1. Choosing the Best Urine Benzo Screen for Monitoring Adherence to Alprazolam or Clonazepam
Maggie L Hopkins MD MBA1, Elisabeth Malmberg MS2, Bella Church ASCP(C)2 , Gwendolyn A McMillin PhD1,2
1Department of Pathology and 2ARUP Laboratories, University of Utah, Salt Lake City, Utah
Introduction
Materials and Methods
Results
Purpose:
Predict detection rates of three common* laboratory-based benzodiazepine immunoassays, for alprazolam or clonazepam in urine
* DRI® (Microgenics), EMIT® II Plus (Beckman Coulter), and CEDIA® (PerkinElmer) benzodiazepine immunoassays
Calculations:
Based on cross-reactivity profiles published for the DRI® (Microgenics), EMIT® II Plus (Beckman Coulter), and CEDIA® (PerkinElmer) benzodiazepine immunoassays, we derived a formula to predict the total “apparent concentration” of alprazolam and clonazepam that each immunoassay would detect. This calculation takes into account the cross-reactivity factor for each analyte (alprazolam and α-hydroxy-alprazolam, clonazepam and 7-amino-clonazepam) and gives a sum of all detected metabolites plus parent drug for the apparent concentration.
Apparent concentration for each drug:
= Sum (reactivity factor for analyte*concentration of analyte)
We compared this calculated apparent level to the cut-off level (200 ng/mL) to estimate detection rates of each immunoassay for urine samples confirmed positive. The percent of positive samples above 200 ng/mL was defined as the sensitivity of an immunoassay to each drug. The percent of samples positive by LC-MS/MS, but negative (below 200 ng/mL) by the immunoassay was defined as the false-negative rate. For the CEDIA assay, cross-reactivity was provided with and without hydrolysis by beta-glucuronidase.
Data Analysis:
The statistical analyses were carried out using SAS 9.3 (SAS Institute, Cary, NC).
TABLE 1.
TABLE 2.
TABLE 3.
Urine samples analyzed by LC-MS/MS n
Total samples analyzed
18,502
Total positive for Alprazolam
5926
Total positive for Clonazepam
4032
Alprazolam detection by Immunoassay +
Sensitivity
CEDIA
4157
70.15%
CEDIA+ glucuronidase
4670
78.81%
EMIT II
4989
84.19%
DRI
2934
49.51%
Clonazepam detection by Immunoassay +
Sensitivity
CEDIA 53
1.31%
CEDIA+ glucuronidase
1609
39.91%
EMIT II 18
0.55%
DRI 20
0.50%
TABLE 4.
TABLE 5.
Analytes detected by LC-MS/MS
Alpraz
H-Alpz
Clonaz
7-AC
Mean
181.64
213.46
39.78
228.39
Median
101.00
129.00
20.00
104.00
Range
1172
1165
1000
Reactivity Factor
Alpraz
H-Alpz
Clonaz
7-AC
CEDIA
1.45
1.23
1.06
0.00
CEDIA+ glucuronidase
1.90
0.40
0.08
EMIT II
3.08
2.00
0.34
DRI 2
1.74
0.89 1
Background:
There are a number of immunoassay platforms on the market for screening benzodiazepines in urine. Cut-off values may be the same, but each immunoassay has a different sensitivity for individual analytes (parent drug and drug metabolites).
Detection rates of individual drugs may be inconsistent between assays, leading to false-negative results. When the purpose of testing is to gauge compliance for medication use, false negatives can greatly disrupt the course of normal care for patients including investigations, medication refill delays and damage to the patient-provider relationship.
Alpraz: Alprazalam, H-Alpz: α-Hydroxy-Alprazolam, Clonaz: Clonazepam, 7AC: 7-Amino-Clonazepam
FIGURE 1.
ALPRAZOLAM
% positive by LC-MS/MS
% positive by LC-MS/MS
% positive by LC-MS/MS
% positive by LC-MS/MS
Apparent concentrations based on DRI sensitivities for Alprazolam
Apparent concentrations based on EMIT II sensitivities for Alprazolam
Apparent concentrations based on CEDIA +G sensitivities for Alprazolam
Apparent concentrations based on CEDIA sensitivities for Alprazolam
CLONAZEPAM
% positive by LC-MS/MS
% positive by LC-MS/MS
% positive by LC-MS/MS
% positive by LC-MS/MS
Materials and Methods
Data Collection:
Results for 18,502 urine samples submitted to ARUP Laboratories for quantitative benzodiazepine testing, by LC-MS/MS.
Analysis includes alprazolam and clonazepam.
Apparent concentrations based on DRI sensitivities for Clonazepam
Apparent concentrations based on EMIT II sensitivities for Clonazepam
Apparent concentrations based on CEDIA +G sensitivities for Clonazepam
Apparent concentrations based on CEDIA sensitivities for Clonazepam
Conclusions
The immunoassays evaluated here are inappropriate for assessing patient compliance with clonazepam, and may be inappropriate for assessing patient compliance with alprazolam. If there is clinical reason to expect clonazepam or alprazolam in a urine drug test, we recommend a definitive testing method such as LC-MS/MS. Pre-analytical hydrolysis may improve detection.