Liquid chromatographytandem mass spectrometry methods for determination of stanozolol and l6β-hydroxy-stanozolol in animal urine

Matraszek-Źuchowska, Iwona; Kłopot, Alicja; Sielska, Katarzyna; Korycińska, Beata; Witek, Sebastian; Zdonek, Paulina; Jedziniak, Piotr

doi:10.2478/jvetres-2023-0030

Figures & Tables

Chemical structures, molecular formulas and weights of molecules of hormones tested

Liquid chromatography-tandem ion trap mass spectrometry selected reaction monitoring (SRM) chromatograms of bovine urine samples spiked with 16β-OH-stanozolol (16β-OH-STAN) and stanozolol (STAN) at 2 μg L−1

Liquid chromatography-tandem triple quadrupole mass spectrometry multiple reaction monitoring chromatograms of A - a pig blank urine sample; B - a pig urine sample spiked with stanozolol (STAN) and 16β-OH-stanozolol (16β-OH-STAN) at 0.5 μg L−1; C - a pig urine sample spiked with STAN and 16β-OH-STAN at 2 μg L−1

LC-MS² (IT)
Compound	SRM transition (m/z)	Collision energy CE (%)	Ion ratio average ± SD	Samples fulfilling the confirmation criteria (%)^*
Compound	SRM transition (m/z)	Collision energy CE (%)	Ion ratio average ± SD	1.00-10.00 μg L⁻¹
STAN	329>311^a	35	-	-
	329>271		0.228 ± 0.032	75.3
	329>229		0.363 ± 0.067	74.1
16β-OH-STAN	345>327	37	0.653 ± 0.212	51.9
	345>309		-	-
	345>227		0.734 ± 0.163	55.6
	345>159		0.735 ± 0.135	58.0
16β-OH-STAN-D3	348>312	36	-	-

Validation parameters of the liquid chromatography-tandem triple quadrupole (LC-MS/MS (QqQ) and -tandem ion-trap (LC-MS2 (IT) mass spectrometry methods for the determination of stanozolol (STAN) and its metabolite 16β-OH-stanozolol (16β-OH-STAN) in bovine urine

	Number of samples	Spiking level (μg L⁻¹)	Compound
	Number of samples	Spiking level (μg L⁻¹)	STAN^*	16β-OH-STAN^*	STAN^**	16β-OH-STAN^**
Apparent recovery (%)	n=18^/21^*	1.00^/0.50^*	125.8	85.4	64.5	98.8
		2.00/1.00	104.7	101.7	65.9	94.0
		3.00/1.50	88.4	90.7	66.8	88.7
	n = 18^/6^*	4.00/2.00	87.4	94.3	98.8	103.2
	n = 18^/6^*	10.00/5.00	81.1	88.0	96.4	97.1
	n= 6^**	CCα^**	NE	NE	91.9	114.6
Repeatability (RSD, %)	n=18^/21^*	1.00^/0.50^*	22.4	24.0	11.2	4.4
		2.00/1.00	22.7	19.2	6.8	6.1
		3.00/1.50	12.9	17.5	7.3	4.4
		4.00/2.00	20.9	11.9	6.7	13.4
	n = 18^/6^*	10.00/5.00	27.7	13.6	7.9	12.1
	n = 6^**	CCα^**	NE	NE	15.3	10.7
Within-lab reproducibility (RSD, %)	n=18^/21^*	2.00^/0.50^*	27.2	22.9	23.8	10.5
		3.00/1.00	22.0	17.6	15.2	17.7
		4.00/1.50	30.2	12.3	10.9	10.9
Decision limit (CCα, μg L⁻¹)			0.44	0.25	0.14	0.08
Detection capability (CCβ, μg L⁻¹)			0.75	0.42	0.24	0.13
Measurement uncertainty at validation level _of ^_{2 μg L}⁻¹_/^*_{1 μg L}⁻¹ (U, k = 2, μg L⁻¹/ %)			0.37/18.5	0.31/15.5	0.20/20.0	0.34/34.0
Matrix effect (ME, %)			NE	NE	7.0	10.0
Standard calibration curve
Slope ± s_b			0.2119 ± 0.0843	0.1058 ± 0.2351	0.2112 ± 0.2456	0.1443 ± 0.0845
y-Intercept ± s_a			0.0812 ± 0.0838	−0.0027 ± 0.0737	0.0570 ± 0.0424	0.0125 ± 0.0096
Correlation coefficient			0.9945	0.9985	0.9866	0.9970
Standard error			0.0662	0.0127	0.0884	0.0285
Matrix matched calibration curve
Slope ± sb			0.1781 ± 0.1220	0.1228 ± 0.0223	0.2516 ± 0.1005	0.1564 ± 0.0554
y-Intercept ± sa			0.1656 ± 0.1199	−0.0102 ± 0.0088	−0.0322 ± 0.0291	−0.0060 ± 0.0103
Correlation coefficient			0.9983	0.9995	0.0291	0.9988
Standard error			0.0426	0.0156	0.0582	0.0148
Additional series of validation^**
Compound	Number of samples	Spiking level (μg L⁻¹)	STAN		16β-OH-STAN
Apparent recovery (%)//	n = 7	0.25	89.4//3.1		102.7//6.7
		0.50	70.2//4.4		108.3//1.8
		0.75	79.8//10.2		101.7//4.5
Repeatability (RSD, %)	n = 4	1.00	80.5//5.5		100.2//4.7
		2.00	86.8//15.9		96.5//3.1
		5.00	81.4//9.0		94.0//2.9
	n = 10	CCα	105.0//4.9		101.3//20.0
Decision limit (CCα, μg L⁻¹)			0.27		0.28
Standard curve: Equation/Correlation coefficient			y = 0.6440x-0.0664/0.9998		y = 0.1570x-0.0168/0.9971
Matrix matched calibration curve: Equation/Correlation coefficient			y = 0.5230x-0.0670/0.9997		y = 0.1453x-0.0033/1.0000

Liquid chromatography-tandem mass spectrometry ion acquisition parameters used for the identification of stanozolol (STAN) and 16β-OH-stanozolol (16β-OH-STAN) using a 16β-OH-stanozolol-D3 internal standard (16β-OH-STAN-D3)

LC-MS/MS (QqQ)
Compound	MRM transition (m/z)	Collision energy CE (V)	Declustering potential DP (V)	Entrance potential EP (V)	Collision cell exit potential CXP (V)	Ion ratio average ± SD	Samples fulfilling the confirmation criteria (%)^*
Compound	MRM transition (m/z)	Collision energy CE (V)	Declustering potential DP (V)	Entrance potential EP (V)	Collision cell exit potential CXP (V)	Ion ratio average ± SD	0.50-5.00 μg L⁻¹	CCα level
STAN	329>121	52	212	10	10	0.346 ± 0.023	98.7	100.0
	329>95	54				0.395 ± 0.019	100.0	100.0
	329>81^a	68				-	-	-
16β-OH-STAN	345>95	61	214	10	10	0.399 ± 0.027	100.0	100.0
16β-OH-STAN	345>81	70	214	10	10	-	-	-
16β-OH-STAN-D3	348>81	72	198	10	5	-	-	-
Additional series of validation						Ion ratio average ± SD	Samples fulfilling the confirmation criteria (%)^**
Additional series of validation						Ion ratio average ± SD	0.25-5.00 μg L⁻¹	CCα level
STAN	329>121		as above			0.218 ± 0.028	100.0	100.0
	329>95					0.273 ± 0.009	100.0	100.0
	329>81^a					-	-	-
16β-OH-STAN	345>95		as above			0.264 ± 0.039	100.0	100.0
16β-OH-STAN	345>8l		as above			-	-	-

Liquid chromatographytandem mass spectrometry methods for determination of stanozolol and l6β-hydroxy-stanozolol in animal urine

Figures & Tables

Fig. 1.

Fig. 2.

Fig. 3.

Validation parameters of the liquid chromatography-tandem triple quadrupole (LC-MS/MS (QqQ) and -tandem ion-trap (LC-MS2 (IT) mass spectrometry methods for the determination of stanozolol (STAN) and its metabolite 16β-OH-stanozolol (16β-OH-STAN) in bovine urine

Liquid chromatography-tandem mass spectrometry ion acquisition parameters used for the identification of stanozolol (STAN) and 16β-OH-stanozolol (16β-OH-STAN) using a 16β-OH-stanozolol-D3 internal standard (16β-OH-STAN-D3)

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