Analysis of Lipoic Acids
R-Lipoic, S-Lipoic and (+/-) Alpha-Lipoic Acid
GeroNova Research provides validated reference standards at our online store for R-(+)-lipoic acid, S-(-)-lipoic acid, R/S-(+/-)-Lipoic Acid and R-(-)-DHLA
HPLC Analysis of Lipoic Acid Products, Non-Chiral
A validated, reverse phase HPLC method for the analysis of total lipoic acid & dihydrolipoic acid. Carlson DA & Smith AR. June 6, 2008.
To date, the simplest, most rapid and reproducible non-chiral method for total lipoic acid content utilizes reverse phase chromatography on a C18 column. GeroNova Research utilizes this method for the analysis of racemic lipoic acid, R-Lipoic Acid, SLA, NaRLA, K-RALA, R-DHLA, S-DHLA, and rac-DHLA. Download PDF.
State-of-the-Art Chiral HPLC
GeroNova Research performs a state-of-the-art analysis with a validated, accurate, reproducible (robust) chiral method. In brief a Chiralcel (Daicel) AD-H column (25 x 0.46 cm) is used. The solvent system is n-hexane: IPA/ TFA. Flow rate: 1.0 ml/min: retention time for R-Lipoic Acid 11.5 min and SLA is 12.4 min. Detection: 215 nm.
To date, this method is the most rapid and reliable for determination of enantiomeric ratios and quality control analysis of R-lipoic Acid and the salts of R-lipoic Acid.
The following validated method may be used by any individual, academic institution or corporation as long as credit is given to GeroNova Research Inc as the source of the method. A Validated, Normal Phase HPLC Method for the Analysis of Lipoic Acid Enantiomers. Carlson DA & Smith AR. Download PDF.
LESS EFFICIENT METHODS BELOW
CHIRAL HPLC METHOD II
Niebch G, Büchele B, Blome J, Grieb S, Brandt G, Kampa P, Raffel HH, Locher M, Borbe HO, Nubert I, Fleischhauer I. Enantioselective high-performance liquid chromatography assay of (+)R- and (-)S-alpha-lipoic acid in human plasma. Chirality. 1997;9 (1):32-6. The original method was modified by researchers at Degussa. Bernkop-Schnürch A, Reich-Rohrwig E, Marschütz M, Schuhbauer H, Kratzel M.
Development of a sustained release dosage form for alpha-lipoic acid. II. Evaluation in human volunteers. Drug Dev Ind Pharm. 2004 Jan; 30(1):35-42.
The method can be used to determine the enantiomeric ratios in plasma or QC samples but involves derivatization of the enantiomers of lipoic acid with o-phthalaldehyde (OPA) in the presence of D-Phenylalanine (D-Phe) after reduction to the dithiol enantiomers. The resulting two diasteromeric derivatives are separated by reverse phase HPLC on a LiChrospher 60 SelectB column (25cm x 4mm I.D.: 5 um particle size at 35 deg C) utilizing FL detection with an excitation wavelength of 230nm and an emission filter of > 418 nm. The standard and sample are eluted with a mixture of 55% 0.2M K2HPO4 (pH 5.8) and 45% acetonitrile/methanol (1:1) as the mobile phase (1.7ml/min). Identification and quantification have been performed by utilizing both internal and external standards method.
GeroNova Research has published results utilizing this method (Carlson et al 2008) See our In-House Research Page but it is time consuming, requires additional chemicals and numerous controls and calibration runs in order to use it quantitatively.
Commercial analytical labs have reported results using this method without report of any controls which renders the results meaningless. This method has been replaced by the normal phase method above.
CHIRAL HPLC METHOD III
Bethge et al of the former Asta Medica (US Patent 5,621,117) reported another enantioselective HPLC method. In this assay, a 100mm x 4 mm, Chiral AGP column with a 100mm x 3mm Guard Column (ICT) was utilized. The mobile phase was 1 liter 0.01M disodium hydrogen phosphate adjusted to pH 4.95-5.05 with dilute phosphoric acid with 150 ml methanol added and thoroughly mixed. Flow rate was 0.5ml/min, 35 bar, 20 deg C, and 210 nm. 1.5-2.0 mg of the enantiomers of ALA are accurately weighed, dissolved in 1 ml MeOH, and diluted to 20 ml with the mobile phase. The solution is filtered through a 0.45 um membrane filter. 20 µl is injected onto the column.
Villani et al of Labochim (US 6,670,484) reported essentially the same method as Bethge et al. Chromatographic Conditions for Determining the Enantiomeric Excess. Chromatographic column: chiral AGP column, size 100 mm x 4 mm, with pre-column; Mobile phase: 1 liter of 0.01 M solution of Na2HPO4, brought to pH=5 with diluted phosphoric acid, mixed with 150 ml of methanol. Flow speed: 0.4 ml/minute Pressure: 35 bar Temperature: 20.degree. C. Wave length: 220 nm. Sample: 1.5-2 mg of substance dissolved in 20 ml of mobile phase.
CHIRAL HPLC METHOD IV
The use of Daicel OD columns was reported by Fadnavis NW and Koteshwar K. Remote Control of Stereoselectivity: lipase catalyzed enantioselective esterification of racemic a-lipoic acid. Tetrahedron Asymmetry Vol 8, No 2 pp 337-339 (1997) and N. W. Fadnavis, Ravi Luke Babu, S. Kumara Vadivel, Ashlesha A. Deshpande, U. T. Bhalerao. Lipase catalyzed regio- and stereospecific hydrolysis: chemoenzymatic synthesis of both (R)- and (S)-enantiomers of a-lipoic acid. Tetrahedron Asymmetry Volume 9, Issue 23, 4099-4284 (1998).
In brief, a Chiralcel (Daicel) column (25 x 0.46 cm) is used. The solvent system is 2% IPA, and 0.1% TFA in n-hexane. Flow rate is 0.9 ml/min: retention time for SLA is 17.8 min and R-Lipoic Acid 18.5 min. Detection at 330 nm. We were unable to reproduce the results reported in this method. We obtained a single peak with the OD-H column, indicating no separation of the enantiomers.
Other Methods of Analysis of racemic-ALA
Determination of lipoic acid and dihydrolipoic acid in human plasma and urine by high-performance liquid chromatography with fluorimetric detection. Haj-Yehia AI, Assaf P, et al. Chromatogr A. 2000 Feb 18;870(1-2):381-8
Satoh S, Toyo'oka T, Fukushima T, Inagaki S. Simultaneous determination of alpha-lipoic acid and its reduced form by high-performance liquid chroma-tography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jul 1;854(1-2):109-15.
Durrani AI, Schwartz H, Schmid W, Sontag G. alpha-Lipoic acid in dietary supplements: development and comparison of HPLC-CEAD and HPLC-ESI-MS methods. J Pharm Biomed Anal. 2007 Nov 30; 45(4):694-9.
Determination of lipoic acid by precolumn derivitization with monobromobimane and reversed-phase high-performance liquid chromatography. Witt W, Rustow B. J Chromatogr B Biomed Sci Appl. 1998 Jan 23; 705(1):127-31
For a review of chromatographic methods for the analysis of lipoic acid: Chromatographic analysis of lipoic acid and related compounds Kataoka, H. J Chrom B, 717 (1998) 247-262