CN106290275A

CN106290275A - Molecular fluorescence difference mark-on is utilized to measure egg yolk, VB2the method of riboflavin in tablet

Info

Publication number: CN106290275A
Application number: CN201610611269.2A
Authority: CN
Inventors: 高向阳; 张娜; 高遒竹; 郭楠楠; 焦健侠; 魏龙; 岳希举
Original assignee: Zhengzhou University of Science and Technology
Current assignee: Zhengzhou University of Science and Technology
Priority date: 2016-07-30
Filing date: 2016-07-30
Publication date: 2017-01-04
Anticipated expiration: 2036-07-30
Also published as: CN106290275B

Abstract

One utilizes molecular fluorescence difference mark-on to measure egg yolk, VB₂The method of riboflavin in tablet, without drawing curve, only need to add target test solution by two differences can quickly obtain the mass fraction of component to be measured, sample background interference is completely eliminated during mensuration, simultaneously, use in two same volume test solutions, add different amounts of standard solution, in sample because of tested concentration of component different may because being polymerized, dissociate, the introduced evaluated error of the side reaction such as photodissociation can obtain and compensate or deduct, accuracy of measurement is protected further and improves.The inventive method detection is limited to 5.76ng/mL, is quantitatively limited to 1.92 × 10^‑2μg/mL。

Description

Molecular fluorescence difference mark-on is utilized to measure egg yolk, VB2The method of riboflavin in tablet

Technical field

The invention belongs to technical field of analysis and detection, be specifically related to one utilize molecular fluorescence difference mark-on measure egg yolk, VB₂The method of riboflavin in tablet.

Background technology

Vitamin B₂, also known as riboflavin, easily decompose under illumination and ultraviolet irradiation, acidproof the most alkaline-resisting, dissolve in Sodium-chloride water solution, is soluble in dilute sodium hydroxide solution.Riboflavin is extremely important to human body, and when lacking, metabolism is easily sent out Raw obstacle.Riboflavin is water miscible, will not accumulate at human body, often supplement, in egg yolk, Semen Glycines, milk, yeast etc. Content is more.

Egg yolk contains abundant fat and protein, including helping the lecithin of synthesis of acetyl choline, contains simultaneously The mineral such as abundant calcium ferrophosphorus, and abundant fatsoluble vitamin, riboflavin content is the highest, and therefore, egg yolk is very Suitable for baby eats；Vitamin B₂Tablet is yellow tablet, is used for treating angular cheilitis, cheilosis, glossitis, scrotitis, conjunctiva Inflammation, seborrheic dermatitis etc..General common specification vitamin B₂Tablet contains 5 milligrams of riboflavin, possibly together with other adjuvants in medicine, as Starch, sucrose, magnesium stearate.Due to the riboflavin importance to human body, and riboflavin easily destroys, and therefore originates it The mensuration of middle content is particularly significant.

At present, the assay method of riboflavin is more, such as electrochemical process, high performance liquid chromatography, high performance capillary electrophoresis, chemistry Luminesceence analysis method, oscilloscopic polarography, fluorescent spectrometry etc..These methods have respective pluses and minuses, as electrochemical methods Although assay method is simple, sensitivity is higher, but accuracy and capacity of resisting disturbance are poor.High performance liquid chromatography has mensuration knot Fruit accurately, reliable, but sample pretreatment process is relatively complicated, measures the most time-consuming, instrument costly, trivial operations.Hair Cons electrophoresis method has good sensitivity, but cost of determination is higher.

Molecule nutriology is a kind of to apply more mulecular luminescence Analytical Methods of Trace, and highly sensitive, detection limit is low.Due to Riboflavin solution, under 430-440nm blue light illumination, sends stronger green fluorescence, and molecule nutriology therefore can be utilized to measure The content of riboflavin, but owing to molecule nutriology is Analytical Methods of Trace, fluorescence pollution, environmental factors and sample Coexisting component Impact be that analysis accurate, quick can not irrespective subject matter.In measuring at present, more quantitative analysis method is work Standard curve method and relative method；Working curve method is that the standard solution of known quantity is configured to the standard of a series of variable concentrations is molten Liquid, and outside test solution to be determined, measure the relative luminous intensity of its standard solution under certain condition, with luminous intensity to mark Quasi-solution concentration drawing curve, it is provided that the dependency between regression equation and parameter, sample test solution and standard solution are identical Under conditions of measure, comparison working curve or substitute into regression equation obtain the content of component to be measured in sample.The advantage of this method is Being applicable to the mensuration of batch samples, shortcoming is that 1. drawing curve need to prepare 5-7 standard solution, expends the time long, surveys Determine cost high；2. working curve needs repaint after one period of working time or correct；3. be due in standard solution not Containing sample to be tested, therefore background during Specimen Determination has larger difference, even replacing with distilled water with the background of standard solution Test solution does overall process blank and gives " button blank ", can not deduct the ambient interferences that the coexisting substances of sample own may introduce, only Can partly deduct distilled water, agents useful for same, the interference of instrument introducing, because the background of standard solution and test solution is different, point Ce Ding be it cannot be guaranteed that complete background correction interference under identical condition determination, the accuracy of mensuration will be had a greatly reduced quality； Mensuration must be carried out, if the concentration of substance to be measured crosses dilute or overrich in the range of linear, it is possible to occurs in various degree Dissociation, polymerization, photodissociation or other side reaction and departs from linear relation, introduce bigger error and even cannot measure.Relative method is: take One standard solution, measures under conditions of identical with test solution, measures reagent blank, according under the conditions of identical simultaneously During mensuration, the relation that concentration is directly proportional to luminous value, the concentration of test solution can be calculated, this law is applicable to the quick survey of single sample Fixed, require concentration of standard solution the most close with liquid concentration to be measured simultaneously, the advantage of this method is quick, easy, analysis cost phase To cheap, it is not necessary to mapping；Although shortcoming is 1. to have deducted the luminous value of reagent blank, but test solution and standard solution background background feelings Condition is not consistent, if system occurs chemical side reactions in various degree or photochemistry side reaction, measurement result during measuring Will introducing error in various degree；2. measure and require that standard solution is the most close with the concentration of test solution to be measured, to unknown sample Actually it is difficult to accomplish, generally requires pre-doing experiment and determine；The dense of signal deciding test solution to be measured is measured by a standard solution Degree, standard solution measures the accuracy of signal and has a strong impact on measurement result.Egg yolk and vitamin B₂Tablet as it has been described above, except Riboflavin, possibly together with the coexisting substances that other are more, especially egg yolk matrix composition is complicated, there is the albumen of high level The material such as matter, aminoacid, either uses working curve method or relative method, is all easily subject to, when measuring, the protein etc. that coexists The impact that the fluorescence interference of non-mensuration component and various fluorescence pollute；Certain error is brought to measurement result；So, Ren Menxi Hope that setting up matrix fluorescence disturbs little or can effectively eliminate interference and the novel fluorescence analysis side measuring riboflavin of fluorescence pollution Method.

Summary of the invention

The purpose of the present invention is that offer one utilizes molecular fluorescence difference mark-on to measure egg yolk, VB₂Tablet center is yellow The method of element, this assay method is quick, effectively eliminates test solution background and the introduced interference of concentration difference, and accuracy obtains Ensureing, work efficiency is improved.

It is an object of the invention to realize in the following manner:

One utilizes molecular fluorescence difference mark-on to measure egg yolk, VB₂The method of riboflavin in tablet, comprises the following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs m_Sample, add the matter of 0.09-0.11moL/L hydrochloric acid, egg yolk and hydrochloric acid solution Amount volume ratio (g/mL) is 1:(2.6-4), VB₂Tablet is 1:(1200-1600 with the mass volume ratio (g/mL) of hydrochloric acid solution), Pressure hydrolysis is extracted, and pressure is (8.0-12.0) × 10⁴Pa, the time is 20-40min, then with NaOH solution regulation pH extremely 4.5-7.5, adds papain solution in egg yolk test solution, papain addition is the 0.13-0.16% of egg yolk quality, VB₂Adding amylase solution in tablet test solution, amylase addition is VB₂The 50-70% of tablet quality, then 37-39 DEG C of guarantor Temperature 15-17h carries out enzymolysis；Enzymolysis solution filters, and filtrate is settled to V_{Sample 1}；

(2) measure liquid to prepare: from V_{Sample 1}In take four parts of same volumes V_{Sample 2}Filtration test solution, different with second part of addition to first part The riboflavin standard solution of volume, riboflavin standard solution mass body volume concentrations is, second part of standard solution volume V_{Mark 2}It is more than First part of V_{Mark 1}, the 4th part adds Hydros and riboflavin is reduced to unstressed configuration material, and as blank, the 3rd part was both not added with Standard solution, is also not added with Hydros, for former filtration test solution；Each pipe adds water to V, and the glacial acetic acid then adding 3-3.5% V mixes Even, it is subsequently adding the potassium permanganate solution of 3-3.5% V, potassium permanganate solution concentration is 25-35g/L, mixing, stands, and dropping is double Oxygen aqueous solution, to potassium permanganate color fade, shaking, makes unnecessary oxygen escape, and constant volume is V₁；

(3) measure: use riboflavin standard solution scanning spectra under spectrofluorophotometer, obtain maximum excitation according to collection of illustrative plates Wavelength and maximum emission wavelength, the test solution (1) handled well is surveyed under maximum excitation wavelength and maximum emission wavelength Fixed, first part of mark-on test solution relative light unit is I_{Mark 1}, second part of mark-on test solution relative light unit is I_{Mark 2}, the 3rd part of testing sample Test solution relative light unit is I_Sample, the 4th part of background blank background relative light unit is I₀, egg yolk or VB₂The matter of riboflavin in tablet Amount mark ω such as formula (1)；

Preferably, comprise the following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs m_Sample, add the quality volume of 0.1moL/L hydrochloric acid, egg yolk and hydrochloric acid solution It is 1:3.5 than (g/mL), VB₂Tablet is 1:1400 with the mass volume ratio (g/mL) of hydrochloric acid solution, and pressure hydrolysis is extracted, pressure It is 10.3 × 10⁴Pa, the time is 30min, then regulates pH to 5.5 by NaOH solution, adds papain in egg yolk test solution Solution, papain addition is the 0.15% of egg yolk quality, VB₂Adding amylase solution in tablet test solution, amylase adds Amount is VB₂The 60% of tablet quality, then carries out enzymolysis at 39 DEG C of insulation 16h；Enzymolysis solution filters, and takes filtrate and is settled to V_{Sample 1}；

(2) measure liquid to prepare: from V_{Sample 1}In take at least four parts of same volumes V_{Sample 2}Filtration test solution, to first part and second part addition The riboflavin standard solution of different volumes, riboflavin standard solution mass body volume concentrations is, second part of standard solution volume V_{Mark 2} More than first part of V_{Mark 1}, the 4th part adds Hydros that riboflavin is reduced to unstressed configuration material is blank as background, the 3rd part Neither add standard solution, be also not added with Hydros, for former filtration test solution；Each pipe adds water to V, adds the glacial acetic acid mixing of 3.3%V, Being subsequently adding the potassium permanganate solution of 3.3%V, potassium permanganate solution concentration is 30g/L, mixing, stands, and drips hydrogen peroxide solution To potassium permanganate color fade, finally add water and be settled to V₁Rear mensuration；

(3) measure: use riboflavin standard solution scanning spectra under spectrofluorophotometer, obtain maximum excitation according to collection of illustrative plates Wavelength and maximum emission wavelength, test solution step (2) handled well is at maximum excitation wavelength and maximum emission wavelength Lower mensuration.

Further, the described molecular fluorescence difference mark-on method that utilizes measures egg yolk, VB₂The method of riboflavin in tablet, bag Include following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs 18-22g egg yolk or 0.04-0.06gVB₂Tablet, adds 0.09- 0.11moL/L hydrochloric acid 60-80 mL, highly pressured hydrolysis extracts, and pressure is (8-12) × 10⁴Pa, the time is 20-40min, then uses NaOH solution regulation pH to 4.5-7.5, adds 2.5-3.5mL 9-11g/L papain solution, VB in egg yolk test solution₂Tablet Test solution adds 2.5-3.5mL 9-11g/L amylase solution, then carries out enzymolysis at 37-39 DEG C of insulation 15-17h；Enzymolysis solution Filter, take filtrate and be settled to V_{Sample 1}；

(2) measure liquid to prepare: from V_{Sample 1}In take at least four parts of same volumes V_{Sample 2}Filtrate, volume is 0.5-10mL, to first Part and the riboflavin standard solution of second part of addition different volumes, riboflavin standard solution mass body volume concentrations is, second part Standard solution volume V_{Mark 2}It is first part of V_{Mark 1}1.2-2 times；4th part adds Hydros and riboflavin is reduced to unstressed configuration thing Matter is blank as background；3rd part neither adds standard solution, is also not added with Hydros, for former filtration test solution；Each pipe adds water to 14-16mL, then adds the mixing of 0.4-0.6mL glacial acetic acid in every part of filtrate, is subsequently adding the potassium permanganate of 0.4-0.6mL Solution, potassium permanganate solution concentration is 25-35g/L, mixing, stands, and dropping hydrogen peroxide solution is to potassium permanganate color fade, Rear each pipe adds water and is settled to 24-26mL；

(3) measure: use riboflavin standard solution scanning spectra under spectrofluorophotometer, obtain maximum excitation according to collection of illustrative plates Wavelength and maximum emission wavelength, sample test solution step (2) handled well is in maximum excitation wavelength and maximum fluorescence emission Measure under wavelength.

Further, comprise the following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs 20g egg yolk or 0.05gVB₂Tablet, adds 0.1moL/L hydrochloric acid 70mL,

Pressure hydrolysis is extracted, and pressure is 10.3 × 10⁴Pa, the time is 30min, then regulates pH to 5.5, egg yolk by NaOH solution Test solution adds 3mL 10g/L papain solution, VB₂Tablet test solution adds 3mL 10g/L amylase solution, then exists 39 DEG C of insulation 16h carry out enzymolysis；Enzymolysis solution filters, and takes filtrate and is settled to 100mL；

(2) measure liquid to prepare: taking the filtration test solution of at least four parts of same volumes from 100mL filtrate, egg yolk filtrate volume is 10.00mL, VB₂Tablet filtrate volume is 1.00mL, be separately added in first part and second part of extracting solution 1.00mL and 2.00mL concentration is the riboflavin standard solution of 1 μ g/mL；4th part adds Hydros and riboflavin is reduced to unstressed configuration Material is as background blank, and the 3rd part neither adds standard solution, be also not added with Hydros, for former filtration test solution；Each pipe adds water To 15mL, in every part of filtrate, then add the mixing of 0.5mL glacial acetic acid, add the potassium permanganate solution of 0.5mL, potassium permanganate Solution concentration is 30g/L, mixing, stands, and dropping hydrogen peroxide solution, to potassium permanganate color fade, finally adds water and is settled to 25mL；

Molecular fluorescence difference mark-on method is utilized to measure egg yolk, VB as above₂The method of riboflavin, step in tablet (1) in, NaOH solution concentration is 1moL/L, and in step (2), hydrogen peroxide mass body volume concentrations is 3%；Egg yolk test solution adds low Asia Metabisulfite solution at least 0.5mL, VB₂Tablet test solution adds Hydros solution at least 5mL；Hydros solution concentration The preparation of 2.5moL/L sodium acetate solution is used for 20g/100mL, described papain and amylase solution,

Described riboflavin standard solution is divided into concentration to be 25 μ g/mL riboflavin standard reserving solutions and concentration is 1 μ g/mL riboflavin mark Quasi-use liquid；Riboflavin standard reserving solution process for preparation is as follows: the crystallization of standard substance riboflavin powder is placed in vacuum desiccator In, after phosphorus pentoxide drying tube and silica-gel desiccant combined drying 24h, accurately weigh 50mg, be placed in 2L volumetric flask, add Enter 2.4mL glacial acetic acid and 1.5mL water, volumetric flask is placed in 30-40 DEG C of warm water shake, treats that it dissolves, be cooled to room temperature, add water fixed Hold to 2L, move in brown bottle, add toluene and be covered in solution surface, preserve in 4 DEG C of refrigerators；Riboflavin standard solution is prepared Process is: draws 25 μ g/mL riboflavin standard reserving solution 2.00mL, is placed in 50mL brown volumetric flask, is settled to scale with water, 4 DEG C of Refrigerator stores, the holding time is 1 week.

Egg yolk also uses direct drying method to carry out the mensuration of moisture.

The present invention establishes a kind of quantitative determination egg yolk, vitamin B quick, easy₂Novel point of riboflavin in tablet Analysis method, it is not necessary to drawing curve, only need to can calculate component to be measured matter in the sample by using two mark-on test solutions Amount mark, completely eliminates the ambient interferences in sample test solution, uses two simultaneously and add various criterion concentration during mensuration Mark-on test solution, because of the different issuable tested component of concentration be polymerized, the side reaction such as dissociation can partly obtain compensate or Deduction, accuracy of measurement is protected further and improves.The inventive method detection is limited to 5.76ng/mL, is quantitatively limited to 1.92 ×10^-2μg/mL.Comparing with national standard method, through F method of inspection and t method of inspection, the precision of two kinds of methods and meansigma methods are all There is not significant difference；But assay method of the present invention is easy, quickly, deducted background luminescence value and concentration in sample test solution The part interference that difference may be brought, improves accuracy of measurement and work efficiency, method without drawing curve and Cross post decontamination, be to be suitable for the novel analytical technology of riboflavin assay in sample, have stronger novelty.

Accompanying drawing explanation

Fig. 1 is the impact on egg yolk test solution relative fluorescence of the hydrochloric acid volume；

Fig. 2 is the pH impact on egg yolk test solution relative fluorescence；

Fig. 3 is the impact on egg yolk test solution relative fluorescence of the Hydros volume；

Fig. 4 is that hydrochloric acid volume is to vitamin B₂The impact of tablet test solution relative fluorescence；

Fig. 5 is that pH is to vitamin B₂The impact of tablet test solution relative fluorescence；

Fig. 6 is that Hydros volume is to vitamin B₂The impact of tablet test solution relative fluorescence.

Detailed description of the invention

Embodiment 1

One utilizes molecular fluorescence mark-on method to measure egg yolk, VB₂The method of riboflavin in tablet, comprises the following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs m_Sample, add the matter of 0.09-0.11moL/L hydrochloric acid, egg yolk and hydrochloric acid solution Amount volume ratio (g/mL) is 1:(2.6-4), VB₂Tablet is 1:(1200-1600 with the mass volume ratio (g/mL) of hydrochloric acid solution), Pressure hydrolysis is extracted, and pressure is (8.0-12.0) × 10⁴Pa, the time is 20-40min, then regulates by 1moL/L NaOH solution PH to 4.5-7.5, adds papain solution in egg yolk test solution, papain addition is the 0.13-of egg yolk quality 0.16%, VB₂Adding amylase solution in tablet test solution, amylase addition is VB₂The 50-70% of tablet quality, then at 37- 39 DEG C of insulation 15-17h carry out enzymolysis；The dry filter paper filtering of enzymolysis solution, takes filtrate and is settled to V_{Sample 1}；Hydrolyzed by hydrochloric acid and enzymolysis, The riboflavin of combined state in sample materials can be allowed free out, participate in measuring, improve reliability and the accuracy of analytical data；Chicken Egg yolk adds papain and can go out the riboflavin with protein bound with enzymolysis；

(2) measure liquid to prepare: from V_{Sample 1}In take at least four parts of same volumes V_{Sample 2}Filtration test solution, to first part and second part addition The riboflavin standard solution of different volumes, riboflavin standard solution mass body volume concentrations is, second part of standard solution volume V_{Mark 2} More than first part of V_{Mark 1}, the 4th part adds Hydros that riboflavin is reduced to unstressed configuration material is blank as background, the 3rd part Neither add standard solution, be also not added with Hydros, for former filtration test solution；Each pipe adds water to V, then adds the glacial acetic acid of 3.3%V Mixing, is subsequently adding the potassium permanganate solution of 3.3%V, and potassium permanganate solution concentration is 30g/L, mixing, stands, and drips hydrogen peroxide Solution to potassium permanganate color fade, finally add high purity water (quartz sub-boiling deionization redistilled water, when 25 DEG C, pH6.63 ± 0.02, Electrical conductivity 1.183 μ s/cm) it is settled to V₁Rear mensuration；Adding glacial acetic acid can maintain solution to be in weakly acidic condition, because riboflavin is weak In acid environment relatively stable；Add the interference of the oxidable removing of potassium permanganate relatively strong reducible agent more that may be present, fall Low background blank value；

(3) measure: use riboflavin standard solution scanning spectra under spectrofluorophotometer, obtain maximum excitation according to collection of illustrative plates Wavelength and maximum emission wavelength, sample step (2) handled well is under maximum excitation wavelength and maximum emission wavelength Measuring, first part of mark-on test solution relative light unit is I_{Mark 1}, second part of mark-on test solution relative light unit is I_{Mark 2}, the 3rd part of testing sample Test solution relative light unit is I_Sample, the 4th part of background blank background relative light unit is I₀, during said determination, four parts of test solutions exist Measure under conditions of identical, the relation being directly proportional to luminous value according to the concentration of component to be measured, it may be assumed that,For the mass body volume concentrations of constant volume test solution to be measured,It it is the difference of the mass body volume concentrations of two parts of mark-on constant volume test solutions；Can push away Calculate egg yolk or VB₂The mass fraction ω such as formula (1) of riboflavin in tablet；

The difference mark-on assay method that the present invention uses, wherein I_{Mark 1,}I_{Mark 2}Cause and I₀Use identical auxiliary reagent, therefore, I_{Mark 1,} I_{Mark 2}Also I is contained₀Value, butRear I₀Offset, owing to after test solution to be measured and mark-on, the mensuration background of test solution to be measured is consistent, Therefore, use the method can eliminate the interference to measurement result of the test solution background, improve the accuracy of mensuration.So, the method Only need to measure the relative light unit of solution under the same conditions, can quickly try to achieve measurement result, it is not necessary to drawing curve and Mapping evaluation, quickly, easy, improve work efficiency greatly, use the mark-on test solution of two variable concentrations to determine component to be measured Concentration, because of the different issuable tested component of sample concentration be polymerized, the side reaction such as dissociation can partly obtain compensate and Deduction, accuracy of measurement is protected further and improves.

Embodiment 2

Molecular fluorescence mark-on method is utilized to measure egg yolk, VB₂The method of riboflavin in tablet, comprises the following steps:

(1) solution preparation:

Riboflavin standard reserving solution (25 μ g/mL): by standard substance riboflavin (Jin Sui bio tech ltd, Shanghai) powder Crystallization be placed in vacuum desiccator, after 24h, accurately weigh 50mg, be placed in 2L volumetric flask, add 2.4mL glacial acetic acid and 1.5mL water.Volumetric flask is placed in warm water shake, treats that it dissolves, be cooled to room temperature, be diluted to 2L, move in brown bottle, add a little Toluene is placed on solution surface, preserves in refrigerator.

Riboflavin standard solution (1 μ g/mL)；Draw 2.00mL riboflavin standard reserving solution, be placed in 50mL brown capacity In Ping, it is diluted with water to scale, lucifuge, stores in 4 DEG C of refrigerators, one week can be preserved；It is yellow that this solution every milliliter is equivalent to 1.00 μ g cores Element.

Papain (10g/L): papain (Pangbo Bioengineering Co Ltd, Nanning), uses 2.5mol/L acetic acid Sodium solution is prepared；During use, existing preparation.

Amylase (10g/L): amylase (Xingtai Wanda's biological engineering company limited), joins with 2.5mol/L sodium acetate solution System.Now prepare during use.

Hydros solution (20g/100mL): (analytical pure, Tianjin causes the limited public affairs of remote chemical reagent to Hydros Department), this liquid used time, (boiled deionization redistilled water, when 25 DEG C, pH6.63 ± 0.02, electrical conductivity 1.183 μ s/ in quartz Asia with high purity water Cm) now join, be saved in ice-water bath, in 4h effectively.

(2) extracting solution is prepared (in this embodiment, sample is egg yolk):

Take the egg yolk sample boiled finely ground, weigh 20.0000g and be placed in 250mL conical flask, add 0.1moL/L hydrochloric acid 70mL (egg yolk is 1:3.5 with the mass volume ratio (g/mL) of hydrochloric acid solution), stirring, until particulate matter is uniformly dispersed, uses 40mL porcelain crucible Living bottleneck for cover buckle, be placed in pressure cooker hydrolysis and extract, pressure is 12.0 × 10⁴Pa, the time is 30min, after hydrolyzed solution cooling, uses 1moLNaOH solution regulation pH to 5.5, (papain adds to add 3mL 10g/L papain solution in egg yolk test solution Amount is the 0.15% of egg yolk quality), then carry out enzymolysis at 39 DEG C of insulation 16h；The dry filter paper filtering of enzymolysis solution, takes filtrate constant volume To 100mL；

(3) measure liquid to prepare

From V_{Sample 1}In take the filtration test solution of four parts of same volumes, volume is 10.00mL, in first part and second part of extracting solution point Jia Ru 1.00mL and 2.00mL concentration be not the riboflavin standard solution of 1 μ g/mL；4th part adds 0.5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration material by 100mL Hydros solution, blank as background；3rd part neither to add standard molten Liquid, is also not added with Hydros, for former filtration test solution；Each pipe adds water to 15mL, then adds 0.5mL in every part of extracting solution (3.3%V) glacial acetic acid mixing, be subsequently adding 0.5mL(3.3%V) potassium permanganate solution, potassium permanganate solution concentration is 30g/L, Mixing, stands, and drips 3% hydrogen peroxide solution to potassium permanganate color fade, acutely shakes sample, make unnecessary oxygen overflow, After add water and be settled to 25mL；

(4) determination of water in egg yolk

For ease of the results contrast of different sample rooms, measurement result is preferably contents on dry basis；Egg yolk will be boiled finely ground, weigh matter Amount is about 5.0000g and carries out determination of water according to direct drying method in GB/T 5009.3-2010, calculates chicken according to formula (2) The percentage composition of egg yolk moisture.

In formula: ω₀Moisture for egg yolk；m₁For weighing botle before sample drying and the quality of sample, g；m₂For claiming Quality after measuring bottle and sample drying, g；m₃For the quality of weighing botle, g.

(5) fluoremetry

Use riboflavin standard solution scanning spectra under spectrofluorophotometer, according to collection of illustrative plates obtain maximum excitation wavelength and Maximum emission wavelength, respectively 452nm and 530nm, sample step (3) handled well is in maximum excitation wavelength and maximum Carry out three parallel assays under fluorescence emission wavelengths, then take its meansigma methods, calculate butt egg yolk center according to formula (3) The mass fraction ω (μ g/100g) of flavin；

Embodiment 3

(1) solution preparation:

Riboflavin standard solution (1 μ g/mL)；Draw 2.00mL riboflavin standard reserving solution, be placed in 50mL brown capacity In Ping, it is diluted with water to scale, lucifuge, stores in 4 DEG C of refrigerators, one week can be preserved.It is yellow that this solution every milliliter is equivalent to 1.00 μ g cores Element.

Papain (10g/L): papain (Pangbo Bioengineering Co Ltd, Nanning), uses 2.5mol/L acetic acid Sodium solution is prepared.During use, existing preparation.

(2) extracting solution prepares that (in this embodiment, sample is VB₂Tablet):

Take VB₂Tablet is finely ground to be dried, and weighs 0.0500g and pours in 250mL conical flask, adds 0.1moL/L hydrochloric acid 70 mL(VB₂ Tablet is 1:1400 with the mass volume ratio (g/mL) of hydrochloric acid solution), stirring, until particulate matter is uniformly dispersed, uses 40mL porcelain crucible Living bottleneck for cover buckle, be placed in pressure cooker hydrolysis and extract, pressure is 12.0 × 10⁴Pa, the time is 30min, after hydrolyzed solution cooling, uses 1moLNaOH solution regulation pH to 5.5, (amylase addition is VB to add 3mL 10g/L amylase solution₂Tablet quality 60%), then enzymolysis is carried out at 39 DEG C of insulation 16h；The dry filter paper filtering of enzymolysis solution, takes filtrate and is settled to 100mL；

(3) measure liquid to prepare

Taking the extraction filtrate of four parts of same volumes, volume is 1.00mL, is separately added in first part and second part of extracting solution 1.00mL L and 2.00mL concentration are the riboflavin standard solution of 1 μ g/mL；4th part adds 5mL concentration is 20g/100mL It is blank as background that riboflavin is reduced to unstressed configuration material by Hydros solution；3rd part neither adds standard solution, the most not Add Hydros, for former filtration test solution；Each pipe adds water to 15mL, then extracts to every part and adds 0.5mL(3.3% in filtrate V) glacial acetic acid mixing, be subsequently adding 0.5mL(3.3%V) potassium permanganate solution, potassium permanganate solution concentration is 30g/L, mixing, Standing, dropping mass body volume concentrations 3% hydrogen peroxide solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen Effusion, finally adds water and is settled to 25mL；

(4) fluoremetry

Use riboflavin standard solution scanning spectra under spectrofluorophotometer, according to collection of illustrative plates obtain maximum excitation wavelength and Maximum emission wavelength, respectively 452nm and 530nm, sample step (3) handled well is in maximum excitation wavelength and maximum Carry out three parallel assays under fluorescence emission wavelengths, then take its meansigma methods, calculate VB according to formula (4)₂Tablet center is yellow The mass percent ω (g/100g) of element；

Embodiment 4

(1) extracting solution is prepared (in this embodiment, sample is egg yolk):

Take the egg yolk sample boiled finely ground, weigh 22.0000g and pour in 250mL conical flask, add 0.1moL/L hydrochloric acid 80 ML, stirring, until particulate matter is uniformly dispersed, is that cover buckle lives bottleneck with 40mL porcelain crucible, is placed in pressure cooker hydrolysis and extracts, and pressure is 12.0×10⁴Pa, the time is 30min, after hydrolyzed solution cooling, regulates pH to 5.5 with 1moLNaOH solution, adds in egg yolk test solution 3.5mL 10g/L papain solution, then carries out enzymolysis at 39 DEG C of insulation 16h；The dry filter paper filtering of enzymolysis solution, takes filtrate It is settled to 50mL；

(2) measure liquid to prepare

From V_{Sample 1}In take the filtration test solution of four parts of same volumes, volume is 5.00mL, in first part and second part of extracting solution respectively Add the riboflavin standard solution that 1.00mL and 2.00mL concentration is 1 μ g/mL；4th part adds 0.5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration material by 100mL Hydros solution, blank as background；3rd part neither to add standard molten Liquid, is also not added with Hydros, for former filtration test solution；Each pipe adds water to 16mL, then adds 0.5mL ice in every part of extracting solution Acetic acid mixes, and is subsequently adding the potassium permanganate solution of 0.5mL, and potassium permanganate solution concentration is 30g/L, mixing, stands, and drips 3% Hydrogen peroxide solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen overflow, and finally adding water is settled to 25mL；

(3) fluoremetry

Use riboflavin standard solution scanning spectra under spectrofluorophotometer, according to collection of illustrative plates obtain maximum excitation wavelength and Maximum emission wavelength, respectively 452nm and 530nm, sample step (3) handled well is in maximum excitation wavelength and maximum Carry out three parallel assays under fluorescence emission wavelengths, then take its meansigma methods, copy formula (3) to calculate butt egg yolk center The mass fraction ω of flavin；Remaining is with embodiment 2.

Embodiment 5

(1) extracting solution is prepared (in this embodiment, sample is egg yolk):

Take the egg yolk sample boiled finely ground, weigh 20.0000g and pour in 250mL conical flask, add 0.09moL/L hydrochloric acid 70mL, stirring, until particulate matter is uniformly dispersed, is that cover buckle lives bottleneck with 40mL porcelain crucible, is placed in pressure cooker hydrolysis and extracts, pressure It is 10.0 × 10⁴Pa, the time is 20min, after hydrolyzed solution cooling, regulates pH to 4.5 with 1moLNaOH solution, adds in egg yolk test solution Enter 2.5mL 11g/L papain solution, then carry out enzymolysis at 37 DEG C of insulation 15h；The dry filter paper filtering of enzymolysis solution, takes filter Liquid is settled to 100mL；

(2) measure liquid to prepare

From V_{Sample 1}In take the filtration test solution of four parts of same volumes, volume is 10.00mL, in first part and second part of extracting solution point Jia Ru 4.00mL and 5.00mL concentration be not the riboflavin standard solution of 1 μ g/mL；4th part adds 0.5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration material by 100mL Hydros solution, blank as background；3rd part neither to add standard molten Liquid, is also not added with Hydros, for former filtration test solution；Each pipe adds water to 15mL, then adds 0.6mL ice in every part of extracting solution Acetic acid mixes, and is subsequently adding the potassium permanganate solution of 0.6mL, and potassium permanganate solution concentration is 35g/L, mixing, stands, and drips 3% Hydrogen peroxide solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen overflow, and finally adding water is settled to 24mL；

(3) fluoremetry

Use riboflavin standard solution scanning spectra under spectrofluorophotometer, according to collection of illustrative plates obtain maximum excitation wavelength and Maximum emission wavelength, respectively 452nm and 530nm, sample step (2) handled well is in maximum excitation wavelength and maximum Carry out three parallel assays under fluorescence emission wavelengths, then take its meansigma methods, copy formula (3) to calculate butt egg yolk center The mass fraction ω of flavin；Remaining is with embodiment 2.

Embodiment 6

(1) extracting solution is prepared (in this embodiment, sample is egg yolk):

Take the egg yolk sample boiled finely ground, weigh 18.0000g and pour in 250mL conical flask, add 0.11moL/L hydrochloric acid 60ml, stirring, until particulate matter is uniformly dispersed, is that cover buckle lives bottleneck with 40mL porcelain crucible, is placed in pressure cooker hydrolysis and extracts, pressure It is 8.0 × 10⁴Pa, the time is 40min, after hydrolyzed solution cooling, regulates pH to 7.5 with 1moLNaOH solution, adds in egg yolk test solution Enter 3mL 9g/L papain solution, then carry out enzymolysis at 37 DEG C of insulation 17h；The dry filter paper filtering of enzymolysis solution, takes filtrate It is settled to 100mL；

(2) measure liquid to prepare

From V_{Sample 1}In take the filtration test solution of four parts of same volumes, volume is 10.00mL, in first part and second part of extracting solution point Jia Ru 2.00mL and 3.00mL concentration be not the riboflavin standard solution of 1 μ g/mL；4th part adds 0.5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration material by 100mL Hydros solution, blank as background；3rd part neither to add standard molten Liquid, is also not added with Hydros, for former filtration test solution；Each pipe adds water to 14mL, then adds 0.4mL ice in every part of extracting solution Acetic acid mixes, and is subsequently adding the potassium permanganate solution of 0.4mL, and potassium permanganate solution concentration is 25g/L, mixing, stands, and drips 3% Hydrogen peroxide solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen overflow, and finally adding water is settled to 26mL；

(3) fluoremetry

Use riboflavin standard solution scanning spectra under spectrofluorophotometer, according to collection of illustrative plates obtain maximum excitation wavelength and Maximum emission wavelength, respectively 452nm and 530nm, sample step (2) handled well is in maximum excitation wavelength and maximum Carry out three parallel assays under fluorescence emission wavelengths, then take its meansigma methods, copy formula (3) to calculate butt egg yolk center The mass fraction ω of flavin；Remaining is with embodiment 2..

Embodiment 7

(1) extracting solution prepares that (in this embodiment, sample is VB₂Tablet):

Take VB₂Tablet is finely ground to be dried, and weighs 0.0500g and pours in 250mL conical flask, adds 0.1moL/L hydrochloric acid 80mL, stirring Until particulate matter is uniformly dispersed, it being that cover buckle lives bottleneck with 40mL porcelain crucible, be placed in pressure cooker hydrolysis and extract, pressure is 12.0 × 10⁴Pa, the time is 30min, after hydrolyzed solution cooling, regulates pH to 5.5 with 1moLNaOH solution, adds 3.5mL 10g/L starch Enzymatic solution, then carries out enzymolysis at 39 DEG C of insulation 16h；The dry filter paper filtering of enzymolysis solution, takes filtrate and is settled to 50mL；

(2) measure liquid to prepare

Taking the extraction filtrate of four parts of same volumes, volume is 0.500mL, is separately added in first part and second part of extracting solution 1.00mL L and 2.00mL concentration are the riboflavin standard solution of 1 μ g/mL；4th part adds 5mL concentration is 20g/100mL It is blank as background that riboflavin is reduced to unstressed configuration material by Hydros solution；3rd part neither adds standard solution, the most not Add Hydros, for former filtration test solution；Each pipe adds water to 16mL, then adds 0.5mL glacial acetic acid in every part of extracting solution and mixes Even, it is subsequently adding the potassium permanganate solution of 0.5mL, potassium permanganate solution concentration is 30g/L, mixing, stands, drips 3% hydrogen peroxide Solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen overflow, and finally adding water is settled to 25mL；

(3) fluoremetry

Use riboflavin standard solution scanning spectra under spectrofluorophotometer, according to collection of illustrative plates obtain maximum excitation wavelength and Maximum emission wavelength, respectively 452nm and 530nm, sample step (2) handled well is in maximum excitation wavelength and maximum Carry out three parallel assays under fluorescence emission wavelengths, then take its meansigma methods, calculate VB according to formula (1)₂Tablet center is yellow The mass fraction ω of element.

Embodiment 8

Take VB₂Tablet is finely ground to be dried, and weighs 0.0600g and pours in 250mL conical flask, adds 0.09moL/L hydrochloric acid 70 mL, stir To mix until particulate matter is uniformly dispersed, be that cover buckle lives bottleneck with 40mL porcelain crucible, be placed in pressure cooker hydrolysis and extract, pressure is 10.0 × 10⁴Pa, the time is 20min, after hydrolyzed solution cooling, regulates pH to 4.5 with 1moLNaOH solution, adds 3mL 11g/L amylase Solution, then carries out enzymolysis at 37 DEG C of insulation 15h；The dry filter paper filtering of enzymolysis solution, takes filtrate and is settled to 100mL；

(2) measure liquid to prepare

Taking the extraction filtrate of four parts of same volumes, volume is 1.00mL, is separately added in first part and second part of extracting solution 4.00mL L and 5.00mL concentration are the riboflavin standard solution of 1 μ g/mL；4th part adds 5mL concentration is 20g/100mL It is blank as background that riboflavin is reduced to unstressed configuration material by Hydros solution；3rd part neither adds standard solution, the most not Add Hydros, for former filtration test solution；Each pipe adds water to 15mL, then adds 0.6mL glacial acetic acid in every part of extracting solution and mixes Even, it is subsequently adding the potassium permanganate solution of 0.6mL, potassium permanganate solution concentration is 35g/L, mixing, stands, drips 3% hydrogen peroxide Solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen overflow, and finally adding water is settled to 24mL；

(3) fluoremetry

Embodiment 9

Take VB₂Tablet is finely ground to be dried, and weighs 0.0400g and pours in 250mL conical flask, adds 0.11moL/L hydrochloric acid 60 mL, stir To mix until particulate matter is uniformly dispersed, be that cover buckle lives bottleneck with 40mL porcelain crucible, be placed in pressure cooker hydrolysis and extract, pressure is 8.0 × 10⁴Pa, the time is 40min, after hydrolyzed solution cooling, regulates pH to 7.5 with 1moLNaOH solution, adds 3mL 11g/L amylase Solution, then carries out enzymolysis at 37 DEG C of insulation 15h；The dry filter paper filtering of enzymolysis solution, takes filtrate and is settled to 100mL；

(2) measure liquid to prepare

Taking the extraction filtrate of four parts of same volumes, volume is 2.00mL, is separately added in first part and second part of extracting solution 2.00mL L and 3.00mL concentration are the riboflavin standard solution of 1 μ g/mL；4th part adds 5mL concentration is 20g/100mL It is blank as background that riboflavin is reduced to unstressed configuration material by Hydros solution；3rd part neither adds standard solution, the most not Add Hydros, for former filtration test solution；Each pipe adds water to 14mL, then adds 0.4mL glacial acetic acid in every part of extracting solution and mixes Even, it is subsequently adding the potassium permanganate solution of 0.4mL, potassium permanganate solution concentration is 25g/L, mixing, stands, drips 3% hydrogen peroxide Solution, to potassium permanganate color fade, acutely shakes sample, makes unnecessary oxygen overflow, and finally adding water is settled to 26mL；

(3) fluoremetry

Test example

1, the selection of wavelength

No matter how riboflavin concentration of standard solution changes, and launches wavelength and excitation wavelength is the most constant.Take the riboflavin configured Standard solution (1 μ g/mL), scanning spectra, maximum excitation wavelength can be obtained according to collection of illustrative plates and maximum emission wavelength is respectively 452nm And 530nm.Therefore, this experiment is chosen maximum excitation wavelength and maximum emission wavelength are respectively 452nm and 530nm.

2, single factor experiment

2.1 egg yolk

2.1.1 the determination of hydrochloric acid extraction liquor capacity

The egg yolk that boils is finely ground, and weighing quality is that 20.0000g egg yolk is placed in 250mL conical flask, is separately added into 0. 1mol/L hydrochloric acid 50mL, 60mL, 70mL, 80mL and 90mL, stirring is until particulate matter is uniformly dispersed, then according to embodiment 2 is remembered Support method is hydrolyzed, enzymolysis, filter, aoxidize decontamination after, be settled to 25mL, parallel assay three times, average, experiment knot Fruit is as shown in Figure 1.

As shown in Figure 1, it is separately added into 0.1mol/L hydrochloric acid 50mL, 60mL, 70mL, 80mL, 90mL, the relative fluorescence recorded Value is more or less the same.When adding 50mL, 60mL, 70mL hydrochloric acid, relative fluorescence is in rising trend, illustrates along with hydrochloric acid volume Increasing, riboflavin extracts the most；And relative fluorescence is on a declining curve when adding 70mL, 80mL, 90mL hydrochloric acid, show along with The increase of hydrochloric acid volume, acidity increases, and stability declines, and illustrates that riboflavin is only stable in the solution that acidity is suitable.Therefore, Add 0. 1mol/L hydrochloric acid 70mL extracting solution the most suitable.

2.1.2 the selection of enzymolysis pH

Under conditions of 0.1mol/L hydrochloric acid is 70mL, between pH4～8, carry out enzymolysis.The egg yolk that boils is finely ground, weigh Quality be 20.0000g egg yolk in 250mL conical flask, be separately added into 0.1mol/L hydrochloric acid 70mL, record according to embodiment 2 Method is hydrolyzed extraction, after hydrolyzed solution cooling, drips 1moL/L sodium hydroxide, respectively regulation pH be 4.500,5.500, 6.500、7.500、8.500.Then according to after embodiment 2 record method carries out enzymolysis, filters, aoxidizes decontamination, parallel assay Three times, averaging, experimental result is as shown in Figure 2.

As shown in Figure 2, when pH is respectively 4.500,5.500,6.500,7.500,8.500, the relative fluorescence recorded without Significant change, and when pH is equal to 5.500, the relative fluorescence recorded is maximum.Therefore, this experimental selection pH=5.500 is optimal real Test condition.

2.1.3 the selection of Hydros liquor capacity

The egg yolk that boils is finely ground, and weighing quality is that 20.0000g egg yolk is placed in 250mL conical flask, then according to implement Example 2 record method be hydrolyzed extraction, enzymolysis, filter and be settled to 100mL after, respectively at the scale test tube with cover of 6 25mL Middle addition 10mL egg yolk extracting solution, carries out aoxidizing decontamination according to embodiment 2 record method, is settled to 25mL, after mixing, adds Enter 0.10mL, 0.20mL, 0.30mL, 0.40mL, 0.50mL, 0.60mL 20g/100mL Hydros solution, after mixing, flat Row measures three times, averages, and measurement result is as shown in Figure 3.

Fig. 3 is it can be seen that work as and add 0.10mL, 0.20mL, 0.30mL, 0.40mL, 0.50mL, 0.60mL Hydros Time, relative fluorescence is gradually lowered along with the increase of Hydros volume, illustrates along with the increase of Hydros volume, In egg yolk, riboflavin is gradually reduced to unstressed configuration material；When adding 0.40mL, 0.50mL, 0.60mL Hydros, Relative fluorescence is held essentially constant along with the increase of Hydros volume, illustrates that the riboflavin in egg yolk is whole It is reduced to unstressed configuration material, and when adding 0.40mL Hydros, in egg yolk, riboflavin has been completely reduced.Cause This, experimental selection Hydros volume is 0.50mL.

2.1.4 the determination of riboflavin standard solution volume

The egg yolk that boils is finely ground, weigh quality be 20.0000g egg yolk in 250mL conical flask, remember according to embodiment 2 Support method is hydrolyzed after extraction, enzymolysis, filtration, constant volume, adds 10.00mL in the scale test tube with cover of 8 25mL Egg yolk extracting solution, be separately added in 3,4,5,6,7, No. 8 pipes 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL1 μ g/mL riboflavin standard solution, No. 1 pipe and No. 2 pipes are not added with standard solution, add 0.50mL in No. 1 pipe 20g/100mL Hydros solution, each pipe adds water to 16mL, carries out aoxidizing decontamination, constant volume according to embodiment 2 record method To 25mL, mixing, respectively manage parallel assay three times under the same conditions, average, copy formula (3) to calculate butt egg yolk The mass fraction of riboflavin, then compares with national standard method, result such as table 1.

As shown in Table 1, riboflavin content in the butt egg yolk that different volumes riboflavin standard solution records is added close. But mark-on 1.00mL, 2.00mL record egg yolk riboflavin content and National Standard Method is closest, therefore, this experimental selection mark-on 1.00mL, 2.00mL riboflavin standard solution is optimum experimental condition.

2.2 vitamin B₂Tablet

2.2.1 the determination of hydrochloric acid extraction liquor capacity

Weigh quality and be about the finely ground dry vitamin B of 0.0500g₂Tablet, in 250mL conical flask, is separately added into 0.1mol/L Hydrochloric acid 50mL, 60mL, 70mL, 80mL and 90mL, stirring is until particulate matter is uniformly dispersed, then according to embodiment 3 record method Be hydrolyzed, enzymolysis, filter, aoxidize decontamination after, be settled to 25mL, parallel assay three times, average, experimental result such as figure Shown in 4.

As shown in Figure 4, being separately added into 50mL, 60mL, 70mL, 80mL, 90mL hydrochloric acid, the relative fluorescence recorded differs not Greatly.When adding 50mL, 60mL, 70mL hydrochloric acid, relative fluorescence is in rising trend, illustrates that, along with the increase of hydrochloric acid volume, core is yellow Element extracts the most；And relative fluorescence is on a declining curve when adding 80mL, 90mL hydrochloric acid, show along with the increasing of hydrochloric acid volume Adding, acidity increases, and stability declines, and illustrates that riboflavin is only stable in the solution that acidity is suitable.Therefore, 0.1mol/L is added Hydrochloric acid 70mL is optimum experimental condition.

2.2.2 the selection of enzymolysis pH

Weigh the finely ground dry vitamin B of 0.0500g₂Tablet, in 250mL conical flask, is separately added into 70mL0. 1mol/L salt Acid, is hydrolyzed according to embodiment 3 record method, after hydrolyzed solution cooling, drips 1moL/L sodium hydroxide, and regulation pH is respectively 4.500、5.500、6.500、7.500、8.500.Then according to embodiment 3 record method carries out enzymolysis, filters, aoxidizes decontamination After, parallel assay three times, experimental result is as shown in Figure 5.

As shown in Figure 5, when enzymolysis solution pH is equal to 5.500, the relative fluorescence recorded is maximum.Therefore, this experimental selection pH= 5.500 be optimum experimental condition.

2.2.3 the determination of Hydros liquor capacity

Weigh the finely ground dry vitamin B of 0.0500g₂Tablet is poured in 250mL conical flask, enters according to embodiment 3 record method Row hydrolysis extractions, enzymolysis, filter and be settled to 100mL after, in the scale test tube with cover of 6 25mL add 1.00mL tie up Raw element B₂Tablet extracting solution, carries out aoxidizing decontamination according to embodiment 3 record method, is settled to 25mL, after mixing, adds 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL 20g/100mL Hydros solution, after mixing, parallel Measuring three times, average, measurement result is as shown in Figure 6.

Fig. 6 can be seen that, add 1.00mL, 2.00mL, 3.00mL, 4.00mL Hydros time, relative fluorescence along with The increase of Hydros volume and be gradually lowered, illustrate along with the increase of Hydros volume, vitamin B₂Tablet center Flavin is gradually reduced to unstressed configuration material；When adding 4.00mL, 5.00mL, 6.00mL Hydros, relative fluorescence It is held essentially constant along with the increase of Hydros volume, vitamin B is described₂Riboflavin in tablet is the most all gone back Originally being unstressed configuration material, experimental selection Hydros volume is 5.00mL.

2.2.4 the selection of riboflavin standard solution volume

Weigh the finely ground dry vitamin B of 0.0500g₂Tablet, in 250mL conical flask, is carried out according to embodiment 3 record method After hydrolysis extraction, enzymolysis, filtration, constant volume, in the scale test tube with cover of 10 25mL, add 1.00mL vitamin B₂Medicine Sheet extracting solution, be separately added in 3,4,5,6,7,8,9, No. 10 pipes 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL, 7.00mL, 8.00mL1 μ g/mL riboflavin standard solution, No. 1 pipe and No. 2 pipes are not added with standard solution, in No. 1 pipe Add 5.00mL20g/100mL Hydros solution, add water to 15mL, carry out aoxidizing roguing according to embodiment 3 record method Matter, is settled to 25mL, mixing.After mixing, each sample is parallel assay three times under conditions of identical, average, according to Formula (1) calculates vitamin B₂The mass fraction of tablet riboflavin, then compares with national standard method, result such as table 2.

As shown in Table 2, riboflavin content in the tablet that different volumes riboflavin standard solution records is added close.But mark-on .1.00mL, the result that records of 2.00mL and GB closest, therefore, this experimental selection mark-on 1.00mL, 2.00mL riboflavin mark Quasi-use liquid carries out difference mark-on mensuration.

The measurement result of 3 samples

At optimum conditions, to 20.0000g egg yolk and 0.0500g vitamin B₂Tablet parallel assay 6 times respectively, experiment knot Fruit is such as table 3 and table 4.

Can be obtained riboflavin content in butt egg yolk sample by table 3 and table 4 is 402 μ g/100g, vitamin B₂Tablet riboflavin contains Amount is 7.9 g/100g.Egg yolk and vitamin B₂The relative standard deviation that tablet measures is respectively 0.88% and 1.0%, measures essence Density is good.

4 recovery of standard addition

Weigh 20.0000g egg yolk and 0.0500g vitamin B₂Tablet is carried out at sample according to embodiment 2 and embodiment 3 respectively Reason, then carries out recovery of standard addition experiment, experimental result such as following table:

Egg yolk and vitamin B can be obtained by table 5 and table 6₂The tablet response rate is respectively 92%～104% and 90%～96%.

5 detection limits and quantitative limit

Taking fluorescent value close to blank, the relatively low riboflavin standard solution of concentration carries out 11 parallel assays, calculates standard Deviation, 3 times of standard deviations are detection limit, and 10 times of standard deviations are quantitative limit.Obtain riboflavin detection and be limited to 5.79ng/mL, quantitatively It is limited to 1.92 × 10^-2μg/mL。

6 measurement result method validations

Respectively according to embodiment 2 and embodiment 3 sample determination method to egg yolk and VB₂Tablet is measured, to same sample With National Standard Method check analysis, respectively carry out 6 parallel assays, after inspection is without dubious value, report of averaging, result such as table 7.

According to table 7 molecular fluorescence difference mark-on method and the standard deviation of National Standard Method and meansigma methods, carry out F inspection and t check:

F method of inspection

Egg yolk

Vitamin B₂Tablet

Calculate degree of freedom f:f_Greatly=n-1=5, f_Little=n-1=5, looks into correlation table and obtains F_Table=5.05, the F calculated₁Value and F₂It is worth the least F value in table.So, there is not significant difference in the precision of both approaches, the confidence level of conclusion is 95%.Then enter Row t method of inspection.

T method of inspection

Egg yolk

Vitamin B₂Tablet

Calculate degree of freedom: f=6+6-2=10, look into correlation table, when confidence level is 95%, t_Table=2.23, t₁Value and t₂Value both less than t_Table Value.So, there is not significant systematic error in the meansigma methods of two kinds of methods.

Checking through F inspection and t, molecular fluorescence difference mark-on method and the precision of National Standard Method and meansigma methods the most do not exist aobvious Writing sex differernce, the confidence level of conclusion is 95%.Molecular fluorescence difference mark-on method measurement result is accurate, is suitable for the fast of riboflavin content Speed measures.

The present invention have studied that hydrochloric acid extraction liquid is long-pending, enzymolysis pH, adds scalar to egg yolk, vitamin B₂Riboflavin in tablet The impact of fluorescent value, experiment determines egg yolk, vitamin B₂Tablet extracting solution is 0.1mol/L hydrochloric acid 70mL, and enzymolysis is optimal It is 1.00mL, 2.00mL that pH 5.500,1 μ g/mL riboflavin most preferably adds scalar.Measurement result: in butt egg yolk, riboflavin contains Amount is 402 μ g/100g, the raw B of dimension₂In tablet, riboflavin content is 7.9 g/100g；Egg yolk relative standard deviation is 0.88%(n =6), the raw B of dimension₂Tablet relative standard deviation is 1.0% (n=6)；Egg yolk recovery of standard addition is 92.0%～104%, vitamin B₂Medicine Sheet recovery of standard addition is 90.0%～96.0%；Detection is limited to 5.76n g/mL, is quantitatively limited to 1.92 × 10^-2μg/mL.The method with National standard method compares, and through F method of inspection and t method of inspection, the precision of two kinds of methods and meansigma methods the most do not exist significance Difference.The inventive method only need to use the test solution that two spiked levels are different, surveys under the same conditions with four parts of same volume test solutions Fixed luminous signal, quickly obtains measurement result, it is not necessary to drawing curve and excessively post decontamination, method has deducted sample test solution The part interference that middle background luminescence value and concentration difference may be brought, improves accuracy of measurement and work efficiency, letter Just, quickly, it is to be suitable for the novel analytical technology of riboflavin assay in sample, has stronger novelty.

Above-described is only the preferred embodiment of the present invention, it is noted that for a person skilled in the art, Without departing under general idea premise of the present invention, it is also possible to making some changes and improvements, these also should be considered as the present invention's Protection domain, the method also can be used to measure the material of the many mulecular luminescences in other samples, including molecular fluorescence, molecular phosphorus The related substances that can measure in the quantitative analyses such as light, chemiluminescence, bioluminescence and component.

Claims

1. one kind utilizes molecular fluorescence difference mark-on to measure egg yolk, VB₂The method of riboflavin in tablet, it is characterised in that include with Lower step:

(2) measure liquid to prepare: from V_{Sample 1}In take at least four parts of same volumes V_{Sample 2}Filtration test solution, to first part and second part addition The riboflavin standard solution of different volumes, riboflavin standard solution mass body volume concentrations is, second part of standard solution volume V_{Mark 2} More than first part of V_{Mark 1}, the 4th part adds Hydros and riboflavin is reduced to unstressed configuration material, and as blank, the 3rd part both It is not added with standard solution, is also not added with Hydros, for former filtration test solution；Each pipe adds water to V, then adds the glacial acetic acid of 3-3.5% V Mixing, is subsequently adding the potassium permanganate solution of 3-3.5% V, and potassium permanganate solution concentration is 25-35g/L, mixing, stands, dropping Hydrogen peroxide solution, to potassium permanganate color fade, shaking, makes unnecessary oxygen escape, and constant volume is V₁；

。

Molecular fluorescence difference mark-on method is utilized to measure egg yolk, VB the most as claimed in claim 1₂The method of riboflavin in tablet, its It is characterised by comprising the following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs 18-22g egg yolk or 0.04-0.06gVB₂Tablet, adds 0.09- 0.11moL/L hydrochloric acid 60-80 mL, pressure hydrolysis extraction, pressure is (8-12) × 10⁴Pa, the time is 20-40min, then uses NaOH solution regulation pH to 4.5-7.5, adds 2.5-3.5mL 9-11g/L papain solution, VB in egg yolk test solution₂Tablet Test solution adds 2.5-3.5mL 9-11g/L amylase solution, then carries out enzymolysis at 37-39 DEG C of insulation 15-17h；Enzymolysis solution Filter, take filtrate and be settled to V_{Sample 1}；

(2) measure liquid to prepare: from V_{Sample 1}In take at least four parts of same volumes V_{Sample 2}Filtrate, volume is 0.5-10mL, to first part With the riboflavin standard solution of second part of addition different volumes, riboflavin standard solution mass body volume concentrations is, second part of standard Liquor capacity V_{Mark 2}It is first part of V_{Mark 1}1.2-2 times；4th part adds Hydros and riboflavin is reduced to unstressed configuration material work Blank for background；3rd part neither adds standard solution, is also not added with Hydros, for former filtration test solution；Each pipe adds water to 14- 16mL, then adds the mixing of 0.4-0.6mL glacial acetic acid in every part of filtrate, and the potassium permanganate being subsequently adding 0.4-0.6mL is molten Liquid, potassium permanganate solution concentration is 25-35g/L, mixing, stands, and dropping hydrogen peroxide solution is to potassium permanganate color fade, finally Each pipe adds water and is settled to 24-26mL；

Molecular fluorescence difference mark-on method is utilized to measure egg yolk, VB the most as claimed in claim 3₂The method of riboflavin in tablet, its Special

Levy and be to comprise the following steps:

(1) prepared by extracting solution: takes sample and grinds, weighs 20g egg yolk or 0.05gVB₂Tablet, adds 0.1moL/L hydrochloric acid 70mL, Pressure hydrolysis is extracted, and pressure is 10.3 × 10⁴Pa, the time is 30min, then regulates pH to 5.5, egg yolk test solution by NaOH solution Middle addition 3mL 10g/L papain solution, VB₂Tablet test solution adds 3mL 10g/L amylase solution, then at 39 DEG C Insulation 16h carries out enzymolysis；Enzymolysis solution filters, and takes filtrate and is settled to 100mL；

(2) measure liquid to prepare: taking the filtration test solution of four parts of same volumes from 100mL filtrate, egg yolk filtrate volume is 10.00mL, VB₂Tablet filtrate volume is 1.00mL, be separately added in first part and second part of extracting solution 1.00mL and 2.00mL concentration is the riboflavin standard solution of 1 μ g/mL；4th part adds Hydros and riboflavin is reduced to unstressed configuration Material is as background blank, and the 3rd part neither adds standard solution, be also not added with Hydros, for former filtration test solution；Each pipe adds water To 15mL, in every part of filtrate, then add the mixing of 0.5mL glacial acetic acid, add the potassium permanganate solution of 0.5mL, potassium permanganate Solution concentration is 30g/L, mixing, stands, and dropping hydrogen peroxide solution, to potassium permanganate color fade, finally adds water and is settled to 25mL；

5. the molecular fluorescence difference mark-on method that utilizes as described in any one of claim 1-4 measures egg yolk, VB₂Riboflavin in tablet Method, it is characterised in that in step (1), NaOH solution concentration is 1moL/L, and in step (2), hydrogen peroxide mass body volume concentrations is 3%。

6. the molecular fluorescence difference mark-on method that utilizes as described in any one of claim 3-4 measures egg yolk, VB₂Riboflavin in tablet Method, it is characterised in that in egg yolk test solution add Hydros solution at least 0.5mL, VB₂Tablet test solution adds low Asia Metabisulfite solution at least 5mL；Hydros solution concentration is 20g/100mL.

7. the molecular fluorescence difference mark-on method that utilizes as described in any one of claim 3-4 measures egg yolk, VB₂Riboflavin in tablet Method, it is characterised in that described papain and amylase solution use the preparation of 2.5moL/L sodium acetate solution.

8. the molecular fluorescence difference mark-on method that utilizes as described in any one of claim 1-4 measures egg yolk, VB₂Riboflavin in tablet Method, it is characterised in that riboflavin standard solution is divided into concentration to be 25 μ g/mL riboflavin standard reserving solutions and concentration is 1 μ g/ ML riboflavin standard solution；Riboflavin standard reserving solution process for preparation is as follows: the crystallization of standard substance riboflavin powder be placed in In vacuum desiccator, after phosphorus pentoxide drying tube and silica-gel desiccant combined drying 24h, accurately weigh 50mg, be placed in 2L In volumetric flask, add 2.4mL glacial acetic acid and 1.5mL water, volumetric flask is placed in 30-40 DEG C of warm water shake, treats that it dissolves, be cooled to Room temperature, adds water and is settled to 2L, moves in brown bottle, adds toluene and is covered in solution surface, preserves in 4 DEG C of refrigerators；Riboflavin mark Quasi-use liquid process for preparation is: draws 25 μ g/mL riboflavin standard reserving solution 2.00mL, is placed in 50mL brown volumetric flask, uses Water is settled to scale, 4 DEG C of Refrigerator stores, and the holding time is 1 week.

9. the molecular fluorescence difference mark-on method that utilizes as described in any one of claim 1-4 measures egg yolk, VB₂Riboflavin in tablet Method, it is characterised in that egg yolk and VB₂Tablet also uses direct drying method to carry out the mensuration of moisture.