Chapter 2: Methods
2.4 Specimen Extraction From Urine, Blood and Hair Samples
63
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Table 11: Preparation of working standard for BZDs extraction from urine and blood matrix
No Compound Name Supplier & conc Working std (10 µg/ml)
Working std (1 µg/ml)
Working std (100 ng/ml) 1 7-Aminoclonazepam Cerilliant 1 mg/ml
100 µl of each 1 mg/ml analyte &
200 µl of chlordiazepoxide
to 10 ml of Methanol (1.7 ml of std +
8.3 ml of Methanol = 10
ml)
1 ml of 10 µg/ml stock in
9 ml of methanol
1 ml of 1 µg/ml stock in
9 ml of methanol 2 Chlordiazepoxide Cerilliant 500µg/ml
3 Zolpidem Cerilliant 1 mg/ml
4 Midazolam Cerilliant 1 mg/ml
5 Bromazepam Cerilliant 1 mg/ml
6 Lorazepam Cerilliant 1 mg/ml
7 Oxezepam Cerilliant 1 mg/ml
8 Clonazepam Cerilliant 1 mg/ml 9 Nordiazepam Cerilliant 1 mg/ml
10 Clobazam Cerilliant 1 mg/ml
11 Phenazepam Cerilliant 1 mg/ml 12 Flunitrazepam Cerilliant 1 mg/ml 13 Estazolam Cerilliant 1 mg/ml 14 Temazepam Cerilliant 1 mg/ml 15 Triazolam Cerilliant 1 mg/ml 16 Alprazolam Cerilliant 1 mg/ml
17 Diazepam Cerilliant 1 mg/ml
Table 12: Preparation of internal standard for BZDs extraction from urine and blood matrix
No Compound Name Supplier & conc Working std (1µg/ml)
1 Phenazepam-D4 Cerilliant 100 µg/ml 100 µl of each 100 µg/ml analyte to 10 ml of Methanol (1.2 ml of std + 8.8 ml of
Methanol = 10 ml)
2 Triazolam-D4 Cerilliant 100 µg/ml
3 Midazolam-D4 Cerilliant 100 µg/ml
4 Lorazepam-D4 Cerilliant 100 µg/ml
5 Flunitrazepam-D7 Cerilliant 100 µg/ml
6 Clonazepam-D4 Cerilliant 100 µg/ml
7 Alprazolam-D5 Cerilliant 100 µg/ml
8 Temazepam-D5 Cerilliant 100 µg/ml
9 Oxazepam-D5 Cerilliant 100 µg/ml
10 7-Aminoflunitrazepam-D7 Cerilliant 100 µg/ml
11 Diazepam-D5 Cerilliant 100 µg/ml
12 7-Aminoclonazepam-D4 Cerilliant 100 µg/ml
65 Table 13: Preparation of calibrators for generation of calibration curve for BZDs method from urine and blood matrix
Level Blank Urine/Blood Aliquot Spike from stock (100 ng/ml) Final Conc.
1 400 µl 5 µl 5 ng/ml
2 400 µl 10 µl 10 ng/ml
3 400 µl 20 µl 25 ng/ml
4 400 µl 50 µl 50 ng/ml
5 400 µl 100 µl 100 ng/ml
Aliquot Spike from stock (1 µg/ml)
6 400 µl 100 µl 250 ng/ml
7 400 µl 200 µl 500 ng/ml
Table 14: Preparation of working standard for BZDs extraction from hair matrix
No Compound Name Supplier & conc Working std (20 µg/ml) STOCK-A
Working std (1µg/ml) STOCK-B 1. 7-Aminoclonazepam Cerilliant 1 mg/ml 200 µl of each 1 mg/ml &
400 µl of chlordiazepoxide to 10 ml of Methanol.
(3.6 ml of std in 6.4 ml Methanol)
0.5 ml of 20 µg/ml (STOCK-A) in 9.5 ml of methanol 2. Chlordiazepoxide Cerilliant 500µg/ml
3. Zolpidem Cerilliant 1 mg/ml
4. Midazolam Cerilliant 1 mg/ml 5. Bromazepam Cerilliant 1 mg/ml 6. Lorazepam Cerilliant 1 mg/ml
7. Oxezepam Cerilliant 1 mg/ml
8. Clonazepam Cerilliant 1 mg/ml 9. Noradiazepam Cerilliant 1 mg/ml 10. Clobazam Cerilliant 1 mg/ml 11. Phenazepam Cerilliant 1 mg/ml 12. Flunitrazepam Cerilliant 1 mg/ml 13. Estazolam Cerilliant 1 mg/ml 14. Temazepam Cerilliant 1 mg/ml 15. Triazolam Cerilliant 1 mg/ml 16. Alprazolam Cerilliant 1 mg/ml 17. Diazepam Cerilliant 1 mg/ml
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Table 15: Preparation of internal standard for BZDs extraction from hair matrix
No Compound Name Supplier & conc Working std (2 µg/ml)
1 7-Aminoclonazepam- D4 Cerilliant 100 µg/ml
200 µl of each 100 µg/ml analyte to 10 ml of Methanol (2.4 ml of std + 7.6 ml of
Methanol = 10 ml)
2 Clonazepam-D4 Cerilliant 100 µg/ml
3 Diazepam-D5 Cerilliant 100 µg/ml
4 Lorazepam-D4 Cerilliant 100 µg/ml
5 Oxazepam-D5 Cerilliant 100 µg/ml
6 Phenazepam-D4 Cerilliant 100 µg/ml
7 Temazepam-D5 Cerilliant 100 µg/ml
8 Triazolam-D4 Cerilliant 100 µg/ml
9 Midazolam-D4 Cerilliant 100 µg/ml
10 Flunitrazepam-
D7
Cerilliant 100 µg/ml
11 Alprazolam-D5 Cerilliant 100 µg/ml
12 7-Aminoflunitrazepam-D7 Cerilliant 100 µg/ml
Table 16: Preparation of calibrators for generation of calibration curve for BZDs method from hair matrix
(STOCK-B) Concentration
Volume pipetted from (STOCK-B)
Weight of Blank Hair
sample
Concentration (ng/mg)
Concentration (pg/mg) (STOCK-B) (1000
ng/ml)
1 μl (1ng) 100 mg 0.01 ng/mg 10 pg/mg
5 μl (5ng) 100 mg 0.05 ng/mg 50 pg/mg
10 μl (5ng) 100 mg 0.1 ng/mg 100 pg/mg
25 μl (25 ng) 100 mg 0.25 ng/mg 250 pg/mg
50 μl (50ng) 100 mg 0.50 ng/mg 500 pg/mg
100 μl (100 ng) 100 mg 1 ng/mg 1000 pg/mg
67 The opiate method for urine and blood matrix involved the preparation of two working calibrator standards (20 μg/mL and 1 μg/mL) (Table 17) for the preparation of selected calibration points (7 levels) as shown in Table 19. Internal standards (2 µg/mL) preparation steps for the opiate extraction method is explained in Table 18. The
preparation of OPI calibrators can be performed by the addition of a listed volume of blank urine or blood in glass disposable tubes and spiking the various concentrations as per Table 16, followed by the addition of 20 µl of IS in each tube. Treat the resultant fluid as a sample and follow the sample preparation step.
For hair matrix, the preparation of six calibration levels involves the use of 1 μg/mL working standards as shown in Table 20. The internal standard concentrations used for the hair sample were the same as those used for the urine and blood matrix (2 µg/mL). The preparation of OPI calibrators can be performed by the addition of 100 mg of blank hair in a glass disposable tube and spiking the various concentrations followed by the addition of 20 µl of IS in each tube. Treat the resultant fluid as a sample and then follow the sample preparation step.
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Table 17: Preparation of working standard for opiate extraction from urine/blood/hair matrix
No Compound Name Supplier & conc Working std (20 µg/ml) STOCK-A
Working std (1µg/ml) STOCK-B
1 Morphine Cerilliant 1 mg/ml
200 µl of each 1 mg/ml analyte each to 10 ml of
Methanol
(3.4 ml of std + 6.6 ml of Methanol =
10 ml)
0.5 ml of 20 µg/ml (STOCK-A) in
9.5 ml of methanol
2 Oxymorphone Cerilliant 1mg/ml
3 Hydromorphone Cerilliant 1 mg/ml
4 Naloxone Cerilliant 1 mg/ml
5 O-Desmethyltramadol Cerilliant 1 mg/ml 6 6-Acetylmorphine Cerilliant 1 mg/ml
7 Codeine Cerilliant 1 mg/ml
8 Hydrocodone Cerilliant 1 mg/ml
9 Norfentanyl Cerilliant 1 mg/ml
10 Tramadol Cerilliant 1 mg/ml
11 Normeperidine (Norpethidine) Cerilliant 1 mg/ml 12 Meperidine (Pethidine) Cerilliant 1 mg/ml
13 Heroin Cerilliant 1 mg/ml
14 Dextromethorphan (Methorphan) Cerilliant 1 mg/ml
15 Fentanyl Cerilliant 1 mg/ml
16 Buprenorphine Cerilliant 1 mg/ml
17 Methadone Cerilliant 1 mg/ml
69 Table 18: Preparation of internal standard for opiate extraction from urine/blood/hair matrix
Drug Supplier & conc Working Internal Std.
(2 µg/mL) Morphine-D3 Cerilliant 100 µg/mL
200 µl of each 100 µg/ml analyte to 10 mL of Methanol (2 ml of std + 8 ml of Methanol = 10 ml) Oxymorphone-D3 Cerilliant 100 µg/mL
Hydromorphone-D3 Cerilliant 100 µg/mL Codeine-D6 Cerilliant 100 µg/mL Norfentanyl-D5 Cerilliant 100 µg/mL Normeperidine-D4 Cerilliant 100 µg/mL Meperidine-D4 Cerilliant 100 µg/mL Fentanyl-D5 Cerilliant 100 µg/mL Methadone-D3 Cerilliant 100 µg/mL Hydrocodone-D3 Cerilliant 100 µg/mL
Table 19: Preparation of calibrators for generation of calibration curve for opiate method from urine and blood matrix
Aliquot of Working Stock #B (µL) Blank Urine/Blood (µL)
Final Conc (ng/mL)
20 1970 10
50 1950 25
100 1900 50
Aliquot of Working Stock #A (µL)
10 1990 100
25 1975 250
50 1950 500
100 1900 1000
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Table 20: Preparation of calibrators for generation of calibration curve for opiate method from hair matrix
(STOCK-B) Concentration
Volume pipetted from (STOCK-B)
Weight of Blank Hair sample
Concentration (ng/mg)
Concentration (pg/mg) (STOCK-B) (1000
ng/ml)
1 μl (1ng) 100 mg 0.01 ng/mg 10 pg/mg
5 μl (5ng) 100 mg 0.05 ng/mg 50 pg/mg
10 μl (5ng) 100 mg 0.1 ng/mg 100 pg/mg
25 μl (25 ng) 100 mg 0.25 ng/mg 250 pg/mg
50 μl (50ng) 100 mg 0.50 ng/mg 500 pg/mg
100 μl (100 ng) 100 mg 1 ng/mg 1000 pg/mg
71 The CAD method for urine and blood matrix involved the preparation of 10 µg/ml working calibrator standards (Table 21) for the preparation of selected calibration points as shown in Table 23. The internal standard (10 µg/ml) preparation steps for the CAD group is explained in Table 22. The preparation of CAD calibrators can be performed by the addition of 100 µl blank urine or blood in glass disposable tubes and spiking the various concentrations as per Table 23, followed by the addition of 20 µl of IS in each tube; treat the resultant fluids as samples and then follow the sample preparation step.
For the hair matrix, the CAD method involved preparation of two working calibrator standards which are 20 µg/ml and 1 µg/ml (Table 24) for the preparation of selected calibration points as shown in Table 26. Internal standard (2 µg/ml) preparation steps for the CAD group is explained in Table 25. The preparation of CAD calibrators can be performed by the addition of a100 mg blank hair sample in a glass disposable tube and spiking the various concentrations as per Table 26, followed by the addition of 20 µl of IS in each tube; treat the resultant fluids as samples and then follow the sample preparation step.
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Table 21: Preparation of working standard for CAD extraction from urine and blood matrix
No Compound Name Supplier & conc Working std (10 µg/ml)
1 Acetaminophen Cerilliant 1 mg/ml
100 µl of each 1 mg/ml analyte &
200 µl of each 500 µg/ml analyte to 10 ml of Methanol
(900 µl of std + 9.1 ml of Methanol
= 10 ml)
2 Baclofen Cerilliant 500µg/ml
3 Carbamazepine Cerilliant 1 mg/ml
4 Carisoprodal Cerilliant 1 mg/ml
5 Gabapentin Cerilliant 1 mg/ml
6 Levetiracetum Cerilliant 1 mg/ml
7 Meprobamate Cerilliant 1 mg/ml
8 Pregablin Cerilliant 1 mg/ml
Table 22: Preparation of internal standard for CAD extraction from urine and blood matrix
No Compound Name Supplier & conc Internal std (10 µg/ml) 1 Acetaminophem-D4 Cerilliant 100 µg/ml
500 µl of each 100 µg/ml analyte to 5 ml of Methanol
(4 ml of std + 1 ml of Methanol = 5 ml)
2 Meprobamate-D7 Cerilliant 100 µg/ml 3 Carbamazepine-D10 Cerilliant 100 µg/ml 4 Carisoprodol-D7 Cerilliant 100 µg/ml
5 Gabapentin-D4 Cerilliant 100 µg/ml
6 Meprobamate-D7 Cerilliant 100 µg/ml
7 Pregabalin-D6 Cerilliant 100 µg/ml
8 Levetiracetum-D6 Cerilliant 100 µg/ml
Table 23: Preparation of calibrators for generation of calibration curve for CAD method from urine and blood matrix
Level Blank Urine/Plasma Spike from stock (10 µg/ml)
Final Conc.
1 100 µl 5 µl 0.5 µg/ml
2 100 µl 10 µl 1.0 µg/ml
3 100 µl 20 µl 2.0 µg/ml
4 100 µl 50 µl 5.0 µg/ml
5 100 µl 100 µl 10.0 µg/ml
73 Table 24: Preparation of working standard for CAD extraction from hair matrix
No Compound Name Supplier & conc Working std (20 µg/ml) STOCK-A
Working std (1µg/ml) STOCK-B 1. Acetaminophen
(Paracetemol)
Cerilliant 1 mg/ml 200 µl of each 1 mg/ml each analyte to 10 ml of
Methanol
(1.6 ml of std +8.4 ml Methanol = 10 ml)
0.5 ml of 20 µg/ml (STOCK-A) in 9.5 ml of methanol 2. Pregabalin Cerilliant 1 mg/ml
3. Gabapentin Cerilliant 1 mg/ml
4. Baclofen Cerilliant 1 mg/ml
5. Levetiracetam Cerilliant 1 mg/ml 6. Meprobamate Cerilliant 1 mg/ml 7. Carbamazepine Cerilliant 1 mg/ml 8. Carisoprodol Cerilliant 1 mg/ml
Table 25: Preparation of internal standard for CAD extraction from hair matrix
No Compound Name Supplier & conc Working std (2 µg/ml)
STOCK -IS
1 Acetaminophem-D4 Cerilliant 100 µg/ml
200 µl of each 100 µg/ml analyte to 10 ml of Methanol (1.6 ml of std + 8.4 ml of
Methanol = 10 ml)
2 Meprobamate-D7 Cerilliant 100 µg/ml
3 Carbamazepine-D10 Cerilliant 100 µg/ml
4 Carisoprodol-D7 Cerilliant 100 µg/ml
5 Gabapentin-D4 Cerilliant 100 µg/ml
6 Meprobamate-D7 Cerilliant 100 µg/ml
7 Pregabalin-D6 Cerilliant 100 µg/ml
8 Levetiracetum-D6 Cerilliant 100 µg/ml
Table 26: Preparation of calibrators for generation of calibration curve for CAD method from hair matrix
(STOCK-B) Concentration
Volume pipetted from (STOCK-B)
Weight of Blank Hair sample
Concentration Concentration
(STOCK-B) (1000 ng/ml)
1 μl 100 mg 0.01 ng/mg 10 pg/mg
5 μl 100 mg 0.05 ng/mg 50 pg/mg
10 μl 100 mg 0.1 ng/mg 100 pg/mg
25 μl 100 mg 0.25 ng/mg 250 pg/mg
50 μl 100 mg 0.50 ng/mg 500 pg/mg
100 μl 100 mg 1 ng/mg 1000 pg/mg
The BZDs extraction process is summarized in Figure 16. The sample preparation in BZDs involves the transfer of 400 μL of case-specimens to disposable, round-glass
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tubes with the addition of 20 μL of 1 μg/mL working internal standard solution to all tubes, followed by 2 mL of 100 mM phosphate buffer to all tubes; and then vortexing all the samples. SPE is the technique used in BZDs extraction. The conditioning step
involves the use of methanol and phosphate buffer. After loading the samples, the wash and dry step involves the use of 3 ml 5% (v/v) acetonitrile in 100 mM phosphate buffer along with applying high pressure followed by the use of a hexane chemical; and end by the reuse of high pressure. The elution part consists of the use of 3 ml of benzodiazepine elution solvent (39 ml ethyl acetate: 1 ml ammonium hydroxide). The samples would then evaporate to dryness and were reconstituted with 100 µL of 50:50 ACN: DI H2O before sending it to LC-MS/MS analysis.
Figure 16: Schematic diagram of BZDs extraction for urine, blood or hair samples (all the devices/ instrument shown are located in MOL-FML)
Figure 17 represents the procedure for opiate extraction. The sample preparations in opiates involve the transfer of 400 μL of case-specimens to disposable, round-glass tubes with the addition of 20 μL of 1 μg/mL working internal standard solution to all
75 tubes, followed by 1 mL 100 mM phosphate buffer to all tubes and then vortexing all the samples. SPE is the technique used in OPI extraction. The conditioning step involves the use of methanol and phosphate buffer. After loading the samples, the wash and dry step involves the use of DI H2O, 100 mM sodium acetate buffer at pH 4.50 and methanol followed by the application of high pressure. The elution part consists of the use of base elution solvent (methylene chloride: isopropyl alcohol: ammonium hydroxide in a ratio of 77:20:3). The samples were then evaporated to dryness and reconstituted with 100 µL of 50:50 ACN: DI H2O before sending it to LC-MS/MS analysis.
Figure 17: Schematic diagram of OPI extraction for urine, blood or hair samples (all the devices/ instrument shown are located in MOL-FML)
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The CAD extraction procedure for urine, blood, and hair is illustrated in Figures 18-20. The LLE protocol was performed in the CAD extraction. A volume of 100 µl was taken from each specimen, calibrators, and controls. The next step involved addition of the IS into disposable tubes followed by the use of sodium phosphate buffer solution.
Cold acetonitrile was then added, and vortexed properly followed by centrifuging for 5 min at 4,000 rpm. The upper organic layer was transferred in a vial and sent for LC- MS/MS analysis.
Figure 18: Schematic diagram of CAD extraction for urine samples (all the devices/
instrument shown are located in MOL-FML)
77 Figure 19: Schematic diagram of CAD extraction for urine samples (all the devices/
instrument shown are located in MOL-FML)
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Figure 20: Schematic diagram of CAD extraction for hair samples (all the devices/
instrument shown are located in MOL-FML)