Thesis - SBaker

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Forensic Analysis of Amphetamine-like Compounds found in

Pre-workout Supplements

A thesis submitted in partial fulfilment of the requirements of the degree

of

Bachelor of Health Science (Honours)

by

Stephen Edward Baker

Student ID: 13131582

Bond University

29th

November 2013

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Abstract

This study was performed to highlight the need for more stringent regulation and

monitoring of pre-workout supplement content, as many pre-workout supplements

contain amphetamine like compounds and the labels don’t always mention them. We

focused on the non-professional people who take pre-workout supplements as they rely

on the label to tell them what is contained within the supplement. The aim of this study

was to analyse seven different pre-workout supplements; Hydroxycut Hardcore

Proseries, Hyper FX, Black Powder, Black Bombs, Fast Fuel, Mesomorph, and

Thermojet; for the presence of six compounds; caffeine, DMAA, hordenine,

methylsynephrine, phenethylamine, and synephrine. The presumptive tests used

included functional group tests and an immunoassay test, with the presence of the

compounds being confirmed through the use of high performance liquid

chromatography coupled with UV spectrophotometry and also with mass spectrometry.

After analysis it was discovered that all of the seven pre-workout supplements

contained an amphetamine-like compound that was not mentioned on its label. In fact,

five of the supplements; Hydroxycut, Black Powder, Fast Fuel, Mesomorph, and

Thermojet; contained compounds that are currently illegal to be sold in supplements;

with Black Powder, Fast Fuel, and Thermojet containing two such compounds.

Although; Thermojet had been taken off the shelves by the time this study was

performed.

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Table of Contents

Abstract ................................................................................................................................................ 2

Statement of Originality ....................................................................................................................... 6

Acknowledgements .............................................................................................................................. 7

Introduction .......................................................................................................................................... 8

Significance ..................................................................................................................................... 8

Background ...................................................................................................................................... 8

Drug use in Recreational sport ..................................................................................................... 8

Amphetamine & derivatives ...................................................................................................... 11

Presumptive tests ........................................................................................................................... 15

Confirmatory Tests ........................................................................................................................ 20

Sample Preparation .................................................................................................................... 20

Sample Analysis by HPLC and Mass Spectrometry ................................................................. 21

Discussion .......................................................................................................................................... 25


Immunoassay ............................................................................................................................. 25

Functional Group tests ............................................................................................................... 26

Confirmatory tests .......................................................................................................................... 29

Extraction Method ..................................................................................................................... 29

HPLC ......................................................................................................................................... 31

LC-MS ....................................................................................................................................... 38

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Quantification of Synephrine ..................................................................................................... 46

Conclusions and Further Work .......................................................................................................... 47

Experimental ...................................................................................................................................... 48


Immunoassay ............................................................................................................................. 48

Functional Group tests ............................................................................................................... 48

Confirmatory tests .......................................................................................................................... 49

Extraction ................................................................................................................................... 49

HPLC ......................................................................................................................................... 50

LC-MS ....................................................................................................................................... 50

Concentration Curve .................................................................................................................. 52

Compounds .................................................................................................................................... 52

Supplements ................................................................................................................................... 53

References .......................................................................................................................................... 55

Appendices ............................................................................................................................................ i

Appendix 1 – Names ......................................................................................................................... i

Appendix 2 - Extraction Validation ................................................................................................ iii

Appendix 3 – UV Chromatographs ................................................................................................ iv

Appendix 4 – LC/MS Results – Hydroxycut ................................................................................... x

Appendix 5 - LC/MS Results – Hyper FX .................................................................................... xii

Appendix 6 - LC/MS Results – Black Powder .............................................................................. xv

Appendix 7 - LC/MS Results – Black Bombs ............................................................................ xviii

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Appendix 8 - LC/MS Results – Fast Fuel ..................................................................................... xxi

Appendix 9 - LC/MS Results – Mesomorph .............................................................................. xxiv

Appendix 10 - LC/MS Results – Thermojet ............................................................................... xxvi

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Statement of Originality

This work has not previously been submitted for a degree or diploma in any university.

To the best of my knowledge and belief, the dissertation contains no material previously

published or written by another person except where due reference is made in the

dissertation itself.

Stephen Edward Baker

29th

November 2013

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Acknowledgements

This work has been carried out under the supervision of Assistant Professors Dr

Stephanie Schweiker and Dr Anna Lohning. I wish to extend my sincerest gratitude and

thanks for their patience, support and advice throughout this year and also in the years

of study before this year.

I also wish to extend my gratitude also to Faith Rose for her help and assistance in

acquiring HPLC data, Ben Matthews for his help and assistance in acquiring LC-MS

data, as well as Dr Darren Grice for his help and support throughout the project.

Also thank you to Alan White for training in the use of the GC and LC mass

spectrometry

Finally, I would like to thank my family for their support throughout the years and for

making every day enjoyable and mostly stress-free.

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Introduction

Significance

This study highlights the need for more stringent regulation and monitoring of pre-

workout supplement content. Pre-workout supplements are consumed by many people,

especially young people, and it is important that they understand what the pre-workout

supplements contain; as well as their physiological effects and potential health risks. Plant

extracts often listed on the label contain many compounds espoused to provide a

particular physiological effect. Often however, their physiological effects are unknown.

In this study we’ll use confirmative tests, such as liquid chromatography coupled with

mass spectrometry (LC-MS) to analyse for amphetamine-like compounds in common pre-

workout supplements. This study focuses on the non-professional people who take pre-

workout supplements as they rely on the label to tell them what the supplement contains.

Background

Drug use in Recreational sport

The use of performance enhancing substances is not confined to professional

sportspeople. Perhaps stemming from the eminent status with which we hold our sporting

heroes, the use of pre-workout supplements has infiltrated the wider community as

everyday people now strive to improve their performance in recreational sport, as well as

their body image. Pre-workout supplements, such as “Black Powder” and “Fast Fuel”, are

used to increase muscle tone, body mass and strength. This is as opposed to weight loss

supplements, which help the body induce lipolysis.

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Many pre-workout supplements which their manufacturers claim to enhance performance

contain plant extracts. Chemical analysis reveals the presence of amphetamine-like

compounds, such as synephrine, as well as banned compounds such as amphetamine

itself. Many of these compounds indirectly stimulate the release of norepinephrine (Drug

and Alcohol Services South Australia, 2006); which in turn increases the heart rate, blood

pressure, and breathing rate. Performance is enhanced as more oxygenated blood is

delivered to the muscles. Most pre-workout supplements also contain caffeine (1), which

also increases the heart rate; mimicking the physiological effects of these amphetamine-

like compounds. However, caffeine can also cause major side effects when mixed with

some of these compounds (Department of Health and Ageing, 2013). For these reasons

pre-workout supplements are under close scrutiny by government authorities, such as the

Therapeutic Goods Administration (TGA) (Department of Health and Ageing).

The TGA is responsible for regulating therapeutic goods including medicines, medical

devices, blood and blood products. Essentially, any product for which therapeutic claims

are made must be listed, registered or included in the Australian Register of Therapeutic

Goods (ARTG) before it can be supplied in Australia. The majority of the pre-workout

supplements that are controlled by the TGA are classified as ‘listable complementary

medicines’ (Department of Health and Ageing). These products may be made from

anything permitted by Schedule 4 of the TGA; this is the schedule for prescription-only

medication. Any combination of these herbal substances, vitamins, and minerals is

allowed, and they can be present in any amount (Cameron, 1998). Products in the

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‘listable’ category are not tested for efficacy, however they do need to meet advertising

regulations, labeling requirements, and compliance of the manufacturing process with a

recognised code of good manufacturing practice, and they must also comply with relevant

statutory standards.

Many pre-workout supplements contain natural plant extracts, such as Citrus aurantium

and Acacia rigidula, which contain a raft of amphetamine-like compounds that vary in

their physiological effects (Fugh-Berman & Myers, 2004). A review of the potential of C.

aurantium for weight loss by Bent, Padula and Neuhaus in 2004 provided limited

information about the safety of the herb. On the other hand, A. rigidula has undergone no

safety testing. C. aurantium contains compounds that theoretically may carry the same

health risks as those found in ephedra. Little research has been done on these compounds

individually; although a study by Fugh-Berman and Myers in 2004 found that Citrus

aurantium extracts had not been associated with adverse effects. A. rigidula, on the other

hand, has been shown to contain between 6.7 and 11.8 parts per million (ppm) of

amphetamine, depending on when in the season it is harvested (Clement, Goff, & Forbes,

1998). Since pre-workout supplement manufacturers are only required to list the plant

extract, there may be a significant health risk associated with those that contain these

natural plant extracts (Clement, Goff, & Forbes, 1998). Self-administration of

compounds that have not undergone scientific testing for the desired effect or for toxicity

is dangerous (Schultz, 1998).

A recent article stated that banned substances and pharmaceuticals had been detected in

hundreds of purportedly natural pre-workout supplements (Cohen, Travisb, & Venhuis,

2013). This article also mentioned that some athletes tested positive for an analogue of

methamphetamine, their excuse being that they had unknowingly ingested the banned

stimulant in the workout supplement ‘Craze’. This methamphetamine analogue, N,α-

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diethyl-phenylethylamine (2), has never been studied in humans, so its effects are entirely

unknown; however the manufacturers recommended serving of Craze, 5.3g, included 21

to 35mg of this drug (Cohen, Travisb, & Venhuis, 2013). Note, N, N-diethyl-

phenethylamine (3) was listed on the label so athletes need to be more vigilant; however,

lay people also need to know of the health risks. However, some underground pre-

workout supplement makers add obscure steroids or amphetamines to their products and

then list a compound with the same chemical formula on the label (Cohen, Travisb, &

Venhuis, 2013).

The aim of this study is to analyse and quantify the presence of six compounds in seven

different pre-workout supplements. The lack of regulation of pre-workout supplement

composition coupled with the dangers of self-administration is placing a significant

number of people, many of them young teenagers, at risk. Most of the commonly known

dangerous compounds were listed in schedule 9 of the Drugs of Misuse Act 1986, e.g.

amphetamine, and methamphetamine. Under this act there is scope to include compounds

with a high degree of structural similarity to the banned substances which may cause

similar biological activity; i.e. the similarity clause. But more research is required to

characterise their individual physiological effects and identify potential health risks

Amphetamine & derivatives

Amphetamine (4) belongs to a family of drugs known as phenethylamines, of which the

simplest is 2-β-phenethylamine (5). 2-β-phenethylamine is a stimulant found naturally in

chocolate and some other foods and is also used in pre-workout supplements. However,

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while 2-β-phenylethylamine is not very active, the addition of a methyl group (i.e.

producing amphetamine) boosts the physiological activity. This methyl group protects the

amphetamine from enzymes. Attaching two methyl groups to 2-β-phenylethylamine

produces the even more potent methamphetamine. Both of these compounds are well

known psychoactives and stimulants. There are quite a few methylated phenethylamines

specifically named in the 1986 Misuse of Drugs Act.

Common side effects of amphetamine include; anxiety, irritability, hypertension, and

muscle and joint pain (Drug and Alcohol Services South Australia, 2006). It is also

possible for dependence to develop during use of amphetamines, with withdrawal being

associated with both mental and physical depression (Mottram, 2011). Phenethylamines

work well in pre-workout supplements due to their similarity to the monoamines that can

be found in the human body, such as epinephrine, and therefore phenethylamines mimic

the effects of these monoamines. Some pre-workout supplements contain, or used to

contain, amphetamine-like compounds, such as; dimethylamylamine (DMAA) (6),

ephedrine (7), hordenine (8), methylsynephrine (9), and synephrine (10).

All these derivatives have the beneficial effects of stimulating the nervous system; i.e. the

release of norepinephrine; which in turn makes them effective as pre-workout

supplements. However, DMAA, or methylhexanamine, was banned by the TGA in

4 5 6 7

8 9 10

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Australia in August 2012, so it is now illegal to market it as a pre-workout supplement;

however it is still legal to be used as it was originally marketed for, a nasal decongestant.

This resulted from evidence of side effects including headache and nausea. One report

linked over-dosing on DMAA together with alcohol with cerebral haemorrhage (Gee,

Jackson, & Easton, 2010).

Ephedrine is the main active component in plants of the genus Ephedra, and is also

banned from supplement use but still prescribed medication for asthma. Ephedrine was

banned in April 2004 after it was discovered that it could be linked to heart attacks,

strokes, hypertension, and psychiatric problems (Kim & LeBourgeois, 2004). After the

banning of ephedra in supplements, ephedra-free products rapidly replaced them on

drugstore shelves. Many of these ephedra-free products still contained extracts from the

plant Citrus aurantium (Fugh-Berman & Myers, 2004); thus taking advantage of the lay

persons lack of knowledge of chemistry.

The active compounds in C. aurantium include the phenethylamine alkaloids hordenine,

methyltyramine (11), and synephrine (Pellati & Benvenuti, 2007). These compounds may

carry the same health risks as ephedrine. A US based report linking the abuse of tablets

containing synephrine and a large myocardial infarction in a male aged 28 (Keogh &

Baron, 1985) highlights the need for more research.

Another plant extract found in pre-workout supplements is Acacia rigidula. Studies show

that consumption of this plant or of a related species, Acacia berlandieri, by goats and

sheep, has been associated with an inability to control the muscles of the legs. This is also

known as ‘limber leg’ (Clement, Goff, & Forbes, 1998). This study conducted A. rigidula

to rigorous chemical analysises and found that, as well as the four amines already known

to be present in the plant; methylphenethylamine (12), tyramine (13), methyltyramine and

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hordenine; forty other alkaloids and amines were present, including; phenethylamine,

nicotine (14), mescaline (15), amphetamine and methamphetamine (16). A. rigidula

extract can be found in the pre-workout supplements “Black Bombs” and “Mesomorph”.

Table 1 - Regulation of compounds (Department of Health and Ageing, 2013)

Compound Schedule

Amphetamine

8 (Controlled Drug – Substances which should be available for

use but require restriction of manufacture, supply, distribution,

possession and use to reduce abuse, misuse and physical or

psychological dependence)

Caffeine Unregulated

Ephedrine

4 (Prescription Only Medicine)

Appendix D (Poisons for which possession without authority is

illegal [e.g. possession other than in accordance with a legal

prescription])

Appendix F (part 3 – poisons to be labelled with warning

statements or safety directions)

DMAA

Appendix C (Substances, other than those included in schedule 9,

of such danger to health as to warrant prohibition of sale, supply

and use)

Hordenine Unregulated

Methamphetamine

8 (Controlled Drug – Substances which should be available for

use but require restriction of manufacture, supply, distribution,

possession and use to reduce abuse, misuse and physical or

psychological dependence)

Methylsynephrine 4 (Prescription Only Medicine)

Phenethylamine Unregulated

Synephrine 4 (Prescription Only Medicine)

11 12 13

14 15 16

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Methylsynephrine (or oxilofrine) (7) was recently made illegal to be sold in pre-workout

supplements, as well as recently added to the World Anti-Doping Agency’s Prohibited

list (World Anti-Doping Agency, 2013). This is a list of compounds that if detected

would disqualify someone from competing in a semi to professional sporting event. This

banning may in part be due to its structure being almost identical to ephedrine. The only

difference between ephedrine and methylsynephrine is the hydroxyl group para to the

ethyl-amine in methylsynephrine (Fig. 1). However, methylsynephrine also has a methyl

group on its alpha carbon; which all the banned compounds also have. It is thought that

this alpha methyl group is what makes the compounds illegal to be sold in pre-workout

supplements, as these compounds are regulated by the TGA (Department of Health and

Ageing, 2013).

Figure 1 - Ephedrine (5) and Methylsynephrine (7)

Presumptive tests

The initial step when looking for illicit substances is presumptive tests, the simplest of

which are functional group tests. These tests are used to detect functional groups that are

present in illicit substances by causing a colour change (Bell, 2006). One such spot test

uses the Marquis reagent, which reacts with both amphetamine and methamphetamine to

produce an orange-red product, and with morphine to produce a violet product. The

chemistry of this reaction is complex and it is thought that the colour is produced as a

result of a relatively stable carbocation formed through the action of formaldehyde

(Kovar & Laudszun, 1989) (Fig. 2).

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Figure 2 - Mechanism of the Marquis test; R = H for amphetamine, and R = CH3 for methamphetamine

Often after the Marquis reagent gives a positive result for amphetamines, a test is

performed using the Simon reagent. This is because this reagent can tell the difference

between primary and secondary amines, therefore amphetamine and methamphetamine

will react differently (Bell, 2006). In the presence of secondary amines, such as those

present in methamphetamine, the reagent turns blue. The mechanism of this reaction

involves the addition of an ethylene group followed by the removal of this group from an

iron complex. This results in a charged amine. This charged amine is responsible for the

colour change that is noticed for secondary amines (Fig. 3). There is a derivative of the

Simon reagent, known commercially as the ‘Robadope’ reagent. This reagent is thought

to work in much the same way as the Simon reagent, except it turns purple in response to

primary amines (Fig. 4). It is unknown how tertiary amines react with either of these

reagents.

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Figure 3 - Mechanism of the Simon test

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Figure 4 – Possible mechanism of the 'Robadope' test

Another colour test is the Lucas reagent, which differentiates between primary,

secondary, and tertiary alcohols (Lucas, 1930). When the Lucas reagent is reacted with

tertiary alcohol the resulting solution will go cloudy immediately, in the case of

secondary the cloudiness will appear after a few minutes, and in the case of primary the

cloudiness will not appear at all. The mechanism of the Lucas reagent involves the

formation of a carbocation intermediate, which is then quenched by the addition of a

chloride group (Fig. 5). The substitution of the hydroxyl group with the chloride changes

the solubility of the compound resulting in it precipitating out of solution, i.e. going

cloudy. The speed of this reaction is what separates the different types of alcohols.

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Figure 5 - Mechanism of the Lucas test with 2-Methyl-2-propanol

Immunoassay tests are chemical tests used to detect a specific illicit substance easily and

on site. The analyte in an aqueous solution produces an immunological reaction to

produce a noticeable change on the cup (Bell, 2006). A common immunoassay test is the

competitive immunoassay, which works on the basis of competition between the drug in

solution and the ‘tagged’ drug for the limited binding sites of the antibody (Fig. 6).

Figure 6 - Mechanism of the Competitive Lateral flow Immunoassay test

If the drug is present in solution then it binds to the antibody, and therefore there are no

binding sites left for the ‘tagged’ drug, this means that the ‘tag’ isn’t seen. But if the

solution is drug-free, then the antibody binds to the ‘tagged’ drug, and the ‘tag’ is seen.

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Limited information is available regarding what other compounds may produce positive

results in the immunoassay. Some compounds may give false positive results; e.g. it is

known that DMAA gives a positive immunoassay result for amphetamine (Vorce, Holler,

Cawrse, & Magluilo Jr., 2011).

The immunoassay kits, like colour spot tests, are only presumptive indicators of the

presence of the compounds, and are subject to false positives and negatives; confirmative

tests are needed, such as high performance liquid chromatography/mass spectrometry

(HPLC/MS), to confirm the identity of the individual compound and are a requirement for

confirmation in court.

Confirmatory Tests

Confirmatory tests confirm the presence of a substance.

Sample Preparation

First the substance of interest must be separated from its environment. The method of

separating a compound from its surroundings is called sample preparation, and the

process depends on the nature of the compound that needs to be separated, and how much

there is of that compound.

Extraction is a common form of sample prepatation; most commonly liquid-liquid, or

liquid-solid. Liquid-liquid extraction, or partitioning, is used when a large amount of the

substance of interest is present, e.g. a tablet dissolved in water. Liquid-liquid extraction

relies on the solubility of the substance in either a polar/aqueous liquid, e.g. water, or a

non-polar/organic, e.g. chloroform. Solid phase extraction works along the same lines as

filtering; it separates a substance that is suspended, but not dissolved, in a solution from

the rest of the compounds present in the solution.

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Sample Analysis by HPLC and Mass Spectrometry

After sample preparation, the sample is further analysed by chromatography, which

works by separating compounds on the basis of their affinity to either the mobile phase or

the stationary phase, with one phase being polar and the other being non-polar. A type of

chromatography that is commonly utilised by forensic chemists is thin layer

chromatography (TLC); this is used extensively as a screening technique. TLC works by

the use of a liquid that works its way up a thin layer of gel via capillary action taking a

sample that was dotted onto the gel with it. The sample then separates into its components

by the components adsorbing to the gel with different degrees of strength causing them to

move up the gel at different rates (Fig. 7). In the case of TLC the liquid is the mobile

phase and the solid is the stationary phase.

Figure 7 - Example of a TLC readout

High performance liquid chromatography (HPLC) is a conclusive form of

chromatography. HPLC works by using a high pressure liquid to force a sample through a

column containing a solid, with the components that make up the sample moving at

different rates (see Fig. 8). This difference in retention between the components is due to

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their affinity to either the liquid or solid phases; if they have more affinity for the liquid

phase then they will move faster, where as if they have more affinity with the solid phase

they move slower.

Figure 8 - Mechanism of HPLC (Bourne, 2010)

The retention factors of compounds are unique under certain conditions and allow

scientists to confirm compounds. In terms of HPLC the retention factor of a

compound/component is the time it takes for the compound/component to come out the

end of the column and be detected. The retention factor of a compound will always be

constant, as long as the conditions of the chromatography remain the same.

Commonly compounds are detected by UV spectrophotometry, ELSD or Mass

Spectrometry (MS). UV spectrophotometry works by each component giving a response

as it elutes from the HPLC. This response is due to a π-electron from a double bond in the

component being elevated to a higher level by absorbing the UV radiation of a certain

wavelength and then falling back to its ground state by emitting radiation at a different

wavelength. The response for each compound is assumed to be proportional to the

concentration of said compound; so the greater the concentration of the compound in

solution, the higher the peak on the readout (Royal Society of Chemistry Fine Chemicals

and Medicinals Group, 1992). ELSD, or evaporating light scattering detection, works by

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converting the components as they elute from the HPLC into a fine spray and then

heating them so that only the mobile phase of the HPLC is evaporated. Light is directed at

the remaining compounds and the scattered light is detected (Robinson, 2008). Like with

UV, ELSD responses are assumed to be proportional to the concentrations of the

components in the original solution.

MS works by breaking up a compound into all its possible ions, by bombarding

molecules of the compound with high energy electrons. The ions are then accelerated

between two magnets so that they are deflected from a straight line based on their mass to

charge (m/z) ratio. Heavy/large ions are deflected the least, and light ions are deflected

the most; and the magnets can be set to vary in strength so that all of these ions are

detected. By analysing the chemical ‘fingerprints’, given by the mass spectrometer, of

each component of a sample that has already been separated through chromatography and

comparing them to the ‘fingerprints’ of known pure compounds, the identity of a

compound found in the sample can be determined. The most important peaks on the

chemical fingerprint are the heaviest ion and the highest peak. The heaviest ion that is

formed will usually have a mass to charge ratio of the molar mass of the compound, this

is called the parent ion; and the highest peak is called the base peak, this is the most

common ion formed from the compound and so is the one detected most often.

To further differentiate compounds that have identical molar mass and similar mass

spectra; this is called tandem mass spectrometry (MS/MS). This is done by coupling

multiple mass spectrometers in series; so after a compound has been fragmented by the

first mass spectrometer, one of the fragments is selected and enters the next mass

spectrometer to be fragmented again. This technique has some advantages for the analysis

of organic compounds in complex mixtures (McLafferty, 1981). Tandem mass

spectrometry may also be used without a mixture being separated by chromatography

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first; in fact tandem mass spectrometry is thought to be able to achieve specificities and

sensitivities equivalent to those of HPLC coupled with mass spectrometry, while taking

less time to perform the analysis (McLafferty, 1981).

A specific type of Mass Spectrometer that has rapidly been embraced by the analytical

community is quadruple–time-of-flight (Q-TOF) mass spectrometer, although it was only

introduced commercially 6 years ago (Chernushevich, Loboda, & Thomson, 2001). These

are considered powerful and robust instruments with unique capabilities.

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Discussion

This study aimed to analyse seven pre-workout supplements; Hydroxycut Hardcore Pro-

series, Hyper FX, Black Powder, Black Bombs, Fast Fuel, Mesomorph, and Themojet; for

the presence of five amphetamine-like compounds; methylsynephrine, synephrine,

hordenine, phenethylamine, and DMAA; to determine the accuracy of their labels. These

seven pre-workout supplements were chosen by an industry representative due to their

popularity. A standard of each of the key amphetamine-like compounds were used and

compared to the pre-workout supplements.

Presumptive tests

Initially, both the compounds and the pre-workout supplements were examined through

presumptive testing, including both immunoassay and functional group tests.

Immunoassay

The immunoassay is used to determine if amphetamine, methamphetamine, opiates,

cocaine, marijuana/cannabis, or benzodiazepines are present. We were interested to see if

our compounds gave positive results for amphetamine and methamphetamine. When the

standard compounds were tested; DMAA, phenethylamine and hordenine gave a positive

result for both amphetamine and methamphetamine (table 1); whereas methysynephrine

and synephrine gave a positive result for methamphetamine. DMAA and phenethylamie

are both primary amines and methylsynephrine and synephrine are both secondary

amines. These structural similarities influence how they interact in the immunoassay test.

Hordenine is a tertiary amine and gave a positive result for both amphetamine and

methamphetamine.

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Table 2 - Immunoassay results for the pure compounds

Compound Results

Amphetamine Methamphetamine

Methylsynephrine - +

Synephrine - +

Hordenine + +

Phenethylamine + +

DMAA + +

When the pre-workout supplements were tested using the immunoassay; Thermojet,

Black Bombs and Mesomorph gave positive results for both amphetamine and

methamphetamine; while Hydroxycut, Hyper FX, Fast Fuel, and Black Powder gave

negative results for both amphetamine and methamphetamine(table 2). These results

suggest that the four pre-workout supplements that tested negative do not contain any of

the five standard amphetamine-like compounds of interest; however, as this is only a

presumptive test, a confirmative test is required to confirm these results.

Table 3 - Immunoassay results for the Pre-workout supplements

Pre-workout

supplement

Results

Amphetamine Methamphetamine

Hydroxycut - -

Hyper FX - -

Black Powder - -

Thermojet + +

Black Bombs + +

Fast Fuel - -

Mesomorph + +

Functional Group tests

The functional group tests we performed used the Marquis, Simon, Robadope, and Lucas

reagents. The Maquis reagent was used to determine the presence of a primary or

secondary amine. The Simon and Robadope reagents can differentiate between primary

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and secondary amines, and the Lucas reagent differentiates between primary, secondary,

and tertiary alcohols.

When synephrine, hordenine and methylsynephrine were mixed with the Marquis reagent

the white powders changed to a brown-orange colour. This is also the case when

amphetamine or methamphetamine and the Marquis reagent react (National Institute of

Justice, 2000). When DMAA was added to the Marquis reagent no colour change was

observed. This was expected because, apart from the benzene ring, it is structurally

identical to amphetamine (Fig. 9). This result suggests that the benzene ring is critical to

producing the carbocation. The stabilisation of the carbocation is due to the resonance

structures proposed in figure 10, which are not formed with DMAA. Phenethylamine was

a clear liquid and solidified into a white crystal when the Marquis reagent reacted it

turned black. The black colour has been regarded as the colour that the Marquis reagent

turns in the presence of methylenedioxy compounds; such as MDA, and MDMA (Fig.

11); however, phenethylamine is not a methylenedioxy compound.

Figure 9 - Similarity between Amphetamine (1) and DMAA (3)

Figure 10 – Possible resonance structures of the Marquis test’s product

1 3

28 | P a g e

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Figure 11 – Possible mechanism of the Marquis test with MDMA

When DMAA, methylsynephrine, synephrine, phenethylamine, and hordenine were

mixed with the Simon reagent; the Methylsynephrine and synephrine turned blue,

phenethylamine did not react, and hordenine and DMAA both turned pink. Both

methylsynephrine and synephrine are secondary amines and gave the expected colour

change (National Institute of Justice, 2000). Also, hordenine gave the expected colour

change for tertiary amines, as did DMAA; however it is a primary amine.

Phenethylamine was found to react only with the Robadope reagent, turning purple;

which is the expected colour as it is a primary amine (Kovar & Laudszun, 1989). DMAA

is also a primary amine; however, it did not react with the Robadope reagent.

When methylsynephrine, synephrine and hordenine were mixed with the Lucas reagent,

no reaction occurred. This is because, although they are secondary alcohols, the reagent

does not react with phenols. This was discovered when no reaction took place when

hydroxybenzene was mixed with the Lucas reagent. The Lucas reagent has been shown to

react only with alcohols that are hexyl or smaller (Lucas, 1930).

29 | P a g e

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The pre-workout supplements were also tested against these functional group tests, but

their results were found to be too complicated as they are not pure compounds. No

conclusions can be made and the practise of spot testing pre-workout supplements is

impossible due to their nature.

Confirmatory tests

The pre-workout supplements was also analysed in a set of confirmatory steps. In order to

confirm the presence of a compound, chromatography followed by MS is needed.

However, before HPLC analysis the pre-workout supplements needed to be semi-purified.

Extraction Method

The method used in the extraction process is summarised in figure 12. The pH of the

initial solution was increased to 12 so that any amines in the pre-workout supplements are

forced into their neural state. This means that it is less polar and so migrates to the

organic phase. Ethyl acetate was decided on as the organic phase as it is the most polar

solvent that is not water miscible. This extraction method was validated by taking a

sample of each phase of each step of the extraction of synephrine. Then, through the use

of a concentration curve, the concentrations of synephrine in each of these samples were

determined. At a concentration of 0.001mg/mL, the area under the peak at 211nm was

14.83, at 0.01mg/mL the area was 213.31, 0.05mg/mL was 1147.8, 0.1mg/mL was

2346.5, 0.5mg/mL was 12521.7, and finally 1mg/mL had an area of 20804.5. The

equation of the line formed by these points on the concentration curve (Fig. 13) was

found to be .

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Figure 12 - Extraction method

Figure 13 - Concentration curve for Synephrine at 211nm

The 0.5mg/mL sample from the concentration curve was used as the pre-extraction

solution, and when the water phase of the first step of the extraction was analysed the

concentration of synephrine that was found to not have disused into the ethyl acetate

phase was 0.39mg/mL. The hydrochloric acid phase of the second step was found to

contain a synephrine concentration of 0.20mg/mL. The amount of synephrine in the pre-

R² = 0.9909

0

5000

10000

15000

20000

25000

0 0.2 0.4 0.6 0.8 1 1.2

Are

a u

nd

er

the

pe

ak a

t 2

11

nm

Synephrine conc. (mg/mL)

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extraction solution was 5µg, and by the end of the extraction process there was 1.01µg of

synephrine, therefore the extraction method produced a yield of 20.23% (See Appendix

2).

HPLC

Firstly a mixture of standards were analysed using HPLC, coupled with UV

spectrophotometry by Faith Rose of the Griffith University Institute of Glycomics.

Synephrine eluted first with a retention time of 5.06 minutes, then came

methylsynephrine with 6.33 minutes, hordenine with 7.49 minutes, phenethylamine with

9.95 minutes (Fig. 14) and lastly caffeine with 13.08 minutes (Fig. 15). UV

spectrophotometry was used to detect DMAA but the detection was limited as DMAA

does not have a large UV Chromataphore (i.e. is not aromatic). At high concentration

DMAA it eluted as a broad split peak with retention times of 11.98 and 12.29 (Fig. 16).

Figure 14 - Chromatogram of Standards Mixture [synephrine (a), methylsynephrine (b), hordenine (c), and

phenethylamine (d)] at 211nm detection

b

a c d

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Figure 15 - Chromatogram of Caffeine at 211nm detection

Figure 16 - Chromatogram of DMAA at 211nm detection

The extracts of each of the pre-workout supplements were also analysed using HPLC,

coupled with UV spectrophotometry to determine if our standard compounds were

present (See Appendix 3). Analysis of Hydroxycut resulted in only caffeine being present,

which confirmed Hydroxycut’s label. Analysis of Hyper FX also showed only caffeine

(Fig. 17); this contradicts Hyper FX’s label which listed ‘bitter orange extract’ (citrus

aurantium) as one of its ingredients, with ‘30% synephrine’ written in brackets.

m in0 5 10 15 20 25

m AU

0

500

1000

1500

2000

D A D 1 B , S ig=211,4 R ef=360,100 (A M P H E TA M IN E -TYP E \24092013000006.D )

1

3.0

84

C affe ine 0 .27µg /m L 10 µL

211 nm

m in0 5 10 15 20 25

m AU

0

500

1000

1500

2000

2500

D A D 1 B , S ig=211,4 R ef=360,100 (A M P H E TA M IN E -TYP E \E XTR A C T000003.D )

Therm oje t

1

.9

50

2

.7

38

3

.3

63

1

1.4

79

Area: 38511.7

1

3.0

42

211 nm

m in0 5 10 15 20 25

m AU

100

200

300

400

D A D 1 B , S ig=211,4 R ef=360,100 (A M P H E TA M IN E -TYP E \S YN E P R IN E 000004.D )

4

.9

93

S ynephrine 0 .1 m g/m L211 nm

m in0 5 10 15 20 25

mAU

-150

-100

-50

0

DAD1 B, Sig=211,4 Ref=360,100 (AMPHETAMINE-TYPE\EXTRACT000005.D)

211 nm

10 mg/mL 10µL

11

.97

8

12

.28

5

m in0 5 10 15 20 25

mV

0

200

400

600

800

1000

1200

ELS1 A, Signal Voltage (AMPHETAMINE-TYPE\EXTRACT000005.D)

DM

AA

10 mg/mL 10µL

ELSD Are

a: 56755.5

12

.44

7

m in0 5 10 15 20 25

mAU

-175

-150

-125

-100

-75

-50

-25

DAD1 B, Sig=211,4 Ref=360,100 (AMPHETAMINE-TYPE\24092013000005.D)

1 mg/mL 10µL

211 nm

1.9

98

2.9

29

4.1

19

12

.47

8 1

2.7

26

m in0 5 10 15 20 25

mV

0

20

40

60

80

100

120

ELS1 A, Signal Voltage (AMPHETAMINE-TYPE\24092013000005.D)

1 mg/mL 10µL

ELSD Are

a: 3472.7

5

12

.91

5

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Figure 17 - Chromatograms of Hyper FX extract and a mixture of the Hyper FX extract and the Standards

Mixture at 211nm detection

Analysis of Black Powder showed caffeine, hordenine and phenethylamine being present

(Fig. 24). However, as both hordenine and phenethylamine gave positive immunoassay

responses for both amphetamine and methamphetamine on their own, the compounds

must not have been present in high enough concentrations in the pre-workout supplement

to be detected by the immunoassay cup. Each drug compound has a set concentration that

it must be at least for it to be considered a positive result with the immunoassay cup, these

cut off levels are those required by standards set by the Substance Abuse and Mental

Health Service Administration.

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The label of Black Powder only mentioned caffeine. Analysis of Fast Fuel when not

spiked showed caffeine and a compound being at retention time = 5.966 minutes. This

was shown to be methylsynephrine when the extract was spiked and there was only one

peak at 5.049 (Fig. 18). The presence of methylsynephrine in Fast Fuel is very low as

shown by the small peak at 5.966, but it was not mentioned on the label, this and the

immunoassay result did not indicate its presence but this is most likely due to its low

concentration.

Figure 18 - Chromatograms of Fast Fuel extract and a mixture of the Fast Fuel extract and the Standards


The HPLC analysis revealed the presence of caffeine, synephrine, methylsynephrine, and

phenethylamine in the pre-workout supplement Black Bombs (Fig. 19). This confirms the

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immunoassay result and partly confirms Black Bombs’ label, which listed caffeine,

acacia rigidula and citrus aurantium; however, the label didn’t mention

methylsynephrine. Methylsynephrine, caffeine and phenethylamine were also discovered

in Mesomorph, which again confirms the immunoassay result and partly confirms the

label; as the label listed caffeine and acacia rigidula but also didn’t mention

methylsynephrine (Fig. 20).

Figure 19 - Chromatograms of Black Bombs extract and a mixture of the Black Bombs extract and the

Standards Mixture at 211nm detection

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Figure 20 - Chromatograms of Mesomorph extract and a mixture of the Mesomorph extract and the Standards


Thermojet was also analysed; and synephrine, methylsynephrine, hordenine, and

phenethylamine were found to not be present, however caffeine was detected (Fig 21).

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Figure 21 - Chromatogram of Thermojet extract at 211nm detection

To detect for DMAA the detection method was changed to ELSD as the concentration of

DMAA was too low to be detected with UV spectrophotometry. Using ELSD, the broad

peaks of DMAA are at 12.92 and 13.12 minutes (Fig. 22). Analysis of Thermojet showed

possible DMAA peaks at 13.22 and 13.42 minutes (Fig. 23). DMAA was also possibly

detected in Black Powder and Black Bombs at low concentrations (Figures 24, and 19),

and in Mesomorph at a higher concentration (Fig. 20). These peaks in these four pre-

workout supplements could be DMAA; however, it cannot be said for certain from these

results, so MS needed to be conducted to prove the presence of DMAA.

Figure 22 - Chromatogram of DMAA with ELSD

min0 5 10 15 20 25

mAU

0

500

1000

1500

2000

2500

DAD1 B, Sig=211,4 Ref=360,100 (AMPHETAMINE-TYPE\EXTRACT000003.D)

1.950

2.738

3.363

11.47

9

Area

: 38511.7 1

3.04

2

m in10 10.5 11 11.5 12 12.5 13 13.5 14 14.5

m V

0

50

100

150

200

250

300

350

ELS1 A, S ignal Voltage (AM PHETAM INE-TYPE\EXTRACT000003.D )

13

.21

5

13

.41

5

Therm ojet Extract

ELSD

m in10 10.5 11 11.5 12 12.5 13 13.5 14 14.5

m V

0

50

100

150

200

250

300

350

ELS1 A, S ignal Voltage (AM PHETAM INE-TYPE\24092013000005.D )

Are

a: 1668.7

4

12

.91

5

Are

a: 1750.5

1

13

.12

1

DM AA 1 m g/m L

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Figure 23 – Fraction of the Chromatogram of Thermojet with ELSD

Figure 24 - Chromatogram of Black Powder extract at 211nm detection

LC-MS

After the pre-workout supplements were each analysed with HPLC by the Institute of

Glycomics, they were then analysed again using liquid chromatography coupled with

mass spectrometry by Ben Matthews of Griffith University’s School of Biomolecular and

Physical Sciences. The components that made up each of the pre-workout supplements

were identified based on their retention times and mass spectral fragmentation patterns

(Japan Science and Technology Agency, 2013). Fragmentation patterns are unique and so

conclusively prove the presence of the compounds. All of the compounds are amines,

however, and the fragmentation patterns of amines are dominated by α-cleavage at the

Carbon alpha to the Nitrogen.

m in10 10.5 11 11.5 12 12.5 13 13.5 14 14.5

m V

0

50

100

150

200

250

300

350

ELS1 A, S ignal Voltage (AM PHETAM INE-TYPE\EXTRACT000003.D )

13

.21

5

13

.41

5

Therm ojet Extract

ELSD

m in10 10.5 11 11.5 12 12.5 13 13.5 14 14.5

m V

0

50

100

150

200

250

300

350

ELS1 A, S ignal Voltage (AM PHETAM INE-TYPE\24092013000005.D )

Are

a: 1668.7

4

12

.91

5

Are

a: 1750.5

1

13

.12

1

DM AA 1 m g/m L

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Caffeine’s fragmentation pattern showed three distinct peaks with mass to charge ratios

(m/z) of 194.0, 109.0, and 55.0 (Fig. 25) (Japan Science and Technology Agency, 2013).

This fragmentation pattern is due to the breakdown of the parent ion ([M+H]+=195)

(C8H10N4O2+, m/z=194) into smaller ions, e.g. C5H7N3

+ (m/z=109) and C3H5N

+ (m/z= 55)

(Fig26). The fragmentation pattern of synephrine showed three distinct peaks with m/z of

107.2, 91.0, and 77.1 (Fig. 27) (Japan Science and Technology Agency, 2013). This is

due to the breakdown of the parent ion ([M+H]+=168.2) into smaller ions; e.g. C7H7O

+

(m/z=107.2), C7H7+ (m/z=91) and C6H5

+ (m/z=77.1) (Fig. 28).

Figure 25 - Mass spectral Fragmentation pattern of Caffeine (Japan Science and Technology Agency, 2013)

Figure 26 – Possible fragmentation of Caffeine

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Figure 27 - Mass spectral Fragmentation pattern of Synephrine (Japan Science and Technology Agency, 2013)

Figure 28 – Possible fragmentation of Synephrine

Hordenine’s fragmentation pattern showed three distinct peaks with m/z of 103.0, 91.0,

and 77.0 (Fig. 29) (Japan Science and Technology Agency, 2013). This fragmentation

pattern is due to the breakdown of the parent ion ([M+H]+=166) into smaller ions; e.g.

C8H7+ (m/z=103), C7H7

+ (m/z=91) and C6H5

+ (m/z=77) (Fig. 30). The fragmentation

pattern of phenethylamine only showed one distinct peak with an m/z of 30.0 (Fig. 31)

(Japan Science and Technology Agency, 2013). This is due to the breakdown of the

parent ion ([M+H]+=122) into smaller ions; e.g. CH4N

+ (m/z=30) (Fig. 32).

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Figure 29 - Mass spectral Fragmentation pattern of Hordenine (Japan Science and Technology Agency, 2013)

Figure 30 – Possible fragmentation of Hordenine

Figure 31 - Mass spectral Fragmentation pattern of Phenethylamine (Japan Science and Technology Agency, 2013)

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Figure 32 - Possible fragmentation of Phenethylamine

DMAA’s fragmentation pattern showed several distinct peaks with m/z of 116.2, 99.2,

75.2, 57.3, and 43.5 (Li, Chen, & Li, 2012) (Fig. 33). This is due to the breakdown of the

parent ion ([M+H]+=116.2) (C7H18N

+) into smaller ions; e.g. C7H15

+ (m/z=99.2), C4H9

+

(m/z=57.3) and C3H7+ (m/z=43.5) (Fig. 34). However, a paper in 2013 (Lopez-Avila &

Zorio)stated that the highest peak should be the immonium ion (CH3N+, m/z=44).

Methylsynephrine is a new compound to be discovered and its fragmentation pattern has

not been studied in depth. However, in 2010, an article published in the Journal of

Chromatography A mentioned the in-source fragment ion of oxilofrine (methylsynephrine

(C10H15NO2)) having an m/z of 164.11 (C10H14NO+) (Badoud, et al., 2010). This ion is

also described as the precursor ion, with its product ions having m/z of 149.13

(C9H11NO+), 133.07 (C9H9O

+), and 105.12 (C7H5O

+) (Fig. 35).

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Figure 33 - Mass spectral Fragmentation pattern of DMAA (Li, Chen, & Li, 2012)

Figure 34 – Possible fragmentation of DMAA

Figure 35 – Possible fragmentation of Methylsynephrine

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Hydroxycut’s Ms (Appendix 4) showed fragmentation patterns consistent with

methylsynephrine, phenethylamine and caffeine. The MS for Hyper FX (Appendix 5)

showed fragmentation patterns consistent with synephrine, hordenine and caffeine. These

results are summarised in table 3, along with the HPLC results for these two pre-workout

supplements.

Table 4 - Summary of results for Hydroxycut and Hyper FX

Hydroxycut Hyper FX

Methylsynephrine

Label - -

HPLC - -

LCMS + -

Synephrine

Label - +

HPLC - -

LCMS - +

Phenethylamine

Label - -

HPLC - -

LCMS + -

Hordenine

Label - -

HPLC - -

LCMS - +

DMAA

Label - -

HPLC - -

LCMS - -

Caffeine

Label + +

HPLC + +

LCMS + +

Black Powder’s MS (Appendix 6) showed fragmentation patterns consistent with

methylsynephrine, phenethylamine, DMAA and caffeine. The MS of Black Bombs

(Appendix 7) showed fragmentation patterns consistent with hordenine and caffeine.

These results are summarised in table 4, along with the HPLC results for these two pre-

workout supplements.

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Table 5 - Summary of results for Black Powder and Black Bombs

Black Powder Black Bombs

Methylsynephrine

Label - -

HPLC - +

LCMS + -

Synephrine

Label - +

HPLC - +

LCMS - -

Phenethylamine

Label - +

HPLC + +

LCMS + -

Hordenine

Label - +

HPLC + -

LCMS - +

DMAA

Label - -

HPLC ? ?

LCMS + -

Caffeine

Label + +

HPLC + +

LCMS + +

Fast Fuel’s MS (Appendix 8) showed fragmentation patterns consistent with

methylsynephrine, phenethylamine, hordenine, DMAA and caffeine. Fragmentation

patterns consistent with methylsynephrine, phenethylamine, hordenine and caffeine were

found in Mesomorph’s MS (Appendix 9). The MS of Thermojet (Appendix 10) showed

fragmentation patterns consistent with methylsynephrine, phenethylamine, hordenine,

DMAA and caffeine. These results are summarised in table 5, along with the HPLC

results for these three pre-workout supplements.

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Table 6 - Summary of results for Fast Fuel, Mesomorph and Themojet

Fast Fuel Mesomorph Thermojet

Methylsynephrine

Label - - -

HPLC + + -

LCMS + + +

Synephrine

Label - - -

HPLC - - -

LCMS - - -

Phenethylamine

Label - + +

HPLC + + -

LCMS - + +

Hordenine

Label - + -

HPLC - - -

LCMS + + +

DMAA

Label - - +

HPLC - + ?

LCMS + - +

Caffeine

Label + + +

HPLC + + +

LCMS + + +

Quantification of Synephrine

One of the compounds that is of particular interest is synephrine, as there was a report

linking the abuse of tablets containing synephrine and a large myocardial infarction in a

male aged 28 (Keogh & Baron, 1985). Although synephrine was found in one of the pre-

workout supplements, Hyper FX, it was only found using LC-MS. This means that

quantification is not possible.

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Conclusions and Further Work

In conclusion, the regulations governing the contents of pre-workout supplements need to

be better enforced. DMAA, which is not found in plant extracts, is illegal to be sold in

pre-workout supplements, yet it was detected in three of the pre-workout supplements

tested, although Thermojet had already been taken of the market by the time this study

was performed. Methylsynephrine is also listed on a prohibited schedule to be sold in pre-

workout supplements, due to its structural similarity to ephedrine, yet it was detected in

five of the seven pre-workout supplements tested. In future, the qualitative aspects of

these compounds should be tested, i.e. how much of these compounds are in the pre-

workout supplements.

Future work could also analyse a wider range of pre-workout supplements for the

presence and amount of these banned compounds, and if these amounts are enough to

cause effects, and or health risks.

Due to conflicting evidence, methylsynephrine should be investigated further, to

characterise the specific alpha adrenergic receptors with which it interacts or their indirect

mode of action as stimulants. This further study would be best tested in animal models.

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Experimental

Presumptive tests

Immunoassay

A 10mg/mL solution was made up of each of the compounds and they were tested using

DrugCheck’s ‘NxStep Onsite Urinalysis Test Cup’s. A 10mg/mL solution was made up

of each of six of the compounds (Hyper FX, Black Powder, Thermojet, Black Bombs,

Fast Fuel, and Mesomorph) and they were also tested using the ‘NxStep Onsite Urinalysis

Test Cup’. The seventh pre-workout supplement (Hydroxycut) came as a capsule and so

one capsule was dissolved in 10 mL of water, and this solution was tested.

Functional Group tests

Protocol 1 - Marquis – 40% formaldehyde (1mL) and concentrated sulphuric acid

(20mL) (National Institute of Justice, 2000). :

Two drops of Marquis reagent were added to a sample of each powder or, in the case of

phenethylamine, neat solution; and the colour change was noted.

Protocol 2 - Simon – Solution A – sodium nitroprusside (400mg) in distilled water

(20mL) and acetaldehyde (800µL).

– Solution B – sodium carbonate (400mg) in distilled water

(20mL) (National Institute of Justice, 2000).

One drop of solution A of Simon reagent was added to each powder or, in the case of

phenethylamine, neat solution; and then two drops of solution B, and the colour change

was noted.

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Protocol 3 - ‘Robadope’ – Solution A – sodium nitroprusside (400mg) in distilled

water (20mL) and acetone (800µL).

– Solution B – sodium carbonate (400mg) in distilled water

(20mL) (National Institute of Justice, 2000).

One drop of solution A of ‘Robadope’ reagent was added to each powder or, in the case

of phenethylamine, neat solution; and then two drops of solution B, and the colour change

was noted.

Protocol 4 – Lucas – anhydrous zinc chloride (32g) in concentrated hydrochloric acid

(20mL) (Lucas, 1930).

Two drops of 10mg/mL solutions of the powders or, in the case of phenethylamine, a 1%

solution were added to 12 drops of the Lucas reagent in culture tubes, and the clarity was

noted both immediately and at five minutes.

Confirmatory tests

Extraction

Protocol 5 – Powdered Pre-workout supplements

2.5g of each pre-workout supplement in water (25mL) was adjusted to pH 12. The

aqueous phase was extracted with ethyl acetate then the ethyl acetate layer extracted with

HCl (1M). The second aqueous layer was used for analysis.

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Protocol 6 - Hydroxycut

Two capsules of pre-workout supplement in water (25mL) were adjusted to pH 12. The

aqueous phase was extracted with ethyl acetate then the ethyl acetate layer extracted with

HCl (1M). The second aqueous layer was used for analysis.

HPLC

Protocol 7:

Separation was performed using a Phenyl Hexyl (4.6x150 mm) column, with an injection

volume of 12 µL. The mobile phase consisted of Methanol and 0.08%TFA:0.1%TFA;

with the methanol on a gradient of 1-60% in 15 minutes, stay for 7 mins, then back down

in 3 mins. Chromatographic separation was performed at a flow rate of 0.8 mL/min at

40°.

ELSD Conditions: 39°C; Gas flow: 1.4L/min; Gain: 2

UV: detection wavelength at 211nm

LC-MS

Protocol 8:

All samples where centrifuged at 13000 G and the supernatants filtered through 0.22 um

PTFE filters to remove particulates prior to analysis.

Initial separation was performed using 2 µl injections onto an Agilent 1290 HPLC using

an Agilent Eclipse plus C18 (2.1x100 mm with 1.7 um particle size) column. The mobile

phases consisted of water with 0.1% formic acid as the aqueous phase (A) and 95%

acetonitrile with 5% water and 0.1% formic acid as the organic phase (B).

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Chromatographic separation was performed at a flow rate of 0.8 ml/min under the

following conditions:

Time (min)

Concentration of organic phase

(B)

0 - 15 1% to 60% gradient.

15 - 22 60% isocratic

22.0 - 22.1 60% to 90%

22.1 - 25 90% isocratic

25 – 25.1 90% to 1%

25.1 to 28 1% isocratic

High Resolution mass spectrometry data was obtained on an Agilent 6530 QTOF

spectrometer with a jet stream-ESI source. All data was acquired in positive mode using

auto-MSMS. In brief, this acquisition mode performed MSMS fragmentation at 10 and 20

CID on either specified target ions or the three most abundant ions in each spectrum.

Blank injection controls where included to determine background compounds due to

system contaminants. Background compounds detected in the blank samples were

removed from the compound lists of the samples.

The data was analysed using the Agilent Mass Hunter Qualitative analysis software

package. Putative compounds where determined using the “Find by AutoMSMS”

function.

Putative identification of detected compounds was performed by accurate mass screening

against the Forensic Toxicology Database (Agilent Technologies). Accurate mass

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screening is based on the empirical formula of an ion determined using accurate mass,

isotopic spacing and isotopic distribution. This is then compared to a library of known

compounds. Comparison using only accurate mass and empirical formula has no capacity

to discriminate between structural isomers of the same compound.

Concentration Curve

Protocol 9:

HPLC conditions: 10 µL Injection volume; 1-60%Methanol +0.08%TFA:0.1%TFA in 15

mins, stay for 2 mins, then back down in 3 mins. 40ºC; 0.8 mL/min; PDA and ELSD

detection: Phenomenx Phenyl Hexyl column 150x4.6 mm

UV: detection wavelength at 211nm

6 Syneprine standards were prepared and ran under the above conditions and a standard

curve prepared from 0.001-1 mg/mL.

Compounds

Caffeine (1)

Reverse phase HPLC (protocol 7) gave 1 with a retention time of 13.08min. LCMS

(ESI) m/z (%): 194.0000 ([M]+, 100), 109.0000 ([C5H7N3]

+, 71), 55.0000 ([C3H5N]

+, 51)

DMAA (6)

Reverse phase HPLC (protocol 7) gave 6 with a split retention time of 11.98 and

12.29min. LCMS (ESI) m/z (%): 116.2 ([M+H]+, 20), 99.2 ([M+H-NH3]

+, 6), 57.3

([C4H9]+, 100), 43.5 ([C3H7]

+, 5)

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Hordenine (8)


(ESI) m/z (%): 103 ([C8H7]+, 52), 91 ([C7H7]

+, 67), 77 ([C6H5]

+, 100)

Methylsynephrine (9)


(ESI) m/z: 164.11 ([M-OH]+), 149.13 ([M-OH-CH3]

+), 133.07 ([C9H9O]

+), 105.12

([C7H5O]+)

Phenethylamine (5)


(ESI) m/z (%): 30 (CH2NH2]+, 100)

Synephrine (10)


(ESI) m/z (%):107.2 ([C7H7O]+, 100), 91 ([C7H7]

+, 41), 77.1 ([C6H5]

+, 71)

Supplements

Hydroxycut

Reverse phase HPLC (protocol 7) gave 1 (retention time of 13.03min). LCMS (protocol

8) (ESI) m/z (%):182.12 ([9 M+H]+, 18), 122.10 ([5 M+H]

+, 77), 195.08 ([1 M+H]

+, 83).

Hyper FX

Reverse phase HPLC (protocol 7) gave 1 (retention time of 13.07min). LCMS (protocol

8) (ESI) m/z (%):168.10 ([10 M+H]+, 0.8), 166.12 ([8 M+H]

+, 2), 195.08 ([1 M+H]

+,

100).

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Black Powder

Reverse phase HPLC (protocol 7) gave 1 (retention time of 13.08min), 6 (retention time

of 12.3min), 5 (retention time of 10.23min), and 8 (retention time of 7.73min). LCMS

(protocol 8) (ESI) m/z (%):182.12 ([9 M+H]+, 22), 122.09 ([5 M+H]

+, 87), 116.14 ([5

M+H]+, 1), 195.09 ([1 M+H]

+, 100).

Black Bombs


of 11.82min), 5 (retention time of 10.02min), 9 (retention time of 6.33min), and 10

(retention time of 4.97min). LCMS (protocol 8) (ESI) m/z (%):116.12 ([8 M+H]+, 11),

195.09 ([1 M+H]+, 100).

Fast Fuel

Reverse phase HPLC (protocol 7) gave 1 (retention time of 13.07min), and 9 (retention

time of 5.97min). LCMS (protocol 8) (ESI) m/z (%): 182.12 ([9 M+H]+, 0.2), 166.12 ([8

M+H]+, 5), 116.14 ([6 M+H]

+, 6), 195.08 ([1 M+H]

+, 100).

Mesomorph


of 12.07min), 5 (retention time of 10.17min), and 9 (retention time of 6.38min). LCMS

(protocol 8) (ESI) m/z (%): 182.11 ([9 M+H]+, 33), 166.12 ([8 M+H]

+, 0.4), 195.08 ([1

M+H]+, 100).

Thermojet

Reverse phase HPLC (protocol 7) gave 1 (retention time of 13.04min), and 6 (retention

time of 13.22 and 13.42min). LCMS (protocol 8) (ESI) m/z (%): 182.12 ([9 M+H]+, 4),

166.12 ([8 M+H]+, 0.4), 122.09 ([5 M+H]+, 21), 116.14 ([6 M+H]+, 0.1), 195.09(1

M+H]+, 100).

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Appendices

Appendix 1 – Names

Caffeine

o IUPAC – 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione

o 1,3,7-trimethylxanthine

o Trimethylxanthine

o Methylxanthine

DMAA

o IUPAC – 4-Methylhexan-2-amine

o 2-amino-4-methylhexane

o Dimethylamylamine

o 1,3-dimethylamylamine

o 1,3-dimethylpentylamine

o 4-methyl-2-hexanamine

o 4-mehyl-2-hexylamine

Hordenine

o IUPAC – 4-(2-Dimethylaminoethyl)phenol

o N,N-Dimethyltyramine

o Peyocactin

o Anhaline

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Methylsynephrine

o IUPAC – (1S,2R)-(±)-4-(1-Hydroxy-2-methylamino-propyl)phenol

o Oxilofrine

o 4-hydroxyephedrine

o Hydroxyephrine

Phenethylamine

o IUPAC – 2-phenyl-2-ethanamine

o β-phenethylamine

o Phenylethylamine

Synephrine

o IUPAC – 4-[1-hydroxy-2-(methylamino)ethyl]phenol

o P-synephrine

o Oxedrine

o Sympatol

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Appendix 2 - Extraction Validation

Step 1 (Water)

mg/mL( decimal places)

Step 2 (HCl)

mg/mL( decimal places)

step

g

step pre extraction

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Appendix 3 – UV Chromatographs

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Appendix 4 – LC/MS Results – Hydroxycut

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Appendix 5 - LC/MS Results – Hyper FX

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Appendix 6 - LC/MS Results – Black Powder

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Appendix 7 - LC/MS Results – Black Bombs

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Appendix 8 - LC/MS Results – Fast Fuel

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Appendix 9 - LC/MS Results – Mesomorph

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Appendix 10 - LC/MS Results – Thermojet

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