Determination of lipoic acid in rat plasma by LC‐MS/MS with electrospray ionization: Assay development, validation and application to a pharamcokinetic study

Abstract

A simple, sensitive and specific LC-MS/MS method for the determination of lipoic acid was developed and validated over the linearity range 5-1000 ng/mL (r2 > 0.99) with 200 microL rat plasma using rosigliatzone as an internal standard (IS). The assay procedure involved a simple one-step liquid-liquid extraction of lipoic acid and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Hichrom RPB column (4.6 x 250 mm, 5 microm). Separation of lipoic acid and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min. The API-3000 LC-MS/MS was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Positive and negative ion acquisition within the same chromatographic run was used in the present method. For lipoic acid a pseudo-molecular ion transition pair was acquired in negative polarity, whereas for IS the transition pair was acquired in positive polarity. Quantitation was determined for both analyte and IS in MRM scan mode. Absolute recovery of lipoic acid and IS was >70 and 97%, respectively. The lower limit of quantification (LLOQ) of lipoic acid was 5.0 ng/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples 5, 15, 400 and 800 ng/mL were in the range 2.18-5.99% relative standard deviation (RSD) and 0.93-13.77% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.40-114.40% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. Stability of lipoic acid was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats confirmed the utility of the assay.

Simultaneous Determination of the Endogenous Free α-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

“…Several papers have been published on the chromatographic analysis of total, free, or bound a-LA in various sample matrices such as foodstuffs [9], dietary supplements [10], and biological samples [11][12][13][14][15][16]. Similarly, very few methods [17][18][19][20][21][22][23] are available for simultaneous determination of the total a-LA and its reduced form, DHLA, in biological samples after oral administration.…”

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development, validation and optimization of experimental parameters

“…Lipoic acid recycles others antioxidants such as glutathione, vitamins C and E to their reduced state and maintains body antioxidant capacity [4,7]. It is also used as a therapeutic agent in various diseases and was found to be useful in patients with diabetes, mitochondrial cytopathies, cardiovascular diseases, hepatitis, cataract, radiation damage, HIV infections, heavy metal poisoning, neurodegenerative disorders, and neurovascular abnormalities associated with diabetic neuropathy [1,2,8]. Because of its extensive and multidisciplinary therapeutic use, a specific, fast and reliable method of analysis is required that can be easily applied by standard clinical laboratories.…”

“…Alpha-lipoic acid (ALA), usually referred as thioctic acid is chemically known as 1,2-dithiolane-3-valeric acid or 6,8-dithiolane octanoic acid, or 6,8-thioctic acid is a naturally occurring antioxidant compound that is found in plants, animals and various microorganisms [1,2]. The chemical structure of ␣-lipoic acid is shown in Fig.…”

“…Various analytical methods are currently reported for the analysis of lipoic acid in the body fluids including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), polarography, colorimetric and microbiological assay, liquid chromatography-mass spectrometry (LC-MS) [1,11,12], gas chromatography-mass spectrometry (GC-MS), and capillary electrophoresis [13,14]. Although GC-MS is a powerful technique for the quantification of LA in biological fluids but it is expensive and beyond the reach of many laboratories [2,9].…”

Highly Selective and Simple Method for Determination of Polythiols Based on Liquid Chromatography with Postcolumn Excimer Fluorescence Derivatization

“…Therefore, many determination methods for them have been reported: such as gas chromatography 1 and some liquid chromatography (LC) methods with electrochemical detection 2,3 and mass spectrometry. 3,4 In addition, various fluorescence derivatization reagents such as monobromobimane, 5,6 o-phthalaldehyde, 7,8 and 4-(aminosulfonyl) 7-fluoro-2,1,3-benzoxadiazole 9,10 are commercially available and widely used for the sensitive LC determination of related compounds. However, to the best of our knowledge, there are no reports on the selective and simple determination of polythiols.…”