GC/MS Method for the Detection of Terbufos, Diazinon and Parathion in Blood

Preparation of 20 ppm terbufos, diazinon and parathion mixed pesticide standard solution

Record manufacturer, manufacturer part number, lot number, and expiration date for all standards and reagents in laboratory notebook.

Pipet 1.50mL of acetonitrile:toluene (3:1) into a 5 mL glass vial.

Transfer 0.75mL 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.

Cap and vortex mix well.

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.

Preparation of acetonitrile:toluene (3:1) solution:

Transfer 600mL of acetonitrile to 1L graduated cylinder.

Add 200mL of toluene to the 1L graduated cylinder containing the acetonitrile.

Transfer contents to a properly labeled 1L glass storage bottle and store at room temperature.

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.

Preparation of 1 ppm terbufos, diazinon, and parathion mixed standard solution:

Record all pertinent information regarding the standard stock solution in laboratory notebook.

Aliquot2.00mLof acetonitrile:toluene (3:1) into a 5 mL glass vial.

Transfer 106µL 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.

Cap and vortex mix well.

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.

Negative control: Negative control blood must be a blood sample that is absent of pesticides. Transfer 2mL of blood to a properly labeled, 15 mL polypropylene tube. Proceed to step 6.2.

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

Sample Extraction Procedure:

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

Proceed with SPE clean-up procedure.

Add 4mL acetonitrile to the sample and vortex mix for approximately 0h 1m 0s.

Centrifuge sample at approximately 4,000 RPM (~ 1500 RCF) for 0h 10m 0s

Aliquot and transfer supernatant to properly labeled 15 mL polypropylene tube.

Add an additional 4mL of acetonitrile to the remaining pellet from step 6.4. Vortex mix for approximately 0h 1m 0s.

Centrifuge sample at approximately 4,000 RPM (~ 1500 RCF) for 0h 10m 0s

Aliquot supernatant and combine with supernatant from step 6.4.

Add 1 g of Na₂SO₄ to the combined supernatants and vortex mix for approximately 0h 1m 0s to dry the extract. Allow Na₂SO₄ to settle before proceeding.

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).

SPE Clean-up Procedure:

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

Load the acetonitrile extract from above (step 6.9) into the SPE cartridge and collect the eluate in a properly labeled waste beaker.

Elute pesticides from SPE cartridge with 15 mL acetonitrile:toluene (3:1) into a properly labeled 50 mL glass test tube.

Place test tube containing SPE extract in a nitrogen evaporator heating block (set at approximately 40^oC) and evaporate eluate to dryness under low nitrogen gas flow. (Stopping point: Dried residue is stable for 24 hours under refrigeration conditions)

Reconstitute residue with0.5mL acetonitrile:toluene (3:1), vortex well to reconstitute entire residue and transfer to spin-x microcentrifuge tubes with nylon filters.

Vortex tubes for 0h 0m 30s. 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)

Proceed to GC/MS analysis

Instrument Parameters:

Inject 1µL 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

A	B	C
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 MS
	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

Acceptance Criteria to Determine the Presence of the Pesticide:

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

A	B	C	D
	Pesticide	Approximate RT* (Minutes)	Relative RT** (Minutes)
	Terbufos	13.20	1.000
	Diazinon	13.17	1.012
	Parathion	14.60	1.121
	Myristic Acid (FA)	12.73	0.978
	Palmitic Acid (FA)	14.20	1.091

• RT can differ with changing column**Notes: The relative retention time is the comparison of the approximate retention time of one pesticide relative to the retention time of another. This value can serve as a predictor of anticipated retention times for other analytes in a given GC run. For example, if a GC analysis of the pesticides indicated that the retention time of terbufos for that run is 12.98 minutes, then multiplying 12.98 minutes by 1.012 would provide an approximate retention time to be expected for diazinon at 13.14 minutes.

The signal-to-noise (S/N) ratio for pesticide needs to be greater than 3 RMS.

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 | A | B | | --- | --- | | Pesticide | Observed Ions | | Terbufos | 231  103  153 | | Diazinon | 137  179 152  199 | | Parathion | 109  139  291 |

• Major ions monitored to detect each pesticide are listed in bold above.

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.

High concentration of fatty acids may interfere with the method performance and therefore need to be monitored. See Appendix 1 for details.

Terbufos linear fit for analysis

To reduce the false negative observations, expanding the acceptance criteria ranges was explored and resulted in the following:

1. The retention time shall be within 3% of the retention time of the positive controls or the 1 ppm standard solution.
2. The signal-to-noise (S/N) ratio for terbufos shall be greater than 3 RMS.
3. The ions 231 m/z, 103 m/z and 153 m/z shall be present for terbufos.
4. One of the relative intensities for ion 103 m/z or 153 m/z shall be within 30 % of the major ion o

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.

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



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        

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.

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.

<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.j8nlkw83dl5r/k3kfbwgmf2.png" alt="Figure 4: The “Fatty Acid to Terbufos Ratio (SN)” versus “Identification” for Terbufos with one-way analysis. Since all samples were spiked with terbufos, an indicator of "absent" is incorrect while "present" is correct." loading="lazy" title="Figure 4: The “Fatty Acid to Terbufos Ratio (SN)” versus “Identification” for Terbufos with one-way analysis. Since all samples were spiked with terbufos, an indicator of "absent" is incorrect while "present" is correct."/>

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.

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.

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.

GC/MS Instrument Control Method: Pesticide Screen Tox.103 20 min

• Inject 1µL 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.

GC Conditions and Settings

MS Conditions and Settings

Analyzing Samples in System Control (Xcalibur)

Open “Xcalibur” by double-clicking the “Xcalibur” icon. Double-click on “Qual Browser” to create the data file for your analysis.

Injection Volume: 1.0 (1 µL).

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.

To start the analysis and run the entire sequence, select the “Run Sequence” icon. A window will open --> select “OK” to begin the analysis.

With Qual Browser open, go to “File” --> “Open.” The “Open Raw File” window will be displayed.

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

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.

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.

File name: Enter name of sample, standards or control vials used (cannot use spaces).

Path: Select the data folder previously created in Qual browser.

Instrument Method: Select “Pesticide Screen Tox.103 20 min run”

Position: Enter the position number for the vial placed in the auto sampler tray

Viewing and Processing Data using Qual Browser in Xcalibur: Viewing and Processing Data using Qual Browser in Xcalibur

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.

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.

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.

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.”

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.”.

Zoom-in on peak areas of interest by clicking-dragging the mouse over that area

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.

Viewing and Processing Data using Qual Browser in Xcalibur: · Viewing and Processing MS Data

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.

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).

With the MS window still selected and showing the mass spectrum left click and select “Ranges”.

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.

In next window, select "Grid" --> "Insert cells" --> "Below" to add another window below the spectrum window.

Select the third window, right click and select --> “View” --> “Spectrum list” to view the relative intensities in percentage for the major ions monitored.

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”.

Process the MS data for all the analytes by selecting the peak of interest in the chromatogram window as shown in steps above.