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Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica
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Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica Claudia Oellig ∗ , Jessica Radovanovic Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany
a r t i c l e
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Article history: Received 18 September 2017 Accepted 10 October 2017 Available online xxx Keywords: 16-O-Methylcafestol (16-OMC) Coffea canephora Screening High-performance thin-layer chromatography (HPTLC) Fluorescence detection (FLD)
a b s t r a c t 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 highperformance 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/ > 320 nm. Limits of detection and quantitation of 5 and 14 mg 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%. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Coffea arabica and Coffea canephora var. robusta (often simply called “Arabica” and “Robusta”) are the two most important coffee species worldwide regarding the economic significance. Due to difference in quality and price, Coffea arabica is often blended with Coffea canephora. The determination of such admixture is of great importance to detect commercial frauds and to protect consumers. Regarding their chemical composition, Coffea arabica and Coffea canephora significantly diverge. For instance, there are differences in the amount of caffeine, trigonelline and chlorogenic acids, and also the composition and the amount of the lipids differ [1]. In coffee beans, the coffee oil represents an amount between 7 and 17% of the total mass, with an average value of 10% for Coffea canephora and 15% for Coffea arabica [2,3]. Besides 75% triacylglycerides, the coffee oil comprises of a unsaponifiable fraction of about 20% mainly consisting of specific pentacyclic diterpenes of the kaurene family, which are solely present in coffee. Cafestol, kahweol and 16-Omethylcafestol (16-OMC) are the dominant representatives, and are nearly completely esterified with fatty acids [2–5]. The diterpene 16-OMC is exclusively present in Coffea canephora with an
∗ Corresponding author. E-mail address: [email protected] (C. Oellig).
average amount of 1.7 g/kg (0.8–2.4 g/kg) [3]. Thus, 16-OMC is a reliable chemical marker to distinguish between the two coffee species and excellently suitable to detect and determine Coffea canephora in coffee blends [3,4,6] and monitor the authenticity of products. For this purpose, the total amount of 16-OMC is of interest, consisting of about 98% of the esterified and about 2% of the free form [7]. For the determination of the total amount of 16-OMC two strategies are possible. Either the almost exclusively present form of the esterified 16-OMC can directly be analyzed and set as the total 16OMC, or the de facto total amount of 16-OMC can be determined after saponification. The initial extraction of diterpene esters from the coffee matrix was commonly done by extraction of the coffee oil, mainly with diethyl ether or tert-butyl methyl ether (TBME) [2–4]. Acid-catalyzed decomposition for lipid extraction was not recommended, because degradation of coffee diterpenes occurred [8]. For the analysis of the esterified 16-OMC, gel permeation chromatography was suggested to separate the esters from the coffee oil triglycerides followed by semi-preparative high-performance liquid chromatography (HPLC) or solid phase extraction on silica gel and subsequent HPLC separation with ultraviolet (UV) detection [2]. To determine the total amount of 16-OMC, generally the extracted coffee oil was saponified, the unsaponifiable matter extracted, sometimes cleaned on silica gel, and subsequently determined by gas chromatography [4,9,10] or HPLC–UV
https://doi.org/10.1016/j.chroma.2017.10.031 0021-9673/© 2017 Elsevier B.V. All rights reserved.
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[4,8,11]. Based on the method of Speer [4], the German standard method DIN 10799 [12] was published, which is identical to the German § 64 LFGB method [13]. This standard method consists of a Soxhlet extraction with TBME for 5 h, saponification with ethanolic potassium hydroxide for 2 h, liquid-liquid extraction of the unsaponifiable matter with TBME and the determination by HPLC–UV, which in total can last 2 days. Alternatively, the direct saponification of a coffee sample was reported by de Roos et al. [6] and Speer et al. [14] for the analysis of 16-OMC, whereas for kahweol and cafestol the direct saponification was mentioned more frequently [6,15–17]. For the determination of coffee diterpenoids, thin-layer chromatography (TLC) was also reported, when a qualitative method for the analysis of 16-OMC with TLC and vanillin sulphuric acid reagent for derivatization was described by Speer and Mischnick [9]. Based on this method, Speer et al. [14] shortly described a TLC screening approach for the esterified and the free form of 16-OMC in the coffee oil and in the direct saponified extract, respectively, with only few validation data. Recently, Schievano et al. [18] and Finotello et al. [19] suggested a quite different method for the detection of 16-OMC. They used nuclear magnetic resonance (NMR) spectroscopy to analyze 16-OMC in roasted coffee beans including a quite rapid sample treatment. The limited availability of the device, however, is a drawback. Considering the time-consuming sample workup and lengthy HPLC–UV analyses of the reported methods, the aim of the present study was to develop a rapid and efficient screening method for the determination of 16-OMC in roasted coffee by high-performance thin-layer chromatography (HPTLC). Thereby, a simple and reliable method for the determination of Coffea canephora admixtures to Coffea arabica should be offered. Extensive and cumbersome sample preparation steps should be replaced by shortened and straightforward procedures. The hydroxyl group of 16-OMC should be selectively labeled for sensitive fluorescence detection (FLD). The great selection of solvents in normal phase HPTLC should enable to develop a selective chromatography to analyze 16OMC without matrix interferences. With the possibility to analyze numerous samples in parallel and with the automated HPTLC devices that guarantee repeatable procedures, the prerequisites for an efficient screening concept are perfectly given.
water (>18 M cm) was supplied by a Synergy System (Millipore, Schwalbach, Germany). HPTLC silica gel 60 plates from Merck were used without pre-washing. Coffea canephora (roasted beans) was provided by Hochland Kaffee Hunzelmann GmbH (Stuttgart, Germany). Coffea arabica (roasted beans) and blends of Coffea arabica with Coffea canephora (70%, 60, 40% and 25% Coffea canephora content, roasted beans and ground coffee) were purchased from local supermarkets. 2.2. Standard and derivatization reagent solutions For the standard stock solution (100 mg/L), 2 mg 16-OMC was dissolved in 20 mL of acetonitrile/2-propanol (3:2, v/v). The stock solution was stored at −20 ◦ C in amber glassware and was used for several weeks. For the internal standard (ISTD) stock solution, 1 mg Sudan IV was dissolved in 1 mL of TBME. The diluted ISTD solution of 3 g/mL was prepared by diluting the stock 1:25 with TBME. For HPTLC–FLD method development, the standard stock solution was used without dilution. For the determination of limit of detection and quantitation (LOD/LOQ), the standard stock solution was diluted 1:50 with acetonitrile, resulting in a concentration of 2 g/mL. To aliquots of 20–200 L of the diluted 16-OMC solutions 75 L of the diluted ISTD solution were added, followed by evaporation under a gentle stream of nitrogen at ambient temperature. The residue was derivatized as described under Section 2.5, resulting in standard solutions for HPTLC of 0.04–0.4 ng/L for the LOD/LOQ experiments, when the concentration of the ISTD generally was 3 ng/L. Calibration standards for sample analyses were analogously prepared by diluting the 16-OMC stock solution 1:10 with acetonitrile and mixing aliquots of 4–600 L with 75 L of the diluted ISTD solution, followed by evaporation and derivatization, and resulting in 16-OMC standard concentrations of 0.04–6 ng/L. The derivatization reagent solution was prepared in anhydrous acetonitrile, containing DMAP and 2-NCl at a concentration of 40 mg/mL and 7.5 mg/mL, respectively. PEI solution was prepared in anhydrous methylene chloride, comprising PEI at a concentration of 250 mg/mL. Both solutions were freshly prepared before use. 2.3. Samples and extraction (saponification)
2. Material and methods 2.1. Chemicals and materials 16-O-Methylcafestol (16-OMC) (99.2%) was obtained from PhytoLab (Vestenbergsgreuth, Germany). Sudan IV (drying loss 1.2%) was received from TCI (Eschborn, Germany). 2-Naphthoyl chloride (2-NCl) (98%), 4-(dimethylamino)pyridine (DMAP) (≥99%, reagent plus), ammonium formate (>97%, purum), gallic acid monohydrate (>98%, purum), acetonitrile and 2-propanol (both LC–MS, Chromasolv), ethanol absolute (≥99.8%, HPLC, Chromasolv), tert-butyl methyl ether (TBME) (≥99.8%, HPLC, Chromasolv) and methylene chloride anhydrous (99.8%) were purchased from Sigma-Aldrich (Steinheim, Germany). Cyclohexane (99.5%, p.a.) and n-hexane (95%, for pesticide residue analysis) (both Chemsolute) were obtained from Th. Geyer (Renningen, Germany). Petroleum ether (PE) (boiling range 40–60 ◦ C, GC, SupraSolv) was from Merck (Darmstadt, Germany). Paraffin oil low viscosity (Ph. eur.) was purchased from Carl Roth (Karlsruhe, Germany). Acetonitrile anhydrous (≥99.8%) and polyethyleneimine branched (PEI) (99%, M.W. 10,000) were obtained from Alfa Aesar (Karlsruhe, Germany). Potassium hydroxide pellets (≥85%, Ph. eur, Prolabo) were from VWR (Bruchsal, Germany). Solid supported liquid extraction (SLE) tubes (Novum SLE, 3cc tubes) were purchased from Phenomenex (Aschaffenburg, Germany). Ultrapure
As samples, different roasted coffee beans (pure coffee species and blends of Coffea arabica with Coffea canephora) and ground coffee (blends of Coffea arabica with Coffea canephora) were used. Before analysis, roasted coffee beans and ground coffee were finely milled in a Tube Mill control for 60 s at 22,000 min−1 (IKA, Staufen, Germany) (<0.3 mm). For extraction, direct saponification was used according to the method of Dias et al. [16] with some modifications. Finely milled coffee sample (100 mg) was weighted into a 6-mL glass centrifuge tube equipped with a screw cap. One mL of 10% potassium hydroxide solution (water/ethanol, 1:9, v/v) was added, the sample was mixed by vortexing for 5 s, and saponification was performed for 1 h on a thermal mixer (Thermomixer comfort, Eppendorf, Hamburg, Germany) at 90 ◦ C. During the saponification time, the tube was programmably shaken six times with 650 rpm for 10 s. After cooling to ambient temperature, 75 L of the ISTD stock solution were added, the sample was briefly mixed by vortexing and centrifuged at 3200×g at 18 ◦ C for 5 min (Biofuge primo R, Heraeus, Hanau, Germany). The clear supernatant was decanted into a 3-mL volumetric flask, filled up with water and was subjected to SLE. Coffee blends for recovery experiments were prepared by adding 2, 5, 20 and 50 mg of finely milled Coffea canephora to 98, 95, 80 and 50 mg of finely milled Coffea arabica, respectively. The relative quantity of 2, 5, 20 and 50% Coffea canephora in the blend corresponded to a 16-OMC content of 36, 90, 360 and 900 mg/kg
Please cite this article in press as: C. Oellig, J. Radovanovic, Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.10.031
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Fig. 1. Workflow for the determination of 16-O-methylcafestol in roasted coffee.
coffee. The calculation was based on an amount for 16-OMC in Coffea canephora of 1.8 g/kg that was previously analyzed with the developed HPTLC–FLD method. During the sample extraction glassware was wrapped into aluminum foil or amber glassware was used.
2.4. Solid supported liquid extraction (SLE) A 400-L aliquot of the saponified sample extract was loaded onto the SLE cartridge. After 12 min soaking (counted once the extract passed the upper frit), SLE was performed twice with 1.2 mL of PE into a 5-mL volumetric flask. The flask was filled up with PE, a 1.5-mL aliquot was evaporated under a gentle stream of nitrogen to complete dryness, and the residue was subjected to derivatization.
2.5. Derivatization Two hundred L of the derivatization reagent solution was pipetted to the sample extract or standard residue, the tube was tightly sealed and was mixed by vortexing for 10 s, followed by storage in the dark at ambient temperature for 40 min, when the tube was vortexed for 10 s after 20 min. After the addition of 800 L acetonitrile, the tube was briefly vortexed, 50 L of PEI solution were added, and the mixture was shaken under light protection for 10 min at 1500 min−1 on a small-size shaking device before being centrifuged at 3200×g at 8 ◦ C for 15 min. A 700-L aliquot of the clear supernatant was transferred into an HPTLC vial. The final extract for HPTLC had a sample concentration of 4 mg/mL. The whole analytical procedure for the determination of 16OMC in roasted coffee, including sample extraction, SLE and derivatization, is sketched in Fig. 1.
2.6. High-performance thin-layer chromatography–fluorescence detection (HPTLC–FLD) Before application, HPTLC plates were dipped 15 mm deep into a 0.05% gallic acid solution in methanol and dried in a gentle stream of cold air for 5 min. An Automatic TLC Sampler 4 (ATS 4, CAMAG, Muttenz, Switzerland) equipped with a light protecting shell was used to apply samples and standards as 6-mm bands with the following settings leading to 22 tracks on a 20 cm × 10 cm plate: 10 mm distance from the lower edge, 8 mm distance from the left edge, and 8.7 mm track distance. Application parameters were: 15 L/s filling speed, 200 nL predosage volume, 200 nL retraction volume, 150 nL/s dosage speed, 4 s rinsing vacuum time, 1 s filling vacuum time, 2 rinsing cycle and 1 filling cycle. Acetonitrile was used as the rinsing solvent. After application, 4 rinsing cycles were performed with water. The application volume generally was 5 L for sample extracts and 16-OMC calibration standards. For LOD/LOQ determination, the standard application volume resulted in 0.2–2 ng/zone, corresponding to 10–100 mg 16-OMC/kg coffee. For calibration standards, the application volume resulted in 0.2–30 ng/zone, corresponding to 10−1500 mg 16-OMC/kg coffee, calculated for 5-L sample extract application. The amount of the ISTD generally was 15 ng/zone. Directly after application, chromatography was performed in the Automatic Developing Chamber (ADC2, CAMAG) with a 20 cm × 10 cm twin-trough chamber (CAMAG), equipped with a light protecting shell. Before development, the plate activity was controlled to 33% relative humidity with saturated magnesium chloride solution for 5 min. As mobile phase, 10 mL of cyclohexane/TBME/formic acid (86:14:2, v/v/v) was used up to a migration distance of 70 mm and a drying step in a stream of cold air followed for 5 min. For detection of the ISTD, the plate was scanned in the absorption mode at 535 nm (tungsten lamp) by the TLC Scanner 4 (CAMAG) in the range of 40–70 mm with a scanning speed of 20 mm/s, a data resolution of 100 m/step and a slit dimen-
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Fig. 2. Images of HPTLC silica gel plates developed with cyclohexane/TBME/formic acid (86:14:2, v/v/v) up to a migration distance of 70 mm under UV 254 nm illumination after dipping in n-hexane/paraffin: (A) 16-O-methylcafestol (16-OMC), 24 ng/zone, (corresponding to ∼70% Coffea canephora in a coffee blend); (B) from left to right, different Coffea arabica sample extracts (4 mg coffee/mL); (C) extract of Coffea canephora (4 mg/mL) with 1.8 g/kg 16-OMC (corresponding to 36 ng 16-OMC/zone); (D) from left to right, extracts of blends (4 mg/mL) of Coffea arabica with 40% and 70% Coffea canephora (corresponding to 14 and 24 ng 16-OMC/zone); (E) reagent blank for comparison. All samples were prepared according to the developed method, the application volume generally was 5 L.
sion of 4 mm × 0.30 mm. The plate was dipped into a solution of n-hexane/paraffin oil (2:1, v/v) (TLC Chromatogram Immersion Device III (CAMAG), immersion speed 2, immersion time 3) and dried in a stream of cold air for 1 min. Plate images were captured under UV 254 and UV 366 nm illumination (TLC Visualizer 4, CAMAG). Thereafter, the plate was scanned in the fluorescence mode at UV 244/ > 320 nm (mercury lamp) with the same settings for scanning speed, data resolution and slit dimension as mentioned above. The manual detector mode was used applying a quick scan range of 32–42 mm on the track of the highest standard of the acylated 16-OMC. HPTLC instruments were controlled by the software winCATS, version 1.4.6.2002 (CAMAG). Microsoft Excel, version Office Professional Plus 2010 was used for statistical analysis. Calibration curves were calculated by linear regression. For validation, means, standard deviations and relative standard deviations were calculated.
2.8. Analysis of coffee samples from the German market Roasted coffee from the German market with labelled contents of Coffea canephora of 100% (coffee beans), 70% (ground coffee), 60% (coffee beans), 40% (coffee beans), 25% (ground coffee and coffee beans) and 0% (pure Coffea arabica, coffee beans) were used. Sample analysis including saponification, SLE, derivatization, and HPTLC–FLD was performed according to the procedures described above (n = 4). For these samples, the calibration range was extended to include pure Coffea canephora. Thus, 10–800 L of the diluted 16OMC standard solution were derivatized as described under Section 2.5, resulting in amounts of 0.5–40 ng/zone which corresponds to 25–2000 mg 16-OMC/kg coffee. 3. Results and discussion 3.1. Approach
2.7. HPTLC–MS For zone elution, the TLC–MS interface (CAMAG) equipped with a circular elution head plunger (4 mm), was used. The eluent was provided by an Agilent (Waldbronn, Germany) 1100 HPLC pump and the TLC–MS interface was directly connected to the MS. The MS device (Agilent) consisted of a G1956 B MSD single quadrupole MS with an atmospheric pressure ionization electrospray (ESI) interface (G1946), and was operated by ChemStation B.04.03 software (Agilent). For HPTLC–MS measurements, the plate was used without dipping into n-hexane/paraffin. The target zone was marked with a pencil under UV 254 nm illumination before zone elution was done. Acetonitrile/10 mM ammonium formate (9:1, v/v) was used as elution solvent at a flow rate of 0.2 mL/min for 60 s. A blank zone was eluted for 60 s after the elution of the target zone to clean the elution head. Measurements in the ESI+ mode were performed with the following parameters: capillary voltage 5.0 kV, skimmer ◦ voltage 35 V, lens 2.5 V, quadrupole temperature 100 C, drying gas ◦ temperature 300 C, drying gas flow rate 10 L/min and nebulizer gas pressure 40 psig. Total ion chronograms were recorded at m/z 200–1200, using a fragmentor voltage of 60 V, gain 5, threshold 100, and step size 0.1.
In a first step, a suitable derivatization for 16-O-methylcafestol (16-OMC) was selected and optimized before the conditions for HPTLC–FLD were developed. In the second step, the sample workup was evaluated, regarding the efficiency of the saponification and the liquid-liquid partition step for 16-OMC from roasted coffee. Having developed the entire HPTLC–FLD screening method, the performance characteristics were assessed by LOD, LOQ and the precision of the method. Finally, several coffee samples from the German market were analyzed by HPTLC–FLD and the Coffea canephora contents was calculated. 3.2. Method development 3.2.1. Derivatization With the aim to gain high selectivity and sensitivity for 16-OMC, the hydroxyl group of the diterpenoid was used for fluorescence marking. Therefore, the simple acylation with 2-naphthoyl chloride (2-NCl) and 4-(dimethylamino)pyridine (DMAP) in methylene chloride according to the method of Oellig [20] for the determination of ricinoleic acid in rye by HPTLC–FLD was selected. HPTLC–MS measurements of the only product zone at hRF 50 (Fig. 2A) with ESI+
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Fig. 3. (A) HPTLC chromatogram on silica plates under UV 254 nm illumination of 16-O-methylcafestol (16-OMC) standards and extracts of Coffea arabica blends with 20 and 50% Coffea canephora (Coffea arabica sample 1), corresponding to 0.36 and 0.9 g 16-OMC/kg (n = 5), and the pure extract of Coffea arabica sample 1; (B) corresponding 3D densitogram of the fluorescence scan at UV 244/ > 320 nm and the absorption scan at 535 nm, and (C) resulting calibration graph of 16-OMC (0.03–1.5 g/kg coffee). Triangles and circles correspond to the blends with 20 and 50% Coffea canephora, respectively.
in the full scan mode showed mass signals at m/z 453, 485 and 991. They indicated the presence of the acylated 16-OMC with signals for the protonated molecule after methanol elimination (m/z 453), the protonated molecule (m/z 485), and the sodium adduct of the
dimer (m/z 991). According to literature [21–23], the kinetic of the acylation is strongly influenced by the configuration of the analyte. Therefore, we evaluated the influence of the reaction time and temperature, the applied catalyst and the stoichiometric ratio of 16-
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OMC/2-NCl/catalyst for the completeness of the derivatization. The reaction yields were quantitated by HPTLC–FLD with the optimal detection settings for the derivatization product. The reaction conditions according to [20], with an slightly extended reaction time of 40 min and light protection, proved suitable for highest yields. With the intention to replace the toxic reaction solvent, several anhydrous solvents were evaluated and acetonitrile was verified to be likewise suitable. The final derivatization procedure consisted of the addition of 200 L of derivatization reagent (containing of 7.5 mg 2-NCl and 40 mg DMAP/mL acetonitrile) to the dried sample, a reaction time of 40 min under light protection and the addition of 800 L acetonitrile. The use of dispersive solid phase extraction with primary secondary amine for the reduction of the excess of derivatization reagent in the final extract was not possible due to occurrence of an additional fluorescent zone, which interfered with the standard zone. Shaking with 50 L of a polyethyleneimine (PEI) solution (250 mg/mL in methylene chloride) followed by a centrifugation step for 15 min at 8 ◦ C turned out to be a suitable alternative and guaranteed an interference-free chromatography. 3.2.2. HPTLC–FLD A 16-OMC standard, the internal standard Sudan IV and several roasted coffee samples (pure Coffea arabica and Coffea canephora and coffee blends) were applied for HPTLC method development. Coffee extracts were prepared according to the developed sample workup including the derivatization. Silica gel and amino bonded HPTLC plates were checked, when silica gel offered best separation of 16-OMC and Sudan IV from matrix components and from components of the derivatization reagent. Several solvents and solvent mixtures were tested as the mobile phase taking into account the selectivity of the groups referred to Snyder [24]. Separation was visible by the fluorescence under UV 254 nm illumination after dipping in n-hexane/paraffin for fluorescence enhancement and under white light (for Sudan IV). Cyclohexane and TBME (86:14, v/v) as the mobile phase turned out to be selective for the separation of 16-OMC from the coffee matrix and by-products of the fluorescent labeling reagent. The addition of formic acid improved the separation. Finally, a mixture of cyclohexane/TBME/formic acid (86:14:2, v/v/v) was selected on HPTLC silica gel material for best separation and sharp zones for 16-OMC and Sudan IV. Because separation and sharpness of the 16-OMC zone was influenced by the water content of the layer, the plate activity was controlled to 33% relative humidity. For a migration distance of 70 mm, the conditions resulted in a hRF value of 50 for 16-OMC, shown by the 16-OMC standard and a pure Coffea canephora extract under UV 254 nm illumination (Fig. 2A and C). The hRF for Sudan IV was 60 (Fig. 3B). Coffea arabica samples showed no interfering matrix (Fig. 2B), confirmed by coffee blends with 40 and 70% Coffea canephora (Fig. 2D), and the derivatization reagent also did not interfere with the separation (Fig. 2E). Visual detection of 16-OMC was possible down to ∼7 ng/zone (corresponding to a coffee blend with ∼20% Coffea canephora, calculated for a 5-L sample volume) (Fig. 3A). Quantitation of 16-OMC was performed in the fluorescence mode, while the internal standard Sudan IV was measured in the absorption mode. The recorded fluorescence spectrum of the acylated 16-OMC in the remission mode revealed an intensive maximum at 244 nm, the absorption spectrum of Sudan IV indicated a maximum at 535 nm. Therefore, an excitation wavelength of 244 nm (mercury lamp) was selected for the fluorescence scan in combination with the 320 nm edge filter, and a wavelength of 535 nm (tungsten lamp) for the absorption scan. 3.2.3. 16-OMC protection During method development, sometimes the intensity of the 16-OMC zone was significantly reduced, directly seen under UV 254 nm illumination and additionally detected in the fluorescence
scan. According to literature [4,8,13,15,16,25], diterpenes are prone to (photo)oxidation. Therefore, light protection was provided during the entire sample preparation and HPTLC. Reduced signal intensities were still observed, because 16-OMC was also prone to oxidation upon contact with the silica gel. This degradation process already occurred in earlier studies for indole alkaloids (ergot alkaloids) and their derivatives [26,27]. Based on these former experiences, gallic acid was applied as protective substance at the application area before the samples and standards were applied. Dipping the plate into a 0.05% solution of gallic acid in methanol offered an effective protection of the diterpenoid from degradation. 3.2.4. Sample preparation For diterpenoid extraction, time-consuming lipid extraction followed by saponification of the coffee oil and laborious liquid-liquid partition (LLE) for the extraction of the unsaponifiable matter were often reported in literature [3,4,7,8,13]. With the intention to shorten sample preparation, more efficient and straightforward sample workup strategies were evaluated. For method development, a 16-OMC standard, pure Coffea arabica and Coffea arabica blended with 20% Coffea canephora were applied, and analyzed after derivatization with HPTLC−FLD to check the differences in matrix loads and recoveries. To correct volume errors during the entire sample preparation and HPTLC application, Sudan IV was used as internal standard. As Sudan IV was easily detected in a visible absorption scan, it did not interfere with the fluorescent 16OMC and additionally was well separated (hRF 60) from 16-OMC (hRF 50). For extraction, we selected direct saponification of roasted coffee samples, which was already mentioned for cafestol and kahweol [16,17] and also 16-OMC [6], and was proven more effective than the pre-extraction of the coffee oil with subsequent saponification [15]. Based on the reported methods [6,15–17] with little modifications, 100 mg of fine milled roasted coffee was directly saponified with 1 mL of 10% potassium hydroxide solution (ethanol/water, 9:1, v/v) at 90 ◦ C. To check the completeness of saponification of the esterified 16-OMC, the saponification time was varied (0.5–3 h), while all other parameters were kept constant. Determined quantities for 16-OMC in the Coffea arabica blend (20% Coffea canephora) after 1 h and 2 h saponification and LLE in TBME did not show significant differences, but longer saponification time reduced the yields. Significantly shorter saponification (0.5 h), however, reduced the yields considerably. These findings were in accordance to Gross et al. [28], Urgert et al. [17] and Dias et al. [15,16], who used 1 h saponification and reported the possibility of degradation and formation of artifacts for long saponification times. Consequently, saponification time was set to 1 h. Analysis of the coffee blend after lipid extraction with TBME in the ultrasonic bath for 5 h, followed by saponification of the extracted coffee oil and LLE in TBME, revealed equal results for 16-OMC. With the aim to simplify and automate the laborious LLE procedures, solid supported liquid extraction (SLE) was selected as alternative. According to literature performing LLE [2,4,6,8,13,15–17], TBME was first checked as elution solvent, resulting however in interfering matrix in the extract. With the intention to obtain matrix-free extracts and efficient recovery of 16-OMC, several single solvents (diethyl ether, diisopropyl ether, petroleum ether (PE)), solvent mixtures, single and multiple elution, and different elution volumes were tested. Finally, PE as elution solvent turned out to be most suitable; highest 16OMC recoveries were obtained and matrix interferences were not present. The resulting procedure comprises of the application of a diluted aliquot of the saponified extract onto the SLE cartridge, a soaking time of 12 min and a twofold elution with PE (Fig. 1). In the end, the straightforward sample preparation consisted of a direct saponification with 10% ethanolic potassium hydroxide
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Table 1 Recoveries of 16-O-methylcafestol (16-OMC) from Coffea arabica blends with Coffea canephora, quantitated against 16-OMC. 2%a Coffea arabica
5%b
20%c
50%d
95 ± 4 94 ± 5
100 ± 4 102 ± 1
97 ± 4 99 ± 6
Coffea canephora Mean recovery [%] ± SDe (n = 5) Mean recovery [%] ± SDe (n = 5)
Sample 1 Sample 2
96 ± 5 97 ± 5
a,b,c,d e
Corresponding to 36, 90, 360, and 900 mg 16-OMC/kg coffee. Standard deviation.
Table 2 16-O-Methylcafestol (16-OMC) and Coffea canephora contents in seven coffee samples form the German market. Sample
Labelled Coffea canephora content [%]
16-OMC [mg/kg] ± SDa (n = 4)
Calculated Coffea canephora content [%]b
1 2 3 4 5 6 7
70 60 40 25 25 0 100
1140 ± 40 1070 ± 20 710 ± 42 410 ± 20 460 ± 25
67 ± 2 63 ± 1 42 ± 2 24 ± 1 27 ± 1 0 –
a b
Standard deviation. Based on a literature average amount of 1.7 g 16-OMC/kg Coffea canephora.
for 1 h followed by a rapid SLE with PE. After an easy to perform pre-chromatographic derivatization with 2-NCl for a sensitive fluorescence detection of 16-OMC, the samples were analyzed by HPTLC–FLD, allowing to analyze up to 22 samples (including calibration standards) in parallel in less than two hours.
method for the analysis of 16-OMC in roasted coffee is a suitable screening tool for the detection of Coffea canephora admixtures to Coffea arabica.
3.3. Method validation
Seven coffee samples from the local market (with labelled contents of Coffea canephora between 0 and 100%) were analyzed by the HPTLC–FLD screening method, and the Coffea canephora content was calculated taking into account the average value of 1.7 g 16OMC/kg Coffea canephora (Table 2). For all samples the calculated Coffea canephora content correlated very well with the labelled amount and results were well repeatable with %RSD <6% (n = 4). The determined amount of 1.8 g 16-OMC/kg Coffea canephora for the pure Coffea canephora sample (sample 7) agreed well with the average amount of 1.7 g 16-OMC/kg Coffea canephora reported in literature [3,18,19].
3.3.1. Limits of detection and quantitation 16-OMC was used as calibration standard and derivatized with 2-NCl like the sample extracts. Calibrations were performed in the range 0.2–2 ng 16-OMC/zone, calculated to 10–100 mg 16OMC/kg coffee (n = 5) taking into account the sample preparation and the sample application volume of 5 L. Resulting calibration graphs revealed good linearity with high coefficients of correlation (R2 > 0.9976). According to the DIN 32645 calibration method [29], limits of detection (LOD) and limit of quantitation (LOQ) were determined to 0.1 and 0.28 ng 16-OMC/zone, respectively, corresponding to 5 and 14 mg 16-OMC/kg coffee. This is equivalent to 0.3 and 0.8% Coffea canephora admixture to Coffea arabica regarding a literature average amount of 1.7 g 16-OMC/kg Coffea canephora. With relative standard deviations (%RSD) of 5.4% and 4.6% the determination was quite well repeatable. Thus, the developed HPTLC–FLD screening allows the determination of 16-OMC quantities that correspond to Coffea canephora admixtures below 1%. 3.3.2. Recoveries Recovery experiments for 16-OMC in roasted coffee were done in blends of Coffea arabica with 2, 5, 20 and 50% Coffea canephora (n = 5), corresponding to a 16-OMC quantity in the blends of 36, 90, 360, 900 mg 16-OMC/kg. Recovery experiments were carried out with two Coffea arabica samples (sample 1 and 2). The internal standard mode was used for quantitation, when the peak areas ratios 16-OMC/ISTD were calculated for evaluation. Quantitation by HPTLC−FLD with a 16-OMC solvent standard is exemplarily shown in Fig. 3. For all prepared coffee blends, the recovery rates were near 100% (Table 1) independent on the used Coffea arabica sample. Consequently, analysis of the pure Coffea arabica samples (blank sample) revealed no 16-OMC. Precision of recovery was determined by standard deviation of replicates (Table 1). With %RSD less than 5% for all 16-OMC quantities and for both Coffea arabica samples the results were well repeatable. Hence, the developed HPTLC–FLD
3.4. 16-OMC contents in coffee samples from the German market
4. Conclusions HPTLC–FLD was shown an efficient screening tool for a simple and rapid detection of Coffea canephora admixtures to Coffea arabica through the analysis of 16-OMC. Simultaneous separation and quantitation of 22 samples (including calibration standards) was realized by HPTLC, while liquid chromatography requires single runs for each extract and standard. The total HPTLC, including application, separation and detection lasts less than eight minutes per coffee. For rapid sample preparation, direct saponification for one hour and an easy to perform SLE was used. After fluorescent labeling, 16-OMC was selectively determined by HPTLC–FLD without matrix interferences, when high sensitivity was additionally guaranteed. Near to-100% recoveries were obtained for 2–50% Coffea canephora admixtures to Coffea arabica. Acknowledgements The authors express many thanks to Merck (Darmstadt, Germany) for support with plate material. Additionally, the authors express thank to Hochland Kaffee Hunzelmann GmbH (Stuttgart, Germany) providing Coffea canephora samples. Furthermore, the authors express many thanks to Professor Dr. Wolfgang Schwack, University of Hohenheim, for the perfect working conditions at the Institute of Food Chemistry and very helpful discussions.
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Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica Claudia Oellig ∗ , Jessica Radovanovic Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany
a r t i c l e
i n f o
Article history: Received 18 September 2017 Accepted 10 October 2017 Available online xxx Keywords: 16-O-Methylcafestol (16-OMC) Coffea canephora Screening High-performance thin-layer chromatography (HPTLC) Fluorescence detection (FLD)
a b s t r a c t 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 highperformance 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/ > 320 nm. Limits of detection and quantitation of 5 and 14 mg 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%. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Coffea arabica and Coffea canephora var. robusta (often simply called “Arabica” and “Robusta”) are the two most important coffee species worldwide regarding the economic significance. Due to difference in quality and price, Coffea arabica is often blended with Coffea canephora. The determination of such admixture is of great importance to detect commercial frauds and to protect consumers. Regarding their chemical composition, Coffea arabica and Coffea canephora significantly diverge. For instance, there are differences in the amount of caffeine, trigonelline and chlorogenic acids, and also the composition and the amount of the lipids differ [1]. In coffee beans, the coffee oil represents an amount between 7 and 17% of the total mass, with an average value of 10% for Coffea canephora and 15% for Coffea arabica [2,3]. Besides 75% triacylglycerides, the coffee oil comprises of a unsaponifiable fraction of about 20% mainly consisting of specific pentacyclic diterpenes of the kaurene family, which are solely present in coffee. Cafestol, kahweol and 16-Omethylcafestol (16-OMC) are the dominant representatives, and are nearly completely esterified with fatty acids [2–5]. The diterpene 16-OMC is exclusively present in Coffea canephora with an
∗ Corresponding author. E-mail address: [email protected] (C. Oellig).
average amount of 1.7 g/kg (0.8–2.4 g/kg) [3]. Thus, 16-OMC is a reliable chemical marker to distinguish between the two coffee species and excellently suitable to detect and determine Coffea canephora in coffee blends [3,4,6] and monitor the authenticity of products. For this purpose, the total amount of 16-OMC is of interest, consisting of about 98% of the esterified and about 2% of the free form [7]. For the determination of the total amount of 16-OMC two strategies are possible. Either the almost exclusively present form of the esterified 16-OMC can directly be analyzed and set as the total 16OMC, or the de facto total amount of 16-OMC can be determined after saponification. The initial extraction of diterpene esters from the coffee matrix was commonly done by extraction of the coffee oil, mainly with diethyl ether or tert-butyl methyl ether (TBME) [2–4]. Acid-catalyzed decomposition for lipid extraction was not recommended, because degradation of coffee diterpenes occurred [8]. For the analysis of the esterified 16-OMC, gel permeation chromatography was suggested to separate the esters from the coffee oil triglycerides followed by semi-preparative high-performance liquid chromatography (HPLC) or solid phase extraction on silica gel and subsequent HPLC separation with ultraviolet (UV) detection [2]. To determine the total amount of 16-OMC, generally the extracted coffee oil was saponified, the unsaponifiable matter extracted, sometimes cleaned on silica gel, and subsequently determined by gas chromatography [4,9,10] or HPLC–UV
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[4,8,11]. Based on the method of Speer [4], the German standard method DIN 10799 [12] was published, which is identical to the German § 64 LFGB method [13]. This standard method consists of a Soxhlet extraction with TBME for 5 h, saponification with ethanolic potassium hydroxide for 2 h, liquid-liquid extraction of the unsaponifiable matter with TBME and the determination by HPLC–UV, which in total can last 2 days. Alternatively, the direct saponification of a coffee sample was reported by de Roos et al. [6] and Speer et al. [14] for the analysis of 16-OMC, whereas for kahweol and cafestol the direct saponification was mentioned more frequently [6,15–17]. For the determination of coffee diterpenoids, thin-layer chromatography (TLC) was also reported, when a qualitative method for the analysis of 16-OMC with TLC and vanillin sulphuric acid reagent for derivatization was described by Speer and Mischnick [9]. Based on this method, Speer et al. [14] shortly described a TLC screening approach for the esterified and the free form of 16-OMC in the coffee oil and in the direct saponified extract, respectively, with only few validation data. Recently, Schievano et al. [18] and Finotello et al. [19] suggested a quite different method for the detection of 16-OMC. They used nuclear magnetic resonance (NMR) spectroscopy to analyze 16-OMC in roasted coffee beans including a quite rapid sample treatment. The limited availability of the device, however, is a drawback. Considering the time-consuming sample workup and lengthy HPLC–UV analyses of the reported methods, the aim of the present study was to develop a rapid and efficient screening method for the determination of 16-OMC in roasted coffee by high-performance thin-layer chromatography (HPTLC). Thereby, a simple and reliable method for the determination of Coffea canephora admixtures to Coffea arabica should be offered. Extensive and cumbersome sample preparation steps should be replaced by shortened and straightforward procedures. The hydroxyl group of 16-OMC should be selectively labeled for sensitive fluorescence detection (FLD). The great selection of solvents in normal phase HPTLC should enable to develop a selective chromatography to analyze 16OMC without matrix interferences. With the possibility to analyze numerous samples in parallel and with the automated HPTLC devices that guarantee repeatable procedures, the prerequisites for an efficient screening concept are perfectly given.
water (>18 M cm) was supplied by a Synergy System (Millipore, Schwalbach, Germany). HPTLC silica gel 60 plates from Merck were used without pre-washing. Coffea canephora (roasted beans) was provided by Hochland Kaffee Hunzelmann GmbH (Stuttgart, Germany). Coffea arabica (roasted beans) and blends of Coffea arabica with Coffea canephora (70%, 60, 40% and 25% Coffea canephora content, roasted beans and ground coffee) were purchased from local supermarkets. 2.2. Standard and derivatization reagent solutions For the standard stock solution (100 mg/L), 2 mg 16-OMC was dissolved in 20 mL of acetonitrile/2-propanol (3:2, v/v). The stock solution was stored at −20 ◦ C in amber glassware and was used for several weeks. For the internal standard (ISTD) stock solution, 1 mg Sudan IV was dissolved in 1 mL of TBME. The diluted ISTD solution of 3 g/mL was prepared by diluting the stock 1:25 with TBME. For HPTLC–FLD method development, the standard stock solution was used without dilution. For the determination of limit of detection and quantitation (LOD/LOQ), the standard stock solution was diluted 1:50 with acetonitrile, resulting in a concentration of 2 g/mL. To aliquots of 20–200 L of the diluted 16-OMC solutions 75 L of the diluted ISTD solution were added, followed by evaporation under a gentle stream of nitrogen at ambient temperature. The residue was derivatized as described under Section 2.5, resulting in standard solutions for HPTLC of 0.04–0.4 ng/L for the LOD/LOQ experiments, when the concentration of the ISTD generally was 3 ng/L. Calibration standards for sample analyses were analogously prepared by diluting the 16-OMC stock solution 1:10 with acetonitrile and mixing aliquots of 4–600 L with 75 L of the diluted ISTD solution, followed by evaporation and derivatization, and resulting in 16-OMC standard concentrations of 0.04–6 ng/L. The derivatization reagent solution was prepared in anhydrous acetonitrile, containing DMAP and 2-NCl at a concentration of 40 mg/mL and 7.5 mg/mL, respectively. PEI solution was prepared in anhydrous methylene chloride, comprising PEI at a concentration of 250 mg/mL. Both solutions were freshly prepared before use. 2.3. Samples and extraction (saponification)
2. Material and methods 2.1. Chemicals and materials 16-O-Methylcafestol (16-OMC) (99.2%) was obtained from PhytoLab (Vestenbergsgreuth, Germany). Sudan IV (drying loss 1.2%) was received from TCI (Eschborn, Germany). 2-Naphthoyl chloride (2-NCl) (98%), 4-(dimethylamino)pyridine (DMAP) (≥99%, reagent plus), ammonium formate (>97%, purum), gallic acid monohydrate (>98%, purum), acetonitrile and 2-propanol (both LC–MS, Chromasolv), ethanol absolute (≥99.8%, HPLC, Chromasolv), tert-butyl methyl ether (TBME) (≥99.8%, HPLC, Chromasolv) and methylene chloride anhydrous (99.8%) were purchased from Sigma-Aldrich (Steinheim, Germany). Cyclohexane (99.5%, p.a.) and n-hexane (95%, for pesticide residue analysis) (both Chemsolute) were obtained from Th. Geyer (Renningen, Germany). Petroleum ether (PE) (boiling range 40–60 ◦ C, GC, SupraSolv) was from Merck (Darmstadt, Germany). Paraffin oil low viscosity (Ph. eur.) was purchased from Carl Roth (Karlsruhe, Germany). Acetonitrile anhydrous (≥99.8%) and polyethyleneimine branched (PEI) (99%, M.W. 10,000) were obtained from Alfa Aesar (Karlsruhe, Germany). Potassium hydroxide pellets (≥85%, Ph. eur, Prolabo) were from VWR (Bruchsal, Germany). Solid supported liquid extraction (SLE) tubes (Novum SLE, 3cc tubes) were purchased from Phenomenex (Aschaffenburg, Germany). Ultrapure
As samples, different roasted coffee beans (pure coffee species and blends of Coffea arabica with Coffea canephora) and ground coffee (blends of Coffea arabica with Coffea canephora) were used. Before analysis, roasted coffee beans and ground coffee were finely milled in a Tube Mill control for 60 s at 22,000 min−1 (IKA, Staufen, Germany) (<0.3 mm). For extraction, direct saponification was used according to the method of Dias et al. [16] with some modifications. Finely milled coffee sample (100 mg) was weighted into a 6-mL glass centrifuge tube equipped with a screw cap. One mL of 10% potassium hydroxide solution (water/ethanol, 1:9, v/v) was added, the sample was mixed by vortexing for 5 s, and saponification was performed for 1 h on a thermal mixer (Thermomixer comfort, Eppendorf, Hamburg, Germany) at 90 ◦ C. During the saponification time, the tube was programmably shaken six times with 650 rpm for 10 s. After cooling to ambient temperature, 75 L of the ISTD stock solution were added, the sample was briefly mixed by vortexing and centrifuged at 3200×g at 18 ◦ C for 5 min (Biofuge primo R, Heraeus, Hanau, Germany). The clear supernatant was decanted into a 3-mL volumetric flask, filled up with water and was subjected to SLE. Coffee blends for recovery experiments were prepared by adding 2, 5, 20 and 50 mg of finely milled Coffea canephora to 98, 95, 80 and 50 mg of finely milled Coffea arabica, respectively. The relative quantity of 2, 5, 20 and 50% Coffea canephora in the blend corresponded to a 16-OMC content of 36, 90, 360 and 900 mg/kg
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Fig. 1. Workflow for the determination of 16-O-methylcafestol in roasted coffee.
coffee. The calculation was based on an amount for 16-OMC in Coffea canephora of 1.8 g/kg that was previously analyzed with the developed HPTLC–FLD method. During the sample extraction glassware was wrapped into aluminum foil or amber glassware was used.
2.4. Solid supported liquid extraction (SLE) A 400-L aliquot of the saponified sample extract was loaded onto the SLE cartridge. After 12 min soaking (counted once the extract passed the upper frit), SLE was performed twice with 1.2 mL of PE into a 5-mL volumetric flask. The flask was filled up with PE, a 1.5-mL aliquot was evaporated under a gentle stream of nitrogen to complete dryness, and the residue was subjected to derivatization.
2.5. Derivatization Two hundred L of the derivatization reagent solution was pipetted to the sample extract or standard residue, the tube was tightly sealed and was mixed by vortexing for 10 s, followed by storage in the dark at ambient temperature for 40 min, when the tube was vortexed for 10 s after 20 min. After the addition of 800 L acetonitrile, the tube was briefly vortexed, 50 L of PEI solution were added, and the mixture was shaken under light protection for 10 min at 1500 min−1 on a small-size shaking device before being centrifuged at 3200×g at 8 ◦ C for 15 min. A 700-L aliquot of the clear supernatant was transferred into an HPTLC vial. The final extract for HPTLC had a sample concentration of 4 mg/mL. The whole analytical procedure for the determination of 16OMC in roasted coffee, including sample extraction, SLE and derivatization, is sketched in Fig. 1.
2.6. High-performance thin-layer chromatography–fluorescence detection (HPTLC–FLD) Before application, HPTLC plates were dipped 15 mm deep into a 0.05% gallic acid solution in methanol and dried in a gentle stream of cold air for 5 min. An Automatic TLC Sampler 4 (ATS 4, CAMAG, Muttenz, Switzerland) equipped with a light protecting shell was used to apply samples and standards as 6-mm bands with the following settings leading to 22 tracks on a 20 cm × 10 cm plate: 10 mm distance from the lower edge, 8 mm distance from the left edge, and 8.7 mm track distance. Application parameters were: 15 L/s filling speed, 200 nL predosage volume, 200 nL retraction volume, 150 nL/s dosage speed, 4 s rinsing vacuum time, 1 s filling vacuum time, 2 rinsing cycle and 1 filling cycle. Acetonitrile was used as the rinsing solvent. After application, 4 rinsing cycles were performed with water. The application volume generally was 5 L for sample extracts and 16-OMC calibration standards. For LOD/LOQ determination, the standard application volume resulted in 0.2–2 ng/zone, corresponding to 10–100 mg 16-OMC/kg coffee. For calibration standards, the application volume resulted in 0.2–30 ng/zone, corresponding to 10−1500 mg 16-OMC/kg coffee, calculated for 5-L sample extract application. The amount of the ISTD generally was 15 ng/zone. Directly after application, chromatography was performed in the Automatic Developing Chamber (ADC2, CAMAG) with a 20 cm × 10 cm twin-trough chamber (CAMAG), equipped with a light protecting shell. Before development, the plate activity was controlled to 33% relative humidity with saturated magnesium chloride solution for 5 min. As mobile phase, 10 mL of cyclohexane/TBME/formic acid (86:14:2, v/v/v) was used up to a migration distance of 70 mm and a drying step in a stream of cold air followed for 5 min. For detection of the ISTD, the plate was scanned in the absorption mode at 535 nm (tungsten lamp) by the TLC Scanner 4 (CAMAG) in the range of 40–70 mm with a scanning speed of 20 mm/s, a data resolution of 100 m/step and a slit dimen-
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Fig. 2. Images of HPTLC silica gel plates developed with cyclohexane/TBME/formic acid (86:14:2, v/v/v) up to a migration distance of 70 mm under UV 254 nm illumination after dipping in n-hexane/paraffin: (A) 16-O-methylcafestol (16-OMC), 24 ng/zone, (corresponding to ∼70% Coffea canephora in a coffee blend); (B) from left to right, different Coffea arabica sample extracts (4 mg coffee/mL); (C) extract of Coffea canephora (4 mg/mL) with 1.8 g/kg 16-OMC (corresponding to 36 ng 16-OMC/zone); (D) from left to right, extracts of blends (4 mg/mL) of Coffea arabica with 40% and 70% Coffea canephora (corresponding to 14 and 24 ng 16-OMC/zone); (E) reagent blank for comparison. All samples were prepared according to the developed method, the application volume generally was 5 L.
sion of 4 mm × 0.30 mm. The plate was dipped into a solution of n-hexane/paraffin oil (2:1, v/v) (TLC Chromatogram Immersion Device III (CAMAG), immersion speed 2, immersion time 3) and dried in a stream of cold air for 1 min. Plate images were captured under UV 254 and UV 366 nm illumination (TLC Visualizer 4, CAMAG). Thereafter, the plate was scanned in the fluorescence mode at UV 244/ > 320 nm (mercury lamp) with the same settings for scanning speed, data resolution and slit dimension as mentioned above. The manual detector mode was used applying a quick scan range of 32–42 mm on the track of the highest standard of the acylated 16-OMC. HPTLC instruments were controlled by the software winCATS, version 1.4.6.2002 (CAMAG). Microsoft Excel, version Office Professional Plus 2010 was used for statistical analysis. Calibration curves were calculated by linear regression. For validation, means, standard deviations and relative standard deviations were calculated.
2.8. Analysis of coffee samples from the German market Roasted coffee from the German market with labelled contents of Coffea canephora of 100% (coffee beans), 70% (ground coffee), 60% (coffee beans), 40% (coffee beans), 25% (ground coffee and coffee beans) and 0% (pure Coffea arabica, coffee beans) were used. Sample analysis including saponification, SLE, derivatization, and HPTLC–FLD was performed according to the procedures described above (n = 4). For these samples, the calibration range was extended to include pure Coffea canephora. Thus, 10–800 L of the diluted 16OMC standard solution were derivatized as described under Section 2.5, resulting in amounts of 0.5–40 ng/zone which corresponds to 25–2000 mg 16-OMC/kg coffee. 3. Results and discussion 3.1. Approach
2.7. HPTLC–MS For zone elution, the TLC–MS interface (CAMAG) equipped with a circular elution head plunger (4 mm), was used. The eluent was provided by an Agilent (Waldbronn, Germany) 1100 HPLC pump and the TLC–MS interface was directly connected to the MS. The MS device (Agilent) consisted of a G1956 B MSD single quadrupole MS with an atmospheric pressure ionization electrospray (ESI) interface (G1946), and was operated by ChemStation B.04.03 software (Agilent). For HPTLC–MS measurements, the plate was used without dipping into n-hexane/paraffin. The target zone was marked with a pencil under UV 254 nm illumination before zone elution was done. Acetonitrile/10 mM ammonium formate (9:1, v/v) was used as elution solvent at a flow rate of 0.2 mL/min for 60 s. A blank zone was eluted for 60 s after the elution of the target zone to clean the elution head. Measurements in the ESI+ mode were performed with the following parameters: capillary voltage 5.0 kV, skimmer ◦ voltage 35 V, lens 2.5 V, quadrupole temperature 100 C, drying gas ◦ temperature 300 C, drying gas flow rate 10 L/min and nebulizer gas pressure 40 psig. Total ion chronograms were recorded at m/z 200–1200, using a fragmentor voltage of 60 V, gain 5, threshold 100, and step size 0.1.
In a first step, a suitable derivatization for 16-O-methylcafestol (16-OMC) was selected and optimized before the conditions for HPTLC–FLD were developed. In the second step, the sample workup was evaluated, regarding the efficiency of the saponification and the liquid-liquid partition step for 16-OMC from roasted coffee. Having developed the entire HPTLC–FLD screening method, the performance characteristics were assessed by LOD, LOQ and the precision of the method. Finally, several coffee samples from the German market were analyzed by HPTLC–FLD and the Coffea canephora contents was calculated. 3.2. Method development 3.2.1. Derivatization With the aim to gain high selectivity and sensitivity for 16-OMC, the hydroxyl group of the diterpenoid was used for fluorescence marking. Therefore, the simple acylation with 2-naphthoyl chloride (2-NCl) and 4-(dimethylamino)pyridine (DMAP) in methylene chloride according to the method of Oellig [20] for the determination of ricinoleic acid in rye by HPTLC–FLD was selected. HPTLC–MS measurements of the only product zone at hRF 50 (Fig. 2A) with ESI+
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Fig. 3. (A) HPTLC chromatogram on silica plates under UV 254 nm illumination of 16-O-methylcafestol (16-OMC) standards and extracts of Coffea arabica blends with 20 and 50% Coffea canephora (Coffea arabica sample 1), corresponding to 0.36 and 0.9 g 16-OMC/kg (n = 5), and the pure extract of Coffea arabica sample 1; (B) corresponding 3D densitogram of the fluorescence scan at UV 244/ > 320 nm and the absorption scan at 535 nm, and (C) resulting calibration graph of 16-OMC (0.03–1.5 g/kg coffee). Triangles and circles correspond to the blends with 20 and 50% Coffea canephora, respectively.
in the full scan mode showed mass signals at m/z 453, 485 and 991. They indicated the presence of the acylated 16-OMC with signals for the protonated molecule after methanol elimination (m/z 453), the protonated molecule (m/z 485), and the sodium adduct of the
dimer (m/z 991). According to literature [21–23], the kinetic of the acylation is strongly influenced by the configuration of the analyte. Therefore, we evaluated the influence of the reaction time and temperature, the applied catalyst and the stoichiometric ratio of 16-
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OMC/2-NCl/catalyst for the completeness of the derivatization. The reaction yields were quantitated by HPTLC–FLD with the optimal detection settings for the derivatization product. The reaction conditions according to [20], with an slightly extended reaction time of 40 min and light protection, proved suitable for highest yields. With the intention to replace the toxic reaction solvent, several anhydrous solvents were evaluated and acetonitrile was verified to be likewise suitable. The final derivatization procedure consisted of the addition of 200 L of derivatization reagent (containing of 7.5 mg 2-NCl and 40 mg DMAP/mL acetonitrile) to the dried sample, a reaction time of 40 min under light protection and the addition of 800 L acetonitrile. The use of dispersive solid phase extraction with primary secondary amine for the reduction of the excess of derivatization reagent in the final extract was not possible due to occurrence of an additional fluorescent zone, which interfered with the standard zone. Shaking with 50 L of a polyethyleneimine (PEI) solution (250 mg/mL in methylene chloride) followed by a centrifugation step for 15 min at 8 ◦ C turned out to be a suitable alternative and guaranteed an interference-free chromatography. 3.2.2. HPTLC–FLD A 16-OMC standard, the internal standard Sudan IV and several roasted coffee samples (pure Coffea arabica and Coffea canephora and coffee blends) were applied for HPTLC method development. Coffee extracts were prepared according to the developed sample workup including the derivatization. Silica gel and amino bonded HPTLC plates were checked, when silica gel offered best separation of 16-OMC and Sudan IV from matrix components and from components of the derivatization reagent. Several solvents and solvent mixtures were tested as the mobile phase taking into account the selectivity of the groups referred to Snyder [24]. Separation was visible by the fluorescence under UV 254 nm illumination after dipping in n-hexane/paraffin for fluorescence enhancement and under white light (for Sudan IV). Cyclohexane and TBME (86:14, v/v) as the mobile phase turned out to be selective for the separation of 16-OMC from the coffee matrix and by-products of the fluorescent labeling reagent. The addition of formic acid improved the separation. Finally, a mixture of cyclohexane/TBME/formic acid (86:14:2, v/v/v) was selected on HPTLC silica gel material for best separation and sharp zones for 16-OMC and Sudan IV. Because separation and sharpness of the 16-OMC zone was influenced by the water content of the layer, the plate activity was controlled to 33% relative humidity. For a migration distance of 70 mm, the conditions resulted in a hRF value of 50 for 16-OMC, shown by the 16-OMC standard and a pure Coffea canephora extract under UV 254 nm illumination (Fig. 2A and C). The hRF for Sudan IV was 60 (Fig. 3B). Coffea arabica samples showed no interfering matrix (Fig. 2B), confirmed by coffee blends with 40 and 70% Coffea canephora (Fig. 2D), and the derivatization reagent also did not interfere with the separation (Fig. 2E). Visual detection of 16-OMC was possible down to ∼7 ng/zone (corresponding to a coffee blend with ∼20% Coffea canephora, calculated for a 5-L sample volume) (Fig. 3A). Quantitation of 16-OMC was performed in the fluorescence mode, while the internal standard Sudan IV was measured in the absorption mode. The recorded fluorescence spectrum of the acylated 16-OMC in the remission mode revealed an intensive maximum at 244 nm, the absorption spectrum of Sudan IV indicated a maximum at 535 nm. Therefore, an excitation wavelength of 244 nm (mercury lamp) was selected for the fluorescence scan in combination with the 320 nm edge filter, and a wavelength of 535 nm (tungsten lamp) for the absorption scan. 3.2.3. 16-OMC protection During method development, sometimes the intensity of the 16-OMC zone was significantly reduced, directly seen under UV 254 nm illumination and additionally detected in the fluorescence
scan. According to literature [4,8,13,15,16,25], diterpenes are prone to (photo)oxidation. Therefore, light protection was provided during the entire sample preparation and HPTLC. Reduced signal intensities were still observed, because 16-OMC was also prone to oxidation upon contact with the silica gel. This degradation process already occurred in earlier studies for indole alkaloids (ergot alkaloids) and their derivatives [26,27]. Based on these former experiences, gallic acid was applied as protective substance at the application area before the samples and standards were applied. Dipping the plate into a 0.05% solution of gallic acid in methanol offered an effective protection of the diterpenoid from degradation. 3.2.4. Sample preparation For diterpenoid extraction, time-consuming lipid extraction followed by saponification of the coffee oil and laborious liquid-liquid partition (LLE) for the extraction of the unsaponifiable matter were often reported in literature [3,4,7,8,13]. With the intention to shorten sample preparation, more efficient and straightforward sample workup strategies were evaluated. For method development, a 16-OMC standard, pure Coffea arabica and Coffea arabica blended with 20% Coffea canephora were applied, and analyzed after derivatization with HPTLC−FLD to check the differences in matrix loads and recoveries. To correct volume errors during the entire sample preparation and HPTLC application, Sudan IV was used as internal standard. As Sudan IV was easily detected in a visible absorption scan, it did not interfere with the fluorescent 16OMC and additionally was well separated (hRF 60) from 16-OMC (hRF 50). For extraction, we selected direct saponification of roasted coffee samples, which was already mentioned for cafestol and kahweol [16,17] and also 16-OMC [6], and was proven more effective than the pre-extraction of the coffee oil with subsequent saponification [15]. Based on the reported methods [6,15–17] with little modifications, 100 mg of fine milled roasted coffee was directly saponified with 1 mL of 10% potassium hydroxide solution (ethanol/water, 9:1, v/v) at 90 ◦ C. To check the completeness of saponification of the esterified 16-OMC, the saponification time was varied (0.5–3 h), while all other parameters were kept constant. Determined quantities for 16-OMC in the Coffea arabica blend (20% Coffea canephora) after 1 h and 2 h saponification and LLE in TBME did not show significant differences, but longer saponification time reduced the yields. Significantly shorter saponification (0.5 h), however, reduced the yields considerably. These findings were in accordance to Gross et al. [28], Urgert et al. [17] and Dias et al. [15,16], who used 1 h saponification and reported the possibility of degradation and formation of artifacts for long saponification times. Consequently, saponification time was set to 1 h. Analysis of the coffee blend after lipid extraction with TBME in the ultrasonic bath for 5 h, followed by saponification of the extracted coffee oil and LLE in TBME, revealed equal results for 16-OMC. With the aim to simplify and automate the laborious LLE procedures, solid supported liquid extraction (SLE) was selected as alternative. According to literature performing LLE [2,4,6,8,13,15–17], TBME was first checked as elution solvent, resulting however in interfering matrix in the extract. With the intention to obtain matrix-free extracts and efficient recovery of 16-OMC, several single solvents (diethyl ether, diisopropyl ether, petroleum ether (PE)), solvent mixtures, single and multiple elution, and different elution volumes were tested. Finally, PE as elution solvent turned out to be most suitable; highest 16OMC recoveries were obtained and matrix interferences were not present. The resulting procedure comprises of the application of a diluted aliquot of the saponified extract onto the SLE cartridge, a soaking time of 12 min and a twofold elution with PE (Fig. 1). In the end, the straightforward sample preparation consisted of a direct saponification with 10% ethanolic potassium hydroxide
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Table 1 Recoveries of 16-O-methylcafestol (16-OMC) from Coffea arabica blends with Coffea canephora, quantitated against 16-OMC. 2%a Coffea arabica
5%b
20%c
50%d
95 ± 4 94 ± 5
100 ± 4 102 ± 1
97 ± 4 99 ± 6
Coffea canephora Mean recovery [%] ± SDe (n = 5) Mean recovery [%] ± SDe (n = 5)
Sample 1 Sample 2
96 ± 5 97 ± 5
a,b,c,d e
Corresponding to 36, 90, 360, and 900 mg 16-OMC/kg coffee. Standard deviation.
Table 2 16-O-Methylcafestol (16-OMC) and Coffea canephora contents in seven coffee samples form the German market. Sample
Labelled Coffea canephora content [%]
16-OMC [mg/kg] ± SDa (n = 4)
Calculated Coffea canephora content [%]b
1 2 3 4 5 6 7
70 60 40 25 25 0 100
1140 ± 40 1070 ± 20 710 ± 42 410 ± 20 460 ± 25
67 ± 2 63 ± 1 42 ± 2 24 ± 1 27 ± 1 0 –
a b
Standard deviation. Based on a literature average amount of 1.7 g 16-OMC/kg Coffea canephora.
for 1 h followed by a rapid SLE with PE. After an easy to perform pre-chromatographic derivatization with 2-NCl for a sensitive fluorescence detection of 16-OMC, the samples were analyzed by HPTLC–FLD, allowing to analyze up to 22 samples (including calibration standards) in parallel in less than two hours.
method for the analysis of 16-OMC in roasted coffee is a suitable screening tool for the detection of Coffea canephora admixtures to Coffea arabica.
3.3. Method validation
Seven coffee samples from the local market (with labelled contents of Coffea canephora between 0 and 100%) were analyzed by the HPTLC–FLD screening method, and the Coffea canephora content was calculated taking into account the average value of 1.7 g 16OMC/kg Coffea canephora (Table 2). For all samples the calculated Coffea canephora content correlated very well with the labelled amount and results were well repeatable with %RSD <6% (n = 4). The determined amount of 1.8 g 16-OMC/kg Coffea canephora for the pure Coffea canephora sample (sample 7) agreed well with the average amount of 1.7 g 16-OMC/kg Coffea canephora reported in literature [3,18,19].
3.3.1. Limits of detection and quantitation 16-OMC was used as calibration standard and derivatized with 2-NCl like the sample extracts. Calibrations were performed in the range 0.2–2 ng 16-OMC/zone, calculated to 10–100 mg 16OMC/kg coffee (n = 5) taking into account the sample preparation and the sample application volume of 5 L. Resulting calibration graphs revealed good linearity with high coefficients of correlation (R2 > 0.9976). According to the DIN 32645 calibration method [29], limits of detection (LOD) and limit of quantitation (LOQ) were determined to 0.1 and 0.28 ng 16-OMC/zone, respectively, corresponding to 5 and 14 mg 16-OMC/kg coffee. This is equivalent to 0.3 and 0.8% Coffea canephora admixture to Coffea arabica regarding a literature average amount of 1.7 g 16-OMC/kg Coffea canephora. With relative standard deviations (%RSD) of 5.4% and 4.6% the determination was quite well repeatable. Thus, the developed HPTLC–FLD screening allows the determination of 16-OMC quantities that correspond to Coffea canephora admixtures below 1%. 3.3.2. Recoveries Recovery experiments for 16-OMC in roasted coffee were done in blends of Coffea arabica with 2, 5, 20 and 50% Coffea canephora (n = 5), corresponding to a 16-OMC quantity in the blends of 36, 90, 360, 900 mg 16-OMC/kg. Recovery experiments were carried out with two Coffea arabica samples (sample 1 and 2). The internal standard mode was used for quantitation, when the peak areas ratios 16-OMC/ISTD were calculated for evaluation. Quantitation by HPTLC−FLD with a 16-OMC solvent standard is exemplarily shown in Fig. 3. For all prepared coffee blends, the recovery rates were near 100% (Table 1) independent on the used Coffea arabica sample. Consequently, analysis of the pure Coffea arabica samples (blank sample) revealed no 16-OMC. Precision of recovery was determined by standard deviation of replicates (Table 1). With %RSD less than 5% for all 16-OMC quantities and for both Coffea arabica samples the results were well repeatable. Hence, the developed HPTLC–FLD
3.4. 16-OMC contents in coffee samples from the German market
4. Conclusions HPTLC–FLD was shown an efficient screening tool for a simple and rapid detection of Coffea canephora admixtures to Coffea arabica through the analysis of 16-OMC. Simultaneous separation and quantitation of 22 samples (including calibration standards) was realized by HPTLC, while liquid chromatography requires single runs for each extract and standard. The total HPTLC, including application, separation and detection lasts less than eight minutes per coffee. For rapid sample preparation, direct saponification for one hour and an easy to perform SLE was used. After fluorescent labeling, 16-OMC was selectively determined by HPTLC–FLD without matrix interferences, when high sensitivity was additionally guaranteed. Near to-100% recoveries were obtained for 2–50% Coffea canephora admixtures to Coffea arabica. Acknowledgements The authors express many thanks to Merck (Darmstadt, Germany) for support with plate material. Additionally, the authors express thank to Hochland Kaffee Hunzelmann GmbH (Stuttgart, Germany) providing Coffea canephora samples. Furthermore, the authors express many thanks to Professor Dr. Wolfgang Schwack, University of Hohenheim, for the perfect working conditions at the Institute of Food Chemistry and very helpful discussions.
Please cite this article in press as: C. Oellig, J. Radovanovic, Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.10.031
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Please cite this article in press as: C. Oellig, J. Radovanovic, Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.10.031