Mystery of the missing heroin...

Scenario:

“Stop lying! You better start spitting out the truth. Where did you hide that stash of heroin? ANSWER ME.”

Agent Lau demanded with a fierce look in her eyes.

“I swear to god! I am innocent! What are you talking about? What… Heroin…? Who in the world will have the audacity to smuggle illegal drugs in spite of the harsh punishment stipulated by the government?!”

The suspect responded calmly.

Agent Lau whip out a transparent evidence bag and threw it onto the interrogation desk. She leaned forward and stared menacingly into the suspect eyes.

“How do you explain this then? Fine white powder is found at a corner of the kitchen of your house. What powder is that and how did it get there?”

“It.. It’s Talcum powder. It must have been accidentally scattered onto the ground when I am applying them on my body after shower. It’s just some household litter that is left on the floor. What is the big fuss about it?”

The suspect replied hesitantly while averting the interrogator’s gaze.

Agent Lau knew that the suspect must be hiding something.

They have been observing the suspect for quite some time and they noticed that quite a number of suspicious persons have been visiting the suspect’s apartment every now and then. Those people usually enter the apartment for only a short amount of time and leaves hurriedly after around 5 minutes. Before Agent Lau can continue her train of thoughts, her colleague, Agent Chen entered.

“So the suspect still refuses to speak the truth I suppose?”

Agent Lau nodded.

“He is one sly drug dealer.. Still trying to cover up the accidental evidence he left at his apartment. That sly fox still dares to claim that the powder is actually Talcum powder. Guess the only way to make him admit his crimes is to prove that the powder found is indeed Heroin. Send this to the forensics to analyze it.”

Agent Chen nodded and proceeded to her next destination, Ngee Ann Forensic Department.

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Heroin:

Structure of heroin (right)

3D view of heroin (wiki)

• Medicine form: Diacetylmorphine
• Used in treating severe pain
• Currently still used in countries (UK, Germany, Switzerland, etc.)
• Causes HIV & hepatitis due to intravenous uses (Sharing of needle)
• Symptoms: diarrhoea, tarchycardia, paranoia, suicide ideation

Current available methods for heroin identification:

Step 1: Isolation of active compounds from sample

1) High performance liquid chromatography (HPLC)

HPLC is used to analyse and isolate active compounds present in the illict street-grade heroin (sample). The active compounds found in illicit street-grade heroin includes heroin, 6-Monoacetylmorphine (morphine derivatives), codeine and acetylcodeine.

Difference between analytical HPLC and preparative HPLC:

Preparative HPLC can apply much higher amount of sample to the stationary phase. And in the purification system, the sample after travelling through the detector in analytical HPLC will go directly into the waste receptacle, whereas in preparative HPLC will go to the fraction collector first.

Reverse Phase HPLC (RP-HPLC)

Carried out with:

1)      HPLC system with an auto-injector & Diode array detector (210nm)

2)      Mobile phase: 23% methanol/water (0.05% (v/v) trifluoroacetic acid in water)

3)      Flow rate : 1.0mL/min

4)      Column temperature: 35 °C

5)      Column dimension:  250 mm × 4.6 mm I.D., 5 μm

  Normal Phase HPLC (NP-HPLC)

 Carried out with:

1)      HPLC system with auto-injector & Diode array detector (210nm)

2)      Mobile phase: Hexane–tetrahydrofunan/isopropanol–0.75% diethylamine in methanol (75:20:5, v/v)

3)      Flow rate : 1.0mL/min

4)      Column temperature: 35 °C

5)      Column dimension: 250 mm × 4.6 mm I.D., 5 μm

  Preparative RP-HPLC separation

  Performed with:

1)      Preparative HPLC system with automatic injector, Diode array detector (at 210nm wavelength) & automatic fraction collector

2)      Preparative column : 250 mm × 20 mm I.D., 15 μm

3)      Mobile phase: 20% methanol–0.05% trifluoroacetic acid in water

4)      Flow rate: 20 mL/min

5)      Injection volume =  200 μL

Preparative NP-HPLC separation

Performed with:

1)      Preparative HPLC system with automatic injector, Diode array detector (at 210nm   wavelength) & automatic fraction collector

2)      Preparative column : 250 mm × 10 mm I.D., 5 μm

3)      Mobile phase: Hexane–tetrahydrofunan–methanol (60:32:8, v/v) or hexane–isopropanol–methanol (65:28:7, v/v)

4)      Flow rate: 8 or 10 mL/min

5)      Injection volume =  300 μL

2) Liquid Chromatography- Mass Spectrometry (LC-MS)

It can be use for the analysis of various active compounds present in the sample.

Performed with:

1)      LC-MS system with binary pump and autoinjector.

2)      Elecrospray source in positive mode

3)      Ion spray voltage 1500V

4)      Nitrogen gas (1.5L/min) and drying gas (10L/min)

5)      Column dimension: 100 mm × 2.0 mm I.D., 5 μm

6)      Mobile phases: 0.5% formic acid in acetonitrile (linear gradient from 10% to 30% acetonitrile in 10min) and 0.5% formic acid in water

7)      Total flow rate: 0.4mL/min

8)      Column temperature: 40 °C

3) Drowning-out crystallisation

It is used for isolation and separation of compound and can be incorporated with HPLC separation techniques for enhancement of separation.

Drowning-out anti-solvent: Isopropanol–methanol (50:1, v/v)

Salting-out agent: Hexane

Carried out using 3 stages:

1)      Dissolution in drowning-out anti-solvents

2)      Addition of salting-out agents

3)      Precipitation

Step 2: Identification of heroin

Structural identification of the unknown compound can be carried out using IR or NMR and then compared their infrared spectral data or nuclear magnetic resonance results with the street-grade heroin data from Bio-Rad/Sadtler IR Data Collection or other available databases.

·        

Infrared Spectroscopy (IR)

It can be performed on Flourier Transform Infrared Spectrometer (FTIR).

Nuclear Magnetic Resonance (NMR)

1H NMR and 13C NMR spectrometry can be carried out a (500 M Hz) NMR spectrometer.

Selected Method:

1) Isolation of active compounds from sample: Preparative NP-HPLC separation with drowning out crystallisation

Reason:

Analytical HPLC does not resolve as well as preparative HPLC. Furthermore, preparative HPLC is able to elute concentrated and dried fraction containing heroin. However, preparative RP-HPLC is not a suitable method to obtain pure heroin from heroin sample because heroin will hydrolyze into monoacetylmorphine when expose to water and being heated. Although LC-MS system was suitable to isolate pure heroin from street heroin samples, could not obtain it in its crystal form.

The active compounds in the sample can be further purify with drowning-out crystallisation. Moreover, its low cost, high energy efficiency and good sensitivity are that allows incorporation with the preparative HPLC separation techniques.

2) Identification of compound:  Infrared spectroscopy

Reason:

It is cheaper than nuclear magnetic resonance method (NMR) and NMR pose risk of exposure to  nuclear compound.

Materials Needed for Analysis:

1) Methanol
2) Tetrahydrofunan
3) Isopropanol
4) n-hexane
5) Diethylamine
6) Trifluoroacetic acid
7) Ultrapure water
8) White powder found at the crime scene (samples)

Methods:
5 mg of the samples were used to dissolve in methanol by sonication to obtain a concentration of 10 mg/mL. It is furthur diluted to 1 mg/mL with the mobile phase. Due to diacetylmorphine instability, it was immediately injected into the preparative HPLC system.

The fraction of eluate containing the purified heroin sample collected from the preparative HPLC, would be used for infrared spectroscopy identification test to find out the fingerprint regions of the purified sample. Then, its fingerprint regions was compared with the known heroin's fingerprint regions found in databases.

Risk Assessment

Hazard Identification				Risk Evaluation
Ref	Work Activity	Hazard	Possible Injury/ Ill-health	Existing Risk Controls	Severity	Likelyhood	RPN
1	Methanol	Fairly dangerous substance. Flammable liquids (Category 2) Acute toxicity, Oral (Category 3) Acute toxicity, Inhalation (Category 3) Acute toxicity, Dermal (Category 3) Specific target organ toxicity - single exposure (Category 1)	Highly flammable liquid and vapour. Toxic if swallowed; contact with skin; inhaled. Causes damage to organs.	1. Always wear personal protective equipment (PPE) when handling the chemical (safety goggles, gloves, labcoat, and face mask). 2. Store in cool places. Keep container tightly closed in a dry and well-ventilated area. 3. Avoid contact with skin and eyes. 4. Avoid inhalation of vapour or mist.	Moderate (3)	Occasional (3)	High risk (9)
2	Tetrahydrofuran	Not a hazardous substance in lieu to GHS classification.	. Inhalation: May be harmful if inhaled. May cause respiratory tract irritation. Ingestion: May be harmful if swallowed. Skin: May be harmful if absorbed through skin. May cause skin irritation. Eyes: May cause eye irritation.	1. Always wear personal protective equipment (PPE) when handling the chemical (safety goggles, gloves, labcoat, and face mask). 2. Store in cool places. Keep container tightly closed in a dry and well-ventilated area. 3. Recommended storage temperature should be           2 to 8 degree. 4. Avoid contact with skin and eyes. 5. Avoid inhalation of vapour or mist.	Negligible (1)	Occasional (3)	Low risk (3)
3	Isopropanol	Fairly dangerous substance. Flammable liquids (Category 2) Eye irritation (Category 2) Specific target organ toxicity - single exposure (Category 3)	Highly flammable liquid and vapour. Eyes: Causes serious eye irritation. May cause drowsiness or dizziness.	1. Always wear personal protective equipment (PPE) when handling the chemical (safety goggles, gloves, labcoat, and face mask). 2. Store in cool places. Keep container tightly closed in a dry and well-ventilated area. 3. Recommended storage temperature should be           2 to 8 degree. 4. Avoid contact with skin and eyes. 5. Avoid inhalation of vapour or mist.	Negligible (1)	Occasional (3)	Low risk (3)
4	n-Hexane	Fairly substance. Flammable liquids (Category 2) Reproductive toxicity (Category 2) Aspiration hazard (Category 1) Specific target organ toxicity - repeated exposure (Category 2) Skin irritation (Category 2) Specific target organ toxicity - single exposure (Category 3) Chronic aquatic toxicity (Category 2)	Highly flammable liquid and vapour. Swallowed: May be fatal if swallowed and enters airways. Skin: Causes skin irritation. May cause drowsiness or dizziness. May cause damage to organs through prolonged or repeated exposure.	1. Always wear personal protective equipment (PPE) when handling the chemical (safety goggles, gloves, labcoat, and face mask). 2. Store in cool places. Keep container tightly closed in a dry and well-ventilated area. 3. Recommended storage temperature should be           -20 degree 4. Avoid contact with skin and eyes. 5. Avoid inhalation of vapour or mist.	Moderate (3)	Occasional (3)	High risk (9)
5	Diethylamine	Dangerous substance. Flammable liquids (Category 2) Acute toxicity, Oral (Category 4) Acute toxicity, Inhalation (Category 4) Acute toxicity, Dermal (Category 4) Skin corrosion (Category 1) Serious eye damage (Category 1)	Highly flammable liquid and vapour. Swallowed: Harmful if swallowed. Skin: Harmful in contact with skin. Eye: Causes severe skin burns and eye damage. Inhalation: Harmful if inhaled.	1. Always wear personal protective equipment (PPE) when handling the chemical (safety goggles, gloves, labcoat, and face mask). 2. Store in cool places. Keep container tightly closed in a dry and well-ventilated area. 3. Recommended storage temperature should be           -20 degree 4. Avoid contact with skin and eyes. 5. Avoid inhalation of vapour or mist. 6. Always wash hands after the practical with soap and plenty of water. 7. Dispose of contents in an approved waste disposal	Moderate (3)	Occasional (3)	High risk (9)

Analytical results:

1)    Preparative NP-HPLC separation

	Preparative NP-HPLC chromatogram of heroin samples. Preparative conditions: hexane–isopropanol–methanol (65:28:7, v/v). Peaks: 1 = acetylcodeine, 2 = heroin

The peak for heroin is well separated from the acetylcodeine peak and this diagram shows the presents of heroin in the sample. The maximum absorption wavelength of heroin is 205nm.

·        2) Drowning out crystallisation

The drowning-out crystallization method increase the purity of the heroin fraction from 99.13% to 99.52%.

·     3) Infrared spectroscopy

(a) IR spectrum of heroin fraction

(b) IR spectrum of heroin base from the Bio-Rad/Sadtler IR Data Collection

The IR spectrum data of the purified heroin fraction is highly similar to the IR spectrum heroin base from the Bio-Rad/Sadtler IR data collection. This concludes that the white sample obtained from the crime scene contains a high percentage of heroin.

Discussion:

A sample of 280mg of street drugs (Crude Heroin) is recovered from the crime scene. Among the 280mg of samples obtained, 140mg of heroin is used for analysis. The result shows that 110mg of pure heroin is extracted from the 140mg of heroin. Hence upon further calculation, we have derived that the sample contains 78.6% of pure heroin. 

Therefore, the sample that are scattered on the suspect’s apartment floor is indeed heroin and the suspect might be a drug dealer. A thorough search of the suspect's apartment will be conducted again for traces of heroin left.

Conclusion:

*Knock*

"Come in." 

Agent Lau exclaimed.

Agent Chen entered and gave Agent Lau a nod.

"The analysis for the sample were completed. Your instincts were right! The analysis came back positive for heroin. Let's bring the suspect along for his apartment investigation. We will make him spit out the exact spot where he hid the drugs."

Agent Lau brimmed in excitement.

"Let's go then."

She ordered.

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* Drug dealer's Apartment... *

Agent Lau dragged the suspect back to his apartment and asked meekly:

"Tell me where you hid the drugs. Now."

"What are you doing? I have already told you I have no idea what are you talking about!"

Agent Lau waved the analysis results right in front of the suspect's face.

"Read it yourself." Agent Lau told the suspect.

"This... This is not right! How can this be? Someone is definitely framing me! I do not do or deal drugs at all! I swear! I don't even have any heroin with me!"

The suspect shouted with slight irritation.

While the suspect was retaliating back with his speech, Agent Lau noticed that the suspect seem to glance in a certain direction directly behind herself. 



Out of curiousity, she spun around and realize that the suspect was glancing at the brick wall behind 
her. She started analyzing the wall and thought to herself:

'...? Is there something interesting behind me? There isn't even anything out of place on this wal-'

Her thoughts were interrupted when she suddenly noticed something out of place on the wall. A particular area of the lower right area on the wall seems to have different colored cement ,which joins the different bricks together, as compared to the other areas' cement. The cement's colour is a little darker as compared its counterparts.

'Wait a minute... That area's cement is much newer then the other areas. I remember someone said before that fresh cement are always of a darker colour as compared to older cement. This is due to the presence of water mixed in together with the cement powder to form cement paste. Fresh cement are of a very dark greyish colour. That means... That area's cement is still quite recent.'

"Team. Try to remove the bricks from the wall with the darker coloured cement."

The suspect's face immediately turned white and stared at Agent Lau in shock after she gave that command to her fellow forensic team.

Her team immediately sprung into action. After a good 15 minutes, the hole on the wall became wide enough to inspect.

"Oh my! I can see white powder packets hidden inside the wall!"

One of the forensics exclaimed. 

"Do you still have any more worthless excuses now?"

Agent Lau directed her question to the now pale-faced suspect.

====================================================================

"Good job in solving the heroin case, Agent Lau!"

Agent Lau smiled at her superior, Agent Z.

"Thank you for the compliment Sir! I will work even harder for the future cases."

Her superior gave her a nod and went off.



* Once again, Agent Lau have saved the day! *

THE END.

References:

1)  Huber.U, Majors. R.E (2007), Principles in preparative HPLC, 1st edn, Agilent Technologies Inc, Germany

2)  Jan.C, J.Bart (2005) Additives in Polymers: Industrial Analysis and Applications, 1st edn, John Wiley & Sons Ltd

3) Zhen G, Hui Z, Lu Y.Z, Yun W (2012), Isolation and purification of heroin from heroin street samples by preparative high performance liquid chromatography, Forensic Science International, Vol 221, No 1-3, Pg 120 - 124

4) http://en.wikipedia.org/wiki/File:Heroin3Dan.gif#globalusage