This research was supported in part by the intramural research program of the US Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services, National Rabies Management Program and IDT Biologika (Dessau-Rosslau, Germany).

All procedures were approved by the USDA National Wildlife Research Center&#39;s institutional Animal Care and Use Committee under approved research protocol QA-2597.
NOTE: The following protocol describes the analysis procedure to detect methyl-iophenoxic acid in mongoose serum. This method is the final version of an iterative process that began with analysis of ethyl-iophenoxic acid in mongoose serum. During the initial evaluation of ethyl-iophenoxic acid minor modifications were made to the...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracyclines+as+a+fluorescent+marker+in+bones+and+teeth+of+rodents.">Tetracyclines as a fluorescent marker in bones and teeth of rodents.</a> Journal of Wildlife Management. 34 (4), 829-834 (1970).</li><li>Fisher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+using+Rhodamine+B+as+a+marker+for+wildlife+studies.">Review of using Rhodamine B as a marker for wildlife studies.</a> Wildlife Society Bulletin. 27 (2), 318-329 (1999).</li><li>Knowlton, F. K., Savarie, P. J., Wahlgren, C. E., Hayes, D. J., Shumake, S. A., Bullard, R. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excretion+and+distribution+of+iophenoxic+acid.">Excretion and distribution of iophenoxic acid.</a> Journal of Pharmacology and Experimental Therapeutics. 178 (1), 159-172 (1971).</li><li>Jones, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+liquid+chromatographic+determination+of+iophenoxic+acid+in+serum.">High-performance liquid chromatographic determination of iophenoxic acid in serum.</a> Journal of Chromatography B: Biomedical Sciences and Applications. 654 (2), 293-296 (1994).</li><li>Wiles, M. C., Campbell, T. 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Authors AV and SO are fulltime employees of an oral rabies vaccine bait manufacturer.

The LC-MS/MS method developed for the studies utilized the selectivity of multiple reaction monitoring to accurately quantify me-IPA and et-IPA in mongoose serum. The selectivity of MS/MS detection also allowed for a simple clean-up protocol relying solely on acetonitrile to precipitate proteins from serum prior to analysis.
Iophenoxic acids are soluble in ACN but are practically insoluble in water. To exclude water from the ACN extraction, sodium chloride was added to force a clear water:ACN ...

Rabies virus (RABV) is a negative sense single stranded lyssavirus, and circulates among diverse wildlife reservoir species within the orders Carnivora and Chiroptera. Multiple species of mongoose are reservoirs of RABV, and the small Indian mongoose (Herpestes auropunctatus) is the only reservoir in Puerto Rico and other Caribbean islands in the Western Hemisphere1,2,3. The control of RABV circulation in wildlife reservoirs is typically accomplished through a strategy of oral rabies vaccination (ORV). In the United States (US), this management activity is coordinated by the USDA/APHIS/Wildlife Services National Rabies Management Program (NRMP)4. Currently no wildlife ORV program exists in Puerto Rico. Research into rabies vaccines and various bait types for mongooses has been conducted with promising results suggesting an ORV program for mongooses is possible5,6,7,8.
Monitoring the impact of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RV antibody seroprevalence. However, this strategy may be challenging in areas with an active wildlife ORV programs or in areas where RV is enzootic and background levels of RABV neutralizing antibodies (RVNA) are present in reservoir species. In such situations, a biomarker included in the bait or the external bait matrix may be useful.
Various biological markers have been used to monitor bait uptake in numerous species, including raccoons (Procyon lotor)9,10,stoats (Mustela ermine)11,12, European badgers (Meles meles)13, wild boars (Sus scrofa)14, small Indian mongooses15 and prairie dogs (Cynomysludovicianus)16,17, among others. In the US, operational ORV baits often include a 1% tetracycline biomarker in the bait matrix to monitor bait uptake18,19. However, drawbacks to the use of tetracycline include a growing concern over the distribution of antibiotics into the environment and that detection of tetracycline is typically invasive, requiring tooth extraction or destruction of the animal to obtain bone samples20. Rhodamine B has been evaluated as a marker in a variety of tissues and can be detected using ultraviolet (UV) light and fluorescence in hair and whiskers10,21.
Iophenoxic acid (IPA) is a white, crystalline powder that has been used to evaluate bait consumption in coyotes (Canis latrans)22, arctic fox (Vulpes lagopus)23, red fox (Vulpes vulpes)24, raccoons9,25, wild boar14, red deer (Cervus elaphus scoticus)26, European badgers12 and ferrets (M. furo)27, among several other mammalian species. Retention times of IPA varies by species from less than two weeks in some marsupials28,29, to at least 26 weeks in ungulates26 and over 52 weeks in domestic dogs (Canis lupus familiaris)30. Retention times may also be dose-dependent31. Iophenoxic acid binds strongly to serum albumin and was historically detected by measuring blood iodine levels32. This indirect approach was supplanted by high-performance liquid chromatography (HPLC) methods to directly measure iophenoxic acid concentrations with UV detection33, and eventually with liquid chromatography and mass spectrometry (LCMS)34,35. For this study, a highly sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed that utilizes multiple reaction monitoring (MRM) to quantify two analogues of iophenoxic acid. Our objective was to use this LC-MS/MS method to evaluate the marking ability of 2-(3-hydroxy-2,4,6-triiodobenzyl)propanoic acid (methyl-IPA or me-IPA) and 2-(3-hydroxy-2,4,6-triiodobenzyl)butanoic acid (ethyl-IPA or et-IPA) and when delivered in an ORV bait to captive mongooses.
Mongooses were live captured in cage traps baited with commercially available smoked sausages and fish oil. Mongooses were housed in individual 60 cm x 60 cm x 40 cm stainless steel cages and fed a daily ration of ~50 g commercial dry cat food, supplemented twice per week with a commercially available chicken thigh. Water was available ad libitum. We delivered two derivatives of IPA, ethyl-IPA and methyl-IPA, to captive mongooses in placebo ORV baits. All baits were composed of a 28 mm x 20 mm x 9 mm foil blister pack with an external coating (hereafter &#34;bait matrix&#34;) containing powdered chicken egg and gelatin (Table of Materials). Baits contained 0.7 mL of water or IPA derivative and weighed approximately 3 g, of which ~2 g was the external bait matrix.
We offered 16 captive mongooses et-IPA in three concentrations: 0.14% (2.8 mg et-IPA in ~2 g bait matrix; 3 males [m], 3 females [f]), 0.4% (2.8 mg et-IPA in 0.7 mL blister pack volume; 3m, 3f), and 1.0% (7.0 mg ethyl-IPA in 0.7 mL blister pack volume; 2m, 2f). The overall dose of 2.8 mg corresponds to a dose rate of 5 mg/kg27,36 and is based on an average mongoose weight of 560 g in Puerto Rico. We selected 1% as the highest concentration as research suggests taste aversion to some biomarkers may occur at concentrations &#62;1% in some species37. We only offered the 1% dose in the blister pack as flocculation prevented the solute from dissolving in the solvent sufficiently to be evenly incorporated into the bait matrix. One control group (2m, 1f) received baits filled with sterile water and no IPA. We offered baits to mongooses in the morning (~8 a.m.) during or prior to feeding of their daily maintenance ration. Bait remains were removed after approximately 24 hours. We collected blood samples prior to treatment, one day post-treatment and then weekly up to 8 weeks post-treatment. We anesthetized mongooses by inhalation of isoflurane gas and collected up to 1.0 mL of whole blood by venipuncture of the cranial vena cava as described for ferrets38. We centrifuged whole blood samples, transferred sera to cryovials and stored them at -80 &#176;C until analysis. Not all animals were sampled during all time periods to minimize the impacts of repeated blood draws on the health of the animals. Control animals were sampled on day 0, then weekly for up to 8 weeks post-treatment.
We delivered me-IPA in three concentrations: 0.035% (0.7 mg), 0.07% (1.4 mg) and 0.14% (2.8 mg), all incorporated into the bait matrix, with 2 males and 2 females per treatment group. Two males and two females received baits filled with sterile water and no IPA. Bait offering times and mongoose anesthesia are described above. We collected blood samples prior to treatment on day 1, and then weekly up to 4 weeks post-treatment.
We tested serum concentration data for normality and estimated means for serum IPA concentrations of different treatment groups. We used a linear mixed model to compare mean serum et-IPA concentrations pooled across individuals. Bait type (matrix/blister pack) was a fixed effect in addition to experimental day, whereas animal ID was a random effect. All procedures were run using common statistical software (Table of Materials) and significance was evaluated at &#945; = 0.05.

<table border="0" cellpadding="0" cellspacing="0" style="width:1036px;" width="1036">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Acetonitrile, Optima grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A996</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Analytical balance</td>
			<td style="width:193px;">Mettler Toledo</td>
			<td style="width:304px;">XS204</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:380px;">C18 column, 2.1 x 50 mm, 2.5-&#181;m particle size</td>
			<td style="width:193px;">Waters Corp.</td>
			<td style="width:304px;">186003085</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">ESI Source</td>
			<td style="width:193px;">Agilent&#160;</td>
			<td style="width:304px;">G1958-65138</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Ethyl-iophenoxic acid, 97 %</td>
			<td style="width:193px;">Sigma Aldrich</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot MKBP5399V</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Formic acid, LC/MS grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A117</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:380px;">LCMS software</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">MassHunter Data Acquisition and Quantitative Analysis</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Methyl-iophenoxic acid, 97 % (w/w)</td>
			<td style="width:193px;">PR EuroChem Ltd.</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot PR0709514717</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Microanalytical balance</td>
			<td style="width:193px;">Mettler Toledo</td>
			<td style="width:304px;">XP6U</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Microcentrifuge</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">5415C</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">MS/MS</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">G6470A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:380px;">N-Evap</td>
			<td style="width:193px;">Organomation</td>
			<td style="width:304px;">115</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:380px;">Oral Rabies Vaccine Baits</td>
			<td style="width:193px;">IDT Biologika, Dessau Rossleau, Germany</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Propyl-iophenoxic acid, 99 % (w/w)</td>
			<td style="width:193px;">PR EuroChem Ltd.</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot PR100612108RR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:380px;">Repeat pipettor</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">M4</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Screw-top autosampler vial caps, PTFE-lined</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">5190-7024</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:380px;">Sodium chloride, Certified ACS grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">S271</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:380px;">Statistical Software Package</td>
			<td style="width:193px;">SAS Institute, Cary, North Carolina, USA</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:380px;">Trifluoroacetic acid, 99 %</td>
			<td style="width:193px;">Alfa Aesar</td>
			<td style="width:304px;">L06374</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:380px;">UPLC</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">1290 Series</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:380px;">Vortex Mixer</td>
			<td style="width:193px;">Glas-Col</td>
			<td style="width:304px;">099A PV6</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">0.2-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.413</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">0.5-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.421</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">1.5-mL microcentrifuge tubes</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">14-666-325</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:380px;">1250-&#181;L capacity pipette tips</td>
			<td style="width:193px;">GeneMate</td>
			<td style="width:304px;">P-1233-1250</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:380px;">1-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.43</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:380px;">2-mL amber screw-top autosampler vials</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">5182-0716</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">5-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.456</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">80-position microcentrifuge tube rack</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">05-541-2</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:380px;">8-mL amber vials with PTFE-lined caps</td>
			<td style="width:193px;">Wheaton</td>
			<td style="width:304px;">224754</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:380px;">70 % (v/v) isopropanol</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A459</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">100-1000 &#181;L air displacement pipette</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">ES-100</td>
			<td style="width:159px;"></td>
		</tr>
	</tbody>
</table>

analysis of iophenoxic acid analogues in small indian mongoose (herpestes auropunctatus) sera for use as an oral rabies vaccination biological marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Representative ion chromatograms from a me-IPA analysis are presented in Figure 1. The control mongoose serum (Figure 1A) illustrates the retention time of et-IPA (surrogate analyte) and the absence of me-IPA at the indicated retention time. The quality control sample (Figure 1B) illustrates the baseline separation of me-IPA from et-IPA as well as the quantifier and qualifier transitions for me-IPA. ...

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Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

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.

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