16-O-Methylated diterpenes in green Coffea arabica: ultra-high-performance liquid chromatography-tandem mass spectrometry method optimization and validation.

Coffee diterpenes are the main constituents of the coffee oil unsaponifiable fraction. The three most important diterpenes are cafestol, kahweol, and 16-O-methylcafestol (16-OMC), and they are produced, except for cafestol, only by plants of the Coffea genus. Recently, in addition to these three major diterpenes, another 16-O-methylated diterpene (16-O-methylkahweol: 16-OMK) has been identified and quantified, for the first time, in Robusta coffee. For many years, 16-OMC has been considered present exclusively in Robusta, and so it has been reputed an excellent authenticity marker for the presence of Robusta in coffee products. For its quantification, nuclear magnetic resonance (NMR) has proved very useful when compared with other methods. Quite recently, the detection of very low levels of the two 16-O-methylated diterpenes (16-OMD) 16-OMC and 16-OMK in roasted Arabica was reported. This finding makes the use of NMR methods in 16-OMD quantification in Arabica coffee particularly challenging in view of both the trace amounts of 16-OMD and the impossibility to discriminate between 16-OMC and 16-OMK. The ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) method, already used to detect 16-OMC and 16-OMK in Arabica roasted coffee, is then more suitable for quantitative analyses. Up to now however, no quantification of coffee 16-OMD via ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) has been carried out; this largely stimulated the present study. For the first time, a simple procedure for the quantitative detection of 16-OMD in Arabica coffee has been developed, and as far as 16-OMC is concerned, fully validated in terms of specificity, linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), and repeatability following the criteria specified in the EU Commission Decision 2002/675/EC. This method proved to be very specific and sensitive. In order to avoid the chemical complexity generated by the roasting process, the method was optimized and validated on several green Arabica samples from different geographical origins.

Guercia E ，Colomban S ，Navarini L 《-》

Limited genotypic and geographic variability of 16-O-methylated diterpene content in Coffea arabica green beans.

The acknowledged marker of Robusta coffee, 16-O-methylcafestol (16-OMC), can be quantified by NMR as a mixture with 16-O-methylkahweol (16-OMK), which accounts for approximately 10% of the mixture. In the present study, we detected and quantified 16-O-methylated diterpenes (16-OMD) in 248 samples of green Coffea arabica beans by NMR. We did not observe any differences between genotypes introgressed by chromosomal fragments of Robusta and non-introgressed genotypes. Environmental effects suggesting a possible protective role of 16-OMD for adaptation, as well as genotypic effects that support a high heritability of this trait were observed. Altogether, our data confirmed the presence of 16-OMD in green Arabica at a level approximately 1.5% that of a typical Robusta, endorsing the validity of 16-OMD as a marker for the presence of Robusta.

Portaluri V ，Thomas F ，Guyader S ，Jamin E ，Bertrand B ，Remaud GS ，Schievano E ，Mammi S ，Guercia E ，Navarini L ... -  《-》

Rapid authentication of coffee blends and quantification of 16-O-methylcafestol in roasted coffee beans by nuclear magnetic resonance.

Schievano E ，Finotello C ，De Angelis E ，Mammi S ，Navarini L ... -  《-》

Rapid approach to identify the presence of Arabica and Robusta species in coffee using 1H NMR spectroscopy.

NMR spectroscopy was used to verify the presence of Arabica and Robusta species in coffee. Lipophilic extracts of authentic roasted and green coffees showed the presence of established markers for Robusta (16-O-methylcafestol (16-OMC)) and for Arabica (kahweol). The integration of the 16-OMC signal (δ 3.165 ppm) was used to estimate the amount of Robusta in coffee blends with an approximate limit of detection of 1-3%. The method was successfully applied for the analysis of 77 commercial coffee samples (coffee pods, coffee capsules, and coffee beans). Furthermore, principal component analysis (PCA) was applied to the spectra of lipophilic and aqueous extracts of 20 monovarietal authentic samples. Clusters of the two species were observed. NMR spectroscopy can be used as a rapid prescreening tool to discriminate Arabica and Robusta coffee species before the confirmation applying the official method.

Monakhova YB ，Ruge W ，Kuballa T ，Ilse M ，Winkelmann O ，Diehl B ，Thomas F ，Lachenmeier DW ... -  《-》

Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography-fluorescence detection - Determination of Coffea canephora admixtures to Coffea arabica.

16-O-Methylcafestol (16-OMC), the characteristic diterpene exclusively present in Coffea canephora, is an excellent marker for Coffea canephora admixtures to Coffea arabica. Here we show a straightforward, selective and sensitive screening method for the determination of 16-OMC in roasted coffee by high-performance thin-layer chromatography with fluorescence detection (HPTLC-FLD). As internal standard, Sudan IV was used, and a direct saponification with 10% ethanolic potassium hydroxide solution was followed by solid supported liquid extraction with petroleum ether. 16-OMC was selectively derivatized with 2-naphthoyl chloride and analyzed by HPTLC-FLD on silica gel plates with cyclohexane/tert-butyl methyl ether/formic acid (86:14:2, v/v/v) as the mobile phase. The enhanced fluorescence was scanned at UV 244/>320nm. Limits of detection and quantitation of 5 and 14mg 16-OMC/kg coffee allowed the determination of Coffea canephora admixtures to Coffea arabica below 1%. Recoveries for blends of Coffea arabica with Coffea canephora were close to 100%.

Oellig C ，Radovanovic J 《-》