Development and validation of indocyanine green assay in human plasma using High-Performance Liquid Chromatography with PDA detector

Sumith K Mathew; Blessed Winston A; Aswathy Mathew; Jeana Jacob; Ratna Prabha; Binu Susan Mathew

Sumith K Mathew ⁽¹⁾ , Blessed Winston A ⁽²⁾ , Aswathy Mathew ⁽³⁾ , Jeana Jacob ⁽⁴⁾ , Ratna Prabha ⁽⁵⁾ , Binu Susan Mathew ⁽⁶⁾

(1) Department of Pharmacology and Clinical Pharmacology, Clinical Pharmacology Unit, Christian Medical College, Vellore – 632004, Tamil Nadu, India, India ,

(2) Department of Pharmacology and Clinical Pharmacology, Clinical Pharmacology Unit, Christian Medical College, Vellore – 632004, Tamil Nadu, India, India ,

(3) Department of Pharmacology and Clinical Pharmacology, Clinical Pharmacology Unit, Christian Medical College, Vellore – 632004, Tamil Nadu, India, India ,

(4) Department of Pharmacology and Clinical Pharmacology, Clinical Pharmacology Unit, Christian Medical College, Vellore – 632004, Tamil Nadu, India, India ,

(5) Department of Pharmacology and Clinical Pharmacology, Clinical Pharmacology Unit, Christian Medical College, Vellore – 632004, Tamil Nadu, India, India ,

(6) Department of Pharmacology and Clinical Pharmacology, Clinical Pharmacology Unit, Christian Medical College, Vellore – 632004, Tamil Nadu, India, India

Issue
Vol. 10 No. 2 (2019): Volume 10 Issue 2

Keywords:

HPLC Assay, Indocyanine Green, Photodiode Array Detector

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Abstract

A robust and economical assay for routine determination of indocyanine green pharmacokinetics was developed and validated using high-performance liquid chromatography with a photodiode array detector. Plasma specimens from critically ill patients and those with hepatitis on various comedications were used as blanks for validation of this assay. Extraction of indocyanine green was performed by simple protein precipitation with acetonitrile, and the supernatant was separated using an octadecyl column with detection at 784 nm. Blanks were found to have no interference for 40 blanks of patients who were on 56 different medications. The precision for LLOQ (0.5 µg/ml) as determined by the percentage coefficient of variation was 1.19. Stability of plasma calibration standards and stock were determined over a period of 61 days, and ICG was found to be stable for 20 days. Stability of whole blood specimens containing ICG was determined at 4°C for a period of 4 hours.