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. 2023 Aug 8;18(8):e0289261. doi: 10.1371/journal.pone.0289261

Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry

David A Goldade 1,*, Shane R Siers 2,#, Steven C Hess 3,#, Robert T Sugihara 3,#, Craig A Riekena 4,#
Editor: Totan Adak5
PMCID: PMC10409299  PMID: 37552678

Abstract

A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 μm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study.

Introduction

Invasive rodents have been linked to extensive damage to delicate island ecosystems [1]. With no natural predators, populations often grow unchecked, impacting the ability of native flora and fauna to thrive [2,3]. Significant efforts have been undertaken to eradicate these invasive rodents. These efforts often involve broadcast application of large quantities of anticoagulant rodenticide baits. Baits containing either brodifacoum or diphacinone have been used in these projects. Each of these rodenticides has the potential for unintended consequences from their use which often necessitate extensive testing to determine their safety.

Brodifacoum, a second-generation anticoagulant rodenticide, is highly efficacious for the lethal control of rodents. Brodifacoum has a drawback which makes it less attractive for island eradication efforts: it has a longer persistence in the environment than first-generation anticoagulants, such as diphacinone [4]. This difference in environmental half-life makes diphacinone more attractive for eradication efforts when large quantities will be applied in a short span of time, especially in ecologically sensitive areas or where there may be pathways to human exposure [5].

Some evidence suggests that eradications in tropical ecosystems are more prone to eradication failure [6]. For eradications to be successful, the applied baits must be both highly efficacious and palatable to the rodents. To address potential palatability issues, novel bait matrices under development for deployment in eradication operations were formulated at 50 and 100 ppm diphacinone and offered to wild-caught Polynesian rats (Rattus exulans) and house mice (Mus musculus) in a two-choice test with the test animals being offered both the formulated bait and a standard maintenance diet. The livers were harvested from the mice and rats and analyzed for diphacinone residues by liquid chromatography with tandem mass spectrometry to verify bait consumption and inform potential risk assessments from secondary exposures.

Rodenticides have been quantified in a variety of matrices by many different analytical techniques. Liquid chromatography paired with ultra-violet or fluorescence detection was the industry standard for many years. This technique was able to achieve detection limits in the low parts-per-million level in most cases. In more recent years advances in tandem mass spectrometry has resulted in its emergence as the preferred technique for low level determinations of rodenticide residues [7]. Combining both selectivity and sensitivity improvements, LC-MS-MS produces superior performance often achieving detection in the sub part-per-billion range [7]. Due to the enhanced selectivity afforded by tandem mass spectrometry, minimal sample clean-up is often needed to permit the detection of rodenticides in complex biological matrices [8–10]. The method discussed in this work takes advantage of these advancements in sample preparation and analysis to determine the residue levels of diphacinone in field-captured rodents. These results will be used to inform potential decisions on the safety and efficacy of the novel baits being developed for the control of invasive rodents.

Materials and methods

Trapping of rats and mice

Ninety-two Polynesian rats (Rattus exulans) and ninety-two house mice (Mus musculus) of mixed genders were trapped from wild populations in Hawaii with no known exposure to anticoagulant rodenticides and transported to the National Wildlife Research Center’s Hawaii Field Station. The rodents were held in quarantine for not less than 7 days, during which they were maintained in individual housing and given free access to water and a maintenance diet (LabDiet™ Laboratory Rodent Diet 5001, Lab Diet, St. Louis, MO, USA).

Bait testing

The rats and mice were randomly divided into test and control groups. On test day one half of the test animals were offered either a 50 or 100 ppm DPN formulated bait comprised of compressed cereal pellets with a proprietary formulation (Bell Laboratories, Madison, WI, USA). The test animals were also offered a USEPA standard challenge diet [11] in a two-choice test. Control animals received only the standard animal feed. The rodents were allowed to feed ad libitum for 15 days, followed by a 10-day holding period during which the formulated baits were removed, and all animals received only the standard animal feed diet. Consumption of bait and challenge diet (g) was recorded daily. Twice daily, the condition of the rodents was observed and noted. Deceased animals were removed; their weight and the time were recorded. No individuals from the control group expired during the exposure period. All rodents that survived the full 15-day exposure period and 10-day post-exposure period were euthanized with CO2. All animals were euthanized humanely in accordance with American Veterinary Medical Association [12] standards and practices.

Justification for use of study animals

The purpose of this research was to evaluate a new bait formulation for the lethal control of invasive rodents in fragile island ecosystems. Due to potential biological variability within a population as well as potential gender-dependent metabolic difference, the use of in-vivo testing was the only suitable approach for a study of this type. The potential risk of a failed eradication necessitated the inclusion of death as an end-point for the study animals. The use of sedatives, analgesics, or antidotes during the study was rejected on the following basis: DPN is an anti-coagulant which is detoxified in the liver [13]. Any veterinary drug which could enhance or suppress specific metabolic pathways could lead to a false assessment of the efficacy of the bait under evaluation. The number of animals selected were the minimum required to produce adequate efficacy data.

Humane treatment of test animals

All procedures involving animals were carried out with the approval of the NWRC Animal Care and Use Committee (NWRC protocol QA-2546) and in accordance with Good Laboratory Practices (40 CFR Part 160). Despite the need for study animal mortality, significant efforts were made to minimize suffering. Three criteria were established to determine if an animal should be humanely euthanized before the end of the study period. Firstly, the rodent must have consumed bait sufficient to meet the LD100 value for that species. Secondly, the animals were observed twice daily by trained animal care staff as verified by the NWRC Animal Care and Use Committee. If during that observation period the animals displayed any of the following clinical signs, they were immediately euthanized: A body condition score of -2 as defined by Foltz and Ullman-Cullere [14], difficulty breathing or rapid breathing over two successive observations, lateral recumbence over two successive observations, weight loss of greater than 10%, or blood in the feces or urine. Lastly, any animal exhibiting agonal vocalizations or persistent convulsions was immediately euthanized, independent of other criteria. Under the conditions listed above, no study animals required euthanasia prior to the end of the study period.

Tissue collection

All rodents were necropsied to remove the liver for analysis. An incision was made in the skin covering the abdomen and the skin was pulled back. A lateral incision was then made at the base of the breastbone and a pair of scissors used to cut the breastbone on each side. The liver was removed from each rodent and ground to a fine powder using a liquid nitrogen freezer mill (SPEX CertiPrep, Metuchen, NJ, USA) and stored in individual vacuum sealed bags at -30°C until analysis.

Liquid chromatography-mass spectrometry analysis

Water (200 μL) was added to the homogenized liver samples (0.075 to 0.125 g) in a 15-mL polypropylene centrifuge tube and the sample vortexed to produce a slurry. The samples were spiked with 50 μL of surrogate dissolved in acetonitrile (20.0 μg/mL d4-diphacinone; CDN Isotopes, Pointe-Claire, Quebec, Canada) and extracted with 5 mL of 1% ammonium hydroxide in acetonitrile (Fisher Scientific, Waltham, MA, USA). A solvent/water partition was created by the addition of 200 mg of a Quechers salt packet (Agilent Technologies, Santa Clara, CA, USA). An aliquot (1.5 mL) of the acetonitrile layer was transferred to a dispersive solid phase extraction tube (Agilent Technologies, Santa Clara, CA, USA) containing 25 mg C18 and 150 mg magnesium sulfate. A 1-mL aliquot of the sample was evaporated to dryness in a vacuum concentrator (Vacufuge; Eppendorf, Enfield, CT, USA) and reconstituted in 80% 10-mM ammonium acetate:20% acetonitrile. Analysis was performed using an Agilent 1290 Liquid Chromatograph attached to an Agilent 6470A QQQ Mass Spectrometer (Santa Clara, CA, USA). Separation was achieved using a 50 mm x 2.1 mm ID, 2.5 μm, X-Bridge BEH C18 column (Waters Corporation, Milford, MA, USA). Other instrument parameters are listed in Table 1.

Table 1. Liquid chromatograph/mass spectrometer conditions.

Injection Volume: 5 μL
Column Temperature: 40°C
Column Flow: 0.350 mL/minute
Mobile Phase A: 80% 10-mM ammonium acetate buffer/20% acetonitrile
Mobile Phase B: Acetonitrile
Gradient Program:
Time (min) Percent A Percent B
0.00 100 0
0.50 100 0
5.00 40 60
5.10 0 100
Run time = 5.50 minutes
Post-run equilibration time = 0.75 minutes
Ion Source: Agilent Jet-Stream ESI Negative
Gas Temperature: 225°C
Gas Flow: 6 L/minute
Nebulizer: 25 psi
Sheath Gas Temperature: 375°C
Sheath Gas Flow: 12 L/minute
Nozzle Voltage: 0 V
Compound Precursor Ion (m/z) Product Ion (m/z) Collision Energy (V) Fragmentor (V) Dwell (ms)
d4-Diphacinone 343.0 167.0 26 120 100
Diphacinone 339.0 167.0 26 120 100
Diphacinone 339.0 172.0 20 120 100
Diphacinone 339.0 116.0 50 120 100
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Method validation

The method was validated by evaluating the following parameters: selectivity, limits of detection (DL) and quantification (QL), linearity, accuracy, and recovery. Unless otherwise specified, result values were tabulated and statistical analysis was performed using Microsoft Excel 15.0 (Microsoft Corp, Redmond, WA, USA).

Control tissues

The repeatability and performance of the method was assessed using Polynesian rats (Rattus exulans) and house mice (Mus musculus) trapped at the same time as the study animals. These rodents received only the standard maintenance diet for the duration of the study. Their tissues were processed according to the procedures outlined above.

Selectivity

Selectivity of the method was evaluated by analyzing 11 replicate samples of liver tissue from wild-caught Polynesian rats and 8 replicate samples of liver tissue from wild-caught house mice. Potential interferences were assessed at the retention time of DPN by comparing the analytical response in the control samples to samples fortified near the QL.

Detection of quantification limits

The detection limit (DL) for DPN in Polynesian rat and house mouse liver was estimated from the mean chromatographic response of control samples compared to control samples that had been fortified to 20 ng/g with DPN. The DL was defined as the concentration of DPN required to generate a signal equal to 3X the baseline noise (measured peak-to-peak) observed in the baseline at the retention time of DPN in the control samples. The quantitation limit (QL) for DPN in Polynesian rats and house mice liver was estimated in a similar fashion to the DL with the multiplier of 10X baseline noise used instead of 3X.

Linearity

Two sets of eight calibration standard solutions were prepared ranging from 0.470 ng/mL to 375 ng/mL (corresponding to a nominal concentration in liver tissue samples of 23.5 ng/g to 18,750 ng/g) by serial dilution into a solution of 10-mL ammonium acetate in water. Each liver tissue sample of 100 mg results in a final extract of 5 mL which produces a 1:50 dilution factor in the sample results. Each standard was injected in duplicate; the response ratio of DPN area to d4-DPN area was plotted against relative concentration. To improve accuracy of responses at low calibration levels, a weighted (1/x) quadratic regression was performed on the data set.

The accuracy of the calibration standards was determined for each data set by calculating the observed concentration of each standard in the calibration curve using the regression equation. This calculated observed concentration was then compared to the theoretical concentration of the standard to determine the accuracy as a percentage.

Recovery

A minimum of 8 replicates at each fortification level were prepared by adding an appropriate aliquot of a fortification solution prepared in acetonitrile to a 100 mg sample of control liver tissue. Control Polynesian rat and house mouse livers were fortified at nominal concentrations of 15.7, 313, and 18,800 ng/g.

The accuracy of the method was determined by calculating the relative error of the mean observed concentration at each fortification level. This value was calculated by determining the absolute value of the difference between observed concentration and target concentration and dividing by the target concentration.

Statistical methods

The statistical model used to evaluate the variance of the quality control data was SAS’s General Linear Model (GLM; SAS Institute, Inc., Cary, NC). The GLM performs analysis of variance by using least squares regression to fit general linear models. The response was the observed residue level in the liver samples. The fixed effects were species type, gender, concentration of the formulated bait, number of days the rodent survived the test, and the amount of bait consumed. The interaction between fixed effects was also evaluated.

Results and discussion

Method development

A deuterated form of DPN (d4-DPN) was selected as the internal standard (IS) for this method. Multiple reaction monitoring (MRM) transition pairs were selected for both DPN and d4-DPN and optimized with respect to fragmentation and collision energy using Masshunter’s Method Optimizer software (Agilent Technologies, Santa Clara, CA, USA). The quantitative transition for DPN was m/z 339.0→167.0 while the d4-DPN transition was m/z 343.0→167.0.

Initial experiments were conducted using a dSPE cartridge containing primary-secondary amine (PSA) in addition to the C-18 and magnesium sulfate in the validated method. The PSA-containing dSPE tubes resulted in lower overall recovery of both the DPN and d4-DPN. The absolute (not corrected for IS response) recovery from the PSA-containing dSPE cartridge averaged approximately 40%, while that for the dSPE cartridge containing only C-18 and magnesium sulfate was approximately 85%.

A small amount of base (ammonium hydroxide) was added to the extraction solvent to improve clean-up of the sample extract. Significant matrix co-extractants were present when acetonitrile was used without the ammonium hydroxide present.

Method validation

Selectivity

Typical MRM chromatograms of unfortified control tissues, control tissues fortified at 15.7 ng/g for Polynesian rat liver, and from a Polynesian rat which fed on a 50 ppm DPN bait formulation are presented in Figs 1–3. Matrix peaks were observed near the retention time of DPN in most of the matrices tested (Fig 1). These peaks occurred after the retention time for DPN and did not interfere with the accurate detection of DPN. Under the conditions specified, the retention times of d4-DPN and DPN were 2.6 minutes.

Fig 1. Chromatograms of liver tissue from an untreated sample.

Fig 1

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Liver tissue extracts from Polynesian rat (a) and house mouse (b). The m/z 339.0 → 167.0 MRM transition is shown. The approximate retention time of DPN is 2.6 minutes.

Fig 3. Chromatograms of liver tissue from rodents offered a formulated bait containing 50 ppm DPN.

Fig 3

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Liver tissue extracts from (a) Polynesian rat (b) house mouse which had fed on the 50 ppm formulated bait. The m/z 339.0 → 167.0 MRM transition is shown. The approximate retention time of DPN is 2.6 minutes.

Fig 2. Chromatograms of liver tissue from an untreated sample which were spiked with DPN at 15.7 ng/g.

Fig 2

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Spiked liver extracts of control liver tissue from (a) Polynesian rat (b) house mouse. The m/z 339.0 → 167.0 MRM transition is shown. The approximate retention time of DPN is 2.6 minutes.

Detection and quantification limits

The detection limit (DL) for DPN in rodent livers was estimated from the mean chromatographic response of control samples compared to control samples that had been fortified to 15.7 ng/g with DPN. The DL and QL for Polynesian rat and house mouse liver tissue are shown in Table 2.

Table 2. Detection and quantitation limits for rodent liver tissue analyzed using LC/MS/MS.
Species Detection Limit Quantitation Limit
Polynesian Rat 3.5 ng/g 11.5 ng/g
House Mouse 4.8 ng/g 15.9 ng/g
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Linearity

The average of all calibration curves was determined (n = 9) where Y represents the ratio of concentration of DPN to d4-DPN and X represents the ratio of DPN area response to d4-DPN area response (Table 3). Calibration was achieved with a 1/x weighted quadratic regression analysis (Y = aX2 + bX + c) with correlation coefficients of 0.999 (0.99993 ± 0.000033) or better (Fig 4). The calibration curve was linear over the range of 0.47 ng/mL to 375 ng/mL DPN. The accuracy of all concentrations in the calibration curve was <10%.

Table 3. Average regression coefficients (n = 9).
Coefficient Mean
a 0.0083 ± 0.0081
b 1.07 ± 0.017
c 0.00024 ± 0.00012
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Fig 4. Representative calibration curve for DPN.

Fig 4

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Calibration standard solutions were prepared ranging from 0.470 ng/mL to 375 ng/mL (corresponding to a nominal concentration in liver tissue samples of 23.5 ng/g to 18,800 ng/g).

Precision and accuracy

The precision and accuracy values for recovery of DPN from rodent livers demonstrated good method performance across all levels (Table 4). The samples were analyzed over the course of a 1-month period. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97%– 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). Therefore, the validated method provided acceptable precision and accuracy for the determination of DPN in liver tissues.

Table 4. Precision and accuracy of DPN in rodent liver tissues.
Species n Target (ng/g) Observed (ng/g) Precision (RSD,%) Accuracy (RE,%)
Polynesian Rat 11 15.7 19.6 ± 4.0 20.4% 14.7%
11 313 329 ± 12 3.65% 4.72%
9 18800 19100 ± 720 3.77% 1.20%
House Mouse 7 15.7 18.8 ± 2.3 12.2% 10.6%
8 313 321 ± 7.7 2.40% 2.45%
8 18800 18600 ± 180 0.97% 1.06%
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ANOVA results from a GLM analysis (SAS General Linear Model (GLM); SAS Institute, Inc., Cary, NC) demonstrated a significant effect from species when it was included in the model (F = 5.64; p = 0.0214), therefore the recovery results from the two species were examined separately. For the rat samples, fortification level was a significant factor (F = 37.53; p = <0.0001) with low, mid, and high levels all being significantly different from each other (α = 0.05). For the mouse samples, there was no significant difference between fortification levels (F = 0.84; p = 0.4561).

Rodent feeding study

Statistical analysis was performed on the data set using ANOVA and a GLM analysis (SAS General Linear Model (GLM); SAS Institute, Inc., Cary, NC). The results demonstrated a significant effect when species was included in the model (F = 20.98; p = <0.0001), therefore further analysis was performed on each subset of data independent of the other. When testing for the effect of rodent gender on the data sets, the results were significant for rats (F = 8.09; p = 0.0066) but not for mice (F = 3.36; p = 0.0732). Bait formulation level had a significant effect for mice (F = 6.15; p = 0.0167) but not for rats (F = 2.35; p = 0.1319). When considering the impact of the amount of treated bait consumed and the number of days the rodent fed on the bait, the concentration of the formulated bait (F = 11.42; p = 0.0016), the interaction of amount of bait consumed and formulation level (F = 9.13; p = 0.0042), and the interaction of all three terms were significant with respect to mice (F = 3.91; p = 0.0543). For rats only the concentration of the formulated bait had a significant effect on residue level (F = 4.92; p = 0.0320). These results, taken as a whole, indicate that the formulated product had less impact on the observed residue level in rodent liver for the rats than it did the mice in this study.

Both formulation levels proved highly effective at producing mortality in Polynesian rats with all animals succumbing to the rodenticide in the 50 ppm group (Table 5) and 25 of the 26 animals perishing in the 100 ppm group (Table 5). Efficacy was much lower in the house mouse treatment groups. The 50 ppm and 100 ppm treatment groups had 5 and 7 of the 26 mice in each group survive the entire study period, respectively (Table 5). It is possible that some taste aversion could be occurring among the mice. The mice that succumbed to the 50 ppm formulated bait had 30.4% average bait acceptance calculated as the ratio of the mass of treated bait consumed to all feed consumed during the two-choice trial. Mice that survived the entire study period only had 10.2% average acceptance. This indicates potential palatability or bait avoidance problems in the mouse treatments groups.

Table 5. Mortality for two species of rodents fed a diphacinone-treated bait.
Species Formulation Level
50 ppm 100 ppm
Polynesian Rat 26/26 (100%) 25/26 (96.2%)
House Mouse 21/26 (80.8%) 19/26 (73.1%)
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Another possible explanation for the difference in survival between rats and mice is the difference in toxicity for DPN in these two classes of rodents. The published LD50 for rats is 0.3 to 2.3 mg/kg [15] while that for mice is 340 mg/kg [16]. Although the mice were much smaller (average 11.5 g) than the rats (average 55.2 g) in terms of body weight, the difference in LD50 means the mice would have to consume more of the treated baits than the rats to achieve a lethal dose.

Conclusion

A method for the quantitation of DPN in rodent liver tissues was developed and validated. The method was successfully used to quantitate residue levels of DPN in wild-caught Polynesian rats and house mice fed a formulated bait containing DPN at two levels (Table 6).

Table 6. Mean DPN residue levels (μg/g) in rodent livers following feeding on treated baits.

Formulation Level Polynesian Rat House Mouse
50 ppm Mean ± sd = 6.63 ± 5.48 2.00 ± 1.92
Range = 0.613–19.6 0.196–8.28
RSD = 83% 96%
n = 26 26
100 ppm Mean ± sd = 8.96 ± 6.82 4.82 ± 5.53
Range = 0.855–31.4 0.097–18.70
RSD = 76% 115%
n = 24 25
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In a secondary hazard study of multiple rodenticides, including diphacinone, Fisher et al. [17] found a mean liver residue in rats that fed on a 50-ppm diet and succumbed to a lethal dose of 4.7 μg/g with a range of <0.1 to 9.0 μg/g. During two studies performed in Hawaii, the average liver residue observed in rats was 3.7 μg/g for a hand-baiting study [18] and 4.4 μg/g during an arial application [19]. The cited studies were conducted with 50 ppm diphacinone baits. These values compare very well with the results of the current study. While the liver residue levels are quite similar, the amount of treated bait needed to produce fatality in the rats was significantly less for this study with an average of 15.9 g of the 50 ppm bait being consumed while an average of 113 g was consumed in a similar feeding study [19]. The novel baits evaluated were efficacious in rats, produced similar residue levels to currently existing baits, and required significantly less bait to achieve similar results.

Data Availability

The data is available at: https://doi.org/10.2737/NWRC-RDS-2023-002.

Funding Statement

This research was supported by the intramural research program of the U.S. Department of Agriculture, Animal and Plant Health Inspection Service and funding provided by Bell Laboratories, Inc. The funders took part in the study design, sample collection, decision to publish, and preparation of the manuscript.

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Decision Letter 0

Totan Adak

2 May 2023

PONE-D-23-07599Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometryPLOS ONE

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Additional Editor Comments:

Prepare the tables as per the journal format.

Authors should cite the relevant references on Bait testing, Justification for use of study animals, Humane treatment of test animals

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 30.. “The sample was cleaned up with a combination….” Should be stated as “The extracted sample was cleaned up with a combination…”

Line 31.. “Separation was achieved….” Should be stated as “chromatographic separation was achieved…”

Line 35 and 37... State precision and accuracy in range instead of average.

Line 73,74, 77, References were cited in form of numbers as they appeared in the document, but some reference were cited with first author name only. Arrange the manuscript as per journal instructions.

Line 90. “On test day 0 half of the test animals were offered…” should be “On test day one half of the test animals were offered…”

Line 100-102. All animals were euthanized humanely in accordance with American Veterinary Medical Association standards and practices. Cite reference to this statement.

Line 110. DPN is an anticoagulant which is detoxified in the liver. Cite reference to this statement.

Line 138-139. ‘The samples were fortified with 1 μg of surrogate dissolved in acetonitrile” should be stated as The samples were spiked with ____µl of surrogate dissolved in acetonitrile (µg/L).

Line 154. accuracy, recovery. Should be stated as accuracy and recovery.

Line 154-155. What does this statement mean “When not specified”, result values were tabulated and analyzed using Microsoft Excel 15.0 (Microsoft Corp, Redmond, WA,USA). This statement seems irrelevant.

Line 175. Coefficient of determination was not mentioned. Without coefficient of determination, linearity is meaningless.

Line 176. Authors are needed to explain how does 0.470 ng/mL to 375 ng/mL is corresponding to a nominal sample concentration of 4.7 ng/g to 3750 ng/g. Authors have mentioned linearity in ng/mL but DL and QL in ng/g. Is there any specific reason to do this? Similarity authors also used positive controls 20, 350 and 12500 ng/g and linearity range 0.470 ng/mL to 375 ng/mL. Authors are needed to remove this ambiguity.

Line 204. Authors also required to discuss in detail experiments performed during method development regarding instrumental parameters such mobile phase, gradient elution, ion source parameters etc.

Line 229-232. The DL was defined as the concentration of DPN required to generate a

signal equal to 3X the baseline noise (measured peak-to-peak) observed in the baseline at the retention time of DPN in the control samples. The quantitation limit (QL) for DPN in rodent livers was estimated in a similar fashion to the DL with the multiplier 232 of 10X baseline noise used instead of 3X. These lines should be removed as these terms already defined in lines 170-174.

Line 297. a range of <0.1 tp 9.0 μg/g should be written as a range of <0.1 to 9.0 μg/g.

Additional comments.

Authors did not mention validation guidelines they have used for this study. Moreover one of the important validation parameter when ESI is used as ion source is ion suppression which was not discussed. Instrumental plotted calibration curve with coefficient of determinations should be shown in the form of figure. Table 1, the gradient program at 5.10 and 5.50 min is same and needed to be corrected.

To conclude, I would advise against accepting this manuscript for publication unless significant revisions are made.

Reviewer #2: The manuscript "Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry" describes the determination of DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. Some of the the sections are not well described and discussed in a very inefficient manner. I strongly recommend the major revision of the manuscript through the incorporation of the following comments although I found it is a generally interesting story.

1) Abstract, and section headings need to be revised along with more quantitative and background information. The abstract does not summarize the objectives, the principal results, and major conclusions of the present study.

2) The originality of the paper needs to be further clarified. The present form does not have sufficient results to justify the novelty of a high-quality journal paper (there is a lot of similar studies that depicted determination of rodenticide levels). The background of the study, the research gap/scientific novelty, and the state-of-the-art (and purpose) of the study are not clearly illustrated. The hypothesis and justification of the study needs to be elaborated.

3) Have you looked for the chirality of the DPN/and their stereoisomers through chiral column analysis? What about the optimization of the sample preparation process and LC-MS/MS conditions?

4) Any information about matrix-matched standards and fortified liver matrix sample preparation and evaluation of the extraction procedures?

5) What about metabolites and their disposition along with Chromatograms of DPN metabolites?

6) Revise the conclusion section by including a summary of your key findings, future directions, highlights of your hypothesis as well as improvements compared to already reported work.

7) The reference lists are not updated and sufficient references are not cited in the text.

8) Provide MS spectra with the fragmentation mechanisms of DPN and their metabolites?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes

Reviewer #2: No

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Aug 8;18(8):e0289261. doi: 10.1371/journal.pone.0289261.r002

Author response to Decision Letter 0

9 Jun 2023

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

I have attempted to adjust the manuscript to meet these requirements. Please advise if I have overlooked anything.

2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse.

I’m not sure what code you are referring to. Is this in reference to the statistical analysis? If that is the case, I can share that.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

Could you clarify this for me? Look at the response below. There was no grant for this work. It was a cooperative agreement between the USDA and Bell Laboratories.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

This research was not funded through a grant. Funding was provided through a cooperative research agreement between the USDA and Bell Laboratories. Therefore, there is no grant number to provide.

4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

All the residue data generated from this work will be made available.

Reviewer #1 Responses:

Line 30. “The sample was cleaned up with a combination….” Should be stated as “The extracted sample was cleaned up with a combination…”

Correction made.

Line 31. “Separation was achieved….” Should be stated as “chromatographic separation was achieved…”

Correction made.

Line 35 and 37. State precision and accuracy in range instead of average.

Range was added to the average so that both are reported.

Line 73,74, 77, References were cited in form of numbers as they appeared in the document, but some reference were cited with first author name only. Arrange the manuscript as per journal instructions.

Corrections made.

Line 90. “On test day 0 half of the test animals were offered…” should be “On test day one half of the test animals were offered…”

Correction made.

Line 100-102. All animals were euthanized humanely in accordance with American Veterinary Medical Association standards and practices. Cite reference to this statement.

A reference to the AVMA standards (which are available on-line) has been added.

Line 110. DPN is an anticoagulant which is detoxified in the liver. Cite reference to this statement.

A reference for this statement was added.

Line 138-139. ‘The samples were fortified with 1 μg of surrogate dissolved in acetonitrile” should be stated as The samples were spiked with ____µl of surrogate dissolved in acetonitrile (µg/L).

Correction made.

Line 154. accuracy, recovery. Should be stated as accuracy and recovery.

Correction made.

Line 154-155. What does this statement mean “When not specified”, result values were tabulated and analyzed using Microsoft Excel 15.0 (Microsoft Corp, Redmond, WA,USA). This statement seems irrelevant.

This sentence has been reworded to clarify that the bulk of the statistical information (mean, standard deviation, etc) was generated using Excel. The more detailed statistical analysis was performed using SAS.

Line 175. Coefficient of determination was not mentioned. Without coefficient of determination, linearity is meaningless.

This line number is in the methods section of the manuscript; therefore it would not be appropriate to describe the coefficient of determination at this point. The criteria for this parameter is discussed in the results section “Calibration was achieved with a 1/x weighted quadratic regression analysis (Y = aX2 + bX + c) with correlation coefficients of 0.999 or better.” We feel that a discussion of the percent accuracy of the standards is a better representation of fitness of the calibration than correlation, especially over very large calibration curves.

Line 176. Authors are needed to explain how does 0.470 ng/mL to 375 ng/mL is corresponding to a nominal sample concentration of 4.7 ng/g to 3750 ng/g. Authors have mentioned linearity in ng/mL but DL and QL in ng/g. Is there any specific reason to do this? Similarity authors also used positive controls 20, 350 and 12500 ng/g and linearity range 0.470 ng/mL to 375 ng/mL. Authors are needed to remove this ambiguity.

The concentration per volume values (ng/mL) represent the concentration of the final sample extract in the vial as it is injected into the LC/MS/MS. This value is then used to generate the concentration per mass of the liver sample (ng/g). The nominal mass of tissue for each sample was 100 mg. This is extracted into 5 mL of solvent. This results in a dilution factor of approximately 1:50. Therefore, a tissue concentration of 18750 ng/g would produce a response equivalent to 375 ng/mL on the LC/MS/MS. The fortification of controls statement will be made more precise to better reflect this dilution. Calculations could be performed in terms of mass of DPN alone without units of mass or volume, but the industry standard tends to be reporting results in terms of ppb in the tissue (ng/g). A statement making this clearer has been added to the manuscript. Please advise if additional clarification is needed.

Line 204. Authors also required to discuss in detail experiments performed during method development regarding instrumental parameters such mobile phase, gradient elution, ion source parameters etc.

Mobile phase and gradient elution parameters were accepted without modification from previously published works; therefore, no experimental optimization was performed on these parameters. The Masshunter software’s Optimizer function was used to optimize the MRM transitions. A reference to this approach was inserted into the manuscript.

Line 229-232. The DL was defined as the concentration of DPN required to generate a signal equal to 3X the baseline noise (measured peak-to-peak) observed in the baseline at the retention time of DPN in the control samples. The quantitation limit (QL) for DPN in rodent livers was estimated in a similar fashion to the DL with the multiplier 232 of 10X baseline noise used instead of 3X. These lines should be removed as these terms already defined in lines 170-174.

Correction made.

Line 297. a range of <0.1 tp 9.0 μg/g should be written as a range of <0.1 to 9.0 μg/g.

Typographical error was corrected.

Authors did not mention validation guidelines they have used for this study.

I am unsure what this is in reference to? If you mean a particular set of regulatory guidelines, then you are correct. There is no strict regulatory guideline for validation of residue methods of this type. It is a non-food use residue study and does not fall under any specific guideline. The data generated are intended for use in future risk assessments.

Moreover one of the important validation parameter when ESI is used as ion source is ion suppression which was not discussed.

You are correct that no investigation of ion suppression was undertaken as a part of this study. While such a study could be useful, the results of the quality control samples speak for themselves and indicate the performance of the method. The use of a deuterated internal standard also provides a correction for this sort of sample-to-sample variability in method performance and is far more reliable than matrix-matched standards in this case. Furthermore, most of the study samples are of significantly high concentration levels, making such a study less important for this type of data.

Instrumental plotted calibration curve with coefficient of determinations should be shown in the form of figure.

Are you requesting a representative calibration curve, or all the calibration data on a single curve? The study was conducted over several weeks with calibration data generated with each analytical run. The discussion section mentions that all calibration curves were of 0.9999 correlation or better. If the calibration were to fall below this level (and it never did during this study) the run would be rejected and remedial action taken to correct the error. My opinion would be that displaying a straight line has no more value than listing the correlation requirements for the run to be acceptable, but I can add one if required.

Table 1, the gradient program at 5.10 and 5.50 min is same and needed to be corrected.

This is used to indicate the final parameters for the 5.50 minute run time of the gradient. The line will be removed, and a second line added to indicate the final run time.

Reviewer #2 Responses:

1) Abstract, and section headings need to be revised along with more quantitative and background information. The abstract does not summarize the objectives, the principal results, and major conclusions of the present study.

Revision to conform to journal formatting has been completed. I’m unclear what additional quantitative information is required? The results of the study in their entirety are discussed in the manuscript. A revision of the abstract has been performed.

2) The originality of the paper needs to be further clarified. The present form does not have sufficient results to justify the novelty of a high-quality journal paper (there is a lot of similar studies that depicted determination of rodenticide levels). The background of the study, the research gap/scientific novelty, and the state-of-the-art (and purpose) of the study are not clearly illustrated. The hypothesis and justification of the study needs to be elaborated.

The novelty of this research lies in the newly developed bait formulations, not in the analytical method itself. The method is fairly derivative and some modifications to the method presented in the literature have been discussed. As the baits under evaluation are novel and proprietary, a discussion in the literature of their exact formulas and ingredients falls under the umbrella of intellectual property. Additionally, since the baits are a new formulation which has not been widely tested, this is the first report of residue levels in rodents resulting from consumption of the new baits. That is where the novelty of this research lies. Efforts have been made to emphasize this in the manuscript.

3) Have you looked for the chirality of the DPN/and their stereoisomers through chiral column analysis? What about the optimization of the sample preparation process and LC-MS/MS conditions?

Investigation of the isomers of DPN has not been conducted as a part of this study. There is no peak splitting to indicate the separation of isomers using the present analytical technique. As the goal of this research was to quantify the concentration of parent (regardless of any isomeric form) in the livers, examination of isomers was not undertaken.

The sample preparation and LC/MS/MS conditions were investigated as discussed in the manuscript. Previous methods developed in our facility optimized the LC conditions (mobile phase, stationary phase, column flow, etc) for the separation of rodenticides in biological matrices. Therefore, no additional time was spent on these experiments. The primary focus of optimization was in the area of sample extraction and clean-up as discussed in the manuscript.

4) Any information about matrix-matched standards and fortified liver matrix sample preparation and evaluation of the extraction procedures?

Matrix-matched standards are often not possible or feasible for wild-caught animals. Biological and dietary variation for the population is much more significant than in lab strains used for clinical research. In addition, the limited amount of sample material prevented the preparation of matrix-matched standards. A deuterated internal standard was used, which should correct for any matrix variability in the samples.

All the fortified quality control samples detailed in the manuscript were prepared in control liver tissues. This is the primary way in which method performance was evaluated and is reflected in the reported results.

5) What about metabolites and their disposition along with Chromatograms of DPN metabolites?

Metabolism of DPN does occur to a small degree, but there are no commercially available standards for the quantification of such metabolites. Therefore, no quantitative results could be generated. Qualitative results of the presence of metabolites would not be particularly useful as DPN is known to metabolize via the CYP 450 pathways of the liver. Additionally, for comparison to literature values of DPN residues in other studies the metabolites would not be useful as other studies of the DPN residues from field applications do not report the metabolites.

6) Revise the conclusion section by including a summary of your key findings, future directions, highlights of your hypothesis as well as improvements compared to already reported work.

A revision of the conclusion section has been performed. Future directions for this work are not being considered at this time as the research objectives have been achieved. The method was developed and validated and the residue levels in the study animals were reported. We are not trying to improve on already-reported methods in the literature. We do highlight changes to the methods we used to improve performance for the research discussed in the manuscript.

7) The reference lists are not updated and sufficient references are not cited in the text.

The reference list was modified to fit journal standards. I’m not sure what additional references would contribute to the manuscript. How many are required to meet this request? We feel sufficient references have been cited to describe the foundation for the research undertaken.

8) Provide MS spectra with the fragmentation mechanisms of DPN and their metabolites?

MS spectra are usually not very instructive in an MRM method paper as it is a targeted assay. However, one can be generated to meet this request.

Metabolites were not the focus of this research and no data on the metabolites of DPN have been collected.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (22KB, docx)

Decision Letter 1

Totan Adak

17 Jul 2023

Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry

PONE-D-23-07599R1

Dear Dr. Goldade,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Totan Adak

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has been largely revised by Authors and have addressed all of my concerns. Hence got my recommendation for publication.

Reviewer #2: Although authors have provided to some extent justifications for the raised queries still the reviewer felt the following information is missing:

a) Provide MS spectra with the fragmentation mechanisms of DPN and their

metabolites?- Justifications are not clear

b) Toxicity summary of the DPN needs to be addressed properly with the heat map

Above all the responses have been given in a non-polite way which is not acceptable!!!

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Muhammad IMRAN (IMRANFSTOX@GMAIL.COM)

Reviewer #2: No

**********

Acceptance letter

Totan Adak

27 Jul 2023

PONE-D-23-07599R1

Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry

Dear Dr. Goldade:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Totan Adak

Academic Editor

PLOS ONE

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