Design VerificationHumaDrug C
3 Specificity, sensitivity and agreement .2
HumaDrug CANNABINOIDS (THC) has been designed as a rapid 1-step test for the qualitative detection of
cannabinoids (THC) in human urine. The test is based on a competitive immunochromatographic technique,
featuring immobilized drug and dye-labeled anti-THC antibodies. If cannabinoids are present in the urine sample
drug molecules compete with the immobilized drug for limited amounts of dye-labeled anti-drug antibodies.
Above a threshold concentration only one coloured line will appear on the membrane (positive result). Below that
concentration two coloured lines will be observed (negative result). The test has been adjusted to the suggested
screening cut-off for positive specimens set by SAMSHA (Substance Abuse and Mental Health Services
Administration, USA). HumaDrug CANNABINOIDS comes in a convenient package, consisting of test cassettes and
disposable dropper pipettes. The urine sample is pipetted onto the test cassettes disposable plastic dropper
pipettes. 2 Sensitivity and dynamic range
Urine drug controls (Bio-Rad, J&S, others) are employed for testing of sensitivity and dynamic range.
The respective drug concentrations have been standardized by the manufacturer.
Standard dilutions are prepared from either urine controls or pure substances (i.e. Sigma, #N5642) by dilution with
phosphate buffered saline to approx. the following concentrations:Table 1: Standard test concentrations (11-nor-∆8-tetrahydrocannabinol-9-carboxylic acid)
All concentrations were tested in 10-fold determination. Strong test and control lines appeared with negative
urines and zero-standards. In the presence of THC at concentrations of 25 ng/ml a strong control line came up
after 5 minutes incubation, accompanied by a clearly visible test line. At 50 ng/ml, the “new” SAMSHA cut-off
value, a strong control line was observed, sometimes accompanied by a faint test line. At 75 ng/ml only a strong
control line appeared within the incubation period of 5 minutes. The test devices must not be interpreted after 10
minutes as in some cases after that time a faint test line may appear even with weak positive samples, due to
unspecific reactions on the membrane. Prozone phenomenon will not occur with such a competitive test format
and even extremely high concentrations of the analyte will not produce false negative results (two lines). 3 Specificity, sensitivity and agreement
The specificity and sensitivity of HumaDrug CANNABINOIDS have been evaluated by method comparison against
the GC/MS reference method. 300 urine specimens have been employed in the comparison.
Design Verification and Product Data for HumaDrug CANNABINOIDS
From this comparison the relative diagnostic sensitivity and specificity have been calculated according:
A number of potentially interfering substances have been added at a concentration of 100 µg/ml to negative and
positive urine specimens, respectively.). Table 2: Potentially interfering substances
Design Verification and Product Data for HumaDrug CANNABINOIDS
Conclusion: At the tested concentration of 100 µg/ml none of the substances listed in table 2 interfered with
Cross-reactivities of HumaDrug CANNABINOIDS have been studied by adding potentially cross-reacting substances
in various concentrations to negative urine samples. The following substances were found to cross react at levels
Conclusion: Cannabinol, ∆9-THC and ∆8-THC produced positive results only at very high concentrations.
Therefore, it exists no cross-reactivity with these substances at lower concentrations. 4 Reproducibility
Day-to-day and lot-to-lot reproducibility was evaluated, employing the standard concentrations as given in 2.1
(up to 75 ng/ml). 4.1 Within-day reproducibility
Within 10 days the results at 0 and 25 ng/ml were consistenly negative. At 50 ng/ml consistenly borderline results
have been obtained (very faint test line), and at 75 ng/ml consistently positive results appeared (no visible test
line). In no case discrepant results were found.
Design Verification and Product Data for HumaDrug CANNABINOIDS
Similarly, the reproducibility between three independent lots was tested with the same concentrations as
mentioned above. Each concentration was tested with each lot in 10-fold determinations. All results were fully
consistent. Conclusion: HumaDrug CANNABINOIDS has shown to produce excellently reproducible results from day to day
The stability of HumaDrug CANNABINOIDS has been demonstrated on real time stability studies and additional
For real time studies, test devices have been stored for a period of up to 24 months at 25°C and 60% relative
For temperature stress studies the test devices have been stored for up to 73 days at 45°C and 60% relative
Standard concentrations according to 2.1 were employed and each concentration was tested in double. The results
are presented in tables 3 and 4 below. Table 3: Real time stability (mean of double determination)
Table 4: Accelerated stress stability (mean of double determination)
Conclusion: According to the results from both real time as well as accelerated temperature stress HumaDrug
CANNABINOIDS has been shown to be at least stable for 24 months when stored at 25°C.
Design Verification and Product Data for HumaDrug CANNABINOIDS
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