Journal of Analytical Toxicology 2015;39:219 –224 doi:10.1093/jat/bku175 Advance Access publication January 16, 2015

Article

A Validated LC –MS-MS Method for Simultaneous Identification and Quantitation of Rodenticides in Blood Sergei Bidny1*, Kim Gago1, Mark David1, Thanh Duong1, Desdemona Albertyn1 and Naren Gunja2,3 1 Forensic and Analytical Science Service, NSW Health Pathology, Sydney, Australia, 2Clinical Pharmacology and Toxicology, Westmead Hospital, Sydney, Australia, and 3Discipline of Emergency Medicine, Sydney Medical School, Sydney, Australia

*Author to whom correspondence should be addressed. Email: [email protected]

A rapid, highly sensitive and specific analytical method for the extraction, identification and quantification of nine rodenticides from whole blood has been developed and validated. Commercially available rodenticides in Australia include coumatetralyl, warfarin, brodifacoum, bromadiolone, difenacoum, flocoumafen, difethialone, diphacinone and chlorophacinone. A Waters ACQUITY UPLC TQD system operating in multiple reaction monitoring mode was used to conduct the analysis. Two different ionization techniques, ES1 and ES2, were examined to achieve optimal sensitivity and selectivity resulting in detection by MS – MS using electrospray ionization in positive mode for difenacoum and brodifacoum and in negative mode for all other analytes. All analytes were extracted from 200 mL of whole blood with ethylacetate and separated on a Waters ACQUITY UPLC BEH-C18 column using gradient elution. Ammonium acetate (10 mM, pH 7.5) and methanol were used as mobile phases with a total run time of 8 min. Recoveries were between 70 and 105% with limits of detection ranging from 0.5 to 1 ng/mL. The limit of quantitation was 2 ng/mL for all analytes. Calibration curves were linear within the range 2 –200 ng/mL for all analytes with the coefficient of determination 0.98. The application of the proposed method using liquid –liquid extraction in a series of clinical investigations and forensic toxicological analyses was successful.

Introduction Anticoagulant rodenticides (AR) were developed for controlling rodent populations. There are two classes of anticoagulant rodenticides with similar mechanisms of action. Based on their chemical structure, they can be divided into hydroxycoumarins and indandiones. AR disrupt the normal blood-clotting mechanisms by inhibiting the vitamin K cycle. This results in the inability to produce essential blood-clotting agents. The specific antidote for rodenticides toxicity is vitamin Kl. The hydroxycoumarin warfarin was the first widely used anticoagulant rodenticide and later introduced as an effective treatment option for thromboembolic disease in humans during the Second World War. The repeated use of warfarin and some other early anticoagulant rodenticides resulted in rats and mice developing a resistance to them and thereby the need for new rodenticides. This led to the development of second-generation anticoagulants known as ‘superwarfarins’, which are more potent in their action. There are four main differences between the first- and second-generation anticoagulant rodenticides. The secondgeneration AR are active after only a single intake. They have longer body retention times (RT), tend to induce bleeding for a

longer period of time and are mainly eliminated as unchanged compounds. Accidental ingestion by children and pets is of concern to human beings, especially given the bright colors of some products. People have intentionally ingested them when attempting suicide. It also presents an occupational hazard during manufacture and application. Until now various techniques have been used in the detection and quantification of anticoagulant rodenticides (1 –15) and include high-performance liquid chromatography with fluorescence and ultraviolet detectors (1 – 3). More recently, liquid chromatography in combination with electrospray ionization mass spectroscopy has been used for simultaneous screening and quantitation of both types of rodenticides (6 – 15). However, none of the existing methods have been able to analyze for the whole range of commercially available ARs in Australia in limited volumes of blood with satisfactory sensitivity. In addition, no methods have been reported which utilize suitable internal standards. Some analytical methods have reported using warfarin as an internal standard for the analysis of indandiones and diphacinone for the quantitation of pindone (4, 6, 9, 11). The study objective was to develop and validate a highly sensitive and specific analytical method to identify and quantitate nine rodenticides that are commercially available in Australia. These are the hydroxycoumarins (coumatetralyl, warfarin, brodifacoum, bromadiolone, difenacoum, flocoumafen and difethialone) and indandiones (diphacinone and chlorophacinone). Warfarin-d5 was used as the internal standard.

Materials and methods Chemicals and standards Analytical reference standards were purchased from Novachem (Collingwood, VIC, Australia). These standards were bromadiolone, coumatetralyl, chlorophacinone, diphacinone, difenacoum and warfarin, each at a concentration of 0.1 mg/mL in methanol, brodifacoum and flocoumafen, each at a concentration of 0.1 mg/mL in acetonitrile, and difethiolone at a concentration of 0.01 mg/mL in acetonitrile. The internal standard, warfarin-d5 (10 mg), was obtained from PM Separations (Capalaba, QLD, Australia). Ethylacetate (HPLC grade), acetonitrile and methanol (Optima grade) were purchased from Thermo Fisher Scientific (Scoresby, VIC, Australia). Ammonium acetate (MS grade) was purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Potassium dihydrogen orthophosphate (AR grade) from AJAX (Taren Point, NSW, Australia) and orthophosphoric acid (AR grade) from Merck (Kilsyth, VIC, Australia). Ultrapure water was prepared using an ELGA (Purelab flex) Milli-Q Water purification system.

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Specimens Reconstituted blank blood was prepared from expired Red Cross red cells and serum/plasma of the same blood type by mixing in a 1:1 ratio. Postmortem blood samples previously screened for the presence of ARs were used in the determination of method selectivity and matrix effects (ME).

Preparation of standard solutions A mixed working standard (1,000 ng/mL in methanol) containing all nine rodenticide analytes was prepared. In addition, 100 and 10 ng/mL mixed standard solutions were prepared by the serial dilution of the 1,000 ng/mL mixed standard solution with methanol. All solutions were thoroughly mixed and stored at 58C. A working solution of the internal standard, warfarin-d5, was prepared by dissolving 10 mg of powder in 10 mL of methanol and further diluting with methanol to obtain a concentration of 200 ng/mL. A second set of rodenticide stock standard solutions with different lot numbers was purchased from Novachem and diluted as above to prepare mixed working standard solutions for use in the preparation of quality control (QC) samples.

Calibration curves and QC samples Calibrators were prepared by dispensing appropriate volumes of the methanolic working standard solutions into 10 mL polypropylene tubes to obtain final concentrations of 2, 5, 10, 30, 50, 100 and 200 ng/mL. The methanol was evaporated under a gentle stream of nitrogen until just dry. The residues were reconstituted in 5 mL of blank blood and mixed on a roller mixer for 60 min. QC samples at concentrations of 8 and 80 ng/mL were prepared from a different batch of standard stock solutions in the same manner. Calibrators and QC samples were dispensed in 1 mL aliquots into sealable plastic vials and stored in the freezer at 2188C.

Sample preparation Blood samples, including calibrators, QC samples, postmortem and clinical samples, were thawed for 30 –40 min at room temperature and then vortexed for 10 s. Then, 200 mL of each sample was dispensed into a 10-mL disposable polypropylene tube. Furthermore, 1 mL of phosphate buffer ( pH 3) and 50 mL of the internal standard (200 ng/mL) were added to each tube and mixed by vortexing. Ethylacetate (5 mL) was added to

each tube, capped and placed on a roller mixer for 15 min. The tubes were centrifuged for 3 min at 3,500 rpm and any emulsion layer broken by gently tapping the tubes. The tubes were then centrifuged for a further 10 min. The upper organic layer was recovered into a 5-mL disposable tube, then evaporated under nitrogen stream and reconstituted in 100 mL of methanol by vortexing for at least 30 s.

Instrumentation A Waters ACQUITY TQD triple quadrupole mass spectrometer combined with a Waters ACQUITY UPLC system operating in multiple reaction monitoring (MRM) mode was used to conduct the analysis. The electrospray ionization (ESI) probe was operated in both negative (ES2) and positive (ESþ) polarity modes. Instrument parameters included the capillary voltage at 1.0 kV; extractor voltage at 3.0 V; source temperature at 1308C; desolvation temperature at 4008C; the desolvation gas flow (nitrogen) at 800 L/h and the cone gas flow at 20 L/h. Argon, used as the collision gas, was run at a collision gas flow rate of 0.15 mL/min with a collision gas pressure set at 3.5  1023 mbar.

Results and discussion UPLC—MSMS method optimization To establish the appropriate MRM conditions for the individual compounds, standard solutions at 100 ng/mL combined with mobile phase A (1:1) were infused at a flow rate of 10 mL/min. Two different ionization techniques, ESþ and ES2, were examined to achieve optimal sensitivity and selectivity. The best results were obtained using positive ESI mode for brodifacoum and difenacoum and for all other analytes, negative mode. Two MRM transitions were chosen for each analyte—one for quantitation and a second transition and ratio for confirmation. The two MRM transitions used for each analyte with the accompanying optimized cone voltages and collision energies are presented in Table I. Separation of the analytes was performed on a Waters ACQUITY UPLC BEH-C18 column (100  2.1 mm, 1.7 mm particle size) using gradient elution at a flow rate of 0.4 mL/min. The mobile phase was a mixture of 10 mmol/L aqueous ammonium acetate (pH 7.5) (eluent A) and methanol (eluent B). Gradient elution settings were 85% A for 1 min, followed by a gradient to 2% eluent A in 6 min. The column was washed with

Table I Analytes and Polarity Modes, MRM Transitions, Cone Voltages and Collision Energies Used for Quantitation and Confirmation Compound/ES mode

Coumatetralyl Warfarin Diphacinone Chlorophacinone Difenacoum Brodifacoum Bromadiolone Difethiolone Flocoumafen Warfarin-d5

220 Bidny et al.

Quantitation

(ES2) (ES2) (ES2) (ES2) (ESþ) (ESþ) (ES2) (ES2) (ES2) (ES2)

Confirmation

Precursor and product ions (m/z)

Cone voltage (V)

Collision energy (eV)

Precursor and product ions (m/z)

Cone voltage (V)

Collision energy (eV)

291.3 . 141.1 307.3 . 161.1 339.3 . 167.2 373.2 . 201.1 445.4 . 179.1 523.2 . 335.1 527.2 . 250.1 539.3 . 81 541.4 . 161.1 312.3 . 255.2

55 40 60 90 40 50 75 70 55 40

28 20 30 22 30 20 35 42 40 24

291.3 . 247.2 307.3 . 250.1 339.3 . 144.9 373.2 . 145 445.4 . 257.3 523.2 . 178.1 525.2 . 250.1 539.3 . 151.1 541.4 . 117.1 312.3 . 161.1

55 40 60 90 40 50 75 70 55 40

24 20 24 25 18 33 33 40 60 20

98% of eluent B for 1 min and then equilibrated at initial conditions. The column temperature for the assay is isothermal at 408C. The injection volume was 5 mL using a 10 mL sample loop in the partial loop with needle overfill mode. Both classes of ARs were separated using the BEH-C18 column, with RT ranging from 2.4 to 5.3 min. The total run time was 8 min. An MRM chromatogram monitoring the quantification

transition of the lowest blood calibrator (2 ng/mL) containing all rodenticides is shown in Figure 1.

Selectivity Method selectivity was established first by verifying the lack of response in the blank blood matrix. Validation of the analysis

Figure 1. Typical MRM chromatogram monitoring the quantitation transitions of 2 ng/mL standard solution, containing all rodenticides. Split peaks for flocoumafen and bromadiolone indicate the possible presence of stereoisomers. LC –MS-MS Method for Quantitation of Rodenticides 221

was conducted by the extraction of eight blank bloods and four drug-free postmortem bloods without the addition of the isotopically labeled IS. No interferences were observed at the RT for each analyte nor after the addition of the internal standard [zero samples (16, 17)], to four blank blood samples. The second step to establish method selectivity was to analyze blank samples that had been spiked with limited number of possible interferences. A mixture of brompheniramine, citalopram, clomipramine, dothiepin, lignocaine, methylenedioxymethamphetamine, metoclopramide, oxycodone, pholcodine, amylobarbitone, salicylic acid and thiopentone, each at a concentration of 1,000 ng/mL, was added to each of two working solutions of mixed AR standards at 10 and 100 ng/mL, respectively. No interfering peaks nor changes in the sensitivity of MRM transitions for the target analytes were observed.

Linearity The linear range was established in spiked, drug-free blood at eight different concentrations with five replications. The preparation of calibrators was as previously described. The calibration curves were obtained by plotting the ratio of the analyte peak area to internal standard peak area versus the analyte concentration, using Waters MassLynx software. The calibration curves showed good linearity for all nine analytes over the concentration range 2 –200 ng/mL under the optimized MS-MS conditions. Coefficients of determination (r 2) of the weighted (1/X) linear regressions were .0.98 for the selected range (see Table II). For all further experiments, the following concentration levels were used for calibrators: 2, 5, 10, 30, 50, 70 and 100 ng/mL. The selected working range from 2 to 100 ng/mL is deemed to be adequate for most AR concentrations that would be expected in antemortem and postmortem blood samples.

The limit of detection and the lower limit of quantitation The limits of detection (LODs) and the limits of quantitation (LOQs) for all compounds were established by extraction of blank blood samples spiked with decreasing concentrations of the analytes. Seven replicates at six concentrations levels (0.3, 0.5, 0.7, 1.0, 2.0 and 3.0 ng/mL) were analyzed. The LOD was determined as the lowest concentration of the analyte for which the response for both quantifier and qualifier ions could clearly be differentiated from the background noise level, i.e., signal-to-noise ratio (S/N) 3. The maximum permitted

Table II Data for Linearity, Coefficient of Determination (r 2), RT, LOD, LOQ and Recovery Drug

Linear range (ng/mL)

r2

RT (min)

LOD (ng/mL)

LOQ (ng/mL)

Extraction recovery (%)

Coumatetralyl Warfarin Diphacinone Chlorophacinone Difenacoum Brodifacoum Bromadiolone Difethiolone Flocoumafen

2 – 200 2 – 200 2 – 200 2 – 200 2 – 200 2 – 200 2 – 200 2 – 200 2 – 200

0.995 0.997 0.985 0.990 0.981 0.996 0.994 0.982 0.993

2.51 2.45 3.22 3.71 4.43 5.21 4.21 5.34 4.85

0.5 0.5 1 0.5 0.5 0.5 0.5 1 0.5

2 2 2 2 2 2 2 2 2

80 –102 85 –105 70 –80 70 –95 70 –90 70 –105 75 –90 70 –100 75 –103

222 Bidny et al.

tolerances for relative ion intensities are within recommended international requirements (18, 19). The LOD was 0.5 ng/mL for all analytes, except diphacinone and difethialone, where an LOD of 1 ng/mL was observed. The LOD values presented in Table II demonstrate that the developed method provides enough sensitivity to test rodenticides at the expected level in blood samples. The lower limit of quantitation (LLOQ) was defined as the lowest concentration of an analyte in the sample that results in a reproducible measurement of peak areas and serves as the lowest point on the standard curve. Furthermore, a signal-to-noise ratio for analytes above 10 was required and the criteria for accuracy +20% and for precision within 20% (16, 20) were demonstrated. The LLOQ was determined to be 2 ng/mL for all analytes (see Table II).

Accuracy and precision The accuracy of the method is expressed as bias and calculated as a percent deviation of the observed mean values from the respective nominal values. The acceptance criteria for accuracy are within +15% of the reference value and within +20% near the LLOQ (16, 20). The precision of the method was expressed as a percentage relative standard deviation (% RSD), which is the standard deviation of the measured mean/measured mean 100. The acceptance criteria for precision are within 15% RSD at the medium and high concentrations levels, and 20% RSD near the LLOQ (16, 20). ‘Within-run’ and ‘between-run’ accuracy and precision data for all nine analytes were determined using three levels of spiked samples, i.e., low (5 ng/mL), medium (50 ng/mL) and high (100 ng/mL). The ‘within-run’ data were gained by analyzing samples in replicate (n ¼ 7). The ‘between-run’ data were determined with three separate runs (on different days and extracted by different analysts) (n ¼ 21). All accuracy and precision data are presented in Table III. The results shown in Table III indicate that ‘within-run’ data accuracy for all analytes was within the acceptable range and did not exceed 8%. ‘Between-run’ bias values were within the acceptable range, with a maximum deviation of 19.4% observed for difenacoum near LLOQ. Both ‘within-run’ and ‘between-run’ precisions were ,15% RSD at medium and high concentrations and ,20% RSD near LLOQ for all analytes.

ME and recovery The ME were assessed using the principal approach described by Matuszewski et al. (21) that involves the comparison of peak areas of analytes in three different sets of samples. Set 1 consisted of pure standards dissolved in methanol, and Set 2 and Set 3 were prepared using blood samples from different sources which were spiked after and before the extraction procedure. Using peak areas from these data, ME and Recovery (RE) can be calculated as follows: ME (%) ¼ A/STD  100; RE (%) ¼ B/A  100 and reported as percentages, where A is the peak area from post-extraction spiked samples, B is the peak area from pre-extraction spiked samples and STD is the peak area from pure standards (21). ME was investigated at three concentration levels: 10, 50 and 100 ng/mL. Five replicates were prepared at each level.

Table III Within-Run and Between-Run Precision and Bias for Nine Rodenticides in Blood, Spiked at a Concentration of 5, 50 and 100 ng/mL Analyte

Coumatetralyl

Warfarin

Diphacinone

Chlorophacinone

Difenacoum

Brodifacoum

Bromadiolone

Difethialone

Flocoumafen

Concentration (ng/mL)

5 50 100 5 50 100 5 50 100 5 50 100 5 50 100 5 50 100 5 50 100 5 50 100 5 50 100

Mean (ng/mL)

5.5 48.6 98.3 5.3 50.0 97.1 4.7 47.9 100.0 3.5 43.8 113.1 4.7 46.9 106.2 4.7 46.2 104.0 5.4 51.7 96.6 5.3 47.2 101.7 5.3 46.7 101.6

Within-run (n ¼ 7)

Between-run (n ¼ 21)

Bias (%)

RSD (%)

Bias (%)

RSD (%)

8.0 2.5 23.8 0.5 2.1 23.2 23.3 3.3 4.9 27.4 21.3 6.0 26.3 24.5 21.0 25.7 27.5 4.0 7.1 3.4 23.4 26.0 4.7 22.1 21.8 0.2 21.4

4.0 1.8 5.7 6.7 2.4 6.9 5.4 4.7 5.1 17.5 10.5 8.1 12.6 12.8 11.2 7.7 8.4 8.6 3.8 6.7 11.3 7.1 4.3 6.9 4.7 6.4 7.9

9.1 0.6 22.3 2.0 0.8 21.2 21.0 0.7 20.7 3.8 2.7 22.8 19.4 4.9 25.4 18.0 0.8 22.4 16.4 21.3 22.9 22.3 1.7 1.4 0.7 21.5 1.4

4.8 4.2 4.1 8.6 3.2 4.3 9.4 6.7 6.5 18.7 7.7 11.9 17.4 10.7 6.1 17.0 11.9 9.8 12.1 8.5 6.6 9.9 6.2 6.0 4.8 5.3 5.2

Table IV Matrix Effect (%) for Different Analytes (Mean Values Calculated at Three Concentration Levels: 10, 50 and 100 ng/mL) Drug

Blank blood

PM-n-Dec blood

PM-Dec blood

Coumatetralyl Wafarin Diphacinone Chlorophacinone Difenacoum Brodifacoum Bromadiolone Difethialone Flocoumafen

83 + 3 101 + 7 100 + 9 64 + 11 89 + 3 93 + 12 93 + 5 81 + 5 72 + 8

93 + 3 105 + 5 84 + 3 64 + 10 88 + 18 81 + 2 80 + 12 64 + 6 72 + 12

80 + 5 89 + 8 41 + 2 40 + 6 66 + 9 89 + 8 58 + 8 35 + 3 46 + 10

Comparison of relative ME for blank blood, postmortem nondecomposed blood (PM-n-Dec blood) and postmortem decomposed blood (PM-Dec blood) is outlined in Table IV. The results of the ME study indicated that the MS-MS response originating from the same analyte differs in different types of blood. Most analytes demonstrate some degree of ion suppression. The variability of the ME for different blood samples was a result of the variability in composition of forensic samples in the presence of decomposition products. The difference in ion suppression for blank blood and PM-n-Dec blood was negligible and any observed loss of sensitivity did not interfere with accurate quantitation. Ion suppression and loss of sensitivity were more noticeable for diphacinone and difethialone in PM-Dec blood and caution should be used when reporting these results. It is well known that the addition of an appropriate stable isotopically labeled internal standards is the preferred way of compensating for observed ME (21, 22). However, they are

Table V Brodifacoum Levels (ng/mL) in Blood Monitored in Two Patients over 6 Months Months (post-ingestion)

Case 1

Case 2

1 2 3 4 5 6

170 60 35 24 12 5

70 10 4 – – –

unfortunately not always available, hence only warfarin-d5 was used as internal standard for this assay.

Stability For the evaluation of the stability of processed samples, two levels of QC samples—low (8 ng/mL) and high (80 ng/mL) were extracted in replicate (n ¼ 5) and analyzed immediately, while another set of samples was analyzed which had been left in the autosampler at 108C for 24 h. For the evaluation of freeze – thaw stability, two levels of QC samples were analyzed before and after three freeze – thaw cycles. Samples were frozen at 2188C for 1 day and then thawed at room temperature. Once the samples had completely thawed, they were extracted in replicate (n ¼ 5) and then frozen again. After repeating the process three times, the mean concentration of frozen and thawed samples were compared with the mean concentration of the untreated controls. All the changes for low QC and high QC were within +15% of the nominal concentrations as required for validation. No instability was observed on samples stored at 108C for 24 h nor after three freeze – thaw cycles.

Applicability—clinical cases The developed method has been useful in confirming the diagnosis of several rodenticide intoxications in humans. The method was useful in confirming brodifacoum poisoning in our previously published article (23), where a series of brodifacoum levels were measured over time from the blood of two subjects who had ingested large amounts of brodifacoum. Large quantities of brodifacoum had been ingested by both patients resulting in elevated international normalized ratio. Blood tested at our laboratory confirmed the presence of high levels of brodifacoum (see Table V). Both patients were treated for several months with high doses of vitamin K1, which is the antidote, until brodifacoum levels fell below toxic thresholds. Serial testing of brodifacoum levels were used as a form of monitoring and estimating the required duration of vitamin K1 therapy. The analysis proved beneficial both in terms of confirming diagnosis and monitoring therapy.

Conclusions A rapid method which is highly sensitive and specific for the detection and quantification of rodenticides in blood has been developed and validated. The main advantage of the developed method is the ability to identify and quantitate all nine commercially available rodenticides in Australia in limited volumes of LC –MS-MS Method for Quantitation of Rodenticides 223

blood—as little as 200 mL of the sample can be used. The simple extraction method using ethylacetate is efficient and good extraction recoveries are achieved, most exceeding 70%. The method was validated according to international guidelines and has been successfully applied to a series of clinical investigations and forensic toxicological analyses. The method is useful for routine analysis of postmortem and antemortem samples and is sensitive enough to test for anticoagulant rodenticides at levels below 10 ng/mL.

9.

10.

11.

Acknowledgments

12.

The authors thank the staff at the Forensic Toxicology Laboratory, NSW FASS, for technical and laboratory assistance and Jennifer Easson, Cheng Tan, Andrew Pomfret and Daniel Pasin for critical reading and valuable comments. The authors also thank Durand Prasad for his help in preparing graphical presentations of some data. They are thankful to Michael Rennie for providing the standards and for helpful discussions.

13.

14.

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