CN103675163B - Preparation method for quinoline yellow standard sample - Google Patents

Abstract

The invention discloses a preparation method for a quinoline yellow standard sample, and belongs to the technical field of analysis and detection. The preparation method comprises the following steps: using an n-butyl alcohol-10% sulfuric acid solution-water two-phase solvent system as a separating solvent system of a counter-current chromatography; adopting a high-speed counter-current chromatography to perform separation and purification on crude quinoline yellow; dissolving separated target components with water, repeatedly extracting the target components dissolved in water with n-butyl alcohol, combining n-butyl alcohol layers, and performing reduced-pressure evaporation and drying; dissolving obtained product with water, then collecting yellow parts through gel column chromatography separation, performing reduced-pressure concentration to obtain target components, and drying the obtained target components at indoor temperature, so as to obtain the quinoline yellow standard sample. The quinoline yellow standard sample prepared through the method is good in uniformity, precise in quantity value, and good in stability, can be used for the alignment on analytical instruments, the evaluation on detection methods and the measurement on quinoline yellow content.

Description

The preparation method of quinoline yellow standard model

Technical field

The present invention relates to technical field of analysis and detection, especially a kind of preparation method of quinoline yellow standard model.

Background technology

Quinoline yellow (Quinoline Yellow): No. CAS: 8004-92-0, molecular formula: C ₁₈h ₉nO ₈na ₂s ₂, molecular weight: 477.38, be the synthetic edible xanthein (colorant) of water-soluble azo class, be usually used in the painted of the food such as ice cream, fruit, cake, chocolate, bread, cheese spread, soft drink in Britain.Because this pigment may cause attention deficient disorder, Japan, the U.S. and Norway forbid in food, and China only allows to add in pre-rectification.The structural formula of quinoline yellow is as follows:

Quinoline yellow, mainly taking aromatic hydrocarbons chemical products as raw material, forms through a series of organic reaction chemical combination such as oversulfonate, azo, and with not only, without nutritive value, and its chemical component material itself is harmful.The impurity simultaneously producing in building-up process all has toxicity in various degree as arsenic, mercury, phenol, aniline, lead, cadmium, ether, chloride, sulfate etc.Synthetic coloring matter quinoline yellow is understood removing toxic substances material in a large amount of consumer after entering human body, disturbs human homergy's function direct effect target organ, and its toxicity main manifestations is intolerance, carcinogenicity, attention deficient disorder.Based on this, requirement to food security improves day by day, the addition of quinoline yellow is detected and more and more come into one's own, for the standard model of its detection preparation also in critical role, therefore, need badly and prepare a kind of quinoline yellow standard model, its composition is relatively stable over a period to come, for the detection to containing quinoline yellow product.

Summary of the invention

The object of the present invention is to provide a kind of preparation method of quinoline yellow standard model.Quinoline yellow standard model good uniformity prepared by the method, value accurately, good stability, can be for the calibration to analytical instrument, the evaluation of detection method and the mensuration to quinoline yellow content.

The technical solution adopted in the present invention is:

The preparation method of quinoline yellow standard model, is characterized in that, comprises the following steps:

S1: the high speed adverse current chromatogram separation and purification of quinoline yellow

The separation dicyandiamide solution of high speed adverse current chromatogram: normal butyl alcohol-10% sulfuric acid solution-water two phase solvent system;

1. prepare quinoline yellow study;

2. prepare the two phase solvent system of above-mentioned normal butyl alcohol-10% sulfuric acid solution-water, after shake well, leave standstill 10-14h, layering, wherein, upper as fixing phase, lower to mobile phase;

3. quinoline yellow study is dissolved in two phase solvent system, is mixed with sample solution;

4. will fix and pump into mutually separating column, be full of mutually after pipeline until fixing, open main frame speed control, make high-speed counter-current chromatograph spiral tube according to clockwise direction rotation, in the time that rotating speed reaches 700-900r/min, pump into mobile phase, and by six-way injection valve sample introduction, in the time that mobile phase starts to flow out chromatographic column, collect the each peak efflux after high speed adverse current chromatogram separates, then the solution of collection is detected by high performance liquid chromatography, determine target component, detection wavelength is 254nm; From high speed adverse current chromatogram, can obtain the information that maximum components separate with time polycomponent, then on high performance liquid chromatography, analyze judgement, determine target component.

S2: quinoline yellow sample depickling

Get target component definite in step S1, after water dissolves, be placed in separating funnel, then use isopyknic saturated extracting n-butyl alcohol three times, merge n-butanol layer evaporated under reduced pressure, then separate by gel column chromatography (sephadexLH-20) afterwards by the water-soluble solution of 10mL, collect yl moiety, reduced pressure concentration obtains target component, and drying at room temperature obtains sterling quinoline yellow; Be quinoline yellow standard items.

S3: packing and storage

To make quinoline yellow standard items, and carry out packing with brown bottle, the sealing of drying at room temperature lucifuge is preserved.

Preferably, the volume ratio of described normal butyl alcohol, 10% sulfuric acid solution, water is 5:1:4.

Preferably, described step 4. medium speed be 800r/min.

Preferably, described step 4. in the fixing speed that pumps into mutually separating column be 20mL/min; The speed that pumps into of mobile phase is 2.0mL/min.

Preferably, described step, 3. for quinoline yellow study is dissolved in two phase solvent system, is mixed with the sample solution that concentration is 25mg/mL.

Preferred technical scheme of the present invention is:

S1: the high speed adverse current chromatogram separation and purification of quinoline yellow

The separation dicyandiamide solution of high speed adverse current chromatogram: normal butyl alcohol-10% sulfuric acid solution-water two phase solvent system;

1. prepare quinoline yellow study;

2. prepare the two phase solvent system of above-mentioned normal butyl alcohol-10% sulfuric acid solution-water, after shake well, leave standstill 10-14h, layering, wherein, upper as fixing phase, lower to mobile phase, the volume ratio of normal butyl alcohol, 10% sulfuric acid solution, water is 5:1:4;

3. just study 200mg is dissolved in the mixed solvent of phase under the upper phase of 4mL and 4mL, is mixed with sample solution;

4. pump into separating column by fixing with the speed of 20mL/min, be full of mutually after pipeline until fixing, open main frame speed control, make high-speed counter-current chromatograph spiral tube according to clockwise direction rotation, in the time that rotating speed reaches 800r/min, speed with 2.0mL/min pumps into mobile phase, and by six-way injection valve sample introduction, in the time that mobile phase starts to flow out chromatographic column, collect the each peak efflux after high speed adverse current chromatogram separates, then the solution of collection is detected by high performance liquid chromatography, determine target component;

S2: quinoline yellow sample depickling

Get target component definite in step S1, after water dissolves, be placed in separating funnel, then use isopyknic saturated extracting n-butyl alcohol three times, merge n-butanol layer evaporated under reduced pressure, then separate by gel column chromatography (sephadexLH-20) afterwards by the water-soluble solution of 10mL, collect yl moiety, reduced pressure concentration obtains target component, and drying at room temperature obtains sterling quinoline yellow, is quinoline yellow standard items;

Collect the target component after high speed adverse current chromatogram separation and purification, after water dissolves, be placed in separating funnel, then repeatedly extract three times with isopyknic saturated normal butyl alcohol, merge n-butanol layer evaporated under reduced pressure, then separate by gel column chromatography (sephadexLH-20) afterwards by the water-soluble solution of 10mL, collect yl moiety, reduced pressure concentration obtains target component, and drying at room temperature must be quinoline yellow standard items;

S3: packing and storage

To make quinoline yellow standard items, and carry out packing with brown bottle, totally 100 bottles of every bottle of 20mg, preserve in the sealing of drying at room temperature lucifuge after numbering.

Beneficial effect of the present invention: quinoline yellow standard model purity prepared by preparation method of the present invention is high, good uniformity, value accurately, good stability, can be for the calibration to analytical instrument, the evaluation of detection method and the mensuration to quinoline yellow content.

Brief description of the drawings

The liquid chromatogram of accompanying drawing 1 quinoline yellow standard model;

The ultra-violet absorption spectrum of accompanying drawing 2 quinoline yellows;

The infrared absorption spectrum of accompanying drawing 3 quinoline yellows;

The proton nmr spectra (HNMR) of accompanying drawing 4 quinoline yellows;

The carbon-13 nmr spectra (CNMR) of accompanying drawing 5 quinoline yellows;

The mass spectrogram of accompanying drawing 6 quinoline yellows;

Accompanying drawing 7 homogeneity trend maps;

Accompanying drawing 8 stability test test findings scatter diagrams.

Description of reference numerals: in accompanying drawing 1, A is the high-efficient liquid phase chromatogram of quinoline yellow before purifying; B is the high-efficient liquid phase chromatogram of quinoline yellow after purifying.

Embodiment

In order to understand better the present invention, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention is not only confined to the following examples, embodiment should not regard limiting the scope of the present invention as.

Embodiment 1

1 sample preparation

1.1 instrument and equipment

High-speed counter-current chromatograph: TBE-300A(Shanghai Tongtian Biotechnology Co., Ltd.), in instrument, carry chromatographic column; R-3 Rotary Evaporators (Bu Qi experimental apparatus company of Switzerland), Agilent1260 liquid chromatograph is joined diode array detector (Agilent company of the U.S.); TSQquantum ultra liquid chromatography-tandem mass spectrometry instrument (Thermo company of the U.S.); Bruker AV400 nuclear magnetic resonance analyser (German Bruker company); Sensibility reciprocal is the analytical balance (Mei Tele company of Switzerland) of 0.1mg; Milli-Q water purifior (Millipore company of the U.S.); IKA-KS130 type oscillator (German IK company); KUDOS ultrasonic cleaner (domestic); SephadexLH-20 gel column (sephadexLH-20 filler is contained in a glass column, sephadex LH-20 filler purchased from Beijing intelligent easy science and technology limited Company).

1.2 materials and reagent

Methyl alcohol, acetonitrile (chromatographically pure, Merck & Co., Inc.); Quinoline yellow is purchased from Shanghai chemical industry three factories.Ethyl acetate, normal butyl alcohol, methyl alcohol, chloroform, sulfuric acid (analyze pure, Chemical Reagent Co., Ltd., Sinopharm Group), water is the deionized water that Milli-Q water purifior generates.Other reagent, mention as non-be analyze pure.Silica gel 400 orders.

1.3 experimental techniques and step

1.3.1 the separation and purification of high speed adverse current chromatogram

Separation dicyandiamide solution using the two phase solvent system of normal butyl alcohol-10% sulfuric acid solution-water as adverse current chromatogram, the two phase solvent system of the above-mentioned normal butyl alcohol-10% sulfuric acid solution-water of preparation 1500mL, the volume ratio of normal butyl alcohol, 10% sulfuric acid solution, water is 5:1:4.After shake well, leave standstill 12h, layering, gets as fixing phase, lower to mobile phase, study 200mg is dissolved in the mixed solvent of phase under the upper phase of 4mL and 4mL, first pump into separating column by fixing with the speed of 20mL/min, be full of mutually after pipeline until fixing, open main frame speed control, make high-speed counter-current chromatograph spiral tube according to clockwise direction rotation, in the time that rotating speed reaches 800r/min, speed with 2.0mL/min pumps into mobile phase, and by six-way injection valve sample introduction, in the time that mobile phase starts to flow out chromatographic column, collect the each peak efflux after high speed adverse current chromatogram separates, then the solution of collection is detected by high performance liquid chromatography, detection wavelength is 254nm.

1.3.2 quinoline yellow sample depickling

Collect the target component after high speed adverse current chromatogram separation and purification, water is placed in separating funnel after dissolving, and then repeatedly extracts three times with isopyknic saturated normal butyl alcohol, merges n-butanol layer evaporated under reduced pressure.Then separate by gel column chromatography (sephadexLH-20) afterwards by the water-soluble solution of 10mL, collect yl moiety, reduced pressure concentration obtains target component, and drying at room temperature obtains quinoline yellow standard items.

1.4 packing and storage

To make quinoline yellow standard items, and carry out packing with brown bottle, totally 100 bottles of every bottle of 20mg, preserve in the sealing of drying at room temperature lucifuge after numbering.

2 purity tests and assay

In the development of quinoline yellow standard model, select HPLC peak area normalization method to measure.

2.1 instruments and condition

High performance liquid chromatograph (Agilent 1260), joins VWD and DAD; Chromatographic column: Venusil XBPC18 (L) chromatographic column (4.6mm × 150mm, 5 μ m, agela Technologies); Or suitable person.Mobile phase and condition of gradient elution are in table 2.1; Flow velocity: 1.0mL/min; Column temperature: room temperature; Sample size: 10 μ L, detect wavelength: 400nm.

Aforesaid operations parameter is typical, can, according to different instrument traits, make the appropriate adjustments to obtain optimum efficiency to given operating conditions.

The quinoline yellow standard model of preparing in above embodiment is carried out to purity test and assay, specifically adopt HPLC peak area normalization method to measure.

Determining instrument and condition: high performance liquid chromatograph (Agilent 1260), join VWD and DAD; Chromatographic column: Venusil XBP C18 (L) chromatographic column (4.6mm × 150mm, 5 μ m, agela Technologies); Or suitable person.Mobile phase and condition of gradient elution are in table 2.1; Flow velocity: 1.0mL/min; Column temperature: room temperature; Sample size: 10 μ L, detect wavelength: 400nm.Aforesaid operations parameter is typical, can, according to different instrument traits, make the appropriate adjustments to obtain optimum efficiency to given operating conditions.

2.2 quantitative measurement

Accurately take a certain amount of sample, with after methyl alcohol dilution constant volume, being mixed with concentration is the quinoline yellow sample liquid of 0.02mg/mL, under above-mentioned liquid phase chromatography condition, measures, and the retention time of quinoline yellow is about 5.2min and 5.9min.

3 structural confirmations

Adopt ultraviolet spectroscopy to carry out structural confirmation to above-mentioned sample.

Analytical instrument: Shimadzu UV-3100UV-VIS-NIR spectrophotometric determination for ultraviolet-visible spectrum.

Analysis condition: by ultrapure water compound concentration 0.02mg/ml quinoline yellow solution for the quinoline yellow standard model of preparing in above-described embodiment; Measure wavelength coverage 190～800nm.

The quinoline yellow solution uv-vis spectra preparing is carried out to full wavelength scanner, can find out from accompanying drawing 2, quinoline yellow has characteristic absorption at wavelength 285nm and 400nm place, and wherein 400nm absorption value is the strongest, and therefore, 400nm is selected in the analysis of liquid chromatography definite value.

3, Structural Identification

3.1 instrument and equipment

TSQ quantum ultra high efficiency liquid phase-GC-MS (Thermo company of the U.S.)

Vertex70 infrared spectrometer (German Brooker company)

Bruker AV400 nuclear magnetic resonance analyser (German Bruker company)

3.2 interpretation of result

Adopt infrared spectrum (IR) (accompanying drawing 3), mass spectrum (MS) (accompanying drawing 6), nuclear magnetic resonance (NMR) (accompanying drawing 4,5) to carry out structural confirmation to quinoline yellow, from spectrogram, extract characteristic and be shown in Table 1.

The characteristic of table 1 quinoline yellow

The above results shows that molecular weight, IR and the NMR data of compound are all identical with the architectural feature of quinoline yellow, proves that this compound is really quinoline yellow.

4 uniformity testings

Choosing of 4.1 detection methods

Technical basis: GB/T15000.3-2008 the 7th chapter

Assay method: the purity of HPLC area normalization method bioassay standard sample.

4.2 detect the acquisition of data

Adopt random sequence duplicate measurements method, in the sample from packing, randomly draw 10 bottles of samples number respectively 1-10, take respectively 3 parts, 1.0mg sample by three kinds of programs from every bottle, every duplicate samples is used respectively 10.0ml deionized water dissolving, carries out HPLC analysis, calculates its purity.Employing method of analysis of variance is tested, and its homogeneity is judged

For the first time: 1-3-5-7-9-2-4-6-8-10;

For the second time: 10-9-8-7-6-5-4-3-2-1;

For the third time: 2-4-6-8-10-1-3-5-7-9.

Data recording sees the following form 2.

The measurement data of table 2 uniformity testing

4.3 data processing

4.3.1 the trend analysis of quinoline yellow sample

Using the mean value of every bottle of content as the function of bottle sequence number being come to trend in study sample preparation and the logical relation of point process of assembling.Result shows: trend stability in quinoline yellow sample preparation; Packing homogeneous, data stabilization.As shown in Figure 7.

Above-mentioned data are carried out to variance analysis and F inspection, the results are shown in 3, table 4.

Mean value, variance and measurement number of times that table 3 is every bottle

Bottle number	Mean value %	Variance	Measure number of times
				1	98.95	0.0208	3
2	98.90	0.003333	3
				3	99.01	0.000133	3
4	98.94	0.0127	3
				5	98.94	0.003333	3
6	98.95	0.000133	3
				7	98.95	0.004433	3
8	98.92	0.002533	3
				9	98.92	0.000833	3
10	98.79	0.0084	3

The analysis of variance table of table 4 homogeneity research

Variation source	SS	Degree of freedom	MS
				Between bottle	0.0862	9	0.009578
In bottle	0.113267	20	0.005663
				Summation	0.199467	29	?

According to data in table, with υ ₁(between group)=9 and υ ₂f dividing value table is looked in (in group)=20, obtains F _0.05(9,20)=2.94, due to F=MS _between/ MS _in=1.69 < F _0.05(9,20), illustrate that quinoline yellow sample is uniform.

Between bottle, variance is calculated with following formula: ${s^2}_A=\frac{{\mathrm{MS}}_{\mathrm{among}}-{\mathrm{MS}}_{\mathrm{within}}}{n_0}=\frac{0.009578-0.005663}{3}=1.3\&times;{10}^{-3}$

Between bottle, standard deviation is the square root of this variance:

Repeatability standard deviation can be by MS _withincalculate: $s_r=\sqrt{{\mathrm{MS}}_{\mathrm{within}}}=\sqrt{0.005663}=0.08$

5 stability tests

After sample packing, the at room temperature dry airtight preservation of lucifuge.This research scheduled to last with 24 months, and the quinoline yellow standard model of preparation is got to the inspection of sample for every 6 months, and every part of sample is to measure the mean value of 5 times as its measurement result, and test findings sees the following form 5.

Table 5 stability test test findings

From upper table data, the mean value at every turn recording does not raise over time and significantly or reduces in minute, by calculating, measured value should and all in 98.96% ± 0.18%, illustrate that this sample is stable in two years.

Check data carried out to statistical study with t:

Do scatter diagram by quinoline yellow stability test data, as shown in Figure 8, observe dependent variable (purity) and whether there is linear relationship with independent variable (month).Whether can find out from scatter diagram, there is obvious linear relationship in Reinheitszahl and its time of 0-24 month, therefore adopt straight line as empirical model, had significant change the stability of standard model is predicted by observation slope value.Mathematics is processed as follows:

Slope can calculate with following formula:

$b_1=\frac{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}(X_i-\stackrel{\&OverBar;}{X})(Y_i-\stackrel{\&OverBar;}{Y})}{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{(X_i-\stackrel{\&OverBar;}{X})}^2}=\frac{-1.08}{360}=-0.003$

In formula: $\begin{array}{ccc}\stackrel{\&OverBar;}{Y}=98.96& 98.96& \stackrel{\&OverBar;}{X}=12\end{array}$

Intercept: $b_0=\stackrel{\&OverBar;}{Y}-b_1\stackrel{\&OverBar;}{X}=98.96+(0.003\&times;12)=98.99$

The standard deviation of the point on straight line: $s^2=\frac{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{(y_i-b_0-b_1{X}_i)}^2}{n-2}=0.0079$

Get its square root s=0.09, the uncertainty relevant to slope calculated with following formula:

$s\left(b_1\right)=\frac{s}{\sqrt{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}(X_i-\stackrel{\&OverBar;}{X})}}=\frac{0.09}{\sqrt{360}}=0.005$

Degree of freedom is n-2 and p=0.95(95% confidence level) the distribution t factor equal 3.182

t _0.95,n-2＝3.182

Due to:

|b ₁|＜t _0.95,n-2.s(b ₁)＝3.182×0.005＝0.02

That is: ︱ b1 ︱ <0.02

Result of calculation shows that slope variation is not remarkable, thereby quinoline yellow standard model does not observe obvious instability in two years.

Stability test uncertainty calculation:

S _surelyfor the standard deviation of five time point determining data mean values of stability test, S _surely=0.016

$\mathrm{Sr}=\sqrt{\frac{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{S_i}^2}{n}}=0.06(n=5)$

Expanded uncertainty wherein t _{0.05, n-1}=2.776

The definite value of 6 standard models

6.1 valued methods

Adopt HPLC to measure, application peak area normalization method calculated purity value.

Instrument and condition: Venusil XBP C18 (L) chromatographic column (4.6mm × 150mm, 5 μ m, agela Technologies) mobile phase and gradient be in table 2.1: flow velocity: 1.0mL/min; Column temperature: 25 DEG C; Sample size: 10 μ L; Detect wavelength 400nm.

Aforesaid operations parameter is typical, can, according to different instrument traits, make the appropriate adjustments to obtain optimum efficiency to given operating conditions.

Choosing and cooperation plan of 6.2 definite value laboratories

Choosing and cooperation plan of definite value laboratory: this standard sample, according to GB/T15000.3-2008 standard-required, adopts the plan of multiple laboratory cooperation definite value, selects to obtain country or department laboratory accreditation, that possess qualification and carries out sample definite value.8 definite value laboratories are respectively standard model research institute of Shenyang Product Quality Supervision and Inspection Institute, Yantai Entry-Exit Inspection and Quarantine Bureau, Chemistry and Chemical Engineering College of Shandong University, Shandong Forecasting and Analysis Center, Jinan Entry-Exit Inspection and Quarantine Bureau, Shangdong Entry-Exit Inspection And Quarantine Bureau technique center, quality inspection institute of Jinan City food Supervision Test Center, Weifang Entry-Exit Inspection and Quarantine Bureau.

The establishment definite value job instruction that cooperates between laboratory, divides feeding sample, summarized results within two weeks in November, 2013 to cooperation definite value laboratory.Each sample is randomly drawed 48 bottles of samples, and 6 bottles, each laboratory adopts above-mentioned HPLC method to measure, and application area normalization method carries out definite value.

Statistics, processing and the calculating of 7 data

7.1 data statistics processing method overviews

(1) fixed value determining data are gathered by laboratory and assay method, reject technically after the dubious value that ascertains the reason, statistically reject dubious value with Grubbs (Grubbs) method of inspection.Can require if desired former laboratory to recheck dubious value.When data are relatively disperseed or when dubious value is many, should conscientiously check each laboratory assay method used, condition determination and operating process, until ascertain the reason.

(2) total data is carried out to normality investigation, under the condition of Normal Distribution or similar normal state distribution, each laboratory, every kind of measured data mean value of assay method are considered as to unitary determination value, form one group of new determination data, calculate its mean value and standard deviation.

(3) in the time assert the accuracy of determination data in certain or certain several laboratories and precision higher than other several laboratory, the method for precision such as also can adopt not, by weighted mean value and respective standard deviation as standard value and standard deviation.

(4) expression of definite value result

The numerical value revision of the convention should be undertaken by the regulation of GB8170-1987, and standard deviation carries out the revision of the convention by the principle of only entering not give up, and definite value result is represented by standard value and uncertainty.

Sample to dispensing carries out statistics, processing and the calculating of data, specific as follows:

The statistical study of 7.2 data

Following table 6 is the definite value result summary sheet in 8 laboratories.To collected measurement result, adopt Grubbs (Grubbs) method of inspection to test, measurement result is normal distribution.According to GB/T15000.3-2008 standard regulation, definite value result is made up of standard value and uncertainty.The uncertainty of the standard value that standard model characteristic certified value's uncertainty of measurement U is obtained by mensuration the uncertainty U of uniformity testing _bbuncertainty U with stability test _stscomposition.According to whole measurement results, calculate standard value and the uncertainty of quinoline yellow standard model, specifically see the following form shown in 7.

Table 6 definite value result summary sheet

Table 7 definite value result statistical analysis table

7.3 definite value expression of results

By above-mentioned statistical data processing: the definite value result of quinoline yellow standard model is:

Standard value: 98.97%

Degree of confidence is 95% expansion uncertainty value: 0.56%

Laboratory internal standard deviation S _r: 0.20%

Standard deviation S between laboratory _l: 0.36%.

8 labels, mark, packaging

Label substance: title, lot number, molecular formula, purity, weight, the term of validity, storage requirement, production unit title etc.

Mark: the airtight preservation of drying at room temperature lucifuge; There is the distinctive mark of " temperature extremes ", " Keep away from rain is afraid of tide ", " Keep away from radioactive "; There is " this product is chemicals, and non-edible product, are not taken orally " security warning.Without indicative mark.Without Warning Mark (being dangerous mark).

Packaging: net weight 20mg specification, inner packing is Brown Glass Brown glass bottles and jars only, overcoat carton.

Claims (5)

1. the preparation method of quinoline yellow standard model, is characterized in that, comprises the following steps:

S1: the high speed adverse current chromatogram separation and purification of quinoline yellow

The separation dicyandiamide solution of high speed adverse current chromatogram: normal butyl alcohol-10% sulfuric acid solution-water two phase solvent system;

1. prepare quinoline yellow study;

2. prepare the two phase solvent system of above-mentioned normal butyl alcohol-10% sulfuric acid solution-water, after shake well, leave standstill 10-14h, layering, wherein, upper as fixing phase, lower to mobile phase;

The volume ratio of described normal butyl alcohol, 10% sulfuric acid solution, water is 5:1:4;

3. quinoline yellow study is dissolved in two phase solvent system, is mixed with sample solution;

4. will fix and pump into mutually separating column, be full of mutually after pipeline until fixing, open main frame speed control, make high-speed counter-current chromatograph spiral tube according to clockwise direction rotation, in the time that rotating speed reaches 700-900r/min, pump into mobile phase, and by six-way injection valve sample introduction, in the time that mobile phase starts to flow out chromatographic column, collect the each peak efflux after high speed adverse current chromatogram separates, then the solution of collection is detected by high performance liquid chromatography, determine target component;

S2: quinoline yellow sample depickling

Get target component definite in step S1, after water dissolves, be placed in separating funnel, then use isopyknic saturated extracting n-butyl alcohol three times, merge n-butanol layer evaporated under reduced pressure, then separate by sephadexLH-20 gel column chromatography afterwards by the water-soluble solution of 10mL, collect yl moiety, reduced pressure concentration obtains target component, and drying at room temperature obtains sterling quinoline yellow, is quinoline yellow standard items;

S3: packing and storage

To make quinoline yellow standard items brown bottle and carry out packing, the sealing of drying at room temperature lucifuge is preserved.

2. the preparation method of quinoline yellow standard model according to claim 1, is characterized in that, described step 4. medium speed is 800r/min.

3. the preparation method of quinoline yellow standard model according to claim 1, is characterized in that, described step 4. in the fixing speed that pumps into mutually separating column be 20mL/min; The speed that pumps into of mobile phase is 2.0mL/min.

4. the preparation method of quinoline yellow standard model according to claim 1, is characterized in that, described step, 3. for quinoline yellow study is dissolved in two phase solvent system, is mixed with the sample solution that concentration is 25mg/mL.

5. a preparation method for quinoline yellow standard model, is characterized in that, comprises the following steps:

S1: the high speed adverse current chromatogram separation and purification of quinoline yellow

The separation dicyandiamide solution of high speed adverse current chromatogram: normal butyl alcohol-10% sulfuric acid solution-water two phase solvent system;

1. prepare quinoline yellow study;

2. prepare the two phase solvent system of above-mentioned normal butyl alcohol-10% sulfuric acid solution-water, after shake well, leave standstill 10-14h, layering, wherein, upper as fixing phase, lower to mobile phase, the volume ratio of normal butyl alcohol, 10% sulfuric acid solution, water is 5:1:4;

3. study 200mg is dissolved in the mixed solvent of phase under the upper phase of 4mL and 4mL, is mixed with sample solution;

4. pump into separating column by fixing with the speed of 20mL/min, be full of mutually after pipeline until fixing, open main frame speed control, make high-speed counter-current chromatograph spiral tube according to clockwise direction rotation, in the time that rotating speed reaches 800r/min, speed with 2.0mL/min pumps into mobile phase, and by six-way injection valve sample introduction, in the time that mobile phase starts to flow out chromatographic column, collect the each peak efflux after high speed adverse current chromatogram separates, then the solution of collection is detected by high performance liquid chromatography, determine target component;

S2: quinoline yellow sample depickling

Get target component definite in step S1, after water dissolves, be placed in separating funnel, then use isopyknic saturated extracting n-butyl alcohol three times, merge n-butanol layer evaporated under reduced pressure, then separate by sephadexLH-20 gel column chromatography afterwards by the water-soluble solution of 10mL, collect yl moiety, reduced pressure concentration obtains target component, and drying at room temperature obtains sterling quinoline yellow, is quinoline yellow standard items;

S3: packing and storage

To make quinoline yellow standard items, and carry out packing with brown bottle, totally 100 bottles of every bottle of 20mg, preserve in the sealing of drying at room temperature lucifuge after numbering.