Are Methylxanthine levels in my beverage dangerous?

In the gloom and grogginess of the morning, most of us start the day with a little pick-me-up like a nice cup of joe. After enjoying the aromatic coffee, we might drink a can of soda or two and maybe a can or Red Bull to battle the urge for a siesta in the afternoon. All of these drinks contain a certain level of caffeine, a methylxanthine, which acts as a stimulant for our nervous system, warding off drowsiness and restoring alertness albeit temporarily. However, it may have harmful effects when consumed in high concentrations.

▼A methylxan- what?

Methylxanthines are a group of alkaloids that are commonly used in treatment as stimulants or as bronchodilators in the case of asthma medication. However, side effects of methylxanthines like caffiene, theobromine and theophylline can include increased heart rate, force of contraction and cardiac arrhythmias. At high concentrations, symptoms such as tremor, nausea, nervousness, tachycardia or even convulsions that are resistant to anticonvulsants can occur. With the average adult consuming about 250 mg of caffeine a day, it is essential that we study the levels of methylxanthines in our beverages in order to avoid such serious health effects. The most common methylxanthines in food and beverages are caffeine, theobromine and theophylline.

▼Most common methylxanthines.

Being the most common in foods and beverages, calculating their concentrations in beverages would suffice in concluding if the levels of methylxanthine in our beverages are too high. But before doing any analysis, we must first understand their chemical properties.

Chemical Properties

Caffeine

Main functional group: Amine, Amide, Alkene, Purine

IUPAC name: 1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione

Molecular Formula: C8H10N4O2

Molecular Weight: 194.1906

Density: 1.23 g/cm³

Melting point: 238°C (sublimes)

Boiling point: 178°C (theoretical)

State at rtp: White colorless powder

Odour: None

Soluble in Water: 21600mg/L of H2O

Chromophores: Purine

Theobromine

Main functional group: Amine, Amide, Alkene, Purine

IUPAC name: 3,7-dimethyl-1H-purine-2,6-dione

Molecular Formula: C7H8N4O2

Molecular Weight: 180.164

Density: 1.50 g/cm³

Melting point: 290-295°C (sublimes)

Boiling point:  357°C (theoretical)

State at rtp: White colorless powder

Odour: None

Soluble in Water:  7360mg/L of H2O

Chromophores: Purine

Theophylline

Main functional group: Amine, Amide, Alkene, Purine

IUPAC name: 1,3-dimethyl-7H-purine-2,6-dione

Molecular Formula: C7H8N4O2 

Molecular Weight: 180.164

Density: 1.47 g/cm³

Melting point: 271-273°C (sublimes)

Boiling point: 454°C (theoretical)

State at rtp: White colorless powder

Odour: None

Soluble in Water: 330mg/L of H2O

Chromophores: Purine

Methods

Based on the properties of these few methylxanthines, we are able to come up with a few methods of quantifying the amounts of methylxanthine in several sample beverages. 

A) Micellar Electrokinetic Capillary Chromatography (MEKC)

Micellar Electrokinetic Capillary Chromatography (MEKC) is a chromatographic technique that is able to quantify the amount of methylxanthine in the sample. Samples are brewed in hot water and subsequently filtered to remove particulate matter before the chromatography

Pros: It is a very high resolution separation method.

Cons: Long separation time of >30 mins with poor resolution of late eluting peaks.

B) Gas Chromatography (GC)

Gas Chromatography (GC) is another viable method of quantifying the amount of methylxanthine in the sample. Samples are extracted using liquid-liquid extraction method and evaporated under a stream of nitrogen then reconstituted in 100 μL of methanol before analysis by the GC to ensure flash vaporisation. In a similar application, Helium was used as the carrier gas along with a Nitrogen-phosphorus detector (NPD)

Pros: Small amounts of sample is sufficient for analysis. Peak concentration is proportional to peak area, making it suitable for quantitative analysis.

Cons: Most of the methylxanthines are of low volatility hence requiring chemical derivatization. The liquid-liquid extraction of the samples is also a causative factor for more errors.

C) High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC) is also an available option for the quantitation of the amount of methylxanthine in the sample. Samples are dissolved in hot water. Methanol-water-acetic acid should be used as a mobile phase with a suitable detector.
Pros: Fast analysis time without need for chemical derivatization.

Cons: Irreversibly adsorbed compounds not detected, co-elution difficult to detect.

Selected analysis method: High-Perfomance Liquid Chromatography (HPLC)

As the samples require no pre-treatment(aside from dissolving in hot water) or chemical derivatization needed for analysis and the fact that the elution time is extremely short as compared to MEKC, HPLC would be the most preferred analysis method. Some researchers have also favoured HPLC as the method best suited for the analysis and have conducted the experiment as such.

Instruments and reagents used:
Chromatograph
Liquid chromatograph brand: Varian liquid chromatograph Model 2510
Injector type: Rheodyne injector with a 20 μL sample loop
Column length: 15cm
Column internal diameter: 4.0mm
Particle size: 5µm
Column stationary phase: Bondesil C18
Mobile phase: Methanol–Water–Acetic acid (20:75:5, v/v/v)
Mode: Isocratic elution
Flow rate of HPLC: 0.7ml/min
Injected volume for LC: 20 µl
Detector: Variable Wavelength Detector (Model: Varian UV–vis detector Model 2550)
Wavelength used for detection: 273nm

Materials
Stock Solutions of Caffeine, Theobromine and Theophylline
Acetic acid
Methanol (HPLC grade)
Pure Water
0.45µm membrane filter
Filter paper
Dark glass flasks
Varian liquid chromatograph Model 2510

Reagent preparation:

Preparation of methylxanthine stock solutions
1. Stock solutions of caffeine, theobromine, and theophylline were prepared by dissolving 40 mg of each in 200 mL of methanol–water (50:50, v/v)
2. Filter through a 0.45-μm membrane filter
3. Store at 40°C in dark-glass flasks.


Preparation of methylxanthine calibration solutions
1. The standard solutions were prepared by dilution in the water of the methylxanthine mixture stock solutions within the concentration range of 0.25 to 60 μg/mL à At least 50x dilution
2. They were stored in the dark-glass flasks at 4°C. In these conditions they remained stable for 60 days.


Sample preparation
1. The soluble coffee was 150mL hot water per 1g of coffee
2. The tea was brewed with the tea-bag (2 g) and 150mL boiling water for 3 min
3. The cocoa consisted of 0.2 g chocolate per 20 mL water and was cleaned from grease by Soxhlet extraction with hexane
4. Coconut water was filtered

Risk assessment

Department: LSCT
Process: High Performance Liquid Chromatography Tandem UV-Vis Spectrophotometry of Methylxanthine levels in beverage samples

1. Hazard Identification				2. Risk Evaluation & Control
SN	Task	Hazards	Possible Consequences	Existing Risk Control (if any)	S	L	R	Additional / New Risk Control	S	L	R	Deadline
1	Elution with Methanol	Flammable and hazardous	Methanol may catch fire and causes skin burn	Place it far away from fire. eg. do not place it near the spirit lamp	3	2	6	Handled under fume hood	3	1	3	26/01/2014
2	Dissolving of samples using hot water from heater	Physical thermal hazard on skin	May cause burns and scalding of skin	Wear heat resistant gloves when handling	2	4	8	-	-	-	-	26/01/2014
3	Handling of hexane when preparing chocolate sample	Flammable and toxic to body	May cause respiratory tract irritation upon inhalation	Handle under fume hood	3	1	3	Place it away from any naked flame available	3	1	3	26/01/2014
4	Detection using HPLC	Methanol might splash out if there are contaminants present	May cause blindness if splashed on the eye	Wear lab goggles	3	4	12	Some labs have specialised areas for HPLC where only people involved in the experiment are allowed access to it, and they must be in complete protective gear.	3	4	12	26/01/2014
5	Clearing organic waste from the waste collector of HPLC	Highly hazardous and flammable	May catch fire, cause skin burn, causes irritation when inhaled and may cause blindness when splashed on eye	Wear chemical-resistant gloves and lab goggles	5	3	15	Handle in fume hood	5	3	15	26/01/2014

Results

Qualitative

▼Graph A and B

Graph A shows the standard methylxanthines and graph B shows the coffee sample with methanol-water-acetic acid through which by comparison of the location of the peak heights show that the method is able to qualitatively analyse the samples and identify the 3 different methylxanthines in the sample.

Quantitative

▼Table I 

▼Table II

Table I above shows the calibration curves for the 3 methylxanthines that were obtained by analysis of the calibration solutions prepared. 

Table II above shows the concentrations of the 3 methylxanthines in the samples that were tested by comparing peak heights of the readings from the analysis and comparing to the corresponding values in the plotted calibration curves.

Discussion

▼Figure 1

▼Figure 2

Choice of Mobile Phase
Most of the methods described in the literature for methylxanthine analysis used mobile phases containing only methanol–water (pH~ 6.14) which allowed for caffeine quantitation but did not separate theophylline from theobromine. Methylxanthines can suffer protonation, which results in ionic species being stabilized (Figure 2). If the pH decreased below 4, the xanthines became protonated and the interaction with C18 reverse-phase columns increased. Using this knowledge, acetic acid was added to the mobile phase (apparent pH < 3) in order to increase the acidity, which resulted in good separation conditions. The HPLC determination was completed in approximately 12 min with the mobile phase methanol–water–acetic acid (20:75:5, v/v/v).

▼Figure 5

Precision Testing

In order to ensure that the experiment is done accurately, a precision test was done by considering the stability of the standards and samples containing methylxanthines as it is one of the fundamental factors for the warranting of exact results. The study of the stability of the methylxanthine standards was conducted for 60 days. The standards were prepared in methanol and stored in dark glass flasks at 4°C. They showed good stability (Figure 5) and could be used safely for at least two months.

The determination of caffeine, theophylline and theobromine was also tested by using seven different aliquots which provided a relative standard deviation of 0.44%, 1.02%, and 0.64%, respectively, in peak-height variation, showing that the method is sufficiently precise. 

Conclusion

Caffeine Levels

This report shows that common beverages consumed around the world all contained caffeine, theophylline and theobromine from the qualitative analysis albeit in different quantities. Through quantitative analysis, it is seen that the amount of methylxanthine present in beverages ranges from < 0.1pg/mL to 350 mg/mL for caffeine, < 0.1 pg/mL to 32 mg/mL for theobromine, and < 0.1 pg/mL to 47mg/mL for theophylline. 

With recommended daily intake limits of methylxanthines for the average person at 400 mg, 39.05 mg and 900 mg for caffeine, theobromine and throphylline respectively, it is quite plausible to hit the threshold in which overdose symptoms like restlessness, cardiac arrhythmia, insomnia and dizziness can happen. In addition, recommended intake limits of methylxanthines can be even lower under other conditions, like in the case where a venti-size cup (~590ml) of our tested coffee A sample would be too much caffeine for pregnant women who have lowered limits of 200 mg a day! Hence, this calls for more precaution and more monitoring of the methylxanthines levels in our beverages we drink daily.

Method
The proposed method was shown to be appropriate for the separation and simultaneous quantitation of caffeine, theobromine, and theophylline in samples of food products, which presented high sensibility and quickness. The analysed samples needed no pretreatment or derivatization. They needed only filtration and if necessary a suitable dilution. Methanol is internationally one of the main solvents employed in liquid chromatography. Its substitution for the usual ethanol in mobile phases used in HPLC systems results in lower costs and toxicity. In this study, it shows the same effectiveness in the separation of methylxanthines. The application of this method can be applied in the control of formulations of medications because the three methylxanthines in this study are generally used as therapeutic agents, and, in food products, mainly because there exists suspicions that caffeine causes teratogenic activity. 

  References:

Bispo, M., Veloso, M., Pinheiro, H., Oliveira, R., Reis, J. and Andrade, J. 2002. Simultaneous Determination of Caffeine, Theobromine, and Theophylline by High-Performance Liquid Chromatography. 40 (1), pp. 45-48. Available from: doi: 10.1093/chromsci/40.1.45.

Injac, R., Srdjenovic, B., Prijatelj, M., Boskovic, M., Karljikovic-Rajic, K. and Strukelj, B. 2008. Determination of Caffeine and Associated Compounds in Food, Beverages, Natural Products, Pharmaceuticals, and Cosmetics by Micellar Electrokinetic Capillary Chromatography. 46 (2), pp. 137-143. Available from: doi: 10.1093/chromsci/46.2.137.

Mccusker, R., Goldberger, B. and Cone, E. 2003. Caffeine Content of Specialty Coffees. 27 (7), pp. 520-522. Available from: doi: 10.1093/jat/27.7.520.

Scribd.com. 2014. Capillary electrophoresis - eletroforese capilar. [online] Available at: http://www.scribd.com/doc/4086007/13/Micellar-electrokinetic-capillary-chromatography [Accessed: 26 Jan 2014].

wiseGEEK. 2014. How Much Caffeine is Consumed By the Average Person?. [online] Available at: http://www.wisegeek.com/how-much-caffeine-is-consumed-by-the-average-person.htm [Accessed: 26 Jan 2014].

Drugs.com. 2014. Theophylline Dosage - Drugs.com. [online] Available at: http://www.drugs.com/dosage/theophylline.html [Accessed: 29 Jan 2014].

Roxby, P. 2011. How much coffee is safe?. [online] Available at: http://www.bbc.co.uk/news/health-15982904 [Accessed: 29 Jan 2014].

Toxnet.nlm.nih.gov. 2014. THEOBROMINE - National Library of Medicine HSDB Database. [online] Available at: http://toxnet.nlm.nih.gov/cgi-bin/sis/search/r?dbs+hsdb:@term+@DOCNO+7332 [Accessed: 29 Jan 2014].