uplc/msms in the analysis of physiological steroids · ©2012 waters corporation 1 uplc/msms in the...

©2012 Waters Corporation 1

UPLC/MSMS in the analysis of physiological steroids

Anders Feldthus

[email protected]

May 2012


Clinical Chemistry Mass Spectrometry Citations (1955 - 2006)

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Citations in title only (196)

Citations in title and abstract (570)


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

Introduction to LC/MS measurement of steroids


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


Low

Volu

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Hig

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olum

e

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


Clinical Chemistry 49(8), 1250-1, 2003.

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.


Testosterone alone? Multiplexed measurement of steroids


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


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


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


Steroidogenesis*

*Courtesy of Wikipedia


21 hydroxylase deficiency

Progesterone

17 α OH Progesterone

21 α hydroxylase

Aldosterone

Cortisol

Androgens e.g. androstenedione



Progesterone

+ 17 α OH Progesterone

21 α hydroxylase

↓ Aldosterone

↓ Cortisol

+ Androgens e.g. androstenedione



Progesterone

+ 17 α OH Progesterone

21 α hydroxylase

↓ Aldosterone

↓ Cortisol


+ 21 deoxycortisol


Simplified steroidogenic pathway

Progesterone

17OHP

Aldosterone

Cortisol

Androgens e.g. androstenedione

Deoxycorticosterone

11-Deoxycortisol

11 β hydroxylase


11 β hydroxylase deficiency

Progesterone

+ 17OHP

↓ Aldosterone

↓ Cortisol


+ Deoxycorticosterone

+ 11-Deoxycortisol

11 β hydroxylase

21 deoxycortisol


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)


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

Diagnostic challenges: false positive test results


Neonatal screening in DBS?

Earlier diagnosis important, especially in boys, to avoid crisis


•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

Beyond CAH?


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


Example chromatogram (lowest calibrator)


Linearity (8 overlaid 7-point curves and 3 levels of QCs)

Compound name: AldosteroneCorrelation coefficient: r = 0.999677, r^2 = 0.999355Calibration curve: 0.682043 * x + 0.00654052Response type: Internal Std ( Ref 11 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0.0 5.0 10.0 15.0 20.0

Res

pons

e

0.0

5.0

10.0

Compound name: AndrostendioneCorrelation coefficient: r = 0.999838, r^2 = 0.999675Calibration curve: 0.605509 * x + 0.00262052Response type: Internal Std ( Ref 12 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0.0 10.0 20.0 30.0 40.0 50.0 60.0

Res

pons

e

0.0

10.0

20.0

30.0

Compound name: CorticosteroneCorrelation coefficient: r = 0.999358, r^2 = 0.998717Calibration curve: 0.188787 * x + -0.00745606Response type: Internal Std ( Ref 13 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0 25 50 75 100 125 150 175

Res

pons

e

0.0

10.0

20.0

30.0

Compound name: CortisolCorrelation coefficient: r = 0.999617, r^2 = 0.999234Calibration curve: 0.0339563 * x + 0.00774359Response type: Internal Std ( Ref 14 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0 100 200 300 400 500 600 700 800

Res

pons

e

0.0

10.0

20.0

30.0

Compound name: 11-DeoxycortisolCorrelation coefficient: r = 0.999140, r^2 = 0.998281Calibration curve: 0.247126 * x + 0.0126426Response type: Internal Std ( Ref 15 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0.0 10.0 20.0 30.0 40.0 50.0

Res

pons

e

0.0

5.0

10.0

Compound name: DHEACorrelation coefficient: r = 0.997390, r^2 = 0.994787Calibration curve: 0.00692836 * x + -0.00609587Response type: Internal Std ( Ref 18 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0 50 100 150 200 250

Res

pons

e

0.00

0.50

1.00

1.50

Compound name: DHEAS negCorrelation coefficient: r = 0.999556, r^2 = 0.999113Calibration curve: 0.00199494 * x + 0.0129063Response type: Internal Std ( Ref 16 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0 2000 4000 6000

Res

pons

e

0.0

5.0

10.0

15.0

Compound name: ProgesteroneCorrelation coefficient: r = 0.998980, r^2 = 0.997962Calibration curve: 1.04123 * x + -0.00904292Response type: Internal Std ( Ref 17 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0.0 20.0 40.0 60.0

Res

pons

e

0.0

20.0

40.0

60.0

80.0

Compound name: 17-alpha hydroxyprogesteroneCorrelation coefficient: r = 0.998220, r^2 = 0.996442Calibration curve: 0.440317 * x + 0.00113332Response type: Internal Std ( Ref 18 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0.0 20.0 40.0 60.0

Res

pons

e

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10.0

20.0

30.0

Compound name: TestosteroneCorrelation coefficient: r = 0.999759, r^2 = 0.999518Calibration curve: 0.926357 * x + 0.0148422Response type: Internal Std ( Ref 19 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

nmol/L0.0 5.0 10.0 15.0 20.0 25.0

Res

pons

e0.0

10.0

20.0


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


Steroidogenesis*

*Courtesy of Wikipedia


Practical application of kit

Presented at DGE, March 2012


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


Method comparison against in-house RIA


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


Thank you!

Any Questions?

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