Forensic application and evaluation of a commercially available pregabalin immunoassay test in serum on an Olympus AU480

Misuse of pregabalin and its forensic relevance is steadily increasing. The aim of this study was to evaluate the usability of the commercially available ARKTM Pregabalin II Assay (ARK Diagnostics) for serum analysis of forensic samples. Overall, 156 samples were tested by both the immunoassay and a validated liquid chromatographic – tandem mass spectrometric (LC-MS/MS) method. Sensitivity was 100%, and specificity was 98.7% ( n = 79 positive cases confirmed by LC-MS/MS in a range of 380 – 37,000 ng/mL). A good correlation ( R 2 = 0.73) could also be shown between quantitative immunoassay and LC-MS/MS results. In conclusion, the assay shows excellent reliability for screening of forensic serum samples.


| I N T RO DU CT I O N
Misuse of pregabalin, an analog of the neurotransmitter gammaaminobutyric acid (GABA), and its forensic relevance is steadily increasing. 1 Originally designed for the treatment of epilepsy, pregabalin is predominantly used to treat neuropathic pain and partial seizure disorders, but it is also approved for use in the treatment of fibromyalgia in the United States and management of generalized anxiety disorder (GAD) in the European Union (EU). 2,3At the time of market launch in the EU in 2004, the potential for drug abuse or physical dependence for pregabalin was assessed to be low.A clinical study with 15 recreational alcohol/sedative users had found that pregabalin in therapeutic doses of 200-450 mg did not produce the same responses as diazepam, indicating that the drug did not have the profile of a prototypic drug of abuse. 4However, in later clinical trials in patients with central neuropathic pain and in patients with GAD, euphoria as an adverse event was reported to be common for pregabalin.Assessments of withdrawal symptoms in clinical trials of GAD showed a profile similar to that of lorazepam, especially in the 600 mg/day dosage.Therefore, in 2014, the European Medicines Agency has changed abuse, misuse, and dependence from a potential risk to an identified risk. 4Use of nontherapeutic dosages in drug users has been reported ever since then. 1,5In Germany, pregabalin has risen to be the fifth often misused substance class after opiates, benzodiazepines, cannabis, and alcohol. 6Besides euphoria, further side effects of therapeutic and nontherapeutic pregabalin use are dizziness, somnolence, and confusion. 2,3This could lead to a limitation of fitness to drive.Therefore, pregabalin is important to be detected in forensic blood serum samples in forensic laboratories.Liquid chromatographic-mass spectrometric (LC-MS) unknown screening methods mostly focus on the detection of basic drugs, whereas drugs with acidic properties like pregabalin can be lost during sample preparation.However, specific and sensitive detection of pregabalin using solid-phase extraction 7,8 or our herein described method using protein precipitation has been described.An immunoassay could be useful in screening on this relevant substance, giving the possibility of highthroughput analysis without sample preparation.
When using immunochemical methods for a preliminary test according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh), the relevant analyte concentrations obtained by a chromatographic method must show positive immunochemical results for the relevant substance, which have to be documented by an appropriate validation procedure. 9Sensitivity should be nearly 100% (at least 90%), and the rate of false positives simultaneously should be as low as possible due to costs and time.The aim of this study was to evaluate the usability of the commercially available ARK Pregabalin II Assay (ARK Diagnostics), which was originally designed for clinical urine screening, for serum analysis of forensic samples. 2

| Samples
Serum samples collected mainly for forensic purposes were used for the study.All samples were tested in the Institute of Forensic Medicine, Mainz (Germany), with immunoassay as well as with LC-MS/MS.Overall, 156 samples were tested.

| Immunoassay test
The test used was the ARK Pregabalin II Assay from ARK Diagnostics (Fremont, CA, USA).The assay was originally designed for the qualitative and/or semiquantitative determination of pregabalin in human urine using a cutoff concentration of 500 ng/mL.It is a homogeneous immunoassay based on competition between pregabalin in the specimen and pregabalin labeled with recombinant glucose-6-phosphate dehydrogenase (rG6PDH) for antibody binding sites.As the latter binds antibodies, enzyme activity decreases.In the presence of pregabalin, enzyme activity increases relative to the drug concentration.Active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate, resulting in an absorbance change that is measured spectrophotometrically. 10  (QC high), respectively.Regarding the analytical specificity, some drugs relevant in forensic toxicology were tested negative by the manufacturer at high concentrations in urine. 10We confirmed that the analysis of the GABA analog gabapentin did not lead to a positive result even at a concentration of 5,000,000 ng/mL in serum.Because there are no legal requirements for the determination of pregabalin, we decided to choose an immunoassay cutoff of 100 ng/mL immunoassay units (IAU) corresponding to a very low pregabalin serum concentration.Therapeutic concentrations of pregabalin are described to be 2000-8000 ng/mL. 11
Afterward, 100 μL of serum was added, and the solution was vortexed for 30 s and centrifuged for 10 min at 3000 g.The LC-MS/MS system used consists of an Agilent (Santa Clara, CA, USA) 1290 Infinity liquid chromatograph coupled to an Agilent 6490A triple quadrupole mass spectrometer.A 5 μL of the supernatant was injected into the system.
Chromatographic separation was achieved using a raptor biphenyl column (2.

| Calculation of sensitivity and specificity
Sensitivity describes the percentage of positives (positive by LC-MS/ MS) correctly identified to be positive by the immunoassay.Specificity describes the percentage of negatives (negative by LC-MS/MS) correctly identified to be negative by the immunoassay.

| Tested samples
Overall, 156 samples were tested by both methods.All blood samples that came to our laboratory in February 2020 and where pregabalin analysis was requested were introduced in the study.These included plasma and serum samples.Furthermore, samples positive on the immunoassay HESS ET AL.