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User Benefits Application News Simultaneous Determination of Functional Components in Coffee Simultaneous analysis of highly polar compounds from acidic to basic is possible. The peaks of the compounds with basic group that have generally poor retention in reversed phase columns can be retained, and stable analysis can be achieved. N. Iwata Introduction Recently, coffee drinking has been reported to be effective in preventing or improving dementia and lifestyle-related diseases such as diabetes and cancer. 1),2) It has also been found that coffee components such as pyrocatechol and trigonelline contribute to these effects. 3),4) However, trigonelline, a compound with basic group, is difficult to analyze due to poor retention on a C18 column. Therefore, a PFPP column that has pentafluorophenylpropyl as the stationary phase was used for the analyses in this study. This column is expected to provide a unique separation selectivity as a result of π–π interaction and dipole-dipole interaction in addition to hydrophobic interaction. This article describes simultaneous determination of functional components in coffee using a Shim-pack Scepter™ PFPP. Table 1 Analytical Conditions Repeatability Table 2 shows the reproducibilities (%RSD) of the retention time and the peak area of mixed standard solution of 1 mg/L for each compound in six repeated analyses. Analysis of Mixed Standard Solution Fig. 1 shows the structural formula of trigonelline, pyrocatechol, chlorogenic acid, and caffeic acid, which were the targets of the analysis. This article compared two analytical columns, a Shim-pack Scepter C18 and a Shim-pack Scepter PFPP. Fig. 2 shows chromatograms of the mixed standard solution (10 mg/L each) acquired by each column using the analytical conditions indicated in Table 1. Trigonelline was retained by the Shim-pack Scepter PFPP but was only poorly retained by the Shim-pack Scepter C18. Generally, longer retention provides improved separation, whereas shorter retention leads to inadequate separation with co-existing interferences. High Performance Liquid Chromatograph Nexera™ lite O H O H O O O H OH O OH O H N + O CH 3 O – OH OH OH OH O O H Trigonelline Pyrocatechol Chlorogenic acid Caffeic acid System: Nexera lite Column (C18): Shim-pack Scepter C18-120 *1 (150 mm×4.6 mm I.D., 3 µm) Column (PFPP): Shim-pack Scepter PFPP-120 *2 (150 mm×4.6 mm I.D., 3 µm) Flowrate: 1.0 mL/min Mobile Phase: A) 20 mmol/L (Sodium) phosphate buffer (pH 2.6) B) Acetonitrile Time Program: 0 %B (0.00-1.00 min)→10%B (4.00 min) →20 %B (10.00-12.00 min) →70 %B (12.01-13.00 min) →0 %B (13.01-18.00 min) Mixer: 180 µL Column Temp.: 25 °C Injection Volume: 5 µL Vial: SHIMADZU LabTotal™ for LC 1.5 mL, Glass *3 Detection (PDA): Ch1 : 270 nm, Ch2 : 325 nm (SPD-M40) *1 P/N: 227-31016-05 *2 P/N: 227-31057-05 *3 P/N: 227-34001-01 0.0 2.5 5.0 7.5 10.0 min 0 25 50 75 mAU 0.0 2.5 5.0 7.5 10.0 min 0 50 100 150 200 mAU 1 2 270 nm 3 4 5 ■Peaks 1. Trigonelline, 2. Pyrocatechol, 3. Chlorogenic acid, 4. Caffeine 5. Caffeic acid 1 2 3 4 5 3 5 3 5 C18 PFPP C18 PFPP 325 nm Fig. 1 Structure of Four Target Compounds Fig. 2 Chromatograms of Mixed Standard Solution (10 mg/L each)
Application News www.shimadzu.com/an/ Shimadzu Corporation Analytical & Measuring Instruments Division Global Application Development Center © Shimadzu Corporation, 2022 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. First Edition: Feb. 2022 01-00280-EN Conclusion A method for simultaneous analysis of functional component with different physical properties in coffee was developed. Using a Shim-pack Scepter PFPP, even basic compounds such as trigonelline were adequately retained and separated from contaminants. The method described in this article is expected to contribute to research and development in food engineering including the study for functional components. Fig. 5 UV Spectra Nexera, Shim-pack Scepter, and Shimadzu LabTotal are registered trademarks of Shimadzu Corporation in Japan and other countries. References 1) van Dam R. M., Feskens E. J., Lancet 360, 1477-1478 (2002). 2) Poole R., Kennedy O. J., Roderick P., Fallowfield J. A, Hayes P. C., Parkes J., BMJ 359, j5024 (2017). 3) Fukuyama K., Kakio S., Nakazawa Y., Kobata K., Funakoshi-Tago M., Suzuki T., Tamura H., Mol. Nutr. Food Res. 62, e1800238 (2018). 4) Farid M.M., Yang X., Kuboyama T., Tohda C. Scientific Reports 10, 16424 (2020). Compound Conc. range (mg/L) r 2 Trigonelline 1-100 0.99999 Pyrocatechol 0.1-10 0.99999 Chlorogenic acid 1-100 0.99999 Caffeine 1-100 0.99999 Caffeic acid 0.1-10 0.99999 (mg/L) (mg/L) Trigonelline Pyrocatechol 0 50 0.0 2.5 5.0 7.5 (x100,000) 0.0 5.0 0.0 2.5 5.0 (x10,000) Fig. 3 Calibration Curves Compound Retention time Peak area Trigonelline 0.08 0.65 Pyrocatechol 0.05 0.50 Chlorogenic acid 0.07 0.14 Caffeine 0.06 0.15 Caffeic acid 0.05 0.21 Table 2 Repeatability (%RSD) in Six Repeated Analyses Calibration Curves The calibration curves for the five target compounds were highly linear, with coefficients of determination (r 2 ) of 0.99999 or greater. Fig. 3 shows the calibration curves of trigonelline and pyrocatechol. Table 3 shows the concentration ranges of calibration curves and the coefficients of determination for all the target compounds. Peak area Peak area Conc. Conc. Table 3 Concentration Ranges of Calibration Curves and Coefficients of Determination (r 2 ) Compound Concentration (mg/L) %RSD Trigonelline 21.5 0.08 Pyrocatechol 1.0 1.05 Chlorogenic acid 21.3 0.07 Caffeine 79.2 0.05 Caffeic acid N.D. N.D. 1 2 3 4 ■Peaks 1. Trigonelline 2. Pyrocatechol 3. Chlorogenic acid 4. Caffeine 3 2 270 nm 325 nm ■Peak 3. Chlorogenic acid 200 300 nm 0 50 100 150 mAU 200 300 nm 0 100 200 300 400 mAU 200 300 nm 0 25 50 75 mAU 200 300 nm 0 250 500 mAU Trigonelline Pyrocatechol ― STD ― Coffee Chlorogenic acid Caffeine 0.0 2.5 5.0 7.5 10.0 min 0 50 100 150 200 250 mAU 7.0 7.5 8.0 min 0.0 2.5 5.0 7.5 mAU 0.0 2.5 5.0 7.5 10.0 min 0 25 50 75 100 mAU ― STD ― Coffee ― STD ― Coffee ― STD ― Coffee Fig. 4 Chromatograms of Coffee (Solid Line: Coffee, Dashed line: Standard Solution) Analysis of Coffee Ten grams of commercial ground coffee beans were extracted with 150 mL of boiling water to form the sample. The sample was filtered through a 0.2 µm membrane filter and diluted ten- fold with ultrapure water before HPLC analysis. Chromatograms of the coffee are shown in Fig. 4 and the analytical results in Table 4. In this table, “Concentration” means the concentration after sample preparation. Fig. 5 shows UV spectra of the sample and the standard solution. The UV spectral similarity to the reference compounds suggests that the target compounds and respective contaminants in the coffee were appropriately separated. Table 4 Analytical Results (N=6)
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