Forensic Toxicology and Drug Analysis 65010

Forensic Toxicology and Drug Analysis 65010 – Experiment 2 2020 Page 1 of 9
Forensic Toxicology
and Drug Analysis
(65010)
Experiment 2
2020
Forensic Toxicology and Drug Analysis 65010 – Experiment 2 2020 Page 2 of 9
DETECTION OF OPIATES IN BIOLOGICAL MATRICES
MATERIALS
Chemicals
Sodium acetate
Sodium hydroxide (NaOH)
Sodium carbonate
Sodium hydrogen carbonate
Sodium di-hydrogen phosphate
di-Sodium hydrogen phosphate
Sodium sulfate anhydrous
Potassium iodide
Iodine
Iodoplatinate reagent
33% Ammonia solution
Acetic acid glacial
Hydrochloric acid (HCl)
Deionised water
Hexane
Methanol
Isopropanol
Acetonitrile (ACN)
Dichloromethane (DCM)
Triethylamine
Acetic anhydride
Bistrimethylsilyltrifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)
-Glucuronidase (Type H-3, from Helix pomatia)
Standards
Morphine
Morphine-D3
Codeine
Codeine-D3
Other materials
Poppy seeds / poppy rolls
Blank urine (student volunteers)
Urine of poppy seeds eaters (student volunteers)
Urine of Panadeine Forte takers (provided)
SPE cartridges (Clean Screen CSDAU 130 mg/3 cc, UCT)
TLC plates (on aluminum or glass sheets)
Pasteur pipettes
Cotton wool
10mL screw top test tubes
GC-MS; GC-MS vials with inserts
Heating blocks
N2 gas drying manifold
Centrifuge
Glass rod
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EXPERIMENT 2A – CHEMICAL INVESTIGATION OF OPIATES IN POPPY SEEDS
(Week 1)
Aims
The aims of this experiment are:
a) To extract and identify opiates and other alkaloids present in poppy seeds;
b) To investigate whether the opiates in poppy seeds are mainly surface bound or throughout
the seeds; and
c) To find out whether baking process alters the composition of alkaloids in poppy seeds.
Materials
Methanol
Poppy seeds raw (PR)
Poppy seeds baked at 150ºC for 30min (PB)
Morphine spotting standard (500 g/mL)
Codeine spotting standard (500 g/mL)
Glass rods
Iodoplatinate reagent
TLC tanks and TLC plates
Opiate Analysis on TLC
1. Weigh 200 mg PR seeds into a test tube. Add 1 mL methanol and cap.
2. Heat the tube to 50 °C for 10 min.
3. Transfer the methanol extract into a clean tube.
4. Reduce solvent volume to about 100 µL by evaporation under a gentle stream of nitrogen at
50 °C.
5. Apply the fraction, morphine and codeine standards (ask demonstrator for the standards) on
a TLC plate.
Tip: view under UV light for adequacy of quantity applied on TLC first before doing step 6.
Reapply the fraction until the spots are clearly visible.
6. Develop this plate in 10 mL of dichloromethane : methanol (9:1) containing 50 µL NH4OH
(30% ammonia solution). Note: NH4OH (50 µL) needs to be mixed with 1 mL of
methanol first before adding 9 mL of dichloromethane.
7. View TLC plates under short wave ultraviolet light (254 nm) for UV absorbing spots.
8. Also view under long wave UV and note any strongly fluorescent spots.
9. Spray plates lightly with iodoplatinate reagent.
10. Leave plates in a fumehood for 1-2 minutes to dry.

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Opiate Analysis on GC-MS
Raw vs. Baked
12. Weigh 200 mg raw poppy seeds (PR) and 200 mg baked poppy seeds (PB) into test tubes
with caps.
13. Add 1 mL methanol into each tube, and heat the tube to 50 ºC for 15 min.
14. Transfer the methanol extracts into GC vials for GC-MS analysis.
Note: Add your group number before the poppy seed identification code, e.g. W2PR, W3PB etc.
You need to analyse the GC-MS data once available to find out what alkaloids are in the samples.
Surface vs. Inner Opiates
15. Label 4 test tubes as PR1, PR2, PB1 and PB2.
16. Weigh 200 mg of PR seeds into PR1, PR2.
17. Weigh 200 mg of PB seeds into PB1, PB2.
18. Add 1 mL of methanol to all test tubes and cap.
19. Extract the poppy seeds for 10 min at room temperature.
20. Transfer the methanol layer (F1) into a GC-MS vial and cap (following the naming system
described below).
21. Crush poppy seeds in test tubes labelled PR2 and PB2.
22. Add 1 mL of methanol to all test tubes including the non-crushed PR1 and PB1 tubes and
cap.
23. Extract the poppy seeds for another 10 min.
24. Transfer the methanol layer (F2) into another GC-MS vial (following the naming system
described below). The crushed fractions may need filtration through syringe microfilters.
Use inserts if the filtrate volume is too low for the GC-MS vials.
25. Submit all fractions for GC-MS analysis (you should have a total of 8 samples for analysis).
Note:
The following naming system is to be followed. The group number is followed by poppy seeds
number and then fraction number. For example, W2PB1F2 refers to group W2 baked seed No. 1
fraction 2.
GC-MS Analysis
The extracts are analysed by GC-MS in SCAN mode with splitless injection. Injection volume is 1
L. Codeine and morphine standards (5 g/mL) are injected as well. Concentrations of morphine
and codeine are estimated by using the peak areas.
REMINDER!
One student volunteer from each group is to eat poppy seeds containing food and to collect urine
specimens for use at the following week. Two urine specimens are needed: one before ingestion of
poppy seeds and one after ingestion (8 hr after ingestion).
Please talk to your demonstrator and laboratory teaching staff regarding the urine containers, poppy
seed containing food, and urine collection issues.
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EXPERIMENT 2B – DETECTION OF CODEINE AND ITS METABOLITES IN URINE
(Week 1-2)
Heroin is an illicit drug of abuse. Abuse of the licit drug codeine sometimes also takes place among
heroin addicts. Distinguishing heroin abuse from codeine use can be difficult. Firstly, codeine is a
common contaminant in heroin during heroin synthesis. Secondly, both heroin and codeine can be
metabolised to morphine. This experiment is set to measure opiates in urine specimens collected
from people ingesting codeine as a pain killer.
The -glucuronide-bound opiates and the metabolites are to be hydrolysed by an acid-catalysed
method. Opiates from urine are to be extracted into a mixture of dichloromethane and isopropanol
at alkaline pH. The compounds are then derivatised with acetic anhydride and the derivatives
analysed by GC-MS in SCAN mode. Deuterated morphine and codeine are used as internal
standards.
Materials
Mixed morphine and codeine standards at 5 µg/mL
Mixed morphine-D3 and Codeine-D3 internal standards at 5 µg/mL
Acetic anhydride
Triethylamine
Dichloromethane/isopropanol (9:1)
1.5 M bicarbonate buffer pH 9.5
4 M HCl
4 M NaOH
Urine before codeine administration (CT0, provided)
Urine after 10 hr of codeine administration (CT10, provided)
Sample Preparation and Hydrolysis (Week 1)
Warning!
Do not use excessive force when capping glass tubes! Glass tubes might snap and cause injury!
1. Place labelled glass sample tubes (total of 6) in a sample rack for 4 calibrators (calib 1, 2, 3,
4), 1 blank (CT0), and 1 urine specimen after 10 hr of codeine administration (CT10).
2. Add 50 L IS to all sample tubes.
3. Add 40 µL, 60 µL, 100 µL and 200 µL aliquots of mixed morphine/codeine standards to
calib 1, calib 2, calib 3, and calib 4 tubes respectively.
4. Dispense 1 mL CT0 urine to all 4 calibrators and the blank.
5. Dispense 1 mL CT10 urine to the CT10 sample tube.
6. Add 500 L of 4 M HCl to all tubes.
7. Cap all test tubes and heat them at 100 °C for 20 min. Allow to cool to room temperature.
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Liquid-Liquid Extraction (Week 2)
8. Add 3 mL hexane to each tube and cap. Make sure the lid is not leaking
9. Shake (or vortex) tubes so that the two phases are mixed thoroughly for 15 seconds.
10. Aspirate the upper organic phase to waste.
11. Add 0.5 mL of 4 M NaOH and 2 mL of 1.5 M carbonate buffer pH 9.0 to all tubes.
12. Add 4 mL of DCM/isopropanol (9:1) to all tubes and cap. Make sure the lid is not leaking.
13. Shake (or vortex) tubes so that the two phases are mixed thoroughly for 15 seconds.
14. Label a new set of small test tubes. Place a Pasteur pipette which is filled with a small
amount of cotton wool and a layer of sodium sulfate anhydrous (~1 cm thick) into each test
tube.
15. Remove the top aqueous layer to waste and transfer the bottom organic layer using Pasteur
pipettes into the new set of tubes via the above prepared Pasteur pipettes.
16. Remove the Pasteur pipette and evaporate the extract to dryness under a gentle stream of
nitrogen at 55 °C.
Derivatisation (Week 2)
Warning! The following steps involve handling of acetic anhydride, a toxic derivatising agent.
Acetic anhydride needs to be handled in a fume hood. Any pipette or pipette tips used need to be
immersed into water for 5 min (to deactivate the reagent) before disposal.
17. Add 100 µL acetic anhydride and 50 µL triethylamine. Cap all tubes and heat them at 60 °C
for 30 min.
18. Evaporate to dryness under a gentle stream of nitrogen at 50 °C.
19. Reconstitute in 200 µL ethyl acetate.
20. Label GC-MS vials and place 250 µL inserts into the vials.
21. Transfer the derivatives in ethyl acetate into the above prepared GC-MS vials.
GC-MS (Week 2)
22. Inject 1 µL to the GC-MS in SIM mode (also collect one full scan spectrum of calib 4).
23. Construct calibration curves by using the base peak area ratios of target compounds to the
deuterated internal standard counterparts based on the SIM method.
24. Concentrations of morphine and codeine are calculated using the above standard curves.
Forensic Toxicology and Drug Analysis 65010 – Experiment 2 2020 Page 7 of 9
EXPERIMENT 2C – DETECTION OF OPIATES IN URINE AFTER INGESTION OF
POPPY SEEDS (Week 2-3)
Introduction
The presence of both morphine and codeine in most poppy seeds has significant impact on
interpretation of positive opiates results in urine drug testing. In November 1998, the US
government raised the cut-off concentration for morphine from 300 ng/mL to 2000 ng/mL to try to
eliminate the defence that the positive results are a result of ingestion of foods containing poppy
seeds. In Australia, the positive reporting level for morphine is also at 2000 ng/mL, while levels
between 300 ng/mL and 2000 ng/mL are reported as trace amounts. This experiment is aimed at
investigating the level of opiates in the urine of poppy seeds eaters and to raise the awareness of the
impact of foods on interpretation of drug testing results.
In this experiment, a volunteer from each group is to eat food containing poppy seeds and to collect
urine specimens for analysis of opiates. Two urine specimens are needed: one before ingestion of
poppy seeds and one after ingestion (8 hr). Urine samples are hydrolysed with -glucuronidase and
the released free opiates are extracted using a SPE method. The compounds are then derivatised
with BSTFA containing 1% TMCS. The trimethylsilyl (TMS) derivatives thus obtained are
analysed by GC-MS in Selected Ion Monitoring (SIM) mode. Deuterated morphine and codeine are
used as internal standards.
Materials
Mixed morphine and codeine standards at 5 g/mL
Mixed morphine-D3 and Codeine-D3 internal standards at 5 g/mL
Hydrolysis enzyme buffer solution (β-glucuronidase Type H-3, from Helix pomatia, diluted to 5000
units/mL in 1 M acetate buffer pH 4.5)
Urine before poppy seeds ingestion (PT0)
Urine after 8 hr of poppy seeds ingestion (PT8)
SPE cartridges (Clean Screen CSDAU 130 mg/3 cc, UCT)
(mixed mode cartridges which contain both C8 silica and strong cation ion exchange resin)
SPE Solvents
Methanol
0.1 M phosphate Buffer pH 6.0
2% acetic acid in water
2% NH4OH (30% ammonia solution) in ethylacetate (freshly prepared and thoroughly mixed)
Sample Preparation and Hydrolysis (Week 2)
Warning!
Do not use excessive force when capping glass tubes! Glass tubes might snap and cause injury!
1. Place labelled sample tubes (total of 6) in a sample rack for 4 calibrators (calib 1, 2, 3, 4), 1
blank (PT0), and 1 urine specimen after poppy seed ingestion (PT8).
2. Add 50 L IS to all sample tubes (add to the bottom of the tubes instead of to the wall of the
tubes).
Forensic Toxicology and Drug Analysis 65010 – Experiment 2 2020 Page 8 of 9
3. Add 200 L enzyme buffer solution to all sample tubes.
4. Add 40 µL, 60 µL, 100 and 200 µL aliquots of mixed morphine/codeine standards to calib
1, calib 2, calib 3 and calib 4 tubes respectively (into the liquid layers).
5. Dispense 1 mL PT0 urine to all 4 calibrators and the blank.
6. Dispense 1 mL PT8 urine to the PT8 sample tube.
7. Cap all test tubes and leave them in a 55 °C block heater overnight.
Solid-Phase Extraction (SPE) (Week 3)
8. Add 2 mL 0.1 M phosphate buffer pH 6.0 to all hydrolysed urines.
9. Label 6 SPE tubes according to the above prepared urine specimens.
10. Load SPE cartridges onto the SPE manifold.
11. Perform SPE according to the following protocols.
Note: If the elution of solvent by gravity is too slow, say less than 0.5 mL/ min, a small vacuum can
be applied to the unit to increase the elution speed. Do not exceed an elution speed of 1mL/min,
especially at the “Loading” and the “Eluting” steps.
Conditioning: Add 2 mL methanol
Add 2 mL 0.1 M phosphate buffer pH 6.0
(Do not let the solvent run dry before proceeding to the next “loading” step!)
Loading: Load urine sample to the corresponding cartridges (by gravity)

Apply vacuum for 5 min (at around -35 kpa)
Add 3 mL methanol
Drying: Apply vacuum for 5 min

Clean SPE tanks with methanol through the top valves.
12. Add roughly 100 mg sodium sulfate anhydrous into the collected fractions and vortex the
tubes to mix.
13. Carefully pour the solvent into a clean set of labelled test tubes, leaving the drying salts in
the original tubes.
14. Evaporate the eluents to dryness under a gentle stream of nitrogen at 50 °C.
Forensic Toxicology and Drug Analysis 65010 – Experiment 2 2020 Page 9 of 9
Derivatisation (Week 3)
Warning! The following steps involve handling of BSTFA, a very toxic derivatising agent. BSTFA
needs to be handled in a fume hood. Any pipette or pipette tips used needs to be immersed into
water for 5 min (to deactivate BSTFA) before disposal.
15. Add 150 µL acetonitrile and 50 µL BSTFA with 1% TMCS into each tube and cap.
16. Heat the tubes at 75 °C for 30 min.
17. Place 250 µL inserts into GC-MS vials and label the vials.
18. Cool down the samples to room temperature and then transfer the contents into the above
prepared GC-MS vials in a fume hood. (Remember to deactivate any pipettes or tips!)
GC-MS (Week 3)
19. Inject 1 µL to the GC-MS in SIM mode.
20. Construct calibration curves by using the base peak area ratios of target compounds to the
deuterated internal standard counterparts.
21. Concentrations of morphine and codeine are calculated using the above standard curves.
POST-WORK (20 marks)
The report for this practical should be written in the following format.
1. Title page should include title, name, student ID, dates when the practical was carried out.
2. Abstract (<300 words)
3. Introduction
4. Experimental and Methods (be brief, do not reproduce manual)
5. Results
6. Discussion (be critical)
7. Answers to additional questions below
8. Conclusion
9. References
Discussion Questions
1. Suggest the enzymatic pathways that are involved in the metabolism of heroin and codeine.
2. When can you be certain that heroin is the drug of abuse based on urinary opiate testing
results?
3. Compare the advantages and disadvantages of the liquid-liquid extraction method vs. the
solid phase extraction method.
4. Compare the advantages and disadvantages of the two derivatisation methods employed in
this practical.
5. Propose the major MS fragmentation pathways for the acetylated morphine and acetylated
codeine obtained in the second experiment of the practical (chemical structures required).
6. Comment on why some drug test laboratories increased the morphine reporting level from
300 ng/mL to 2000 ng/mL.

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