Identification of markers of thermal processing (“roasting”) in aqueous extracts of Coffea arabica L. seeds through NMR fingerprinting and chemometrics

Roasting of Coffea arabica L. seeds gives rise to chemical reactions that produce more than 800 compounds, some being responsible for the desired organoleptic properties for which the beverage called “coffee” is known. In the industry, the “roasting profile,” that is, the times and temperatures applied, is key to influence the composition of roasted coffee beans and the flavour of the beverage made from them. The impact of roasting on the chemical composition of coffee has been the subject of numerous studies, including by nuclear magnetic resonance (NMR) spectroscopy. However, the roasting equipment and profiles applied in these studies are often far from real industrial conditions. In this work, the effects of two critical technological parameters of the roasting process, namely, the “development time” (the period of time after the “first crack,” a characteristic noise due to seed disruption) and the final roasting temperature on coffee extracts, were investigated. Seeds were roasted at pilot scale according to 13 industrial roasting profiles and extracted in D₂O. The extracts were analysed by ¹H NMR experiments. The NMR spectra were compared using (a) quantitative analysis of main signals by successive orders of magnitude and (b) chemometric tools (principal component analysis, partial least squares and sparse-orthogonal partial least squares analysis). This allowed to identify compounds, which may serve as markers of roasting and showed that changes in chemical composition can be detected even for slight change in final temperature (~1°C) or in total roasting time (~25 s).     

亲水作用色谱法测定胡芦巴中的胡芦巴碱

建立了亲水作用色谱法(HILIC)测定胡芦巴药材中胡芦巴碱含量的方法。采用Waters Atlantis HILIC Silica色谱柱(150 mm×2.1 mm, 3 μm),以乙腈-乙酸铵溶液(pH 4.4)(体积比为70:30)为流动相,流速0.4 mL/min,检测波长265 nm。胡芦巴碱的线性范围为2.50～100 mg/L (r=0.9996);两个加标水平的平均加样回收率为102%,相对标准偏差(RSD)分别为4.17%和2.28%(n=3)。结果表明所建方法分离效果好、快速简易,可以弥补中国药典中离子对色谱法(IPLC)平衡时间过长的缺陷,适用于胡芦巴药材中强极性胡芦巴碱的测定,为胡芦巴的质量控制提供了有效的方法。     

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography–MS/MS analysis: Application to a pharmacokinetic study

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI‐MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile–ammonium formate (10 mm , pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.     

超声波萃取-高效液相色谱测定咖啡粉和速溶咖啡中的葫芦巴碱

建立了高效液相色谱测定咖啡粉和速溶咖啡中葫芦巴碱含量的方法。采用Waters BondPak NH2色谱柱(250 mm×4.6 mm, 5 μm),以甲醇-水(82:18, v/v)为流动相,流速0.8 mL/min,检测波长260 nm。葫芦巴碱线性范围为1～40 mg/L (相关系数为0.9998)。分别进行日内、日间和人员比对测定葫芦巴碱的重复性试验及加标回收率试验。经测定,样品的加标回收率大于90%,相对标准偏差小于3%。比较了超声波萃取和热浸提萃取方法测定葫芦巴碱含量的差异,两个方法的测定结果符合线性关系,相关系数为0.9964。该法简便、快速、灵敏度高,适用于咖啡豆中葫芦巴碱分析以及咖啡豆原料与制品的质量控制。     

Development and optimization of a method for determining betaine and trigonelline in the fruits of Lycium species by using solid‐phase extraction combined with high‐performance liquid chromatography–diode array detector

Betaine is an essential nutrient for humans and a source of methyl donors for methionine and S‐adenosylmethionine formation, and it is used as a biomarker for pharmacological activities and to evaluate the quality of Lycium species and common foods. However, because of its special structural features, poor ultraviolet‐chromophore, and high polarity, the existing methods for betaine extraction and quantification cannot provide higher extraction efficiency, better sensitivity, or resolution degree. A simple, fast, and efficient high‐performance liquid chromatography–diode array detector coupled with solid‐phase extraction was adopted for simultaneous separation and quantification of betaine in four types of Lycium species. The results revealed that after degreasing with dichloromethane, extraction with 80% ethanol (pH adjusted to 1.0 with hydrochloric acid), and elution with aluminum oxide (OH^? form), the improvement in the average yield rate of betaine was thrice of that of the existing methods. In addition, trigonelline was identified as the interfering substance of betaine for the first time in Lycium species, and betaine and trigonelline were simultaneously separated and quantified. Furthermore, their chemical characteristics and content distribution in different Lycium species were carried out.     

毛细管电泳用于中药白鲜皮中生物碱的分析

建立了毛细管电泳同时分离白鲜皮中3种生物碱(胡芦巴碱、白鲜碱和胆碱)的的定量分析方法.以缓冲液H3BO3—Na2B4O7(pH=8.4,5 mmol/L)、添加剂3 mmol/L SDS和0.1%triton X-100为电泳运行液,24 kV为分离电压,分离在5 min内就可以快速完成.在230 nm检测波长处,3种组分的线性范围为:胡芦巴碱5～100μg/mL、白鲜碱5～75μg/mL、胆碱50～300μg/mL,检测限分别为1.4、1.4和16.0μg/mL.方法用于白鲜皮实际样品的测定,回收率范围在92.0%～101.0%之间。     

Selective determination of caffeine and trigonelline in aqueous extract of green coffee beans by FT-MIR-ATR spectroscopy

A non-destructive, fast, simple and reliable Fourier transform mid-infrared attenuated total reflectance spectroscopy (FT-MIR-ATR) method for the selective determination of caffeine and trigonelline in the aqueous extract of green coffee beans was developed and validated. The calibration curves were linear in the range 2000 − 7000 mg/L for caffeine and trigonelline with R² ≥ 0.9997. The limits of detection (LOD) were 140 and 100 mg/L and the limits of quantification (LOQ) were 470 and 330 mg/L for caffeine and trigonelline, respectively. The precision (% RSD) was 3.0% and 4.3% for caffeine and trigonelline, respectively. The developed method was applied to 20 samples of green coffee beans to determine the two alkaloids. The amount of caffeine and trigonelline in the green coffee beans were found in the range 0.84 − 1.15% (w/w) and 0.83 − 1.13% (w/w), respectively. The accuracy of the developed analytical method was evaluated by spiking standard caffeine and trigonelline to green coffee beans and the average recoveries were 93 ± 5% and 98 ± 4%, respectively. Therefore, the developed FT-MIR-ATR methods can be used for direct determination of the two alkaloids in the green coffee beans.