Malaysian Journal of Analytical Sciences Vol 23 No 2 (2019): 336 - 344

DOI: 10.17576/mjas-2019-2302-17

 

 

 

METHOD DEVELOPMENT AND VALIDATION FOR QUANTIFICATION OF CORTISOL AND CORTISONE USING ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

 

(Pembangunan dan Pengesahan Kaedah untuk Kuantifikasi Kortisol dan Kortison Menggunakan Kromatografi Cecair Berprestasi Ultra Tinggi-Spektrometri Jisim Gabungan)

 

Hazirah Abd Azhar^1,2, Mohd Salleh Rofiee¹, Mohammad Zulfadhly Jan Jam¹, Muhammad Hisyam Jamari¹, Roziah Mohd Janor^1,3, Richard Johari James^1,2*, Teh Lay Kek^1,2, Mohd Zaki Salleh^1,2

 

¹Integrative Pharmacogenomics Institute (iPROMISE)

²Faculty of Pharmacy

Universiti Teknologi MARA Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia

³Faculty of Computer & Mathematical Sciences,

Universiti Teknologi MARA Shah Alam Campus, 40450 Shah Alam, Selangor, Malaysia

*Corresponding author:  richard@uitm.edu.my

 

 

Received: 30 May 2018; Accepted: 14 April 2019

 

 

Abstract

Cortisol and its metabolite cortisone have been used as a biological marker of stress in human psychobiological studies. Cortisol and cortisone quantitation may help to examine physiological responses towards stress. A highly sensitive and selective ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantitation of cortisol and cortisone in human serum. These two compounds were chromatographically separated using Agilent ZORBAX Eclipse Plus C₁₈ Rapid Resolution HD column (2.1 mm × 50 mm, 1.8 µm) and were eluted with (A) 0.1% formic acid in water and (B) methanol mobile phase. The sample injection volume was 2 µL and the flow rate was set at 0.3 mL/min. The analytes were determined in positive ionization mode and quantitated by multiple-reaction monitoring (MRM) mode. The method was linear from 7.8 to 500 ng/mL for cortisol (r² = 0.986) and cortisone (r² = 0.990). The precision, accuracy, and recovery of the method for cortisol ranged from 1.98-10.46%, 90.68-91.94%, 90.38-93.88%, and cortisone ranged from 1.01-11.36%, 93.12-94.79% and 90.48-95.90%, respectively. The developed method has met the quality standards of European Medicines Agency (EMA).

 

Keywords:  cortisol, cortisone, serum, ultra-high performance liquid chromatography-tandem mass spectrometry

 

Abstrak

Kortisol dan metabolitnya kortison telah digunakan sebagai penanda tekanan biologi  untuk mengkaji psikobiologi manusia. Pengkuantitian kortisol dan kortison boleh membantu untuk menilai tindak balas fisologi terhadap tekanan. Kaedah kromatografi cecair berprestasi ultra tinggi -spektrometri jisim gabungan yang sangat sensitif dan selektif telah dibangunkan untuk pengkuantitian kortisol dan kortison secara serentak di dalam serum manusia. Kedua-dua sebatian ini telah dipisahkan secara kromatografi menggunakan turus Agilent ZORBAX Eclipse Plus C₁₈ Resolusi Pantas HD (2.1 mm × 50 mm, 1.8 μm) dengan campuran fasa bergerak (A) 0.1% asid formik dalam air dan (B) metanol. Jumlah suntikan sampel adalah 2 μL dan kadar aliran ditetapkan pada 0.3 mL/min. Analisis ini telah ditentukan dalam mod pengionan positif dan dikira oleh mod pemantauan pelbagai tindak balas. Kaedah ini adalah linear dari 7.8 hingga 500 ng/mL untuk kortisol (r² = 0.986) dan kortison (r² = 0.990). Ketepatan, kejituan dan kadar pemulihan untuk kortisol adalah antara 1.98-10.46%, 90.68-91.94%, 90.38-93.88%, dan kortison terdiri daripada 1.01-11.36%, 93.12-94.79% dan 90.48-95.90%. Kaedah yang dibangunkan telah memenuhi piawaian kualiti agensi perubatan eropah (EMA).

 

Kata kunci:  kortisol, kortison, serum, kromatografi cecair berprestasi ultra tinggi - spektrometri jisim gabungan

 

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