Enzyme Multiplied Immunoassay Technique
Cross-reactivities of various phenethylamine-type designer drugs to immunoassays for amphetamines, with special attention to the evaluation of the one-step urine drug test Instant-View™, and the Emit® assays for use in drug enforcement.
Keiko Nakanishi, Akihiro Miki, Kei Zaitsu, Hiroe Kamata, Noriaki Shima, Tooru Kamata, Munehiro Katagi, Michiaki Tatsuno, Hitoshi Tsuchihashi, Koichi Suzuki
Forensic Science Laboratory, Osaka Prefectural Police Headquarters, 1-3-18 Hommachi, Chuo-ward, Osaka 541-0053, Japan. email@example.com
Cross-reactivities of 76 kinds of phenethylamine-type designer drugs and related compounds to the urine drug tests Instant-View ™ (IV)(the Methamphetamine (MA) test, the Amphetamine 300 test, and the MDMA test) have been investigated. An on-site urine test kit consisting of these three IV tests has been evaluated for the on-site screening of MA users, and the kit has been found to have satisfactory specificity for drug enforcement purposes by separately detecting both MA and its metabolite amphetamine. The cross-reactivity profiles of Emit(®) II Plus Amphetamines Assay, Emit(®) II Plus Ecstasy assay, and Emit(®) d.a.u.(®) Amphetamine Class assay have also been investigated and discussed.
Most cited papers:
Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit.
Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universität Göttingen, Germany. firstname.lastname@example.org
BACKGROUND The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. METHODS The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. RESULTS The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L),<4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%,<4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. CONCLUSIONS The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.
P J Gregoor, R G de Sévaux, R J Hené, C J Hesse, L B Hilbrands, P Vos, T van Gelder, A J Hoitsma, W Weimar
Department of Internal Medicine I, University Hospital Rotterdam, The Netherlands. email@example.com
BACKGROUND: Triple drug treatment consisting of mycophenolate mofetil (MMF), in a standard dose of 2 g daily, combined with cyclosporine (CsA) and prednisone, has become the standard immunosuppressive regimen after kidney transplantation in many centers. The need for therapeutic drug monitoring of mycophenolic acid (MPA) has not yet been established. Several drug interactions with MMF are known. We investigated the influence of CsA withdrawal on MPA trough levels in renal transplant patients. METHODS: Fifty-two patients were treated with 1 g of MMF twice daily, and prednisone and CsA targeted between 125 and 175 ng/ml for 6 months after transplantation. At 6 months after transplantation, 19 patients were randomized for continuation of triple therapy (group A), 19 patients discontinued CsA (group B), and 14 patients discontinued prednisone (group C). We compared 12-hr fasted MPA trough levels at 6 and 9 months after transplantation within and between these groups. RESULTS: MPA trough levels during treatment with CsA, MMF, and prednisone were significantly lower than those during treatment with MMF and prednisone only (group B); median levels were 1.87 mg/L (range: 0.56-5.27) vs. 3.16 mg/L (range: 0.32-7.78), respectively (P=0.002). MPA trough levels in groups A and C did not change between 6 and 9 months after transplantation; group A median levels were 1.87 (range: 0.31-4.32) vs. 1.53 mg/L (range: 0.36-3.70), and group C median levels were 1.62 (range: 0.69-10.34) vs. 1.79 mg/L (range: 0.54-6.00), respectively. At 9 months after transplantation, patients in whom CsA was discontinued had higher MPA trough levels as compared with patients who continued the use of triple therapy (P=0.001) or patients in whom steroids were withdrawn (P=0.014). CONCLUSION: A significant increase of MPA trough levels was found after discontinuation of CsA (6 months after transplantation), resulting in almost a doubling of MPA trough levels at 9 months after transplantation. This resulted in increased MPA levels in patients without CsA as compared to MPA levels in patients continuing triple therapy or discontinuing prednisone.
Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay.
Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n=102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/-standard deviation [SD]) bias (EMIT-HPLC) was 1.88+/-0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.
Therapeutic drug monitoring of mycophenolic acid: comparison of HPLC and immunoassay reveals new MPA metabolites.
E Schütz, M Shipkova, V W Armstrong, P D Niedmann, L Weber, B Tönshoff, K Pethig, T Wahlers, F Braun, B Ringe, M Oellerich
Abteilung für Klinische Chemie, Georg-August Universität, Goettingen, Germany.
Department of Clinical Pharmacology, Christchurch Hospital, Christchurch, New Zealand. firstname.lastname@example.org
AIMS To review the basis and optimal use of therapeutic drug monitoring of antimicrobial agents. METHODS Antimicrobial agents for which a reasonable case exists for therapeutic drug monitoring are reviewed under the following headings: pharmacokinetics, why monitor, therapeutic range, individualization of therapy, sampling times, methods of analysis, interpretative problems and cost-effectiveness of monitoring. RESULTS There is a strong historical case for monitoring aminoglycosides. The recent move to once-daily dosing means that criteria for therapeutic drug monitoring need to be redefined. Vancomycin has been monitored routinely but many questions remain about the most appropriate approach to this. A case can be made for monitoring teicoplanin, flucytosine and itraconazole in certain circumstances. CONCLUSIONS The approach to monitoring aminoglycosides is being redefined in the light of once daily dosing. It may be that less stringent monitoring is required in some circumstances but toxicity, especially ototoxicity, remains a problem with these drugs. Monitoring to avoid high AUCs (areas under the concentration-time curve) is recommended. The ideal method for monitoring vancomycin remains to be defined although a reasonable case exists for measuring trough concentrations, mainly to ensure efficacy. Teicoplanin is sometimes monitored to ensure efficacy while flucytosine may be monitored to avoid high concentrations associated with toxicity. Itraconazole has various pharmacokinetic problems and monitoring has been suggested to ensure that adequate concentrations are achieved.
Division of Clinical Epidemiology, Brigham and Women's Hospital, Boston, MA 02115, USA.
OBJECTIVES To develop explicit, reliable appropriateness criteria for antiepileptic drug level monitoring and to assess the appropriateness of monitoring in one tertiary care institution. DESIGN Appropriateness criteria derived from the literature and through expert opinion were used to evaluate a stratified random sample of antiepileptic drug level determinations obtained from chart review. SETTING Tertiary care center performing more than 10,000 antiepileptic drug level determinations per year. PATIENTS A total of 330 inpatients in whom antiepileptic drug levels were measured a total of 855 times. METHODS Drug levels were assessed at least 200 times for each of four antiepileptic drugs (phenytoin, carbamazepine, phenobarbital, and valproic acid). MAIN OUTCOME MEASURES The proportion of antiepileptic drug levels with an appropriate indication and, of those, the proportion sampled appropriately. RESULTS Overall, 27%(95% confidence interval, 24% to 30%) of levels had an appropriate indication. Interrater agreement for appropriateness was substantial (kappa = 0.61). There was no significant difference in the appropriateness rate among the four drugs (range, 25% to 29%). Of the 624 antiepileptic drug level determinations considered inappropriate (73%), only four (0.6%) were more than 20% higher than the upper limit of normal, and none of the four patients had clinical signs of drug toxicity. A median of six levels (range, one through 69) was determined per patient, and the median interval between level determinations was 24 hours. Of the 27% of level determinations with an appropriate indication, 51% were sampled correctly, resulting in an overall appropriateness rate of 14%. CONCLUSIONS Only 27% of antiepileptic drug level determinations had an appropriate indication, and half of these were not sampled correctly. Routine daily monitoring without pharmacological justification accounted for most of the inappropriate drug level determinations. Efforts to decrease inappropriate monitoring may result in substantial cost reductions without missing important clinical results.
Abteilung Klinische Chemie, Georg-August-Universität, Göttingen, Germany.
Marijuana use among HIV-positive and high-risk adolescents: a comparison of self-report through audio computer-assisted self-administered interviewing and urinalysis.
Department of Psychiatry, University of California, Los Angeles 90025-7510, USA. email@example.com
The validity of self-report of drug use has been found to vary widely. Moreover, previous research has focused on samples of adults. In 1996-1998, human immunodeficiency virus (HIV)-infected adolescents and high-risk, noninfected adolescents (n = 182) were recruited at 16 locations in 13 US cities into the Reaching for Excellence in Adolescent Care and Health (REACH) project, to the authors' knowledge, the first national study of disease progression among HIV-positive adolescents who were infected through sexual behavior or injection drug use. Self-report of marijuana use was assessed through audio computer-assisted self-administered interviewing (ACASI). Urines were tested for marijuana at a certified laboratory by using the enzyme-multiplied immunoassay technique. Conditional kappas for 2-, 5-, and 7-day self-reports were 0.57, 0.71, and 0.69, respectively. Maximum sensitivity was obtained from a combination of ACASI and urine drug testing. Contrary to previous studies, the data suggest that if a single evaluative instrument is to be used for prevalence, ACASI is more sensitive than urine drug testing for marijuana overall, but particularly for HIV-infected adolescents.
Determination of morphine-3-glucuronide in human urine by capillary zone electrophoresis and micellar electrokinetic capillary chromatography.
Department of Clinical Pharmacology, University of Bern, Switzerland.
Attempts to determine morphine-3-glucuronide (MO3G) by high-performance capillary electrophoresis and micellar electrokinetic capillary chromatography are reported. Using direct injection of urine, it was possible to achieve a limit of detection of about 20 micrograms/ml, which is poor compared with high-performance liquid chromatography and immunoassays. However, employing sample extraction with C8 cartridges, the presence of MO3G in urines that tested positive for opioids using a commercial enzyme-multiplied immunoassay technique could be successfully confirmed. The limit of detection with unambiguous identification of MO3G via spectral analysis was about 1 microgram/ml.
Department of Clinical Pharmacology, St Bartholomew's and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. A.Johnston@mds.qmw.ac.uk