CN109932468A - A kind of synchronous method for detecting free state CML and 5-HMF in liquid or semisolid flavouring - Google Patents
A kind of synchronous method for detecting free state CML and 5-HMF in liquid or semisolid flavouring Download PDFInfo
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Abstract
The invention discloses the methods of free state CML and 5-HMF in a kind of synchronous detection liquid or semisolid flavouring.Firstly, carrying out the detection pre-treatment of free state CML and 5-HMF in liquid or semisolid flavouring system, the sample to be tested Jing Guo pre-treatment is then subjected to CML detection synchronous with 5-HMF's；The pre-treatment step are as follows: (1) be diluted with water, be uniformly mixed, centrifugal treating into survey liquid or semisolid flavouring；(2) supernatant after taking centrifugal treating adds water to elute using PolyClean X-MCX Solid Phase Extraction column purification, then ammoniates water-methanol solution elution, collects eluent；(3) eluent is dry, it then redissolves in methanol-water solution, through micro-filtration film process, sample to be tested is made.The method of the invention have the characteristics that reduce matrix effect, exclusive PCR, it is easy to operate efficiently, safe and reliable, detection efficiency is high.
Description
Technical field
The invention belongs to harmfulness component detection method fields in food, and in particular to a kind of synchronous detection liquid or half
The method of free state CML and 5-HMF in solid seasoning.The effective streamline operation of this method improves detection efficiency, can fit
With the detection of plurality of liquid or semisolid flavouring.
Background technique
CML (carboxylic first lysine) is one of product of Maillard reaction, is widely present in all kinds of conventional foods, can be
It is absorbed and accumulates in human body, covalent cross-linking easily occurs with amino acid, peptide and protein etc., have to human body potentially hazardous
Property.5-HMF (5 hydroxymethyl furfural) is a kind of furfuran compound, can equally be generated by Maillard reaction, it is existing research shows that
The 5-HMF of high concentration has cytotoxicity, and to eyes, the upper respiratory tract, skin and mucous membrane are irritant.5-HMF master in diet
Will be by urine tachymetabolism and excretion, while having a small amount of covalent bond in kidney, bladder and liver, there are carcinogenic risks.
It mainly include ultraviolet light photometry (UV), gas chromatography at present for the detection of harmfulness small-molecule substance
(GC), combined gas chromatography mass spectrometry (GC-MS, GC-MS/MS), liquid chromatography (HPLC) and liquid chromatography mass are combined method
(HPLC-MS, HPLC-MS/MS).Wherein furfural etc. has the methods of substance migration UV and HPLC of UV absorption relatively broad, and
The substance without UV absorption such as CML, acrylamide and imidazoles is then mainly combined method as detection using gas phase or liquid chromatography mass spectrometric
Method.In contrast mass spectrometry system has higher accuracy, and HPLC-MS/MS is not necessarily to spread out to sample progress volatility
Biochemical treatment reduces operation link, is conducive to improve Detection accuracy.
Conventional process of the food samples before HPLC-MS/MS detection includes removing macromolecular fat, protein, shallow lake
Powder etc., this sequence of maneuvers process very complicated, takes a long time.Main component in soy sauce includes water, amino acid, peptide, carbohydrate
Deng, and be in a liquid state.At present for the harmfulness composition detection in soy sauce, it is related to the less of CML, and 5-HMF is mostly with UV absorption
Based on detection, since the color of soy sauce itself is deeper, if decoloration not exclusively has a certain impact to testing result；And it many grinds
Study carefully it has been shown that free state CML content compared with reference state is higher by the 1-2 order of magnitude in soy sauce, it is known that CML is mainly with free in soy sauce
State form exists.
There are not the HPLC-MS/MS detection method of free state CML and 5-HMF in synchronous detection soy sauce also at present, for two
The exploration of the pretreatment process and testing conditions of person is few.
Summary of the invention
To solve the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of synchronous detection
The method of free state CML and 5-HMF in liquid or semisolid flavouring.
This method only needs a pretreatment process, high efficient detection can go out two in liquid or semisolid flavouring simultaneously
The content of the common harmfulness ingredient of kind；The content of two kinds of common harmfulness ingredients especially in soy sample, while can be used for depth
Enter to analyze the generating process of CML and 5-HMF in liquid or semisolid flavouring, for the life of regulation liquid or semisolid flavouring
It produces and establishes Research foundation with the content of CML and 5-HMF in preparation process, simultaneously for liquid or semisolid production of condiments mistake
Maillard reaction correlation harmfulness ingredient has directive significance in journey, and this method especially has directive significance to soy sauce.
The object of the invention is achieved through the following technical solutions:
A kind of synchronous method for detecting free state CML and 5-HMF in liquid or semisolid flavouring, firstly, carrying out liquid
Or in semisolid flavouring system free state CML and 5-HMF detection pre-treatment, then by the sample to be tested Jing Guo pre-treatment
Carry out CML detection synchronous with 5-HMF's；
The detection pre-treatment of free state CML and 5-HMF in the liquid or semisolid flavouring system, including following step
It is rapid:
(1) it is diluted with water, is uniformly mixed, centrifugal treating into liquid or semisolid flavouring；
(2) supernatant after taking centrifugal treating adds water to elute using PolyClean X-MCX Solid Phase Extraction column purification,
Then plus volume ratio elutes for the ammonia water-methanol solution of 10:90, collects eluent；Every 1-2mL supernatant corresponds to 10-20mL water
With 10-20mL ammonia water-methanol solution；
(3) eluent is dry, it then redissolves in methanol-water solution, through micro-filtration film process, sample to be tested is made.
Herein described first alcohol and water is chromatographic grade.
Step (1) liquid or semisolid flavouring are preferably soy sauce.
Preferably every 1mL liquid flavoring is diluted with water described in step (1) or 1g semisolid flavouring is diluted to 5-
10mL。
Step (1) the preferably vortex that is uniformly mixed mixes.
The centrifugal force of step (1) described centrifugal treating is preferably 7000g；Centrifugation time is preferably 15min；Centrifuging temperature
Preferably 4 DEG C.
Step (2) the PolyClean X-MCX solid-phase extraction column is activated using preceding through 5-10mL methanol, then plus 5-
10mL pure water equilibrium.
Step (3) the methanol-water solution dosage is preferred are as follows: supernatant described in every 1mL step (2) corresponds to 5-10mL
Step (3) described methanol-water solution, more preferably 10mL.
The volume ratio of methanol and water is preferably 10:90 in step (3) described methanol-water solution.
Step (3) drying means is preferably dried with nitrogen.
The aperture of step (3) described microfiltration membranes is preferably 0.22-0.45 μm；More preferably 0.22 μm.
The detection method is preferably High Performance Liquid Chromatography/Mass Spectrometry (HPLC-MS/MS) method.
The high performance liquid chromatography uses 1260 type of Agilent, and testing conditions are as follows:
Column model: ACE Proshell 120 SB-C18 chromatographic column 50 × 3.0mm, 2.7 μm；
Column temperature: 30 DEG C；
Mobile phase: methanol: 0.1% formic acid water=10:90；
Flow velocity: 0.5mL/min；
Sample volume: 10 μ L；
Mass spectrum is triple level four bars mass spectrums, using 6460 mass spectrograph of Agilent, testing conditions are as follows:
Ion source: ESI+；
Ion source temperature: 300 DEG C；
Capillary voltage: 3.5kV；
Taper hole throughput: 9L/min；
MSI level four bars temperature: 100 DEG C；
MS2 level four bars temperature: 100 DEG C；
Desolventizing temperature: 300 DEG C；
Nebulizer pressure: 15psi；
Detection mode: multiion reaction monitoring (MRM).
The MRM parameter such as table 1:
The Mass Spectrometer Method condition of table 1 CML and HMF
Note: wherein the ion of band " * " is quantitative daughter ion
Compared with prior art, the present invention has the following advantages and beneficial effects:
(1) present invention is separated using PolyClean X-MCX Solid Phase Extraction (SPE) column, can be efficiently separated out and be detected
Object CML, 5-HMF, to achieve the purpose that free state CML and 5-HMF in synchronous detection soy sauce.
(2) pre-treating method of the present invention is simple, easy to operate, avoids complicated pretreatment process bring experimental error；
It is cleaned by specific ion column, reduces the interference of Mechanism and effect；Free state CML and 5-HMF can be detected simultaneously, improve
Detection efficiency saves plurality of reagents and instrument loss, reduces testing cost.
(3) the method for the invention can be widely applied to free state CML and 5- in plurality of liquid and semisolid flavouring
The qualitative and quantitative detection of HMF.
(4) the method for the invention operates accurate, high sensitivity；It detects during limit is surveyed with hybrid standard product examine 3 times
Signal-to-noise ratio (S/N=3) is minimum detection limit (LOD), 10 times of signal-to-noise ratio (S/N=3) are minimum quantitative limit (LOQ).
Detailed description of the invention
The second order ms detection gained fragment ion and abundance that Fig. 1 is CML.
The second order ms detection gained fragment ion and abundance that Fig. 2 is 5-HMF.
Fig. 3 is the chromatogram of CML and HMF hybrid standard product under different mobile phases.
Fig. 4 is that different chromatographic columns influence the chromatogram of CML and HMF.
Fig. 5 is the standard curve of CML concentration and mass signal peak area relationship.
Fig. 6 is the standard curve of 5-HMF concentration and mass signal peak area relationship.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are not
It is limited to this.
1, the establishment of herein described method accuracy
The determination of mass spectrum and chromatographic parameter is all made of standard items (CML, 5 μ g/mL；5-HMF, 5 μ g/mL, the mixing of the two
Solution) carry out verifying detection.
The establishment of mass spectrometry parameters and liquid phase chromatogram condition
Mass spectrum module is triple level four bars mass spectrographs；Instrument mode is electrospray ionisation cation mould (ESI+)；Monitor mould
Formula: more reaction monitoring modes (MRM)；Ion source temperature: 300 DEG C；Atomization gas pressure: 0.1 MPa；Capillary voltage: 4kV；MS1
Level four bars temperature: 100 DEG C；MS2 level four bars temperature: 100 DEG C.
Liquid chromatogram parameter: it is middle polarity molecule according to CML and HMF, selects 120 EC-C18 (50mm of Proshell
× 3.0mm, 2.7 μm)；120 SB-C18 of Proshell (50mm × 3.0mm, 2.7 μm)；ZORBAX Eclipse XDB-C18
The chromatographic column of the different fillers of chromatographic column (250mm × 4.6mm, 5 μm) three and type is analyzed, observe the variation of its peak type and
Retention time.Mobile phase A is mutually methanol, and B phase is 0.1% aqueous formic acid, uses 100% respectively；90%；80%；70%；
50%；30%；20%；The methanol of 10% and 5% different proportion compares its peak type and appearance time situation, examines as mobile phase
Examine the feasibility and optimal flow matched of isocratic elution；Flow velocity: 0.5mL/min；Column temperature: 30 DEG C；Sample volume: 10 μ L.
(1) Mass Spectrometer Method condition
According to the chemical structural formula and relative molecular mass of CML and HMF, successively to standard items using sweeping (Scan) entirely；It is single
Channel (SIM)；Daughter ion scans (Product Ion Scan)；Multiple reaction monitors (MRM) and Optimizer software pair
CML and HMF carries out qualitative detection and testing conditions optimization.According to the second order ms of CML and HMF detect gained fragment ion and
Abundance infers that the two structural break mode is as shown in Figure 1 and Figure 2.
The qualitative and quantitative ion pair of determinand is established with this, and optimizes the collision voltage and impact energy of response, makes its sound
It should be most strong.Qualitative parent ion (Q1 mass) ion of gained CML and HMF, quantitative daughter ion (Q3 mass), optimal broken electricity
Press (Fragmentor Volatage) and impact energy (Collision Energy) as shown in table 1.
The Mass Spectrometer Method condition of table 1 CML and HMF
Note: wherein the ion of band " * " is quantitative daughter ion
(2) chromatographic separation condition
To hybrid standard product (CML, 5 μ g/mL under Mass Spectrometry Conditions after optimization；5-HMF, 5 μ g/mL, the mixing of the two
Solution) verifying measurement is carried out, the separate condition of both detections and reservation under the conditions of mobile phase (methanol-water) of different proportion
Time, gained chromatogram as shown in figure 3, by comparison it is found that methanol ratio be greater than or equal to 30% under conditions of, target
Detectable substance CML and 5-HMF are unable to reach preferably separating effect, and methanol ratio should be lower than 30% in mobile phase.In mobile phase
When ratio is 20%, although the two has certain separating degree, in view of having the dry of magazine during detection actual sample
It disturbs, easily overlaps with object mass peak, thus preferably consider bigger separating degree.When methanol-water volume ratio is 10:90, two peaks
There is preferable separating effect, and peak type response is higher.When mobile phase methanol ratio is 5%, it is seen that object 5-HMF's
Response peak broadens, becomes short, this peak type is unfavorable for quantitative integration when sample detection, is also easy to produce error, reduces the standard of detection process
True property.
Under conditions of mobile phase methanol ratio is 10%, two substance appearance times have certain distance, respectively
0.53min and 1.72min, the interval time of the two 1.2min, can be improved distinguish both detect respectively it is accurate calmly, made with this
To detect optimal flow Phase Proportion.
Since separating degree and peak type and quantitative integration process accuracy are closely related in the detection process, and and chromatographic column
Filler it is in close relations, therefore selected 120 SB-C18 of proshell (50mm × 3.0mm, 2.7 μm) and proshell
120 EC-C18 (50mm × 3.0mm, 2.7 μm)；Tri- kinds of ZORBAX Eclipse XDB-C18 different liquid chromatogram post detections 5
μ g/mL CML and 5-HMF mixed sample, observes its peak type；The variation of response intensity and retention time, result such as Fig. 4 institute
Show.
As shown in Figure 4, EC-C18 and SB-C18 within 5min can appearance finish, and XDB-C18 is then needed 20 minutes,
This is because the appearance efficiency that the above two fillers of its core-shell structure and the column length of 50mm both cause will be much higher than 250mm
XDB-C18 column.The theoretical cam curve of CML and HMF, theoretical tray under different chromatographic columns are calculated according to peak type and appearance time
Highly, the parameters such as separating degree, the results are shown in Table 2.
The different chromatographic columns of table 2 and corresponding object detection parameters
Table 2 Compare of different chromatographic columns for standards
Therefore, comprehensive detection efficiency and accuracy in detection, 120 SB-C18 column of final choice proshell is as target
The separation detection chromatographic column of object, final detection time are 3min.
2, the manner of formulation of standard curve used in the embodiment of the present application is as follows:
10mg CML and 5-HMF standard items are accurately weighed respectively, are dissolved in 100mL volumetric flask with ultrapure water, exact value
0.1mg obtains 100 μ g/mL CML standard solutions and 100 μ g/mL 5-HMF standard solutions.Respectively take 100 μ g/mL of 1mL
CML and 5-HMF standard solution is settled to 50mL in same volumetric flask, using ultrapure water, and it is molten to obtain 2 μ g/mL hybrid standard product
Liquid.It is diluted to 0.01 μ g/mL step by step again；0.05 μg/mL；0.1μg/mL；0.25μg/mL；0.5μg/mL；1μg/mL；2μg/
CML the and HMF hybrid standard product of mL7 various concentration.
Measured respectively with HPLC-MS/MS 0.01 μ g/mL, 0.05 μ g/mL, 0.1 μ g/mL, 0.25 μ g/mL, 0.5 μ g/mL,
1 μ g/mL, 2 μ g/mL CML and 5-HMF hybrid standard product be with standard concentration using quota ion peak area as ordinate
Abscissa, gained standard curve such as Fig. 5 and Fig. 6.
Wherein, the high performance liquid chromatography uses 1260 type of Agilent, and testing conditions are as follows:
Column model: ACE Proshell 120 SB-C18 chromatographic column 50 × 3.0mm, 2.7 μm；
Column temperature: 30 DEG C；
Mobile phase: methanol: 0.1% formic acid water=10:90；
Flow velocity: 0.5mL/min；
Sample volume: 10 μ L.
Mass spectrum is triple level four bars mass spectrums, using 6460 mass spectrograph of Agilent, testing conditions are as follows:
Ion source: ESI+；
Ion source temperature: 300 DEG C；
Capillary voltage: 3.5kV；
Taper hole throughput: 9L/min；
MSI level four bars temperature: 100 DEG C；
MS2 level four bars temperature: 100 DEG C；
Desolventizing temperature: 300 DEG C；
Nebulizer pressure: 15psi；
Detection mode: multiion reaction monitoring (MRM).
The MRM parameter such as table 1.
Known to Figures 5 and 6 under the testing conditions, in 0.01-2 μ g/mL concentration range, chromatography is rung by CML and HMF
It answers intensity and concentration that there is good linear relationship (R2 > 0.998), further ensures the accuracy of experimental result.
Under this condition, using S/N=3 as minimum detection limit (LOD), S/N=10 is minimum quantitative limit (LOQ).It obtains
The minimum detection limit (LOD) of CML is 0.3ng/mL, and minimum quantitative limit (LOQ) is 1ng/mL；The minimum detection limit of 5-HMF
It (LOD) is 6ng/mL, minimum quantitative limit (LOQ) is 20ng/mL, shows this method sensitivity with higher.
3, the rate of recovery of CML and 5-HMF in herein described method is determined
The preparation of sample: 1mL liquid soy sauce sample is diluted with water to 5mL, after whirlpool concussion mixes, with the speed of 7000g
It spends and is centrifuged 15min at 4 DEG C.
The preparation of mark-on sample: 1mL liquid soy sauce sample adds the hybrid standard liquid of 1mL known concentration, adds water dilute
It releases to 5mL and 15min is centrifuged at 4 DEG C with the centrifugal force of 7000g after whirlpool concussion mixes.
The supernatant of 1mL sample obtained above and mark-on sample is taken respectively, carries out identical pretreatment process, specifically
It is: crosses PolyClean X-MCX solid-phase extraction column (after the activation of 5mL methanol, 5mL pure water equilibrium) removal of impurities, 10mL water is added to drench
It washes, 10mL 10% (i.e. volume ratio is 10:90) ammonia water-methanol solution elution is then added, collects eluent.Eluent is through nitrogen
Dry up it is dry after, redissolve in 5mL volume ratio be 10:90 methanol-water solution, it is to be measured to get two kinds through 0.22 μm of micro-filtration film process
Sample.
Above two sample to be tested is subjected to HPLC-MS/MS detection respectively, it is calculated according to scalar quantity and testing result
Recovery of standard addition.Simultaneously with 3 times of signal-to-noise ratio (S/N=3) in standard items detection process for minimum detection limit (LOD), 10 times of noises
It is minimum quantitative limit (LOQ) than (S/N=3).
By above-mentioned experimentation, demonstrate CML and 5-HMF in herein described method minimum detection limit (LOD),
Minimum quantitative limit (LOQ) and calculate CML and 5-HMF the rate of recovery such as table 3:
Detection limit, quantitative limit and the rate of recovery of table 3 CML and 5-HMF
Wherein, the high performance liquid chromatography uses 1260 type of Agilent, and testing conditions are as follows:
Column model: ACE Proshell 120 SB-C18 chromatographic column 50 × 3.0mm, 2.7 μm；
Column temperature: 30 DEG C；
Mobile phase: methanol: 0.1% formic acid water=10:90；
Flow velocity: 0.5mL/min；
Sample volume: 10 μ L；
Mass spectrum is triple level four bars mass spectrums, using 6460 mass spectrograph of Agilent, testing conditions are as follows:
Ion source: ESI+；
Ion source temperature: 300 DEG C；
Capillary voltage: 3.5kV；
Taper hole throughput: 9L/min；
MSI level four bars temperature: 100 DEG C；
MS2 level four bars temperature: 100 DEG C；
Desolventizing temperature: 300 DEG C；
Nebulizer pressure: 15psi；
Detection mode: multiion reaction monitoring (MRM).
The MRM parameter such as table 1.
4, the comparative experiments of solid-phase extraction column (SPE column)
1mL liquid soy sauce sample is taken, 5mL is diluted with water to, after whirlpool concussion mixes, with the centrifugal force of 7000g in 4 DEG C
Lower centrifugation 15min.
Take the above-mentioned supernatant of 1mL, respectively cross PolyClean X-MCX,C18 solid-phase extraction column is (through 5mL first
After alcohol activation, 5mL pure water equilibrium) removal of impurities, add 10mL water or methanol to elute and collects eluent.Eluent through nitrogen dry up it is dry after,
It redissolves in the methanol-water solution that 5mL volume ratio is 10:90, through 0.22 μm of micro-filtration film process to get required sample to be tested.
With the content of CML and 5-HMF in the sample to be tested of HPLC-MS/MS detecting step 2, wherein test condition is above-mentioned
Determining chromatography, Mass Spectrometer Method condition.
Reservation of the different types of SPE column of table 4 to target
Note :-indicate to be not detected；* indicate that detected level is lower than applied sample amount 10%；* indicates that detected level accounts for applied sample amount 10-90%；
* * indicates that detected level is higher than applied sample amount 90%.
By upper table analysis it is found that only PolyClean X-MCX solid-phase extraction column has consistent suction to CML and 5-HMF
Attached effect has strong adsorptivity to CML and 5-HMF, can reach 100% reservation, is suitable for pre-treatment optimization removal of impurities.
5, the comparative experiments of eluent
1mL liquid soy sauce sample is taken, 5mL is diluted with water to, after whirlpool concussion mixes, with the centrifugal force of 7000g in 4 DEG C
Lower centrifugation 15min.
The above-mentioned supernatant of 1mL is taken, it is (pure through the activation of 5mL methanol, 5mL to cross PolyClean X-MCX solid-phase extraction column respectively
After water balance) removal of impurities, add 10mL water to elute, is then respectively adding two kinds of eluents of 5% ammonium hydroxide methanol and 10% ammonium hydroxide methanol
Concentration is eluted respectively with 5 or 10mL eluent, and eluent is collected.Eluent through nitrogen dry up it is dry after, redissolve in 5mL volume
Than the methanol-water solution for 10:90, through 0.22 μm of micro-filtration film process to get required sample to be tested.
With the content of CML and 5-HMF in the sample to be tested of HPLC-MS/MS detecting step 2, wherein test condition is above-mentioned
Determining chromatography, Mass Spectrometer Method condition.
The percentage of above-mentioned ammonium hydroxide methanol solution refers both to percentage by volume, such as ammonium hydroxide in 10% ammonium hydroxide methanol and methanol
Volume ratio is 10:90.
Elution effect of the different eluents of table 5 to object
After by contrast, select the 5% ammonium hydroxide methanol of volume 5mL when being eluted, the rate of recovery of CML far more than lower, and
When the ammonium hydroxide methanol of 10mL 5% is eluted, the eluting rate of HMF illustrates its result inaccuracy not yet up to 100%；Select body
When the 10% ammonium hydroxide methanol of product 10mL is eluted, CML and 5-HMF elution effect are best, CML the and 5-HMF rate of recovery is 98-
102%.Therefore it is 10% ammonium hydroxide methanol, effluent volume 10mL that eluate concentration is selected in this experiment.
Example 1:
1, material and equipment
The raw materials of soy sauce of different fermentations production link, methanol, water are chromatographic grade, German 224 S of Sartorius BS electricity
Sub- balance, the triple level four bars mass spectrometry systems (Agilent company, the U.S.) of 1260 high performance liquid chromatography of Agilent -6460.
2, sample pre-treatments
1g semisolid or 1mL fluid sample are taken, 5mL is diluted with water to, after whirlpool concussion mixes, with the centrifugal force of 7000g
15min is centrifuged at 4 DEG C.
The above-mentioned supernatant of 1mL is taken, it is (flat through the activation of 5mL methanol, 5mL pure water to cross PolyClean X-MCX solid-phase extraction column
After weighing apparatus) removal of impurities, add 10mL water to elute, 10mL 10% (i.e. volume ratio is 10:90) ammonia water-methanol solution is then added and washes
It is de-, collect eluent.Eluent through nitrogen dry up it is dry after, redissolve in 5mL volume ratio be 10:90 methanol-water solution, through 0.22 μ
M micro-filtration film process is to get required sample to be tested.
3, with the content of CML and 5-HMF in the sample to be tested of HPLC-MS/MS detecting step 2.
The high performance liquid chromatography uses 1260 type of Agilent, and testing conditions are as follows:
Column model: ACE Proshell 120 SB-C18 chromatographic column 50 × 3.0mm, 2.7 μm；
Column temperature: 30 DEG C；
Mobile phase: methanol: 0.1% formic acid water=10:90；
Flow velocity: 0.5mL/min；
Sample volume: 10 μ L；
Mass spectrum is triple level four bars mass spectrums, using 6460 mass spectrograph of Agilent, testing conditions are as follows:
Ion source: ESI+；
Ion source temperature: 300 DEG C；
Capillary voltage: 3.5kV；
Taper hole throughput: 9L/min；
MSI level four bars temperature: 100 DEG C；
MS2 level four bars temperature: 100 DEG C；
Desolventizing temperature: 300 DEG C；
Nebulizer pressure: 15psi；
Detection mode: multiion reaction monitoring (MRM).
The MRM parameter such as table 1.
4, measurement result calculates: carrying out quantitative analysis with external standard method, the resulting object of test sample in step 3 is determined
Ion peak areas is measured, standard curve calculating is brought into, the content of free state CML and 5-HMF in sample can be acquired, the results are shown in Table
6。
Sample described in table 6 is the soy sample that same produces different production links on chain.
The content of free state CML and 5-HMF in the different production link soy samples of table 6
Example 2:
1, material and equipment
The commercial soy sauce of different brands, methanol, water are chromatographic grade, German 224 S electronic balance of Sartorius BS,
The triple level four bars mass spectrometry systems (Agilent company, the U.S.) of 1260 high performance liquid chromatography of Agilent -6460.
2, sample pre-treatments
1mL fluid sample is taken, 5mL is diluted with water to and is centrifuged at 4 DEG C after whirlpool concussion mixes with the speed of 7000g
15min。
The above-mentioned supernatant of 1mL is taken, it is (flat through the activation of 5mL methanol, 5mL pure water to cross PolyClean X-MCX solid-phase extraction column
After weighing apparatus) removal of impurities, add 10mL water to elute, 10mL 10% (i.e. volume ratio is 10:90) ammonia water-methanol solution is then added and washes
It is de-, collect eluent.Eluent through nitrogen dry up it is dry after, redissolve in 5mL volume ratio be 10:90 methanol-water solution, through 0.22 μ
M micro-filtration film process is to get required sample to be tested.
3, with the content of CML and 5-HMF in the sample to be tested of HPLC-MS/MS detecting step 2.
The high performance liquid chromatography uses 1260 type of Agilent, and testing conditions are as follows:
Column model: ACE Proshell 120 SB-C18 chromatographic column 50 × 3.0mm, 2.7 μm；
Column temperature: 30 DEG C；
Mobile phase: methanol: 0.1% formic acid water=10:90；
Flow velocity: 0.5mL/min；
Sample volume: 10 μ L；
Mass spectrum is triple level four bars mass spectrums, using 6460 mass spectrograph of Agilent, testing conditions are as follows:
Ion source: ESI+；
Ion source temperature: 300 DEG C；
Capillary voltage: 3.5kV；
Taper hole throughput: 9L/min；
MSI level four bars temperature: 100 DEG C；
MS2 level four bars temperature: 100 DEG C；
Desolventizing temperature: 300 DEG C；
Nebulizer pressure: 15psi；
Detection mode: multiion reaction monitoring (MRM).
The MRM parameter such as table 1.
4, measurement result calculates: carrying out quantitative analysis with external standard method, the resulting object of test sample in step 3 is determined
Ion peak areas is measured, standard curve calculating is brought into, the content of free state CML and 5-HMF in sample can be acquired, the results are shown in Table
7。
The content of free state CML and 5-HMF in 7 different brands soy sample of table
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by change, modification, substitution, combination, letter
Change, should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. the method for free state CML and 5-HMF in a kind of synchronous detection liquid or semisolid flavouring, which is characterized in that firstly,
Carry out the detection pre-treatment of free state CML and 5-HMF in liquid or semisolid flavouring system, then by by pre-treatment to
Sample carries out CML detection synchronous with 5-HMF's；
The detection pre-treatment of free state CML and 5-HMF in the liquid or semisolid flavouring system, comprising the following steps:
(1) it is diluted with water, is uniformly mixed, centrifugal treating into liquid or semisolid flavouring；
(2) supernatant after taking centrifugal treating adds water to elute using PolyClean X-MCX Solid Phase Extraction column purification, then plus
The ammonia water-methanol solution that volume ratio is 10:90 elutes, and collects eluent；Every 1-2mL supernatant corresponds to 10-20mL water and 10-
20mL ammonia water-methanol solution；
(3) eluent is dry, it then redissolves in methanol-water solution, through micro-filtration film process, sample to be tested is made.
2. a kind of synchronous side for detecting free state CML and 5-HMF in liquid or semisolid flavouring according to claim 1
Method, which is characterized in that step (1) liquid or semisolid flavouring are soy sauce.
3. free state CML and 5-HMF in a kind of synchronous detection liquid according to claim 1 or claim 2 or semisolid flavouring
Method, which is characterized in that step (3) the methanol-water solution dosage are as follows: supernatant described in every 1mL step (2) corresponds to 5-
10mL step (3) described methanol-water solution.
4. a kind of synchronous side for detecting free state CML and 5-HMF in liquid or semisolid flavouring according to claim 3
Method, which is characterized in that the volume ratio of methanol and water is 10:90 in the methanol-water solution.
5. a kind of synchronous side for detecting free state CML and 5-HMF in liquid or semisolid flavouring according to claim 3
Method, which is characterized in that the centrifugal force of step (1) described centrifugal treating is 7000g；Centrifugation time is 15min；Centrifuging temperature is 4
℃。
6. free state CML and 5-HMF in a kind of synchronous detection liquid according to claim 1 or claim 2 or semisolid flavouring
Method, which is characterized in that the aperture of step (3) described microfiltration membranes is 0.22-0.45 μm.
7. free state CML and 5-HMF in a kind of synchronous detection liquid according to claim 1 or claim 2 or semisolid flavouring
Method, which is characterized in that step (2) the PolyClean X-MCX solid-phase extraction column is activated using preceding through 5-10mL methanol, then
Add 5-10mL pure water equilibrium.
8. free state CML and 5-HMF in a kind of synchronous detection liquid according to claim 1 or claim 2 or semisolid flavouring
Method, which is characterized in that described be diluted with water of step (1) is diluted to 5- for every 1mL liquid flavoring or 1g semisolid flavouring
10mL；It is described to be uniformly mixed as vortex mixing；
Step (3) drying means is to be dried with nitrogen.
9. free state CML and 5-HMF in a kind of synchronous detection liquid according to claim 1 or claim 2 or semisolid flavouring
Method, which is characterized in that the detection method is HPLC MS.
10. a kind of synchronous side for detecting free state CML and 5-HMF in liquid or semisolid flavouring according to claim 9
Method, which is characterized in that the high performance liquid chromatography uses 1260 type of Agilent, and testing conditions are as follows:
Column model: ACE Proshell 120 SB-C18 chromatographic column 50 × 3.0mm, 2.7 μm；
Column temperature: 30 DEG C；
Mobile phase: methanol: 0.1% formic acid water=10:90；
Flow velocity: 0.5mL/min；
Sample volume: 10 μ L；
Mass spectrum is triple level four bars mass spectrums, using 6460 mass spectrograph of Agilent, testing conditions are as follows:
Ion source: ESI+；
Ion source temperature: 300 DEG C；
Capillary voltage: 3.5kV；
Taper hole throughput: 9L/min；
MSI level four bars temperature: 100 DEG C；
MS2 level four bars temperature: 100 DEG C；
Desolventizing temperature: 300 DEG C；
Nebulizer pressure: 15psi；
Detection mode: multiion reaction monitoring.
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