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.

Application of an enzyme-multiplied immunoassay technique for determination of caffeine elimination kinetics as a test of liver function in clinically normal dogs.

A commercially available automated enzyme-multiplied immunoassay technique (EMIT) was used to determine serum caffeine concentration after oral and IV administrations of caffeine at dosage of 5 mg/kg of body weight to 12 clinically normal dogs. Dogs were allotted to 2 groups of 6 dogs each; 1 group initially received caffeine orally and the other received caffeine IV. After 72 hours, caffeine administration was repeated in all dogs in the alternate manner. Serum samples were obtained at multiple intervals over 24 hours to determine distribution and elimination kinetics. Analysis of the drug concentration-time data indicated IV elimination half-life (t1/2) of 6.39 +/- 1.87 hours, volume of distribution at steady state of 685.3 +/- 132.2 ml/kg, total body clearance of 1.31 +/- 0.38 ml/min/kg, absorption t1/2 of 1.02 +/- 0.68 hour, oral elimination t1/2 of 6.53 +/- 2.72 hours, lag time after oral administration of 0.0614 +/- 0.0661 hour, highest measured concentration of 5.29 +/- 1.17 micrograms/ml, time to peak concentration of 2.74 +/- 1.30 hours, and bioavailability of 99.4 +/- 19.4%. Data from 6 dogs best fit a 1-compartment open model and those from 6 other dogs best fit a 2-compartment open model. On the basis of data from the 6 dogs that best fit a 2-compartment model, t1/2 of distribution was 0.58 +/- 0.72 hour. Data for oral administration best fit a single absorption phase and a single elimination phase. The increased availability and simplicity of the EMIT offers an opportunity to study the application of caffeine elimination for clinical evaluation of dogs with liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Interference with testing for lysergic acid diethylamide.

We found a high rate (4.2%) of positive results for lysergic acid diethylamide (LSD) by Emit in 1898 urine samples that were submitted primarily from psychiatric patients for drugs-of-abuse (DOA) testing. Specimens that tested positive for LSD by Emit subsequently tested negative for LSD with two RIAs. Furthermore, LSD was not detected in randomly selected Emit-positive urine samples by gas chromatography-mass spectrometry. Normal urine samples tested positive for LSD by Emit when they were supplemented with therapeutic medications that were prescribed for patients with positive urine LSD results by Emit. These therapeutic drugs interfered specifically with the Emit assay for LSD, since other Emit DOA tests were not affected by these medications at the tested concentrations.

Enzyme immunoassay, kinetic microparticle immunoassay, radioimmunoassay, and fluorescence polarization immunoassay compared for drugs-of-abuse screening.

The newest formulation of the Syva EMIT assay for drugs of abuse, EMIT II, and a new immunoassay, OnLine (Roche), utilizing the kinetic interaction of microparticles in solution (KIMS) methodology, RIA tests, and TDx fluorescence polarization immunoassay (FPIA) procedures were compared for marijuana, cocaine, opiates, and barbiturates. Both EMIT II and OnLine immunoassays were performed with a Hitachi 717 analyzer. Calibration curves, the degree of separation between negative and cutoff calibrators, precision, likelihood of carryover from positive to negative samples, and overall ease and speed of analysis were evaluated. RIA and OnLine detected 99% of gas chromatography/mass spectrometry (GC/MS)-confirmed marijuana samples; TDx, 95%; and EMIT II, 88%. All four immunoassays detected approximately 99% of confirmed cocaine-positive urines. RIA, OnLine, and TDx all detected 100% of opiate-confirmed samples; EMIT II, 97%. Barbiturate assays exhibited the greatest disparity, with OnLine and TDx detecting 100% of confirmed positives; EMIT II, 88%; and RIA, 78%. For a variety of reasons, we prefer the fully automated EMIT II and OnLine assays for high-volume urine testing, in comparison with our laboratory's semiautomated RIA tests and the limited-throughput TDx system. The four immunoassays investigated delivered comparable performance in terms of detection rates for GC/MS-confirmed positives for some drugs but not for others.

Evaluation of EMIT Cyclosporine Assay for use with whole blood.

We evaluated the EMIT Cyclosporine Assay, designed for specific quantification of cyclosporine (CsA) in whole blood. The assay was run on the Roche Cobas Mira or Cobas Mira S analyzer. Analytical recovery from both normal donor whole blood and transplant patients' samples was within 17% of the standards. Measurement of diluted out-of-range samples also yielded excellent recovery (101-105%). Within-run, between-run, and total CVs were < or = 8.1%, 10.9%, and 12.1%, respectively. The detection limit was < 32 micrograms/L. The assay was linear from 0 to 500 micrograms/L. No significant cross-reactivity was observed for CsA metabolites AM1, AM19, and AM4N. Slight cross-reactivity occurred with metabolite AM9; 670 micrograms/L AM9 increased the measured CsA concentration by 49 micrograms/L. High concentrations of bilirubin, uric acid, triglycerides, and cholesterol, as well as 54 commonly coadministered drugs did not interfere with CsA quantification. Similarly, neither extreme values of hematocrit nor choice of anticoagulant affected CsA recovery. Sample extract stability was > 4 h, and assay calibration was stable for at least 2 weeks. Patients' samples analyzed by the EMIT assay showed strong correlation with both HPLC and 125I-RIA (r > 0.97). We conclude that the EMIT Cyclosporine Assay provides a convenient, accurate, and precise method for specific quantification of CsA in whole blood.

Decreased signal in Emit assays of drugs of abuse in urine after ingestion of aspirin: potential for false-negative results.

During routine drug analysis with the Syva d.a.u. Emit immunoassays we observed a high frequency of urines with lower rates of changes in absorbance (delta A R) than the rate for a drug-free urine calibrator. Many of these urines contained salicylates. Among 40 urines with apparent salicylate concentrations between 15 and 420 mg/dL tested for benzoylecgonine (BE), 20 had delta A R < -4 (range +2 to -28 mA/min). The rates decreased with increasing salicylate: delta A R = -0.057 x (salicylate, mg/dL) -0.22 mA/min (r = 0.85, n = 40, P < 0.01). Urines from 100 control subjects (no salicylate) had mean +/- SD delta A R values of -1.05 +/- 2.2 mA/min (range +3 to -7; only two were < -4 mA/min). Although direct addition of salicylic acid (200 mg/dL) to urine specimens did not reproduce the negative bias, ingestion of aspirin (acetylsalicylic acid) did by -0.09 mA/min per 1 mg/dL (72.4 mumol/L) salicylate. Negative biases observed for other Emit d.a.u. assays after salicylate ingestion lead us to conclude that ingestion of therapeutic doses of aspirin may cause false-negative results for drug screens in urines by this technology.

Metabolite and matrix interference in phenytoin immunoassays.

The major phenytoin metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin glucuronide (HPPG), was primarily responsible for the positive bias noted when uremic specimens were assayed with the Abbott TDx Free Phenytoin fluorescence polarization immunoassay. The amount of bias depended on both HPPG and phenytoin concentration, increasing with increases in either concentration. The new Abbott TDx II assays for phenytoin and free phenytoin exhibited no significant cross-reactivity with HPPG and no bias in clinical specimens from uremic patients. Both assays correlated well with Emit-based assays (r >0.98), had CVs of <3.5%, and had minimum detection limits of <0.1 mg/L. Calibration curves were stable for at least 6 weeks. All of the TDx assays cross-reacted with another metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), but expected HPPH concentrations are too low to cause a clinically significant bias. The Emit-based phenytoin assay exhibited a significant matrix effect when calibrators were prepared in defibrinated plasma processed to resemble serum.

Electrochemical homogeneous enzyme immunoassay of theophylline in hemolyzed, icteric, and lipemic samples.

We demonstrate here an electrochemical homogeneous enzyme immunoassay for theophylline, which can be performed in hemolyzed, lipemic, and icteric samples. The assay used an unmodified Syva EMIT theophylline kit. One of the enzymatic reaction products, NADH, reacted with 2,6-dichloroindophenol (DCIP) to reduce DCIP to DCIPH2, which was detected electrochemically with flow-injection analysis. The inter- and intraassay coefficients of variation of this manual technique were < 9% at theophylline concentrations of 14 to 34 mg/L. The CVs were 9-15% at low concentrations (6.3 mg/L), which is below the therapeutic range. Analytical recoveries were 91-97% for normal serum and 92-111% for hemolyzed, icteric, or lipemic sera. The measured concentrations (y) were compared with those obtained by the fluorescence polarization immunoassay (x); a scatter plot of the results showed a linear relationship of y = 1.00 x - 0.57 mg/L (r = 0.966, Sy/x = 1.51). This alternative way to measure the serum concentration of theophylline overcomes the shortcomings of spectrophotometric methods, by which it is difficult to measure theophylline in severely hemolyzed, icteric, or lipemic sera.

Cross-reactivity assessment of carbamazepine-10,11-epoxide, oxcarbazepine, and 10-hydroxy-carbazepine in two automated carbamazepine immunoassays: PETINIA and EMIT 2000.

This study was conducted to compare the cross-reactivity of two commercially available carbamazepine (CBZ) immunoassays (PETINIA and EMIT 2000) with carbamazepine-10,11-epoxide (CBZ-E), the active metabolite of CBZ. Oxcarbazepine (OCBZ) and its main metabolite 10-hydroxy-carbazepine (HCBZ) have a chemical structure closely related to that of CBZ. The cross-reactivities of these two drugs were also investigated. In the first part of the study, Lyphocheck blank human serum and Chemonitor quality controls (containing CBZ without CBZ-E) were spiked with variable amounts of CBZ-E. The apparent CBZ levels were measured by PETINIA and EMIT 2000 methods. The interference from OCBZ and HCBZ was directly assessed by measuring the apparent CBZ levels in Chromsystems Trileptal quality controls (containing OCBZ and HCBZ). In the second part of the study, the CBZ levels of serum samples from 49 patients, including 2 patients with massive CBZ ingestion, were measured by immunoassays and compared with a high-pressure liquid chromatography (HPLC) reference technique allowing the simultaneous measurement of CBZ and CBZ-E. The antibody used in the PETINIA assay cross-reacts (about 90%) with CBZ-E. In one case of CBZ poisoning (CBZ and CBZ-E levels measured by HPLC were 26.2 and 18.2 mg/L, respectively), CBZ level measured by PETINIA was falsely elevated (42.5 mg/L). In contrast, the specificity of EMIT 2000 was satisfactory (29.5 mg/L). The two immunoassays tested showed low cross-reactivity with OCBZ and HCBZ. In conclusion, it appears that the CBZ-E metabolite present in samples can falsely increase CBZ levels measured by the PETINIA assay.

Mechanism and elimination of aspirin-induced interference in Emit II d.a.u. assays.

The presence of salicylates in urine reduces the signal in Emit assays (Syva), potentially yielding false-negative drugs-of-abuse screening results. We demonstrate that the principal urinary metabolite of salicylate, salicyluric acid (SUA; 2-hydroxybenzoylaminoacetic acid), interferes with the measurement of NADH formed in the assay by reducing the molar absorptivity of NADH at 340 nm. Thus, for a given concentration of d.a.u. analyte the change in absorbance over the assay time interval is less in the presence of SUA. With the Emit cocaine assay on the Hitachi 704 analyzer, the rate of absorbance change (delta AR) monitored at 340 nm for a specimen containing approximately 270 micrograms/L benzoylecgonine (BE) was 57 +/- 1.9 mA/min without SUA and 29 +/- 2.7 mA/min with 5 g/L SUA (n = 20). In contrast, delta AR determined at 376 nm was 18.6 +/- 0.5 mA/min with and 17.9 +/- 0.8 mA/min without 5 g/L SUA (n = 20). Measuring the Emit assay signal at wavelengths where SUA has no absorbance (376 nm) eliminates the interference due to SUA while maintaining the precision of the assay near the cutoff concentration for BE (300 micrograms/L).

Adsorption losses from urine-based cannabinoid calibrators during routine use.

The major metabolite of cannabis found in urine, 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (delta 9-THC), is the compound most often used to calibrate cannabinoid immunoassays. The hydrophobic delta 9-THC molecule is known to adsorb to solid surfaces. This loss of analyte from calibrator solutions can lead to inaccuracy in the analytical system. Because the calibrators remain stable when not used, analyte loss is most probably caused by handling techniques. In an effort to develop an effective means of overcoming adsorption losses, we quantified cannabinoid loss from calibrators during the testing process. In studying handling of these solutions, we found noticeable, significant losses attributable to both the kind of pipette used for transfer and the contact surface-to-volume ratio of calibrator solution in the analyzer cup. Losses were quantified by immunoassay and by radioactive tracer. We suggest handling techniques that can minimize adsorption of delta 9-THC to surfaces. Using the appropriate pipette and maintaining a minimum surface-to-volume ratio in the analyzer cup effectively reduces analyte loss.

Analytical performance evaluation of EMIT II monoclonal amphetamine/methamphetamine assay: more specificity than EMIT d.a.u. monoclonal amphetamine/methamphetamine assay.

We evaluated a new EMIT II monoclonal amphetamine/methamphetamine assay for screening human urine by comparing it with the EMIT d.a.u. monoclonal amphetamine/methamphetamine assay and a fluorescence polarization assay. The EMIT II assay has a cutoff of 1 mg/L d-methamphetamine. The EMIT II and EMIT d.a.u. assays were run on a BM/Hitachi 704 analyzer; for the fluorescence polarization assay we used a TDx analyzer. All EMIT II positive samples were also analyzed by the fluorescence polarization assay. We used gas chromatography/mass spectrometry (GC/MS) for confirmation of the presence of amphetamine or methamphetamine. Within-run CVs for the Level 1 (1 mg/L) and Level 2 (3 mg/L) calibrators for the EMIT II assay were 0.47% and 0.53%, respectively. Corresponding between-run CVs were 1.48% and 1.60%, respectively. Of the 1007 samples screened for amphetamines, 50 were positive by the EMIT d.a.u. assay; 21 samples (not a subset of the 50 samples) were positive by the EMIT II assay. However, 19 samples that tested positive by EMIT II also tested positive by the EMIT d.a.u. assay. Subsequent testing of the EMIT II positive samples by the fluorescence polarization assay detected in six positive samples. By means of chiral derivatization wer identified two specimens containing primarily l-isomers of amphetamine and methamphetamine. Sympathomimetic amines were identified in several of the samples not containing amphetamine or methamphetamine.

Effect of Chinese medicines Chan Su and Danshen on EMIT 2000 and Randox digoxin immunoassays: wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin measurement by different brands of the same product.

Chan Su is a Chinese medicine prepared from the skin gland of a Chinese toad and is used in treating arrhythmia and other heart diseases. Danshen is prepared from the Chinese medicinal plant and is used for various cardiovascular diseases including angina pectoris. The authors studied the potential interference of such medicines with the widely used EMIT 2000 (Dade Behring; Deerpark, IL) digoxin assay and the recently marketed Randox digoxin assay (Randox Laboratories Ltd, Antrim, United Kingdom) (both run on the Bayer ADVIA 1650 analyzer) (Bayer Diagnostics, Tarrytown, NY) and compared their results with an FPIA (Abbott Laboratories) and a chemiluminescent immunoassay (CLIA; Bayer Diagnostics) for digoxin. Aliquots of drug-free serum were supplemented with 1 microL ethyl acetate extract of Danshen or aqueous extract of Chan Su, and apparent digoxin concentrations were measured by all four digoxin immunoassays (FPIA, EMIT, Randox, CLIA). The authors also supplemented aliquots of several different serum pools prepared from patients taking digoxin with very small amounts of Chan Su or Danshen extract and compared digoxin values with the control digoxin values (serum pool containing no Chinese medicine). The authors observed no interference of Danshen in either EMIT, Randox, or CLIA assay but observed an interference with the FPIA assay. On the other hand, the authors observed high interference of Chan Su in the FPIA assay but moderate interference with the EMIT 2000 and Randox digoxin assays. CLIA assay was again free from any interference. The authors also observed a wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin immunoassay in different brands of Chan Su and Danshen, indicating poor quality control in manufacturing of these Chinese medicines. Taking advantage of the high protein binding of digoxin-like immunoreactive components of Chan Su, the authors further demonstrated that interference of Chan Su in EMIT 2000 and Randox assays can be mostly eliminated by monitoring free digoxin.

Endogenous serum antibodies that interfere with a common thyroid hormone uptake assay: characterization and prevalence.

We identified individuals whose serum contained a substance that produced falsely decreased thyroid hormone (T)-uptake values determined by the Emit (Syva) procedure. Investigation of this interference was prompted by identification of a patient with T-uptake values inconsistent with clinical assessment. IgG depletion and supplementation studies with this patient's serum suggested that the interference was due to endogenous antibodies with specificity for the thyroxine-glucose-6-phosphate dehydrogenase conjugate in the Emit T-uptake assay. The prevalence of the interference was examined by prospectively comparing routine Emit T-uptake values of 1710 patients' samples to T-uptake values obtained by another method. Discrepant samples were also assayed by a radioactive binding triiodothyronine-uptake assay. We identified eight samples that had falsely decreased T-uptake values by Emit, for an overall prevalence of 0.46%. Among 45 consecutive patients with a T-uptake value < 20%, five patients, or 11%, were falsely decreased by Emit and three of these were clearly due to an interfering IgG. We suggest that samples with abnormally low T-uptake values determined by the Emit method be confirmed by an alternative method.

Amphetamines and 3,4-methylendioxymetamphetamine (MDMA): evaluation of KIMS (kinetic interaction of microparticles in solution) assay at two cut-off levels.

Two screening methods for the assay of amphetamines and their derivatives have been applied to the same analytical instrument for their evaluation. In addition to an assay at a cut-off of 1000 microg/l, a new specific reagent was evaluated for an ultra-sensitive assay of amphetamines and 3,4-methylendioxymetamphetamine with a cut-off of 300 microg/l. The assay confirmation was performed using high-performance liquid chromatography and gas chromatography/ mass spectrometry techniques. The results were positive for both screening methods, confirming the efficacy of two simultaneous methods with different cut-off levels.