Simultaneous determination of lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma using high-performance liquid chromatography coupled with electrochemical detection

Abstract

A fast, simple, and a reliable high-performance liquid chromatography linked with electrochemical detector (HPLC–ECD) method for the assessment of lipoic acid (LA) and dihydrolipoic acid (DHLA) in plasma was developed using naproxen sodium as an internal standard (IS) and validated according to standard guidelines. Extraction of both analytes and IS from plasma (250 μl) was carried out with a single step liquid–liquid extraction applying dichloromethane. The separated organic layer was dried under stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both compounds and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 9 min using acetonitrile: 0.05 M phosphate buffer (pH 2.4 adjusted with phosphoric acid) (52:48, v/v) as a mobile phase pumped at flow rate of 1.5 ml min⁻¹ using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification for lipoic acid were 500 pg/ml and 3 ng/ml, and for dihydrolipoic acid were 3 ng/ml and 10 ng/ml, respectively. The absolute recoveries of lipoic acid and dihydrolipoic acid determined on three nominal concentrations were in the range of 93.40–97.06, and 93.00–97.10, respectively. Similarly coefficient of variations (% CV) for both intra-day and inter-day were between 0.829 and 3.097% for lipoic acid and between 1.620 and 5.681% for dihydrolipoic acid, respectively. This validated method was applied for the analysis of lipoic acid/dihydrolipoic acid in the plasma of human volunteers and will be used for the quantification of these compounds in patients with oxidative stress induced pathologies.

Introduction

Alpha lipoic acid (ALA), is a derivative of octanoic acid and is also known as thioctic acid. Chemically it is, 1,2-dithiolane-3-valeric acid or 6,8-dithiolane octanoic acid, or 6,8-thioctic acid. It is found naturally in both plants and animals [1], [2], [3], [4]. Alpha-lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) is the crucial universal antioxidant redox couple of human body [4], [5]. The chemical structures of LA and DHLA are presented in Fig. 1a and b, respectively.

The universal antioxidant redox couple of α-lipoic acid/dihydrolipoic acid plays the key role in human health by scavenging free radicals, chelating having metals, restoration of other antioxidants, and controlling regulatory proteins and genes essential for normal healthy life [6]. It is a cofactor for mitochondrial alphaketo-acid dehydrogenase, crucial prosthetic group of various cellular enzymes, chelating agent for heavy metals poisoning, and a scavenger of various free radicals such as hydroxyl radicals, hypochlorous acid, peroxyradicals, superoxide radicals, and singlet oxygen [1], [2], [5], [7], [8], [9], [10], [11]. Lipoic acid restores others antioxidants of the body antioxidant network including glutathione, coenzyme Q10 and vitamins C and E to their reduced state and maintains body antioxidant capacity [4], [12], [13]. It is an effective therapeutic agent in so many diseases including diabetes, mitochondrial cytopathies, cardiovascular diseases, hepatitis, cataract, radiation damage, HIV infections, heavy metal poisoning, neurodegenerative disorders, and neurovascular abnormalities associated with diabetic neuropathy [3], [14]. Due to its extensive and multidisciplinary role in human health, a specific, quick and robust method of analysis is required that can be easily applied by standard research and clinical laboratories for simultaneous determination of its oxidized and reduced forms. The quantification of lipoic acid (both oxidized and reduced forms) is helpful in biochemical, nutritional and pharmacokinetic studies especially related to its homeostatic, and antioxidant role in human health [5].

Several chromatographic methods have been reported for the quantification of lipoic acid and its metabolites in pharmaceutical dosage forms [10], [15], body fluids [1], [5], [16], and food samples [17]. Among the reported analytical methods for the determination of lipoic acid in the body fluids include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), polarography, colorimetric and microbiological assay, liquid chromatography mass spectrometry (LC-MS) [1], [3], [18], [19], gas chromatography mass spectrometry (GC-MS) and capillary electrophoresis [20], [21]. Though GC-MS has been reported to be a suitable and powerful tool for the quantification of LA in body fluids but it is expensive and its use is not common in most of the laboratories [5], [15]. HPLC is one of the most suitable and proper method for quantification of lipoic acid in biological fluids owing to its high accuracy, sensitivity, and simplicity regarding sample preparation and treatment procedures in routine laboratory practice [22]. HPLC linked with ultraviolet [23], fluorescence [5], [24], [25] and electrochemical detectors [16], [22], [26], [27] has been used for the evaluation of lipoic acid in food supplements and biological samples. The major demerits associated with HPLC-UV method are the absence of strong chromophore in lipoic acid and its comparatively lower sensitivity [15]. HPLC with fluorescence detection, offers a good method for the determination of α-lipoic acid in biological fluids but its lengthy and time consuming derivitization and sample preparation steps are laborious [5], [24]. HPLC with electrochemical detection method is therefore seems to be an ultimate choice in terms of sensitivity, shorter analysis time and lower cost for most of the standard research laboratories to quantify α-lipoic acid in biological fluids in comparison with other literature reported methods [3], [5], [15], [18], [19], [20], [21], [23], [24]. To our knowledge only a few HPLC methods with fluorescence detection have been reported for the simultaneous determination of LA and DHLA in biological fluids and pharmaceuticals samples [5], [13], and no HPLC–ECD method for the simultaneous determination of LA and DHLA in biological fluids has been reported so for.

The aim of our presented work was to develop a fast, sensitive and robust HPLC–ECD method for the simultaneous determination of LA and DHLA in human plasma with internal standard calibrated (IS) method. This suggested method is fast and inexpensive in comparison with other literature reported HPLC–ECD methods for the determination of lipoic acid in human body fluids [16], [22]. The method was found suitable for the quantification of LA and DHLA in human plasma using a simple single step liquid–liquid extraction. The method can also be applied for routine analysis of LA and DHLA in pharmaceutical dosage forms and dietary supplements with suitable adjustment in the extraction technique.