5.2.1. Chromatographic Methods
Going back to the 20th century, the chromatographic methods used included paper and thin layer chromatography, most often only to detect the components present in the samples. Swain identified coumarins in 1952, using filter-paper chromatography, since it was shown that coumarins are involved in enzymatic browning in potatoes [
82]. Various mobile phases, as well as reagents for spraying paper, were used to identify coumarin by making them noticeable on paper and display R
F (retention factor) values. R
F actually represents the distance traveled by the compound, divided by the distance traveled by the solvent; the number is distinctive, thus representing how the component can be identified. Among the various mobile phases used, the combinations of n-butanol-acetic acid-water and amyl alcohol-acetic acid-water proved to be the most appropriate, since the glycosides and aglycones could also be separated. The most common problems encountered with other mobile phases were elliptical and elongated spots with tailing. After developing the chromatogram, the components were detected using ultraviolet light, and various paper spraying reagents were tested. For the detection of coumarin, a paper spray prior to detection with 2N NaOH was required. Displaying the R
F values for all components tested, indicates the suitability of the above method for separating and identifying the coumarins and other components tested from different sources. In addition to paper chromatography, thin-layer chromatography (TLC) is also one of the earliest used chromatographic techniques. This method, in the context of coumarin determination in foods, has been most commonly used to determine vanilla flavors [
74,
83]. For the qualitative as well as the quantitative determination of coumarin in the vanilla flavorings, the pre-adsorbent silica gel plates were used as stationary phase, while the toluene-methanol solvent mixture (97:3,
v/
v) was used as the mobile phase. The plate was sprayed with NaOH and then placed under UV light to spot component stains. Coumarin was identified with respect to fluorescence, or the appearance of color, and quantified using a densitometer [
83]. In addition to detecting coumarin, TLC has also been used to detect a number of components such as vanillin, ethyl vanillin, 4-hydroxy-benzaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, vanillic acid, coumarin, piperonal, anisic acid and anisaldehyde in vanilla flavor as well as in foods containing vanilla flavors. To make the separation process selective and reproducible, an automated multiple development (AMD) system was used. Silica gel plates were used as a stationary phase, and to ensure good separation and resolution without tailoring of polar components, a stepwise mobile phase including chloroform, ethyl acetate, 1-propanol, acetic acid and hexane was used. By developing such a method that allowed the separation of different components from food, beverages and confectionery products, it was shown how TLC can be used in this direction and how it is a suitable technique for determining different components. The main advantages of TLC for testing the composition of vanilla extracts and vanilla-containing foods are the simplicity of sample preparation, fast analysis and a high sample throughput at reduced costs compared to the more advanced analytical techniques. Considering all the above recommendations, this method could be a suitable routine method for quality control [
74]. In addition, TLC is a method that has evolved over the years, so high-performance thin layer chromatography (HPTLC) was created, which enables the automation of certain steps, increases resolution, and can make quantitative measurements more accurately. Krüger et al. [
17] have determined the amount of coumarin in 43 commercially available cinnamon and cinnamon-containing foods using HPTLC and HPTLC-MS while developing a method that combines several techniques called high-pressure thin layer chromatography-fluorescence detection-mass spectrometry/effect-direct analysis (HPTLC-FLD-MS/EDA). Considering the positive characteristics of TLC, and applying a more advanced version, the idea was to develop a quicker and simpler method that would allow simultaneous screening and quantification at low concentrations for a large number of samples, which is especially important for fast and routine analysis of multiple products. The problem of the food analysis is the complexity and variability of the matrix, so the composition analysis are difficult and often involves different pretreatments. The aforementioned developed method for the analysis of various food products, such as tea, breakfast cereals, milk rice, jam, cinnamon stars and cinnamon buns, shows a high specificity for coumarin, without the first additional sample processing. The samples were extracted and diluted and then, after the completion of the chromatography process, derivatized and as such were detected using fluorescence, 17 samples at a time. In order to show the efficiency of the method, the precision and repeatability of the method was determined, even for foods such as cereals and milk rice whose matrix is complex and affects the results, precision is between 4%–7% and repeatability is below 10%. Given the low LOD (limit of detection) and LOQ, the method indicated allows coumarin in different foods to be determined at low concentrations, such as 200 pg/6-mm band and 400 pg/6-mm band, respectively. In addition, HPLTC was combined with effect-directed analysis, as a powerful bioanalytical tool, to provide additional information on bioactive components in the samples and to make a complete risk assessment [
17].
Today, by using chromatography, rapid and effective methods for the detection and quantification of coumarin in various samples can be developed, such as the method by Solaiman Al-Zehouri [
76], where they extracted Cinnamomum cassia Blume by ultrasound and then analyzed obtained extracts by HPLC. It is important to emphasize that despite the numerous papers, it is problematic to precisely separate the simple coumarins because of the similarity of the chemical structure as well as the polarity [
77]. In China, the amount of cinnamaldehyde, cinnamic acid, cinnamyl alcohol, and coumarin were examined as important components in 44 samples of Cassia bark from fields and markets using HPLC. In addition to the quantification of the components themselves, fingerprinting was performed for five different types of collected cassia bark and it was shown that five significant peaks were sufficient to distinguish the original Cassia bark and as such could be used in quality control testing as a rapid and reliable method. The content of coumarin, depending on the type of cassia bark, ranged from 0.01–12.18 mgg
−1 among the collected 44 samples. [
72].
In addition to HPLC, UHPLC can be used to develop a shorter method with less consumption of organic solvents, but with equally good peak separation. The consumption of organic solvent is directly related to the duration of the methods that, by using UHPLC, can take several minutes compared to 10–90 min with HPLC [
77].
Using UHPLC-DAD, the content of coumarin was analyzed in 74 samples of various cinnamon-containing foods on the market in Denmark [
64]. Internal calibration with 4-methylumbelliferone was carried out, and this analysis confirmed that the problematic category of food was a fine bakery in which EU limit for coumarin was exceeded in almost 50% of cases. These results confirm that this method can be used for quick and routine determination of coumarins in foods. UHPLC-DAD is also a method that can also be used to determine components such as coumarin, trans-cinnamic acid, trans-cinnamaldehyde and eugenol in encapsulated cinnamon flavoring powder. This powder is actually cinnamon essential oil encapsulated with different carriers, which improves product characteristics, such as stability, oxidation, and thus ensures a longer shelf life of the product. On the other hand, this affects the way the sample is prepared so the efficiency of extraction, it this case is even more important. That is why the authors optimized the extraction process, applying different extraction parameters, to ensure maximum process efficiency. One of the important features of the UHPLC method is the shorter analysis time compared to HPLC, and therefore the method developed and validated here lasted 12 min, with concentrations of coumarin, trans-cinnamic acid, trans-cinnamaldehyde and eugenol determined in the samples [
78].
Since some of the coumarins contain fluorophore in their structure it is possible to use fluorescence detectors (FL), in addition to UV and DAD detectors, which Machyňáková and Hroboňová [
77], used in their work to detect coumarin derivatives and metabolites from Meliloti herb, propolis tincture and crude propolis. One of the important characteristics of coumarins for determination using UV/DAD and FL detectors is their absorbance and fluorescence in the UV range. Two different wavelengths appeared to be suitable for the determination of several coumarins, such as 4-hydroxycoumarin, coumarin and scoparone with detection at a wavelength of 280 nm, while a wavelength of 323 nm proved suitable for esculin, daphnetin, fraxetin, umbelliferone, 4-methylumbelliferone and herniarin. The use of FL detectors, in this case, improved the sensitivity and thus the detection of coumarins exhibiting fluorescence such as esculin, umbelliferone, 4-methylumbelliferone, scoparone and herniarin, especially in the case of detection of umbelliferone and scoparone. Hroboňová et al. [
57] examined the number of coumarins such as sculin, daphnetin, fraxetin, umbelliferone, 4-methylumbelliferone, 4-hydroxycoumarin, scoparone, coumarin, herniarin and cinnamyl alcohol in propolis samples for which they developed an appropriate HPLC method. During the development of the method, different mobile phases with different proportions of acetonitrile and acetic acid were tested, with an optimal ratio of acetonitrile content and 0.3% acetic acid in water which was 10:90 (
v/
v). The lower amount of acetic acid in the mobile phase (0.1%) during isocratic elution had a negative effect on peak tailing, that is, on the shape and symmetry of the peak, which is improved by increasing the acetic acid content up to 0.3%.
In addition to the FL detector which has improved sensitivity and thus the detection of coumarins exhibiting fluorescence, mass spectrometry in gas and liquid chromatography can also be used to detect coumarin. Raters and Matissek [
84] developed a selective and sensitive isotope dilution LC-MS/MS method which was compared with the HPLC-UV method. The developed method showed increased selectivity and sensitivity compared to the HPLC-UV method. That is important since these characteristics of the method are significant in cases of determination of components in food that are not represented in higher concentrations, such as coumarin. Such methods enable the detection of lower concentrations of coumarin in different foods and provide a more realistic picture of the presence of coumarins in the daily diet. Nevertheless, Sproll et al. [
19] have developed the HPLC-DAD method which they claim is as suitable for determination and quantification of coumarin in food as the LC-MS/MS method developed by Raters and Matissek [
84]. Certainly, the advantage of the HPLC method lies in the simplicity of sample preparation, simpler analysis and processing of results as well as in the lower cost of equipment and labor. According to the authors, in interlaboratory testing, the results obtained are excellent compared to more complex methods, such as LC-MS/MS with deuterium-labeled coumarin as the internal standard.
As mentioned, vanilla extract is one of the most commonly used flavoring ingredients in the diet, including food and beverages. Given the high cost of authentic vanilla extract, artificial vanilla flavor containing synthetically produced vanillin and/or ethyl vanillin is often used, while some manufacturers also add coumarin to enhance the vanilla flavor. Therefore, vanillin, ethyl vanillin and coumarin of twenty-four vanilla extract products on Mexican market was tested using the LC-MS method, as the only chromatographic method to determine the abovementioned components simultaneously in only 13 min and which, in addition, provides a qualitative mass spectral confirmation. The method used was compared with the use of UV detectors, showing that, in most cases, the results obtained were similar, with more accurate results obtained using LC-MS. Such a conclusion is not surprising given the higher level of specificity of LC-MS method. Applying the above method, it has been shown that the addition of coumarin to vanilla products is not common, assuming that such a trend has been influenced by laws and the public [
59]. In addition to the above method, the GC method was also used to identify synthetic vanillin as well as other contaminants as early as 1965 [
83]. The retention time and peak area were determined to identify the contamination, and the results were shown as relative peak area response, relative to vanillin. Gas chromatography continued to be used to determine contaminants in flavors, and so a simple gas chromatography method in combination with a flame ionization detector was used to detect the prohibited flavors, such as diethylene glycol (DEG), diethylene glycol monoethyl ether (DEGME) and coumarin in food samples. The method developed was validated by determining the limit of detection, linear range, recovery, and reproducibility of the retention time, as soon as it was shown that this method was appropriate for the determination of coumarin in solid and liquid food samples. It is important to note that none of the soft drink and juice tested contain coumarin [
73].
Tonka beans, as the first plant from which coumarin was extracted and isolated since they contain 1–3% coumarin, and in rare cases up to 10%, were also investigated. In addition to coumarin, they contain umbelliferone, o-coumaric acid and diterpenoids, as well as the other 138 components that have been detected in the tonka beans extract. The tonka beans extract was analyzed using GC-MS, and then an HPLC method was developed for routine analysis. The following components of tonka beans such as coumarin, o-coumaric acid, 5-hydroxymethylfurfural, melilotic acid, methyl melilotate, ethyl melilotate and dihydrocoumarin in ethanol extract were detected using GC-MS, and then using the HPLC method, these components were detected and quantified not only in ethanol but and the extract obtained by using methanol and acetonitrile as solvent. Accordingly, methanol has been shown to be the most suitable solvent for the extraction of o-coumaric and melilotic acid as well as for coumarin, whose values for extracts obtained under the same conditions are different, with large deviations. A possible reason for this lies in the different and irregular distribution of coumarin crystals within tonka beans. Coumarin values ranged from 1.12–2.33 g/100 g for the extracts obtained with ethanol, methanol and acetonitrile. This method is a simple and convenient method for detecting components in tonka beans extracts [
71].
In cases where breast milk is not available or sufficient, the use of the infant formula is necessary for the normal maintenance of infant growth and development. A study conducted in China found that vanillin or coumarin, or sometimes both, are present in some products. Considering that infants are a risk group that needs adequate nutrient intake, without the intake of substances that may have genotoxic and carcinogenic effects, Shen et al. [
85] developed and validated a fast and accurate liquid chromatography-quadrupole linear ion trap mass spectrometry method for determination of vanillin, ethyl vanillin, and coumarin. Given the low levels of contaminants in food, conventional analytical techniques are sometimes not sensitive enough to detect and quantify such components. Therefore, more sensitive techniques, such as LC-MS/MS or LC-MS/MS with a triple quadrupole, are increasingly used in the analysis of complex matrices such as food. Given that food is a complex matrix, it is important to keep in mind that matrix effects could influence the error in quantitative LC-MS/MS analysis of food samples. Therefore, Shen et al. [
85] added internal standards such as vanillin-13C6 and coumarin-D4 in the sample to correct the influence of the matrix. Similar method, ultra-performance liquid chromatography- linear ion trap triple quadrupole mass spectrometry method (UHPLC-ESI-QqQLIT-MS/MS) was developed, validated and then used to determine coumarin and other phenolics not only in authentic Cinnamomum verum bark samples, but also in samples from south India market. Despite the fact that south India is one of the centers of cinnamon trade, the quality and composition of cinnamon origin from this region has not been tested. In addition, because of the price of cinnamon present on the market, it is very common to mix Cassia cinnamon which has lower-price with Cinnamomum verum, ultimately leading to a high content of coumarin. As for the method itself, applying the UHPLC method over conventional HPLC has led to better resolution in a shorter time. Of the validation parameters tested, it is important to mention the LOD and LOQ given that they were less than 0.36 and 0.81 ng/mL, respectively, which tells us that coumarin can not only be detected but also quantified at very low amounts, which makes this method very sensitive and selective. Of the five cinnamon samples tested from the market, four had high amounts of coumarin, ranging from 819 to 3462 mg/kg, while only one had 19.6 mg/kg. Compared to authentic bark samples containing 12.3 to 143.0 mg/kg, there is a significant difference likely due to mixing with cheaper
C. cassia and
C. burmanii. These situations require frequent and constant quality control of cinnamon and cinnamon products on the market, since cinnamon intake with such a high dose of coumarin may exceed the TDI. In addition to the above methods, LC-ESI-QTOF-MS/MS method can be used to identify and determine components, with more accurate quantification including coumarins such as scopolin and scopoletin. The above method was used to determine the composition of Artemisia annua, originally determined by validated HPLC-DAD, which shows efficacy in determining not only coumarin but also other groups of active compounds such as flavonoids and sesquiterpenes [
86]. One of the more advanced analytical methods is certainly the UHPLC-QqQ-MS/MS, which was developed to simultaneously identify multiple groups of components including coumarin in Pummelo Fruits. This method, like other chromatographic methods, ensures good separation, selectivity, sensitivity and quantification with respect to good results with validation parameters, but in as short a time as possible, this method takes only 13 min, separating as many as 47 components including isomers [
87].
Although sample pretreatment was not used in most of these methods, food, like many natural products, contains many different interfering components, thus making it a complex material that should undergo pre-treatment before chromatographic techniques to ensure reliable results. Considering time-consuming pre-treatments that consume a lot of solvents and loses some of the analytes, it is important to find a pre-treatment that is fast and accurate and.
Machyňáková et al. [
80] therefore propose on-line solid-phase extraction (SPE), as a technique that is versatility and reproducibility, at a lower cost and cost of solvents and human labor, which reduces the error of operation [
88,
89]. What makes this technique even more compelling is the easy connection between SPE and HPLC using on-line column switching devices, which enables reliable treatment of complex matrices such as food. With regard to sorbents, molecularly imprinted polymers (MIPs) were used in the aforementioned work with respect to stability under different operating conditions and high specificity and selectivity for the desired analyte [
90,
91]. The reason why they are not used often is the problem of transferring to a column-switching system without loss of extraction selectivity since non-polar solvents and buffers that are not suitable for the conditions of operation of the chromatographic column are often used. In this work, 7-hydroxycoumarin molecularly imprinted polymer was used which proved effective for extracting and separating different coumarins from different sources, such as Cassia cinnamon, chamomile tea and Tokaj specialty wines. The developed method is validated by applying parameters such as LOD and LOQ, linearity, accuracy and precision. According to the results, on-line MISPE-HPLC is a satisfactory method for the determination of coumarin in different food samples, with a simpler sample preparation procedure and faster analysis since the whole process, from sample cleanup to equilibration, takes 13.5 min. However, the efficiency of the sample cleaning process depends on the sample itself, ie its complexity, as well as the accuracy and precision of the results obtained [
80].
5.2.2. Spectrophotometric and Spectrofluorimetric Determination
Fluorescence spectroscopy is one of the commonly used methods right because of its selectivity and sensitivity at a low cost. In this area, there is a difference between synchronous fluorescence spectroscopy (SFS) and conventional fluorescence spectroscopy, with the former having several advantages such as simplicity and efficiency in quantitative determination. For this reason, Poláček et al. [
58] (developed a rapid, simple and inexpensive method for the determination of coumarin, 4-hydroxycoumarin and dicoumarol in herbal tea (Melilotus Officinalis), whose accuracy and efficacy were then compared with the HPLC method. This synchronous fluorescence method in a combination with PLS multivariate calibration provides excellent results for the calibration and prediction, thus providing a good screening method for herbal tea samples. The spectrofluorimetric method was also used for determination of total coumarins, expressed as scoparone in propolis and propolis products, and the developed method was compared by HPLC with spectrophotometric and fluorescent detection method, by which coumarins from a group of simple coumarins such as esculin, daphnetin, fraxetine, umbelliferone, 4-methylumbelliferone, 4-hydroxycoumarin, scoparone, coumarin, herniarin were determined. In the case of the spectrofluorimetric method, excitation wavelength was set at 370 nm, and the fluorescence intensity was recorded at 423 nm compared to the blank solution. In order to compare the two methods, Hroboňová et al. [
57] determined for both methods validation parameters such as the correlation coefficient of the calibration graph, LOD, LOQ, intra-day precision, inter-day precision, and recovery. In both methods, the achieved linear relationship between peak area or fluorescence intensity with the concentration of test components is satisfactory (R
2 = 0.999), with a wider range of linearity observed by HPLC. On the other hand, fluorescence spectrometry resulted in better LOD and LOQ. However, for both methods, the LOQ for the components is lower than expected for the actual samples, which makes both methods acceptable for simple coumarin determinations. As for the other parameters, intra-day precision was similar for both methods, while better results of inter-day precision were obtained by HPLC. Also, the results for recovery are slightly lower with fluorescence spectrometry, which the authors consider to be the reason for the influence of the matrix. The sample preparation for both methods is similar, while the duration of the test is shorter using fluorescence spectrometry. From all the above it can be concluded that both methods are suitable for the determination of coumarins in real samples. However, by determining total coumarins in propolis samples, 15% higher results were obtained compared to HPLC, with the authors attributing higher matrix effects on the fluorescence of the spectrum [
57].
Spectrofluorimetry, together with high-performance liquid chromatography has been tested for the determination of coumarins such as umbelliferone, scopoletin and 4-methylumbelliferone in distilled beverages. As is the problem with most foods, coumarins are also present in very small quantities in distilled beverages, making it difficult to detect and quantify them using common analytical techniques. In the work of Fernández Izquierdo et al. [
56], they compared in detail two analytical methods using different statistical methods, spectrofluorometry and high-performance liquid chromatography, both of which show the possibility of application in the determination of coumarin in distilled beverages, with certain differences, especially in accuracy and precision. In addition, the authors suggested that by modifying the HPLC method, even better accuracy and precision can be achieved, and given that the method is faster and simpler, it often has the advantage of determining coumarin. However, analysts can choose between the two methods given that they are both sensitive enough and give accurate and accurate results for umbelliferone, scopoletin and 4-methylumbelliferone in distilled beverages.
Vanilla extracts, in addition to chromatographic methods, were also investigated using Fourier transform mid-infrared (MID-FTIR) spectroscopy coupled with chemometric analysis. This method, compared to the chromatographic methods that are more expensive and require longer sample preparation, is simple and fast, without the use of reagents and pre-treatment. For this reason, the aim of Moreno-Ley et al. [
92] was to conduct an analysis of the content of coumarin and ethyl vanillin in vanilla extracts using the MID-FTIR spectroscopy and the HPLC-DAD method to design chemometric models that can then be applied to commercial samples. Samples that were adulterated were tested, ie to which a certain amount of ethyl vanillin and coumarin were added and commercially available vanilla extracts, and a predictive model was thus constructed using several algorithms, the best of which was partial least squares with single y-variables. Although the predictions are better for ethyl vanillin compared to coumarin, given that the spectrum of samples forged with coumarin is similar to the control sample, with the above algorithm ethyl vanillin and coumarin were determined in amounts of 0.20–10% and 0.1–10 ppm, respectively. Given that there were no significant differences between the developed model and the HPLC analysis, as well as very small detectable quantities, the developed method and model are a viable method, especially in conditions where results are required immediately [
92].
A similar method involving the use of fluorescence spectrometry, and the chemometric approach, as well as extraction using a molecularly imprinted solid-phase was also used to analyze coumarin in wine. Unlike the work of Machyňáaková et al. [
80], MIP SPE used here was offline type, used as well as online to clean and prepare samples for analysis. Samples were first analyzed using a validated HPLC method with UV–VIS and fluorescence detectors to determine coumarin content such as esculin, coumarin, herniarin, 4-methylumbelliferone, scoparone, scopoletin, and then analyzed using fluorescence spectrometry. Data collected from both analyzes, such as coumarin concentrations in the samples as well as emission spectral data and synchronous fluorescence spectra, were then used to develop the PLS model. Comparing the model developed with the results obtained by HPLC, a good correlation is observed, which supports the above method and model for further use in the analysis of coumarins in wine [
93].
Table 6.
Overview of newer methods of detection and quantification of coumarin and related components.
Table 6.
Overview of newer methods of detection and quantification of coumarin and related components.
Sample | Method | Work Conditions | Detected Compounds | Literature |
---|
Cinnamons and cinnamon containing foods | HPTLC | HPTLC plates silica gel 60 mixture of n-hexane—ethyl acetate—ammonia (3.8:1.3:0.05, v/v/v) 10% ethanolic KOH solution for detection 10% methanolic PEG 400 solution - stabilization of the fluorescence 366 nm | coumarin | [17] |
HPTLC-MS | 100% methanol 0.1 mL min−1 capillary and source gas temperature 250 °C capillary voltage 150 V source voltage offset 10 V 100–600 m/z |
Cinnamon-containing food products | HPLC-DAD | 5 mM ammonium acetate buffer, 0.2% acetic acid: ACN/MeOH (1:2) gradient elution 0.2 mL min−1 40 °C 279.8 nm | coumarin | [19] |
Distilled beverages | HPLC-FL | 3% glacial acetic acid in water:ACN with 3% glacial acetic acid gradient elution 1 mL min−1 room temp. excitation and emission 340 and 425 nm | umbelliferone, scopoletin and 4-methylumbelliferone | [56] |
Spectrofluorometry | excitation and emission wavelengths 340 and 425 nm 25 ± 1 °C slits set at an aperture of 3.0 nm |
Propolis | Fluorescence spectrometry | excitation and emission slits 5 nm fluorescence emission spectra from 380 to 600 nm excitation wavelength 370 nm emission spectrum at 23 °C | sculin, daphnetin, fraxetin, umbelliferone, 4-methylumbelliferone, 4-hydroxycoumarin, scoparone, coumarin, herniarin and cinnamyl alcohol | [57] |
HPLC | ACN with 0.3% acetic acid:ACN gradient elution 1 mL min−1 23 °C 280 and 323 nm |
Herbal tea from sweet clover plant (Melilotus officinalis) | SFS | excitation and emission splits 5 nm scan speed 200 nm/min PMT Voltage 500 Volts the excitation wavelength range at 200–400 nm Δλ = 90 nm | coumarin, 4-hydroxycoumarin and dicoumarol | [58] |
HPLC | 0.3% acetic acid in methanol: 0.3% acetic acid in water gradient method 0.5 mL min−1 23 °C 280 nm |
Vanilla extract products | LC-UV-MS | ACN: 0.1% formic acid (35:65) isocratic elution 0.25 mL min−1 20 °C 254 nm quantification was based on a peak area ratio of the SIM signals of the analyte and the IS | coumarin, vanillin and ethly vanillin | [59] |
Cinnamon-containing food products | UHPLC-DAD | 5% MeOH in demineralized water/ACN Gradient elution 0.6 mL min−1, 45 °C 278.1 nm | coumarin | [64] |
Cinnamon samples | UHPLC-ESI-QqQLIT-MS/MS | 0.1% formic acid in water:ACN gradient elution 0.3 mL min−1 30 °C both positive and negative ESI modes Source temperature 550°C range of m/z 100–1000 | coumarin, scopoletin, o-coumaric acid, cinnamic acid and cinnamaldehyde | [65] |
The seeds of Dipteryx odorata (tonka beans) | HPLC | 0.0001% 85% o-phosphoric acid:ACN:MeOH gradient elution 1 mL min−1 45 °C 260–275 nm | coumarin, o-coumaric acid, 5-hydroxymethylfurfural, melilotic acid, methyl melilotate and ethyl melilotate and dihydrocoumarin | [71] |
GC-MS | Helium 70–280 °C 70 eV 0.2 mA mass range m/z 50–380 |
Cassia Bark (Cortex Cinnamomi) | HPLC quantitative analyses | ACN:0.04% acetic acid (25:75) isocratic elution 1 mL min−1 20 °C 250, 280 nm | cinnamaldehyde, cinnamic acid, coumarin and cinnamyl alcohol | [72] |
HPLC fingerprint analysis | ACN:0.02% acetic acid gradient elution 1 mL min−1 20 °C 280 nm | coumarin, cinnamic acid, cinnamaldehyde, unknown, and eugenol |
Food containing soft drinks and juice, infant formula and food, cereals, flours and snacks | GC-FID | Oxygen free nitrogen 1 mL min−1 100–260 °C | diethylene glycol, diethylene glycol monoethyl ether, coumarin and caffeine | [73] |
Food flavored with vanilla | TLC | Ethyl acetate, Chloroform, Propanol, Acetic acid, Hexane gradient elution AMD chamber 280 nm—densitometer | vanillin, ethyl vanillin, 4-hydroxy-benzaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, vanillic acid, coumarin, piperonal, anisic acid, and anisaldehyde | [74] |
Cinnamomum cassia Blume | HPLC | ACN:0.5% acetic acid in water (25:75, v/v) isocratic elution 1.0 mL min−1 25 °C detection at 278 nm | Coumarin | [76] |
Spice/spice mixture; Cinnamon cookies; Gingerbread | HPLC–UV | Ammonium acetate (5 mmol/L)/ACN:MeOH (1:2) gradient elution 0.8 mL min−1 40 °C 279 nm | coumarin | [78] |
LC–MS/MS | MeOH:ACN:0.1% formic acid (80:0.1:19.9) isocratic elution 0.25 mL min−1 20 °C identified by selected-reaction monitoring (SRM) positive electrospray ionization mode (ESI+) |
Cassia cinnamon, chamomile tea | HPLC | ACN: 0.3% acetic acid gradient elution 1 mL min−1 20 µL 30 °C 280 and 323 nm | 6,7-dihydroxycoumarin 7,8-dihydroxy-6-methoxy-coumarin 7-hydroxycoumarin 7-hydroxy-4-methylcoumarin 6,7-dimethoxycoumarin coumarin 7-methoxycoumarin | [80] |
On-line MISPE-HPLC system | 20 mg of MIP sorbent switching sstem washing with H2O HPLC conditions as above |
Infant formula | LC-QqLIT-MS | 0.1% formic acid/ACN gradient elution 0.25 mL min−1 30 °C ESI positive mode Source temperature 500°C Ionization voltage 5000 V
| vanillin, ethyl vanillin, and coumarin | [85] |
Artemisia annua | HPLC-DAD | 0.1% formic acid:ACN gradient elution 0.6 mL min−1 30 °C 10 µL 210 and 360 nm | rutin, cynaroside, isorhamnetin, chrysosplenol D and casticin, scopolin and scopoletin, arteannuinB, artemisinin, dihydroartemisinic acid and artemisinic acid | [86] |
LC-ESI-QTOF-MS/MS | positive and negative mode 50 to 1000 Da 180°C the end plate offset was−500V capillary voltages were 4500 V and −3500 V |
Pummelo Fruits | UHPLC-QqQ-MS/MS | 0.1% formic acid:MeOH gradient elution 0.3 mL min−1 2 µL 40 °C positive and negative modes curtain gas 20.0; ionspray voltage (IS) ±4500.0 temperature (TEM) 500.0; ion source gas 1 (GS1) 50.0; ion source gas 2 (GS2) 50.0
| 47 components, 12 coumarins and furocoumarins; umbelliferone, scoparone, psoralen, bergaptol, xanthotoxin, Limettin, bergapten, isomeranzin, 6′,7′dihydroxybergamottin, imperatorin, isoimperatorin, 6′,7′epoxybergamottin | [87] |
Cassia cinnamon, chamomile tea | HPLC | ACN: 0.3% acetic acid gradient elution 1 mL min−1 20 µL 30 °C 280 and 323 nm | 6,7-dihydroxycoumarin 7,8-dihydroxy-6-methoxy-coumarin 7-hydroxycoumarin 7-hydroxy-4-methylcoumarin 6,7-dimethoxycoumarin coumarin 7-methoxycoumarin | [80] |
On-line MISPE-HPLC system | 20 mg of MIP sorbent switching sstem washing with H2O HPLC conditions as above |
Vanilla extracts | MID-FTIR spectroscopy | range of 4000–550 cm−1 resolution of 4 cm−1 64 scans | ethyl vanillin, coumarin | [92] |
HPLC-DAD | ACN: acid water (pH 2.3) gradient elution 30 °C 1µL 230, 260, 280 nm |
Tokaj wine | HPLC-DAD HPLC-FL | MeOH/acetic acid:1% of acetic acid gradient elution 23 °C 20 µL 280, 320, 450 nm | esculin, coumarin, herniarin, 4-methylumbelliferon, scoparone, scopoletin | [93] |
fluorescence spectroscopy | excitation and emission slit width 5 nm excitation wavelength 320 nm synchronous fluorescence spectra → 250–400 nm range |