TITLE: Quantitation of Eight Anticoagulant Rodenticides in Liver
AUTHORS: megan.romano

[DESCRIPTION]
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:
 https://pubs.acs.org/doi/10.1021/acs.jafc.7b02280

[STEPS]
SECTION: Prepare Solutions
1. 10 % (v/v) Methanol in Acetonitrile: Dilute 25 mL methanol to 250 mL with acetonitrile.

SECTION: Prepare Solutions
2. Primary Stock Solutions – 1000 ug/mL: For each anticoagulant rodenticide, dissolve 5.0 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 
 
 
 


SECTION: Prepare Solutions
3. 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.         

 
                                                                                                                                                                                                   
SECTION: Prepare Solutions
4. Mobile Phase Solutions: De-gas mobile phase solutions by helium sparging as needed.
SECTION: Prepare Solutions
4.1. a.  0.01M Ammonium Acetate, pH 9  – Dissolve 0.77 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. 

SECTION: Prepare Matrix-Matched Calibrants and Quality Control (QC) Samples
5. Transfer 1.0 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.
SECTION: Sample Homogenization and Extraction
9. Transfer 1.0 g unknown sample liver to a 50-mL disposable centrifuge tube.

SECTION: Sample Homogenization and Extraction
10. Homogenize the calibrants, QC samples, and unknown samples as follows:
SECTION: Sample Homogenization and Extraction
10.1. Add a 9.5mm steel ball bearing to each tube and cap. 

SECTION: Sample Homogenization and Extraction
11. Add 6 mL 10 % (v/v) methanol in acetonitrile to each tube and vortex mix for 10 s to mix thoroughly.

SECTION: Sample Homogenization and Extraction
12. Repeat the homogenization process as described in step 10.2.
A single repetition of impact grinding at 650 rpm for 5 min is usually sufficient.
SECTION: Sample Homogenization and Extraction
13. Load all calibrants, QC samples, and unknown samples on the reciprocating shaker for 30 min.

SECTION: Sample Homogenization and Extraction
14. Centrifuge at 829g (2000 RPM using the specified centrifuge) for 5 min  

SECTION: Sample Clean-up with d-SPE
15. Transfer the supernatant of each extraction mixture to the corresponding d-SPE tube by pouring carefully and recap the d-SPE tube.
SECTION: Sample Clean-up with d-SPE
16. Vortex for 10 s to wet the sorbent material completely.

SECTION: Sample Clean-up with d-SPE
17. Load all calibrants, QC samples, and unknown samples on the multi-mixer & rotator for 30 min.

SECTION: Sample Clean-up with d-SPE
18. Centrifuge at 829g (2000 RPM using the specified centrifuge) for 5 min.

SECTION: Solvent Exchange / Concentrating Extracts
19. Transfer the supernatant of the d-SPE clean-up mixture to a 15-mL disposable centrifuge tube.

 

SECTION: Solvent Exchange / Concentrating Extracts
20. Evaporate to dryness under a gentle stream of nitrogen at 45°C.         

 
                                                   
SECTION: Solvent Exchange / Concentrating Extracts
21. Transfer 1.0 mL methanol to each tube and vortex for 10 s. Sonicate in a water bath for no more than 5 minto completely dissolve the residue. Required time for sonication may vary based on unique sample characteristics.

SECTION: Solvent Exchange / Concentrating Extracts
22. Transfer each prepared solution to a clean disposable microbeaker.
SECTION: Solvent Exchange / Concentrating Extracts
23. Draw up the entire volume into a clean 5-mL Luer-lok syringe.
SECTION: Solvent Exchange / Concentrating Extracts
24. Filter the sample through a 0.22 um PTFE syringe filter into an autosampler vial for analysis.
SECTION: UHPLC – MS/MS Analysis
25. 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  
     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: UHPLC – MS/MS Analysis
25.1. Flow Rate: 0.300 mL/min 

SECTION: UHPLC – MS/MS Analysis
25.2. Column Temperature: 25 °C 

SECTION: UHPLC – MS/MS Analysis
25.3. Injection Volume: 1 µL 

SECTION: UHPLC – MS/MS Analysis
25.4. Total Run Time: 34 min 

SECTION: UHPLC – MS/MS Analysis
26. MS/MS Detection
These parameters are suggested settings and may need to be optimized for different MS instruments
SECTION: UHPLC – MS/MS Analysis
26.1. 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:   
e. Auxillary Gas Pressure:   
f. Ion Sweep Gas Pressure:   
g. Capillary Temperature: 300 °C 
h. Skimmer Offset: (Not used)
SECTION: UHPLC – MS/MS Analysis
26.2. Other Parameters
a. Collision Gas Pressure:   
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

 



SECTION: UHPLC – MS/MS Analysis
27. Post-Acquisition Data Analysis


 
                                                                                                 
SECTION: UHPLC – MS/MS Analysis
27.1. 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.
SECTION: Prepare Solutions
4.2. b.     Methanol, HPLC grade - This solution may be stored at room temperature for up to 5 years.
SECTION: Sample Homogenization and Extraction
10.2. Grind tissue at a rate of 650 rpm for 5 min. One round of impact
grinding may be sufficient, however a second round of grinding may be performed
if needed.
SECTION: Prepare Matrix-Matched Calibrants and Quality Control (QC) Samples
6. 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

 
 
SECTION: Prepare Matrix-Matched Calibrants and Quality Control (QC) Samples
7. Cap tubes and vortex mix the spiked liver for 10 s to mix thoroughly.

SECTION: Prepare Matrix-Matched Calibrants and Quality Control (QC) Samples
8. Proceed to step 10
SECTION: UHPLC – MS/MS Analysis
27.2. 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.

 

d.      The intensity of the quantifying ion is greater than the intensity of the same ion in the least concentrated calibrant.