Mathematical equations to calculate true mycophenolic acid concentration in human plasma by using two immunoassays with different cross-reactivities with acyl glucuronide metabolite: comparison of calculated values with values obtained by using an HPLC-UV method.

BACKGROUND: Both immunoassays and chromatographic methods are available for therapeutic drug monitoring of mycophenolic acid (MPA). Although chromatographic methods are more precise, immunoassays are widely used in clinical laboratories due to ease of adopting such assays on automated analyzers. We studied the possibility of using mathematical equations to calculate true MPA concentration by accounting for acyl glucuronide cross-reactivities with immunoassays by using two immunoassays with widely different cross-reactivities with the metabolite. METHODS: We determined MPA concentrations in 20 specimens obtained from transplant recipients using cloned enzyme donor immunoassay (CEDIA) assay and a new particle enhanced turbidimetric inhibition immunoassay (PETINIA) assay. Then we developed mathematical equations to calculate true MPA concentration using values obtained by both immunoassays and reported cross-reactivity of acyl glucuronide with respective immunoassays. Calculated concentrations were compared with values obtained by using a high-performance liquid chromatography combined with ultraviolet detection (HPLC-UV) method. RESULTS: We obtained good correlation between calculated MPA concentrations and corresponding MPA level obtained by using HPLC-UV method. Using x-axis as the MPA concentrations determined by the HPLC-UV method and y-axis as the calculated MPA level, we observed the following regression equation: y = 1.083x - 0.0995 (r = 0.99, n = 20). CONCLUSIONS: Mathematical equations can be used to calculate true MPA concentrations using two immunoassays with different cross-reactivities with acyl glucuronide metabolite.

Comparison of mycophenolic acid concentrations determined by a new PETINIA assay on the dimension EXL analyzer and a HPLC-UV method.

OBJECTIVE: Mycophenolic acid requires routine therapeutic drug monitoring. We evaluated the suitability of a new PETINIA (particle enhanced turbidimetric inhibition immunoassay) assay on the Dimension EXL analyzer for monitoring of mycophenolic acid by comparing values obtained by this assay with a HPLC-UV method. DESIGN AND METHODS: Mycophenolic acid concentrations determined in sera of 60 organ transplant recipients using high performance liquid chromatography combined with ultraviolet detection (HPLC-UV, reference method) and the new immunoassay on the Dimension RxL analyzer. RESULTS: The within and between run precision of the new PETINIA assay was <10%. The assay was linear for a mycophenolic acid concentration up to 30µg/mL. When mycophenolic acid concentrations in 60 transplant recipients obtained by the HPLC-UV (x-axis) method were compared with corresponding values obtained by the PETINIA assay (y-axis), the following regression equation was obtained: y=1.1204 x+0.0881 (r=0.983, n=60). CONCLUSIONS: If PETINIA assay is used for therapeutic drug monitoring of mycophenolic acid, caution must be exercised in interpreting serum mycophenolic acid level due to observed positive bias.

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.

Hydroxyzine and cetirizine interfere with the PENTINA carbamazepine assay but not with the ADVIA CENTEUR carbamazepine assay.

Because of a published report indicating significant interference of hydroxyzine with the particle-enhanced turbidimetric inhibition immunoassay (PENTINA) carbamazepine assay, we investigated whether such interference can be avoided by using the ADVIA Centaur carbamazepine assay. Both the Dimension Vista analyzer and ADVIA Centaur analyzer are available from Siemens Diagnostics. Aliquots of a drug-free serum pool were supplemented with various concentrations of hydroxyzine or cetirizine (0.05 microg/mL to 20 microg/mL covering therapeutic and toxic levels in serum) followed by analysis using both assays. We observed significant apparent carbamazepine concentrations using the PENTINA assay but no apparent carbamazepine level using the ADVIA Centaur assay. Because crossreactivity should be studied in the presence of the primary analyte, we also prepared a serum carbamazepine pool from patients receiving carbamazepine and then supplemented aliquots of this pool with various amounts of hydroxyzine or cetirizine followed by reanalyzing carbamazepine concentration using both assays. We observed falsely elevated carbamazepine values using the PENTINA assay but no interference was observed using the ADVIA Centaur assay. However, the falsely elevated carbamazepine values using the PENTINA assay were clinically significant at hydroxyzine or cetirizine concentrations expected in patients with severe overdoses with these drugs. We conclude that the ADVIA Centaur carbamazepine assay is free from interference of both hydroxyzine and cetirizine.

Effect of spironolactone, potassium canrenoate and their common metabolite canrenone on serum digoxin measurement by digoxin III, a new digoxin immunoassay.

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. It has been well documented in the literature that spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with several digoxin immunoassays at concentrations expected after therapeutic usage of these drugs and falsely elevate or lower serum digoxin concentrations. Recently, Abbott Laboratories marketed a new Digoxin III immunoassay for application on the AxSYM analyzer. We studied the potential interference of these compounds with this new digoxin assay. The Tina-quant assay was used as the reference method because spironolactone, potassium canrenoate, and canrenone do not interfere with serum digoxin measurement using this assay. Aliquots of drug-free serum were supplemented with therapeutic and above therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured using the Digoxin III assay and Tina-quant assay. Significant apparent digoxin concentrations were observed when the Digoxin III digoxin assay was used, but no apparent digoxin levels was observed using the Tina-quant assay. When serum pools prepared from patients receiving digoxin were further supplemented with these compounds in concentrations expected in sera of patients receiving these medications, falsely elevated digoxin levels were observed using Digoxin III assay, but no statistically significant change was observed using the Tina-quant assay. We conclude that spironolactone, potassium canrenoate, and their common metabolite canrenone interfere with the serum digoxin measurements using the new Digoxin III assay.

Effect of Asian ginseng, Siberian ginseng, and Indian ayurvedic medicine Ashwagandha on serum digoxin measurement by Digoxin III, a new digoxin immunoassay.

Asian ginseng, Siberian ginseng, and Indian Ayurvedic medicine Ashwagandha demonstrated modest interference with serum digoxin measurements by the fluorescent polarization immunoassay (FPIA). Recently, Abbott Laboratories marketed a new digoxin immunoassay, Digoxin III for application on the AxSYM analyzer. We studied potential interference of these herbal supplements on serum digoxin measurement by Digoxin III assay in vitro and compared our results with the values obtained by Tina-quant assay. Aliquots of drug-free serum pool were supplemented with various amounts of Asian ginseng, Siberian ginseng, or Ashwagandha approximating expected concentrations after recommended doses and overdoses of these herbal supplements in serum. Then digoxin concentrations were measured by the Digoxin III and Tina-quant (Roche Diagnostics) assay. We also supplemented aliquots of a digoxin pool prepared from patients receiving digoxin with various amounts of these herbal supplements and then measured digoxin concentrations again using both digoxin immunoassays. We observed modest apparent digoxin concentrations when aliquots of drug-free serum pool were supplemented with all three herbal supplements using Digoxin III assay (apparent digoxin in the range of 0.31-0.57 ng/ml), but no apparent digoxin concentration (except with the highest concentration of Ashwagandha supplement for both brands) was observed using the Tina-quant assay. When aliquots of digoxin pool were further supplemented with these herbal supplements, digoxin concentrations were falsely elevated when measured by the new Digoxin III assay. For example, we observed 48.2% (1.63 ng/ml digoxin) increase in digoxin concentration when an aliquot of Digoxin pool 1 (1.10 ng/ml digoxin) was supplemented with 50 microl of Asian ginseng extract (Brand 2). Measuring free digoxin does not eliminate the modest interferences of these herbal supplements in serum digoxin measurement by the Digoxin III assay.

St. John's wort does not interfere with therapeutic drug monitoring of 12 commonly monitored drugs using immunoassays.

St. John's wort, a popular herbal remedy for depression, is known to interact with many Western drugs because of the ability of its components to induce liver enzymes. Lower concentrations of various drugs due to increased clearance have been reported. Because immunoassays are commonly used in clinical laboratories for therapeutic drug monitoring, we studied the potential interference of St. John's wort with commonly monitored therapeutic drugs. Drug-free serum pools were supplemented with St. John's wort to achieve in vitro St. John's wort concentrations mimicking in vivo concentrations after both recommended use and overdose. Concentrations of digoxin, tricyclic antidepressants (TCAs), phenytoin, carbamazepine, theophylline, valproic acid, quinidine, phenobarbital, procainamide, and N-acetyl procainamide were measured in serum. Pooled serum specimens from patients who were taking a particular drug were also supplemented in vitro with concentrations of St. John's wort to investigate whether observed concentrations changed after supplementation with St. John's wort. The effect of St. John's wort on cyclosporine and tacrolimus (FK 506) was studied in whole blood. We found no significant interference from St. John's wort with any assay studied. Moreover, when drug-free serum was supplemented with very high concentrations of hypericin (2 microg/mL) and hyperforin (2 microg/mL) pure standard, we observed no apparent drug level with any immunoassay. The presence of both hypericin and hyperforin was also confirmed by thin layer chromatography (TLC) in both preparations of St. John's wort. We conclude that immunoassays may be used to measure levels of therapeutic drugs in patients who self-medicate with St. John's wort.