Determination of Alpha-Lipoic Acid in a Nutritional Supplement Using High Performance Liquid Chromatography

Alpha lipoic acid has the ability to react and neutralize reactive oxygen species (ROS) such as superoxide radicals, simple oxygen, hydroxyl radicals, hypochlorous acid and peroxyl radicals. A rapid high-performance liquid chromatographic method for determination of lipoic acid in a nutritional supplement was developed. The method involved sample preparation and the mobile phase comprised of 50 mM disodium hydrogen phosphate buffer (pH 2.5 adjusted with 1 M H 3 PO 4 ): acetonitrile in the ratio of 50:50. The separation was done using a C18 column (150 mm) and detection was carried out using UV detection at 201 nm. The assay was found to be linear in the range of 1.56 - 50 µg/mL with the correlation coefficient of 0.9997. Method precision was determined while LOD was 0.05 μg/mL and LOQ 0.15 μg/mL. The chromatographic peak LA retention time was 6 min.


Introduction
Alpha lipoic acid is an organosulfur compound derived from octanoic acid and shows a strong antioxidant activity against various conditions related to oxidative stress. Its synthesis is found endogenously in the human body, specifically in the mitochondria [1]. Alpha-lipoic acid contains two sulfur (S) atoms at C6 and C8 carbons joined by a disulfide bond resulting in oxidation. The carbon atom (C6) is asymmetric and two enantiomers are found on it: (R)-(+)-Lipoic Acid (RLA) and (S)-(-)-Lipoic Acid (SLA) (Figure 1) [2].
It acts as a cofactor for a variety of mitochondrial enzymes, catalyzing the oxidative decarboxylation of pyruvic, acetoglutarate and branched-chain α-keto acids. In both its reduced and oxidized form, it has the ability to react and neutralize reactive oxygen species (ROS) such as superoxide radicals, simple oxygen, hydroxyl radicals, hypochlorous acid and peroxyl radicals [3]. Free radicals have a dual role in the body. On the one hand, they have beneficial properties necessary for the proper functioning of the organism since, within normal limits, they protect against viruses, parasites and microbes, shielding its defenses, while at the same time participating in signal transduction, redox homeostasis, gene expression, cell differentiation and cell proliferation. On the other hand, when free radicals are present in high concentrations in the body they tend to cause problems with damaging effects on biomolecules such as lipids, proteins and DNA in general. The consequence of this is both the onset of premature aging and a number of diseases. This condition is called oxidative stress and is what is caused when there is an excess of free radicals in the body. In biological systems, the disruption of the balance between the production of reactive oxygen species (ROS) and the availability and action of antioxidant systems constitutes a condition called oxidative stress [4].
The occurrence of oxidative stress is multifactorial and is influenced by UV radiation, tissue oxygen deficiency, chemotherapy, radiotherapy and aging factors [5] [6] [7]. In addition, it is responsible for the manifestation of pathophysiological diseases and skin conditions. Some of them are cancer, Parkinson's and Alzheimer's disease, rheumatoid arthritis, asthma, diabetes and arteriosclerosis, heart diseases and hypertension, premature aging, inflammatory diseases, phototoxicity, photo allergy, and immune system diseases [8] [9] [10] [11]. Concerning Diabetes mellitus, it is a multi-faceted metabolic disorder where there is increased oxidative stress that contributes to the pathogenesis of the disease.
This has stimulated many investigations into the use of antioxidants as a complementary therapeutic approach. Alpha lipoic acid prevents beta cell destruction, improves glucose uptake, and its antioxidant effects may be helpful in reducing the development of diabetic complications such as diabetic neuropathy [11].
Alpha-lipoic acid is the only antioxidant substance with a dual nature as a partially water-soluble and fat-soluble antioxidant, with the consequence that its antioxidant action affects the whole body, inside and outside the cells. In particular, its solubility is low in water and oils but particularly high in ethyl alcohol [12] [13].
Alpha lipoic acid as an antioxidant: 1) It neutralizes oxygen free radicals and interacts with active radicals in all cells of the body which demonstrates it as a universal antioxidant [14].
2) Acts as a chelating agent and removes heavy metals responsible for oxidative stress [7].
3) Reduces blood glucose levels during metabolic activity. This has the effect of maintaining the antioxidant capacity of the human body.
Alpha-lipoic acid is added to various cosmetics because of its properties. It is added to anti-aging cosmetics as an active ingredient against aging, creams, emulsions both for its anti-wrinkle action and improvement of facial wrinkles as well as for its antioxidant action and protection of collagen [20]. In addition, alpha lipoic acid has been found to exhibit photoprotective activity and can be incorporated into sunscreen products [21] [22]. At the same time, it may protect the skin barrier due to its moisturizing properties [23].

Reagents and Standards
Alpha-LA pure powder was purchased from Sigma Chemical Company, aceto-

Instrumentation
Chromatographic

Preparation of Standard Solution
Stock standard solution of (1000 μg/mL) of LA was prepared by dissolving the pure powder in ACN. The concentrations of LA in the working standards were 1.56, 3.125, 6.25, 12.5, 25 and 50 μg/mL. The solutions were kept frozen at −20˚C until analysis.

Sample Preparation
Weigh accurately 0.162 g of α-lipoic acid and dissolve with 100 mL ACN, fol-

Results
The detector at which the analyte showed optimal response was selected as the optimized range for the determination of LA. The UV detector at 201 nm showed an optimal response and it was used for measurement of LA content.
This was performed using photodiode array (PDA), though LA can be detected using UV detector, as the detection wavelength falls within the range of UV detector. The chromatographic peak LA retention time was 6 min.
The peak for LA was observed linear at different concentrations. The calibration curve of LA standard solutions constructed using six concentrations shows good linearity in the range 1.56 to 50.0 µg/mL as shown in Figure 2 (correlation coefficient (r) of 0.9997). The peak area is measured into mAu/min.
Precision is the most used quality attribute of an analytical method. Precision was attributed through repeatability. Repeatability is presented in Figure 2, where standard solutions 1.56, 12.5, 50 μg/mL were measured three times and The assessment of detectability was made by calculating the limit of Detection (LOD) and the Limit of Quantification (LOQ). Therefore, it was measured a series of samples (after dilution the standard solution 1.56 μg/mL 1/2) (n=10) and the standard deviation was calculated ( Table 2).
The Limit of Detection LOD, was LOD = 3 × SD = 0.05 μg/mL and the Limit   of Quantification LOQ, was LOQ = 10 × SD = 0.15 μg/mL. In Table 3, the measurements of solutions A and B and the estimated concentrations of them, were calculated. The calculated concentrations were equivalent to those reported in the specific nutritional supplement used.