TITLE: Quantitation of Anticoagulant Rodenticides in Serum
AUTHORS: kyle.francis, megan.romano, Rupam Sarma

[DESCRIPTION]
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/

[STEPS]
SECTION: Prepared Reagents
2. 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.

SECTION: Prepared Reagents
1. 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   
 
SECTION: Prepared Reagents
3. 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.       


 
                                                                                                                             
SECTION: Prepared Reagents
4. 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.

SECTION: Prepared Reagents
5. 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.
SECTION: Prepared Reagents
6. Mobile Phase Solutions: De-gas mobile phase solutions by helium sparging
SECTION: Prepared Reagents
6.1. 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.
SECTION: Prepared Reagents
6.2. Methanol, LC-MS grade
SECTION: Sample Treatment
7. Matrix-Matched Calibrants and Quality Control Samples

 

SECTION: Sample Treatment
7.1. 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.

SECTION: Sample Treatment
7.2. Cap tubes and vortex mix for 10 s to mix thoroughly.

SECTION: Sample Treatment
7.3. Proceed to step 8.2


 Table 2: Preparation of Matrix-Matched Calibrants and Quality Control Samples
       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      

SECTION: Sample Treatment
8. Sample Extraction
SECTION: Sample Treatment
8.1. Transfer 250 µL unknown sample serum to a labelled 1.5-mL disposable micro-centrifuge tube. 


SECTION: Sample Treatment
8.2. 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 10 s.

SECTION: Sample Treatment
8.3. Centrifuge the samples at 16,000 g and 4°C for 10 min

SECTION: Sample Treatment
8.4. Decant the supernatant for each sample into a new, labelled 1.5-mL disposable micro-centrifuge tube.
SECTION: Sample Treatment
8.5. 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 10 s.

SECTION: Sample Treatment
8.6. Place the precipitate-containing samples into the micro-centrifuge tube flotation rack and place in the sonication bath.
SECTION: Sample Treatment
8.7. Sonicate the samples for 5 min

SECTION: Sample Treatment
8.8. Centrifuge the samples at 16,000 g and 4°C for 10 min

SECTION: Sample Treatment
8.9. Transfer, via glass pipette, the supernatant for each sample and combine with the previous corresponding supernatant in step 8.4. Vortex mix thoroughly for 10 s

SECTION: Sample Treatment
8.10. Centrifuge the combined supernatant tubes at 16,000 g and 4°C for 10 min.
SECTION: Sample Treatment
8.11. 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:                                                                                                                                                                                                                                                                                     

 
 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.

SECTION: HPLC – MS/MS Analysis
9. HPLC Settings
SECTION: HPLC – MS/MS Analysis
9.1. 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   
 
SECTION: HPLC – MS/MS Analysis
9.2. Flow Rate: 0.400 mL/min
SECTION: HPLC – MS/MS Analysis
9.3. Column Temperature: 25°C
SECTION: HPLC – MS/MS Analysis
9.4. Injection Volume:10 µL

SECTION: HPLC – MS/MS Analysis
9.5. Total Run Time: 34 min

SECTION: HPLC – MS/MS Analysis
9.6. Autosampler temperature: +24C (room temperature). Note, pesticides are usually very stable
SECTION: HPLC – MS/MS Analysis
10. 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

     Anticoagulant   Rodenticide      Retention Time   (min)      Precursor Ion  ((M-H+)-;   u)      Fragment Ion  (u)      Collision Energy   (eV)      Tube Lens  (V)        Warfarin      3.70      307      161a      22      70        307      250      25      70        Coumachlor      7.50      341      284      26      71        341      161      23      71        Diphacinone      10.06      339      167      28      77        339      165      48      77        Dicoumarol      10.30      335      161      21      47        335      117      47      47        Chlorophacinone      13.26      373      201      24      76        373      145      25      76        Bromadioloneb      15.33      525      250      38      97        525      273      40      97        Brodifacoum      17.92      521      135      40      101        521      143      57      101        Difethialone      18.22      537      151      41      100        537      371      35      100   

SECTION: HPLC – MS/MS Analysis
10.1. 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)
SECTION: HPLC – MS/MS Analysis
10.2. 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
SECTION: Post-Acquisition Data Analysis
11.  
 


SECTION: Post-Acquisition Data Analysis
12. Qualitative Identification- The respective analyte is considered to be qualitatively identified in the unknown sample if the following criteria are met:
SECTION: Post-Acquisition Data Analysis
12.1. 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.

SECTION: Post-Acquisition Data Analysis
12.2. 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
SECTION: Post-Acquisition Data Analysis
12.3. The quantifying ion:confirming ion ratio is within +/- 20% of the expected ratio (typically the average of the batch standards’ ion ratios).
SECTION: Post-Acquisition Data Analysis
13. Quantitative Analysis – The following parameters should be used to generate calibration curves to determine quantitative results
SECTION: Post-Acquisition Data Analysis
13.1. Perform quadratic least squares regression using peak areas for all calibrants versus concentration, ranging from 2.5 to 500 ppb

SECTION: Post-Acquisition Data Analysis
13.2. Weighting: 1/x2
SECTION: Post-Acquisition Data Analysis
13.3. Ignore Origin
SECTION: Post-Acquisition Data Analysis
13.4. Correlation coefficients (R2) are expected to be greater than or equal to 0.95
SECTION: Post-Acquisition Data Analysis
13.5. The peak area of the quantifying ion is greater than the peak area of the same ion in the least concentrated calibrant.