Nov 15, 2023
Quantitation of Anticoagulant Rodenticides in Serum
- kyle.francis1,
- Megan Romano1,
- Rupam Sarma1
- 1University of Kentucky
Protocol Citation: kyle.francis, Megan Romano, Rupam Sarma 2023. Quantitation of Anticoagulant Rodenticides in Serum. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly752mlx9/v1
Manuscript citation:
Comprehensive Evaluation of HPLC-MS/MS Method for Quantitation of Seven Anticoagulant Rodenticides and Dicoumarol in Animal Serum. J Anal Toxicol.2023 Mar 3;bkad017. doi: 10.1093/jat/bkad017. Kyle A Francis 1, Andriy Tkachenko 2, Joseph T Johnson 1, Lori L Smith 1, Robyn T Noonan 3, Michael S Filigenzi 3, L Cynthia Gaskill 1, Megan C Romano. https://pubmed.ncbi.nlm.nih.gov/36869712/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 22, 2023
Last Modified: November 15, 2023
Protocol Integer ID: 79271
Keywords: Anticoagulant, dicoumarol, rodenticides, chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone, warfarin, LC-MS/MS, animal, serum, pesticides, diagnostics
Funders Acknowledgement:
U.S. FDA's Vet-LIRN
Grant ID: 1U18FD005015
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
This SOP describes the extraction and sample clean-up method for the quantitative determination of eight anticoagulant rodenticides in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography–tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. Limits of quantitation at 2.5 ng/mL for all analytes.
Method validation/evaluation/verification:
In-house method validation data and evaluation by an independent laboratory (Vet-LIRN) in collaborative multi-laboratory studies are published: https://pubmed.ncbi.nlm.nih.gov/36869712/
Attachments
Materials
I.Materials / Equipment
A.Supplies
Micropipettors and corresponding pipette tips (0.5 uL to 1000 uL)
Disposable 5-mL syringes with Luer-lok tips (BD Syringe, #309646)
Disposable syringe filters, 0.45 μm, PVDF membrane (MicroSolv Filters, #58045-V04-C)
Disposable micro-centrifuge tubes, 1.5 mL, polypropylene (VWR, #89000-028)
Silanized autosampler vials (2-mL; silanized; amber; Fisher Scientific #03-377F)
Vial closures and septa (Fisher Scientific #03-379-113)
Glass Pasteur pipettes
B.Chemicals (Higher grade chemicals may be substituted)
Distilled, deionized (DDI) water
Methanol, Acetonitrile, Acetone, Chloroform (HPLC or LC-MS grade; Fisher Scientific)
Ammonium acetate, HPLC grade (Fisher Scientific #A639-500)
Ammonium hydroxide, ACS grade (ACS grade, #BDH3014-500MLP, VWR International)
Standard Reference Materials:
a .Coumachlor, 98% (Sigma-Aldrich #189219-1G)
b. Dicoumarol, 98.0% (Sigma-Aldrich #M1390-5G)
c. Brodifacoum, 98.1% (US EPA National Pesticide Standard Repository)
d. Bromadiolone, 98.9% (US EPA National Pesticide Standard Repository)
e. Chlorophacinone, 99.8% (US EPA National Pesticide Standard Repository)
f. Difethialone, 99.7% (US EPA National Pesticide Standard Repository)
g. Diphacinone, 99.3% (US EPA National Pesticide Standard Repository)
h. Warfarin, 99.5% (US EPA National Pesticide Standard Repository)
C.Equipment / Instrumentation (Equivalent may be substituted)
Analytical Balance (Model PB303-S, Mettler Toledo)
Vortex (Vortex Genie 2, Fisher Scientific)
Centrifuge w/ cooling capability suitable for 1.5 mL microcentrifuge tubes (Micromax RF, Thermo Electron)
Ultrasonic Cleaner (FS30, Fisher Scientific) & micro-centrifuge floatation rack (VWR)
Thermo Scientific / Dionex UltiMate 3000 Rapid Separation Liquid Chromatography (UPLC) system with autosampler, binary pump and thermostatted column compartment
Thermo Scientific Quantum Access Max triple quadrupole mass analyzer with heated electrospray ionization
Analytical Chromatography Column (Zorbax XDB-C18 Eclipse, 2.1 x150 mm, 5μm; Agilent, #993700-902)
Guard Column Cartridge System (Syncronis C18 cartridges, 2.1 x 10 mm, 5 μm, Thermo Scientific, #97105-012101)
Prepared Reagents
Prepared Reagents
Primary Stock Solutions – 1000 ug/mL: For each anticoagulant rodenticide, dissolve 5.0 ± 0.1 mg standard reference material in 5 mL of the appropriate solvent (as per Table 1), using 5-mL volumetric flasks. These eight solutions should be stored at -20°C for up to one year.
Table 1: Solvents for Anticoagulant rodenticides
| Anticoagulant Rodenticide | Solvent | |
| Bromadiolone, Coumachlor, Warfarin | Methanol | |
| Brodifacoum, Chlorophacinone, Difethialone, Diphacinone | Acetone | |
| Dicoumarol | Chloroform |
10% (v/v) Acetone in Methanol: Transfer 25 mL acetone to a 250-mL graduated cylinder and bring to a total volume of 250 mL with methanol.
Secondary Stock Solution – 10 µg/mL: Transfer 50 µL of each primary stock solution to a single 5-mL volumetric flask. Complete the volume with methanol to prepare a single solution that is 10 µg/mL of each AR. This solution should be stored at -20°C for up to one month.
Note
The use of a positive displacement pipette may be necessary for accurate transfer of acetone- and chloroform-containing solutions.
Working Solution A – 1.25 µg/mL: Transfer 625 µL of the secondary stock solution to a single 5-mL volumetric flask. Complete the volume with methanol to prepare a single solution that is 1.25 µg/mL of each AR. This solution should be stored at -20°C for up to one month.
Working Solution B – 0.125 µg/mL: Transfer 62.5 µL of the secondary stock solution to a single 5-mL volumetric flask. Complete the volume with methanol to prepare a single solution that is 0.125 µg/mL of each AR. This solution should be stored at -20°C for up to one month.
Mobile Phase Solutions: De-gas mobile phase solutions by helium sparging
0.01M Ammonium Acetate, pH 9 – Dissolve 0.77 ± 0.01g ammonium acetate in ~750ml distilled, deionized water (DDI water) in a 1-L volumetric flask. Adjust pH to 9 by adding ammonium hydroxide dropwise. Complete volume with DDI water.
Methanol, LC-MS grade
Sample Treatment
Sample Treatment
Matrix-Matched Calibrants and Quality Control Samples
Note
Prepare 7 calibrants and 3 QC samples in labelled 1.5-mL disposable micro-centrifuge tubes using control serum.
Pipette the appropriate volumes of AR Standard Solutions followed by controlled serum into the corresponding tube, as described in Table 2, yielding a final volume of 250 µL .
Cap tubes and vortex mix for 00:00:10 to mix thoroughly.
10s
Proceed to step 8.2
Table 2: Preparation of Matrix-Matched Calibrants and Quality Control Samples
| A | B | C | D | E | F | |
| Calibrant / QC Sample | Concentration (ppb; ng/g) | Volume of Secondary Stock Solution (μL) | Volume of Working Solution A (μL) | Volume of Working Solution B (μL) | Volume of Control Serum (μL) | |
| Cal 1 | 2.5 | --- | --- | 5.0 | 245 | |
| Cal 2 | 5.0 | --- | --- | 10 | 240 | |
| Cal 3 | 10 | --- | --- | 20 | 230 | |
| Cal 4 | 25 | --- | 5.0 | --- | 245 | |
| Cal 5 | 50 | --- | 10 | --- | 240 | |
| Cal 6 | 250 | 6.25 | --- | --- | 243.75 | |
| Cal 7 | 500 | 12.5 | --- | --- | 237.50 | |
| QC Blank | 0 | --- | --- | --- | 250 | |
| QC 5.0PPB | 5.0 | --- | --- | 10 | 240 | |
| QC 400PPB | 400 | 10 | --- | --- | 240 |
Sample Extraction
Transfer 250 µL unknown sample serum to a labelled 1.5-mL disposable micro-centrifuge tube.
To all calibrants, QC samples, and unknown samples, add 250 µL 10% (v/v) acetone in methanol pre-chilled at 4°C using an accurate pipettor. Vortex mix thoroughly for 00:00:10 .
10s
Centrifuge the samples at 16,000 g and 4°C for 00:10:00
10m
Decant the supernatant for each sample into a new, labelled 1.5-mL disposable micro-centrifuge tube.
Into the decanted tubes with precipitate, add 250 µL 10% (v/v) acetone in methanol pre-chilled at 4°C using an accurate pipettor to all calibrants, QC samples, and unknown samples. Vortex mix thoroughly for 00:00:10 .
10s
Place the precipitate-containing samples into the micro-centrifuge tube flotation rack and place in the sonication bath.
Sonicate the samples for 00:05:00
5m
Centrifuge the samples at 16,000 g and 4°C for 00:10:00
10m
Transfer, via glass pipette, the supernatant for each sample and combine with the previous corresponding supernatant in step 8.4. Vortex mix thoroughly for 00:00:10
10s
Centrifuge the combined supernatant tubes at 16,000 g and 4°C for 00:10:00 .
10m
Filter each supernatant by syringe filtering:
- Remove the syringe plunger and attach a PVDF luer-lok syringe filter.
- Transfer the supernatant to the syringe, re-insert the plunger and filter into:
Note
If the filtered sample’s volume is below or approximately equal to the minimum volume needed for proper autosampler syringe aliquoting (i.e. syringe height) when following i., then follow ii. to ensure proper syringe aliquoting and injection onto column.
i. a labelled glass silanized autosampler vial or
ii. a 1.5-mL micro-centrifuge tube, then pipet 150 µL of the filtered sample into a labelled glass silanized autosampler vial containing a vial insert.
HPLC – MS/MS Analysis
HPLC – MS/MS Analysis
HPLC Settings
Gradient Elution Profile: Profile parameters may be adjusted slightly at the discretion of the chemist to achieve baseline resolution of brodifacoum and difethialone at 500ppb (Cal 7). The recommended gradient profile when using a Zorbax XDB-C18 Eclipse, 2.1 x150 mm, 5μm analytical column along with Syncronis C18 guard column is in Table 3.
Table 3. Recommended Gradient Profile
| Time (min) | 0.01M Ammonium Acetate, pH 9 (%) | Methanol (%) | |
| 0 | 60 | 40 | |
| 1 | 60 | 40 | |
| 9 | 43 | 57 | |
| 15 | 23 | 77 | |
| 18 | 19 | 81 | |
| 19 | 10 | 90 | |
| 24 | 10 | 90 | |
| 25 | 60 | 40 | |
| 34 | 60 | 40 |
Flow Rate: 0.400 mL/min
Column Temperature: 25°C
Injection Volume:10 µL
Total Run Time: 00:34:00
34m
Autosampler temperature: +24C (room temperature). Note, pesticides are usually very stable
MS/MS Detection These parameters are suggestions and may need to be optimized for different MS instruments. Multiple reaction monitoring transition parameters are listed in Table 4.
Table 4. MRM Transitions and Approximate Expected Retention Times
| A | B | C | D | E | F | |
| Anticoagulant Rodenticide | Retention Time (min) | Precursor Ion ((M-H+)-; u) | Fragment Ion* | Collision Energy (eV) | Tube Lens (V) | |
| Warfarin | 3.70 | 307 | 161 | 22 | 70 | |
| 250 | 25 | 70 | ||||
| Coumachlor | 7.50 | 341 | 284 | 26 | 71 | |
| 161 | 23 | 71 | ||||
| Diphacinone | 10.06 | 339 | 167 | 28 | 77 | |
| 165 | 48 | 77 | ||||
| Dicoumarol | 10.30 | 335 | 161 | 21 | 47 | |
| 117 | 47 | 47 | ||||
| Chlorophacinone | 13.26 | 373 | 201 | 24 | 76 | |
| 145 | 25 | 76 | ||||
| Bromadiolone** | 15.33 | 525 | 250 | 38 | 97 | |
| 273 | 40 | 97 | ||||
| Brodifacoum | 17.92 | 521 | 135 | 40 | 101 | |
| 143 | 57 | 101 | ||||
| Difethialone | 18.22 | 537 | 151 | 41 | 100 | |
| 371 | 35 | 100 |
*Transitions in bold are used for quantitation
**Two isomers are present for Bromadiolone; only the earliest eluting (and most abundant) isomer is used for detection and quantitation.
ESI Source Conditions: Optimized on the basis of direct infusion of solvent-diluted reference standards
- Negative ion mode
- Spray Voltage: 4000 V
- Vaporizer Temperature: 380°C
- Sheath Gas Pressure: 50 psi
- Auxiliary Gas Pressure: 45 psi
- Ion Sweep Gas Pressure: 0 psi
- Capillary Temperature: 300°C
- Skimmer Offset:(Not used)
Other Parameters:
- Collision Gas Pressure: 1.7 mTorr
- Collision Energy: Ion-Dependent; see Table 4 (MRM Transitions)
- Tube Lens: Ion-Dependent; see Table 4 (MRM Transitions)
- Q1 / Q3 Peak Width (FWHM): 0.70 u
- Cycle Time: 0.300 s
Post-Acquisition Data Analysis
Post-Acquisition Data Analysis
Note
Peak area integration is performed using pre-selected software parameters (i.e. smoothing, S/N, etc.) as a starting point. The baseline setting and the peak integration start and stop points are then visually inspected in each chromatogram and manually adjusted as needed.
Qualitative Identification- The respective analyte is considered to be qualitatively identified in the unknown sample if the following criteria are met:
The quantifying ion and the corresponding confirming ion co-elute within 0.1 min of one another, each with a signal-to-noise ratio > 3.
The retention times of the quantifying and confirming ions are within 2% of the mean retention time for the same analyte in all calibrants and QC samples acquired within the same batch analysis
The quantifying ion:confirming ion ratio is within +/- 20% of the expected ratio (typically the average of the batch standards’ ion ratios).
Quantitative Analysis – The following parameters should be used to generate calibration curves to determine quantitative results
Perform quadratic least squares regression using peak areas for all calibrants versus concentration, ranging from 2.5 to 500 ppb
Weighting: 1/x2
Ignore Origin
Correlation coefficients (R2) are expected to be greater than or equal to 0.95
The peak area of the quantifying ion is greater than the peak area of the same ion in the least concentrated calibrant.