Shuhuan ZHAO, Yunxia WANG, Lijun LIU, Cuizhi LI*, Zhiyong LU, Zhijun LI

1. Quality Management Department, Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China; 2. Quality Management Department of Yogurt Division, Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China

Abstract [Objectives] To determine the content of organic pollutants by CALUX bioassay. [Methods] According to JJF 1059.1-2012 Technical Specification for Evaluation and Expression of Uncertainty in Measurement, the determination results of organic pollutants were analyzed, various sources of uncertainty that may be introduced in the detection process were analyzed, and the mathematical model of uncertainty was established. Type A and B evaluation methods were used to calculate the components of uncertainty and extended uncertainty. [Results] In the range of 95% confidence interval, the determination result of organic pollutants was 6.59 pg/g fat, and the extended uncertainty was 0.076 4 pg/g fat, so the determination results of organic pollutants could be expressed as (6.59±0.076 4) pg/g fat (k=2). [Conclusions] This study can provide a basis for more accurate determination of organic pollutants.

Key words Uncertainty, Organic pollutants, CALUX bioassay

1 Introduction

Organic pollutants are mainly by-products of human industrial activities[1-3]. Organic pollutants are highly toxic substances, among which 2,3,7,8-TCDD is the most toxic substance. For ordinary people, daily exposure to organic pollutants basically comes from food, accounting for 90%, mainly fish, followed by dairy products and meat. The European Union clearly stipulates the content of organic pollutants in food, and limits the allowable residues of organic pollutants[4].

The uncertainty measurement is used to characterize the dispersion reasonably assigned the measured value, and it is a parameter contained in the measurement result[5-6]. Uncertainty evaluation is of great significance in laboratory data comparison, method confirmation, standard equipment calibration and numerical traceability, and international parties related to conformity assessment also attach great importance to uncertainty measurement[7-8]. CNAS-CL07:2011RequirementsforMeasurementofUncertainty[9]clearly stipulates that testing laboratories should be able to evaluate the uncertainty of each measurement result with numerical requirements, and different evaluation methods can be used for different testing items and objects. The measurement of uncertainty is an important index to evaluate the quality of the test data, which indicates the proximity between the result and the measured true value, and reflects the credibility of the test data[10-14]. Therefore, in order to ensure the accuracy and effectiveness of the test results, this experiment evaluated the uncertainty in the determination results of organic pollutants by CALUX biological detection according toEvaluationandExpressionofUncertaintyMeasurement(JJF 1059.1-2012)[15].

2 Materials and methods

2.1 Materials and reagentsN-hexane (agricultural residue grade, J. T. Baker); dichloromethane (agricultural residue grade, Thermo Fisher Life); toluene (chromatographic purity, Knowles); Dimethyl sulfoxide (AMRESCO); CALUX Cell (American XDSI); RPMI Medium 1640 medium (Gibco); PBS (Gibco); Penicillin Streptomycin (Gibco); 0.05% Trypsin-EDTA (Gibco); Luciferase Cell Culture Lysis (Promega).

2.2 Instruments and equipmentElectronic balance, Mettler-Toledo Instruments Shanghai Co., Ltd.; automatic rapid solvent extraction instrument, Beijing Jitian Instruments Co., Ltd.; sample purification system, CAPE; parallel evaporation instrument, Swiss BUCHI Labortechnik AG; microwell plate luminescence detector, Germany Berthold Technologies; carbon dioxide incubator, Germany Memmert Co., Ltd.; inverted microscope, Carl Zeiss (Shanghai) Management Co., Ltd.; cell counter, Shanghai Ruiyu Biotechnology Co., Ltd.

2.3 Methods

2.3.1Sample pretreatment. About 10 g (accurate to 0.01 g) of the sample was precisely weighed, fully mixed with diatomite of equal weight, and filled into a stainless steel extraction tank. The stainless steel cover was tightened and placed in a rapid solvent extractor for extraction. Extraction reference conditions: extraction solvent (n-hexane∶dichloromethane 1∶1); pressure (10.3 MPa); temperature (120 ℃); heating time (7 min); static extraction time (8 min); number of cycles (3); elution volume (40%).

The weight of the evaporation bottle was measured, and the organic solvent in the collection bottle was transferred to the evaporation bottle and placed in a parallel evaporator for evaporation. After the organic solvent was evaporated, the evaporation bottle was weighed and the fat value of the sample was obtained. The extracted fat was purified and separated by acid silica gel column and activated carbon column. The eluate of organic pollutants was collected, and 40 μL of DMSO was added to the dry eluent.

2.3.2Determination of organic pollutants by cell method. According to the concentration points of the standard curve and the number of samples, the number of wells in the microplate was calculated. 200 μL of cell fluid with a concentration of 7.5×105cell/mL was collected into microplate and cultured in CO2incubator at 37 ℃ for 24 h. 400 μL of complete culture medium was suctioned into corresponding wells, and 4 μL of standard substance and sample were added, and shaken and mixed evenly. The cell microwell plate cultured for 24 h was taken out and the medium in the plate was discarded. 190 μL of standard substance and sample diluents were added to the corresponding wells and cultured in CO2incubator at 37 ℃ for 24 h. After culture, the microwell plate was removed, the medium in wells was discarded. 50 μL of PBS was added, shaken for 2 min, and PBS was discarded. 30 μL of cell lysate was added and shaken for 10 min. The microporous plate was placed on the microporous plate photoluminescence meter, and the fluorescence value was measured. The fluorescence value results were inputted into the CALUX sample data processing software to calculate the content of organic pollutants.

3 Results and analysis

3.1 Establishment of mathematical model of uncertaintyThe expression formula for the determination of organic pollutant content (X) by biological method is as follows:

(1)

whereXis the content of organic pollutants in the sample, pg/g fat;Cis the concentration of organic pollutants in the test solution of the sample on the plate, pg/mL;mis fat weight of the sample, g;Vis the volume of DMSO, mL.

The similar influencing factors were combined. The input variablesC,V,mand repeatability factors were combined together, and classified as the repeatability factors of output variableX. Therefore, it is not necessary to evaluate the uncertainty components introduced by repeated measurement of the input variablesC,Vandm, respectively, and we can directly evaluate the uncertainty components introduced by the repeated measurement results (organic pollutant contentX).

Formula (1) was changed as follows:

(2)

wherefrepis the correction factor for measuring the factors affecting repeatability, and its value is 1.

According to formula (2), the input variablesC,V,mwas not correlated withfrep, and the square sum root method was used to measure the combined standard uncertainty. Moreover, the formula for calculating the content of organic pollutants only involves the product and quotient of the input variablesC,V,m, so the combined standard uncertainty was calculated in the following simplified way:

(3)

where the sensitivity coefficientc1=1,c2=1,c3=-1,c4=1.

3.2 Analysis of the source of uncertaintyAccording to the analysis of the detection process and mathematical model, the main sources of uncertainty in the determination of organic pollutants are as follows:

(i) the uncertainty introduced by weighing the fat; (ii) the uncertainty introduced by the displacement volume of solution; (iii) the uncertainty introduced by the concentration of the liquid to be measured on the plate; (iv) the uncertainty introduced by repeatability of measurement.

3.3 Evaluation of uncertainty components

So the standard uncertainty caused by the electronic analytical balance is as follows:

The weight of fat measured at this time wasm=3.17 g, and the relative standard uncertainty introduced by fat weighing is as follows:

Relative standard uncertainty is as follows:

3.3.3Relative standard uncertainty introduced by the concentration of the solution to be measured on the plate. In the process of the solution to be tested on the plate, 400 μL of cells were transferred by a 1 mL pipette and cultured on a 48-well plate. Then a 10 μL pipette was used to transfer 4 μL of the tested solution into the cell plate. After shaking and mixing, 190 μL of diluted liquid was transferred twice with a 200 μL pipette into the cells cultured for 24 h. The uncertainty of this process consists of the allowable errors of 1 mL, 10 μL and 200 μL pipettes.

Relative standard uncertainty is as follows:

In summary, the relative standard uncertainty introduced by the pipette in the process of the liquid to be tested on the plate is as follows:

=0.028 16.

3.3.4Uncertainty introduced by repeated measurement. Six parallel samples were measured, and the results of the determination of organic pollutants in the samples are as follows: when measured once, the result was 6.24 pg/g fat; when measured twice, the result was 7.10 pg/g fat; when measured for 3 times, the result was 6.58 pg/g fat; when measured 4 times, the result was 5.81 pg/g fat; when measured 5 times, the result was 6.76 pg/g fat; when measured 6 times, the result was 7.05 pg/g fat. The average value was 6.59 pg/g fat.

Then the relative standard uncertainty introduced by the repeated measurement of organic pollutant content is as follows:

3.4 Relative combined standard uncertaintyBased on the above calculation, the relative combined standard uncertainty of the content of organic pollutants in the sample with a measured value of 6.59 pg/g fat is as follows:

=0.005 802.

3.5 Expanded uncertaintyThe coverage factork(x)=2 and the coverage probabilityp≈95%, so the relative expanded uncertainty (Urel) of the organic pollutant content measurement result isUrel=Urel(X)×k(x)×C0(C0is the average content of the sample)=0.005 802×2×6.59×0.076 4 pg/g fat, and the detection result indicated that the content of organic pollutant in the sampleX=(6.59±0.076 4) pg/g fat,k=2.

4 Conclusion

In this experiment, CALUX bioassay was used to determine the content of organic pollutants. The determination results were evaluated according to the main sources of uncertainty. In accordance with the experimental process and evaluation results, we can know that the influencing factors of measurement uncertainty in this method mainly come from sample weighing, the use of liquid pipette and sample repeatability experiment. Among them, the uncertainty of measurement introduced by the fat weighing process has the least influence, while the uncertainty from the use of the pipette and the repeated sample measurement has a great influence. In order to ensure the accuracy of the measurement results, it is necessary to strictly do a good job in the regular calibration and verification of the instrument, strengthen the maintenance of the instrument, reduce the transfer deviation of trace solution as far as possible, and use quality control measures such as adding standards to monitor the accuracy of the process during the testing, reduce the uncertainty as much as possible, improve the reliability and accuracy of the test results, and obtain more accurate experimental results.