Some standard content:
NY438---2001
Clenbuterol hydrochloride, its chemical name is α-[(tert-butylamino)methyl}-4-amino-3,5-cyanobenzyl alcohol hydrochloride, commonly known as clenbuterol, is a β, adrenergic receptor agonist, and is a drug prohibited by the state for use in feed. This standard is formulated after a large number of experimental studies based on reference to the relevant recommended methods of the European Union and domestic and foreign literature. This standard specifies the method for determining clenbuterol hydrochloride in feed by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), and stipulates that GC-MS is the arbitration method. This standard is proposed and managed by the National Feed Standardization Promotion Technical Committee. This standard is drafted by the National Feed Product Quality Supervision and Inspection Center (Beijing), and the Beijing Normal University Analysis and Testing Center participated in the drafting. The main drafters of this standard are: Chang Biying, Fan Li, Yan Huiwen, Song Rong, Li Xinxin, and Li Lan. 29
1 Scope
Agricultural Industry Standard of the People's Republic of China
Determination of clenbuterol hydrochloride in feed
Determination of clenbuterol hydrochloride in feedNY438--2001
This standard specifies the method for determining clenbuterol hydrochloride in feed by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), and specifies GC-MS as the arbitration method. This standard is applicable to the determination and arbitration of clenbuterol hydrochloride in compound feed, concentrated feed and premixed feed. The minimum detection limit of HPLC method is 0.5ng (when sampling 5g, the minimum detection concentration is 0.05mg/kg). The minimum detection limit of GC-MS method is 0.025ng (when sampling 5g, the minimum detection concentration is 0.01 mg/kg). 2 Referenced Standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised. Parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T6682-1992 Specifications and test methods for water in analytical laboratories 3 HPLC method
3.1 Principle of the method
Clenbuterol hydrochloride in feed is dissolved with dilute acid solution with methanol, the solution is alkalized, and after liquid-liquid extraction and solid phase extraction column purification, it is separated and determined on an HPLC instrument. 3.2 Reagents and materials
The following reagents and water are analytical reagents unless otherwise specified, and water is grade 3 water in accordance with the provisions of GB/T6682. 3.2.1 Methanol: chromatographically pure, filtered through a 0.45μm filter membrane. 3.2.2 B: chromatographically pure, filtered through a 0.45μm filter membrane. 3.2.3 Extraction solution: 0.5% metaphosphoric acid solution (14.29g metaphosphoric acid dissolved in water and diluted to 1L): methanol = 80:20. 3.2.4 Sodium hydroxide solution c (NaOH) about 2mol/L: 20g sodium hydroxide dissolved in 250mL water. 3.2.5 Reagents for liquid-liquid extraction
3.2.5.1 Ether.
3.2.5.2 Anhydrous sodium sulfate.
3.2.6 Nitrogen.
3.2.7 Hydrochloric acid solution c (HCL) about 0.02mol/L: 1.67mL hydrochloric acid diluted to 1L with water. 3.2.8 Reagents for solid phase extraction (SPE)
3.2.8.130mg/lcc Oasis?HLB solid phase extraction cartridge\or purification cartridge with equivalent effect. 1) Oasis? HLB solid phase extraction cartridge is the trade name of a product provided by Waters Corporation (34 Maple Street Milford MA, USA). This information is provided for the convenience of users of this industry standard, not for the approval of this product by the standard administration department. Approved by the Ministry of Agriculture of the People's Republic of China on February 12, 2001 300
Implementation on May 1, 2001
3.2.8.2 SPE eluent
NY 438—2001
3.2.8.2.1 Eluent-1: 5% methanol aqueous solution containing 2% ammonia. 3.2.8.2.2 Eluent-2: 30% methanol aqueous solution containing 2% ammonia. 3.2.9 HPLC Special Reagents
3.2.9.1 HPLC Mobile Phase: 1mL 1:1 phosphoric acid (super pure) diluted to 1L with laboratory secondary water, and mixed with acetonitrile (3.2.2) at a ratio of 100:12, and ultrasonically degassed for 5min before use. 3.2.9.2 Clenbuterol Hydrochloride Standard Solution
3.2.9.2.1 Stock Solution: 200μg/mL: 10.00mg clenbuterol hydrochloride (containing C12H1gCl2N.0·HCl not less than 98.5%) dissolved in 0.02mol/L hydrochloric acid solution and dilute to 50mL, stored in the refrigerator. Valid for 1 month. 3.2.9.2.2 Working Solution, 2.00μg/mL: Use a micropipette to take 500uL of the stock solution, dilute to 50mL with 0.02mol/L hydrochloric acid solution, and store in the refrigerator.
3.2.9.2.3 Standard series: Use a micropipette to transfer 25, 50, 100, 500, 1000ul of the working solution (3.2.9.2.2), and dilute to 2mL with 0.02mol/mL hydrochloric acid solution. The corresponding concentrations of clenbuterol hydrochloride in this standard series are: 0.025, 0.050, 0.1000.500, 1.00μg/mL, and store in a refrigerator. 3.3 Instruments and equipment
, 3.3.1 Commonly used laboratory instruments and equipment. 3.3.2 Analytical balance: sensitivity 0.0001g; sensitivity 0.00001g. 3.3.3 Ultrasonic water bath.
3.3.4 Centrifuge: capable of reaching 4000r/min. 3.3.5 Separatory funnel: 150mL.
3.3.6 Electric heating block or sand bath: temperature can be controlled at (50±5)℃. 3.3.7 Oven: temperature can be controlled at (70±5)℃. 3.3.8 High performance liquid chromatograph: with C18 column 4μm (such as 150mm×3.9mmID) or similar analytical column and UV detector or diode array detector.
3.4 Sample preparation
Take a representative feed sample, reduce it to about 200g by quartering, crush it through a 0.45mm pore size sieve, mix it thoroughly, and put it into a ground-mouth bottle for later use.
3.5 Analysis steps
3.5.1 Extraction: Weigh an appropriate amount of sample (5g of compound feed, 2g of premix and concentrate) accurately to 0.0001g, place in a 100mL conical flask, accurately add 50mL of extracting solution (3.2.3), vibrate to moisten all, place in an ultrasonic water bath for ultrasonic extraction for 15min, take out and shake by hand every 5min. After the end of the ultrasound, shake by hand for at least 10s, and take the upper layer of liquid and centrifuge it at 4000r/min for 10min. 3.5.2 Purification: Accurately draw 10.00mL of supernatant, place in a 150mL separatory funnel, add sodium hydroxide solution (3.2.4), shake thoroughly, and adjust the pH to 11-12. This process reacts slowly. After leaving for 3-5min, check the pH. If the pH is lower, add alkali to adjust it. The solution was extracted twice with 30.25 mL of ether, and the ether layer was dried over anhydrous sodium sulfate. A small amount of ether was used to elute the separatory funnel and anhydrous sodium sulfate, and the volume was adjusted to 50 mL with ether. Accurately pipette 25.00 mL into a 50 mL beaker, and evaporated to dryness in a fume hood, on a heating block or sand bath (3.3.6) below 50 °C. The residue was dissolved in 2.00 mL of hydrochloric acid solution (3.2.7), and 1.00 mL was placed on an SPE column that had been treated with 1 mL of methanol and 1 mL of deionized water, respectively. The syringe was slightly pressurized so that the column flow rate did not exceed 1 mL/min, and then eluted with 1 mL of SPE eluent-1 (3.2.8.2.1) and eluent-2 (3.2.8.2.2) respectively, and finally eluted with methanol. The eluent was placed on a (70 ± 5) °C heating block or sand bath, and dried with nitrogen.
3.5.3 Determination
3.5.3.1 Accurately add 1.00-2.00mL hydrochloric acid solution (3.2.7) to the purified and dried sample residue, shake thoroughly, sonicate, dissolve the shallow residue, filter through a 0.45um filter membrane if necessary, and measure the clear solution on the machine, using the clenbuterol hydrochloride standard series (3.2.9.2.3) for single-point or multi-point calibration.
3.5.3.2 HPLC determination parameter setting:
NY 438—2001
Chromatographic column: Cl: column, 150mm×3.9mmID, particle size 4μm or similar analytical column. Column temperature: room temperature.
Mobile phase: 0.05% phosphoric acid aqueous solution: acetonitrile = 100:12 (3.2.9.1), flow rate: 1.0mL/min. Detector: diode array or UV detector. Detection wavelength: 210nm or 243nm.
Injection volume: 20~50μL.
3.5.3.3 Qualitative and quantitative methods:
Qualitative method: In addition to using retention time for qualitative analysis, the characteristic spectrum of clenbuterol hydrochloride in the ultraviolet region can also be measured by diode array, that is, there are three absorption peaks at 210, 243 and 296nm with lower peaks in turn. Quantitative method: Integrate to obtain the peak area, and then use single-point or multi-point calibration method for quantitative analysis. 3.6 Expression of analysis results
The mass of clenbuterol hydrochloride contained in each dry gram of sample is calculated according to formula (1): X=m×D
Wherein: X---the mass of clenbuterol hydrochloride in each gram of sample, mg; m--the mass of clenbuterol hydrochloride corresponding to the area of HPLC chromatogram, μg; D--dilution factor;
m--the mass of the weighed sample, g.
The results are expressed to one decimal place.
3.7 Allowable difference
The arithmetic mean of the parallel determination results is taken as the determination result, and the relative deviation of two parallel determinations shall not exceed 10%. 4GC-MS method (arbitration method)
4.1 Principle of the method
(1)
Use dilute acid solution with methanol to dissolve clenbuterol hydrochloride in feed, alkalize the solution, and separate and determine it on a GC-MS instrument after liquid-liquid extraction and solid phase extraction column purification. 4.2 Reagents and materials
The following reagents and water are analytically pure reagents unless otherwise specified, and the water is grade 3 water in accordance with GB/T6682. 4.2.1 Extraction and purification reagents: Same as 3.2.1~3.2.8 in HPLC method. 4.2.2 Derivatization agent: N,O-bistrimethylsilyltrifluoroacetamide (BSTFA). 4.2.3 Toluene.
4.2.4 Clenbuterol hydrochloride standard solution
4.2.4.1 Stock solution, 200μg/mL: 10.00mg of clenbuterol hydrochloride (containing C12Hl:Cl, Nz0·HCl not less than 98.5%) is dissolved in methanol and diluted to 50mL, and stored in a refrigerator. Valid for 3 months. 4.2.4.2 Working solution, 2.00ug/mL: Use a micropipette to transfer 500μL of the stock solution (4.2.4.1), dilute to 50mL with methanol, and store in a refrigerator.
4.2.4.3 Standard series: Use a micropipette to take 25, 50, 100, 500, 1000 μL of the working solution (4.2.4.2), dilute to 2 mL with methanol, the corresponding concentrations of clenbuterol hydrochloride in this standard series are: 0.025, 0.050, 0.100, 0.500, 1.00 ug/ml., store in a refrigerator.
4.3 Instruments and equipment
4.3.1 Sample pretreatment equipment is the same as HPLC method 3.3.1 to 3.3.7. 4.3.2 GC-MS coupling instrument
NY 438--2001
Gas chromatograph equipped with a weakly polar or non-polar capillary column and an electron bombardment ion source and detector. 4.4 Sample preparation
Same as HPLC method 3.4.
4.5 Analysis steps
4.5.1 Extraction: Same as HPLC method 3.5.1.
4.5.2 Purification: Same as HPLC method 3.5.2.
4.5.3 Determination
4.5.3.1 Derivatization: Add 50uL of the biodegradant BSTFA (4.2.2) to the purified and dried sample residue, vortex thoroughly, place in a (70±5)℃ supply box, and derivatize for 30min. Blow dry with nitrogen, add 100uL of toluene (4.2.3), mix well, and measure on GC-MS. Simultaneously use the clenbuterol hydrochloride standard series (4.2.4.3) for synchronous derivatization. 4.5.3.2 GC-MS determination parameter setting:
Chromatographic column: DB-5MS, 30 mX0.25 mm2ID0.25 μm. Carrier gas: hydrogen; column head pressure: 50 kPa.
Injection port temperature: 260℃.
Injection volume: 1 tuL, no splitting.
Column temperature program: 70℃ for 1 min, increase to 200℃ at 25℃/min, maintain at 200℃ for 6 min, then increase to 280℃ at 25℃/min and maintain for 2 min.
EI source electron bombardment energy: 70 eV.
Detector temperature: 200℃.
Interface temperature: 250℃.
Mass scanning range: 60～400 AMU.
Solvent delay: 7 min.
The characteristic mass spectrum peaks of the trimethylsilane derivative of clenbuterol for detection are: m/Z=86, 187, 243, 262. 4.5.3.3 Qualitative and quantitative methods
Qualitative method: The relative deviation of the retention time of the sample and the standard is not more than 0.5%. The characteristic ion base peak percentage is not more than 20% different from that of the standard.
Quantitative method: Selected ion monitoring (SIM) method is used to calculate the peak area, and single-point or multi-point calibration method is used for quantitative analysis. 4.6 Expression of analytical results
The mass of clenbuterol hydrochloride contained in each kilogram of sample is calculated according to formula (2): Xm×D
Wherein: X-
The mass of clenbuterol hydrochloride in each kilogram of sample, mg; The mass of clenbuterol hydrochloride corresponding to the area of the GC-MS chromatographic peak, ug; Dilution factor;
The mass of the weighed sample, g.
The results are expressed to 1 decimal place.
4.7 Allowable difference
The arithmetic mean of the parallel determination results is taken as the determination result. The relative deviation of two parallel determinations shall not exceed 20%. (2)4 Sample preparation
Same as HPLC method 3.4.
4.5 Analysis steps
4.5.1 Extraction: Same as HPLC method 3.5.1.
4.5.2 Purification: Same as HPLC method 3.5.2.
4.5.3 Determination
4.5.3.1 Derivatization: Add 50uL of the biodegradant BSTFA (4.2.2) to the purified and dried sample residue, vortex thoroughly, place in a (70±5)℃ supply box, and derivatize for 30min. Dry with nitrogen, add 100uL of toluene (4.2.3), mix well, and determine on GC-MS. Simultaneously derivatize with the clenbuterol hydrochloride standard series (4.2.4.3). 4.5.3.2 GC-MS determination parameter setting:
Chromatographic column: DB-5MS, 30 mX0.25 mm2ID0.25 μm. Carrier gas: hydrogen; column head pressure: 50 kPa.
Injection port temperature: 260℃.
Injection volume: 1 tuL, no split.
Column temperature program: 70℃ for 1 min, increase to 200℃ at a rate of 25℃/min, maintain at 200℃ for 6 min, then increase to 280℃ at a rate of 25℃/min and maintain for 2 min.
EI source electron bombardment energy: 70 eV.
Detector temperature: 200℃.
Interface temperature: 250℃.
Mass scanning range: 60～400 AMU.
Solvent delay: 7 min.
The characteristic mass spectrum peaks of the trimethylsilane derivative of clenbuterol for detection are: m/Z=86, 187, 243, 262. 4.5.3.3 Qualitative and quantitative methods
Qualitative method: The relative deviation of the retention time of the sample and the standard is not more than 0.5%. The characteristic ion base peak percentage is not more than 20% different from that of the standard.
Quantitative method: Selected ion monitoring (SIM) method is used to calculate the peak area, and single-point or multi-point calibration method is used for quantitative analysis. 4.6 Expression of analytical results
The mass of clenbuterol hydrochloride contained in each kilogram of sample is calculated according to formula (2): Xm×DbzxZ.net
Wherein: X-
The mass of clenbuterol hydrochloride in each kilogram of sample, mg; The mass of clenbuterol hydrochloride corresponding to the area of the GC-MS chromatographic peak, ug; Dilution factor;
The mass of the weighed sample, g.
The results are expressed to 1 decimal place.
4.7 Allowable difference
The arithmetic mean of the parallel determination results is taken as the determination result. The relative deviation of two parallel determinations shall not exceed 20%. (2)4 Sample preparation
Same as HPLC method 3.4.
4.5 Analysis steps
4.5.1 Extraction: Same as HPLC method 3.5.1.
4.5.2 Purification: Same as HPLC method 3.5.2.
4.5.3 Determination
4.5.3.1 Derivatization: Add 50uL of the biodegradant BSTFA (4.2.2) to the purified and dried sample residue, vortex thoroughly, place in a (70±5)℃ supply box, and derivatize for 30min. Dry with nitrogen, add 100uL of toluene (4.2.3), mix well, and determine on GC-MS. Simultaneously derivatize with the clenbuterol hydrochloride standard series (4.2.4.3). 4.5.3.2 GC-MS determination parameter setting:
Chromatographic column: DB-5MS, 30 mX0.25 mm2ID0.25 μm. Carrier gas: hydrogen; column head pressure: 50 kPa.
Injection port temperature: 260℃.
Injection volume: 1 tuL, no split.
Column temperature program: 70℃ for 1 min, increase to 200℃ at a rate of 25℃/min, maintain at 200℃ for 6 min, then increase to 280℃ at a rate of 25℃/min and maintain for 2 min.
EI source electron bombardment energy: 70 eV.
Detector temperature: 200℃.
Interface temperature: 250℃.
Mass scanning range: 60～400 AMU.
Solvent delay: 7 min.
The characteristic mass spectrum peaks of the trimethylsilane derivative of clenbuterol for detection are: m/Z=86, 187, 243, 262. 4.5.3.3 Qualitative and quantitative methods
Qualitative method: The relative deviation of the retention time of the sample and the standard is not more than 0.5%. The characteristic ion base peak percentage is not more than 20% different from that of the standard.
Quantitative method: Selected ion monitoring (SIM) method is used to calculate the peak area, and single-point or multi-point calibration method is used for quantitative analysis. 4.6 Expression of analytical results
The mass of clenbuterol hydrochloride contained in each kilogram of sample is calculated according to formula (2): Xm×D
Wherein: X-
The mass of clenbuterol hydrochloride in each kilogram of sample, mg; The mass of clenbuterol hydrochloride corresponding to the area of the GC-MS chromatographic peak, ug; Dilution factor;
The mass of the weighed sample, g.
The results are expressed to 1 decimal place.
4.7 Allowable difference
The arithmetic mean of the parallel determination results is taken as the determination result. The relative deviation of two parallel determinations shall not exceed 20%. (2)
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