CN103149312A - Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry - Google Patents
Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry Download PDFInfo
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Abstract
The ineveniton discloses an analyzing method of sialic acid in infant milk powders with an ultra-high performance liquid chromatography tandem quadrupole mass spectrometry method. On the basis of an original detecting of the sialic acid in milk powders with high performance liquid chromatography, the ultra-high performance liquid chromatography (UPLC) technology is combined, the sialic acid in three forms in a sample is extracted, and therefore the method of detecting the sialic acid in infant formula milk with the UPLC tandem mass spectrometry, wherein the method is higher in sensitivity and faster. According to the method, the average recovery of the sialic acid is within 84.3%-98.9%, the relative standard deviation (RSD) is 4.9%-8.2%, and the lowest detection limit of the sialic acid is 0.01 microgram/milliliter. The analyzing method of the sialic acid in infant milk powders with the UPLC tandem quadrupole mass spectrometry is simple and convenient to operate, a better separation mode is adopted, matrix effects are relatively small, the process of high-speed centrifugation after hydrolysis is adopted in pretreatment and simple, easy and convenient to operate, the loss of the sialic acid in a pretreatment process is effectively avoided, and the recovery rate is improved.
Description
Technical field
The present invention relates to a kind of sialic method in analysis infant milk powder, particularly a kind ofly utilize sialic method in Ultra Performance Liquid Chromatography series connection level Four bar mass spectrophotometry infant milk powder, belong to the food inspection field.
Background technology
Sialic acid refers to a series of general designations that contain the carboxylated monose acylated derivatives of nine carbon atoms, and its systematic naming method is 5-amino-3,5-dideoxy-D-glycerine-D-gala ketononose.It is in human milk and infant formula Ruzhong, exist with free, compound sugar combination and protein combination form respectively, the human milk oligosaccharides bound sialic acid accounts for 73%, infant formula milk protein combination sialic acid accounts for 70%, mainly N-acetyl-neuraminate (N-acetylneuraminic acid, Neu5Ac), so often N-acetyl-neuraminate is called sialic acid, cow's milk and infant formula Ruzhong sialic acid content are all very low.Latest report proof sialic acid is also a kind of natural brain nutrient, can promote infant's memory and intelligence development, the high-load with it in breast milk and being paid close attention to widely, but the content in milk and infant formula is very low.This is also indicating in baby formula milk powder, particularly can effectively promote their nervous system and the growth of brain for adding sialic acid in premature's baby formula milk powder, further improves simultaneously it in the early stage intelligence development level of growth.
The method that is used for the sialic acid analysis is a lot, and enzyme reaction, antibody, agglutinin and virus have been used for definite total sialic acid content or have detected the sialic acid of particular type; Orcin, resorcinol, regularly/thiobarbituricacidα-colourimetry, spectrophotometric method also has been widely used in sialic detection for many years.The common limitation of these methods is that they can't distinguish sialic type and require sample purification.The classic methods such as colourimetry and chromatogram photometric method are with the sialic acid after ion exchange column purifying acid hydrolysis sample, in this method sample the sialic acid loss larger, the accuracy that impact detects.Sialic detection method in food, bibliographical information have high performance liquid chromatography (HPLC), but its pre-treatment is loaded down with trivial details, need to ask sialic acid is carried out measuring after derivatization, have increased the complicacy of experiment, and sensitivity and accuracy are not also very desirable.
Due to sialic acid use in food at home fewer, and the matrix that exists of Ruzhong self, as protein, vitamin, calcium, potassium, the mineral matters such as magnesium can impact analysis result, are not therefore also very ripe about the correlation technique to its detection.Therefore, study a kind of method that efficiently detects fast sialic acid content in baby formula milk powder, do further optimization process for the lower content sample, improve sialic content in its prescription emulsifiable powder, make it more near breast milk, thereby better promote the healthy development of infant's brain, the physiology nutritional need that satisfies infant growth becomes a kind of exigence.At present, the existing infant formula milk in home market adds sialic acid, makes it more near people's milk composition.Along with the increase of adding sialic acid food, food quality supervision department and manufacturing enterprise's quality control need to also be on the increase, and certainly will require more accurately and detection method fast.Along with the development of chromatographic column filler technology, have higher separating power based on the Ultra Performance Liquid Chromatography (UPLC) of 1.7 μ m granule technology than traditional HPLC, mass spectrometry can carry out the Accurate Measurement of molecular weight.
Li Hui etc. utilize liquid chromatography-mass spectrography (Li Hui, Jin Yibao, Liu Hongxia, in managing state affairs etc., the analytical test journal, 2008,11 (27): 193-194) sialic acid content in milk powder is measured, carry out gradient elution take tetrabutylammonium aqueous solution-methyl alcohol as mobile phase, triple quadrupole bar mass spectrum adopts negative ion mode, detects and is limited to 0.06mg/L, and relative standard deviation is less than 10%.Applied chromatogram and mass spectrum condition are as follows: chromatographic column: XBridgeC
18(3.5 μ m, 2.1mm * 100mm) (Waters, Massachusetts, USA); Mobile phase: 4mmol/L tetrabutylammonium aqueous solution-methyl alcohol gradient elution; Flow velocity: 0.2mL/min; Sample size 10 μ L; Triple quadrupole bar mass spectrum adopts negative ion mode, and ion source temperature is 110 ° of C, and the ion gun ionization voltage is 3kV, and the atomization gas flow velocity is 800L/h
-1, the ion acquisition mode adopts multiple-reaction monitoring pattern (MRM), gathers ion 308.1〉and 87.1, retention time is 6.1min.
The method is utilized the mass spectral:mass spectrographic method of the liquid chromatography triple level Four bars of series connection, adopt many reaction negative ion monitoring patterns, mobile phase selects tetrabutylammonium aqueous solution-methanol solution to carry out gradient elution, sialic acid in milk powder has been realized good separation, retention time is 6.1min, sialic minimum detectability is 0.06mg/L, and relative standard deviation is less than 10%.But separate at mass spectrum, when using electric spray ion source to the parent ion full scan, we find, under negative ion multiple-reaction monitoring pattern, the Abundances of parent ion is not very high, for this reason the chemical structural formula of bound sialic acid we courageously suppose, can use positive ion multiple-reaction monitoring pattern fully the parent ion of sialic acid standard items is carried out full scan, find the higher reliable mass spectrum condition of Abundances under negative source and positive source condition, for sensitivity and the accuracy that improves detection method lays the foundation; Secondly in conjunction with chromatographic column selective flow phase time, we think that mobile phase should make sialic acid well be separated on chromatographic column, obtain best response aspect retention time, therefore to abandon numerous conforming to the principle of simplicity when research eluent gradient wash-out, select excellent definite mobile phase and proportioning thereof in excellent, and then sensitivity and the detection limit of raising this method, do the basis for setting up industry standard.
The employing high performance liquid chromatographies such as Feng Jun have carried out measuring (Feng Jun, Li Hongji, Han Liqiang to the sialic content in Ruzhong, Li Weihua, Li Ping, Yang Guoyu, modern food science and technology, 2007,12:23), utilize acid-hydrolysis method that the sialic acid in Ruzhong is discharged, take o-dihydroxy ammon hydrochloride (10mg/mL) as derivatization reagent, derivative 40min, adopt Symmetry C in 80 ℃ of water-baths
18Post (250mm * 4.6mm, 5 μ m); Mobile phase is 1.0% tetrahydrofuran aqueous solution (containing 0.2% phosphoric acid)-acetonitrile (92:8); Flow velocity is 1.0mL/min, and column temperature is 35 ℃; Ultraviolet detects wavelength 230nm.Result shows, sialic acid is good in 10 ~ 320 μ g/mL scope internal linear, and the lowest detection of N-acetyl-neuraminate is limited to 0.25 μ g/mL, and average recovery rate is 95.7%.
The method is utilized conventional high performance liquid chromatography, because most saccharide compound does not have chromophoric group, sialic acid is no exception, so separation determination after the analysis of HPLC need to derive sialic acid has increased the complicacy of experiment and possible disturbing factor.Derivatization reagent is considered relatively costly from economic angle, the derivatization process relatively bothers, and is loaded down with trivial details; Sensitivity and the accuracy of high performance liquid chromatography detection simultaneously are limited, therefore research method should try every possible means to prevent the loaded down with trivial details of pre-treatment, reduce the complicacy of experiment and possible disturbing factor, further determine the simplest Pretreatment of optimization, adopt rational separation and detection technology, and then improve sialic detection technique and standard in Milk Powder Formula For Infants.Although but the method Accurate Determining Ruzhong sialic acid content, sensitivity and accuracy are still waiting further raising.
Summary of the invention
For present problems of the prior art, the present invention detects in milk powder on sialic basis in original high performance liquid chromatography, based on the 1.7 short grained Ultra Performance Liquid Chromatography of μ m (UPLC) technology, three kinds of form sialic acids in sample are extracted, sensitivity is higher, the Ultra Performance Liquid Chromatography tandem mass spectrum detects the sialic method in baby formula Ruzhong, actual test sample successful faster thereby set up.
In order to reach above purpose, the technological means that the present invention adopts is:
A kind of sialic method in Ultra Performance Liquid Chromatography series connection level Four bar mass spectrophotometry Milk Powder Formula For Infants of utilizing of the present invention is characterized in that comprising the following steps:
(1) drawing standard curve;
(2) determination: three kinds of form sialic acids in sample are extracted, and described three kinds of form sialic acids are respectively free sialic acid, compound sugar bound sialic acid and protein combination sialic acid;
Three kinds of form sialic acid solution that extraction obtains adopt respectively the Solid-Phase Extraction column purification of HLB polymer filler, adopt ultrapure water as leacheate, and ammoniacal liquor-methyl alcohol mixed solution is as eluent;
(3) after the extract solution after the purification that step (2) is obtained mixes, regulate pH value to 2.5 ~ 3, use the acetonitrile constant volume, the 0.22 organic membrane filtration of μ m is measured with UPLC-ESI-MS/MS;
The selection of (4) mass spectrophotometry condition and parameter:
Ion gun: electric spray ion source; Scan mode: positive ion scanning; Detection mode: multiple-reaction monitoring system; Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 ℃; Desolventizing temperature: 450 ℃; Desolventizing airshed: 650L/hr; Taper hole blowback air flow: 50L/hr; Collision airshed: 0.24mL/min; Photomultiplier cell voltage: 650V;
(5) chromatographic separation condition:
Chromatographic column and gradient elution: specification is 50mm * 2.1mm, the Waters ACQUITY Ultra BEH HILIC Ultra Performance Liquid Chromatography post of 1.7 μ m; Column temperature: 30 ℃; Sample chamber temperature: 30 ℃; Sample size: 5 μ L; Flow velocity: 0.25mL/min; Mobile phase A: acetonitrile; Mobile phase B: 0.1% formic acid (v/v) aqueous solution.
In the present invention, preferred, three kinds of form sialic acids described in step (2) are to extract in accordance with the following methods:
The extraction of free and compound sugar bound sialic acid: precision takes a certain amount of Milk Powder Formula For Infants, adds trichloroacetic acid solution to dissolving fully, reaches the purpose of protein precipitation, gets supernatant, under 4 ℃, 15000r/min condition, and centrifugal 10min, standby;
The sialic extraction of protein combination: add hydrochloric acid solution in the precipitation after the extraction of compound sugar bound sialic acid " free and " processed, put into 80 ℃ of water-bath 1h, cooling after taking out, under 4 ℃, 15000r/min condition, centrifugal 10min, standby.
Wherein, preferred, the concentration of described trichloroacetic acid solution is 20%(v/v), described trichloroacetic acid solution (ml) is 1:6-10 with the volume mass ratio of Milk Powder Formula For Infants (g); The concentration of described hydrochloric acid solution is 0.1%(v/v), described hydrochloric acid solution (ml) is 1:2-5 with the volume mass ratio of Milk Powder Formula For Infants (g).
In the present invention, preferred, the solid-phase extraction column of the HLB polymer filler described in step (2) is in Oasis HLB solid phase extraction column, and in ammoniacal liquor-methyl alcohol mixed liquor, the volume ratio of ammoniacal liquor and methyl alcohol is 25:75.
In the present invention, preferred, be settled to 2-5 times of former extract mixings liquor capacity with acetonitrile in step (3).
In the present invention, preferred, the desolventizing gas described in step (4) is nitrogen, and described collision gas is argon gas.
In the present invention, preferred, the elution program of the gradient elution described in step (5) is: in initial eluent, the percent by volume of mobile phase A is 90%, and the percent by volume of Mobile phase B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A is down to 12%, B by 90% and is upgraded to 88% by 10%, and is maintained until 1.8min; 1.8 ~ 2.2min, A is upgraded to 90%, B by 12% and is down to 10% by 88%.
Experiment showed, that using the standard interpolation average recovery rate that the inventive method is measured is 84.3%～98.9%, sialic minimum detectability is 0.01 μ g/mL, and the method for the present invention's foundation has the higher recovery and sensitivity as can be known.
Compared to prior art, the invention has the advantages that:
1, set up faster, the higher Ultra Performance Liquid Chromatography tandem mass spectrometry of sensitivity and accuracy detects the sialic method in Milk Powder Formula For Infants;
2, adopt the multiple-reaction monitoring pattern, the negative ion scan mode is determined the mass spectrum condition, and then the accuracy of raising method;
3, when chromatographic resolution, choose by the analysis to chromatographic column, determine to adopt BEH HILIC (50mm * 2.1mm, 1.7 μ m) chromatographic column, choose 0.1% aqueous formic acid and acetonitrile as mobile phase, adopt the gradient elution mode, guarantee the successful reservation on sialic chromatographic column, lay the foundation for further analyzing its content.
This product basic mechanical design feature index:
1, sialic acid is good with the linear relationship of sialic acid peak area in 0.05～5.0 μ g/mL scope;
2, sialic average recovery rate is between 84.3%～98.9%, and relative standard deviation is 4.9%～8.2%;
3, sialic lowest detection is limited to 0.01 μ g/mL.
Description of drawings
Fig. 1 is the MRM chromatogram of typical 1.0 μ g/mL sialic acid standard items;
Fig. 2 is the MRM chromatogram of blank powdered milk sample;
Fig. 3 is the MRM chromatogram of mark-on powdered milk sample.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
The mensuration of sialic acid content in embodiment 1 Milk Powder Formula For Infants
Method:
1, the drafting of typical curve
Accurately pipette appropriate sialic acid standard items storing solution, dilution obtains the standard solution that concentration is 0.05,0.1,0.2,0.5,1.0 and 5.0 μ g/mL successively.The peak area (Y) of measuring according to sialic quota ion carries out linear regression with sialic mass concentration (X, μ g/mL), the drawing standard curve, and sialic equation of linear regression is:
Y=8514.5X－811.09，R
2=0.9989
Wherein Y is the chromatographic peak peak area of mensuration, and X is sialic mass concentration (μ g/mL), learns that the linear relationship of this typical curve is good.
2, determination
Get baby formula milk powder sample A1, each 1.0g of B2, C3 of 3 parts of different brands, carry out in accordance with the following methods respectively three kinds of sialic extractions of form, 3 parallel analysiss of each sample, upper machine carries out determination experiment, carries out quantitatively with the matrix matching curve.
2.1 the extraction of free and compound sugar bound sialic acid
Precision takes 1.0g baby formula milk powder, adds 8mL, 20% trichloroacetic acid solution to dissolving fully, reaches the purpose of protein precipitation, getting supernatant packs in the centrifuge tube of 4 2mL, under 4 ℃, 15000r/min condition, centrifugal 10min merges supernatant standby.
2.2 the sialic extraction of protein combination
Add 2mL, 0.1%(v/v in precipitation after the extraction of compound sugar bound sialic acid " free and " processed) hydrochloric acid solution, put into 80 ℃ of water-bath 1h, cooling after taking out, under 4 ℃, 15000r/min condition, centrifugal 10min.
2.3 the purification of sample supernatant
The centrifugal sample solution that obtains is loaded into Oasis HLB solid phase extraction column (shake Xiang company of Beijing, 3mL/60mg), experiment adopts ultrapure water as leacheate, ammoniacal liquor-methyl alcohol (volume ratio 25:75) solution is as eluent, and wherein 4mL leacheate and 3mL eluent can satisfy the purification requirement.
2.4 after at last two parts extract solution after purifying being mixed, regulate pH value to 2.5 ~ 3 left and right, be settled in the 20mL volumetric flask with acetonitrile, with the 0.22 organic membrane filtration of μ m, measure with UPLC-ESI-MS/MS.
The selection of 3, mass spectrophotometry condition and parameter
Ion gun: electric spray ion source (ESI); Scan mode: positive ion (ESI+) scanning; Detection mode: multiple-reaction monitoring system (MRM); Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 ℃; Desolventizing temperature: 450 ℃; Desolventizing gas (nitrogen) flow: 650L/hr; Taper hole blowback air flow: 50L/hr; Collision gas (argon gas) flow: 0.24mL/min; Photomultiplier cell voltage: 650V;
4, Ultra Performance Liquid Chromatography condition
Chromatographic column: Waters ACQUITY Ultra BEH HILIC Ultra Performance Liquid Chromatography post (U.S. Waters company) (50mm * 2.1mm, 1.7 μ m); Column temperature: 30 ℃; Sample chamber temperature: 30 ℃; Sample size: 5 μ L; Flow velocity: 0.25mL/min; Mobile phase A: acetonitrile; Mobile phase B: 0.1% aqueous formic acid; The gradient elution program: initial ratio A is that 90%, B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A is down to 12%, B by 90% and is upgraded to 88% by 10%, and is maintained until 1.8min; 1.8 ~ 2.2min, A is upgraded to 90%, B by 12% and is down to 10% by 88%.
Result:
The conclusive evidence analysis of actual sample:
Get baby formula milk powder sample A1, each 1.0g of B2, C3 of 3 parts of different brands, after processing according to the method for setting up, 3 parallel analysiss of each sample, upper machine carries out determination experiment, carry out quantitatively with the matrix matching curve, the sialic content that obtains 3 parts of different samples is respectively 107.7 μ g/g, 72.9 μ g/g and 55.8 μ g/g.With existing method (yellow Hua Jun, sialic assay method in the milk powder such as Xi Xinglin. analytical test journal, 2006,25 (4): 129～131; Chen Haijiao, sialic acid content in the high effective liquid chromatography for measuring breast milks such as Wang Ping. Food Science, 2011,32 (16): 1002～1008) measurement result is made comparisons, and learns that the sample size of checking the method is in rational scope.
The selection of test example 1 mass spectrophotometry condition and the optimization of parameter
Get sialic standard solution (mass concentration is 0.5 μ g/mL) and under the negative source of ESI and positive source condition, parent ion is carried out full scan.Obtain sialic parent ion bar graph under the different ions source.Experimental result shows: the abundance of sialic acid parent ion bar graph under the condition of ESI positive source is more than the height in negative source, therefore selected ESI positive source to do ionization source.
Under positive source module, sialic standard solution (mass concentration is 0.5 μ g/mL) is carried out the parent ion full scan, determine various sialic quasi-molecular ions and optimize respectively taper hole voltage (cone voltage, CV), determine that final taper hole voltage is 45V; Then take its quasi-molecular ion as parent ion, parent ion is 310.1, and its daughter ion is carried out full scan, and two ions that in the selection fragmention, abundance is higher are as qualitative, quantitative characteristic ion, and daughter ion is 274.1 and 292.1, and wherein 274.1 is quota ion.Sialic acid quantitative and qualitative analysis ion current chromatogram is seen Fig. 1.
Optimize various mass spectrum tuner parameters with positive ion multiple-reaction monitoring pattern at last, farthest improved detection sensitivity, the results are shown in Table 1.
The sialic chromatography-mass spectroscopy parameter of table 1
*: the feature daughter ion
Final mass spectrophotometry condition and the parameter of determining:
Ion gun: electric spray ion source; Scan mode: positive ion scanning; Detection mode: multiple-reaction monitoring system; Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 ℃; Desolventizing temperature: 450 ℃; Desolventizing airshed: 650L/hr; Taper hole blowback air flow: 50L/hr; Collision airshed: 0.24mL/min; Photomultiplier cell voltage: 650V.
The optimization of test example 2 chromatographic separation conditions
The selection of chromatographic column:
The physicochemical property of bound sialic acid and sialic acid are selected respectively Waters ACQUITY Ultra BEH HILIC(2.1 * 50mm and 2.1 * 100mm), BEH C based on 1.7 μ m granule technology at the partition characteristic of chromatographic column
18With four kinds of chromatographic columns of phenyl post, sialic acid is separated, learn that sialic retention time has larger difference.Adopt BEH C
18Separate with the phenyl post, after optimizing, sialic retention time is respectively 0.51min and 0.60min, and retention time is too short, and when standard items and sample detection, signal strength differences is larger, and sample sensitivity is all low, is unsuitable for sample detection.Employing BEH HILIC(2.1 * 100mm), after optimizing, sialic retention time is 2.15min, and retention time is more reasonable, but the hangover of peak type, chromatographic behavior is undesirable, and sensitivity is lower.Select at last BEH HILIC(2.1 * 50mm, 1.7 μ m) carry out determination and analysis, find that this post has good separating effect and higher sensitivity to sialic acid, retention time 1.22min is optimum value, illustrate that this post has more separating power preferably than traditional chromatographic column, response and sensitivity all can reach the requirement of experiment, and blank sample and mark-on sample drawing are seen Fig. 2 and shown in Figure 3.
The selection of mobile phase:
Sialic acid is a kind of carbohydrates, usually exists with free state or in the end of glycoprotein or the glycolipid form with glucosides, belongs to carbohydrate.Adding 90% acetonitrile with 10%5mM formic acid ammonia solution, water, 0.1% formic acid solution respectively in experiment tests, with standard items sample introduction under different mobile phase conditions of 1.0 μ g/mL, result shows that when 0.1% formic acid solution is made mobile phase, signal is the strongest, chromatographic behavior is better, and retention time is desirable.And acetonitrile has good solvent nature because of it, and polarity is larger, can dissolve most samples, infinitely dissolves each other with water and alcohol, and can improve the peak type of chromatographic peak.Therefore, mobile phase is selected 0.1% aqueous formic acid and acetonitrile.Contrasting aspect isocratic elution and gradient elution, when finding isocratic elution, the hangover of chromatogram peak-to-peak type, undesirable.Finally determine to choose 0.1%(v/v) aqueous formic acid and acetonitrile be as mobile phase, thereby adopt the mode of gradient elution to realize sialic good separation, significantly improve the distribution of sialic acid in chromatographic column, and had higher Ionization Efficiency, strengthened the sensitivity that detects.Sialic acid gradient elution separable programming is as shown in table 2.
Table 2UPLC/MS/MS sialic acid gradient elution separable programming
Initial ratio A is that 90%, B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A is down to 12%, B by 90% and is upgraded to 88% by 10%, and is maintained until 1.8min; 1.8 ~ 2.2min, A is upgraded to 90%, B by 12% and is down to 10% by 88%.
The optimization of test example 3 Pretreatments
The optimization of extraction conditions:
The sialic method of separation and Extraction is had nothing in common with each other from different raw materials, after sialic acid discharges, mostly adopts ion-exchange chromatographies to separate with gel permeation chromatography and utilizes the step such as HPLC purifying.Because separating effect chromatographic column is preferably selected in this experiment, the rear ultracentrifugal process of hydrolysis has been adopted in pre-treatment, and method is easy, effective.The focus of paying close attention in the hydrolysis program is that carbohydrates is subject to minimum destruction and sialic maximum release, studies show that, use 0.1%(v/v under 80 ℃ of conditions) hydrochloric acid hydrolysis 1h, all sialic acid chains are ruptured and make these residue deacylated tRNA decarboxylation, guarantee sialic complete hydrolysis.
The optimization of purification condition:
Because the comparison of ingredients in milk powder is complicated, the supernatant after centrifugal need carry out Solid-Phase Extraction to remove the impurity in sample liquid.The Solid-Phase Extraction method that the sample of milk powder class is commonly used at present mainly adopts anti-phase and ion-exchange SPE post, also has the HLB solid-phase extraction column.Studies show that, adopt the Solid-Phase Extraction column purification of HLB polymer filler, can make sample reach good clean-up effect, and method is simple, favorable reproducibility.Sample solution is loaded in Oasis HLB solid phase extraction column, and experiment adopts ultrapure water as leacheate, and ammoniacal liquor-methyl alcohol (volume ratio 25:75) solution is as eluent, and wherein 4mL leacheate and 3mL eluent can satisfy the purification requirement.
Test example 4 methodology checkings
Linear relationship is investigated:
Choose the blank powdered milk sample that does not contain component to be measured, process the blank matrix liquid of preparation through pre-treating method, accurately pipette appropriate sialic acid standard items storing solution, add in blank matrix liquid, join the matrix standard solution that concentration is followed successively by 0.05,0.1,0.2,0.5,1.0 and 5.0 μ g/mL.Upper machine is measured, and records peak area.The peak area (Y) of measuring according to sialic quota ion carries out linear regression with sialic mass concentration (X, μ g/mL), the drawing standard curve, and through Masslynx v4.1 data processing software analyzing and processing, sialic equation of linear regression is:
Y=8114.5X－811.09，R
2=0.9989
Wherein Y is the chromatographic peak peak area of mensuration, and X is sialic mass concentration (μ g/mL).
The recovery, precision and detection limit:
Take the infant milk powder sample of the unknown sialic acid concentration of 1.0g, measure after processing according to the method for embodiment 1, another claim the 1.0g sample on the pre-treatment basis, get the approximately supernatant of 7mL, and to add concentration be the standard solution of 5.0 μ g/mL, and in the volumetric flask of 10mL, upper machine is measured with the acetonitrile constant volume, the selection of mass spectrophotometry condition and parameter, Ultra Performance Liquid Chromatography condition are set according to embodiment 1 is described, obtain sialic concentration in unknown sample.Add respectively successively the sialic acid standard specimen of 0.1,0.5,2.5 and 5.0 μ g/mL on the basis of former content sample, after detecting, deduction is blank, the recovery of assay method, and data are as shown in table 3.
The sialic interpolation recovery of table 3, precision and detection limit (LOD) are (n=3)
It is 84.3%～98.9% that this method is added average recovery rate at bioassay standard, has the good recovery; Be limited to 0.05 μ g/mL under the range of linearity of this method, its signal to noise ratio (S/N ratio) (S/N) is greater than 10, therefore determine quantitatively to be limited to 0.05 μ g/mL; With the detection limit of 3 times of S/N computing method, learn that sialic minimum detectability is 0.01 μ g/mL, the method for setting up as can be known has higher sensitivity.
Claims (7)
1. one kind is utilized sialic method in Ultra Performance Liquid Chromatography series connection level Four bar mass spectrophotometry Milk Powder Formula For Infants, it is characterized in that comprising the following steps:
(1) drawing standard curve;
(2) determination: three kinds of form sialic acids in sample are extracted, and described three kinds of form sialic acids are respectively free sialic acid, compound sugar bound sialic acid and protein combination sialic acid;
Three kinds of form sialic acid solution that extraction obtains adopt respectively the Solid-Phase Extraction column purification of HLB polymer filler, adopt ultrapure water as leacheate, and ammoniacal liquor-methyl alcohol mixed solution is as eluent;
(3) after the extract solution after the purification that step (2) is obtained mixes, regulate pH value to 2.5 ~ 3, use the acetonitrile constant volume, the 0.22 organic membrane filtration of μ m is measured with UPLC-ESI-MS/MS;
The selection of (4) mass spectrophotometry condition and parameter:
Ion gun: electric spray ion source; Scan mode: positive ion scanning; Detection mode: multiple-reaction monitoring system; Capillary voltage: 3.0KV; Taper hole voltage: 45V; Source temperature: 150 ℃; Desolventizing temperature: 450 ℃; Desolventizing airshed: 650L/hr; Taper hole blowback air flow: 50L/hr; Collision airshed: 0.24mL/min; Photomultiplier cell voltage: 650V;
(5) chromatographic separation condition:
Chromatographic column and gradient elution: specification is 50mm * 2.1mm, the Waters ACQUITY Ultra BEH HILIC Ultra Performance Liquid Chromatography post of 1.7 μ m; Column temperature: 30 ℃; Sample chamber temperature: 30 ℃; Sample size: 5 μ L; Flow velocity: 0.25mL/min; Mobile phase A: acetonitrile; Mobile phase B: 0.1% formic acid (v/v) aqueous solution.
2. in accordance with the method for claim 1, it is characterized in that three kinds of form sialic acids described in step (2) are to extract in accordance with the following methods:
The extraction of free and compound sugar bound sialic acid: precision takes a certain amount of Milk Powder Formula For Infants, adds trichloroacetic acid solution to dissolving fully, reaches the purpose of protein precipitation, gets supernatant, under 4 ℃, 15000r/min condition, and centrifugal 10min, standby;
The sialic extraction of protein combination: add hydrochloric acid solution in the precipitation after the extraction of compound sugar bound sialic acid " free and " processed, put into 80 ℃ of water-bath 1h, cooling after taking out, under 4 ℃, 15000r/min condition, centrifugal 10min, standby.
3. in accordance with the method for claim 2, the concentration that it is characterized in that described trichloroacetic acid solution is 20%(v/v), described trichloroacetic acid solution is 1:6-10 with the volume mass ratio of Milk Powder Formula For Infants; The concentration of described hydrochloric acid solution is 0.1%(v/v), described hydrochloric acid solution is 1:2-5 with the volume mass ratio of Milk Powder Formula For Infants.
4. in accordance with the method for claim 1, the solid-phase extraction column that it is characterized in that the HLB polymer filler described in step (2) is in Oasis HLB solid phase extraction column, and in ammoniacal liquor-methyl alcohol mixed liquor, the volume ratio of ammoniacal liquor and methyl alcohol is 25:75.
5. the 2-5 that in accordance with the method for claim 1, it is characterized in that in step (3) being settled to former extract mixing liquor capacity with acetonitrile doubly.
6. in accordance with the method for claim 1, it is characterized in that the desolventizing gas described in step (4) is nitrogen, described collision gas is argon gas.
7. in accordance with the method for claim 1, it is characterized in that the elution program of the gradient elution described in step (5) is: in initial eluent, the percent by volume of mobile phase A is 90%, and the percent by volume of Mobile phase B is 10%, and is maintained until 0.2min; 0.2 ~ 0.4min, A is down to 12%, B by 90% and is upgraded to 88% by 10%, and is maintained until 1.8min; 1.8 ~ 2.2min, A is upgraded to 90%, B by 12% and is down to 10% by 88%.
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