CN105738525A - High performance liquid chromatographic detection method for squalene contained in tobacco leaves - Google Patents

Abstract

The invention discloses a high-performance liquid chromatography detection method for squalene contained in tobacco leaves. For tobacco leaf samples, the invention uses chloroform as an extraction solvent, combined with a Soxhlet extraction method, to ensure a higher extraction efficiency. Direct injection for high performance liquid chromatography detection. The invention further optimizes the detection conditions of high-performance liquid chromatography, and when it is used to detect squalene in tobacco leaves, the chromatographic peaks are well separated, and it has the advantages of accuracy, speed, high sensitivity, good reproducibility, and high recovery rate.

Description

High performance liquid chromatography detection method of squalene in tobacco leaves

technical field

The invention relates to the technical field of detection of components in tobacco leaves, in particular to a high-performance liquid chromatography detection method for squalene contained in tobacco leaves.

Background technique

Squalene was first discovered from the liver oil of sharks and was named Squalene in 1914, and its chemical name is 2,6,10,15,19,23--hexamethyl--2,6,10,14, 18,22--Tetracosahexaene, which belongs to open-chain triterpenoids. Squalene has various physiological functions such as improving the activity of superoxide dismutase (SOD) in the human body, enhancing the body's antioxidant and immune capabilities, improving sexual function, anti-aging, anti-fatigue, and anti-tumor. It is a non-toxic Biologically active substances that have the functions of preventing and curing diseases. Recent studies have found that squalene also has a detoxifying effect on the human body, removing fat-soluble toxins from tissues. In April 2014, the State Tobacco Monopoly Administration issued the “Notice on Accelerating the Promotion and Application of Cigarette Harm Reduction Technology Achievements” (Guoyanbanzong [2014] No. 218), proposing to increase the major special research results of cigarette harm reduction technology To promote the application and promotion of cigarettes, targeted technical measures should be taken to speed up the pace of cigarette tar reduction and harm reduction.

The applicant's research work on the ecological accumulation and harm reduction of squalene, a natural antioxidant in tobacco leaves, found that squalene can selectively reduce harmful components such as benzopyrene, crotonaldehyde, and NH3 in smoke, and reduce the hazard index of cigarettes. At the same time, squalene has the function of removing gas-phase and solid-phase free radicals in smoke. Because squalene has unique physiological functions, the development and utilization of squalene has become a research hotspot in the scientific and technological circles.

However, there are currently no qualitative and quantitative detection methods for squalene in tobacco leaves that can be referenced at home and abroad. Therefore, it is very necessary and prospective to establish a qualitative and quantitative detection method for squalene in flue-cured tobacco in the tobacco industry.

Contents of the invention

The technical problem to be solved by the present invention is to fill in the deficiency of existing detection technology of squalene in tobacco leaves, and provide a high-performance liquid chromatography detection method for squalene contained in tobacco leaves.

Technical scheme of the present invention is realized by following technical scheme:

The present invention uses methanol and acetonitrile as the mobile phase, adopts the column chromatographic separation technology filled with a high-efficiency stationary phase with a particle size of 5 μm, optimizes the detection conditions of high-performance liquid chromatography, and injects the sample pretreated by the method of the present invention into a high-performance liquid chromatograph Carry out chromatographic separation, and detect squalene with an ultraviolet absorption detector.

Specifically, a high-performance liquid chromatography detection method for squalene contained in tobacco leaves is provided, comprising the following steps:

S1. The sample is pre-treated to obtain a sample solution, and the sample solution is purified by a purifier to obtain a sample solution to be tested;

S2. preparing a standard solution and establishing a standard curve;

S3. Detection: draw the sample to be tested and inject it into a high-performance liquid chromatograph for chromatographic separation, and use an ultraviolet absorption detector to detect squalene;

Wherein, the sample pretreatment described in step S1 includes the following steps:

S11. Tobacco leaves are cut into shreds, using chloroform as an extraction solvent, and the extract is obtained through Soxhlet extraction;

S12. Extract the extract with methanol: water with an extractant whose volume ratio is 70:30, collect the extract, perform rotary evaporation to near dryness, then dissolve with acetonitrile, pass through a 0.22 μm organic film, and collect the filtrate;

The detection condition described in step S3 is:

High performance liquid chromatography HPLC (Agilent1100) with UV detector;

Chromatographic column: C18, 5μm, 4.6×250mm;

Column temperature: 30°C;

Mobile phase type: methanol: acetonitrile (volume ratio is 75/25);

Mobile phase flow rate: 1mL/min;

Detection wavelength: 210nm;

Injection volume: 10 μL.

The method for preparing the standard solution described in step S2 is to accurately weigh 0.1g squalene standard substance, accurate to 0.0001g, dissolve the squalene with acetonitrile (chromatographically pure) and settle to 100mL to prepare the squalene standard solution of 1000mg/L. Shake well by shaking. The prepared standard solution was stored at 4°C until use.

The method for establishing the squalene standard curve described in step S2 is: adopt the serial dilution method, prepare the squalene standard solution that concentration is 0.002, 0.01, 0.05, 0.2, 0.5mg/mL respectively and inject in high performance liquid chromatography Detection; take the injection concentration as the abscissa and the peak area as the ordinate to establish a standard curve, and obtain the linear regression equation between the squalene concentration and the corresponding peak area.

Preferably, in step S11, before adding chloroform, the shredded tobacco is vortexed first, and the vortex treatment is vortexed for 1 min and then rested for 10 min.

Further preferably, the condition of the vortex treatment is 2000r/min.

Preferably, the dosage of chloroform in step S11 is determined according to the ratio of 5.0 g shredded tobacco: 200 mL chloroform.

Preferably, the conditions of the Soxhlet extraction described in step S11 are water bath at 40°C and reflux for 9 hours.

Preferably, the amount of the extractant used in step S12 is determined according to the volume ratio of the extraction agent to the extraction solution is 1:1.

Preferably, the number of extractions in step S12 is 3 times.

The present invention has the following beneficial effects:

Existing studies have shown that smoke after burning cigarettes contains nearly 5,000 compounds. According to the chemical analysis of the chloroform extract of tobacco leaves, there are more than 300 compounds in it, and the content of squalene is very low. For rapid detection, it is necessary to establish a scientific and systematic detection method. The present invention uses trichloromethane as the extraction solvent for the tobacco leaf sample, combined with the Soxhlet extraction method, to ensure a higher extraction efficiency, and further optimizes the detection conditions under the technical conditions of the scientific pretreatment of the sample liquid to be tested. Targetedly solve the problem of impurity peak processing in samples, effectively utilize the advantages of high performance liquid chromatography, such as accuracy, speed, and high sensitivity, and provide a chromatographic peak separation, accuracy, speed, high sensitivity, and A detection method with good reproducibility and high recovery rate.

Description of drawings

Fig. 1 squalene standard curve diagram.

Fig. 2 Chromatogram of squalene standard sample.

Figure 3 Chromatogram of unspiked target sample extracted with chloroform.

Fig. 4 Chromatogram of the added target sample extracted with chloroform.

Fig. 5 squalene (50mg/L) standard solution chromatogram.

Fig. 6 Background sample chromatogram.

Figure 7. Background added sample chromatogram.

Fig. 8 squalene standard solution (5mg/L) chromatogram.

Figure 9 Chromatogram of the sample.

detailed description

The method of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The following embodiments and drawings are used for illustrative purposes only, and should not be construed as limiting the present invention. Unless otherwise specified, the reagent raw materials used in the following examples are commercially available or commercially obtained reagent raw materials. Unless otherwise specified, the methods and equipment used in the following examples are methods and equipment routinely used in the art. For the convenience of description, the instruments and reagents used in the examples of the present invention are described as follows, but the present invention is not limited thereby.

instrument:

(1) High-performance liquid chromatography HPLC (Agilent1100), equipped with: degassing pump, quaternary pump, automatic sampler, column thermostat and ultraviolet detector (Agilent Corporation of the United States)

(2) Shaker oscillator: SHZ-82AB type (Jiangsu Jintan City Ronghua Instrument Manufacturing Co., Ltd.)

(3) Platform scale: T-10000 type, d=0.1g (Shuangjie Brothers (Group) Co., Ltd. of the United States)

(4) Electronic balance: BSA224S type, d=0.1mg (Sartorius Scientific Instruments (Beijing) Co., Ltd.)

(5) Rotary vacuum evaporator: RE52-99 type (Shanghai Yarong Biochemical Instrument Factory)

(6) Vacuum filter pump: SHZ-DⅢ type (Gongyi City Yuhua Instrument Co., Ltd.)

(7) Ultrasonic cleaner: KQ-50 type (Kunshan Ultrasonic Instrument Co., Ltd.)

(8) Vortex instrument: MS3BS25 (IKA company)

(9) Constant temperature water bath: 501 digital display type (Honghua Instrument Factory, Jintan City, Jiangsu Province)

(10) All kinds of laboratory glassware

Reagent:

(1) 99.8% squalene reference substance (National Drug Control Institute)

(2) Methanol (chromatographically pure and analytically pure)

(3) Acetonitrile (chromatographically pure)

(4) n-Hexane (analytical pure)

(5) Chloroform (chloroform, analytically pure)

(6) Petroleum ether (analytical pure)

(7) 0.22μm organic microporous membrane (UK original membrane)

(8) Acetone (analytical pure)

(9) Cleaning agent: PSA, C18, silica gel (welchrom company)

(10) Shredded tobacco (Guangxi China Tobacco Industry Co., Ltd., randomly selected)

Example 1

The present embodiment provides a kind of detection method of squalene in tobacco leaf, comprises the following steps:

S1. Sample pretreatment to obtain the sample solution to be tested;

S2. preparing a standard solution and establishing a standard curve;

S3. Detection: draw the sample to be tested and inject it into a high-performance liquid chromatograph for chromatographic separation, and use an ultraviolet absorption detector to detect squalene;

S4. adopt external standard method, calculate its content according to absorption peak area.

Wherein, the sample pretreatment method described in step S1 is:

Sample pretreatment method: take 0.05mL, 10000mg/L squalene standard sample in 10mL n-hexane to obtain external standard base solution, after cutting tobacco leaves, take 5.0g shredded tobacco (take 0.05mL, 10000mg/L squalene standard sample In 10mL of n-hexane, then put 5.0g of shredded tobacco into it, vortex in a 2000r/min vortex for 1min, let it stand still, evaporate naturally to dryness (about 24h), and then wrap it with filter paper with a diameter of 15cm) in Soxhlet In the extractor, put 200mL of n-hexane, chloroform, and acetone into a round-bottomed flask respectively, put the Soxhlet extractor in a 40°C water bath, reflux for 9 hours, collect the extract, and take 30mL from the extract, Put it into a separatory funnel, add 30mL of extract solution methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract solution, carry out rotary evaporation to near dryness, then dissolve it with 5mL chromatographic grade acetonitrile, pass 0.22μm organic filter membrane, to be tested.

The detection condition of described detection is:

High performance liquid chromatography HPLC (Agilent1100) with UV detector;

Chromatographic column: C18, 5μm, 4.6×250mm;

Column temperature: 30°C;

Mobile phase type: methanol: acetonitrile (volume ratio is 75/25);

Mobile phase flow rate: 1mL/min;

Detection wavelength: 210nm;

Injection volume: 10 μL.

The method for establishing the squalene standard curve described in step S2 is: adopt the serial dilution method, prepare the squalene standard solution that concentration is 0.002, 0.01, 0.05, 0.2, 0.5mg/mL respectively and inject in high performance liquid chromatography Detect; adopt external standard method to quantify, take injection concentration (mg/L) as abscissa, and peak area (UV S) is ordinate (shown in Table 1) and establish standard curve, as shown in accompanying drawing 1, find The linear regression equation of the squalene concentration and the corresponding peak area was obtained, and the regression equation was y=40.092x+52.046, R ² =0.9999.

Table 1 Corresponding relationship between squalene standard solution concentration and peak area

The results showed that the correlation coefficient of the standard curve was 0.9999, the linearity of the method was good, and it could be used for the determination of the detection limit and the calculation of the quantitative target.

Chromatogram of squalene standard sample, chromatogram of unadded target sample extracted with chloroform, chromatogram of added target sample extracted with chloroform, chromatogram of squalene (50mg/L) standard solution, background sample chromatogram, The chromatograms of the background added samples are shown in accompanying drawings 2 to 7 respectively.

Embodiment 2 Detection method of the present invention and conditional superiority verification test

For further verifying the superiority of detection method of the present invention and detection condition, carry out following experiment respectively:

2.1 Comparison of extraction efficiency of different extractants (C18 column)

2.1.1 Sample pretreatment method

Control group (CK): Take 5.0 g of shredded tobacco (wrapped with filter paper with a diameter of 15 cm) in a Soxhlet extractor, put 200 mL of each extraction solvent into a round-bottomed flask, and put the Soxhlet extractor into a 40°C water bath Reflux for 9 hours, collect the extract, take 30mL from the extract, put it into a separatory funnel, add 30mL of extract methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract The solution was evaporated to nearly dryness by rotary evaporation, then dissolved in 5 mL of chromatographic grade acetonitrile, passed through a 0.22 μm organic film, and then tested.

Add sample pre-treatment: Take 5.0g of shredded tobacco (take 0.05mL, 10000mg/L squalene standard sample in 10mL of n-hexane, then put 5.0g of shredded tobacco into it, vortex for 1min in a vortex instrument at 2000r/min, stand still, Evaporate naturally to dryness (about 24h), then wrap it with a filter paper with a diameter of 15cm) in a Soxhlet extractor, put 200mL of extraction solvents in a round bottom flask respectively, and put the Soxhlet extractor into a water bath at 40°C , reflux for 9 hours, collect the extract, take 30mL from the extract, put it into a separatory funnel, add 30mL of extract methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract , rotatively evaporated to nearly dryness, and then dissolved in 5 mL of chromatographic grade acetonitrile, passed through a 0.22 μm organic mesembrane, and stored at 4°C until testing.

To avoid repeating them one by one, this embodiment takes the extraction solvent n-hexane, chloroform or acetone as an example for illustration.

2.1.2 Results of comparison of extraction efficiency with different solvents

The results of the comparison of extraction efficiencies of different solvents are shown in Table 2. It can be seen from Table 2 that the extraction efficiency of chloroform is the highest.

The extraction efficiency comparison of different extractants in table 2

"-" in the table means "no addition or no recovery"

2.1.3 Discussion on the comparison results of different solvent extraction efficiencies

Using the Soxhlet extraction method, the efficiency of squalene in different extractants was compared. From the recovery rate of the results, it can be seen that the extraction efficiency of chloroform is the highest, reaching 62.27%, followed by n-hexane, reaching 36.36%. Acetone was the worst at 18.59%. Although the extraction recovery rate of chloroform as an extraction solvent is higher than that of n-hexane and acetone, it has not yet met the extraction requirements, and further exploration and combination of new extraction means and methods are needed.

2.2 Comparison of different mobile phase ratios (C18 column)

In order to improve the analysis efficiency, in the experiment of this example, the influence of different ratios of mobile phase methanol: acetonitrile on the peak time was designed, including but not limited to: methanol: acetonitrile = 50:50, methanol: acetonitrile = 70:30, Methanol: Acetonitrile = 90:10, Methanol: Acetonitrile = 10:90, Methanol: Acetonitrile = 85:15, Methanol: Acetonitrile = 80:20, Methanol: Acetonitrile = 30:70, Methanol: Acetonitrile = 40:60, Methanol: Acetonitrile=75:25 and other different ratios. The peak eluting time of different ratios and the separation of the target peak and the impurity peak are shown in Table 3:

Table 3 Effects of different mobile phase ratios on the separation of target and impurity peaks

The best mobile phase conditions under optimized conditions (C18 column): methanol: acetonitrile = 75: 25 (30min) → methanol 100% (19min). Flow rate = 1 mL/min, detection wavelength = 210 nm, column temperature = 30°C.

Test result sees squalene (50mg/L) standard solution chromatogram, background sample chromatogram, background added sample chromatogram shown in accompanying drawing 5 to accompanying drawing 7.

When the ratio of the mobile phase is different, the separation effect of the target substance and the impurity peak in the sample is ideal when methanol: acetonitrile = 75:25.

2.3 Comparison of extraction efficiency of different extraction methods (C18 column)

Under the above-mentioned different optimized conditions, chloroform was used as the extractant for Soxhlet extraction, and the extraction efficiency of the sample was greatly improved when the extractant methanol-water (V:V=70:30) was used for extraction. The results are shown in Table 4:

Table 4 Comparison of extraction efficiency of target substance by different extraction methods

Background sample pretreatment method (CK—straight): Accurately weigh 3 parts of 1.0g shredded tobacco, vortex in a 2000r/min vortex meter for 1min, rest for 10min, evaporate to dryness naturally (about 24h), and then use a diameter of 15cm filter paper) in a Soxhlet extractor, put 200mL of chloroform into a round-bottomed flask respectively, put the Soxhlet extractor into a 40°C water bath, reflux for 9 hours, collect the extract, and take 30mL, put it into a separatory funnel, add 30mL of extract solution methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract solution, carry out rotary evaporation to near dryness, and then dissolve with 5mL chromatography grade acetonitrile , over 0.22μm organic film to be tested.

Sample pretreatment method (straight): Accurately weigh three portions of 1.0g cut tobacco (add 1mL100ppm squalene standard solution respectively), vortex in a vortex instrument at 2000r/min for 1min, stand still for 10min and evaporate to dryness naturally (about 24h) , then wrap it with filter paper with a diameter of 15cm) in a Soxhlet extractor, put 200mL of chloroform into a round-bottomed flask respectively, put the Soxhlet extractor into a 40°C water bath, reflux for 9 hours, and collect the extract. Take 30mL from the extract, put it into a separatory funnel, add 30mL of extract methanol-water (V:V=70:30) for extraction, repeat 3 times, collect the extract, carry out rotary evaporation to nearly dryness, and then use 5mL Dissolve in chromatographic grade acetonitrile, pass through a 0.22 μm organic membrane, and wait for the test.

2.4 High performance liquid chromatography conditions (C18 column)

Methanol: acetonitrile = 75:25 (30min) → methanol 100% (19min). Flow rate = 1 mL/min, detection wavelength = 210 nm, column temperature = 30°C.

Test result is shown in accompanying drawing 8 and accompanying drawing 9, wherein, accompanying drawing 8 is the chromatogram of squalene standard solution (5mg/L), and accompanying drawing 9 is the chromatogram of sample. It can be seen from accompanying drawing 9 that, adopting chloroform as extraction solvent, through Soxhlet extraction, the high performance liquid chromatography qualitative analysis of squalene in tobacco leaf was successfully realized.

Claims (9)

1. a high performance liquid chromatography detection method of squalene contained in tobacco leaves, is characterized in that, comprises the following steps: S1. The sample is pre-treated to obtain a sample solution, and the sample solution is purified by a purifier to obtain a sample solution to be tested; S2. preparing a standard solution and establishing a standard curve; S3. Detection: draw the sample to be tested and inject it into a high-performance liquid chromatograph for chromatographic separation, and use an ultraviolet absorption detector to detect squalene; Wherein, the sample pretreatment described in step S1 includes the following steps: S11. Tobacco leaves are cut into shreds, using chloroform as an extraction solvent, and the extract is obtained through Soxhlet extraction; S12. Extract the extract with methanol: water with an extractant whose volume ratio is 70:30, collect the extract, perform rotary evaporation to near dryness, then dissolve with acetonitrile, pass through a 0.22 μm organic film, and collect the filtrate; The detection condition described in step S3 is: Agilent1100 high performance liquid chromatography HPLC with UV detector; Chromatographic column: C18, 5μm, 4.6×250mm; Column temperature: 30°C; Mobile phase type: methanol: acetonitrile, the volume ratio is 75/25; Mobile phase flow rate: 1mL/min; Detection wavelength: 210nm; Injection volume: 10 μL. 2. according to the high performance liquid chromatography detection method of squalene contained in the described tobacco leaf of claim 1, it is characterized in that, the method for preparing standard solution described in step S2 is to accurately take by weighing 0.1g squalene standard substance, accurate to 0.0001g, dissolved in acetonitrile and then adjusted to 100mL to prepare a 1000mg/L squalene standard solution, shake well. 3. according to the high performance liquid chromatography detection method of squalene contained in the described tobacco leaf of claim 1, it is characterized in that, the method for establishing of squalene standard curve described in step S2 is: adopt serial dilution method, be mixed with respectively Squalene standard solutions with concentrations of 0.002, 0.01, 0.05, 0.2, and 0.5 mg/mL were injected and detected by high performance liquid chromatography. 4. according to the high performance liquid chromatography detection method of squalene contained in the described tobacco leaf of claim 1, it is characterized in that, step S11 is before adding chloroform, earlier carries out vortex treatment to shredded tobacco, and described vortex treatment is Vortex for 1 min and let stand for 10 min. 5. the high performance liquid chromatography detection method of squalene contained in the tobacco leaf according to claim 4, is characterized in that, the condition of described vortex treatment is 2000r/min. 6. according to the high performance liquid chromatography detection method of squalene contained in the described tobacco leaf of claim 1, it is characterized in that, the consumption of chloroform described in step S11 is 5.0g shredded tobacco according to the ratio with shredded tobacco: 200mL chloroform Sure. 7. The high-performance liquid chromatography detection method for squalene contained in tobacco leaves according to claim 1, characterized in that the Soxhlet extraction condition in step S11 is 40° C. water bath, reflux for 9 hours. 8. The high-performance liquid chromatography detection method of squalene contained in the tobacco leaf according to claim 1, characterized in that, the consumption of the extractant described in step S12 is determined as 1:1 according to its volume ratio with the extract. 9. The high-performance liquid chromatography detection method of squalene contained in the tobacco leaf according to claim 1, characterized in that, the number of extractions described in step S12 is 3 times.