Quantitation of Anticoagulant Rodenticides in Serum

Primary Stock Solutions – 1000 ug/mL : For each anticoagulant rodenticide, dissolve 5.0 ± 0.1 mg standard reference material in 5mL 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

A	B
Bromadiolone, Coumachlor, Warfarin	Methanol
Brodifacoum, Chlorophacinone, Difethialone, Diphacinone	Acetone
Dicoumarol	Chloroform

10% (v/v) Acetone in Methanol : Transfer 25mL acetone to a 250-mL graduated cylinder and bring to a total volume of 250mL with methanol.

Secondary Stock Solution – 10 µ g/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 µg/mL of each AR. This solution should be stored at -20°C for up to one month.

Working Solution A – 1.25 µ g/mL : Transfer 625µL 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 µ g/mL : Transfer 62.5µL 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

Matrix-Matched Calibrants and Quality Control Samples

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 250µL.

Cap tubes and vortex mix for 0h 0m 10s to mix thoroughly.

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 250µL unknown sample serum to a labelled 1.5-mL disposable micro-centrifuge tube.

Centrifuge the combined supernatant tubes at 16,000 g and 4°C for 0h 10m 0s.

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 150µL of the filtered sample into a labelled glass silanized autosampler vial containing a vial insert.

To all calibrants, QC samples, and unknown samples, add 250µL 10% (v/v) acetone in methanol pre-chilled at 4°C using an accurate pipettor. Vortex mix thoroughly for 0h 0m 10s.

Centrifuge the samples at 16,000 g and 4°C for 0h 10m 0s

Decant the supernatant for each sample into a new, labelled 1.5-mL disposable micro-centrifuge tube.

Into the decanted tubes with precipitate, add 250µL 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 0h 0m 10s.

Place the precipitate-containing samples into the micro-centrifuge tube flotation rack and place in the sonication bath.

Sonicate the samples for 0h 5m 0s

Centrifuge the samples at 16,000 g and 4°C for 0h 10m 0s

Transfer, via glass pipette, the supernatant for each sample and combine with the previous corresponding supernatant in step 8.4. Vortex mix thoroughly for 0h 0m 10s

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

A	B	C
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:10µL

Total Run Time: 0h 34m 0s

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

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.