The need for supervised urine collection in drug testing programmes has prompted efforts to identify new, less intrusive sample types including dried blood spots. In parallel, mass spectrometry is gaining prominence over immunoassay methods to meet the growing demand for multi-panel drug testing. A recent study carried out in Sweden explored the possibility of combining dried blood spots with liquid chromatography-mass spectrometry using clinical samples collected with Capitainer’s quantitative DBS device. The work led to a multi-panel drug test that was validated to forensic standards, with excellent agreement in drug measurements obtained from urine and quantitative dried blood spot samples.

Drug testing programmes based on liquid urine samples and immunoassay screening are well established in medical and forensic settings. Within the clinical setting, such programmes are routine in clinics that administer methadone to treat opioid dependence. Drug and alcohol testing is also performed in certain workplaces, e.g., air traffic control centres, prisons, and construction sites.

Regardless of where and why testing is performed, the intrusive nature of supervised urine sample collection has sparked a demand for alternative sample types that are more discrete and also amenable to multi-panel testing.

Alternative sample types explored so far include dried blood spots (DBS), saliva and exhaled breath. However, despite their attractiveness over urine, they are not widely compatible with immunoassay testing, which by itself cannot meet the demand for the increasing number of substances required in drug testing panels. So, in addition to alternative sample formats, efforts have intensified to develop new analytical workflows based on liquid chromatography-mass spectrometry (LC-MS).

The benefits of using LC-MS over immunoassay in drugs of abuse (DoA) testing include the increased sensitivity and specificity needed to fulfil forensic requirements, and flexibility as the identity and numbers of target analytes evolves.

Swedish team explores quantitative DBS and LC-MS analysis for multi-panel drug testing

DBS sampling offers many advantages to the testing facility and the person being tested, e.g., it is less intrusive, samples can be shipped as regular post at ambient temperature, and it eliminates the cost of having a phlebotomist present to take venous blood samples. In addition, while urine offers a longer detection window than blood – because of the slower metabolism of drugs through the kidney than through the liver – blood tests generally lead to more accurate results, since it is a more consistent liquid than urine, which is a variable sample matrix with regard to natural dilution factors.

DBS sampling for DoA testing is not a new concept (reviewed in 1). However, while previous studies have shown that it is possible to detect certain drugs in DBS samples collected and stored on filter paper, this approach uses blood spots of unknown and inconsistent volumes and is thus not quantitative. The emergence of volumetric DBS collection devices such as Capitainer®B offers the possibility to collect fixed volumes of blood for quantitative and accurate analysis.

Realising the need for a quantitative alternative to liquid urine DoA testing, a team of researchers from The Karolinska Institute, Stockholm County Council and ABC Labs in Sweden developed a multi-panel testing workflow that combined qDBS collected via Capitainer®B with a LC-MS method capable of detecting a panel of 35 analytes in qDBS samples and 37 analytes in liquid urine samples.

Their findings, published recently in Journal of Pharmaceutical and Biomedical Analysis, support the use of Capitainer®B in multi-panel DoA testing, with excellent overall agreement between qDBS and standard liquid urine samples obtained in a clinical setting (2).

Drug testing in qDBS vs. liquid urine in clinical samples – the workflow

• A total of 99 patients were recruited from a drug dependence clinic in Stockholm, all of whom were in methadone maintenance treatment for opioid dependence. Two drops of capillary blood from a fingertip were collected from each patient using a Capitainer®B device, and for comparison, an aliquot of a urine sample collected under supervision was included for each patient.

• qDBS samples were extracted from the 6 mm pre-cut filter discs within Capitainer®B using a two-step solvent extraction procedure.

• Urine samples (50 μL aliquot per patient) were extracted in 96-well plates using an automated protocol to hydrolyse and precipitate the analytes of interest. As a side note, creatinine testing is performed in routine workplace drug testing as a means to detect deliberate urine dilution: the higher the water content in urine, the lower the creatinine concentration. In the present study, creatinine was measured using a commercially available reagent and instrumentation.

• A solution of acetonitrile containing internal standards for the majority of the analytes being tested was added to each qDBS and each urine sample during the extraction procedure.

• Following extraction, all samples were dried and redissolved in 25 % methanol in preparation for analysis on a Waters LC-MS/MS instrument. The analytical workflow and analyte identification steps were performed in accordance with acceptance limits laid out in the European Guidelines for Workplace Drug Testing in Urine. All methods used were validated following recommendations by the US Scientific Working Group for Forensic Toxicology.

Agreement between urine and qDBS samples validates use of Capitainer®B in DoA testing

The study tested a panel of DoA in clinical samples and included representative drugs or drug derivatives from the following categories: amphetamine, cathinone, opiates, benzodiazepines, and others.

As expected, methadone and its primary metabolite EDDP were detected in all urine and qDBS samples, indicating 100 % agreement between the sample types with respect to sensitivity and specificity. In most cases (97 patients), these two analytes were detected in urine above the upper limit of quantification, but this was solved by diluting the urine samples with saline. For the remaining two patients, methadone was detected in urine and qDBS below the standard cutoff level for immunoassay.

For several other drugs, detection in qDBS samples was more sensitive than in urine samples, with a higher number of positives recorded in qDBS samples, e.g., amphetamine, cocaine, morphine and 6-AM. The applied cutoffs for the newly-developed LC-MS method were the same or somewhat lower than the standard cutoffs used in immunoassay screening, which results in increased sensitivity and specificity for drug testing. In addition, the study revealed that qDBS samples offer improved stability over fresh urine samples. Most analytes tested in qDBS samples were stable for 28 days at −20 °C, while many of the analytes were stable at room temperature.

Validated qDBS-LCMS method for DoA testing provides adaptable alternative for patients

The present study demonstrated the feasibility of using volumetric DBS in clinically-relevant drug testing, with excellent agreement between qDBS and urine for methadone testing, and increased sensitivity over urine samples for several other drugs of major importance. These findings are echoed in an unrelated study by researchers in Greece who recently reported LC-MS-based detection of 26 DoA in qDBS samples, also collected using Capitainer®B (3).

Importantly, when asked for their opinion regarding sample type for drug testing, 97 of the 99 patients included in this study reported a need for an alternative sample type. An additional advantage of qDBS over urine is the possibility to combine DoA testing with PEth testing, which is the gold standard approach to detecting long-term alcohol consumption, and which must be performed in blood.

In summary, the optimised LC/MS-MS workflow for qDBS and urine samples offers a high-throughput method with a short reporting time of less than 24 hours, which is a more useful guide to clinical decision-making than unconfirmed positives from immunoassay screening. In addition, an alternative to supervised urine sampling will increase privacy and dignity for those individuals being tested. Finally, the new method meets forensic standards and can be adapted to meet the evolving requirements within drug and alcohol testing.

References

1. Ververi C, Vincenti M, Salomone A. Recent advances in the detection of drugs of abuse by dried blood spots. Biomed Chromatogr. 2023 Jul;37(7):e5555.
2. Guterstam J, Tavic C, Barosso M, Beck O. A multicomponent LC-MS/MS method for drugs of abuse testing using volumetric DBS and a clinical evaluation by comparison with urine. J Pharm Biomed Anal. 2024 Jun 15;243:116075.
3. Meikopoulos T, Gika H, Theodoridis G, Begou O. Detection of 26 Drugs of Abuse and Metabolites in Quantitative Dried Blood Spots by Liquid Chromatography-Mass Spectrometry. Molecules. 2024 Feb 23;29(5):975.