CN106442840B - The assay method of biogenic amine in a kind of cigarette mainstream flue gas - Google Patents

Abstract

The invention belongs to a kind of assay methods of biogenic amine in cigarette mainstream flue gas, comprise the following steps：（1）Prepare the serial standard working solution of mixing：It prepares with concentration gradient and the serial standard working solution of mixing containging interior traget；（2）Sample pre-treatments：The cambridge filter for having trapped cigarette mainstream smoke total particulate matter is added to 2% aqueous formic acid, ultrasonic extraction 30 minutes；（3）Solid Phase Extraction column purification：Take step（2）Obtained extract liquor crosses mixed type cation exchange solid-phase extraction column, after elution, eluent is placed under nitrogen and is dried up, then is redissolved using 2mL acetonitrile solutions, obtains sample purification liquid；（4）By step（1）Obtained mixing series standard working solution containging interior traget and step（3）Obtained sample purification liquid carries out HPLC MS/MS measure under the same conditions；（5）As a result calculate：It is quantified by the ratio between target analytes peak area and internal standard peak area using internal standard method.

Description

A method for the determination of biogenic amines in cigarette mainstream smoke

technical field

The invention relates to the technical field of detection of components in cigarette smoke, in particular to a method for measuring biogenic amines in mainstream smoke of cigarettes.

Background technique

Biogenic amine is a general term for a class of biologically active nitrogen-containing low-molecular-weight basic organic compounds. They are precursors for the synthesis of hormones, nucleotides, and proteins by organisms themselves. They can be regarded as 1-3 hydrogens in ammonia molecules Low-molecular-weight alkaloids formed after atoms are substituted by alkyl or aryl groups. Biogenic amines can be divided into three categories according to their structures: aliphatic (putrescine, cadaverine, spermine, spermidine, etc.), aromatic amines (tyramine, β-phenylethylamine, etc.) and heterocyclic amines (histamine, tryptamine, etc.).

Biogenic amines are mainly formed by the decarboxylation of amino acids, and a small part is formed by the amination of aldehydes or ketones. Various animal and plant tissue cells contain a small amount of biogenic amines. There are three main conditions for their production: First, there are biogenic amines that can be formed The second is the existence of microorganisms that can metabolize amino acid decarboxylase, and the third is the environment suitable for the growth of the microorganisms. Biogenic amines are commonly found purely in a variety of foods and alcoholic fermented beverages. Biogenic amines are not only important sources of food aroma, but also precursors for the synthesis of N-nitroso compounds. When the human body ingests excessive biogenic amines, it is very easy to cause damage to the nervous system and cardiovascular system. In 2008, my country issued relevant standards to limit requirements for biogenic amines in food.

At present, high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), thin-layer chromatography (TLC) and gas chromatography (GC) are generally used for the detection of biogenic amines. The above-mentioned detection methods all need to derivatize the sample, and the operation is relatively complicated. . At present, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is more and more used in the detection of trace high boiling point substances because it does not require derivatization of samples and has high sensitivity and accuracy.

As a special food, tobacco also contains a large amount of protein and amino acids, and the raw materials need to be purified in the production process, which is easy to produce biogenic amines. Biogenic amines are compounds with a medium boiling point. During cigarette smoking, biogenic amines in tobacco leaves are likely to migrate directly into cigarette smoke, and the decarboxylation of amino acids is also likely to produce biogenic amines. Therefore, the qualitative analysis of biogenic amines in cigarette smoke is established. and quantitative detection methods are necessary.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings in the prior art and provide a method for determining biogenic amines in cigarette mainstream smoke. The method uses liquid chromatography-tandem mass spectrometry to simultaneously measure 7 kinds of biogenic amines in cigarette mainstream smoke , can quickly and effectively detect the content of biogenic amines in cigarette mainstream smoke.

The object of the present invention is achieved by the following technical solutions: a method for assaying biogenic amines in cigarette mainstream smoke, comprising the following steps:

(1) Prepare a mixed series of standard working solutions: prepare a mixed series of standard working solutions with a concentration gradient and containing an internal standard;

(2) Sample pretreatment: Place the cigarette samples (with a weight within the range of ±0.02g and an average draw resistance within the range of ±49Pa/cigarette) in an environment at 21-23°C and a relative humidity of 58-62% to balance 48-48 96h, use a rotary smoking machine, take one puff every 60 seconds, each puff has a suction volume of 35ml, and the puff duration is 2s. Cambridge filters with a diameter of 92mm are used to capture the total particulate matter in mainstream cigarette smoke. To collect the total particulate matter of 20 cigarettes, take one piece of filter piece that collected the total particulate matter of 20 cigarettes, place it in a 250mL Erlenmeyer flask, add 30mL of 2vol% formic acid aqueous solution, and perform ultrasonic extraction for 30 minutes;

(3) Purification of solid-phase extraction column: take the extract obtained in step (2) and pass it through a mixed-type cation-exchange solid-phase extraction column (DIKMA ProElut PXC 150mg/6mL), after elution, place the eluent to dry under nitrogen , and then redissolved with 2 mL of acetonitrile aqueous solution to obtain a sample purification solution;

(4) The mixed series standard working solution containing the internal standard obtained by step (1) and the sample purification solution obtained by step (3) are carried out under the same conditions for HPLC-MS/MS determination;

(5) Calculation of results: the ratio of the peak area of the target analyte to the peak area of the internal standard is used for quantification by the internal standard method.

Said step (1) prepares the concrete process of mixed series standard working solution as follows: preparation concentration gradient is the mixed standard working solution of 0.5,1,2,5,10,25ng/mL, the content of the mixed standard working solution of each concentration The concentration of the standard 1,7-heptanediamine is 10ng/mL; in the mixed standard working solution with a concentration gradient of 0.5, 1, 2, 5, 10, 25ng/mL, putrescine, cadaverine, histamine, tyramine, The concentrations of spermine, spermidine, and tryptamine are 0.5, 1, 2, 5, 10, and 25 ng/mL respectively (for example, when the gradient of the mixed standard working solution is 0.5 ng/mL, the mixed standard working solution Putrescine, cadaverine, histamine, tyramine, spermine, spermidine, tryptamine seven biogenic amine concentrations were 0.5ng/mL).

The conditions of HPLC-MS/MS determination are as follows:

Chromatographic conditions:

A hydrophilic interaction chromatographic column is selected, the flow rate is 0.2-0.5mL/min, the injection volume is 5-10μL; the analysis time is 14min; the column temperature is 40°C; the mobile phase is a mixed system of A and B, A is ammonium formate and A mixed aqueous solution of formic acid, wherein the volume ratio of formic acid to water is 0.1:100, and the content of ammonium formate is 15-25mmol/L; B is a formic acid acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.1:100; using gradient elution, The volume percentage of B in different time periods is: 0-1min, 95%; 1-12min, 95%-45%; 12-14min, 95%;

Mass Spectrometry Conditions:

Ion source, electrospray ion source (ESI); scan mode, positive ion scan; detection method, multiple reaction monitoring (MRM); electrospray voltage, 5500V; curtain gas pressure, 0.124Mpa; auxiliary gas 1 (N ₂ ) pressure, 0.379Mpa; auxiliary gas 2 (N ₂ ) pressure, 0.345Mpa; ion source temperature, 600.00°C.

The elution process in the described step (3) is as follows: use 2mL formic acid aqueous solution and 2mL methanol to rinse successively and then use 10mL ammonia water methanol solution to elute, wherein the volume fraction of formic acid in the formic acid aqueous solution is 1～2%, ammonia water in the ammonia water methanol solution (25wt%) volume concentration of 3% to 7%.

The hydrophilic interaction chromatographic column is an Agilent Hilic column with a specification of 3.5 μm; 2.1 mm×100 mm.

The beneficial effects produced by the present invention are: the present invention first adopts Cambridge filter to capture mainstream smoke, uses solid-liquid extraction-tandem solid-phase extraction as pretreatment means, and adopts high performance liquid chromatography-tandem mass spectrometry (HPLC-MS /MS) method for the determination of seven biogenic amines (putrescine, cadaverine, histamine, tyramine, spermine, spermidine and tryptamine) in tobacco. Compared with the literature, the present invention establishes a method suitable for measuring biogenic amines in cigarette mainstream smoke, the established method is simple and reliable, and can be applied to the detection of biogenic amines in cigarette mainstream smoke; the method is simple and convenient for sample analysis , low detection limit, high sensitivity, good repeatability and recovery.

Description of drawings

Fig. 1 is the MRM figure of putrescine in the standard sample;

Fig. 2 is the MRM figure of cadaverine in the standard sample;

Figure 3 is the MRM diagram of histamine in the standard sample

Figure 4 is the MRM diagram of tyramine in the standard sample

Figure 5 is the MRM diagram of spermine in the standard sample

Fig. 6 is the MRM figure of spermidine in the standard sample;

Fig. 7 is the MRM figure of tryptamine in the standard sample;

Figure 8 is the MRM diagram of the internal standard 1,7-heptanediamine in the standard sample.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example 1

A method for assaying biogenic amines in tobacco, comprising the following steps,

(1) Prepare a mixed series of standard working solutions: prepare a mixed series of standard working solutions with a concentration gradient and containing an internal standard;

(2) Sample pretreatment: place the cigarette sample in an environment with a relative humidity of 60% at 22°C for 48 hours, and then select cigarettes whose average weight is within the range of ±0.02g and average draw resistance within the range of ±49Pa/cigarette As a test cigarette. A rotary smoking machine is used to take a puff every 60 seconds, with a suction volume of 35ml per puff, and a puff duration of 2s. Use a Cambridge filter with a diameter of 92mm to capture the total particulate matter of mainstream cigarette smoke. Each filter piece collects the total particulate matter of 20 cigarettes. Take one filter piece that collects the total particulate matter of 20 cigarettes and place it in a 250mL Accurately add 30mL 2vol% formic acid aqueous solution to the Erlenmeyer flask, and ultrasonically extract for 30 minutes.

(3) Solid-phase extraction column purification: 10 mL of the extract obtained in step (2) was passed through a PCX cation-exchange solid-phase extraction column (DIKMA ProElut PXC 150 mg/6 mL), and 2 mL of formic acid aqueous solution (volume fraction 2%) and 2 mL of methanol After washing successively, use 10mL ammonia water methanol solution (the volume concentration of 25wt% ammonia water in the ammonia water methanol solution is 3%～7%) to elute, and the eluent is dissolved in 2mL acetonitrile aqueous solution (the volume ratio of acetonitrile and water is 95:5), to obtain the sample purification solution;

(4) The mixed series standard working solution containing internal standard that step (1) obtains and the sample purification solution that step (3) obtains carry out HPLC-MS/MS measurement under identical conditions; The condition that HPLC-MS/MS measures is as follows:

The chromatographic conditions are:

A hydrophilic interaction chromatographic column (Agilent Hilic column, specification: 3.5 μm; 2.1 mm×100 mm) was selected, the flow rate was 0.3 mL/min, the injection volume was 10 μL; the analysis time was 14 min; the column temperature was 40 ° C; the mobile phase was A mixed system of A and B, A is a mixed aqueous solution of ammonium formate and formic acid, wherein the volume ratio of formic acid to water is 0.1:100, and the content of ammonium formate is 25mmol/L; B is a formic acid acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile It is 0.1:100; using gradient elution, wherein the volume percentage of B in different time periods is: 0～1min, 95%; to 45%); 12-14min, 95%;

Mass Spectrometry Conditions:

Ion source: electrospray ion source (ESI); scan mode, positive ion scan; detection mode, multiple reaction monitoring (MRM); drying gas (N ₂ ) temperature and flow rate: 290 ° C, 11 L/min; nebulizer pressure, 206.85kPa (30psi); sheath gas (N ₂ ) temperature and flow rate, 400°C, 12L/min; capillary voltage (Capillary voltage), 3500V; fragmentation voltage (Fragmentor voltage), 380V; collision gas: N ₂ .

(5) Calculation of results: the ratio of the peak area of the target analyte to the peak area of the internal standard is quantified by the internal standard method; the details are as follows: the ratio of the peak area of the target analyte to the peak area of the internal standard in the mixed series of standard working solutions containing the internal standard The ratio is the ordinate, the concentration of the target analyte is the abscissa, and the working curve is drawn to obtain the standard curve regression equation and correlation coefficient of the target; according to the ratio of the peak area of the target analyte in the sample purification solution to the internal standard peak area, substitute the curve to obtain the content of the sample purification solution. The release amount of heterocyclic amine compounds in mainstream smoke is calculated by the following formula:

In the formula: m——the amount of heterocyclic amine compounds released in mainstream smoke of each cigarette, in nanograms per cigarette (ng/cig); A——the amount of heterocyclic amine compounds in the sample purification solution Concentration in nanograms per milliliter (ng/mL).

Said step (1) prepares the concrete process of mixed series standard working solution as follows: preparation concentration gradient is the mixed standard working solution of 0.5,1,2,5,10,25ng/mL, the content of the mixed standard working solution of each concentration The concentration of the standard 1,7-heptanediamine is 10ng/mL; in the mixed standard working solution with a concentration gradient of 0.5, 1, 2, 5, 10, 25ng/mL, putrescine, cadaverine, histamine, tyramine, The concentrations of spermine, spermidine, and tryptamine are 0.5, 1, 2, 5, 10, and 25 ng/mL respectively (for example, when the gradient of the mixed standard working solution is 0.5 ng/mL, the mixed standard working solution Putrescine, cadaverine, histamine, tyramine, spermine, spermidine, tryptamine seven biogenic amine concentrations were 0.5ng/mL). Determination was carried out by HPLC-MS/MS.

The MRM parameters of seven biogenic amines (putrescine, cadaverine, histamine, tyramine, spermine, spermidine, tryptamine) and their internal standard 1,7-heptanediamine are shown in Table 1 (Figure 1-8 is MRM diagrams of seven biogenic amines and internal standard 1,7-heptanediamine).

Table 1 MRM ^parameters of seven biogenic amines and internal standard 1,7-heptanediamine①

Note: ①CE is the collision energy; DP is the declustering voltage; * is the quantitative ion.

The mixed series standard working solution containing the internal standard at the lowest concentration was used for 10 determinations, and the standard deviations of 7 biogenic amines (putrescine, cadaverine, histamine, tyramine, spermine, spermidine, and tryptamine) were calculated, as Three times the standard deviation was used as the detection limit. The results showed (Table 2) that the method had good linearity and low detection limit.

Table 2 The linear regression equation, linear correlation coefficient and detection limit of seven biogenic amines

In order to investigate the reproducibility of the method of the present invention, a standard solution of seven kinds of biogenic amines was added to the tobacco sample to prepare three levels of high (40ng/mL), medium (10ng/mL), and low (1ng/mL) levels. standard sample, then analyze it with the assay method of the present invention, deduct the content of blank test biogenic amine in the sample, calculate its measurement amount, and calculate the recovery rate by the addition amount and measurement amount, each concentration level is done three parallels (results see table 3). The results showed that the recoveries of the three levels were between 88.3-106%.

Table 3 The recovery rate of seven kinds of biogenic amines

The above-mentioned low-concentration standard sample (1ng/mL) was subjected to 6 intra-day and inter-day parallel determinations according to the assay method of the present invention. The results showed that the intra-day and inter-day relative standard deviations of each compound were between 0.8-4.1%, indicating that the method had good reproducibility and stability (results are shown in Table 4).

Table 4 Intra-day and inter-day precision of seven biogenic amines

Example 3

Get cigarette sample 1-5#, carry out sample analysis according to the assay method of the present invention, sample analysis result is shown in Table 5.

Content of biogenic amines in table 5 tobacco leaf samples

Note: "-" means not detected in the sample.

While particular embodiments of the invention have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (3)

1. a method for assaying biogenic amines in cigarette mainstream smoke, is characterized in that, comprises the following steps: (1) Prepare a mixed series of standard working solutions: Prepare a mixed series of standard working solutions with concentration gradients and internal standards; (2) Sample pre-treatment: add the Cambridge filter which has captured the total particulate matter in mainstream cigarette smoke to 2% formic acid aqueous solution, and perform ultrasonic extraction for 30 minutes; (3) Solid-phase extraction column purification: take the extract obtained in step (2) and pass it through a mixed-type cation-exchange solid-phase extraction column. Dissolved to obtain sample purification solution; (4) The mixed series standard working solution containing internal standard obtained in step (1) and the sample purification solution obtained in step (3) are subjected to HPLC-MS/MS determination under the same conditions; (5) Calculation of results: Quantification is performed by the internal standard method based on the ratio of the peak area of the target analyte to the peak area of the internal standard; In step (2), the method for capturing the total particulate matter in mainstream cigarette smoke by the Cambridge filter is as follows: place the cigarette sample in an environment with a temperature of 21-23°C and a relative humidity of 58-62% for 48-96 hours, and use a turntable to Smoking machine, take a puff every 60 seconds, each puff has a suction volume of 35ml, and the puff duration is 2s. The Cambridge filter with a diameter of 92mm is used to capture the total particulate matter in mainstream cigarette smoke, and each filter collects 20 cigarettes. Total particulate matter of cigarettes; The elution process in the step (3) is as follows: wash with 2mL formic acid aqueous solution and 2mL methanol sequentially, and then elute with 10 mL ammonia water methanol solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 1~2%, and the ammonia water methanol solution The volume concentration of ammonia water is 3%~7%; The conditions of HPLC-MS/MS determination are as follows: Chromatographic conditions: A hydrophilic interaction chromatographic column was selected, the flow rate was 0.2-0.5 mL/min, the injection volume was 5-10 μL; the analysis time was 14 min; the column temperature was 40°C; the mobile phase was a mixed system of A and B, and A was formic acid A mixed aqueous solution of ammonium and formic acid, wherein the volume ratio of formic acid to water is 0.1:100, and the content of ammonium formate is 15-25 mmol/L; B is a formic acid acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.1:100; gradient Elution, wherein the volume percentage of B in different time periods is: 0~1min, 95%; 1~12min, 95%~45%; 12~14min, 95%; Mass Spectrometry Conditions: Ion source, electrospray ion source (ESI); scan mode, positive ion scan; detection method, multiple reaction monitoring (MRM); electrospray voltage, 5500V; curtain gas pressure, 0.124Mpa; auxiliary gas 1, N ₂ , pressure, 0.379 Mpa; auxiliary gas 2, N ₂ , pressure, 0.345 Mpa; ion source temperature, 600.00°C. 2. The method for determining biogenic amines in mainstream cigarette smoke according to claim 1, wherein the specific process of preparing a mixed series of standard working solutions containing an internal standard in the step (1) is as follows: the preparation concentration gradient is 0.5, 1 , 2, 5, 10, 25ng/mL mixed standard working solution, the concentration of the internal standard 1,7-heptanediamine of each concentration of mixed standard working solution is 10ng/mL; the concentration gradient is 0.5, 1, 2, In the mixed standard working solution containing internal standard at 5, 10, and 25 ng/mL, the concentrations of seven biogenic amines including putrescine, cadaverine, histamine, tyramine, spermine, spermidine, and tryptamine were all 0.5, 1, 2, 5, 10, 25 ng/mL. 3 . The method for determining biogenic amines in mainstream cigarette smoke according to claim 1 , wherein the hydrophilic interaction chromatographic column is an Agilent Hilic column, and the specifications are: 3.5 μm; 2.1 mm×100 mm.