B-296 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Buprenorphine and its Major Metabolites in Urine

Background Buprenorphine is a synthetic opioid derived from thebaine. It is structurally and pharmacologically similar to morphine, but is 20-30 times more potent. It is a partial agonist receptor modulator and has a longer duration of action relative to morphine due to its unusual slow rate of dissociation from its receptor. Buprenorphine produces a variety of symptoms including, but not limited to addiction, life-threatening respiratory depression, neonatal opioid withdrawal syndrome, severe hypotension. After administration in humans, buprenorphine is primarily metabolized through N-dealkylation to form norbuprenorphine. Both buprenorphine and norbuprenorphine undergo further metabolism via conjugation with D-glucuronic acid to form buprenorphine-glucuronide and norbuprenorphine-glucuronide. Pharmaceutical buprenorphine is a medication used to treat opioid use disorders (OUDs) and manage severe pain that necessitates an opioid analgesic when other treatments are insufficient. Furthermore, it has occasionally been used off-label (i.e., for purposes not approved by the FDA) via injection, including applications in perineural anesthesia and managing withdrawal in hospitalized patients dependent on heroin. In 2002, buprenorphine, including its salts, isomers and salts of isomers, became a Schedule III narcotic substance under the Controlled Substances Act for its potential for abuse and risk of dependence. ARK Diagnostics has developed the ARK Buprenorphine Assay to detect buprenorphine and its metabolites at a cutoff concentration of 5 ng/mL of buprenorphine with high cross-reactivity to its metabolites, norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide without additional treatment of glucuronidase. Methods The ARK Buprenorphine Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 5 ng/mL and semi-quantitative range up to 100 ng/mL. The performance of this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ± 40% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 4.0 to 8.0 % CV. Spiked recovery ranged from 94.0% to 103.4 % for the samples spanning 2.0 to 100.0 ng/mL. The major metabolites, norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide, showed approximate equivalences to the 5 ng/mL buprenorphine cutoff at 10.0 ng/mL (50.0 % cross-reactivity), 5.8 ng/mL (86.2 % cross-reactivity), and 8.5 ng/mL (58.8% cross-reactivity), respectively. Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with specificity and sensitivity. Conclusion The ARK Buprenorphine Assay measures buprenorphine and its major metabolites, norbuprenorphine, buprenorphine glucuronide, and Norbuprenorphine glucuronide in human urine with good analytical performance. The assay has superior specificity as other opiates and opioids do not cross react. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.