Validation of the Cozart microplate ELISA for detection of opiates in hair

The purpose of this study was to determine the performance characteristics of the Cozart Opiates Microplate ELISA assay for the detection of opiates in hair specimens. One hundred and six hair specimens were collected from volunteers and from drug-related deaths. The hair samples were extracted by sonication followed by overnight extraction in methanol at 60 degrees C. The methanol extract was dried, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture and 0.1M HCl were added to 20 mg of specimen or spiked blank hair and sonicated for 1 h. The opiates were extracted by solid-phase and derivatized with BSTFA + 1% TMS for GC-MS analysis. Fifty-one hair specimens were confirmed positive by GC-MS. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results as the reference method. The optimum cutoff for the Cozart Opiate Microplate ELISA was determined to be between 200 and 300 pg morphine equivalents/mg hair using a 20-mg hair sample. The Cozart Opiates Microplate EIA for opiates in hair using a cutoff of 200 pg/mg hair with a 20-mg hair sample had a sensitivity of 98% +/- 2% and a specificity of 92.7% +/- 3.5% versus GC-MS.     

Validation of the Cozart microplate EIA for cocaine and metabolites in oral fluid

The purpose of these studies was to determine the performance characteristics of the Cozart microplate EIA for the detection of cocaine and cocaine metabolites in oral fluid. A total of 217 samples were collected using the Cozart RapiScan oral fluid Collection System from donors who were receiving treatment and being monitored for their addiction. The samples were screened in the laboratory using the Cozart microplate EIA for cocaine and metabolites and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. Of the 217 samples tested, 116 were positive for cocaine and/or cocaine metabolites. The Cozart microplate EIA for cocaine, using a cutoff of 30 ng/mL benzoylecgonine equivalents in neat oral fluid, had a sensitivity of 95.7% +/- 2.0% and specificity of 100% +/- 0.7%. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Validation of the immunalysis microplate ELISA for the detection of methamphetamine in hair

The object of this study was to validate the Immunalysis Methamphetamine Microplate ELISA for detecting methamphetamine in hair. Twenty-nine scalp hair samples were obtained as routine cases submitted to the National Institute of Scientific Investigation in Seoul by the police. The hair samples were washed with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. The samples were screened using the Immunalysis Methamphetamine Microplate ELISA and confirmed using gas chromatography-mass spectrometry (GC-MS). Twenty-eight hair samples were screened and confirmed as positive for methamphetamine. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h at 60 degrees C. For GC-MS, the samples were extracted for 20 h in methanol containing 1% hydrochloric acid. The methanol/acid solution was evaporated to dryness and the resulting residue was derivatized with trifluoroacetic anhydride. Methamphetamine and amphetamine were detected using selective ion monitoring (SIM) mode. The Immunalysis Methamphetamine Microplate ELISA demonstrated a sensitivity and specificity of 97% and 100%, respectively, using a cut-off concentration of 0.5 ng/mg d-methamphetamine. The ELISA kit showed 63% cross-reactivity with d,l-methamphetamine and did not cross-react to any significant extent with the licit l-methamphetamine isomer. The intra- and interassay precisions were 2.5% and 3.7%, respectively.     

Gas chromatography-mass spectrometry confirmation of Cozart RapiScan saliva methadone and opiates tests

The object of this study was to determine the sensitivity and specificity of the Cozart RapiScan onsite saliva test for methadone and opiates versus laboratory-based enzyme immunoassay (EIA) and gas chromatography-mass spectrometry (GC-MS) confirmation. Fifty saliva specimens were obtained from 28 volunteers among persons entering a substance abuse clinic. Specimens were tested onsite using the Cozart RapiScan Saliva test and Cozart RapiScan Reader. Specimens were retested by Cozart Microplate EIA assays on receipt at the laboratory and then frozen for later confirmation by GC-MS. For GC-MS, deuterated internal standards were added to specimen aliquots which were extracted using solid-phase columns at pH 6 and eluted with dichloromethane/isopropanol/ammonia (80:19:2). The dry residues were derivatized with PFOH and PFPA and dried, and the reconstituted extract was injected and quantitated by GC-MS. The Cozart RapiScan Methadone Saliva Assay was found to have a sensitivity and specificity of 100% +/- 12% versus GC-MS (2-ng/mL cutoff) and a sensitivity of 100% +/- 11% and a specificity of 95% +/- 2.4% versus the Microplate EIA for methadone (30-ng/mL cutoff). The Cozart RapiScan Saliva Opiate test had a sensitivity of 100% +/- 12% and a specificity of 92% +/- 3.2% versus GC-MS (2-ng/mL cutoff) and a sensitivity of 96% +/- 2.2% and specificity of 95% +/- 2.4% versus the Microplate EIA for opiates (30-ng/mL cutoff).     

Analysis of methadone and its metabolites in meconium by enzyme immunoassay (EMIT) and GC-MS

An EMIT-ETS d.a.u. immunoassay screening method for methadone in meconium and a gas chromatography-mass spectrometry (GC-MS) method for methadone and its metabolites including 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) in meconium were described. The GC-MS method showed good linearity (r2 > or = 0.998) over a concentration range of 25-2000 ng/g with limits of detection of 10, 25, and 10 ng/g for methadone, EDDP, and EMDP, respectively, and a limit of quantitation of 25 ng/g for all three analytes. Fifty pooled meconium samples were screened using a cutoff of 200 ng/g, and all samples screened negative. GC-MS analysis of all samples showed four samples to contain methadone (35.2 to 79.9 ng/g), EDDP (28.5 to 557.2 ng/g), or both, with no detectable amount of EMDP. The negative results on the four specimens at the cutoff used may be explained by the fact that EMIT-ETS d.a.u. antibody for methadone was specific to the parent drug. The results point to the fact that immunoassays should be directed to EDDP for detection of prenatal exposure of methadone through analysis of meconium specimens.     

Cozart RapiScan Oral Fluid Drug Testing System: an evaluation of sensitivity, specificity, and efficiency for cocaine detection compared with ELISA and GC-MS following controlled cocaine administration

Oral fluid has become a widely accepted alternative matrix for drugs of abuse detection. Immunoassays have been developed for on-site testing of cocaine and metabolites in oral fluid. The performance of the Cozart RapiScan Oral Fluid Drug Testing System (CRS) was evaluated in comparison with Cozart Microplate Enzyme Immunoassay Cocaine Oral Fluid Kit (COC ELISA) and gas chromatography-mass spectrometry (GC-MS) at several screening and confirmation cutoffs, including those proposed by SAMHSA and those currently in use in the U.K. Oral fluid samples (n = 1271) were collected prior to and following controlled clinical cocaine administration. CRS provides a qualitative screen at a preset cutoff of 30 microg/L. Sensitivity, specificity, and efficiency for CRS (30 microg/L) as compared with COC ELISA with a cutoff of 30 microg/L were 92.1%, 91.8%, and 92.0%. The comparison of CRS (30 microg/L) with the 8-mg/L proposed SAMHSA confirmation cutoffs for cocaine and/or benzoylecgonine exhibited a sensitivity of 82.7%, a specificity of 94.5%, and an efficiency of 87.6%. For this study, an alternative CRS cutoff of 20 microg/L was also evaluated. Performance characteristics of CRS (20 microg/L) at the proposed SAMHSA confirmation cutoffs were 89.9%, 89.7%, and 89.8%, respectively. At cutoffs in use in the U.K., 30- micro g/L CRS screen and 15- microg/L GC-MS cutoffs for cocaine, benzoylecgonine, and/or ecgonine methyl ester sensitivity, specificity, and efficiency were 89.4%, 92.2%, and 90.7%, respectively. Cozart RapiScan had performance similar to the COC ELISA assay for the detection of cocaine exposure and suitable sensitivity and specificity at the proposed SAMHSA cutoffs.     

Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine

The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%.     

Phenprocoumon metabolites in human plasma; characterization by HPLC and GC-MS

Summary Pooled plasma from patients receiving phenprocoumon anticoagulant therapy was extracted and the following substances were characterized: phenprocoumon, and its 7-hydroxy,4-hydroxy and 6-hydroxy derivatives; they were identified by HPLC and after methylation by quartz capillary GC-MS using the electron impact and selective ion monitoring modes. This is the first occasion when phenprocoumon metabolites have been identified in plasma; they were unconjugated and in much lower concentrations (43.2 and 2 ng/ml for the 7,4 and 6-hydroxy derivatives, respectively) than the original compound (2000 ng/ml).     

Screening of clostebol and its metabolites in bovine urine with ELISA and comparison with GC-MS results in an interlaboratory study

An extraction procedure for clostebol metabolites in urine is developed including enzymatic hydrolysis of conjugated metabolites with Helix pomatia juice (SHP) and solid-phase extraction (SPE) with further cleanup of sample extracts. For the enzymatic deconjugation step, variables such as buffer pH, amount of enzyme, incubation time, and temperature are examined. For the SPE step, different wash solutions and combinations with subsequent liquid-liquid extractions are examined. Incurred bovine urine samples, obtained through oral and intramuscular administration of clostebol acetate to animals, are used to test the performance of the developed method. In addition to the optimization of the sample pretreatment procedure, an interlaboratory study for the analysis of the incurred urine samples with ELISA and GC-MS is performed and good agreements are observed.     

A new GC-MS method for the determination of five amphetamines in human hair

A new gas chromatography-mass spectrometry method for the simultaneous identification and quantitation of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethamphetamine (MDEA) in hair is proposed. Hair was hydrolyzed in 1 M NaOH at 40 degrees C, subjected to extraction with 4:1 (v/v) methylene chloride/isopropanol, and derivatized with pentafluoropropionic anhydride (PFPA) and ethyl acetate. Calibration curves for the five analytes were constructed over the concentration range 0.5-25.0 ng/mg, using their pentadeuterated analogues as internal standards. The limits of detection and quantitation obtained were 0.045 and 0.151 ng/mg for AP; 0.014 and 0.048 ng/mg for MA; 0.013 and 0.043 ng/mg for MDA; 0.017 and 0.057 ng/mg for MDMA; and 0.007 and 0.023 ng/mg for MDEA. The accuracy of the method was found to be in the range +/- 9%, and the coefficients of variation were less than 8%. Overall, 24 hair specimens tested positive for one or more amphetamines, with average concentrations of 0.88 ng/mg for AP, 10.14 ng/mg for MA, 1.30 ng/mg for MDA, and 8.87 ng/mg for MDMA. Only one specimen tested positive for MDEA with a concentration of 0.84 ng/mg.     

Detection of methadone, LAAM, and their metabolites by methadone immunoassays.

l-Alpha-acetylmethadol (LAAM) was recently approved as a substitute for methadone. LAAM, methadone, and their common metabolite, methadol, are extensively N-demethylated. The structural similarities of LAAM and its metabolites to methadone suggest that they may cross-react in methadone immunoassays. To test this hypothesis, drug-free urine was fortified with LAAM, norLAAM, dinorLAAM, methadol, normethadol, dinormethadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), or 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) at 12 concentrations (0.03 to 100 microg/mL). Samples were analyzed using two enzyme immunoassays (Behring Diagnostics, EIA-b; Diagnostic Reagents, EIA-d); a fluorescent polarization immunoassay (Abbott, FPIA); two enzyme-linked immunosorbant immunoassays (Diagnostix, ELISA-d; STC Technologies, ELISA-s); a kinetic microparticles in solution immunoassay (Roche Diagnostic Systems, KIMS); and a radioimmunoassay (Diagnostic Products, RIA). LAAM had high cross-reactivity with ELISA-d (318.3%), RIA (249.5%), EIA-d (100.8%), KIMS (91.1%), and ELISA-s (75.3%). Methadol also displayed relatively high cross-reactivity as follows: EIA-d (97.8%), KIMS (85.4%), ELISA-d (70.3%), and FPIA (37.7%). Successive N-demethylations of LAAM and methadol were associated with loss of cross-reactivity. The methadone metabolites EDDP and EMDP showed little cross-reactivity. These findings suggest that LAAM use could result in positive immunoassay test results when using many of the commercially available methadone immunoassay kits and that confirmation of LAAM and its metabolites should be considered.     

Detection of benzodiazepines in hair using ELISA and LC-ESI-MS-MS

This study was designed to validate an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of nine benzodiazepines in hair. Sixteen hair case samples were tested from drug-related deaths where a positive benzodiazepine blood result was obtained. The case samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h and then neutralized with dibasic phosphate buffer. They were diluted 1:5 with phosphate buffer saline (PBS) prior to analysis. For LC-MS-MS, the samples were incubated overnight in methanol/25% ammonium hydroxide (20:1). The benzodiazepines were extracted by solid phase. Thirteen samples were confirmed positive by LC-MS-MS. The benzodiazepines detected included diazepam, nordiazepam, temazepam, oxazepam, nitrazepam, and lorazepam. Using a cut-off concentration of 0.1 ng/mg oxazepam, the Immunalysis Benzodiazepine Microplate ELISA demonstrated a sensitivity and specificity of 100% and 81%, respectively, compared with LC-MS-MS results.     

Interindividual dose/concentration relationship for methadone in hair

Hair samples were collected from 60 patients receiving long-term methadone maintenance: 50 were taking the drug orally and 10 were receiving the drug by intravenous injection. The amount of methadone present in the hair samples was measured using methanolic extraction, derivatization of the extracts with MTBSTFA, followed by electron impact gas chromatography-mass spectrometry operating in selected ion monitoring mode. The limit of quantitation for the assay was 0.4 ng/mg hair. The dose/concentration relationship for methadone in hair was investigated. No interindividual correlation between prescribed dose and concentration of methadone in hair was observed.     

PCR和ELISA检测不同人群TTV的感染状况

目的　检测湛江地区不同人群TTV的感染情况。方法　应用PCR和ELISA法同时检测正常献血员血清标本 5 12份 ,HBsAg阳性血清标本 60份 ,抗 HCV阳性血清标本 48份。结果　正常献血员、HBsAg阳性、抗 HCV阳性标本用PCR法检测TTVDNA阳性率分别为 10 .94%、13 .3 3 %和 16.67%;ELISA检测抗 TTVIgG ,阳性率分别为 7.42 %、10 .0 0 %和 10 .42 %。两种检测方法结果差异无显著性 (P>0 .0 5 ) ,不同人群TTVDNA和TTVIgG阳性率差异也无显著性 ( P >0 .0 5 )。结论　该地区献血员人群TTVDNA、TTVIgG与HBsAg阳性、抗 HCV阳性无明显相关性     

Validation of an ELISA method for screening methadone in postmortem blood

In this study, we evaluate Venture Labs' enzyme-linked immunosorbent assay (ELISA) for the detection of methadone in postmortem specimens. Sixty-one postmortem specimens that previously screened positive for methadone along with 59 specimens which screened negative for methadone were included. All specimens were screened using the Venture Labs methadone assay in conjunction with a liquid-liquid basic extraction and gas chromatographic-mass spectrometric (GC-MS) analysis. All cases screening positive by either method were confirmed for methadone and its metabolite 2-ethylidene-1,5-dimethyl- 3,3-diphenylpyrrolidine by a solid-phase extraction utilizing deuterated internal standards and GC-MS-SIM. Twenty-four postmortem samples that screened negative by both methods were also extracted and analyzed using the confirmation method to demonstrate the validity of both screening methods. The intra- and interassay precision for the ELISA method was evaluated at the cut-off concentration used for the analysis (50 ng/mL). True positives, true negatives, false positives, and false negatives were calculated for the ELISA results as compared to the GC-MS screening data. The Venture Labs methadone assay demonstrated a sensitivity of 96.7%+/-2.3% and a specificity of 98.3%+/-1.7% relative to the GC-MS method.     

Immunoassays for the detection of nicergoline and its metabolites in human plasma

In order to determine nicergoline pharmacokinetics after oral administration to humans, we have developed two radioimmunoassays, one directed against nicergoline and the other directed against known nicergoline metabolites. The assays were validated according to the recommendations of international regulatory agencies and their limits of quantification were 40 and 10 pg/ml, respectively. In order to further validate the methods, a chromatographic separation of immunoreactive entities was performed with samples from healthy volunteers who were given 15 mg of Sermion (nicergoline orally administered). Chromatographic determination of assay specificity showed that the metabolite radioimmunoassay recognised known nicergoline metabolites but also a new metabolite. Using the antibodies directed against nicergoline, we were unable to detect nicergoline in the human plasma. This suggests that nicergoline is absent in the circulation because of complete metabolism through its first-pass effect.     

Analysis of clomiphene isomers in human plasma and detection of metabolites using reversed-phase chromatography and fluorescence detection

A method is described for the quantitative clinical analysis of plasma concentrations of the E and Z isomers of clomiphene, which is used in the induction of ovulation. The isomers of clomiphene, in addition to metabolites, are extracted from plasma with tert-butyl methyl ether (MTB). The MTB layer is dried, reconstituted and an aliquot subjected to chromatography. The drug and metabolites are separated by reversed-phase high-performance liquid chromatography. The eluent is fed into a knitted or braided, cylindrical reaction coil made of Teflon, into which is inserted a low-energy mercury lamp. This results in a photoinduced stilbene-to-phenanthrene oxidation yielding highly fluorescent analytes; this provides excellent sensitivity for the quantitation of the intact drug isomers and the detection of presently uncharacterized metabolites. Use of the reversed-phase chromatographic mode results in elution of the polar metabolites prior to the intact drug isomers. A combination of reversed-phase chromatography and an in-line post-column reaction coil results in a sensitive method that is more reliable and rapid than those previously reported and is applicable to the routine analysis of clinical samples. The method has been applied to individual isomers of clomiphene in plasma at concentrations of 0.06-600 ng/ml.     

Qualitative screening for drugs of abuse in hair using GC-MS

A previously described method for the analysis of hair has been modified to include analysis for amphetamines, benzodiazepines, cocaine and its metabolites, methadone and its metabolite, and phencyclidine in addition to opiates on a sample of hair. The samples of hair were washed twice with dichloromethane and cut into 1-mm segments prior to extraction with methanol at 45 degrees C for 18 h. The extracts were split into two parts; both were evaporated to dryness. One half of the extract was derivatized using MBTFA for analysis of amphetamines, and the other half was derivatized using MTBSTFA for analysis of the remaining drugs. The extracts were analyzed using electron impact gas chromatography-mass spectrometry operating in selected ion monitoring mode. In total, 18 drugs of abuse/metabolites could be detected. The method was used to screen 20 hair samples from patients attending a methadone-maintenance clinic.     

Quantitative analysis of cocaine in human hair by HPLC with fluorescence detection

Cocaine is currently one of the most widespread abuse drugs in the world. Since hair cocaine concentrations are a reliable marker of exposition to the drug, an original liquid chromatographic method has been developed for the determination of cocaine in human hair. The chromatographic analysis was carried out on a Hydro-RP C18 column, using a mobile phase containing a phosphate buffer (pH 3.0)-acetonitrile-methanol (75:15:10, v/v/v). Native cocaine fluorescence was monitored at 315nm while exciting at 230nm. Mirtazapine was used as the internal standard. Sample pre-treatment was carried out by incubative extraction with 0.1M HCl followed by solid-phase extraction with C2 cartridges. Good linearity was obtained over a working range of 0.3-100.0ng/mg. Both extraction yield (>89%) and precision values (R.S.D.<6.2%) were highly satisfactory. The method was successfully applied to hair samples collected from cocaine users. Thus, the method is suitable for the long-term monitoring of cocaine use by means of hair testing.