METHOD DEVELOPMENT AND VALIDATION OFMETANEPHRINE AND NORMETANEPHRINE ON LIQUIDCHROMATOGRAPHY-TANDEM MASS SPECTROMETRY
Main Article Content
Keywords
Liquid chromatography-mass spectrometry, metanephrine, normetanephrine, Pheochromocytoma
Abstract
Catecholamines and its metabolites play significant physiological roles in the human body.
Paraganglioma and pheochromocytoma are rare tumors of an adrenal region that affect the
catecholamines and their metabolites (metanephrine (MN) and normetanephrine (NMN))
concentration. Given the significance of these metabolites for diagnosing, managing, and
monitoring adrenal tumors, a method has been developed and validated using Liquid
Chromatography-Tandem Mass Spectrometry (LC-MS/MS). This method is specifically designed
for quantification of MN and NMN in urine. The objective of developing and validating this
analytical approach is to provide physicians with a reliable tool for assisting in the management of
adrenal tumors.
For sample extraction, 2-aminoethyl-diphenylborinate and ethyl acetate were used. The organic
layer was separated and dried under nitrogen at 50-60°C and reconstituted with mobile phase. The
chromatographic separation was done using the Agilent Eclipse C-18 column with two mobile
phases: (A) 5% Methanol in 0.1% Formic acid and (B) 100% Methanol in an isocratic flow. The run
times for MN and NMN were 1.259 and 1.268 min, respectively. The validated analytical measuring
range (AMR) was 7.8-1000 ng/mL with lower limits of quantification (LLOQ) 7.8 ng/mL for MN
and NMN, and the recovery was 95.11% to 105.6% for MN and 95.87% to 104.78% for NMN. The
method showed an excellent correlation (r2) of 0.997 for MN and 0.998 for NMN. The method was
found steady after inter-day and intra-day analysis with coefficients of variation (CV) <3% and
accuracy <6%. This validated method can be used for metabolite screening in clinical and research
settings.
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Hafiza Monaza Batool
*School of Chemistry, University of the Punjab, Lahore, Pakistan
Madeeha Batool
School of Chemistry, University of the Punjab, Lahore, Pakistan
Muhammad Qaiser Alam Khan
Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology
(AFIP), Rawalpindi, Pakistan.
Muhammad Usman Munir
Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology
(AFIP), Rawalpindi, Pakistan.
Muhammad Aamir
Shifa International Hospital, Islamabad, Pakistan.
Sobia Irum Kirmani
Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology
(AFIP), Rawalpindi, Pakistan.