Chapter 3: Results and Discussions
3.2 Urine, Blood and Hair Samples Results for Drugs Users
3.2.1 Immunoassay Results for Samples Collected from Drug Users
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Figure 21: Failed calibration curve of meprobamate drug for DOA CS T 52 kit in Randox Evidence Plus immunoassay instrument
Figure 22: Accepted calibration curve of buprenorphine drug for DOA CS T 52 kit in Randox Evidence Plus immunoassay instrument
125 Figure 23: Accepted calibration curve of pregabalin drug for DOA CS T 52 kit in
Randox Evidence Plus immunoassay instrument
The two levels of QC (low and high) should be run along with any suspected sample on IA. These two QCs are provided by the company along with the calibrators. Low QC is defined as the negative control where values of all analytes are negative. High QC is a positive control where all analytes are positive. QCs play a crucial role in determining the performance of the instrument and detects how well the measurement system is in producing results within the selected calibration range. The acceptable performance of quality controls is defined as ± 3 standard deviation (SD). Figures 24 and 25 represent an example of accepted low- and high-QC, respectively, for 6-acetylmorphine.
If there is a control failure, two scenarios should be followed. The first scenario is preparing and running the new QCs to overcome any error that might result during the preparation process. If the QCs still fail, it is recommended to prepare a fresh calibration curve followed by QCs then your samples. In one case where we faced a failure in QCs, the issue behind it was the differences in lot number between the QCs used and calibrator stock. This issue led us to follow a different path and start using in-house QCs where lot number-related issues would no longer be involved.
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Figure 24: Low quality control of 6-acetylmorphine drug for DOA CS T 52 kit in Randox Evidence Plus immunoassay instrument
Figure 25: High quality control of 6-acetylmorphine drug for DOA CS T 52 kit in Randox Evidence Plus immunoassay instrument
127 The urine and blood samples with the confiscated drug samples, were obtained from Ajman and Umm Al Quwain drug enforcement department and were first screened by Randox Evidence Plus immunoassay. We were able to analyze 500 urine samples as well as 500 blood samples from various suspects by IA. The results showed that 302 cases tested positive in urine for one or more drugs of abuse (BENZ and/or OPIAT and/or CAD).
Whereas 283 blood samples were positive for BENZ and/or OPIAT and/or CAD (Appendix). Figure 26 displays an example of a negative IA result for one case of urine matrix, while a negative IA result for a blood sample is shown in Figure 27. The drug groups detected most commonly in urine were opiate (63.2%), CAD (55.9%), and BZD (19.2%). In contrast, the drug groups detected most commonly in blood samples were opiate (60.8%), CAD (53.7%), and BZD (19.4%).
Figure 28 represents the IA result for a urine case (TOX-U-153) which tested positive for monoacetylmorphine, amphetamine, BZD, methamphetamine, opiate, PGB, and tramadol. The opiate group was detected as a positive result by IA for a suspect’s urine sample. A positive blood case (TOX-B-207) is shown in Figure 29 which was positive for amphetamine, methamphetamine, PGB, and tramadol. In addition, Table 49 and 50 summarize some of the IA results obtained from a suspects’ urine and blood samples, respectively.
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Figure 26: Evidence Plus IA result for a negative urine sample (MAM:
monoacetylmorphine, ABCHM: AB-CHIMINACA, ABPIN: AB-PINACA, AMPH:
Amphetamine, BARB: Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP:
Buprenorphine, BZG: Cocaine metabolite, ETG: Ethyl glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine, MODONE: Methadone, MPB:
Meprobamate, OPIAT: Opiate, PGB: Pregabalin, TCA: Nortriptyline, THC:
Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids, CREAT: Creatinine)
129 Figure 27: Evidence Plus IA result for a negative blood sample (MAM:
monoacetylmorphine, ABCHM: AB-CHIMINACA, ABPIN: AB-PINACA, AMPH:
Amphetamine, BARB: Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP:
Buprenorphine, BZG: Cocaine metabolite, ETG: Ethyl glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine, MODONE: Methadone, MPB:
Meprobamate, OPIAT: Opiate, PGB: Pregabalin, TCA: Nortriptyline, THC:
Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids)
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Figure 28: Evidence Plus IA result for a positive urine sample (MAM:
monoacetylmorphine, ABCHM: AB-CHIMINACA, ABPIN: AB-PINACA, AMPH:
Amphetamine, BARB: Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP:
Buprenorphine, BZG: Cocaine metabolite, ETG: Ethyl glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine, MODONE: Methadone, MPB:
Meprobamate, OPIAT: Opiate, PGB: Pregabalin, TCA: Nortriptyline, THC:
Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids, CREAT: Creatinine)
131 Figure 29: Evidence Plus IA result for a positive blood sample (MAM:
monoacetylmorphine, ABCHM: AB-CHIMINACA, ABPIN: AB-PINACA, AMPH:
Amphetamine, BARB: Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP:
Buprenorphine, BZG: Cocaine metabolite, ETG: Ethyl glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine, MODONE: Methadone, MPB:
Meprobamate, OPIAT: Opiate, PGB: Pregabalin, TCA: Nortriptyline, THC:
Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids)
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Table 49: Some of immunoassay results for urine samples using Evidence Plus Analyzer Serial # Sample ID Gender Age Positive IA results
1 TOX-U-01 M 21-30 OPI/TRM/BNZ
2 TOX-U-02 M 21-30 PRG
3 TOX-U-03 M 31-40 OPI
4 TOX-U-04 M 21-30 OPI/TRM
5 TOX-U-05 M 31-40 PRG/TRM
6 TOX-U-06 M 41-50 OPI/MPB
7 TOX-U-07 M 31-40 MPB
8 TOX-U-08 M 31-40 PRG/MPB
9 TOX-U-09 M 31-40 OPI/TRM
10 TOX-U-10 M 31-40 OPI/PRG
11 TOX-U-11 M 41-50 OPI
12 TOX-U-12 F 21-30 BNZ
13 TOX-U-13 M 31-40 OPI/BNZ
14 TOX-U-14 F 21-30 BNZ/TRM
15 TOX-U-15 F 21-30 PRG
16 TOX-U-16 F 21-30 TRM
133 Table 50: Some of immunoassay results for positive blood samples using Evidence Plus Analyzer
Serial # Sample ID Gender Age Positives IA result
1 TOX-B-01 M 31-40 PRG
2 TOX-B-02 F 21-30 OPI/TRM
3 TOX-B-03 M 21-30 BNZ/CAD
4 TOX-B-04 F 21-30 OPI/BNZ
5 TOX-B-05 M 31-40 BNZ
6 TOX-B-06 M 41-50 BNZ
7 TOX-B-07 M 31-40 OPI
8 TOX-B-08 F 31-40 TRM
9 TOX-B-09 M 31-40 OPI/CAD
10 TOX-B-10 M 31-40 OPI/CAD
11 TOX-B-11 M 41-50 OPI/BNZ/CAD
12 TOX-B-12 F 21-30 OPI/TRM
13 TOX-B-13 M 31-40 OPI
14 TOX-B-25 M 31-40 TRM
15 TOX-B-26 M 31-40 BNZ
16 TOX-B-27 M 41-50 CAD
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Failed IA test or error in IA data results were noticed in four samples of urine.
Figure 30 illustrates the error obtained while running the TOX-U-011 sample. Error code 6011 can be defined, based on the manufacturer’s error code list, as the inability of the instrument to read the selected sample. Sample turbidity was the common feature in all the four samples that showed the error. This led us to conclude that centrifugation of urine samples is required before processing to IA when sample turbidity is suspected.
Figure 30: Error obtained in Evidence Plus IA result for a urine sample (MAM:
monoacetylmorphine, ABCHM: AB-CHIMINACA, ABPIN: AB-PINACA, AMPH:
Amphetamine, BARB: Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP:
Buprenorphine, BZG: Cocaine metabolite, ETG: Ethyl glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine, MODONE: Methadone, MPB:
Meprobamate, OPIAT: Opiate, PGB: Pregabalin, TCA: Nortriptyline, THC:
Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids, CREAT: Creatinine)
135 Due to the lack of selectivity in the IA instrument, it can result in various false positives as well as false negatives. A variety of factors can cause false-negative or false- positive results, including the cross-reactivity of the antibody in the IA, the cutoff concentration that yields a positive result, and/or the time between drug ingestion. The selected cutoffs lower the risk of a false negative and provide better accuracy with drug monitoring. As discussed, in certain cases (Raouf et al., 2018), the opiate panel in most of the IA instruments test for metabolites of morphine, codeine, and heroin, and may consequently lead to semisynthetic/synthetic opioids not being detected.
Six suspects tested positive for PGB in urine samples by our IA; however, when confirmed using LC-MSMS, GBP only was detected. This indicated the presence of a cross-reactivity where GBP is able to bind to the assay antibodies in a manner similar to the PGB molecule. A similar situation has been indicated in a study which demonstrated that cross-reactive compounds in immunoassays tend to have high similarity to the target molecule and limit testing of the target compounds (Krasowski, Siam, Iyer, & Ekins, 2009).
Furthermore, I have analyzed thirteen diluted urine samples where the detected creatinine levels were bellow 20 mg/dL (as shown in Figure 31). Out of these samples, 7 samples belonged to female suspects, which may represent a degree of impairment in observing the urine collection process. These samples were tested additionally with the adulteration cup which showed pH values less than 3 and a relative density less than 1.003.
Interestingly, two urine samples obtained from male suspects reported as positive for creatinine (level lower than 20 mg/dL); however, these were still positive for PGB in one case (above 500 ng/ml) and amphetamine with methamphetamine in the other case.
Figure 32 represents the IA result of the first case which was positive for PGB even though the creatinine level was below 20 mg/dL. The clarification of this case can fall between medical conditions resulting in overhydration or water-loading (consuming a large volume of water right before sample collection) based on the clinical studies that were performed to study the validity of random urine specimens submitted for DOA analysis (Cook, Caplan, LoDico, & Bush, 2000).
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Figure 31: IA result of urine sample from female suspect with positive creatinine (less than 20 mg/dL) (MAM: monoacetylmorphine, ABCHM: AB-CHIMINACA, ABPIN:
AB-PINACA, AMPH: Amphetamine, BARB: Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP: Buprenorphine, BZG: Cocaine metabolite, ETG: Ethyl
glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine,
MODONE: Methadone, MPB: Meprobamate, OPIAT: Opiate, PGB: Pregabalin, TCA:
Nortriptyline, THC: Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids, CREAT: Creatinine)
137 Figure 32: IA result of urine sample from male suspect with positive creatinine (less than 20 mg/dL) and positive pregabalin (>500 ng/mL) (MAM: monoacetylmorphine,
ABCHM: AB-CHIMINACA, ABPIN: AB-PINACA, AMPH: Amphetamine, BARB:
Barbiturates, BNZ1: Benzodiazepines, BS1: Bath Salt, BUP: Buprenorphine, BZG:
Cocaine metabolite, ETG: Ethyl glucuronide, FENT: Fentanyl, KET: Norketamine, MAMP: Methamphetamine, MODONE: Methadone, MPB: Meprobamate, OPIAT:
Opiate, PGB: Pregabalin, TCA: Nortriptyline, THC: Cannabinoids, TRM: Tramadol, UR144: Synthetic Cannabinoids, CREAT: Creatinine)
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