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A novel method for the determination of isoxazoline derivatives in plasma by ultra-high performance liquid chromatography–tandem mass spectrometry: validation and applicability to screening tests Cover

A novel method for the determination of isoxazoline derivatives in plasma by ultra-high performance liquid chromatography–tandem mass spectrometry: validation and applicability to screening tests

Journal of Veterinary Research
Volume 69 (2025): Issue 3 (September 2025)
By: Paulina Markowska-BuńkaORCID,  Jerzy J. JaroszewskiORCID,  Bartosz RasińskiORCID and  Hubert ZiółkowskiORCID  
Open Access
|Sep 2025

Figures & Tables

Fig 1.

Liquid chromatography–tandem mass spectrometry traces of separation of four isoxazoline derivatives from plasma (fluralaner, sarolaner, lotilaner and afoxolaner)
Liquid chromatography–tandem mass spectrometry traces of separation of four isoxazoline derivatives from plasma (fluralaner, sarolaner, lotilaner and afoxolaner)

Validation results for total recovery by ultra-high performance liquid chromatography–tandem mass spectrometry for the determination of isoxazoline derivatives in plasma from clinically healthy dogs, cats and humans and validation results for matrix effect

Validation testDogCatHuman
FluralanerSarolanerLotilanerAfoxolaner FluralanerSarolanerLotilanerAfoxolanerFluralanerSarolanerLotilanerAfoxolaner
Total recovery (%)Mean13.1013.9010.1013.9011.1514.109.8013.612.1013.0011.9310.70
SD2.101.801.182.001.431.122.502.202.900.800.901.10
Matrix effect (%)Mean8.9013.9013.3012.1012.209.809.5011.9013.6013.708.939.60
SD1.541.891.042.032.122.342.121.071.142.011.081.71

Validation results of an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of sarolaner in clinically healthy laying hen plasma

Linearityr2IIIIIIIVVMean
0.990.990.990.990.990.99
Precision (%) and accuracy (%)Intra-day, n = 6; 3 replications LLOQLQCIQCMQCHQC
PrecisionI9.676.071.672.342,04
Accuracy5.004.671.353.861,36
PrecisionII14.483.873.954.227,49
Accuracy11.673.002.674.665,16
Inter-day, n = 18PrecisionIII10.951.792.086.715,10
Accuracy6.671.331.657.083,33
Precision 11.703.912.576.024.88
Accuracy 7.783.001.893.333.28
Concentration (ng/mL)S/N
LLOQ and LODLLOQ overall mean, n = 181.0091.48
LLOQ overall SD, n = 180.124.74
LOD overall mean, n = 180.54.01
LOD overall SD, n = 180.360.02
SampleTotal recovery (%)
Total recoveryMean1 ng/mL99.78
1,500 ng/mL96.21
SD1 ng/mL14.09
1,500 ng/mL7.47
Matrix effect (%)
Matrix effectMean1 ng/mL14.40
1,500 ng/mL6.97
SD1 ng/mL8.09
1,500 ng/mL7.00
Stability test results
StabilityPeriod (h)Changes in the concentration of QCs (%)
LQCIQCMQCHQC
Working standard, 2°C2413.9712.2412.3513.31
4812.0814.3813.5514.44
1207.602.8511.643.20
Autosampler, 4°C1685.028.8114.9012.42
2413.5113.932.306.93
247.1214.1114.927.17
Freeze and thaw, –75°C489.5812.844.523.91
7212.5613.0012.4014.90
4807.162.302.6610.25
Sample processing, 21°C313.3814.623.214.07

Results of screening tests (concentrations in ng/mL)

SpeciesBreedFluralanerSarolanerLotilanerAfoxolaner
DogGerman shepherd-434.6--
German shepherd-12.1--
German shepherd----
German shepherd414.3---
German shepherd---1.3
German Shepherd-19.0--
German shepherd346.5---
Yorkshire terrier---1.0
Yorkshire terrier---1.0
Yorkshire terrier---1.0
Schnauzer388.3---
Schnauzer76.7---
Schnauzer----
Golden retriever----
Golden retriever-225.0--
Siberian husky----
Siberian husky-196.0--
Beagle----
Beagle-5.6--
Labrador retriever-100.6--
CatEuropean cat----
European cat-23.8--
European cat-233.3--
European cat----
European cat----
Persian cat----
Persian cat----
Persian cat----
Persian cat----
Persian cat----

Calculation methods and acceptance criteria for parameters for validation of an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of isoxazoline derivatives in plasma

ParameterAcceptance criteria
LinearityCalibration pointsBack-calculated concentrations should be within ± 15% of the nominal concentration, and at least 75% of the calibration points, but no fewer than 6, must fulfil this criterion
Coefficient of determination (r2)≥ 0.99
Relative residuals (Yi)| yi−y^iy^i |×100%≤20%\left| {{{{y_i} - {{\hat y}_i}} \over {{{\hat y}_i}}}} \right| \times 100\% \le 20\%
SD of relative residuals (SYi)∑ (Yi−Y¯)2n−2≤0.1\sqrt {{{\sum {{{\left( {{Y_i} - \bar Y} \right)}^2}} } \over {n - 2}}} \le 0.1
StabilityStock and working standardStS0×1100%=within±15%ofS0{{{S_t}} \over {{S_0}}} \times 1100\% = {\rm{ within }} \pm 15\% {\rm{ of }}{S_0}
Autosampler
Freeze and thaw
Sample processing temperature
Precision (RSD or CV)SDCmean ×100%= within ±15% of the nominal concentration{{SD} \over {{C_{mean{\rm{ }}}}}} \times 100\% = {\rm{ }}within{\rm{ }} \pm 15\% {\rm{ }}of the nominal concentration
Accuracy (deviation) (for at least 5 points per group/day)| (Ct−Cn) |Cn×100%= within ±15% of the  nominal concentration{{\left| {\left( {{C_t} - {C_n}} \right)} \right|} \over {{C_n}}} \times 100\% = {\rm{ }}within{\rm{ }} \pm 15\% {\rm{ }}of the nominal concentration
LOD3 × SDCfortified where S/N ≥ 3: 1
LLOQ with accuracy and precision6 × Cfortified where S/N ≥ 10: 1
Matrix effect100−(XiX×100%)=±15% relative to the sample without the matrix100 - \left( {{{{X_i}} \over X} \times 100\% } \right) = \pm 15\% \;relative to the sample without the matrix
Total recoveryXzXi×100%{{{X_z}} \over {{X_i}}} \times 100\%
Selectivity/SpecificityNo endogenous peaks in the retention time of the analyte
Carry-overArea of carry-over peaks: ≤ 20% of LLOQ and 5% of IS area

Validation results of an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of lotilaner in clinically healthy laying hen plasma

Linearityr2IIIIIIIVVMean
0.990.990.990.990.990.99
Precision (%) and accuracy (%)Intra-day, n = 6; 3 replications LLOQLQCIQCMQCHQC
PrecisionI14.971.793.674.467.82
Accuracy11.671.332.586.725.60
PrecisionII7.404.104.955.024.61
Accuracy5.003.293.277.033.76
Inter-day, n = 18PrecisionIII6.322.335.998.221.42
Accuracy3.331.674.039.111.19
Precision 9.572.744.875.154.61
Accuracy 6.672.103.294.993.52
Concentration (ng/mL)S/N
LLOQ and LODLLOQ overall mean, n = 181.0073.23
LLOQ overall SD, n = 180.095.95
LOD overall mean, n = 180.55.55
LOD overall SD, n = 180.392.87
SampleTotal recovery (%)
Total recoveryMean1 ng/mL95.50
1,500 ng/mL93.16
SD1 ng/mL14.76
1,500 ng/mL5.07
Matrix effect (%)
Matrix effectMean1 ng/mL14.10
1,500 ng/mL7.09
SD1 ng/mL5.98
1,500 ng/mL4.51
Stability test results
StabilityPeriod (h)Changes in the concentration of QCs (%)
LQCIQCMQCHQC
Working standard, 2°C246.327.4010.905.91
488.2413.256.078.71
1209.5813.602.9611.92
Autosampler, 4°C1688.4514.8711.2912.82
2413.5013.999.5414.94
2412.307.2712.889.35
Freeze and thaw, –75°C488.755.6011.6312.44
7213.032.373.956.73
48013.3411.4211.269.70
Sample processing, 21°C34.215.4210.182.61

Validation results of an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of afoxolaner in clinically healthy laying hen plasma

Linearityr2IIIIIIIVVMean
0.990.990.990.990.990.99
Precision (%) and accuracy (%)Intra-day, n = 6; 3 repetitions LLOQLQCIQCMQCHQC
PrecisionI7.6610.283.042.473.45
Accuracy5.008.672.483.222.00
PrecisionII13.224.204.306.668.16
Accuracy10.002.673.275.546.39
Inter-day, n = 18PrecisionIII9.673.102.265.234.52
Accuracy5.002.671.608.023.65
Precision 10.185.863.206.075.05
Accuracy 6.674.672.456.014.01
Concentration (ng/mL)S/N
LLOQ and LODLLOQ overall mean, n = 181.0092.30
LLOQ overall SD, n = 180.044.8
LOD overall mean, n = 180.50.9
LOD overall SD, n = 180.441.11
SampleTotal recovery (%)
Total recoveryMean1 ng/mL85.19
1,500 ng/mL87.05
SD1 ng/mL14.14
1,500 ng/mL7.88
Matrix effect (%)
Matrix effectMean1 ng/mL13.77
1,500 ng/mL14.69
SD1 ng/mL9.56
1,500 ng/mL10.52
Stability test results
StabilityPeriod (h)Changes in the concentration of QCs (%)
LQCIQCMQCHQC
Working standard, 2°C245.743.074.961.16
4814.9114.5812.0114.28
1209.827.8211.7614.80
Autosampler, 4°C16812.793.3111.2813.84
2413.158.109.5514.14
2410.0012.996.473.44
Freeze and thaw, –75°C4814.1013.7814.0512.39
7214.843.1213.4810.89
48012.5310.9012.668.37
Sample processing, 21°C35.6614.3713.2613.68

Validation results of an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of fluralaner in clinically healthy laying hen plasma

Linearityr2I 0.99II 0.99III 0.99IV 0.99V 0.99Mean 0.99
Precision (%) and accuracy (%)Intra-day, n = 6; 3 replications LLOQLQCIQCMQCHQC
PrecisionI8.945.591.943.891.66
Accuracy6.674.331.622.641.29
PrecisionII4.156.812.347.812.47
Accuracy1.675.331.904.651,99
Inter-day, n = 18PrecisionIII7.669.385.543.256.16
Accuracy5.007.334.606.014,87
Precision 7.232.191.753.033.16
Accuracy5.701.674.912.902.60
Concentration (ng/mL)S/N
LLOQ and LODLLOQ overall mean, n = 181.0090.00
LLOQ overall SD, n = 180.145.91
LOD overall mean, n = 180.56.22
LOD overall SD, n = 180.333.91
Total recoverySampleTotal recovery (%)
Mean1 ng/mL91.19
1,500 ng/mL85.39
SD1 ng/mL12.73
1,500 ng/mL5.94
Matrix effect (%)
Matrix effectMean1 ng/mL10.15
1,500 ng/mL14.81
SD1 ng/mL8.22
1,500 ng/mL2.94
Stability tests results
StabilityPeriod (h)Changes in the concentration of QCs (%)
LQCIQCMQCHQC
2413.253.074.961.16
Working standard, 2°C488.9214.5812.0114.28
12013.2510.909.0212.24
16814.3113.9511.1511.10
Autosampler, 4°C247.019.8014.487.72
2414.7113.915.302.77
Freeze and thaw, –75°C4814.0714.9312.604.65
7212.3314.3913.9813.49
48014.4914.7710.308.52
Sample processing, 21°C314.049.234.013.73

Selected parameters of ultra-high performance liquid chromatography–tandem mass spectrometry (MS/MS) for the determination of isoxazoline derivatives in plasma

MS/MS parameters Compound
FluralanerSarolanerLotilanerAfoxolaner
Precursor ions (m/z) 556.06581.03595.98625.73
Product ions (m/z)Quantification ions457.0457.0439.75469.89
Confirmation ions400.0415.9166.03195.97
Collision energy (eV)Quantification ions18253026
Confirmation ions12203548
Retention time (min) 2.552.553.333.18
Capillary voltage (kV) 3.0
Cone voltage (V) 20
Desolvation gas Nitrogen
Desolvation gas temperature (°C) 600
Desolvation gas flow (L/h) 1000
Collision gas flow (mL/min) 0.2
Collision gas Argon
Source temperature (°C) 120
Electrospray mode Positive
Dwell (s) 0.1
DOI: https://doi.org/10.2478/jvetres-2025-0050 | Journal eISSN: 2450-8608
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Language: English
Page range: 405 - 415
Submitted on: Mar 7, 2025
Accepted on: Sep 17, 2025
Published on: Sep 24, 2025
Published by: National Veterinary Research Institute in Pulawy
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
afoxolaner,
fluralaner,
lotilaner,
sarolaner,
UPLC-MS/MS
Related subjects:
Life sciences,
Molecular biology,
Microbiology and virology,
Life sciences, other,
Medicine,
Veterinary medicine

© 2025 Paulina Markowska-Buńka, Jerzy J. Jaroszewski, Bartosz Rasiński, Hubert Ziółkowski, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution 4.0 License.

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