Determination of cortisol and cortisone in human mother’s ... - The Matrix - vd Voorn.pdf ·...
Transcript of Determination of cortisol and cortisone in human mother’s ... - The Matrix - vd Voorn.pdf ·...
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Determination of cortisol and cortisone in human mother’s milk.
Bibian van der Voorn
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DISCLOSURE
PAOKC – nov 2015
(Potentiële) Belangenverstrengeling Geen
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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.
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Glucocorticoids have been found in mother’s milk
Van der Voorn 2015 Clin. Chim. Acta
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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
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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.
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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
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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%
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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
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Stability of human milk cortisol and cortisone
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Stability of human milk cortisol and cortisone
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Stability of milk cortisol and cortisone
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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
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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
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Diurnal variation milk glucocorticoids
(hour)(hour)
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Correlation breast-milk, saliva glucocorticoid levels
Slope 1.97Intercept -1.71R 0.92
Slope 1.03Intercept 4.64R 0.93
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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).
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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?
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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
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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
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Correlation breast-milk, saliva glucocorticoid levelsBland-Altman plot
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