Chapter 3: Results and Discussions
3.2 Urine, Blood and Hair Samples Results for Drugs Users
3.2.2 LC-MS/MS Confirmatory Results
138
139 Figure 34: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (benzodiazepine method)
140
Figure 34: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (benzodiazepine method) (Continued)
141 Figure 34: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (benzodiazepine method) (Continued)
142
Figure 34: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (benzodiazepine method) (Continued)
143 Figure 34: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (benzodiazepine method) (Continued)
In this study, 302 suspects tested positive for one or more drugs within the BZD, OPI, and CAD groups. Twenty different drugs were detected with eight of them belonging to the BZD group. Bromazepam, alprazolam, midazolam, nordiazepam, diazepam, oxazepam, 7-aminoclonazepam, and clonazepam were detected and confirmed in various ranges in the suspects’ urine samples. The BZD drug most commonly detected was diazepam, which was detected in 22 different cases with a concentration range varying from 12.3 ng/ml to 112.6 ng/ml ( 0.1). Diazepam alone without its major metabolites was detected in 7 samples representing the recent exposure to diazepam as reported by Umezawa (Umezawa et al., 2008). Drugs that were detectable but not present in quantifiable amounts such as in one case was the midazolam and it’s level was below 5
144
ng/ml which can be explained as traces during production, distribution, or storage of these street drugs (Whitehead et al., 2023).
In contrast, eight different drugs were detected in the suspects’ urine samples that belong to the opiates group. Detected opiate drugs were codeine, morphine, buprenorphine, 6-acetylmorphine (6-AM), dextromethorphan, tramadol, O- desmethyltramadol, and methadone. The opiate drug detected most commonly was morphine in 113 cases. Of the 8 opiate compounds only 8 cases with morphine exposure above its quantification limit (1,000 ng/ml) were found, whereas one case with the presence of O-desmethyltramadol below the detection limit (10 ng/ml) was found. In urine, approximately 30% of tramadol is excreted as is, while approximately 60% is excreted as metabolites (N- and O-desmethyltramadol). Tramadol has a half-life of 5–6 h, and O-desmethyltramadol has a slightly longer half-life of 8 h (Yilmaz & Erdem, 2015).
This might indicate that has been a while since the last use of tramadol for a person with traces amount of O-desmethyltramadol. As seen in other studies, following heroin administration, 6-AM was excreted rapidly with an average half-life of 0.6 h before it was hydrolyzed to morphine (Ellis, McGwin, Davis, & Dye, 2016). Therefore, being able to detect 6-AM in 14 cases indicate the recent use of heroin.
From combined acid drugs group, pregabalin, carisoprodol and gabapentin were detected with high concentration in suspects’ urine samples. As expected, PGB was found most frequently compared to the other CADs due to the extensive usage of Lyrica in the UAE as a result of the extensive and careless prescription of this medicine from psychiatrists as recently noticed (Dajani, 2017). The range of drugs found in urine and their frequencies are given in Table 51 and Figure 35.
Figure 36 represents the total ion chromatogram (TIC) in MRM mode of a suspect’s urine sample (TOX-U-112) that is positive to oxazepam and diazepam drugs. Figures 37 and 38 illustrate the MRM-LC-MS/MS chromatogram of oxazepam and diazepam drugs, respectively, in the TOX-U-112 case.
145 Table 51: The quantitative range and frequency of occurrence of confirmed drugs in suspects’ urine samples
Drugs Frequency of occurrences Range of drugs found in urine (ng/ml) ( 0.1)
Codeine 88 10.7 − 762.1
Morphin 113 17.8 − >HLOQ (1000)
Pregabalin 98 501.2 − 7539.6
Buprenorphine 20 304.8 − 875.9
Meprobamate 57 501.4 − 7732.4
6 acetylmorphine 14 103.2 − 246.6
Dextromethorphan 16 60.5 − 642.1
Bromazepam 12 41.8 − 102.1
Tramadaol 15 11.7 − 789.7
O-Desmethyltramadol 22 <LLOQ (10) − 971.2
Alprazolam 14 51.6 − 113.7
Metahdone 4 403.4 − 752.9
Midazolam 6 <LLOQ (5) – 214.5
Nordiazepam 22 15.11− 201.7
Diazepam 12 12.3 − 112.6
Oxazepam 17 26.9 – 410.3
7-aminoclonazepam 13 208.6 − 432.4
Clonazepam 3 14.1− 21.7
Carisoprodol 11 679.1− 3061.7
Gabapentin 4 6798.4 − 9945.7
146
Figure 35: Mean concentrations of analytes in urine samples along with the frequency of positive occurrences (shown in brackets). The error bars represent standard deviation
-1000 0 1000 2000 3000 4000 5000 6000 7000 8000 9000
Mean Conc. (ng/ml)
Analytes
147 Figure 36: Total ion chromatogram of a positive urine sample TOX-U-112
(benzodiazepine method)
(Where: 1)7-Aminoclonazepam-D4; 2) 7-Aminoflunitrazepam-D7; 3) Midazolam-D4; 4) Lorazepam-D4; 5) Oxazepam; 6) Oxazepam-D5; 7) Clonazepam-D4; 8) Phenazepam- D4; 9) Flunitrazepam-D7; 10) Triazolam-D4; 11) Temazepam-D5; 12) Alprazolam-D5;
13) Diazepam; 14) Diazepam-D5)
148
Figure 37: MRM-LC-MS/MS chromatogram showing oxazepam drug from TOX-U-112 positive urine sample (benzodiazepine method); A) MRM 287.1->269, B) MRM 287.1->
241.1, C) MRM 287.1-> 104.1, D) Tandem mass spectrometry analysis of oxazepam
Figure 38: MRM-LC-MS/MS chromatogram showing diazepam drug from TOX-U-112 positive urine sample (benzodiazepine method)
149 3.2.2.2 Blood Matrix Confirmatory Results Using LC-MSMS for BZDs, OPI, CAD
The applicability of the BZD, OPI, and CAD methods were also tested on blood samples. Overall, 283 blood samples were analyzed using these methods and compared to results from the IA. The range of drugs found in the blood matrix and their frequencies are given in Table 52 and Figure 39.
The blood samples are considered as the major sample matrix of choice for quantitation analysis compared to urine. As seen in several studies, one of the common features of using the blood matrix compared to urine is the ability of detecting the parent drugs after intake and before metabolism and filtration by the body (Borriello, Carfora, Cassandro, & Petrella, 2015; Thaulow et al., 2018). We noticed in the results of tested samples where parent drugs such as tramadol, diazepam, and clonazepam were detected in blood samples in levels higher than their metabolites in contrast to the results obtained from urine samples.
From the BZD group, the compound detected most frequently in blood matrix was alprazolam followed by diazepam reflecting the wide use of anxiety medication in the UAE. Hydromorphone, which is commonly used as pain medicine that can only be prescribed by a physician, was detected significantly from the opiate group. However, it has been reported that hydromorphone can also be produced from morphine elimination.
Although it can be produced from morphine in small concentrations of ∼1%, high-dose morphine treatments, may lead to concentrations exceeding 1,000 ng/mL, leading to finding a high dose of hydromorphone in individuals (Milone, 2012). Similar to urine matrix, PGB from the CAD group was most frequently found in suspects’ blood samples but in concentrations higher than that in urine cases.
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Table 52: The quantitative range and frequency of occurrence of confirmed drugs in blood samples
Drugs Frequency of occurrences Range of drugs found in blood (ng/ml) (
0.1)
Codeine 5 10.7 − 762.1
Morphine 14 17.8 − 392.4
Hydrocodone 1 10.4
Hydromorphone 23 98.1 − 304.8
oxycodone 2 15.9 − 213.4
Pregabalin 49 661.8 − 9865.2
Buprenorphine 6 41.8 − 102.1
Meprobamate 3 111.7 − 789.7
6 acetylmorphine 1 9.2
Bromazepam 9 56.1 − 113.7
Tramadaol 31 609.8 − 897.4
O-Desmethyltramadol 8 15.11 − 96.9
Alprazolam 41 12.3 − 224.7
Metahdone 11 26.9 − 410.3
Midazolam 7 114.5 − 208.6
Nordiazepam 7 14.7 − 21.7
Diazepam 21 201.1 − 779.1
Oxazepam 3 98.3 − 458.1
7-aminoclonazepam 4 22.9 − 127.4
Clonazepam 17 492.2 − 654.2
Carisoprodol 25 553.5 − 2345.5
Gabapentin 37 898.9 − 4039.4
Acetaminophen 4 198.9 − 632.1
151 Figure 39: Mean concentrations of analytes in blood samples along with the frequency of positive occurrences (shown in brackets). The error bars represents standard deviation
As performed with urine matrix, a blank blood sample was run along with each batch to ensure that no contamination or carry-over is present. Figure 40 represents a blank blood sample with no peaks of analytes present indicating the clearance of extraction procedure performed. Figure 41 displays the positive control used for the opiate method.
LC-MS/MS chromatogram of a suspects’ blood sample (TOX-B-065) that is positive for morphine and 6-AM is shown in Figure 42. MRM scans of morphine and 6-AM shown corresponding to TOX-B-065 are displayed in Figure 43.
-1000 0 1000 2000 3000 4000 5000 6000
Mean Con. (ng/ml)
Analytes
152
Figure 40: Total ion chromatogram of a blank blood sample (opiate method)
153 Figure 41: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (opiate method)
154
Figure 41: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (opiate method) (Continued)
155 Figure 41: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (opiate method) (Continued)
156
Figure 41: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (opiate method) (Continued)
157 Figure 41: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (opiate method) (Continued)
Figure 42: Total ion chromatogram of a positive blood sample TOX-B-065 (opiate method) (Where: 1- Morphine; 2-Morphine-D3; 3-Oxymorphone-D3; 4-Hydrocodone- D3; 5-Hydromorphone-D3; 6- Codeine-D6; 7- 6-acetylmorphine; 8-Norfentanyl-D5; 9- Normeperidine-D4; 10-Meperidine-D4; 11-Fentanyl-D5; 12-Methadone-D3)
158
Figure 43: Typical LC-MS/MS chromatograms of TOX-B-065 blood sample for demonstration of intensities of positive drugs MRM transitions (opiate method) 3.2.2.3 Hair Matrix Confirmatory Results Using LC-MSMS for BZDs, OPI, CAD
The developed methods was then applied to the analysis of 492 authentic hair samples obtained from the aforementioned drug enforcement departments. The demographic data of the samples was as follows: 57 samples were from females (21–40 years old) and 191 samples from males (21–51 years old). All the samples were black or brown color head hair. The range of drugs found in hair and their frequencies are given in Table 53 and Figure 44.
The BZD drugs detected in hair were alprazolam, diazepam, zolpidem, midazolam, oxazepam, and clonazepam. For clonazepam, there were 4 samples with concentrations lower than LOQ. In addition, there were six cases with oxazepam concentrations lower than LOQ. These results indicate the need for a high sensitivity method to analyze very low concentrations (at least in the order of 1 pg/mg).
The detected opiate drugs were buprenorphine, methadone, tramadol, O- desmethyltramadol, codeine, morphine and 6-AM. Cases with positive buprenorphine (BUP) drugs were basically from former heroin users who were all in the process of detoxification following a BUP. Concentration of codeine, morphine, and 6-AM were
159 higher with longer hair segments (2–3 cm) compared to those in short segments (1–2 cm).
This finding supported the study which investigated the appearance of 6-AM, MOR and COD from human hair after the discontinuation of opiate consumption (M. Shen, Xiang, Sun, & Shen, 2013). Moreover, detected drugs from the CAD group in hair samples were only PGB and meprobamate, with a higher frequency of PGB.
Information about drug history was only obtained from 4 cases. The concentrations obtained in these hair samples related to their prescribed drugs were as follows: Case 1 (clonazepam, 1 mg/wk): clonazepam (< LOQ), 7-aminoclonazepam (33 pg/mg). Case 2 (diazepam, 5 mg/d): diazepam (13 pg/mg), nordiazepam (15 pg/mg). Case 3 (clonazepam, 10 mg/d): clonazepam (15 pg/mg) and 7-aminoclonazepam (597 pg/mg). Case 4 (alprazolam, 0.25 mg/d): negative results. One study suggested that the concentrations found in hair matrix may represent prescription use of an individual (Del Mar Ramirez Fernandez et al., 2015); however, reporting the concentrations of drugs in hair is usually difficult. It is often not possible with BZDs and other drugs to determine when an analyte is a metabolite or parent drug or both, so the levels will likely just be reported, and interpretation discussed. For example, nordiazepam may be a metabolite of diazepam or a parent drug; therefore, it may be difficult to suggest the possible medication ingested.
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Table 53: The quantitative range and frequency of occurrence of confirmed drugs in hair samples
Drugs Frequency of occurrences Range of drugs found in hair (pg/mg) ( 0.1)
Codein 14 61.2 − 280.4
Morphin 12 72.3 − 140.7
Pregabalin 103 998.4 − >LOQ
Buprenorphine 7 17.8 − 37.5
Meprobamate 12 532.8 − >LOQ
6 acetylmorphine 8 36.1 − 342.4
Tramadaol 35 170.3 − 642.1
O-Desmethyltramadol 41 13.2 − 45.1
Alprazolam 26 17.3 − 49.5
Metahdone 5 >LOQ
Midazolam 6 10.2 − 16.9
Nordiazepam 10 13 − 32.9
Diazepam 16 12.6 − 51.2
Oxazepam 11 <LOQ − 15.3
7-aminoclonazepam 19 33.1 − 597.2
Clonazepam 21 <LOQ − 21.3
161 Figure 44: Mean concentrations of analytes in hair samples along with the frequency of positive occurrences (shown in brackets). The error bars represents standard deviation.
Figure 45 shows the TIC in MRM mode of a blank hair sample which was run along with each batch. A positive control for the CAD method is shown in Figure 46. An example of a positive hair sample (positive to PGB and carisoprodol) is shown in Figures 47 and 48.
-20 0 20 40 60 80 100 120
Mean Conc. (pg/mg)
Analytes
162
Figure 45: Total ion chromatogram of a blank hair sample (CAD method)
163 Figure 46: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (CAD method)
164
Figure 46: MRM-LC-MS/MS chromatogram showing drugs list in a single extract of a positive control (CAD method) (Continued)
165 Figure 47: Total ion chromatogram of a suspects hair sample TOX-H-012 (CAD
method) (Where: 1- Acetaminophem-D4; 2- Pregabalin; 3- Pregabalin-D6; 4-
Gabapentin-D4; 5-Levetiracetam-D6; 6-Meprobamate-D7; 7-Carbamazepine-D10; 8- Carisoprodol; 9-Carisoprodol-D7)
166
Figure 48: MRM-LC-MS/MS chromatogram showing positive drugs listed in suspects’
hair sample TOX-H-012 (CAD method) (MRM of pregabalin, panels A) and (MRM of carisoprodol, panels B)
167 3.2.2.4 Comparison of Drugs in Urine, Blood and Hair Results from Same Individual
In this section, we compared the findings of IA data to LC-MS/MS of urine, blood, and hair samples of the same individual. Urine, blood, and hair samples were collected from each case with a total of 12 cases. Any recent exposure to drugs (like in case 3, 5 and 10 for BZD drugs) would not be detected in hair samples where drugs will be found 10 to 15 days after drug exposure. Codeine in case 1 was not detected in the hair matrix and this is explained by its low concentration (M. Shen et al., 2013). The presence of drugs/
metabolites in hair matrices as well as in urine and blood such as in cases 1, 3, 4, 6, 7, and 10 might indicate the regular use of these drugs.
Interestingly, three cases were analyzed in IA and showed negative results in all.
These cases belong to routine drug analysis. This means the users were caught once for drug usage and the court listed them within the routine drug analysis whereby, they will be tested monthly for 2 years for drugs. These people tend to re-use drugs the day after the collection, as their body eliminates the drugs before next collection (next month). We were able to detect heroin in one case although results of urine and blood samples were negative.
In the other case, a low concentration of morphine was detected in the urine sample as well as in the hair matrix. These findings support the need for using hair as an alternative matrix for cases such as routine drugs analysis where adulteration of samples and false negative results could be eliminated.
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Table 54: The IA and LC-MS/MS results from same individual in urine, blood and hair
Case
#
Age Gender IA result Urine Blood Hair Hair
length (cm)
1 21-30 M OPI COD
MOR
COD MOR 6AM
ND MOR 6AM
4
2 21-30 M BUP
PRG
BUP PRG
BUP PRG
BUP PRG
3
3 21-30 M PRG PRG PRG ND 3
4 31-40 M BENZ 7-AC CLN
7-AC
CLN 7-AC
2
5 31-40 F MPB MPB MPB ND 7
6 21-30 M TRM
PRG
TRM O-DM
PRG GAB
TRM PRG GAB
TRM PRG
3
7 21-30 F OPI COD
MOR
COD MOR
COD MOR 6-AM
9
8 31-40 M NG NG NG MOR/6AM 4
9 31-40 M MDONE MDONE MDONE + 3
10 31-40 M BENZ
MPB PRG
NOR OX MPB PRG CAR
DIZ NOR MPB PRG CAR
PRG CAR
3
11 21-30 F NG NG NG TRM 10
12 21-30 M NG MOR NG MOR 4
NG: negative; OPI: opiate; COD: codeine; MOR: morphine; 6-AM: 6-acetylmorphine;
PRG: pregabalin; BUP: buprenorphine; BENZ: benzodiazepines; CLN: clonazepam; 7- AC: 7-aminoclonazepam; MPB: meprobamate; TRM: tramadol; O-DM: O-
desmethyltramadol; GAB: gabapentin; MDONE: methadone; NOR: nordiazepam; OX:
oxazepam; CAR: carisoprodol; DIZ: diazepam; +>LOQ; ND: not detected; M: male; F:
female
169 3.2.2.5 Hair Segmental Analysis
This study presents the segmental results of drugs in hair samples in six suspects.
The suspects were four women and two men ranging in age from 2.5 to 38 years. Young women, particularly in the age range of 21–38 years, predominated among these drug users. Three suspects had dyed or bleached hair, as assessed by the color difference along the hair shafts and discolouration of the wash solvent or extraction medium. The remaining four suspects had natural black hair color. Drug concentrations in the hair segments for each suspect are reported in Table 55.
The concentrations of FLU in the hair segments of a suspect ranged from 10.2 to 21.7 pg/mg ( 0.1), and those of 7AF ranged from 11.3 to 28.1 pg/mg ( 0.1). The results showed that the concentration of the 7-amino-metabolite was higher than that of its parent compound, which concurs with the results in previous publications (H. Kim et al., 2018;
A Negrusz et al., 2001; Xiang, Shen, & Drummer, 2015). The reason could be that the 7- amino-metabolite is more readily incorporated into hair than its parent drug, owing to the increased basicity of the 7-amino-metabolite.
Concentrations of 6-AM, MOR and COD in the hair segments ranged from 10.7 to 229.5, 61.5 to 531.3 and 103.1 to 511.3 pg/mg ( 0.1), respectively. The average ratio of 6-AM versus MOR in the proximal 3 cm segment was much lower than that in the 5–6 cm segments. This finding is similar to that of previous studies (M. Shen et al., 2013; Zhuo et al., 2020) showing a correlation between 6-AM and MOR in the proximal segment (0–3 cm) of current heroin users. These results can be explained by various environmental factors. After 4–5 months, the drugs present in hair may gradually be affected by repeated shampooing or be decomposed due to the effect of ultraviolet light or heat (M. Shen et al., 2013).
Interestingly, a postmortem, suspected accidental overdose case was received from the MOJ forensic medicine department. A 2.5-year-old female was found dead in the living room with patches of prescribed tramadol tablets that belong to her mother. As the mother claimed, her child accidentally swallowed a bunch of the prescribed tramadol pills and when the ambulance arrived, the child has already passed away. The overdose of tramadol (>1,000 ng/ml) in her blood sample was confirmed and reported as the cause of death.
While analyzing the child’s hair sample (case 3 in Table 55), we noticed a history of
170
tramadol exposure in the child. When reporting these findings, the mother admitted the fact that she used to administer tramadol tablets in her child’s milk to avoid hyperactivity of her child. The MOJ forensic drug chemistry was able to detect traces of tramadol in the feeding bottle as well. This resulted in charging the mother with child abuse.
171 Table 55: The concentrations of drugs in each hair segment of suspects (pg/mg) ( 0.1)
No. Age (years)
Gender Hair colour
Hair length
(cm)
Drug S1 S2 S3 S4 S5 S6
1 38 F Brown
(dyed)
4 COD
MOR 6-AM
103.1 ND ND
162.1 61.5 47.3
511.3 102.6 92.4
/ / /
2 23 M Black 7 FLU 10.2 11.3 15.2 16.1 13.8 21.7
7AF 11.3 10.8 17.4 17.3 19.4 28.1
3 2.5 F Black 4 TRM 964.8 345.8 639.1 + / /
4 28 F 0–3 cm:
black; 3–
6 cm:
brown (dyed)
6 CLN 10.3 11.8 21.5 17.4 39.4 /
7-AC 176.2 97.3 75.8 84.3 209.1 /
5 21 F Black 14 MOR ND ND 131.7 219.2 327.2 531.3
6-AM ND 10.7 72.3 109.7 213.6 229.5
6 32 M Brown
(dyed)
3 PRG ND 765.3 + / / /
TRM: tramadol; COD: codeine; MOR: morphine; 6-AM: 6-acetylmorphine; PRG:
pregabalin; CLN: clonazepam; 7-AC: 7-aminoclonazepam; FLU: flunitrazepam; 7AF: 7 - aminoflunitrazepam; S1: 0-1 cm; S2: 1-2cm; S3: 2-3cm; S4: 3-4cm; S5: 4-5cm; S6: 5- 6cm; /: No hair; +: > LOQ; ND: not detected; M: male; F: female.
172