High performance liquid chromatography rapid detection method for low-content nicotine in PBS buffer salt system

Abstract

The invention discloses a high performance liquid chromatography rapid detection method for low-content nicotine in a PBS buffer salt system, and the method comprises the following steps: obtaining a sample to be detected, the sample to be detected being a PBS buffer salt solution containing nicotine; the sample to be detected is subjected to isocratic elution or gradient elution by adopting high performance liquid chromatography so as to detect the nicotine content, acetonitrile is adopted as a mobile phase A in the isocratic elution or gradient elution, and a 10-20 mM acidic aqueous solution is adopted as a mobile phase B in the isocratic elution or gradient elution. The method has the advantages of low cost, no damage to a sample, excellent low-content detection capability, successful overcoming of matrix interference of a salt solution, realization of accurate qualitation and quantification of nicotine as low as 1 [mu] g / mL in the solution, detection sensitivity and repeatability superior to those of a traditional method, and wide application prospect. The method has wide application prospects in the fields of tobacco industry quality control, biological medicine detection, environment monitoring and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmaceutical analysis, and particularly relates to a high-performance liquid chromatography rapid detection method for low-content nicotine in a PBS buffer salt system. BACKGROUND

[0002] Nicotine, commonly known as nicotine, has a chemical name of 3-(N-methylpyrrolidin-2-yl)pyridine, a molecular formula of C 10 H 14 N2, and is a unique alkaloid in tobacco. The pyridine ring and N-methylpyrrolidine group in its molecular structure constitute a key pharmacophore, enabling it to act on N-choline receptors (including neuronal N1 receptors and muscle N2 receptors) with high efficiency, and also having a significant excitatory or paralytic effect on the central nervous system. The content of nicotine in the body determines its effect. Therefore, accurate monitoring of its content is of great significance in the fields of tobacco product quality control, research and development of new tobacco products (such as electronic cigarettes), environmental exposure assessment, and clinical pharmacology research.

[0003] At present, the commonly used methods in the field of trace nicotine analysis are gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, the purchase and maintenance costs of mass spectrometry detectors are high, and professional technical personnel are required for operation, resulting in high overall detection costs. At the same time, for low-content nicotine in salt solutions and other complex matrices, matrix effects can seriously interfere with ionization efficiency, leading to a decrease in signal-to-noise ratio and an increase in background interference, and a significant reduction in detection accuracy. Existing methods are often only effective for nicotine content higher than 0.1 mg / mL, and cannot meet the urgent need for accurate determination of extremely low concentration samples. SUMMARY

[0004] The purpose of the embodiments of the present application is to provide a high-performance liquid chromatography rapid detection method for low-content nicotine in a PBS buffer salt system, to solve the detection limitations in the field of biological application of nicotine, and to provide a stable, rapid, low-cost and efficient detection scheme.

[0005] To achieve the above-mentioned purpose, the embodiments of the present application provide a high-performance liquid chromatography rapid detection method for low-content nicotine in a PBS buffer salt system, comprising: obtaining a sample to be tested, which is a PBS buffer salt solution containing nicotine; using high-performance liquid chromatography to perform isocratic elution or gradient elution on the sample to be tested, so as to detect the content of nicotine, wherein the isocratic elution or gradient elution uses acetonitrile as mobile phase A and 10-20 mM acid aqueous solution as mobile phase B.

[0006] Further, in the high performance liquid chromatography, Chromatographic conditions: Waters E2695 high performance liquid chromatograph, ultraviolet detector was used; Detection wavelength: 260 ± 20 nm; Column temperature: 20-50℃; Injection volume: 5-20μL; Chromatographic column: Agilent Poroshell 120 HILIC column, 2.1x150mm, 2.7μm; Mobile phase flow rate: 0.1-0.4mL / min.

[0007] Further, the column temperature is 30℃, the mobile phase flow rate is 0.3mL / min, and the detection wavelength is 260nm.

[0008] Further, the gradient elution is as follows: 0-6min, linear gradient elution, mobile phase A remains 80-100% unchanged, mobile phase B remains 0-20% unchanged; 6-6.5min, linear gradient elution, mobile phase A changes from 80-100% to 60-70%, mobile phase B changes from 0-20% to 30-40%; 6.5-12min, linear gradient elution, mobile phase A remains 60-70% unchanged, mobile phase B remains 30-40% unchanged; 12-12.1min, linear gradient elution, mobile phase A changes from 60-70% to 80-100%, mobile phase B changes from 30-40% to 0-20%; 12.1-15min, linear gradient elution, mobile phase A remains 95-100% unchanged, mobile phase B remains 0-5% unchanged.

[0009] Further, the isocratic elution is as follows: mobile phase A remains 65-85% unchanged, mobile phase B remains 15-35% unchanged.

[0010] Further, the 10-20mM acidic aqueous solution is prepared by the following process: weigh 31.528-63.056g of ammonium formate, add to 950mL of ultrapure water, adjust the pH to 2.0-3.5 with formic acid, and dilute to 1000mL with ultrapure water.

[0011] Further, the 10-20mM acidic aqueous solution is prepared by the following process: weigh 38.541-77.082g of ammonium acetate, add to 950mL of ultrapure water, adjust the pH to 2.0-3.5 with glacial acetic acid, and dilute to 1000mL with ultrapure water.

[0012] Further, the reagents used in the preparation of acetonitrile and the acidic aqueous solution are HPLC grade.

[0013] The technical scheme provided by the embodiments of the present application can include the following beneficial effects: The detection method of the present application innovatively uses an acidic aqueous solution and an organic solvent complex system, and by adjusting the ratio, pH value and ionic strength of the two, the retention behavior and separation effect of nicotine on the chromatographic column are effectively improved. The acidic environment not only enhances the polar dissociation of nicotine and avoids irreversible adsorption of nicotine and the stationary phase of the chromatographic column, but also inhibits the interference of other impurities in the sample matrix, significantly improving the separation degree and peak shape symmetry of the target peak. Compared with the traditional mobile phase system, the complex mobile phase has the advantages of strong compatibility, high stability and low toxicity, and the reagents are easy to obtain and low in cost, laying a foundation for the large-scale application of the method.

[0014] The detection method is optimized for the specific application scenario of the buffer salt system, and in terms of detection performance, the method exhibits multiple core advantages: first, the detection range is wide, and it can accurately cover the nicotine concentration range of 1-150 μg / mL, which can not only meet the detection needs of low-content samples (such as trace nicotine in biological fluids and environmental water samples), but also adapt to the quantitative analysis of high-content samples (such as tobacco product extracts and nicotine preparations), without the need for complex sample dilution or concentration treatment; second, the analysis efficiency is high, by optimizing the key parameters such as chromatographic column specifications, mobile phase flow rate and detection wavelength, the single analysis period is strictly controlled within 20 minutes, compared with the traditional HPLC method (the analysis period is usually 30-60 minutes), the detection efficiency is improved by more than 50%, which greatly shortens the sample detection turnaround time and is suitable for rapid screening of batch samples; third, the detection accuracy and stability are excellent, through multiple parallel experiments, the relative standard deviation (RSD) of the method is ≤2%, and the standard addition recovery rate is between 95%-105%, which meets the strict requirements of trace analysis field on data reliability, and can effectively avoid the detection result fluctuation caused by instrument error and operation deviation; fourth, the operation is simple, the sample only needs to be filtered and centrifuged for pretreatment before being analyzed, without complex derivatization reaction or pretreatment steps, which reduces the requirement for professional skills of the operator, and ordinary experimenters can master the operation after short-term training, and the human cost and time investment are reduced.

[0015] The detection method of the present application solves the problems of traditional nicotine detection technology in PBS buffer salt system, such as being easily interfered by matrix, long detection period, insufficient low content detection ability, etc. It is simple to operate, controllable in cost, rapid and accurate in detection, and has wide application prospects in tobacco industry quality control, biological medicine detection, environmental monitoring and other fields, such as batch detection of nicotine content in tobacco products, dynamic monitoring of nicotine metabolites in human biological samples (such as urine and saliva) during smoking cessation treatment, trace analysis of nicotine residues in public place environment air, etc., which provides efficient and reliable technical support for related field research and application.

[0016] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are incorporated into and form part of the specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the application.

[0018] Figure 1 A chromatogram obtained by a high performance liquid chromatography rapid detection method for low content nicotine in PBS buffer salt system (PBS buffer salt solution containing nicotine as the detection object) proposed by the present application.

[0019] Figure 2 A chromatogram obtained by a high performance liquid chromatography rapid detection method for low content nicotine in PBS buffer salt system (no detection object) proposed by the present application.

[0020] Figure 3 A chromatogram obtained by a high performance liquid chromatography rapid detection method for low content nicotine in PBS buffer salt system (PBS buffer salt solution as the detection object) proposed by the present application.

[0021] Figure 4 Linear fitting graph in Example 3. DETAILED DESCRIPTION

[0022] The exemplary embodiments will be described in detail herein with reference to the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0023] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this application and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term "and / or," as used herein, refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0024] It should be understood that, although the terms first, second, third, etc. can be used herein to describe various information, these terms are not intended to denote a particular order or hierarchy. These terms are used only to distinguish one type of information from another. For example, a first information can be termed a second information, and similarly, a second information can be termed a first information, without departing from the scope of the present application. The word "if" as used herein means "when" or "upon" or "in response to the determination" depending on the context.

[0025] The present application provides a high-performance liquid chromatography rapid detection method for low content nicotine in PBS buffer salt system, comprising: (1) obtaining a sample to be tested, the sample to be tested is a PBS buffer salt solution containing nicotine; (2) using high-performance liquid chromatography, the sample to be tested is subjected to isocratic elution or gradient elution, so as to detect the content of nicotine, wherein the isocratic elution or gradient elution uses acetonitrile as mobile phase A and 10-20 mM acid aqueous solution as mobile phase B.

[0026] During the detection process, it should be noted that: Specifically, the chromatographic conditions are as follows: Waters E2695 high-performance liquid chromatograph, ultraviolet detector, detection wavelength: 260±20nm; column temperature: 20-50℃; injection volume: 5-20μL; chromatographic column: Agilent Poroshell 120 HILIC column, 2.1x150mm, 2.7μm; mobile phase flow rate: 0.1-0.4mL / min.

[0027] Specifically, the gradient elution is as follows: 0-6min, linear gradient elution, mobile phase A remains at a proportion of 80-100%, and mobile phase B remains at a proportion of 0-20%; 6-6.5min, linear gradient elution, the proportion of mobile phase A changes from 80-100% to 60-70%, and the proportion of mobile phase B changes from 0-20% to 30-40%; 6.5-12min, linear gradient elution, the proportion of mobile phase A remains at 60-70%, and the proportion of mobile phase B remains at 30-40%; 12~12.1 min, linear gradient elution, mobile phase A proportion changes from 60~70% to 80~100%, mobile phase B proportion changes from 30~40% to 0~20%; 12.1~15 min, linear gradient elution, mobile phase A proportion remains 95~100%, mobile phase B proportion remains 0~5%.

[0028] Specifically, the 0~20 mM acidic aqueous solution is prepared by the following process: accurately weighing 31.528~31.528~63.056 g of ammonium formate, adding to 950 mL of ultrapure water, adjusting the pH to 2.0~3.5 with formic acid, and then diluting to 1000 mL with ultrapure water.

[0029] Specifically, the 0~20 mM acidic aqueous solution is prepared by the following process: accurately weighing 31.528~31.528~63.056 g of ammonium formate, adding to 950 mL of ultrapure water, adjusting the pH to 2.0~3.5 with formic acid, and then diluting to 1000 mL with ultrapure water.

[0030] Specifically, the acetonitrile, ammonium formate, formic acid, ammonium acetate, and glacial acetic acid used in the method are HPLC grade.

[0031] The application will be further described below in conjunction with the accompanying drawings and specific examples. It should be noted that the specific examples are an explanation of the application rather than a limitation.

[0032] Example 1 Step 1: Take the nicotine reference substance, add ultrapure water to mix, prepare a 9.80 mg / mL solution, and obtain a concentrated stock solution.

[0033] Step 2: Dilute the concentrated stock solution to a sample solution of 1~150 μg / mL with PBS buffer solution (pH=5.0~7.4) as the diluent; Step 3: Use high performance liquid chromatography to inject the sample solution prepared in Step 1, record the analysis data and spectrum (HPLC-MS / MS) Figure 1Chromatographic conditions: Waters E2695 high performance liquid chromatograph, ultraviolet detector, detection wavelength: 260 nm; column temperature: 30 °C; injection volume: 10 μL; column: Agilent Poroshell 120 HILIC column, 2.1 x 150 mm, 2.7 μm; mobile phase A: acetonitrile; mobile phase B: 20 mM ammonium formate aqueous solution (pH adjusted to 3.0 with formic acid); flow rate: 0.3 mL / min; gradient elution was performed, 0-6 min, linear gradient elution, mobile phase A kept at 85% proportion, mobile phase B kept at 15% proportion; 6-6.5 min, linear gradient elution, mobile phase A proportion changed from 85% to 70%, mobile phase B proportion changed from 15% to 30%; 6.5-12 min, linear gradient elution, mobile phase A kept at 70% proportion, mobile phase B kept at 30% proportion; 12-12.1 min, linear gradient elution, mobile phase A proportion changed from 70% to 85%, mobile phase B proportion changed from 30% to 15%; 12.1-15 min, linear gradient elution, mobile phase A kept at 85% proportion, mobile phase B kept at 15% proportion.

[0034] In step 3 above, 20 mM acid aqueous solution is accurately weighed 63.056 g of ammonium formate, added to 950 mL of ultrapure water, adjusted to pH 2.0-3.5 with formic acid, and then diluted to 1000 mL with ultrapure water.

[0035] In addition, two cases of no detection object and PBS buffer salt solution as detection object were set respectively, and the same method was used for detection, and the corresponding chromatograms were obtained (Figures 2 and 3) Figure 2 、 Figure 3 It can be seen that nicotine has obvious characteristic peaks, and ultrapure water and PBS buffer solution do not interfere with the detection of nicotine.

[0036] Example 2 Precision experiment According to the solution preparation method specified in the analysis method, 6 test sample solutions were prepared in parallel to investigate the precision of the test results. The solution was prepared: 6 test sample solutions were prepared in parallel with PBS as the diluent, and the concentration was 20 μg / mL. The above solutions were injected, and the test results were recorded.

[0037] Table 1 Reproducibility test results As can be seen from Table 2, the relative standard deviation (RSD) of the determination results of the 6 test samples is 0.12%, which meets the determination standard of RSD not greater than 2% for reproducibility results, and the tightness test is qualified.

[0038] Example 3 Linear experiment Eight concentration points were selected within the sample concentration range of 1% to 150%. A curve was plotted with concentration on the x-axis and peak area on the y-axis. The standard curve should be linear, and the linear correlation coefficient R²(R) should be the square of the value of the peak area. 2 ≥0.99.

[0039] Table 2 Results of linearity experiments like Figure 4 As shown, R 2 =0.99992, r=0.99994, greater than 0.99; y-intercept is 0.43% of 100% response value, less than 2.0%. Within the concentration range of 1-150 μg / mL, the nicotine injection amount and peak response value show a significant linear relationship.

[0040] Example 4 Stability test The changes in detection results at five time points after the test solution was left at room temperature for 0, 2, 6, 12, and 24 hours were investigated.

[0041] Table 3 Stability test results The RSD calculated based on the peak area is 0.52%, which is less than 2.0%, and meets the requirements.

[0042] Example 5 Accuracy Prepare test solutions of high, medium and low concentrations, inject them separately, and determine the recovery rate and RSD of each group.

[0043] Table 4 Accuracy Measurement Results The average recovery rate was 99.5%, which is within the limit range of 95.0% to 102.0%, and the RSD was 0.65%, which is less than 2.0%, thus meeting the requirements.

[0044] Example 6 Flow rate durability Make small changes to the flow rate and observe the changes in retention time, peak area and other parameters.

[0045] Table 5 Results of flow rate durability test When the flow rate is changed, the higher the flow rate, the shorter the retention time of the principal component peak and the smaller the peak area of ​​the principal peak. The stability of the method is not affected by small changes in the flow rate.

[0046] Example 7 Column temperature durability The column temperature was slightly changed and the changes in retention time, peak area and other parameters were observed.

[0047] Table 6 Results of column temperature durability determination The column temperature had no significant effect on the peak area when the column temperature was changed, and the slight change in the column temperature did not affect the method stability.

[0048] Example 8 Wavelength durability The wavelength was slightly changed and the changes in retention time, peak area and other parameters were observed.

[0049] Table 7 Results of wavelength durability determination The column temperature had no significant effect on the peak area when the wavelength was changed within a certain range (260±2nm), and the slight change in the column temperature did not affect the method stability.

[0050] In summary, the present method is based on the separation and quantitative advantages of high performance liquid chromatography (HPLC) technology. Through scientific optimization of the mobile phase composition, chromatographic operation parameters and sample pretreatment process, accurate and efficient detection of nicotine in PBS buffer salt system is achieved, providing a reliable solution for the analysis of low content nicotine in complex matrix.

[0051] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the application cover any variations, uses or adaptive changes of the application following the general principles thereof and including such departures from the present disclosure as come within known or customary practice in the art to which the application pertains or can relate.

Claims

1. A rapid high-performance liquid chromatography method for the detection of low levels of nicotine in a PBS buffer system, characterized in that, include: Obtain the sample to be tested, which is a PBS buffer solution containing nicotine; The nicotine content is detected by isocratic elution or gradient elution of the sample under test using high performance liquid chromatography (HPLC). Acetonitrile is used as mobile phase A and 10-20 mM acidic aqueous solution is used as mobile phase B for isocratic elution or gradient elution.

2. The method according to claim 1, characterized in that, In the high performance liquid chromatography method described above Chromatographic conditions: Waters E2695 high performance liquid chromatograph with ultraviolet detector; Detection wavelength: 260±20nm; Column temperature: 20~50℃; Injection volume: 5–20 μL; Chromatographic column: Agilent Poroshell 120 HILIC column, 2.1 x 150 mm, 2.7 μm; Mobile phase flow rate: 0.1-0.4 mL / min.

3. The method according to claim 1, characterized in that, The column temperature was 30℃, the mobile phase flow rate was 0.3 mL / min, and the detection wavelength was 260 nm.

4. The method according to claim 1, characterized in that, The gradient elution is as follows: From 0 to 6 minutes, linear gradient elution was performed, with mobile phase A maintained at 80-100% and mobile phase B maintained at 0-20%. From 6 to 6.5 min, linear gradient elution was performed, with the proportion of mobile phase A changing from 80-100% to 60-70% and the proportion of mobile phase B changing from 0-20% to 30-40%. From 6.5 to 12 minutes, linear gradient elution was performed, with mobile phase A maintained at 60-70% and mobile phase B maintained at 30-40%. From 12 to 12.1 min, linear gradient elution was performed, with the proportion of mobile phase A changing from 60-70% to 80-100% and the proportion of mobile phase B changing from 30-40% to 0-20%. From 12.1 to 15 min, linear gradient elution was performed, with mobile phase A maintained at 95-100% and mobile phase B maintained at 0-5%.

5. The method according to claim 1, characterized in that, The isocratic elution is performed as follows: mobile phase A is kept at a constant proportion of 65-85%, and mobile phase B is kept at a constant proportion of 15-35%.

6. The method according to claim 1, characterized in that, The 10-20 mM acidic aqueous solution is prepared by the following process: Weigh 31.528-63.056 g of ammonium formate, add it to 950 mL of ultrapure water, adjust the pH to 2.0-3.5 with formic acid, and bring the volume to 1000 mL with ultrapure water.

7. The method according to claim 1, characterized in that, The 10-20 mM acidic aqueous solution is prepared by the following process: Weigh 38.541-77.082 g of ammonium acetate, add it to 950 mL of ultrapure water, adjust the pH to 2.0-3.5 with glacial acetic acid, and bring the volume to 1000 mL with ultrapure water.

8. The method according to claim 1, characterized in that, The reagents used in the preparation of the acetonitrile and the acidic aqueous solution are all HPLC grade.

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