Analysis of sodium monofluoracetate (compound 1080) in animal kidney tissue by LC-MS/MS

5% Ammonium hydroxide in water

Combine 10mL of ammonium hydroxide with 190mL of water'

0.5% Trifluoroacetic acid in acetonitrile

Combine5mL of trifluoroacetic acid with 995mL of water

5 mM Ammonium formate, 0.01% formic acid in water (Mobile Phase A)

Dissolve 0.315g of ammonium formate in approximately 950mL ofwater in a 1 L volumetric flask. 

Add 0.100mL of formic acid, and fill to a final volume of 1.00L with

water. 

Store in an amber bottle.

Working standard solutions of sodium monofluoroacetate are prepared from neat material or

solution at 1, 0.1, and 0.01 µg/mL levels.

Calibration standards are prepared as shown in Table 1 below.  Calibration standard solutions are preparedin from working standard solutions, diluting into 0.5% trifluoroacetic acid in

acetonitrile solvent.

A	B
Calibration Level	[Sodium Monofluoroacetate] ppb (ng/mL)
1	0.10
2	0.25
3	0.50
4	1.00
5	2.50
6	5.00
7	10.0
8	25.0
9	50.0

Table 1.  Calibration standard solutions

Working internal standard (IS) solutions are prepared from neat material or solution at 0.5 and 0.25 µg/mL levels.

Internal standard (IS) is added to each calibration standard at a concentration of 1.25

ppb (ng/mL).

Ensure all samples are finely chopped

Weigh 1g (+/- 0.05 g) of sample into a 50 mL plastic centrifuge tube

Add 0.020mL of 0.50 µg/mL ¹³C₂, d₂-sodium monofluoroacetate (IS) solution

Add 4mL of water and 2 ball bearings

Genogrind at 750 rpm for 0h 5m 0s

Remove ball bearings using a magnet

Centrifuge to pellet at 4,000 x g, 0h 10m 0s, 10 °C

Filter supernatant using a 6 mL syringe and a Whatman 1 µm GMF-150 syringe filter into a Pierce protein concentrator, PES, 3 kDa MWCO, 5 – 20 mL size

Centrifuge to obtain flow-through of a least 2 mL volume.  Centrifuge set to 4,000 x g, 60 min, 10

°C.  If insufficient flow-through obtained, repeat step with a new protein concentrator.

Further sample clean-up is next done using Waters Oasis MAX SPE cartridges, 500 mg, 6 cc:

Cartridges are positioned atop an SPE vacuum manifold

Pooled eluent is next analyzed by LC-MS/MS

Cartridges are first conditioned by rinsing with 4mL of methanol, followed by rinsing with3mL of water, and then an additional 3mL of water

2mL of the flow-through from the protein concentrator is next loaded onto the resin, taking care to load at a flow rate of no more than 1 drop per second

Cartridge is washed with 4mL of 5% ammonium hydroxide in water

Cartridge is washed with 4mL of methanol, and then pulled dry under vacuum for 0h 0m 30s

Cartridge is washed with 3 mL of 0.5% trifluoroacetic acid in acetonitrile, and then pulled dry under vacuum for 0h 0m 30s

mL Cartridge is washed another time with 3 mL of 0.5% trifluoroacetic acid in acetonitrile, and then pulled dry under vacuum for 0h 0m 30s

A 15 mL centrifuge is placed under each SPE column as a collection tube, and then the cartridge is eluted with 2 mL of 0.5% trifluoroacetic acid in acetonitrile at a flow rate of no more than 1 drop per second, and then pulled dry under vacuum for 0h 0m 30s, collecting the eluent

The cartridge is eluted again with2mL of 0.5% trifluoroacetic acid in acetonitrile at a flow rate of no more than 1 drop per second, and then pulled dry under vacuum for 0h 0m 30s, collecting the eluent in the same collection tube, for a total volume of 4mL pooled eluent

UHPLC Setting

Mobile Phase A:  5 mM Ammonium formate, 0.01% formic acid in water

MobilePhase B:  Acetonitrile

Injection volume:  5 µL

Autosampler temperature:  10 °C

Column temperature:  ambient room temperature

Gradient:

A	B	C	D
Time (min)	%A	%B	Flow Rate (mL/min)
0	10	90	0.45
3	10	90	0.45
4	60	40	0.45
5.5	60	40	0.45
5.6	10	90	0.45
16	10	90	0.45

Mass spectrometer settings

Polarity:  Negative

Ionization:  ESI

Curtain gas:  40 psi

Ion source gas 1:  90 psi

Ion source gas 2:  40 psi

Temperature:  500 °C

Scan type:  MRM

Ion spray voltage:  4000 volts

Mass table

A	B	C	D	E	F	G
Compound	Q1 mass (Da)	Q3 mass (Da)	Dwell Time (ms)	EP (V)	CE (V)	CXP (V)
Quantifier Ion	77	57	100	-10	-14	-10
Qualifier Ion	77	33	100	-10	-17	-10
IS Ion	81	60	100	-10	-16	-6

Data is acquired using SciEx OS software

All calculations are performed using the Analyst software within SciEx OS software

The sequence should begin and end with analysis of the calibration standards

The beginning set of standards are followed by a fortified control sample (spike) or overspiked sample

All sample and QC sample injections are done in duplicate

Sets of sample injections should be preceded by a reagent blank or an unfortified control sample (check) to demonstrate that no carryover from standards or fortified control is present

The analysis is acceptable if the reagent blank and/or check sample are negative, and reporting limit (RL) spike results for the analyte meets the criteria for positive identification (sec. 2, below).

An analyte is considered positively identified if it meets the following criteria:

The peak height signal to noise is greater than 3:1

The retention time of the analyte in the sample does not differ from that of the midpoint standard in the calibration curve by more than 0.25 min

The ratio of the peak area of the quantifier ion to that of the qualifier ion does not differ more

than 20% from either that of the average of the ion ratios from the calibration standards –-or- from that of the midpoint standard from the opening calibration curve.

The group leader, chief chemist or the toxicologist may approve deviations from these guidelines to meet the diagnostic utility of the test.

Criteria for Data Acceptance (Quantitation)- The following conditions must be met for a concentration to be reported for the analyte:

The peak area ratio of the analyte to that of its internal standard in the sample must fall within

the range of peak area ratios determined by the calibration curve

The spike recovery for the analyte should be between 70% and 120%.

If the sequence ends with a standard curve, then the two standardcurves are combined and the coefficient of determination (R²) for the combined curve must be >0.99.

If the sequence ends with a midpoint standard from the calibration curve, then the R² for the analyte in the calibration curve must be 0.99 and the calculated concentration of analyte in the midpoint standard must fall within +/- 20%.

The group leader and/or chief chemist can approve any deviations from these guidelines to meet the diagnostic utility of this test.