Analysis of Cocaine

Brief Intro and Background Information:

DAY 1
We have received a phone call from Detective John. A month ago, a high school student was found dead in the toilet suspected due to cocaine overdose. He mentioned that they have a possible suspect, Alvin,  responsible of dealing cocaine in the school. However, there is a catch. They could not find any stash of cocaine in Alvin's personal belongings. This caused a halt in the investigation. Thus, Detective John then decided to consult us about this matter.

"I need you guys to analyse some evidences for me. We need to prove that he's responsible! Just because he's the son of the Senator, that doesn't mean he can walk away a free man! We need concrete evidence or this investigation will be dropped!" Detective John exclaimed.

He passed us all of Alvin's belongings from the school locker. Bags, books, wallet and etc. We decided to get to work. IT'S ANALYSIS TIME!

Fictional story inspired from: http://news.nationalgeographic.com/news/2009/08/090816-cocaine-money.html

DAY 2
Before we have fun with analysis, let's get to know a little bit more about COCAINE!

benzoylmethylecgonine

Colourless/White solid crystal

Molecular Weight= 303.4

Molecular formula: C17 H21 O4 N
Melting Point: 98 °C
Boiling Point: 187 °C
Slightly volatile, anhydrous, bitter tasting crystals

Solubility In water:  0.17 grams per 100 mL

Cocaine is an alkaloid ester. It is a triple reuptake inhibitor: Serotonin–norepinephrine–dopamine reuptake inhibitor and has pocket with both high hydrophilic and lipophilic efficiency. This causes it to diffuse through the blood–brain barrier much better than other psychoactive chemicals and may lead to blood brain barrier breakdown. This is why cocaine has such a powerful effect on our central nervous system, causing symptoms such as anxiety, feeling of euphoria and so on.

Why is cocaine detected on banknotes?

Cocaine traces can be detected on banknotes as the notes are rolled and used to snort the drug powder. In addition, high traces of cocaine can contaminate banknotes during transaction between a drug dealer and a buyer. This is so because most cocaine dealers are the ones responsible for producing the drugs. Thus their hands may hold high amount of cocaine powder and cross-contamination happens.

Furthermore, once the banknotes are contaminated with cocaine, it is likely to remain contaminated. Cocaine binding studies with tritium-labeled cocaine have shown that the banknote matrix has the capacity to bind cocaine in a specific and reversible manner.

DAY 3
After getting a little bit more familiar with cocaine, this are some of the important properties of cocaine taken into consideration to decide the right method of analysis

• Presence of chromophore
• Presence of NO4
• Organic compound with high boiling point and thermally stable
• Slightly volatile

Here a the list of methods that not only able to detect cocaine, but also taking note that the source of extraction is from bank notes and not the usual urine/hair sample!

Methods
 a) Gas Chromatography (GC)
 The analytical method most frequently used for determination of cocaine on banknotes is GC–MS. However, GC allows many analytical methods and they could be separated into 3 groups based on the detector used:                         
                        (i) nitrogen-phosporous detection (GC-NPD)

(ii) single Mass Spectrophotometry (GC-MS)

(iii) MS2 (GC-MS2).

In recent years, researches have been coupling GC with tandem mass spectrometry to increase the sensitivity and so lowers the limit of detection (LOD). This method is able to provide an LOD of 0.15 ng/note. It is therefore a much selective and sensitive method to determine cocaine on banknotes.

    b) Liquid chromatography-Tandem mass spectrometry 
For the separation of cocaine from other substances present in the matrix, a multi-step
linear gradient of ammonium acetate (pH 4.5) and methanol was used as mobile phase. Iit was determined that a 20-cm monolithic column, which offered the highest sample peak at acceptable mobile-phase flow rates and column backpressures, and had a higher overall capacity than a 10-cm monolithic column (thus allowing increased injection volumes for trace analysis), provided the best performance for the proposed application. The LOD and the limit of quantitation (LOQ) were 4 pg/note and 14 pg/note, respectively. Recovery values obtained from the above procedure were 87 ± 8%. The elution time of cocaine using the above conditions was 10 min.

    c) Capillary electrophoresis (CE)
CE with electrochemiluminescence (ECL) detection was
proposed for the determination of cocaine on banknotes. Cocaine was extracted
from banknotes by using 0.01 mM acetic acid for 15 min. A high-efficiency, on-column, field amplified, sample stacking (FASS) technique was optimized to increase the detector’s sensitivity. An LOD of 60 nM for cocaine was achieved.

    d) Thermal desorption tandem mass spectrometry
TD-MS2 is the second most commonly used method for the analysis of cocaine on banknotes. It is a rapid, sensitive technique in detection and in discriminating between
innocent money from general circulation and drug money. However, it cannot be considered a quantitative approach, because precise quantification has not been attempted due to the difficulty of comparing responses. To analyse, dust from the banknotes is trapped on filters using a vacuum system, or cocaine is directly desorbed from the banknotes into the source of the mass
spectrometer. This method provides high sensitivity with little contamination, so 50 banknotes can be analysed in a 4-min period. The LOD of the technique depends upon the total amount of material desorbed, as that may affect the ionization of any controlled substances present, but it is typically below 1 ng. In conclusion, TD-MS2 methods provide the possibility of analyzing a great number of banknotes in a short time, but the main disadvantage is that the desorption procedure is not completely effective, so it does not provide an accurate reflection of the levels of drug contamination on currency.

    e) Ion-mobility spectrometry (IMS)
IMS can reveal traces of controlled substances collected onto a filter paper, heated to vaporization, and ionized by electrons emitted by a 63Ni source. The time required for
the ions to drift through an electric field and impinge on a detector is measured, and the substances are identified. It allows the analysis of cocaine in large quantities of banknotes using a vacuum sampling method, but it also allows the determination of drugs in individual banknotes by collecting surface swabs, which are analyzed by insertion into the vaporizer unit of the IMS, or by directly inserting the banknotes themselves (about 1000 times more sensitive than vacuum collection). IMS provides a sophisticated screening method capable of detecting nano grams amounts of drugs and is ideally suited to operating in the field. However, the desorption procedure is not completely effective and it is difficult to obtain precise quantitative results. However, due to the versatility, robustness, portability and little need for maintenance, IMS is widely used in airport and other security settings.

DAY 4
Now that we have a clear understanding of the methods, let's move on to ANALYSIS!
Recently, an article had mentioned that traces of cocaine exists up to 90% of bank notes in US. In order to make our investigation reliable, we decided to use GCMS to analyse bank notes from 14 cities in US for presence of cocaine contamination. We will also analyse the evidence from Alvin's belonging including his money to see if he is involve in drug transaction.

The steps taken are as follow:

Materials and Methods

Instrumentation
 
Analysis was performed on a HP 5890A gas chromatograph with an autosampler interfaced with a mass selective detector (MSD).
A split-splitless capillary inlet system and fused-silica capillary column (12 m x 0.2-ram i.d., 0.33-profilm thickness) was used for cocaine analysis.




Materials
US paper currency
 
Sample: ten $1 bank notes collected from 14 cities in the US
Control: new, uncirculated and drug-free control bank notes from the United States Treasury Department








Extraction and analysis of currency
Sample preparation:
Each currency were treated by deuterated internal standards*
Samples were extracted by Solid Phase Extraction (SPE) followed by GCMS


For SPE, the sample were mixed with sodium acetate buffer and filtered. The eluents were decanted onto preconditioned SPE columns. Next, the analytes were eluted with elution solvent and the extract were evaporated to dryness under nitrogen. Last but not least it was reconstituted in acetonitrile. The sample was ready for GC-MS analysis:


·  Derivatizing reagent was added ( to make the analytes more suitable for analysis to increase   respond and improve resolution)
· The vials were sealed and heated at 60oC for 30 min
· 1uL was injected, using autosampler, for GC-MS analysis
· The samples were analyzed in the scan mode for identification purposes and in the selected ion monitoring mode for quantitation.
· Cocaine was measured in currency extracts by internal standardization and compared to Standard curves** of unused, drug-free currency.
· Duplicate control samples were also analyzed with each batch (to increase accuracy and minimize gross errors)
· Control samples were prepared with drug-free currency and contained cocaine and benzoylecgonine at two amounts (100 ng and 500 ng per extract).


*deurated: It is a form of the actual drug of interest. The deuterate is different by + or - molecular weight (MW). In the case of GC/MS it has characteristics so similar to the drug of interest that it elutes (comes out) from the column as a peak with characteristic ions.
**Standard curves were run in duplicate with each batch of currency and contained cocaine, ecgonine methyl ester, and benzoylecgonine in amounts of 0, 12.5, 25, 50, 100, 250, and 500 ng and anhydroecgonine methyl ester, ecgonine ethyl ester, cocaethylene, norcocaine, and norcocaethylene in amounts of 0, 6.25, 12.5, 25, 50, 125, and 250 ng.


Result
After cocaine was extracted from the bank notes and identified via GC-MS, let’s take a closer look at the result of the chromatogram (fig.1)

Figure 1A showed the response of cocaine analogues extracted from a single uncirculated currency bill (without internal standard). The inset contains the mass spectrum of standard cocaine at a retention time of 6.6 min that was obtained in the same run. Figure 1B showed the response from an extract of a single piece of circulated currency collected in Spanish Fort, AL.


Qualitative analysis of GCMS
Cocaine on the bank notes was identified by comparing the peaks with retention time of 6.6min with that of the cocaine standard. The characteristic ions of cocaine at m/z 303, 182, and 82 provided clear evidence of the identity of cocaine in this currency. The graph also shows the presence of traces of benzoylecgonine on the currency extract with retention time of 7.1min.

Quantitative analysis of GCMS
Calibration curves were linear across the range from 0 to 500ng of cocaine extracted from the standard. The limit of quantitation was arbitrarily selected as 12.5 ng, the lowest point on the calibration curve.
By comparing with the known standard, we were able to estimate that there was a total of 70.2ug cocaine and 3.6ug benzoylecgonine present in a single piece of bank note.


Altogether, 79% of bank notes showed presence of cocaine above 0.1ug, and 54% above 1.0ug of cocaine. Contamination was extensive and was detected in bank notes from all collection sites. The amount of cocaine was inconsistent, from ng to mg.
The highest amount of cocaine found was 1327ug on a single note from Portsmount, OH.



Discussion
This study has illustrated that bank notes circulated around US are highly contaminated with cocaine. It was found in every bank notes from all collection sites. The only cocaine derivatives present as well was benzoylecgonine, a hydrolysis product of cocaine. In addition, the only obvious trend for the degree of contamination was that currencies from less populous areas (e.g., Pittsburgh, PA and Whitefish, MT) are more likely to have lower amount

The results from this study suggest that the presence of cocaine in amounts up to 1mg per bill does not signify that the currency was involved in a drug transaction. When the bank notes has been contaminated, it is likely to remain so due to the high binding affinity and capacity of the United States bank notes. 

In relation to Alvin's case, we found traces of cocaine in his wallet and money, ranging from 0.2ug up to the highest we found was 891ug on a $50 bill.

So back to our story!
After a week of our phone call with Detective John, he came to the lab to see the results of our analysis.
"So what do you guys have? Make it quick and straight to the point! No need for jargon!" He snapped at us while rubbing the back of his neck.
Someone is extra snarly today. We did a brief report on the analysis, making sure to tell him that while most US Dollars are indeed contaminated with cocaine and other drugs like heroine, Alvin's cash seems to retain quite a huge amount.
"What do you guys think? Is this money involved in drug circulation?" He glanced at us before his eyes scanned the report once more.
"Possibly." Selina answered.
"He might be a drug abuser, or received the money from someone else. We can't tell you if he's responsible for that girl's death." I said while pushing the crate full of Alvin's belongings back to him.
"Girls, I just want a clear answer. Do you think he's guilty or not?" He muttered while pinching his nose bridge.

Well Detective. Evidence, we provide. Guilty or innocent, YOU decide!

END OF STORY.

But wait, there's more!

Bonus Interesting facts: 
1) Cocaine level of banknotes around the world
   

The bar chart shows the statistics of cocaine contamination on bank notes around the world.
Top Contamination:
1st: US Dollar - 2.86–28.75 ug cocaine/note
2nd: Spain Euro - 1.55 ug cocaine/note
3rd: German and Irish Euro - roughly 0.576 ug cocaine/note

Reports have also shown that at least 51% of UK Pounds are contaminated with cocaine while Swiss currencies have 6% contamination that is of 1ng cocaine/note.

2) Do you know if you handle bank notes contaminated with cocaine, it may appear in your urine? 
An experiment has been done to evaluate if casual contact with contaminated money would test positive by urinalysis. Two $1 bills were immersed in powdered cocaine and one individual were asked to handle the money for several times. Analysis of urine samples collected over a period of approximately 24hr showed that the individual excreted up to 72 ng/mL of benzoylecgonine 12.5 h after!
But no worries, it was concluded that casual handling of articles contaminated with cocaine would not result in a positive urine test at a cutoff concentration of 300 ng/mL of benzoylecgonine.

Reference:

Oyler J, D. Darwin, and J.E Cone. 1996, Cocaine Contamination of United States Paper Currency. Available at:<http://www.ncbi.nlm.nih.gov/pubmed/8835657> [Accessed on 25/1/2013]

Loralie J. Langman1, Matthew W. Bjergum1, Christopher L. Williamson, and Frank W. Crow. 2009. Sensitive Method For Detection Of Cocaine And

Associated Analytes By Liquid Chromatography–

Tandem Mass Spectrometry In Urine. Available at:<http://jat.oxfordjournals.org/content/33/8/447.full.pdf> [Accessed on 25/1/2013]

  S. Armenta, M. de la Guardia, 2008, Analytical methods to determine cocaine contamination of banknotes from across the world, Available at:<http://www.sciencedirect.com/science/article/pii/S0165993608000149> [Accessed on 25/1/2013]

E. J. Nestler, 2005, The Neurobiology of Cocaine Addiction, Available at:<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851032/> [Accessed on 25/1/2013]