Benchtop (60 MHz) proton NMR spectroscopy for quantification of 16-O-methylcafestol in lipophilic extracts of ground roast coffee

Abstract

We present a method for analysing the lipophilic fraction extracted from ground coffee beans using 60 MHz proton (¹H) NMR spectroscopy. In addition to the triglycerides from coffee oil, spectral features are seen from a range of secondary metabolites, such as various diterpenes. We demonstrate quantitation of a peak attributed to one such compound, 16-O-methylcafestol (16-OMC), which is of interest as a coffee species marker. It is present in low concentrations (<<50 mg/kg) in Coffea arabica L. ('Arabica') beans, but in orders of magnitude greater concentrations in other coffees, in particular the other commercially grown species C. canephora Pierre ex A. Froehner (commonly known as 'robusta'). A series of coffee extracts spiked with 16-OMC analytical standard are used to establish a calibration, and to estimate 16-OMC concentrations in a range of different coffees (Arabicas and blends with robustas). To validate the method, values obtained are compared with an analogous quantitation method that uses high field (600 MHz) NMR spectroscopy. •Quantitation of 16-O-methylcafestol in ground roast coffee extracts using benchtop (60 MHz) NMR spectroscopy•Validated by comparison with quantitative high field (600 Mz) NMR spectroscopy•Detection limit is sufficient for discovering adulteration of Arabica coffee with non-Arabica species.

Keywords: Benchtop (60 MHz) proton NMR spectroscopy for quantification of 16-O-methylcafestol in lipophilic extracts of ground roast coffee; Coffee; Metabolites; NMR; Quantitation; Spectroscopy; Triglycerides.

Graphical abstract

Fig. 1

The ¹H 60 MHz spectrum of a sample of ground roast robusta coffee, compared with the 600 MHz spectrum of the same extract. Both spectra are dominated by features from triglycerides. In the benchtop spectrum there is considerable band overlap, much more so than at the high field strength. However, some of the spectral features can be attributed to specific functional groups, as indicated. Although peaks are wider at the lower field strength, note that the central peak positions of multiplets as well as singlets are field-independent, so when making assignments it is helpful to draw on relevant high-field studies in the literature for example Schievano, Finotello, De Angelis, Mammi, and Navarini and Monakhova et al. .

Fig. 2

(a) The 3.16 ppm peak in a series of extracts from an Arabica coffee spiked with increasing amounts of 16-OMC. The data are shown after normalization to the area of the glyceride region (4.02 - 4.58 ppm) and local baseline correction of the region of interest. Peak integrals are calculated using region limits as shown (3.13 – 3.19 ppm) and the values obtained are plotted in (b) versus the 16-OMC spike concentration.

Fig. 3

(a) 16-OMC concentration predictions by low-field NMR for duplicate analyses of 32 coffees, from a range of either pure Arabica coffees or blends with <10% Robusta. (b) Probability plot between the half-normal distribution and the absolute differences of the outcomes of the pairwise duplicate analyses.

Fig. 4

Predictions of 16-OMC concentrations by low-field NMR versus those from the reference method (high-field NMR) for a collection of 25 coffees (pure Arabicas and blends with robusta). The expected error with low-field NMR is indicated with error bars. The regression line for the low upon high field outcomes, its equation and R² value are also shown.