The document summarizes the methods used to test local water samples for caffeine and theobromine. A LC/MS/MS instrument was used to analyze 500 ml water samples that underwent solvent extraction. No significant amounts of caffeine or its derivatives were detected in any samples. While below detection limits, caffeine may still be present at low levels.

1. Data and Results
References
The main instrument used for analysis in this study was a LC/MS/MS produced
by waters, which is a two-part instrument containing a Waters e2695 separations
module, and a Quattro micro micromass mass spectrometer. The two components
were connected by a Phenomenex Kinetex 5µ C18 100Å column that was 150 x
2.1mm. The instrument contained an auto sampler, and the wash used in between
sample collections was a mixture of 50% ASC grade methanol and 50% R.O. water.
There were several different solvent gradients tried, and the primary method
developed used a eluent of 80% R.O. water and 20% ACS grade methanol. The
primary method was also optimized for caffeine and its derivatives, and the MRM
component was programmed to look for all expected mass transitions of
theobromine, caffeine, and theophylline, as indicated by the NIST online database.
The water samples were collected either from public access points, or with
permission of the property owner. Approximately 1000 ml of each sample was
collected using plastic quart Ziploc bags, and were stored in the dark for no longer
than 10 days before undergoing the extraction procedure.
500 ml of each sample was used for the extraction, which was performed by first
using heat to reduce the samples to 100ml, and then adding ACS grade 2-propanol
as the organic phase, and mixing in ACS grade sodium chloride to increase the
Materials and Methods
Discussion
Conclusions
Introduction
After water, coffee and tea are two of the most consumed beverages on the
planet. According to a study done by the FDA, the average American
consumes 300mg of caffeine per day1, and metabolize about 95-98% of it into
paraxanthine, theobromine, and theophylline. In highly populated areas, the
excreted caffeine then goes through standardized waste water treatment where,
on average, 60-70% of the remaining caffeine is removed, and the rest enters
the local streams and rivers intact.
There is growing concern among environmentalists about modern
consumption of caffeine, because for muscles, tadpoles, snails, and other
aquatic life, the caffeine molecule is significantly more toxic than it is to
humans. Caffeine has been shown to be harmful at a mere 0.097mg/L2,
which, to put in perspective, is only 0.045mg/16oz. A recent study on inter-
tidal salt water muscles discovered that they produce significant amounts of
stress proteins when exposed to concentrations as low as 50ng/L, and while
caffeine does not bio-accumulate, reduced populations of these organisms
could cause major shifts in the entire ecosystem.
Multiple previous studies have shown higher than expected amounts of
caffeine in both salt and fresh water, and in North America, there are published
numbers for caffeine content from multiple states. However, since there was
little to no data found in the literature regarding the concentration of caffeine
or its metabolites in the water systems in and around Shepherdstown, this
study examined the levels of caffeine in local rivers, streams, and drinking
water sources.
Acknowledgments
This project was funded by the WV EPSCoR Summer
Undergraduate Research Experience Grant
Testing of Local Water Samples for Caffeine and Theobromine:
Potential Aquatic Pollutants
Megan Behrmann; Jacquelyn Cole, PhD
Shepherd University; Shepherdstown, WV 25443
1) Somogyi, L. P. Caffeine Intake by US population. USDA report,
Subcontract Number: 70000073494. completed Sept. 2009,
revised Aug. 2010.
2) Stephanie L. Fraker, Feoffrey R. Smith. Interactive Effects of
Ecologically Relevant Concentrations of Organic Wastewater
Contaminants on Rana pipiens Tadpoles. Environ Toxicol 19:
250–256, 2004.
While no significant amounts of caffeine or its derivatives were
detected in this study, it is still possible that there are measurable
amounts of caffeine in the water sources that were examined. To
further study this, an instrument with a lower detection threshold could
be used, or a larger sample of water could be treated with solvent in
the extraction. Additionally, alternate extraction methods could be
experimented with.
While caffeine itself can be toxic, some scientists speculate that the
molecule may also be a marker for even more concerning pollutants
like pharmaceuticals and industrial waste. Future studies may want to
examine water samples for other pollutants as well, and observe if
there is any correlation between caffeine and other waste water
molecules.
Other future work might include looking at the change in
concentration of caffeine overtime, to see if there is ecological
accumulation and what the possible sources may be. To gain a better
estimation of concentration levels in the area, more samples could be
collected, and at more regular distance intervals. To obtain more
accurate approximations of the concentration at each location, samples
could be collected at regular time intervals from each location, and the
data averaged.
As can clearly be seen by the MRM spectra shown, the theophylline
internal standard is clearly visible around the 4.8 minute mark. Since
the internal standard was not added before the extraction procedure, a
standard deviation test was performed to ensure accuracy of the
theophylline volumes. The percent standard deviation was 7.0%.
All samples were run first under the MS basic scan, and then run
through the instrument a second time using the MRM scan. For both
trials, no caffeine was visible and the internal standard was clearly
visible. While it is possible that one may expect slight variance in the
caffeine levels due to extraction efficiency, the total lack of caffeine in
all samples suggests that the results are accurate and consistent.
Therefore, since no caffeine was detected by the instrument, it is
clear that there is most likely no caffeine present in the local water
systems tested, and if there is, it is below the detectable limit of the
instrument.
Fig. 1
Initially, to ensure instrument
precision and to help with optimization,
a solution was mixed containing all
three of the compounds of interest. This
solution was run using the same primary
method as the water samples, and as you
can see in the figure to the left, all three
molecules are clearly visible and
separated, and the retention time is listed
above the peaks.
The figure shown is a product of the
multiple reaction monitoring (MRM)
capabilities of the type of instrument
used. MRM spectra are valuable,
because they allow for narrowing down
of the MS parameters to only look for
specific mass transitions. The
transitions used for this experiment were
taken from the NIST online data base for
mass spectrometry, and were optimized
based on the data.
The water samples collected were from a variety of
sources, and the locations of the sample collections are shown
on the map to the left by stars at the collection sites. Care was
taken to ensure that samples were not collected on or right
after days when precipitation occurred.
density of the aqueous layer to create
clear separation. After extraction, the
organic solvent was evaporated using a
heated water bath, until only crystals
remained. It was then reconstituted in
10 ml of a mixture of 80% R.O. water
and 20% ACS grade methanol, and
theophylline was added as an internal
standard.
The reconstituted sample was stored
in glass bottles at 40*C for less than 24
hours before being analyzed.
Molecule images courtesy of Discovery Studio Visualizer
Shown to the left is the data collected from each sample.
The data was collected using the MRM capabilities of the
instrument, and the mass transitions selected were those that
had been optimized during the course of the study.
As can clearly be seen, there is a large peak that
corresponds to the theophylline internal standard located
around 4.7 minutes, and this is consistent with the
theophylline peak shown above in the mixed solution. It is
also apparent that there is no other peaks visible.
When the MRMs were analyzed using the computer, no
significant caffeine or theobromine was detected.
Fig. 2
Fig. 3