B-281 Performance Characteristics Evaluation of the ARK Diagnostics Immunoassay for Xylazine/4-HydroxyXylazine in Urine
Authors: Kyle P B , Mattiello C J , Hua A , Toohey J M , Korn W R , Saldana-Reed A , Stickle D F 
Published: October 2024

Abstract

Background
Xylazine exposure is currently common in some US cities, but routine laboratory testing is not available. We evaluated the ARK Diagnostics immunoassay for qualitative detection of xylazine/4-hydroxy Xylazine in urine, prior to its anticipated release as a Forensic Use Only assay. Evaluation of performance characteristics of the ARK assay were conducted at two university hospitals, using either the semi-quantitative application of the assay, as performed on the Roche Cobas 503 analyzer (Institution A), or the qualitative application of the assay, as performed on the Beckman Coulter AU480 analyzer (Institution B).

Methods
Xylazine (X) and 4-hydroxy Xylazine (4-OHX) were obtained from Cayman Chemical (Ann Arbor, MI). Pooled xylazine-free urine was spiked either with X or 4-OHX in the range of 0-2000 ng/mL. De-identified, to-be-discarded patient urine samples were obtained from among those submitted for routine drugs-of-abuse panel testing. X measurements on all samples were performed by LC-MS/MS; samples were segregated into X-NEGATIVE (X&lt;10 ng/mL) and X-POSITIVE (X&gt;=10 ng/mL). The ARK assay was conducted according to manufacturer’s instructions, either as a semi-quantitative assay (Institution A, Roche analyzer) or as a qualitative assay (Institution B, Beckman Coulter analyzer), with 10 ng/mL as the manufacturer-specified cutoff for ARK POSITIVE, using manufacturer-supplied calibration standards ranging from 0-500 ng/mL X.

Results
The ARK semi-quantitative analysis (Roche analyzer) showed essentially equivalent curvilinear response curves for either X or 4-OHX at concentrations below 1000 ng/mL; for the semi-quantitative ARK assay, we use “&gt;1200 ng/mL” for X-ARK &gt;1200 ng/mL. By mixing studies (Roche), there were neither positive nor negative interferences observed as assessed for common drugs of abuse or for common therapeutic drugs. The presence of hemoglobin, bilirubin or moderate lipemia were devoid of assay interference. Precision studies for manufacturer-supplied controls were as follows: intra-assay CVs were 6.9% for the NEGATIVE control (5.1±0.35 ng/mL) and 5.9% for the POSITIVE control (15.7±0.53 ng/mL) (n=20); interassay CVs were 7.8% for the NEGATIVE control (5.1±0.40 ng/mL) and 5.7% for the POSITIVE control (15.9±0.90 ng/mL) (n=20). For the qualitative ARK assay as performed on the Beckman analyzer, POSITIVE and NEGATIVE controls ran invariably according to their designations. For the semi-quantitative assay application (Roche analyzer): among 78 X-NEGATIVE samples by LC-MS/MS, there were 0% ARK-POSITIVE results (false-positive rate=0%); among 74 X-POSITIVE samples by LC-MS/MS, there was one ARK-NEGATIVE result (false-negative rate=1.4%); this sample (X=65 ng/mL) was found to have a high amorphous crystalline content, and was unable to be reanalyzed. For the qualitative assay application (Beckman Coulter analyzer): among 78 X-NEGATIVE samples there was one ARK-POSITIVE sample (false-positive rate=1.3%) for a sample with xylazine=8.8 ng/mL by LC-MS/MS, with undetectable 4-OHX (&lt;10 ng/mL); among 74 X-POSITIVE samples, there were no ARK-NEGATIVE results (false-negative rate=0%).

Conclusions
The ARK xylazine immunoassay demonstrated acceptable analytical performance with respect to detection of xylazine in urine at 10 ng/mL. With additional consideration of the assay’s cross reactivity with the major xylazine metabolite, 4-OH-xylazine, the assay was judged to be highly suitable for routine use as a screening test for detection of xylazine exposure via urine testing.