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

Iwona Matraszek-Źuchowska
Iwona Matraszek-Źuchowska
, 
Alicja Kłopot
Alicja Kłopot
, 
Katarzyna Sielska
Katarzyna Sielska
, 
Beata Korycińska
Beata Korycińska
, 
Sebastian Witek
Sebastian Witek
, 
Paulina Zdonek
Paulina Zdonek
 and 
Piotr Jedziniak
Piotr Jedziniak
   | Jun 16, 2023
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Published Online: Jun 16, 2023
Page range: 275 - 287
Received: Oct 18, 2022
Accepted: May 16, 2023
DOI: https://doi.org/10.2478/jvetres-2023-0030
Keywords
© 2023 Iwona Matraszek-Źuchowska° et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

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

Fig. 2.

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 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

Fig. 3.

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
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 (%)*
1.00-10.00 μg L−1
STAN 329>311a 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
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
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 ± sb 0.2119 ± 0.0843 0.1058 ± 0.2351 0.2112 ± 0.2456 0.1443 ± 0.0845
y-Intercept ± sa 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 (%)*
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>81a 68 - - -
16β-OH-STAN 345>95 61 214 10 10 0.399 ± 0.027 100.0 100.0
345>81 70 - - -
16β-OH-STAN-D3 348>81 72 198 10 5 - - -
Additional series of validation Ion ratio average ± SD Samples fulfilling the confirmation criteria (%)**
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>81a - - -
16β-OH-STAN 345>95 as above 0.264 ± 0.039 100.0 100.0
345>8l - - -
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