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

Fig 1.

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
		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
	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
	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

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
			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		5.00	4.67	1.35	3.86	1,36
		Precision	II	14.48	3.87	3.95	4.22	7,49
		Accuracy		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		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
		1,500 ng/mL	96.21
	SD	1 ng/mL	14.09
		1,500 ng/mL	7.47
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	14.40
		1,500 ng/mL	6.97
	SD	1 ng/mL	8.09
		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

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	-	-	-	-

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)	| 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
Stability	Stock and working standard	StS0×1100%=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
LOD		3 × SDCfortified where S/N ≥ 3: 1
LLOQ with accuracy and precision		6 × Cfortified where S/N ≥ 10: 1
Matrix effect		100−(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 recovery		XzXi×100%{{{X_z}} \over {{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

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
			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		11.67	1.33	2.58	6.72	5.60
		Precision	II	7.40	4.10	4.95	5.02	4.61
		Accuracy		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		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
		1,500 ng/mL	93.16
	SD	1 ng/mL	14.76
		1,500 ng/mL	5.07
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	14.10
		1,500 ng/mL	7.09
	SD	1 ng/mL	5.98
		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

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
			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		5.00	8.67	2.48	3.22	2.00
		Precision	II	13.22	4.20	4.30	6.66	8.16
		Accuracy		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		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
		1,500 ng/mL	87.05
	SD	1 ng/mL	14.14
		1,500 ng/mL	7.88
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	13.77
		1,500 ng/mL	14.69
	SD	1 ng/mL	9.56
		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

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		6.67	4.33	1.62	2.64	1.29
		Precision	II	4.15	6.81	2.34	7.81	2.47
		Accuracy		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		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
		1,500 ng/mL	85.39
	SD	1 ng/mL	12.73
		1,500 ng/mL	5.94
			Matrix effect (%)
Matrix effect	Mean	1 ng/mL	10.15
		1,500 ng/mL	14.81
	SD	1 ng/mL	8.22
		1,500 ng/mL	2.94
Stability tests results
Stability	Period (h)		Changes in the concentration of QCs (%)
			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
	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

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
		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
	Confirmation ions	400.0	415.9	166.03	195.97
Collision energy (eV)	Quantification ions	18	25	30	26
	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