Malaysian Journal of Analytical Sciences, Vol 27 No 4 (2023): 840 - 848

VALIDATION OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHOD FOR DELPHINIDIN-3-O-GLUCOSIDE CHLORIDE IN RAT PLASMA

(Pengesahan Kaedah Kromatografi Cecair Berprestasi Tinggi (KCPT) bagi Delfinidin-3-O-Glukosida Klorida dalam Plasma Tikus)

Nur Atielah Nabila Mohd Shafawi¹, Ruzilawati Abu Bakar², Liza Noordin³, Shafreena Shaukat Ali¹,

and Wan Amir Nizam Wan Ahmad¹

¹School of Health Sciences, Health Campus, Universiti Sains Malaysia Kubang Kerian, 16150 Kota Bharu, Kelantan, Malaysia

²Department of Phamacology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia Kubang Kerian, 16150 Kota Bharu, Kelantan, Malaysia

³Department of Physiology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia Kubang Kerian, 16150 Kota Bharu, Kelantan, Malaysia

^*Corresponding author: wanamir@usm.my

Received: 7 April 2023; Accepted: 23 July 2023; Published: 22 August 2023

Abstract

This study involved the development and validation of a high-performance liquid chromatography (HPLC) assay for the determination of delphinidin-3-O-glucoside chloride, specifically to be applied in future pharmacokinetic and bioavailability studies in rats supplemented with roselle extract. The HPLC method was validated following solid-phase extraction, with six validation parameters (linearity, recovery, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) assessed according to Food and Drug Administration guidelines. Chromatographic separations were performed using Agilent Reverse Phase EC-C18 (4.6×150 mm; 2.7 µm particle size) and mobile phase acetonitrile with 0.1% trifluoracetic acid in an aqueous solution (81:19, v/v). The calibration curve of delphinidin-3-O-glucoside chloride had a coefficient determination (R²) of ≥95.0% and recovery percentage of 80.37%. It also showed good precision and accuracy, and its method detection and quantification limits were 47 ng/mL and 213 ng/mL, respectively. In conclusion, the validated HPLC method in this study can be used to determine the pharmacokinetic of delphinidin-3-O-glucoside chloride or other type of anthocyanins in the future.

Keywords: delphinidin-3-o-glucoside chloride, rat plasma, high-performance liquid chromatography, method validation

Abstrak

Kajian ini melibatkan pembangunan dan pengesahan kaedah kromatografi cecair berprestasi tinggi (KCPT) bagi pengesanan sejenis antosianin, iaitu delfinidin-3-O-glukosida klorida, khususnya bertujuan untuk digunakan dalam kajian lanjutan untuk farmakokinetik dan bioavailibiliti antosianin di dalam tikus yang disuplementasi dengan ekstrak rosel. Kaedah KCPT ini disahkan menggunakan kaedah pengekstrakan fasa pepejal dan enam parameter pengesahan dinilai mengikut garis panduan Pentadbiran Makanan dan Dadah iaitu kelinearan, pemulihan, ketepatan, kejituan, had pengesanan, dan had kuantifikasi. Pengasingan kromatografik dilakukan menggunakan Agilent Fasa Berbalik EC-C18 (4.6×150 mm; 2.7µm saiz partikel) manakala fasa gerak adalah asetonitril bersama 0.1% trifluoroasetik asid di dalam air (81:19, v/v). Lengkung penentukuran delfinidin-3-0-glukosida klorida mempunyai penentuan pekali (R²) ≥95.0% dan peratusan pemulihan delfinidin-3-0-glukosida klorida adalah 80.37%. Delfinidin-3-0-glukosida klorida juga menunjukkan ketepatan dan kejituan yang baik dan had pengesanan dan kuantifikasi kaedah untuk delfinidin-3-0-glukosida klorida masing-masing adalah 47 ng/mL dan 213 ng/mL. Kesimpulannya, kaedah HPLC yang disahkan dalam kajian ini boleh digunakan untuk menentukan tahap antosianin dalam kajian farmakokinetik pada masa depan.

Kata kunci: delfinidin-3-o-glukosida klorida, plasma tikus, kromatografi cecair berprestasi tinggi, pengesahan kaedah

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