Comparison of several immunoassays used in drugs of abuse screening: Assessment against gold standard methods and calculation of measurement uncertainty

Journal Pre-proof Comparison of several immunoassays used in drugs of abuse screening: Assessment against gold standard methods and calculation of measurement uncertainty

Ashraf Mina PII:

S1056-8719(19)30407-1

DOI:

https://doi.org/10.1016/j.vascn.2019.106649

Reference:

JPM 106649

To appear in:

Journal of Pharmacological and Toxicological Methods

Received date:

29 May 2019

Revised date:

27 August 2019

Accepted date:

5 November 2019

Please cite this article as: A. Mina, Comparison of several immunoassays used in drugs of abuse screening: Assessment against gold standard methods and calculation of measurement uncertainty, Journal of Pharmacological and Toxicological Methods (2019), https://doi.org/10.1016/j.vascn.2019.106649

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© 2019 Published by Elsevier.

Journal Pre-proof Comparison of Several Immunoassays Used in Drugs of Abuse Screening: Assessment against Gold Standard Methods and Calculation of Measurement Uncertainty Ashraf Mina1,2,* [email protected], Dr Ashraf Mina1,2 1

NSW Health Pathology, Forensic & Analytical Science Service (FASS), Sydney, Australia.

2

Affiliated Senior Clinical Lecturer, Faculty of Medicine and Health, Sydney University, Australia.

*

Corresponding author at: NSW Health Pathology - Forensic & Analytical Science Service, PO Box 53, North Ryde Mail Centre, North Ryde NSW 1670.

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No previous presentation of the manuscript

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Keywords: CEDIA; EMIT; DRI; EIA; Drugs of Abuse; DOA Screening; DOA Immunoassays, Measurement Uncertainty.

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List of abbreviation

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6-MAM: 6-monoacetylmorphine (heroin) CEDIA: Cloned Enzyme Donor Immunoassay. DRI: Diagnostic Reagents Inc.

EDDP: 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine. EIA: Enzyme Immunoassay. EA: Enzyme acceptor. ED: Enzyme donor. ED-LC: ED-ligand conjugate. 1

Journal Pre-proof EMIT: Enzyme Multiplied Immunoassay Technique. MU: Measurement Uncertainty. N: Number of specimens. rG6PDH: Recombinant glucose-6-phosphate dehydrogenase. SD: Standard Deviation.

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SDx: Specialty Diagnostix.

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Background

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Abstract

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Immunoassays provide simple, powerful and inexpensive screening methods for urine drug screening. Other substances and/or factors may interfere with the test and cause false or positive results. It is essential to understand the differences between methods to be able to evaluate their impact on the results. All evaluated immunoassays were assessed in comparison with GC-MS or LC-MS/MS, which are generally accepted as vigorous confirmation gold standard techniques. Methods CEDIA, DRI, EIA and EMIT II Plus screening immunoassays were evaluated on BeckmanCoulter AU5810 analyser. All results were confirmed using GC-MS or LC-MS/MS methods.

2

Journal Pre-proof Measurement Uncertainty for immunoassays was calculated by using standard deviation multiplied by 1.96 to cover 95% confidence interval of tested samples. Results No discrepancy was found between CEDIA and EMIT II Plus for cocaine, methadone, heroin, and benzodiazepines assays. No discrepancy was found between oxycodone DRI assay and Immunalysis enzyme immunoassay (EIA). Cannabinoids EMIT II Plus assay

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performed better than DRI assay. EMIT II Plus assays for amphetamine and ecstasy performed better than CEDIA amphetamine/ecstasy combined assay. Opiates EMIT II Plus

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assay performed better than CEDIA assay. Fentanyl Ark EIA method performed slightly

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better than the DRI method. Buprenorphine CEDIA second-generation assay performed

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better than CEDIA first-generation assay, EMIT II Plus assay and EMIT II Plus with added Beta-Glucuronidase assay. Measurement Uncertainty for immunoassays was calculated and

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Conclusions

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tabulated.

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This study covered a fundamental gap in available knowledge by evaluating the performance of screening the current new generation of immunoassays methods for drugs of abuse in urine against gold standard methods.

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Introduction

A literature review performed at the time of writing this article failed to identify any

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publication that covered the analytes and the scope of methods compared herein. Screening immunoassays for all of the major drugs of abuse are widely used and effective for large-volume urine screening programs.(1) When using immunoassays for drugs of abuse, interfering antibodies and some drugs or interfering substances may cause a false positive or negative result.(2) When immunoassays semi-quantitative data was compared with LCMS/MS results, some drugs were overestimated, such as benzodiazepine. This draws attention to the problem of the careless use of immunoassay tests for forensic purposes as they may provide false positive and/or false-negative results that can lead to errors of great severity.(3) Sympathomimetic amines were identified in several of the samples not containing

amphetamine

or

methamphetamine.(4)

Insufficient

cross-reactivity of the 4

Journal Pre-proof antibodies towards current synthetic cannabinoids in combination with relatively high detection limits of the immunoassays strongly recommended not to rely only on the evaluated immunoassays for synthetic cannabinoids in clinical or forensic settings.(5) Oxidizing reagents (H2O2 or CrO3) are most effective as adulterants in interfering in the measurement of benzoylecgonine,

EDDP,

ethyl-D-glucuronide,

and

morphine sulphate. NaN3 also

generated false-negative results for a number of drugs of abuse.(6) Some amphetamines immunoassays unevenly detect amphetamine-like drugs.(7) Designer benzodiazepines can be

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detected in standard urine immunoassay drug screening and this should be taken into consideration when performing confirmation analysis.(8)

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Since a positive result could have medical/legal consequences, all positive results, if not

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justified or expected, need to be confirmed by using a reference method for confirmation and

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quantitation.(9) It is worthwhile mentioning that the published studies in this area are not up to date with the second generations of immunoassays and the enhancements introduced to

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improve the current generation of immunoassay methods. Many assays of the second

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generation of immunoassays are able to detect the targeted drug and its metabolites,

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increasing the specificity of the assay. Therefore a study was needed to evaluate the current immunoassays available so laboratories can make informative decisions. This study was undertaken to evaluate the performance of the drugs of abuse screening on urine samples using Beckman-Coulter AU5810 analyser. CEDIA and EMIT methods compared

for

cocaine,

heroin,

amphetamine,

ecstasy,

opiates,

benzodiazepines and

buprenorphine. DRI EIA and Immunalysis EIA methods were also compared for oxycodone. CEDIA and EIA methods were compared for methadone metabolite. DRI EIA and EMIT II Plus methods were compared for Cannabinoids. DRI EIA method for fentanyl was compared with the LC-MS/MS method.

5

Journal Pre-proof Routine urine specimens and specimens with drugs concentrations close to the cut-off values were also selected for this study. All results were confirmed by using GC-MS or LC-MS/MS methods, as indicated, to determine the validity of the immunoassays outcome. Unlike immunoassays methods, the GC-MS and LC-MS/MS methods are considered the gold standard methods because of their ability to detect the drug and its metabolites without possible interference from interfering antibodies.

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Materials and Methods

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The CEDIA immunoassay is based on the use of two polypeptides that are created by

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separating the bacterial enzyme ß-galactosidase into two inactive fragments; the enzyme acceptor (EA) and the enzyme donor (ED). These two fragments can spontaneously

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recombine to create an active enzyme. To detect an analyte in a patient sample, the ED fragment is first chemically coupled to the target analyte. The labelled ED fragment is

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referred to as an ED-ligand conjugate (ED-LC). The labelling of the ED fragment does not

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restrict its ability to recombine (complement) with the EA fragment and create an active

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enzyme. When the reagents and the sample fluid are brought together, the labelled fragments (ED-LC) and the free analyte in the sample fluid, if present, compete in binding to a limited number of analyte-specific antibody binding sites. If the analyte is not present in the sample, the ED-LC will bind to the antibody, allowing fewer active enzymes to form. However, if the analyte is present in these analyte molecules, it will successfully compete for analyte binding sites, resulting in greater complementation of ED-LC and EA fragments. When these fragments spontaneously recombine,

the active enzymes cleave the assay substrate,

generating a colour change that can be measured spectrophotometrically. The extent of the colour change is proportional to the analyte concentration in the sample.(10)

6

Journal Pre-proof The DRI, EIA, and Emit II Plus share the same scientific principle. This is based on competition between drug in the specimen and drug labelled with the recombinant glucose-6phosphate dehydrogenase (rG6PDH) for antibody binding sites. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the specimen can be measured in terms of enzyme activity. The active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate (G6P), resulting in an absorbance change that is measured spectrophotometrically. Endogenous serum G6PDH does not

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interfere because the coenzyme NAD functions only with the bacterial Leuconostoc

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mesenteroides enzyme employed in the assay.

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For this study, patient urine specimens close or at the cut-off values were targeted as well as other specimens that tested positive or negative comprised the evaluation data set. CEDIA,

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DRI, EIA and EMIT II Plus immunoassays were evaluated on Beckman-Coulter AU5810 analysers. LC-MS/MS (AB Sciex QTRAP 5500) was used to determine the validity of the

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outcome for Buprenorphine and Fentanyl. GC-MS (Agilent 6890) was used to determine the

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validity of the outcome of the other analytes.

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The reagents obtained from Specialty Diagnostix and their part numbers were EMIT II Plus amphetamine (10445421), EMIT II Plus ecstasy (10445478), EMIT II Plus benzodiazepine (10445431), EMIT II Plus cocaine (10445439), EMIT II Plus opiate (10445417), EMIT II Plus THC (10445471), EMIT II Plus 6-AM (10470440), EMIT II Plus buprenorphine (10720047), Immunalysis oxycodone (10991156), ARK EDDP (5051-0001-01), ARK fentanyl immunoassay (5031-0001-01), EMIT II multidrug calibrator (110445407), EMIT II multidrug calibrator 2 (10445408), EMIT II multidrug calibrator 3 (10445409),

EMIT

multidrug

(10470442),

calibrator

4

(10445410),

6-AM/ecstasy

calibrator

Level 2

II

Immunalysis oxycodone calibrator (10991158), ARK EDDP calibrator (5051-0002-02), EMIT II special drug calibrator level 2 (10720050), fentanyl cut-off calibrator set (50317

Journal Pre-proof 0002-02), standard control set (18003225), advanced control set (18003226), fentanyl control set (5031-0003-00). The reagents obtained from Thermo Fisher and their part numbers were CEDIA benzodiazepine (CDF1775561), Beta-glucuronidase for benzodiazepine (CDK030707598), CEDIA cocaine (CDF1661230), THC cannabinoid DRI immunoassay (CDF0186), CEDIA heroin metabolite immunoassay (CDF100108), buprenorphine II (CDF10020849), DRI oxycodone (CDF100248), CEDIA Sample Check (CDF1815555), CEDIA methadone

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metabolite (CDF1868217), CEDIA heroin metabolite cut-off calibrator (CDF100031), CEDIA multi-drug optional calibrator (CDF100033), CEDIA Negative calibrator II (CDF

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10021390), CEDIA buprenorphine II calibrator (CDF10020799), DRI oxycodone calibrator

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(CDF100250), DRI THC calibrator (CDF0042), DRI THC calibrator (CDF0044), DRI THC

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calibrator (CDF0235), CEDIA buprenorphine II controls (CDF10020804). The immunoassays evaluated between CEDIA and EMIT II Plus methods were cocaine, 6-MAM (6-monoacetylmorphine),

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heroin

opiates,

benzodiazepines,

and

buprenorphine.

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Amphetamine/ecstasy (combined) CEDIA method was evaluated against both EMIT II Plus

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amphetamine and EMIT II Plus ecstasy methods. Oxycodone method was compared between DRI EIA kit and Immunalysis EIA kit. Methadone metabolite EDDP (2-ethylidine-1,5dimethyl-3,3-diphenylpyrrolidine) methods were evaluated between CEDIA and both ARK EIA and Immunalysis EIA methods. Cannabinoids (11-nor-Δ9-THC-COOH) method was compared with DRI EIA and EMIT II Plus methods. Fentanyl DRI EIA was compared with ARK EIA method. Fentanyl: 15 spiked specimens were tested because the half-life time of the drug is 3 to 12 hours. Fentanyl can be detected in urine for 8 to 24 hours, depending on a variety of factors including age and weight.(5) Buprenorphine CEDIA first-generation, CEDIA second-generation, EMIT II Plus, and EMIT II Plus with added Beta-Glucuronidase methods were all compared against LC-MS/MS. Few

8

Journal Pre-proof different studies were performed to evaluate different generations of the buprenorphine assays. Also, some of the assays were optimised by adding Beta-glucuronidase to improve the detection of the other metabolites. Additionally, 41 specimens were analysed concurrently for

the

following

Generation),

CEDIA

buprenorphine

assays;

CEDIA

(2nd

generation)

buprenorphine

buprenorphine immunoassay,

immunoassay EMIT

II

(1st Plus

buprenorphine immunoassay, and EMIT II Plus buprenorphine immunoassay with Betaglucuronidase added to the reaction.

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The measurement uncertainty was calculated for assays by using assays standard deviations

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multiplied by 1.96 to cover 95% confidence interval of tested samples.(11)

supplemental data for comparison results. All results obtained by

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Please refer to

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Results

immunoassay methods are confirmed using GC-MS or LC-MS/MS as indicated.

Table 1

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The outcome is summarised in Table 1.

Cocaine: Cut-off = 0.3 µg/mL (300 ng/mL), number of specimens analysed (N) = 34. EMIT II Plus and CEDIA methods were evaluated. No discrepancy was found. Methadone metabolite EDDP (2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine): Cut-off = 0.10 µg/mL (100 ng/mL), N= 32. Two-ways comparison assessments were performed between CEDIA and ARK EIA methods and also between CEDIA and Immunalysis EIA methods. No discrepancy was found. Oxycodone: Cut-off = 100 ng/mL, N= 22. No discrepancy was found between DRI EIA and Immunalysis EIA methods.

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Journal Pre-proof Heroin 6-MAM (6-monoacetylmorphine): Cut-off = 10 ng/mL, N= 61. No discrepancy was found between CEDIA and EMIT II Plus Methods. Cannabinoids (11-nor-Δ9-THC-COOH): Cut-off = 50 ng/mL, N= 57. DRI EIA method gave two false-negative results when compared to the EMIT II Plus method. Amphetamine/Ecstasy:

Cut-off

=

0.3

µg/mL

(300

ng/mL),

N=

62.

CEDIA

amphetamine/ecstasy method gave 6 false-positive results when compared against EMIT II Plus amphetamine and EMIT II Plus ecstasy methods.

results when compared with the EMIT II Plus method.

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Opiates: Cut-off = 0.3 µg/mL (300 ng/mL), N= 59. CEDIA method gave six false-positive

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Benzodiazepines: Cut-off= 0.2 µg/mL (200 ng/mL), N= 60. CEDIA method gave one false-

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positive result. EMIT II Plus method also gave one other false-positive result, but not on the

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same sample. Fentanyl:

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Fentanyl DRI EIA method: Cut-off = 2 ng/mL, N= 15. DRI EIA method gave one false

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negative result of the 15 specimens when compared with the LC-MS/MS method.

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Fentanyl ARK EIA method: Cut-off = 1.0 ng/mL, N=15. No discrepancy was found when compared with LC-MS/MS results. The Ark fentanyl assay is more sensitive and can detect norfentanyl at 300 ng/mL while DRI assay detects it at 10,000 ng/mL concentration (cross-react by 0.02%), which makes norfentanyl detectable by DRI assay only at 10,000 ng/mL concentration or above. Buprenorphine: CEDIA Buprenorphine “First Generation” immunoassay: Cut-off= 5.0 ng/mL, N= 54. CEDIA buprenorphine “First Generation” immunoassay gave 12 false-positive results out of the 54 specimens when compared to EMIT II Plus immunoassay.

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Journal Pre-proof CEDIA Buprenorphine II “Second Generation” immunoassay: Cut-off= 10.0 ng/mL, N= 37. CEDIA buprenorphine II “Second Generation” immunoassay gave one false-negative result out of 37 patient specimens when compared to LC-MS/MS. EMIT II Plus Buprenorphine II immunoassay: Cut-off= 5.0 ng/mL, N= 37. Buprenorphine EMIT II Plus immunoassay gave 13 false-negative results out of 37 patient specimens when compared to LC-MS/MS. CEDIA Buprenorphine immunoassay (1st Generation), CEDIA Buprenorphine (2nd

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generation) immunoassay, EMIT II Plus Buprenorphine immunoassay, EMIT II Plus Buprenorphine immunoassay with added Beta-Glucuronidase:

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Additional 41 specimens were analysed concurrently. CEDIA “Second Generation” assay

Measurement Uncertainty (MU)

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outperformed all other assays.

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The decision limits for Measurement Uncertainty were calculated from the quality control

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materials at two different levels around the cut-off values for each assay.

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MU at two different levels of EMIT II, ARK and Immunalysis immunoassays The data is summarised in Table 1.

Table 2

MU at two different levels of CEDIA and DRI immunoassays The data is summarised in Table 2. Table 3

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Journal Pre-proof Discussion The literature reviewed showed that there was no publication that covered the scope of assays evaluated in this article.(1, 3, 4, 8, 12-19) Additionally, the current generations of immunoassays are optimised and improved than the first generation of immunoassays, therefore a current study was needed to address this area of investigation. Because immunoassays are widely used for drug screening, it is essential not only to understand the

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to be able to select a suitable solution for a laboratory.

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difference between methods but also to assess these methods against gold standard methods

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The study showed that the immunoassay performance was similar for CEDIA and EMITT II

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plus immunoassay for cocaine, CEDIA and ARK EIA for EDDP, CEDIA and Immunalysis EIA for EDDP, DRI EIA and Immunalysis EIA for Oxycodone, CEDIA and EMIT II Plus

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for Heroin 6-MAM, and CEDIA and EMIT II Plus for Benzodiazepine. EMIT II Plus performed better than DRI RIA for Cannabinoids. EMIT II Plus performed

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better than CEDIA for both Amphetamine and Ecstasy. EMIT II Plus performed better than

Generation” immunoassay performed better than CEDIA “First

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CEDIA II “Second

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CEDIA for Opiates. ARK EIA performed better than DRI EIA for Fentanyl. Buprenorphine

Generation” and EMIT II Plus with and without added Beta-Glucuronidase Reagent. Manufacturers usually provide a list of cross-reactivity from other drugs that can affect the employed method. Cross-reactivity testing is performed by spiking the appropriate compound into negative urine. This information should be used as a general guideline.(20) It is not intended as a complete reference. Several new psychoactive substances were demonstrated to display cross-reactivity in the immunoassays

since

they

have

amphetamine-like

structure

and

activity.

CEDIA

Amphetamine/Ecstasy and EMIT Amphetamine Class tests showed the highest reactivity towards these new drugs.(12) The specificity of the CEDIA buprenorphine assay increased to 12

Journal Pre-proof 99.7% when the cut-off was increased from 5 ng/mL to 10 ng/mL. Intake of a therapeutic dose of codeine can yield a false-positive CEDIA buprenorphine result. Raising the cut-off from 5 ng/mL to 10 ng/mL increased the diagnostic accuracy.(20) Human metabolism patterns vary, and the effect of conjugation and other metabolic processes cannot be completely replicated. Positive cross-reactivity means the concentration of a drug that gives an assay response equal to the cut-off calibrator. Structurally related negative cross-reactivity means the concentration of a drug that did not produce a positive result

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equivalent to the cut-off calibrator. Structurally unrelated negative cross-reactivity means the concentration of drug that did not yield a false response for two levels of controls at ± 25% of

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the cut-off calibrator.

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There is also a sample validity issue to consider when interpreting results. Sample validity

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testing is integral to both screening and confirmatory methods. Each laboratory should have a valid scheme in place to address this important issue, which is complicated further by

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synthetic drugs and synthetic urines.(5, 21)

Acknowledgments

The author would like to thank Mrs. Stephanie Burmuzoska for her work in preparing the samples, Dr. Santiago Vazquez his support, and Dr. Emmanuel Favaloro for reviewing the article.

13

References

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1. Armbruster DA, Hubster EC, Kaufman MS, Ramon MK. Cloned enzyme donor immunoassay (CEDIA) for drugs-of-abuse screening. Clin Chem. 1995;41(1):92-8. 2. Henniona M, Barcelob D. Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: A review. Anal Chim Acta. 1998;362(1):03-34. 3. Bertol E, Vaiano F, Borsotti M, Quercioli M, Mari F. Comparison of immunoassay screening tests and LC-MS-MS for urine detection of benzodiazepines and their metabolites: results of a national proficiency test. J Anal Toxicol. 2013;37(9):659-64. 4. Dasgupta A, Saldana S, Kinnaman G, Smith M, Johansen K. Analytical performance evaluation of EMIT II monoclonal amphetamine/methamphetamine assay: more specificity than EMIT d.a.u. monoclonal amphetamine/methamphetamine assay. Clin Chem. 1993;39(1):104-8. 5. Franz F, Angerer V, Jechle H, Pegoro M, Ertl H, Weinfurtner G, et al. Immunoassay screening in urine for synthetic cannabinoids - an evaluation of the diagnostic efficiency. Clin Chem Lab Med. 2017;55(9):1375-84. 6. Matriciani B, Huppertz B, Keller R, Weiskirchen R. False-negative results in the immunoassay analysis of drugs of abuse: can adulterants be detected by sample check test? Ann Clin Biochem. 2018;55(3):348-54. 14

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7. Petrie M, Lynch KL, Ekins S, Chang JS, Goetz RJ, Wu AH, et al. Cross-reactivity studies and predictive modeling of "Bath Salts" and other amphetamine -type stimulants with amphetamine screening immunoassays. Clin Toxicol (Phila). 2013;51(2):83-91. 8. Pettersson Bergstrand M, Helander A, Hansson T, Beck O. Detectability of designer benzodiazepines in CEDIA, EMIT II Plus, HEIA, and KIMS II immunochemical screening assays. Drug Test Anal. 2017;9(4):640-5. 9. Schumann GB, Schweitzer SC. Examination of Urine in Clinical Chemistry:Theory, Analysis and Correlation. 2nd Edition ed: Kaplan LA, and Pesce AJ; 1989. 10. Fisher T. One Source-Multiple Technologies: Thermo Fisher; 2012 [Available from: https://www.analis.be/site/objects/media/0/0/7/5/2/0075211_media/media1.pdf. 11. Limited ASoCIaA. Uncertainty of Measurement (inter-batch/lot precision): Australasian Society of Clinical Immunology and Allergy Limited; 2005 [Available from: https://www.allergy.org.au/hp/papers/guidelines-uncertainty-of-measurement. 12. Beck O, Rausberg L, Al-Saffar Y, Villen T, Karlsson L, Hansson T, et al. Detectability of new psychoactive substances, 'legal highs', in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse. Drug Test Anal. 2014;6(5):492-9. 13. Hino Y, Ojanpera I, Rasanen I, Vuori E. Performance of immunoassays in screening for opiates, cannabinoids and amphetamines in post-mortem blood. Forensic Sci Int. 2003;131(2-3):14855. 14. Regester LE, Chmiel JD, Holler JM, Vorce SP, Levine B, Bosy TZ. Determination of designer drug cross-reactivity on five commercial immunoassay screening kits. J Anal Toxicol. 2015;39(2):14451. 15. Wiegand RF, Klette KL, Stout PR, Gehlhausen JM. Comparison of EMIT II, CEDIA, and DPC RIA assays for the detection of lysergic acid diethylamide in forensic urine samples. J Anal Toxicol. 2002;26(7):519-23. 16. Zhao H, Brenneisen R, Scholer A, McNally AJ, ElSohly MA, Murphy TP, et al. Profiles of urine samples taken from Ecstasy users at Rave parties: analysis by immunoassays, HPLC, and GC -MS. J Anal Toxicol. 2001;25(4):258-69. 17. Kohler KM, Hammer R, Riedy K, Auwarter V, Neukamm MA. Evaluation of CEDIA and DRI Drugs of Abuse Immunoassays for Urine Screening on a Thermo Indiko Plus Analyzer. J Clin Lab Anal. 2017;31(1). 18. Hsu J, Liu C, Liu CP, Tsay WI, Li JH, Lin DL, et al. Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens. J Anal Toxicol. 2003;27(7):471-8. 19. Wu AH, Forte E, Casella G, Sun K, Hemphill G, Foery R, et al. CEDIA for screening drugs of abuse in urine and the effect of adulterants. J Forensic Sci. 1995;40(4):614-8. 20. Berg JA, Schjott J, Fossan KO, Riedel B. Cross-reactivity of the CEDIA buprenorphine assay in drugs-of-abuse screening: influence of dose and metabolites of opioids. Subst Abuse Rehabil. 2015;6:131-9. 21. Begeman A, Franssen EJF. Lack of Detection of New Amphetamine -Like Drugs Using Conventional Urinary Immunoassays. Ther Drug Monit. 2018;40(1):135-9.

15

Journal Pre-proof

METHODS

FALSE

TRUE

TRUE

POSTIVE

NEGATIVE

POSITIVE

NEGATIVE

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CUT-OFF VALUE

FALSE

rn

SAMPLES

ANALYTE AND

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NUMBER OF

Table 1: Summary of The Comparison Between Immunoassays 16

Journal Pre-proof

62

62

59

60

15

Fenta nyl Cut-off = 1.0 ng/mL

54

Buprenorphi ne Cut-off= 5.0 ng/mL

37

Buprenorphi ne Cut-off= 10.0 ng/mL

37

Buprenorphi ne Cut-off= 5.0 ng/mL

41

-

-

20

12

CEDIA a nd Immuna l ys i s EIA (Res ults confi rmed by GC-MS)

-

-

20

12

DRI EIA a nd Immuna l ys i s EIA (Res ults confi rmed by GC-MS)

-

-

10

12

-

-

22

39

-

2 (DRI)

26

29

6 (CEDIA)

-

38

18

6 (CEDIA)

-

38

18

CEDIA a nd EMIT II Pl us (Res ults confi rmed by GC-MS)

-

6 (CEDIA)

22

31

CEDIA a nd EMIT II Pl us (Res ults confi rmed by GC-MS)

2 (CEDIA a nd EMIT II Pl us )

-

22

36

DRI EIA a nd LC-MS/MS (Res ults confi rmed by GC-MS)

-

1 (DRI)

6

8

-

-

7

8

12 (CEDIA 1s t Genera ti on)

-

15

27

35

1

22

2

39

-

41

-

28

-

38

-

CEDIA a nd EMIT II Pl us (Res ults confi rmed by GC-MS) DRI RIA a nd EMIT II Pl us (Res ults confi rmed by GC-MS) CEDIA (Amphetamine/Ecs ta s y) a nd EMIT II Pl us Amphetami ne (Res ults confi rmed by GC-MS) CEDIA Ampheta mi ne/Ecs ta s y a nd EMIT II Pl us Ecs ta s y (Res ults confi rmed by GC-MS)

ARK EIA a nd LC-MS (Res ults confi rmed by GC-MS) CEDIA “Fi rs t Genera ti on” a nd EMIT II Pl us (Res ults confi rmed by GC-MS) Buprenorphine: CEDIA “Second Genera ti on” a nd LC-MS/MS

-

EMIT II Pl us a nd LC-MS/MS

-

CEDIA “Fi rs t Genera ti on” a nd LC-MS/MS

1 (CEDIA)

1 (CEDIA 2nd Generation) 13 (EMIT II Pl us ) 1 (CEDIA)

CEDIA II “Second Genera ti on” a nd LC-MS/MS

-

-

Jo u

41

CEDIA a nd ARK EIA (Res ults confi rmed by GC-MS)

rn

15

23

Buprenorphi ne Cut-off= 5.0 ng/mL Buprenorphi ne Cut-off= 10.0 ng/mL

f

54

11

oo

61

-

pr

22

-

e-

32

CEDIA a nd EMITT II pl us (Res ults confi rmed by GC-MS)

Pr

32

Coca i ne Cut-off = 0.3 µg/mL (300 ng/mL) EDDP Cut-off = 0.10 µg/mL (100 ng/mL) EDDP Cut-off = 0.10 µg/mL (100 ng/mL) Oxycodone Cut-off = 100 ng/mL Heroi n 6-MAM Cut-off = 10 ng/mL Ca nna binoids Cutoff = 50 ng/mL Amphetamine/Ecst asy Cut-off = 0.3 µg/mL (300 ng/mL), Amphetamine/Ecst asy Cut-off = 0.3 µg/mL (300 ng/mL) Opi a tes Cut-off = 0.3 µg/mL (300 ng/mL) Benzodi a zepi ne Cut-off= 0.2 µg/mL (200 ng/mL) Fenta nyl Cut-off = 2 ng/mL

al

34

41

Buprenorphi ne Cut-off= 5.0 ng/mL

EMIT II Pl us a nd LC-MS/MS

-

41

Buprenorphi ne Cut-off= 5.0 ng/mL

EMIT II Pl us (with a dded Beta Gl ucuroni da s e Rea gent) a nd LC-MS/MS

-

13 (EMIT II Pl us ) 3 (EMIT II Plus + β Gl uc)

Measurement Uncertainty (MU) Level 1 of EMIT II, ARK and Immunalysis Immunoassays

17

384 346

258

397 378 66 115

10

11

15

14 9 2 5 4

20 22 30 28 17 5 10

3

3

6

4

2

4

6

6

12

8

4

8

Measurement Uncertainty (MU) Level 2 of EMIT II, ARK and Immunalysis Immunoassays ARK Fentanyl ng/mL EMIT II Heroin (6MAM) ng/mL

139 1.31 12.54

0.24 7 0.05 0.65

8 0.48 13 0.11 1.29

4

3

3.52

5

4.05

5.16

8

6

6.91

10

7.94

10.12

EMITT II Buprenorphine ng/mL

Immualysis Methadone (EDPP) ng/mL

Immunalysis Oxycodone ng/mL

EMITT II Cannabinoids (11-nor-Δ9-THC-COOH) ng/mL ARK Methadone (EDPP) ng/mL

8 19 7 3 5 3 0.19 5 0.06 0.5

17 22 16 38 14 6 9 6 0.36 10 0.12 1.0

4 5 5 9 3 8 6.5

8 10 10 18 6 16

f

11

oo

9

pr

e-

Pr

EMIT II Opiates ng/mL

ARK Fentanyl ng/mL EMIT II Heroin (6MAM) ng/mL

156 225 229 39 76 71 3.20 80 0.52 7.7

EMIT II Opiates ng/mL EMITT II Cannabinoids (11-nor-Δ9-THC-COOH) ng/mL ARK Methadone (EDPP) ng/mL Immualysis Methadone (EDPP) ng/mL

EMIT II Cocaine ng/mL

Immunalysis Oxycodone ng/mL

EMIT II Amphetamines ng/mL EMIT II Ecstasy (MDMA) ng/mL EMIT II Benzodiazepine ng/mL

Level 1

EMITT II Buprenorphine ng/mL

232 228

al

EMIT II Cocaine ng/mL

rn

EMIT II Benzodiazepine ng/mL

Combined Mean Combined SD Combined SD x 1.96 (MU in units) Combined CV% CV% x 1.96 (MU in %)

Jo u

Combined Mean Combined SD Combined SD x 1.96 (MU in units) Combined CV% CV% x 1.96 (MU in %)

EMIT II Amphetamines ng/mL EMIT II Ecstasy (MDMA) ng/mL

Level 2

Journal Pre-proof

6

4

5.68 6 11.92

12

8

11.13 12 23.37 12.8

114 6.78

Table 2: Measurement Uncertainty (MU) at Two Different Levels of EMIT II, ARK and

Immunalysis Immunoassays

18

Journal Pre-proof

33

79

17

49

8

15

4

0

33

96

16

29

7

9

7

3

8

17

13

6

16

3.6

75

1.2

7.6

0.8

11

0.2

0.8

1.6

22

0.3

1.6

10

25.3

15

14.2

10.8

19

49.6

30

27.7

21.1

oo

f

CEDIA Heroin (6-MAM) ng/mL

180

DRI Fentanyl ng/mL

CEDIA Methadone (EDPP) ng/mL

245

DRI Oxycodone ng/mL

DRI Cannabinoids (11-norΔ9-THC-COOH) ng/mL

148

pr

0

11

e-

Level 1

21

Pr

Combined Mean Combined SD Combined SD x 1.96 (MU in units) Combined CV% CV% x 1.96 (MU in %)

CEDIA Buprenorphine II ng/mL

CEDIA Opiates ng/mL

210

CEDIA Amphetamines/Ecstasy ng/mL CEDIA Benzodiazepine ng/mL

CEDIA Cocaine ng/mL

Measurement Uncertainty (MU) Level 1 of CEDIA and DRI Immunoassays

CEDIA Benzodiazepine ng/mL

CEDIA Cocaine ng/mL

CEDIA Opiates ng/mL

DRI Cannabinoids (11-nor-Δ9THC-COOH) ng/mL

CEDIA Methadone (EDPP) ng/mL

CEDIA Buprenorphine II ng/mL

DRI Oxycodone ng/mL

DRI Fentanyl ng/mL

CEDIA Heroin (6-MAM) ng/mL

350

236

384

371

56

132

7.8

124

2.9

13.7

38

96

18

19

5

12

1.4

13

0.2

1.5

74

188

35

37

10

24

2.8

26

0.4

3.0

11

16

5

5

9

9

18.4

10

7.5

11.0

21

32

9

10

17

18

36.0

20

14.8

21.6

Jo u

rn

al

CEDIA Amphetamines/Ecstasy ng/mL

Level 2

Measurement Uncertainty (MU) Level 2 of CEDIA and DRI Immunoassays

Combined Mean Combined SD Combined SD x 1.96 (MU in units) Combined CV% CV% x 1.96 (MU in %)

Table 3: Measurement Uncertainty (MU) at Two Different Levels of CEDIA and DRI Immunoassays 19