CN103743849B - Chromatography of ions-high resolution mass spectrum the method for combined use of multiple organic acid during rapid screening identifies milk product while of a kind of - Google Patents
Chromatography of ions-high resolution mass spectrum the method for combined use of multiple organic acid during rapid screening identifies milk product while of a kind of Download PDFInfo
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Abstract
The chromatography of ions high resolution mass spectrum method for combined use of multiple organic acid during rapid screening identifies milk product while of the invention discloses a kind of, extract including by testing sample potassium hydroxide solution, the little column purification of extracting solution reverse phase filler pre-treatment, IonPac AS19 chromatographic column is used to separate, KOH leacheate eluting, the most in the negative ion mode by Orbitrap high resolution detection device full scan mode detection, the data recorded carry out examination and qualification with available data contrast；This method reducing the requirement to sample pre-treatments, effectively save time for sample pretreatment, shorten the retention time measuring measured object greatly, can get rid of false positive, the data of collection can be called repeatedly according to detection demand.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of rapid screening simultaneously multiple in identifying milk product
The chromatography of ions of organic acid-high resolution mass spectrum method for combined use.
Background technology
Organic acid in milk product has the breeding of suppression harmful bacteria, provides differently flavoured effect, in breast system
Product product is widely present.Its main source is divided into production to add and two kinds of approach of natural product: production and processing
Process often uses malic acid, tartaric acid, citric acid, fumaric acid, phosphoric acid etc. as acidity regulator；Natural
Organic acid is mainly produced free fatty acid such as acetic acid, butanoic acid by butter oil hydrolysis, and the physiological metabolism of cattle produces
The catabolism of citric acid, orotic acid, uric acid, antibacterial and lactic acid bacteria produce lactic acid, acetic acid, acetone acid,
Propanoic acid, formic acid, most carbon source is converted into lactic acid through metabolism of pyruvate by homofermentative lactic, it is possible to
To produce other organic acid and local flavor metabolite.
Due to the safety that it is of a relatively high, in addition to fumaric acid, to these organic acid in the national standard of China
Use do not limit the quantity regulation, depending on maximum usage amount be " according to production needs ".But experimentation table
Bright, it is eaten for a long time or eats excess organic acid and human body can be produced infringement equally, Qin Shuzhen etc. enters in mice
The acute toxicity tests of row shows: the toxicity of citric acid own is slight, but can cause after heavy metal chelating
Hepatocyte diffusivity is downright bad, and lobules of liver disappears.
For avoiding unrestricted, the immeasurable use organic acid of illegal retailer in food production, the most very
The range of organic acid has all been made respective specified with limitation by many tissues and country: international food code is entrusted
Citric acid limitation 3000mg/kg in member's meeting (ACA) regulation fruice, tartaric acid limitation 4000mg/kg,
Lactic acid limitation 2000mg/kg in the supplement of infant, acetic acid limitation 5000mg/kg, fresh pasta,
Fumaric acid limitation 700mg/kg in noodles；European Union specifies fumaric acid limitation in drying powdery sweets mixture
4000g/kg, cookies and rusk mesotartaric acid limitation 5000g/kg；China Taiwan the most individually specifies baby
Food must not use malic acid.
At present, in the detection technique in the field such as food, environment, chemical industry, medical science, for anion, sun from
Son, water solublity and polar micromolecules compound are frequently with ion chromatography technology.But, ion chromatograph
The detection time is longer, usually 40-60min, and only with retention time as qualitative, quantitative foundation, it is impossible to carry
For structural information, the confirmation in structure cannot be carried out further for the material that retention time is identical, thus easily
Cause false positive results.
Mass-spectrometric technique can provide qualitative and quantitative detection more accurately, in recent years about ion during analyzing
Hydrolysis and condensation it has been reported that method rue etc. utilizes capillary ion chromatography mass spectrometry analytical equipment to enter
The analysis of the water-soluble components such as row organic acid, sugar, alcohol, but its Low Resolution Mass Spectra used is only capable of having been provided that
The given compound of standard substance is analyzed, it is impossible to be difficult to obtain standard substance or without laws and regulations requirement
Unvested compound carries out examination, it is difficult to the detection meeting various places, world today laws and regulations frequent updating is wanted
Ask；And Low Resolution Mass Spectra setup parameter the most before collection starts, once gather and cannot repeatedly call many numbers of plies
According to.
High resolution mass spectrum has the high function with accurate molecular weight of mass resolution, therefore to sample pre-treatments
Require to reduce, effectively save time for sample pretreatment；And due to its high-resolution advantage, it is not necessary at color
Carrying out segmentation separation in spectrum separation can be by component identification to be measured, and the chromatographic isolation time is thus by original
50-60min shortens to about 30min；The data that high resolution mass spectrum gathers under full scan pattern can basis
Detection demand is called repeatedly, remains able to carry out Screening analysis, thus in the case of not providing standard substance
Make up the deficiency of tradition Low Resolution Mass Spectra method；The scanning of its data dependency can be passed through while examination
Fragment ion carries out qualitative confirmation.In recent years by with liquid chromatography tandem by the way of at food inspection, albumen
The aspects such as matter group have started to gradually apply, but still belong to empty at the detection field applied of connecting with chromatography of ions
In vain.
Summary of the invention
Chromatography of ions-the high score of multiple organic acid during rapid screening identifies milk product while that the present invention providing a kind of
Distinguish mass spectrometry combination method.
The chromatography of ions of multiple organic acid-high resolution mass spectrum combination during rapid screening identifies milk product while of a kind of
Method, comprises the following steps:
Testing sample potassium hydroxide solution is extracted, the little column purification of extracting solution reverse phase filler pre-treatment, make
Separate by IonPac AS19 chromatographic column, KOH leacheate eluting, use Orbitrap the most in the negative ion mode
High resolution detection device full scan mode detection, carries out examination and qualification according to the data recorded；
Preferably, chromatographic condition is:
Column temperature: 35 DEG C；
Flow velocity: 0.25mL/min；
Flowing phase: 30mM KOH；
Mass Spectrometry Conditions is:
Electric spray ion source；
Spray voltage: 5.00kV
Mass charge ratio range: 50-300
Capillary voltage :-21.00V
Pipe lens voltage :-128.70V
Capillary temperature: 350 DEG C；
Sheath gas (N2) flow velocity: 17L/min (i.e. 30arb)；
Auxiliary gas (N2) flow velocity: 8L/min (i.e. 10arb)；
Resolution R=60000.
Further, the mass concentration of potassium hydroxide solution is 1%.
Further, described reverse phase filler pre-treatment pillar is RP pre-treatment pillar.
RP pre-treatment pillar is Thermo RP pre-treatment pillar.Further, RP pre-treatment pillar uses
Before successively with 10mL water, 10mL methanol, 10mL water activation, the most again by extracting solution loading.Actual behaviour
In work, after discarding front 5mL liquid, collecting filtrate, filtrate chromatographic column separates.
Further, chromatographic column uses IonPac AS19,250mm × 2.1mm, 2.5 μm.
Further, in chromatographic condition, sample room temperature: 10 DEG C；Sample size: 10 μ L；Analysis time:
30min。
KOH leacheate gradient elution is that automatic generator on-line automatic generation KOH leacheate carries out gradient and washes
De-.
Xcalibar software can be used the molecular formula of compound each in sample, retention time, first mass spectrometric matter
The informations such as lotus ratio, natural isotope distribution.
The instrument used is: ion chromatography system: Dionex ICS-2000；High resolution mass spectrum system: Thermo
LTQ-Orbitrap XL。
Beneficial effects of the present invention is as follows:
1, the while that the present invention establishing a kind of rapid screening identify the chromatography of ions of multiple organic acid in milk product-
High resolution mass spectrum method for combined use, the novel analytical equipment of chromatography of ions-high resolution mass spectrum of application has high-resolution
Rate, the feature of high sensitivity, reduce the requirement to sample pre-treatments, effectively save sample pre-treatments
Time.
2, the present invention can be by component identification to be measured without carrying out segmentation separation in chromatographic isolation, greatly
Shorten the retention time measuring measured object, shorten to 30min from original ion chromatograph 40-60min
Left and right.
3, the present invention can provide accurate molecular weight while providing retention time, is combined by this bidirectional information
Applied analysis, can utilize its structural information to get rid of false positive, utilizes its retention time to differentiate isomers.
4, the data that the present invention gathers under full scan pattern can be called according to detection demand, thus repeatedly
Make up setup parameter the most before tradition Low Resolution Mass Spectra collection starts, once gather and cannot repeatedly call many numbers of plies
According to deficiency.
5, the present invention can carry out the Screening analysis of unvested object in the case of not providing standard substance,
It it is the extremely effective application of the testing requirement of reply various places, world today laws and regulations frequent updating.
6, the data dependency scanning of the present invention can realize carrying out determining by fragment ion while examination
Property confirmation purpose.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in further detail；
Fig. 1 is the full scan high resolution mass spectrum figure of sample in example 1；
Fig. 2 is the XIC(a of sulphuric acid) and high resolution mass spectrum qualification diagram (b)；
Fig. 3 is the XIC(a of phosphoric acid) and high resolution mass spectrum qualification diagram (b)；
Fig. 4 is the XIC(a of lactic acid) and high resolution mass spectrum qualification diagram (b)；
Fig. 5 be sample full scan high resolution mass spectrum figure (a) of the multiple samples in example 2 and tartaric acid (b),
The XIC figure of lactic acid (c), citric acid (d) and malic acid (e)；
Fig. 6 is the high resolution mass spectrum figure of example 2 mesotartaric acid；
Fig. 7 is the high resolution mass spectrum figure of lactic acid in example 2；
Fig. 8 is the high resolution mass spectrum figure of wine citric acid in example 2；
Fig. 9 is the high resolution mass spectrum figure of malic acid in example 2.
Detailed description of the invention
For being more fully understood that the present invention, the side of the present invention will be further illustrated by specific embodiment below
Case, protection scope of the present invention should include the full content of claim, but be not limited to this.
Embodiment 1
The chromatography of ions of multiple organic acid-high resolution mass spectrum combination during rapid screening identifies milk product while of a kind of
Method, comprises the following steps:
Sample extraction: milk product 1mL 10mL1% potassium hydroxide solution to be measured extracts and is sufficiently mixed,
4 DEG C, 12000r/min be centrifuged 10min.Take supernatant, to be clean；
Pre-treatment column purification: Thermo RP pre-treatment pillar, uses front RP pre-treatment pillar to use 10mL successively
Water, 10mL methanol, 10mL water activate；The most again by supernatant loading；
Collect: discard the front 5mL filtrate that RP post flows out, collect rear end clear liquid, to be measured；
Upper machine testing: sample is carried out examination and qualification by chromatography of ions-high resolution mass spectrum combined instrument；
Chromatographic condition is:
Ion chromatography system: Dionex ICS2000
Chromatographic column: Dionex IonPac AS19,250mm × 2.1mm, 2.5 μm；
Column temperature: 35 DEG C；
Sample room temperature: 10 DEG C；
Sample size: 10 μ L；
Flow velocity: 0.25mL/min；
Flowing phase: 30mMKOH；
Analysis time: 30min.
Mass Spectrometry Conditions:
High resolution mass spectrum system: Thermo LTQ-Orbitrap XL
Electric spray ion source；Negative ion mode；
Spray voltage: 5.00kV
Mass charge ratio range: 50-300
Capillary voltage :-21.00V
Pipe lens voltage :-128.70V
Capillary temperature: 350 DEG C；
Sheath gas (N2) flow velocity: 17L/min (i.e. 30arb)；
Auxiliary gas (N2) flow velocity: 8L/min (i.e. 10arb)；
Scan pattern: Orbitrap detector full scan (Full Scan) mode detection；
Resolution R=60000 of full scan.
By the XIC manager of Xcalibar software to the molecular formula of compound each in sample, retention time,
The informations such as first mass spectrometric mass-to-charge ratio and natural isotope distribution.Can by with this method to several
The sample with certain acid known does positive control, and data measured is compared with these data, carries out examination
And qualification.Or, with these data and existing database resource, such as Chinese Academy of Sciences's compound reference type number
According to storehouse etc., in data carry out comparison.Through comparison, embodiment 1 obtains three kinds of organic acid: sulphuric acid, phosphoric acid,
Lactic acid.Experimental result is as shown in Figure 1, Figure 2, Figure 3 and Figure 4.
Fig. 1 is the full scan high resolution mass spectrum figure of sample in example 1；
Fig. 2 is the XIC(a of sulphuric acid) and high resolution mass spectrum qualification diagram (b)；
Fig. 3 is the XIC(a of phosphoric acid) and high resolution mass spectrum qualification diagram (b)；
Fig. 4 is the XIC(a of lactic acid) and high resolution mass spectrum qualification diagram (b).
Embodiment 2
28 commercial milk product are used and carries out sample treatment, apparatus measures, number with the method for embodiment 1
According to analysis, obtain 4 kinds of organic acid: the screening results of tartaric acid, lactic acid, citric acid and malic acid.Experiment
Result is as shown in Fig. 5～9.
Fig. 5 be sample full scan high resolution mass spectrum figure (a) of the multiple samples in example 2 and tartaric acid (b),
The XIC figure of lactic acid (c), citric acid (d) and malic acid (e)；
Fig. 6 is the high resolution mass spectrum figure of example 2 mesotartaric acid；
Fig. 7 is the high resolution mass spectrum figure of lactic acid in example 2；
Fig. 8 is the high resolution mass spectrum figure of wine citric acid in example 2；
Fig. 9 is the high resolution mass spectrum figure of malic acid in example 2.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and also
Non-is the restriction to embodiments of the present invention, for those of ordinary skill in the field, above-mentioned
Can also make other changes in different forms on the basis of explanation, here cannot be to all of enforcement
Mode gives exhaustive, every belongs to the obvious change or variation that technical scheme extended out
Row still in protection scope of the present invention.
Claims (4)
1. chromatography of ions-high resolution mass spectrum connection of multiple organic acid in the milk product of rapid screening qualification simultaneously
By method, it is characterised in that comprise the following steps:
Testing sample potassium hydroxide solution is extracted, the little column purification of extracting solution reverse phase filler pre-treatment, make
Separate by IonPac AS19 chromatographic column, KOH leacheate eluting, use Orbitrap the most in the negative ion mode
High resolution detection device full scan mode detection, carries out examination and qualification according to the data recorded；
Chromatographic condition is:
Column temperature: 35 DEG C；
Flow velocity: 0.25mL/min；
Flowing phase: 30mM KOH；
Mass Spectrometry Conditions is:
Electric spray ion source；
Spray voltage: 5.00kV；
Mass charge ratio range: 50-300；
Capillary voltage :-21.00V；
Pipe lens voltage :-128.70V；
Capillary temperature: 350 DEG C；
Sheath gas velocity: 17L/min；
Secondary air speed: 8L/min；
Resolution R=60000；
Described extraction by testing sample potassium hydroxide solution refers to: by milk product 1mL 10mL to be measured
1% potassium hydroxide solution extract be sufficiently mixed, 4 DEG C, 12000r/min be centrifuged 10min, taking supernatant is
Extracting solution；
The specification of described IonPac AS19 chromatographic column is 250mm × 2.1mm, 2.5 μm；
Described organic acid includes: sulphuric acid, phosphoric acid, lactic acid, tartaric acid, citric acid and malic acid；
In chromatographic condition, sample room temperature: 10 DEG C；Sample size: 10 μ L；Analysis time: 30min.
2. chromatography of ions-high resolution mass spectrum method for combined use as claimed in claim 1, it is characterised in that institute
The mass concentration stating potassium hydroxide solution is 1%.
3. chromatography of ions-high resolution mass spectrum method for combined use as claimed in claim 1, it is characterised in that institute
Stating reverse phase filler pre-treatment pillar is RP pre-treatment pillar.
4. chromatography of ions-high resolution mass spectrum method for combined use as claimed in claim 3, it is characterised in that institute
State and activate, the most again with 10mL water, 10mL methanol, 10mL water successively before RP pre-treatment pillar uses
By extracting solution loading.
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| CN105784908B (en) * | 2016-04-13 | 2017-10-10 | 广东中烟工业有限责任公司 | The chromatography of ions detection method of 6 kinds of anion in cigarette paper |
| CN107064404B (en) * | 2017-01-13 | 2019-08-06 | 伊犁师范学院 | The method of ion-chromatographic determination hawthorn mesotartaric acid, oxalic acid and citric acid content |
| CN114755341A (en) * | 2022-04-24 | 2022-07-15 | 中国科学院微生物研究所 | Ion chromatography and mass spectrometry combined method for detecting lactic acid and short-chain fatty acid |
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