comparison of immunoassay and hplc-ms/ms used to measure urinary metabolites of atrazine, metolachlor, and chlorpyrifos from farmers and non-farmers in iowa - dry ice packs for coolers

by:Demi     2019-11-24
comparison of immunoassay and hplc-ms/ms used to measure urinary metabolites of atrazine, metolachlor, and chlorpyrifos from farmers and non-farmers in iowa  -  dry ice packs for coolers
In a study to investigate pesticide exposure at farm homes in Iowa, 51 participants collected urine samples.
The equal sample of the sample was sent to two different laboratories and analyzed the metabolites of atrazine Dejin (
Mercapturate), metolachlor (
Methyl amine ester)
Chlorfos (TCP)
Two different analytical methods, immune analysis and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). HPLC-
The method of MS/MS is often very specific, but it is costly and time consuming.
The immune analysis method is cheaper and faster, but it may not be very sensitive due to cross reactivity and matrix effects.
Three statistical methods were used to compare the two analytical methods.
Each statistical method has a difference in the sample where the result is below the detection limit (LOD)were treated.
The first two approaches involve a filling procedure and the third one uses maximum likelihood estimation (MLE).
Using the fourth statistical method, each laboratory was modeled and compared using MLE, respectively.
Immune analysis and HPLC-
The method of MS/MS is moderately related (correlation 0. 40–0. 49)
, But the geometric mean value of the immune analysis method is significantly higher (GM)
The estimated value of each pesticide metabolites.
The GM estimate of atrazine Dejin Ester, methyl phenol Ester and TCP by immune analysis is between 0. 16–0. 98u2009μgu2009l−1, 0. 24–0.
Respectively with HPLC determination the 45 mu g ll-1 and 14 mu g 1 L-1
MS/MS from 0. 0015–0. 0039u2009μgu2009l−1, 0. 12–0.
16 μg ll-1 and 2. 9–3.
0 μ g u2009 l-1 respectively.
Immune analysis is often cheaper and faster than HPLC
However, MS/MS may lead to an upward deviation in the level of pesticide metabolites in urine.
50 households in the spring and summer of 2001 (
25 farms, 25 non-farmsfarm)
Iowa participated in a study to investigate agricultural pesticide contamination in the home and exposure to pesticides in the home.
Participant recruitment has been described earlier ().
In order to be eligible for this study
Farm families have to live on land that is not used for agriculture, and there are no families that are engaged in agricultural or commercial pesticide applications.
The farm family must use at least one of the seven target pesticides: atrazine to go to Tianjin, B-chlorol, A. A. , chlorfos, glyphosate, or 3, 4-D.
All of these pesticides, except chlorfos, are corn or soybean herbicides, a pesticide used in corn, soybeans and other crops.
During June, July and August 2001, each family was visited twice.
The first visit to the farm family was shortly after the application for the event (1-5 days)
Visit non
Plan the farm family that will be carried out at the same time as the farm visit.
A second visit after about 4 weeks (
Average 4 weeks, 3-5 weeks).
Two samples of live urine were collected from participants at each visit, one on that evening and the other on the morning of the following day.
Urine samples were collected in a 500 na gene bottle and participants were asked to store their urine in a refrigerator or in a cooler with ice bags.
Investigators collected samples the day after the visit, 25-
Take out ml equal samples, store them on dry ice and ship them to the laboratory.
Record the total volume of each empty urine.
A total of 178 urine gaps from a subset of 51 farms and non-farms
Family members of the farm (
89 samples from 24 farm fathers, 2 samples from a farm mother, 80 samples from 23 non-farm fathers
Seven samples of the farm father and three non-farm fathersfarm mothers)
There is enough volume to be separated and analyzed by two different laboratories.
The first lab.
First laboratory of National Environmental Health Center)
Use of HPLC-analysis of the separation of atrazine Dejin, alpha-chlorine and chlorfos urine metabolitesMS/MS;
Laboratory 2 (
Second Laboratory, National Institute of Occupational Safety and Health)
The separation of urinary metabolites of atrazine Dejin and chlorfos was analyzed by ELISA, and the Alpha chlorine was analyzed by FCMIA method.
Limit of detection (LOD)
Change by Analyte and Method ();
The LODs of lab 1 were lower compared to Lab 2, especially for atrazine detianjin ester. A 25-
Ml equal samples of each urine sample were sent to laboratory 1 by HPLC-
MS/MS of immune analysis and laboratory 2.
Metabolic products of three pesticidesatrazine (
Mercapturate), metolachlor (
Methyl amine ester)
Chlorfos (3,5,6-
Chlorpyrazole (TCP))were measured.
The average LOD of each method and pesticide metabolites is given in.
In some cases, the values reported by the laboratory are lower than the average LOD and are used based on the statistical analysis used.
The value reported at average LOD or below has more uncertainty than the value greater than LOD, however, this uncertainty is considered to be less than the use of estimates for samples below LOD.
Analysis of samples by HPLC
The MS/MS used by the recently released Method (; ). Briefly, a 2-
Ml equal sample was added with the standard marked in the same position and then diluted with 1. 5u2009ml 0.
2 m acetate buffer for 800 active units-
Glucose gourd enzyme/sulfuric acid enzyme was added.
The solution is allowed to be incubated overnight at 37 °c to release glucose gourd acid-and sulfate-
Application of hydrolysis to the oasis hlb solid extraction box (
United States, MA, Milford, Waters).
The specl box was cleaned with 2 ml 5% methanol in 1% acetic acid and was eluted with 1. 5u2009ml methanol.
Methanol was diluted with 2 ml of methanol and then evaporated to dry.
The residue was reformulated in acetone of 50 µL.
Determination of pesticide metabolites in sample extracts using HPLC
MS/MS with atmospheric pressure chemical ionization.
A multi-reaction monitoring experiment was used to separate specific precursor and product ion pairs for each analyte under test.
Prepare calibration standards, quality control materials and blank samples and analyze them at the same time as unknown samples.
The concentration of three pesticide metabolites was calculated using isotope dilution quantification.
At zero concentration, the LOD is calculated to triple the noise SD.
Samples analyzed by immune analysis were used for ELISA analysis of atrazine Dejin Ester and TCP, and FCMIA analysis of nail music ester.
The LODs of the immunotherapy method are defined as 90% binding antibodies divided by unbinding antibodies (90% B/B0).
Immunization of Atrazine Dejin (A00071)
Rapid detection of ELISA kit (
Strategic diagnosis, Newtown, PA, United States)
Used to determine the metabolites atrazine Dejin Ester according to the manufacturer's instructions, with the following exceptions: Calibration criteria (0. 0, 0. 1, 0. 5, 1. 0, 2. 0, and 5. 0 p. p. b. )
Anonymous volunteers were prepared to strengthen the combined urine dilution 1:10 UriSub (
CST Technologies Inc. A synthetic buffer solution that is physically equivalent to normal urine
Big Neck New York, United States of America)
Dejin ester with synthetic mercap.
Urine samples from all participants were diluted by 1: 10 with UriSub.
The previously published TCP assay was used to determine the urinary metabolites of chlorfos ().
TCP Assay Kit (A00208)
Rapid detection of ELISA (
Strategic diagnosis, Newtown, PA, United States)
Use according to manufacturer's instructions with the following exceptions: Calibration Standard (0. 0, 0. 0156, 0. 3125, 0. 625, 1. 25, 2. 5, 5. 0, and 10. 0 p. p. b. )
It is prepared with TCP to strengthen UriSub.
Urine samples from all participants were diluted by 1: 10 with UriSub.
The method described uses a minimum of 1:50 urine dilution to avoid the urinary matrix effect.
However, considering the concentration range of TCP in urine found in these samples, 1:10 dilution is considered appropriate to bring the sample into the detection range of the method.
In addition, 20 liters per sample-glucuronidase (
Roche diagnosis, part one1-585-
665, Mannheim, Germany)
At room temperature for 30 min, TCP was then analyzed to be separated from its glucose ester-bound form.
Using the recently developed FCMIA (). A pesticide-
The protein-bound compounds of pesticides are coupled to a group of addressable microbeads. The conjugate-
Coupling beads are then used for competitive analysis of pesticide metabolites.
Pesticide metabolites in solution compete with beads
Binding complex of fluorescent labeling-
Pesticide antibody;
Therefore, an increase in the concentration of a given pesticide in the urine results in a decrease in the fluorescent signal of the beads of that pesticide.
Calibration Standard (0. 0, 0. 1, 0. 3, 1. 0, 3. 0, 10. 0, 30.
0,100,300 p. p. b. )were prepared.
These criteria were prepared from a mixed urine diluted at 1: 10 (
Abraxis LLC harsboro, PA, USA)and UriSub(1:3)
Strengthened with methyl amine ester.
All statistical analysis using SAS 9 software (
SAS Institute LimitedCary, North Carolina, United States).
Percent agreement and Cohen's kappa were originally used to flatly compare the two analytical methods, which measure agreements that are expected to be impossible (i. e. , non-detect, detect).
Since the two methods have different LODs, kappa is calculated using four categories (non-
Detection, value
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