CN109991259A - The method for detecting a variety of small molecule water soluble substances in milk powder product simultaneously - Google Patents

Abstract

The present invention provides a kind of methods for detecting a variety of small molecule water soluble substances in milk powder product simultaneously, and described method includes following steps: a. detects the dairy products sample using quantitative magnetic nuclear resonance method, and obtains¹The step of H-NMR map；B. described in determining¹The ownership at the quantifiable signal peak of each small molecule water soluble components in H-NMR map；C. to being determined that each quantifiable signal peak of ownership carries out deconvolution processing, and the content of each small molecule water soluble components is calculated.Its precision, stability, reproducibility and rate of recovery for detecting are superior to traditional qNMR quantitative approach, and formula research and development, production for baby from now on milk powder, quality Quality Control, safety detection provide technical support.

Description

The method for detecting a variety of small molecule water soluble substances in milk powder product simultaneously

Technical field

Technical solution of the present invention belongs to field of food detection, particular belongs to the composition detection field of milk powder based article, Especially, belong to milk power for infant and young children composition detection field.

Background technique

Milk power for infant and young children is to be properly added nutrient with cow's milk, sheep cream etc. for raw material, so that it is summed up composition and supplies A kind of artificial foods required for infant physical growth and development.For breast milk can be simulated to the greatest extent, meet the growth hair of infant Needs are educated, it is water-soluble that production firm can design additives, these small molecules such as addition nucleotide, organic acid, amino acid according to formula Property substance infant immunological regulation, improve memory, improve intestinal flora and promote in terms of all play weight The effect wanted.Therefore, particularly important to the quality monitoring of prescription emulsifiable powder small molecular water-soluble substances.

Currently, national standard mostly uses the methods of high performance liquid chromatography, spectrophotometry using standard items as reference, to wherein One or a substance detected, its shortcoming is that: (1) just for several compounds in a substance detected, Be difficult to react milk powder real quality comprehensively: such as, nucleotide is absorbed and used in the form of nucleosides in vivo, breast milk and baby children Contain nucleosides and nucleotide in youngster's prescription emulsifiable powder, but specify only the detection method of 5 kinds of nucleotide in national standard, lacks nucleosides Detection method, it is difficult to the true magnitude of reaction milk powder nucleotide constituents comprehensively；(2) detection efficiency is low: being detected by instrument The limitation of the reasons such as principle, high performance liquid chromatography can only detect certain class or a few class chemical components under same spectrogram, It is difficult to realize detection while many kinds of substance；(3) detection relies on standard items and does reference, it sometimes appear that the result of false positive.Mesh Before, the report detected in bibliography [1-2] to soluble small molecular in prescription emulsifiable powder mainly has the side such as liquid phase, gas phase, mass spectrum Method, but these methods are also both for one or the detection of a substance, it is difficult to realize the complete of a variety of small molecule water soluble substances Face detection.But prescription emulsifiable powder, especially milk power for infant and young children are a COMPLEX MIXED objects systems, because it is milk-derived by its raw material, Formula design, production technology, adding ingredient and condition of storage Different Effects, the material composition contained have difference, therefore, right The method that one or several chemical components are detected is difficult to be suitable for polygamy side, multicomponent milk power for infant and young children quality control System and safety evaluation, qualitative, quantitative analysis method can disposably be carried out to Multiple components by needing to develop.

Quantitative nuclear magnetic resonance (quantitative nuclear magneticresonance, qNMR) analysis method is to tie Based on structure measurement, have pretreatment process simple, sampling amount is small, and specificity is strong, and doing reference without reference substance can determine simultaneously Property, it is quantitative the advantages that, be widely used in the research fields such as Pharmaceutical Analysis.Nuclear magnetic resonance technique is the lossless inspection of unbiasedness It surveys, one-time detection can measure many kinds of substance ingredient, to the quality control of raw material, product stability, production process in food production The identification of quality in the novel substance ingredient and R & D of complex of middle generation has detection advantage well.Theoretically,¹H-NMR Spectral peak and sample in the hydrogen atom of each compound correspond, each of institute's sample hydrogen atom all has it in map Related spectral peak, and in the relatively strong and weak reflection sample of signal therefore the relative amount of each component detects universality height, is very suitable to It is detected while Multiple components.

Traditional qNMR method is that the compound of known quantity is added in the sample as correction reference internal standard, and sample is determined The signal peak area of amount proton is compared with internal standard compound quantifiable signal peak area, the final concentration for determining sample.Therefore, qNMR The accuracy of method quantifies peak integral area dependent on complete clearly quantifiable signal peak and accurately.However, prescription emulsifiable powder product, The especially complicated component of milk power for infant and young children, small molecule water soluble components structure is similar, content is not high, causes¹H-NMR letter There is signal overlap of peaks in number summit, therefore can be difficult to recognize, and have an impact the accuracy of subsequent integral calculation.For example, When carrying out quantitative analysis using traditional qNMR method, it can encounter and be unable to get complete signal integral, be influenced by phase, baseline adjusted The problems such as big, to influence qualitative, quantitative accuracy.

Full spectrum deconvolute (Global Spectral Deconvolution, GSD) be a kind of NMR signal processing method, Deconvolution processing can be carried out to entire map in seconds, integrate complicated overlap of peaks signal, thus provide complete peak shape, Chemical shift, coupling constant and integration information, are very suitable to¹The not high substance quantitative analysis of H-NMR signal overlap, signal-to-noise ratio. Meanwhile GSD processing can reduce¹H-NMR map is integrated to baseline and phase-adjusted requirement, keeps integrated value more stable and quasi- Really, referring to bibliography [3].Currently, this signal processing technology has been applied to the Analysis of quality control neck of natural drug extract Domain reports in the document referring to bibliography [4] and quantifies nmr for the determination astragalus injection freeze-dried powder using deconvolution In 8 kinds of nascent Metabolites, but have no the application in the increasingly complex field of food of matrix especially milk powder product field Report.Contain a large amount of polarity, nonpolarity element and protein in (1) prescription emulsifiable powder compared with natural drug water extract, Aqueous solution is emulsion, and nucleus magnetic hydrogen spectrum is increasingly complex, and needs to carry out sample scientific and reasonable pre-treatment, with retention analysis object Remove interfering substance；(2) prescription emulsifiable powder contains a large amount of acid, alkaline components, and the pH value of different formulations milk powder sample solution becomes Change larger, nucleus magnetic hydrogen spectrum chemical shift is influenced very big by the pH value of solution, it is therefore desirable to buffer solution be added to stablize sample The fine difference of pH value and counter-ion concentrations, but the water in buffer can generate huge water peak and embed other protons Signal, different from natural drug water extract analysis use zg30 pulse, need using increasingly complex water suppression pulse into The spectrum experiment of row hydrogen；(3) the small-molecule substance composition in prescription emulsifiable powder and/or structure are increasingly complex, especially institute's needle in the present invention Pair five kinds of nucleotide and its corresponding nucleotide structure it is very much like, the difficulty of signals assignment and quantitative analysis is bigger.

Thus, it will be seen that for the prescription emulsifiable powder emulsion that existing polar component has nonpolarity element again, on It states astragalus injection, forms relatively simple, and the molecular structure of 8 kinds of substances to be measured is also relatively simple, and characteristic peak is also more Obviously.Accordingly it is also possible to think current existing trial, it is still limited to forming relatively simple water extract or it is mixed The test separation of zoarium system.However, due to food compositions, especially in formula dairy products, soluble small molecular ingredient type is more To be various, complicated, also, the map overlapping of certain components in these compositions or mixed system is even more serious, for example, right In existing simultaneously a variety of nucleosides and nucleotide in product to be measured, detection is difficult simultaneously, therefore, such composition Complexity, degree of difficulty and the above-mentioned astragalus injection of detection be not on a level.In turn, by existing in group At detection means used in the relatively simple sample test of situation, applied to the detection of above-mentioned composition, testing result is still So it is filled with uncertainty.Therefore, for the detection of various milk power for infant and young children products, there is presently no occur attempting to make The report successfully detected with above-mentioned detection method.

Thus, it will be seen that in currently available technology, for using detection means how is extremely complex to composition to match The exploration quickly detected that square dairy products carry out Multiple components simultaneously can not say it is sufficient.Further, for having at present The exploitations of applicable object of various detection techniques also have the leeway further expanded.

Bibliography:

Bibliography [1]:

Sabater C,Prodanov M,Olano A,et al.Quantification of prebiotics in commercial infant formulas[J].Food Chemistry,2016,194(1):6-11.

Bibliography [2]:

Liu Z Q,Cocks B,Patel A,et al.Identification and quantification of phosphatidylinositol in infant formulas by liquid chromatography–mass spectrometry[J].Food Chemistry,2016,205(15):178-186.

Bibliography [3]:

Bernstein M A,S,Peng C,et al.Optimization and automation of quantitative NMR data extraction.[J].Analytical Chemistry,2013,85(12):5778- 5786.

Bibliography [4]: CN104698021 (A)

Summary of the invention

Problems to be solved by the invention

It is formulated in dairy products, contains a large amount of natural material milk compositions, and the nutritional ingredient manually added, therefore, group Substance classes and quantity at ingredient are extremely complex and numerous.Technical problem to be solved by the present invention lies in provide one Kind simultaneously, quickly detects the various small molecule water soluble substances of above-mentioned prescription emulsifiable powder product, especially milk power for infant and young children product Method, and this method testing result have high repeatability, accuracy and the rate of recovery.

Meanwhile technical problem to be solved of the invention lies also in how to expand existing quantitative nuclear magnetic resonance and complete Compose the purposes for the joint technology deconvoluted.

The solution to the problem

The present invention is by continuously attempting to, it is determined that can successfully solve above-mentioned technical problem by the following method.

It is described present invention firstly provides a kind of method for detecting a variety of small molecule water soluble substances in milk powder product simultaneously Method includes the following steps:

A. the dairy products sample is detected using quantitative magnetic nuclear resonance method, and obtained¹The step of H-NMR map；

B. described in determining¹The ownership at the quantifiable signal peak of each small molecule water soluble components in H-NMR map；

C. to being determined that each quantifiable signal peak of ownership carries out deconvolution processing, and each small molecule water soluble components are calculated Content.

According to the process described above, before the step a, further include the steps that carrying out sample pre-treatment, it is described The step of pre-treatment includes ultrafiltration centrifugal treating, and in the ultrafiltration centrifugal treating, revolving speed is 9000~10000r/min, when centrifugation Between be 15~25min, centrifuging temperature be 1~10 DEG C.

According to the process described above, in the step a in quantitative magnetic nuclear resonance method, internal standard compound is 3- (trimethyl silicane Base) deuterated sodium propionate (TMSP), while buffer solution is added, so that the pH value of system is 7.2~7.6.

According to method described in any of the above item, the small molecule water soluble components are selected from one or more selected from following 23 The ingredient of kind: valine, lactic acid, alanine, acetic acid, citric acid, creatine, creatinine, choline, L- L-carnitine, orotic acid, secondary Huang Purine nucleosides, adenosine, cytidine, hippuric acid, uridine, guanosine, cytidylic acid are urinated phonetic Pyridine nucleotide, guanylic acid, formic acid, inosinic acid, adenylic acid and niacin.

According to the process described above, the small molecule water soluble components include 23 kinds of ingredients described in whole.

According to method described in any of the above item, recycling of the method for small molecule water soluble components described in each Rate is 85% or more.

Further, the present invention also provides a kind of method of quickly detection milk power for infant and young children ingredient, the method packets The method for including described in any of the above item while detecting a variety of small molecule water soluble substances in milk powder product.

The effect of invention

Effect acquired by above-mentioned technical proposal of the present invention is as follows:

The target user of milk power for infant and young children is special, belongs to special diet food, and quality problem can not be ignored. Soluble small molecular organic matter in milk power for infant and young children is most important to the growth and development of infant, to water in prescription emulsifiable powder Dissolubility small-molecule substance fast quantitative analysis, has important practical significance to the quality control and evaluation of prescription emulsifiable powder.

Implementation for above-mentioned technical proposal can simultaneously, quickly detect prescription emulsifiable powder product, especially infant and match The presence and content of a variety of small molecule water soluble substances in square milk powder product.In particular, the GSD-qNMR that the present invention establishes is quantitative Method can include 23 small molecule water dissolubilities including five kinds of nucleotide and five kinds of nucleosides with measurement in milk powder in baby simultaneously The content of object is closed, precision, stability, reproducibility and the rate of recovery of detection are superior to traditional qNMR quantitative approach, for from now on Formula research and development, production, quality Quality Control, safety detection offer technical support of the baby with milk powder.

Further, above scheme provided by the invention actually not only realizes the integrated optimization to existing detection means, The test object range that existing detection technique is applicable in also has been widened simultaneously, and has been demonstrated conscientiously for new, composition is more complicated Test object the result is that reliable and comprehensive.

Detailed description of the invention

Fig. 1: GSD-qNMR quantitative detection flow chart in the present invention

Fig. 2: milk power for infant and young children sample¹H-NMR map (A) and deconvolute signal extraction figure (B)

Fig. 3-1 and Fig. 3-2: milk power for infant and young children¹H-NMR High-Field amplification map (A) is deconvoluted with corresponding region High-Field Quantitative peak-to-peak signal extracts figure (B)

1.TMSP；2. valine；3. lactic acid；4. alanine；5. acetic acid；6. citric acid；7. creatine；8. creatinine；9. choline； 10.L- L-carnitine；11. cytidine；12. adenosine；13. inosine；14. orotic acid；15. horse urinates Acid；16. uridine；17. guanosine；18. cytidylic acid；19. uridylate；20. guanosine Acid；21. formic acid；22. inosinic acid；23. adenylic acid；24. niacin

Specific embodiment

A specific embodiment of the invention will be described below, such as without Special Statement, unit used in the present invention It is SI units, and the numerical value occurred in the present invention, numberical range should all be interpreted as containing in industrial production The inevitable Systematic Errors of institute.

<prescription emulsifiable powder product>

The targeted object of detection method is various prescription emulsifiable powder products.The prescription emulsifiable powder product is for maximum Limit close to breast milk, and a series of transformations are carried out to fresh cow milk and comply with a kind of milk replacer of digestion and absorption and nutritional need.

Especially, for milk power for infant and young children product, the security requirement for its formula is very high.The present invention Detection and quality evaluation of the provided detection method particularly suitable as these milk powder products, can in time, be quickly obtained Reliable testing result.

For the above prescription emulsifiable powder product, currently, mostly using the methods of liquid phase, gas phase, mass spectrum to baby in national standard and document One or a kind of soluble small molecular material progress quantitative detection in baby formulas milk powder.Also, these existing methods are all Using standard items as reference, one of those or a substance are detected, cumbersome, detection efficiency is low, easily occurs false The positive, therefore, progress is comprehensive, evaluation is difficult fast and accurately.

In addition, in the present invention, the specific form of above-mentioned prescription emulsifiable powder product is not required particularly, in some embodiment party In case, these products are such as the formula milks with powdered existing.In other embodiments, these products are to paste Shape object perhaps these paste or paste existing for the form of paste be actually also based on powdered basic products into Obtained from the processing of one step.

<pretreatment of sample>

In some preferred embodiments of the present invention, pre-treatment or pre- place have been carried out for the sample of prescription emulsifiable powder product Reason, to obtain pretreated sample.

Further, heretofore described pretreatment purpose is enrichment to be carried out for main soluble small molecular and to non- Main component is separated and is removed.In some embodiments, using the processed sample of pre-treatment step, for subsequent Detection can be played the role of significantly improving accuracy.

In the present invention, preferred pretreatment mode is to carry out low temperature ultrafiltration centrifugal treating.For example, using ultrafiltration centrifugation apparatus Carry out pre-treatment step.In some embodiments, sample is dissolved in pure water or deionized water, is dissolved.It is right It is not limited in the means of dissolution, common dissolution supplementary means can be used, typically, such as assisted using ultrasonic oscillation Carry out the dissolution of sample.

For sufficiently having dissolved the solution of sample, centrifugal treating is carried out using ultrafiltration centrifugation apparatus.In some preferred realities Apply the revolving speed that centrifugal treating process setting is centrifuged in scheme for 9000~10000r/min, preferably 9200~9700r/min, more Preferably 9400~9600r/min.Further, for the time of centrifugal treating, preferably 10~30min, preferably 15~ 25min.Centrifuging temperature is 1~10 DEG C, preferably 2~8 DEG C, further preferably 3~6 DEG C.In some cases, when centrifugation temperature When degree is more than 10 DEG C, it is possible to the undesirable dissolution for causing fat constituent, it is molten for it is expected when centrifuging temperature is lower than 1 DEG C The dissolution degree of the ingredient of solution may be insufficient.Further, in a preferred embodiment of the present invention, it is centrifuged in above-mentioned ultrafiltration In processing, the ultra-filtration centrifuge tube used is that can be realized the ultra-filtration centrifuge tube that molecular cut off is 3000 dalton, such In the case of, it can be relatively easy to separate desired detection ingredient.

In the present invention, pass through the selection of above-mentioned centrifugal rotational speed and centrifugation time, centrifuging temperature and centrifuge tube characteristic, energy Removal of enough ideal realizations for nonpolarity elements and protein such as grease types in prescription emulsifiable powder emulsion, remains each Kind small molecule water soluble substance, it is obvious for the improvement effect of subsequent detection reliability.In addition, in addition to pre- place disclosed above Reason method, it is unrestricted, before this, other preprocess methods can also be used in the present invention, for example, for laboratory milk The drying of powder product sample, grinding, filtering, dissolution-distillation, the mutually means such as separation.For equipment used by these means, do not have It is particularly limited to, uses the common equipment in this field.

<quantitative magnetic nuclear resonance method>

Quantitative nuclear magnetic resonance (quantitative nuclear magneticresonance, qNMR) analysis method is to tie Based on structure measurement, have pretreatment process simple, sampling amount is small, and specificity is strong, simple operation and other advantages.

It must use reference subject can be simultaneously like that in addition, quantitative magnetic nuclear resonance method does not need traditional detection method Carry out qualitative, quantitative determination.

In the present invention, quantitative magnetic nuclear resonance method can be carried out using internal standard method or external standard method.Of the invention preferred Embodiment in, use internal standard method.Water-soluble internal standard compound can be used in selection for internal standard compound, some excellent in the present invention In the embodiment of choosing, internal standard compound uses 3- (trimethyl silicon substrate) deuterated sodium propionate (TMSP).TMSP dissolubility is good, chemical property Stablize, do not chemically reacted with the small molecule water soluble components in prescription emulsifiable powder product, and has content accurately with high purity The characteristics of.Meanwhile TMSP can preferably avoid overlapping with prescription emulsifiable powder product small molecular water-soluble substances.Therefore, In the preferred embodiment of the invention, use TMSP that can play the role of improving testing precision as internal standard compound.

In the present invention, the sample that pretreatment obtains is dissolved in heavy water, and at the same time quantitative internal standard compound is added to In the solution, so that the solution is uniformly mixed to configure sample to be tested.

In addition, in a preferred embodiment of the present invention, the configuration for sample to be tested, in addition to sample, above-mentioned internal standard Other than object, heavy water, buffer solution is also used.The addition of buffer solution can stablize the pH value of sample, make different formulations milk powder sample Product solution map is stablized.Usually in configuration, sample: PBS: the volume ratio of heavy water can be 6~7.5:1.8~2.1:1, and The addition of usual buffer solution enables to the pH value of system to maintain between 7.2~7.6.

Specifically, the sample to be tested configuration mode that can be enumerated are as follows: take the sample of 420 μ L, the PBS of 120 μ L, the weight of 60 μ L Water (TMSP containing 11.208 μ g) is mixed, and last volume is 600 μ L.

In some embodiments of the present invention, in sample to be tested the content of TMSP between 20~10 μ g.Actual In detection, the amount of the sample to be tested of last actually machine testing is between 540~600 μ L.

For instrument used in quantitative magnetic nuclear resonance method, there is no particular limitation by the present invention, and this field can be used Typically Bruker Nuclear Magnetic Resonance can be used for example in usually used test equipment.

In the present invention, obtained using quantitative magnetic nuclear resonance method by Multiple-Scan¹H-NMR map.For scanning times, It is not particularly limited, can be set according to test equipment concrete condition, generally can be 4~128 times.It is some preferably in the present invention Embodiment in, by what is directly measured¹H-NMR map is handled using software or program with can be relatively clear variable Mode presents final as a result, typically, 3.2 software of Bruker Topspin can be used and handled.

It is obtaining¹After H-NMR map, it is thus necessary to determine that, compound corresponding to the characteristic peak in map determines¹H-NMR The ownership at the quantifiable signal peak of each small molecule water soluble components in map.Method for determining ownership, the present invention be not special Restriction, such as can combine¹H-NMR data result simultaneously carries out ownership determination with reference to existing document.Typically, according to map The signal characteristics such as chemical shift, the coupling constant of middle proton signal, in conjunction with¹The information such as H-NMR data and pertinent literature report, altogether It is right¹Each small molecule water soluble components carry out signals assignment in H-NMR map.

<data processing of deconvoluting>

To obtained above in the present invention¹The data processing that H-NMR map deconvolutes.As previously mentioned, full spectrum goes to roll up Product (Global Spectral Deconvolution, GSD) is the signal processing method of NMR a kind of, can be right in seconds Entire map carries out deconvolution processing, integrates complicated overlap of peaks signal.

The subsequent content for accurately calculating each small molecule water soluble components is enabled to by GSD data processing.

In GSD Data processing, typically, the progress of MestReNova (version 12.0, Spain) software can be used Processing.By the correction to signal peak, deconvolute to the quantitative peak of each small molecule water soluble substance and quantitative internal standard compound Processing, the final peak area for obtaining each object substance and quantifying peak.

Each small molecule water soluble components to be measured are obtained referring to internal standard compound by the Integral Processing to the above peak area Content.

<small molecule water soluble components>

The present invention using the method for quantitative nuclear magnetic resonance, acquires milk powder aqueous solution first¹H-NMR map, in map 23 small molecule water soluble substances such as nucleotide, nucleosides, organic acid, amino acid carry out signals assignments, specifically wherein wrap The five kinds of nucleotide and corresponding five kinds of nucleosides contained；And use GSD method pair¹23 quantitative combination objects of H-NMR map and interior The quantitative peak of mark object has carried out integral and has extracted.

For 23 kinds of above-mentioned small molecule water soluble substances, belong to the important component in prescription emulsifiable powder product, for these The accurate measurement of small molecule water soluble substance type and content can provide section for the quality of thoroughly evaluating prescription emulsifiable powder product Foundation.

Further, above-mentioned small molecule water soluble substance includes below one or more: valine, lactic acid, alanine, second Acid, citric acid, creatine, creatinine, choline, L- L-carnitine, orotic acid, inosine, adenosine, cytimidine core Glycosides, hippuric acid, uridine, guanosine, cytidylic acid, uridylate, guanylic acid, formic acid, Inosinic acid, adenylic acid and niacin.

Detection method provided by the present invention can measure one of above-mentioned various small molecule water soluble substances or simultaneously Measure the presence and content of many kinds of substance ingredient.Especially, detection method provided by the present invention can be detected quickly simultaneously The presence and content situation of above-mentioned 23 kinds of small molecule water soluble substances in prescription emulsifiable powder product, wherein for using conventional method The a variety of nucleosides and nucleotide of be difficult to quantitative detection are also able to carry out accurately quantitative analysis.

Embodiment

<prescription emulsifiable powder product detection method>

Hereinafter, in detection method, first to the aqueous solution of milk power for infant and young children by the way of ultrafiltration centrifugation Pre-treatment has been carried out, has stablized the pH value of sample solution by the way that buffer is added, acquisition milk powder aqueous solution¹H-NMR map, to figure 23 small molecule water soluble substances such as nucleotide, nucleosides, organic acid, amino acid in spectrum carry out signals assignments, specifically its In include five kinds of nucleotide and corresponding five kinds of nucleosides；Common integration method and GSD method pair is respectively adopted¹H-NMR figure The quantitative peak of 23 quantitative combination objects and internal standard compound has carried out integral extraction in spectrum, has carried out methodological study to two methods； Finally using establish GSD-qNMR method, to a variety of small molecule water soluble components in the milk power for infant and young children of 6 brands into Quantitative determination is gone.

Material and reagent

The milk power for infant and young children of different batches producer is purchased from this city supermarket, within the shelf-life, closed container after unlatching Interior 4 DEG C of preservations；Heavy water (D₂O, deuterated degree: 99.8%), CIL Corp., 3- (trimethyl silicon substrate) deuterated sodium propionate (TMSP) U.S., Thermo Fisher company, the U.S. PBS solution (pH=7.4)；Millipore company, the U.S. ultra-filtration centrifuge tube (3KD), Norell Norell company, the 5mm nuclear magnetic tube U.S..

Instrument and equipment

Bruker AVANCE 600MHZ superconduction fourier transform NMR instrument (it pops one's head in equipped with CPBBO, Topspin 3.2 processing softwares and 60 autosamplers)；Mettler Toledo company, XS204 electronic balance Switzerland；Centrifuge Eppendorf company, 5424R centrifuge Germany.

Experimental method

The preparation-of sample solution

2.00g powdered milk sample is weighed in 10mL centrifuge tube with cover, pure water 5mL, vortex 3min, ultrasonic 15min is added Afterwards, then the 1min that is vortexed.It takes the liquid 3mL after being vortexed in the ultra-filtration centrifuge tube of 3KD, is centrifuged under 4 DEG C and 9500r/min revolving speed 20min.In 5mm nuclear magnetic tube, the heavy aqueous solution for adding 60 μ L (contains the 420 μ L of sample liquids for taking ultrafiltration to be centrifuged The TMSP of 11.208 μ g) and 120 μ L PBS (pH=7.40) solution, it is to be measured after mixing.

Instrument condition-

Qualitative determination:¹H-NMR experiment uses Bruker instrument pulse protocol noesypr1d, detects temperature 298K, setting Parameter¹90 ° of pulse widths of H are 11.90 μ s, spectrum width 12019.230Hz, centre frequency 2811.01Hz, and scanning times are 32 times.J- resolution,¹H-¹H COSY, HSQC, HMBC use Bruker calibration pulse program, and detection temperature is 298K, scanning time Number is respectively 32,16,8,16 times.

Quantitative determination:¹H-NMR experiment uses Bruker instrument pulse protocol noesypr1d, detects temperature 298K, setting Parameter¹90 ° of pulse widths of H are 11.90 μ s, spectrum width 12019.230Hz, centre frequency 2811.01Hz.When pulse daley Between be 4s, mixed sweep number be 96.

Sample measurement and spectrogram processing-

Under the experiment condition of instrument condition item, adjustment instrument parameter, tuning, temperature control, shimming, sampling and Fourier become It changes, obtains¹H-NMR map.It measures¹H-NMR spectrum is handled using Bruker Topspin3.2 software, and transformation points are 64K, LB It for 1.00Hz, is handled with exponential window function, baseline and phasing are all made of manual mode progress, and TMSP is internal standard signal (δ 0.00)。

- GSD data processing-

Treated¹H-NMR map imports MestReNova (version 12.0, Spain) software, selects integral correction Method is that signal peak corrects (Peaks), and setting refinement levels are grade 1, is determined 23 quantitative materials and quantitative internal standard TMSP It measures peak and carries out deconvolution processing, obtain the peak area at each quantitative peak.

Quantitative calculation formula-

Be quantitative internal standard with TMSP, the map integration method after traditional integration method and deconvolution processing is respectively adopted by Following formula calculates the content of each substance in sample.

Formula:

In formula (1): W is the quality of substance；A is quantitative peak integral area；The proton number that N is included for quantitative peak；M is object Matter relative molecular mass；Subscript u and t are respectively analyte and internal standard compound.

<methodological study is carried out to the method that inventive formulation milk powder product detects using standard sample>

In the present invention, the investigation or verifying of methodology are carried out to prescription emulsifiable powder product detection method by following methods.

Standard sample and standard curve-

It weighs 23 kinds of plasmid standards for quantitation to be placed in right amount in 10mL volumetric flask, be shaken up after adding water to graduation mark, be configured to mixed mark Stock solution.Suitable mixed mark stock solution is drawn, pure water is added, is diluted to the mixed mark solution of 5 concentration respectively.Take mixed mark solution 420 μ L add the PBS (pH=of heavy aqueous solution (TMSP containing 11.208 μ g) and 120 μ L of 60 μ L in 5mm nuclear magnetic tube 7.40) solution mixes.It is carried out according to identical determination condition above¹H-NMR map quantifies peak to integrate obtained standard items Area is Y-axis, does linear regression as X-axis so that standard items quality is added.

Precision verifying-

Test sample portion is prepared by method in " preparation of sample solution " above, by the test sample prepared " instrument above It is measured in parallel 6 times under conditions of condition ", calculates the relative standard deviation (RSD) that 23 compounds in sample quantify peak integral area.

Repeatability verifying-

It takes same brand, with a batch of powdered milk sample, prepares 6 parts in parallel by method in " preparation of sample solution " above Test solution measures under conditions of " instrument condition " above¹H-NMR map calculates 23 compounds in sample and quantifies peak The RSD of integral area.

Stability-

Take same test solution, respectively 0 after preparation, 2,4,8,12, for 24 hours, measured under the conditions of same as above¹H- NMR spectra, 23 compounds for calculating each time point map quantify the RSD of peak area.

The rate of recovery-

9 parts of powdered milk sample of same brand are weighed in parallel, prepare test solution according to same procedure above, wherein 3 parts As control, other 6 parts of each mixed mark solution being added after 100 μ L dilution, wherein in mixed mark solution containing 59.00 μ g/mL of valine, 878.75 μ g/mL of lactic acid, 56.25 μ g/mL of alanine, 23.75 μ g/mL of acetic acid, 1786.23 μ g/mL of citric acid, creatine 153.60 μ g/mL, 41.75 μ g/mL of creatinine, 325.75 μ g/mL of choline, 4.25 μ g/mL of L- L-carnitine, 34.5 μ g/mL of cytidine, 36.75 μ g/mL of adenosine, 27.88 μ g/mL of inosine, 108.65 μ g/mL of orotic acid, 98.00 μ g/ of hippuric acid ML, 15.45 μ g/mL of uridine, 32.98 μ g/mL of guanosine, 113.66 μ g/mL of cytidylic acid, uridine diphosphate 116.98 μ g/mL of thuja acid, 163.25 μ g/mL of guanylic acid, 6.75 μ g/mL of formic acid, 36.75 μ g/ of inosinic acid ML, 89.87 μ g/mL of adenylic acid, 44.65 μ g/mL of niacin.Condition measurement same as above¹H-NMR map calculates each The recovery of standard addition of compound.

The result and analysis-of standard sample

Choose the standard sample detection and interpretation of result of above-mentioned preparation.

1.Quantitative magnetic nuclear resonance method detection sample to be tested simultaneously obtains¹The qualitative ownership result of signal peak after H-NMR map

According to the signal characteristics such as the chemical shift of proton signal, coupling constant in map, in conjunction with¹H-NMR data and correlation The information such as document report, total pair¹23 small molecule water soluble components carry out signals assignment in H-NMR map, its acceptance of the bid that see Table 1 for details Thick data are the quantitative proton information of compound.

23 small molecule water soluble components in 1. milk power for infant and young children of table¹H-NMR map signals assignment table

Compound name	δH(J/Hz)
		Valine	0.99 (d, 7.00), 1.04 (d, 7.00), 2.26 (m)
Lactic acid	1.33 (d, 6.90), 4.13 (q, 6.90)
		Alanine	1.48(d)
Acetic acid	1.92(s)
		Citric acid	2.54 (d, 16.26), 2.70 (d, 16.26)
Creatine	3.04 (s), 3.93 (s)
		Creatinine	3.05 (s), 4.05 (s)
Choline	3.20 (s), 4.08 (s)
		L- L-carnitine	3.22 (s), 3.43 (m)
Orotic acid	6.20(s)
		Inosine	6.10 (d, 5.76), 8.25 (s), 8.36 (s)
Adenosine	6.08 (d, 6.36), 8.27 (s), 8.36 (s)
		Cytidine	6.06 (d, 7.70)
Hippuric acid	7.56 (m), 7.65 (m), 7.84 (d, 7.31)
		Uridine	7.88 (d, 8.30)
Guanosine	8.01(s)
		Cytidylic acid	8.10 (d, 7.75)
Uridylate	8.11 (d, 8.07)
		Guanylic acid	8.21(s)
Formic acid	8.46(s)
		Inosinic acid	8.58(s)
Adenylic acid	8.61(s)
		Niacin	7.61 (m), 8.26 (m), 8.72 (m), 8.94 (d, 2.36)

Note: the quantitative proton information that thick data are compound is marked

2.Quantitative approach based on GSD data processing is established

The deconvolution processing-at quantifiable signal peak

According to " GSD data processing " step above, using Deconvolution Method from sample¹23 changes are extracted in H-NMR map Close the quantitative peak-to-peak signal of object, sample¹H-NMR map and signal extraction figure are shown in Fig. 2, Fig. 3, carry out content according to above-mentioned formula (1) It calculates.

3. methodology validation

Adopt map by Fourier transform, calibration, phase and baseline adjustment after, GSD method and traditional quadrature is respectively adopted Two ways integrates the quantitative peak of internal standard and compound, and the methodology validation of two kinds of integral ways the results are shown in Table 2, table 3.

Table 2 is using 23 compound quantitation methodology results after traditional quadrature method processing map

Table 3 is using 23 compound quantitation methodology results after GSD method processing map

The results show that using the related coefficient of traditional quadrature mode quantitative approach between 0.9843~0.9990, it is accurate The RSD of degree between 2.03~8.28, repeated RSD between 2.53~7.23, the RSD of stability 2.19~5.93 it Between, the rate of recovery is between 72.6%~129.41%；Using the related coefficient of GSD integral way quantitative approach 0.9960~ Between 0.9997, the RSD of precision between 1.02~3.72, repeated RSD between 3.68~1.34, stability RSD is between 1.62~3.20, and the rate of recovery is between 85.02%~110%.It can be seen that the precision of GSD quantitative approach, Stability, reproducibility and the rate of recovery will be better than traditional integration method, and therefore, the qNMR method based on GSD processing is more suitable for The complete detection of milk power for infant and young children small molecular water-soluble substances.It is fixed to reduce especially with this integral way The dependence adjusted to baseline is measured, when interior scalar quantity peak, which quantifies peaking displacement study with compound, differs larger, effect is become apparent.

That is, using the test of standard sample above, the reliability of the method for the present invention is demonstrated.

<detection of sample to be tested detects>

Precision weighs the milk power for infant and young children sample of 6 brands, provides test product according to " preparation of sample solution ", according to Conditions described above measures sample¹H-NMR map integrates quantitative peak using GSD method, calculates according to formula (1) The content of each quantitative material, the results are shown in Table 4.

The content (mg/100g, n=3) of 46 brand milk powder sample analytes of table

Serial number

Compound

No. 1 sample

No. 2 samples

No. 3 samples

No. 4 samples

No. 5 samples

No. 6 samples

1

Valine

1.47±0.05

0.88±0.02

4.7±0.17

0.69±0.03

0.62±0.02

7.34±0.27

2

Lactic acid

12.55±0.34

36.57±0.99

111.96±1.6

38.85±0.97

4.64±0.09

116.9±2.64

3

Alanine

1.76±0.05

2.71±0.07

5.86±0.14

2.41±0.09

2.44±0.08

7.41±0.18

4

Acetic acid

2.44±0.05

4.01±0.09

11.97±0.26

9.39±0.20

2.04±0.03

2.45±0.05

5

Citric acid

287.3±8.00

431.98±9.98

280.16±7.63

552.93±15.55

620.25±12.06

600.61±13.99

6

Creatine

19.11±0.24

37.9±0.61

47.14±0.98

32.89±0.78

40±0.67

21.99±0.52

7

Creatinine

5.93±0.24

10.09±0.35

13.31±0.48

7.66±0.38

8.75±0.22

5.23±0.18

8

Choline

39.23±1.10

29.85±0.78

14.83±0.53

66.87±2.02

52.05±2.16

56±1.47

9

L- L-carnitine

20.99±0.69

13.58±0.31

28.89±0.80

19.13±0.66

14.55±0.53

7.64±0.23

10

Cytidine

2.24±0.09

2.29±0.08

2.6±0.09

1.14±0.04

-

-

11

Adenosine

-

-

-

-

-

-

12

Inosine

-

3.24±0.08

7.5±0.17

-

-

-

13

Orotic acid

2.23±0.03

8.45±0.09

3.32±0.06

11.65±0.28

27.46±0.34

16.16±0.13

14

Hippuric acid

8.53±0.25

14.34±0.32

18.81±0.59

7.82±0.23

17.23±0.53

12.88±0.29

15

Uridine

8.97±0.25

5.58±0.15

48.99±1.09

4.86±0.11

4.51±0.14

1.31±0.04

16

Guanosine

-

-

-

-

-

-

17

Cytidylic acid

9.33±0.24

15.81±0.41

4.99±0.14

18.23±0.52

17.39±0.49

25.43±0.65

18

Uridylate

17.43±0.47

20.67±0.56

19.86±0.59

10.85±0.29

19.64±0.49

13.39±0.37

19

Guanylic acid

1.14±0.03

7.27±0.21

1.88±0.05

2.26±0.06

-

11.11±0.24

20

Formic acid

0.39±0.01

0.68±0.01

1.28±0.01

0.45±0.01

0.64±0.01

1.3±0.02

21

Inosinic acid

2.73±0.08

4.24±0.13

-

1.56±0.05

1.28±0.04

-

22

Adenylic acid

6.96±0.17

8.23±0.22

6.77±0.19

3.63±0.11

3.05±0.09

8.2±0.24

23

Niacin

3.29±0.11

1.83±0.06

5.4±0.17

4.09±0.14

2.33±0.08

5.01±0.17

Industrial availability

Detection method provided by the present invention is verified, be it is accurate and reliable, can with acting formulations dairy products products at Sorting is surveyed and evaluation.

Claims (7)

1. a kind of method for detecting a variety of small molecule water soluble substances in milk powder product simultaneously, which is characterized in that the method packet Include following steps:

A. the dairy products sample is detected using quantitative magnetic nuclear resonance method, and obtained¹The step of H-NMR map；

B. described in determining¹The ownership at the quantifiable signal peak of each small molecule water soluble components in H-NMR map；

C. to being determined that each quantifiable signal peak of ownership carries out deconvolution processing, and containing for each small molecule water soluble components is calculated Amount.

2. the method according to claim 1, wherein further including carrying out pre-treatment to sample before the step a The step of, the step of pre-treatment includes ultrafiltration centrifugal treating, and in the ultrafiltration centrifugal treating, revolving speed is 9000~ 10000r/min, centrifugation time are 15~25min, and centrifuging temperature is 1~10 DEG C.

3. method according to claim 1 or 2, which is characterized in that interior in the step a in quantitative magnetic nuclear resonance method Mark object is 3- (trimethyl silicon substrate) deuterated sodium propionate (TMSP), while buffer solution is added so that the pH value of system be 7.2~ 7.6。

4. method according to claim 1-3, which is characterized in that the small molecule water soluble components are selected from one kind Or a variety of selected from following 23 kinds of ingredient: valine, lactic acid, alanine, acetic acid, citric acid, creatine, creatinine, choline, L- are left-handed Carnitine, orotic acid, inosine, adenosine, cytidine, hippuric acid, uridine, guanosine, Cytidylic acid, uridylate, guanylic acid, formic acid, inosinic acid, adenylic acid and cigarette Acid.

5. according to the method described in claim 4, it is characterized in that, the small molecule water soluble components include 23 described in whole Kind ingredient.

6. method according to claim 1-5, which is characterized in that the method is for small molecule described in each The rate of recovery of water soluble ingredient is 85% or more.

7. a kind of method of quickly detection milk power for infant and young children ingredient, which is characterized in that the method includes being wanted according to right The method asked described in any one of 1-6 while detecting a variety of small molecule water soluble substances in milk powder product.