A method for detecting octadecyl vinyl ethers and its application

Abstract

This invention belongs to the field of component detection technology and discloses a method and application for the detection of octadecyl vinyl ether. Using ginseng prepared by co-fermentation of ginseng powder and bacterial enzymes as the sample, a three-step pretreatment method is employed: ethanol extraction combined with liquid-liquid extraction of a toluene:n-hexane mixture (5:1 volume ratio) and PDMS solid-phase microextraction. This is combined with gas chromatography-tandem mass spectrometry (GC-MS) to achieve qualitative and quantitative analysis of octadecyl vinyl ether. The three-step pretreatment method uses ethanol extraction combined with liquid-liquid extraction of a toluene:n-hexane mixture and PDMS solid-phase microextraction. The ethanol extractant is consistent with the solvent used in the preparation of fermented ginseng, ensuring sufficient dissolution of the target analyte. The toluene-n-hexane mixed solvent efficiently separates the target analyte from polar matrix impurities, and the boiling point difference facilitates subsequent solvent removal. PDMS solid-phase microextraction further enriches trace amounts of the target analyte. Compared with the reduced pressure concentration method, the detection sensitivity is improved by more than 2 times, and matrix interference is removed more thoroughly.

Description

Technical Field

[0001] This invention belongs to the field of component detection technology, and in particular to a method and application for detecting octadecyl vinyl ether. Background Technology

[0002] Ginseng is an important flavor additive in cigarette products, and its quality directly determines the sensory experience of cigarette products. Octadecyl vinyl ether is a key off-odor compound that causes ginseng to have an earthy taste. This compound is a "small amount, large aroma" type of substance. Even a very low content can produce a noticeable earthy taste, which seriously affects the quality of ginseng and cigarette products.

[0003] Existing technologies have developed a method for the synergistic fermentation of ginseng powder using *Acetobacter flavum* (CGMCC No. 18664), cellulase, and amylase. This method, through a specific fermentation process and subsequent extraction with a 45%–55% ethanol aqueous solution, can reduce the content of octadecyl vinyl ether in ginseng by more than 80%. The resulting ginseng, when applied to cigarettes, can significantly reduce the earthy odor of the smoke. Therefore, there is an urgent need for a highly sensitive and accurate detection method adapted to the water-alcohol matrix characteristics of this fermented ginseng to achieve precise quantification of trace amounts of octadecyl vinyl ether in ginseng and ginseng powder before and after fermentation, in order to evaluate the effectiveness of this fermentation process in reducing the earthy odor.

[0004] Existing octadecyl vinyl ether detection technologies have the following three core shortcomings, failing to meet the needs for precise trace detection in fermented ginseng: 1. Poor compatibility of pretreatment: Conventional detection methods often use single organic solvent extraction or vacuum concentration, which are incompatible with the water-alcohol matrix of fermented ginseng. The target substance is not fully dissolved, the extraction recovery rate is only 60% to 75%, and it cannot effectively remove the interference of polar matrix such as polysaccharides and polyphenols in ginseng, resulting in severe suppression of the detection signal.

[0005] 2. Insufficient detection sensitivity: The detection limit of the existing method is only 0.01 to 0.05 μg / L, which cannot accurately quantify octadecyl vinyl ethers with a content of less than 0.01 μg / L after fermentation, making it difficult to evaluate the effect of fermentation on reducing earthy odor.

[0006] 3. Low accuracy in qualitative and quantitative analysis: External standard method is often used for quantification, which does not eliminate pretreatment losses, instrument drift and matrix effects. The relative standard deviation (RSD) is as high as 10% to 20%, and false positives are easy to occur in qualitative analysis, making the quantitative results unreliable. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for the detection of octadecyl vinyl ether.

[0008] The technical solution adopted by this invention to solve its technical problem is: A method for detecting octadecyl vinyl ether, wherein the method uses ginseng powder and bacterial enzyme co-fermentation prepared as the test sample, and employs a three-step pretreatment method of ethanol extraction combined with liquid-liquid extraction of a mixture of toluene:n-hexane (v / v) = 5:1 and solid-phase microextraction with polydimethylsiloxane (PDMS). The qualitative and quantitative analysis of octadecyl vinyl ether is achieved by combining gas chromatography-tandem mass spectrometry (GC-MS / MS).

[0009] Furthermore, it includes the following steps: (1) Sample pretreatment: If it is liquid ginseng, measure it directly as the extract; if it is solid ginseng powder, add 45% to 55% ethanol aqueous solution (preferably 50% ethanol aqueous solution) to the liquid at a weight ratio of 1:15 to 25 to obtain the extract. (2) Ethanol extraction: Place the liquid to be extracted in a shaking device and stir at room temperature. Filter and collect the supernatant to obtain the ethanol extract. (3) Liquid-liquid extraction of toluene: n-hexane = 5:1 mixture: Transfer the ethanol extract to a stoppered separatory funnel, add a liquid-liquid extraction of toluene and n-hexane in a volume ratio of 5:1, collect the upper organic phase after standing and separating the layers, repeat the extraction 1 to 3 times, and combine the organic phases extracted multiple times. (4) PDMS solid-phase microextraction: The combined organic phase was purged with nitrogen and concentrated to 5% to 10% of the original volume, preferably to 5 mL. Solid-phase microextraction was performed using a polydimethylsiloxane (PDMS) coated fiber head. After adsorption of the target analyte, the sample was directly injected for analysis. (5) GC-MS / MS detection: The SPME fiber head of the adsorbed target analyte was injected into the gas chromatograph-tandem mass spectrometer for GC-MS / MS detection. The qualitative analysis was completed using multiple reaction monitoring mode (MRM), and the quantitative analysis was completed using the internal standard method combined with the standard curve. (6) Result determination: Confirm whether the sample contains octadecyl vinyl ether based on the qualitative ion pair matching results detected by GC-MS / MS, and calculate the content of octadecyl vinyl ether in the sample based on the peak area ratio of the quantitative ion pairs.

[0010] Further, the ginseng prepared by the co-fermentation of bacteria and enzymes in step (1) is the supernatant obtained by extracting and filtering ginseng powder after co-fermentation of ginseng powder with Acetobacter pomorum (CGMCC No. 18664), cellulase and amylase, and the supernatant obtained by filtration; the weight ratio of the solid ginseng powder to the 45% to 55% ethanol aqueous solution is preferably 1:20.

[0011] Furthermore, in step (2), the stirring speed for room temperature stirring extraction is 80-120 r / min, and the extraction time is 24 h; filtration is performed using filter paper or a 0.45 μm aqueous filter membrane; Alternatively, in step (3), the volume ratio of the ethanol extract to the toluene and n-hexane mixture in a volume ratio of 5:1 is 1:1 to 3, the shaking time for liquid-liquid extraction is 15 to 30 min, and the settling time is 20 to 40 min.

[0012] Furthermore, the operating conditions for solid-phase microextraction in step (4) are: extraction temperature 30-60℃, extraction time 20-40min, and stirring speed 200-500r / min.

[0013] Further, the chromatographic conditions for GC-MS / MS detection in step (5) are as follows: Column: HP-5 or DB-5 weakly polar capillary column, 30 m × 0.25 mm × 0.25 μm; Temperature program: initial temperature 60℃, hold for 1 min, increase to 280℃ at a rate of 8~12℃ / min, hold for 5~10 min; Carrier gas: high purity helium, purity ≥99.999%, constant flow mode, flow rate 1.0~1.2 mL / min; Injector temperature: 250~280℃, split ratio 10:1~20:1, SPME injection resolution time 2~5 min; The column outlet is directly connected to the mass spectrometer ion source; Alternatively, the mass spectrometry conditions for GC-MS / MS detection in step (5) are: ion source: EI source, electron energy 70 eV, ion source temperature 230~250℃; transfer line temperature: 280~300℃; Detection mode: Multiple reaction monitoring (MRM); Qualitative ion pairs for octadecyl vinyl ether are m / z 296→281 and m / z 296→253, quantitative ion pairs are m / z 296→281, collision energy 15–25 eV; Collision gas: High-purity nitrogen, purity ≥99.999%; Further, in step (5), the internal standard used in the internal standard method is eicosane. The qualitative ion pairs of the internal standard are m / z 284→269 and m / z 284→241, and the quantitative ion pair is m / z 284→269. The collision energy is 10~20 eV. The standard curve is prepared as follows: prepare octadecyl vinyl ether standard solutions with a concentration gradient of 0.001~0.1 μg / L, add an equal amount of eicosane internal standard to each solution, process according to the method in steps (3)~(4), and then perform GC-MS / MS detection. Plot the standard curve with the peak area ratio of the quantitative ion pairs of octadecyl vinyl ether and the internal standard as the abscissa and the concentration ratio as the ordinate. The correlation coefficient of the standard curve is r>0.999.

[0014] Furthermore, the detection method has a limit of detection (LOD) of 0.0005–0.003 μg / L, a limit of quantitation (LOQ) of 0.002–0.01 μg / L, a spiked recovery rate of 85%–110%, and a relative standard deviation (RSD) of <5%.

[0015] The above-described detection method is applied to the detection of octadecyl vinyl ether content in ginseng powder and / or ginseng products co-fermented with microorganisms and / or ginseng for cigarette use.

[0016] The above-described detection method is applied to reducing the earthy smell during the fermentation process of ginseng powder.

[0017] The advantages and positive effects of this invention are as follows: 1. This invention employs a three-step pretreatment method combining ethanol extraction with liquid-liquid extraction using a toluene:n-hexane (5:1) mixture and PDMS solid-phase microextraction: The ethanol extractant is consistent with the solvent used in the preparation of fermented ginseng, which can fully dissolve the target analyte; the toluene-n-hexane mixed solvent efficiently separates the target analyte from polar matrix impurities, and the difference in boiling points facilitates subsequent solvent removal; PDMS solid-phase microextraction further enriches trace amounts of the target analyte, improving detection sensitivity by more than 2 times compared to the reduced pressure concentration method, and removing matrix interference more thoroughly.

[0018] 2. This invention clarifies the basis for the solvent ratio of toluene:n-hexane = 5:1: this ratio balances high extraction recovery rate with low matrix interference, while avoiding solvent residue affecting SPME adsorption; it also clarifies the basis for selecting the PDMS fiber head: it perfectly matches the long-chain weakly polar structure of octadecyl vinyl ether, achieving high selective enrichment and effectively eliminating interference from complex ginseng matrices.

[0019] 3. This invention combines multiple reaction monitoring (MRM) mode of GC-MS / MS technology, and achieves micro-level detection at the μg / L level through the specific selection of characteristic mother ion-daughter ion pairs. It can accurately detect low levels of octadecyl vinyl ether in fermented ginseng. It uses dual ion pairs for qualitative analysis and characteristic ion pairs for quantitative analysis, combined with the internal standard method to eliminate systematic errors. The correlation coefficient of the standard curve is r>0.999, the spiked recovery rate is 85%~110%, and the relative standard deviation is RSD<5%, demonstrating excellent qualitative and quantitative accuracy and reproducibility.

[0020] 4. The quantitative advantages of the internal standard method used in this invention: This invention uses eicosane as an internal standard for quantitative analysis via the internal standard method, which has the following significant advantages compared to the external standard method: (1) Eliminate systematic errors: The internal standard and the target substance undergo the entire process of ethanol extraction, liquid-liquid extraction, SPME enrichment and GC-MS / MS detection simultaneously, which can effectively compensate for the loss, injection volume fluctuation and instrument response drift in the pretreatment process, and significantly improve the quantitative accuracy and reproducibility.

[0021] (2) Strong resistance to matrix interference: In complex ginseng matrix, the matrix effect (ion inhibition / enhancement) of internal standard and target is highly consistent, which can effectively counteract the matrix interference on the detection signal and ensure the quantitative reliability at low concentration (μg / L level).

[0022] (3) Wide linear range: Within the concentration gradient of 0.001 to 0.1 μg / L, the correlation coefficient of the standard curve r>0.999, which shows excellent linearity and can cover the range of changes in the content of octadecyl vinyl ether in ginseng samples before and after fermentation.

[0023] 5. Synergistic effect of the three-step pretreatment method of this invention Compared to existing vacuum concentration methods, this invention employs a three-step pretreatment method consisting of ethanol extraction + toluene:n-hexane = 5:1 liquid-liquid extraction + PDMS solid-phase microextraction, achieving a step-by-step purification process of "extraction-separation-enrichment". The first step, ethanol extraction, is a solvent that is perfectly matched to the preparation solvent of fermented ginseng, maximizing the dissolution of the target substance while maintaining the compatibility of the water-ethanol matrix.

[0024] The second step is liquid-liquid extraction: the synergistic effect of toluene and n-hexane is used to efficiently separate the target analyte from polar impurities (such as polysaccharides, polyphenols, and small molecule alcohols), and the extraction recovery and selectivity are balanced by a volume ratio of 5:1, providing a pure organic phase for subsequent SPME enrichment.

[0025] The third step, PDMS solid-phase microextraction, targets the long-chain, weakly polar structure of octadecyl vinyl ether, achieving high selectivity and high enrichment, further reducing the detection limit to the 0.0005 μg / L level, which is far superior to the reduced pressure concentration method and meets the needs of trace detection.

[0026] 6. The methodological performance of this invention is comprehensively improved. Compared with existing technologies, this invention represents a significant technological advancement: the detection limit is reduced from 0.01–0.05 μg / L to 0.0005–0.003 μg / L, resulting in a 2–100-fold increase in sensitivity; the quantitation limit is reduced from 0.04–0.2 μg / L to 0.002–0.01 μg / L, a 20–100-fold decrease in the lower quantitation limit. The spiked recovery rate is increased from 60–75% to 85–110%, an improvement of 20–35 percentage points; the relative standard deviation is reduced from 10–20% to below 5%, resulting in a 2–4-fold improvement in reproducibility. The matrix interference peak area is reduced from 8.6 × 10⁻⁶. 6 Reduced to 2.8×10 6Interference was reduced by 67.4%; the signal-to-noise ratio (SNR) increased from 12.3 to 36.9, a 2.0-fold improvement. Furthermore, this invention is simpler to operate, eliminating the need for complex vacuum concentration equipment; SPME injection can be directly coupled with GC-MS / MS for automated and high-throughput detection. Details are shown in Table 1.

[0027] Table 1. Differences between prior art and the present invention

[0028] 7. The method of the present invention can accurately quantify the content of octadecyl vinyl ether in ginseng and ginseng powder before and after synergistic fermentation of bacteria and enzymes, providing reliable technical support for the optimization of fermentation process, quality control of ginseng and flavor enhancement of cigarette products.

[0029] 8. The method of the present invention can be applied to the detection of octadecyl vinyl ether content in ginseng powder, ginseng fermented with bacterial enzymes, and deep-processed ginseng products for cigarettes, such as qualitative and quantitative detection. It can be used to evaluate the effect of reducing earthy odor in the process of ginseng powder fermented with Acetobacter fructose (CGMCC No. 18664), cellulase and amylase, and to verify the effect of reducing the octadecyl vinyl ether content in ginseng by more than 80% after fermentation.

[0030] 9. This invention addresses the need for precise detection of trace amounts of octadecyl vinyl ether in ginseng, ginseng powder, and ginseng deep-processed products for cigarettes prepared by ginseng fermentation with bacterial enzymes. A three-step pretreatment method is employed: ethanol extraction combined with liquid-liquid extraction using a toluene:n-hexane mixture (5:1 volume ratio) and polydimethylsiloxane (PDMS) solid-phase microextraction for sample enrichment and purification. This is followed by gas chromatography-tandem mass spectrometry (GC-MS / MS) to achieve qualitative and quantitative analysis of the target analyte. This method uses eicosane as an internal standard to eliminate systematic errors and matrix interference, further improving the accuracy and reproducibility of quantitative analysis of trace target analytes. This method features highly targeted pretreatment, is well-suited to the characteristics of ginseng and its aqueous-alcoholic matrix, achieves high extraction efficiency of the target substance, thoroughly removes matrix interference, exhibits high detection sensitivity, excellent qualitative and quantitative accuracy and reproducibility, and has a detection limit as low as 0.0005 μg / L. It can be efficiently applied to the detection of octadecyl vinyl ether content in ginseng powder and ginseng co-fermented with bacteria and enzymes, providing reliable technical support for ginseng quality control, quantitative evaluation of the effect of fermentation process on reducing earthy odor, and flavor enhancement of cigarette products. Detailed Implementation

[0031] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0032] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0033] This invention provides a method for detecting octadecyl vinyl ether, employing a three-step pretreatment method combining ethanol extraction with liquid-liquid extraction using a toluene:n-hexane (5:1) mixture and PDMS solid-phase microextraction. Combined with GC-MS / MS technology, it achieves qualitative and quantitative analysis of the target analyte. This method addresses the problems of low extraction efficiency, significant matrix interference, and insufficient accuracy and sensitivity in existing detection methods. It is adapted to the water-alcohol matrix characteristics of ginseng co-fermented with bacteria and enzymes, enabling precise detection of octadecyl vinyl ether at the μg / L level in ginseng powder and ginseng co-fermented with bacteria and enzymes. Simultaneously, it provides a reliable technical means to evaluate the effect of Acetobacter fructus (CGMCC No. 18664) co-fermentation of ginseng powder on reducing earthy odor.

[0034] The selection of the toluene:n-hexane = 5:1 mixture was based on the following criteria: 1. Toluene is a high-boiling-point aromatic hydrocarbon solvent (boiling point 110.6℃), with a boiling point difference of about 42℃ from n-hexane (boiling point 68.7℃). It can be efficiently separated in subsequent nitrogen purging, avoiding interference from solvent residues in solid-phase microextraction. At the same time, toluene has a very strong solubility for long-chain, weakly polar octadecyl vinyl ethers, which can efficiently extract the target analyte. 2. Hexane is a non-polar alkane solvent, which can enhance the affinity of the mixed solvent for long-chain alkyl groups, reduce the polarity of the mixed solvent, reduce the co-extraction of polar impurities in the matrix (such as polysaccharides, polyphenols, and small molecule alcohols), and improve the extraction selectivity. 3. Setting the volume ratio to 5:1 can ensure a high extraction recovery rate of octadecyl vinyl ether while removing polar interfering substances to the greatest extent, and avoid solvent peak interference during SPME adsorption caused by an excessively high toluene ratio.

[0035] The specific ratio optimization experiment is as follows: Using extraction recovery and matrix interference peak area as indicators, the toluene:n-hexane (volume ratio) ratios of 3:1, 4:1, 5:1, 6:1, and 7:1 were investigated, with each group tested in parallel six times. As shown in Table 2, the volume ratio of 5:1 exhibited the highest recovery, lowest interference, and best signal-to-noise ratio, making it the optimal ratio.

[0036] The specific testing methods are as follows: Using extraction recovery rate, matrix interference peak area, and signal-to-noise ratio as indicators, the volume ratios of toluene to n-hexane (toluene: chromatographic grade, commercially available conventional reagent, purchased from Sinopharm Chemical Reagent Co., Ltd., for liquid-liquid extraction; n-hexane: chromatographic grade, commercially available conventional reagent, purchased from Sinopharm Chemical Reagent Co., Ltd., for liquid-liquid extraction) were investigated at 3:1, 4:1, 5:1, 6:1, and 7:1, a total of 5 ratios, with 6 parallel experiments per group. The test sample was the ginseng liquid sample co-fermented by bacteria and enzymes as described in Example 1; the detection methods were all performed according to steps (1) to (6) recorded in the "Invention Content" section of this invention, only changing the volume ratio of toluene to n-hexane, while keeping the other pretreatment conditions, GC-MS / MS detection conditions, and internal standard quantification conditions consistent. According to Table 2, the volume ratio of 5:1 had the highest recovery rate, the lowest matrix interference, and the best signal-to-noise ratio, making it the optimal ratio.

[0037] Table 2. Effect of different ratios on detection results

[0038] The basis for selecting polydimethylsiloxane (PDMS) coated fiber heads is as follows: The specific testing methods are as follows: Test sample: ginseng liquid sample co-fermented with bacteria and enzymes as described in Example 1; Experimental Methods: The detection was carried out according to steps (1) to (6) recorded in the "Invention Content" section of this invention. Solid phase microextraction was compared using PDMS, PA, and CAR / PDMS coated fiber heads (PDMS, PA, and CAR / PDMS coated solid phase microextraction (SPME) fiber heads: all are commercially available analytical instrument accessories, purchased from Supelco, USA, for solid phase microextraction enrichment of octadecyl vinyl ether in samples). Except for the different extraction coatings, the other pretreatment conditions, GC-MS / MS detection conditions, and internal standard quantitative conditions were completely consistent. Each group was tested in parallel for 6 times. The peak area, detection limit, and relative standard deviation (RSD) were used as evaluation indicators to screen the optimal extraction coating.

[0039] Table 3 shows a comparison of the detection effects of different coatings, indicating that the PDMS coating has the strongest extraction and enrichment ability for the target analyte, with a peak area reaching 15.6 × 10⁻⁶. 6The adsorption capacity of the PDMS coating was significantly higher than that of the PA and CAR / PDMS coatings, indicating a higher adsorption capacity for the target analyte. Furthermore, the detection limit of the PDMS coating was as low as 0.0005 μg / L, far superior to the other two coatings, resulting in higher method sensitivity. In addition, its relative standard deviation (RSD) was only 2.8%, demonstrating the best precision and reproducibility. Considering extraction capacity, detection sensitivity, and method stability, the PDMS coating performed best; therefore, PDMS was selected as the coating for solid-phase microextraction.

[0040] Table 3. Detection results of different coatings

[0041] To comprehensively examine the effects of different internal standards on the quantitative results of octadecyl vinyl ether, this invention uses eicosane, octadecane, and docosane as internal standards to systematically verify the linearity, recovery rate, and precision of the method.

[0042] The specific detection method is as follows: The test sample is the ginseng liquid sample co-fermented by bacteria and enzymes as described in Example 1; chromatographically pure eicosane, octadecane, and docosane (eicosane, octadecane, and docosane: all are chromatographically pure standards, commercially available reagents, purchased from Sinopharm Chemical Reagent Co., Ltd. or Shanghai Yuanye Biotechnology Co., Ltd.) are selected as internal standards, and the detection is carried out according to the steps (1) to (6) recorded in the "Invention Content" section of this invention. Only the type of internal standard is changed, and the other pretreatment conditions, GC-MS / MS detection conditions, standard curve preparation and quantitative methods are kept consistent. Each group is tested in parallel 6 times. The correlation coefficient of the standard curve, the spiked recovery rate and the relative standard deviation (RSD) are used as evaluation indicators to screen the optimal internal standard.

[0043] As shown in Table 4, when eicosane is used as the internal standard, the correlation coefficient r of the standard curve reaches 0.9996, indicating excellent linearity. The method recovery rate is (94.2±2.1)%, which falls within the ideal range of 90%~110%, demonstrating good accuracy. The relative standard deviation (RSD) is only 2.5%, showing significantly better precision than octadecane (7.8%) and docosane (5.6%). Furthermore, eicosane has similar structural properties and chromatographic behavior to the target analyte, effectively correcting systematic errors in the pretreatment and injection processes, resulting in the best overall quantitative effect. Therefore, eicosane was ultimately chosen as the internal standard for the determination of octadecyl vinyl ether content.

[0044] Table 4 Quantitative effects of different internal standards

[0045] Example 1 A method for detecting octadecyl vinyl ether includes the following steps: Test sample: Ginseng co-fermented with bacteria and enzymes (prepared according to the method disclosed in Chinese Patent Publication CN121362710A, using Acetobacter fruitis CGMCC No.18664, cellulase and amylase to co-ferment ginseng powder, followed by extraction and filtration with 50% ethanol aqueous solution). Detection steps: (1) Accurately measure 100 mL of ginseng sample co-fermented with bacteria and enzyme as the extract; (2) Place the extract in a constant temperature stirrer, set the stirring speed to 100 r / min, and stir and extract at room temperature for 24 h. Filter through a 0.45 μm aqueous filter membrane to obtain a clear ethanol extract; (3) Transfer the ethanol extract to a 250 mL stoppered separatory funnel, add 100 mL of a mixture of toluene and n-hexane in a volume ratio of 5:1, vortex for 20 min, let stand for 30 min to separate the layers, and collect. Collect the upper organic phase; add 50 mL of toluene:n-hexane = 5:1 (volume ratio) mixture to the aqueous alcohol phase again, repeat the above liquid-liquid extraction operation once, and combine the two organic phases; (4) Purge the combined organic phase with nitrogen to concentrate to 5 mL, transfer to a 15 mL headspace vial, insert a PDMS-coated SPME fiber head, and extract for 30 min at 40℃ and 300 r / min stirring conditions, then remove the fiber head and directly inject the sample for analysis; (5) GC-MS / MS detection: the chromatographic column is HP-5 (30 m×0.25 mm×0.25 μm), heating program: 60℃ for 1 min, then increase to 280℃ at 10℃ / min and hold for 5 min; injection port temperature 270℃, SPME resolution time 3 min; mass spectrometry uses MRM mode, the quantitative ion pair of octadecyl vinyl ether is m / z 296→281, collision energy 20eV; the quantitative ion pair of internal standard eicosane is m / z 284→269, collision energy 15eV; (6) result judgment: the qualitative ion pair matching degree ≥95%, confirming that the sample contains octadecyl vinyl ether, and the content is calculated according to the peak area ratio of the quantitative ion pair.

[0046] The detection results of the method of this invention are as follows: the content of octadecyl vinyl ether in the sample is 0.0032±0.0015 μg / L; the limit of detection (LOD) is 0.0005 μg / L, the limit of quantitation (LOQ) is 0.002 μg / L; the spiked recovery rate is 94.5±2.3% (spiking concentration 0.002 μg / L); the relative standard deviation (RSD) is 2.8% (n=6); and the matrix interference peak area is 2.8×10⁻⁶. 6 The signal-to-noise ratio was 36.9. Compared with the unfermented ginseng powder sample (content 0.0318±0.0062 μg / L), the content was reduced by 89.9%, verifying the effect of the bacterial-enzyme co-fermentation process in reducing the earthy smell.

[0047] Comparative Example 1 (Existing technical detection method: single organic solvent extraction + vacuum concentration + external standard method) Test sample: Ginseng extract from the same batch of bacteria and enzymes co-fermentation as in Example 1 (theoretical content of octadecyl vinyl ether 0.0035 μg / L). Detection steps: (1) Take 100 mL of the above liquid sample as the extract; (2) Extract with a single organic solvent (n-hexane), add n-hexane at a volume ratio of 1:2 to the extract, shake for 25 min, let stand for 30 min to separate the layers, collect the upper organic phase, repeat the extraction twice, and combine the organic phases; (3) Place the combined organic phase in a rotary evaporator, concentrate it to near dryness under reduced pressure at 40 °C, redissolve the residue with 5 mL of methanol to obtain the sample solution; (4) Detect using a GC-MS / MS instrument, the chromatographic and mass spectrometric conditions are the same as in Example 1, and quantitative analysis is performed using the external standard method; (5) The result determination is the same as in Example 1.

[0048] Current technical detection results: The content of octadecyl vinyl ether in the sample was 0.0011±0.0008 μg / L; the limit of detection was 0.01 μg / L, and the limit of quantitation was 0.04 μg / L; the recovery rate was 68.2±4.5%; the relative standard deviation (RSD) was 12.5% ​​(n=6); and the peak area of ​​matrix interference was 8.6×10⁻⁶. 6 The signal-to-noise ratio is 12.3.

[0049] Comparative Example 2 (Comparative Example with the same proportions as Example 1) The specific preparation methods are the same as in Example 1, except that in step (3), the toluene:n-hexane = 5:1 (volume ratio) mixture is replaced with a toluene:n-hexane = 3:1 (volume ratio) mixture.

[0050] Results: The content of octadecyl vinyl ether in the sample was 0.0012 ± 0.0009 μg / L; the limit of detection was 0.004 μg / L, and the limit of quantitation was 0.013 μg / L; the recovery rate was 72.5 ± 3.1%; the relative standard deviation (RSD) was 7.6% (n=6); the area of ​​matrix interference peaks was 8.6 × 10⁻⁶. 6 The signal-to-noise ratio was 12.3. Compared with Example 1, the detection sensitivity decreased by 8 times, the spiked recovery rate decreased by 22 percentage points, and the RSD increased by 4.8 percentage points, showing significantly worse performance than Example 1.

[0051] Comparative Example 3 (Comparative example of the coating corresponding to Example 1) The specific preparation methods are the same as in Example 1, except that in step (4), PDMS solid-phase microextraction is replaced with PA (polyamide) coated SPME fiber head.

[0052] Results: The content of octadecyl vinyl ether in the sample was 0.0008 ± 0.0006 μg / L; the limit of detection was 0.006 μg / L, and the limit of quantitation was 0.02 μg / L; the recovery rate was 65.3 ± 3.8%; the relative standard deviation (RSD) was 8.5% (n=6); the peak area of ​​matrix interference was 7.9 × 10⁻⁶. 6 The signal-to-noise ratio was 13.8. Compared with Example 1, the detection sensitivity decreased by 12 times, the spiked recovery rate decreased by 29.2 percentage points, and the RSD increased by 5.7 percentage points, showing significantly worse performance than Example 1.

[0053] Comparison and discussion of the results of Example 1 with Comparative Examples 1, 2, and 3 1. Comparison with existing technologies (Comparative Example 1): ① Detection sensitivity: The detection limit of this invention is 0.0005 μg / L, while that of the existing technology is 0.01 μg / L, representing a 20-fold increase in sensitivity. This invention can accurately detect a low concentration of the target substance at 0.0032 μg / L, while the existing technology, due to insufficient sensitivity, only measures a concentration of 0.0011 μg / L, severely underestimating the target substance concentration and failing to accurately evaluate the effect of fermentation on reducing earthy odor; ② Extraction recovery rate: This invention achieves 94.5%, while the existing technology achieves 68.2%, representing an improvement of 26.3 percentage points. This is because this invention uses ethanol extraction + toluene:n-hexane = 5:1 mixed extraction, which is suitable for water-alcohol matrix and efficiently separates polar impurities, while the existing technology uses only n-hexane extraction, resulting in poor compatibility and insufficient extraction; ③ Qualitative and quantitative accuracy: The RSD of this invention is 2.8%, while that of the existing technology is 12.5%. This invention eliminates systematic errors through the internal standard method, and its reproducibility is far superior to the external standard method of the existing technology. Furthermore, the dual ion pair qualitative method can avoid false positives, while the existing technology is easily affected by matrix interference, leading to large quantitative deviations; ④ Matrix interference: The matrix interference peak area of ​​this invention is 2.8 × 10⁻⁶. 6 Existing technology 8.6×10 6 Interference was reduced by 67.4%, the signal-to-noise ratio was improved by 2.0 times, and the detection signal was more stable.

[0054] 2. Comparison with the comparative example (Comparative Example 2): Comparative Example 2 used a toluene:n-hexane ratio of 3:1. Due to the excessively high proportion of n-hexane, the mixed solvent's ability to dissolve the target analyte was insufficient, resulting in a decrease in the extraction recovery rate to 72.5%. Matrix interference increased, and the detection sensitivity and reproducibility decreased significantly. This further proves the rationality and superiority of the toluene:n-hexane ratio of 5:1. Only this ratio can balance the extraction recovery rate and selectivity, providing a pure organic phase for subsequent SPME enrichment.

[0055] 3. Comparison with the coating control example (Comparative Example 3): Comparative Example 3 used a PA coating. Due to poor matching with the weakly polar target analyte, the adsorption capacity was small and the interference was large, resulting in a spiked recovery rate of 65.3% and a detection limit of 0.006 μg / L. This could not meet the requirements for low-content detection. This proves that the PDMS coating is the best choice for the target analyte and can achieve high selectivity and high enrichment.

[0056] Meanwhile, by comparing Example 1, Comparative Example 2 and Comparative Example 3, it can be seen that the mixture of toluene and n-hexane in a volume ratio of 5:1 in step (3) of the method of the present invention and the PDMS-coated SPME fiber head in step (4) have a synergistic effect, which can synergistically improve the sensitivity, reproducibility, detection limit and qualitative and quantitative accuracy of the detected octadecyl vinyl ether content.

[0057] Example 2 A method for detecting octadecyl vinyl ether includes the following steps: Test sample: Ginseng powder (commercially available, pulverized and passed through an 80-mesh sieve); Detection steps: (1) Take 5g of ginseng powder and mix it with a material-liquid ratio of 1:20 (5g of ginseng powder + 100mL of 50% ethanol aqueous solution) to obtain the extract; (2) to (5) are the same as steps (2) to (5) in the detection steps of Example 1; (6) The result determination is the same as in Example 1.

[0058] The results of the method of this invention are as follows: the content of octadecyl vinyl ether in the sample is 0.0321±0.0058 μg / L; the limit of detection (LOD) is 0.0005 μg / L, the limit of quantitation (LOQ) is 0.002 μg / L; the recovery rate is 92.3±2.5% (spiking concentration 0.01 μg / L); the relative standard deviation (RSD) is 3.2% (n=6); and the peak area of ​​matrix interference is 2.9×10⁻⁶. 6 The signal-to-noise ratio was 35.7. All methodological parameters met the requirements of the claims of this invention (LOD 0.0005–0.003 μg / L, LOQ 0.002–0.01 μg / L, spiked recovery 85%–110%, RSD < 5%), demonstrating that the method of this invention can effectively detect the target analyte in solid ginseng powder.

[0059] Comparative Example 4 (Existing technical detection method: ethanol extraction + vacuum concentration + external standard method) Test sample: Ginseng powder from the same batch as in Example 2 (theoretical content of octadecyl vinyl ether 0.032 μg / L); Detection steps: (1) Take 5g of ginseng powder, add 50% ethanol aqueous solution at a material-to-liquid ratio of 1:20, stir evenly to obtain the extract; (2) Stir and extract at room temperature for 24h, filter with a 0.45μm aqueous filter membrane, and take the supernatant to obtain the ethanol extract; (3) Concentrate the ethanol extract under reduced pressure at 40℃ to near dryness, redissolve the residue with 5mL of n-hexane to obtain the sample solution; (4) Detect using GC-MS / MS instrument, the chromatographic and mass spectrometric conditions are the same as in Example 1, and the external standard method is used for quantitative analysis; (5) The result judgment is the same as in Example 1.

[0060] Current technical detection results: The content of octadecyl vinyl ether in the sample was 0.0115±0.0022 μg / L; the limit of detection was 0.008 μg / L, and the limit of quantitation was 0.025 μg / L; the recovery rate was 73.8±3.8%; the relative standard deviation (RSD) was 8.2% (n=6); and the matrix interference peak area was 8.5×10⁻⁶. 6 The signal-to-noise ratio is 12.8.

[0061] Comparative Example 5 (Comparative Example with the same proportions as Example 2) The specific preparation methods are the same as in Example 2, except that in step (3), the toluene:n-hexane = 5:1 (volume ratio) mixture is replaced with a toluene:n-hexane = 7:1 (volume ratio) mixture.

[0062] Results: The content of octadecyl vinyl ether in the sample was 0.0152±0.0028 μg / L; the limit of detection was 0.003 μg / L, and the limit of quantitation was 0.01 μg / L; the recovery rate was 88.6±3.3%; the relative standard deviation (RSD) was 6.8% (n=6); the peak area of ​​matrix interference was 7.3×10⁻⁶. 6 The signal-to-noise ratio was 15.7. Compared with Example 1, the detection sensitivity decreased by 6 times, the spiked recovery rate decreased by 3.7 percentage points, and the RSD increased by 3.6 percentage points, showing significantly worse performance than Example 1.

[0063] Comparative Example 6 (Comparative example of the coating corresponding to Example 2) The specific preparation methods are the same as in Example 2, except that in step (4), PDMS solid-phase microextraction is replaced with CAR / PDMS (activated carbon / polydimethylsiloxane) coated SPME fiber head.

[0064] Results: The content of octadecyl vinyl ether in the sample was 0.0186±0.0032 μg / L; the limit of detection was 0.004 μg / L, and the limit of quantitation was 0.013 μg / L; the recovery rate was 82.1±3.5%; the relative standard deviation (RSD) was 6.3% (n=6); the area of ​​matrix interference peaks was 6.8×10⁻⁶. 6The signal-to-noise ratio was 18.9. Compared with Example 1, the detection sensitivity decreased by 8 times, the spiked recovery rate decreased by 10.2 percentage points, and the RSD increased by 3.1 percentage points, showing significantly worse performance than Example 1.

[0065] Comparison and discussion of the results of Example 2 with Comparative Examples 4, 5, and 6 1. Comparison with existing technologies (Comparative Example 4): ① Limit of detection and limit of quantitation: The limit of detection of this invention is 0.0005 μg / L and the limit of quantitation is 0.002 μg / L, while the existing technologies are 0.008 μg / L and 0.025 μg / L, respectively, representing a 16-fold and 12.5-fold increase in sensitivity. This invention can accurately detect 0.0321 μg / L of the target substance in solid ginseng powder, while the existing technology only measures 0.0115 μg / L, which is a serious underestimation; ② Spike recovery rate: This invention has a recovery rate of 92.3%, while the existing technology has a recovery rate of 73.8%, an improvement of 18.5 percentage points. This is because this invention removes polar impurities such as polysaccharides and polyphenols through a toluene:n-hexane mixture extraction at a ratio of 5:1, resulting in higher extraction efficiency. In contrast, the existing technology cannot remove impurities through vacuum concentration, and the target substance is easily lost; ③ Reproducibility: The RSD of this invention is 3.2%, while that of the existing technology is 8.2%. The internal standard method of this invention effectively eliminates pretreatment and instrument errors, and the reproducibility of this invention is far superior to that of the existing technology; ④ Matrix interference: The matrix interference peak area of ​​this invention is 2.9 × 10⁻⁶. 6 Existing technology 8.5×10 6 Interference is reduced by 65.9%, the signal-to-noise ratio is improved by 2.8 times, and the detection accuracy is higher.

[0066] 2. Comparison with the ratio comparison example (Comparative Example 5): Comparative Example 5 uses a toluene:n-hexane ratio of 7:1. Due to the excessively high toluene ratio, the polarity of the mixed solvent increases, which increases the co-extraction of polar impurities, resulting in increased matrix interference and a decrease in signal-to-noise ratio. The spiked recovery rate and reproducibility are inferior to those of Example 1, further proving that the 5:1 ratio can balance the extraction recovery rate and selectivity and is the optimal ratio.

[0067] 3. Comparison with the coating control example (Comparative Example 6): Comparative Example 6 uses a CAR / PDMS coating. Due to its strong adsorption capacity for polar impurities, it leads to increased interference, reduced peak area, and a spiked recovery rate of 82.1%, with the RSD rising to 6.3%, exceeding the acceptable threshold of 5%. This proves that the PDMS coating can effectively avoid interference from polar impurities and achieve high selective enrichment of the target analyte.

[0068] Meanwhile, by comparing Example 2, Comparative Example 5 and Comparative Example 6, it can be seen that the mixture of toluene and n-hexane in a volume ratio of 5:1 in step (3) of the method of the present invention and the PDMS-coated SPME fiber head in step (4) have a synergistic effect, which can synergistically improve the sensitivity, reproducibility, detection limit and qualitative and quantitative accuracy of the detected octadecyl vinyl ether content.

[0069] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for detecting octadecyl vinyl ether, characterized in that: The method uses ginseng prepared by ginseng powder and bacterial enzyme co-fermentation as the test sample. It adopts a three-step pretreatment method of ethanol extraction combined with liquid-liquid extraction of a mixture with a volume ratio of toluene:n-hexane = 5:1 and solid-phase microextraction with polydimethylsiloxane PDMS. The qualitative and quantitative analysis of octadecyl vinyl ether is achieved by gas chromatography-tandem mass spectrometry (GC-MS / MS).

2. The detection method according to claim 1, characterized in that: Includes the following steps: (1) Sample pretreatment: If it is liquid ginseng, measure it directly as the extract; if it is solid ginseng powder, add 45% to 55% ethanol aqueous solution at a weight ratio of 1:15 to 25 to obtain the extract. (2) Ethanol extraction: Place the liquid to be extracted in a shaking device and stir at room temperature. Filter and collect the supernatant to obtain the ethanol extract. (3) Liquid-liquid extraction of toluene: n-hexane = 5:1 mixture: Transfer the ethanol extract to a stoppered separatory funnel, add a liquid-liquid extraction of toluene and n-hexane in a volume ratio of 5:1, collect the upper organic phase after standing and separating the layers, repeat the extraction 1 to 3 times, and combine the organic phases extracted multiple times. (4) PDMS solid phase microextraction: The combined organic phase was concentrated to 5% to 10% of the original volume by purging with nitrogen, and solid phase microextraction was performed using a polydimethylsiloxane coated fiber head. After adsorption of the target analyte, it was directly injected for analysis. (5) GC-MS / MS detection: The fiber head of the adsorbed target analyte was injected into a gas chromatograph-tandem mass spectrometer for GC-MS / MS detection. Qualitative analysis was performed using multiple reaction monitoring (MRM) mode, and quantitative analysis was performed using the internal standard method combined with the standard curve. (6) Result determination: Confirm whether the sample contains octadecyl vinyl ether based on the qualitative ion pair matching results detected by GC-MS / MS, and calculate the content of octadecyl vinyl ether in the sample based on the peak area ratio of the quantitative ion pairs.

3. The detection method according to claim 1, characterized in that: The ginseng prepared by the co-fermentation of bacteria and enzymes in step (1) is the supernatant obtained by extracting and filtering ginseng powder after co-fermentation of ginseng powder with Acetobacter pomorum (CGMCC No.18664), cellulase and amylase, and then filtering it.

4. The detection method according to claim 2, characterized in that: In step (2), the stirring speed for room temperature stirring extraction is 80-120 r / min, and the extraction time is 24 h; filtration is performed using filter paper or a 0.45 μm aqueous filter membrane. Alternatively, in step (3), the volume ratio of the ethanol extract to the toluene and n-hexane mixture in a volume ratio of 5:1 is 1:1 to 3, the shaking time for liquid-liquid extraction is 15 to 30 min, and the settling time is 20 to 40 min.

5. The detection method according to claim 2, characterized in that: The operating conditions for solid-phase microextraction in step (4) are: extraction temperature 30-60℃, extraction time 20-40min, and stirring speed 200-500r / min.

6. The detection method according to claim 2, characterized in that: The chromatographic conditions for GC-MS / MS detection in step (5) are as follows: Column: HP-5 or DB-5 weakly polar capillary column, 30 m × 0.25 mm × 0.25 μm; Temperature program: initial temperature 60℃, hold for 1 min, increase to 280℃ at a rate of 8~12℃ / min, hold for 5~10 min. Carrier gas: high-purity helium, purity ≥99.999%, constant flow mode, flow rate 1.0~1.2 mL / min; injection port temperature: 250~280℃, split ratio 10:1~20:1, SPME injection and resolution time 2~5 min; column outlet directly connected to mass spectrometry ion source; Alternatively, the mass spectrometry conditions for GC-MS / MS detection in step (5) are: ion source: EI source, electron energy 70 eV, ion source temperature 230~250℃; transfer line temperature: 280~300℃; Detection mode: Multiple reaction monitoring (MRM); Qualitative ion pairs for octadecyl vinyl ether are m / z 296→281 and m / z 296→253, quantitative ion pairs are m / z 296→281, collision energy 15–25 eV; Collision gas: High-purity nitrogen, purity ≥99.999%.

7. The detection method according to claim 2, characterized in that: In step (5), the internal standard used in the internal standard method is eicosane. The qualitative ion pairs of the internal standard are m / z 284→269 and m / z 284→241, and the quantitative ion pair is m / z 284→269. The collision energy is 10~20 eV. The standard curve is prepared as follows: prepare octadecyl vinyl ether standard solutions with a concentration gradient of 0.001~0.1 μg / L, add an equal amount of eicosane internal standard to each solution, process according to the method in steps (3)~(4), and then perform GC-MS / MS detection. Plot the standard curve with the peak area ratio of the quantitative ion pairs of octadecyl vinyl ether and the internal standard as the abscissa and the concentration ratio as the ordinate. The correlation coefficient of the standard curve is r>0.

999.

8. The detection method according to any one of claims 1 to 6, characterized in that: The detection method has a limit of detection (LOD) of 0.0005–0.003 μg / L, a limit of quantitation (LOQ) of 0.002–0.01 μg / L, a recovery rate of 85%–110%, and a relative standard deviation (RSD) of <5%.

9. The application of the detection method according to any one of claims 1 to 6 in the detection of octadecyl vinyl ether content in ginseng powder and / or ginseng deep-processed products for cigarette use produced through co-fermentation of ginseng and / or ginseng with bacterial enzymes.

10. The application of the detection method according to any one of claims 1 to 6 in reducing the earthy smell during the fermentation process of ginseng powder.

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