Comparison of EMIT II, CEDIA and DPC RIA Assays for the Detection of Lysergic Acid Diethylamide in Forensic Urine Samples*

Transcription

1 Journal of Analytical Toxicology, Vol, 26, October 2002 Comparison of EMIT II, CEDIA and DPC RIA Assays for the Detection of Lysergic Acid Diethylamide in Forensic Urine Samples* Russell F. Wiegand, Kevin L. Klette ~, Peter R. Stout, and Jay M. Gehlhausen Navy Drug Screening Laboratory, Naval Air Station, H-2033, Jacksonville, Florida t Abstract In an effort to determine a practical, efficient, and economical alternative for the use of a radioimmunoassay (RIA) for the detection of lysergic acid diethylamide (LSD) in human urine, the performance of two photometric immunoassays (Dade Behring EMIT II and Microgenics CEDIA) and the Diagnostics Products Corp. (DPC) RIA were compared. Precision, accuracy, and linearity of the 3 assays were determined by testing 60 replicates (10 for RIA) at 5 different concentrations below and above the 500.pg/mt LSD cut-off. The CEDIA and RIA exhibited better accuracy and precision than the EMIT II immunoassay. In contrast, the EMIT II and CEDIA demonstrated superior linearity r 2 = and , respectively, as compared with the RIA (~- = ). The specificity of the three assays was assessed using compounds that have structural and chemical properties similar to LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the 144 compounds studied, the EMIT II cross.reacted with twice as many compounds as did the CEDIA and RIA. Specificity was also assessed in 221 forensic human urine specimens that previously screened positive for LSD by the EMIT II assay. Of these, only 11 tested positive by CEDIA, and 3 were positive by RIA. This indicated a comparable specificity performance between CEDIA and RIA. This also was consistent with a previously reported high false-positive rate of EMIT II (low specificity). Each of the immunoassays correctly identified LSD in 23 out of 24 human urine specimens that had previously been found to contain LSD by gas chromatography-mass spectrometry at a cut-off concentration of 200 pg/ml The CEDIA exhibited superior precision, accuracy, and decreased cross-reactivity to compounds other than LSD as compared with the EMIT II assay and does not necessitate the handling of radioactive materials. * Disclaimer: The opinions contained in the publication are not to be construed as official or as reflecting the views of the Department of Defense or the Department of the Navy. f Author to whom correspondence should be addressed. kklette@dlj0ndsl.med.navy.mil. Introduction Lysergic acid is a naturally occurring alkaloid found in the ergot fungus, Claviceps pururea, which grows on rye, barley, and wheat (1). Lysergic acid diethylamide (LSD) is an indole derivative synthesized from lysergic acid and diethylamine (2). The synthesized LSD is a clear or white, odorless, water-soluble compound (3). Adverse effects of LSD use include impairment of behavioral control, visual and auditory hallucinations, and difficulty in performing simple tasks (3,4). Accurate irnmunoassay (IA) detection of LSD in human urine is difficult due partially to the combined effects of the very low doses ingested ( I.tg) and to rapid metabolism resulting in less than 1% of the LSD excreted unchanged in urine (5). Several commercial LSD IA screening technologies are available for the screening of human urine. Among these is the Dade Behring enzyme-multiplied IA technique (EMIT II) (3) and the Microgenics cloned enzyme donor (CEDIA) IA (6). Both technologies are photometric IAs that use an enzyme kinetic response to determine analyte concentration. A third type of LSD screening assay, the Diagnostic Products Corp. (DPC), radioimmunoassay (RIA) involves the counting of radioactivity from nuclide-labeled (Iodine-125) LSD to determine analyte concentration (3). Currently, all U.S. Navy Drug Screening Laboratories assay for the presence of LSD using the EMIT II LSD kit as the initial screening technique. All LSD presumptive positive samples are then rescreened for LSD using the DPC RIA to eliminate LSD false-positive samples resulting from cross-reacting compounds. The cross-reactivity of the EMIT II LSD assay with therapeutic drugs has been previously reported (7,8). Nonetheless, the use of RIA also presents several disadvantages that include the increased costs of purchasing RIA kits, the need for specific instrumentation associated with RIA analysis, and a multitude of Nuclear Regulatory Commission (NRC) requirements for the safe transport, handling, use, and disposal of radioactive material. A non-radioactive LSD screening assay with Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 519

2 equivalent specificity for LSD would facilitate an improvement in the LSD confirmation rate and eliminate the use of radioactive materials in the laboratory. Thus, the purpose of this study was to compare commercially available LSD immunoassay screening reagents for use as an alternative to the RIA assay. Materials and Methods LSD IA screening was accomplished on a Roche/Hitachi Modular automated analyzer (Indianapolis, IN) using the Dade Behring (Cupertino, CA) EMIT II and the Microgenics (Freemont, CA) CEDIA assay. The assays were calibrated using Roche (Indianapolis, IN) certified drug-free urine as a zero point and an LSD cut-off standard prepared with 500 pg/ml LSD. LSD testing was also accomplished using a Tecan pipetting system (Research Triangle Park, NC) and the Coat-a- Count radioimmunoassay (RIA) purchased from DPC (Los Angeles, CA). A Packard (Downers Grove, IL) gamma counter model # E5005 calibrated using four replicate cut-off standards (500 pg/ml LSD) with the mean of those being used as the denominator in the ratio calculation (a ratio of 1.00 or less indicating a positive sample) was used for sample analysis. All Table I. Interference Compounds Tested in Negative Urine Drug (mg/ml) Drug (mg/ml) Drug (mg/ml) Caffeine 0.1 Glutethimide 50 Phenylpropanolamine Lidocaine 0.1 Meperidine 50 Cotinine EDDP 0.1 Methamphetamine 50 Caffeine Methadone 0.1 Oxycodone 50 Doxylamine Desipramine 0.1 Pentobarbital 50 Carbamazepine Glutethimide 0.1 Phenobarbital 50 Doxepin Phenobarbital 0.1 Amobarbital 50 Azacyclonol Methaqualone 0.1 Butabarbital 50 Proadifen (IS) Cocaine 0.1 Codeine 50 Oxycodone Carbamazepine 0.1 Doxepin 50 Amoxapine Codeine 0,1 Imipramine 50 Quinidine Hydromorphone 0.1 Methadone 50 Hydroxyzine Nalorphine 0,1 Methaqualone 50 Verapamil Morphine 0,I Pentazocine 50 Acetaminophen 0,05 Oxycodone 0,1 Phencyclidine 50 Brompheniramine maleate 0.05 Diacetylmorphine 0.1 Secobarbital 50 Caffeine 0.05 Barbital 1.0 Amphetamine Cimetidine ,11-Dihydrocarbamazepine 1.0 Methamphetamine 0,0004 Dextromethorphan hydrobromide 0.05 Ethosuximide 1.0 Nicotine Doxylamine Succinate 0.05 Mephenytoin 1.0 Ephedrine d-ephedrine HCI 0.05 Metharbital 1.0 Meperidine d-pseudoephedrine HCI Methylprimidone 1.0 Fluoxetine 0,0004 Imipramine HCI 0.05 Methsuximide 1.0 Lidocaine Lidocaine HCI monohydrate 0.05 PEMA 1.0 Phencyclidine Meperdine 0.05 Phensuximide 1.0 Chlorpheniramine Methaqualone HCI 0.05 Carbamazepine 1.0 Methadone Methylphenidate HCI ,5-Diphenylhydantoin 1.0 Amitriptyline Naproxen 0.05 Ethotoin 1.0 Nortriptyline Oxazepam 0.05 Mephobarbital 1.0 Codeine 0,0005 Phenylpropanolamine HCI 0.05 Methyl PEMA 1.0 Diazepam Dextropropoxyphene HCI 0.05 (~-Methyl-~-propylsuccinimide 1.0 Nordiazepam Ranitidine HCl 0.05 N-Normethsuximide 1.0 Chloroquine Salicylic Acid 0.05 Phenobarbital 1.0 Flurazepam Phenethylamine 0.05 Primidone 1.0 Alprazolam Acebuto]ol 0.05 Medazepam 0.1 Thioridazine Ergonovine Lorazepam 0.1 Diphenhydramine Lysergol 0.05 Temazepam 0,1 Brompheniramine Ergotamine tartrate 0.05 Oxazepam 0.1 Dextromethorphan Dihydroergotamine tartrate 0.05 Diazepam 0,1 Promethazine Lysergic Acid 0.05 Bromazepam 0.1 Chlorpromazine Tryptophan ethyl ester 0.05 Amitriptyline 50 Oxycodone Nor-LSD and Nor-iso-LSD Amphetamine 50 Amphetamine Iso-LSD Cocaine 50 Methamphetamine Oxo-3-hydroxy-LSD Desipramine 50 Pseudoephedrine (z-ergocryptine

3 IA and RIA reagents, standards, and controls were utilized in accordance with the manufacturer's instructions for the analysis of urine samples. An LSD solution purchased from Alltech Associates, Inc. (Deerfield, IL) spiked into certified drug-free urine was used to manufacture the LSD cut-off calibrator (500 pg/ml) and a low LSD control prepared at 325 pg/ml. A positive LSD control (750 pg/ml) was obtained from Biopool International (Ventura, CA). In order to assess the precision, accuracy, and linearity of the 3 assays, 60 replicates prepared at 5 concentrations of LSD (200 pg/ml, 375 pg/ml, 500 pg/ml, 625 pg/ml, and 1000 pg/ml) were analyzed by the EMIT II and CEDIA assay. Only 10 replicates at these concentrations were analyzed by RIA because of cost constraints. The mean and standard deviation were calculated for each concentration tested. A one-way, crossed, fixed-effect analysis of variance (ANOVA) test followed by least significant difference means testing was used to determine significant differences between concentration levels using Statgraphics 6.0 (Manugistics, Rockville, MD). Linear regression analysis was performed using Origin 6.0 (Microcal Software, Inc., Northhampton, MA) to determine the linearity of each assay. Between assay precision and accuracy was assessed utilizing open LSD controls prepared at 750 pg/ml and 325 pg/ml which were assayed with all samples and precision replicates. Over-the-counter (OTC) drug and pharmaceutical multiconstituent standards consisting of the following drug categories: barbiturates, benzodiazepines, cocaine and cocaine metabolites, tricyclic antidepressants, stimulants, hallucinogens, opiates, ergots, and compounds structurally related to LSD were donated by the Armed Forces Institute of Pathology (AFIP, Rockville, MD) or purchased from Alltech Associates, Inc. (Deerfield, IL), Sigma Chemical, (St. Louis, MO), International Chemical Nexus, (Costa Mesa, CA), Cerilliant (Austin, TX), and Research Diagnostics Inc. (RDI, Flanders, N J). Table I lists the compounds assessed and their final concentrations. Urine samples (19,000), collected under forensic conditions Table II. Mean and Standard Deviation of Precision Replicates for CEDIA, EMIT II, and RIA* LSD Assay (pg/ml) (pg/ml) (pg/ml) (pg/ml) CEDIA Mean StdDev % CV EMIT II Mean StdDev % CV RIA Mean StdDev % CV * Values are in arbitrary units for CEDIA and EMIT II (cut-off = 100). RIA values are ratio of sample count/standard mean count. from active-duty United States Navy and Marine Corps personnel were screened by the laboratory's current LSD screening assay (EMIT II) using a cut-off concentration of 500 pg/ml. All analyses included 3% blind quality control material and 4% open quality controls with two concentrations (325 and 750 pg/ml). Results and Discussion ANOVA analysis confirmed that the five concentrations of standards prepared at 200 pg/ml, 375 pg/ml, 500 pg/ml, 625 pg/ml, and 1000 pg/ml were significantly resolved at a 95% confidence level of (p < 0.01) with F-ratios of 4142, 6010 and 222 for EMIT II, CEDIA, and RIA, respectively. The performance of the three assays (means, standard deviations, and coefficients of variation [% CV]) is summarized in Table II. The lowest within assay CVs were obtained with the RIA, followed by the CEDIA, and, lastly, the EMIT II assay. However, excluding the 40% cut-off control, the CEDIA produced CVs equivalent to those of the RIA. The within assay accuracy of the CEDIA assay was superior to that of the EMIT II for the standards prepared at 200, 375, 500, and 625 pg/ml (Table II). For example, the mean of the 500-pg/mL cut-off control for EMIT II was 84.7 (theoretical value = 100 arbitrary units) as compared with a mean value of for the CEDIA. The EMIT II assay lost accuracy after calibration resulting in the lower value for the 100% cut-off control. This could present a significant problem for detection of LSD in samples near the cut-off because the mean was significantly less than the arbitrary cut-off of 100. The between assay precision and accuracy of the IAs was assessed using open controls prepared at 750 pg/ml and 325 pg/ml, assayed over 14 days with 18 separate batches for the photometric assays and over 13 days with 13 batches for the RIA. Of the three assays, the RIA exhibited the lowest CVs followed by the CEDIA and, lastly, the EMIT II assay. The mean plus or minus standard deviation and % CV for the 750 pg/ml and 325 pg/ml controls as assayed by the RIA were , 7.1% and , 6.1%, respectively. The CEDIA produced CVs nearly equivalent to the RIA and exhib- looo ited normalized accuracy data that was (pg/mt) closer to the expected values as compared with the EMIT II. The mean plus or minus standard deviation and % CV for the pg/ml and 325 pg/ml controls as assayed by the EMIT II was , 11.1% 7.7 and , 29.2%, respectively. The 4.2 corresponding data for the CEDIA was , 8.5% and , 8.8 % o.o33 for the 750 pg/ml and 325 pg/ml con- 4.3 trois. Many workplace drug-testing laboratories include an above-threshold and sub-threshold quality-control samples to demonstrate the assay's ability to properly 521

4 differentiate a sample as positive and negative, respectively. Thus, assays exhibiting better precision and accuracy at these concentrations would decrease screening batch failure rates. There was a linear relationship between the expected and measured concentrations for all of the assays. The EMIT II _= ~" loo. 50- O- 18o ~ 120 o g: loo 80 A f ~ ~ confidence limit 9 [... Lower 95% confidence limit! 1 t! / B 80 Linear fit of CEDIA response Upper 95% confidence limit 40 Lower 95% confidence limit 20 i i i i , 1 - v Linear regression of RIA response ratio. [ ~_-~ Upper 95~ confidence limit 12- ",,'..~ Lower confidence limit i i i i /, Figure 1. Linear regression for LSD EMIT II assay, r 2 = (A) and CEDIA, r 2 = (B) using 60 replicates each at 200, 375, 500, 625, and 1000 pg/ml LSD. Linear regression for the LSD RIA assay, r 2 = (C) was obtained from 10 replicates at the same concentrations. and CEDIA demonstrated excellent linearity from 0 to 1000 pg/ml (r 2 = , , respectively) (Figures 1A and 1B). The lower linearity exhibited by the RIA (r 2 = ) (Figure 1C) may result from the use of a one-point calibration method rather than a two-point method as used in the EMIT II and CEDIA assay. The three IAs were evaluated for interferences from compounds that are structurally similar to LSD, common over-thecounter (OTC) products, prescription drugs and some of their metabolites, and other drugs commonly tested in-workplace drug-testing laboratories at concentrations exceeding the assays cut-off concentration9 Of the LSD-related compounds, 2- oxo-3-hydroxy-lsd, iso-lsd, nor-lsd, and nor-iso-lsd each screened positive with all three assays at the concentrations listed in Table III. Ergonovine (at 4000 ng/ml) was not positive by CEDIA but was by EMIT II and RIA. Lysergol was positive by EMIT II and CEDIA but not by RIA. Of the 133 non-lsd-related standards examined, none screened positive for LSD except by EMIT II at the concentrations tested (Table I). These compounds were tested in multi-constituent mixtures and the concentrations were chosen as easily achieved physiological concentrations. Some drugs were present at several concentrations as they were components of more than one commercial mixture preparation at variable concentrations. Specifically, brompheniramine, imipramine, and methylphenidate screened positive by EMIT II at 0.05 mg/ml (Table III). Imipramine and methylphenidate have previously been reported to interfere with the LSD EMIT assay (7,8). Assay specificity was also assessed in 221 forensic human urine specimens previously identified as presumptive positive for LSD using the EMIT II assay. These specimens were rescreened in parallel using the EMIT II, CEDIA, and RIA reagents. Of the 221 EMIT II samples that screened positive for LSD, 11 samples tested positive by the CEDIA assay (6%) and three tested positive by RIA (2%). The three RIA positives were analyzed by GC-MS for LSD using a previously reported method (9). LSD was not detected in any of the three samples. These results are consistent with a long-running observation in this laboratory of a confirmation rate of less than 1% when compared with EMIT II (number confirming positive/number EMIT II screening positive). Likewise these results were con- Table III. Compounds Tested as Positive (at or above 500-pg/mL LSD cut-off) by EMIT II, CEDIA, and RIA in Negative Urine Minimum Concentration with Positive Screen (ng/ml) Drug EMIT II CEDIA RIA Ergonovine 3000 > Lysergol 25,000 50,000 > 50,000 Nor-LSD and Nor-iso-LSD Iso-LSD Oxo-3-hydroxy-LSD Brompheniramine maleate 25,000 > 50,000 > 50,000 Imipramine HCI 50,000 > 50,000 > 50,000 Methylphenidate HCI 50,000 > 50,000 > 50,

5 sistent with high rates of cross-reactivity (low specificity) of EMIT II to extraneous compounds. Thus, CEDIA and RIA exhibited a comparable specificity and false-positive rate that was far lower than EMIT II. The specificity of the interference was further investigated by analyzing the forensic human urine specimens for other illicit drugs typically screened for in this laboratory including marijuana, cocaine, amphetamines/amphetamine designer drugs, methamphetamine, barbiturates, phencyclidine, and opiates. Of the 221 EMIT II positive samples, 10 samples (4.5%) screened positive for amphetamine using the Roche Online Kinetic Interaction of Microparticles in Solution (KIMS) reagent at a cutoff concentration of 500 ng/ml. When analyzed by GC-MS using a cutoff concentration of 500 ng/ml only one of the 10 samples confirmed positive for amphetamine at 7000 ng/ml. The remaining nine samples tested negative for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4- methylenedioxyethylamphetamine (MDEA). Additionally, 2 of the 221 samples screened positive for barbiturates (0.5%) and opiates (0.5%) using the Roche Online KIMS reagent at a cutoff concentration of 200 ng/ml and 2000 ng/ml, respectively. These data suggest the EMIT II assay may be cross-reacting with metabolites of OTC or prescription medications. To evaluate the accuracy of the IAs, 24 human urine specimens that had previously been found to contain LSD by GC-MS (9) were reanalyzed using the EMIT II, CEDIA, and RIA reagents. The GC-MS concentrations of these specimens ranged from 227 to 1400 pg/ml using a 200-pg/mL cutoff concentration. Twenty-three of the 24 specimens produced positive LSD results by each assay. The inability to detect LSD in the one discrepant sample may have occurred due to sample degradation because the specimen was stored for two years at-20~ with an original GC-MS quantitation of 227 pg/ml, near the 200-pg/mL LSD cut-off concentration. These results confirm particularly that CEDIA and RIA have low rates of false-negative results. Conclusions All three assays significantly resolved control materials prepared at five concentrations. The EMIT II and CEDIA demonstrated better linearity than the RIA under the conditions tested. The CEDIA and RIA demonstrated better precision (both within and between assay) than the EMIT II. The CEDIA and RIA cross-reacted with fewer compounds than the EMIT II and produced fewer extraneous positives than the EMIT II. The CEDIA reagent has additional advantages for the Department of Navy Drug Testing Program in that it does not necessitate the handling and administration of radioactive materials and can be performed on the same equipment as other assays used in the laboratory. Acknowledgments The authors wish to recognize and thank the efforts of Mr. John Dubay, Ms. Mary K. Carter, and Mr. Erasmo Villaluz in processing samples for this study. References 1. W.H. Foye, T.L. Lemke, and D.A. Williams. Principles of Medicinal Chemistry, 4th ed. William and Wilkins, Baltimore, MD, 1995, pp R.C. Baselt. Disposition of Toxic Drugs and Chemicals in Man, 5th ed. Chemical Toxicology Institute, Foster City, CA, 2000, pp B. Levine. Principles of Forensic Toxicology. American Association for Clinical Chemistry, Washington, D.C., 1999, pp , J. Cai and J. Henion. Elucidation of LSD in vitro metabolism by liquid chromatography and capillary electrophoresis coupled with tandem mass spectrometry. J. Anal. Toxicol. 20:27-37 (1996). 5. B.D. Paul, J.M. Mitchell, R. Burbage, M. Moy, and R. Sroka. Gas chromatographic-electron-impact mass fragmentation determination of lysergic acid diethylamide in urine.j. Chromatogr. 529: (1990). 6. D.R. Henderson, S.B. Friedman, J.D. Harris, W.B. Manning, and M.A. Zoccoli. CEDIA, a new homogeneous IA system. Clin. Chem. 32: (1986). 7. D. Ritter, C.M. Cortese, L.C. Edwards, J.L. Barr, H.D. Chung, and C. Long. Interference with testing for lysergic acid diethylamide. Clin. Chem. 43(4): (1997). 8. K.L. Klette, C.K. Horn, RR. Stout, and C.J. Anderson. LC-MS analysis of human urine specimens for 2-oxo-3-hydroxy LSD: method validation for potential interferants and stability study of 2-oxo-3-hydroxy LSD under various storage conditions. J. Anal Toxicol. 26: (2002). 9. G.K. Poch, K.L. Klette, D.A. Hallare, M.G. Manglicmot, R.J. Czamy, L.K. McWhorter, and C.J. Anderson. The detection of metabolites of LSD in human urine specimens: 2-oxo-3-hydroxy LSD--a prevalent metabolite of LSD. J. Chromatogr. 724(1): (1999). 523