Fluorescence Assay for the Determination of d-Panthenol Based on Novel Ring-Fused 2-Pyridone Derivative.

Wiktor Kasprzyk,Filip Koper,Tomasz Świergosz

doi:10.3390/ijms21218386

Wiktor Kasprzyk, Filip Koper + Show 1 more

Open Access

https://doi.org/10.3390/ijms21218386

Copy DOI

Abstract

Herein, a novel fluorescent method for the determination of d-panthenol (DP) level in solutions with no separate hydrolysis step has been revealed based on the utilization of citric acid (CA) as a derivatizing agent. Consequently, the essential parameters of the derivatization process were established, resulting in the development of sensitive, repeatable, and accurate determination of panthenol. The method was approved, and its usefulness in characterizing the concentration of DP in pharmaceutical formulations and selectivity in the determination of DP were validated. The chemical structure of the new fluorophore formulating in the reaction in DP with CA, i.e., 6-oxo-3,4-dihydro-2H,6H-pyrido[2,1-b][1,3]oxazine-8-carboxylic acid (ODPC), was elucidated using detailed NMR experiments: one-dimensional (1H, 13C) as well as two-dimensional NMR spectra (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, 1H-15N HSQC, 1H-15N HMBC).

Highlights

On the LC-DAD chromatogram of the methanolic solutions of reaction mixtures, one main peak and several minor fractions absorbing around 350 nm were observed, indicating that the fluorophore was formed as a major component during the reaction (Figure 1A)
Comparing m/z 196 [M + H]+ peak areas of chromatograms of samples prepared at various molar ratios of Citric acid (CA):DP, it was found that for the efficient synthesis of the fluorophore, the molar ratio of reagents 1:1 was the most suitable (Figure 1D)
The chemical structure of the new fluorophore (ODPC) was elucidated using detailed NMR experiments, allowing us to suggest a possible mechanism for its formation involving the formation of six-membered anhydride originated from dehydration of CA, thermal hydrolysis of amide linkage of DP resulting in the release of a 3-amino-1-propanol moiety, and inter- and intramolecular condensation of CA anhydride with 3-amino-1-propanol

Summary

Introduction

The above methods use HPLC separation or tedious DP extraction steps before the actual reaction with a derivatizing agent in order to reach an appropriate level of selectivity The latter fact is associated with the ability of NBD and ninhydrine to react with all α-amine-containing components of a studied sample and 3-amino-1-propanol [20,21]. CA has been investigated in terms of possible use as an inexpensive carbon source for bottom-up, hydrothermal synthesis of carbon dots [23,24,25] which resulted in the development of a series of novel highly photoluminescent organic fluorophores, i.e., derivatives of ring-fused 2-pyridones [26,27,28,29,30,31,32,33] The latter fluorophores were found to be a product of the condensation of CA and specific β-amines [29]. In this research, a novel spectrofluorimetric method for DP determination in various pharmaceutical and cosmetic formulations without a separate hydrolysis step of panthenol was introduced

Methods

Results

Conclusion