Development of HPLC method for estimation of glyoxylic acid after pre-column fluorescence derivatization approach based on thiazine derivative formation: A new application in healthy and cardiovascular patients’ sera

https://doi.org/10.1016/j.jchromb.2020.122054Get rights and content

Highlights

  • A highly sensitive pre-column fluorescence derivatization is described for determination of GA.

  • The method based on reaction of GA with 2-ABT in acidic medium to form highly fluorescent thiazine derivative.

  • The method showed high sensitivity and utilized to monitor GA in healthy and cardiovascular patients’ sera.

Abstract

Glyoxylic acid (GA) is the intermediate metabolite in various mammalian metabolic pathways. GA showed high reactivity towards formation of advanced glycation end-products (AGEs); the main cause of pathogenesis and complications of many diseases. The presented study aimed to detect GA in healthy and cardiovascular patients’ (CV) sera; however analysis of GA in biological fluid is a challenge and requires chemical derivatization. Hence, a new, highly sensitive, time saving and reproducible pre-column fluorescence derivatization procedure coupled with high performance liquid chromatography (HPLC) method was developed. The derivatization method was based on reaction of 2-aminobenzenthiol (2-ABT), a fluorogenic reagent, with GA in acidic medium to form highly fluorescent thiazine derivative (290 and 390 nm for excitation and emission wavelengths respectively). The fluorescent derivative was separated within 6 min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol–water (70:30, v/v%). The proposed method parameters were optimized and the method was validated. A good linearity in the concentration range (0.05–5.0 µM) was obtained with detection limit (LOD) of 10 nM (200 fmol/injection), which is more sensitive than several previous methods. Moreover, the recovery results were within the range of 85.0–95.5 % and the intra- and inter-day precision results were ≤3.5%. It should be emphasized that this method is the first one for monitoring of GA in CV patients; to investigate its role for diagnosis and monitoring the severity and complications of this disease in clinical laboratory.

Introduction

Glyoxylic acid (GA) is α-dicarbonyl compound and the intermediate metabolite in different mammalian metabolic pathways [1]. GA can be produced in vivo from deamination of glycine in peroxisomes, by oxidation of glycolate and through catabolism of hydroxyproline in liver mitochondria [1], [2]. GA is oxidized into oxalate in mammals as a final metabolite by lactate dehydrogenase resulting in high concentration of oxalate which is susceptible to urinary calculus formation [1]. GA has an aldehydic group which is highly reactive towards formation of advanced glycation end-products (AGEs) [1], [2]. Meanwhile, AGEs have been involved in the pathogenesis of different diseases such as diabetes mellitus (DM) [1], [2], cardiovascular (CV) [3], [4], [5], [6], [7], neurodegenerative and renal diseases [5]. Studies revealed that GA was found to be more reactive toward formation of AGEs than D-glucose, D-fructose and DL-glyceraldehyde [1], [2].

Several analytical methods were reported for determination of GA in different matrices including high performance liquid chromatography (HPLC) [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], gas chromatography (GC) [12], [18], [19], thin layer chromatography (TLC) [20], electrophoresis [21], voltammetry [22], polarography [20] and electrochemiluminescence [23]. However, most of these methods have several drawbacks either low sensitivity [9], [10], [11], [13], [14], [16], [20], [23], utility of expensive and sophisticated equipment that are not available in most laboratories [12], [18], [19] or long analysis time [11], [15], [16], [17].

Till now, few HPLC with fluorescence detection were reported [8], [15], [17], two of them were performed in our laboratory based on Petasis reaction [15], [17], where GA reacted with a fluorescence labeling reagent: 1-pyreneboronic acid. Although high sensitivity of this method, it suffered from having long analysis time (1hr), besides a crowded chromatogram had been obtained due to excess reagent [15]. In order to clear up the chromatogram of GA to be free from reagent peaks, a clean up procedure using solid phase extraction (SPE) method was conducted [17].

Owing to the high importance in detection and quantitation of GA in biological fluids, 2-aminobenzenthiol (2-ABT), a fluorogenic reagent, was utilized for derivatization of GA. The presented reagent 2-ABT has no fluorescence itself, however by reaction with GA in acidic medium it forms a highly fluorescent thiazine derivative after 25 min.

Different parameters affecting the derivatization reaction and chromatographic separation were optimized. In addition, the proposed method was validated according to ICH guidelines where linearity, sensitivity parameters and reproducibility were studied. The proposed method showed higher sensitivity over previously reported one following a simple and time saving derivatization procedure and utilizing instruments available in many laboratories. Further, the proposed method was applied for the first time to determine GA in healthy and cardiovascular (CV) patients’ sera samples in order to ensure its suitability to diagnose and monitor severity of CV disease in clinical laboratories.

Section snippets

Chemicals and reagents

Glyoxylic acid monohydrate (GA) was purchased from Sigma Aldrich (Osaka, Japan). 1,2-Di(2-furyl)-1,2-ethanedione used as an internal standard (I.S.) was purchased from TCI Chemical company (Tokyo, Japan). 2- Aminobenzenthiol (2-ABT), hydrochloric, acetic and perchloric acids were obtained from Nacalai Tesque Chemical Company (Tokyo, Japan). Nitric, phosphoric acids, methanol and acetonitrile (HPLC grade solvent) were obtained from Kanto Chemical Company (Tokyo, Japan). All reagents used for

Results and discussion

Owing to the absence of chromophore and fluorophore in GA structure, derivatization reaction is mandatory for its determination either in biological or environmental samples. In the presented method, simple and highly sensitive fluorescence derivatization method was developed based on reaction of GA with 2-ABT; a fluorogenic reagent in acidic medium after heating at 90 °C for 30 min. Fig. S1 (Supplementary Material) shows the fluorescence spectra of the derivatized product obtained from the

Conclusion

A simple pre-column fluorescence derivatization process was developed for determination of GA. The method based on reaction of GA with 2-ABT, a fluorogenic reagent, to form fluorescent derivative separated on HPLC-FL system within 6 min. The proposed method was more sensitive than several previous methods, cost–effective and time saving one. All experimental parameters were optimized; the method was validated and showed good linearity in the concentration range 0.05–5.0 µM with correlation

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgment

The authors would like to thank Dr. Takahiro Imazato (Sasebo Chuo Hospital, Sasebo, Japan) for his valuable help in this article.

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