Chemical and archaeological evidence for the earliest cacao beverages

Henderson et al. 10.1073/pnas.0708815104.

Supporting Information

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SI Figure 5
SI Figure 6
SI Figure 7
SI Methods

Fig. 5. MS Chromatogram showing total ion chromatogram (TIC) of theobromine and caffeine standards. Peak at 3 min is theobromine. Peak at 5.9 min is caffeine. The equipment used was a Shimadzu HPLC, equipped with a Shimadzu QP8000 MS Detector. The MS was operated in the positive ion mode, monitoring ions at m/z 181 for theobromine and m/z 195 for caffeine. The column used was a uBondapak C-18, 3.9 mm ID ´ 150 mm, with a mobile phase of 69/30/1 (vol/vol/vol) water/methanol/acetic acid flowing at 1 ml/min.

Fig. 6. UV Chromatogram of theobromine and caffeine standards, with UV detection at 270 nm.

Fig. 7. Mass spectrum of peak at 3 min from SI Fig. 6, providing identification of theobromine (m/z 181).

SI Methods

The LC-MS was carried out on a HPLC, coupled to an atmospheric-pressure chemical-ionization mass spectrometer (APCI MS). The samples were extracted in the Penn Laboratory by immersing pottery sherds in distilled water, boiling at »90°C for 30 min, and then filtering the extract through 8 mm of paper. A second extraction with fresh water was performed, and the two extracts combined. The extracts were concentrated to smaller volume, then filtered through 0.2 mm of paper. These aqueous samples were provided to the Hershey Laboratory for LC-MS.

Before LC-MS analysis, each sample was passed through membrane filters to eliminate particulate matter. For APCI MS, the probe was operated in positive-ion mode to monitor for peaks at m/z 181 (theobromine) and m/z 195 (caffeine), with the UV detector set at 270 nm. The theobromine peak of UV and MS chromatograms at 3 min for the ancient samples coincided with that of the standard (SI Fig. 5 a and b) and the selected ion-monitoring trace at m/z 181 (SI Fig. 5c) confirmed this peak as due to theobromine. Caffeine, whose concentration is about 10% of that of theobromine in the cacao bean, was not detected in any of the samples run by LC-MS, because of its low concentration. One sample run by LC-MS was assigned as "borderline positive," because its chromatogram peak at 3 min was minimal.

Two additional samples, not tested by the Hershey Laboratory, and two samples already run at Hershey, were also analyzed by GC-MS at the Winterthur Museum Laboratory, by using a Hewlett-Packard 6890 instrument equipped with a 5973 mass selective detector. The first pair of samples was extracted with 2/1 methane/methanol, and the second pair was extracted with chloroform and methanol. The samples were derivatized using the Alltech MethPrep reagent before injection, to enable detection of less volatile and/or more polar components. They were then injected splitless onto a HP-5MS column (5% phenyl methyl siloxane) in a method designed to separate components by boiling point. The derivatization method converts theobromine (3, 7 dimethylxanthine) into caffeine (1, 3, 7 trimethylxanthine). Identification of the resulting caffeine signals was by retention time and mass spectrum.

The four samples analyzed by GC-MS showed small amounts of caffeine. Because neither of the samples already run by LC-MS at Hershey had given evidence for caffeine, this compound must be a derivatized product from theobromine present in the original sample. Therefore, the low levels of caffeine in the two additional samples not tested by the Hershey Laboratory can also be attributed to theobromine.

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