Jan 24, 2024
Quantitation of Eight Anticoagulant Rodenticides in Liver
- 1University of Kentucky
External link: https://pubs.acs.org/doi/10.1021/acs.jafc.7b02280
Protocol Citation: Megan Romano 2024. Quantitation of Eight Anticoagulant Rodenticides in Liver. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn26w6g5d/v1
Manuscript citation:
Development and Validation of Quantitative Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Assay for Anticoagulant Rodenticides in Liver. Lori L Smith, Boying Liang, Marcia C Booth, Michael S Filigenzi, Andriy Tkachenko, Cynthia L Gaskill. J Agric Food Chem. 2017 Aug 9;65(31):6682-6691. doi: 10.1021/acs.jafc.7b02280. Epub 2017 Jul 27.
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: December 01, 2022
Last Modified: January 24, 2024
Protocol Integer ID: 73399
Keywords: rodenticides, electrospray, UPLC−MS/MS, poisoning, hydroxycoumarins, indanediones, dispersive SPE, warfarin, coumachlor, dicoumarol, bromadiolone, brodifacoum, difethialone, diphacinone, chlorophacinone
Funders Acknowledgement:
U.S. FDA's Veterinary Laboratory Investigation and Response Network (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
Target analytes comprised two chemical classes: hydroxycoumarins (warfarin, coumachlor, dicoumarol, bromadiolone, brodifacoum, and difethialone) and indanediones (diphacinone and chlorophacinone).Liver extracts were cleaned using dispersive solid phase extraction (d-SPE) to remove matrix interferences and analyzed by reverse phase ultraperformance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS). Electrospray ionization in negative ion mode combined with multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer provided simultaneous confirmation and quantitation. Detection limits spanned 0.75−25 ng/g, and lower quantitation limits were established as 50 ng/g.
Method validation/evaluation/verification:
In-house method validation data and evaluation by an independent laboratory (Vet-LIRN) are published:
Attachments
Materials
A. Equipment / Instrumentation
- Analytical balance (Sartorius AG BP221S)
- Geno/Grinder and corresponding ball bearings (SPEX Sample Prep)
- Vortex (Vortex Genie 2, Fisher Scientific)
- Reciprocating Shaker (MaxQ 2500, Fisher Scientific)
- Multi-Mixer & Rotator (MTR 22, United Products & Instruments, Inc.)
- Centrifuge with rotors suitable for 15-mL and 50-mL centrifuge tubes (Sorvall Legend RT+, Thermo Scientific)
- Evaporation System with water bath (N-EVAP, Organomation Associates)
- Tabletop Ultrasonic Cleaner (Model FS-30, Fisher Scientific)
- Thermo Scientific / Dionex UltiMate 3000 Rapid Separation Liquid Chromatography (UPLC) system with autosampler, binary pump and thermostatted column compartment
- Thermo Scientific TSQ Quantum Access Max triple quadrupole mass analyzer with electrospray ionization
- Analytical Chromatography Column (Accucore C18, 2.1 x 100 mm; 2.6 um; Thermo Scientific #17126-102130)
- Guard Column Cartridge System (UNIGUARD Defender Accucore C18 cartridges; 2.1x10mm, 2.6 um, Thermo Scientific #17126-012105)
B. Supplies
- Volumetric glassware including flasks (5-mL) and graduated cylinders (10-, 25-, 250-mL)
- Sharp scissors and forceps
- Micropipettors and corresponding pipette tips (0.5 uL to 1000 uL)
- Disposable centrifuge tubes (15-mL; polypropylene; Fisher Scientific #14-959-70C)
- Disposable centrifuge tubes (50-mL; polypropylene; VWR International #89401-562)
- Disposable Luer-lok syringes, (5-mL; Fisher Scientific #14-829-45)
- Disposable microbeakers (5-mL; polystyrene; Fisher Scientific #08-732-119)
- Syringe filters (PTFE; 0.22 um, 13 mm diameter; Fisher Scientific #50-014-52582)
- Autosampler vials (2-mL; silanized; amber glass; Fisher Scientific #03-377F)
- Vial closures and septa (Fisher Scientific #03-379-113)
C. Reagents and Chemicals
- Organic solvents(HPLC grade; Fisher Scientific):
a. Methanol (#A452-4),
b. Acetonitrile (#A298-4),
c. Acetone (#A949-4),
d. Chloroform (#C606-4)
2. Ammonium acetate, HPLC grade (Fisher Scientific #A639-500)
3. Ammonium hydroxide, ACS grade (#BDH3014-500MLP, VWR International)
4. 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)
5. d-SPE sample clean-up tubes containing 175mg MgSO4,100mg Florisil, 50mg Alumina Basic and 50mg Primary Secondary Amine (UCT #ECQUUS1215CT)
Prepare Solutions
Prepare Solutions
10 % (v/v) Methanol in Acetonitrile: Dilute 25 mL methanol to 250 mL with acetonitrile.
Primary Stock Solutions – 1000 ug/mL: For each anticoagulant rodenticide, dissolve 5.0 mg +/-0.1 mg standard reference material in 5 mL of the appropriate solvent (see Table 1), using a 5-mL volumetric flask. These eight solutions should be stored at -20°C and can be stored for up to one year.
Table 1: Solvent Selection Guide
| A | B | |
| Anticoagulant Rodenticide | Solvent | |
| Bromadiolone, Coumachlor, Warfarin | Methanol | |
| Brodifacoum, Chlorophacinone, Difethialone, Diphacinone | Acetone | |
| Dicoumarol | Chloroform |
Secondary Stock Solution – 10 ug/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 ug/mL of each ACR. This solution should be stored at -20°C and can be stored 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
Mobile Phase Solutions: De-gas mobile phase solutions by helium sparging as needed.
a. 0.01M Ammonium Acetate, pH 9 – Dissolve 0.77 g +/- 0.01 g 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. This solution may be stored at room temperature for up to one month.
b. Methanol, HPLC grade - This solution may be stored at room temperature for up to 5 years.
Prepare Matrix-Matched Calibrants and Quality Control (QC) Samples
Prepare Matrix-Matched Calibrants and Quality Control (QC) Samples
Transfer 1.0 g +/- 0.1 g pre-homogenized control liver to a 50-mL disposable centrifuge tube compatible with the Geno/Grinder. Repeat this step for a total of 7 calibrants and 3 QC samples.
Pipette out the appropriate volume of Secondary Stock Solution, as described in Table 2, into the corresponding tube and directly into the homogenized liver.
Table 2: Calibrant and Control Sample Preparation Guide
| A | B | C | |
| Calibrant / QC Sample | Concentration (ppb; ng/g) | Volume of Secondary Stock Solution (uL) | |
| Cal 1 | 25 | 2.5 | |
| Cal 2 | 50 | 5 | |
| Cal 3 | 75 | 7.5 | |
| Cal 4 | 100 | 10 | |
| Cal 5 | 500 | 50 | |
| Cal 6 | 1000 | 100 | |
| Cal 7 | 2500 | 250 | |
| QC Blank | 0 | 0 | |
| QC 50 ppb | 50 | 5 | |
| QC 1000 ppb | 1000 | 100 |
Cap tubes and vortex mix the spiked liver for 00:00:10 to mix thoroughly.
10s
Proceed to step 10
Sample Homogenization and Extraction
Sample Homogenization and Extraction
Transfer 1.0 g +/- 0.1 g unknown sample liver to a 50-mL disposable centrifuge tube.
Homogenize the calibrants, QC samples, and unknown samples as follows:
Add a 9.5mm steel ball bearing to each tube and cap.
Grind tissue at a rate of 650 rpm for 00:05:00 . One round of impact
grinding may be sufficient, however a second round of grinding may be performed
if needed.
5m
Add 6 mL 10 % (v/v) methanol in acetonitrile to each tube and vortex mix for 00:00:10 to mix thoroughly.
10s
Repeat the homogenization process as described in step 10.2.
A single repetition of impact grinding at 650 rpm for 00:05:00 is usually sufficient.
5m
Load all calibrants, QC samples, and unknown samples on the reciprocating shaker for 00:30:00 .
30m
Centrifuge at 829g (2000 RPM using the specified centrifuge) for 00:05:00
5m
Sample Clean-up with d-SPE
Sample Clean-up with d-SPE
Transfer the supernatant of each extraction mixture to the corresponding d-SPE tube by pouring carefully and recap the d-SPE tube.
Vortex for 00:00:10 to wet the sorbent material completely.
10s
Load all calibrants, QC samples, and unknown samples on the multi-mixer & rotator for 00:30:00 .
30m
Centrifuge at 829g (2000 RPM using the specified centrifuge) for 00:05:00 .
5m
Solvent Exchange / Concentrating Extracts
Solvent Exchange / Concentrating Extracts
Transfer the supernatant of the d-SPE clean-up mixture to a 15-mL disposable centrifuge tube.
Note
The supernatant may be carefully poured from the d-SPE tube to the clean tube.
Evaporate to dryness under a gentle stream of nitrogen at 45°C.
Note
Near the end of the drying process, the solution may foam. As long as no bulk solution exists and foam is of negligible volume, the sample preparation process can continue.
Transfer 1.0 mL methanol to each tube and vortex for 00:00:10 . Sonicate in a water bath for no more than 00:05:00 to completely dissolve the residue. Required time for sonication may vary based on unique sample characteristics.
5m 10s
Transfer each prepared solution to a clean disposable microbeaker.
Draw up the entire volume into a clean 5-mL Luer-lok syringe.
Filter the sample through a 0.22 um PTFE syringe filter into an autosampler vial for analysis.
UHPLC – MS/MS Analysis
UHPLC – MS/MS Analysis
34m
UHPLC Settings
Gradient Elution Profile: Profile parameters may be adjusted slightly at the discretion of the analyst to achieve baseline resolution of brodifacoum and difethialone at 2500ppb (Cal 7). The suggested gradient profile is in Table 3.
Table 3: Gradient elution profile
| A | B | C | |
| 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: 0.300 mL/min
Column Temperature: 25 °C
Injection Volume: 1 µL
Total Run Time: 00:34:00
34m
MS/MS Detection
These parameters are suggested settings and may need to be optimized for different MS instruments
ESI Source Conditions:Optimized on the basis of direct infusion of solvent-diluted reference standards
a. Negative ion mode
b. Spray Voltage: 4000 V
c. Vaporizer Temperature: 380 °C
d. Sheath Gas Pressure: 50 psi
e. Auxillary Gas Pressure: 45 psi
f. Ion Sweep Gas Pressure: 0 psi
g. Capillary Temperature: 300 °C
h. Skimmer Offset: (Not used)
Other Parameters
a. Collision Gas Pressure: 1.7 mTorr
b. Collision Energy: Ion-Dependent; see MRM Transitions Table (Table 4)
c. Tube Lens: Ion-Dependent; see MRM Transitions Table (Table 4)
d. Q1 / Q3 Peak Width (FWHM): 0.70 u
e. Cycle Time: 0.300 s
Table 4. MRM Monitored Transitions / Expected Retention Times
| A | B | C | D | E | F | |
| Anticoagulant Rodenticide | Retention Time (min) | Precursor Ion ((M-H+)-; u) | Fragment Ion (u) | Collision Energy (eV) | Tube Lens (V) Tube Lens (V) | |
| Warfarin | 2.00 | 307 | 161* | 22 | 70 | |
| 307 | 250 | 25 | 70 | |||
| Coumachlor | 4.87 | 341 | 284 | 26 | 71 | |
| 341 | 161 | 23 | 71 | |||
| Diphacinone | 7.48 | 339 | 167 | 28 | 77 | |
| 339 | 165 | 48 | 77 | |||
| Dicoumarol | 7.68 | 335 | 161 | 21 | 47 | |
| 335 | 117 | 47 | 47 | |||
| Chlorophacinone | 11.08 | 373 | 201 | 24 | 76 | |
| 373 | 145 | 25 | 76 | |||
| Bromadiolone** | 13.76 | 525 | 250 | 38 | 97 | |
| 525 | 273 | 40 | 97 | |||
| Brodifacoum | 16.28 | 521 | 135 | 40 | 101 | |
| 521 | 143 | 57 | 101 | |||
| Difethialone | 16.53 | 537 | 151 | 41 | 100 | |
| 537 | 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
Post-Acquisition Data Analysis
Note
Automated peak integration should be used as much as possible to minimize
manual data integration. Automated integration parameters should be determined based upon those that provide accurate peak integration in sample and calibrant chromatograms and may vary between analytes.
Qualitative Identification: Comparisons of unknown chromatograms to those of calibrants and QC samples are used to determine the presence or absence of each analyte. For an analyte to be considered present in a sample, the following requirements must be met:
a. Retention Time:
- The retention times of the primary (Quan) and secondary (Confirming) ions are within 0.25 min of the mean retention time for the corresponding analyte in all calibrants and overspiked QC samples acquired within the same batch.
- The primary (Quan) ion and the corresponding secondary (Confirming) ion co-elute within 0.1 min of one another.
b. Signal Intensity:
- The peak areas in the primary and secondary ion channels must have a S/N ratio of 3 or greater.
- If there are detectable AR levels in any of the associated blank samples (e.g. instrument blanks, reagent blanks, and negative control samples), then the peak area in the unknown sample must be at least 10 times that measured in the blank sample.
c. Ion Ratio Measurement: The ion ratios of the primary to secondary ions for a given analyte, expressed as a percentage, must be within 20% of the average ion ratio for the associated spiked matrix-matched samples (e.g. calibrants, QC samples) acquired concurrently.
Quantitative Analysis: Recommended method
Perform linear least squares regression using peak areas for all calibrants versus concentration, ranging from 25 to 2500 ppb. The following parameters should be used to generate calibration curves to determine quantitative results:
a. Weighting 1/x2
b. Ignore Origin
c. The coefficients of determination (R2) for each calibration are expected to be ≥0.985 when the fit is linear and the origin (0,0) is ignored.
Note
Occasionally, a quadratic instrument response is observed at concentrations > 1000 ppb. It is permissible to use a quadratic trendline, with or without weighting, in these situations with an R2 value of 0.985 or better.
d. The intensity of the quantifying ion is greater than the intensity of the same ion in the least concentrated calibrant.
Note
Overspiked QC samples (e.g., 50 ppb and 1,000 ppb) must yield AR concentration results that are within acceptable range (e.g., +/- 15%) of the expected AR concentrations