论文部分内容阅读
Abstract ［Objectives］ Uncertainty in liquid chromatography-mass spectrometry detection of nitrofuran metabolite residues in chicken bone was analyzed to find out the influencing factors.
［Methods］ According to relevant theories such as Evaluation and expression of uncertainty in measurement , the uncertainty in the results of nitrofuran metabolites in chicken bone was analyzed and calculated combining with mathematical models.
［Results］ Under the addition amount of 1.0 μg/kg , the uncertainty of the four nitrofuran metabolites was as follows: nitrofurazone metabolites CSEM =0.981 μg/kg, U=0.070 μg/kg, k =2; furazolidone metabolites CAOZ =1.032 μg/kg, U=0.061 μg/kg, k =2; furaltadone metabolites CAMOZ =1.068 μg/kg, U=0.076 μg/kg, k =2; furadantin metabolites CAHD =1.007 μg/kg, U=0.046 μg/kg, k =2.
［Conclusions］ The uncertainty in the measurement process mainly comes from the purity of the standards, the preparation process, the sample weight, the number of repeated measurement and the recovery of spiked sample. The research results are of great significance for reducing uncertainty and improving data accuracy and reliability during actual operation.
Key words Liquid chromatography-tandem mass spectrometry; Chicken bone; Nitrofuran metabolite; Uncertainty
Received: February 23, 2020Accepted: April 5, 2020
Xiaolin ZHANG (1980-), female, P. R. China, intermediate engineer, devoted to research about food safety and quality control.
*Corresponding author. E-mail: zx18567@163.com.
Nitrofuran drugs are a class of broad-spectrum antibacterial drugs, named after the nitro group in molecules. Because of their advantages such as low price and strong sterilization effect, nitrofuran drugs are widely used in aquaculture and livestock product breeding［1-2］. Nitrofurans are easily metabolized in the body, and their metabolites bind with proteins in the body to form stable compounds, which have a long storage time in the body, can induce gene mutations, and result in the hazards of high teratogenicity, carcinogenesis, and mutagenesis［3-5］. In 1997, the European Union lists nitrofuran drugs as Class A illicit drugs, and Japan and the United State also issued regulations［6］. Announcement 235 of the Ministry of Agriculture and Announcement 560 of the Ministry of Agriculture［7-8］ list nitro-furaltadone, nitro-furazolidone, nitro-furadantin and nitrofurazone as prohibited veterinary drugs, which must not be detected in all food animals. At present, the detection methods for nitrofuran metabolites mainly include immunocolloid gold method［9］, enzyme-linked immunoassay［10］, high performance liquid chromatography［11］ and liquid chromatography-tandem mass spectrometry (LC-MS/MS)［12-13］. The immuno-colloidal gold method can only perform qualitative detection and cannot perform quantitative detection. Although enzyme-linked immunoassay can perform quantitative detection, the data error is large. Although high-performance liquid chromatography also can perform qualitative detection, the precision is not high. In general, HPLC-MS/MS is the one achieving highest precision and obtains most accurate data among the several detection methods. Measurement uncertainty is an important parameter representing the dispersion of measured values［14］, as well as an important indicator for evaluating results and levels. GB / T 27025-2008 clearly stipulates the requirement of having uncertainty or the procedures for evaluating uncertainty［15］. With the gradual improvement of testing capability and accuracy of testing data, the uncertainty in the qualification process has become a task that must be completed in the testing process. The study of uncertainty is of great significance to the accuracy of the test results. Zhu et al. ［13］ and Guo et al. ［16］ used liquid chromatography-mass spectrometry to evaluate the uncertainty of nitrofuran metabolites in aquatic products according to Announcement 783-1-2006 of the Ministry of Agriculture. Based on JJF1059-1999 Evaluation and expression of uncertainty in measurement and CNAS-GL06 Guidance on Evaluatinng the Uncertainnty in Chemical Analysis , uncertainty analysis was carried out on the internal standard method of nitrofuran metabolite residues in chicken bone to find out the influencing factors, so as to ensure the accuracy of the data and improve the quality of test data.
Materials and Methods
Experimental materials
Instruments and equipment
Ultra-high performance liquid chromatography-tandem mass spectrometer (1260-6460, Agilent, USA); water-bathing constant temperature vibrator; MS3 Digital vortex oscillator (German IKA company); electronic balance; high-speed freezing centrifuge; pure water machine; pH meter; 5 ml , 1 ml and 100 μl pipettes.
Reagents and materials
Nitrofurazone metabolite hydrochloride SEM·HCL (purity 99.8%), Germany Dr; furadantin metabolite hydrochloride AHD·HCL (purity 99.3%), Germany Dr; furazolidone metabolite AOZ (purity 99.3%), German Dr.; furaltadone metabolite AMOZ (purity 99.6%), German Dr; nitrofurazone metabolite isotope standard SEM-13 C-15 N2 (hydrochloride), WITEGA company; furadantin metabolite isotope standard AHD-13 C3, WITEGA company; furazolidone metabolite deuteride AOZ-D4, Germany Dr.; furaltadone metabolite deuteride AMOZ-D5, Germany Dr; methanol, acetonitrile, ethyl acetate, n-hexane, formic acid and o-nitrobenzaldehyde, all chromatographically pure, purchased from Fisher Scientific; Concentrated hydrochloric acid, sodium hydroxide, ammonium acetate and dipotassium hydrogen phosphate, all analytically pure, purchased from Shanghai Sinopharm Group; ultrapure water. The experimental sample was from Tianbo Food Ingredients Co., Ltd.
Experimental methods
Standard solution preparation
Standard stock solution: Certain amounts of AOZ (10.06 mg), SEM HCL (14.9 mg), AMOZ (10.04 mg), AHD HCL (13.26 mg), AOZ-D4 (10.0 mg), SEM-13 C-15 N2 10.0 mg, AMOZ-13 C3 10.04 mg and AHD-13 C3 (10.0 mg) were accurately weighed, respectively. Each weighed sample was dissolved and diluted with acetonitrile to constant weight in a 100 ml volumetric flask, obtaining 1.0 mg/ml standard stock solutions.
Mixed standard stock solution: A certain amount of each 1.0 mg/ml standard stock solutions (1.0 ml) was transferred using 1 000 μl pipettes into the same 10 ml volumetric flask, and the mixed solution was diluted with acetonitrile to constant volume. A 10.0 μg/L mixed standard solution was prepared in the same way.
Mixed internal standard working solution: A 10.0 μg/L mixed internal standard working solution was prepared by dilution according to the above method.
Preparation of mixed standard solutions: Certain amounts of the 10.0 μg/L mixed standard working solution (50, 100 μl) and 100.0 μg/L mixed standard solution (20, 50, 100 μl) to five 50 ml centrifuge tubes, respectively. Into each centrifuge tube, 20 μl of mixed internal standard working solution was added, and 0.50, 1.0, 2.0, 5.0 and 10.0 μg/L standard solutions were prepared according to the steps of pretreatment and determined on the machine.
Pretreatment
A 2.0 g of sample was weighed and added into a mortar, into which 2.5-4.0 g of diatomaceous earth was added for fine grinding. The ground sample was transferred to a 50 ml centrifuge tube and added with 20 μl of the 10.0 μg/L internal standard. A certain amount of external standard solution in addition to the internal standard was added during sample recovery, and then 10 ml of 0.2 mol/L hydrochloric acid and o-nitrobenzaldehyde (0.015 g of o-nitrobenzaldehyde was weighed and dissolved with methanol, obtaining a solution, which was diluted with methanol to 1 ml, and 100 μl was corresponding to a sample) were added. The sample was homogenized at 11 500 r/min for 1 min , and was placed in a 37 ℃ thermostatic oscillator for 16 h in the dark. The centrifuge tube was taken out and cooled to room temperature, and added with dipotassium hydrogen phosphate and sodium hydroxide to adjust the pH to about 7.5. The obtained mixture was added with 5 ml of ethyl acetate, shaken for 20 min, centrifuged at 9 000 r/min for 5 min. The supernatant was transferred to a 10 ml centrifuge tube and extracted twice repeatedly. The supernatants were merged and blown to near dryness at 40 ℃. The extract was added with 1 ml of 0.1% formic acid solution and 2 ml of n-hexane, vortex-treated for 2 min, and centrifuged at 5 000 r/min for 5 min. The lower layer of liquid was filtered through a 0.22 μm water-based membrane, and then tested. Sources of measurement uncertainty
The uncertainty mainly includes the following aspects: the uncertainty introduced by the purity of standards (including internal and external standards), the uncertainty introduced during the preparation of standard solutions, the uncertainty introduced during the preparation process of mixed standard solutions (including the mixed solutions of internal and external standards), the uncertainty introduced by weighed sample amount, the uncertainty introduced by repeated measurements, the uncertainty introduced by the resolution of equipment, the uncertainty introduced by reproducibility limit R , and the uncertainty introduced by instrument resolution.
Results and Analysis
Uncertainty assessment
Mathematical model building
In this study, the residues of nitrofuran metabolites in chicken bone were calculated according to the following formula (1):
X=R×c×VRS×m (1)
Wherein R represents the ratio of the peak area of the analyte to that of the internal standard in the sample solution; c represents the concentration of the analyte in the mixed matrix standard solution (ng/ml); V is the final constant volume of the sample liquid in milliliters (ml); RS represents the ratio of the peak area of the analyte to that of the internal standard in the mixed matrix standard solution; and m represents sample mass (g).
Combined uncertainty formula
The combined uncertainty formula was calculated as follows:
urel (c) =u2rel (1) +u2rel (2) +u2rel (3) +u2rel (4) +u2rel (5) +u2rel (6) +u2rel (7) (2)
Wherein urel (1) is the uncertainty introduced by the purity of standards; urel (2) is the uncertainty introduced during the preparation of the standard solutions; urel (3) is the uncertainty introduced by standard solution preparation; urel (4) is the uncertainty introduced by internal standard solution preparation; urel (5) is the uncertainty introduced by weighed sample amount; urel (6) is the uncertainty introduced by repeated measurements; and urel (7) is the uncertainty introduced by recovery of spiked sample.
Component calculation of uncertainty
Uncertainty introduced by standard purity
Because the expanded uncertainty of SEM·HCL u =0.92% was k times the standard uncertainty, and included the factor k =2, the standard uncertainty introduced by its purity was: urel (SEM·HCL) = u /k=0.92%/2 =0.004 6. The expanded uncertainty of AMOZ was u =3.73%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AMOZ) = u /k=3.73%/2=0.018 65.
The expanded uncertainty of AOZ was u =1.73%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AOZ) = u /k=1.73%/2=0.008 65.
The expanded uncertainty of AHD was u =2.99% including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AHD) = u /k=2.99%/2=0.014 95.
Uncertainty introduced by internal standard purity
The expanded uncertainty of AOZ-D4 was u =1.95%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AOZ-D4) = u /k=1.95%/2=0.009 75.
The expanded uncertainty of AMOZ-D5 was u =1.15%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AMOZ-D5) = u /k=1.15%/2=0.005 75.
The purity of AHD-13 C3 was 99.8%±0.2%, and the standard uncertainty introduced by it was:
urel (AHD-13 C3) =0.2%99.8%×3=0.001 16.
The purity of SEM-13 C-5N2 was 99.98%±0.2%, and the standard uncertainty introduced by it was:
urel (SEM-13 C-5N2) =0.2%3×99.8%=0.001 16.
Uncertainty introduced by standard solution preparation
Uncertainty introduced by weighing reference materials
The weighing uncertainty of m 1 comes from three aspects: a, the uncertainty caused by repeated weighing, which can be expressed by uA ( m 1) through 10 times of repeated weighing, b, the uncertainty caused by inaccurate weighing of balance, and c, the uncertainty introduced by the readability (digital resolution) of the balance scale.
a: m 1 was measured for 10 times repeatedly (making m1 consistent with actual weight as much as possible). The Besser method was used to calculate its standard deviation, while the standard uncertainty is equal to 1 time of the standard deviation:
uA(m1)=s(m1)∑10i=1(m1i -m1 )210-1=0.029 mg.
b: The maximum allowable error of electronic balance was ± 0.03 mg, so the half-width interval a 1 ( m 1)=0.03 mg, and the distribution belonged to uniform distribution. The part of uncertainty included factor k1 (m1)=3, and it standard uncertainty was: uB1 (m1)=s(m1)=a1(m1)k1(m1)=0.033 mg=0.017 3 mg.
C: The standard uncertainty component introduced by balance resolution was uB2 ( m 1)=0.005 mg, including k2( m 1)=3, and its standard uncertainty was:
uB2 (m1)=a2(m1)k2(m1)=0.0053 mg=0.002 9 mg.
The combined uncertainty was:
urel (m1)=u2A(m1)+u2B1 (m1)+u2B2 (m1) =0.033 9 mg.
The standard uncertainty of each compound was:
urel (SEM·HCL) =0.033 9 mg/14.9 mg=0.002 275,
urel (AMOZ) =0.033 9 mg/10.04 mg=0.003 376,
urel (AOZ) =0.033 9 mg/10.06 mg=0.003 370,
and urel (AHD·HCL) =0.033 9 mg/13.26 mg=0.002 557.
Uncertainty introduced by the constant volume of the standard stock solutions
The uncertainty introduced by the constant volume of the stock solution preparation comes from three aspects: first, the uncertainty caused by repeatability uA (V), second, the uncertainty caused by tolerance of volumetric flask uB1 (V) , and third, the uncertainty aused by temperature coefficient uB2 (V).
The operation of diluting to constant volume was performed with a 100 ml volumetric flask for 10 times. The Besser method was used to calculate its standard deviation, while the standard uncertainty was equal to 1 time of the standard deviation:
uA(V)=s(V)=∑10i=1［(mi-m/ρ)］210-1 =0.02 ml
The standard stock solutions were prepared by dissolving and diluting with acetonitrile to a 100 ml volumetric flask. According to the verification regulation of working glass container JJG196-2006 Commonly Used Glass Measuring Tools , 100 ml volumetric flasks are A-class volumetric flasks with a tolerance of ± 0.10 ml, so the half-width interval a 1(V)=0.10 ml. Suppose the uncertainty caused by the tolerance of volumetric flasks obeyed a triangular distribution, and included factor k 1(V)=6, the standard uncertainty uB1 (V) was:
uB1 (V)=a1(V)k1(V)=0.1 ml 6 =0.04 ml
According to the verification regulation, the verification of volumetric flasks is carried out at 20℃. The experiment was supposed to be carried out at (20±4) ℃. Because the volume expansion coefficient of liquid is much larger than that of glass, only the volume expansion coefficient of liquid needs to be considered. The volume expansion coefficient of acetonitrile is 1.37×10-3 /℃ , and the resulting volume change was ±(100×4×1.37×10-3 ) =±0.548 ml, so the half-width interval was a 2(V)=0.548 ml. Suppose the uncertainty caused by the temperature coefficient was in interval distribution, and included factor k 2=3, the standard uncertainty was: uB2 (V)=a2(V)k2(V)=0.5483 ml =0.316 4 ml
The combined uncertainty was: uc(V)=u2A(V)+u2B1 (V)+u2B2 (V) =0.319 5 ml
Its relative uncertainty was:
urel (V)=uc(V)V=0.319 5100 =0.003 195
Xiaolin ZHANG et al. Uncertainty Analysis of Residues of Nitrofuran Metabolites in Chicken Bone
Uncertainty introduced by standard solution dilution
The mixed intermediate solutions were obtained by stepwise dilution, from 1.0 mg/ml to 10.0 μg/L using 10 ml volumetric flasks and 1 000 μl pipettes. The maximum allowable error of the 10 ml volumetric flasks is ± 0.020 ml, and the maximum allowable error of the 1 000 μl pipettes is ± 1.0%. According to the uniform distribution, k =3, the uncertainty was:
urel (a)=0.02010×3=0.001 15
urel (b)=1.0 %3=0.005 77
The combined uncertainty was:
urel (X)=5u2rel (a)+5u2rel (b) =0.013 16
Uncertainty introduced by internal standard solution preparation
The uncertainty introduced by weighed amounts of internal standards during internal standard solution preparation was:
urel (SEM-13 C-15 N2)=0.033 9 mg/10.0 mg=0.003 39
urel (AMOZ-13 C3)=0.033 9 mg/10.04 mg=0.003 376
urel (AHD-13 C3)=0.033 9 mg/10.0 mg=0.003 39
urel (AOZ-D4)=0.033 9 mg/10.0 mg=0.003 39
The uncertainty introduced by diluting to constant weight in stock solution preparation and the dilution process in working solution preparation was the same as "Uncertainty introduced by the constant volume of the standard stock solutions" and "Uncertainty introduced by standard solution dilution".
Uncertainty introduced by weighed amount of sample
The resolution of the balance is 0.1 mg, and the half-width interval was a2=0.05 mg. The uncertainty included k 3=3, and the sample weight was 2.00 g, so its standard uncertainty was:
urel(5) =a3k3= 0.05 mg2.00×3=0.014 4
Uncertainty introduced by repeated measurement
A certain concentration was repeatedly measured for 10 times, and the uncertainty was calculated according to the Bessel formula as follows:
ufrep =S(X)n (3)
The formula for standard uncertainty:
urel(6) =ufrep X (4)
Relative uncertainty introduced by recovery of spiked sample urel (R) A recovery test was carried out on the sample for 6 times at the level of 2 μg/kg, and the uncertainty was calculated according to formula (5). The uncertainty caused by recovery of spiked sample was in uniform distribution, including k =3.
urel (R)=(1-Rk)2+(S6)2 (5)
Combined relative standard uncertainty and uncertainty
The above uncertainties were independent of each other, and regardless of the correlation between the components, the combined uncertainty urel (c) was calculated according to formula (2) and the uncertainty uc (X) was calculated according to formula (6).
uc(X)=urel (c).X (6)
At confidence level of 95%, the expanded uncertainty ( k =2) was U=kuc (X). The calculated results were as follows: nitrofurazone metabolites CSEM =0.981 μg/kg, U=0.070 μg/kg, k =2; furazolidone metabolites CAOZ =1.032 μg/kg, U=0.061 μg/kg, k =2; furaltadone metabolites CAMOZ =1.068 μg/kg, U=0.076 μg/kg, k =2; furadantin metabolites CAHD =1.007 μg/kg, U=0.046 μg/kg, k =2.
Conclusions and Discussion
According to the measurement uncertainty evaluation method, the sources of uncertainty of nitrofuran metabolites in chicken bone were analyzed. By establishing mathematical models, each uncertainty component was evaluated and calculated. The uncertainty in the measurement process mainly comes from the purity of the standards, the preparation process, the sample weight, the number of repeated measurement and the recovery of spiked sample. Therefore, in the daily testing, we should choose standards with higher purity, and use high-precision glass measuring devices or pipettes and balances in the preparation process. Meanwhile, we should make multiple measurements during the determination process to reduce the uncertainty generated during the detection process, thereby improving the accuracy of the detection.
References
［1］ ZHU WX, LIU YF, LIANG W. Progress on nitrofuran drug residues in animal-borne food［J］. Progress in Veterinary Medicine, 2010, 31(2): 99-102. (in Chinese)
［2］ TANG HM, ZENG F, LI CH. Progress in analytical methods of nitrofuran antibiotics and their metabolites in food: A review［J］. Food Safety and Quality Detection Technology, 2016, 7(10): 3952-3959. (in Chinese)
［3］ WANG Q, MA B, LYU HY, et al. Evaluation of the safety of nitrofurans and their metabolites in food to human health［J］. Chinese Fishery Quality and Standards, 2013, 3(2): 4-10. (in Chinese) ［4］ JIANG Y, DING T, XU JZ. Study on the elimination rule of nitrofuran drugs in the tissue of Procambarus clarkii［J］. Animal Husbandry and Veterinary Medicine, 2008, 40(2): 34-37. (in Chinese)
［5］ HOOGENBOOM LA, VAN BRUCHEM GD, et al. Absorption of amutagenic metabolite released from protein-bound residues of furazolidone［J］. Environ Toxicol Pharmacol, 2002, 11(3): 273-287.
［6］ European Food Safety Authority. Opinion of the scientific panel on food additives,flavourings,processing aids and materials in contact with food on a request from the Com-mission related to Semicarbazide in food［J］. EFSA J,2005(219): 1-36．
［7］ Ministry of Agriculture of the Peoples Republic of China. Announcement 235 of the Ministry of Agriculture of the Peoples Republic of China［EB/OL］. (2002-12-24) ［2017-03-05］. http:/ /www. Moa. Gov. cn/zwllm/nybz/200803/t20080304_1028649. htm. (in Chinese)
［8］ Ministry of Agriculture of the Peoples Republic of China. Announcement 560 of the Ministry of Agriculture of the Peoples Republic of China ［EB/OL］. (2005-10-28) ［2017-03-05］. http:/ /www. Moa. Gov. cn/zwllm/tzgg/gg/200511/t20051117_496523.htm. (in Chinese)
［9］ LIU AC, LIU C, ZHAO Y, et al. Rapid determination of nitrofuran metabolites in aquatic products by colloidal gold method［J］. Acta Agriculturae Zhejiangensis,2013,25(1): 95-102. (in Chinese)
［10］ JIANG HW. ELISA Detection of Residues of the nitro-furan chemicals in the Animal Products［J］. Shaanxi Journal of Agricultural Sciences, 2006(5): 53-55. (in Chinese)
［11］ WANG Y, CAI YQ, JIA DF, et al. Determination of nitrofuran metabolite residues in aquatic products by high performance liquid chromatography［J］. Chinese Journal of Analysis Laboratory,2009,28(12): 86-90. (in Chinese)
［12］ LI XP, PENG JJ, HU HG, et al. Uncertainty analysis of determination of nitrofuran metabolites in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry［J］. Agriculture and Technology, 2016, 36(23): 1-4. (in Chinese)
［13］ ZHU ZT, LEI MK, PENG F, et al. Uncertainty evaluation in determination of nitrofuran metabolites residues in aquatic product by liquid chromatography-tandem mass spectrometry［J］. Food Safety and Quality Detection Technology, 2015, 6(7): 2857-2862. (in Chinese)
［14］ CORDERO R, SECKMEYER G, PISSULLA D, et al. Uncertainty evaluation of the spectral UV irradiance evaluated by u-sing the UVSPEC radiative transfer model［J］. Opt Com-mun, 2007, 276(12): 44-53. (in Chinese)
［15］ Technical Committee on Certification and Accreditation of Standardization. GB/T 27025-2008: General requirements for the competence of testing and calibration laboratories［S］. Beijing: China Standard Press, 2008. (in Chinese)
［16］ GUO LN, LIU CW, FU YC. Evaluation of the uncertainty for the determination of nitrofuran metabolites in aquatic products by LC-MS/MS［J］. Modern Food, 2017(2): 101-105. (in Chinese)
Editor: Yingzhi GUANGProofreader: Xinxiu ZHU
［Methods］ According to relevant theories such as Evaluation and expression of uncertainty in measurement , the uncertainty in the results of nitrofuran metabolites in chicken bone was analyzed and calculated combining with mathematical models.
［Results］ Under the addition amount of 1.0 μg/kg , the uncertainty of the four nitrofuran metabolites was as follows: nitrofurazone metabolites CSEM =0.981 μg/kg, U=0.070 μg/kg, k =2; furazolidone metabolites CAOZ =1.032 μg/kg, U=0.061 μg/kg, k =2; furaltadone metabolites CAMOZ =1.068 μg/kg, U=0.076 μg/kg, k =2; furadantin metabolites CAHD =1.007 μg/kg, U=0.046 μg/kg, k =2.
［Conclusions］ The uncertainty in the measurement process mainly comes from the purity of the standards, the preparation process, the sample weight, the number of repeated measurement and the recovery of spiked sample. The research results are of great significance for reducing uncertainty and improving data accuracy and reliability during actual operation.
Key words Liquid chromatography-tandem mass spectrometry; Chicken bone; Nitrofuran metabolite; Uncertainty
Received: February 23, 2020Accepted: April 5, 2020
Xiaolin ZHANG (1980-), female, P. R. China, intermediate engineer, devoted to research about food safety and quality control.
*Corresponding author. E-mail: zx18567@163.com.
Nitrofuran drugs are a class of broad-spectrum antibacterial drugs, named after the nitro group in molecules. Because of their advantages such as low price and strong sterilization effect, nitrofuran drugs are widely used in aquaculture and livestock product breeding［1-2］. Nitrofurans are easily metabolized in the body, and their metabolites bind with proteins in the body to form stable compounds, which have a long storage time in the body, can induce gene mutations, and result in the hazards of high teratogenicity, carcinogenesis, and mutagenesis［3-5］. In 1997, the European Union lists nitrofuran drugs as Class A illicit drugs, and Japan and the United State also issued regulations［6］. Announcement 235 of the Ministry of Agriculture and Announcement 560 of the Ministry of Agriculture［7-8］ list nitro-furaltadone, nitro-furazolidone, nitro-furadantin and nitrofurazone as prohibited veterinary drugs, which must not be detected in all food animals. At present, the detection methods for nitrofuran metabolites mainly include immunocolloid gold method［9］, enzyme-linked immunoassay［10］, high performance liquid chromatography［11］ and liquid chromatography-tandem mass spectrometry (LC-MS/MS)［12-13］. The immuno-colloidal gold method can only perform qualitative detection and cannot perform quantitative detection. Although enzyme-linked immunoassay can perform quantitative detection, the data error is large. Although high-performance liquid chromatography also can perform qualitative detection, the precision is not high. In general, HPLC-MS/MS is the one achieving highest precision and obtains most accurate data among the several detection methods. Measurement uncertainty is an important parameter representing the dispersion of measured values［14］, as well as an important indicator for evaluating results and levels. GB / T 27025-2008 clearly stipulates the requirement of having uncertainty or the procedures for evaluating uncertainty［15］. With the gradual improvement of testing capability and accuracy of testing data, the uncertainty in the qualification process has become a task that must be completed in the testing process. The study of uncertainty is of great significance to the accuracy of the test results. Zhu et al. ［13］ and Guo et al. ［16］ used liquid chromatography-mass spectrometry to evaluate the uncertainty of nitrofuran metabolites in aquatic products according to Announcement 783-1-2006 of the Ministry of Agriculture. Based on JJF1059-1999 Evaluation and expression of uncertainty in measurement and CNAS-GL06 Guidance on Evaluatinng the Uncertainnty in Chemical Analysis , uncertainty analysis was carried out on the internal standard method of nitrofuran metabolite residues in chicken bone to find out the influencing factors, so as to ensure the accuracy of the data and improve the quality of test data.
Materials and Methods
Experimental materials
Instruments and equipment
Ultra-high performance liquid chromatography-tandem mass spectrometer (1260-6460, Agilent, USA); water-bathing constant temperature vibrator; MS3 Digital vortex oscillator (German IKA company); electronic balance; high-speed freezing centrifuge; pure water machine; pH meter; 5 ml , 1 ml and 100 μl pipettes.
Reagents and materials
Nitrofurazone metabolite hydrochloride SEM·HCL (purity 99.8%), Germany Dr; furadantin metabolite hydrochloride AHD·HCL (purity 99.3%), Germany Dr; furazolidone metabolite AOZ (purity 99.3%), German Dr.; furaltadone metabolite AMOZ (purity 99.6%), German Dr; nitrofurazone metabolite isotope standard SEM-13 C-15 N2 (hydrochloride), WITEGA company; furadantin metabolite isotope standard AHD-13 C3, WITEGA company; furazolidone metabolite deuteride AOZ-D4, Germany Dr.; furaltadone metabolite deuteride AMOZ-D5, Germany Dr; methanol, acetonitrile, ethyl acetate, n-hexane, formic acid and o-nitrobenzaldehyde, all chromatographically pure, purchased from Fisher Scientific; Concentrated hydrochloric acid, sodium hydroxide, ammonium acetate and dipotassium hydrogen phosphate, all analytically pure, purchased from Shanghai Sinopharm Group; ultrapure water. The experimental sample was from Tianbo Food Ingredients Co., Ltd.
Experimental methods
Standard solution preparation
Standard stock solution: Certain amounts of AOZ (10.06 mg), SEM HCL (14.9 mg), AMOZ (10.04 mg), AHD HCL (13.26 mg), AOZ-D4 (10.0 mg), SEM-13 C-15 N2 10.0 mg, AMOZ-13 C3 10.04 mg and AHD-13 C3 (10.0 mg) were accurately weighed, respectively. Each weighed sample was dissolved and diluted with acetonitrile to constant weight in a 100 ml volumetric flask, obtaining 1.0 mg/ml standard stock solutions.
Mixed standard stock solution: A certain amount of each 1.0 mg/ml standard stock solutions (1.0 ml) was transferred using 1 000 μl pipettes into the same 10 ml volumetric flask, and the mixed solution was diluted with acetonitrile to constant volume. A 10.0 μg/L mixed standard solution was prepared in the same way.
Mixed internal standard working solution: A 10.0 μg/L mixed internal standard working solution was prepared by dilution according to the above method.
Preparation of mixed standard solutions: Certain amounts of the 10.0 μg/L mixed standard working solution (50, 100 μl) and 100.0 μg/L mixed standard solution (20, 50, 100 μl) to five 50 ml centrifuge tubes, respectively. Into each centrifuge tube, 20 μl of mixed internal standard working solution was added, and 0.50, 1.0, 2.0, 5.0 and 10.0 μg/L standard solutions were prepared according to the steps of pretreatment and determined on the machine.
Pretreatment
A 2.0 g of sample was weighed and added into a mortar, into which 2.5-4.0 g of diatomaceous earth was added for fine grinding. The ground sample was transferred to a 50 ml centrifuge tube and added with 20 μl of the 10.0 μg/L internal standard. A certain amount of external standard solution in addition to the internal standard was added during sample recovery, and then 10 ml of 0.2 mol/L hydrochloric acid and o-nitrobenzaldehyde (0.015 g of o-nitrobenzaldehyde was weighed and dissolved with methanol, obtaining a solution, which was diluted with methanol to 1 ml, and 100 μl was corresponding to a sample) were added. The sample was homogenized at 11 500 r/min for 1 min , and was placed in a 37 ℃ thermostatic oscillator for 16 h in the dark. The centrifuge tube was taken out and cooled to room temperature, and added with dipotassium hydrogen phosphate and sodium hydroxide to adjust the pH to about 7.5. The obtained mixture was added with 5 ml of ethyl acetate, shaken for 20 min, centrifuged at 9 000 r/min for 5 min. The supernatant was transferred to a 10 ml centrifuge tube and extracted twice repeatedly. The supernatants were merged and blown to near dryness at 40 ℃. The extract was added with 1 ml of 0.1% formic acid solution and 2 ml of n-hexane, vortex-treated for 2 min, and centrifuged at 5 000 r/min for 5 min. The lower layer of liquid was filtered through a 0.22 μm water-based membrane, and then tested. Sources of measurement uncertainty
The uncertainty mainly includes the following aspects: the uncertainty introduced by the purity of standards (including internal and external standards), the uncertainty introduced during the preparation of standard solutions, the uncertainty introduced during the preparation process of mixed standard solutions (including the mixed solutions of internal and external standards), the uncertainty introduced by weighed sample amount, the uncertainty introduced by repeated measurements, the uncertainty introduced by the resolution of equipment, the uncertainty introduced by reproducibility limit R , and the uncertainty introduced by instrument resolution.
Results and Analysis
Uncertainty assessment
Mathematical model building
In this study, the residues of nitrofuran metabolites in chicken bone were calculated according to the following formula (1):
X=R×c×VRS×m (1)
Wherein R represents the ratio of the peak area of the analyte to that of the internal standard in the sample solution; c represents the concentration of the analyte in the mixed matrix standard solution (ng/ml); V is the final constant volume of the sample liquid in milliliters (ml); RS represents the ratio of the peak area of the analyte to that of the internal standard in the mixed matrix standard solution; and m represents sample mass (g).
Combined uncertainty formula
The combined uncertainty formula was calculated as follows:
urel (c) =u2rel (1) +u2rel (2) +u2rel (3) +u2rel (4) +u2rel (5) +u2rel (6) +u2rel (7) (2)
Wherein urel (1) is the uncertainty introduced by the purity of standards; urel (2) is the uncertainty introduced during the preparation of the standard solutions; urel (3) is the uncertainty introduced by standard solution preparation; urel (4) is the uncertainty introduced by internal standard solution preparation; urel (5) is the uncertainty introduced by weighed sample amount; urel (6) is the uncertainty introduced by repeated measurements; and urel (7) is the uncertainty introduced by recovery of spiked sample.
Component calculation of uncertainty
Uncertainty introduced by standard purity
Because the expanded uncertainty of SEM·HCL u =0.92% was k times the standard uncertainty, and included the factor k =2, the standard uncertainty introduced by its purity was: urel (SEM·HCL) = u /k=0.92%/2 =0.004 6. The expanded uncertainty of AMOZ was u =3.73%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AMOZ) = u /k=3.73%/2=0.018 65.
The expanded uncertainty of AOZ was u =1.73%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AOZ) = u /k=1.73%/2=0.008 65.
The expanded uncertainty of AHD was u =2.99% including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AHD) = u /k=2.99%/2=0.014 95.
Uncertainty introduced by internal standard purity
The expanded uncertainty of AOZ-D4 was u =1.95%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AOZ-D4) = u /k=1.95%/2=0.009 75.
The expanded uncertainty of AMOZ-D5 was u =1.15%, including the factor k =2, and the standard uncertainty introduced by its purity was: urel (AMOZ-D5) = u /k=1.15%/2=0.005 75.
The purity of AHD-13 C3 was 99.8%±0.2%, and the standard uncertainty introduced by it was:
urel (AHD-13 C3) =0.2%99.8%×3=0.001 16.
The purity of SEM-13 C-5N2 was 99.98%±0.2%, and the standard uncertainty introduced by it was:
urel (SEM-13 C-5N2) =0.2%3×99.8%=0.001 16.
Uncertainty introduced by standard solution preparation
Uncertainty introduced by weighing reference materials
The weighing uncertainty of m 1 comes from three aspects: a, the uncertainty caused by repeated weighing, which can be expressed by uA ( m 1) through 10 times of repeated weighing, b, the uncertainty caused by inaccurate weighing of balance, and c, the uncertainty introduced by the readability (digital resolution) of the balance scale.
a: m 1 was measured for 10 times repeatedly (making m1 consistent with actual weight as much as possible). The Besser method was used to calculate its standard deviation, while the standard uncertainty is equal to 1 time of the standard deviation:
uA(m1)=s(m1)∑10i=1(m1i -m1 )210-1=0.029 mg.
b: The maximum allowable error of electronic balance was ± 0.03 mg, so the half-width interval a 1 ( m 1)=0.03 mg, and the distribution belonged to uniform distribution. The part of uncertainty included factor k1 (m1)=3, and it standard uncertainty was: uB1 (m1)=s(m1)=a1(m1)k1(m1)=0.033 mg=0.017 3 mg.
C: The standard uncertainty component introduced by balance resolution was uB2 ( m 1)=0.005 mg, including k2( m 1)=3, and its standard uncertainty was:
uB2 (m1)=a2(m1)k2(m1)=0.0053 mg=0.002 9 mg.
The combined uncertainty was:
urel (m1)=u2A(m1)+u2B1 (m1)+u2B2 (m1) =0.033 9 mg.
The standard uncertainty of each compound was:
urel (SEM·HCL) =0.033 9 mg/14.9 mg=0.002 275,
urel (AMOZ) =0.033 9 mg/10.04 mg=0.003 376,
urel (AOZ) =0.033 9 mg/10.06 mg=0.003 370,
and urel (AHD·HCL) =0.033 9 mg/13.26 mg=0.002 557.
Uncertainty introduced by the constant volume of the standard stock solutions
The uncertainty introduced by the constant volume of the stock solution preparation comes from three aspects: first, the uncertainty caused by repeatability uA (V), second, the uncertainty caused by tolerance of volumetric flask uB1 (V) , and third, the uncertainty aused by temperature coefficient uB2 (V).
The operation of diluting to constant volume was performed with a 100 ml volumetric flask for 10 times. The Besser method was used to calculate its standard deviation, while the standard uncertainty was equal to 1 time of the standard deviation:
uA(V)=s(V)=∑10i=1［(mi-m/ρ)］210-1 =0.02 ml
The standard stock solutions were prepared by dissolving and diluting with acetonitrile to a 100 ml volumetric flask. According to the verification regulation of working glass container JJG196-2006 Commonly Used Glass Measuring Tools , 100 ml volumetric flasks are A-class volumetric flasks with a tolerance of ± 0.10 ml, so the half-width interval a 1(V)=0.10 ml. Suppose the uncertainty caused by the tolerance of volumetric flasks obeyed a triangular distribution, and included factor k 1(V)=6, the standard uncertainty uB1 (V) was:
uB1 (V)=a1(V)k1(V)=0.1 ml 6 =0.04 ml
According to the verification regulation, the verification of volumetric flasks is carried out at 20℃. The experiment was supposed to be carried out at (20±4) ℃. Because the volume expansion coefficient of liquid is much larger than that of glass, only the volume expansion coefficient of liquid needs to be considered. The volume expansion coefficient of acetonitrile is 1.37×10-3 /℃ , and the resulting volume change was ±(100×4×1.37×10-3 ) =±0.548 ml, so the half-width interval was a 2(V)=0.548 ml. Suppose the uncertainty caused by the temperature coefficient was in interval distribution, and included factor k 2=3, the standard uncertainty was: uB2 (V)=a2(V)k2(V)=0.5483 ml =0.316 4 ml
The combined uncertainty was: uc(V)=u2A(V)+u2B1 (V)+u2B2 (V) =0.319 5 ml
Its relative uncertainty was:
urel (V)=uc(V)V=0.319 5100 =0.003 195
Xiaolin ZHANG et al. Uncertainty Analysis of Residues of Nitrofuran Metabolites in Chicken Bone
Uncertainty introduced by standard solution dilution
The mixed intermediate solutions were obtained by stepwise dilution, from 1.0 mg/ml to 10.0 μg/L using 10 ml volumetric flasks and 1 000 μl pipettes. The maximum allowable error of the 10 ml volumetric flasks is ± 0.020 ml, and the maximum allowable error of the 1 000 μl pipettes is ± 1.0%. According to the uniform distribution, k =3, the uncertainty was:
urel (a)=0.02010×3=0.001 15
urel (b)=1.0 %3=0.005 77
The combined uncertainty was:
urel (X)=5u2rel (a)+5u2rel (b) =0.013 16
Uncertainty introduced by internal standard solution preparation
The uncertainty introduced by weighed amounts of internal standards during internal standard solution preparation was:
urel (SEM-13 C-15 N2)=0.033 9 mg/10.0 mg=0.003 39
urel (AMOZ-13 C3)=0.033 9 mg/10.04 mg=0.003 376
urel (AHD-13 C3)=0.033 9 mg/10.0 mg=0.003 39
urel (AOZ-D4)=0.033 9 mg/10.0 mg=0.003 39
The uncertainty introduced by diluting to constant weight in stock solution preparation and the dilution process in working solution preparation was the same as "Uncertainty introduced by the constant volume of the standard stock solutions" and "Uncertainty introduced by standard solution dilution".
Uncertainty introduced by weighed amount of sample
The resolution of the balance is 0.1 mg, and the half-width interval was a2=0.05 mg. The uncertainty included k 3=3, and the sample weight was 2.00 g, so its standard uncertainty was:
urel(5) =a3k3= 0.05 mg2.00×3=0.014 4
Uncertainty introduced by repeated measurement
A certain concentration was repeatedly measured for 10 times, and the uncertainty was calculated according to the Bessel formula as follows:
ufrep =S(X)n (3)
The formula for standard uncertainty:
urel(6) =ufrep X (4)
Relative uncertainty introduced by recovery of spiked sample urel (R) A recovery test was carried out on the sample for 6 times at the level of 2 μg/kg, and the uncertainty was calculated according to formula (5). The uncertainty caused by recovery of spiked sample was in uniform distribution, including k =3.
urel (R)=(1-Rk)2+(S6)2 (5)
Combined relative standard uncertainty and uncertainty
The above uncertainties were independent of each other, and regardless of the correlation between the components, the combined uncertainty urel (c) was calculated according to formula (2) and the uncertainty uc (X) was calculated according to formula (6).
uc(X)=urel (c).X (6)
At confidence level of 95%, the expanded uncertainty ( k =2) was U=kuc (X). The calculated results were as follows: nitrofurazone metabolites CSEM =0.981 μg/kg, U=0.070 μg/kg, k =2; furazolidone metabolites CAOZ =1.032 μg/kg, U=0.061 μg/kg, k =2; furaltadone metabolites CAMOZ =1.068 μg/kg, U=0.076 μg/kg, k =2; furadantin metabolites CAHD =1.007 μg/kg, U=0.046 μg/kg, k =2.
Conclusions and Discussion
According to the measurement uncertainty evaluation method, the sources of uncertainty of nitrofuran metabolites in chicken bone were analyzed. By establishing mathematical models, each uncertainty component was evaluated and calculated. The uncertainty in the measurement process mainly comes from the purity of the standards, the preparation process, the sample weight, the number of repeated measurement and the recovery of spiked sample. Therefore, in the daily testing, we should choose standards with higher purity, and use high-precision glass measuring devices or pipettes and balances in the preparation process. Meanwhile, we should make multiple measurements during the determination process to reduce the uncertainty generated during the detection process, thereby improving the accuracy of the detection.
References
［1］ ZHU WX, LIU YF, LIANG W. Progress on nitrofuran drug residues in animal-borne food［J］. Progress in Veterinary Medicine, 2010, 31(2): 99-102. (in Chinese)
［2］ TANG HM, ZENG F, LI CH. Progress in analytical methods of nitrofuran antibiotics and their metabolites in food: A review［J］. Food Safety and Quality Detection Technology, 2016, 7(10): 3952-3959. (in Chinese)
［3］ WANG Q, MA B, LYU HY, et al. Evaluation of the safety of nitrofurans and their metabolites in food to human health［J］. Chinese Fishery Quality and Standards, 2013, 3(2): 4-10. (in Chinese) ［4］ JIANG Y, DING T, XU JZ. Study on the elimination rule of nitrofuran drugs in the tissue of Procambarus clarkii［J］. Animal Husbandry and Veterinary Medicine, 2008, 40(2): 34-37. (in Chinese)
［5］ HOOGENBOOM LA, VAN BRUCHEM GD, et al. Absorption of amutagenic metabolite released from protein-bound residues of furazolidone［J］. Environ Toxicol Pharmacol, 2002, 11(3): 273-287.
［6］ European Food Safety Authority. Opinion of the scientific panel on food additives,flavourings,processing aids and materials in contact with food on a request from the Com-mission related to Semicarbazide in food［J］. EFSA J,2005(219): 1-36．
［7］ Ministry of Agriculture of the Peoples Republic of China. Announcement 235 of the Ministry of Agriculture of the Peoples Republic of China［EB/OL］. (2002-12-24) ［2017-03-05］. http:/ /www. Moa. Gov. cn/zwllm/nybz/200803/t20080304_1028649. htm. (in Chinese)
［8］ Ministry of Agriculture of the Peoples Republic of China. Announcement 560 of the Ministry of Agriculture of the Peoples Republic of China ［EB/OL］. (2005-10-28) ［2017-03-05］. http:/ /www. Moa. Gov. cn/zwllm/tzgg/gg/200511/t20051117_496523.htm. (in Chinese)
［9］ LIU AC, LIU C, ZHAO Y, et al. Rapid determination of nitrofuran metabolites in aquatic products by colloidal gold method［J］. Acta Agriculturae Zhejiangensis,2013,25(1): 95-102. (in Chinese)
［10］ JIANG HW. ELISA Detection of Residues of the nitro-furan chemicals in the Animal Products［J］. Shaanxi Journal of Agricultural Sciences, 2006(5): 53-55. (in Chinese)
［11］ WANG Y, CAI YQ, JIA DF, et al. Determination of nitrofuran metabolite residues in aquatic products by high performance liquid chromatography［J］. Chinese Journal of Analysis Laboratory,2009,28(12): 86-90. (in Chinese)
［12］ LI XP, PENG JJ, HU HG, et al. Uncertainty analysis of determination of nitrofuran metabolites in aquatic products by ultra performance liquid chromatography-tandem mass spectrometry［J］. Agriculture and Technology, 2016, 36(23): 1-4. (in Chinese)
［13］ ZHU ZT, LEI MK, PENG F, et al. Uncertainty evaluation in determination of nitrofuran metabolites residues in aquatic product by liquid chromatography-tandem mass spectrometry［J］. Food Safety and Quality Detection Technology, 2015, 6(7): 2857-2862. (in Chinese)
［14］ CORDERO R, SECKMEYER G, PISSULLA D, et al. Uncertainty evaluation of the spectral UV irradiance evaluated by u-sing the UVSPEC radiative transfer model［J］. Opt Com-mun, 2007, 276(12): 44-53. (in Chinese)
［15］ Technical Committee on Certification and Accreditation of Standardization. GB/T 27025-2008: General requirements for the competence of testing and calibration laboratories［S］. Beijing: China Standard Press, 2008. (in Chinese)
［16］ GUO LN, LIU CW, FU YC. Evaluation of the uncertainty for the determination of nitrofuran metabolites in aquatic products by LC-MS/MS［J］. Modern Food, 2017(2): 101-105. (in Chinese)
Editor: Yingzhi GUANGProofreader: Xinxiu ZHU