Correlation between enzyme multiplied immunoassay technique and high-performance liquid chromatography in the quantification of voriconazole in a paediatric population.

The enzyme multiplied immunoassay technique (EMIT) is a new method for determining the plasma concentration of voriconazole (VRZ). This study aimed to investigate the correlation between EMIT and high-performance liquid chromatography/ultraviolet rays (HPLC/UV) in determining the plasma VRZ trough concentration in children, in China. A total of 419 blood samples were collected, and plasma VRZ concentrations were detected by the EMIT and HPLC methods. The results of 304 samples were analysed after excluding samples that were undetectable or beyond the quantification limit. A test result value of 0 was defined as undetectable, while concentrations outside the detection range (0.2 - 20.0 µg/ml for HPLC and 0.5 - 16.0 µg/ml for EMIT) were defined as beyond the quantification limit. Results from both methods were compared using the Passing Bablok regression, Bland-Altman plot analysis, and paired Wilcoxon test. The plasma VRZ concentrations determined by EMIT and HPLC showed a strong linear correlation through the linear regression equation YEMIT = 1.310 × HPLC +0.149 (R2 = 0.9082). The Bland-Altman plot analysis showed poor level consistency as measured by the two methods. The paired Wilcoxon-test showed a significant difference between the two methods (p < .0001). Compared to EMIT, HPLC accurately detected plasma VRZ concentration, making it suitable for VRZ therapeutic drug monitoring. The numerical values of the EMIT-measured levels were higher than those of HPLC, which may be related to VRZ metabolites interference and co-administrated drugs.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Pharmacokinetics Evaluation of Mycophenolic Acid and Its Glucuronide Metabolite in Chinese Renal Transplant Recipients Receiving Enteric-Coated Mycophenolate Sodium and Tacrolimus.

BACKGROUND: The aim of this study was to characterize the pharmacokinetics of mycophenolic acid (MPA) and MPA glucuronide (MPAG) in Chinese renal transplant patients taking enteric-coated mycophenolate sodium (EC-MPS). Limited sampling strategies (LSSs) were developed to estimate the area under the concentration curve from 0 to 12 hours (AUC0-12h) of total and free MPA. Another objective was to investigate the correlation between high-performance liquid chromatography (HPLC) and enzyme-multiplied immunoassay technology (EMIT) for total MPA determination. METHODS: Serial blood samples were collected over 12 hours from 15 patients who were administered multiple doses of EC-MPS. LSS was developed by multiple stepwise regression analysis. Measurement by HPLC and EMIT was compared using Passing-Bablok regression and Bland-Altman analysis. RESULTS: Normalized to 720 mg twice daily, the AUC0-12h of total MPA and MPAG was 43.0 ± 17.4 and 653 ± 329 mg·h/L, respectively, whereas the free MPA AUC0-12h was 1.368 ± 0.988 mg·h/L. The free fraction of MPA was 3.01% ± 3.15%. The combination of C2h-C4h-C6h and C2h-C4h-C6h-C8h was found to be superior to estimate total and free MPA simultaneously. The EMIT showed an acceptable correlation with HPLC, with an AUC0-12h overestimation of 11.32% ± 15.77%. CONCLUSIONS: The pharmacokinetic profile of total and free MPA and its main metabolite MPAG was examined in Chinese adult renal transplant patients receiving EC-MPS. The use of LSS to estimate individual free and total MPA exposure could be useful in optimizing patient care.

Comparison of whole-blood tacrolimus concentrations measured by different immunoassay systems.

INTRODUCTION: Different measured values for tacrolimus were obtained with different automated immunoassays. We aimed to examine the differences in the blood tacrolimus concentrations measured by the major immunoassay systems commercially available in Japan. METHODS: Whole-blood samples from 118 patients were assayed by 3 commercial assays: chemiluminescent enzyme immunoassay (CLIA), affinity column-mediated immunoassay (ACMIA), and enzyme-multiplied immunoassay technique (EMIT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for reference. KEY FINDINGS: The correlation coefficient of immunoassay vs LC-MS/MS was excellent for ACMIA (.83) and CLIA (.81) and good for EMIT (.71). The mean error was negative for ACMIA and positive for CLIA and EMIT. The mean absolute error and root-mean-square error were almost the same for ACMIA and CLIA and lower than those for EMIT. CONCLUSIONS: The ACMIA and CLIA yield considerably better results than the EMIT for monitoring blood tacrolimus concentrations.

Evaluation of venous thrombosis and tissue factor in epithelial ovarian cancer.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population. METHODS: We performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF. RESULTS: 35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75-6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001. CONCLUSIONS: TFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Estimation of Mycophenolic Acid Area Under the Curve With Limited-Sampling Strategy in Chinese Renal Transplant Recipients Receiving Enteric-Coated Mycophenolate Sodium.

BACKGROUND: The enteric-coated mycophenolate sodium (EC-MPS), whose active constituent is mycophenolic acid (MPA), has been widely clinically used for organ transplant recipients. However, its absorption is delayed due to its special designed dosage form, which results in difficulty to monitor the exposure of the MPA in patients receiving the EC-MPS. This study was aimed at developing a relatively practical and precise model with limited sampling strategy to estimate the 12-hour area under the concentration-time curve (AUC0-12 h) of MPA for Chinese renal transplant recipients receiving EC-MPS. METHODS: A total of 36 Chinese renal transplant recipients receiving the EC-MPS and tacrolimus were recruited in this study. The time point was 2 weeks after the transplantation for all the patients. The MPA concentrations were measured with enzyme-multiplied immunoassay technique for 11 blood specimens collected predose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 hours after the morning dose of EC-MPS. The measured AUC was calculated with these 11 points of MPA concentrations with the linear trapezoidal rule. Limited sampling strategy was used to develop models for estimated AUC in the model group (n = 18). The bias and precision of different models were evaluated in the validation group (n = 18). RESULTS: C4 showed the strongest correlation with the measured AUC. The best 3 time point equation was 6.629 + 8.029 × C0 + 0.592 × C3 + 1.786 × C4 (R = 0.910; P < 0.001), whereas the best 4 time point equation was 3.132 + 5.337 × C0 + 0.735 × C3 + 1.783 × C4 + 3.065 × C8 (R = 0.959; P < 0.001). When evaluated in the validation group, the 4 time point model had a much better performance than the 3 time point model: for the 4 time point model: R = 0.873, bias = 0.505 [95% confidence interval (CI), -10.159 to 11.170], precision = 13.370 (95% CI, 5.186-21.555), and 77.8% of estimated AUCs was within 85%-115% of the measured AUCs; for the 3 time point model: R = 0.573, bias = 6.196 (95% CI, -10.627 to 23.018), precision = 21.286 (95% CI, 8.079-34.492), and 50.0% of estimated AUCs was within 85%-115% of the measured AUCs. CONCLUSIONS: It demanded at least 4 time points to develop a relatively reliable model to estimate the exposure of MPA in renal transplant recipients receiving the EC-MPS. The long time span needed restricted its application, especially for the outpatients, but it could be a useful tool to guide the personalized prescription for the inpatients.

Evaluation of the area under the curve of enteric-coated mycophenolate sodium using a limited sampling strategy in Chinese kidney transplant recipients
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AIMS: To investigate the area under the curve (AUC) of mycophenolic acid (MPA) in adult Chinese renal allograft recipients receiving concomitant enteric-coated mycophenolate sodium (EC-MPS) and cyclosporine (CsA) during the early post-transplant phase and to develop optimal model equations for estimation of the MPA area under the plasma concentration-time curve from 0 to 12 hours (AUC0-12h) using a limited-sampling strategy (LSS). METHODS: The present study enrolled 24 Chinese renal recipients treated with EC-MPS, CsA, and corticosteroid, from whom 24 serial blood samples were collected over 12 hours. MPA concentration was evaluated with enzyme multiplied immunoassay technique (EMIT). LSS was developed by multiple stepwise regression analysis using a two-group method (test group, n = 12; and validation group, n = 12). RESULTS: The MPA predose concentration had a poor correlation with MPA AUC0-12h, and the best equations obtained from the test group were the following: 25.73 + 0.59 × C1.5 + 0.79 × C2 + 2.03 × C4 (for three time points, r2 = 0.761) and 22.13 + 1.7 × C0.5 + 0.61 × C1.5 + 0.78 × C2 + 1.83 × C4 (for four time points, r2 = 0.853). When these equations were tested in the validation group, there were no significant differences in prediction errors. CONCLUSION: An LSS using time points at 1.5, 2, and 4 hours or 0.5, 1.5, 2, and 4 hours provides the most accurate and reliable estimation of the MPA AUC0-12h in Chinese adult renal recipients treated concomitantly with EC-MPS and CsA during the early post-transplant phase.
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Therapeutic drug monitoring of caffeine in preterm infants: Could saliva be an alternative to serum?

OBJECTIVE: Evaluate whether saliva could be a useful alternative to serum for routine therapeutic drug monitoring of caffeine in preterm infants using the enzyme multiplied immunoassay technique (EMIT) assay. METHODS: We conducted a prospective study including preterm infants (less than 34 weeks' amenorrhea) admitted to the intensive care and neonatal medicine department. All infants received 5, 10, 15, 20 and 25mg/kg/day of citrate caffeine intravenously from the first to the fifth day of birth, respectively. For each patient, two concomitant blood and saliva samples corresponding to the trough concentrations were collected 24hours after each caffeine dose. The caffeine concentrations were determined using the EMIT®2000 caffeine assay. RESULTS: Thirteen preterm infants were included. The saliva and the serum caffeine concentration increased proportionally to the administered dose. Saliva and serum kinetics were comparable and the saliva caffeine concentrations were correlated to the serum ones (r2=0.76). CONCLUSION: Saliva caffeine monitoring by EMIT is a valid, useful and safe alternative to serum in preterm infants.

A Comparison of the Immunochemical Methods, PETINIA and EMIT, With That of HPLC-UV for the Routine Monitoring of Mycophenolic Acid in Heart Transplant Patients.

BACKGROUND: The aim of this study was to evaluate particle enhanced turbidimetric inhibition immunoassay (PETINIA) recently developed for mycophenolic acid (MPA) determination in plasma and to compare it with a reference high-performance liquid chromatography (HPLC) method, using samples from heart transplant recipients. The results are presented in the context of PETINIA being compared with enzyme multiplied immunoassay technique (EMIT). METHODS: PETINIA evaluation was performed using 194 routine trough plasma samples at steady state. EMIT was evaluated using 677 samples from 61 steady-state 12-hour profiles obtained from 35 heart transplant patients. Evaluation was undertaken on a Dimension EXL 200 analyzer (PETINIA) and on a Viva-E analyzer (EMIT). RESULTS: The mean MPA concentration measured by PETINIA was significantly higher than that measured by high-performance liquid chromatography combined with UV detector (2.36 ± 1.30 mcg/mL versus 1.82 ± 1.23 mcg/mL, respectively, P < 0.0001). Bland-Altman analysis revealed a mean bias of 0.54 mcg/mL [95% confidence interval (CI), 0.49-0.59] comprising 33.48% (95% CI, 30.34-36.61). Passing-Bablok regression was: y = 1.100x + 0.38 (95% CI for slope: 1.044-1.154 and for intercept: 0.30-0.47). Regardless of a significant observed correlation (r = 0.9230, P < 0.0001), the statistical analyses showed a significant difference between PETINIA and the reference chromatographic method. The mean MPA concentration measured by EMIT was significantly higher than that measured by HPLC (7.48 ± 8.34 mcg/mL versus 5.57 ± 6.61 mcg/mL, respectively, P < 0.0001) with a mean bias of 1.91 mcg/mL (95% CI, 1.75-2.07) comprising 35.91% (95% CI, 34.37-37.45). The significant difference between EMIT and HPLC was confirmed by Passing-Bablok regression: y = 1.300x + 0.24 (95% CI for slope: 1.279-1.324 and for intercept: 0.18-0.29). The analysis of the determinations, grouped by sampling time, revealed positive bias between EMIT and HPLC ranging from 24.54% to 42.77% and inversely proportional to MPA concentrations with r = 0.9122 (P < 0.001). CONCLUSIONS: The new immunochemical PETINIA method was associated with significantly higher MPA concentrations in routine therapeutic drug monitoring samples from heart transplant patients. The magnitude of the MPA overestimation was similar to that observed by use of the EMIT method.

Limitations of EMIT benzodiazepine immunoassay for monitoring compliance of patients with benzodiazepine therapy even after hydrolyzing glucuronide metabolites in urine to increase cross-reactivity: comparison of immunoassay results with LC-MS/MS values.

BACKGROUND: Benzodiazepines are widely prescribed, and compliance of patients with benzodiazepine therapy is often monitored using urine specimens. Although various commercially available benzodiazepines immunoassays are widely used for compliance monitoring, such immunoassays usually have low cross-reactivity with glucuronide metabolites. We studied the effect of hydrolyzing such glucuronide before analysis to reevaluate suitability of Enzyme multiplied immunoassay technique benzodiazepine immunoassay for monitoring compliance with benzodiazepine therapy. METHODS: In 31 urine specimens collected from patients taking benzodiazepines, the true analyte concentrations were determined (after hydrolyzing glucuronide metabolites using beta-glucuronidase) using liquid chromatography-tandem mass spectrometry. These urine specimens were reanalyzed using EMIT benzodiazepine assay (Flex Reagent Cartridge; Siemens Diagnostics) and Vista analyzer. RESULTS: We observed false negative test results with EMIT in 11 of 31 specimens analyzed where liquid chromatography-tandem mass spectrometry values were above the 200 ng/mL cutoff concentration, but EMIT benzodiazepine assay showed a negative result, indicating that despite hydrolysis of the specimen to liberate parent drug (glucuronide metabolite often has poor cross-reactivity), the false negative rate using EMIT assay was 35.5%. CONCLUSIONS: Patient compliance with benzodiazepine therapy must be monitored using a chromatographic method.

Limited sampling strategy for predicting area under the concentration-time curve for mycophenolic Acid in Chinese adults receiving mycophenolate mofetil and tacrolimus early after renal transplantation.

BACKGROUND: The objective of the study was to investigate the pharmacokinetics of mycophenolate mofetil (MMF) in Chinese adults early after renal transplantation by an enzyme multiplied immunoassay technique and to establish a limited sampling strategy to predict the area under the concentration-time curve for plasma levels of mycophenolic acid (MPA-AUC). METHODS: Fifty-eight recipients who underwent renal transplantation with an organ donated after cardiac death used a triple immunosuppressant strategy of MMF, tacrolimus, and prednisone. On the seventh day posttransplantation, plasma samples were collected at 0 hours (pre-dose) and at 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 hours postdose (C0h, C0.5h, C1h, C1.5h, C2h, C4h, C6h, C8h, C10h, and C12h, respectively). Enzyme multiplied immunoassay technique was used to measure mycophenolic acid concentration, and model equations were generated by multiple stepwise regression analysis to determine MPA-AUC0-12h. RESULTS: The 3-point equation obtained by multiple linear regression analysis was MPA-AUC = 7.951 + 4.04C6h + 1.893C2h + 4.542C10h (adjusted r = 0.863); the 4-point equation was MPA-AUC = 4.272 + 4.074C6h + 1.896C2h + 4.680C10h + 0.859C0.5h (adjusted r = 0.918). The % mean prediction error, % mean absolute error, and % root mean squared prediction error for the best-fit formula using C6h, C2h, C10h, and C0.5h were -0.2%, 8.7%, and 14.2%, respectively. CONCLUSIONS: In Chinese adults receiving MMF and tacrolimus early after renal transplantation, the best equation for predicting MPA-AUC0-12h is 4.272 + 4.074C6h + 1.896C2h + 4.680C10h + 0.859C0.5h.

LC-MS/MS and EMIT measure the whole blood concentration of cyclosporine A: The two methods yield concordant results within the dynamic range of the latter, but the former shows broader application scenarios.

Cyclosporine A (CsA) is a widely used immunosuppressive drug with a narrow therapeutic index and large individual differences. Its therapeutic and toxic effects are closely related to blood drug concentrations, requiring routine therapeutic drug monitoring (TDM). The current main methods for TDM of CsA are enzyme multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, few study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children has been reported. In this study, we developed a simple and sensitive LC-MS/MS assay for the determination of CsA, and 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by the validated in-house LC-MS/MS method on the same batch of samples, aimed to address the aforementioned concern. Consistency between the two assays was evaluated using linear regression and Bland-Altman analysis. The linear range of LC-MS/MS was 0.500-2000 ng/mL and that of the EMIT was 40-500 ng/mL, respectively. Overall, the correlation between the two methods was significant (r-value ranging from 0.8842 to 0.9441). Unsatisfactory consistency was observed in the concentrations < 40 ng/mL (r = 0.7325) and 200-500 ng/mL (r = 0.6851). Bland-Altman plot showed a mean bias of -18.0 % (±1.96 SD, -73.8 to 37.8 %) between EMIT and LC-MS/MS. For Passing-Bablok regression between EMIT and LC-MS/MS did not differ significantly (p > 0.05). In conclusion, the two methods were closely correlated, but the CsA concentration by LC-MS/MS assay was slightly higher than that by EMIT method. Switching from the EMIT assay to the LC-MS/MS method was acceptable, and the LC-MS/MS method will receive broader application in clinical settings due to its better analytical capabilities, but the results need to be further verified in different laboratories.

Amperometric homogeneous competitive immunoassay in a perfluorocarbon emulsion oxygen therapeutic (PEOT).

The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on the detection of the drugs theophylline and phenytoin was explored using a commercial enzyme multiplied immunoassay technique (EMIT®). The EMIT technique is based on the enzymatic production of NADH, which is typically detected in serum samples spectrophotometrically. Here, amperometry using the rotating disk electrode on a single drop of solution is demonstrated to detect theophylline and phenytoin in the presence of PEOT. In the study, 2,6-dichloroindophenol (DCIP) added to the immunoassay mixture is reduced by the NADH to DCIPH2. Oxidation of DCIPH2 is monitored electrochemically at +200 mV using a glassy carbon rotating disk electrode. Slopes of amperograms are proportional to the concentration of drug in the immunoassay sample. This technique yields excellent quantitative data in the therapeutic range for both drugs in 2-20% PEOT.

The effects of smoking cessation and a programme intervention on birth and other perinatal outcomes among rural pregnant smokers.

OBJECTIVE: The purpose of this study was to evaluate the perinatal outcomes of rural pregnant smokers enrolled in the Smoke Free Baby & Me trial. METHODS: Data on smoking status and other pre-natal variables were collected during pregnancy. Outcomes were retrieved from a review of hospital records of 161 singleton births (79 from the control group, 82 from the intervention group). RESULTS: The results show that, after adjusting for gender and gestational age, the more self-reported cigarettes at the first pre-natal visit, the less the infant birth weight (p = 0.033), the less maternal weight gain (p = 0.042) and the shorter the labour length (p = 0.041). Infants of women with positive urinary cotinine at the first pre-natal visit in the intervention group had higher 1 minute Apgar scores than those with negative cotinine (p = 0.022). Smokers also had a preponderance of male infants (64% vs 36%), while non-smokers had more females (59% vs 41%) (p = 0.006). CONCLUSIONS: Smoking during pregnancy affects perinatal outcomes. Assuming a foetal origin of chronic disease morbidity, implementing smoking cessation during pregnancy would not only improve maternal and foetal health, but also might contribute to an improvement in the incidence of adult chronic disease morbidity.

Improved sensitivity for methotrexate analysis using enzyme multiplied immunoassay technique on the Siemens Viva-E instrument.

BACKGROUND: The available assay kit for methotrexate (MTX) using the Syva enzyme multiplied immunoassay technique (EMIT) reagents on the Siemens Viva-E instrument allows for the detection of MTX in serum or plasma to concentrations as low as 0.3 µmole/L. Current clinical decision points for MTX therapeutic drug monitoring and leucorvorin rescue exist at concentrations below that limit. OBJECTIVE: The goal of this study was to lower the limit of MTX quantitation to 0.05 µmole/L using the EMIT assay technology. METHODS: EMIT MTX assay parameters were modified on the Viva-E instrument to increase the sample volume, alter the calibration method, and employ an alternate calibrator set created to achieve lower detection. Intraassay and interassay precision was assessed for MTX controls. RESULTS: We observed a CV of 9.4% for intraassay precision with a bias of <0.01% and a CV of 15.7% for interassay precision with a bias of 22.5% for the 0.05 µmole/L control. Precision data for all other controls were <4%. The modified EMIT MTX assay and the unmodified approved assay were compared with a high sensitivity fluorescence polarization immunoassay method. Linear regression of correlation data revealed that both the modified and the commercial EMIT assays produced positive bias compared with the high sensitivity fluorescence polarization immunoassay method (y-int = 0.03 and 0.08, respectively). However, the modified EMIT assay had the best correlation in the low range (0.03-2 µmole/L). Additionally, endogenous and chemical interference testing demonstrated that the modified assay was not affected to a clinically significant extent. CONCLUSIONS: The described modifications have enhanced the sensitivity of the Syva EMIT assay for MTX measurements down to 0.05 µmole/L with acceptable precision that can be used in clinical practice for monitoring MTX therapy.

Numerical analysis of an immunochromatographic test strip reader in abused drugs screening.

A point-of-care immunoassay strip reader, Uniscan™, was applied to detect methamphetamine, opiate, and marijuana in human urine by providing numerical apparent drug concentrations. Calibration curves were determined by a nonlinear regression. The cutoff was verified using spiked controls. Clinical samples were analyzed and compared with enzyme multiplied immunoassay technique (EMIT). The discrepant results were confirmed by gas chromatography-mass spectrometry (GC-MS). The impacts of interference and cross-reactivity were determined for numerous compounds. The coefficients of the calibration curves had a high correlation coefficient. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and total recovery all had high values for spiked controls. For the 19 discrepant results of clinical samples, GC-MS confirmed that Uniscan and EMIT were correct for 11 and eight samples, respectively. For both methamphetamine and opiate, Uniscan had a lower false positive rate, a higher true negative rate, and a higher total recovery rate than EMIT. For marijuana, Uniscan had a higher true positive rate and a lower false negative rate than EMIT. The Uniscan performed excellently when compared to EMIT. It is advantageous for Uniscan to interpret the test result based on digital read-out, rather than subjective visual judgment.

Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry.

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.

Development and evaluation of a simulation procedure to take into account various assays for the Bayesian dose adjustment of tacrolimus.

BACKGROUND AND OBJECTIVES: Several analytical techniques with different performances are available for the measurement of tacrolimus blood concentrations. The performance of Bayesian estimators (MAP-BEs) allowing dose adjustments of tacrolimus is dependent on the precision of the analytical technique. Hence, any Bayesian estimator should only be used for concentration data obtained with the same assay employed for its development. The present study aimed at evaluating the feasibility of developing Bayesian estimators dedicated to different immunoassays, using the concentrations obtained with the reference high-performance liquid chromatography with mass spectrometric detection (LC-MS/MS) method and a simulation approach. PATIENTS AND METHODS: One hundred thirty-five full pharmacokinetic profiles of tacrolimus were obtained from 45 renal transplant patients using 3 different analytical techniques: 2 immunoassays [enzyme-multiplied immunoassay technique (EMIT) and chemiluminescent microparticle immunoassay (CMIA)] and LC-MS/MS. In a first step, 3 MAP-BEs were developed using the concentrations measured with the 3 techniques. Taking into account the correlation equations between the concentrations obtained with each of the immunoassays and LC-MS/MS, as well as the analytical error of the techniques, 2 hybrid MAP-BEs dedicated to the immunoassays were then developed after simulation of 100 pharmacokinetic profiles. Their performances were compared with those of the respective MAP-BEs developed using the actual immunoassay concentrations. RESULTS: The mean concentrations measured over the dosing interval using EMIT and CMIA were significantly higher than those measured using LC-MS/MS (+15% and +11% in the AUC0₋24 h value, respectively, P < 0.0001), leading to differences in dose recommendations of -0.9 ± 1.1 and -0.7 ± 0.9 mg, respectively. When applying the MAP-BE developed from LC-MS/MS data for the EMIT or CMIA concentrations, tacrolimus AUC0₋24 h was estimated with an imprecision >20% in 33% and 27% of the patients, respectively. In contrast, the "CMIA" and "EMIT" hybrid MAP-BEs provided a good AUC0₋24 h estimation in 85%-93% of the patients. CONCLUSIONS: This study showed the impact of the analytical technique on the performance of Bayesian estimators dedicated to tacrolimus dose adjustment and the feasibility to develop MAP-BE for a specific assay using results from a different assay, based on a limited method comparison study. This methodology could offer clinicians the opportunity to dose adjust tacrolimus whatever the assay used in their center.

An influenza hemagglutinin A peptide assay based on the enzyme-multiplied immunoassay technique.

A practical approach for constructing enzyme-multiplied immunoassay technique (EMIT)-based protein/peptide assays is described. Normally used in small-molecule drug testing, EMIT is a homogeneous assay method that is attractive for its simplicity, sensitivity, and rapidity. The EMIT-based peptide/protein assay was developed by conjugating a cysteine-modified HA peptide (from influenza hemagglutinin A) to the reporter enzyme, glucose-6-phosphate dehydrogenase. The 13-min assay gave a free HA limit of detection of 10 nM and proved effective for detection of a high-molecular-weight model protein tagged with HA. Similar EMIT-based assay approaches may be developed for applications in biotoxin and infectious disease detection.

An investigation into the bias between liquid chromatography-tandem mass spectrometry and an enzyme multiplied immunoassay technique for the measurement of mycophenolic acid.

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 microL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and gamma-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.