A liquid chromatography analysis method for detecting N-nitrososarcosine content

Abstract

The present disclosure relates to a liquid chromatography analysis method for detecting the content of N-nitrososarcosine, and the liquid chromatography analysis method comprises the following steps: (1) preparing a standard solution, and determining a standard curve of N-nitrososarcosine, in which Y is the sum of the peak areas corresponding to 3.00-3.50min and 3.50-4.00min; (2) using a liquid chromatography analysis method to detect the sample to be tested, in the obtained chromatogram, the peak area corresponding to the target is the sum of the peak areas corresponding to 3.00-3.50min and 3.50-4.00min. The analysis method provided by the present disclosure creatively incorporates the detection of isomer content into the detection of N-nitrososarcosine content, which can greatly avoid the phenomenon of "false qualification".

Description

Technical Field

[0001] The present invention relates to the technical field of compound content detection, and in particular to a method for detecting N -Liquid chromatography analysis method for the content of nitroso-sarcosine, particularly a method for detecting nitroso-sarcosine in oral smoke N -Liquid chromatography analysis method for nitrososarcosine content. Background Art

[0002] Tobacco has high economic value and a long history of application. In addition to being made into cigarettes and dry tobacco for people to smoke, it also has a variety of medical uses. The "National Compilation of Chinese Herbal Medicines" records that tobacco is warm in nature, sweet in taste, and poisonous. It has the effects of reducing swelling, detoxifying, and killing insects. It is mainly used for carbuncle, tinea capitis, white ringworm, baldness, snake bites, etc. Buccal tobacco is an important new tobacco product that does not produce smoke and can meet the demand for nicotine intake. Due to its unique way of use, buccal tobacco has gradually become an important supplementary form of tobacco consumption and an important development trend in the international tobacco field. The economic value of buccal tobacco is relatively high, but its ingredients contain a certain amount of N -Nitrososarcosine, its structural formula is as follows:

[0003]

[0004] According to the list of carcinogens published by the World Health Organization, N-nitrososarcosine is a Class 2B carcinogen. Therefore, the content of N-nitrososarcosine in products needs to be limited. At present, there are few tests on the content of N-nitrososarcosine, and the phenomenon of "false qualification" is prone to occur.

[0005] Therefore, it is particularly important to provide a detection method that can avoid the false acceptance of N-nitrososarcosine content. Summary of the invention

[0006] In order to solve the above technical problems, the present disclosure provides a liquid chromatography analysis method for detecting the content of N-nitrososarcosine.

[0007] In a first aspect, the present disclosure provides a liquid chromatography analysis method for detecting the content of N-nitrososarcosine, the liquid chromatography analysis method comprising the following steps:

[0008] (1) preparing a standard solution and determining a standard curve of N-nitrososarcosine, wherein Y is the sum of the peak areas corresponding to 3.00-3.50 min and 3.50-4.00 min;

[0009] (2) The sample to be tested is tested using a liquid chromatography analysis method. In the obtained chromatogram, the peak area corresponding to the target substance is the sum of the peak areas corresponding to 3.00-3.50 min and 3.50-4.00 min.

[0010] In the chromatogram obtained by liquid chromatography detection of the standard solution, in view of the normal fluctuations within the allowable range caused by different liquid chromatographs, different detection times and different detection personnel, the present disclosure limits the elution time of the chromatographic peak of N-nitrososarcosine to 3.00-3.50 min, and the elution time of its isomers to 3.50-4.00 min.

[0011] In the current detection of N-nitrososarcosine, usually only the content of the target substance itself is detected. However, the present disclosure finds that N-nitrososarcosine and its isomers can be converted into each other under certain conditions, and the conversion conditions are extremely simple. Therefore, in the detection of the content of N-nitrososarcosine, the present disclosure creatively incorporates the content of N-nitrososarcosine isomers into the detection method provided by the present disclosure, thereby avoiding the situation where the detection result is inaccurate if the isomers are converted into N-nitrososarcosine after the detection is completed.

[0012] In the present disclosure, the liquid chromatography detection parameters include: the chromatographic column is InfinityLab Poroshell120PFP, the mobile phase is an aqueous solution containing acetonitrile and formic acid, the flow rate is 0.1-0.5mL / min, for example, 0.2mL / min, 0.3mL / min, 0.4mL / min, the detection wavelength is 233nm, and the column temperature is 35-45°C, for example, 38°C, 40°C, 42°C, 44°C.

[0013] The present disclosure found that the maximum absorption wavelength of N-nitrososarcosine is 234.2 nm, and the maximum absorption wavelength of its isomer is 232.5 nm. Therefore, the present disclosure prefers a detection wavelength of 233 nm. When the detection wavelength is 233 nm, simultaneous detection of N-nitrososarcosine and its isomers can be achieved.

[0014] As a preferred technical solution of the present disclosure, the volume ratio of acetonitrile, formic acid and water in the mobile phase is 500:1:9500.

[0015] As a preferred technical solution of the present disclosure, the standard solution contains N-nitrososarcosine standard products at 5 levels of concentration.

[0016] The five levels of concentration described in the present disclosure are 1.18 μg / mL, 2.35 μg / mL, 5.88 μg / mL, 23.54 μg / mL, and 58.85 μg / mL, respectively.

[0017] As a preferred technical solution of the present disclosure, the pre-treatment method of the sample to be tested includes: extracting the sample to be tested, eluting with an extraction column, concentrating, and drying to obtain a sample that can be used for detection and analysis.

[0018] As a preferred technical solution of the present disclosure, the present disclosure now provides the following pre-treatment method:

[0019] For gum-based oral cigarettes, dissolve and disperse the sample in n-hexane and transfer it to a 50mL extraction centrifuge tube; for American moist oral cigarettes and Swedish oral cigarettes, take 1g or 1 bag and add it directly to a 50mL extraction centrifuge tube. Then add 10mL of deionized water, vortex at 2500rpm for 30min, take 5mL of the extract and place it on an ISOLUTE diatomaceous earth solid phase supported liquid-liquid extraction column (BIOTAGE isolute SLE), elute with 30mL of ethyl acetate (containing 2% acetic acid) three times, 10mL each time, collect the eluate, concentrate to dryness at 45℃, and re-dissolve and mix with 0.5mL of 25% acetonitrile.

[0020] The analytical method provided by the present disclosure is capable of detecting extremely small amounts of target substances. As a preferred technical solution of the present disclosure, in the analytical method, the detection limit concentration corresponding to 3 times the noise is 0.24 μg / mL.

[0021] Compared with the prior art, the technical solution provided by the embodiments of the present disclosure has the following advantages:

[0022] The analytical method provided in the present disclosure creatively incorporates the detection of isomer content into the detection of N-nitrososarcosine content, thereby being able to avoid to a great extent the "false pass" phenomenon caused by the conversion of isomers into N-nitrososarcosine after the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0025] Figure 1 This is the chromatogram obtained by detecting the standard solution (11.77 μg / mL) in the embodiment of the present disclosure. DETAILED DESCRIPTION

[0026] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0028] Example 1

[0029] This embodiment provides a liquid chromatography analysis method for detecting the content of N-nitrososarcosine, as follows:

[0030] (1) using 25% acetonitrile solvent to dissolve N-nitrososarcosine standard to prepare standard solutions with concentrations of 1.18 μg / mL, 2.35 μg / mL, 5.88 μg / mL, 23.54 μg / mL, and 58.85 μg / mL, respectively;

[0031] (2) The standard solution was measured by liquid chromatography, and each concentration was measured twice. The peak area Y was linearly regressed against the concentration X to draw a standard curve;

[0032] The liquid chromatograph used in this example is an UltiMate 3000 liquid chromatograph, the chromatographic column is an Infinity Lab Poroshell 120 PFP, the mobile phase is an aqueous solution containing acetonitrile and formic acid (the volume ratio of acetonitrile, formic acid and water is 500:1:9500), the flow rate is 0.3 mL / min, the detection wavelength is 233 nm, and the column temperature is 40°C.

[0033] Figure 1 This is the chromatogram obtained by detecting the standard solution with a concentration of 11.77 μg / mL in this example, wherein the chromatographic peak at 3.303 min is N-nitrososarcosine, and the chromatographic peak at 3.740 min is its isomer;

[0034] When determining the standard curve, Y is the sum of the peak areas of the chromatographic peak at 3.00-3.50 min and the chromatographic peak at 3.50-4.00 min, X is the concentration of the standard solution, and the standard curve obtained is Y=0.801613X-0.117503, with a correlation coefficient of r=0.9999;

[0035] (3) Pretreatment of samples to be tested:

[0036] Weigh 1g of gum-based oral cigarette, add 1mL of n-hexane to dissolve and disperse the sample, and then transfer it to a 50mL extraction centrifuge tube, then add 10mL of deionized water, vortex at 2500rpm for 30min, take 5mL of the extract, place it on an ISOLUTE diatomaceous earth solid phase supported liquid-liquid extraction column (BIOTAGE isolute SLE), and elute it with 30mL of ethyl acetate (containing 2% acetic acid) three times, 10mL each time, collect the eluate, concentrate it to dryness at 45°C, re-dissolve it with 0.5mL of 25% acetonitrile, mix it evenly, and keep it for analysis;

[0037] (4) The sample to be tested is tested by liquid chromatography analysis. The peak area corresponding to the target substance is the sum of the peak area at 3.00-3.50 min and the peak area at 3.50-4.00 min. The peak area is substituted into the standard curve to obtain the concentration of the target substance (N-nitrososarcosine) in the sample to be tested.

[0038] Comparative Example 1

[0039] This comparative example provides a liquid chromatography analysis method for detecting the content of N-nitrososarcosine.

[0040] The difference from Example 1 is that in this comparative example, the peak area corresponding to Y is the peak area at 3.00-3.50 min, and at the same time, the peak area corresponding to the target is the peak area at 3.00-3.50 min.

[0041] Performance Analysis 1: Accuracy of the Analysis Method

[0042] N-nitrososarcosine was added to the blank matrix to prepare N-nitrososarcosine concentrations of 4.71 μg / mL, 9.42 μg / mL, and 47.08 μg / mL. The target content was detected using the methods provided in Example 1 and Comparative Example 1, respectively. The results are shown in Table 1:

[0043] Table 1

[0044]

[0045] As can be seen from Table 1, the method provided by the present disclosure can accurately detect the concentration of the target N-nitrososarcosine. If the target isomer is not included in the detection method, the content of N-nitrososarcosine in the sample to be tested may be lower than the actual content due to the mutual conversion between the target and the isomer, which may lead to a "false pass" phenomenon.

[0046] Performance Test 2

[0047] (1) Detection limit

[0048] The signal-to-noise ratio of the N-nitrososarcosine isomer at a concentration of 1.18 μg / mL was 15.00. Based on the standard curve obtained in Example 1, the concentration corresponding to the signal-to-noise ratio of 3 was calculated. From the above performance test, it can be seen that when the detection limit is determined by the noise method, the detection limit concentration corresponding to 3 times the noise is 0.24 μg / mL.

[0049] (2) Precision

[0050] The sample was prepared into a solution with a mass concentration of 5.88 μg / mL, and the above method was used for 6 consecutive injections. The RSD was 1.66% based on the peak area.

[0051] Performance Test 3

[0052] The N-nitrososarcosine standard was dissolved in a deuterated reagent to prepare a sample that could be tested by nuclear magnetic resonance. The samples to be tested were randomly divided into two groups. Each group was tested by nuclear magnetic resonance at 30°C and 50°C. After equilibration at each temperature for at least 10 minutes, three independent samples were collected continuously to obtain 1 H-NMR, 13 C-NMR spectrum, statistically test whether there are isomers of N-nitrososarcosine in the test results, confirm the ratio between the two, and calculate the average value. The results are shown in Table 2:

[0053] Table 2

[0054]

[0055] As can be seen from Table 2, the obtained NMR spectrum shows that the sample includes two substances, N-nitrososarcosine and its isomers, indicating that even if the sample to be tested is an N-nitrososarcosine standard, some components are still converted into isomers during the detection. Moreover, from the detection data at different temperatures, it can be seen that the conversion conditions of N-nitrososarcosine and its isomers are extremely simple.

[0056] From this, we can see that if during the test, only the content of the target substance is tested and the content of its isomers is ignored to determine the product is qualified, if the isomer is converted into the target substance after the test, it is very easy to form a "false qualified" phenomenon, causing certain adverse consequences.

[0057] Therefore, the analytical method provided in the present disclosure creatively incorporates the isomer content into the detection of N-nitrososarcosine content, which can avoid the phenomenon of "false pass" to a great extent.

[0058] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0059] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A test N -Liquid chromatography analysis method for nitrososarcosine content, characterized in that, The liquid chromatography analysis method comprises the following steps: (1) Prepare standard solution and determine N - a standard curve of nitrososarcosine, in which Y is the sum of the peak areas corresponding to 3.00-3.50 min and 3.50-4.00 min; (2) The sample to be tested is tested by liquid chromatography analysis. In the obtained chromatogram, the peak area corresponding to the target substance is the sum of the peak areas corresponding to 3.00-3.50 min and 3.50-4.00 min; The liquid chromatography detection parameters include: The chromatographic column was InfinityLabPoroshell 120 PFP, the mobile phase was an aqueous solution containing acetonitrile and formic acid, the flow rate was 0.1-0.5 mL / min, the detection wavelength was 233 nm, and the column temperature was 35-45°C; The volume ratio of acetonitrile, formic acid and water in the mobile phase is 500:1:9500.

2. The liquid chromatography analysis method according to claim 1, characterized in that: The standard solution contains 5 levels of concentration N -Nitrososarcosine standard.

3. The liquid chromatography analysis method according to claim 1, characterized in that: The pretreatment method of the sample to be tested includes: extracting the sample to be tested, eluting with an extraction column, concentrating, and drying to obtain a sample that can be used for detection and analysis.

4. The liquid chromatography analysis method according to claim 1, characterized in that: In the analytical method, the detection limit concentration corresponding to 3 times the noise is 0.24 μg / mL.

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