TITLE: GC/MS Method for the Detection of Terbufos, Diazinon and Parathion in Blood
AUTHORS: Christina Wilson-Frank, Michael Ewbank

[DESCRIPTION]
Scope
This is a multi-residue method for the detection of the organophosphate pesticides, terbufos, diazinon and parathion in blood by gas chromatography and mass spectrometry (GC/MS). This is a qualitative screening method intended to isolate and concentrate organophosphate pesticides from sample extracts for GC/MS confirmation. This method also allows for adequate separation and detection of 42 other organophosphorus, carbamate and organochlorine pesticides.

In this multi-residue method, pesticides are extracted from blood samples in acetonitrile and isolated using a dual-layer, ENVI-Carb-II‱/PSA solid-phase extraction procedure. The pesticides are subsequently separated and detected using gas chromatography and detected using electron-impact mass spectrometry.

Table 1. Instrument and Method Limits of Detection 
  
 
 

 
Method validation/evaluation/verification:

In-house method validation data and evaluation by an independent laboratory (Vet-LIRN) in collaborative multi-laboratory studies

[STEPS]
SECTION: Extraction Solution and Standard Diluent Preparation
2. Preparation of acetonitrile:toluene (3:1) solution:

                                                                                                                                                                                                                        
SECTION: Extraction Solution and Standard Diluent Preparation
2.1. Transfer 600 mL of acetonitrile to 1L graduated cylinder. 

SECTION: Extraction Solution and Standard Diluent Preparation
2.2. Add 200 mL of toluene to the 1L graduated cylinder containing the acetonitrile. 

SECTION: Extraction Solution and Standard Diluent Preparation
2.3. Transfer contents to a properly labeled 1L glass storage bottle and store at room temperature. 
SECTION: Extraction Solution and Standard Diluent Preparation
2.4. Label should include contents, expiration date, laboratory notebook reference, analyst initials and hazard communication designations. The expiration date for the solution should be the earliest expiration date of the two reagents/standards used.
SECTION: Preparation of Pesticide Standards and Spiking Solutions
1. Preparation of 20 ppm terbufos, diazinon and parathion mixed pesticide standard solution 

SECTION: Preparation of Pesticide Standards and Spiking Solutions
1.1. Record manufacturer, manufacturer part number, lot number, and expiration date for all standards and reagents in laboratory notebook. 
SECTION: Preparation of Pesticide Standards and Spiking Solutions
1.2. Pipet 1.50 mL of acetonitrile:toluene (3:1) into a 5 mL glass vial. 
SECTION: Preparation of Pesticide Standards and Spiking Solutions
1.3. Transfer 0.75 mL of each of the 100 ppm terbufos, 100 ppm diazinon and 100 ppm parathion. Label on the 5 mL glass vial should include contents, expiration date, laboratory notebook reference, analyst initials and hazard communication designations.  The expiration date for the solution should be the earliest expiration date of the two reagents/standards used.

SECTION: Preparation of Pesticide Standards and Spiking Solutions
1.4. Cap and vortex mix well.
SECTION: Preparation of Pesticide Standards and Spiking Solutions
1.5. The 20 ppm mixed standard solution is stable for up to 5 weeks when stored at refrigeration temperature and for 24 hours at room temperature.
SECTION: Preparation of Pesticide Standards and Spiking Solutions
3. Preparation of 1 ppm terbufos, diazinon, and parathion mixed standard solution: 
SECTION: Preparation of Pesticide Standards and Spiking Solutions
3.1. Record all pertinent information regarding the standard stock solution in laboratory notebook. 

SECTION: Preparation of Pesticide Standards and Spiking Solutions
3.2. Aliquot2.00 mLof acetonitrile:toluene (3:1) into a 5 mL glass vial. 

SECTION: Preparation of Pesticide Standards and Spiking Solutions
3.3. Transfer 106 µL of the 20 ppm terbufos, diazinon, and parathion mixed standard solution prepared above. Label on the 5 mL glass vial should include contents, expiration date, laboratory notebook reference, analyst initials and hazard communication designations.  The expiration date for the solution should be the earliest expiration date of the two reagents/standards used.

SECTION: Preparation of Pesticide Standards and Spiking Solutions
3.4. Cap and vortex mix well.
SECTION: Preparation of Pesticide Standards and Spiking Solutions
3.5. The 1 ppm mixed standard solution is stable for up to 3 weeks when stored at refrigeration temperature and for 24 hours at room temperature.
SECTION: Control Samples
4. Negative control:  Negative control blood must be a blood sample that is absent of pesticides. Transfer  2 mL of blood to a properly labeled, 15 mL polypropylene tube. Proceed to step 6.2. 

SECTION: Control Samples
5. Positive control: Positive control blood is negative control blood that is fortified with a known concentration of pesticides.  Transfer 2 mL of blood to a properly labeled test tube and add  106 µL of the 20 ppm terbufos, diazinon, and parathion mixed standard solution to obtain a spike level of 1 ppm.  Proceed to step 6.2.

SECTION: Procedure for Samples
6. Sample Extraction Procedure:

SECTION: Procedure for Samples
6.1. Aliquot 2 mL of blood sample into a properly labeled 15 mL polypropylene tube. Record sample ID, sample volume and any other pertinent information in laboratory notebook.

SECTION: Procedure for Samples
6.2. Add 4 mL acetonitrile to the sample and vortex mix for approximately 1 min.

SECTION: Procedure for Samples
6.3. Centrifuge sample at approximately 4,000 RPM (~ 1500 RCF) for 10 min

SECTION: Procedure for Samples
6.4. Aliquot and transfer supernatant to properly labeled 15 mL polypropylene tube.
SECTION: Procedure for Samples
6.5. Add an additional 4 mL of acetonitrile to the remaining pellet from step 6.4. Vortex mix for approximately 1 min.

SECTION: Procedure for Samples
6.6. Centrifuge sample at approximately 4,000 RPM (~ 1500 RCF) for 10 min

SECTION: Procedure for Samples
6.7. Aliquot supernatant and combine with supernatant from step 6.4.
SECTION: Procedure for Samples
6.8. Add 1 g of Na2SO4 to the combined supernatants and vortex mix for approximately 1 min to dry the extract. Allow Na2SO4 to settle before proceeding.

SECTION: Procedure for Samples
6.9. Using a disposable glass pipet, transfer all of dried acetonitrile extract to a 15 mL polypropylene tube. (Stopping point: This extract is stable for 24 hours under refrigeration conditions).
SECTION: Procedure for Samples
6.10. Proceed with SPE clean-up procedure. 
SECTION: Procedure for Samples
7. SPE Clean-up Procedure:

SECTION: Procedure for Samples
7.1. Condition a multi-layer supelclean ENVI-Carb-II/PSA SPE cartridge (20 or 6 mL capacity) with 5 mLacetonitrile:toluene (3:1). Collect the eluate as waste in a properly labeled waste beaker.

SECTION: Procedure for Samples
7.2. Load the acetonitrile extract from above (step 6.9) into the SPE cartridge and collect the eluate in a properly labeled waste beaker.
SECTION: Procedure for Samples
7.3. Elute pesticides from SPE cartridge with 15 mL acetonitrile:toluene (3:1) into a properly labeled 50 mL glass test tube.
SECTION: Procedure for Samples
7.4. Place test tube containing SPE extract in a nitrogen evaporator heating block (set at approximately 40oC) and evaporate eluate to dryness under low nitrogen gas flow. (Stopping point: Dried residue is stable for 24 hours under refrigeration conditions)
SECTION: Procedure for Samples
7.5. Reconstitute residue with0.5 mL acetonitrile:toluene (3:1), vortex well to reconstitute entire residue and transfer to spin-x microcentrifuge tubes with nylon filters. 

SECTION: Procedure for Samples
7.6. Vortex tubes for 30 s. Transfer the filtrate to a properly labeled GC/MS autosampler vial containing a vial insert. (Stopping point: This solution is stable for 24 hours under refrigeration conditions)

SECTION: Procedure for Samples
7.7. Proceed to GC/MS analysis
SECTION: GC/MS Analysis for the Detection of Pesticides
8. Instrument Parameters:


SECTION: GC/MS Analysis for the Detection of Pesticides
8.1. Inject 1 µL of the reconstituted sample extract into a GC/MS that is equipped with a triple quadrupole mass spectrometer under conditions found in Table 1.

Initial results have shown that the use of an ion-trap mass spectrometer has significantly higher instrument and method LODs when compared to using triple quadrupole MS.

Table 1: Instrument Parameters for GC/MS for the Detection of Terbufos, Diazinon, and Parathion in Blood Samples
     GC Parameters                 Initial Oven Temp and Hold      40 C for 0.5 min               Final Oven Temp and Hold      300 C for 1.0 min               Injector Temp      275 C (splitless)               Carrier Gas Flow      1.4 ml/min               Injection Volume      1 µL               Column      ThermoFisher TR-5               Column Size      30 m X 0.25 mm               Column Film Size      0.25 µL               Rinse for Autosampler      Toluene        MS Parameters                 Transfer Line Temp      325 C               Ion Source Temp      310 C               Ionization Mode      EI               Scan Range      50-650 m/z               Quadrupole Temp                      MS Type      Triple Quadrupole   

 
 
SECTION: GC/MS Analysis for the Detection of Pesticides
9. Acceptance Criteria to Determine the Presence of the Pesticide:
 



SECTION: GC/MS Analysis for the Detection of Pesticides
9.1. The retention time needs to be within 3% of the retention time of the positive controls or the 1 ppm standard solution. (See Table 2 for more information)


Table 2: Retention Time (RT) Information for Pesticides
            Pesticide      Approx. RT*      **Relative RT               Terbufos      13.02 min      1.000               Diazinon      13.17 min      1.012               Parathion      14.60 min      1.121               Myristic Acid (FA)      12.73 min      0.978               Palmitic Acid (FA)      14.20 min      1.091   

SECTION: GC/MS Analysis for the Detection of Pesticides
9.2. The signal-to-noise (S/N) ratio for pesticide needs to be greater than 3 RMS. 
SECTION: GC/MS Analysis for the Detection of Pesticides
9.3. All observed ions need to be present (See Table 3 for more information). 

Table 3: Ions Monitored for the Detection of Terbufos, Diazinon, and Parathion 
            Pesticide      Observed Ions               Terbufos      231  103  153               Diazinon      137  179 152  199               Parathion      109  139  291   

SECTION: GC/MS Analysis for the Detection of Pesticides
9.4. The relative ion intensities for secondary ions monitored need to be within 30% of the major ion observed for each pesticide when compared to ions in the positive control sample or the 1 ppm mixed standard
solution. 
SECTION: GC/MS Analysis for the Detection of Pesticides
9.5. High concentration of fatty acids may interfere with the method performance and therefore need to be monitored. See Appendix 1 for details.
11. To reduce the false negative observations, expanding the acceptance criteria ranges was explored and resulted in the following:
The retention time shall be within 3% of the retention time of the positive controls or the 1 ppm standard solution.
The signal-to-noise (S/N) ratio for terbufos shall be greater than 3 RMS.
The ions 231 m/z, 103 m/z and 153 m/z shall be present for terbufos.
One of the relative intensities for ion 103 m/z or 153 m/z shall be within 30 % of the major ion o
12. An “Alert Criteria” was established to determine if a false positive result could be suspected for this method. This concern stems from the possibility of co-eluting peaks with m/z values of 103 and 153 from
blood matrix interferences. For this particular method that could not be successfully achieved due to co-elution peaks (103 and 153) from the blood matrix.

1.  The “Alert Criteria” is the ratio of the sum of the S/N ratios of the fatty acids myristic and palmitic acid divided by the S/N of terbufos peak. See equation 1 below.

Equation 1: Calculated Ratio for “Alert Criteria”

                               S/N for Myristic Acid peak + S/N for Palmitic Acid peak
Alert Criteria=     ---------------------------------------------------------
                                                                S/N for Terbufos peak

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
SECTION: Appendix 1
10. Terbufos linear fit for analysis

 
13. Studies were completed to determine what could influence intensities or the amount of terbufos after extraction and clean-up procedures. The first study included examiningdifferent lot
numbers of blood determine their effects on terbufos intensities. These findings were significant.

 
The intensities were then correlated with the S/N ratio for myristic acid. This correlation was also found to be significant. 

14. 
 


15. A main effects screening design for water and its removal was completed to determine their influences on the process. All samples were spiked with 0.3 ppm of terbufos, diazinon, and parathion. However, the identification of terbufos was the focus. This study showed the amount of water and the amount of Na2SO4 used was significant to fatty acid removal. The lot number of the blood control and time the Na2SO4 was added were found not to be significant in the removal of fatty acids.

Further statistical examination revealed differences in identification probabilities related to the sum of the S/N ratio of the fatty acids, however there were samples that did not correlate with these criteria, apparently due to baseline variations. To account for these differences in the baseline, the S/N ratio for terbufos was introduced into the equation to eliminate baseline variability.

 

16. In summary, the removal of key saturated fatty acids appears to be important in identifying terbufos at low levels in bovine, blood samples. Levels of fatty acids can fluctuate by the amount of water, amount of sodium sulfate, and the blood from the particular animal. The levels can fluctuate from sample to sample due to what may carry forward into the clean-up step of the work-up procedure.

17. Possible solutions to help reduce the fatty acid concentrations during the preparation of the sample:
Switch from sodium sulfate to magnesium sulfate as previous research has shown sodium sulfate is not as effective drying agent for acetonitrile solutions.
Change the extraction/elution solvents during the process.Solutions in published literature include using acetone, acetone:toluene (65:35), and acetone:cyclohexane (various compositions) with the current SPE cartridges containing PSA.
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
18. The figures are examples for using Xcalibur at the Purdue University ADDL. These screen shots are subject to change and are not representative of other software.
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
19. GC/MS Instrument Control Method: Pesticide Screen Tox.103 20 min
Inject 1 µL of reconstituted sample extract into GC/MS (GC/MS operating system is equipped with Trace 1310 gas chromatography/TSQ 8000 mass spectrometer and TriPlus RSH autosampler) under the conditions found in Table 1 using the Xcalibur software system. These conditions should currently be in the method entitled "TOX.103 20 min."
If method "TOX.103 20 min" cannot be found or needs to be modified, then follow the instructions below.

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
19.1. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
19.2. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
19.3. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
20. GC Conditions and Settings
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
20.1. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
20.2. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
21. MS Conditions and Settings

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
21.1. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22. Analyzing Samples in System Control (Xcalibur)
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.1. Open “Xcalibur” by double-clicking the “Xcalibur” icon. Double-click on “Qual Browser” to create the data file for your analysis. 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.2. With Qual Browser open, go to “File” --> “Open.” The “Open Raw File” window will be displayed. 
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.3. Right click inside the list of files and create a new folder (“New” --> “Folder”). Name the data file with the date and any descriptors. Once the data folder is created, exit out of Qual Browser
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.4. To enter samples into the sample set, click on “Sequence Setup”. Enter the pertinent information about the samples to be analyzed. See Figure 11 for placement details.
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.5. Sample type: Select one from the drop-down box
Unknown: This is selected for all samples and extracted samples.
Blank: This is for all blank vials (e.g. containing acetonitrile: toluene (3:1) method blank vials). 
QC: This is for positive, negative, and internal control samples. 
Std Bracket: This is for neat/pure standard injections. 
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.6. File name: Enter name of sample, standards or control vials used (cannot use spaces).

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.7. Path: Select the data folder previously created in Qual browser.
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.8. Instrument Method: Select “Pesticide Screen Tox.103 20 min run”
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.9. Position: Enter the position number for the vial placed in the auto sampler tray
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.10. Injection Volume: 1.0 (1 µL).
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.11. Once sample set information has been entered, go to “File” --> “Save As” --> and type in the name of the method to save the method file in XCalibur --> Methods --> FDA Vet-LIRN Grant Method Files. Make sure to include the date, description, and notebook number and pages. 
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
22.12. To start the analysis and run the entire sequence, select the “Run Sequence” icon. A window will open --> select “OK” to begin the analysis.
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23. Viewing and Processing Data using Qual Browser in Xcalibur: Viewing and Processing Data using Qual Browser in Xcalibur
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.1. Go to “Road Map View” in XCalibur and double-click on the “Qual Browser” icon. Select “File" --> “Open Sequence” --> then select the sequence file for that sample set.
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.2. The vials injected for that sequence will be listed. Select the file you want to open and the chromatogram and mass spectrum window will open. 

 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.3. Click and select the pin (turns green on selection) on the top right corner of the chromatogram to allow viewing and zooming of the chromatographic data. 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.4. In order to process the data, right click in the chromatogram pane -->select “Peak Detection” --> “Set Peak Detection Algorithm and Detect in this Plot” --> “ICIS.”

 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.5. Then, right click in the top window (chromatogram) and select “Display Options.” Another window will be opened for the display options --> select the tab labeled “Labels.” Check the boxes for “Signal-to-Noise,” and “Retention Time” --> Then select “OK.”. 

 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.6.  Zoom-in on peak areas of interest by clicking-dragging the mouse over that area
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
23.7. When peak detection is selected, “Detection tab” button appears (letter ‘I’ in the button stands for ICIS detection algorithm selected earlier). Select the detection tab, check “Manual noise region” and enter the retention time range to be used for calculating signal-to-noise region.
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24. Viewing and Processing Data using Qual Browser in Xcalibur: ·Viewing and Processing MS Data
SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.1. Select the MS window by clicking on the pin (turns green on selection) on the top right corner of the MS window (bottom window) to allow matching the spectrum and the major ions in the spectral list for confirming identity of the analytes of interest.

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.2. Left click over the peak of interest from the chromatogram and match the major ions to be monitored for the analyte of interest from the spectral window. For example, peak seen at RT 12.72 min, selection of this peak shows the spectrum with presence of the major ions m/z 231, m/z 103 and m/z 153 (circled in blue in the bottom window of the image below). 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.3. With the MS window still selected and showing the mass spectrum left click and select “Ranges”.
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.4. The “Spectrum Ranges” window open up. Change the lower mass range from “60.00 – 600.00” to “100.00 – 600.00” and select “OK” to eliminate any low m/z ions interfering with analyte identification. 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.5. In next window, select "Grid" --> "Insert cells" --> "Below" to add another window below the spectrum window.
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.6. Select the third window, right click and select --> “View” --> “Spectrum list” to view the relative intensities in percentage for the major ions monitored.
 
 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.7. Print the data in the peak windows for chromatogram, spectrum and spectral list by selecting “File”
--> “Print” --> “All cells in selected window” --> "One Page” --> “OK”.

 

SECTION: Appendix 2. Pesticide SOP TOX.103 Software and Instrument Details
24.8. Process the MS data for all the analytes by selecting the peak of interest in the chromatogram window as shown in steps above.