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.
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.
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 "bait matrix") 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 >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 °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 α = 0.05.
All procedures were approved by the USDA National Wildlife Research Center'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 methods, resulting in the final protocol presented below. Representative results include those obtained during both iterations.
1. Preparation of solutions and standards
2. Sample preparation
CAUTION: Personnel performing this procedure must have received the full series of rabies pre-exposure prophylaxis and have a documented rabies antibody titer above 0.5 IU from a Federal Occupational Health designated medical facility. Personnel must wear lab coats and eye protection at all times while performing the extraction. CAUTION: Perform steps 2.3−2.6 in a class II biosafety cabinet.
3. Quality control samples
CAUTION: Follow the cautionary statements described in section 2.
NOTE:The following procedure describes the minimum number of quality control (QC) samples required for an analysis. Replicates at each level are recommended if sufficient control mongoose serum is available.
4. LC-MS/MS analysis
5. Quantification
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. Figure 1C shows a representative sample from the study with an observed serum concentration of 33.5 µg/mL me-IPA.
A representative calibration curve from a me-IPA analysis is presented in Figure 2. The 7-level, 14-point standard curve ranges from 0.00202 to 8.27 µg/mL me-IPA with a correlation coefficient (r2) of 0.9998. Correlation coefficients ranged from 0.9998 to 0.99997 for the five me-IPA analyses. The surrogate analyte et-IPA concentration was 0.502 µg/mL in all standards.
Table 6 presents the accuracy and precision results for control mongoose serum fortified with 0, 1.3, 31, and 82 µg/mL me-IPA (n = 10 at each level). The results were collected from five separate analyses. Percent recoveries ranged from 96.9% to 109%. The percent relative standard deviation (% RSD) at the three fortification levels was 3.4%, 1.7%, and 2.3%, respectively.
The signal-to-noise ratio (S/N) observed in quality control samples (n = 10 negative controls; n = 10 at 1.3 µg/mL me-IPA) was used to determine the detection limit (DL; 3 x S/N) and quantitation limit (QL; 10 x S/N). The DL and QL for me-IPA in mongoose serum were 0.012 µg/mL and 0.042 µg/mL, respectively.
The peak area responses of the qualifier MRM transition divided by the quantifier MRM transition ( was calculated for all standards and samples. This ratio for each sample was then divided by the average ratio observed in the calibration standards to determine the qualifier percent match. The qualifier ratio for the sample shown in Figure 1C was 0.439, with a 96.3% match.
The recovery of et-IPA surrogate analyte was calculated for all QC samples and unknown samples by dividing the et-IPA peak area response by the average et-IPA peak area response observed in the calibration standards. Average surrogate analyte recoveries were 91.0% (negative controls), 91.4% (1.3 µg/mL), 92.8% (31 µg/mL), and 95.4% (82 µg/mL).
No interference peaks for either the quantifier or qualifier transitions of me-IPA were observed in control mongoose serum (Figure 1A).
The extraction procedure and instrumental conditions used to determine et-IPA in mongoose serum (Figure 3 and Table 7) was identical to the me-IPA method, but with the following changes. Propyl-iophenoxic acid (pr-IPA) was used as the surrogate analyte and an older, less sensitive LC-MS/MS was used. The source drying gas temperature was 350 °C with a flow of 12 L/min and a nebulizer pressure of 35 psi. The capillary voltage was -2,500 V. The source had no sheath gas or means to adjust nozzle voltage. The quantifier MRM transition for et-IPA was 570.7 → 442.8 (584.7 → 456.8 for pr-IPA). The fragmentor was 80 V and the collision energy was 10 V for both analytes. The qualifier MRM for et-IPA was 570.7 → 126.8 with a collision energy of 40 V.
The testing of all mongoose serum samples for et-IPA was performed over eight analyses. The 7-level calibration curve ranged from 0.00207 to 8.48 µg/mL with correlation coefficients (r2) ranging from 0.9990 to 0.9999. The surrogate analyte pr-IPA concentration was 0.512 µg/mL. Table 7 presents the accuracy and precision results for control mongoose serum fortified with 0, 1.3, 13, 32, 85, and 170 µg/mL me-IPA. The results were collected from eight separate analyses. Percent recoveries ranged from 89.5% to 115%. The % RSD at the five fortification levels was 4.3%, 1.5%, 2.3%, 5.6%, and 1.1%, respectively. The S/N observed in quality control samples (n = 21 negative controls; n = 21 at 1.3 µg/mL me-IPA) was used to determine the DL and QL. The DL and QL for me-IPA in mongoose serum were 0.12 µg/mL and 0.42 µg/mL, respectively. The average surrogate analyte recovery from quality control samples was 86.8% (n = 75). No interference peaks for either the quantifier or qualifier transitions of et-IPA were observed in control mongoose serum.
All mongooses offered et-IPA baits consumed ≥25% of the bait within the 24 hour time constraint and had quantifiable levels of et-IPA in their sera (Table 8). From the mixed model analysis, overall mean serum IPA concentrations were marginally higher from baits with 2.8 mg of biomarker in the bait matrix (17.5 µg/mL, 95% CI 11.7−23.3 µg/mL) in comparison to the blister pack (9.8 µg/mL, 95% CI 4.0−15.6 µg/mL) (F = 3.6, P = 0.07). Concentrations for both bait types decayed with experimental day (β = -0.15 ± 0.04, F = 14.4, P = 0.0005). Individual level variability in serum IPA concentrations was observed (animal ID covariance parameter estimate = 46.6 ± 20.7). All mongooses consumed 100% of control baits with only the empty foil blister pack remaining. Mean concentration of et-IPA residue in serum was variable by time period and did not appear to decrease consistently over time (Figure 3).
All mongooses offered me-IPA baits consumed ≥ 25% of the bait within the 24 hour time limit and had quantifiable levels of me-IPA in their sera (Table 9). Mean serologic concentration of et- and me-IPA in mongoose sera appeared dependent upon the concentration of IPA in the bait. Higher concentrations of IPA in the bait resulted in higher serum residues even in cases where the overall dose (2.8 mg in the case of et-IPA) remained the same. Interestingly, me-IPA appeared to produce a more even degradation pattern over time with an initial spike at day 1, followed by a steady decline until day 14 where concentrations appeared to plateau (Figure 4).
Figure 1: Representative ion chromatograms. (A) Representative MRM ion chromatogram of control mongoose serum fortified with surrogate analyte ethyl-iophenoxic acid (et-IPA). The arrow indicates the retention time for methyl-iophenoxic acid (me-IPA). (B) Representative MRM ion chromatogram of control mongoose serum fortified with 31 µg/mL me-IPA. The relative intensities of the quantifier (Quant) and qualifier (Qual) transitions for me-IPA are shown. (C) Representative MRM ion chromatogram of a mongoose serum sample with an observed me-IPA concentration of 33.5 µg/mL. Please click here to view a larger version of this figure.
Figure 2: Representative calibration curve. An original calibration curve generated by the data analysis software for methyl-iophenoxic acid (me-IPA). Please click here to view a larger version of this figure.
Figure 3: Mean serum ethyl IPA (et-IPA) concentration by bait type and concentration over time. Please click here to view a larger version of this figure.
Figure 4: Mean serum methyl IPA (me-IPA) concentration by bait concentration over time. All me-IPA concentrations were incorporated into the external bait matrix. Please click here to view a larger version of this figure.
me-IPA Concentration (µg/mL) | ||
ID | ||
25x-7 | Refer to step 1.6 | 200 |
25x-6 | Combine 1.000 mL 25x-7 Stock with 3.000 mL ACN | 50 |
25x-5 | Combine 1.000 mL 25x-6 Stock with 3.000 mL ACN | 13 |
25x-4 | Combine 1.000 mL 25x-5 Stock with 3.000 mL ACN | 3.1 |
25x-3 | Combine 1.000 mL 25x-4 Stock with 3.000 mL ACN | 0.78 |
25x-2 | Combine 1.000 mL 25x-3 Stock with 3.000 mL ACN | 0.2 |
25x-1 | Combine 1.000 mL 25x-2 Stock with 3.000 mL ACN | 0.049 |
Table 1: Preparation of 25x me-IPA stocks in ACN (in 8-mL amber glass vials).
Concentration (µg/mL) | |||
ID | me-IPA | et-IPA | |
4x-7 | 0.200 mL 25x-7 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 32 | 1.6 |
4x-6 | 0.200 mL 25x-6 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 8 | 1.6 |
4x-5 | 0.200 mL 25x-5 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 2 | 1.6 |
4x-4 | 0.200 mL 25x-4 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 0.5 | 1.6 |
4x-3 | 0.200 mL 25x-3 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 0.12 | 1.6 |
4x-2 | 0.200 mL 25x-2 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 0.031 | 1.6 |
4x-1 | 0.200 mL 25x-1 + 0.200 mL 25x Surrogate + 0.850 mL ACN | 0.008 | 1.6 |
4x-0 | 0.200 mL 25x Surrogate + 1.050 mL ACN | 0 | 1.6 |
Table 2: Preparation of 4x IPA stocks in ACN (in 2-mL autosampler vials).
Concentration (µg/mL) | |||
ID | me-IPA | et-IPA | |
Std 7 | 0.200 mL 4x-7 + 0.600 mL DI water | 8 | 0.4 |
Std 6 | 0.200 mL 4x-6 + 0.600 mL DI water | 2 | 0.4 |
Std 5 | 0.200 mL 4x-5 + 0.600 mL DI water | 0.5 | 0.4 |
Std 4 | 0.200 mL 4x-4 + 0.600 mL DI water | 0.13 | 0.4 |
Std 3 | 0.200 mL 4x-3 + 0.600 mL DI water | 0.03 | 0.4 |
Std 2 | 0.200 mL 4x-2 + 0.600 mL DI water | 0.0078 | 0.4 |
Std 1 | 0.200 mL 4x-1 + 0.600 mL DI water | 0.002 | 0.4 |
Std 0 | 0.200 mL 4x-0 + 0.600 mL DI water | 0 | 0.4 |
Blank | 0.200 mL ACN + 0.600 mL DI water | 0 | 0 |
Table 3: Preparation of me-IPA standards in 75/25 water/ACN (in 2-mL autosampler vials).
Concentration (µg/mL) in serum | |||
ID | me-IPA | et-IPA | |
Negative Control | 0.050 mL 25x Surrogate + 0.950 mL Diluent | 0 | 10 |
Low Fortification | 0.050 mL 25x Surrogate + 0.020 mL 25x-4 + 0.930 mL Diluent | 1.2 | 10 |
Mid Fortification | 0.050 mL 25x Surrogate + 0.030 mL 25x-6 + 0.920 mL Diluent | 30 | 10 |
High Fortification | 0.050 mL 25x Surrogate + 0.020 mL 25x-7 + 0.930 mL Diluent | 80 | 10 |
Table 4: Quality control sample fortification (prepare in 1.5-mL microcentrifuge tubes).
Liquid Chromatography: | |||||||||||||||||||||
Column: | C18, 2.1 x 50 mm, 2.5 µm particle size | ||||||||||||||||||||
Column temperature: | 70 °C | ||||||||||||||||||||
Injection volume: | 5 µL | ||||||||||||||||||||
Flow rate: | 0.800 mL/min | ||||||||||||||||||||
Mobile Phase: | Solvent A: | 0.1% formic acid in water | |||||||||||||||||||
Solvent B: | 0.1% formic acid in ACN | ||||||||||||||||||||
Gradient program: | |||||||||||||||||||||
Time (min): | 0 | 0.25 | 2.25 | 2.26 | 3.4 | 3.41 | 4.75 | ||||||||||||||
% B: | 40 | 40 | 55 | 100 | 100 | 40 | 40 | ||||||||||||||
Needle Wash: ACN, 3 s | |||||||||||||||||||||
MS/MS Source: ESI (negative mode) | |||||||||||||||||||||
Gas temperature: | 300 °C | ||||||||||||||||||||
Gas flow: | 5 L/min | ||||||||||||||||||||
Nebulizer: | 45 psi | ||||||||||||||||||||
Capillary: | -4000 V | ||||||||||||||||||||
Nozzle voltage: | -500 V | ||||||||||||||||||||
Sheath gas temperature: | 250 °C | ||||||||||||||||||||
Sheath gas flow: | 7 L/min | ||||||||||||||||||||
MRM Transitions: | |||||||||||||||||||||
Analyte | Precursor Ion (m/z) | Product Ion (m/z) * | Fragmentor (V) | Collision Energy (V) | Dwell Time (ms) | Time Segment | |||||||||||||||
Me-IPA | 556.6 | 428.7 | 74 | 12 | 60 | 2 | |||||||||||||||
126.9 | 65 | 60 | |||||||||||||||||||
126.8 | 61 | 60 | |||||||||||||||||||
Et-IPA | 570.7 | 442.7 | 87 | 16 | 60 | ||||||||||||||||
Time Segments: | |||||||||||||||||||||
Segment | Start (min) | End (min) | Type | Diverter | Delta EMV | Polarity | Data Stored | ||||||||||||||
1 | 0 | 0.6 | MS2 Scan | To Waste | 0 | Negative | No | ||||||||||||||
2 | 0.6 | 2 | MRM | To MS | -100 | Negative | Yes | ||||||||||||||
3 | 2 | 4.75 | MS2 scan | To Waste | 0 | Positive | No | ||||||||||||||
* Quantifier transitions are bolded. |
Table 5: LC-MS/MS parameters. Quantifier product ions are bolded.
Target | Observed | Percent | ||||
ID | Day | me-IPA | me-IPA | Recovery | ||
QC-1 | 1 | 0 | ND | N/A | ||
QC-2 | 1 | 0 | ND | N/A | ||
QC-11 | 2 | 0 | ND | N/A | ||
QC-12 | 2 | 0 | ND | N/A | ||
QC-21 | 3 | 0 | ND | N/A | ||
QC-22 | 3 | 0 | ND | N/A | ||
QC-31 | 4 | 0 | ND | N/A | ||
QC-32 | 4 | 0 | ND | N/A | ||
QC-3 | 5 | 0 | ND | N/A | ||
QC-4 | 5 | 0 | ND | N/A | ||
QC-13 | 1 | 1.25 | 1.26 | 101% | Ave (10) = | 102% |
QC-14 | 1 | 1.25 | 1.23 | 98.40% | SD = | 3.50% |
QC-23 | 2 | 1.25 | 1.26 | 101% | % RSD = | 3.40% |
QC-24 | 2 | 1.25 | 1.28 | 102% | ||
QC-33 | 3 | 1.29 | 1.3 | 101% | ||
QC-34 | 3 | 1.29 | 1.25 | 96.90% | ||
QC-5 | 4 | 1.29 | 1.37 | 106% | ||
QC-6 | 4 | 1.29 | 1.41 | 109% | ||
QC-15 | 5 | 1.29 | 1.3 | 101% | ||
QC-16 | 5 | 1.29 | 1.34 | 104% | ||
QC-25 | 1 | 30.1 | 30.2 | 100% | Ave (10) = | 103% |
QC-26 | 1 | 30.1 | 31.2 | 104% | SD = | 1.80% |
QC-35 | 2 | 30.1 | 31.1 | 103% | % RSD = | 1.70% |
QC-36 | 2 | 30.1 | 31.1 | 103% | ||
QC-7 | 3 | 31 | 31.6 | 102% | ||
QC-8 | 3 | 31 | 31.4 | 101% | ||
QC-17 | 4 | 31 | 32.5 | 105% | ||
QC-18 | 4 | 31 | 32.8 | 106% | ||
QC-27 | 5 | 31 | 31.7 | 102% | ||
QC-28 | 5 | 31 | 32.1 | 104% | ||
QC-37 | 1 | 80.2 | 77.8 | 97.00% | Ave (10) = | 101% |
QC-38 | 1 | 80.2 | 78.9 | 98.40% | SD = | 2.30% |
QC-9 | 2 | 80.2 | 81.8 | 102% | % RSD = | 2.30% |
QC-10 | 2 | 80.2 | 79.8 | 99.50% | ||
QC-19 | 3 | 82.7 | 83.5 | 101% | ||
QC-20 | 3 | 82.7 | 84 | 102% | ||
QC-29 | 4 | 82.7 | 84.7 | 102% | ||
QC-30 | 4 | 82.7 | 87.2 | 105% | ||
QC-39 | 5 | 82.7 | 83 | 100% | ||
QC-40 | 5 | 82.7 | 84.1 | 102% |
Table 6: QC results for me-IPA in mongoose serum (µg/mL).
Target et-IPA (µg/mL) | N | Mean (%) | SD (%) | % RSD |
0 | 21 | |||
1.3 | 12 | 103 | 4.5 | 4.3 |
13 | 9 | 91.7 | 1.4 | 1.5 |
32 | 12 | 106 | 2.4 | 2.3 |
85 | 12 | 105 | 5.8 | 5.6 |
170 | 9 | 106 | 1.1 | 1.1 |
Table 7: QC results for et-IPA in mongoose serum (µg/mL).
Bait type and ethyl IPA bait concentration (µg/mL) | ||||||||
Time Period | 0.4% – Blister | 1.0% – Blister | 0.14% – Matrix | Control | ||||
Mean (SD) | N | Mean (SD) | N | Mean (SD) | N | Mean (SD) | N | |
Day 0 | ND | 5 | ND | 4 | ND | 6 | ND | 3 |
Day 1 | 13.7 (10.9) | 2 | 11.2 (10.4) | 4 | 20.6 (17.3) | 2 | NA | NA |
Day 7 | 11.5 (6.3) | 6 | 9.9 (9.6) | 4 | 21.4 (10.9) | 6 | ND | 3 |
Day 14 | 10.2 (5.2) | 6 | 5.7 (2.5) | 3 | 17.8 (10.2) | 6 | ND | 3 |
Day 21 | 8.9 (4.1) | 4 | 10.0 (9.5) | 4 | 23.8 (5.3) | 3 | ND | 2 |
Day 28 | 8.5 (3.9) | 5 | 11.1 (10.6) | 3 | 17.7 (9.3) | 4 | ND | 3 |
Day 35 | 17.0 (NA) | 1 | 9.1 (9.0) | 4 | 21.7 (9.3) | 2 | ND | 1 |
Day 42 | NA | 0 | 8.4 (8.5) | 4 | 16.5 (NA) | 1 | ND | 2 |
Day 49 | 10.2 (5.8) | 2 | 8.1 (8.4) | 4 | 17.5 (4.7) | 2 | ND | 2 |
Day 56 | 10.9 (5.4) | 2 | 3.8 (1.1) | 3 | 17.6 (6.1) | 2 | ND | 2 |
Table 8: Mean (stand deviation [SD]) ethyl-IPA serum concentration by bait type. ND = not detected, NA = Not applicable.
Dose and methyl IPA concentration (µg/mL) | ||||||||
Time Period | 0.04% | 0.07% | 0.14% | Control | ||||
Mean (SD) | N | Mean (SD) | N | Mean (SD) | N | Mean (SD) | N | |
Day 0 | ND (NA) | 4 | ND (NA) | 4 | ND (NA) | 4 | ND (NA) | 4 |
Day 1 | 7.8 (7.1) | 4 | 22.2 (9.2) | 4 | 29.1 (16.0) | 4 | ND (NA) | 4 |
Day 7 | 4.4 (4.8) | 4 | 14.4 (4.6) | 4 | 21.3 (12.0) | 4 | ND (NA) | 4 |
Day 14 | 5.7 (5.9) | 4 | 12.0 (3.5) | 4 | 19.6 (10.6) | 4 | ND (NA) | 4 |
Day 21 | 4.9 (4.5) | 4 | 12.0 (0.8) | 3 | 18.3 (9.9) | 4 | ND (NA) | 4 |
Day 28 | 5.4 (5.0) | 4 | 9.8 (2.4) | 4 | 16.4 (8.1) | 4 | ND (NA) | 4 |
Table 9: Mean (SD) methyl-IPA serum concentration by dose. ND = Not detected, NA = Not applicable. All methyl IPA concentrations were incorporated into the external bait matrix.
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 phase separation by increasing the ionic strength of the aqueous (serum) phase. The volatile acid trifluoracetic acid (TFA) was also added to ensure that iophenoxic acids were protonated during the extraction and would be more readily solubilized in the ACN phase. TFA is removed during the dry-down step prior to LC-MS/MS analysis.
Blood draws from mongoose were approximately 1 mL, yielding 0.5 mL or less of sera. To perform replicate analyses of each sample, a micro-scale sample preparation procedure was required that used microcentrifuge tubes rather than typical analytical laboratory glassware such as Class A volumetric pipettes and flasks. To achieve accurate and precise results it is necessary that analysts be proficient in the use of glass microliter syringes and positive-displacement repeat pipettors with microliter-size tips.
A limitation of this method is that it requires costly LC-MS/MS instrumentation and analysts trained in its use and maintenance. However, because me-IPA and et-IPA are baseline resolved using the HPLC conditions described, the method could potentially be adapted to a single-quadrupole LCMS or HPLC with UV detector provided no interferences were observed.
The difference in mean serum concentrations between mongooses that consumed baits with et-IPA in the blister pack and bait matrix at the 2.8 mg dose suggests spillage when the marker is contained in the blister pack, rather than incorporated into the bait matrix. This may have implications for the purpose of estimating vaccine as opposed to bait uptake. For example, incorporating the marker into the external bait matrix may inflate the estimation of vaccine coverage if the animal eats the matrix but vaccine spills out. However, regulatory restrictions may preclude the ability of mixing a biological marker directly with a vaccine within the blister pack. In the cases in which <100% consumption was recorded, three cases were with baits containing et-IPA in the blister pack, two of which were the higher 1% concentration. This suggests potential taste aversion when et-IPA is at higher concentrations. When baits containing 1% (20.0 mg) et-IPA incorporated into the bait matrix were offered to mongooses, all animals rejected the bait.
Mean IPA concentrations for et- and me-IPA at day 14 were approximately 17 and 19 µg/mL, respectively. Similar research performed at Instituto de Investigación en Recursos Cinegéticos, Ciudad Real, Spain, using butyl and pentyl-IPA found day 14 concentrations of approximately 45 and 10 µg/mL in mongoose sera when delivered at the same concentration and bait formulation as in our study. These differences suggest that different derivatives of IPA may metabolize at different rates in mongooses, which can be useful when marker retention (either short or long-term retention) is a concern.
Differences in the physiology of the gastrointestinal system may affect absorption and excretion of IPA in different species. The plasma elimination half-life of IPA in domestic cats (Felis catus) when delivered at 1.5 mg/kg was 107 days whereas the rate in brushtail possums at the same dose rate was approximately one day28. When IPA was given to domestic goats (Capra aegagrus hircus) at a dose rate of 1.5 mg/kg the terminal elimination half-life of IPA was 81 days, although investigators continued to find elevated iodine concentrations up to 160 days following administration39. The gastrointestinal system of members of Vivveridae (the family to which mongooses had previously been assigned40) is described as similar to that of the domestic cat41, which may explain the retention time of IPA in mongooses. Research also suggests IPA may be metabolized differently in marsupials, allowing for more rapid excretion than in eutherian species29.
Both derivatives of IPA evaluated in this study provided long-term (4−8 weeks) marking ability in mongooses. The use of IPA as a biological marker in mongooses should take into consideration the study objectives and the desired duration of marking. In cases where animals are to be marked from the same study sites during consecutive time periods, different derivatives of IPA could be used to ensure results from one marking event are not confounded by marking during previous events. As part of operational ORV for wildlife in the US, sampling the target species and testing for the presence of RVNA and biomarker is typically conducted 4−6 weeks following ORV distribution42. From a practical standpoint, it appears either et- or me-IPA can be readily detected during this time period. Future research to evaluate the residue decay function in mongooses offered various concentrations of both congeners of IPA evaluated in this study would be useful.
The authors have nothing to disclose.
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).
Acetonitrile, Optima grade | Fisher | A996 | |
Analytical balance | Mettler Toledo | XS204 | |
C18 column, 2.1 x 50 mm, 2.5-µm particle size | Waters Corp. | 186003085 | |
ESI Source | Agilent | G1958-65138 | |
Ethyl-iophenoxic acid, 97 % | Sigma Aldrich | N/A | Lot MKBP5399V |
Formic acid, LC/MS grade | Fisher | A117 | |
LCMS software | Agilent | MassHunter Data Acquisition and Quantitative Analysis | |
Methyl-iophenoxic acid, 97 % (w/w) | PR EuroChem Ltd. | N/A | Lot PR0709514717 |
Microanalytical balance | Mettler Toledo | XP6U | |
Microcentrifuge | Eppendorf | 5415C | |
MS/MS | Agilent | G6470A | |
N-Evap | Organomation | 115 | |
Oral Rabies Vaccine Baits | IDT Biologika, Dessau Rossleau, Germany | N/A | |
Propyl-iophenoxic acid, 99 % (w/w) | PR EuroChem Ltd. | N/A | Lot PR100612108RR |
Repeat pipettor | Eppendorf | M4 | |
Screw-top autosampler vial caps, PTFE-lined | Agilent | 5190-7024 | |
Sodium chloride, Certified ACS grade | Fisher | S271 | |
Statistical Software Package | SAS Institute, Cary, North Carolina, USA | N/A | |
Trifluoroacetic acid, 99 % | Alfa Aesar | L06374 | |
UPLC | Agilent | 1290 Series | |
Vortex Mixer | Glas-Col | 099A PV6 | |
0.2-mL pipettor tips | Eppendorf | 30089.413 | |
0.5-mL pipettor tips | Eppendorf | 30089.421 | |
1.5-mL microcentrifuge tubes | Fisher | 14-666-325 | |
1250-µL capacity pipette tips | GeneMate | P-1233-1250 | |
1-mL pipettor tips | Eppendorf | 30089.43 | |
2-mL amber screw-top autosampler vials | Agilent | 5182-0716 | |
5-mL pipettor tips | Eppendorf | 30089.456 | |
80-position microcentrifuge tube rack | Fisher | 05-541-2 | |
8-mL amber vials with PTFE-lined caps | Wheaton | 224754 | |
70 % (v/v) isopropanol | Fisher | A459 | |
100-1000 µL air displacement pipette | Eppendorf | ES-100 |