CN106290275B - Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet - Google Patents

Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet Download PDF

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CN106290275B
CN106290275B CN201610611269.2A CN201610611269A CN106290275B CN 106290275 B CN106290275 B CN 106290275B CN 201610611269 A CN201610611269 A CN 201610611269A CN 106290275 B CN106290275 B CN 106290275B
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solution
riboflavin
added
tablet
yolk
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CN106290275A (en
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高向阳
张娜
高遒竹
郭楠楠
焦健侠
魏龙
岳希举
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Zhengzhou University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

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Abstract

It is a kind of to measure yolk, VB using molecular fluorescence difference mark-on2The method of riboflavin in tablet, without drawing working curve, the mass fraction of component to be measured need to can be only quickly obtained by the test solution of two difference mark-ons, sample background interference is completely eliminated in continuous mode, simultaneously,, may be because the available compensation of the introduced evaluated errors of side reactions such as polymerization, dissociation, photodissociation occur or deduct because being tested concentration of component difference in sample using different amounts of standard solution is added into two same volume test solutions, accuracy of measurement further be protected and improve.The method of the present invention detection is limited to 5.76ng/mL, is quantitatively limited to 1.92 × 10‑2μg/mL。

Description

Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet
Technical field
The invention belongs to technical field of analysis and detection, and in particular to it is a kind of using molecular fluorescence difference mark-on measurement yolk, VB2The method of riboflavin in tablet.
Background technique
Vitamin B2, also known as riboflavin is easy to decompose under illumination and ultraviolet irradiation, and it is acidproof not alkaline-resisting, it dissolves in Sodium-chloride water solution is soluble in dilute sodium hydroxide solution.Riboflavin is extremely important to human body, when lacking, is metabolized and is easy hair Raw obstacle.Riboflavin be it is water-soluble, will not accumulate, supplemented often in human body, in egg yolk, soya bean, milk, yeast etc. Content is more.
Egg yolk fat rich in and protein, the lecithin including that can help to synthesize acetylcholine contain simultaneously The minerals such as calcium ferrophosphorus abundant and liposoluble vitamin abundant, core yellow cellulose content is also relatively high, and therefore, egg yolk is very Suitable for baby is edible；Vitamin B2Tablet is yellow tablet, for treating angular stomatitis, cheilosis, glossitis, scrotitis, conjunctiva Inflammation, seborrhea etc..General common specification vitamin B2Tablet contains 5 milligrams of riboflavin, other auxiliary materials is also contained in drug, such as Starch, sucrose, magnesium stearate.Since riboflavin is to the importance of human body, and riboflavin easily destroys, therefore to its source The measurement of middle content is particularly significant.
Currently, the measuring method of riboflavin is more, such as electrochemical process, high performance liquid chromatography, capillary electrophoresis, chemistry Luminesceence analysis method, oscilloscopic polarography, fluorescent spectrometry etc..These methods have respective advantage and disadvantage, as electrochemical methods Although measuring method is simple, sensitivity is higher, and accuracy and anti-interference ability are poor.High performance liquid chromatography has measurement knot Fruit is accurate, reliable, but sample pretreatment process is relatively complicated, measure it is more time-consuming, instrument costly, trivial operations.Hair Cons electrophoresis method has good sensitivity, but cost of determination is relatively high.
Molecule nutriology is a kind of using more mulecular luminescence Analytical Methods of Trace, high sensitivity, and detection limit is low.Due to Riboflavin solution issues stronger green fluorescence under 430-440nm blue light illumination, therefore can use molecule nutriology measurement The content of riboflavin, but since molecule nutriology is Analytical Methods of Trace, fluorescence pollution, environmental factor and sample Coexisting component Influence be that accurate, quick analysis can not irrespective main problem.Quantitative analysis method more in measurement is work at present Calibration curve method and comparison method；Working curve method is that a series of standard that the standard solution of known quantity is configured to various concentrations is molten Liquid, and except test solution to be determined, the relative luminous intensity of its standard solution is measured under certain condition, with luminous intensity to mark Quasi- solution concentration draws working curve, provides the correlation between regression equation and parameter, sample test solution is with standard solution identical Under conditions of measure, compare working curve or substitute into the content that regression equation finds out component to be measured in sample.The advantages of method is Suitable for the measurement of batch samples, the disadvantage is that 5-7 standard solution need to be prepared by 1. drawing working curve, consuming time is long, surveys It is fixed at high cost；2. working curve needs were repainted or are corrected after one section of working time；3. be due in standard solution not The background of background and standard solution containing sample to be tested, therefore when Specimen Determination has larger difference, even being replaced with distilled water Test solution does overall process blank and gives " button blank ", can not deduct the background interference that sample coexisting substances itself may introduce, only The interference that distilled water, agents useful for same, instrument introduce can be partially deducted, because the background of standard solution and test solution is different, point Ce Ding not be it cannot be guaranteed that complete background correction to be interfered under identical determination condition, the accuracy of measurement will have a greatly reduced quality；④ Measurement must carry out in the linear range, if the concentration of substance to be measured is excessively dilute or overrich, it is possible to occur different degrees of Dissociation, polymerization, photodissociation or other side reactions and deviate linear relationship, introducing large error can not even measure.Comparison method are as follows: take One standard solution measures under conditions of identical with test solution, while measuring reagent blank, according under the conditions of identical When measurement, the concentration relationship directly proportional to luminous value can calculate the concentration of test solution, and this law is suitable for the quick survey of single sample Fixed, while requiring concentration of standard solution and prepare liquid concentration close as far as possible, the advantages of method is quick, easy, analysis cost phase To cheap, without mapping；Although the disadvantage is that 1. deducted the luminous value of reagent blank, test solution and standard solution background background feelings Condition is not consistent, if different degrees of chemical side reactions or photochemistry side reaction, measurement result occur in continuous mode for system Introducing error that will be different degrees of；2. measurement requires the concentration of standard solution and test solution close as far as possible, to unknown sample Actually it is difficult to accomplish, generally requires pre- determination of doing experiment；3. measuring the dense of signal deciding test solution by a standard solution The accuracy of degree, standard solution measurement signal seriously affects measurement result.Egg yolk and vitamin B2Tablet as described above, in addition to Riboflavin, also complicated containing other more coexisting substances, especially egg yolk matrix composition, there are the albumen of high level The substances such as matter, amino acid either use working curve method or comparison method, are easy that protein etc. is coexisted in measurement The influence of fluorescence interference and the pollution of various fluorescence of non-measurement component；Certain error is brought to measurement result；So Ren Menxi It hopes and establishes the novel fluorescence analysis side that matrix fluorescence interferes measurement riboflavin that is small or can effectively eliminating interference and fluorescence pollution Method.
Summary of the invention
The object of the invention is that providing a kind of utilization molecular fluorescence difference mark-on measurement yolk, VB2Core yellow in tablet The method of element, the measuring method is quick, effectively eliminates test solution background and the introduced interference of concentration difference, accuracy obtain Guarantee, working efficiency is improved.
The object of the present invention is achieved in the following manner:
It is a kind of to measure yolk, VB using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) prepared by extracting solution: taking sample to grind, weighs mSample, 0.09-0.11moL/L hydrochloric acid, yolk and hydrochloric acid solution is added Mass volume ratio (g/mL) be 1:(2.6-4), VB2The mass volume ratio (g/mL) of tablet and hydrochloric acid solution is 1:(1200- 1600), pressure hydrolysis is extracted, and pressure is (8.0-12.0) × 104Pa, time 20-40min, is then adjusted with NaOH solution PH to 4.5-7.5, papain solution is added in yolk test solution, and papain additional amount is the 0.13- of yolk quality 0.16%, VB2Amylase solution is added in tablet test solution, amylase additional amount is VB2The 50-70% of tablet quality, then exists 37-39 DEG C of heat preservation 15-17h is digested；Enzymolysis liquid filtering, filtrate are settled to VSample 1；
(2) measurement liquid preparation: from VSample 1In take four parts of same volume VSample 2Filtering test solution, to first part and second part be added The riboflavin standard solution of different volumes, riboflavin standard solution mass-volume concentration are ρ, second part of standard solution volume VMark 2 Greater than first part VMark 1, riboflavin is reduced to unstressed configuration substance by the 4th part of addition Hydros, and as blank, third part was both Standard solution is not added, Hydros are also not added, for former filtering test solution；Then plus the glacial acetic acid of 3-3.5%V each pipe adds water to V, It mixes, the liquor potassic permanganate of 3-3.5%V is then added, liquor potassic permanganate concentration 25-35g/L is mixed, and is stood, and is added dropwise Hydrogen peroxide solution to potassium permanganate color fade, shaking escapes extra oxygen, constant volume V1；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, obtained according to map maximum Excitation wavelength and maximum emission wavelength, the test solution that (1) is handled well is in maximum excitation wavelength and maximum emission wavelength Lower measurement, first part of mark-on test solution relative light unit are IMark 1, second part of mark-on test solution relative light unit is IMark 2, third part is to be measured Sample test solution relative light unit is ISample, the 4th part of background blank background relative light unit is I0, yolk or VB2Riboflavin in tablet Mass fraction ω such as formula (1)；
Preferably, comprising the following steps:
(1) prepared by extracting solution: taking sample to grind, weighs mSample, 0.1moL/L hydrochloric acid, the quality of yolk and hydrochloric acid solution is added Volume ratio (g/mL) is 1:3.5, VB2The mass volume ratio (g/mL) of tablet and hydrochloric acid solution is 1:1400, and pressure hydrolysis is extracted, Pressure is 10.3 × 104Then Pa, time 30min adjust pH to 5.5 with NaOH solution, pawpaw egg are added in yolk test solution White enzyme solutions, papain additional amount are 0.15%, VB of yolk quality2Amylase solution, starch are added in tablet test solution Enzyme additional amount is VB2Then the 60% of tablet quality is digested in 39 DEG C of heat preservation 16h；Enzymolysis liquid filtering, takes filtrate to be settled to VSample 1；
(2) measurement liquid preparation: from VSample 1In take at least four parts of same volume VSample 2Filtering test solution, to first part and second part The riboflavin standard solution of different volumes is added, riboflavin standard solution mass-volume concentration is ρ, second part of standard solution volume V Mark 2Greater than first part VMark 1, riboflavin is reduced to unstressed configuration substance as background blank by the 4th part of addition Hydros, and Three parts neither add standard solution, and Hydros are also not added, for former filtering test solution；Each pipe adds water to V, adds the glacial acetic acid of 3.3%V It mixes, the liquor potassic permanganate of 3.3%V is then added, liquor potassic permanganate concentration 30g/L is mixed, and is stood, and dioxygen is added dropwise Aqueous solution is to potassium permanganate color fade, and finally plus water is settled to V1After measure；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, obtained according to map maximum Excitation wavelength and maximum emission wavelength, the test solution that step (2) is handled well is in maximum excitation wavelength and maximum fluorescence emission It is measured under wavelength.
Further, the utilization molecular fluorescence difference mark-on measures yolk, VB2The method of riboflavin in tablet, including Following steps:
(1) prepared by extracting solution: taking sample to grind, weighs 18-22g egg yolk or 0.04-0.06gVB2Tablet is added 0.09- 0.11moL/L hydrochloric acid 60-80mL, highly pressured hydrolysis extract, and pressure is (8-12) × 104Pa, time 20-40min, so PH to 4.5-7.5 is adjusted with NaOH solution afterwards, 2.5-3.5mL 9-11g/L papain solution is added in yolk test solution, VB22.5-3.5mL 9-11g/L amylase solution is added in tablet test solution, is then digested in 37-39 DEG C of heat preservation 15-17h； Enzymolysis liquid filtering, takes filtrate to be settled to VSample 1；
(2) measurement liquid preparation: from VSample 1In take at least four parts of same volume VSample 2Filtered fluid, volume 0.5-10mL, to A and second part of addition different volumes riboflavin standard solution, riboflavin standard solution mass-volume concentration are ρ, second part Standard solution volume VMark 2For first part of VMark 11.2-2 times；Riboflavin is reduced to unstressed configuration object by the 4th part of addition Hydros Matter is as background blank；Third part neither adds standard solution, and Hydros are also not added, for former filtering test solution；Each pipe adds water to Then 14-16mL 0.4-0.6mL glacial acetic acid is added into every part of filtered fluid and mixes, the potassium permanganate of 0.4-0.6mL is then added Solution, liquor potassic permanganate concentration 25-35g/L are mixed, and are stood, dropwise addition hydrogen peroxide solution to potassium permanganate color fade, most Each Guan Jiashui is settled to 24-26mL afterwards；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, obtained according to map maximum Excitation wavelength and maximum emission wavelength, the sample test solution that step (2) is handled well is in maximum excitation wavelength and maximum fluorescence It is measured under launch wavelength.
Further, comprising the following steps:
(1) prepared by extracting solution: taking sample to grind, weighs 20g egg yolk or 0.05gVB20.1moL/L hydrochloric acid is added in tablet 70mL,
Pressure hydrolysis is extracted, and pressure is 10.3 × 104Then Pa, time 30min adjust pH to 5.5 with NaOH solution, 3mL 10g/L papain solution, VB are added in yolk test solution23mL 10g/L amylase solution is added in tablet test solution, so It is digested afterwards in 39 DEG C of heat preservation 16h；Enzymolysis liquid filtering, takes filtrate to be settled to 100mL；
(2) the filtering test solution of at least four parts same volumes, egg yellow filtrate body measurement liquid preparation: are taken from 100mL filtrate Product is 10.00mL, VB2Tablet filtrate volume be 1.00mL, be separately added into first part and second part of extracting solution 1.00mL with 2.00mL concentration is the riboflavin standard solution of 1 μ g/mL；Riboflavin is reduced to unstressed configuration object by the 4th part of addition Hydros Matter neither adds standard solution, Hydros is also not added as background blank, third part, for former filtering test solution；Each pipe adds water to Then 15mL 0.5mL glacial acetic acid is added into every part of filtered fluid and mixes, the liquor potassic permanganate of 0.5mL is added, potassium permanganate is molten Liquid concentration is 30g/L, is mixed, and is stood, and hydrogen peroxide solution is added dropwise to potassium permanganate color fade, finally plus water is settled to 25mL；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, obtained according to map maximum Excitation wavelength and maximum emission wavelength, the sample test solution that step (2) is handled well is in maximum excitation wavelength and maximum fluorescence It is measured under launch wavelength.
Yolk, VB are measured using molecular fluorescence difference mark-on as described above2The method of riboflavin in tablet, step (1) Middle NaOH solution concentration is 1moL/L, and hydrogen peroxide mass-volume concentration is 3% in step (2)；Low sulfurous is added in yolk test solution Acid sodium solution at least 0.5mL, VB2Hydros solution at least 5mL is added in tablet test solution；Hydros solution concentration is 20g/100mL, the papain and amylase solution are prepared using 2.5moL/L sodium acetate solution,
The riboflavin standard solution is divided into that concentration is 25 μ g/mL riboflavin standard reserving solutions and concentration is 1 μ g/mL core yellow Plain standard solution；Riboflavin standard reserving solution process for preparation is as follows: standard items riboflavin powdery crystal being placed in vacuum and is done In dry device, with phosphorus pentoxide drying tube and silica-gel desiccant combined drying for 24 hours after, accurately weigh 50mg, be placed in 2L volumetric flask In, 2.4mL glacial acetic acid and 1.5mL water is added, volumetric flask is placed in 30-40 DEG C of warm water and is shaken, to its dissolution, is cooled to room temperature, Add water to be settled to 2L, move in brown bottle, toluene is added to be covered in solution surface, is saved in 4 DEG C of refrigerators；Riboflavin standard uses Liquid process for preparation are as follows: draw 25 μ g/mL riboflavin standard reserving solution 2.00mL, be placed in 50mL brown volumetric flask, with water constant volume To scale, 4 DEG C of refrigerators are saved, and the holding time is 1 week.
Yolk also uses the measurement of direct drying method progress moisture content.
The present invention establishes a kind of quick, easy quantitative determination yolk, vitamin B2Novel point of riboflavin in tablet Analysis method only need to can calculate the matter of component to be measured in the sample by using two mark-on test solutions without drawing working curve Score is measured, the background interference in sample test solution is completely eliminated in continuous mode, while using two addition various criterion concentration Mark-on test solution, because the different issuable tested component of concentration occur the side reactions such as polymerization, dissociation can partially be compensated or It deducts, accuracy of measurement is further protected and improves.The method of the present invention detection is limited to 5.76ng/mL, is quantitatively limited to 1.92 ×10-2μ g/mL.It is compared with national standard method, by F method of inspection and t method of inspection, the precision and average value of two methods Significant difference is all not present；But measuring method of the present invention is easy, quickly, has deducted in sample test solution background luminous value and dense The possible a part interference of difference institute is spent, improves accuracy of measurement and working efficiency, method is without drawing working curve With column decontamination excessively, it is the novel analytical technology for being suitble to riboflavin assay in sample, there is stronger novelty.
Detailed description of the invention
Fig. 1 is influence of the hydrochloric acid volume to egg yolk test solution relative fluorescence；
Fig. 2 is influence of the pH to egg yolk test solution relative fluorescence；
Fig. 3 is influence of the Hydros volume to egg yolk test solution relative fluorescence；
Fig. 4 is hydrochloric acid volume to vitamin B2The influence of tablet test solution relative fluorescence；
Fig. 5 is pH to vitamin B2The influence of tablet test solution relative fluorescence；
Fig. 6 is Hydros volume to vitamin B2The influence of tablet test solution relative fluorescence.
Specific embodiment
Embodiment 1
It is a kind of to measure yolk, VB using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) prepared by extracting solution: taking sample to grind, weighs mSample, 0.09-0.11moL/L hydrochloric acid, yolk and hydrochloric acid solution is added Mass volume ratio (g/mL) be 1:(2.6-4), VB2The mass volume ratio (g/mL) of tablet and hydrochloric acid solution is 1:(1200- 1600), pressure hydrolysis is extracted, and pressure is (8.0-12.0) × 104Pa, time 20-40min, it is then molten with 1moL/L NaOH Liquid adjusts pH to 4.5-7.5, and papain solution is added in yolk test solution, and papain additional amount is yolk quality 0.13- 0.16%, VB2Amylase solution is added in tablet test solution, amylase additional amount is VB2The 50-70% of tablet quality, so It is digested afterwards in 37-39 DEG C of heat preservation 15-17h；Enzymolysis liquid is filtered with dry filter paper, and filtrate is taken to be settled to VSample 1；Pass through hydrochloric acid water Solution and enzymatic hydrolysis, can allow the riboflavin separate out of reference state in sample materials, participate in measurement, improve analysis data reliability and Accuracy；Papain is added in egg yolk can digest the riboflavin in conjunction with protein；
(2) measurement liquid preparation: from VSample 1In take at least four parts of same volume VSample 2Filtering test solution, to first part and second part The riboflavin standard solution of different volumes is added, riboflavin standard solution mass-volume concentration is ρ, second part of standard solution volume V Mark 2Greater than first part VMark 1, riboflavin is reduced to unstressed configuration substance as background blank by the 4th part of addition Hydros, and Three parts neither add standard solution, and Hydros are also not added, for former filtering test solution；Then plus 3.3%V each pipe adds water to V, Glacial acetic acid mixes, and the liquor potassic permanganate of 3.3%V is then added, and liquor potassic permanganate concentration 30g/L is mixed, and stands, drop Add hydrogen peroxide solution to potassium permanganate color fade, finally increase pure water (the sub- boiling deionization redistilled water of quartz, at 25 DEG C, PH6.63 ± 0.02,1.183 μ s/cm of conductivity) it is settled to V1After measure；Glacial acetic acid, which is added, can maintain solution to be in weak acid ring Border, because riboflavin is relatively stable in weakly acidic condition；The oxidable removing stronger reduction more that may be present of potassium permanganate is added Property substance interference, reduce background blank value；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, obtained according to map maximum Excitation wavelength and maximum emission wavelength, the sample that step (2) is handled well is in maximum excitation wavelength and maximum fluorescence emission It is measured under wavelength, first part of mark-on test solution relative light unit is IMark 1, second part of mark-on test solution relative light unit is IMark 2, third part Sample to be tested test solution relative light unit is ISample, the 4th part of background blank background relative light unit is I0, four during said determination Part test solution measures under conditions of identical, according to the concentration of the component to be measured relationship directly proportional to luminous value, it may be assumed thatρSampleFor the mass-volume concentration of constant volume test solution to be measured, Δ ρMarkFor the mass body of two parts of mark-on constant volume test solutions The difference of product concentration；Yolk or VB can be extrapolated2The mass fraction ω such as formula (1) of riboflavin in tablet；
The difference mark-on measuring method that the present invention uses, wherein IMark 1、IMark 2Cause and I0Using identical auxiliary reagent, therefore, I Mark 1、IMark 2Also contain I0Value, but IMark 2-IMark 1I afterwards0It has been offset that, due to the measurement background one of test solution after test solution and mark-on It causes, therefore, interference of the test solution background to measurement result can be eliminated using this method, improve the accuracy of measurement.So the party Method need to only measure the relative light unit of solution under the same conditions, measurement result can quickly be acquired, without drawing working curve It is quickly, easy with mapping evaluation, working efficiency is greatly improved, determines to be measured group using the mark-on test solution of two various concentrations The concentration of part can be compensated partially because the side reactions such as polymerization, dissociation occur for the different issuable tested components of sample concentration And deduction, accuracy of measurement are further protected and improve.
Embodiment 2
Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) solution is prepared:
Riboflavin standard reserving solution (25 μ g/mL): by standard items riboflavin (Shanghai Jin Sui Biotechnology Co., Ltd) powder Last shape crystallization is placed in vacuum desiccator, and after for 24 hours, is accurately weighed 50mg, is placed in 2L volumetric flask, 2.4mL ice second is added Acid and 1.5mL water.Volumetric flask is placed in warm water and is shaken, to its dissolution, is cooled to room temperature, is diluted to 2L, move in brown bottle, add A little toluene is placed on solution surface, saves in refrigerator.
Riboflavin standard solution (1 μ g/mL)；2.00mL riboflavin standard reserving solution is drawn, 50mL brown capacity is placed in In bottle, it is diluted with water to scale, is protected from light, 4 DEG C of refrigerators are stored in, can be reserved for one week；Every milliliter of this solution is equivalent to 1.00 μ g core yellows Element.
Papain (10g/L): papain (Pangbo Bioengineering Co Ltd, Nanning), with 2.5mol/L acetic acid Sodium solution is prepared；In use, existing prepare.
Amylase (10g/L): amylase (Xingtai Wanda's bioengineering Co., Ltd) is matched with 2.5mol/L sodium acetate solution System.It is now prepared when use.
Hydros solution (20g/100mL): (analysis is pure, and Tianjin causes the remote limited public affairs of chemical reagent for Hydros Department), this liquid used time with high purity water (the sub- boiling deionization redistilled water of quartz, at 25 DEG C, pH6.63 ± 0.02,1.183 μ s/ of conductivity Cm) now match, be stored in ice-water bath, in 4h effectively.
(2) extracting solution prepares (sample is yolk in this embodiment):
It takes cooked egg yolk sample finely ground, weighs 20.0000g and be placed in 250mL conical flask, 0.1moL/L hydrochloric acid is added 70mL (mass volume ratio (g/mL) of yolk and hydrochloric acid solution is 1:3.5), stirring is uniformly dispersed until particulate matter, uses 40mL Porcelain crucible is that cover buckle lives bottleneck, is placed in pressure cooker hydrolysis and extracts, pressure is 12.0 × 104Pa, time 30min hydrolyze liquid cooling But after, pH to 5.5 is adjusted with 1moLNaOH solution, 3mL 10g/L papain solution (Papain is added in yolk test solution Enzyme additional amount is the 0.15% of yolk quality), then digested in 39 DEG C of heat preservation 16h；Enzymolysis liquid is filtered with dry filter paper, takes filter Liquid is settled to 100mL；
(3) measurement liquid preparation
From VSample 1In take the filtering test solution of four parts of same volumes, volume 10.00mL, to first part and second part of extracting solution In be separately added into 1.00mL and 2.00mL concentration be 1 μ g/mL riboflavin standard solution；4th part of addition 0.5mL concentration is Riboflavin is reduced to unstressed configuration substance by 20g/100mL Hydros solution, as background blank；Third part neither mark-on is quasi- Hydros are also not added in solution, for former filtering test solution；Each pipe adds water to 15mL, is then added into every part of extracting solution 0.5mL (3.3%V) glacial acetic acid mixes, and the liquor potassic permanganate of 0.5mL (3.3%V), liquor potassic permanganate concentration is then added It for 30g/L, mixes, stands, 3% hydrogen peroxide solution is added dropwise to potassium permanganate color fade, acutely shakes sample, makes extra oxygen Gas overflows, and finally plus water is settled to 25mL；
(4) determination of moisture in yolk
To compare convenient for the result of different sample rooms, measurement result is preferably contents on dry basis；Cooked egg yolk is finely ground, claim Taking quality is about 5.0000g according to direct drying method progress determination of moisture in GB/T 5009.3-2010, is calculated according to formula (2) The percentage composition of egg yolk moisture out.
In formula: ω0For the moisture content of egg yolk；m1For the quality of weighing bottle before sample drying and sample, g；m2To weigh Quality after bottle and sample drying, g；m3For the quality of weighing bottle, g.
(5) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (3) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, butt egg yolk is calculated according to formula (3) The mass fraction ω (μ g/100g) of middle riboflavin；
Embodiment 3
Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) solution is prepared:
Riboflavin standard reserving solution (25 μ g/mL): by standard items riboflavin (Shanghai Jin Sui Biotechnology Co., Ltd) powder Last shape crystallization is placed in vacuum desiccator, and after for 24 hours, is accurately weighed 50mg, is placed in 2L volumetric flask, 2.4mL ice second is added Acid and 1.5mL water.Volumetric flask is placed in warm water and is shaken, to its dissolution, is cooled to room temperature, is diluted to 2L, move in brown bottle, add A little toluene is placed on solution surface, saves in refrigerator.
Riboflavin standard solution (1 μ g/mL)；2.00mL riboflavin standard reserving solution is drawn, 50mL brown capacity is placed in In bottle, it is diluted with water to scale, is protected from light, 4 DEG C of refrigerators are stored in, can be reserved for one week.Every milliliter of this solution is equivalent to 1.00 μ g core yellows Element.
Papain (10g/L): papain (Pangbo Bioengineering Co Ltd, Nanning), with 2.5mol/L acetic acid Sodium solution is prepared.In use, existing prepare.
Amylase (10g/L): amylase (Xingtai Wanda's bioengineering Co., Ltd) is matched with 2.5mol/L sodium acetate solution System.It is now prepared when use.
Hydros solution (20g/100mL): (analysis is pure, and Tianjin causes the remote limited public affairs of chemical reagent for Hydros Department), this liquid used time with high purity water (the sub- boiling deionization redistilled water of quartz, at 25 DEG C, pH6.63 ± 0.02,1.183 μ s/ of conductivity Cm) now match, be stored in ice-water bath, in 4h effectively.
(2) extracting solution prepare (in this embodiment sample be VB2Tablet):
Take VB2The finely ground drying of tablet weighs 0.0500g and pours into 250mL conical flask, and 0.1moL/L hydrochloric acid 70mL is added (VB2The mass volume ratio (g/mL) of tablet and hydrochloric acid solution is 1:1400), stirring is uniformly dispersed until particulate matter, uses 40mL Porcelain crucible is that cover buckle lives bottleneck, is placed in pressure cooker hydrolysis and extracts, pressure is 12.0 × 104Pa, time 30min hydrolyze liquid cooling But after, pH to 5.5 is adjusted with 1moLNaOH solution, 3mL 10g/L amylase solution is added, and (amylase additional amount is VB2Tablet The 60% of quality), then digested in 39 DEG C of heat preservation 16h；Enzymolysis liquid is filtered with dry filter paper, and filtrate is taken to be settled to 100mL；
(3) measurement liquid preparation
The extraction filtered fluid of four parts of same volumes is taken, volume 1.00mL distinguishes into first part and second part of extracting solution The riboflavin standard solution that 1.00mL L and 2.00mL concentration is 1 μ g/mL is added；4th part of addition 5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration substance as background blank by 100mL Hydros solution；Third part neither adds standard solution, Also Hydros are not added, for former filtering test solution；Each pipe adds water to 15mL, and 0.5mL then is added into every part of extraction filtered fluid (3.3%V) glacial acetic acid mixes, and the liquor potassic permanganate of 0.5mL (3.3%V), liquor potassic permanganate concentration 30g/ is then added L is mixed, and is stood, and 3% hydrogen peroxide solution of mass-volume concentration is added dropwise to potassium permanganate color fade, acutely shakes sample, makes more Remaining oxygen evolution, finally plus water is settled to 25mL；
(4) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (3) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, VB is calculated according to formula (4)2In tablet The mass percent ω (g/100g) of riboflavin；
Embodiment 4
Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) extracting solution prepares (sample is yolk in this embodiment):
It takes cooked egg yolk sample finely ground, weighs 22.0000g and pour into 250mL conical flask, 0.1moL/L hydrochloric acid is added 80mL, stirring are uniformly dispersed until particulate matter, are that cover buckle lives bottleneck with 40mL porcelain crucible, are placed in pressure cooker hydrolysis and extract, pressure It is 12.0 × 104Pa, time 30min after hydrolyzate is cooling, adjust pH to 5.5 with 1moLNaOH solution, in yolk test solution 3.5mL 10g/L papain solution is added, is then digested in 39 DEG C of heat preservation 16h；Enzymolysis liquid is filtered with dry filter paper, is taken Filtrate is settled to 50mL；
(2) measurement liquid preparation
From VSample 1In take the filtering test solution of four parts of same volumes, volume 5.00mL, into first part and second part of extracting solution It is separately added into the riboflavin standard solution that 1.00mL and 2.00mL concentration is 1 μ g/mL；4th part of addition 0.5mL concentration is Riboflavin is reduced to unstressed configuration substance by 20g/100mL Hydros solution, as background blank；Third part neither mark-on is quasi- Hydros are also not added in solution, for former filtering test solution；Each pipe adds water to 16mL, is then added into every part of extracting solution 0.5mL glacial acetic acid mixes, and the liquor potassic permanganate of 0.5mL is then added, and liquor potassic permanganate concentration 30g/L is mixed, quiet It sets, 3% hydrogen peroxide solution is added dropwise to potassium permanganate color fade, acutely shakes sample, overflows extra oxygen, finally plus water It is settled to 25mL；
(3) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (3) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, formula (3) is copied to calculate butt egg yolk The mass fraction ω of middle riboflavin；Remaining is the same as embodiment 2.
Embodiment 5
Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) extracting solution prepares (sample is yolk in this embodiment):
It takes cooked egg yolk sample finely ground, weighs 20.0000g and pour into 250mL conical flask, 0.09moL/L salt is added Sour 70mL, stirring are uniformly dispersed until particulate matter, are that cover buckle lives bottleneck with 40mL porcelain crucible, are placed in pressure cooker hydrolysis and extract, pressure Power is 10.0 × 104Pa, time 20min after hydrolyzate is cooling, adjust pH to 4.5 with 1moLNaOH solution, in yolk test solution 2.5mL 11g/L papain solution is added, is then digested in 37 DEG C of heat preservation 15h；Enzymolysis liquid is filtered with dry filter paper, is taken Filtrate is settled to 100mL；
(2) measurement liquid preparation
From VSample 1In take the filtering test solution of four parts of same volumes, volume 10.00mL, to first part and second part of extracting solution In be separately added into 4.00mL and 5.00mL concentration be 1 μ g/mL riboflavin standard solution；4th part of addition 0.5mL concentration is Riboflavin is reduced to unstressed configuration substance by 20g/100mL Hydros solution, as background blank；Third part neither mark-on is quasi- Hydros are also not added in solution, for former filtering test solution；Each pipe adds water to 15mL, is then added into every part of extracting solution 0.6mL glacial acetic acid mixes, and the liquor potassic permanganate of 0.6mL is then added, and liquor potassic permanganate concentration 35g/L is mixed, quiet It sets, 3% hydrogen peroxide solution is added dropwise to potassium permanganate color fade, acutely shakes sample, overflows extra oxygen, finally plus water It is settled to 24mL；
(3) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (2) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, formula (3) is copied to calculate butt egg yolk The mass fraction ω of middle riboflavin；Remaining is the same as embodiment 2.
Embodiment 6
Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet, comprising the following steps:
(1) extracting solution prepares (sample is yolk in this embodiment):
It takes cooked egg yolk sample finely ground, weighs 18.0000g and pour into 250mL conical flask, 0.11moL/L salt is added Sour 60ml, stirring are uniformly dispersed until particulate matter, are that cover buckle lives bottleneck with 40mL porcelain crucible, are placed in pressure cooker hydrolysis and extract, pressure Power is 8.0 × 104Pa, time 40min after hydrolyzate is cooling, adjust pH to 7.5 with 1moLNaOH solution, in yolk test solution 3mL 9g/L papain solution is added, is then digested in 37 DEG C of heat preservation 17h；Enzymolysis liquid is filtered with dry filter paper, takes filter Liquid is settled to 100mL；
(2) measurement liquid preparation
From VSample 1In take the filtering test solution of four parts of same volumes, volume 10.00mL, to first part and second part of extracting solution In be separately added into 2.00mL and 3.00mL concentration be 1 μ g/mL riboflavin standard solution；4th part of addition 0.5mL concentration is Riboflavin is reduced to unstressed configuration substance by 20g/100mL Hydros solution, as background blank；Third part neither mark-on is quasi- Hydros are also not added in solution, for former filtering test solution；Each pipe adds water to 14mL, is then added into every part of extracting solution 0.4mL glacial acetic acid mixes, and the liquor potassic permanganate of 0.4mL is then added, and liquor potassic permanganate concentration 25g/L is mixed, quiet It sets, 3% hydrogen peroxide solution is added dropwise to potassium permanganate color fade, acutely shakes sample, overflows extra oxygen, finally plus water It is settled to 26mL；
(3) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (2) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, formula (3) is copied to calculate butt egg yolk The mass fraction ω of middle riboflavin；Remaining is the same as embodiment 2.
Embodiment 7
(1) extracting solution prepare (in this embodiment sample be VB2Tablet):
Take VB2The finely ground drying of tablet weighs 0.0500g and pours into 250mL conical flask, and 0.1moL/L hydrochloric acid 80mL is added, Stirring is uniformly dispersed until particulate matter, is that cover buckle lives bottleneck with 40mL porcelain crucible, is placed in pressure cooker hydrolysis and extracts, pressure 12.0 ×104Pa, time 30min after hydrolyzate is cooling, adjust pH to 5.5 with 1moLNaOH solution, 3.5mL 10g/L are added and forms sediment Then powder enzyme solutions are digested in 39 DEG C of heat preservation 16h；Enzymolysis liquid is filtered with dry filter paper, and filtrate is taken to be settled to 50mL；
(2) measurement liquid preparation
The extraction filtered fluid of four parts of same volumes is taken, volume 0.500mL distinguishes into first part and second part of extracting solution The riboflavin standard solution that 1.00mL L and 2.00mL concentration is 1 μ g/mL is added；4th part of addition 5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration substance as background blank by 100mL Hydros solution；Third part neither adds standard solution, Also Hydros are not added, for former filtering test solution；Each pipe adds water to 16mL, and 0.5mL ice second is then added into every part of extracting solution Acid-mixed is even, and the liquor potassic permanganate of 0.5mL is then added, and liquor potassic permanganate concentration 30g/L is mixed, and is stood, and is added dropwise 3% Hydrogen peroxide solution acutely shakes sample, overflows extra oxygen, finally plus water is settled to potassium permanganate color fade 25mL；
(3) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (2) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, VB is calculated according to formula (1)2In tablet The mass fraction ω of riboflavin.
Embodiment 8
(1) extracting solution prepare (in this embodiment sample be VB2Tablet):
Take VB2The finely ground drying of tablet weighs 0.0600g and pours into 250mL conical flask, and 0.09moL/L hydrochloric acid 70mL is added, Stirring is uniformly dispersed until particulate matter, is that cover buckle lives bottleneck with 40mL porcelain crucible, is placed in pressure cooker hydrolysis and extracts, pressure is 10.0×104Pa, time 20min after hydrolyzate is cooling, adjusts pH to 4.5 with 1moLNaOH solution, 3mL 11g/L are added Then amylase solution is digested in 37 DEG C of heat preservation 15h；Enzymolysis liquid is filtered with dry filter paper, and filtrate is taken to be settled to 100mL；
(2) measurement liquid preparation
The extraction filtered fluid of four parts of same volumes is taken, volume 1.00mL distinguishes into first part and second part of extracting solution The riboflavin standard solution that 4.00mL L and 5.00mL concentration is 1 μ g/mL is added；4th part of addition 5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration substance as background blank by 100mL Hydros solution；Third part neither adds standard solution, Also Hydros are not added, for former filtering test solution；Each pipe adds water to 15mL, and 0.6mL ice second is then added into every part of extracting solution Acid-mixed is even, and the liquor potassic permanganate of 0.6mL is then added, and liquor potassic permanganate concentration 35g/L is mixed, and is stood, and is added dropwise 3% Hydrogen peroxide solution acutely shakes sample, overflows extra oxygen, finally plus water is settled to potassium permanganate color fade 24mL；
(3) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (2) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, VB is calculated according to formula (1)2In tablet The mass fraction ω of riboflavin.
Embodiment 9
(1) extracting solution prepare (in this embodiment sample be VB2Tablet):
Take VB2The finely ground drying of tablet weighs 0.0400g and pours into 250mL conical flask, and 0.11moL/L hydrochloric acid 60mL is added, Stirring is uniformly dispersed until particulate matter, is that cover buckle lives bottleneck with 40mL porcelain crucible, is placed in pressure cooker hydrolysis and extracts, pressure 8.0 ×104Pa, time 40min after hydrolyzate is cooling, adjusts pH to 7.5 with 1moLNaOH solution, 3mL 11g/L starch are added Then enzyme solutions are digested in 37 DEG C of heat preservation 15h；Enzymolysis liquid is filtered with dry filter paper, and filtrate is taken to be settled to 100mL；
(2) measurement liquid preparation
The extraction filtered fluid of four parts of same volumes is taken, volume 2.00mL distinguishes into first part and second part of extracting solution The riboflavin standard solution that 2.00mL L and 3.00mL concentration is 1 μ g/mL is added；4th part of addition 5mL concentration is 20g/ Riboflavin is reduced to unstressed configuration substance as background blank by 100mL Hydros solution；Third part neither adds standard solution, Also Hydros are not added, for former filtering test solution；Each pipe adds water to 14mL, and 0.4mL ice second is then added into every part of extracting solution Acid-mixed is even, and the liquor potassic permanganate of 0.4mL is then added, and liquor potassic permanganate concentration 25g/L is mixed, and is stood, and is added dropwise 3% Hydrogen peroxide solution acutely shakes sample, overflows extra oxygen, finally plus water is settled to potassium permanganate color fade 26mL；
(3) fluoremetry
Using riboflavin standard solution under sepectrophotofluorometer scanning spectra, according to map obtain maximum excitation wave Long and maximum emission wavelength, respectively 452nm and 530nm, the sample that step (2) is handled well in maximum excitation wavelength and It is measured in parallel three times under maximum emission wavelength, then takes its average value, VB is calculated according to formula (1)2In tablet The mass fraction ω of riboflavin.
Test example
1, the selection of wavelength
No matter how riboflavin concentration of standard solution changes, and launch wavelength and excitation wavelength are all constant.Take configured core Flavine standard solution (1 μ g/mL), scanning spectra can obtain maximum excitation wavelength according to map and maximum emission wavelength is respectively 452nm and 530nm.Therefore, this experiment choose maximum excitation wavelength and maximum emission wavelength be respectively 452nm and 530nm。
2, single factor experiment
2.1 yolk
2.1.1 the determination of hydrochloric acid extraction liquor capacity
Cooked egg yolk 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 uniformly dispersed until particulate matter, then according to embodiment 2 After record method is hydrolyzed, digests, filtering, aoxidizing decontamination, it is settled to 25mL, is measured in parallel three times, is averaged, is tested As a result as shown in Figure 1.
As shown in Figure 1,0.1mol/L hydrochloric acid 50mL, 60mL, 70mL, 80mL, 90mL, the relative fluorescence measured are separately added into Value is not much different.When 50mL, 60mL, 70mL hydrochloric acid is added, relative fluorescence is in rising trend, illustrates with hydrochloric acid volume Increase, riboflavin extracts more；And relative fluorescence is on a declining curve when 70mL, 80mL, 90mL hydrochloric acid is added, show with The increase of hydrochloric acid volume, acidity increase, stability decline, illustrate that riboflavin is only stablized in the solution that acidity is suitable for.Therefore, 0.1mol/L hydrochloric acid 70mL extracting solution, which is added, is more suitable for.
2.1.2 the selection of pH is digested
Under conditions of 0.1mol/L hydrochloric acid is 70mL, digested between pH4~8.Cooked egg yolk is finely ground, Weighing quality is 20.0000g egg yolk in 250mL conical flask, 0.1mol/L hydrochloric acid 70mL is separately added into, according to embodiment 2 Record method is hydrolyzed extraction, and after hydrolyzate is cooling, 1moL/L sodium hydroxide is added dropwise, adjust respectively pH be 4.500, 5.500,6.500,7.500,8.500.Then it after being digested according to 2 record method of embodiment, filter, aoxidize decontamination, puts down Row measurement three times, is averaged, 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 that measures without Significant change, and when pH is equal to 5.500, the relative fluorescence that measures is maximum.Therefore, this experimental selection pH=5.500 is best Experiment condition.
2.1.3 the selection of Hydros liquor capacity
Cooked egg yolk is finely ground, and weighing quality is that 20.0000g egg yolk is placed in 250mL conical flask, then according to After 2 record method of embodiment is hydrolyzed extraction, enzymatic hydrolysis, filters and be settled to 100mL, respectively at the scale with cover of 6 25mL 10mL egg yolk extracting solution is added in test tube, carries out oxidation decontamination according to 2 record method of embodiment, is settled to 25mL, mixes Afterwards, 0.10mL, 0.20mL, 0.30mL, 0.40mL, 0.50mL, 0.60mL 20g/100mL Hydros solution is added, mixes Afterwards, it is measured in parallel three times, is averaged, measurement result is as shown in Figure 3.
Fig. 3 can be seen that when addition 0.10mL, 0.20mL, 0.30mL, 0.40mL, 0.50mL, 0.60mL Hydros When, relative fluorescence is gradually decreased with the increase of Hydros volume, illustrate the increase with Hydros volume, Riboflavin is gradually reduced to unstressed configuration substance in egg yolk；When 0.40mL, 0.50mL, 0.60mL Hydros are added, Relative fluorescence is held essentially constant with the increase of Hydros volume, illustrates that the riboflavin in egg yolk is whole When being reduced to unstressed configuration substance, and 0.40mL Hydros are added, riboflavin has been completely reduced in egg yolk.Cause This, experimental selection Hydros volume is 0.50mL.
2.1.4 the determination of riboflavin standard solution volume
Cooked egg yolk is finely ground, and weighing quality is 20.0000g egg yolk in 250mL conical flask, according to embodiment After extraction, enzymatic hydrolysis, filtering, constant volume is hydrolyzed in 2 record methods, it is added respectively in the scale test tube with cover of 8 25mL 10.00mL egg yolk extracting solution, be separately added into 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 standard solution, are added into No. 1 pipe 0.50mL 20g/100mL Hydros solution, each pipe add water to 16mL, carry out oxidation impurity elimination according to 2 record method of embodiment Matter is settled to 25mL, mixes, and each pipe is measured in parallel three times under the same conditions, is averaged, copies formula (3) to calculate dry The mass fraction of base egg yolk riboflavin, is then compared with national standard method, as a result such as table 1.
1 egg yolk core yellow cellulose content of table, μ g/100g
As shown in Table 1, riboflavin content phase in the butt egg yolk that different volumes riboflavin standard solution measures is added Closely.But mark-on 1.00mL, 2.00mL measure egg yolk core yellow cellulose content and National Standard Method is closest, therefore, this experimental selection mark-on 1.00mL, 2.00mL riboflavin standard solution are optimum experimental condition.
2.2 vitamin B2Tablet
2.2.1 the determination of hydrochloric acid extraction liquor capacity
Weigh the vitamin B that quality is about the finely ground drying of 0.0500g2Tablet is separately added into 250mL conical flask 0.1mol/L hydrochloric acid 50mL, 60mL, 70mL, 80mL and 90mL, stirring is uniformly dispersed until particulate matter, then according to embodiment 3 After record method is hydrolyzed, digests, filtering, aoxidizing decontamination, it is settled to 25mL, is measured in parallel three times, is averaged, is tested As a result as shown in Figure 4.
As shown in Figure 4, it is separately added into 50mL, 60mL, 70mL, 80mL, 90mL hydrochloric acid, the relative fluorescence difference measured is not Greatly.When 50mL, 60mL, 70mL hydrochloric acid is added, relative fluorescence is in rising trend, illustrates the increase with hydrochloric acid volume, core yellow Element extracts more；And relative fluorescence is on a declining curve when 80mL, 90mL hydrochloric acid is added, and shows the increasing with hydrochloric acid volume Add, acidity increases, stability decline, illustrates that riboflavin is only stablized in the solution that acidity is suitable for.Therefore, 0.1mol/L is added Hydrochloric acid 70mL is optimum experimental condition.
2.2.2 the selection of pH is digested
Weigh the vitamin B of the finely ground drying of 0.0500g2Tablet is separately added into 70mL0.1mol/L in 250mL conical flask Hydrochloric acid is hydrolyzed according to 3 record method of embodiment, and after hydrolyzate is cooling, 1moL/L sodium hydroxide is added dropwise, and adjusts pH respectively and is 4.500,5.500,6.500,7.500,8.500.Then it digested according to 3 record method of embodiment, filtered, aoxidize decontamination Afterwards, it is measured in parallel three times, experimental result is as shown in Figure 5.
As shown in Figure 5, when enzymolysis liquid pH is equal to 5.500, the relative fluorescence measured is maximum.Therefore, this experimental selection pH =5.500 be optimum experimental condition.
2.2.3 the determination of Hydros liquor capacity
Weigh the vitamin B of the finely ground drying of 0.0500g2Tablet pours into 250mL conical flask, according to 3 side of record of embodiment After method is hydrolyzed extraction, enzymatic hydrolysis, filters and be settled to 100mL, it is added respectively in the scale test tube with cover of 6 25mL 1.00mL vitamin B2Tablet extracting solution carries out oxidation decontamination according to 3 record method of embodiment, is settled to 25mL, after mixing, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL, 6.00mL 20g/100mL Hydros solution is added, after mixing, It is measured in parallel three times, is averaged, measurement result is as shown in Figure 6.
Fig. 6 can be seen that, when 1.00mL, 2.00mL, 3.00mL, 4.00mL Hydros are added, relative fluorescence with The increase of Hydros volume and gradually decrease, illustrate the increase with Hydros volume, vitamin B2Tablet center Flavine is gradually reduced to unstressed configuration substance；When 4.00mL, 5.00mL, 6.00mL Hydros are added, relative fluorescence It is held essentially constant with the increase of Hydros volume, illustrates vitamin B2Riboflavin in tablet is all gone back It originally was unstressed configuration substance, experimental selection Hydros volume is 5.00mL.
2.2.4 the selection of riboflavin standard solution volume
Weigh the vitamin B of the finely ground drying of 0.0500g2Tablet is in 250mL conical flask, according to 3 record method of embodiment After extraction, enzymatic hydrolysis, filtering, constant volume is hydrolyzed, respectively at addition 1.00mL vitamin in the scale test tube with cover of 10 25mL B2Tablet extracting solution, be separately added into 3,4,5,6,7,8,9, No. 10 pipes 1.00mL, 2.00mL, 3.00mL, 4.00mL, Standard solution is not added in 5.00mL, 6.00mL, 7.00mL, 8.00mL1 μ g/mL riboflavin standard solution, No. 1 pipe and No. 2 pipes, to 5.00mL20g/100mL Hydros solution is added in No. 1 pipe, adds water to 15mL, carries out oxygen according to 3 record method of embodiment Change decontamination, be settled to 25mL, mixes.After mixing, each sample is measured in parallel three times under conditions of identical, is averaged Value, calculates vitamin B according to formula (1)2The mass fraction of tablet riboflavin, is then compared with national standard method, as a result Such as table 2.
2 vitamin B of table2Tablet core yellow cellulose content, g/100g
As shown in Table 2, it is close that riboflavin content in the tablet that different volumes riboflavin standard solution measures is added.But add The result and national standard that mark .1.00mL, 2.00mL are measured are closest, therefore, this experimental selection mark-on 1.00mL, 2.00mL riboflavin Standard solution carries out the measurement of difference mark-on.
The measurement result of 3 samples
At optimum conditions, to 20.0000g egg yolk and 0.0500g vitamin B2Tablet is measured in parallel 6 times respectively, real Test result such as table 3 and table 4.
The measurement of 3 egg yolk core yellow cellulose content of table
The measurement of 4 vitamin B2 tablet core yellow cellulose content of table
It is 402 μ g/100g, vitamin B that riboflavin content in butt egg yolk sample, which can be obtained, by table 3 and table 42Tablet core yellow Cellulose content is 7.9g/100g.Egg yolk and vitamin B2The relative standard deviation of tablet measurement is respectively 0.88% and 1.0%, It is good to measure precision.
4 recovery of standard addition
Weigh 20.0000g egg yolk and 0.0500g vitamin B2Tablet carries out sample according to embodiment 2 and embodiment 3 respectively Product processing, then carries out recovery of standard addition experiment, and experimental result is as follows:
The recovery of standard addition of 5 egg yolk of table
6 vitamin B of table2The recovery of standard addition of tablet
Egg yolk and vitamin B can be obtained by table 5 and table 62The tablet rate of recovery is respectively 92%~104% and 90%~ 96%.
5 detection limits and quantitative limit
Take fluorescent value close to blank, the lower riboflavin standard solution of concentration carries out 11 parallel determinations, calculates Standard deviation, 3 times of standard deviations are detection limit, and 10 times of standard deviations are quantitative limit.It obtains riboflavin detection and is 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 3 sample measuring method of embodiment to egg yolk and VB2Tablet is measured, to same Sample and National Standard Method check analysis, 6 parallel determinations of each progress are averaged report after examining without dubious value, as a result such as table 7.
The blank determination result of table 7 molecular fluorescence mark-on method and National Standard Method
According to the standard deviation and average value of 7 molecular fluorescence difference mark-on method and National Standard Method of table, carries out F and examines and t inspection:
F method of inspection
Egg yolk
Vitamin B2Tablet
Calculate freedom degree f:fGreatly=n-1=5, fIt is small=n-1=5 looks into correlation table and obtains FTable=5.05, calculated F1Value and F2Value is both less than the F value in table.So significant difference is not present in the precision of both methods, the confidence level of conclusion is 95%.Then t method of inspection is carried out.
T method of inspection
Egg yolk
Vitamin B2Tablet
Freedom degree: f=6+6-2=10 is calculated, correlation table is looked into, when confidence level is 95%, tTable=2.23, t1Value and t2Value is all Less than tTableValue.So significant systematic error is not present in the average value of two methods.
It is examined by F and t is examined, the precision and average value of molecular fluorescence difference mark-on method and National Standard Method are all not present aobvious Sex differernce is write, the confidence level of conclusion is 95%.Molecular fluorescence difference mark-on method measurement result is accurate, is suitble to the fast of core yellow cellulose content Speed measurement.
The present invention has studied hydrochloric acid extraction liquid product, enzymatic hydrolysis pH, scalar quantity to egg yolk, vitamin B2Riboflavin in tablet The influence of fluorescent value, experiment have determined egg yolk, vitamin B2Tablet extracting solution is 0.1mol/L hydrochloric acid 70mL, digests Optimal pH 5.500, the 1 best scalar quantities of μ g/mL riboflavin are 1.00mL, 2.00mL.Measurement result: riboflavin content is in butt egg yolk 402 μ g/100g tie up raw B2Riboflavin content is 7.9g/100g in tablet；Egg yolk relative standard deviation is 0.88% (n= 6) raw B, is tieed up2Tablet relative standard deviation is 1.0% (n=6)；Egg yolk recovery of standard addition is 92.0%~104%, vitamin B2Tablet recovery of standard addition is 90.0%~96.0%；Detection is limited to 5.76n g/mL, is quantitatively limited to 1.92 × 10-2μ g/mL。 This method is compared with national standard method, and by F method of inspection and t method of inspection, the precision and average value of two methods are not deposited In significant difference.The test solution that the method for the present invention need to only use two spiked levels different, with four parts of same volume test solutions identical Under the conditions of the luminous signal that measures, be quickly obtained measurement result, without drawing working curve and crossing column decontamination, method is deducted The possible a part interference of background luminous value and concentration difference institute, improves accuracy of measurement and work in sample test solution Efficiency, it is easy, quick, it is the novel analytical technology for being suitble to riboflavin assay in sample, there is stronger novelty.
What has been described above is only a preferred embodiment of the present invention, it is noted that for those skilled in the art, Without depart from that overall concept of the invention, several changes and improvements can also be made, these also should be considered as of the invention Protection scope, this method can also be used to measure the substance of many mulecular luminescences in other samples, including molecular fluorescence, molecular phosphorus The related substances and component that can be measured in the quantitative analyses such as light, chemiluminescence, bioluminescence.

Claims (9)

1. a kind of measure yolk, VB using molecular fluorescence difference mark-on2The method of riboflavin in tablet, it is characterised in that including with Lower step:
(1) prepared by extracting solution: taking sample to grind, weighs mSample, 0.09-0.11moL/L hydrochloric acid, the matter of yolk and hydrochloric acid solution is added Amount volume ratio is 1:2.6-4, unit g/mL, VB2The mass volume ratio of tablet and hydrochloric acid solution is 1:1200-1600, unit For g/mL, pressure hydrolysis is extracted, and pressure is 8.0 × 104-12.0×104Pa, time 20-40min, then with NaOH solution tune PH to 4.5-7.5 is saved, papain solution is added in yolk test solution, papain additional amount is the 0.13- of yolk quality 0.16%, VB2Amylase solution is added in tablet test solution, amylase additional amount is VB2The 50-70% of tablet quality, then exists 37-39 DEG C of heat preservation 15-17h is digested；Enzymolysis liquid filtering, filtrate are settled to VSample 1；
(2) measurement liquid preparation: from VSample 1In take at least four parts of same volume VSample 2Filtering test solution, to first part and second part be added The riboflavin standard solution of different volumes, riboflavin standard solution mass-volume concentration are ρ, second part of standard solution volume VMark 2 Greater than first part VMark 1, riboflavin is reduced to unstressed configuration substance by the 4th part of addition Hydros, and as blank, third part was both Standard solution is not added, Hydros are also not added, for former filtering test solution；Then plus the glacial acetic acid of 3-3.5%V each pipe adds water to V, It mixes, the liquor potassic permanganate of 3-3.5%V is then added, liquor potassic permanganate concentration 25-35g/L is mixed, and is stood, and is added dropwise Hydrogen peroxide solution to potassium permanganate color fade, shaking escapes extra oxygen, constant volume V1；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, maximum excitation is obtained according to map Wavelength and maximum emission wavelength survey the test solution that (1) is handled well under maximum excitation wavelength and maximum emission wavelength Fixed, first part of mark-on test solution relative light unit is IMark 1, second part of mark-on test solution relative light unit is IMark 2, third part sample to be tested Test solution relative light unit is ISample, the 4th part of background blank background relative light unit is I0, yolk or VB2The matter of riboflavin in tablet Measure score ω such as formula (1)；
(1).
2. measuring yolk, VB using molecular fluorescence difference mark-on as described in claim 12The method of riboflavin in tablet, it is special Sign be the following steps are included:
(1) prepared by extracting solution: taking sample to grind, weighs mSample, 0.1moL/L hydrochloric acid, the quality volume of yolk and hydrochloric acid solution is added Than for 1:3.5, unit g/mL, VB2The mass volume ratio of tablet and hydrochloric acid solution is 1:1400, unit g/mL, pressure water Solution is extracted, and pressure is 10.3 × 104Then Pa, time 30min adjust pH to 5.5 with NaOH solution, are added in yolk test solution Papain solution, papain additional amount are 0.15%, VB of yolk quality2It is molten that amylase is added in tablet test solution Liquid, amylase additional amount are VB2Then the 60% of tablet quality is digested in 39 DEG C of heat preservation 16h；Enzymolysis liquid filtering, takes filter Liquid is settled to VSample 1；
(2) measurement liquid preparation: from VSample 1In take at least four parts of same volume VSample 2Filtering test solution, to first part and second part be added The riboflavin standard solution of different volumes, riboflavin standard solution mass-volume concentration are ρ, second part of standard solution volume VMark 2 Greater than first part VMark 1, riboflavin is reduced to unstressed configuration substance as background blank, third part by the 4th part of addition Hydros Neither plus standard solution, Hydros are also not added, for former filtering test solution；Each pipe adds water to V, adds the glacial acetic acid of 3.3%V mixed It is even, the liquor potassic permanganate of 3.3%V is then added, liquor potassic permanganate concentration 30g/L is mixed, and is stood, and hydrogen peroxide is added dropwise Solution is to potassium permanganate color fade, and finally plus water is settled to V1After measure；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, maximum excitation is obtained according to map Wavelength and maximum emission wavelength, the test solution that step (2) is handled well is in maximum excitation wavelength and maximum emission wavelength Lower measurement.
3. measuring yolk, VB using molecular fluorescence difference mark-on as described in claim 12The method of riboflavin in tablet, it is special Sign be the following steps are included:
(1) prepared by extracting solution: taking sample to grind, weighs 18-22g egg yolk or 0.04-0.06gVB20.09- is added in tablet 0.11moL/L hydrochloric acid 60-80mL, pressure hydrolysis are extracted, and pressure is 8 × 104-12×104Pa, time 20-40min, is then used NaOH solution adjusts pH to 4.5-7.5, and 2.5-3.5mL 9-11g/L papain solution, VB is added in yolk test solution2Tablet 2.5-3.5mL 9-11g/L amylase solution is added in test solution, is then digested in 37-39 DEG C of heat preservation 15-17h；Enzymolysis liquid Filtering, takes filtrate to be settled to VSample 1；
(2) measurement liquid preparation: from VSample 1In take at least four parts of same volume VSample 2Filtered fluid, volume 0.5-10mL, to first part The riboflavin standard solution of different volumes is added with second part, riboflavin standard solution mass-volume concentration is ρ, second part of standard Liquor capacity VMark 2For first part of VMark 11.2-2 times；Riboflavin is reduced to unstressed configuration substance and made by the 4th part of addition Hydros For background blank；Third part neither adds standard solution, and Hydros are also not added, for former filtering test solution；Each pipe adds water to 14- Then 16mL 0.4-0.6mL glacial acetic acid is added into every part of filtered fluid and mixes, the potassium permanganate that 0.4-0.6mL is then added is molten Liquid, liquor potassic permanganate concentration 25-35g/L are mixed, and are stood, dropwise addition hydrogen peroxide solution to potassium permanganate color fade, finally Each Guan Jiashui is settled to 24-26mL；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, maximum excitation is obtained according to map Wavelength and maximum emission wavelength, the sample test solution that step (2) is handled well is in maximum excitation wavelength and maximum fluorescence emission It is measured under wavelength.
4. measuring yolk, VB using molecular fluorescence difference mark-on as claimed in claim 32The method of riboflavin in tablet, it is special Sign be the following steps are included:
(1) prepared by extracting solution: taking sample to grind, weighs 20g egg yolk or 0.05gVB20.1moL/L hydrochloric acid 70mL is added in tablet, Pressure hydrolysis is extracted, and pressure is 10.3 × 104Then Pa, time 30min adjust pH to 5.5, yolk test solution with NaOH solution Middle addition 3mL 10g/L papain solution, VB23mL 10g/L amylase solution is added in tablet test solution, then at 39 DEG C Heat preservation 16h is digested；Enzymolysis liquid filtering, takes filtrate to be settled to 100mL；
(2) measurement liquid preparation: the filtering test solution of four parts of same volumes is taken from 100mL filtrate, egg yolk filtrate volume is 10.00mL VB2Tablet filtrate volume be 1.00mL, be separately added into first part and second part of extracting solution 1.00mL with 2.00mL concentration is the riboflavin standard solution of 1 μ g/mL；Riboflavin is reduced to unstressed configuration object by the 4th part of addition Hydros Matter neither adds standard solution, Hydros is also not added as background blank, third part, for former filtering test solution；Each pipe adds water to Then 15mL 0.5mL glacial acetic acid is added into every part of filtered fluid and mixes, the liquor potassic permanganate of 0.5mL is added, potassium permanganate is molten Liquid concentration is 30g/L, is mixed, and is stood, and hydrogen peroxide solution is added dropwise to potassium permanganate color fade, finally plus water is settled to 25mL；
(3) it measures: using riboflavin standard solution scanning spectra under sepectrophotofluorometer, maximum excitation is obtained according to map Wavelength and maximum emission wavelength, the sample test solution that step (2) is handled well is in maximum excitation wavelength and maximum fluorescence emission It is measured under wavelength.
5. according to any one of claims 1-4 measure yolk, VB using molecular fluorescence difference mark-on2Riboflavin in tablet Method, it is characterised in that NaOH solution concentration is 1moL/L in step (1), and hydrogen peroxide mass-volume concentration is in step (2) 3%.
6. measuring yolk, VB using molecular fluorescence difference mark-on as claim 3-4 is described in any item2Riboflavin in tablet Method, it is characterised in that Hydros solution at least 0.5mL, VB are added in yolk test solution2Low sulfurous is added in tablet test solution Acid sodium solution at least 5mL；Hydros solution concentration is 20g/100mL.
7. measuring yolk, VB using molecular fluorescence difference mark-on as claim 3-4 is described in any item2Riboflavin in tablet Method, it is characterised in that the papain and amylase solution are prepared using 2.5moL/L sodium acetate solution.
8. according to any one of claims 1-4 measure yolk, VB using molecular fluorescence difference mark-on2Riboflavin in tablet Method, it is characterised in that riboflavin standard solution is divided into that concentration is 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: standard items riboflavin powdery crystal is placed in very In empty drier, with phosphorus pentoxide drying tube and silica-gel desiccant combined drying for 24 hours after, accurately weigh 50mg, be placed in 2L appearance In measuring bottle, 2.4mL glacial acetic acid and 1.5mL water is added, volumetric flask is placed in 30-40 DEG C of warm water and is shaken, to its dissolution, is cooled to room Temperature adds water to be settled to 2L, moves in brown bottle, and toluene is added to be covered in solution surface, saves in 4 DEG C of refrigerators；Riboflavin standard Use liquid process for preparation are as follows: draw 25 μ g/mL riboflavin standard reserving solution 2.00mL, be placed in 50mL brown volumetric flask, use water It is settled to scale, 4 DEG C of refrigerators save, and the holding time is 1 week.
9. according to any one of claims 1-4 measure yolk, VB using molecular fluorescence difference mark-on2Riboflavin in tablet Method, it is characterised in that yolk and VB2Tablet also uses the measurement of direct drying method progress moisture content.
CN201610611269.2A 2016-07-30 2016-07-30 Yolk, VB are measured using molecular fluorescence difference mark-on2The method of riboflavin in tablet Expired - Fee Related CN106290275B (en)

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