UPLC/MSMS in the analysis of physiological steroids. May Anders Feldthus Waters Corporation 1

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1 UPLC/MSMS in the analysis of physiological steroids Anders Feldthus May Waters Corporation 1

2 Clinical Chemistry Mass Spectrometry Citations ( ) 70 Number of Citations Citations in title and abstract (570) Citations in title only (196) Year (02/ /2006) 2012 Waters Corporation 2

3 Introduction to LC/MS measurement of steroids This is one of the fastest growing clinical application area today for LC/MS/MS Selectivity and sensitivity of LC/MS/MS offers the potential for more reliable measurement compared to other detection systems, such as immunoassays Challenges Present at Low levels <ng/ml Matrix interference Lack of commercial calibration materials Sample pre-treatment requirements Reference methods 2012 Waters Corporation 3

4 Testosterone Measurement GC/MS is the Gold Standard LC/MS/MS demonstrates excellent correlation with GC/MS L. Theinpont et al, Clinical Chemistry 54:8, (2008) Widely reported problems with Immunoassays Analysis of levels < 0.3ng/mL (1nM) : CV s >40% Inconsistent analysis for female samples 2012 Waters Corporation 4

5 Testosterone Measurement Normal Ranges Females: ng/ml ( nm) Males : ng/ml ( nm) High Female Testosterone (Hirsutism) Adrenal or ovarian tumours: acne, infertility CAH: virilization Low Testosterone Males Hypogonadism Andropause Paediatric: <0.1ng/mL (0.3 nm) Low volume assay, specialised labs High Volume Low Volume 2012 Waters Corporation 5

6 Recent developments in the field of mass spectrometry have provided the accuracy and sensitivity to evaluate very-low-abundance steroids such as testosterone in female and pediatric patients.. Taieb et al compared 10 commercially available immunoassays with isotope-dilution gas chromatography mass spectrometry (ID-GC/MS) and reached the inescapable conclusion that testosterone immunoassay results for specimens from females are inaccurate. Taieb et al. are the first to show that for every commercially available testosterone assay studied, the values are in error by a factor of 2 on average and in some cases by a factor of almost 5. Are assays that miss target values by % meaningful? Guessing would be more accurate and additionally could provide cheaper and faster testosterone results for females without even having to draw the patient s blood. Laboratory professionals should not be associated with a test where an educated guess would provide an equivalent or better result. Clinical Chemistry 49(8), , Waters Corporation 6

7 Testosterone alone? Multiplexed measurement of steroids 2012 Waters Corporation 7

8 What is congenital adrenal hyperplasia? A group of inherited disorders causing impaired adrenal hormone synthesis CAH can result in decreased circulating concentrations of glucocorticoids and mineralcorticoids, as well as excessive levels of androgens The biochemical picture depends on the underlying enzyme deficiency 2012 Waters Corporation 8

9 Clinical presentations of 21OHD type CAH in females Severe virilizing CAH Severe 21OHD ambiguous genitalia in the newborn Incorrect gender assignment When identified, treatment with steroids usually protects against salt wasting crisis Simple virilizing CAH Less severe 21OHD is not identified until childhood, puberty or adulthood Precocious puberty or accelerated skeletal growth Non-classical CAH Mild 21OHD causing hirsuitism, oligomennorhoea and infertility Many differential diagnoses to exclude e.g. polycystic ovarian syndrome 2012 Waters Corporation 9

10 Clinical presentations of 21OHD in males Classical salt-wasting CAH No genital abnormality so not easily detected in neonates unless in salt-losing crises Severe 21OHD presents at 1 4 weeks of life with failure to thrive, hypotension, vomiting, hyponatraemia with hyperkalaemia Simple virilizing CAH Less severe 21OHD presents later in childhood Precocious puberty, behavioural problems and accelerated skeletal growth 2012 Waters Corporation 10

11 Steroidogenesis* *Courtesy of Wikipedia 2012 Waters Corporation 11

12 21 hydroxylase deficiency Progesterone Aldosterone 21 α hydroxylase 17 α OH Progesterone Cortisol Androgens e.g. androstenedione 2012 Waters Corporation 12

13 21 hydroxylase deficiency Progesterone Aldosterone 21 α hydroxylase + 17 α OH Progesterone Cortisol + Androgens e.g. androstenedione 2012 Waters Corporation 13

14 21 hydroxylase deficiency Progesterone Aldosterone 21 α hydroxylase + 17 α OH Progesterone Cortisol + 21 deoxycortisol + Androgens e.g. androstenedione 2012 Waters Corporation 14

15 Simplified steroidogenic pathway Progesterone Deoxycorticosterone Aldosterone 11 β hydroxylase 17OHP 11-Deoxycortisol Cortisol Androgens e.g. androstenedione 2012 Waters Corporation 15

16 11 β hydroxylase deficiency Progesterone + Deoxycorticosterone Aldosterone 11 β hydroxylase + 17OHP + 11-Deoxycortisol Cortisol 21 deoxycortisol + Androgens e.g. androstenedione 2012 Waters Corporation 16

17 Screening for congenital adrenal hyperplasia Newborn screening for CAH highly important steroid replacement therapy when initiated early enables a substantial reduction in morbidity and mortality Quantification of 17-OHP as a marker Immunoassays Radioimmunoassay ELISA Existing reference ranges are variable and reflect differences in assay technique (with or without solvent extraction) and antibody specificity (cross reactivity) 2012 Waters Corporation 17

18 Diagnostic challenges: false positive test results Specificity of screening for CAH by immunoassays is low and the risk of a false-positive result is high Cross-reactivity of antibodies with steroids other than 17-OHP (metabolites), especially in preterm neonates and in critically ill newborns Premature newborns are often subjected to unnecessary follow up investigations for secondary 17-OHP increases that could be due to stress or physiologically delayed expression of 11-hydroxylase 2012 Waters Corporation 18

19 Neonatal screening in DBS? Earlier diagnosis important, especially in boys, to avoid crisis 2012 Waters Corporation 19

20 Beyond CAH? Many female testosterone requests require follow-up. Androgen index (testo + androstenedione) PCOS? Adrenal insufficiency? Late onset CAH? Multiplexed measurement of steroids? 11-Deoxycortisol 17-Hydroxyprogesterone Testosterone Androstendione DHEAS 2012 Waters Corporation 20

21 ACQUITY UPLC/Xevo TQ-S The application of the Xevo TQ-S mass spectrometer to the measurement of physiological steroids using the Perkin Elmer CHS steroid kit 2012 Waters Corporation 21

22 Example chromatogram (lowest calibrator) 2012 Waters Corporation 22

23 Linearity (8 overlaid 7-point curves and 3 levels of QCs) Compound name: Aldosterone Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 11 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Compound name: Androstendione Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 12 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Compound name: Corticosterone Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 13 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Response Response Response nmol/l Compound name: Cortisol Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 14 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None nmol/l Compound name: 11-Deoxycortisol Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 15 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None nmol/l Compound name: DHEA Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 18 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 30.0 Response Response Response nmol/l 0.0 nmol/l nmol/l Compound name: DHEAS neg Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 16 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Compound name: Progesterone Compound name: 17-alpha hydroxyprogesterone Correlation coefficient: r = , r^2 = Correlation coefficient: r = , r^2 = Calibration curve: * x Calibration curve: * x Response type: Internal Std ( Ref 17 ), Area * ( IS Conc. / IS Area ) Response type: Internal Std ( Ref 18 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: NoneCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Compound name: Testosterone Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 19 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Response Response Response Response nmol/l 0.0 nmol/l 0.0 nmol/l 0.0 nmol/l Waters Corporation 23

24 Linearity and sensitivity Overlay of 8 calibration curves, for the full concentration ranges yield coefficients of determination of: Steroid Lowest cal (nmol/l) Highest Cal (nmol/l) r^2 S:N ratio on lowest cal Aldosterone > :1 Androstendione > :1 Corticosterone > :1 Cortisol > :1 11-Deoxycortisol > :1 DHEA > :1 DHEAS > :1 Progesterone > :1 17-alpha hydroxyprogesterone > :1 Testosterone > : Waters Corporation 24

25 Steroidogenesis* *Courtesy of Wikipedia 2012 Waters Corporation 25

26 Practical application of kit Presented at DGE, March Waters Corporation 26

27 Panel of 5 for PCOS Testosterone and androstendione Androgen profile supports virilisation observation 17-OHP and 11-deoxycortisol Exclude late onset CAH DHEAS Adrenal/Ovarian tumour Adrenal insufficiency 2012 Waters Corporation 27

28 Method comparison against in-house RIA 2012 Waters Corporation 28

29 Conclusions Steroid analysis can be done by UPLC/MSMS Robust Simple sample pretreatment Better accuracy less interference Multiplexed measurement o Greater diagnostic certainty o Simpler requesting fewer repeat requests Commercial standards o Better standardisation o Easier troubleshooting o Better support Versatile platform 2012 Waters Corporation 29

30 Thank you! Any Questions? 2012 Waters Corporation 30