Liquid Chromatography-Tandem Mass Spectrometry in Studies of Steroid Hormones and Steroid Glucuronide Conjugates in Brain and Urine

Sirkku Jäntti

Tutkimustuotos: OpinnäyteVäitöskirja

Kuvaus

Steroids are endogenous compounds, which are present in the body tissues and fluids in free and conjugated forms, e.g. as steroid glucuronide conjugates. First goal of the study was to develop methodologies for the synthesis of steroid glucuronides. An enzyme-assisted synthesis was carried out to produce glucuronide-conjugated steroids at milligram levels. Hepatic microsomal preparations of bovine, porcine, and Arochlor-induced rats were compared with respect to specificity and efficiency of uridine diphosphate glucuronosyl transferase (UGT) enzymes in steroid glucuronidation. Both bovine and porcine liver microsomes efficiently produced the steroid glucuronides of all eight steroids studied, whereas rat liver microsomes produced glucuronides efficiently only for three steroids. Synthesized steroid glucuronides were purified with solid-phase extraction (SPE) or LC fractionation and characterized by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution MS. Steroid glucuronides were obtained in milligram amounts with good yields. The synthesised glucuronides were used in method development and as reference material in the analysis.

As a second goal of the study, two methods were developed, one for brain samples using capillary liquid chromatography-electrospray-tandem mass spectrometry (CapLC-ESI-MS/MS) and the other for the urine samples using ultra performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS).The methods were carefully optimized to obtain good selectivity and maximum sensitivity. Linear range of 3-4 magnitudes was obtained (R2> 0.996) with good precision (RSD < 15%). Detection limits of 6-100 pmol/l were obtained for steroid glucuronides, 10-30 pmol/l for steroid sulphates and 0.03-22 nmol/l for free steroids, respectively. The developed methods were applied to the analysis of mouse brain extracts, to study in vitro metabolism of steroids in rat and mouse brain, and to the analysis of free and glucuronide conjugated steroids during pregnancy.

Using the CapLC-MS/MS method steroid glucuronides were observed for the first time in brain. The UPLC-MS/MS method employed in determination of urinary profiles of steroids and steroid glucuronide conjugates steroids during pregnancy. The concentrations of 36 targeted steroids and steroid glucuronides as well as the mostly unidentified C21-steroid glucuronides clearly altered during the pregnancy. In general, the concentrations of the steroids and steroid glucuronides gradually increased during pregnancy, decreased rapidly just before or during delivery, and returned to control sample level five days after the delivery.
Alkuperäiskielienglanti
JulkaisupaikkaHelsinki
Kustantaja
Painoksen ISBN978-952-10-9191-9
Sähköinen ISBN978-952-10-9192-6
TilaJulkaistu - 2013
OKM-julkaisutyyppiG5 Tohtorinväitöskirja (artikkeli)

Tieteenalat

  • 317 Farmasia

Lainaa tätä

@phdthesis{9d3f3b96d8d4469a9bb17140c274bd38,
title = "Liquid Chromatography-Tandem Mass Spectrometry in Studies of Steroid Hormones and Steroid Glucuronide Conjugates in Brain and Urine",
abstract = "Steroids are endogenous compounds, which are present in the body tissues and fluids in free and conjugated forms, e.g. as steroid glucuronide conjugates. First goal of the study was to develop methodologies for the synthesis of steroid glucuronides. An enzyme-assisted synthesis was carried out to produce glucuronide-conjugated steroids at milligram levels. Hepatic microsomal preparations of bovine, porcine, and Arochlor-induced rats were compared with respect to specificity and efficiency of uridine diphosphate glucuronosyl transferase (UGT) enzymes in steroid glucuronidation. Both bovine and porcine liver microsomes efficiently produced the steroid glucuronides of all eight steroids studied, whereas rat liver microsomes produced glucuronides efficiently only for three steroids. Synthesized steroid glucuronides were purified with solid-phase extraction (SPE) or LC fractionation and characterized by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution MS. Steroid glucuronides were obtained in milligram amounts with good yields. The synthesised glucuronides were used in method development and as reference material in the analysis. As a second goal of the study, two methods were developed, one for brain samples using capillary liquid chromatography-electrospray-tandem mass spectrometry (CapLC-ESI-MS/MS) and the other for the urine samples using ultra performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS).The methods were carefully optimized to obtain good selectivity and maximum sensitivity. Linear range of 3-4 magnitudes was obtained (R2> 0.996) with good precision (RSD < 15{\%}). Detection limits of 6-100 pmol/l were obtained for steroid glucuronides, 10-30 pmol/l for steroid sulphates and 0.03-22 nmol/l for free steroids, respectively. The developed methods were applied to the analysis of mouse brain extracts, to study in vitro metabolism of steroids in rat and mouse brain, and to the analysis of free and glucuronide conjugated steroids during pregnancy. Using the CapLC-MS/MS method steroid glucuronides were observed for the first time in brain. The UPLC-MS/MS method employed in determination of urinary profiles of steroids and steroid glucuronide conjugates steroids during pregnancy. The concentrations of 36 targeted steroids and steroid glucuronides as well as the mostly unidentified C21-steroid glucuronides clearly altered during the pregnancy. In general, the concentrations of the steroids and steroid glucuronides gradually increased during pregnancy, decreased rapidly just before or during delivery, and returned to control sample level five days after the delivery.",
keywords = "317 Pharmacy",
author = "Sirkku J{\"a}ntti",
year = "2013",
language = "English",
isbn = "978-952-10-9191-9",
series = "Dissertationes Biocentri Viikki Universitatis Helsingiensis",
publisher = "University of Helsinki",
number = "19/2013",
address = "Finland",

}

Liquid Chromatography-Tandem Mass Spectrometry in Studies of Steroid Hormones and Steroid Glucuronide Conjugates in Brain and Urine. / Jäntti, Sirkku.

Helsinki : University of Helsinki, 2013. 118 s.

Tutkimustuotos: OpinnäyteVäitöskirja

TY - THES

T1 - Liquid Chromatography-Tandem Mass Spectrometry in Studies of Steroid Hormones and Steroid Glucuronide Conjugates in Brain and Urine

AU - Jäntti, Sirkku

PY - 2013

Y1 - 2013

N2 - Steroids are endogenous compounds, which are present in the body tissues and fluids in free and conjugated forms, e.g. as steroid glucuronide conjugates. First goal of the study was to develop methodologies for the synthesis of steroid glucuronides. An enzyme-assisted synthesis was carried out to produce glucuronide-conjugated steroids at milligram levels. Hepatic microsomal preparations of bovine, porcine, and Arochlor-induced rats were compared with respect to specificity and efficiency of uridine diphosphate glucuronosyl transferase (UGT) enzymes in steroid glucuronidation. Both bovine and porcine liver microsomes efficiently produced the steroid glucuronides of all eight steroids studied, whereas rat liver microsomes produced glucuronides efficiently only for three steroids. Synthesized steroid glucuronides were purified with solid-phase extraction (SPE) or LC fractionation and characterized by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution MS. Steroid glucuronides were obtained in milligram amounts with good yields. The synthesised glucuronides were used in method development and as reference material in the analysis. As a second goal of the study, two methods were developed, one for brain samples using capillary liquid chromatography-electrospray-tandem mass spectrometry (CapLC-ESI-MS/MS) and the other for the urine samples using ultra performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS).The methods were carefully optimized to obtain good selectivity and maximum sensitivity. Linear range of 3-4 magnitudes was obtained (R2> 0.996) with good precision (RSD < 15%). Detection limits of 6-100 pmol/l were obtained for steroid glucuronides, 10-30 pmol/l for steroid sulphates and 0.03-22 nmol/l for free steroids, respectively. The developed methods were applied to the analysis of mouse brain extracts, to study in vitro metabolism of steroids in rat and mouse brain, and to the analysis of free and glucuronide conjugated steroids during pregnancy. Using the CapLC-MS/MS method steroid glucuronides were observed for the first time in brain. The UPLC-MS/MS method employed in determination of urinary profiles of steroids and steroid glucuronide conjugates steroids during pregnancy. The concentrations of 36 targeted steroids and steroid glucuronides as well as the mostly unidentified C21-steroid glucuronides clearly altered during the pregnancy. In general, the concentrations of the steroids and steroid glucuronides gradually increased during pregnancy, decreased rapidly just before or during delivery, and returned to control sample level five days after the delivery.

AB - Steroids are endogenous compounds, which are present in the body tissues and fluids in free and conjugated forms, e.g. as steroid glucuronide conjugates. First goal of the study was to develop methodologies for the synthesis of steroid glucuronides. An enzyme-assisted synthesis was carried out to produce glucuronide-conjugated steroids at milligram levels. Hepatic microsomal preparations of bovine, porcine, and Arochlor-induced rats were compared with respect to specificity and efficiency of uridine diphosphate glucuronosyl transferase (UGT) enzymes in steroid glucuronidation. Both bovine and porcine liver microsomes efficiently produced the steroid glucuronides of all eight steroids studied, whereas rat liver microsomes produced glucuronides efficiently only for three steroids. Synthesized steroid glucuronides were purified with solid-phase extraction (SPE) or LC fractionation and characterized by nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution MS. Steroid glucuronides were obtained in milligram amounts with good yields. The synthesised glucuronides were used in method development and as reference material in the analysis. As a second goal of the study, two methods were developed, one for brain samples using capillary liquid chromatography-electrospray-tandem mass spectrometry (CapLC-ESI-MS/MS) and the other for the urine samples using ultra performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS).The methods were carefully optimized to obtain good selectivity and maximum sensitivity. Linear range of 3-4 magnitudes was obtained (R2> 0.996) with good precision (RSD < 15%). Detection limits of 6-100 pmol/l were obtained for steroid glucuronides, 10-30 pmol/l for steroid sulphates and 0.03-22 nmol/l for free steroids, respectively. The developed methods were applied to the analysis of mouse brain extracts, to study in vitro metabolism of steroids in rat and mouse brain, and to the analysis of free and glucuronide conjugated steroids during pregnancy. Using the CapLC-MS/MS method steroid glucuronides were observed for the first time in brain. The UPLC-MS/MS method employed in determination of urinary profiles of steroids and steroid glucuronide conjugates steroids during pregnancy. The concentrations of 36 targeted steroids and steroid glucuronides as well as the mostly unidentified C21-steroid glucuronides clearly altered during the pregnancy. In general, the concentrations of the steroids and steroid glucuronides gradually increased during pregnancy, decreased rapidly just before or during delivery, and returned to control sample level five days after the delivery.

KW - 317 Pharmacy

M3 - Doctoral Thesis

SN - 978-952-10-9191-9

T3 - Dissertationes Biocentri Viikki Universitatis Helsingiensis

PB - University of Helsinki

CY - Helsinki

ER -

Jäntti S. Liquid Chromatography-Tandem Mass Spectrometry in Studies of Steroid Hormones and Steroid Glucuronide Conjugates in Brain and Urine. Helsinki: University of Helsinki, 2013. 118 s. (Dissertationes Biocentri Viikki Universitatis Helsingiensis; 19/2013).