TITLE: Standard Operating Procedure (SOP) for the Analysis of benoxacor in subcellular fraction incubations by gas chromatography mass spectrometry/electron capture detector
AUTHORS: Derek Simonsen, Hans-Joachim Lehmler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This standard operating procedure uses liquid-liquid extraction for the determination of benoxacor in incubations with liver microsomes, liver cytosol, and other samples using a gas chromatography-mass spectrometry (GC-MS) and gas chromatography with electron capture detection (GC-ECD).</div></div>

[STEPS]
?. [Sample preparation]
Label disposable culture tubes and GC vials. Tubes and vials that need to be labeled include quality assurance/quality control (QA/QC) samples (i.e., buffer, cofactor with no benoxacor, inactivated subcellular fractions, subcellular fractions without c-substrate, and DMSO spiked buffer with cofactor), experimental samples and the Reference Standard.Important: Use unique sample numbers that include your initials and identifiers for the experiment (e.g., notebook number) and the sample. The goal is to avoid identical sample numbers across experiments.
?. [Sample preparation]
At least one tube for method blanks (buffer only).
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At least one tube per sample blank (matrix samples from species/sex analyzed).
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Three tubes for Ongoing  Precision and Recovery (OPR) samples (matrix samples spiked with known amounts of the target analyte(s), i.e., benoxacor).
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Tubes for all samples (depends on number of samples to be analyzed; it is recommended that 20% of the samples are QA/QC samples; increase the number of blank samples accordingly).
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One tube for the Reference Standard (will include known amounts of all analytes, including the surrogate recovery standards and internal standard) (Note: Solutions of all analytes are added to the tube with the Reference Standard at the same time when the samples are spiked)
?. [Protein degradation]
Immediately after incubation is completed place samples in furnace for 10 minutes at 110 oC(Note: Leaving samples in furnace longer to 10 minutes will reduce the recovery of the analytes)
?. [Protein degradation]
Cool samples to room temperature before continuing extraction.
?. [Protein degradation]
Spike samples with 25 μL of 20 μg/mL trifluralin dissolved in ethyl acetate
?. [Extraction]
Add 2 mL pesticide grade ethyl acetate to all samples(Note: Do not add extra ethyl acetate to reference)
?. [Extraction]
Put samples on tube rotator for 1 minute at 40 rpm to mix buffer and ethyl acetate.(Note:)  Make sure caps are tight so they do not leak
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Transfer the samples to a centrifuge and centrifuge at 3,000 rpm (1811 x g) for 5 minutes to facilitate the phase separation
?. [Extraction]
Add 2 mL of 1% KCl to the 4 mL of ethyl acetate to remove residual proteins.
?. [Extraction]
Put samples on tube rotator to mix the ethyl acetate and 1% KCl
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Transfer the samples to a centrifuge and centrifuge at 3,000 rpm (1811 x g) for 5 minutes to facilitate the phase separation
?. [Extraction]
Transfer 2 mL of ethyl acetate into a clean culture tube
?. [Extraction]
Repeat steps 3-5.1(Note: Combine the 2 mL of ethyl acetate to the previous 2 mL ethyl acetate generated from the first extraction)
?. [Extraction]
Transfer the 4 mL ethyl acetate to a clean culture tube  containing 0.25 mg of magnesium sulfate
?. [Extraction]
Briefly vortex each tube.
?. [Extraction]
Transfer the samples to a centrifuge and centrifuge at 3,000 rpm (1811 x g) for 5 minutes to facilitate precipitation of magnesium sulfate
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Carefully transfer the ethyl acetate into a clean tube while attempting to not transfer any magnesium sulfate(Note: if small amounts of magnesium sulfate does get transferred it can be removed in subsequent steps)
?. [Extraction]
Spike each tube with 25 μL with 20 μg/mL acenaphthene
?. [Extraction]
Evaporate each tube under nitrogen to approximately 500 μL
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Transfer samples into a GC vial and dilute to 1 mL
?. [GC/MS Analysis]
Run samples on GC/MS followed by GC/ECD.
?. [GC/MS Analysis]
?. [GC/MS Analysis]
GC/MS Parameters Samples were injected by splitless injection at 280 oC. We used a Supelco SLB-5MS column (30 m length, 0.25 mm inner diameter, 0.25 µm film thickness) with a column helium flow rate of 1 mL/min. The oven parameters were programmed as follows: Initial temperature, 50 oC hold for 1 min, 15 oC/min to 240 oC, hold for 13 min, 15 oC/min to 300 oC, hold for 10 min. The temperatures of the MS source and the MS quad were 230 oC and 150 oC, respectively.CompoundAvg. Rention timeQuantQualAcenaphthene11.35153154Trifluralin12.74264306Benoxacor15.57120259
CompoundAvg. Rention timeQuantQualAcenaphthene11.35153154Trifluralin12.74264306Benoxacor15.57120259
?. [GC/MS Analysis]
GC-ECD Parameters A 63Ni-µECD detector was used to monitor enantioselective metabolism.  We used an Agilent CP-Chrialsil Dex CB (CB) column (25 m length, 0.25 mm inner diameter, 0.25 µm film thickness) to determine the enantiomeric fraction of benoxacor using the following temperature program: 50 oC, hold for 1 min, 10 oC/min to 145 oC, hold for 50 min, 15 oC/min to 200 oC, hold for 63Ni-µECD detector 12 min. The injector temperature was set at 250 oC, and a constant helium flow rate of 2 mL/min was used.