TITLE: Analysis of sodium monofluoracetate (compound 1080) in animal kidney tissue by LC-MS/MS
AUTHORS: Jim Langston

[DESCRIPTION]
This diagnostic method provides for the identification and quantitative analysis of sodium monofluoroacetate (Compound 1080) from kidney tissue using LC-MS/MS

Sodium monofluoroacetate (Compound 1080) is an EPA Category I toxin.  In the US, its use is restricted to licensed applicators who may use it in bait collars to protect small ruminants.  Animal intoxication and death
are reported, giving a need for a rapid, sensitive diagnostic method for detection and quantitation of Compound 1080 in animal tissues, including kidney.  This SOP describes an LC-MS/MS method for the identification and quantitative analysis of Compound 1080 in kidney tissue at low ppb levels (e.g. ~5ppb).  The method is rapid, sensitive, and selective.

Validation data (in-house and by an independent laboratory via collaborative study such as Blinded Method Test) are available upon request. 

[BEFORE_START]
Calibration and spiking standard solutions are prepared using certified sodium
monofluoroacetate standard materials, provided either neat or in solution.  Certified standard materials are available
from Cambridge Isotope Laboratories and other vendors.  Isotopically labeled sodium monofluoroacetate
(13C2, d2-sodium monofluoroacetate) is available as an internal standard (IS) from Cambridge Isotope Laboratories and other vendors.




[GUIDELINES]
DEFINITIONS AND ACRONYMS 

Compound 1080 = Sodium monofluoroacetate
ESI = Electrospray ionization
UHPLC = Ultra high performance liquid chromatograph
IS = Internal standard
LC-MS/MS = Liquid Chromatography/Mass Spectrometry/Mass
Spectrometry
MRM = Multiple reaction monitoring
RL = Reporting limit
SPE = Solid phase extraction


SPECIMEN INFORMATION

5 g of kidney tissue (optimal).  Sub-optimal sample amounts and quality may require modifications to the method and elevation of the method reporting limit (RL). Non-representative samples may affect results.


Quality Control and Reference Material
Internal standard (IS) is added to each reagent blank, control sample, and sample prior to extraction.  The IS used is 13C2, d2-sodium monofluoroacetate, added at a concentration of 10 ppb.   
Each set of samples must include a negative, unfortified control (check) sample from an appropriate control matrix.
It is recommended, at the discretion of the analyst, to include a reagent blank with each set of samples 
Each set of samples must include a spiked (fortified) sample from an appropriate control matrix or an over spike of the sample material.  The following spike levels are suggested:
                  5 ppb level:  add 0.005 mL of 1 µg/mL spiking standard into 1 g of control kidney     
                  10 ppb level:  add 0.010 mL of 1 µg/mL spiking standard into 1 g of control kidney 
For quantitative results, a set of calibrations standards must be prepared and included
The group leader, chief chemist or the toxicologist may approve deviations from these guidelines to meet the diagnostic utility of the test.


METHOD LIMITATIONS

The sample clean-up by SPE requires Compound 1080 be present as an anion.  With a pKa value of 2.66, this
requires that the extract be at a pH value of 4.66 or greater prior to loading onto the MAX SPE resin.  It is
recommended to test the pH of the extract prior to loading onto the MAX SPE resin to verify this.  During validation,
extracts from spiked kidneys and incurred kidneys were shown to be ~pH 7.  


DOCUMENT AND RECORD REQUIREMENTS

The following information, as applicable, must be recorded in the lab notebook or other sample preparation records:
Name of analyst prepping the samples; 
 Date manipulations were started (extraction date);
 Method extraction description (ex SOP number)
 Sample ID (full accession number)
  QC sample(s) IDs (QC ID or Lot Number)
  Physical manipulations performed on samples and QC samples including: 
            amount measured
            dilutions
            aliquot amount
            final volumes
            Spiking information: ID of spiking solution used, initials and date of person performing spiking.
            Pipet ID #
            Lot #s of SPEs 
            Signature/Date of analyst upon completion

This is the minimum amount of information that should be recorded.  If certain of the above data elements are not relevant then they may be excluded.







[STEPS]
SECTION: Prepare Reagents
1. 5% Ammonium hydroxide in water
SECTION: Prepare Reagents
1.1.  Combine 10 mL of ammonium hydroxide with 190 mL of water'

SECTION: Prepare Reagents
2. 0.5% Trifluoroacetic acid in acetonitrile
SECTION: Prepare Reagents
2.1. Combine5 mL of trifluoroacetic acid with 995 mL of water

SECTION: Prepare Reagents
3. 5 mM Ammonium formate, 0.01% formic acid in water (Mobile Phase A)
SECTION: Prepare Reagents
3.1. Dissolve 0.315 g of ammonium formate in approximately 950 mL ofwater in a 1 L volumetric flask.  

SECTION: Prepare Reagents
3.2. Add 0.100 mL of formic acid, and fill to a final volume of 1.00 L with
water.  

SECTION: Prepare Reagents
3.3. Store in an amber bottle.

 

SECTION: Calibration standard solutions
4. Working standard solutions of sodium monofluoroacetate are prepared from neat material or
solution at 1, 0.1, and 0.01 µg/mL levels.

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



SECTION: Calibration standard solutions
5.1. 

Internal standard (IS) is added to each calibration standard at a concentration of 1.25
ppb (ng/mL).



SECTION: Calibration standard solutions
4.1. 

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.    

     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   

 


SECTION: Sample Preparation
6. Ensure all samples are finely chopped 

 

SECTION: Sample Preparation
7. Weigh 1 g (+/- 0.05 g) of sample into a 50 mL plastic centrifuge tube



SECTION: Sample Preparation
8. Add 0.020 mL of 0.50 µg/mL 13C2, d2-sodium monofluoroacetate (IS) solution



SECTION: Sample Preparation
9. Add 4 mL of water and 2 ball bearings



SECTION: Sample Preparation
10. Genogrind at 750 rpm for 5 min



SECTION: Sample Preparation
11. Remove ball bearings using a magnet



SECTION: Sample Preparation
12.   Centrifuge to pellet at 4,000 x g, 10 min, 10 °C



SECTION: Sample Preparation
13. 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



SECTION: Sample Preparation
14. 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.



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



SECTION: Sample Preparation
15.1. Cartridges are positioned atop an SPE vacuum manifold



SECTION: Sample Preparation
15.2. Cartridges are first conditioned by rinsing with 4 mL of methanol, followed by rinsing with3 mL of water, and then an additional 3 mL of water 



SECTION: Sample Preparation
15.3. 2 mL 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



SECTION: Sample Preparation
15.4. Cartridge is washed with 4 mL of 5% ammonium hydroxide in water



SECTION: Sample Preparation
15.5. Cartridge is washed with 4 mL of methanol, and then pulled dry under vacuum for 30 s



SECTION: Sample Preparation
15.6. Cartridge is washed with 3 mL of 0.5% trifluoroacetic acid in acetonitrile, and then pulled dry under vacuum for 30 s



SECTION: Sample Preparation
15.7.   Cartridge is washed another time with 3 mL of 0.5% trifluoroacetic acid in acetonitrile, and then pulled dry under vacuum for 30 s


SECTION: Sample Preparation
15.8. 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 30 s, collecting the eluent



SECTION: Sample Preparation
15.9. The cartridge is eluted again with2 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 30 s, collecting the eluent in the same collection tube, for a total volume of 4 mL pooled eluent



SECTION: Sample Preparation
15.10. Pooled eluent is next analyzed by LC-MS/MS



SECTION: Instrument Parameters
16. 
 

SECTION: Instrument Parameters
17. UHPLC Setting


SECTION: Instrument Parameters
17.1.   Mobile Phase A:  5 mM Ammonium formate, 0.01% formic acid in water

SECTION: Instrument Parameters
17.2. MobilePhase B:  Acetonitrile


SECTION: Instrument Parameters
17.3. Injection volume:  5 µL


SECTION: Instrument Parameters
17.4.  Autosampler temperature:  10 °C

SECTION: Instrument Parameters
17.5. Column temperature:  ambient room temperature


SECTION: Instrument Parameters
17.6.   Gradient:

     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   

SECTION: Instrument Parameters
18. Mass spectrometer settings


SECTION: Instrument Parameters
18.1.  Polarity:  Negative

SECTION: Instrument Parameters
18.2. Ionization:  ESI

SECTION: Instrument Parameters
18.3. Curtain gas:  40 psi

SECTION: Instrument Parameters
18.4. Ion source gas 1:  90 psi

SECTION: Instrument Parameters
18.5.  Ion source gas 2:  40 psi


SECTION: Instrument Parameters
18.6. Temperature:  500 °C

SECTION: Instrument Parameters
18.7. Scan type:  MRM
SECTION: Instrument Parameters
18.8.  Ion spray voltage:  4000 volts

SECTION: Instrument Parameters
18.9. Mass table
     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   

SECTION: Instrument Parameters
19. Data is acquired using SciEx OS software

SECTION: Analytical Sequence
21.    The sequence should begin and end with analysis of the calibration standards 


SECTION: Analytical Sequence
22. The beginning set of standards are followed by a fortified control sample (spike) or overspiked sample


SECTION: Analytical Sequence
23.     All sample and QC sample injections are done in duplicate

SECTION: Analytical Sequence
24. 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



SECTION: Instrument Parameters
20. All calculations are performed using the Analyst software within SciEx OS software

 



SECTION: INTERPRETATION OF RESULTS
25. 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).


SECTION: INTERPRETATION OF RESULTS
26. An analyte is considered positively identified if it meets the following criteria:


SECTION: INTERPRETATION OF RESULTS
26.1. The peak height signal to noise is greater than 3:1


SECTION: INTERPRETATION OF RESULTS
26.2. 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

SECTION: INTERPRETATION OF RESULTS
26.3. 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.

SECTION: INTERPRETATION OF RESULTS
26.4. The group leader, chief chemist or the toxicologist may approve deviations from these guidelines to meet the diagnostic utility of the test.



SECTION: INTERPRETATION OF RESULTS
27. Criteria for Data Acceptance (Quantitation)- The following conditions must be met for a concentration to be reported for the analyte:



SECTION: INTERPRETATION OF RESULTS
27.1. 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

SECTION: INTERPRETATION OF RESULTS
27.2. The spike recovery for the analyte should be between 70% and 120%.


SECTION: INTERPRETATION OF RESULTS
27.3.  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.



SECTION: INTERPRETATION OF RESULTS
27.4. 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%.



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