Performance Evaluation of Multi-Drug Rapid Test for Rapid Detection of Multiple Drugs and Drug Metabolites in Human Blood Samples

Objective: The Multi-Drug Rapid Test Cassette, developed by Hangzhou AllTest Biotech Co., Ltd, is a rapid chromatographic immunoassay designed for the qualitative detection of multiple drugs and drug metabolites in human whole blood, serum, or plasma. It aims to simultaneously detect various abused substances, including Amphetamine (AMP), Barbiturates (BAR), Benzodiazepines (BZO), Buprenorphine (BUP), Cocaine (COC), Cannabis (THC), Methadone (MTD), Methamphetamine (MET), 3,4-Methylenedioxymethamphetamine (MDMA), Morphine (MOP/OPI), Propoxyphene (PPX), Tricyclic antidepressants (TCA), Oxycodone (OXY), 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), Cotinine (COT), Tramadol (TML), Fentanyl (FYL), 3,4-Methylenedioxy pyrovalerone (MDPV), Synthetic cannabinoids(K2), Phencyclidine(PCP), Ketamine(KET), Lysergic acid diethylamide(LSD), 3,4-Methylenedioxyamphetamine(MDA), Acetaminophen(ACE), Ketamine(CAT), 6-Monoacetylmorphine (6-MAM), Zolpidem (ZOL), AB-PINACA (ABP/K3). Therefore, the present research was done to evaluate the diagnostic performance of this test.


INTRODUCTION
According to the latest data from the World Drug Report 2023 by the United Nations Office on Drugs and Crime, the estimated number of global injectable drug users was approximately 13.2 million in 2021, which is 18% higher than previous estimates.On a global scale, the number of drug users exceeded 296 million in 2021, marking a 23% increase compared to a decade ago.Moreover, the number of people suffering from drug addiction has skyrocketed to 39.5 million, indicating a 45% increase over the past decade [1].Given the widespread nature of this drug abuse phenomenon, screening for illegal drug use is necessary.The most sophisticated drug-testing approach is gas chromatography coupled with mass spectrometry (GC/MS), which is regarded as a "gold standard"; it is used in confirmatory testing.
Typically, GC/MS is preceded by a rapid immunoassay method to eliminate the majority of the specimens/individuals with negative drug test results, thereby preventing an excessive burden on more complex and time-consuming confirmation processes [2].
There are several biological samples that can be used for testing.These include blood or serum, sweat, hair, oral fluid, nails, and urine.The most commonly used biological sample is urine, as it is non-invasive, and the concentration of a given xenobiotic is generally higher when compared to other samples.This usually results in a higher sensitivity.Additional considerations include how long a xenobiotic remains detectable in various matrices.This consideration becomes particularly significant when determining the appropriate testing approach based on the specific objectives and reasons for conducting the test [3].In emergency situations, blood testing offers the advantage of providing a precise assessment of a specific level [4].The detection window is usually one to two days [5].

Sample Materials
The samples used in this study include whole blood, serum, or plasma obtained through either a venipuncture or a fingerstick procedure.
To collect fingerstick whole blood specimens, the patient's hand should be washed with soap and warm water or cleaned with an alcohol swab.After drying, the hand is gently massaged without touching the puncture site.The skin is then punctured with a sterile lancet, and the first sign of blood is wiped away.The hand is gently rubbed to form a rounded drop of blood over the puncture site.

Precision
This study was performed at three hospitals using three different batches of a product.The purpose of the study was to evaluate the precision of the measurements within a single run, between different runs, and between different operators.
To assess the precision, a card with coded specimens was prepared.These specimens contained drugs at concentrations that were either higher or lower than the predetermined cut-off level by up to 50%.The card was labeled and its contents were concealed to ensure unbiased testing.The card was then tested at each of the three hospital sites ( Lei Z

Sensitivity
A drug-free mixture of whole blood/serum/plasma was subjected to a drug-spiking experiment with drugs at concentrations of 0%, -50%, 100%, 150%, and 300%.Now, the sensitivity of the drug detection assay in this experiment needs to be calculated.The results are summarized below (Table 3).

(
MAM), Zolpidem (ZOL), AB-PINACA (ABP/K3).Lei Z Each test line contains anti-drug mouse monoclonal antibody and corresponding drug-protein conjugates.The control line system contains goat anti-rabbit IgG polyclonal antibodies and rabbit IgG.During the test, a specimen of whole blood, serum, or plasma migrates upward through capillary action.If a drug is present in the specimen below its designated cut-off concentration, it will not saturate the binding sites of its specific antibody.In this case, the antibody will react with the drug-protein conjugate, resulting in a visible colored line in the test region.If the drug concentration exceeds the cut-off level, it will saturate all the binding sites of the antibody, preventing the formation of a colored line in the test region.A drug-positive specimen will not generate a colored line in the specific test region due to drug competition, while a drug-negative specimen will produce a line in the test region due to the absence of drug competition.To ensure proper procedure, a colored line will always appear at the control region, indicating that the correct volume of the specimen has been added and that membrane wicking has occurred.This serves as a procedural control.

Table 1 :
Clinic Result of Whole Blood/Serum/Plasma.
Prior to conducting the test, ensure that the test kit, specimen, buffer, and controls have reached room temperature (15-30°C).Place the cassette on a clean and level surface.For serum or plasma specimens,

Table 2 :
Test Results from Three Sites.

Table 3 :
Analytical Sensitivity.Thirdly, a positive result from the test indicates the presence of the drug or its metabolites but does not provide information about the level of intoxication, administration route, or concentration in the specimen.To overcome this limitation, the test results should be used in conjunction with additional clinical data to make informed decisions.Furthermore, a negative result does not necessarily indicate drug-free whole blood, serum, or plasma.Negative results can occur when the drug is present but below the test's cut-off level.It is important to be