Development of A Liquid Chromatography-Tandem Mass Spectrometry Method for Salivary Cortisol

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Development of A Liquid Chromatography-Tandem
Mass Spectrometry Method for Salivary Cortisol
Can Salivary Cortisol be used in Short Synacthen
Tests?

• Shonagh Haslam
• University Hospital of South Manchester

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Introduction

• Salivary cortisol is minimally invasive.
• Present use for salivary cortisol includes
  screening for Cushings.
• Clinically significant levels in saliva upto
  100 nmol/L. No binding protein.
• Current methods are predominantly immunoassays -
  poor specificity.

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LC-MS/MS Method Development
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LC-MS/MS Method

• 200 µl saliva, std or QC plus 25 µl IS
  (d2-cortisol) added to 96 well plate.
• 50 µl injected onto a C8 guard column for on-line
  sample extraction.
• Analytical separation C18 4µm phenomenex
  column, isocratic elution.
• Transitions (m/z) used for detection
• Cortisol 363.2gt121.1
• d2-cortisol 365.1gt122.2.

Specific for cortisol
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Chromatography
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Linearity
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Precision Profile
LLOQ 2 nmol/L
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Comparison Studies

• LC-MS/MS method compared to RIA. (n 120)

Bias -2.6
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Interference

• 17 structurally related compounds were analysed
  at 1000 nmol/L, including
• Prednisolone
• Cortisone
• Dexamethosone
• No interference - showing the assay is highly
  specific.

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Saliva and Short Synacthen Tests
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Can Salivary Cortisol be used for SSTs?

• 42 Patients were recruited all had suspected
  adrenal pathology.
• SSTs were performed using 250 µg synacthen
  serum and saliva were taken at 0, 30 and 60 mins.
• Considered normal if
• gt550 nmol/L at 30 min and
• gt200 nmol/L increment at 30 min

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Materials and Methods

• Serum cortisol Siemens Centaur IA
• Salivary cortisol LC-MS/MS
• Saliva collected in Sarstedt salivette tubes

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Correlation Between Serum and Saliva All Time
Points
Spearman-Rank correlation r 0.88, p lt0.0001
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Saliva SSTs
Mann-Whitney U test - lt0.05
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Relative Increase
Significant difference with Wilcoxons test p
lt0.0001
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Case - Mrs Y

• Presented with ?adrenal insufficiency

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Conclusions

• Development of a simple and specific method for
  salivary cortisol.
• Salivary cortisol correlates well with serum.
  Abnormal patients tend to have a lower 30 min
  saliva.
• Saliva may confer advantages
• Ease of sample collection
• Useful in patients with CBG deficiency

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Further Work

• Salivary cortisone to be added to LC-MS/MS
  method.
• Salivary cortisol stability studies.
• SST in healthy people for reference range.
• Further uses for Salivary cortisol - IST,
  hydrocortisone day curves.

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Acknowledgments

• Wythenshawe Hospital Biochemistry Dept
• Brian Keevil, Laura Owen
• Christie Hospital Endocrine Dept
• Ilias Perogamvros
• Southampton General Hospital Biochemistry Dept