CN104634897B - A kind of method detecting cigarette mainstream flue gas middle and advanced stage advanced glycation end products - Google Patents

A kind of method detecting cigarette mainstream flue gas middle and advanced stage advanced glycation end products Download PDF

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CN104634897B
CN104634897B CN201510077987.1A CN201510077987A CN104634897B CN 104634897 B CN104634897 B CN 104634897B CN 201510077987 A CN201510077987 A CN 201510077987A CN 104634897 B CN104634897 B CN 104634897B
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flue gas
products
mainstream flue
cigarette
solution
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CN104634897A (en
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余晶晶
颜权平
王昇
赵阁
王冰
蔡君兰
丁丽
于永杰
谢复炜
张晓兵
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Zhengzhou Tobacco Research Institute of CNTC
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Zhengzhou Tobacco Research Institute of CNTC
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Abstract

A kind of method of detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products (AGEs), specifically includes carboxymethyl-lysine (CML) and lysopine (CEL)) analysis mensuration.After the method includes using cambridge filter trapping cigarette smoke condensates, use hydrochloric acid solution ultrasonic extraction filter disc, be analyzed in conjunction with cation exchange solid-phase extraction column purification method and liquid chromatography-tandem mass spectrometry technology (LC-MS/MS).It is a feature of the present invention that: (1) sets up the detection method of AGEs in cigarette mainstream flue gas first;(2) according to the feature of cigarette smoke sample substrate, establishing sample-pretreating method, it is not necessary to derivative reaction, pre-treatment is the most efficient;(3) the method accuracy highly sensitive, qualitative is good, it is adaptable to the analysis of batch samples.

Description

A kind of method detecting cigarette mainstream flue gas middle and advanced stage advanced glycation end products
Technical field
The invention belongs to harmful substance analysis and detection technology research in cigarette mainstream flue gas, be specifically related to a kind of liquid phase color Spectrum-tandem mass spectrometry detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products (AGEs) (includes carboxymethyl-lysine (CML) With lysopine (CEL)) method.
Background technology
Advanced glycation endproducts (AGEs) is that sugar generates with protein or fat reaction, is primarily present in food The food that high-temperature heating is cooked.When AGEs content is higher, human immune system is in inflammatory status, has promotion inflammation, the heart The illeffectss such as angiopathy, diabetes and acceleration are old and feeble[1].At present the AGEs of most study be carboxymethyl-lysine (CML) and Lysopine (CEL).The generation of AGEs has certain associating with Maillard reaction, is reducing sugar and protein, fat etc. Reaction is formed, and intermediate product is schiff base compounds and the Amadori product of series, forms more stable AGEs afterwards[2].Nicotiana tabacum L. rich in proteins and recuding sugars compound, during modulating, be aged, process and burning and sucking, the most to some extent There is Maillard reaction, generate multiple tobacco flavor composition.Medicated cigarette is in combustion, it may occur that Maillard reaction, possesses late The Production conditions of phase advanced glycation end products.This provides Potential feasibility for the formation of AGEs in Nicotiana tabacum L..But, cigarette product Final consumption form be to suck flue gas.Therefore, investigate the ten points of necessity of AGEs in cigarette smoke, have not yet to see report Medicated cigarette The analyzing detecting method of AGEs in flue gas.
The detection method of AGEs mainly includes euzymelinked immunosorbent assay (ELISA), high performance liquid chromatography, gas chromatography-mass spectrography, liquid phase Chromatograph-tandem mass spectrometry (LC-MS/MS).Compared with other analysis method, LC-MS/MS method sensitivity is higher, and second order ms Can more effectively get rid of false positive, qualitative accuracy is higher, has been widely used for the detection of AGEs in processed food.Due to for The particularity of food samples substrate, first needs sample carries out the steps such as defat, reduction, protein precipitation, proteolysis Suddenly[3], then carry out solid phase extraction techniques[3]Or derivative reaction[4]To improve detection sensitivity, pre-treatment is the most loaded down with trivial details to take Time.The present invention is directed to cigarette smoke substrate and establish sample-pretreating method, and directly use LC-MS/MS technology to be analyzed, Without derivative reaction, pre-treatment is the most efficient.
Summary of the invention
The purpose of the present invention is based on above-mentioned prior art situation, includes carboxylic first for AGEs(in cigarette mainstream flue gas Base lysine (CML) and lysopine (CEL)) detection and a kind of LC-MS/MS of providing analyzes method.
It is an object of the invention to be achieved through the following technical solutions:
A kind of method detecting cigarette mainstream flue gas middle and advanced stage advanced glycation end products, is by liquid chromatography-tandem matter Spectrometry detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products (AGEs) includes that carboxymethyl-lysine (CML) and carboxyethyl rely The method of propylhomoserin (CEL), its concrete steps include:
The preparation of a, sample and process: smoking cigarette sample, and trap cigarette smoke condensates with cambridge filter.Will suction The filter disc crossed adds in conical flask, adds the hydrochloric acid solution of certain volume and mixes deuterated inner mark solution, carrying out ultrasonic extraction.Take Partial extraction solution is loaded on the solid-phase extraction column that activated, successively with hydrochloric acid solution and the water wash of certain volume, The ammonia water-methanol solution of rear employing certain volume carries out eluting to solid-phase extraction column.In solid phase extraction procedure, liquid keeps certain Flow velocity.Eluent uses nitrogen to dry up, and is subsequently adding 1mL water and redissolves, and takes the solution after redissolution in chromatogram bottle, enters LC-MS/ MS Instrumental Analysis.
It is C18 liquid-phase chromatographic column that b, sample analysis condition: LC-MS/MS analyze chromatographic column used;Column temperature: 30oC;Flowing Phase A: the water (volume ratio) containing 0.1% formic acid, Mobile phase B: the acetonitrile (volume ratio) containing 0.1% formic acid;Flow velocity 300 μ L/ min;Sampling volume 10 μ L;Condition of gradient elution is shown in Table 1.
Table 1: condition of gradient elution
Time (min) Mobile phase A (%) Mobile phase B (%)
0 100 0
5 100 0
5.5 0 100
14 0 100
14.5 100 0
20 100 0
Ion source: electron spray ionisation source (ESI);Scan mode: cation scans;Detection mode: multiple-reaction monitoring MRM; Electron spray voltage: 5000V;Gas curtain atmospheric pressure: 30 psi;Auxiliary gas 1 pressure: 70 psi;Auxiliary gas 2 pressure: 70 psi;Ion Source temperature: 550oC.The internal standard of each compound, parent ion, daughter ion, residence time, collision energy (CE) and remove a bunch voltage (DP) table 2 is seen.
Table 2: the internal standard of each compound, parent ion, daughter ion, residence time, collision energy and remove a bunch voltage
aQuota ion pair
In the present invention, the tool plug ground conical flask that container is 50 mL is collected described in step a;Taking out described in step a Inhaling Medicated cigarette is 4 ~ 10;Extraction solution described in step a is HCl solution, and concentration range is 0.1M ~ 0.5M, and amount ranges is 20 ~ 60 mL, ultrasonic extraction time range is 15 ~ 60 min;Taking partial extraction liquor capacity in step a is 15 ~ 30mL; Step a mixes deuterated in be designated as d4-CML and d4-CEL, consumption is 100 L;In step a, solid-phase extraction column is that cation is handed over Changing solid-phase extraction column, model is Dikma ProElut PXC (150mg/6mL/30pkg);Solid-phase extraction column activation in step a Step for being sequentially added into 3mL methanol, 3mL0.1M hydrochloric acid solution;In step a, solid-phase extraction column rinsing step is for being sequentially added into 3mL0.1M hydrochloric acid, 3mL water;In step a, solid-phase extraction column eluent consists of 5% ammonia water-methanol solution, and elution volume is 5 ~ 10mL;In step a in solid phase extraction procedure flow rate of liquid for naturally to flow down by gravity;In step a, nitrogen dries up employing automatically Nitrogen blows concentrating instrument, and bath temperature is 35oC。
In the present invention, the C18 liquid-phase chromatographic column model described in step b is Agilent Proshell 120 SB- C18 (2.7 μm, 3.0 × 150mm).
The standard conditions that the present invention uses straight line smoking machine to specify according to YC/T29-1996 at cigarette smoking are aspirated, every time Experiment 4 Medicated cigarette of suction.
Use the water preparation mixing mother solution of CML, CEL, deuterated mixing inner mark solution and standard working solution.Standard work is molten Liquid containing the internal standard d4-CML and d4-CEL concentration are 1 ng/mL.Respectively standard working solution is carried out LC-MS/MS analysis, and with CML, CEL carry out linear regression with internal standard substance peak area ratio to concentration ratio, obtain the standard working curve of each target compound.Separately Outward, using the solution after cigarette smoke sample pre-treatments is substrate, adds standard solution and deuterated mixing inner mark solution, carries out base Plasmogamy mark is tested, and sample uses LC-MS/MS to analyze, and it is as shown in table 3 that gained substrate joins mark linear equation.As known from Table 3, two groups The slope of linear equation is close, shows the impact using deuterated internal standard can effectively reduce matrix effect in this method, can use molten Agent is joined mark and is carried out sample analysis.Take least concentration standard working solution, 10 parallel assays, calculate standard deviation, 3 times of standard deviation Difference is detection limit, and 10 times of standard deviations are quantitative limit, the results are shown in Table 3.
The solvent of table 3:CML, CEL join mark and substrate join mark concentration range, linear equation,
Detection limit and quantitative limit
Same cigarette sample is all added 10 ng CML and CEL, in one day, carries out 6 parallel assays and point 5 days mensuration, The relative standard deviation (RSD) of measurement result represents withinday precision and the day to day precision of the method.Carry out mark-on to return simultaneously Yield is tested, and result is as shown in table 4.Visible, the precision of the inventive method experiment is good, recovery of standard addition is high, it is adaptable to large quantities of The analysis of amount sample.
Withinday precision, day to day precision and the response rate of table 4 method
Beneficial effects of the present invention is as follows:
(1) establish AGEs(in cigarette mainstream flue gas first and include CML, CEL) analyzing detecting method;(2) present invention Employed in LC-MS/MS method, compare in document euzymelinked immunosorbent assay (ELISA), LC method and the GC-MS method etc. of report, highly sensitive, and two grades Mass spectrum can more effectively get rid of false positive, and qualitative accuracy is higher;(3) according to the feature of cigarette smoke sample substrate, establish Sample-pretreating method, it is not necessary to derivative reaction, pre-treatment is the most efficient.
Accompanying drawing explanation
Fig. 1 is LC-MS/MS multiple-reaction monitoring mass spectrum (MRM) figure of CML.
Fig. 2 is LC-MS/MS multiple-reaction monitoring mass spectrum (MRM) figure of d4-CML.
Fig. 3 is LC-MS/MS multiple-reaction monitoring mass spectrum (MRM) figure of CEL.
Fig. 4 is LC-MS/MS multiple-reaction monitoring mass spectrum (MRM) figure of d4-CEL.
Detailed description of the invention
The present invention is described further below with reference to embodiment:
Embodiment 1
The standard conditions suction specified according to YC/T29-1996 by Medicated cigarette after balance and screening, uses cambridge filter to catch Collect 4 Cigarette grain phases.Filter disc is placed in 50 mL tool plug ground conical flasks, adds 100 Ld4-CML and d4-CEL molten Liquid, adds 30mL0.1M hydrochloric acid solution, ultrasonic extraction 30min.Take 15mL extraction solution and be loaded to Dikma ProElut PXC (150mg/6mL/30pkg) (3mL methanol and 3mL0.1M hydrochloric acid before loading, is used to live successively on cation exchange solid-phase extraction column Change), use 3mL0.1M hydrochloric acid and 3mL water wash successively, finally with 10mL5% ammonia water-methanol solution eluting.Solid phase extraction procedure Middle liquid flows down naturally by action of gravity.Eluent uses nitrogen to blow concentrating instrument, and (bath temperature is 35oC) dry up, be subsequently adding 1mL Water redissolves, and solution adds employing LC-MS/MS Instrumental Analysis in chromatogram bottle.
Embodiment 2
Choose Kentucky with reference to Medicated cigarette 3R4F and 10 kinds of commercial cigarettes sample, the mark specified according to YC/T 29-1996 respectively Floating screed part aspirates, and uses cambridge filter 4 Cigarette grain phases of suction, then according to the described step of embodiment 1 carries out sample Treatment Analysis.Experimental result is all not detect in Kentucky is with reference to the main flume of Medicated cigarette 3R4F and 10 kinds of commercial cigarettes samples CML、CEL。
List of references
[1] Cai Chenggang, Zhang Hui, Li He, Lu Yin etc., food research and development, 2013,34,280-282.
[2] Vlassara HMD, Striker GMD, Current diabetes reports, 2007, 7(3): 235-241.
[3] Hegele J, Buetler T, Delatour T, Analytica Chimica Acta, 2008, 617, 85-96.
[4] Hashimoto C, Iwaihara Y, Chen SJ, et.al, Analytical chemistry, 2013, 85, 4289-4295。

Claims (6)

1. the method detecting cigarette mainstream flue gas middle and advanced stage advanced glycation end products, it is characterised in that: it is by liquid phase color Spectrum-tandem mass spectrometry detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products includes that carboxymethyl-lysine and carboxyethyl rely ammonia The method of acid, concrete steps include:
The preparation of a, sample and process: smoking cigarette sample, and trap cigarette smoke condensates with cambridge filter, by aspirated Filter disc adds in conical flask, adds hydrochloric acid solution and the deuterated inner mark solution of mixing of 100 L of 20 ~ 60 mL, carries out ultrasonic extraction Take;Take on the solid-phase extraction column that partial extraction solution is loaded to activate, successively with hydrochloric acid solution and the 3mL water of 3mL 0.1M Drip washing, finally uses the ammonia water-methanol solution of 5 ~ 10mL that solid-phase extraction column is carried out eluting, and in solid phase extraction procedure, liquid keeps Naturally the flow velocity flowed down by gravity, eluent uses nitrogen to dry up, and is subsequently adding 1mL water and redissolves, takes the solution after redissolution to color In spectrum bottle, enter LC-MS/MS Instrumental Analysis;
It is C18 liquid-phase chromatographic column that b, sample analysis condition: LC-MS/MS analyze chromatographic column used, and chromatographic column model is Agilent Proshell 120 SB-C18, specification 2.7 μm, 3.0 × 150mm;Column temperature: 30oC;Mobile phase A: containing 0.1% formic acid Water, Mobile phase B: containing the acetonitrile of 0.1% formic acid, flow velocity 300 μ L/min, sampling volume 10 μ L, gradient elution;Ion source: Electron spray ionisation source (ESI);Scan mode: cation scans;Detection mode: multiple-reaction monitoring MRM;Electron spray voltage: 5000V;Gas curtain atmospheric pressure: 30 psi;Auxiliary gas 1 pressure: 70 psi;Auxiliary gas 2 pressure: 70 psi;Ion source temperature: 550oC;
Condition of gradient elution is as follows:
Time m-min mobile phase A-% Mobile phase B-%
0 100 0
5 100 0
5.5 0 100
14 0 100
14.5 100 0
20 100 0。
The method of detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products the most according to claim 1, its feature exists In: the smoking cigarette described in step a is 4 ~ 10, and cigarette smoking uses straight line smoking machine to specify according to YC/T29-1996 Standard conditions are aspirated.
The method of detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products the most according to claim 1, its feature exists In: the extraction solution described in step a is hydrochloric acid solution, and concentration range is 0.1M ~ 0.5M, ultrasonic extraction time range is 15 ~ 60 min。
The method of detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products the most according to claim 1, its feature exists In: step a mixes deuterated in be designated as d4-carboxymethyl-lysine and d4-lysopine.
The method of detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products the most according to claim 1, its feature exists In: in step a, solid-phase extraction column is that cation exchanges solid-phase extraction column, and model is Dikma ProElut PXC, specification 150mg/6mL/30pkg, is sequentially added into 3mL methanol, and 3mL0.1M hydrochloric acid solution activates.
The method of detection cigarette mainstream flue gas middle and advanced stage advanced glycation end products the most according to claim 1, its feature exists In: in step a, solid-phase extraction column eluent consists of 5% ammonia water-methanol solution.
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CN104965040B (en) * 2015-05-22 2016-08-10 武汉轻工大学 Free state and the detection method of combined state carboxymethyl-lysine in milk and milk products
CN106124641B (en) * 2016-05-13 2019-01-29 中华全国供销合作总社杭州茶叶研究所 A kind of synchronous method for detecting carboxymethyl-lysine and lysopine in green tea
CN106353434B (en) * 2016-10-21 2018-10-09 中国烟草总公司郑州烟草研究院 A kind of analysis method quantitative determining Amadori compounds in tobacco
CN108776131B (en) * 2018-03-19 2020-06-09 深圳市南山区疾病预防控制中心 Method for detecting AGEs content in food
CN108982700B (en) * 2018-08-15 2021-03-23 江苏省原子医学研究所 Method for studying inhibition effect of PQQ on AGEs
CN111122733A (en) * 2019-12-27 2020-05-08 江南大学 Method for synchronously measuring three late glycosylation products

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