We outlined a procedure to detect and quantify iophenoxic acid in mongoose serum. Results suggest that iophenoxic acid may be used as a biological marker to verify beta uptake in the species. The main advantage of this method is that it allows for the quantification of iophenoxic acid analogs at parts per billion levels, using a simple to perform protein precipitation technique.
For mobile phase A, combine one milliliter of formic acid with one liter of ultra pure water. For mobile phase B, combine one milliliter of formic acid with one liter of acetonitrile. For the diluent, combine one milliliter of trifluoroacetic acid with 200 milliliters of acetonitrile.
Next, weigh approximately 10 milligrams of methyl-IPA on microbalance, and record the mass to plus or minus 0001 milligrams. Quantitatively transfer the methyl-IPA to a 10 milliliter class A volumetric flask, using four to five milliliters of acetonitrile. Sonicate for one minute to dissolve all solids and then bring to volume with acetonitrile.
Transfer approximately eight milliliters of each stock to eight milliliter amber glass vials with PTFE-lined caps and store the samples at room temperature. Then, transfer the remaining stock to hazardous waste. For the 25X stock seven methyl-IPA stock prepare a stock of methyl-IPA and acetonitrile at approximately 200 micrograms per milliliter.
Transfer one milliliter of the stock to a five milliliter flask and dilute to volume with acetonitrile. Then, transfer the stock to an eight milliliter amber glass vial with a PTFE-lined cap and store at room temperature. Using a repeat pipettor, prepare the six additional 25X methyl-IPA stocks described in the text protocol in eight milliliter amber glass vials with PTFE-lined caps and store at room temperature.
For the 25X surrogate stock, transfer 0.1 milliliter of the concentrated ethyl-IPA stock to a 10 milliliter class A volumetric flask using a 100 microliter glass syringe and then dilute to volume with acetonitrile. Transfer approximately eight milliliters of the surrogate stock to an eight milliliter amber glass vial with PTFE-lined cap and store at room temperature. Transfer the remaining stock to hazardous waste.
Next, prepare 4X stocks containing both methyl-IPA and ethyl-IPA in 10 milliliter screw-top glass auto sampler vials, as described in the text protocol. For example, to prepare stock 4X7, add 0.2 milliliters of the 25X stock seven methyl-IPA stock to a two milliliter vial using the repeat pipettor with a 0.5 milliliter capacity tip. Add 0.2 milliliters of the 25X surrogate ethyl-IPA stock using a repeat pipettor with a 0.5 milliliter capacity tip.
Add 0.85 milliliters of acetonitrile using a repeat pipettor with a one milliliter capacity tip. Cap the vial securely and invert five times to mix. Following this, prepare the standard curve in two milliliter screw-top auto sampler vials as described in the text protocol.
For example, to prepare standard seven, add 0.2 milliliters of the 4X7 stock to a two milliliter vial, using a repeat pipettor with a 0.5 milliliter capacity tip. Add 0.6 milliliters of ultra purity ionized water using a repeat pipettor with a one milliliter capacity tip. Cap the vials securely and invert five times to mix.
For each sample, prepare a 1.5 milliliter micro centrifuge tube containing 200 to 300 milligrams of sodium chloride and arrange the tubes in an 80 position plastic rack. For each sample, label one 1.5 milliliter micro centrifuge tube as A and a second micro centrifuge tube as B.Arrange the tubes in an 80 position plastic rack. Place the following materials and equipment needed for serum extraction in a class two biosafety cabinet:the micro centrifuge tubes, a vortex mixer, a repeat pipettor with 0.5 milliliter and five milliliter capacity tips, a 100 to 1000 microliter air displacement pipette with 1000 microliter tips, containers with an approximately 100 milliliters each of diluent and ultra purity ionized water, and biohazard waste container.
Next, remove serum samples from frozen storage and warm to room temperature in the biosafety cabinet. Vortex mix each serum sample prior to sampling. Using a repeat pipettor with a 0.5 milliliter capacity tip, dispense 05 milliliters of mongoose serum into tube A and add 05 milliliters of the 25X surrogate stock.
Then add 0.95 milliliters of diluent to tube A using a repeat pipettor with a five milliliter capacity tip. Cap the tubes securely and vortex mix for 10 to 15 seconds. Next, add the control mongoose serum to tube A.Fortify each of the 4QC samples with me-IPA using a repeat pipettor with a 0.5 milliliter capacity tip.
Add 25X surrogate stock to the QC samples. Then, add the diluent to the QC samples. Cap the tubes and vortex mix.
Dispense the pre-weighed sodium chloride into tube A and vortex mix three times for eight to 12 seconds. Then, wipe down the outside surfaces of the vial rack containing tube A using 70%isopropanol. After removing the samples from the biosafety cabinet, centrifuge tube A at 1200 times G for one minute to separate the aqueous and acetonitrile phases.
Pipette 0.8 milliliters of the upper acetonitrile phase to tube B, using a 100 to 1000 microliter air displacement pipette. Transfer the remaining solution in tube A to hazardous waste and discard the empty tube in a biohazardous waste container. Now, remove acetonitrile and trifluoroacetic from tube B with a gentle flow of nitrogen gas in a 45 degrees Celsius water bath.
Add 0.25 milliliters of acetonitrile to tube B using a repeat pipettor and vortex mix for four to five seconds. Then, centrifuge at 1200 times G for two to four seconds to collect the liquid in the bottom of the tube. Following centrifugation, add 0.75 milliliters of ultra pure deionized water to tube B using a repeat pipettor with a five milliliter capacity tip and vortex mix for four to five seconds.
Centrifuge at 1200 times G for one minute to clarify the sample. Then, transfer 0.75 milliliters of the supernatant to an auto sampler vial using a 1000 microliter air displacement pipette and discard the pipette tips in the biohazard waste container. Cap each auto sampler vial securely for LC-MS/MS analysis.
Transfer the remaining solution in tube B to hazardous waste and discard the empty tube in a biohazardous waste container. Configure the LC-MS/MS with all parameters described in the text protocol. Power on the LC-MS/MS and allow the column to reach 70 degrees Celsius before setting the flow rate to 0.8 milliliters per minute.
Set up a sequence in the data acquisition software to inject the standard curve before and after each batch consisting of quality control samples and unknown samples. Inject all standards and samples and acquire MR ion chromatograms using parameters listed in the text protocol. After sequence completion, turn off the LC-MS/MS and dispose of all auto sampler vials in hazardous waste.
The ion chromatogram of the control mongoose serum illustrates the retention time of ethyl-IPA and the absence of methyl-IPA at the indicated retention time. The ion chromatogram of the quality control sample illustrates the baseline separation of methyl-IPA from ethyl-IPA, as well as the quantifier and qualifier transitions for methyl-IPA. The ion chromatogram of the representative sample from the study shows an observed serum concentration of 33.5 micrograms per microliter of methyl-IPA.
The accuracy and precision results for control mongoose serum fortified with methyl-IPA are shown here. Percent recoveries ranged from 96.9 to 109%The percent relative deviation at the three fortification levels was 3.4%1.7%and 2.3%respectively. The accuracy and precision results for control mongoose serum fortified with ethyl-IPA are shown here.
Percent recoveries ranged from 89.5 to 115%The percent relative standard deviation at the five fortification levels was 4.3%1.5%2.3%5.6%and 1.1%respectively. All mongooses offered ethyl-IPA and methyl-IPA baits consumed at least 25%of the bait within the 24 hour time constraint and had quantifiable levels of ethyl-IPA and methyl-IPA in their sera, respectively. The potential utility of the method could be increased by configuring the LC-MS to collect data for other iophenoxic acid analogs, such as propyl and butyl iophenoxic acid.
We encouraged personnel to consult the most recent recommendations of the advisory committee on immunization practices, or their institutional biosafety committee for guidance on whether preexposure rabies vaccination is required. Sections 2.3 through 2.6 of this protocol involving working with undiluted serum should be performed in a class two biosafety cabinet.
