Determination of cortisol and cortisone in human mother’s milk.

Bibian van der Voorn

DISCLOSURE

PAOKC – nov 2015

(Potentiële) Belangenverstrengeling Geen

Very preterm birth (gestational age<32 weeks)

and the HPA axis

Extra-uterine life with immature HPA-axis 1

• Inadequate CRH secretion• ↓ activity 11β hydroxylase• Cortisol-cortisone

interconversion favors cortisone

Human mother’s milk benefits:

• ↓ mortality and morbidity• Dynamic composition

1. Bolt 2002 Pediatric Research & Clin. Endocrinol.

Glucocorticoids have been found in mother’s milk

Van der Voorn 2015 Clin. Chim. Acta

Aim: to develop a valid LC–MS/MS method to determine cortisol and cortisone in mother's milk.

Validation:• Intra- and inter-assay CV • Recovery• Linearity• LLOQ

Stability • Different storing conditions• Freeze-thaw cycles

Physiological variation

Theoretical background

Milk glucocorticoids are mainly:

• bound to CBG and albumin 1, 2

• found in the unconjugated phase 3

Glucocorticoids are very stable in other matrices 4

1. Kulski & Hartmann 1981 Aust J Exp Biol Med Sci

2. Tucker & Schwalm 1977 J of Animal Science

3. Sahlberg & Axelson 1986 J of steroid biochemistry

4. Florey 1983 BOOK: Profiles of drug substances,

excipients and related methodology.

Method measurement of milk cortisol and cortisone

Donor mother's milk from 13 healthy mothers • Donated between 8 to 28 weeks postpartum

• Stored in polypropylene vials at −20 °C

Preparation 200 μL milk:• Addition of 2H4 labeled cortisol and 2H8 labeled cortisone • 3x washing with hexane to remove undesired lipids

40 μL of washed milk purified by online SPE

LC-MS/MS analysis: • By a Quattro Premier XE tandem mass spectrometer, operating

in the electrospray positive ionization mode

Cortisol

concentration (nmol/L)

N Intra-assay

CV %

Cortisone

concentration (nmol/L)

N Intra-assay

CV %

0.56 13 13% 3.1 10 7%

3.3 38 8% 7.7 8 5%

7.0 77 4% 15.0 36 6%

23.0 48 5% 33.0 74 5%

Cortisol

concentration (nmol/L)

N Inter-assay

CV %

Cortisone

concentration (nmol/L)

N Inter-assay

CV %

6.3 18 9% 31.9 16 9%

22.0 5 4%

Intra- assay CV%

Inter-assay CV%

Recovery (of spiked analytes)

• 97–102% for cortisol

• 98–106% for cortisone

Linearity (by 2-, 4- and 8-fold dilutions)

• 93–106% for cortisol

• 97–106% for cortisone

LLOQ (allowable intra-assay CV of 15%)

• 0.5 nmol/L for cortisol

• 0.25 nmol/L for cortisone

Stability of human milk cortisol and cortisone

Stability of human milk cortisol and cortisone

Stability of milk cortisol and cortisone

Pilot preterm vs term milk

(nm

ol/

L)

1. Primary outcome:

Concentrations are lower in preterm milk

2. Post-hoc analysis:

Concentrations are associated with time of collection

Healthy term mothers:

10 paired milk and saliva samples during 24h

Pearson correlation and lineair regressionLongitudinal data analysis: GEE

Aim : exploring physiological variation in breast-milk glucocorticoid concentrations

Diurnal variation milk glucocorticoids

(hour)(hour)

Correlation breast-milk, saliva glucocorticoid levels

Slope 1.97Intercept -1.71R 0.92

Slope 1.03Intercept 4.64R 0.93

Physiological variation in breast-milk glucocorticoid concentrations

Breast-milk glucocorticoïd levelsCortisol: 0.2 – 97.4 nmol/L Cortisone: 1.6 – 95.0 nmol/L

Diurnal rhythmicity in breast-milk glucocorticoïdconcentrations (p≤0.001)

Cortisol and cortisone levels in breast milk and saliva are highly correlated (p≤0.001).

Conclusion

Reliable LC-MS/MS method to measurebreast-milk glucocorticoïd levels

Good

• Intra- and inter-assay CV

• Recovery

• Linearity

• LLOQ, in respect to the physiological ranges

• Stability under different storing conditions

Physiological ranges based on 141 samples of 39 mothers

• Reference ranges per time moment?

• Breast-milk glucocorticoid levels in pathological cases?

Acknowledgements

Annemieke C. Heijboer Department of Clinical ChemistryFrans Martens Department of Clinical Chemistry

Joost Rotteveel Department of PediatricsMartijn J.J. Finken Department of Pediatrics

VU University Medical Center, Amsterdam

Cortisol and cortisone in breast milk

• Cortisol: 0.2 – 97.4 nmol/L• Cortison: 1.6 – 95.0 nmol/L

Mean values (nmol/L)

Brook’s Clinical

Pediatric Endocrinology

6th edition, 2009

Correlation breast-milk, saliva glucocorticoid levelsBland-Altman plot

0 2 0 4 0 6 0

-1 0

0

1 0

2 0

3 0

C o rtis o n e c o n c . in s a liv a (n m o l/L )D

iffe

ren

ce

be

twe

en

mil

k a

nd

sa

liv

a (

nm

ol/

L)

m e a n

-1  S D

+ 1 S D

0 5 1 0 1 5 2 0 2 5

-1 0

0

1 0

2 0

3 0

C o rtis o l c o n c . in s a liv a (n m o l/L )

Re

sid

ua

ls b

rea

st

mil

k (

nm

ol/

L)

m e a n

-1  S D

+ 1  S D