A method for analyzing 20 amino acids in tobacco and flavors and fragrances

Abstract

The present invention provides a method for analyzing 20 kinds of amino acids in tobacco and flavors and spices, comprising the following steps: 1) sample pretreatment: adding a solvent and an internal standard solution to a tobacco or flavor and spices sample, mixing, ultrasonically extracting, centrifuging, and filtering to obtain a sample pretreatment solution; 2) sample pre-column derivatization: mixing the sample pretreatment solution obtained in step 1) with a buffer reagent and a derivatization reagent in a derivatization kit, respectively, and heating to perform derivatization to obtain a sample test solution; 3) sample analysis: using a multi-heart-cutting two-dimensional liquid chromatography method to measure the sample test solution obtained in step 2) to determine the content of at least one of the 20 kinds of amino acids in the sample test solution. The present invention provides a method for analyzing 20 kinds of amino acids in tobacco and flavors and spices, which effectively removes impurity interference through multidimensional chromatographic separation, and realizes simultaneous quantitative analysis of 20 kinds of amino acids in tobacco and flavors and spices.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical component analysis in tobacco and relates to a method for analyzing 20 kinds of amino acids in tobacco. Background Art

[0002] Amino acid is one of the important chemical components in tobacco and flavors and fragrances, and is also a key indicator for measuring their quality. During the process of tobacco leaf baking, curing, aging and smoking, free amino acids and reducing sugars will undergo enzyme or non-enzyme catalyzed browning reactions to generate a variety of heterocyclic compounds. At the same time, the thermal decomposition of amino acids will also produce many chemical components. For example, phenylalanine will produce compounds such as benzyl alcohol and phenylacetaldehyde through thermal decomposition, and other amino acids will produce compounds such as pyrrole, indole, pyridine, phenol, and hydrogen cyanide through thermal decomposition. Some of these compounds are flavor components, some are irritating components, and some are harmful components, and their effects on product quality are completely different. Therefore, analyzing the amino acids in tobacco and tobacco flavors and fragrances, and in-depth research on their effects on the quality of tobacco products can provide important theoretical guidance for cigarette formulation, tobacco leaf curing, aging, etc.

[0003] Due to the important role of amino acids in tobacco, their analysis and research have also received attention from researchers in the tobacco industry. At present, the analysis methods at home and abroad include amino acid automatic analyzer method, capillary electrophoresis method, gas chromatography and liquid chromatography. The tobacco industry currently mainly uses amino acid automatic analyzer method and gas chromatography to detect amino acids. However, the detection of amino acid automatic analyzer method is easily interfered by co-elution peaks for complex matrix samples such as tobacco, and the sensitivity is not high. It can only accurately quantify several amino acids with high content, and the accuracy of other amino acid quantitative results is not ideal. The gas chromatography method for detecting amino acids has complex pretreatment, large differences in amino acid derivatization efficiency, and high toxicity of the organic reagents used. It is not an ideal amino acid detection method.

[0004] Multidimensional liquid chromatography can optimize the combination of liquid chromatography with different separation principles, has high selectivity and sensitivity, and is an ideal choice for analyzing complex matrix samples. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for analyzing 20 amino acids in tobacco and flavors and fragrances, so as to solve the problem that there is a lack of a method based on multi-heart-cutting two-dimensional liquid chromatography that can simultaneously determine 20 amino acids in the prior art.

[0006] To achieve the above object and other related objects, the present invention provides a method for analyzing 20 kinds of amino acids in tobacco and flavors and fragrances, comprising the following steps:

[0007] 1) Sample pretreatment: Add the tobacco or flavor sample to the solvent and the internal standard solution, mix, ultrasonically extract, centrifuge, and filter to obtain a sample pretreatment solution;

[0008] 2) Sample pre-column derivatization: the sample pretreatment solution obtained in step 1) is mixed with the buffer reagent and the derivatization reagent in the derivatization kit respectively, and then heated for derivatization to obtain a sample test solution;

[0009] 3) Sample analysis: The sample solution obtained in step 2) is measured by multiple heart-cutting two-dimensional liquid chromatography to determine the content of at least one of the 20 amino acids in the sample solution.

[0010] Preferably, the tobacco is selected from at least one of tobacco leaves, tobacco dust or tobacco shreds.

[0011] The above-mentioned tobacco dust or shredded tobacco is crushed to obtain a powder sample.

[0012] Preferably, the tobacco leaves are selected from at least one of green tobacco leaves and flue-cured tobacco leaves.

[0013] The green tobacco leaves are freeze-dried and then ground to obtain a powder sample;

[0014] The flue-cured tobacco leaves are dried and then crushed to obtain a powder sample.

[0015] Preferably, the flavor is a commonly used flavor. Specifically, the flavor is a mixture of multiple spices for flavoring products.

[0016] Preferably, the fragrance is a natural fragrance raw material. The natural fragrance raw material is a conventionally used natural raw material, including essential oils, extracts, absolute oils, ointments, tinctures and the like.

[0017] Preferably, the amino acid is selected from histidine (His, CAS No. 71-00-1), glutamine (Gln, CAS No. 56-85-9), arginine (Arg, CAS No. 74-79-3), asparagine (Asn, CAS No. 70-47-3), serine (Ser, CAS No. 56-45-1), glycine (Gly, CAS No. 56-40-6), aspartic acid (Asp, CAS No. 56-84-8), glutamic acid (Glu, CAS No. 56-86-0), threonine (Thr, CAS No. 72-19-5), alanine (Ala, CAS No. 56-41-1), 7), at least one of proline (Pro, CAS No. 147-85-3), methionine (Met, CAS No. 63-68-3), valine (Val, CAS No. 72-18-4), cysteine ​​(Pro, CAS No. 52-90-4), lysine (Lys, CAS No. 56-87-1), tyrosine (Tyr, CAS No. 60-18-4), isoleucine (Ile, CAS No. 73-32-5), leucine (Leu, CAS No. 61-90-5), tryptophan (Trp, CAS No. 73-22-3), and phenylalanine (Phe, CAS No. 63-91-2).

[0018] Preferably, the amino acids are 20 kinds, including histidine, glutamine, arginine, asparagine, serine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, methionine, valine, cysteine, lysine, tyrosine, isoleucine, leucine, tryptophan, and phenylalanine.

[0019] Preferably, in step 1), the solvent is selected from at least one of aqueous hydrochloric acid solution and aqueous methanol solution.

[0020] Preferably, the solvent is an aqueous hydrochloric acid solution. More preferably, the aqueous hydrochloric acid solution is an aqueous hydrochloric acid solution with a concentration of 0.05-0.15 mol / mL. Further preferably, the aqueous hydrochloric acid solution is an aqueous hydrochloric acid solution with a concentration of 0.1 mol / mL.

[0021] Preferably, the methanol aqueous solution is a methanol aqueous solution with a concentration of 50%-80%. Further preferably, the methanol aqueous solution is a methanol aqueous solution with a concentration of 70%.

[0022] Preferably, in step 1), the ratio of the added mass g of the tobacco or flavor and fragrance sample to the added volume mL of the solvent is 1:10-20.

[0023] Preferably, in step 1), the internal standard solution is a hydrochloric acid aqueous solution containing norvaline (Nval, CAS No. 6600-40-4). Preferably, the concentration of norvaline in the internal standard solution is 2-3nmol / μL. More preferably, the concentration of norvaline in the internal standard solution is 2.5nmol / μL.

[0024] Preferably, the concentration of the aqueous hydrochloric acid solution in the internal standard solution is 0.05-0.15 mol / mL. More preferably, the concentration of the aqueous hydrochloric acid solution in the internal standard solution is 0.1 mol / mL.

[0025] Preferably, in step 1), the ratio of the volume μL of the internal standard solution to the volume of the solvent added is 1:30-70.

[0026] Preferably, in step 1), the mixing is performed until uniform, generally by shaking.

[0027] Preferably, in step 1), the ultrasonic extraction time is 20-40 min.

[0028] Preferably, in step 1), the centrifugal speed is 2800-3200 r / min.

[0029] Preferably, in step 1), the filtration is to filter the supernatant after centrifugation through a membrane, and the membrane is a 0.22 μm aqueous membrane.

[0030] Preferably, in step 2), the derivatization kit is a commercial AccQ-TagUltra kit produced by Waters Corporation of the United States. The buffer reagent is a borate buffer solution provided in the AccQ-Tag Ultra kit. The derivatization reagent is an AccQ-Tag Ultra reagent provided in the AccQ-Tag Ultra kit, the main component of which is 6-aminoquinoline-N-hydroxysuccinimidyl formate.

[0031] Preferably, in step 2), the volume ratio of the sample pretreatment solution to the buffer reagent added is 10:50-90.

[0032] Preferably, in step 2), the volume ratio of the sample pretreatment solution to the derivatization reagent is 10:10-40.

[0033] Preferably, in step 2), the derivatization is to mix the sample pretreatment solution with the buffer reagent for a first time, then add the derivatization reagent for a second mixing and then heat for derivatization.

[0034] Preferably, the first mixing and the second mixing are vortex mixing.

[0035] Preferably, the mixing time of the first mixing is 5-15 s.

[0036] Preferably, the mixing time of the second mixing is 2-10s.

[0037] Preferably, the second mixing is followed by standing still at room temperature, which is 20-30°C.

[0038] More preferably, the standing time is 50-70 seconds, preferably 60 seconds.

[0039] Preferably, in step 2), the heating temperature is 50-60° C. The heating is performed in a thermostatic box.

[0040] Preferably, in step 2), the heating time is 5-11 minutes.

[0041] Preferably, in step 3), the multi-heart-cutting two-dimensional liquid chromatography method comprises the following steps:

[0042] A) adding a solvent to a standard substance of at least one of the 20 amino acids, and then adding an internal standard solution, mixing the prepared standard working solution with a buffer reagent and a derivatization reagent in a derivatization kit, and then heating to perform derivatization, to obtain a standard solution to be tested;

[0043] B) The sample test solution and the standard test solution obtained in step A) are measured using a multi-heart-cutting two-dimensional liquid chromatography system, at least one of the 20 amino acids is separated by first-dimensional liquid chromatography according to different retention times, captured on different capture columns, and then the captured components are eluted and separated and detected by second-dimensional liquid chromatography, the retention times are compared for qualitative analysis, and the internal standard method is used for quantitative analysis to determine the content of at least one of the 20 amino acids in the sample test solution.

[0044] Preferably, in step A), the standard working solution is obtained by adding a standard of at least one of the 20 amino acids to a solvent to obtain a mixed stock solution, diluting it step by step, adding an internal standard solution, and then adding a solvent to dissolve and fix the volume.

[0045] More preferably, the concentration of each amino acid in the mixed stock solution is 20-30 nmol / μL, preferably 25 nmol / μL.

[0046] More preferably, the concentration of the internal standard in the standard working solution is 20-100 pmol / μL, preferably 50 pmol / μL.

[0047] Preferably, in step A), the solvent is selected from at least one of aqueous hydrochloric acid solution and aqueous methanol solution.

[0048] More preferably, the solvent is an aqueous hydrochloric acid solution. Further preferably, the aqueous hydrochloric acid solution is an aqueous hydrochloric acid solution with a concentration of 0.05-0.15 mol / mL. Even more preferably, the aqueous hydrochloric acid solution is an aqueous hydrochloric acid solution with a concentration of 0.1 mol / mL.

[0049] More preferably, the methanol aqueous solution is a methanol aqueous solution with a concentration of 50%-80%. Further preferably, the methanol aqueous solution is a methanol aqueous solution with a concentration of 70%.

[0050] Preferably, in step A), the internal standard solution is an aqueous hydrochloric acid solution containing norvaline (Nval, CAS No. 6600-40-4). More preferably, the concentration of norvaline in the internal standard solution is 2-3nmol / μL. Further preferably, the concentration of norvaline in the internal standard solution is 2.5nmol / μL.

[0051] More preferably, the concentration of the aqueous hydrochloric acid solution in the internal standard solution is 0.05-0.15 mol / mL. Further preferably, the concentration of the aqueous hydrochloric acid solution in the internal standard solution is 0.1 mol / mL.

[0052] Preferably, in step A), the derivatization kit is a commercial AccQ-TagUltra kit produced by Waters Corporation of the United States. The buffer reagent is a borate buffer solution provided by the AccQ-Tag Ultra kit. The derivatization reagent is an AccQ-Tag Ultra reagent provided by the AccQ-Tag Ultra kit, and its main component is 6-aminoquinoline-N-hydroxysuccinimidyl formate.

[0053] Preferably, in step A), the volume ratio of the standard working solution to the buffer reagent added is 10:50-90.

[0054] Preferably, in step A), the volume ratio of the standard working solution to the derivatization reagent is 10:10-40.

[0055] Preferably, in step A), the derivatization is performed by first mixing the standard working solution with a buffer reagent, then adding a derivatization reagent, mixing for a second time, and then heating for derivatization.

[0056] More preferably, the first mixing and the second mixing are vortex mixing.

[0057] More preferably, the mixing time of the first mixing is 5-15 s.

[0058] More preferably, the mixing time of the second mixing is 2-10 s.

[0059] More preferably, the second mixing is followed by standing still at room temperature, which is 20-30°C.

[0060] More preferably, the standing time is 50-70 seconds, preferably 60 seconds.

[0061] Preferably, in step A), the heating temperature is 50-60° C. The heating is performed in a thermostatic box.

[0062] Preferably, in step A), the heating time is 5-11 min.

[0063] Preferably, in step B), the multi-heart-cutting two-dimensional liquid chromatography system is a conventionally used multi-heart-cutting two-dimensional liquid chromatography system, and its specific structure is shown in Figure 1 .

[0064] Preferably, in step B), at least one of the 20 amino acids is separated by the first-dimensional chromatography using a multi-heart cutting method.

[0065] The multi-heart cutting method is a fraction cutting and transfer method commonly used in multidimensional chromatography. In the two-dimensional liquid chromatography of the present invention, the eluent corresponding to the chromatographic peaks of multiple target objects separated by the first-dimensional liquid chromatography is transferred to the second-dimensional liquid chromatography in batches, and the other eluent parts do not enter the second-dimensional liquid chromatography.

[0066] Preferably, in step B), the measurement conditions of the first dimension liquid chromatography are:

[0067] Detector: at least one selected from a diode array detector, a fluorescence detector or a mass spectrometer detector; First separation column: selected from C 18 One of the chromatographic column or CN chromatographic column; column temperature: 45-55°C; injection volume: 1-10 μL; flow rate: 0.1-0.5 mL / min; detection wavelength: 255-265 nm; mobile phase A: water containing 0.05-0.1% formic acid; mobile phase B: methanol; analysis time: 100 min; gradient elution.

[0068] Further preferably, the measurement conditions of the first dimension liquid chromatography are:

[0069] Detector: diode array detector (DAD); first separation column: CN chromatographic column (2.1 mm × 100 mm, 5 μm) (see the product official website http: / / www.agela.com.cn / product / detail / 500 for details); column temperature: 50 ° C; injection volume: 2 μL; flow rate: 0.3 mL / min; detection wavelength: 260 nm; mobile phase A: water containing 0.1% formic acid; mobile phase B: methanol; analysis time: 100 min; gradient elution.

[0070] Further preferably, the specific procedure of the gradient elution is:

[0071] 0-2min, volume ratio of phase A:phase B was 95:5-95:5;

[0072] 2-12min, volume ratio of phase A:phase B is 95:5-5:95;

[0073] 12-20min, volume ratio of phase A:phase B is 5:95-5:95;

[0074] 20-20.01min, volume ratio of phase A:phase B is 5:95-50:50;

[0075] 20.01-75min, volume ratio of phase A:phase B is 50:50-50:50;

[0076] 75-75.01min, volume ratio of phase A:phase B is 50:50-95:5;

[0077] 75.01-100min, the volume ratio of phase A:phase B is 95:5-95:5.

[0078] Preferably, in step B), the trapping conditions of the trapping column are:

[0079] Capture column: C 18 Chromatographic column, preferably XBridge C 18 Chromatographic column (4.6 mm×30 mm, 5 μm); compensation pump mobile phase: at least one selected from deionized water, phosphate buffer, acetate buffer, formate buffer, and ammonia solution, preferably deionized water; the flow rate of the compensation pump mobile phase is 1000-1500 μL / min, preferably 1200 μL / min. The compensation pump mobile phase is used to reduce the organic phase ratio of the mobile phase entering the capture column.

[0080] More preferably, the determination conditions of the second dimension liquid chromatography are:

[0081] Detector: at least one selected from a diode array detector, a fluorescence detector or a mass spectrometer detector; Second separation column: selected from C 18 At least one of a chromatographic column, an AQ chromatographic column, a T3 chromatographic column or an RP18 chromatographic column; column temperature: 50-60°C; flow rate: 0.5-2mL / min; excitation wavelength: 264-268nm, emission wavelength: 471-475nm; mobile phase A: water containing 0.05-1% formic acid; mobile phase B: acetonitrile; analysis time: 100min; gradient elution.

[0082] Further preferably, the measurement conditions of the second dimension liquid chromatography are:

[0083] Detector: fluorescence detector (FLD); second separation column: AQ chromatographic column (4.6 mm×100 mm, 2.7 μm); column temperature: 55°C; flow rate: 1.0 mL / min; excitation wavelength: 266 nm, emission wavelength: 473 nm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; analysis time: 100 min; gradient elution.

[0084] Further preferably, the specific procedure of the gradient elution is:

[0085] 0-15min, volume ratio of phase A:phase B was 98:2-98:2;

[0086] 15-16min, the volume ratio of phase A:phase B is 98:2-96:4;

[0087] 16-20min, volume ratio of phase A:phase B is 96:4-96:4;

[0088] 20-25min, volume ratio of phase A:phase B is 96:4-80:20;

[0089] 25-25.01min, volume ratio of phase A:phase B is 80:20-10:90;

[0090] 25.01-30min, volume ratio of phase A:phase B is 10:90-10:90;

[0091] 30-30.01min, volume ratio of phase A:phase B is 10:90-100:0;

[0092] 30.01-35min, volume ratio of phase A:phase B is 100:0-100:0;

[0093] 35-45min, volume ratio of phase A:phase B is 100:0-80:20;

[0094] 45-45.01min, volume ratio of phase A:phase B is 80:20-10:90;

[0095] 45.01-50min, volume ratio of phase A:phase B is 10:90-10:90;

[0096] 50-50.01min, volume ratio of phase A:phase B is 10:90-98:2;

[0097] 50.01-55min, volume ratio of phase A:phase B is 98:2-98:2;

[0098] 55-57min, the volume ratio of phase A:phase B is 98:2-90:10;

[0099] 57-65min, volume ratio of phase A:phase B is 90:10-90:10;

[0100] 65-70min, the volume ratio of phase A:phase B is 90:10-75:25;

[0101] 70-70.01min, volume ratio of phase A:phase B is 75:25-10:90;

[0102] 70.01-75min, volume ratio of phase A:phase B is 10:90-10:90;

[0103] 75-75.01min, volume ratio of phase A:phase B is 10:90-98:2;

[0104] 75.01-80min, the volume ratio of phase A:phase B is 98:2-98:2;

[0105] 80-90min, the volume ratio of phase A:phase B is 98:2-40:60;

[0106] 90-90.01min, volume ratio of phase A:phase B is 40:60-10:90;

[0107] 90.01-95min, volume ratio of phase A:phase B is 10:90-10:90;

[0108] 95-95.01min, volume ratio of phase A:phase B is 10:90-98:2;

[0109] 95.01-100min, the volume ratio of phase A:phase B is 98:2-98:2.

[0110] Preferably, in step B), the internal standard method refers to adding a solvent and an internal standard solution to a standard of at least one of the 20 amino acids and then derivatizing it, and preparing a series of standard test solutions of different concentrations by using a multi-heart-cutting two-dimensional liquid chromatography system for analysis, obtaining a linear relationship between the chromatographic peak area ratio of at least one of the 20 amino acids / internal standard and the corresponding concentration ratio, drawing a corresponding standard working curve, and calculating the regression equation of the standard working curve; then analyzing the sample test solution by using a multi-heart-cutting two-dimensional liquid chromatography system to obtain the chromatographic peak area ratio of at least one of the 20 amino acids to the internal standard, substituting it into the regression equation of the standard working curve, and calculating the content of the corresponding amino acid in the sample test solution.

[0111] More preferably, in the standard working curve, the chromatographic peak area ratio of at least one of the 20 amino acids to the internal standard is used as the ordinate (Y-axis), and the concentration ratio of the corresponding amino acid to the internal standard is used as the abscissa (X-axis).

[0112] As described above, the method for analyzing 20 kinds of amino acids in tobacco and flavors and fragrances provided by the present invention can realize simultaneous quantitative analysis of 20 kinds of amino acids in tobacco and flavors and fragrances, targeting the characteristics of tobacco and flavors and fragrances with complex matrices, such as many kinds of amino acids and large interference from impurities. The method effectively removes the interference of impurities through multidimensional chromatographic separation, reduces the interference on amino acid separation, is suitable for the simultaneous detection of multiple amino acids, and provides a more accurate and sensitive analysis method for the detection of amino acids in tobacco and flavors and fragrances. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 Schematic diagram of the connection structure of the multi-heart-cutting two-dimensional liquid chromatography system.

[0114] Figure 2 It is the second dimension liquid chromatogram of histidine (His), glutamine (Gln), arginine (Arg), asparagine (Asn), serine (Ser), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala) and proline (Pro) standards.

[0115] Figure 3 This is the second dimension liquid chromatogram of the second dimension of methionine (Met), valine (Val) and norvaline (Nval) standards.

[0116] Figure 4 It is the second dimension liquid chromatogram of cysteine ​​(Cys), lysine (Lys), tyrosine (Tyr), isoleucine (Ile) and leucine (Leu) standards.

[0117] Figure 5 The second dimension liquid chromatogram of tryptophan (Trp) and phenylalanine (Phe) standards.

[0118] Figure 6 This is the second-dimensional liquid chromatogram of amino acids in actual samples.

[0119] Reference numerals:

[0120] 1 Injector;

[0121] 2 One-dimensional pump;

[0122] 3 One-dimensional chromatography column;

[0123] 4 One-dimensional detector;

[0124] 5. Compensation pump;

[0125] 6 Three-way interface;

[0126] 7 Multi-way valve;

[0127] 71 Multi-way valve first position;

[0128] 72 Multi-way valve second position;

[0129] 73 Multi-way valve third position;

[0130] 74 Multi-way valve fourth position;

[0131] 75 Multi-way valve fifth position;

[0132] 76 Multi-port valve sixth position;

[0133] 8 2D pump;

[0134] 9 Two-dimensional chromatography column;

[0135] 10 2D detector;

[0136] 11. Multi-chromatographic selection switching valve;

[0137] 111 a first inlet of a multi-chromatographic selection switching valve;

[0138] 112 a first outlet of a multi-chromatographic selection switching valve;

[0139] 113 a second inlet of a multi-chromatographic selection switching valve;

[0140] 114 a second outlet of the multi-chromatographic selection switching valve;

[0141] 115 a third inlet of the multi-chromatographic selection switching valve;

[0142] 116 a third outlet of the multi-chromatographic selection switching valve;

[0143] 117 a fourth inlet of the multi-chromatographic selection switching valve;

[0144] 118 a fourth outlet of the multi-chromatographic selection switching valve;

[0145] 119 the fifth inlet of the multi-chromatographic selection switching valve;

[0146] 120, a fifth outlet of the multi-chromatographic selection switching valve;

[0147] 121 a sixth inlet of the multi-chromatographic selection switching valve;

[0148] 122 a sixth outlet of the multi-chromatographic selection switching valve;

[0149] 123 Multi-chromatographic selection switching valve "IN" inlet;

[0150] 124 Multi-chromatographic selection switching valve "OUT" outlet;

[0151] 12 first trapping column;

[0152] 13 second trapping column;

[0153] 14 third trapping column;

[0154] 15 fourth trapping column;

[0155] 16 fifth trapping column;

[0156] 17 Sixth trapping column. DETAILED DESCRIPTION

[0157] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0158] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0159] The reagents and instruments used in the following examples are as follows:

[0160] 1. Reagents

[0161] Tobacco leaves, tobacco dust and shredded tobacco are provided by Shanghai Tobacco Group Co., Ltd.; flavors and fragrances are provided by Shanghai Peony Flavors and Fragrances Co., Ltd.

[0162] 20 kinds of amino acids: histidine (His), glutamine (Gln), arginine (Arg), asparagine (Asn), serine (Ser), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala), proline (Pro), methionine (Met), valine (Val), cysteine ​​(Cys), lysine (Lys), tyrosine (Tyr), isoleucine (Ile), leucine (Leu), tryptophan (Trp) and phenylalanine (Phe) were purchased from Sigma-Aldrich; norvaline (Nval) was purchased from Sigma-Aldrich.

[0163] Acetonitrile, methanol (HPLC grade, TEDIA, USA); formic acid (purity not less than 98%, Merck, Germany); concentrated hydrochloric acid (analytical grade, Sinopharm Group); AccQ-TagTM kit (Waters, USA).

[0164] 2. Instruments

[0165] The 1290 liquid chromatograph was equipped with an automatic sample injector (1), a quaternary mixing pump, a binary pump, a monopump, a column oven, a two-position six-way valve, a G4234A / C fast switching valve, a diode array detector (DAD), and a fluorescence detector (FLD) (Agilent, USA); an electronic balance (accuracy: 0.0001 g, Mettler Toledo, Switzerland); an SW12H ultrasonic analyzer (SonoSwiss, Switzerland); a Milli-Q water purifier (Millpore, USA); and an Eppendorf 5810R high-speed centrifuge (Eppendorf, Germany).

[0166] 3. Build system

[0167] The multi-heart-cutting two-dimensional liquid chromatography system for performing multi-heart-cutting two-dimensional liquid chromatography analysis in the present invention is a conventional multi-heart-cutting two-dimensional liquid chromatography system, that is, a multi-chromatographic column selection switching valve is used as a key component of the multi-heart-cutting system. The specific structure is shown in Figure 1 .

[0168] Specifically, the system is connected in sequence to a one-dimensional pump 2, an injector 1, a column oven, a one-dimensional chromatographic column 3, a one-dimensional detector 4, a three-way interface 6, a multi-way valve 7, a two-dimensional chromatographic column 9 and a two-dimensional detector 10, the multi-way valve 7 is connected to the two-dimensional pump 8, the three-way interface 6 is also connected to a compensation pump 5 and a multi-chromatographic column selection switching valve, the capture column unit is connected to different interfaces of the multi-chromatographic selection switching valve 11, the multi-way valve 7 can be a two-position six-way valve or a two-position ten-way valve, the multi-way valve 7 includes a first point 71 of the multi-way valve, a second point 72 of the multi-way valve, a third point 73 of the multi-way valve, a fourth point 74 of the multi-way valve, a fifth point 75 of the multi-way valve, and a sixth point 76 of the multi-way valve, the multi-chromatographic column selection switching valve includes a first inlet 111 of the multi-chromatographic selection switching valve, a first outlet 112 of the multi-chromatographic selection switching valve, and a second inlet 113 of the multi-chromatographic selection switching valve. 13. a second outlet 114 of a multi-chromatogram selection switching valve, a third inlet 115 of a multi-chromatogram selection switching valve, a third outlet 116 of a multi-chromatogram selection switching valve, a fourth inlet 117 of a multi-chromatogram selection switching valve, a fourth outlet 118 of a multi-chromatogram selection switching valve, a fifth inlet 119 of a multi-chromatogram selection switching valve, a fifth outlet 120 of a multi-chromatogram selection switching valve, a sixth inlet 121 of a multi-chromatogram selection switching valve, a sixth outlet 122 of a multi-chromatogram selection switching valve, an “IN” inlet 123 of a multi-chromatogram selection switching valve and an “OUT” outlet 124 of a multi-chromatogram selection switching valve. The interfaces of the multi-way valve 7 and the multi-chromatogram column selection switching valve described above can be increased or decreased according to actual needs. The capture column unit includes a first capture column 12, a second capture column 13, a third capture column 14, a fourth capture column 15, a fifth capture column 16 and a sixth capture column 17.

[0169] The present invention switches the valve on the two-position multi-way valve 7, so that the outflow of the first dimension can be selectively captured by the capture column unit or directly enter the waste liquid, effectively removing the interference of impurities and improving the accuracy and sensitivity. By switching the multi-chromatographic column selection switching valve, the target of the first dimension can be divided into multiple sections and captured on different capture columns. The multi-heart cutting two-dimensional liquid chromatography system in the present invention is divided into a capture mode and an analysis mode, which are as follows:

[0170] In the capture mode, the sample is first separated by the one-dimensional chromatographic column 3, and the chromatographic elution time of the target object is analyzed by the one-dimensional detector 4. Then, the compensation mobile phase is introduced by the compensation pump 5, and mixed with the one-dimensional chromatographic effluent through the three-way interface 6 and then enters the two-position multi-way valve 7. The mixture flows from the first point 71 of the multi-way valve to the second point 72 of the multi-way valve on the two-position multi-way valve 7, and then enters the multi-chromatographic column selection switching valve from the second point 72 of the multi-way valve of the two-position six-way valve, enters from the "IN" inlet (123) of the multi-chromatographic selection switching valve, flows out from the first inlet 111 of the multi-chromatographic selection switching valve, enters the first capture column 12 for capture, and the effluent flows into the multi-chromatographic column selection switching valve again from the first outlet 112 of the multi-chromatographic selection switching valve, and flows out from the "OUT" outlet 124 of the multi-chromatographic selection switching valve. Subsequently, the effluent after capture flows into the fifth point (75) of the multi-port valve, flows from the fifth point 75 of the two-position multi-port valve to the sixth point 76 of the multi-port valve, and then enters the waste liquid. In this mode, the outflow of the first-dimensional chromatographic column 3 can be divided into multiple sections according to the retention time of the target analyte. By switching the position of the multi-chromatographic column selection switching valve, the first section can be captured by the first inlet of the multi-chromatographic switching valve, the first outlet 112 of the multi-chromatographic selection switching valve, and the first capture column 12, and the second section can be captured by the second inlet 113 of the multi-chromatographic switching valve, the second outlet 114 of the multi-chromatographic selection switching valve, and the second capture column 13. By analogy, the target outflow of the one-dimensional chromatographic column 3 can be captured on the first capture column 12 to the sixth capture column 17 respectively by switching the multi-chromatographic column selection switching valve. At the same time, in this mode, the mobile phase of the two-dimensional pump 8 flows from the third position 73 of the multi-way valve to the fourth position 74 of the multi-way valve, and then flows into the two-dimensional chromatographic column 9 to balance the chromatographic column.

[0171] In the analysis mode, the one-dimensional chromatographic effluent is mixed with the mobile phase of the compensation pump 5 through the three-way interface 6, flows from the first point 71 of the multi-way valve to the sixth point 76 of the multi-way valve, and then enters the waste liquid. The mobile phase of the two-dimensional pump 8 flows from the third site 73 of the multi-way valve to the second site 72 of the multi-way valve, and then flows out from the second site 72 of the multi-way valve to enter the "IN" inlet 123 of the multi-chromatogram selection switching valve. The position of the multi-chromatogram column selection switching valve is switched from the first inlet 111 of the multi-chromatogram selection switching valve to the first outlet 112 of the multi-chromatogram selection switching valve. The mobile phase flows from the "IN" inlet 123 of the multi-chromatogram selection switching valve to the first inlet 111 of the multi-chromatogram selection switching valve. The first segment of target analytes are eluted from the first capture column 12 by the change of the mobile phase gradient. The effluent flows into the multi-chromatogram column selection switching valve through the first outlet 112 of the multi-chromatogram selection switching valve, flows from the first outlet 112 of the multi-chromatogram selection switching valve to the "OUT" outlet 124 of the multi-chromatogram selection switching valve, and then flows from the "OUT" outlet 124 of the multi-chromatogram selection switching valve into the fifth site 75 of the multi-way valve, flows from the fifth site 75 of the multi-way valve to the fourth site 74 of the multi-way valve, and then flows into the two-dimensional chromatogram column 9 for further separation, and finally analyzed by the two-dimensional detector 10. In this mode, by switching the position of the multi-chromatogram column selection switching valve (respectively, the first inlet 111 of the multi-chromatogram selection switching valve to the first outlet 112 of the multi-chromatogram selection switching valve, the second inlet 113 of the multi-chromatogram selection switching valve to the second outlet 114 of the multi-chromatogram selection switching valve, the third inlet 115 of the multi-chromatogram selection switching valve to the third outlet 116 of the multi-chromatogram selection switching valve, the fourth inlet 117 of the multi-chromatogram selection switching valve to the fourth outlet 118 of the multi-chromatogram selection switching valve, the fifth inlet 119 of the multi-chromatogram selection switching valve to the fifth outlet 120 of the multi-chromatogram selection switching valve, and the sixth inlet 121 of the multi-chromatogram selection switching valve to the sixth outlet 122 of the multi-chromatogram selection switching valve), the target analytes on the first capture column 12 to the sixth capture column 17 can be eluted in turn and enter the two-dimensional chromatogram column 9 for fine separation, and then detected by a fluorescence detector.

[0172] In the above-mentioned multi-heart-cutting two-dimensional liquid chromatography system, a multi-chromatographic column selection switching valve is used as a key component of the multi-heart-cutting system. In the capture mode, the sample is first separated by the first-dimensional chromatographic column 3, and then the compensation flow path is introduced by the compensation pump 5 to reduce the organic phase ratio and improve the retention efficiency of the target on the capture column. Through the switching of the two-position six-way valve, the effluent of the first dimension can be selectively captured by the capture column or directly enter the waste liquid. This method can remove impurities in certain non-cutting segments in the first dimension to reduce interference with the detection of the target and improve the accuracy and sensitivity of the method. Through the switching of the multi-chromatographic column selection switching valve, the target of the first dimension can be divided into multiple sections and captured on the capture column. Then the system switches to the analysis mode, and the mobile phase of the two-dimensional pump 8 flows through the capture column, eluting the analyte on the capture column section by section, entering the second-dimensional analysis column for fine separation, and then using a fluorescence detector for detection.

[0173] Example 1

[0174] 1. Sample pretreatment

[0175] Accurately weigh 1g of tobacco leaf powder in a 50mL centrifuge tube, add 19.6mL of 0.1mol / mL hydrochloric acid aqueous solution and 400μL of 0.1mol / mL hydrochloric acid aqueous solution containing 2.5nmol / μL norvaline, shake well. Then ultrasonically extract for 30min, centrifuge at 3000r / min, take the supernatant, filter through a 0.22μm aqueous filter membrane, and obtain the sample pretreatment solution.

[0176] 2. Sample pre-column derivatization

[0177] Pipette 70 μL of borate buffer solution as a buffer reagent into a clean sample bottle, add 10 μL of the sample pretreatment solution obtained in step 1, and vortex mix for the first time. Then use a pipette to transfer 20 μL of AccQ-Tag Ultra reagent as a derivatization reagent into the sample bottle for a second vortex mix. After standing at room temperature for 1 minute, place the sample bottle in a constant temperature box and heat at 55°C for 10 minutes. Convert the amino acid into a stable derivative through 6-aminoquinoline-N-hydroxysuccinimidyl formate to obtain a sample test solution. Transfer the sample test solution to a chromatographic bottle and wait for injection.

[0178] 3. Prepare the standard test solution

[0179] The standard samples of 20 amino acids were added to 0.1 mol / mL hydrochloric acid aqueous solution to prepare a mixed stock solution. The concentration of each amino acid in the mixed stock solution was 25 nmol / μL.

[0180] Accurately pipette a series of mixed stock solutions of different volumes into a 10mL volumetric flask, add 200μL of 0.1mol / mL hydrochloric acid aqueous solution containing 2.5nmol / μL norvaline, and then add 0.1mol / mL hydrochloric acid aqueous solution to dissolve and fix the volume to obtain a standard working solution. The standard working solution is derivatized according to the method in the sample column pre-derivation in step 2 to obtain a standard test solution. That is, 70μL of borate buffer solution is pipetted as a buffer reagent into a clean sample bottle, and 10μL of standard working solution is added respectively, and the first vortex mixing is performed. Then, 20μL of CCQ-Tag Ultra reagent is transferred to the sample bottle as a derivatization reagent with a pipette for a second vortex mixing. After standing at room temperature for 1 minute, the sample bottle is placed in a thermostat and heated at 55°C for 10min. The amino acid is converted into a stable derivative by 6-aminoquinoline-N-hydroxysuccinimidyl formate to obtain a standard test solution. The standard test solution is transferred to a chromatographic bottle and waits for injection.

[0181] 4. Determination

[0182] The sample test solution and the standard test solution were measured by a multi-heart-cutting two-dimensional liquid chromatography system. 20 kinds of amino acids were separated by the first-dimensional liquid chromatography according to different retention times and captured on different capture columns. The captured components were eluted and then separated and detected by the second-dimensional liquid chromatography. The retention times were compared for qualitative analysis and the internal standard method was used for quantitative analysis to determine the contents of the 20 kinds of amino acids in the sample test solution.

[0183] The first dimension liquid chromatography conditions are as follows: detector: diode array detector (DAD); first separation column: XBP CN chromatographic column (2.1mm×100mm, 5μm); column temperature: 50℃; injection volume: 2μL; flow rate: 0.3mL / min; detection wavelength: 260nm; mobile phase A: water containing 0.1% formic acid; mobile phase B: methanol; analysis time: 100min; gradient elution.

[0184] The specific procedure of the gradient elution is:

[0185] 0-2min, volume ratio of phase A:phase B was 95:5-95:5;

[0186] 2-12min, volume ratio of phase A:phase B is 95:5-5:95;

[0187] 12-20min, volume ratio of phase A:phase B is 5:95-5:95;

[0188] 20-20.01min, volume ratio of phase A:phase B is 5:95-50:50;

[0189] 20.01-75min, volume ratio of phase A:phase B is 50:50-50:50;

[0190] 75-75.01min, volume ratio of phase A:phase B is 50:50-95:5;

[0191] 75.01-100min, the volume ratio of phase A:phase B is 95:5-95:5.

[0192] The trapping conditions of the trapping column are: Trapping column: XBridge C 18 Chromatographic column (4.6 mm×30 mm, 5 μm); compensation pump mobile phase: deionized water; the flow rate of the compensation pump mobile phase is 1200 μL / min.

[0193] The measurement conditions of the second-dimensional liquid chromatography are as follows: detector: fluorescence detector (FLD); second separation column: Poroshell 120SB-AQ chromatographic column (4.6mm×100mm, 2.7μm); column temperature: 55°C; flow rate: 1.0mL / min; excitation wavelength: 266nm, emission wavelength: 473nm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; analysis time: 100min; gradient elution.

[0194] The specific procedure of the gradient elution is:

[0195] 0-15min, volume ratio of phase A:phase B was 98:2-98:2;

[0196] 15-16min, the volume ratio of phase A:phase B is 98:2-96:4;

[0197] 16-20min, volume ratio of phase A:phase B is 96:4-96:4;

[0198] 20-25min, volume ratio of phase A:phase B is 96:4-80:20;

[0199] 25-25.01min, volume ratio of phase A:phase B is 80:20-10:90;

[0200] 25.01-30min, volume ratio of phase A:phase B is 10:90-10:90;

[0201] 30-30.01min, volume ratio of phase A:phase B is 10:90-100:0;

[0202] 30.01-35min, volume ratio of phase A:phase B is 100:0-100:0;

[0203] 35-45min, volume ratio of phase A:phase B is 100:0-80:20;

[0204] 45-45.01min, volume ratio of phase A:phase B is 80:20-10:90;

[0205] 45.01-50min, volume ratio of phase A:phase B is 10:90-10:90;

[0206] 50-50.01min, volume ratio of phase A:phase B is 10:90-98:2;

[0207] 50.01-55min, volume ratio of phase A:phase B is 98:2-98:2;

[0208] 55-57min, the volume ratio of phase A:phase B is 98:2-90:10;

[0209] 57-65min, volume ratio of phase A:phase B is 90:10-90:10;

[0210] 65-70min, the volume ratio of phase A:phase B is 90:10-75:25;

[0211] 70-70.01min, volume ratio of phase A:phase B is 75:25-10:90;

[0212] 70.01-75min, volume ratio of phase A:phase B is 10:90-10:90;

[0213] 75-75.01min, volume ratio of phase A:phase B is 10:90-98:2;

[0214] 75.01-80min, the volume ratio of phase A:phase B is 98:2-98:2;

[0215] 80-90min, the volume ratio of phase A:phase B is 98:2-40:60;

[0216] 90-90.01min, volume ratio of phase A:phase B is 40:60-10:90;

[0217] 90.01-95min, volume ratio of phase A:phase B is 10:90-10:90;

[0218] 95-95.01min, volume ratio of phase A:phase B is 10:90-98:2;

[0219] 95.01-100min, the volume ratio of phase A:phase B is 98:2-98:2.

[0220] From the above determination results, it can be seen that the 20 amino acids are divided into 4 sections according to the retention time of the first-dimension liquid chromatography separation for capture, and then finely separated and determined by the second-dimension liquid chromatography column. The determination results are shown in Figure 2-5 .from Figure 2-5 It can be seen that, except for Glu and Thr, other amino acids are effectively separated, and the derivatization byproducts do not interfere with the analysis of amino acids. The signal response of Glu and Thr is the same under ACCQ reagent derivatization, and the combined total amount of Glu and Thr can be calculated.

[0221] Example 2

[0222] 1. Sample pretreatment

[0223] Accurately weigh 1g of flavor in a 50mL centrifuge tube, add 19.6mL of 0.1mol / mL hydrochloric acid aqueous solution and 400μL of 0.1mol / mL hydrochloric acid aqueous solution containing 2.5nmol / μL norvaline, shake well. Then ultrasonically extract for 30min, centrifuge at 3000r / min, take the supernatant, pass through a 0.22μm aqueous filter membrane, and obtain the sample pretreatment solution.

[0224] 2. Sample pre-column derivatization

[0225] Pipette 70 μL of borate buffer solution as a buffer reagent into a clean sample bottle, add 10 μL of the sample pretreatment solution obtained in step 1, and vortex mix for the first time. Then use a pipette to transfer 20 μL of AccQ-Tag Ultra reagent as a derivatization reagent into the sample bottle for a second vortex mix. After standing at room temperature for 1 minute, place the sample bottle in a thermostat and heat at 55°C for 10 minutes. Convert the amino acid into a stable derivative through 6-aminoquinoline-N-hydroxysuccinimidyl formate to obtain the test solution. Transfer the test solution to a chromatographic bottle and wait for injection.

[0226] 3. Prepare the standard test solution

[0227] The standard samples of 20 amino acids were added to 0.1 mol / mL hydrochloric acid aqueous solution to prepare a mixed stock solution. The concentration of each amino acid in the mixed stock solution was 25 nmol / μL.

[0228] Accurately pipette a series of mixed stock solutions of different volumes into a 10mL volumetric flask, add 200μL of 0.1mol / mL hydrochloric acid aqueous solution containing 2.5nmol / μL norvaline, and then add 0.1mol / mL hydrochloric acid aqueous solution to dissolve and fix the volume to obtain a standard working solution. The standard working solution is derivatized according to the method in the sample column pre-derivation in step 2 to obtain a standard test solution. That is, 70μL of borate buffer solution is pipetted as a buffer reagent into a clean sample bottle, and 10μL of standard working solution is added respectively, and the first vortex mixing is performed. Then, 20μL of CCQ-Tag Ultra reagent is transferred to the sample bottle as a derivatization reagent with a pipette for a second vortex mixing. After standing at room temperature for 1 minute, the sample bottle is placed in a thermostat and heated at 55°C for 10min. The amino acid is converted into a stable derivative by 6-aminoquinoline-N-hydroxysuccinimidyl formate to obtain a standard test solution. The standard test solution is transferred to a chromatographic bottle and waits for injection.

[0229] 4. Determination

[0230] The sample solution and the standard solution are measured by a multi-heart-cutting two-dimensional liquid chromatography system, and 20 kinds of amino acids are separated by the first-dimensional liquid chromatography according to different retention times, and captured on different capture columns, and then the captured components are eluted and separated and detected by the second-dimensional liquid chromatography, and the retention time is compared for qualitative analysis, and the internal standard method is used for quantitative analysis to determine the content of the 20 kinds of amino acids in the solution to be tested. The measurement conditions of the first-dimensional liquid chromatography and the second-dimensional liquid chromatography are shown in step 4 in Example 1, and the capture conditions of the capture column are shown in step 4 in Example 1.

[0231] Example 3

[0232] 1. Sample pretreatment

[0233] Accurately weigh 1g of tobacco powder into a 50mL centrifuge tube, add 20mL of 0.05mol / mL hydrochloric acid aqueous solution and 450μL of 0.05mol / mL hydrochloric acid aqueous solution containing 2.0nmol / μL norvaline, shake well, then perform ultrasonic extraction for 40min, centrifuge at 3200r / min, take the supernatant, filter through a 0.22μm aqueous filter membrane, and obtain the sample pretreatment solution.

[0234] 2. Sample pre-column derivatization

[0235] Pipette 75 μL of borate buffer solution as a buffer reagent into a clean sample bottle, add 10 μL of the sample pretreatment solution obtained in step 1, and vortex mix for the first time. Then use a pipette to transfer 25 μL of AccQ-Tag Ultra reagent as a derivatization reagent into the sample bottle for a second vortex mix. After standing at room temperature for 65 seconds, place the sample bottle in a constant temperature box and heat it at 60°C for 8 minutes. Convert the amino acid into a stable derivative through 6-aminoquinoline-N-hydroxysuccinimidyl formate to obtain the test solution. Transfer the test solution to a chromatographic bottle and wait for injection.

[0236] 3. Prepare the standard test solution

[0237] The standard samples of 20 kinds of amino acids were added to 0.05 mol / mL hydrochloric acid aqueous solution to prepare a mixed stock solution. The concentration of each amino acid in the mixed stock solution was 25 nmol / μL.

[0238] Accurately pipette a series of mixed stock solutions of different volumes into a 10mL volumetric flask, add 250μL of 0.05mol / mL hydrochloric acid aqueous solution containing 2.0nmol / μL norvaline, and then add 0.05mol / mL hydrochloric acid aqueous solution to dissolve and fix the volume to obtain a standard working solution. The standard working solution is derivatized according to the method in the sample column pre-derivation in step 2 to obtain a standard test solution. That is, 75μL of borate buffer solution is pipetted as a buffer reagent into a clean sample bottle, and 10μL of standard working solution is added respectively, and the first vortex mixing is performed. Then, 25μL of AccQ-Tag Ultra reagent is transferred to the sample bottle as a derivatization reagent with a pipette for a second vortex mixing. After standing at room temperature for 1 minute, the sample bottle is placed in a thermostat and heated at 60℃ for 8min. The amino acid is converted into a stable derivative by 6-aminoquinoline-N-hydroxysuccinimidyl formate to obtain a standard test solution. The standard test solution is transferred to a chromatographic bottle and waits for injection.

[0239] 4. Determination

[0240] The sample test solution and the standard test solution were measured by a multi-heart-cutting two-dimensional liquid chromatography system. 20 kinds of amino acids were separated by the first-dimensional liquid chromatography according to different retention times and captured on different capture columns. The captured components were eluted and then separated and detected by the second-dimensional liquid chromatography. The retention times were compared for qualitative analysis and the external standard method was used for quantitative analysis to determine the contents of the 20 kinds of amino acids in the test solution.

[0241] The determination conditions of the first dimension liquid chromatography are:

[0242] Detector: diode array detector (DAD); first separation column: Venusi XBP CN chromatographic column (2.1 mm×100 mm, 5 μm); column temperature: 55°C; injection volume: 6 μL; flow rate: 0.5 mL / min; detection wavelength: 262 nm; mobile phase A: water containing 0.05% formic acid; mobile phase B: methanol; analysis time: 100 min; gradient elution. The specific procedure of gradient elution is shown in step 4 in Example 1.

[0243] The trapping conditions of the trapping column are: Trapping column: XBridge C 18 Chromatographic column (4.6 mm×30 mm, 5 μm); compensation pump mobile phase: deionized water; the flow rate of the compensation pump mobile phase is 1300 μL / min.

[0244] The conditions for the second-dimensional liquid chromatography are as follows: detector: fluorescence detector (FLD); second separation column: Poroshell 120SB-AQ chromatographic column (4.6 mm×100 mm, 2.7 μm); column temperature: 58° C.; flow rate: 1.5 mL / min; excitation wavelength: 268 nm, emission wavelength: 475 nm; mobile phase A: water containing 0.05% formic acid; mobile phase B: acetonitrile; analysis time: 100 min; gradient elution. The specific procedure of gradient elution is shown in step 4 in Example 1.

[0245] Example 4

[0246] According to step 3 of Example 1, a series of standard test solutions of different concentrations were prepared, and the determination was carried out according to step 4 of Example 1 to obtain the linear relationship between the chromatographic peak area ratio of the standard / internal standard of 20 amino acids and the corresponding concentration ratio. The chromatographic peak area ratio of at least one of the 20 amino acids to the internal standard was used as the ordinate (Y axis), and the concentration ratio of the corresponding amino acid to the internal standard was used as the abscissa (X axis). The corresponding standard working curve was drawn, and the regression equation of the standard working curve was calculated. The standard working curve was continuously diluted, and 3 times and 10 times the signal-to-noise ratio (S / N) were used as the detection limit and quantitative limit of the analytical method, respectively. The results are shown in Table 1, and the method has a good linear relationship (r 2 >0.993), the detection limit was between 0.06-12.25μg / g, and the quantification limit was between 0.18-34.07μg / g.

[0247] Table 1 Linear equations, correlation coefficients, detection limits and quantification limits of amino acids

[0248]

[0249] Example 5

[0250] Select tobacco samples, add standard test solutions with known amino acid concentrations, and perform determination according to the pretreatment, derivatization and determination steps in Example 1. Determine the spiked recovery rate, and the corresponding recovery rates are shown in Table 2. As can be seen from Table 2, except for cystine and tryptophan, which have low responses and partial recoveries of about 75%, the spiked recoveries of most amino acids are between 80.16-108.98%, which is a good recovery rate. At the same time, select tobacco samples, perform 6 parallel determinations according to the pretreatment, derivatization and determination steps in Example 1, and the precision of the method is measured to be between 0.05-9.11%. It can be seen that this method has good precision, meets the detection requirements, and can be used for the quantitative analysis of amino acids in tobacco leaves.

[0251] Table 2 Spiked recovery, intra-day and inter-day precision of amino acids in flue-cured tobacco samples

[0252]

[0253] Example 6

[0254] Select tobacco leaf samples and perform determination according to the pretreatment, derivatization and determination steps in Example 1. Perform analysis. The determination results are shown in Figure 6 .Depend on Figure 6 It can be seen that even for complex samples such as tobacco, the multi-heart-cutting two-dimensional liquid chromatography method can effectively separate 20 amino acids with little interference from impurities, thus laying the foundation for the accurate quantification of amino acids in complex matrix samples.

[0255] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for analyzing amino acids in tobacco or flavors and fragrances, comprising the following steps: 1) Sample pretreatment: Add the tobacco or flavor sample to the solvent and internal standard solution, mix, ultrasonically extract, centrifuge, and filter to obtain the sample pretreatment solution; 2) Sample pre-column derivatization: the sample pretreatment solution obtained in step 1) is mixed with the buffer reagent and the derivatization reagent in the derivatization kit respectively, and then heated for derivatization to obtain a sample test solution; 3) Sample analysis: The sample solution obtained in step 2) is measured by multiple heart-cutting two-dimensional liquid chromatography to determine the content of at least one of the 20 amino acids in the sample solution; The 20 amino acids are histidine, glutamine, arginine, asparagine, serine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, methionine, valine, cysteine, lysine, tyrosine, isoleucine, leucine, tryptophan and phenylalanine; In step 2), the derivatization kit is an AccQ-Tag Ultra kit, the buffer reagent is a borate buffer solution provided in the AccQ-Tag Ultra kit, and the derivatization reagent is an AccQ-Tag Ultra reagent provided in the AccQ-Tag Ultra kit, the main component of which is 6-aminoquinoline-N-hydroxysuccinimide carbamate; In step 3), the multi-heart-cutting two-dimensional liquid chromatography method comprises the following steps: A) adding a solvent to a standard of at least one of the 20 amino acids, and then adding an internal standard solution, the prepared standard working solution is mixed with a buffer reagent and a derivatization reagent in a derivatization kit, and then heated for derivatization to obtain a standard test solution; B) the sample solution and the standard solution obtained in step A) are measured by a multi-heart-cutting two-dimensional liquid chromatography system, at least one of the 20 amino acids is separated by first-dimensional liquid chromatography according to different retention times, captured on different capture columns, and then the captured components are eluted and separated and detected by second-dimensional liquid chromatography, the retention times are compared for qualitative analysis, and the internal standard method is used for quantitative analysis to determine the content of at least one of the 20 amino acids in the sample solution; In step B), the determination conditions of the first dimension liquid chromatography are: detector: diode array detector; first separation column: CN chromatographic column; column temperature: 45-55°C; injection volume: 1-10µL; flow rate: 0.1-0.5mL / min; detection wavelength: 255-265nm; mobile phase A: water containing 0.05-0.1% formic acid; mobile phase B: methanol; analysis time: 100min; gradient elution; The specific procedure of the gradient elution of the first dimension liquid chromatography is: 0-2min, volume ratio of phase A:phase B was 95:5-95:5; 2-12min, volume ratio of phase A:phase B is 95:5-5:95; 12-20min, volume ratio of phase A:phase B is 5:95-5:95; 20-20.01min, volume ratio of phase A:phase B is 5:95-50:50; 20.01-75min, volume ratio of phase A:phase B is 50:50-50:50; 75-75.01min, volume ratio of phase A:phase B is 50:50-95:5; 75.01-100min, the volume ratio of phase A:phase B is 95:5-95:5; In step B), the trapping conditions of the trapping column are: trapping column: C 18 Chromatographic column; compensation pump mobile phase: at least one selected from deionized water, phosphate buffer, acetate buffer, formate buffer, and ammonia solution; the flow rate of the compensation pump mobile phase is 1000-1500 μL / min; In step B), the determination conditions of the second dimension liquid chromatography are as follows: detector: fluorescence detector; second separation column: AQ chromatographic column; column temperature: 50-60°C; flow rate: 0.5-2 mL / min; excitation wavelength: 264-268 nm, emission wavelength: 471-475 nm; mobile phase A: water containing 0.05-1% formic acid; mobile phase B: acetonitrile; analysis time: 100 min; gradient elution; The specific procedure of the gradient elution of the second dimension liquid chromatography is: 0-15min, volume ratio of phase A:phase B was 98:2-98:2; 15-16min, the volume ratio of phase A:phase B is 98:2-96:4; 16-20min, volume ratio of phase A:phase B is 96:4-96:4; 20-25min, volume ratio of phase A:phase B is 96:4-80:20; 25-25.01min, volume ratio of phase A:phase B is 80:20-10:90; 25.01-30min, volume ratio of phase A:phase B is 10:90-10:90; 30-30.01min, volume ratio of phase A:phase B is 10:90-100:0; 30.01-35min, volume ratio of phase A:phase B is 100:0-100:0; 35-45min, volume ratio of phase A:phase B is 100:0-80:20; 45-45.01min, volume ratio of phase A:phase B is 80:20-10:90; 45.01-50min, volume ratio of phase A:phase B is 10:90-10:90; 50-50.01min, volume ratio of phase A:phase B is 10:90-98:2; 50.01-55min, volume ratio of phase A:phase B is 98:2-98:2; 55-57min, the volume ratio of phase A:phase B is 98:2-90:10; 57-65min, volume ratio of phase A:phase B is 90:10-90:10; 65-70min, the volume ratio of phase A:phase B is 90:10-75:25; 70-70.01min, volume ratio of phase A:phase B is 75:25-10:90; 70.01-75min, volume ratio of phase A:phase B is 10:90-10:90; 75-75.01min, volume ratio of phase A:phase B is 10:90-98:2; 75.01-80min, the volume ratio of phase A:phase B is 98:2-98:2; 80-90min, the volume ratio of phase A:phase B is 98:2-40:60; 90-90.01min, volume ratio of phase A:phase B is 40:60-10:90; 90.01-95min, volume ratio of phase A:phase B is 10:90-10:90; 95-95.01min, volume ratio of phase A:phase B is 10:90-98:2; 95.01-100min, the volume ratio of phase A:phase B is 98:2-98:

2.

2. The method for analyzing amino acids in tobacco or flavors and fragrances according to claim 1, characterized in that: Step 1) includes any one or more of the following conditions: A1) the tobacco is selected from at least one of tobacco leaves, tobacco dust or tobacco shreds; A2) the solvent is selected from at least one of a hydrochloric acid aqueous solution and a methanol aqueous solution; A3) The ratio of the added mass of the tobacco or flavor sample to the added volume of the solvent is 1:10-20, g / mL; A4) the internal standard solution is a hydrochloric acid aqueous solution containing norvaline; A5) the volume ratio of the internal standard solution to the solvent added is 1:30-70; A6) The ultrasonic extraction time is 20-40 minutes; A7) The centrifugal speed is 2800-3200 r / min; A8) The filtration is to filter the supernatant after centrifugation using a membrane.

3. The method for analyzing amino acids in tobacco or flavors and fragrances according to claim 1, characterized in that: Step 2) includes any one or more of the following conditions: B1) the volume ratio of the sample pretreatment solution to the buffer reagent added is 10:50-90; B2) the volume ratio of the sample pretreatment solution to the derivatization reagent is 10:10-40; B3) The derivatization is to mix the sample pretreatment solution with the buffer reagent for a first time, then add the derivatization reagent for a second mixing and then heat for derivatization; B4) The heating temperature is 50-60°C; B5) The heating time is 5-11 minutes.

4. The method for analyzing amino acids in tobacco or flavors and fragrances according to claim 3, characterized in that: Item B3 includes any one or more of the following conditions: C1) the first mixing and the second mixing are vortex mixing; C2) The mixing time of the first mixing is 5-15s; C3) The mixing time of the second mixing is 2-10s; C4) The second mixing is followed by standing.

5. The method for analyzing amino acids in tobacco or flavors and fragrances according to claim 1, characterized in that: Step A) includes any one or more of the following conditions: D1) The standard working solution is obtained by adding a standard of at least one of the 20 amino acids to a solvent to obtain a mixed stock solution, adding an internal standard solution after stepwise dilution, and then adding a solvent to dissolve and fix the volume; D2) the solvent is selected from at least one of a hydrochloric acid aqueous solution and a methanol aqueous solution; D3) the internal standard solution is a hydrochloric acid aqueous solution containing norvaline; D4) the derivatization kit is an AccQ-Tag Ultra kit, the buffer reagent is a borate buffer solution provided in the AccQ-Tag Ultra kit, and the derivatization reagent is an AccQ-Tag Ultra reagent provided in the AccQ-Tag Ultra kit; D5) The volume ratio of the standard working solution to the buffer reagent added is 10:50-90; D6) the volume ratio of the standard working solution to the derivatization reagent is 10:10-40; D7) The derivatization is to first mix the standard working solution with the buffer reagent, then add the derivatization reagent for a second mixing and then heat for derivatization; D8) The heating temperature is 50-60°C; D9) The heating time is 5-11 minutes.

Images