Analysis method for simultaneously detecting capsaicin and sanshoamides in beef tallow hotpot condiment

Abstract

The invention discloses an analysis method for simultaneously detecting capsaicin and sanshoamides in beef tallow hotpot condiment, which belongs to the technical field of analytical chemistry and comprises the following steps: (1) sample preparation, (2) sample extraction, (3) extract purification and (4) chromatographic analysis. The invention establishes a high performance liquid chromatography detection method for simultaneously detecting capsaicin and sanshoamides in the beef tallow hotpot condiment, the total eight components are high in sensitivity and good in accuracy, accurate detection of spicy components can be realized, and a technical means is provided for accurate control of the spicy components in the beef tallow hotpot condiment.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to an analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef oil hot pot base. Background Art

[0002] Chili pepper and Sichuan pepper are important spices in my country, which can add stimulating flavor to food. The numbing taste is composed of a series of prickly ash amide substances in Sichuan pepper, and the spicy taste is composed of a type of capsaicin in chili pepper. These two types of components determine the spiciness of chili pepper and the spiciness of Sichuan pepper. The spicy taste with Chinese characteristics comes from Sichuan pepper and chili pepper respectively. The numbing monomer components of Sichuan pepper mainly include four types (hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool), and the spicy monomer components of chili pepper mainly include three types (capsaicin, dihydrocapsaicin and nordihydrocapsaicin). Synthetic capsaicin is chemically synthesized, and has a very similar structure and spicy feeling to capsaicin. Due to its high cost-effectiveness, unscrupulous vendors use it to replace capsaicin to make huge profits.

[0003] At present, there are many high-performance liquid chromatography methods for the content of numbing or spicy components in pepper or chili pepper and their products at home and abroad, but the sample matrices are mostly pepper, chili pepper or vegetable oil. For beef oil hot pot base, which is widely used in the catering market, there is no method reported for the simultaneous detection of all spicy components in this type of matrix. Patent CN201810233267.3 discloses a method for simultaneously determining zanthoxylum bungeanum and capsaicin in food. The zanthoxylum bungeanum components include zanthoxylum bungeanum 1, zanthoxylum bungeanum 2, and zanthoxylum bungeanum 3, and the capsaicin components include capsaicin and dihydrocapsaicin. The present invention measures more spicy substances and can simultaneously detect 4 zanthoxylum bungeanum-like substances and 4 capsaicin-like substances. Patent CN201810233267.3 uses an ultraviolet detector for high performance liquid chromatography conditions for detecting the contents of zanthoxylum bungeanum and capsaicin. However, the content of spicy ingredients in beef oil hot pot base on the market is usually at the level of 0.01%. Capsaicin-like substances can only be detected by fluorescence detectors to achieve lower instrument sensitivity. The present invention uses ultraviolet and fluorescence detectors in series, which can not only simultaneously detect 8 types of zanthoxylum bungeanum and capsaicin, but also accurately detect low-content samples.

[0004] The matrix composition of beef hot pot base is complex, containing beef tallow, ginger, chili, pepper, garlic, seasonings and other ingredients. It is not easy to prepare the sample evenly. In addition, the complex matrix pretreatment is difficult and must be purified for accurate detection. Compared with patent CN201810233267.3, the present invention adds pretreatment steps such as purification, which reduces the interference of the sample matrix on the target component and improves the detection accuracy.

[0005] The present invention can simultaneously detect 8 kinds of spicy substance components (hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, hydroxy-ε-sanshool, capsaicin, dihydrocapsaicin, nordihydrocapsaicin and synthetic capsaicin) in hot pot soup base, with good liquid phase separation, thereby realizing precise control of product quality. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef tallow hot pot base.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: an analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef oil hot pot base, the analytical method comprising the following steps:

[0008] (1) Sample preparation: Heat the sample at 40-60°C for 20-40 min to fully melt it into a liquid. Use a homogenizer to crush it, then quickly transfer it to a -80°C freezer and freeze it for more than 30 min. Return it to room temperature and weigh the sample before testing.

[0009] (2) Sample extraction: Weigh 1-5 g of sample, add 10-20 mL of n-hexane, ultrasonicate at 40°C for 10 min, then add 10-20 mL of methanol, vortex for 5-15 min, ultrasonicate at 40°C for 30-50 min, then place at 4-6°C for refrigerated centrifugation for 10-15 min, transfer the lower methanol extract to a heart-shaped bottle, repeat the extraction 1-2 times, combine the extracts, and concentrate at 35-40°C in a water bath with rotary evaporation until nearly dry;

[0010] (3) Extraction solution purification: add 10 mL of methanol to the heart-shaped bottle in step (2), re-dissolve by ultrasonication, transfer to a 50 mL centrifuge tube, and then add N-propylethylenediamine (PSA), octadecylsilane bonded silica gel (C 18 ), graphitized carbon black (GCB), vortex mixing and centrifugation, and the liquid after centrifugation is filtered through an organic microporous filter membrane;

[0011] (4) Chromatographic analysis: The filtrate was injected into a high performance liquid chromatograph to detect the capsaicin and zanthoxylum bungeanum content in the beef hot pot base.

[0012] The conditions of the high performance liquid chromatography in step (4) of the present invention are as follows: the chromatographic column is: C 18 , 100×3.0mm, 2.7μm or other equivalent columns.

[0013] In the step (4) of the present invention, the mobile phase of the high performance liquid chromatograph is B (acetonitrile) and A (1% acetic acid water), the flow rate is 1.5 mL / min, and the mobile phase elution program is 0 min to 1 min, 75% A, 1 min to 29 min, from 75% A to 60% A, 29 min to 33 min, from 60% A to 75% A, 33 min to 36 min, keeping 75% A unchanged.

[0014] In the step (4) of the present invention, the column temperature of the high performance liquid chromatograph is 40° C., the injection volume is 10 μL, and the detector is an ultraviolet and fluorescence detector in series, wherein: hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool are quantitatively analyzed by an ultraviolet detector with a detection wavelength of 270 nm; capsaicin, dihydrocapsaicin, nordihydrocapsaicin and synthetic capsaicin are quantitatively analyzed by a fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 325 nm.

[0015] In the step (3) of the present invention, GCB, C 18 , the mass ratio of PSA purification materials is 1:10-15:3-5. Vortex mixing and centrifugation are performed, and the liquid after centrifugation is filtered through a 0.45 μm organic microporous filter membrane;

[0016] The organic microporous filter membrane in step (3) of the present invention is a 0.45 μm filter membrane.

[0017] The beneficial effects of the above technical solution are as follows: 1. The ingredients of beef hot pot base are complex. The special preparation method of fully heating and melting, crushing with a homogenizer, and quickly freezing can make the sample mixed evenly and improve the accuracy of detection. 2. The fat is removed by n-hexane extraction, the oil is precipitated by freezing centrifugation, and the extract is then subjected to GCB, C 18 , PSA composed of purification materials, remove pigments and fats, reduce the interference of sample matrix on target components, in addition, the purification material cost is low, the operation is simple, and the detection accuracy and efficiency are improved. 3. Screening chromatographic columns and optimizing mobile phase conditions make the 8 spicy substances (hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, hydroxy-ε-sanshool, natural capsaicin, dihydrocapsaicin, nordihydrocapsaicin and synthetic capsaicin) have good liquid phase separation, and the peak time is controlled within 40min. Capsaicin is detected by fluorescence detector, and Zanthoxylum numbing factor is detected by ultraviolet detector. The use of ultraviolet and fluorescence detectors in series can not only detect 8 kinds of Zanthoxylum numbing factor and capsaicin at the same time, but also accurately detect low-content samples, which improves the detection sensitivity. The method quantitative limits of 4 kinds of Zanthoxylum numbing factor and 4 kinds of capsaicin are all 0.05mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1: UV-liquid chromatograms of 4 kinds of capsaicinoids and 4 kinds of Zanthoxylum bungeanum components;

[0019] Figure 2 : Fluorescence liquid chromatograms of 4 capsaicin component standard samples. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with specific embodiments.

[0021] Example 1

[0022] A liquid chromatography quantitative analysis method for detecting the contents of capsaicin and Zanthoxylum bungeanum in beef hot pot base.

[0023] The reagents and materials used in this example include chromatographically pure methanol, chromatographically pure acetonitrile, high-grade glacial acetic acid, chromatographically pure n-hexane, capsaicin, dihydrocapsaicin, nordihydrocapsaicin, synthetic capsaicin, hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool standards, N-propylethylenediamine (PSA), octadecylsilane bonded silica gel (C 18 ), graphitized carbon black (GCB).

[0024] The instruments used in this example are a high performance liquid chromatograph equipped with a UV-visible detector and a fluorescence detector, an analytical balance (accurate to 0.0001 g), an ultrasonic cleaner, a refrigerated centrifuge, an ultrapure water machine, and a 50 mL centrifuge tube.

[0025] The sample of this example is a three-fresh beef hot pot base (blank), to which 0.05 mg / kg, 0.1 mg / kg, and 0.5 mg / kg of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, synthetic capsaicin, hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, and hydroxy-ε-sanshool were added respectively to prepare the beef hot pot base sample of this example.

[0026] (1) Sample preparation: Heat the sample at 50°C for 30 min to fully melt it into a liquid. Use a homogenizer to crush it, then quickly transfer it to a -80°C freezer and freeze it for more than 30 min. Return it to room temperature and weigh it before testing.

[0027] (2) Sample extraction: Weigh 5 g of sample, add 20 mL of n-hexane, ultrasonicate at 40°C for 10 min, then add 20 mL of methanol, vortex for 10 min, ultrasonicate at 40°C for 40 min, then centrifuge at 4°C at 10,000 rpm for 10 min, transfer the lower layer of methanol extract to a heart-shaped bottle, repeat the extraction twice, combine the extracts, and concentrate in a 40°C water bath rotary evaporator to near dryness.

[0028] (3) Extraction solution purification: Add 10 mL of methanol to the heart-shaped bottle, re-dissolve by ultrasonication, transfer to a 50 mL centrifuge tube, and then add 10 mg of GCB, 100 mg of C 18 , 50 mg PSA purification material, vortex mix for 5 minutes, centrifuge at 4000 r / min for 5 minutes, and filter the liquid after centrifugation through a 0.45 μm organic microporous filter membrane.

[0029] (4) Chromatographic analysis: The filtrate was tested using a high performance liquid chromatograph.

[0030] The linear range of the calibration curve is a series of working solutions of 0.01 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, and 10 mg / L. The linear equations and correlation coefficients of the four capsaicinoids and four Zanthoxylum bungeanum components are shown in Table 1.

[0031] Table 1 Linear equations and correlation coefficients of 8 components of 4 capsaicinoids and 4 Zanthoxylum bungeanum

[0032]

[0033]

[0034] In Example 1, a liquid chromatography analysis method of the present invention is used to quantitatively detect the capsaicin and zanthoxylum bungeanum content in beef hot pot base. The detected spiked recovery rate data and detection precision data are shown in Table 2.

[0035] Table 2 The recoveries and precision of 4 capsaicinoids and 4 Zanthoxylum bungeanum components

[0036]

[0037] Example 2

[0038] A liquid chromatography quantitative analysis method for detecting the contents of capsaicin and Zanthoxylum bungeanum in beef hot pot base.

[0039] The reagents and materials used in this example include chromatographically pure methanol, chromatographically pure acetonitrile, high-grade glacial acetic acid, chromatographically pure n-hexane, capsaicin, dihydrocapsaicin, nordihydrocapsaicin, synthetic capsaicin, hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool standards, N-propylethylenediamine (PSA), octadecylsilane bonded silica gel (C 18 ), graphitized carbon black (GCB).

[0040] The instruments used in this example are a high performance liquid chromatograph equipped with a UV-visible detector and a fluorescence detector, an analytical balance (accurate to 0.0001 g), an ultrasonic cleaner, a refrigerated centrifuge, an ultrapure water machine, and a 50 mL centrifuge tube.

[0041] The sample in this example is beef hot pot base A

[0042] (1) Sample preparation: Heat the sample at 40°C for 20 min to fully melt it into a liquid. Use a homogenizer to crush it, then quickly transfer it to a -80°C freezer and freeze it for more than 30 min. Return it to room temperature and weigh the sample before testing.

[0043] (2) Sample extraction: Weigh 5 g of sample, add 20 mL of n-hexane, sonicate at 40°C for 10 min, then add 20 mL of methanol, vortex for 10 min, sonicate at 40°C for 30 min, then place at 6°C for refrigerated centrifugation for 10 min, transfer the lower methanol extract to a heart-shaped bottle, repeat the extraction once, combine the extracts, and concentrate by rotary evaporation at 40°C in a water bath until nearly dry;

[0044] (3) Extraction solution purification: Add 10 mL of methanol to the heart-shaped bottle in step (2), re-dissolve by ultrasonication, transfer to a 50 mL centrifuge tube, and then add 10 mg of GCB, 150 mg of C 18 , 50 mg PSA purification material, vortex mix for 5 minutes, centrifuge at 4000 r / min for 5 minutes, and filter the liquid after centrifugation through a 0.45 μm organic microporous filter membrane.

[0045] (4) Chromatographic analysis: The filtrate was injected into a high performance liquid chromatograph to detect the capsaicin and zanthoxylum bungeanum content in the beef hot pot base.

[0046] The test results of the samples in Example 2 are shown in Table 3.

[0047] Table 3 Capsaicin and Zanthoxylum bungeanum test results of beef oil hot pot base A

[0048]

[0049]

[0050] Example 3

[0051] A liquid chromatography quantitative analysis method for detecting the contents of capsaicin and Zanthoxylum bungeanum in beef hot pot base.

[0052] The reagents and materials used in this comparative example include chromatographically pure methanol, chromatographically pure acetonitrile, high-grade glacial acetic acid, chromatographically pure n-hexane, capsaicin, dihydrocapsaicin, nordihydrocapsaicin, synthetic capsaicin, hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool standards, N-propylethylenediamine (PSA), octadecylsilane bonded silica gel (C 18 ), graphitized carbon black (GCB).

[0053] The instruments used in this example are a high performance liquid chromatograph equipped with a UV-visible detector and a fluorescence detector, an analytical balance (accurate to 0.0001 g), an ultrasonic cleaner, a refrigerated centrifuge, an ultrapure water machine, and a 50 mL centrifuge tube.

[0054] The sample in this example is beef hot pot base B

[0055] (1) Sample preparation: Heat the sample at 60°C for 40 min to fully melt it into a liquid. Use a homogenizer to crush it, then quickly transfer it to a -80°C freezer and freeze it for more than 30 min. Return it to room temperature and weigh the sample before testing.

[0056] (2) Sample extraction: Weigh 1 g of sample, add 10 mL of n-hexane, sonicate at 40 °C for 10 min, then add 20 mL of methanol, vortex for 15 min, sonicate at 40 °C for 30 min, then place in a refrigerated centrifuge at 4 °C for 15 min, transfer the lower methanol extract to a heart-shaped bottle, repeat the extraction twice, combine the extracts, and concentrate by rotary evaporation at 35 °C in a water bath until nearly dry;

[0057] (3) Extraction solution purification: Add 10 mL of methanol to the heart-shaped bottle in step (2), re-dissolve by ultrasonication, transfer to a 50 mL centrifuge tube, and then add 10 mg of GCB, 100 mg of C 18 , 30 mg PSA purification material, vortex mix for 5 minutes, centrifuge at 4000 r / min for 5 minutes, and filter the liquid after centrifugation through a 0.45 μm organic microporous filter membrane.

[0058] (4) Chromatographic analysis: The filtrate was injected into a high performance liquid chromatograph to detect the capsaicin and zanthoxylum bungeanum content in the beef hot pot base.

[0059] The test results of the samples in Example 3 are shown in Table 4.

[0060] Table 4 Capsaicin and Zanthoxylum bungeanum test results of beef hot pot base B

[0061]

[0062] The above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention is described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef hot pot soup base, characterized in that: The analytical method comprises the following steps: (1) Sample preparation: Heat the sample at 40-60°C for 20-40 min to fully melt it into a liquid. Use a homogenizer to crush it, then quickly transfer it to a -80°C freezer and freeze it for more than 30 min. Return it to room temperature and weigh the sample before testing. (2) Sample extraction: Weigh 1-5 g of sample, add 10-20 mL of n-hexane, ultrasonicate at 40°C for 10 min, then add 10-20 mL of methanol, vortex for 5-15 min, ultrasonicate at 40°C for 30-50 min, then place in a refrigerated centrifuge at 4-6°C for 10-15 min, transfer the lower methanol extract to a heart-shaped bottle, repeat the extraction 1-2 times, combine the extracts, and concentrate in a water bath rotary evaporator at 35-40°C until nearly dry; (3) Extraction solution purification: Add 10 mL of methanol to the heart-shaped bottle in step (2), re-dissolve by ultrasonication, transfer to a 50 mL centrifuge tube, and then add N-propylethylenediamine (PSA), octadecylsilane bonded silica gel (C 18 ), graphitized carbon black (GCB), vortex mixing and centrifugation, and the liquid after centrifugation is filtered through an organic microporous filter membrane; (4) Chromatographic analysis: The filtrate was injected into a high performance liquid chromatograph to detect the capsaicin and zanthoxylum bungeanum content in the beef hot pot base.

2. The analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef hot pot soup base according to claim 1, characterized in that: The conditions of the high performance liquid chromatography in step (4) are as follows: the chromatographic column is: C 18 , 100×3.0mm, 2.7μm.

3. The analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef hot pot soup base according to claim 1, characterized in that: In the step (4), the mobile phase of the HPLC is B (acetonitrile) and A (1% acetic acid water), the flow rate is 1.5 mL / min, and the mobile phase elution program is 0 min to 1 min, 75% A, 1 min to 29 min, from 75% A to 60% A, 29 min to 33 min, from 60% A to 75% A, 33 min to 36 min, keeping 75% A unchanged.

4. The analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef hot pot soup base according to claim 1, characterized in that: In the step (4), the column temperature of the high performance liquid chromatograph is 40° C., the injection volume is 10 μL, and the detector is a series connection of ultraviolet and fluorescence detectors, wherein: hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool are quantitatively analyzed by ultraviolet detector with a detection wavelength of 270 nm; capsaicin, dihydrocapsaicin, nordihydrocapsaicin and synthetic capsaicin are quantitatively analyzed by fluorescence detector with an excitation wavelength of 280 nm and an emission wavelength of 325 nm.

5. The method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef hot pot soup base according to any one of claims 1 to 4, characterized in that: In step (3), GCB, C 18 The mass ratio of the PSA purification material is 1:10-15:3-5. Vortex mixing and centrifugation are performed, and the liquid after centrifugation is filtered through a 0.45 μm organic microporous filter membrane.

6. The analytical method for simultaneously detecting capsaicin and zanthoxylum bungeanum in beef hot pot base according to any one of claims 1 to 4, characterized in that: The organic microporous filter membrane in step (3) is a 0.45 μm filter membrane.

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