NY/T 727-2003 Determination of furazolidone in feed by high performance liquid chromatography
Some standard content:
NY/T727--2003
This standard is modified to adopt ISO14797:1999 (E) "Determination of furazolidone content in feed by high performance liquid chromatography" (English version). This standard is redrafted based on ISO14797:1999 (E). Taking into account my country's national conditions and laboratory equipment conditions, this standard has made some modifications when adopting ISO14797:1999 (E). The relevant technical differences and editorial changes have been incorporated into the text and marked with a vertical single line in the margins of the clauses they involve. A list of these technical differences and editorial changes is given in Appendix B for reference. Appendices A and B of this standard are both informative appendices. This standard is proposed by the Ministry of Agriculture of the People's Republic of China. This standard is under the jurisdiction of the National Feed Standardization Technical Committee. The responsible drafting unit of this standard is the National Feed Product Quality Supervision and Inspection Center (Beijing), and the participating drafting unit is the Feed Industry Center of the Ministry of Agriculture.
Main drafters of this standard: Yan Huiwen, Yang Shuming, Zhao Xiaoyang, Wang Tong, Yang Wenjun. 1 Scope
Determination of furazolidone in feed
High performance liquid chromatography
NY/T 727-2003
This standard specifies the method for the determination of furazolidone in compound feed, premixed feed and concentrated feed by high performance liquid chromatography (HPLC). This standard can be used for compound feed containing 10mg/kg~5000mg/kg furazolidone and premixed feed and concentrated feed with a content of 0.5%~20%.
2 Normative references
The clauses in the following documents become clauses of this standard through reference in this standard. For any dated referenced document, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, the latest version shall apply to this standard. GB/T6682 Specifications and test methods for water used in analytical laboratories GB/T14699.1 Feed sampling methods
3 Principle
Moisten the compound feed with a small amount of water, and extract the oxadiazine with a mixture of acetonitrile and methanol. For premixed feed and concentrated feed, directly extract the furazolidone with a mixture of acetonitrile and methanol. The extract is purified by a short alumina column, diluted with diluent, separated on a reversed-phase HPLC, and measured at 365 nm by a UV detector. 4 Reagents and materials
Unless otherwise specified, only reagents that are confirmed to be of premium grade or chromatographic grade are used in the analysis. 4.1 Water: in accordance with the provisions of GB/T6682 for grade water 4.2 Extractant: acetonitrile: methanol - 1:1. Mix thoroughly and bring to room temperature before use. 4.3 Diluent Mix 350 mL of extractant (4.2) with 650 mL of water (4.1). 4.4 10% acetic acid solution: dilute 10 mL of glacial acetic acid with water to 100 mL 4.5 sodium acetate buffer c(CH,COaNa) = 0.01 rmol/L. pH = 6.0. Dissolve 0.82 g of sodium acetate in about 700 mL of water, adjust the pH to 6.0 with Zn solution (4.4), dilute to 1000 mL with water, and mix.
4.6 HPLC mobile phase: take 800 mL of sodium acetate buffer (4.5) and 200 mL of acetonitrile, mix well, filter through a 0.2um filter membrane, and degas for 10 min by ultrasonication before use.
4.7 Furazolidone standard: N-(5-nitro-2-furylmethylidene)-3-nitro-2-oxazolidinone. Note: Since furazolidone is very sensitive to light, all operations should be performed under light-proof conditions. During operation, inhalation and contact with toxic furazolidone standards and solutions should be avoided. The solution should be prepared in a fume hood. Wear glasses and work clothes for protection. 4.8 Furazolidone solution (about 250 mg/ml): weigh 25 mg ± 1 mg furazolidone standard (4.7) to an accuracy of 0.1 mg, dissolve with the extract (4.2), dilute to 100 ml, mix, and store in a refrigerator at 0℃~8℃. The purity of the standard should be taken into account when calculating the solution concentration. The solution is valid for months.
4.9 Furazolidone working solution (about 5 g/mL and 12.5 g/mL): Accurately pipette 2.0 mL and 5.0 mL of stock solution (4.8) into 100 mL volumetric flasks, add 65 mL of water, dilute to scale with extract (4.2) and mix well. Fresh working solution should be prepared for each batch of samples.
4.10 Neutral alumina, activity 1 (for fully deactivated alumina, there can be 0% to 1% water). The treatment method is as follows: Place neutral alumina (100 mesh to 200 mesh) in a Muffle furnace, burn at 550℃ for 3 hours, transfer to a desiccator to cool, and store in a sealed container.
5 Instruments and equipment
Common laboratory instruments and equipment, especially the following instruments and equipment: 5.1 pH meter (with temperature compensation, accurate to 0.01). 5.2 Solvent filtration system, all-glass filter, pore size 0.2μm. 5.3 Ultrasonic water bath.
5.4 Oscillator, horizontal oscillation, frequency 250r/min～300r/min. 5.5 Filter device, use medium-speed filter paper.
5.6 Glass fiber.
5.7 Glass chromatography column, 30cm long, 10mm inner diameter, narrowed at the end to accommodate glass wool. Filter system, capable of accommodating polyvinyl difluoride (PVDF) or polytetrafluoroethylene (PTFE) filter membrane with a pore size of 0.2μm. 5.8
5.9 HPLC system, consisting of the following components: 5.9.1 Pump, pulse-free, capable of maintaining a flow rate of 0.1mL/min～2.0mL/min. 5.9.2 Injection system, injection loop volume 20μL~～50μI. 5.9.3 UV detector, suitable for determination at a wavelength of 365nm. If possible, a diode array detector can be used, which can also be used for confirmation of furazolidone.
5.9.4 Recorder.
5.9.5 Guard column: 20 mm long, 3.9 mm inner diameter, filled with Ci8 bonded silica gel with a particle size of 37 μm to 100 μm or other guard columns of equivalent quality.
5.9.6 Analytical column: 200 mm long, 3.0 mm inner diameter, filled with C18 bonded silica gel with a particle size of 5 μm or other analytical columns with equivalent performance.
The performance of the analytical column should ensure the following conditions: The capacity factor K' of furazolidone ≥ 1.
Note: The calculation of capacity factor is shown in formula (1)
K'=R-1
Wherein:bzxz.net
K'--capacity factor;
tr--retention time of furazolidone, in minutes (min); t--retention time of unretained components, in minutes (min). 5.10 Microsyringe.
6 Sample preparation
Collect laboratory samples according to GB/T14699.1. Grind the laboratory sample (usually 500g) so that it all passes through a 1mm sieve and mix thoroughly. Store in a ground-mouth bottle for later use.
7 Analysis steps
7.1 General requirements
Each measurement should be carried out in parallel with the blank sample and blank spiked sample, and if possible, the reference sample should also be measured. Note: Blank sample is a uniform mixture of samples with furazolidone content less than 10 mg/kg, and blank spiked sample is a blank sample with furazolidone added. Blank sample and reference sample can be stored for one year at 0℃～8℃. If the measured spiked recovery is less than 94% or greater than 106%, the analysis should be repeated. 7.2 Preparation of blank spiked sample
NY/T 727—2003
The furazolidone content in the spiked sample should be equivalent to the expected furazolidone content in the test sample. The preparation method is as follows: To prepare a spiked sample with a furazolidone content of 250 mg/kg, accurately pipette 5.0 mL of furazolidone stock solution (4.8) into a 250 mL conical flask, blow with nitrogen until about 0.5 mL remains, add 5 g of blank feed sample, mix thoroughly, let stand for at least 10 minutes, and then extract.
7.3 Extraction
Weigh an appropriate amount of the prepared sample, place it in an appropriate conical flask, accurately add a certain volume of water (if necessary), mix and place for 5 minutes, accurately add the extractant (4.2), cover the lid, place it on a cyclotron and shake vigorously for 30 minutes, filter it with a filter device (5.5), and perform column chromatography on the filtrate according to the provisions of 7.4.
Dilute the filtrate with diluent (4.3) so that the furazolidone content in the final solution reaches 5ug/mL~10μg/mL. Mix thoroughly, filter it with a filter system (5.8), and the filtrate is filtered according to 7.HPLC analysis was performed as described in 5. The specific extraction conditions are shown in Table 1.
Furazolidone content in sample
10 mg/kg~~2 500 mg/kg
2 500 mg/kg~5 000 mg/kg
0. 5% ~7%
7%～10%
10% ~20%
7.4 Column chromatography
Sample weight
5g±50mg
5 g±100 mg
1g±50mg
1 g±10 mg
0. 5g±5mg
Water/ml
Extractant/mL
For each sample extract, a dry-filled glass chromatography column is required. A small ball of glass fiber (5.6) is placed at the bottom of the glass chromatography column, which is filled with 4g of neutral alumina (4.10). 20mL of the sample extract prepared according to 7.3 is added to the column, the first 4mL of the effluent is discarded, and the subsequent 8mL of the effluent is collected in a graduated test tube. If necessary, the effluent is diluted with a diluent (4.3) to make the furazolidone content reach 5μg/mL to 10μg/ml, and the dilution factor is f. Filter with a filtration system (5.8), and the filtrate is subjected to HPLC analysis as described in 7.5. 7.5HPLC analysis
7.5.1Chromatographic conditions
a) Mobile phase flow rate: 0.6mL/min;
b) Injection volume: 20 μL;
c) Detection wavelength: 365nm.
7.5.2 Analytical Procedure
7.5.2.1 Continuously inject furazolidone working solution (4.9) into the HPLC analyzer until a furazolidone peak with a stable baseline, symmetrical peak shape and reproducible peak height or peak area is obtained, that is, the difference between the maximum and minimum values of the three consecutive peak heights or peak areas is less than 5% of the average value.
The furazolidone peak should be symmetrical (f..<2). Note: fa is the quotient of the peak width of the tail half and the peak width of the front half at 10% of the peak height of the furazolidone peak. There should be a positive proportional relationship between the peak height and concentration of the two furazolidone working solutions. If the deviation is greater than 5%, a new working solution needs to be prepared.
Inject the blank sample and the extraction and purification solution with the blank sample added. If the furazolidone peak shape is asymmetrical or cannot be separated from the impurities in the matrix, it is necessary to replace the analytical column or change the ratio of organic phase to aqueous phase in the mobile phase. Inject furazolidone working solution (4.9), five sample extraction (purification) solutions and furazolidone working solution (4.9) in sequence, and observe the peak height of the working solution. The difference in peak area shall not exceed 5% of the mean. The samples to be tested may be added continuously in this order. 7.5.2.2 If the measured furazolidone content is significantly lower than the expected value, it is necessary to re-extract the sample with 50 mL of extraction solution (4.2) more than the volume listed in 7.3 and analyze it on the machine.
If the new analysis result is more than 15% higher than the previous result, it is necessary to re-extract the sample with another 50 mL of extraction solution (4.2) and analyze it on the machine, and repeat this process until the difference between the two determination results is less than 15%. 8 Confirmation
8.1 General
If the peak shape and measured value of furazolidone are suspected, or the measured furazolidone content is less than 25 mg/kg, it must be confirmed by overlapping chromatography (8.2) or diode array detector (8.3). 8.2 Overlapping chromatography
Add an appropriate amount of furazolidone working solution (4.9) to the sample extract. The amount added should be equivalent to the amount of furazolidone in the extract. Inject the sample extract, furazolidone working solution and the sample extract with furazolidone added in sequence. If the half-peak width of the sample peak after adding the extract does not change by more than 10%, and the peak height or peak area changes proportionally, it can be confirmed that the original furazolidone peak is furazolidone.
8.3 Diode array detector
8.3.1 Conditions
The determination conditions are the same as those specified in 7.5.1, except that the UV detector is replaced by a diode array detector. The specific parameters are as follows: Parameters
Determination wavelength
Bandwidth
Reference wavelength
Reference bandwidth
Spectral range
Spectral value
8.3.2 Steps
4nm (i.e., wavelength 365±2nm)
100 nm
225 nm～400nm
Baseline, peak maximum, rising slope and falling slope inflection point After the HPLC system is stable, inject 5μg/mL furazolidone working solution (4.9), the sample extract in question and another 5ug/mL furazolidone working solution (4.9) in sequence. Record and store the baseline, maximum value and inflection point data on both sides of each chromatographic peak. 8.3.3 Evaluation
Normalize the different spectral values of the sample peak (sample-baseline) and draw a graph, record the peak value and the inflection point values before and after the peak. Normalize the sample peak spectrum and the spectrum of furazolidone working solution respectively and draw a graph. Mark at the peak top. 8.3.4 Confirmation Standard
The sample peak can only be confirmed to be furazolidone if it meets the following conditions: a) The retention time of the sample peak should be the same as that of the standard peak (difference ≤ 5%). If there is any doubt, a standard addition experiment (i.e., adding the standard to the sample) is required.
b) The purity evaluation of the sample peak is based on the degree of conformity between the recorded peak top, the inflection point of the rising slope and the inflection point of the falling slope and the spectrum. The relative absorbance value of each wavelength in all spectra should be equal (difference ≤ 15%). There is no obvious difference between the sample spectrum and the standard spectrum in the part with wavelength greater than 220nm and relative absorption of at least 10%. The maximum absorption wavelength of the sample peak and the standard peak are the same, that is, the difference is not greater than the range determined by the resolution of the detection system (generally 2nm~～4nm), and the deviation of any observation point of the two spectra cannot exceed 15% of the absorption value of the standard determination substance at the specific wavelength.
9 Result calculation
The content of furazolidone in the sample extract is calculated by comparing the peak height or peak area of the sample extraction chromatographic peak and the average peak height or peak area of 4
furazolidone working solution injected before and after the sample. 9.1 Samples with furazolidone content of 10 mg/kg to 5000 mg/kg: The furazolidone content Wt in the compound feed sample can be calculated using formula (2): Wt
Wherein:
furazolidone content in the sample, in milligrams per kilogram (mg/kg); A
chromatographic peak area measured by the sample extract; chromatographic peak area measured by the furazolidone standard working solution; furazolidone content in the standard working solution, in micrograms per milliliter (μg/ml); volume of extract added to the sample, in milliliters (mL); sample mass, in grams (g);
dilution factor of the extract.
Note: Peak height can also be used instead of peak area for calculation. The result is retained to 1 mg/kg.
9.2 Samples with a furazolidone mass fraction of 0.5% to 20%. The mass fraction Wip of furazolidone in premixed feed or concentrated feed can be calculated by formula (3): A×C. ×
×f× 10
Wherein:
The content of furazolidone in the sample, %;
The chromatographic peak area measured by the sample extract; A
The chromatographic peak area measured by the furazolidone standard working solution; C.-The content of furazolidone in the standard working solution, in micrograms per milliliter (μg/mL); V
The volume of the extract added to the sample, in milliliters (mL); The mass of the sample, in grams (g);
The dilution factor of the extract.
Note: Peak height can also be used instead of peak area for calculation. The result is retained to 0.01%.
10 Precision
10.1 Repeatability
The results of two parallel determinations completed by the same operator in the same laboratory shall meet the following requirements: a) When the furazolidone content is 10mg/kg~5000mg/kg, the relative difference is ≤8%; b) When the furazolidone content is 0.5%~~20%, the relative difference is ≤10%. 10.2 Reproducibility
NY/T 727—2003
·(2)
The results of two determinations completed by different operators using different instruments in different laboratories shall meet the following requirements: a) When the furazolidone content is 10mg/kg~5000mg/kg, the relative difference is ≤17%; b) When the furazolidone content is 0.When the relative difference is 5%~~20%, the relative difference is ≤20%. 5
NY/T 727—2003
Appendix A
(Informative Appendix)
Standard chromatogram and spectrum of furazolidone
Standard chromatogram of furazolidone
20.0022.00
260.00280.00300.00320. 00340.00360.00380.00400.00420.00440.00Figure 2 Standard spectrum of furazolidone
(Informative Appendix)
Technical and editorial differences between this standard and IS014797:1999 (E) and their causesNY/T 727.---2003
Table B.1 Technical and editorial differences between this standard and ISO14797:1999(E) and their reasons Article number of this standard
Technical and editorial differences
Added the test for concentrated feed.
Revised the minimum detection limit from 25 mg/kg in the original standard to 10 mg/kg.
Added GB/T14699.1--1993.
Added GB/T 6682.
Added GB/T14699.1-1993.
Except ISO 6498.
Modified the format of the original extraction steps. This is an editorial change.
Added "or the measured furazolidone content is less than 25 mg/kg\.
Changed "available" to "necessarily used"
Changed the bee height in the calculation formula to bee area. Originally
This modification was made to increase the applicability of the method in view of the high production of concentrated feed in my country.
According to experimental data, there are active and passive additions of furazolidone in feed production in my country, and the content of the latter is generally high. The method strives to effectively control the two systems.
To suit my country's national conditions.
Strive to make this standard compatible with other relevant standards in my country.
Simplify the standard format for easy operation and use. Strive to achieve accuracy in determination.
Strive to be close to other standards in my country.
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.