A method for detecting multiple components in liqueur

Abstract

This invention discloses a method for detecting multiple components in liqueur. The liqueur sample is extracted four times with water-saturated n-butanol, evaporated to dryness, and reconstituted with a 50% (v / v) methanol aqueous solution. A C18 column (250 mm × 4.6 mm, 5 μm particle size, 12 nm pore size) is used for gradient elution with acetonitrile-water. The detection wavelength is switched according to the retention time intervals of 14 target components, and quantification is performed using an external standard curve. After detection, the mobile phase is restored to 12% acetonitrile and 88% water, and the column is equilibrated for 10 min. This method matches the chromatographic retention time sequence formed by gradient elution with the detection wavelength sequence, enabling simultaneous detection and quantification of 14 target components in the same high-performance liquid chromatography (HPLC) analysis.

Description

Technical Field

[0001] This invention relates to the field of alcoholic beverage analysis and testing technology, specifically to a method for simultaneously determining multiple target components in liqueurs using high performance liquid chromatography in the same analytical process. Background Technology

[0002] Liqueurs are typically made by using distilled spirits, fermented wines, or edible alcohol as a base, adding one or more food ingredients, medicinal and edible ingredients, or their extracts, and then processing them through extraction, blending, and other techniques. Due to the complexity of the raw material sources and processing, liqueurs can contain multiple types of components, including flavonoids, isoflavones, flavonoid glycosides, and saponins. These different components exhibit differences in polarity, chromatographic retention behavior, and ultraviolet absorption response.

[0003] For liqueurs containing multiple target components, establishing separate detection methods for multiple single components or a few components typically requires multiple sample preparations and injections. Conversely, using a single fixed detection wavelength may not simultaneously address the response intensity of different target components and interference from complex matrices. Therefore, achieving suitable quantitative separation and detection responses for multiple target components within the same sample treatment and chromatographic analysis process is a key technical challenge for the simultaneous detection of multiple components in liqueurs.

[0004] Sample pretreatment, gradient elution procedures, and detection wavelengths in high-performance liquid chromatography (HPLC) all affect the final analytical results. For specific multi-component systems, simply setting up conventional extraction, gradient elution, and multi-wavelength detection separately may not create analytical conditions suitable for the simultaneous quantification of all target components. Therefore, it is necessary to establish a detection method that coordinates pretreatment, chromatographic retention time, and detection wavelength timing to meet the common detection needs of multiple target components in liqueurs. Summary of the Invention

[0005] In view of this, the present invention aims to provide a method for detecting multiple components in liqueurs, so as to solve the problem that it is difficult to achieve separation effect, detection response and matrix interference for multiple target components in complex liqueur matrices under the same analysis process.

[0006] The detection method provided by this invention is used to simultaneously determine the following in liqueurs: isoflavone glucoside, hyperoside, astragaloside, gentianoside, ginsenoside Rg1, ginsenoside Re, isoflavone, astragalin B, astragalin C, icariin, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2 and ginsenoside Rd.

[0007] The technical solution of the present invention includes: evaporating the liquor sample to dryness and then re-dissolving it in water, extracting it four times with water-saturated n-butanol, combining the n-butanol extracts and evaporating them to dryness, re-dissolving them with a 50% (v / v) methanol aqueous solution and filtering to obtain the sample test solution; preparing single-component standard solutions of the above 14 components respectively, and preparing a series of mixed standard solutions of different concentrations.

[0008] High-performance liquid chromatography (HPLC) was used for separation. A C18 column with specifications of 250 mm × 4.6 mm, a packing particle size of 5 μm, and a packing pore size of 12 nm was used. The column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Water was used as mobile phase A and acetonitrile was used as mobile phase B. The acetonitrile volume fraction increased linearly from 12% to 27% within 0–60 min, and from 27% to 41% within 60–110 min.

[0009] During the gradient elution process, an ultraviolet detector with programmed wavelength switching function is used, and the detection wavelength is switched according to the chromatographic running time: 360nm for 0-20min, 260nm for 20-29min, 380nm for 29-35min, 350nm for 35-45min, 251nm for 45-50min, 203nm for 50-58min, 240nm for 58-70min, and 203nm for 70-110min.

[0010] In some embodiments, 10 mL of the liqueur sample is evaporated to dryness, the residue is dissolved in 25 mL of water, and then extracted with 20 mL of water-saturated n-butanol each time, for a total of 4 extractions; the n-butanol extracts are combined and evaporated to dryness in a water bath at 85–92 °C, and the residue is transferred to a 5 mL volumetric flask with a 50% (v / v) methanol aqueous solution and diluted to volume, and then filtered through a 0.22 μm PVDF syringe filter.

[0011] In some embodiments, a YMC Triart C18 column with an organic / inorganic hybrid silica matrix, measuring 250 mm × 4.6 mm, a packing particle size of 5 μm, and a pore size of 12 nm, is used; the column temperature is 30 °C, the flow rate is 1.0 mL / min, and the injection volume is 10 μL. After each gradient elution, the mobile phase is restored to the initial mobile phase of 12% acetonitrile and 88% water, and the column is equilibrated for 10 min.

[0012] In some implementations, a standard curve is established with the concentration of each target component as the abscissa and the corresponding chromatographic peak area as the ordinate, and the content of each target component in the sample test solution and the original liqueur sample is calculated using the external standard method.

[0013] In this invention, sample pretreatment is used to reduce the influence of the liqueur matrix and allow different types of target components to enter a unified test sample system. The gradient elution program utilizes the differences in the distribution behavior of each target component between the mobile phase and the stationary phase to form a relatively defined peak order and retention time range. The detection wavelength program is then matched with the retention time range, so that the target components in different time ranges are acquired at the corresponding wavelengths. Column specifications, column temperature, and flow rate all affect the retention time of the target components. Therefore, this invention sets wavelength switching nodes under corresponding chromatographic conditions to ensure that the chromatographic retention time sequence formed by gradient elution corresponds to the detection wavelength sequence. After each gradient elution, the initial mobile phase is restored and the column is equilibrated, which helps to ensure that subsequent injections are performed under the same initial column conditions. Thus, sample pretreatment, chromatographic separation conditions, gradient program, and programmed detection wavelength together constitute a continuous analysis program.

[0014] The present invention has the following advantages over the prior art: First, the simultaneous detection of 14 target components can be completed in the same sample processing and the same high-performance liquid chromatography analysis process, reducing the need for repeated sample duplication and injection required to establish multiple detection methods separately; Second, four water-saturated n-butanol extractions were used to ensure sufficient extraction of different target components. In Example 2, the average spiked recoveries of the 14 components ranged from 96.2% to 101.9%. Third, by generating retention time series of target components through specific chromatographic conditions and gradient programs, and switching detection wavelengths within corresponding time intervals, the problems of insufficient response of some target components or matrix interference that occur when using a single fixed wavelength can be reduced; Fourth, the correlation coefficient R of the standard curves of 14 target components within their respective linear ranges is [missing information]. 2 All values ​​are greater than 0.999, and can be used for quantitative analysis of the liqueur samples. Attached Figure Description

[0015] Figure 1 The chromatogram of the mixed standard detection in Example 1 is shown below. Figure 2 The chromatogram of sample S1 of the ginseng and deer antler liquor from Example 1 is shown below. Figure 3 The chromatogram of sample S1 of the ginseng and deer antler liquor from Comparative Example 2 is shown below. Figure 4 The chromatogram of sample S1 of Ginseng and Deer Antler Wine in Comparative Example 3 at a fixed wavelength of 203 nm is shown. Figure 5 The chromatogram of sample S1 of Ginseng and Deer Antler Liquor in Comparative Example 3 at a fixed wavelength of 240 nm is shown. Figure 6 The chromatogram of sample S1 of ginseng and antler velvet liquor in Comparative Example 3 at a fixed wavelength of 251 nm is shown. Figure 7 The chromatogram of sample S1 of ginseng and antler velvet liquor in Comparative Example 3 at a fixed wavelength of 260 nm is shown. Figure 8 The chromatogram of sample S1 of ginseng and antler velvet liquor in Comparative Example 3 at a fixed wavelength of 350 nm is shown. Figure 9 The chromatogram of sample S1 of ginseng and antler velvet liquor in Comparative Example 3 at a fixed wavelength of 360 nm is shown. Figure 10 The chromatogram of sample S1 of Ginseng and Deer Antler Wine at a fixed wavelength of 380 nm is shown in Comparative Example 3. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to embodiments and comparative examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise stated, the reagents, standards, and chromatographic consumables used can all be obtained through conventional commercial channels; the concentration of each standard is calculated according to the content indicated on its reference standard certificate.

[0017] Example 1: Simultaneous Detection of 14 Target Components in Ginseng and Deer Antler Wine In this embodiment, commercially available ginseng and deer antler liquor was used as the sample to be tested, and it was designated as sample S1. The 14 target components in sample S1 were simultaneously detected according to the following method. For ease of description, the following components are sequentially designated as target components 1 to 14: verbascoside, hyperoside, astragaloside, gentianoside, ginsenoside Rg1, ginsenoside Re, verbascoside, astragalin B, astragalin C, icariin, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd.

[0018] (1) Preparation of the sample test solution Accurately measure 10 mL of sample S1 and place it in an evaporating dish. Evaporate to dryness in a water bath. Dissolve the residue in 25 mL of water and transfer it to a separatory funnel. Extract with 20 mL of water-saturated n-butanol each time, for a total of 4 extractions. Combine the n-butanol extracts and evaporate to dryness in a water bath at 85–92 °C. Transfer the residue to a 5 mL volumetric flask with a 50% (v / v) methanol aqueous solution and dilute to volume. Mix well and filter through a 0.22 μm PVDF syringe filter to obtain the sample test solution.

[0019] (2) Preparation of standard solutions Fourteen standards were weighed and placed in 10 mL volumetric flasks, dissolved in methanol and diluted to volume to obtain 14 single-component standard solutions. The weighed amounts of each standard, the content indicated on the reference standard certificate, and the converted concentrations of the single-component standard solutions are shown in Table 1.

[0020] Table 1. Preparation of single-component standard solutions

[0021] Sequentially measure 0.6 mL, 0.2 mL, 0.2 mL, 0.3 mL, 2.0 mL, 3.0 mL, 0.2 mL, 0.2 mL, 0.4 mL, 4.0 mL, 5.0 mL, 2.0 mL, and 0.6 mL of each of the single-component standard solutions 1-14, and place them in 20 mL volumetric flasks. Dilute to volume with methanol to obtain mixed standard solution 1#. Then measure 1.0 mL, 2.0 mL, 4.0 mL, and 6.0 mL of mixed standard solution 1#, and place them in four 10 mL volumetric flasks respectively. Dilute to volume with methanol to obtain mixed standard solutions 2#-5#. Determine the concentration of each target component in the series of mixed standard solutions 1#-5# based on the concentration of each single-component standard solution and the corresponding dilution factor.

[0022] (3) High performance liquid chromatography conditions A YMC Triart C18 column with an organic / inorganic hybrid silica matrix (250 mm × 4.6 mm, 5 μm particle size, 12 nm pore size) was used. The column temperature was 30 °C. Water was used as mobile phase A and acetonitrile as mobile phase B. The flow rate was 1.0 mL / min and the injection volume was 10 μL. A UV detector with programmed wavelength switching was used for signal acquisition. Gradient elution and equilibration conditions are shown in Table 2.

[0023] Table 2 Gradient elution program

[0024] During the 0–60 min period, the acetonitrile volume fraction increased linearly from 12% to 27%, and during the 60–110 min period, it increased linearly from 27% to 41%. After the detection, the mobile phase was restored to the initial mobile phase with an acetonitrile volume fraction of 12% and a water volume fraction of 88%, and the column was equilibrated for 10 min under this initial mobile phase condition. The detection wavelength was switched according to the procedure shown in Table 3.

[0025] Table 3. Programmed Detection Wavelengths

[0026] (4) Establishment of the standard curve Mixed standard solutions of different concentrations (1# to 5#) were taken and injected for detection under the chromatographic conditions described above. Linear regression was performed with the concentration of each target component as the x-axis and the peak area as the y-axis to obtain the linear range, retention time, standard curve, and correlation coefficient for each target component. The results are shown in Table 4. The chromatogram for the mixed standard solution detection is shown below. Figure 1 .

[0027] Table 4. Linear range and standard curves of each target component

[0028] Table 4 shows that the correlation coefficient R of the standard curves for the 14 target components within the listed linear ranges is... 2 All values ​​are greater than 0.999, indicating a good linear relationship.

[0029] (5) Detection of sample S1 of ginseng and deer antler liquor Take sample S1, prepare the sample test solution according to step (1), and inject it for detection according to the chromatographic conditions in step (3). Calculate the concentration of each target component in the sample test solution based on the standard curve established in step (4), and convert the content of each target component in sample S1 according to the volume relationship of 10 mL sample diluted to 5 mL after processing. The chromatogram of sample S1 is shown below. Figure 2 The test results are shown in Table 5.

[0030] Table 5. Detection results of 14 target components in Ginseng and Deer Antler Tonic Wine Sample S1

[0031] Example 2: Method Accuracy Verification This embodiment is used to verify the quantitative accuracy of the detection method established in Example 1. Using mixed standard solution 1# as the spiking standard, 5 mL of sample S1 was accurately measured to prepare 9 aliquots, numbered 7# to 15#. 2.0 mL of mixed standard solution 1# was added to each of aliquots 7# to 9#, 3.0 mL to each of aliquots 10# to 12#, and 4.0 mL to each of aliquots 13# to 15#. After evaporation to dryness, each sample was reconstituted with 25 mL of water and extracted with 20 mL of water-saturated n-butanol for a total of 4 extractions. The n-butanol extracts were combined, evaporated to dryness in a water bath at 85–92°C, and diluted to 5 mL with 50% methanol aqueous solution. The solution was then filtered through a 0.22 μm PVDF syringe filter. Detection was performed according to the chromatographic conditions of Example 1, and the recovery rate was calculated based on the background content of the samples, the spiking amount, and the detection results. The average recovery rates of the 14 target components are shown in Table 6.

[0032] Table 6. Average spiked recoveries of 14 target components

[0033] As shown in Table 6, the average spiked recoveries of the 14 target components ranged from 96.2% to 101.9%, indicating that the sample pretreatment and chromatographic detection method established in Example 1 can be used for the quantitative detection of the above-mentioned target components.

[0034] Comparative Example 1 This comparative example examines the number of extractions in sample pretreatment. Using the same batch of sample S1 as the detection object, except that the number of extractions of water-saturated n-butanol was changed to 2 and 3 times respectively, the other sample preparation, standard preparation, and chromatographic detection conditions were the same as in Example 1. The detection results and their deviations from Example 1 are shown in Table 7.

[0035] Table 7 Comparison of detection results for different extraction times

[0036] As shown in Table 7, reducing the number of extractions of water-saturated n-butanol to 2 or 3 times resulted in varying degrees of decrease in the detection results of multiple target components. Specifically, after 2 extractions, target components 1-5 decreased by more than 10% compared to Example 1, while after 3 extractions, target components 1, 2, 4, and 5 decreased by approximately 5% or more. Combined with the spiked recovery results of Example 1, it can be concluded that in the common detection system of the 14 target components described in this application, using 4 extractions is beneficial in reducing the lower detection results caused by insufficient extraction.

[0037] Comparative Example 2 This comparative example uses a different set of chromatographic conditions to detect the same batch of sample S1. Sample preparation and standard solution preparation are the same as in Example 1; the chromatographic column used is an Agilent SB-Aq C18 with dimensions of 250 mm × 4.6 mm and a packing particle size of 5 μm; the column temperature is 30 °C; the mobile phase is acetonitrile-water, the flow rate is 1.0 mL / min, the injection volume is 10 μL, the detection wavelength is 203 nm, and the gradient elution conditions are shown in Table 8.

[0038] Table 8 Gradient elution procedure for Comparative Example 2

[0039] Sample S1 was tested under the conditions described above, and the chromatogram is shown below. Figure 3 Compared with Example 1 Figure 2 In contrast, under the conditions of column, gradient program, and fixed detection wavelength of 203 nm used in Comparative Example 2, the impurity peaks in the sample caused significant interference, the separation of some target components was poor, and several target components failed to produce effective detection results suitable for quantification. These results indicate that for the simultaneous detection of the 14 target components described in this application, the chromatographic separation conditions and detection wavelength program need to be compatible.

[0040] Comparative Example 3 This comparative study investigated the effect of a fixed single detection wavelength on the simultaneous detection of 14 target components. Using the same batch of sample S1 as the detection target, the sample preparation, standard solution preparation, chromatographic column, gradient elution, and column equilibration conditions were identical to those in Example 1. The only difference was that the programmed detection wavelength in Example 1 was replaced with fixed single wavelengths of 203 nm, 240 nm, 251 nm, 260 nm, 350 nm, 360 nm, and 380 nm, respectively. The resulting chromatograms are shown below. Figures 4 to 10 .

[0041] At a fixed wavelength of 203 nm, target components 1, 2, 3, and 7 were significantly interfered with by impurity peaks, resulting in poor separation. At fixed wavelengths of 240 nm, 251 nm, and 260 nm, the target components that could be effectively observed and separated were mainly 1, 2, 4, 7, 8, 9, and 10, while the remaining target components were interfered with or did not receive an effective response. At fixed wavelengths of 350 nm, 360 nm, and 380 nm, the target components that could be effectively observed and separated were mainly 2, 3, 8, 9, and 10, with some target components showing a small response, and many other target components not being effectively detected. The results of different detection methods are summarized in Table 9.

[0042] Table 9 Comparison of detection results between fixed single wavelength and programmed wavelength

[0043] From Table 9 and Figures 4 to 10 It is known that in the ginseng and antler liquor sample S1 and the 14 target component system described in this application, it is difficult to simultaneously quantify all 14 target components using any of the investigated fixed single wavelengths. Example 1, based on specific chromatographic conditions and retention time intervals formed by gradient elution, programmatically switches the detection wavelengths to enable each target component to obtain a detection signal for quantification within its corresponding time interval, while reducing matrix interference in some time intervals.

[0044] In summary, this invention uses water-saturated n-butanol for multiple extractions to form a sample solution suitable for uniform chromatographic analysis. Retention time sequences of 14 target components are established using specific column specifications, column temperature, flow rate, and acetonitrile-water gradient. Programmed wavelength switching is then performed based on these retention time sequences. After detection, the initial mobile phase is restored and the column is equilibrated to ensure that subsequent injections maintain the same initial column state, thereby achieving simultaneous separation and quantification of the aforementioned 14 target components in the ginseng and antler liquor sample S1.

Claims

1. A method for detecting multiple components in liqueur, used to simultaneously determine the following in liqueur: guanylic acid glucoside, hyperoside, astragaloside, gentianoside, ginsenoside Rg1, ginsenoside Re, guanylic acid, astragalin B, astragalin C, icariin, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd, characterized in that... Includes the following steps: (1) Evaporate the liquor sample to dryness, redissolve it with water, extract it 4 times with water-saturated n-butanol, combine the n-butanol extracts and evaporate them to dryness, redissolve them with 50% methanol aqueous solution and filter them to obtain the sample test solution. (2) Prepare single-component standard solutions of the 14 components respectively, and prepare a series of mixed standard solutions of different concentrations from the single-component standard solutions; (3) The sample test solution and mixed standard solution were detected by high performance liquid chromatography (HPLC). A C18 column with a specification of 250 mm × 4.6 mm, a packing particle size of 5 μm, and a packing pore size of 12 nm was used. The column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Water was used as mobile phase A and acetonitrile was used as mobile phase B. The acetonitrile volume fraction was linearly increased from 12% to 27% within 0–60 min, and linearly increased from 27% to 41% within 60–110 min. Ultraviolet detection with programmed wavelength switching function was used. The detector was used to acquire signals at 360 nm for 0–20 min, 260 nm for 20–29 min, 380 nm for 29–35 min, 350 nm for 35–45 min, 251 nm for 45–50 min, 203 nm for 50–58 min, 240 nm for 58–70 min, and 203 nm for 70–110 min. After 110 min of signal acquisition, the mobile phase was restored to the initial mobile phase of 12% acetonitrile and 88% water, and the column was equilibrated for 10 min. (4) Establish standard curves for each target component based on the detection data of the mixed standard solution, and calculate the content of each target component in the liqueur sample based on the standard curves.

2. The method for detecting multiple components of liqueur according to claim 1, characterized in that, In step (1), 10 mL of the liqueur sample is precisely measured and evaporated to dryness. The resulting residue is dissolved in 25 mL of water and then extracted with water-saturated n-butanol.

3. The method for detecting multiple components of liqueur according to claim 2, characterized in that, Extraction was performed using 20 mL of water-saturated n-butanol each time, for a total of 4 extractions. The n-butanol extracts obtained from the 4 extractions were combined and evaporated to dryness in a water bath at 85–92 °C. The residue was transferred to a 5 mL volumetric flask and diluted to volume with a 50% (v / v) methanol aqueous solution. The solution was then filtered through a 0.22 μm PVDF syringe filter to obtain the sample test solution.

4. The method for detecting multiple components of liqueur according to claim 1, characterized in that, The C18 column uses organic / inorganic hybrid silica gel as its matrix.

5. The method for detecting multiple components of liqueur according to claim 1, characterized in that, In step (2), the amount of each standard is calculated according to the content indicated on the corresponding reference standard certificate, and then prepared into single-component standard solutions with methanol.

6. The method for detecting multiple components of liqueur according to claim 5, characterized in that, Sequentially measure 0.6 mL, 0.2 mL, 0.2 mL, 0.3 mL, 2.0 mL, 3.0 mL, 0.2 mL, 0.2 mL, 0.4 mL, 4.0 mL, 5.0 mL, 2.0 mL, and 0.6 mL of single-component standard solutions of verbascoside, hyperoside, astragaloside, gentianoside, ginsenoside Rg1, ginsenoside Re, verbascoside, astragaloside B, astragaloside C, icariin, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd into 20 mL volumetric flasks and dilute to volume with methanol to obtain mixed standard solution 1#.

7. The method for detecting multiple components of liqueur according to claim 6, characterized in that, Measure 1.0 mL, 2.0 mL, 4.0 mL and 6.0 mL of the mixed standard solution 1# respectively, place them in 10 mL volumetric flasks and dilute to volume with methanol to obtain mixed standard solutions 2# to 5#. The mixed standard solutions 1# to 5# constitute a series of mixed standard solutions with 5 concentration levels.

8. The method for detecting multiple components of liqueur according to claim 7, characterized in that, The linear ranges of verbascoside, hyperoside, astragaloside, gentianoside, ginsenoside Rg1, ginsenoside Re, verbascoside, astragaloside B, astragaloside C, icariin, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd were 3.090–30.899 mg / L, 0.790–7.898 mg / L, 0.459–4.586 mg / L, 1.476–14.756 mg / L, and 9.6 mg / L, respectively. 43–96.432 mg / L, 8.983–89.826 mg / L, 0.587–5.870 mg / L, 0.517–5.174 mg / L, 0.649–6.494 mg / L, 1.057–10.568 mg / L, 24.951–249.508 mg / L, 13.769–137.690 mg / L, 9.358–93.575 mg / L, and 5.301–53.009 mg / L.

9. The method for detecting multiple components of liqueur according to claim 1, characterized in that, In step (4), a linear standard curve is established with the concentration of each target component as the abscissa and the corresponding chromatographic peak area as the ordinate, and the concentration of each target component in the sample test solution is calculated using the external standard method.

10. The method for detecting multiple components of liqueur according to claim 2, characterized in that, Based on the volume relationship of a 10mL liqueur sample being processed and then diluted to 5mL, the concentration of each target component in the sample test solution is multiplied by 0.5 to calculate the content of each target component in the original liqueur sample.

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