Stroke Biochip
• Results in 30 minutes on the Evidence MultiSTAT
• Unique solution for stroke classification
• Differentiation of ischemic and
hemorrhagic stroke
• Guarantees rapid thrombolytic therapy ensuring better patient outcomes

John Lamont, R&D Director for Randox Laboratories, explained:
“While patients undergo a CT scan to confirm either the presence or lack of a hemorrhagic stroke, a blood test on the Randox Biochip can be run on our innovative point-of-care analyser, the Evidence MultiSTAT, to identify the same for an ischemic stroke.”
“For the almost 90% of stroke patients who are ruled out for hemorrhagic stroke(1), the Randox Biochip will then accelerate decision making for clinicians with regards to thrombolytic therapy.”
 “Any treatment is most effective if started as soon as possible after the stroke occurs, and so every minute that passes without a diagnosis is likely to leave a permanent mark on a stroke patient’s future health and lifestyle. The vitally important diagnostic information from the Randox Stroke Biochip facilitates accurate stroke classification, directs the appropriate patient care pathway, and enables rapid thrombolytic therapy, ensuring a better patient outcome for ischemic stroke sufferers, for whom time is of the essence.”

Whilst the Randox Stroke Biochip is currently being used as complementary testing in parallel with CT scanning, Mr Lamont is confident of a more prominent role for the test in the future patient pathway:
“The Biochip has the potential to really revolutionize the stroke diagnosis pathway as we currently know it. The accessibility of this type of blood testing could potentially extend its use beyond the A&E department, to ambulances and even the home, in the form of a hand-held testing device.”
Emergency Drugs of Abuse screening
Randox are world leaders in clinical toxicology solutions with the capability of detecting over 500 drugs and drug metabolites using innovative Biochip Technology. The Evidence MultiSTAT Drugs of Abuse panels have been specifically selected to detect levels of the most common drug types, covering over 200 drugs and drug metabolites.

Designed to work across a wide variety of matrices, this revolutionary multi-analyte testing platform allows clinicians to achieve a complete immunoassay profile in the initial screening phase. Offering the most advanced screening technology on the market, Randox has transformed the landscape of drugs of abuse testing. The Randox DoA toxicology panel simultaneously detects 21 classical, prescription and synthetic drugs from a single patient sample suitable for use with urine and blood samples with as little as 200μl sample volume required.
References
1. Intercollegiate Stroke Working Party. National clinical guideline for stroke, 5th edition. London: Royal
College of Physicians 2016
The author
Martin Conway, Marketing Executive
Martin.Conway@randox.com
Randox Laboratories
55 Diamond Road
Crumlin,
County Antrim,
BT29 4QY
United Kingdom