Methanol in Acetonitrile 10% (v/v) Methanol in Acetonitrile: Dilute 25mL methanol to 250mL with acetonitrile.

Primary Stock Solutions – 1000 ug/mL : For each anticoagulant rodenticide, dissolve 5.0mg standard reference material in 5mL 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 50µL 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.

Phase Solutions : De-gas phase solutions by helium sparging as needed.

a. 0.01M Ammonium Acetate, pH 9 – Dissolve 0.77g 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.

Transfer 1.0g 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 0h 0m 10s to mix thoroughly.

Proceed to step 10

Transfer 1.0g 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 0h 5m 0s. One round of impact

grinding may be sufficient, however a second round of grinding may be performed

if needed.

Add 6mL 10% (v/v) methanol in acetonitrile to each tube and vortex mix for 0h 0m 10s to mix thoroughly.

Repeat the homogenization process as described in step 10.2.

A single repetition of impact grinding at 650 rpm for 0h 5m 0s is usually sufficient.

Load all calibrants, QC samples, and unknown samples on the reciprocating shaker for 0h 30m 0s.

Centrifuge at 829g (2000 RPM using the specified centrifuge) for 0h 5m 0s

Transfer the supernatant of each extraction mixture to the corresponding d-SPE tube by pouring carefully and recap the d-SPE tube.

Vortex for 0h 0m 10s to wet the sorbent material completely.

Load all calibrants, QC samples, and unknown samples on the multi-mixer & rotator for 0h 30m 0s.

Centrifuge at 829g (2000 RPM using the specified centrifuge) for 0h 5m 0s.

Transfer the supernatant of the d-SPE clean-up mixture to a 15-mL disposable centrifuge tube.

Evaporate to dryness under a gentle stream of nitrogen at 45°C.

Transfer 1.0mL methanol to each tube and vortex for 0h 0m 10s. Sonicate in a water bath for no more than 0h 5m 0sto completely dissolve the residue. Required time for sonication may vary based on unique sample characteristics.

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 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: 0.300mL/min

Column Temperature: 25°C

Injection Volume: 1µL

Total Run Time: 0h 34m 0s

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: 4000V

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

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 :

1. 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.
2. The primary (Quan) ion and the corresponding secondary (Confirming) ion co-elute within 0.1 min of one another.

b. Signal Intensity :

1. The peak areas in the primary and secondary ion channels must have a S/N ratio of 3 or greater.
2. 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.

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