Estimation of carbamazepine and carbamazepine-10,11-epoxide concentrations in plasma using mathematical equations generated with two carbamazepine immunoassays.

Carbamazepine is metabolized to an active metabolite known as carbamazepine-10,11-epoxide, or simply the "epoxide" metabolite. The presence of this metabolite can have clinically significant implications in therapeutic drug monitoring of carbamazepine, but accurate quantification of the epoxide metabolite is currently limited to chromatographic techniques. In this study, mathematical equations are proposed for the estimation of carbamazepine and epoxide metabolite concentrations based on values generated by common carbamazepine immunoassays. Three immunoassays were studied: particle-enhanced turbidimetric inhibition immunoassay (PETINIA, Siemens Healthcare Diagnostics, Deerfield, IL), ADVIA Centaur (Siemens Healthcare Diagnostics), and a cloned enzyme donor immunoassay (CEDIA; Roche, Indianapolis, IN). Equations were based on observed cross-reactivity of epoxide with the PETINIA (average, 96.2%; range, 86.6%-105.7%) and epoxide cross-reactivity with the ADVIA Centaur assay (average, 6.5%; range, 5.3%-7.7%). In addition, equations were developed using average cross-reactivity of epoxide with the PETINIA and with the CEDIA. Values determined by calculation correlated well with carbamazepine and epoxide concentrations in supplemented and patient samples, for which values of carbamazepine (2.2-12.0 microg/mL [9-51 micromol/L]) and the epoxide metabolite (0.6-2.4 microg/mL) were also verified by liquid chromatography-tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry assay for topiramate analysis in plasma and cerebrospinal fluid: validation and comparison with fluorescence-polarization immunoassay.

SUMMARY: The authors report the development and validation of a liquid chromatography tandem mass spectrometry assay (LC/MS/MS assay) for the analysis of topiramate (2,3:4,5-bis-o-(-1-methyl)-beta-D-fructopyranose sulfamate) in plasma and cerebrospinal fluid (CSF). Comparison is made with the commercially available fluorescence-polarization immunoassay (FPIA). LC/MS/MS ASSAY: Using the internal standard, 1,2:3,4-bis-o-(1-methylethylidene-alpha-D-galactopyranose sulfamate), a structural isomer, the calibration curve in plasma was linear in the concentration range of 0.02-20.0 mg/L (r(2) = 0.9998). The coefficients of variation in plasma were < or = 3%, and the accuracy ranged from 100% to 101% in the therapeutically relevant concentration range of 0.4-16.0 mg/L. In CSF, the mean recovery was 98%, and there was linearity between the nominal and the estimated concentration in the range of 1.5-20.0 mg/L (r(2)= 0.9996). FPIA: The calibration curve was linear in the concentration interval of 1.6-24.3 mg/L (r(2) = 0.9994), and the mean recovery was 96%. Accuracy in plasma was 99- 104%, and precision was 3.2-6.0%. In CSF, there was linearity between the nominal concentration and the estimated concentration in the range of 1.5-20.0 mg/L (r(2) = 0.9995), and the mean recovery was 100%. COMPARISON BETWEEN FPIA AND LC/MS/MS: There was a high correlation between the FPIA and the LC/MS/MS assay (r(2) = 0.9965 in plasma and r(2) = 0.9996 in CSF, P < 0.001 for both). In plasma and CSF, the two methods showed equal results, evaluated as the ratio between the two methods (plasma: median ratio = 1.00; 95% confidence interval [CI], 0.98-1.02, paired-sample test, P = 0.79; and CSF: median ratio = 1.00, 95% CI, 0.99-1.02, paired-sample test, P = 0.75). The coefficient of variation on the ratios between the two methods had similar levels: 5% in plasma and 3% in CSF. CONCLUSION: The new LC/MS/MS assay has favorable characteristics, being highly precise and accurate. FPIA also proved precise and accurate, and there was a high agreement with the LC/MS/MS assay in plasma and CSF. Either method displayed sufficient precision and accuracy and may thus be implemented in daily routine.

Express detection of pentachlorophenol as dioxins precursor in natural water.

A rapid detection method for the pesticide pentachlorophenol (PCP)--polarization fluoroimmunoassay (PFIA)--in the dynamic range of 10-9,000 ppb was developed. PCP may form polychlorinated dibenzo-p-dioxins, making environmental monitoring of this compound an issue of great importance. In order to optimize the PFIA procedure, a number of fluorescein-labeled PCP derivatives and similar compounds (tracers) were synthesized, and the influence of their structure on PFIA characteristics was studied. Also, two antisera were tested in developing PFIA for PCP. The developed method is highly specific for PCP and can be used for its determination in water samples at a level down to 10 ppb. Total time of the assay for 10 samples is about 7 min. The assay provides a useful and a highly practical screening tool for the processing of large numbers of samples and for the preliminary estimation of potential dioxins contamination in water resources.

Comparison of high-performance liquid chromatography with fluorescence polarization immunoassay for the analysis of vancomycin in patients with chronic renal failure.

Eighty-two plasma samples from patients with chronic renal failure undergoing vancomycin treatment and hemodialysis (HD) were analyzed with fluorescence polarization immunoassay (FPIA) and high-performance liquid chromatography (HPLC). Vancomycin was infused once and the samples were collected during three subsequent HD sessions at 2 h, 3 days and 5 days post-infusion. The HPLC method, modified from an earlier assay, was simple. There was a wide variation in the estimated concentration between the two assay methods. The results obtained by HPLC were 69% lower than those obtained by FPIA. This difference in vancomycin concentration was independent of the sampling time after vancomycin infusion. HPLC analysis commenced approximately 1.5 year after that of FPIA. To study the effect of in vitro degradation, the vancomycin concentration in ten of the samples was redetermined with FPIA during HPLC analysis. The concentrations of those samples decreased to 78-98% (average 92%) of the original concentration. Because FPIA appears to lack specificity, there is a need of other methods such as HPLC for vancomycin measurements, particularly in samples from patients with end-stage renal failure.

Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer.

We have developed a totally automated fluorescence polarization immunoassay for homocyst(e)ine with no pretreatment or chromatographic steps. Comparison with four well-established chromatographic methods yielded r values ranging from 0.980 to 0.997 and slopes from 1.030 to 1.493. Inter- and intraassay CVs ranged from 0.0% to 8.0% and from 0.0% to 6.4%, respectively. Imprecision (CV) of measuring six plasma samples on three instruments ranged from 6.3% to 10.2%. The assay was linear for plasma samples diluted with buffer from 0 to 8-fold. Mean recovery of homocysteine added to two plasma samples was 97.1% and 99.9%. The assay exhibited almost no cross-reactivity towards cysteine and methionine, and a batch of 20 samples can be processed in 60 min.

Effect of beta-glucuronidase on urinary benzodiazepine concentrations determined by fluorescence polarization immunoassay.

In samples from patients treated with oxazepam, beta-glucuronidase increased the immunoreactivity of urinary benzodiazepines analyzed by fluorescence polarization immunoassay (FPIA). Increasing concentrations of beta-glucuronidase added to samples from drug-free controls did not influence the results. In the absence of beta-glucuronidase, 22 of 35 samples from patients undergoing detoxification gave positive results at a cutoff concentration of 200 micrograms/L. Pretreatment with beta-glucuronidase increased the number of drug-positive samples to 33. The drug-negative samples were obtained from two patients who had been oxazepam-free for at least 1 week. Thus, beta-glucuronidase can be used to increase the sensitivity of the urinary benzodiazepine FPIA without reducing the specificity of the method.

Direct stopped-flow fluorescence polarization immunoassay of abused drugs and their metabolites in urine.

Kinetic methodology was applied to the direct determination of abused drugs (amphetamines, cocaine, and cannabinoids) in urine by stopped-flow fluorescence polarization immunoassay (SF-FPIA). This technique provides analytical data within a few seconds by measuring the variation of polarized fluorescence with time during development of immunochemical reactions. Methods based on this principle are particularly suitable for routine screening of these drugs in urine, being more expeditious than conventional FPIA methods. The dynamic ranges of the calibration curves were 20-300 micrograms/L for d,l-amphetamine, 15-300 micrograms/L for benzoylecgonine (a cocaine metabolite), and 10-400 micrograms/L for 11-nor-delta 8-tetrahydrocannabinol-9-carboxylic acid (a cannabinoid metabolite). The detection limits and within- and between-assay precision were better than those provided by conventional FPIA. Analytical recoveries ranged between 97.5% for d,l-amphetamine and 102.4% for the cannabinoid metabolite. The results for the three analytes were consistent with those obtained by conventional FPIA.

Monitoring tricyclic antidepressant concentrations in serum by fluorescence polarization immunoassay compared with gas chromatography and HPLC.

The fluorescence polarization immunoassay (FPIA) developed by Abbott to diagnose intoxication with tricyclic antidepressants was adapted for therapeutic drug monitoring and validated with chromatograpic methods to investigate its potential for this use. We compared serum concentrations of tricyclic antidepressants in vivo and in vitro obtained by FPIA with those by gas chromatography and HPLC. For amitriptyline, imipramine, clomipramine, and doxepin, the detection limit of the FPIA was 72, 71, 64, and 72 nmol/L (approximately 20 micrograms/L), respectively; that by gas chromatography was 18, 18, and 16 nmol/L (approximately 5 micrograms/L) for amitriptyline, imipramine and clomipramine, respectively; with HPLC the lower limit of detection for doxepin was 36 nmol/L (10 micrograms/L). The intra- and interassay CVs ranged from 3% to 6%. In patients being treated with amitriptyline, imipramine, clomipramine, and doxepin, at steady-state the correlation coefficients between FPIA and GC/HPLC results for split samples were 0.95, 0.92, 0.90 and 0.70, respectively. However, the slopes were close to unity only for amitriptyline and doxepin, being 0.6 for imipramine and 1.9 for clomipramine.

Steady-state concentration of cyclosporin G (OG37-325) and its metabolites in renal transplant recipients.

The steady-state concentrations of cyclosporin G (OG37-325) (CsG) and six of its metabolites (GM1, GM9, GM4N, GM1c, GM1c9, GM19) were measured throughout the 12-h dosing interval in six renal transplant recipients receiving CsG as prophylaxis against acute cellular rejection. The mean 12-h whole-blood trough concentrations (micrograms/L) were CsG, 131 +/- 26; GM1, 79 +/- 55; GM9, 110 +/- 114; GM4N, 28 +/- 18; GM1c, 31 +/- 18; GM1c9, 216 +/- 145; and GM19, 303 +/- 217. The relative concentration of the primary metabolites (GM1, GM9, GM4N) remained stable with respect to CsG throughout the dosing interval, whereas that of the secondary metabolites increased. The secondary metabolites GM19 and GM1c9 exhibited extensive between-patient variation. We investigated the effect of these metabolites on commercially available monoclonal antibody-based fluorescence polarization immunoassays (FPIA) and RIAs adapted for measurement of CsG. The 12-h whole-blood trough concentrations measured by FPIA and RIA exceed those measured by HPLC by 19% and 36%, respectively. These measured biases corresponded closely with the calculated biases (FPIA 19%, RIA 28%) based on the known cross-reactivities of CsG metabolites and their concentrations. These results suggest that cross-reactivity with metabolites account for a large part of the bias observed in immunoassays of CsG.

Free cortisol in serum assayed by temperature-controlled ultrafiltration before fluorescence polarization immunoassay.

A method is described for a temperature-controlled ultrafiltration procedure to measure free cortisol in serum. A special thermometer with a sensor was developed, measuring the temperature directly in the ultrafiltration device. The sensor is screwed on the axis of the centrifuge rotor, and the centrifuge is placed in a temperature-controlled box so that the temperature of the sample is kept at 37 degrees C +/- 0.1 degrees C. The overall CV of the free cortisol assay ranges from 2.2% to 11.4%, of which the ultrafiltration contributes only 2.2-3.6%. Increasing amounts of cortisol-binding protein, as found in women using estrogen-containing oral contraceptives, have minor but significant effects on the free cortisol concentrations in serum. Serum free cortisol concentrations in a reference population (n = 114; central 95 percentiles) were 12-43 nmol/L (4-9.5% of total cortisol); in the group of the oral-contraceptive users (n = 27), the reference interval was 11-53 nmol/L (1.5-4.5%).

Whole-blood cyclosporin G in renal transplant recipients determined by two immunoassays and liquid chromatography.

Cyclosporin G (CsG) is less nephrotoxic than cyclosporin A (CsA) and is undergoing clinical trials for use as an immunosuppressive agent after renal transplantation. Three assays for whole-blood CsA-HPLC, RIA (INCSTAR, Cyclo-Trac SP), and FPIA (Abbott TDx)--were adapted for use with CsG and were assessed for analytical suitability and to determine which assay was capable of deriving CsG values rapidly after transplantation. The assays were acceptable in terms of sensitivity, linearity, analytical recovery, and precision. When considering blood samples (n = 107) from renal transplant recipients receiving a low dose of CsG (5 mg/kg per day) and a high dose (10 mg/kg per day), we obtained the following correlation data: RIA = 0.974HPLC + 27.89 (r = 0.9798, Sy/x = 39.24); FPIA = 0.964HPLC + 33.59 (r = 0.9819, Sy/x = 36.66); and FPIA = 0.977RIA + 9.50 (r = 0.9894, Sy/x = 28.12). The FPIA of CsG is recommended as the most rapid method, although it is the most expensive. HPLC, RIA, and FPIA were capable of accurately deriving projected CsG concentrations at various stages of the clinical trial when the low- and high-dose regimes were tapered over a period of 16 weeks.

Measurement of cyclosporine by liquid chromatography and three immunoassays in blood from liver, cardiac, and renal transplant recipients.

In an effort to replace HPLC for whole-blood determination of cyclosporine (CsA), we compared HPLC with radioimmunoassay (RIA; INCSTAR, Cyclo-Trac SP assay), fluorescence polarization immunoassay (FPIA; Abbott TDx), and in-house modified enzyme-multiplied immunoassay technique (EMIT; Syva Co.). For blood samples from 200 various transplant recipients, RIA = 1.262 (HPLC) - 8.16, r = 0.983; FPIA = 1.200 (HPLC) + 19.90, r = 0.981; and EMIT = 1.038 (HPLC) + 11.28, r = 0.985. For segregation by transplant type, RIA, FPIA, and EMIT demonstrated positive biases of 27%, 12%, and 3%, respectively, for liver transplant recipients (n = 50) when compared with HPLC. Heart transplant recipients (n = 50) gave positive bias values of 23%, 14%, and 4% for RIA, FPIA, and EMIT, respectively. Adult renal transplant recipients (n = 50) demonstrated positive bias values of 30%, 31%, and 0% for RIA, FPIA, and EMIT, respectively. For pediatric renal transplant recipients (n = 50), positive biases of 40%, 31%, and 9% were obtained for RIA, FPIA, and EMIT, respectively. We conclude that the modified EMIT represents the best replacement for HPLC.

Monoclonal fluorescence polarization immunoassay evaluated for monitoring cyclosporine in whole blood after kidney, heart, and liver transplantation.

In a prospective study we evaluated a novel fluorescence polarization immunoassay (FPIA) for determining cyclosporine (CsA) in whole blood. FPIA uses a monoclonal antibody and is performed on the TDx (Abbott). The within-series (CV less than 2%) and between-days (CV less than 3.3%) precision of the assay was excellent. The results obtained by the monoclonal FPIA in samples from transplant patients (n = 100) averaged 31.9% and 20.2% higher than those by HPLC and a specific radioimmunoassay (INCStar), respectively. Results by all three methods correlated well. Follow-up studies during the early course after liver transplantation, however, suggested that high metabolite concentrations affect FPIA results. This is explained by previously described cross-reactions of the monoclonal antibody with some CsA metabolites. The FPIA results in samples of such patients should be interpreted cautiously.