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

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
MS/MS parameters		Fluralaner	Sarolaner	Lotilaner	Afoxolaner
Precursor ions (m/z)		556.06	581.03	595.98	625.73
Product ions (m/z)	Quantification ions	457.0	457.0	439.75	469.89
Product ions (m/z)	Confirmation ions	400.0	415.9	166.03	195.97
Collision energy (eV)	Quantification ions	18	25	30	26
Collision energy (eV)	Confirmation ions	12	20	35	48
Retention time (min)		2.55	2.55	3.33	3.18
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

Time (min)	Flow rate (mL/min)	Mobile phase (%)		Curve
Time (min)	Flow rate (mL/min)	A	B	Curve
0.00	0.35	35.0	65.0	Initial
2.70	0.35	28.0	72.0	6
3.00	0.35	0.0	100.0	6
3.50	0.35	35.0	65.0	11
Mobile phase		0.1% formic acid in acetonitrile	0.1% formic acid in water
Column	ACQUITY HSS T3, 1.8 μm 2.1 × 100 mm

Table 2.

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

	Parameter	Acceptance criteria
Linearity	Calibration points	Back-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)	$\| \frac{y_{i} - {\hat{y}}_{i}}{{\hat{y}}_{i}} \| \times 100 % \leq 20 %$
	SD of relative residuals (S_Yi)	$\sqrt{\frac{\sum {(Y_{i} - \bar{Y})}^{2}}{n - 2}} \leq 0.1$
Stability	Stock and working standard	$\frac{S_{t}}{S_{0}} \times 1100 % = w i t h i n \pm 15 % o f S_{0}$
	Autosampler
	Freeze and thaw
	Sample processing temperature
Precision (RSD or CV)		$\frac{S D}{C_{m e a n}} \times 100 % = w i t h i n \pm 15 % o f t h e n o m i n a l c o n c e n t r a t i o n$
Accuracy (deviation) (for at least 5 points per group/day)		$\frac{\| (C_{t} - C_{n}) \|}{C_{n}} \times 100 % = w i t h i n \pm 15 % o f t h e n o m i n a l c o n c e n t r a t i o n$
LOD		3 × SD_C_fortified where S/N ≥ 3: 1
LLOQ with accuracy and precision		6 × C_fortified where S/N ≥ 10: 1
Matrix effect		$100 - (\frac{X_{i}}{X} \times 100 %) = \pm 15 % r e l a t i v e t o t h e s a m p l e w i t h o u t t h e m a t r i x$
Total recovery		$\frac{X_{z}}{X_{i}} \times 100 %$
Selectivity/Specificity		No endogenous peaks in the retention time of the analyte
Carry-over		Area of carry-over peaks: ≤ 20% of LLOQ and 5% of IS area

1 y_i – experimental signal; ŷ_i – calculated signal; SD – standard deviation; Y_i – relative residual; Ȳ – mean value of relative residuals; S_t – peak area when the analysis was paused for time t; S₀ – initial peak area without additional pauses during the analysis (freshly prepared standards); RSD – relative standard deviation; CV – coefficient of variation; C_mean – mean concentration (ng/mL); C_t – individually calculated concentration (ng/mL); C_n – nominal concentration (ng/mL); LOD – limit of detection; SD_Cfortified – standard deviation calculated from fortified samples with the lowest acceptable concentration; S/N – signal-to-noise; LLOQ – lower limit of quantification; C_fortified – minimal fortified concentration which meets the requirements; X_i – peak area of the analyte added to the matrix after extraction; X – peak area of the analyte in the final solvent; X_z – peak area of the analyte added to the matrix before extraction

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

Table 3.

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 test		Dog				Cat				Human
Validation test		Fluralaner	Sarolaner	Lotilaner	Afoxolaner	Fluralaner	Sarolaner	Lotilaner	Afoxolaner	Fluralaner	Sarolaner	Lotilaner	Afoxolaner
Total recovery (%)	Mean	13.10	13.90	10.10	13.90	11.15	14.10	9.80	13.6	12.10	13.00	11.93	10.70
Total recovery (%)	SD	2.10	1.80	1.18	2.00	1.43	1.12	2.50	2.20	2.90	0.80	0.90	1.10
Matrix effect (%)	Mean	8.90	13.90	13.30	12.10	12.20	9.80	9.50	11.90	13.60	13.70	8.93	9.60
Matrix effect (%)	SD	1.54	1.89	1.04	2.03	2.12	2.34	2.12	1.07	1.14	2.01	1.08	1.71

1 SD – standard deviation

Table 4.

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

Linearity	_r2		I 0.99	II 0.99	III 0.99	IV 0.99	V 0.99	Mean 0.99
Precision (%) and accuracy (%)	Intra-day, n = 6; 3 replications			LLOQ	LQC	IQC	MQC	HQC
		Precision	I	8.94	5.59	1.94	3.89	1.66
		Accuracy	I	6.67	4.33	1.62	2.64	1.29
		Precision	II	4.15	6.81	2.34	7.81	2.47
		Accuracy	II	1.67	5.33	1.90	4.65	1,99
	Inter-day, n = 18	Precision	III	7.66	9.38	5.54	3.25	6.16
		Accuracy	III	5.00	7.33	4.60	6.01	4,87
		Precision		7.23	2.19	1.75	3.03	3.16
		Accuracy		5.70	1.67	4.91	2.90	2.60
			Concentration (ng/mL)			S/N
LLOQ and LOD	LLOQ overall mean, n = 18		1.00			90.00
	LLOQ overall SD, n = 18		0.14			5.91
	LOD overall mean, n = 18		0.5			6.22
	LOD overall SD, n = 18		0.33			3.91
Total recovery	Sample		Total recovery (%)
	Mean	1 ng/mL	91.19
	Mean	1,500 ng/mL	85.39
	SD	1 ng/mL	12.73
	SD	1,500 ng/mL	5.94
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	10.15
	Mean	1,500 ng/mL	14.81
	SD	1 ng/mL	8.22
	SD	1,500 ng/mL	2.94
Stability tests results
Stability	Period (h)		Changes in the concentration of QCs (%)
	Period (h)		LQC	IQC		MQC		HQC
	24		13.25	3.07		4.96		1.16
Working standard, 2°C	48		8.92	14.58		12.01		14.28
	120		13.25	10.90		9.02		12.24
	168		14.31	13.95		11.15		11.10
Autosampler, 4°C	24		7.01	9.80		14.48		7.72
Autosampler, 4°C	24		14.71	13.91		5.30		2.77
Freeze and thaw, –75°C	48		14.07	14.93		12.60		4.65
	72		12.33	14.39		13.98		13.49
	480		14.49	14.77		10.30		8.52
Sample processing, 21°C	3		14.04	9.23		4.01		3.73

1 I–V – replicate number; LLOQ – lower limit of quantification; LQC – low quality control; IQC – intermediate quality control; MQC – medium quality control; HQC – high quality control; LOD – limit of detection; SD – standard deviation

Table 5.

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

Linearity	_r2		I	II	III	IV	V	Mean
Linearity	_r2		0.99	0.99	0.99	0.99	0.99	0.99
Precision (%) and accuracy (%)	Intra-day, n = 6; 3 replications			LLOQ	LQC	IQC	MQC	HQC
		Precision	I	9.67	6.07	1.67	2.34	2,04
		Accuracy	I	5.00	4.67	1.35	3.86	1,36
		Precision	II	14.48	3.87	3.95	4.22	7,49
		Accuracy	II	11.67	3.00	2.67	4.66	5,16
	Inter-day, n = 18	Precision	III	10.95	1.79	2.08	6.71	5,10
		Accuracy	III	6.67	1.33	1.65	7.08	3,33
		Precision		11.70	3.91	2.57	6.02	4.88
		Accuracy		7.78	3.00	1.89	3.33	3.28
			Concentration (ng/mL)				S/N
LLOQ and LOD	LLOQ overall mean, n = 18		1.00				91.48
	LLOQ overall SD, n = 18		0.12				4.74
	LOD overall mean, n = 18		0.5				4.01
	LOD overall SD, n = 18		0.36				0.02
	Sample		Total recovery (%)
Total recovery	Mean	1 ng/mL	99.78
	Mean	1,500 ng/mL	96.21
	SD	1 ng/mL	14.09
	SD	1,500 ng/mL	7.47
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	14.40
	Mean	1,500 ng/mL	6.97
	SD	1 ng/mL	8.09
	SD	1,500 ng/mL	7.00
Stability test results
Stability	Period (h)		Changes in the concentration of QCs (%)
			LQC	IQC		MQC		HQC
Working standard, 2°C	24		13.97	12.24		12.35		13.31
	48		12.08	14.38		13.55		14.44
	120		7.60	2.85		11.64		3.20
Autosampler, 4°C	168		5.02	8.81		14.90		12.42
	24		13.51	13.93		2.30		6.93
	24		7.12	14.11		14.92		7.17
Freeze and thaw, –75°C	48		9.58	12.84		4.52		3.91
	72		12.56	13.00		12.40		14.90
	480		7.16	2.30		2.66		10.25
Sample processing, 21°C	3		13.38	14.62		3.21		4.07

Table 6.

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

Linearity	r²		I	II	III	IV	V	Mean
Linearity	r²		0.99	0.99	0.99	0.99	0.99	0.99
Precision (%) and accuracy (%)	Intra-day, n = 6; 3 replications			LLOQ	LQC	IQC	MQC	HQC
		Precision	I	14.97	1.79	3.67	4.46	7.82
		Accuracy	I	11.67	1.33	2.58	6.72	5.60
		Precision	II	7.40	4.10	4.95	5.02	4.61
		Accuracy	II	5.00	3.29	3.27	7.03	3.76
	Inter-day, n = 18	Precision	III	6.32	2.33	5.99	8.22	1.42
		Accuracy	III	3.33	1.67	4.03	9.11	1.19
		Precision		9.57	2.74	4.87	5.15	4.61
		Accuracy		6.67	2.10	3.29	4.99	3.52
			Concentration (ng/mL)				S/N
LLOQ and LOD	LLOQ overall mean, n = 18		1.00				73.23
	LLOQ overall SD, n = 18		0.09				5.95
	LOD overall mean, n = 18		0.5				5.55
	LOD overall SD, n = 18		0.39				2.87
	Sample		Total recovery (%)
Total recovery	Mean	1 ng/mL	95.50
	Mean	1,500 ng/mL	93.16
	SD	1 ng/mL	14.76
	SD	1,500 ng/mL	5.07
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	14.10
	Mean	1,500 ng/mL	7.09
	SD	1 ng/mL	5.98
	SD	1,500 ng/mL	4.51
Stability test results
Stability	Period (h)		Changes in the concentration of QCs (%)
			LQC	IQC		MQC		HQC
Working standard, 2°C	24		6.32	7.40		10.90		5.91
	48		8.24	13.25		6.07		8.71
	120		9.58	13.60		2.96		11.92
Autosampler, 4°C	168		8.45	14.87		11.29		12.82
	24		13.50	13.99		9.54		14.94
	24		12.30	7.27		12.88		9.35
Freeze and thaw, –75°C	48		8.75	5.60		11.63		12.44
	72		13.03	2.37		3.95		6.73
	480		13.34	11.42		11.26		9.70
Sample processing, 21°C	3		4.21	5.42		10.18		2.61

Table 7.

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

Linearity	r²		I	II	III	IV	V	Mean
Linearity	r²		0.99	0.99	0.99	0.99	0.99	0.99
Precision (%) and accuracy (%)	Intra-day, n = 6; 3 repetitions			LLOQ	LQC	IQC	MQC	HQC
		Precision	I	7.66	10.28	3.04	2.47	3.45
		Accuracy	I	5.00	8.67	2.48	3.22	2.00
		Precision	II	13.22	4.20	4.30	6.66	8.16
		Accuracy	II	10.00	2.67	3.27	5.54	6.39
	Inter-day, n = 18	Precision	III	9.67	3.10	2.26	5.23	4.52
		Accuracy	III	5.00	2.67	1.60	8.02	3.65
		Precision		10.18	5.86	3.20	6.07	5.05
		Accuracy		6.67	4.67	2.45	6.01	4.01
			Concentration (ng/mL)				S/N
LLOQ and LOD	LLOQ overall mean, n = 18		1.00				92.30
	LLOQ overall SD, n = 18		0.04				4.8
	LOD overall mean, n = 18		0.5				0.9
	LOD overall SD, n = 18		0.44				1.11
	Sample		Total recovery (%)
Total recovery	Mean	1 ng/mL	85.19
	Mean	1,500 ng/mL	87.05
	SD	1 ng/mL	14.14
	SD	1,500 ng/mL	7.88
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	13.77
	Mean	1,500 ng/mL	14.69
	SD	1 ng/mL	9.56
	SD	1,500 ng/mL	10.52
Stability test results
Stability	Period (h)		Changes in the concentration of QCs (%)
			LQC	IQC		MQC		HQC
Working standard, 2°C	24		5.74	3.07		4.96		1.16
	48		14.91	14.58		12.01		14.28
	120		9.82	7.82		11.76		14.80
Autosampler, 4°C	168		12.79	3.31		11.28		13.84
	24		13.15	8.10		9.55		14.14
	24		10.00	12.99		6.47		3.44
Freeze and thaw, –75°C	48		14.10	13.78		14.05		12.39
	72		14.84	3.12		13.48		10.89
	480		12.53	10.90		12.66		8.37
Sample processing, 21°C	3		5.66	14.37		13.26		13.68

Table 8.

Results of screening tests (concentrations in ng/mL)

Species	Breed	Fluralaner	Sarolaner	Lotilaner	Afoxolaner
Dog	German shepherd	-	434.6	-	-
	German shepherd	-	12.1	-	-
	German shepherd	-	-	-	-
	German shepherd	414.3	-	-	-
	German shepherd	-	-	-	1.3
	German Shepherd	-	19.0	-	-
	German shepherd	346.5	-	-	-
	Yorkshire terrier	-	-	-	1.0
	Yorkshire terrier	-	-	-	1.0
	Yorkshire terrier	-	-	-	1.0
	Schnauzer	388.3	-	-	-
	Schnauzer	76.7	-	-	-
	Schnauzer	-	-	-	-
	Golden retriever	-	-	-	-
	Golden retriever	-	225.0	-	-
	Siberian husky	-	-	-	-
	Siberian husky	-	196.0	-	-
	Beagle	-	-	-	-
	Beagle	-	5.6	-	-
	Labrador retriever	-	100.6	-	-
Cat	European cat	-	-	-	-
	European cat	-	23.8	-	-
	European cat	-	233.3	-	-
	European cat	-	-	-	-
	European cat	-	-	-	-
	Persian cat	-	-	-	-
	Persian cat	-	-	-	-
	Persian cat	-	-	-	-
	Persian cat	-	-	-	-
	Persian cat	-	-	-	-

References

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Articles in this issue

DOI: https://doi.org/10.2478/jvetres-2025-0050 | Journal eISSN: 2450-8608 (formerly 2300-3235)

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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

Keywords:

Related subjects:

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.

Volume 69 (2025): Issue 3 (September 2025)

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

Figures & Tables

Table 1.