GB/T 5009.192-2003 Determination of clenbuterol residues in animal foods
Some standard content:
ICS67.040
National Standard of the People's Republic of China
GB/T5009.192—2003
Determination of 4-amino-3,5-dichloro-α［(tert-butylamino)methyl]-benzyl alcoho)(clenbuterol) residues in animal foods foods2003-08-11issued
Ministry of Health of the People's Republic of China
Standardization Administration of China
2004-01-01implemented
GB/T5009.192-2003
This standard modifies and adopts EUR15127-EN "Veterinary drug residues, animal foods and products-reference substances and analytical methods, 2nd Ed, Sg2.1.Cg2.3, Sg2.4 and Cy2.3 EU drug residue method: method for the determination of veterinary drug residues in animal foods" (English second edition, 1994).
The difference between this standard and the EU "Determination of Veterinary Drug Residues in Animal Food" Sg2.1, Sg2.3, Sg2.4 and Cy2.3 is that Sg2.1.Sg2.3.Sg2.4 and Cy2.3 are multi-component residue detection methods for β-agonists in animal food. This standard only proposes a detection method for single component residues of clenbuterol in animal food. Sg2.1 is a GC-MS screening method for β-agonists in bovine urine, Sg2.3 is an ELISA screening method for β-agonists in bovine urine, Sg2.4 is a GC-MS screening method for β-agonists in bovine liver, kidney and meat, and Cy2.3 is a GC-MS confirmation method for β-agonists in bovine urine. This standard proposes a set of methods from ELISA screening, HPLC quantification to GC-MS confirmation and verification to meet the needs of monitoring clenbuterol residues in animal foods in my country.
The determination principle, operation process and requirements, main technical parameters and detection sensitivity of ELISA screening and GC-MS in this standard are consistent with the EU method. The HPLC in this standard is proposed based on experimental data and verification results.
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard: Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine, China Meat Food Comprehensive Research Center, Beijing Center for Disease Control and Prevention.
The main drafters of this standard: Wu Yongning, Miao Hong, Zhao Yunfeng, Zhao Jingling, Zhao Rong, Wu Guohua, Wang Lingyan. 521
GB/T5009.192-2003
Clenbuterol is a potent selective 3: receptor agonist with a strong and lasting effect of relaxing bronchial smooth muscle and is used to treat asthma. Clenbuterol can promote animal growth, improve fat distribution in animals, and increase lean meat rate. In the 1990s, my country mistakenly introduced and promoted it as a feed additive as a scientific research result, and it was commonly known as "epidemic meat essence". After a series of Zhongsen incidents caused by eating foods containing clenbuterol, clenbuterol became a feed additive that is generally banned in the world. Since 1997, relevant administrative departments in my country have repeatedly issued clear orders to prohibit the production, sale and use of clenbuterol hydrochloride in the animal husbandry industry. However, clenbuterol poisoning incidents still occur frequently in various parts of my country, indicating that the illegal use of clenbuterol still exists. In order to monitor clenbuterol in livestock products, strengthen market supervision and inspection, and prevent the occurrence of poisoning incidents, it is necessary to establish an effective detection method. my country started late in this area of testing. With the ban and monitoring requirements for clenbuterol internationally and domestically, it is urgent to develop a set of testing methods from screening to confirmation that are suitable for my country's national conditions. To this end, this standard proposes a set of methods from enzyme-linked immunosorbent assay (ELISA) screening, high-performance liquid chromatography (HPLC) quantification to gas chromatography-mass spectrometry (GC-MS) confirmation and quantification to meet the needs of monitoring clenbuterol residues in animal foods in my country.
1 Scope
Determination of clenbuterol residues in animal foods This standard specifies the determination method of clenbuterol in animal foods. GB/T5009.192--2003
This standard applies to the determination of clenbuterol residues in fresh or frozen livestock and poultry meat and viscera and their products. This standard also applies to the determination of clenbuterol in biological materials (human or animal blood, urine). The detection limit of this method is: 0.5μg/kg for the first method of gas chromatography-mass spectrometry; 0.5pg/kg for the second method of high performance liquid chromatography; 0.5pg/kg for the third method of enzyme-linked immunosorbent assay. Linear range: 0.025ng~2.5ng for the first method of gas chromatography-mass spectrometry; 0.5ng~4ng for the second method of high performance liquid chromatography; 0.004ng~0.054ng for the third method of enzyme-linked immunosorbent assay. Principle of the first method of gas chromatography-mass spectrometry (GC-MS) 2
Solid samples are cut into pieces and homogenized with perchloric acid solution. Liquid samples are added to perchloric acid solution, ultrasonically heated and extracted, extracted with isopropanol + ethyl acetate (40 + 60), the organic phase is concentrated, separated by a weak cation exchange column, eluted with ethanol + concentrated ammonia (98 + 2) solution, the eluent is concentrated, and after derivatization with N, O-bistrimethylsilane trifluoroacetamide (BSTFA), it is determined on a gas chromatography-mass spectrometer. Metoprolol was used as internal standard for quantification.
3 Reagents
3.1 Clenbuterol hydrochloride, purity ≥99.5%. 3.2 Metoprolol, purity ≥99%. 3.3 Sodium dihydrogen phosphate.
3.4 Sodium hydroxide.
3.5 Sodium azide.
3.6 Perfluoric acid.
Concentrated ammonia.
Isopropanol.
Ethyl acetate.
Methanol: HPLC grade.
Toluene: chromatographic grade.
Ethanol.
Derivatizing agent: N,O-bistrimethylsilyl trifluoroacetamide (BSTFA). Perchloric acid solution (0.1mol/L).
Sodium hydroxide solution (1mol/L).
Sodium dihydrogen phosphate buffer (0.1mol/L, pH=6.0). Isopropanol + ethyl acetate (40 in 60).
Ethanol + concentrated ammonia (98+2).
Metoprolol internal standard solution: Accurately weigh the metoprolol standard, dissolve it in methanol to make an internal standard reserve solution with a concentration of 240mg/L, store it in a refrigerator, and dilute it with methanol to a 2.4mg/L internal standard solution when used. 3.20 Clenbuterol standard solution: Accurately weigh the clenbuterol standard, dissolve it in methanol to make a standard reserve solution with a concentration of 250mg/L, store it in a refrigerator, and dilute it with methanol to a 0.5mg/L clenbuterol standard solution when used. 523
GB/T5009.192—2003
3.21 Weak cation exchange column (LC-WCX) (3 mL). 3.22 Syringe microporous filter membrane (0.45 μm, aqueous phase). 4 Apparatus
4.1 Gas chromatograph-mass spectrometer (GC/MS). 4.2 Ground glass centrifuge tube: 11.5 cm (length) × 3.5 cm (inner diameter), with stopper. 4.35 mL glass centrifuge tube.
4.4 Ultrasonic cleaner.
Acidity meter.
Centrifuge.
Oscillator.
Rotary evaporator.
Vortex mixer.
Thermostatic heater.
N2-evaporator.
2Homogenizer.
5 Analysis steps
5.1 Extraction
5.1.1 Muscle, liver, and kidney samples
Weigh 10g of muscle, liver, or kidney sample (accurate to 0.01g), homogenize with 20mL of 0.1mol/I perchloric acid solution, and place in a ground-mouth glass centrifuge tube: then ultrasonicate in an ultrasonic cleaner for 20min, take out and place in an 80℃ water bath for heating for 30min. Take out and cool, then centrifuge (4500r/min) for 15min. Pour out the supernatant, wash the precipitate with 5mL of 0.1mol/L pernitrogen acid solution, centrifuge again, and combine the two supernatants. Adjust the pH value to 9.5±0.1 with 1mol/L sodium hydroxide solution. If there is precipitation, centrifuge again (4500r/min) for 10min, transfer the supernatant to a ground-mouth glass centrifuge tube, add 8g of sodium chloride, mix, and add 25ml.Isopropanol + ethyl acetate (40 + 60), shake and extract on an oscillator for 20 minutes. After extraction, place for 5 minutes (if there is an emulsion layer, centrifuge it slightly). Use a pipette to carefully transfer the upper organic phase to a rotary evaporation bottle, and repeat the extraction once with 20mL of isopropanol + ethyl acetate (40 + 60), combine the non-organic phase, and concentrate it to near dryness on a rotary evaporator at 60℃. Use 1mL 0.1mol/L sodium dihydrogen phosphate grade buffer (pH6.0) to fully dissolve the residue, filter it through a syringe microporous filter membrane, wash it three times, and transfer it completely to a 5ml. glass centrifuge tube, and use 0.1mol/l. sodium dihydrogen phosphate buffer (pH6.0) to make up to the scale. 5.1.2 Urine sample
Use a pipette to measure 5mL of urine, add 20mL of 0.1mol/1. peroxy acid solution, and mix it by ultrasonic for 20 minutes. Place it in an 80℃ water bath and heat it for 30 minutes. The following steps are as follows: 5.1.1, starting from "adjusting pH to 9.5 ± 0.1 with 1 mol/L sodium hydroxide solution". 5.1.3 Blood sample
Centrifuge the blood at 4500 r/min, take 1 mL of the upper serum with a pipette and place it in a 5 mL glass centrifuge tube, add 2 mL of 0.1 mol/L perchloric acid solution, mix well, place in an ultrasonic cleaner for 20 min, heat in a 80°C water bath for 30 min. Take out, cool, and centrifuge (4500 r/min) for 15 min. Pour out the supernatant, wash the precipitate with 1 ml of 0.1 mol/l perchloric acid solution, centrifuge (4500 r/min) for 10 min, combine the supernatants, repeat the washing steps, and combine the supernatants. Add about 1g of sodium chloride to the clear solution, add 2mL of isopropanol + ethyl acetate (40+60), oscillate and extract on a vortex mixer for 5min, let stand for 5min (centrifuge for a while if there is an emulsion layer), carefully remove the organic phase to a 5mL glass centrifuge tube, repeat the above extraction steps twice, and combine the organic phases. Blow dry the organic phase on an N-concentrator. Use 1mL of 0.1mol/L sodium dihydrogen phosphate buffer (pH6.0) to fully dissolve the residue, transfer it completely to a 5mL glass centrifuge tube through a simple microporous filter membrane, and make up to the scale with 0.1mol/l. sodium dihydrogen phosphate buffer (pH6.0). 5.2 Purification
Use 10mL of ethanol, 3mL of water, 3mL of 0.1mol/L phosphoric acid in sequence. Sodium dihydrogen buffer (pH 6.0), 3ml. water to rinse the weak cation exchange column, take appropriate amount of 5.1.1, 5.1.2 and 5.1.3 extracts to the weak cation exchange column, discard the effluent, rinse the column with 4mL water and 524
GB/T5009.192—2003
4mL ethanol respectively, discard the effluent, rinse the column with 6ml. ethanol + concentrated ammonia water (98 + 2), collect the effluent. Concentrate the effluent to dryness on a N2-evaporator.
5.3 Derivatization
For purification, add 100μL~500uL methanol and 50μL2.4mg/L internal standard working solution to the dried sample residue, concentrate to as low as 40% on a Nz-evaporator, and quickly add 40uμL derivatization agent (BSTFA), cover the stopper, mix on a vortex mixer for 1min, and derivatize in a constant temperature heater at 75℃ for 90min. After the derivatization reaction is completed, take it out and cool it to room temperature, mix it on a vortex mixer for 30s, and place it on a N2-evaporator to concentrate it to dryness. Add 200L toluene, mix it thoroughly on a vortex mixer, and wait for injection into the gas chromatography-mass spectrometer. At the same time, use the clenbuterol standard solution for series synchronous derivatization. 5.4 Gas chromatography-mass spectrometry determination
5.4.1 Gas chromatography-mass spectrometry determination parameter setting Gas chromatography column: DB-5MS column, 30mX0.25mm×0.25μm. Carrier gas: He, column head pressure: 8psi.
Injection port temperature: 240℃.
Injection volume: 1μL, no splitting. bZxz.net
Column temperature program: 70℃ for 1 min, then increase to 200℃ at 18℃/min, then increase to 245℃ at 5℃/min, then increase to 280℃ at 25℃/min and maintain for 2 min. EI source
Electron variable impact energy: 70eV.
Ion source temperature: 200℃.
Interface temperature: 285℃.
Solvent delay: 12min.
EI source detection of characteristic mass spectrum peaks: Clenbuterol: m/z86, 187, 243, 262; Metoprolol: m/z72, 223. 5.4.2 Determination
Pipette 1ul, derivatized sample solution or standard solution and inject it into the gas chromatography-mass spectrometer. The relative retention time of the sample peak (m/286, 187, 243, 262, 264, 277, 333) and the internal standard peak (m/z72, 223) is used for qualitative analysis. It is required that the relative intensity of at least 3 pairs of selected ions in the sample peak (ratio to the base peak) does not exceed ±20% or 3 times the standard deviation of the average relative intensity of the corresponding selected ions of the standard. The peak area ratio of the sample peak (m/z86) to the internal standard peak (m/z72) is used for single-point or multi-point calibration and quantitative analysis. 5.4.3 The total ion flow chart and mass spectrum of the selected ions after derivatization of the clenbuterol standard and internal standard are shown in Figures 1 to 3.
Clenbute
Cenbuterol
Afetoprolol
m/z72.223
m/z86,187,243,262,264,277,333Metoprolol
Figure 1 Selective ion total ion chromatogram of clenbuterol and internal standard derivativesT
GB/T5009.192—2003
5.5 Result calculation||tt ||243262364277
TUTTTTTIEI
160180200220240260280300
Figure 2 Selected ion mass spectrum of clenbuterol derivatives 223
10011012013014015016017018019020021022080
Figure 3 Selected ion mass spectrum of internal standard derivatives The content of clenbuterol in the sample was calculated by single-point or multi-point calibration using the internal standard method. See formula (1):
Wherein:
The content of clenbuterol in the sample, in micrograms per kilogram (or micrograms per liter) [ug/kg (or ug/L); the mass of clenbuterol corresponding to the peak area ratio of the sample chromatographic peak to the internal standard chromatographic peak, in nanograms (ng), f——sample dilution factor;
m-sample sampling volume, in grams (or milliliters) [g or mL)]. The calculation result is expressed to two decimal places. 6 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. Method 2 High Performance Liquid Chromatography (HPLC) 7 Principle
(1)
Cut the solid sample into pieces and homogenize it with perchloric acid solution. Add pernitrogenous acid solution to the liquid sample and extract it with isopropanol + ethyl acetate (40 + 60) after ultrasonic heating. Concentrate the organic phase and separate it with a weak cation exchange column. Elute it with ethanol + ammonia (98 + 2) solution. Concentrate the eluate, make it constant with the mobile phase and measure it on a high performance liquid chromatograph. Quantify it with the external standard method. 526
8 Reagents and Materials
8.1 Clenbuterol (clenbuterolhydrochloride), purity ≥ 99.5%. 8.2 Sodium dihydrogen phosphate.
8.3 Sodium hydroxide.
8.4 Sodium chloride.
8.5 Pernitrogenous acid.
Concentrated ammonia water.
Isopropanol.
Ethyl acetate.
Methanol: HPLC grade.
Artificial alcohol.
Perfluoric acid solution (0.1mol/L).
Sodium hydroxide solution (1mol/L).
Sodium dihydrogen phosphate buffer (0.1mol/L, pH=6.0). Isopropanol + ethyl acetate (40+60).
Ethanol + concentrated ammonia (98+2).
Methanol + water (45+55).
CB/T5009.192—2003
8.17 Preparation of Clenbuterol Standard Solution: Accurately weigh the Clenbuterol standard substance and use methanol to prepare a standard stock solution with a concentration of 250mg/L, and store it in a refrigerator: When using, dilute it with methanol to a 0.5mg/L Clenbuterol standard solution, and further dilute it appropriately with methanol decahydrate (45+55).
Weak cation exchange column (LC-WCX) (3mL). 9Instruments
Water bath ultrasonic cleaner.
Ground glass centrifuge tube: 11.5cm (length) × 3.5cm (inner diameter), with stopper. 9.35mL glass centrifuge tube.
Acidity meter.
Centrifuge.
Oscillator.
Rotary evaporator.
Vortex mixer.
Needle type microporous filter membrane (o.45μm, aqueous phase). 9.10
N-evaporator.
Homogenizer.
High performance liquid chromatograph.
Analysis steps
10.1 Extraction
10.1.1 Muscle, liver, kidney samples
Same as 5.1.1.
10.1.2 Urine samples
Same as 5.1.2.
GB/T5009.192—2003
10.1.3 Blood sample
Same as 5.1.3.
10.2 Purification
Same as 5.2.
10.3 Preparation before sample measurement
Add 100μL~500μL mobile phase to the sample residue after purification and blowing, shake it fully on a vortex mixer to dissolve the residue, filter it with a 0.45μm needle-type microporous filter membrane when the liquid is turbid, and the supernatant is ready for liquid chromatography measurement. 10.4 Measurement
10.4.1 Reference conditions for liquid chromatography measurement
Chromatographic column: BDS or ODS column, 250mmX4.6mm, 5m Mobile phase: methanol + water (45+55).
Flow rate: 1mL/min.
Injection volume: 20μ~50.
Column box temperature: 25℃.
Ultraviolet detector: 244nm.
10.4.2 Determination
Pipette 20μL~50μL standard calibration solution and sample solution into the liquid chromatograph for qualitative determination by retention time and quantitative determination by external standard method single point or multi-point calibration method.
10.4.3The HPLC chromatogram of the clenbuterol standard is shown in Figure 4.
Clenbuterot
Figure 4 HPLC chromatogram of clenbuterol standard (100μg/L) 10.5 Result calculation
Calculate the clenbuterol content in the sample by the external standard method. See formula (2):
Wherein:
X——the content of clenbuterol in the sample, in micrograms per kilogram (or micrograms per liter) [Lg/kg (or μg/L)]; A——the mass of clenbuterol corresponding to the peak area ratio of the sample chromatographic peak to the standard chromatographic peak, in nanogram (ng); f——the dilution factor of the sample:
——the sampling volume of the sample, in grams (or milliliters) [g (or mL)]. The calculation result is expressed to two decimal places. 11 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 528
12 Principle
Method 3 Enzyme-linked immunosorbent assay (ELISA screening method) GB/T5009.192-2003
It is forbidden to conduct competitive inhibition determination in antigen-antibody reaction. The microplate is coated with a coating antibody against clenbuterol IgG. Clenbuterol antibody is added, and after incubation and washing steps, competitive enzyme markers, standards or sample solutions are added. Clenbuterol competes with the competitive enzyme marker for clenbuterol antibody, and the clenbuterol marker enzyme not linked to the antibody is removed during the washing step. Substrate (urea peroxide) and chromogen (tetramethylbenzidine) are added to the wells for incubation, and the bound marker enzyme converts the colorless chromogen into a blue product. Adding the reaction stop solution changes the color from blue to yellow. The absorbance is measured at 450nm, and the absorbance ratio is inversely proportional to the natural logarithm of the clenbuterol concentration.
13 Reagents
13.1 Sodium diammonium phosphate.
13.2 Perchloric acid.
3 Isopropanol.
13.4 Ethyl acetate.
13.5Perfluoric acid solution (0.1mol/1.). Sodium hydroxide solution (1mol/L).
Sodium dihydrogen phosphate buffer (0.1mol/LpH=6.0). 13.7
Isopropanol + ethyl acetate (40+60).
Needle simple microporous filter membrane (0.45m, aqueous phase). 13.9
Clenbuterol enzyme-linked immunosorbent assay kit.
13.10.196-well plate (12 strips × 8 wells) coated with anti-antibody against clenbuterol IgG. 13.10.2Clenbuterol series standard solution (at least 5 doubling dilution concentration levels, plus 1 blank). Peroxidase marker (concentrated solution). 13.10.3
Clenbuterol antibody (concentrated solution).
Enzyme substrate: urea peroxide.
Coloring agent: tetramethylbenzidine.
Reaction stop solution: 1mol/L sulfuric acid.
Buffer: for diluting enzyme markers and antibody concentrates. 13.10.8
14Instruments
Ultrasonic cleaner.
Ground-mouth glass centrifuge tube: 11.5cm (length) × 3.5cm (inner diameter), with stopper. 14.3
Acidity meter.
14.4Centrifuge.
14.5Oscillator.
14.6Rotary evaporator.
14.7Vortex mixer.
14.8Homogenizer.
14.9Microplate reader (equipped with 450nm filter). 14.10 Micropipette: single channel 20μL, 50μl, 100μL and multi-channel 50μL~250L adjustable. 529
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15 Sample determination
15.1.1 Muscle, liver and kidney samples
Same as 5.1.1.
15.1.2 Urine sample
If the urine is turbid, centrifuge it (3000r/min) for 10min, dilute the supernatant appropriately and then load it onto the ELISA plate for ELISA screening experiment. 15.1.3 Blood sample
Centrifuge serum or plasma (3000r/min) for 10min, take the serum and dilute it appropriately and then load it onto the ELISA plate for ELISA screening experiment. 15.2 Assay
15.2.1 Preparation of Reagents
15.2.1.1 Competitive Enzyme Markers
Competitive enzyme markers are provided as concentrates. Due to the poor stability of diluted enzyme markers, only the amount of enzyme markers actually needed should be diluted. Shake carefully before aspirating the concentrate. Dilute the enzyme marker concentrate with buffer at a ratio of 1:10 (e.g. 400μL concentrate + 4.0mL buffer, enough for 4 microplate strips with 32 wells). 15.2.1.2 Clenbuterol Antibody
The clenbuterol antibody is provided as a concentrate. Due to the poor stability of diluted clenbuterol antibody, only the amount of clenbuterol antibody actually needed should be diluted. Shake carefully before aspirating the concentrate. Dilute the antibody concentrate with buffer at a ratio of 1:10 (e.g. 400μL concentrate + 4.0mL buffer, enough for 4 microplate strips with 32 wells). 15.2.1.3 Microwell strips coated with anti-antibody Cut the tin foil bag along the outer edge of the horizontal wrinkle, take out the required number of microwell plates and frames, put the unused microwell plates into the original tin foil bag and reseal it with the provided desiccant, and store at 2℃～8℃. 15.2.2 Sample preparation
Take 20μL of the extract in 10.1 for analysis. Samples with high residues should be further diluted with distilled water. 15.2.3 Determination
Put the kit at room temperature (19℃～25℃) for 1h2h before use. 15.2.3.1
Insert the number of strips used for standards and samples (at least calculated by two parallel experiments) into the microwell rack, and record the position of the standards and samples.
15.2.3.2 Add 100μl of the diluted antibody solution to each microwell. Mix well and incubate at room temperature for 15min. 15.2.3.3 Pour out the liquid in the wells, turn the microwell rack upside down on absorbent paper and tap (tap 3 times for each row) to ensure that the liquid in the wells is completely removed. Fill the wells with 250uL of distilled water, pour out the liquid in the microwells again, and repeat the operation two more times. 15.2.3.4 Add 20uL of standard or treated sample to each microwell. At least two parallel experiments are performed for standards and samples. 15.2.3.5 Add 100uL of diluted enzyme marker and incubate at room temperature for 30 minutes. 15.2.3.6 Pour out the liquid in the wells, turn the microwell rack upside down on absorbent paper and tap (tap 3 times for each row) to ensure that the liquid in the wells is completely removed. Fill the wells with 250uL of distilled water, pour out the liquid in the microwells again, and repeat the operation two more times. 15.2.3.7 Add 50 μL of enzyme substrate and 50 μL of colorimetric reagent to the microwells, mix thoroughly and incubate at room temperature in the dark for 15 min. 15.2.3.8 Add 100 μL of reaction stop solution to the microwell. Mix well and measure the absorbance at 450 nm as soon as possible. 15.3 Calculation of results
Calculate the ratio of the absorbance of the standard solution and sample solution to the blank solution. See formula (3): Relative absorbance (%) = B/B. ×100 Where:
B——the absorbance of the standard (or sample) solution; Bo——the absorbance of the blank (standard solution with a concentration of 0). 530
（3）
GB/T5009.192—2003
The calculated relative absorbance value (%) is plotted against the natural logarithm of the clenbuterol concentration (ng/L) in a semi-logarithmic coordinate system. The calibration curve is linear in the range of 0.004ng~0.054ng (200ng/L～2000ng/L). The corresponding sample concentration can be calculated from the calibration curve.
See formula (4):
Wherein:
mx1000
-The content of clenbuterol in the sample, in micrograms per kilogram (or microgram per liter) [μg/kg (or μg/1) lA-The clenbuterol content corresponding to the relative absorbance value (%) of the sample, in nanograms per liter (ng/L); f-sample dilution factor:
m-sample sampling volume, in grams (or milliliters) g (or mL)]. The calculation result is expressed to two decimal places. Positive results need to be confirmed by the first method. Precision
The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. (4)
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