Kinetic Interaction Of Microparticles Research Articles

Investigation into the linearity of the Roche c 702 carbamazepine assay.

Carbamazepine is an anticonvulsant drug which is monitored in patients due to toxic side effects. At Manchester University NHS Foundation Trust (MFT), carbamazepine is measured using Roche's Kinetic Interaction of Microparticles in Solution (KIMS) method on the c 702 platform. The assay has an upper limit of linearity of 20mg/L. Samples with concentrations above this limit should be identified and manually diluted. However, a poor EQA return from UK NEQAS for Tox and TDM Distribution 456 has highlighted an issue with the Roche KIMS assay. Sample A of the distribution had a carbamazepine concentration of 36mg/L but was underreported by several Roche users. This indicated that the assay was not consistently identifying high concentration samples which required a dilution. In this investigation, fresh frozen plasma was spiked with carbamazepine concentrations ranging from 15 to 40mg/L. The spiked samples and EQA material were analysed at two clinical laboratories using the Roche KIMS assay. Samples spiked with concentrations 20-30mg/L were not consistently identified for dilution by the analyser. This was observed at both hospital sites. Spike samples and EQA with concentrations >30mg/L were correctly identified at both sites. The manual dilution policy has been changed at MFT, so all samples with a carbamazepine level ≥15mg/L will be manually diluted. The problem was reported to Roche who are investigating the issue further. We would suggest that other laboratories look at validating their dilution protocols.

B-262 Validation of a point-of-care lateral flow immunoassay for urine drug testing prior to application in an outpatient rapid access addiction medicine clinic

Abstract Background Point-of-care (POC) urine drug testing is a useful adjunct to self-reporting in rapid access addiction medicine settings to immediately guide patient management. However, available POC immunoassays have limitations including cross-reactivity with unrelated compounds or low sensitivity that may cause false results. Here, we assessed the performance of a multi-drug test panel by comparing against gold-standard liquid chromatography tandem mass spectrometry (LC-MS/MS) testing. Methods 102 residual urine specimens were assayed using a competitive lateral flow immunoassay (LFA) for 10 drugs: 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP, methadone metabolite), buprenorphine, morphine, hydromorphone, oxycodone, fentanyl, cocaine, methamphetamine, amphetamine, and benzodiazepines (BTNX Rapid ResponseTM Multi-Drug Panel). Results were compared to those obtained by LC-MS/MS (n=67, 66%) or kinetic interaction of microparticles in solution automated immunoassay (KIMS) (Roche Diagnostics, n=35, 33%). Broad spectrum LC-MS/MS results were reviewed in entirety for discordant cases, particularly in false positives to identify the presence of any known cross-reacting compounds. Results Of 10 drugs evaluated, four demonstrated ≥95% agreement with LC-MS/MS or KIMS immunoassay (EDDP, buprenorphine, oxycodone, methamphetamine). Fentanyl demonstrated the highest false negative rate of 44% (LFA cut-off: 10 ng/mL) followed by amphetamines (22%, cut-off: 1000 ng/mL). Morphine and hydromorphone demonstrated false positive rates of 14% and 18%, respectively, with most cases likely due to cross-reacting opiate or opioid metabolites. Benzodiazepines (target: Oxazepam) demonstrated false positive and negative rates of 13% and 24%, respectively. Conclusions To our knowledge, this is the first study to evaluate the performance of the BTNX multi-drug test panel against definitive testing in urine samples. While good concordance was observed for most drugs, high rates of discordant results for fentanyl and others emphasize the need for confirmatory testing by methods with higher sensitivity and specificity. Careful consideration prior to implementation would be essential, including physician education, interpretative comments, and training resources.

Comparison of vancomycin assays in patients undergoing hemodialysis

Vancomycin is a glycopeptide antibiotic mainly excreted by glomerular filtration. Therefore, patients undergoing hemodialysis tend to accumulate its crystalline degradation product, which has been associated with cross-reaction in commercial immunoassays. The aim of this study was to assess the performance of two commercial immunoassays for measuring vancomycin levels in patients undergoing hemodialysis. This method-comparison study enrolled patients undergoing hemodialysis at two hospitals in Porto Alegre, Brazil. Vancomycin serum concentrations measured by Chemiluminescent Microparticle Assay (CMIA) and measured by Kinetic Interaction of Microparticles in Solution (KIMS) were compared with Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). A total of 64 samples from 42 patients and 54 samples from 23 patients were included in CMIA and KIMS groups. Both measurements were highly correlated with LC-MS/MS, with Spearman rank correlation coefficient r = 0.840 (p < 0.001) and r = 0.926 (p < 0.001), respectively. No deviation of linearity was observed (p = 0.81 and p = 0.49, respectively). The mean difference between CMIA and LC-MS/MS was -1.19 μg/mL and between KIMS and LC-MS/MS was -2.28 μg/mL. LC-MS/MS measured levels were, on average, 2.64 % higher than CMIA and 8.81 % higher than KIMS. CMIA and KIMS revealed accurate commercial methods to measure vancomycin serum concentrations in patients undergoing hemodialysis.

Comparison of the immunoassay method with the commercial and in-house LC-MS/MS methods for substance abuse in urine

Abstract Objectives The aim of this study was to compare the analytical performance of the KIMS (kinetic interaction of microparticles in solution) immunochemical method with a validated in-house and a commercial LC-MS/MS method. Methods The urine samples of the 100 subjects were included in the present study. The urine samples were analysed with Roche DAT immunochemical method based on KIMS method. In-house LC-MS/MS method was validated for 58 parameters according to the CLSI C62-A recommendations with the following parameters: matrix effect, lower limit of quantification (LLOQ), linearity, intra-day and inter-day precision and accuracy. Eureka Lab Division Drugs of Abuse kit was used as the commercial LC-MS/MS method. Results The immunochemical method had a satisfactory performance with specificity, sensitivity and accuracy values above 80 % and met the DRUID recommendation except benzodiazepines. The sensitivity and specificity of the immunochemical method were between 97–100 % and 84–100 %, respectively (except for benzodiazepines). The bias obtained for THC-COOH, morphine and codeine parameters were −17.5, 24.6 and 43.6 between two LC-MS/MS methods. The commercial method had a tendency to have a negative bias except for cannabinoids. Conclusions The analytical performance of the KIMS-based urine immunochemical method was found to be satisfactory for the intended use, except for benzodiazepines. The validated urine in-house LC-MS/MS method was found to be a good alternative for confirmation of substance abuse.

Precision and accuracy of commercial assays for vancomycin therapeutic drug monitoring: evaluation based on external quality assessment scheme.

Vancomycin remains a mainstay of the treatment of Gram-positive bacterial infections. It is crucial to accurately determine vancomycin serum concentration for adequate dose adjustment. To evaluate the precision and accuracy of commercial assay techniques for vancomycin concentration and to assess the comparability of vancomycin detection methods in Chinese laboratories. Human serum samples spiked with known concentrations of vancomycin were provided to laboratories participating in the external quality assessment scheme (EQAS). Assay methods included chemiluminescence, enzyme immunoassay (EIA) and so on. The dispersion of the measurements was analysed and the robust coefficient of variation (rCV), relative percentage difference (RPD) and satisfactory rate for method groups were calculated. Moreover, performance of the Chinese laboratories was assessed. A total of 657 results from 75 laboratories were collected, including 84 samples from 10 Chinese laboratories. The median rCV, median RPD and satisfactory rates classified by methods ranged from 1.85% to 15.87%, -14.75% to 13.34% and 94.59% to 100.00%, respectively. Significant differences were seen in precision, between kinetic interaction of microparticles in solution (KIMS) and other methods, and in accuracy, between enzyme-multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA) and other techniques. Vancomycin detection in China mainly depended on the chemiluminescence and EMIT methods, which tended to result in lower measurements. Although almost all assays in this study achieved an acceptable performance for vancomycin serum concentration monitoring, obvious inconsistencies between methods were still observed. Chinese laboratories were more likely to underestimate vancomycin concentrations. Thus, recognizing inconsistencies between methods and regular participation in vancomycin EQAS are essential.

Positive Propoxyphene Test Result for Drug-Free Serum or Plasma Obtained When Using the Roche Cobas c502 Urine Propoxyphene Screening Test

Abstract Introduction During a recent autopsy case, we were asked to investigate possible surreptitious inpatient use of drugs of abuse (DOA). As serum was the only sample type available for this subject, we initially ran serum samples on our urine drugs of abuse (DOAU) assays. Results were unexpectedly positive for propoxyphene (PPX). We immediately determined, however, that all plasma or serum samples, selected randomly from the hospital laboratory as presumed-negative controls, produced PPX-positive test results on the urine PPX assay. We report our investigation of this finding. Methods The PPX urine assay (Roche Cobas c502, Indianapolis, IN) is a semiquantitative screening test based on kinetic interaction of microparticles in solution (KIMS), with a cutoff concentration of 300 ng/mL. The assay was conducted according to manufacturer’s instructions, other than the substitution of plasma for urine as described below. Plasma or urine samples were obtained from to-be-discarded samples residual from routine testing in the hospital laboratory. Results Pooled plasma required a 1:4 dilution with drug-free urine to produce a PPX-negative test result. Bovine serum albumin (7 g/dL), mimicking total protein of plasma, produced a PPX-negative test result. Pretreatment of plasma using heterophilic antibody blocking reagent (Scantibodies Laboratory, Santee, CA) did not alter the PPX-positive test result. However, pretreatment of plasma by polyethylene glycol (PEG) to precipitate immunoglobulins altered the test result to PPX negative. PEG pretreatment did not alter PPX-positive results for PPX-spiked urine. Conclusions An autopsy case produced an incidental finding of false-positive test results for plasma tested on the Roche urine PPX assay. Based on PEG pretreatment data, it appears that some common component of immunoglobulins in plasma produces a positive interference in this assay. In principle, rare circumstances of severe renal dysfunction to produce plasma-like urine could potentially lead to false-positive results with the Roche urine PPX screening test.

Validación de un método inmunológico para la determinación de vancomicina en líquido cefalorraquídeo

Vancomycin (VAN) is an antibiotic used to treat serious infections. Its use is related to adverse effects such as acute facial hyperemia, nephrotoxicity and ototoxicity. By having a very narrow therapeutic range, its monitoring is necessary to maximize efficiency and minimize toxic effects. It is estimated that its concentration in CSF is approximately 10% of the plasma level in patients who receive intravenous treatment and who have meninges inflammation. Plasma concentrations of VAN are not a reliable indicator of those present in CSF. The aim of this study was to validate an immunological method based on the kinetic interaction of microparticles in solution (KIMS) for the determination of VAN in CSF. Materials and Methods: KIMS was validated for the evaluation of VAN in CSF. For this, the parameters of linearity, precision, accuracy, limit of detection, limit of quantification, interference, selectivity and specificity were determined. Results: The method was linear in a range between 0 and 15 µg/mL, the CV% obtained oscillated between 0.7 and 2.5% on the linear range. The LOD and LOQ were 0.4 µg/mL and 1.4 µg/mL respectively. The equation of the line obtained based on the correlation of methods between KIMS and HPLC-UV was y = 0.9151x + 1.1695, R² = 0.9453. Conclusion: The KIMS method demonstrated to have an adequate sensitivity and specificity to determine VAN in CSF and being a useful tool for monitoring patients who present complicated infections at CNS level. KIMS was validated for the evaluation of VAN in CSF. For this, the parameters of linearity, precision, accuracy, limit of detection, limit of quantification, interference, selectivity and specificity were determined. The method was linear in a range between 0 and 15 µg/mL, the CV% obtained oscillated between 0.7 and 2.5% on the linear range. The LOD and LOQ were 0.4 µg/mL and 1.4 µg/mL respectively. The equation of the line obtained based on the correlation of methods between KIMS and HPLC-UV was y = 0.9151x + 1.1695, R² = 0.9453. The KIMS method demonstrated to have an adequate sensitivity and specificity to determine VAN in CSF and being a useful tool for monitoring patients who present complicated infections at CNS level.

Bufotoxin poisoning that showed the sign of acute digitalis overdose in the patient of Kyushin® intoxication

Introduction: Kyushin® is a widely used herbal medicine in East Asia for heart failure. Toad venom in present in Kyushin®, which has positive inotropic effect as digitoxin. Case presentation: An 81-year-old, female patient presented with decreased mental status after overdose of Kyushin®. The first electrocardiogram showed junctional tachycardia with 142/min, suddenly dropped to 27/min and followed by ventricular fibrillation. After one cycle of CPR, spontaneous circulation returned but junctional bradycardia, tachycardia, and ventricular fibrillation appeared. After six times of defibrillation, spontaneous circulation returned, and mechanical ventilator and transcutaneous pacing were applied. Plasma toxicology test revealed digitoxin 66.90 ng/mL by cloned enzyme donor immunoassay and digoxin 0.76 ng/mL by kinetic interaction of microparticles in solution immunoassay. After 8 h from presentation, the patient’s mental status came to be alert, and then transcutaneous pacing was removed. Discussion: Cloned enzyme donor immunoassay has been reported to be highly cross-reactive with digoxin-like substances, which strongly supports bufotoxin to be responsible for arrhythmia of the patient. Conclusion: Poisoning of bufotoxin in Kyushin® can cause cardiac arrhythmia, even arrest, but without digoxin-specific Fab, conventional therapy could be successful.

KIMS, CEDIA, and HS-CEDIA Immunoassays Are Inadequately Sensitive for Detection of Benzodiazepines in Urine from Patients Treated for Chronic Pain

Background: Patients treated for chronic pain may frequently undergo urine drug testing to monitor medication compliance and detect undisclosed prescribed or illicit drug use. Due to the increasing use and abuse of benzodiazepines, this class of medications is often included in drug screening panels. However, immunoassay-based methods lack the requisite sensitivity for detecting benzodiazepine use in this population primarily due to their poor cross-reactivity with several major urinary benzodiazepine metabolites. A High Sensitivity Cloned Enzyme Donor Immunoassay (HS-CEDIA), in which betaglucuronidase is added to the reagent, has been shown to perform better than traditional assays, but its performance in patients treated for chronic pain is not well characterized. Objectives: To determine the diagnostic accuracy of HS-CEDIA, as compared to the Cloned Enzyme Donor Immunoassay (CEDIA) and Kinetic Interaction of Microparticles in Solution (KIMS) screening immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS), for monitoring benzodiazepine use in patients treated for chronic pain. Study Design: A study of the diagnostic accuracy of urine benzodiazepine immunoassays. Setting: The study was conducted at an academic tertiary care hospital with a clinical laboratory that performs urine drug testing for monitoring medication compliance in pain management. Methods: A total of 299 urine specimens from patients treated for chronic pain were screened for the presence of benzodiazepines using the HS-CEDIA, CEDIA, and KIMS assays. The sensitivity and specificity of the screening assays were determined using the LC-MS/MS results as the reference method. Results: Of the 299 urine specimens tested, 141 (47%) confirmed positive for one or more of the benzodiazepines/metabolites by LC-MS/MS. All 3 screens were 100% specific with no false-positive results. The CEDIA and KIMS sensitivities were 55% (78/141) and 47% (66/141), respectively. Despite the relatively higher sensitivity of the HS-CEDIA screening assay (78%; 110/141), primarily due to increased detection of lorazepam, it still missed 22% (31/141) of benzodiazepine-positive urines. The KIMS, CEDIA, and HS-CEDIA assays yielded accuracies of 75%, 79%, and 90%, respectively, in comparison with LC-MS/MS. Limitations: This study was limited by its single-site location and the modest size of the urine samples utilized. Conclusions: While the HS-CEDIA provides higher sensitivity than the KIMS and CEDIA assays, it still missed an unacceptably high percentage of benzodiazepine-positive samples from patients treated for chronic pain. LC-MS/MS quantification with enzymatic sample pretreatment offers superior sensitivity and specificity for monitoring benzodiazepines in patients treated for chronic pain. Key words: High sensitivty immunoassay, benzodiazepine, beta-glucoronidase, pain management, compliance, LC-MS/MS, screening

EPA-1097 - False positive phencyclidine drug screenings during psychopharmacologic treatment

False-positive drug screenings have been reported for several drugs and can affect the therapeutic relationship. We wanted to find out, which medication can cause false-positive phencyclidine drug screenings. We systematically looked at all psychiatric inpatients with phencyclidine positive urine drug screenings using a kinetic interaction of microparticles in a solution (KIMS) based system treated in our psychiatric department between 2008 and 2013. 39 of 40 positive phencyclidine urine drug screenings could plausibly be explained as false-positives by psychopharmacologic medication. The most frequent common medication in our case series was chlorprothixene, which has not been reported as a cause for any false-positive drug screenings so far. We also found trimipramine as a medication in three cases, being structurally similar to imipramine, which has been reported before to potentially cause cross-reactivity. Other false-positive results could be explained by venlafaxine, lamotrigine, imipramine and tramadol, which have been reported to have the capacity for cross-reactivity. Chlorprothixene and venlafaxine accounted for almost 75 % of the positive screenings. Confirmation by a second method like gas chromatography/mass spectrometry should follow positive drug screenings for phencyclidine.

Comparison of Immunoassay Screening Tests and LC-MS-MS for Urine Detection of Benzodiazepines and Their Metabolites: Results of a National Proficiency Test

For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA®; Enzyme-Multiplied Immunoassay Technique, EMIT®; Fluorescent Polarization ImmunoAssay, FPIA®; Kinetic Interaction of Microparticles in Solution, KIMS® and Immunochromatographic Techniques, IMC) with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC-MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity.

Prescription Compliance or Illicit Designer Drug Abuse?

A 62-year-old Caucasian woman underwent evaluation for possible liver transplantation. She had end-stage cirrhosis secondary to hepatitis C and alcohol abuse. Among the tests ordered were urine screens for various drugs of abuse, including amphetamines, for which the patient's urine sample was positive. Given the patient's history of alcohol abuse and possible other drug abuse, her physicians questioned her in detail about recent use of any illicit substances. The patient stated emphatically that she had used no recreational drugs for the past 15 years. The physicians wondered what could be the cause of a false-positive result and relayed to the laboratory that the patient was on several medications. The laboratory arranged to have the initial sample analyzed for amphetamines by GC-MS. The result was reported as negative for amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine (MDMA),3 and 3,4-methylenedioxy-amphetamine (MDA). In addition, the laboratory contacted the manufacturer of the screening immunoassay used [Amphetamines II, a kinetic interaction of microparticles in solution (KIMS) assay; Roche Diagnostics] and was informed that the medications that the patient had been prescribed, namely trazodone and bupropion, had been tested and did not exhibit any cross-reactivity. Amphetamines are a group of compounds that include amphetamine, methamphetamine, MDMA, and a large number of “designer drugs.” Widespread abuse has been reported worldwide and has increased over the last decade (1). In the clinical laboratory, the detection of amphetamine abuse has become increasingly important because of the potential for adverse drug–drug interactions involving existing prescription pharmaceuticals. Many antidepressants and antipsychotics function by affecting concentrations of dopamine, norepinephrine, and/or serotonin. Likewise, amphetamines increase concentrations of these biogenic amines by blocking their uptake by plasma membrane and/or vesicular transporters. Thus, in addition to their well-documented habit-forming properties and toxicities (2), amphetamines are of concern to clinicians because of their potentially toxic or fatal drug–drug interactions …

Is Dextromethorphan a Concern for Causing a False Positive During Urine Drug Screening?

The initial screening for drugs of abuse invariably poses the risk for false positives due to cross-reactivity. The over-the-counter antitussive medication dextromethorphan (DXM) can cause false-positive results for phencyclidine (PCP) upon urine drug screening (UDS). It is believed that DXM can also cause a false positive for opiates, presumably due to structural similarities. This could have serious implications, as DXM abuse is reported to be increasing. However, many of these claims are anecdotal, as no fully comprehensive studies exist. Furthermore, what studies have been completed reveal that DXM does not cause a false positive for opiates, despite their similarity in structure. Laboratories have established cut-off limits for opiates for the initial screen, and like all presumptive positives, confirmation with more sensitive methods are strongly recommended to rule out false positives. * DXM : dextromethorphan PCP : phencyclidine UDS : urine drug screening DXO : dextrorphan CNS : central nervous system NMDA : N-methyl-D-aspartate DAU : drugs of abuse-urine EMIT : enzyme multiplied immunoassay technique FPIA : fluorescence polarization immunoassay RIA : radioimmunoassay KIMS : kinetic interaction of microparticles in solution CEDIA : cloned enzyme donor immunoassay SAMHSA : Substance Abuse and Mental Health Services Administration GC-MS : gas chromatography-mass spectrometry LC-MS : liquid chromatography-mass spectrometry HPLC : high-performance liquid chromatography

Development and Validation of a High-Performance Liquid Chromatography Method for the Evaluation of Niflumic Acid Cross-Reactivity of Two Commercial Immunoassays for Cannabinoids in Urine

Niflumic acid is a nonsteroidal, anti-inflammatory drug widely prescribed in Greece. We recently noticed that this drug cross-reacts for cannabinoids in a kinetic interaction of microparticles in a solution (KIMS) immunoassay method but does not in an enzyme multiplied immunoassay technique (EMIT) immunoassay method. The objective of the study was to develop and validate a high-performance liquid chromatographic method in order to evaluate niflumic acid cross-reactivity in two commercial immunoassays for cannabinoids in urine, both in niflumic acid standards as well as in urine specimens obtained from subjects receiving niflumic acid. Urine niflumic acid standards were prepared in drug-free urine at 13 concentrations ranging from 1.25 to 1000 microg/mL. The standards gave presumptive positive cannabinoids results when analyzed by the KIMS immunoassay method when the concentration was above 2.5 microg/mL. None of the prepared standards gave a false-positive cannabinoid result when analyzed by the EMIT immunoassay method. By applying a 50 ng/mL cutoff for cannabinoids in these assays, all 55 urine specimens collected from the 5 subjects who participated gave negative results by the EMIT and false-positive results by the KIMS immunoassay method. It is concluded that KIMS is more prone to cross-reactions by niflumic acid compared to EMIT. Therefore, all positive screening tests for cannabinoids obtained by KIMS should be confirmed by another technique.

Hypothesis on interferences in kinetic interaction of microparticles in solution (KIMS) technology

The present paper describes an evaluation of the interferences found with an immunochemical drug test performed during a workplace control. During the period 1993-2003 more than 200,000 urine samples from workers were examined by the Italian Air Force. Samples were screened for drugs of abuse (opiates, cocaine, cannabinoids, amphetamines, methadone) using an immunochemical technique (Roche, kinetic interaction of microparticles in solution, KIMS). A total of 520 positive samples were sent to the Forensic Toxicology Laboratory for confirmation by gas chromatography/mass spectrometry (GC/MS). Approximately 39% of these were found to be true positives. For the remaining samples, pharmacological therapy in subjects was estimated to evaluate possible interferences due to medicine intake. Our study showed a high frequency of false-positive results with this immunochemical technique, mainly for the cannabinoid and amphetamine groups. Recurrent references to some medicines during subject anamnesis were noted.

Urine Benzodiazepine Screening using Roche Online(R) KIMS Immunoassay with -Glucuronidase Hydrolysis and Confirmation by Gas Chromatography-Mass Spectrometry

Performance of the Roche Online KIMS (kinetic interaction of microparticles in solution) benzodiazepine (BZD) immunoassay (IA) with and without beta-glucuronidase treatment was evaluated on a Hitachi Modular automated IA analyzer calibrated using nordiazepam at 100 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences were evaluated. Precision of the assay (percent coefficient of variation (%CV)) with and without addition of the enzyme was less than 6% and 9%, respectively, with linearity (r(2) value of 0.9578 and 0.9746), respectively. Between-run precision of a 125 ng/mL nordiazepam control (n = 287) over 67 days, produced a %CV of 13.6% for the hydrolytic assay. Modification of the BZD assay to include automated hydrolysis of urinary BZD glucuronide conjugates was evaluated using three glucuronidated BZD standards prepared at concentrations ranging from 250 to 10,000 ng/mL. With hydrolysis, temazepam, oxazepam, and lorazepam glucuronides, produced cross-reactivities of 25%, 15%, and 20%, respectively. Without hydrolysis, the glucuronidated BZD standards produced less than 1% cross-reactivity in the assay. The ability of the assay to differentiate between positive and negative samples was evaluated by assaying 20 negative urine samples and serial dilutions of certified drug-free urine fortified with 28 different BZDs. All of the negative and positive urine samples produced the appropriate screening result. Cross-reactivities of 27 different BZDs, calculated as the normalized IA response divided by the BZD concentration that produced a response approximately equivalent to the response of a 100 ng/mL nordiazepam standard and multiplied by 100, ranged from 15% to 149%. Human urine samples (n = 28) that were previously found to contain BZDs by gas chromatography-mass spectrometry (GC-MS) also produced a positive BZD IA result. The IA was challenged with 78 potentially interfering compounds, and none produced a positive BZD response. As a part of the validation, a large number of human urine samples (29,500) were assayed using the modified Online BZD IA method to evaluate the performance of the method in production. Of the 29,500 samples tested, 80 produced a positive IA result. Analysis by GC-MS confirmed the presence of at least 1 BZD compound in 61 of the samples corresponding to a confirmation rate of 76%. The Online BZD IA modified by the automatic addition of beta-glucuronidase appears well adapted for the rapid detection of BZDs and their metabolites in human urine.

Rifampicin causes false-positive immunoassay results for urine opiates.

The treatment of tuberculosis usually includes the antibiotic rifampicin, especially in patients with concomitant human immunodeficiency virus infection. Some of these patients are in withdrawal therapy for drug abuse. When opiate screening is carried out in patients receiving rifampicin, false positive results are detected with the kinetic interaction of microparticles in solution method. We evaluated this interference in a Cobas-Integra analyzer and found a 12% cross-reactivity of rifampicin for antibiotic concentrations ranging from 0.19 to 6.08 mumol/l (156 to 5000 micrograms/l). This effect is not explained by the colour of the rifampicin solutions. Calculations assuming first order kinetics of elimination show that more than 18 hours after a single oral dose of 600 mg of rifampicin, a false positive result for opiates could be obtained. This indicates that the risk of a false positive result must always be considered when urine samples from these patients are analyzed.

Synthesis of new d-propoxyphene derivatives and the development of a microparticle-based immunoassay for the detection of propoxyphene and norpropoxyphene

The synthesis of [S-(R,S)]-4-[[methyl[2-methyl-3-(1-oxopropoxy)-3, 4-diphenylbutyl]amino]-1-oxobutoxy]-2,5-pyrrolidinedione+ ++ (propoxyphene active ester, 2) is described. This was used as an intermediate to prepare a propoxyphene immunogen, [S-(R,S)]-4-[methyl][2-methyl-3-(1-oxopropoxy)-3,4-diphenylbuty l]-amino]- 1-oxobutyl-Bovine Thyroglobulin (3). This immunogen was then used to generate antibodies which demonstrate good cross-reactivity to d-propoxyphene, d-norpropoxyphene, and other propoxyphene metabolites. In addition, these antibodies were shown to have very low cross-reactivity to methadone, a structurally related compound. The introduction of an aminomethyl benzoate spacer into the propoxyphene active ester (2), followed by the activation of the carboxylic acid, provided for a more stable active ester (5). This stable active ester, together with the antibodies generated from the propoxyphene immunogen, has led to the development of an immunoassay based on the Kinetic Interaction of Microparticles in Solution (KIMS).

Synthesis of New d-Propoxyphene Derivatives and the Development of a Microparticle-Based Immunoassay for the Detection of Propoxyphene and Norpropoxyphene

The synthesis of [S-(R,S)]-4-[[methyl[2-methyl-3-(1-oxopropoxy)-3, 4-diphenylbutyl]amino]-1-oxobutoxy]-2,5-pyrrolidinedione+ ++ (propoxyphene active ester, 2) is described. This was used as an intermediate to prepare a propoxyphene immunogen, [S-(R,S)]-4-[methyl][2-methyl-3-(1-oxopropoxy)-3,4-diphenylbuty l]-amino]- 1-oxobutyl-Bovine Thyroglobulin (3). This immunogen was then used to generate antibodies which demonstrate good cross-reactivity to d-propoxyphene, d-norpropoxyphene, and other propoxyphene metabolites. In addition, these antibodies were shown to have very low cross-reactivity to methadone, a structurally related compound. The introduction of an aminomethyl benzoate spacer into the propoxyphene active ester (2), followed by the activation of the carboxylic acid, provided for a more stable active ester (5). This stable active ester, together with the antibodies generated from the propoxyphene immunogen, has led to the development of an immunoassay based on the Kinetic Interaction of Microparticles in Solution (KIMS).

Method Comparison of EMIT II and OnLine with RIA for Drug Screening

The newest formulation of the EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine, using the kinetic interaction of microparticles in solution methodology, were evaluated for marijuana, cocaine, opiates, barbiturates, and phencyclidine. Both types of immunoassays were performed on an Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, probability of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. EMIT II and OnLine were compared with RIA tests for the five drugs to determine each assay's ability to detect samples which confirm positive by GC/MS. The RIA and OnLine marijuana tests detected > 99% of confirmed positive samples while EMIT II detected about 90%. All three immunoassays performed equivalently for cocaine and opiates, each assay detecting at least 98% of positives. Barbiturates showed the greatest disparity with OnLine detecting 96%, EMIT II 85%, and RIA 79% of confirmed positive samples. Too few phencyclidine positive samples were detected for a method comparison study. The fully automated EMIT II and OnLine assays are preferable for a variety of reasons to our laboratory's current semi-automated RIA tests for large volume urine testing. The immunoassays offer comparable performance for some drugs but not for others.