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SOURCE MOLECULAR CORPORATION
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Trace determination of caffeine by liquid chromatography-atmospheric
pressure chemical ionization-mass spectrometry
Submitter: Blue River
Submitter #’s: BR-1, BR-2, BR-3, BR-4
Source Molecular #’s: SM 0056, SM 0057, SM 0058, SM 0059
Samples Received: January 3rd, 2011
Date Reported: January 10th, 2011
Mass Spectrometry Results
* < LOD indicates a detected concentration below the 4 ng/L detection limit.
Four 1-liter water samples were filtered and analyzed for the presence of unmetabolized caffeine.
The analytical method used for these samples is able to detect the presence of unmetabolized
caffeine in as low as 4 ng/L. Research has shown that caffeine can be used as a stable and good
marker for human activities, and in particular as a proxy for human fecal contamination.
Samples BR-2 (Our Ref: SM 0057) and BR-3 (Our Ref: SM 0058) were below the detection limit
of 4 ng/L, therefore the samples were classified as negative for the presence of caffeine. Negative
samples should be analyzed with other molecular and genetic methods described below in order
to demonstrate conclusively that there is no presence of human forms of contamination in the
Samples BR-1 (Our Ref: SM 0056) and BR-4 (Our Ref: SM 0059) were positive for the presence
of unmetabolized caffeine suggesting that human forms of contamination are present in these
water samples. Nonetheless, as with the negative samples, additional tests should be conducted
to strengthen the results. The Human Enterococcus IDTM, Human Bacteroidetes IDTM and Human
Fecal Virus IDTM services can be used to validate the presence of human contamination.
All samples were filtered through a combusted 0.45-Am glass fiber filter (Whatman) before analysis. A one liter volume of filtered
water sample was transferred to a two liter separatory funnel, and the appropriate amount of internal standard (atrazine
added. After raising the pH of the sample to 8–9 with a 0.1 M sodium hydroxide solution, the sample was extracted using liquid
liquid extraction with three portions of 50 ml methylene chloride. The organic extracts were concentrated to a volume of 2
using a water bath and a Snyder column and further evaporated to dryness using a gentle stream of purified nitrogen. After
reconstituting the samples to 500 Al of 1:1 methanol/water, they were analyzed by LC/MS.1
Quantitative determination of caffeine in the final extracts was carried out by HPLC–APCI–MS in positive mode under selected
ion monitoring (SIM) using a Navigator aQa system (Finnigan). A Luna HPLC column (150 X4.6 mm I.D.) packed with a bonded
C18 phase (5 Am) was used for the chromatographic separation (Phenomenex). The mobile phase was programmed to run in a
linear gradient from 30% methanol / 70% water to 100% methanol at a flow
-rate of 1 ml/min. The total run time required for the
analysis was 10 min with caffeine and atrazine-d5 eluting at 3.97 and 8.83 min, respectively. Internal standard linear calibration
plots were constructed using eight points at 2.5, 5, 10, 25, 50,100, 200 and 400 pg/ml.
Caffeine, 1,3,7-trimethylxanthine is ranked number one drug worldwide and is usually employed as a st
in coffee, tea, cocoa, soft drinks and chocolate. It is also a component in hundreds of prescription and over
ranging from analgesics to cold medicines.3 The average human consumes considerable amounts of caffeine. From coffee
alone, an average American consumes 131 mg of caffeine per day. However, caffeine is extensively metabolized by humans
with only approximately 3% excreted unchanged in the urine. Nevertheless, there is far more caffeine introduced to the sewag
system by disposal of unconsumed coffee, tea or soft drinks down the sink, and rinsing of coffee pots and cups.
Research has suggested that the presence of caffeine in the environment can serve as an indicator of the presence of human
sewage. Levels of caffeine in domestic wastewater have been measured to be between 20 and 300 µg/liter.
waters can however be much lower due to significant dilution. Consequently, any analysis must be able to determine caffeine in
the low part parts per trillion levels (4 ng/L LOD) in both freshwater and saltwater. The method described in the section above
overcomes many of the previous limitations in detecting caffeine and is able to detect caffeine as low as 4ng/L.1, 2
Caffeine is considered a good, stable, dissolved marker directly related to human activities with no potential biogenic sources
because of its high solubility (13.5 g/l), low octanol-water partition coefficient (log Kow = 0.01) and negligible volatility.1 This is of
particular importance in environments where septic tanks contribute large amounts of the wastewater discharges in comparison
with treated municipal wastewaters. As such, the detection of caffeine can demonstrate the presence of failing septic systems
1 Gardinali PR, Zhao X. Trace determination of caffeine in surface water samples by liquid chromatography --
atmospheric pressure chemical ionization--mass spectrometry (LC-APCI-MS).
Environ Int. 2002 Dec; 28(6):521-8.
2 Bendriss E, Markoglou N, Wainer IW. Liquid chromatographic method for the simultaneous determination of caffeine and
fourteen caffeine metabolites in urine.
J Chromatogr, B 2000;746:331–8.
3 Seiler RL, Zaugg SD, Thomas JM, Howcroft DL. Caffeine and pharmaceuticals as indicators of wastewater contamination
Ground Water 1999;37:405– 10.
4 Scott, Troy M., Rose, Joan B., Jenkins, Tracie M., Farrah, Samuel R., Lukasik, Jerzy Microbial Source Tracking: Current
Methodology and Future Directions.
Appl. Environ. Microbiol. (2002) 68: 5796-5803.
Limitation of Damages – Repayment of Service Price
It is agreed that in the event of breach of any warranty or breach of contract, or negligence of the Source
Molecular Corporation, as well as its agents or representatives, the li ability of the Source Molecular
Corporation shall be limited to the repayment, to the purchaser (submitter), of the individual analysis
price paid by him/her to the Source Molecular Corporation. The Source Molecular Corporation shall not
be liable for any damages, either direct or consequential. The Source Molecular Corporation provides
analytical services on a PRIME CONTRACT BASIS ONLY. Terms are available upon request.
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