The analysis of Glucocorticoid Steroids in Plasma, Urine and Saliva by UPLC/MS/MS

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1 The analysis of Glucocorticoid Steroids in Plasma, Urine and Saliva by UPLC/MS/MS Brett McWhinney, Supervising Scientist, HPLC Section, Pathology Central, Pathology Queensland

2 Overview 1. Overview of Pathology Queensland 2. Glucorticoid production 3. Cushing s Syndrome 4. Testing and diagnosis 5. Case History

3 1. Overview of Pathology Queensland

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6 Pathology Queensland Specimens / Day Patients / Day 1800 Staff 33 Laboratories Reports Printed / Day

7 Distance Covering 1,727,000 square kilometres, it is Australia s second largest State. It represents more than a quarter of the country s total area with a current population of over three million people. To put Queensland in perspective, it s more than seven times the size of the United Kingdom, more than four and half time the size of Japan, around six and half times the size of New Zealand, more than five times the size of Texas.

8 AUSLAB State-wide Laboratory and Reporting System Largest public pathology network in Southern Hemisphere On every hospital clinical computer in Queensland Health

9 2. Glucorticoid Production

10 Any of a group of steroid hormones, such as cortisol, that are produced by the adrenal cortex, are involved in carbohydrate, protein, and fat metabolism, and have anti-inflammatory properties

11 Adrenal glands

12 Adrenal glands Functionally consist of three tissues under independent control. Outer cortex (ZG), mainly controlled by the reninangiotensin system regulates release of aldosterone (sodium homeostasis). Inner cortex (ZF&R), mainly controlled by the CRH - ACTH system regulates the responses to stress via the actions of cortisol. Also produces adrenal androgens. Medulla, which is part of the sympathetic nervous system produces adrenaline & noradrenaline.

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20 3. Cushing s Syndrome

21 CUSHING S SYNDROME large group of signs and symptoms chronic exposure to excessive concentrations of glucocorticoids challenging diagnosis Adrenal tumour, Pituitary tumour (ACTH) or Ectopic ACTH production

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25 4. Testing and Diagnosis

26 Diagnosis Disease suggested by signs and symptoms Many non-specific None are truly characteristic of Cushing s Must confirm findings with biochemical tests Medications Glucocorticoids injested, inhaled, topical Factitious

27 Cortisol Assay 1 Tandem mass spectroscopy gold standard method lab logistical issues assay frequency, availability of instrumentation Immunoassay readily available for cortisol assay issues cross-reactivity, binding protein interference Random access and run 24/7

28 Cortisol Assay 2 Other issues biological variability diurnal variation plus episodic secretion total vs free >95% circulates bound to CBG and albumin nonactive free - biologically active

29 Recommendations for Initial Testing Urinary Free Cortisol (UFC) x 2 ~midnight salivary cortisol 1 mg o/n dexamethasone suppression test (DST) Longer low-dose DST (2 mg/d for 48 hrs. Extended Liddles test

30 Adrenal Insufficiency (Addison s disease) tiredness, weakness, lethargy anorexia, nausea, vomiting weight loss BP, postural hypotension, dizziness loss of body hair pigmentation

31 Conclusion Syndromes due to cortisol excess or deficiency cause significant morbidity and mortality and may be difficult to diagnose. Modulation of hypothalamic-pituitary-adrenal feedback with synthetic hormone analogues is often required for diagnosis. Synacthen is most commonly used to stimulate the HPA axis and dexamethasone to suppress.

32 5. Overview of UPLC MS/MS Method

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34 Clinical states requiring multianalyte analysis Metyrapone Cortisol and 11 deoxycortisol Cushing s Sydrome Cortisone and Cortisol Congenital Adrenal Hyperplasia (CAH) Cortisol and 11 deoxycortisol Dexamethasone Suppression Test Cortisol and Dexamethasone Fictious Cushing s Syndrome Cortisol, Cortisone, Prednisolone, Methyl Prednisolone and Dexamethasone

35 Advantages of UPLC MS/MS Multi component analysis Sensitivity (LOQ < 1nmol/L) Small sample volume (200 ul) Minimal sample prep, commonality across matrices High throughput (Cycle time 4 min) Specificity

36 Sample preparation 1 Plasma total steroids Incubated with 1M HCl for 15 min to release from binding proteins Plasma free steroids Ultrafiltration under temperature controlled conditions Urine Saliva

37 Sample preparation 2 200ul sample + Internal standards + water Extracted using 50mg bed OASIS HLB SPE columns Washed and eluted with ethyl acetate Dried under air and reconstituted in Mobile phase Inject 20ul

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39 Liquid chromatography solvent gradients for the UPLC MS/MS method. Table 4. Method comparison with ULC MS/MS (linear regression). Urine and plasma free Plasma total Time %A %B Curve Time %A %B Curve Initial Initial u urine, p - plasma Time measured in minutes, Curve 6 refers to a linear change between initial and final conditions A: 2 mmol/l ammonium acetate in water with 0.1% formic acid B: 2 mmol/l ammonium acetate in methanol with 0.1% formic acid

40 Compound MRM Dwell time Cone V Collision energy Cortisol 363.1> s 32 V 25 ev Cortisone 361.1> s 32 V 25 ev Fludrocortisone 381.1> s 35 V 25 ev Prednisolone 361.1> s 18 V 20 ev 11-deoxycortisol 347.1> s 32 V 25 ev Dexamethasone 373.1> s 25 V 25 ev Mass spectrometer conditions: Capillary charge 0.6 kv, source temperature 120 C, desolvation temperature 450 C, desolvation gas flow 800 L/h, cone gas flow 50 L/h and collision cell gas flow 0.15 ml/min.

41 Imprecision results for urine, free plasma and total plasma glucocorticoids Intra-assay Inter-assay Mean nmol/l CV % Mean nmol/l CV % u-cortisol u-cortisone u-prednisolone pf-cortisol pf-cortisone p-cortisol p-cortisone p-11-deoxycortisol p-dexamethasone p-prednisolone u urine, pf- plasma ultrafiltrate, p - plasma

42 Method comparison with UPLC MS/MS (linear regression) Compare slope intercept R 2 n u-cortisol HPLC u-cortisone HPLC p-cortisol Beckman p-cortisol HPLC p-cortisone HPLC p-11-deoxycortisol HPLC u urine, p - plasma

43 6. Case History

44 36y Female referred from gynaecologist Weight gain, mood swings, rounded face and hirsuitism UFC 945 nmol/24hrs (< 150) Plasma Cortisol 850 nmol/l (< 750) DST basal cortisol 850 to 156 nmol/l (<50) Dexamethasone 8 nmol/l (> 3) Cyclical Cushing s UFC ( nmol/24hrs) Disruption of usual circadian rhythm, raised midnight serum and salivary cortisol IPSS revealed a pituitary source of ACTH Patient underwent transphenoidal pituitary surgery and UFC and plasma cortisol returned to normal ranges