Method for identifying saffron crocus producing area by extracting crocin and measuring stable isotope ratio of crocin

Abstract

The invention discloses a method for identifying the producing area of saffron crocus by extracting saffron crocus aldehyde and measuring the stable isotope ratio of the saffron crocus aldehyde. The invention discloses a method for extracting crocetin from saffron crocus and measuring the stable isotope ratio of carbon to hydrogen to oxygen. The method comprises the following steps: S1, determining the stable isotope ratio of carbon to hydrogen to oxygen of a crocetin standard substance by using an elemental analysis-stable isotope mass spectrometer; s2, extracting crocus sativus aldehyde from the crocus sativus sample to obtain a crocus sativus aldehyde extracting solution; s3, determining the carbon, hydrogen and oxygen stable isotope ratio of crocetin in the crocetin extracting solution by using a gas chromatography-stable isotope mass spectrometer; and S4, calibrating the carbon, hydrogen and oxygen stable isotope ratio of crocetin in the crocetin extracting solution by adopting the carbon, hydrogen and oxygen stable isotope ratio of the crocetin standard substance. The standard substance is used for calibrating the stable isotope ratio of carbon, hydrogen and oxygen of the single compound crocetin, and the method can be used for identifying the producing area of crocetin.

Description

Technical Field

[0001] This invention belongs to the field of stable isotope analysis technology, specifically involving a method for extracting saffron aldehyde and measuring its stable isotope ratio to identify the origin of saffron. In particular, it involves extracting saffron aldehyde from dried saffron and determining the stable isotope ratio of carbon, hydrogen, and oxygen in saffron aldehyde to identify the origin of saffron. Background Technology

[0002] Saffron, specifically the stigma of the saffron crocus, is one of the most prized spices, used as a food coloring and flavoring agent. Furthermore, saffron is widely recognized for its broad pharmacological activities, including antidepressant, anti-inflammatory, analgesic, antioxidant, anti-acetylcholinesterase, antitussive, and antihypertensive effects. Due to its high market value and scarcity, saffron is easily adulterated by adding inferior and similar-looking plant materials, such as: 1. processed safflower or marigold products; 2. processed saffron stamens; 3. soaked saffron; 4. lotus stamens; 5. processed daylily products; 6. corn florets and their processed products; 7. processed chrysanthemum ray florets; 8. processed Indian saffron; 9. saffron mixed with gardenia, etc. In addition, saffron is produced globally, each region having its unique climate and soil conditions, thus affecting the quality and flavor of the saffron. Therefore, a technological means is needed to control the quality of saffron on the market and trace its origin.

[0003] Saffron aldehyde is a monoterpene aldehyde and one of the main volatile components of saffron, considered a beneficial component. ISO 3632 defines saffron aldehyde as a compound that affects the quality of saffron. ISO 3632-2:2010 defines a method for determining saffron aldehyde content using a UV-Vis spectrophotometer. However, due to the difficulty in distinguishing saffron aldehyde from other components that absorb near its measurement wavelength, accurate determination of saffron aldehyde content is not possible. Other methods for quantitative analysis of saffron aldehyde have been reported, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC). Some reports use commercially available saffron aldehyde reagents or saffron aldehyde with a purity exceeding 88%, while others use reagents without purity information or use inaccurately pure saffron aldehyde standards, leading to deviations in quantitative values. Furthermore, crocin is not present in fresh stigmas; it is produced through the hydrolysis of the bitter glycoside "crocin" during the drying process and storage. Drying conditions vary greatly between countries, affecting the crocin content. Therefore, quantitative detection of crocin in saffron alone is insufficient for tracing the origin of saffron.

[0004] There are existing reports on the determination of multi-element content and stable isotope ratios of saffron for the identification of its place of origin (Journal, Journal of Mass Spectrometry. Volume 55, Issue 11. 2020. PP e4595). However, this method is based on the premise that the sample itself is genuine saffron and is determined by high-temperature combustion of whole saffron samples. If the sample is counterfeited, this method cannot be used. Summary of the Invention

[0005] The purpose of this invention is to provide a method for identifying the origin of saffron by extracting saffron aldehyde and measuring its stable isotope ratios. This method utilizes an elemental analyzer-stable isotope mass spectrometer (EA-IRMS) and a gas chromatography-stable isotope mass spectrometer (GC-IRMS) to determine the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde, a characteristic substance in saffron aldehyde standards and dried saffron samples. This provides a technical approach for tracing the origin of saffron and combating counterfeiting.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for extracting saffron aldehyde from saffron and determining its carbon, hydrogen, and oxygen stable isotope ratios, comprising the following steps: S1. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde standard were determined using elemental analysis-stable isotope mass spectrometry. S2. Extract saffron aldehyde from the saffron sample to obtain saffron aldehyde extract; S3. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract were determined using gas chromatography-stable isotope mass spectrometry. S4. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract are calibrated using the carbon, hydrogen, and oxygen stable isotope ratios of the saffron aldehyde standard.

[0008] In the above method, further, in step S1, the elemental analyzer parameters for determining the carbon stable isotope ratio are as follows: carrier gas flow rate: 180 mL / min, oxygen flow rate: 180 mL / min, reference gas flow rate: 50 mL / min, right furnace temperature: 960℃, furnace temperature: 50℃, sample transport delay time: 13 seconds, oxygen passage time: 3 seconds, operating mode: solid C; the stable isotope ratio mass spectrometer parameters are as follows: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶ -6 mBar, High voltage: 9.45KV, Current: 1.5mA; In step S1, the elemental analyzer test conditions for determining the oxygen / hydrogen stable isotope ratio are as follows: carrier gas flow rate: 10 mL / min, oxygen flow rate: 0 mL / min, reference gas flow rate: 100 mL / min, left furnace temperature: 1380℃, furnace temperature: 45℃, sample delay time: 0 seconds, oxygen flow time: 0 seconds, operating mode: liquid H₂O, dilution ratio: CO 0%, H₂ 0%; the stable isotope ratio mass spectrometer test parameters are as follows: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶ -6 mBar, High voltage: 9.47KV, Current: 1.5 mA.

[0009] In the above method, the step of extracting saffron aldehyde further includes: grinding dried saffron into powder, adding organic reagents for extraction, and collecting the extract.

[0010] Preferably, the ratio of saffron powder to the organic reagent is (0.95-1.05) g: 15 mL; the organic reagent is n-hexane; the extraction is carried out under ultrasonic conditions, with an ultrasonic power of 300 W, an extraction temperature of 20-25 °C, and an extraction time of 60 min.

[0011] Furthermore, the step of extracting saffron aldehyde also includes the following purification step: 1) After pre-concentrating the extract, add it to a silica gel column pretreated with n-hexane, elute with a n-hexane solution of ethyl acetate of the first concentration, and collect the first target eluent; 2) After pre-concentrating the first target eluent, add it to a silica gel column pretreated with n-hexane. First, elute with a n-hexane solution of ethyl acetate of the second concentration and collect the second target eluent. Then, elute with a n-hexane solution of ethyl acetate of the third concentration and collect all of the third eluent. 3) Combine the second target eluent and the third eluent, and concentrate them.

[0012] Preferably, in step 1), 1 g of the silica gel column is added to every 2 mL of the pre-concentrated extract, the filtrate is discarded, and 14 mL of the first concentration of ethyl acetate in hexane is added for elution, the first target eluent being the subsequent 8 mL of eluent. In step 1), the first concentration is 5% by mass. In step 2), 1 g of the silica gel column is added to every 0.2 mL of the pre-concentrated first target eluent, the filtrate is discarded, and 14 mL of the second concentration of ethyl acetate in n-hexane is added for elution. The second target eluent is the 7 mL eluent that follows. In step 3), 7 mL of the third concentration of ethyl acetate in n-hexane is added for elution. In step 2), the second concentration is 0.5% by mass, and the third concentration is 1% by mass.

[0013] In the above method, the gas chromatographic conditions in the gas chromatograph-stable isotope mass spectrometer are further as follows: Non-polar capillary column; Temperature program: Initial temperature 70℃, hold for 1 min, ramp to 100℃ at a rate of 3℃ / min, hold for 1 min, ramp to 300℃ at a rate of 20℃ / min, hold for 2 min; Splitless mode; Carrier gas: High-purity helium; Flow rate: 1.5 ml / min; Split ratio: 10:1; Injector temperature 250℃; δ-measurement 13 The injection volume for C was 0.2 μL, and the δ was measured. 18 O and δ 2 The H injection volume was 0.5 μL; The online combustion conditions in the gas chromatograph-stable isotope mass spectrometer are as follows: Measurement of δ 13 C: Use an oxidation combustion tube, set the temperature to 1000℃; Measurement of δ 2 H: Use a high-temperature pyrolysis tube, with the temperature set at 1420℃; Measurement of δ 18 O: Use a high-temperature pyrolysis tube, set the temperature to 1280℃; The stable isotope mass spectrometer conditions in the gas chromatography-stable isotope mass spectrometer are as follows: In the determination of the carbon stable isotope ratios, an EI ion source was used with a vacuum level of 1.6 × 10⁻⁶. -6 mBar, High voltage: 9.45KV, Current: 1.5mA; In the determination of the ratio of stable oxygen and hydrogen isotopes, the vacuum degree was 1.6 × 10⁻⁶. -6 mBar, High voltage: 9.47KV, Current: 1.5 mA.

[0014] In the above method, the calibration step further includes: The saffron aldehyde standard, after being set, is diluted to the same concentration as that in the saffron sample, and the diluted saffron aldehyde standard is used as the calibration sample.

[0015] In the above method, further, the method includes the following step before step S3: The concentration of crocin in the saffron sample was determined using gas chromatography-mass spectrometry.

[0016] Secondly, the present invention provides a method for identifying the origin of saffron, comprising determining the carbon, hydrogen, and oxygen stable isotope ratio of saffron aldehyde in a saffron sample using any of the methods described above, and identifying the origin of saffron based on the ratio.

[0017] This invention provides a technical method for tracing the origin of saffron and combating counterfeiting, and has the following advantages: (1) The method of the present invention uses elemental analysis-stable isotope mass spectrometry to determine the carbon, hydrogen and oxygen stable isotope ratio of purchased saffron aldehyde standard, which is used for the calibration of the saffron aldehyde determination value in the sample extract by gas chromatography-stable isotope mass spectrometry to ensure the reliability of the value.

[0018] (2) The method of the present invention uses gas chromatography-mass spectrometry to determine saffron aldehyde in dried saffron extract. The parameters of the gas chromatography equipment in the experiment are initially determined, such as the injection temperature, column type, furnace temperature gradient, peak time and position of saffron aldehyde, and concentration of extracted saffron aldehyde. The gas chromatography instrument parameters and column type used in the gas chromatography-mass spectrometry are applied to the gas chromatography-stable isotope mass spectrometer. According to the requirements of the stable isotope mass spectrometer, the gas chromatography instrument parameters and the amount of dried saffron sample required for the experiment are improved. Then, the carbon, hydrogen and oxygen stable isotope ratio of saffron aldehyde in the sample extract is determined by the stable isotope mass spectrometer. The present invention has optimized the determination conditions of gas chromatography-stable isotope mass spectrometer through a large number of experiments to realize the determination of carbon, hydrogen and oxygen stable isotope ratio of saffron aldehyde.

[0019] (3) This invention utilizes elemental analysis-stable isotope mass spectrometry (EA-IRMS) to determine the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde standard, which is then diluted with sample extract. The diluted concentration needs to be close to the sample concentration, and the determination is performed under the same conditions as the sample. This is used to calibrate the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in saffron sample extract. Stable isotope standards for a single compound, saffron aldehyde, are not commercially available. This invention uses certified standards to calibrate the carbon, hydrogen, and oxygen stable isotope ratios of a single compound, saffron aldehyde, using EA-IRMS. This establishes a method that can unify the stable isotope standards for saffron aldehyde, laying the foundation for the subsequent establishment of a saffron sample library.

[0020] (4) The method of the present invention does not require complicated extraction steps. The sample after impurity removal and concentration can be used to determine the carbon, hydrogen and oxygen stable isotope ratio of saffron aldehyde in dried saffron samples.

[0021] (5) Existing methods for determining the multi-element content and stable isotope ratios of saffron for origin identification rely on the premise that the sample itself is genuine saffron. These methods involve high-temperature combustion of whole saffron samples. If the sample is adulterated, for example, by using other plants as substitutes, this method is unusable. This invention utilizes a combination of gas chromatography-mass spectrometry (GC-MS), elemental analyzer-stable isotope mass spectrometry (EA-IRMS), and gas chromatography-stable isotope mass spectrometry (GC-IRMS) to determine the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde, a characteristic substance in dried saffron samples. This provides a technical method for saffron traceability and anti-counterfeiting. Further testing of saffron samples from different origins confirms the reliability of this method for saffron traceability. Attached Figure Description

[0022] Figure 1 shows the gas chromatography-mass spectrometry results of crocin in the extract of dried saffron A sample determined in Example 1 of this invention: Figure 1A -Chromatogram (peak position 16.45 min); Figure 1B - Primary mass spectrum; Figure 1C and Figure 1D-2 Mass spectrum.

[0023] Figure 2 The δ-caryophyllin content in the extract of dried saffron sample A, as determined in Example 1 of this invention. 13 The ion current spectrum of C, where the vertical axis is intensity ([V]) and the horizontal axis is time ([S]), red: 44.0 m / z, blue: 45.00 m / z, green: 46.00 m / z.

[0024] Figure 3 The δ-caryophyllin content in the extract of dried saffron sample A, as determined in Example 1 of this invention. 2 The ion current spectrum of H, where the vertical axis is intensity ([V]) and the horizontal axis is time ([S]).

[0025] Figure 4 The δ-caryophyllin content in the extract of dried saffron sample A, as determined in Example 1 of this invention. 18 The ion flux spectrum of O is shown, where the vertical axis represents intensity ([V]) and the horizontal axis represents time ([S]). Detailed Implementation

[0026] Some counterfeit saffron or saffron that has already been extracted contains low or even no saffron aldehyde content. The method of this invention measures the carbon, hydrogen, and oxygen stable isotope ratio of saffron aldehyde in commercially available dried saffron. Taking advantage of the fact that saffron aldehyde is a marker of the active ingredient in saffron, this method can be used to trace the origin and control the quality of saffron.

[0027] The first part of this invention provides a method for extracting saffron aldehyde from saffron and determining its carbon, hydrogen, and oxygen stable isotope ratios, comprising the following steps: S1. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde standard were determined using elemental analysis-stable isotope mass spectrometry. S2. Extract saffron aldehyde from the saffron sample to obtain saffron aldehyde extract; S3. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract were determined using gas chromatography-stable isotope mass spectrometry. S4. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract are calibrated using the carbon, hydrogen, and oxygen stable isotope ratios of the saffron aldehyde standard.

[0028] Based on the above technical solution, this invention first utilizes an elemental analyzer-stable isotope mass spectrometer (EA-IRMS) to determine the carbon, hydrogen, and oxygen stable isotope ratios (δ¹⁸) of a saffron aldehyde standard with only a percentage content. 13 C、δ 2 H and δ 18 (O); Then, organic reagents are added to the dried saffron sample. After pre-concentration and purification, a test solution containing saffron aldehyde is obtained. The elution time and position of saffron aldehyde are determined using gas chromatography-mass spectrometry (GC-MS). Then, referring to the instrument conditions of the GC section of GC-MS, the test solution is heated to a gaseous state using gas chromatography-isotope mass spectrometry (GC-IRMS). Saffron aldehyde is then separated from other substances in the test solution using a capillary column. Finally, the sample is sent to a stable isotope mass spectrometer to determine the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the sample. Finally, the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the dried saffron sample extract are calibrated using saffron aldehyde standards determined by EA-IRMS. This invention provides a reliable method for tracing the origin of saffron and combating counterfeiting.

[0029] According to an embodiment of the present invention, in step S1, the elemental analyzer parameters for determining the carbon stable isotope ratio are as follows: carrier gas flow rate: 180 mL / min, oxygen flow rate: 180 mL / min, reference gas flow rate: 50 mL / min, right furnace temperature: 960℃, furnace temperature: 50℃, sample transport delay time: 13 seconds, oxygen passage time: 3 seconds, operating mode: solid C; the stable isotope ratio mass spectrometer parameters are as follows: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶ -6 mBar, High voltage: 9.45KV, Current: 1.5mA; In step S1, the elemental analyzer test conditions for determining the oxygen / hydrogen stable isotope ratio are as follows: carrier gas flow rate: 10 mL / min, oxygen flow rate: 0 mL / min, reference gas flow rate: 100 mL / min, left furnace temperature: 1380℃, furnace temperature: 45℃, sample delay time: 0 seconds, oxygen flow time: 0 seconds, operating mode: liquid H₂O, dilution ratio: CO 0%, H₂ 0%; the stable isotope ratio mass spectrometer test parameters are as follows: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶ -6 mBar, High voltage: 9.47KV, Current: 1.5 mA.

[0030] In step S1 of this invention, the carbon, hydrogen, and oxygen stable isotope ratios of the saffron aldehyde standard are determined using an elemental analysis-stable isotope mass spectrometer. This value is then used for calibration of the subsequent gas chromatography-stable isotope mass spectrometry determination of the saffron aldehyde value in the sample extract, ensuring the reliability of the numerical values.

[0031] Compared to gas chromatography-mass spectrometry (GC-MS), gas chromatography-instantaneous isotope ratio mass spectrometry (GC-IRMS) has higher requirements for sample purity. Samples that can be directly introduced into GC-MS may not be directly introduced into stable isotope mass spectrometry. Therefore, it is necessary to accurately purify crocin in the extract. The sample entering GC-IRMS must have high purity and a high extraction rate. Furthermore, crocin must not undergo chemical transformation during the extraction process. Therefore, the extraction steps and conditions, as well as the purification steps and conditions in step S2 of this invention, were obtained through extensive experimental exploration.

[0032] Regarding extraction methods, there are currently many publicly available methods for extracting saffron aldehyde, such as steam distillation, supercritical carbon dioxide extraction, and solvent extraction. However, the high temperature and water in steam distillation can cause fractionation of saffron aldehyde, and the presence of water also prevents its injection into GC-IRMS. Supercritical carbon dioxide extraction produces chemical reactions and generates kinetic isotope effects, which may affect the final determination of stable isotope values. Therefore, this invention preferably uses solvent extraction.

[0033] According to an embodiment of the present invention, the step of extracting saffron aldehyde includes: grinding dried saffron into powder, adding an organic reagent for extraction, and collecting the extract. Further, considering safety, the inventors found through experiments that n-hexane provides the best selectivity. In a specific embodiment of the present invention, the ratio of saffron powder to the organic reagent is (0.95–1.05) g:15 mL, such as 1 g:15 mL; the organic reagent is n-hexane; the extraction is performed under ultrasonic conditions, with an ultrasonic power of 300 W, an extraction temperature of 20–25 °C, and an extraction time of 60 min. The inventors found that unsuitable extraction conditions affect the recovery rate of saffron aldehyde and lead to variations and fractionation of saffron aldehyde, affecting subsequent determination of saffron aldehyde on GC-IRMS.

[0034] For the purification step, after the dried saffron sample is extracted with n-hexane and concentrated by nitrogen blowing, it can be directly injected into GC-MS for the determination of saffron aldehyde without considering the sample matrix or interference from other substances. However, it cannot be injected into GC-IRMS. Direct injection will clog the capillary tube with just one injection, and the high residue in the GC-IRMS injection liner, coupled with the small injection volume, will cause the residue in the liner to adsorb and vaporize the sample, resulting in unstable sample signals and affecting the final parameters. Therefore, this invention requires further purification of the extracted saffron aldehyde. The final solution after extraction and purification needs to be determined by GC-MS to confirm whether it is saffron aldehyde and its approximate concentration. This is because if the concentration of the solution in the GC-IRMS sample is too low, it will not meet the determination requirements of GC-IRMS. This is because GC-IRMS and GC-MS mainly require consistent injection conditions and furnace temperature gradients, while the injection volumes of the two instruments are different.

[0035] The inventors discovered that the saffron aldehyde extract contained many complex substances that required further purification. In addition to the target substance, the extract obtained after ultrasonic extraction also contained a large amount of lipids. Therefore, the inventors attempted to use an extraction column for separation and purification.

[0036] According to an embodiment of the present invention, the step of extracting crocin aldehyde further includes the following purification steps: 1) After pre-concentrating the extract, it is added to a silica gel column pretreated with n-hexane, eluted with a n-hexane solution of ethyl acetate of a first concentration, and the first target eluent is collected; 2) After pre-concentrating the first target eluent, it is added to a silica gel column pretreated with n-hexane, eluted first with a n-hexane solution of ethyl acetate of a second concentration, the second target eluent is collected, and then eluted with a n-hexane solution of ethyl acetate of a third concentration, and all of the third eluent is collected; 3) The second target eluent and the third eluent are combined and concentrated. The purification steps and conditions, such as the type and size of the extraction column and the concentration of the extract in the present invention, were also obtained by the inventors through a large number of experiments. Taking the extraction column as an example, GC-MS showed that the recovery rate decreased after passing through a Floris silica column and neutral alumina, and that crocin aldehyde with an isomer structure was generated after passing through a neutral alumina column. Therefore, the final method of using two silica gel columns with different weights and performing two extractions was adopted to obtain samples that met the GC-IRMS injection conditions without affecting the determination of stable isotopes in the samples.

[0037] In a specific embodiment of the present invention, in step 1), 1g of the silica gel column is added to every 2mL of the pre-concentrated extract, the filtrate is discarded, and 14ml of the first concentration of ethyl acetate in n-hexane is added for elution, the first target eluent being the subsequent 8ml eluent; in step 1), the first concentration is 5% by mass. In step 2), 1g of the silica gel column is added to every 0.2mL of the pre-concentrated first target eluent, the filtrate is discarded, and 14ml of the second concentration of ethyl acetate in n-hexane is added for elution, the second target eluent being the subsequent 7ml eluent, and in step 3), 7ml of the third concentration of ethyl acetate in n-hexane is added for elution; in step 2), the second concentration is 0.5% by mass, and the third concentration is 1% by mass. Similarly, the inventors tried various concentrations of ethyl acetate in n-hexane solutions and finally optimized the above scheme.

[0038] According to an embodiment of the present invention, the gas chromatographic conditions in the gas chromatograph-stable isotope mass spectrometer are as follows: non-polar capillary column; temperature program: initial temperature 70℃, hold for 1 min, increase to 100℃ at a rate of 3℃ / min, hold for 1 min, increase to 300℃ at a rate of 20℃ / min, hold for 2 min; splitless mode; carrier gas: high-purity helium; flow rate: 1.5 ml / min; split ratio: 10:1; injection port temperature 250℃; δ-measurement 13 The injection volume for C was 0.2 μL, and the δ was measured. 18 O and δ 2 The H injection volume was 0.5 μL; The online combustion conditions in the gas chromatography-stable isotope mass spectrometer are as follows: Determination of δ 13 C: Using an oxidation combustion tube, the temperature was set to 1000℃; δ was measured. 2 H: A high-temperature pyrolysis tube was used, with the temperature set at 1420℃; δ was measured. 18 O: Use a high-temperature pyrolysis tube, set the temperature to 1280℃; The stable isotope mass spectrometer conditions in the gas chromatography-stable isotope mass spectrometer are as follows: For the determination of the carbon stable isotope ratio, the EI ion source is used, and the vacuum degree is 1.6 × 10⁻⁶. -6 mBar, high voltage: 9.45KV, current: 1.5mA; in the determination of the ratio of the oxygen and hydrogen stable isotopes, the vacuum degree was 1.6×10⁻⁶. -6 mBar, High voltage: 9.47KV, Current: 1.5mA.

[0039] According to an embodiment of the present invention, the saffron aldehyde standard after being set is diluted to the same concentration as that in the saffron sample, and the diluted saffron aldehyde standard is used as the calibration sample.

[0040] According to an embodiment of the present invention, the method further includes, before step S3: testing the concentration of crocin in the saffron sample using gas chromatography-mass spectrometry (GC-MS). GC-MS is mainly used in experimental research and development to verify extraction methods and approximate concentrations when exploring extraction conditions. The structural diagram and signal intensity of the GC-MS are used to determine whether it is crocin and the extraction effect. In actual testing, the concentration of crocin determined by GC-MS can be used to determine or assist in determining the authenticity of saffron. As an example, the conditions of the gas chromatography-mass spectrometry (GC-MS) instrument are as follows: Column: Non-polar capillary column (0.25 mm × 30.0 m, 0.25 mm); Temperature program: Initial temperature 70 °C, hold for 1 min, increase to 100 °C at a rate of 3 °C / min, hold for 1 min, increase to 280 °C at a rate of 20 °C / min, hold for 2 min; Split mode; Carrier gas: High-purity helium; Flow rate: 2 ml / min; Split ratio: 10:1; Injector temperature 250 °C; Injection volume: 0.2 ml. EI ionization source, energy 70 eV; Ion source temperature 230 °C; Transfer line temperature 270 °C; Solvent delay 2.5 min; Scan range m / z 50~500; Ion rod temperature 150 °C. Mass spectrometry search: NIST standard library. Injection volume is 0.2 μL; the needle is washed with n-hexane before injection and with ethanol after injection.

[0041] In the second part, the present invention provides a method for identifying the origin of saffron, comprising determining the carbon, hydrogen, and oxygen stable isotope ratio of saffron aldehyde in a saffron sample using any of the methods described above, and identifying the origin of saffron based on the ratio.

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0044] Example 1 1. Materials and Standards B2191 water (δ) 2 H V-SMOW =843.43‰±2.6‰、δ 18 O V-SMOW =108.63‰±0.33‰), B2192 water (δ 2 H V-SMOW =+11.26‰±1.34‰、δ 18 O V-SMOW =-0.41‰±0.11‰), B2193 water (δ 2 H V-SMOW =-61.97‰±2.10‰、δ 18 O V-SMOW =-10.18‰±0.20‰), B2205 ( 13 C V-PDB =-28.19‰±0.14‰) All standard samples above were purchased from Elemental Microanalysis (UK). 8536 water (δ 2 H V-SMOW =-189.5‰±0.1‰、δ 18 O V-SMOW =-24.78‰±0.01‰) purchased from NIST, USA. IA-R004 (δ 13 C V-PDB =-10.99‰±0.06‰) from the Iso-Analytical Laboratory. IAEA-CH-7 (δ 13 C V-PDB=-32.‰±0.05‰) was obtained from IAEA. Crocin (HPLC, ≥98%) standard was purchased from Xinyang Zhongjian Metrology Biotechnology Co., Ltd. n-Hexane (chromatographic grade) was purchased from Merck, Germany. Ethanol (chromatographic grade, ≥99.8%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Ethyl acetate (HPLC grade, ≥99.8%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Dried saffron sample A was purchased online (sample A was labeled as originating from Chongming, Shanghai).

[0045] 2. Instruments The Thermo Fisher Scientific 253PLUS stable isotope mass spectrometer was purchased from Thermo Fisher Scientific, and its elemental analyzer was a FLASH IRMS model, while the gas chromatograph was a TRACE 1610 model. The gas chromatograph-mass spectrometer (Agilent 7890B model) was purchased from Agilent Technologies, Inc., USA. The ultrasonic cleaner (SD-250H) was purchased from Beijing Zhongcheng Technology Co., Ltd. The equilibrium concentrator (AutoEVA-20L) was purchased from Ruike Group Co., Ltd.

[0046] 3. Experimental methods and conditions Step S1 δ-values ​​of saffron aldehyde standard were determined using an elemental analyzer-stable isotope mass spectrometer. 18 O and δ 2 At time H, liquid sampling mode was used, with 3 μL of saffron aldehyde standard extracted directly into the pyrolysis tube by an autosampler. A standard curve was constructed using the measured values ​​and specified values ​​of four water standards (B2191, B2192, B2193, and 8536). Seven samples of saffron aldehyde standard were continuously measured as parallel samples, and the average value was used as the measured value. After calibration with the standard curve, the δ-value of the saffron aldehyde standard was determined. 18 O and δ 2 H value. Wherein, in this experiment, δ... 18 O and δ 2 H was determined using the EA-IRMS method, employing a Thermo Fisher 253PLUS / Flash IRMS instrument. Oxygen in the sample was converted to CO gas in the elemental analyzer unit, and hydrogen was converted to H2 gas in the same unit. The gas was then introduced into the 253PLUS stable isotope ratio mass spectrometer via a ConFlo IV continuous flow interface for detection. Key parameters of the elemental analyzer were: carrier gas flow rate: 10 mL / min, oxygen flow rate: 0 mL / min, reference gas flow rate: 100 mL / min, left furnace temperature: 1380℃, furnace temperature (column): 45℃, sample delay time: 0 seconds, oxygen flow time: 0 seconds, operating mode: liquid H2O, dilution ratio: CO 0%, H2 0%. Stable isotope ratio mass spectrometer parameters were: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶.-6 mBar, High voltage: 9.47KV, Current: 1.5 mA. δ 18 Continuous determination of O <0.06‰ (n=10), δ 2 The continuous measurement of H is <0.4‰ (n=10).

[0047] δ-values ​​of saffron aldehyde standard were determined using an elemental analyzer-stable isotope mass spectrometer. 13 At time C, a solid sample mode was used. 0.2-0.3 μL of saffron aldehyde standard was drawn into a tin cup using a liquid syringe, quickly sealed, and used as a solid sample for injection. Considering the volatility of the sample, it could not be pre-packaged and needed to be prepared immediately after packaging. Standard curves were constructed using the measured values ​​and specified values ​​of three solid standards: IAEA-CH-7, B2205, and IA-R004. Seven consecutive measurements of the saffron aldehyde standard were used as parallel samples, and the average value was taken as the measured value. After calibration with the standard curve, the δ of the saffron aldehyde standard was determined. 13 C value. Wherein, in this experiment δ 13 Carbon (C) was determined using the EA-IRMS method, employing a Thermo Fisher 253PLUS / Flash IRMS instrument. After the carbon in the sample was converted to CO2 gas in the elemental analyzer unit, it was introduced into the 253PLUS stable isotope ratio mass spectrometer via a ConFlo IV continuous flow interface for detection. Key parameters of the elemental analyzer were: carrier gas flow rate: 180 mL / min, oxygen flow rate: 180 mL / min, reference gas flow rate: 50 mL / min, right furnace temperature: 960℃, furnace temperature (column): 50℃, sample transport delay time: 13 seconds, oxygen inlet time: 3 seconds, operating mode: solid C. Stable isotope ratio mass spectrometer parameters were: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶. -6 mBar, High voltage: 9.45KV, Current: 1.5mA. δ 13 The continuous measurement of C was <0.06‰ (n=10).

[0048] The experimental results are as follows: The δ value of saffron aldehyde standard was determined after calibration with standard curves. 18 O and δ 2 The H values ​​were 24.73‰±0.13‰ and 87.34‰±0.15‰, respectively.

[0049] The δ value of saffron aldehyde standard was determined after calibration with standard curves. 13 The C value is -29.87‰ ± 0.02‰.

[0050] Step S2 Grind dried saffron sample A through a 40-mesh sieve. Accurately weigh six portions of powder (1 ± 0.05 g) each and place them in 25 ml glass test tubes. Add 15 ml of n-hexane, wrap with aluminum foil to protect from light, and sonicate (300 W power, 40 kHz frequency). Control the water temperature at 20-25 ℃ for 60 min. Filter with a 0.45 µm hydrophobic PTFE filter. Blow the collected filtrate to 2 ml under nitrogen at less than 30 ℃ in the dark. Add this 2 ml solution to a 2 g silica gel column pretreated with n-hexane. Discard the filtrate. Elute with 14 ml of 5% ethyl acetate in n-hexane solution. Discard the first 6 ml of eluent and collect the last 8 ml of eluent. Blow with nitrogen to 1 ml and add 1 ml of n-hexane to rinse the tube wall. Continue blowing with nitrogen until completely dry, and then add 0.2 ml of n-hexane to redissolve. Add 0.2 ml of the obtained solution to a 1 g silica gel column (the silica gel column needs to be pretreated with n-hexane). Discard the filtrate. Elute the 1 g silica gel column with 14 ml of 0.5% ethyl acetate in n-hexane solution. Discard the first 7 ml and collect the last 7 ml. Then add 7 ml of 1% ethyl acetate in n-hexane solution to elute the same 1 g silica gel column again. Collect the eluent and combine it with the last 7 ml collected from the previous elution with 0.5% ethyl acetate in n-hexane solution. Purge 0.2 ml of the eluent with nitrogen at below 30°C, protected from light, and store in a brown glass bottle with an inner liner at -20°C until analysis. Avoid strong light exposure throughout the entire process and perform the procedure as dark as possible.

[0051] During the experiment, the concentration of crocin in the saffron samples was determined using gas chromatography-mass spectrometry (GC-MS). The GC-MS conditions were as follows: Chromatographic column: Non-polar capillary column (0.25 mm × 30.0 m, 0.25 mm); Temperature program: Initial temperature 70℃, hold for 1 min, ramp to 100℃ at a rate of 3℃ / min, hold for 1 min, ramp to 280℃ at a rate of 20℃ / min, hold for 2 min; Split mode; Carrier gas: High-purity helium; Flow rate: 2 ml / min; Split ratio: 10:1; Injector temperature 250℃; Injection volume: 0.2 ml. EI ionization source, energy 70 eV; Ion source temperature 230℃; Transfer line temperature 270℃; Solvent delay 2.5 min; Scan range m / z 50~500; Ion rod temperature 150℃. Mass spectrometry search: NIST standard library. Injection volume: 0.2 μL; needle washed with n-hexane before injection and with ethanol after injection.

[0052] The experimental results are as follows: the content of saffron aldehyde in saffron sample A is approximately 500 ppm. Figure 1A and Figure 1B -C).

[0053] Step S3 The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the extract of saffron aldehyde sample A were determined using gas chromatography-stable isotope mass spectrometry. The test conditions were as follows: Gas chromatography conditions Non-polar capillary column; Temperature program: Initial temperature 70℃, hold for 1 min, ramp to 100℃ at a rate of 3℃ / min, hold for 1 min, ramp to 300℃ at a rate of 20℃ / min, hold for 2 min; Splitless mode; Carrier gas: High-purity helium; Flow rate: 1.5 ml / min; Split ratio: 10:1; Injector temperature 250℃; δ-measurement 13 The injection volume for C was 0.2 μL, and the δ was measured. 18 O and δ 2 The H injection volume was 0.5 μL; Online combustion conditions Measurement of δ 13 C: Use an oxidation combustion tube, set the temperature to 1000℃. The oxidation combustion tube needs to be oxidized for 2 hours before testing to regenerate the tube.

[0054] Measurement of δ 2 H: Use a high-temperature pyrolysis tube, set at 1420℃. Before sampling in the high-temperature pyrolysis tube, inject 0.5 μL of n-hexane four times for carbon coating of the reaction tube.

[0055] Measurement of δ 18 O: Use a high-temperature pyrolysis tube, set the temperature to 1280℃. Before testing the sample in the high-temperature pyrolysis tube, inject 0.5 μL of n-hexane four times for carbon coating of the reaction tube.

[0056] Stable isotope mass spectrometer conditions EI ion source, vacuum level: 1.6 × 10⁻⁶ -6 mBar, High voltage: 9.45-9.47KV, Current: 1.5mA.

[0057] The experimental results are as follows: The original values ​​of sample A without standard calibration are shown in Table 1.

[0058] Table 1. Original stable isotope ratios of saffron sample A

[0059] Step S4 The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract were calibrated using the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde standard. The specific steps are as follows: Sample A has a concentration of approximately 500 ppm. The saffron aldehyde standard needs to be diluted to 500 ppm as a calibrator. 1 μL of saffron aldehyde standard equals 1 mg. Take 10 μL of saffron aldehyde standard and add it to 1990 μL of n-hexane to prepare a 5000 μg / mL stock solution. Dilute the stock solution with n-hexane to the required concentration based on the actual sample concentration. For example, if sample A has a concentration of approximately 500 ppm, dilute the stock solution with n-hexane to 500 ppm. When testing, first measure the diluted 500 ppm standard solution. Then, input the stable isotope ratios of each element in the EA-IRMS calibrated saffron aldehyde standard into the software. Specify the measured 500 ppm standard as the calibration sample. Subsequent samples will then be calculated using the calibration sample to obtain the calibrated sample's true value, which is the final result.

[0060] The values ​​of sample A after standard calibration are shown in Table 2.

[0061] Table 2. Stable isotope ratios of saffron sample A after calibration

[0062] 5. Methodological Validation 5.1 Precision determination The extract of saffron sample A (purchased from Taobao, with the sample origin labeled as Chongming, Shanghai) was concentrated, and the δ-value of saffron aldehyde was repeatedly determined using the gas chromatography stable isotope technique employed in this invention. 13 C、δ 2 H, δ 18 O value (n=6), the measurement results are shown in Table 3, δ of saffron aldehyde in sample A 13 C、δ 2 H, δ 18 The O values ​​were -34.62, -241.75, and 23.48, respectively. Ion chromatograms are shown below. Figures 2-4 The relative standard deviations (RSDs) were 0.31, 0.23, and 1.46, respectively, all less than 2%, indicating that the measurement accuracy meets the needs of routine measurements.

[0063] Table 3. Precision determination of stable isotope ratios of saffron aldehyde (C, H, O) in dried saffron sample A

[0064] According to the determination method of the present invention, the δ of crocin in this dried saffron sample A is... 13 C、δ 2 H, δ 18 The O values ​​were -34.62, -241.75, and 23.48, respectively.

[0065] Example 2 According to the determination method in Example 1, a dried saffron sample B (B purchased online, origin indicated as Tibet) was determined, and the results are as follows: the content of saffron aldehyde in saffron sample B is approximately 400 ppm. The stable isotope ratios of the three elements C, H, and O in saffron aldehyde are shown in Table 4. The δ¹⁸O of saffron aldehyde in sample B was measured. 13 C、δ 2 H, δ 18 The O values ​​were -32.04, -230.61, and 21.42, respectively. The relative standard deviations (RSDs) were 0.68, 1.04, and 0.94, respectively, all less than 2%, indicating that the measurement accuracy meets the needs of routine measurements.

[0066] Table 4. Precision determination of stable isotope ratios of saffron aldehyde (C, H, O) in dried saffron sample B.

[0067] Example 3 According to the determination method in Example 1, a dried saffron sample C (C was purchased online, and the sample was labeled as originating from Iran) was determined. The results are as follows: the content of saffron aldehyde in saffron sample C was approximately 400 ppm. The stable isotope ratios of saffron aldehyde C, H, and O are shown in Table 5. The δ¹⁸O content of saffron aldehyde in sample C was determined. 13 C、δ 2 H, δ 18 The O values ​​were -30.85, -218.33, and 19.78, respectively. The relative standard deviations (RSDs) were 0.25, 0.68, and 0.88, respectively, all less than 2%, indicating that the measurement accuracy meets the needs of routine measurements.

[0068] Table 5. Precision determination of stable isotope ratios of saffron aldehyde (C, H, O) in dried saffron C sample.

[0069] As can be seen from Examples 1-3, the stable isotope ratios of saffron aldehyde in saffron from three different origins show significant differences in numerical values. The method of the present invention can be used to identify the origin of saffron.

[0070] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for extracting saffron aldehyde from saffron and determining its carbon, hydrogen, and oxygen stable isotope ratios, characterized in that, Includes the following steps: S1. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde standard were determined using elemental analysis-stable isotope mass spectrometry. S2. Extract saffron aldehyde from the saffron sample to obtain saffron aldehyde extract; S3. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract were determined using gas chromatography-stable isotope mass spectrometry. S4. The carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in the saffron aldehyde extract are calibrated using the carbon, hydrogen, and oxygen stable isotope ratios of the saffron aldehyde standard.

2. The method according to claim 1, characterized in that: In step S1, the elemental analyzer parameters for determining the carbon stable isotope ratio are as follows: carrier gas flow rate: 180 mL / min, oxygen flow rate: 180 mL / min, reference gas flow rate: 50 mL / min, right furnace temperature: 960℃, furnace temperature: 50℃, sample transport delay time: 13 seconds, oxygen flow time: 3 seconds, operating mode: solid C; the stable isotope ratio mass spectrometer parameters are as follows: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶ - 6 mBar, high voltage: 9.45KV, current: 1.5mA; In step S1, the elemental analyzer test conditions for determining the oxygen / hydrogen stable isotope ratio are as follows: carrier gas flow rate: 10 mL / min, oxygen flow rate: 0 mL / min, reference gas flow rate: 100 mL / min, left furnace temperature: 1380℃, furnace temperature: 45℃, sample delay time: 0 seconds, oxygen flow time: 0 seconds, operating mode: liquid H₂O, dilution ratio: CO 0%, H₂ 0%; the stable isotope ratio mass spectrometer test parameters are as follows: ionization method: EI ion source, vacuum degree: 1.6 × 10⁻⁶ -6 mBar, High voltage: 9.47KV, Current: 1.5mA.

3. The method according to any one of claims 1-2, characterized in that: The steps for extracting saffron aldehyde include: grinding dried saffron into powder, adding organic reagents for extraction, and collecting the extract.

4. The method according to claim 3, characterized in that: The ratio of saffron powder to the organic reagent is (0.95–1.05) g: 15 mL; The organic reagent is n-hexane; The extraction was performed under ultrasonic conditions, with an ultrasonic power of 300W, an extraction temperature of 20–25°C, and an extraction time of 60 min.

5. The method according to any one of claims 3-4, characterized in that: The step of extracting saffron aldehyde also includes the following purification step: 1) After pre-concentrating the extract, add it to a silica gel column pretreated with n-hexane, elute with a n-hexane solution of ethyl acetate of the first concentration, and collect the first target eluent; 2) After pre-concentrating the first target eluent, add it to a silica gel column pretreated with n-hexane. First, elute with a n-hexane solution of ethyl acetate of the second concentration and collect the second target eluent. Then, elute with a n-hexane solution of ethyl acetate of the third concentration and collect all of the third eluent. 3) Combine the second target eluent and the third eluent, and concentrate them.

6. The method according to claim 5, characterized in that: In step 1), 1 g of the silica gel column is added to every 2 mL of the pre-concentrated extract, the filtrate is discarded, and 14 mL of the first concentration of ethyl acetate in n-hexane is added for elution. The first target eluent is the following 8 mL of eluent. In step 1), the first concentration is 5% by mass. In step 2), 1 g of the silica gel column is added to every 0.2 mL of the pre-concentrated first target eluent, the filtrate is discarded, and 14 mL of the second concentration of ethyl acetate in n-hexane is added for elution. The second target eluent is the 7 mL eluent that follows. In step 3), 7 mL of the third concentration of ethyl acetate in n-hexane is added for elution. In step 2), the second concentration is 0.5% by mass, and the third concentration is 1% by mass.

7. The method according to any one of claims 1-6, characterized in that: The gas chromatography conditions in the gas chromatography-stable isotope mass spectrometer are as follows: Non-polar capillary column; Temperature program: Initial temperature 70℃, hold for 1 min, increase to 100℃ at a rate of 3℃ / min, hold for 1 min, increase to 300℃ at a rate of 20℃ / min, hold for 2 min; Splitless mode; Carrier gas: high-purity helium; flow rate: 1.5 ml / min; split ratio: 10:1; injection port temperature: 250℃; δ measurement 13 The injection volume for C was 0.2 μL, and the δ was measured. 18 O and δ 2 The H injection volume was 0.5 μL; The online combustion conditions in the gas chromatograph-stable isotope mass spectrometer are as follows: Measurement of δ 13 C: Use an oxidation combustion tube, set the temperature to 1000℃; Measurement of δ 2 H: Use a high-temperature pyrolysis tube, with the temperature set at 1420℃; Measurement of δ 18 O: Use a high-temperature pyrolysis tube, set the temperature to 1280℃; The stable isotope mass spectrometer conditions in the gas chromatography-stable isotope mass spectrometer are as follows: In the determination of the carbon stable isotope ratios, an EI ion source was used with a vacuum level of 1.6 × 10⁻⁶. -6 mBar, high voltage: 9.45KV, current: 1.5mA; In the determination of the ratio of stable oxygen and hydrogen isotopes, the vacuum degree was 1.6 × 10⁻⁶. -6 mBar, High voltage: 9.47KV, Current: 1.5mA.

8. The method according to any one of claims 1-7, characterized in that: The calibration steps include: The saffron aldehyde standard, after being set, is diluted to the same concentration as that in the saffron sample, and the diluted saffron aldehyde standard is used as the calibration sample.

9. The method according to any one of claims 1-8, characterized in that: The method further includes the following steps before step S3: The concentration of crocin in the saffron sample was determined using gas chromatography-mass spectrometry.

10. A method for identifying the origin of saffron, characterized in that, This includes using the method described in claims 1-9 to determine the carbon, hydrogen, and oxygen stable isotope ratios of saffron aldehyde in saffron samples, and identifying the origin of saffron based on the ratios.

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