UPLC/MSMS in the analysis of physiological steroids Anders Feldthus Anders_Feldthus@Waters.com May 2012 2012 Waters Corporation 1
Clinical Chemistry Mass Spectrometry Citations (1955-2006) 70 Number of Citations 60 50 40 30 20 Citations in title and abstract (570) Citations in title only (196) 10 0 1955 1958 1961 1964 1967 1972 1975 1978 1981 1984 1987 1990 1993 1996 1999 2002 2005 Year (02/1955-10/2006) 2012 Waters Corporation 2
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
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, 1290 1297 (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
Testosterone Measurement Normal Ranges Females: 0.08-0.6 ng/ml (0.24-1.8 nm) Males : 2.4-9.5 ng/ml (7.2-28.5 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
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 200 500% 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), 1250-1, 2003. 2012 Waters Corporation 6
Testosterone alone? Multiplexed measurement of steroids 2012 Waters Corporation 7
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
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
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
Steroidogenesis* *Courtesy of Wikipedia 2012 Waters Corporation 11
21 hydroxylase deficiency Progesterone Aldosterone 21 α hydroxylase 17 α OH Progesterone Cortisol Androgens e.g. androstenedione 2012 Waters Corporation 12
21 hydroxylase deficiency Progesterone Aldosterone 21 α hydroxylase + 17 α OH Progesterone Cortisol + Androgens e.g. androstenedione 2012 Waters Corporation 13
21 hydroxylase deficiency Progesterone Aldosterone 21 α hydroxylase + 17 α OH Progesterone Cortisol + 21 deoxycortisol + Androgens e.g. androstenedione 2012 Waters Corporation 14
Simplified steroidogenic pathway Progesterone Deoxycorticosterone Aldosterone 11 β hydroxylase 17OHP 11-Deoxycortisol Cortisol Androgens e.g. androstenedione 2012 Waters Corporation 15
11 β hydroxylase deficiency Progesterone + Deoxycorticosterone Aldosterone 11 β hydroxylase + 17OHP + 11-Deoxycortisol Cortisol 21 deoxycortisol + Androgens e.g. androstenedione 2012 Waters Corporation 16
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
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
Neonatal screening in DBS? Earlier diagnosis important, especially in boys, to avoid crisis 2012 Waters Corporation 19
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
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
Example chromatogram (lowest calibrator) 2012 Waters Corporation 22
Linearity (8 overlaid 7-point curves and 3 levels of QCs) Compound name: Aldosterone Correlation coefficient: r = 0.999677, r^2 = 0.999355 Calibration curve: 0.682043 * x + 0.00654052 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 = 0.999838, r^2 = 0.999675 Calibration curve: 0.605509 * x + 0.00262052 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 = 0.999358, r^2 = 0.998717 Calibration curve: 0.188787 * x + -0.00745606 Response type: Internal Std ( Ref 13 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 30.0 30.0 Response 10.0 5.0 Response 20.0 10.0 Response 20.0 10.0 0.0 0.0 5.0 10.0 15.0 20.0 nmol/l Compound name: Cortisol Correlation coefficient: r = 0.999617, r^2 = 0.999234 Calibration curve: 0.0339563 * x + 0.00774359 Response type: Internal Std ( Ref 14 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 0.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 nmol/l Compound name: 11-Deoxycortisol Correlation coefficient: r = 0.999140, r^2 = 0.998281 Calibration curve: 0.247126 * x + 0.0126426 Response type: Internal Std ( Ref 15 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 0.0 0 25 50 75 100 125 150 175 nmol/l Compound name: DHEA Correlation coefficient: r = 0.997390, r^2 = 0.994787 Calibration curve: 0.00692836 * x + -0.00609587 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 20.0 10.0 Response 10.0 5.0 Response 1.50 1.00 0.50 0.0 0 100 200 300 400 500 600 700 800 nmol/l 0.0 nmol/l 0.0 10.0 20.0 30.0 40.0 50.0 0.00 nmol/l 0 50 100 150 200 250 Compound name: DHEAS neg Correlation coefficient: r = 0.999556, r^2 = 0.999113 Calibration curve: 0.00199494 * x + 0.0129063 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 = 0.998980, r^2 = 0.997962 Correlation coefficient: r = 0.998220, r^2 = 0.996442 Calibration curve: 1.04123 * x + -0.00904292 Calibration curve: 0.440317 * x + 0.00113332 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 = 0.999759, r^2 = 0.999518 Calibration curve: 0.926357 * x + 0.0148422 Response type: Internal Std ( Ref 19 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Response 15.0 10.0 5.0 Response 80.0 60.0 40.0 20.0 Response 30.0 20.0 10.0 Response 20.0 10.0 0.0 nmol/l 0.0 nmol/l 0.0 nmol/l 0.0 nmol/l 0 2000 4000 6000 0.0 20.0 40.0 60.0 0.0 20.0 40.0 60.0 0.0 5.0 10.0 15.0 20.0 25.0 2012 Waters Corporation 23
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 0.087 21.50 >0.999 20:1 Androstendione 0.290 63.60 >0.999 627:1 Corticosterone 0.844 190.00 >0.998 94:1 Cortisol 3.960 853.00 >0.999 185:1 11-Deoxycortisol 0.190 50.60 >0.998 95:1 DHEA 1.290 254.00 >0.994 10:1 DHEAS 34.300 7564.00 >0.999 469:1 Progesterone 0.364 77.20 >0.997 227:1 17-alpha hydroxyprogesterone 0.376 70.70 >0.996 250:1 Testosterone 0.113 27.80 >0.999 30:1 2012 Waters Corporation 24
Steroidogenesis* *Courtesy of Wikipedia 2012 Waters Corporation 25
Practical application of kit Presented at DGE, March 2012 2012 Waters Corporation 26
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
Method comparison against in-house RIA 2012 Waters Corporation 28
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
Thank you! Any Questions? 2012 Waters Corporation 30