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GB/T 14700-2018

Chinese Standard: 'GB/T 14700-2018'
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Detail Information of GB/T 14700-2018; GB/T14700-2018
Description (Translated English): Determination of thiamine in feed
Sector / Industry: National Standard (Recommended)
Classification of Chinese Standard: B46
Classification of International Standard: 65.120
Word Count Estimation: 9,915
Date of Issue: 2018-05-14
Date of Implementation: 2018-12-01
Older Standard (superseded by this standard): GB/T 14700-2002
Drafting Organization: Institute of Agricultural Quality Standards and Testing Technology, Chinese Academy of Agricultural Sciences [National Feed Quality Supervision and Inspection Center (Beijing)]
Administrative Organization: National Feed Industry Standardization Technical Committee (SAC/TC 76)
Regulation (derived from): National Standards Announcement No. 6 of 2018
Proposing organization: National Feed Industry Standardization Technical Committee (SAC/TC 76)
Issuing agency(ies): State Administration of Markets and China National Standardization Administration

GB/T 14700-2018
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 14700-2002
Determination of thiamine in feed
饲料中维生素 B1的测定
ISSUED ON. MAY 14, 2018
IMPLEMENTED ON. DECEMBER 1, 2018
Issued by. State Administration for Market Regulation of the People's
Republic of China;
Standardization Administration of the People's Republic of
China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Method I. fluorescence spectrophotometry ... 4
4 Method II. high performance liquid chromatography ... 10
Annex A (Informative) chromatogram and spectrogram of thiamine B1 ... 15
Determination of thiamine in feed
1 Scope
This Standard specifies two methods for the determination of thiamine B1 in feed,
including fluorescence spectrophotometry and high performance liquid
chromatography.
Method I of this Standard applies to the determination of thiamine B1 in feed material,
compound feed and concentrated feed. The quantitation limit of this method is 1 mg/kg
(in case of the existence of any interfering substance which absorbs thiamine or
influences thiamine fluorescence, this method is not applicable). Thiamine B1
determined in this Method includes the total of intrinsic and additive amounts.
Method II of this Standard specifies the determination of compound premixed feed and
thiamine premixed feed. The detection limit of method II is 3 mg/kg; the quantitation
limit is 15 mg/kg.
2 Normative references
The following referenced documents are indispensable for the application of this
document. For dated references, only the edition dated applies to this document. For
undated references, the latest edition of the referenced documents (including all
amendments) applies to this document.
GB/T 6682, Water for analytical laboratory use – Specification and test methods
GB/T 14699.1, Feeding Stuffs – Sampling
GB/T 20195, Animal feeding stuffs – Preparation of test samples
3 Method I. fluorescence spectrophotometry
3.1 Principle
After thiamine B1 in sample is digested by diluted acid and digestive enzyme and
absorbed-separated-purified by adsorbent, it is oxidized by potassium ferricyanide
under alkaline conditions to generate fluorochrome-thiochrome and then extracted by
normal butanol. The fluorescence intensity of thiochrome in normal butanol is in direct
for use within 6 months.
Before use, check the recovery of thiamine B1 standard solution by zeolite; if it is less
than 92%, re-activate zeolite.
NOTE. Check of the recovery of thiamine B1 by zeolite. transfer 2 mL of thiamine B1 standard
medium solution (3.2.13.2) and add acidic potassium chloride solution (3.2.6) to make up to
100 mL. Carry out oxidization in accordance with the procedures specified in 3.5.4.1 ~ 3.5.4.3
as the external standard. Transfer 25 mL of thiamine B1 standard working solution (3.2.13.3) to
repeat the operation of column chromatography isolation specified in 3.5.3.1 ~ 3.5.3.3; carry
out oxidization in accordance with the procedures specified in 3.5.4.1 ~ 3.5.4.3. Measure the
fluorescence intensity of both solutions at the same time; carry out calculation in accordance
with Equation (1); convert the result into a percentage, which is the recovery value of thiamine
B1 by zeolite.
3.2.13 Thiamine B1 standard solution
3.2.13.1 Thiamine B1 standard stock solution. take the standard substance of thiamine
nitrate (with purity greater than 99%) and dry in a phosphorus pentoxide desiccator for
24 h. Weigh 0.01 g (accurate to 0.0001 g); dissolve in acidic 20% ethanol solution
(3.2.11) and make up to 100 mL; load in a brown bottle; store in a refrigerator at 2°C ~
8°C for use within 3 months. This solution contains 0.1 mg/mL of thiamine B1.
3.2.13.2 Thiamine B1 standard medium solution. take 10 mL of thiamine B1 standard
stock solution (3.2.13.1) before adding acidic 20% ethanol solution (3.2.11) to make
up to 100 mL; load in a brown bottle; store in a refrigerator at 2°C ~ 8°C for use within
48 h. The solution contains 10 μg/mL of thiamine B1.
3.2.13.3 Thiamine B1 standard working solution. take 2 mL of thiamine B1 medium
solution (3.2.13.2) to mix with 65 mL of hydrochloric acid solution (3.2.1) and 5 mL of
sodium acetate solution (3.2.3); make up to 100 mL; prepare before analysis. The
solution contains 0.2 μg/mL of thiamine B1.
3.2.14 Quinine sulfate solution
3.2.14.1 Quinine sulfate stock solution. weigh 0.1 g of quinine sulfate (accurate to
0.001 g); use sulfuric acid solution (3.2.2) to dissolve and make up to 1,000 mL. Store
in a brown bottle for cold storage. If the solution is turbid, then prepare it once again.
3.2.14.2 Quinine sulfate working solution. take 3 mL of stock solution (3.2.13.1); use
sulfuric acid solution (3.2.2) to make up to 1,000 mL. Store in a brown bottle for cold
storage. The solution contains 0.3 μg/mL of quinine sulfate.
3.2.15 Normal butanol. the fluorescence intensity is not greater than that of 4% of
quinine sulfate working solution (3.2.14.2). Or else, it shall be re-distilled using distilling
glassware; take the fraction at 114°C ~ 118°C.
a brown volumetric flask of 100 mL; use water to make up to 100 mL; shake up.
3.5.2.3 Filtration. filter all preparation through ashless filter paper; discard 5 mL of
primary filtrate; collect the filtrate as sample solution.
3.5.3 Purification of sample solution
3.5.3.1 Preparation of absorption column. weigh 1.5 g of activated artificial zeolite
(3.2.11) to place in a small beaker of 50 mL; add 3% glacial acetic acid solution (3.2.10)
for soaking, with the liquid level of the solution above zeolite. Place degreasing cotton
at the bottom of the absorption-separation column (3.3.5); use a glass rod to press
gently. Then wash all zeolite soaked in acetic acid into the column (do not let the
absorption column be dehydrated). The flow rate passing the column is preferably
controlled at 1 mL/min. Then use 10 mL of nearly-boiling water to wash the column
once.
3.5.3.2 Absorb 25 mL of sample solution (3.5.2.3); add slowly into the absorption
column prepared; discard the filtrate; use 5 mL of nearly-boiling water each time to
wash the column for 3 times; discard the washings. Meanwhile, prepare parallel
sample.
3.5.3.3 Use 25 mL of acidic potassium solution (3.2.6) at 60°C ~ 70°C to add into the
absorption column continuously three times; collect the eluant in a volumetric flask of
25 mL; use acidic potassium chloride solution to make up after cooling; mix up.
3.5.3.4 Meanwhile, use 25 mL of thiamine B1 standard working solution (3.2.13.3).
Repeat the operation specified in 3.5.3.1 ~ 3.5.3.3 as the external standard.
3.5.4 Oxidization and extraction
Caution – The following operation is carried out in a dark place.
3.5.4.1 Add respectively 5 mL of eluant (3.5.3.3) into two centrifugal tubes with stopper
(3.3.6); mark them as A and B.
3.5.4.2 Add 3 mL of sodium hydroxide solution (3.2.7) into tube B; add 3 mL of alkaline
potassium ferricyanide solution (3.2.9) into tube A; rotate to shake gently. In succession,
immediately add 15 mL of normal butanol (3.2.15) into tube A and put on stopper;
vibrate to shake violently for 15 s; add 15 mL of normal butanol into tube B and put on
stopper; vibrate to shake for 90 s together; place aside for layering.
3.5.4.3 Use an injection syringe (3.3.8) to remove the lower water phase; add about 2
g of anhydrous sodium sulfate into all reaction tubes; rotate to shake as sample to be
tested.
3.5.4.4 Meanwhile, pour 5 mL of eluant (3.5.3.4) as the external standard into another
3.7 Repeatability
For feed whose thiamine B1 content is less than 5 mg/kg, under repeatable conditions,
the difference between the results measured independently two times and their
arithmetical mean value shall not be greater than 15% of the arithmetical mean value
of the two values measured;
For feed whose thiamine B1 content is greater than 5 mg/kg and less than 50 mg/kg,
under repeatable conditions, the difference between the results measured
independently two times and their arithmetical mean value shall not be greater than
10% of the arithmetical mean value of the two values measured;
For feed whose thiamine B1 content is greater than 50 mg/kg, under repeatable
conditions, the difference between the results measured independently two times and
their arithmetical mean value shall not be greater than 5% of the arithmetical mean
value of the two values measured.
4 Method II. high performance liquid chromatography
4.1 Principle
After the ultrasonic extraction of sample by acidic extracting solution, inject the test
solution filtered and centrifuged into the inverse phase of the high performance liquid
chromatographic system for separation; use ultraviolet (or diode matrix detector) for
testing; calculate the content of thiamine B1 using the external standard method.
4.2 Reagents or solutions
Unless specified otherwise, all reagents used are analytically pure and the water for
chromatography shall comply with the specification for grade one water as specified in
GB/T 6682.
4.2.1 Ammonium chloride. guaranteed reagent.
4.2.2 Sodium heptanesulfonate (PICB7). guaranteed reagent.
4.2.3 Glacial acetic acid. guaranteed reagent.
4.2.4 Triethylamine. chromatographically pure.
4.2.5 Methanol. chromatographically pure.
4.2.6 Acidic ethanol solution. 20%, see 3.2.11 for its preparation.
4.2.7 Ethylenediaminetetraacetic acid disodium (EDTA). guaranteed reagent.
4.3.5 High performance liquid chromatograph which is equipped with an ultraviolet or
diode matrix detector.
4.4 Sample
Take representative feed sample as specified in GB/T 14699.1; reduce sample by
quartering. Prepare sample as specified in GB/T 20195; fully mix up.
4.5 Test procedures
Caution – Protect from exposure to direct strong light.
4.5.1 Extraction
4.5.1.1 Extraction of thiamine premixed feed
Weigh 0.25 g ~ 0.5 g (accurate to 0.0001 g) of sample to place in a brown volumetric
flask of 100 mL; add about 70 mL of extracting solution (4.2.8); add while shaking up;
place on an ultrasonic water bath for ultrasonic extraction for 15 min; shake twice
during the period; cool; use extracting solut......
Related standard:   GB/T 14701-2019  GB/T 14702-2018
   
 
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