Integration prospects for the multiplex phosphorescence immunoassay of pooled dry urine samples into screening examinations in dispensary drug control

Introduction. The worsening problem of drug abuse in Russia and the growing number of hidden users of narcotic drugs (ND) require the list of screening examinations for ND identification to be extended by including more economical approaches that reduce costs at the stages of collection, transportation, storage, and analytical examination of biological samples.

Objective. Development of a multiplex immunoassay method based on the PHOSPHAN technology for detecting the main groups of narcotic and psychotropic substances in pools of paper-dried urine samples, followed by an assessment of its potential for identifying drug addicts as part of an extended drug control program.

Materials and methods. Dry urine samples (n = 31) were prepared on paper test strips from liquid samples containing (n = 30) or non-containing (n = 1) cocaine, cannabinoids, amphetamines, opiates, benzodiazepines, barbiturates, methamphetamine, or methadone, according to toxicology screening (TS). The samples were studied as pools containing 1–40 fragments (0.45×0.45 cm) of test strips. The luminescent signal was recorded on a microplate immunochip using an IFI-05 photoluminescence pulsed indicator. The ND presence in the samples was assessed by the inhibition rate of antibody binding in the related microplate test zone (B/B₀ ratio). Statistical processing of the results was carried out using the standard Microsoft Office package.

Results. The inclusion of dry urine samples in the pools (up to 10), where only one contained the target ND, had no significant effect on the capability of the method to detect NDs with sensitivity levels that meet the TS requirements. The following substances were detected: cocaine (2 samples), cannabinoids (11 samples), amphetamines (6 samples), opiates (9 samples), benzodiazepines (7 samples), barbiturates (10 samples), methamphetamine (7 samples), and methadone (6 samples), including samples with high concentrations of opiates and amphetamines.

Conclusions. A method of multiplex phosphorescence microplate immunoassay has been developed for the detection of eight main groups of NDs and psychotropic substances in pools of paper-dried urine samples (dried urine spot, DUS). The detection limits of the studied NDs in extracts from DUS test-strips were 2–8 ng/mL, which is significantly lower than the detection limits recommended for screening examination. The proposed approach can form the basis of a new screening methodology that includes collection of urine samples, their application onto filter paper test-strips, and transportation to a laboratory for the examination of individuals at industrial facilities of critical importance. The use of the developed multiplex phosphorescence immunoassay and pooled urine samples will significantly reduce the test cost (by more 10-fold) compared to conventional immunochromatographic assays.

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