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

Abad Khan, Muhammad I. Khan, Zafar Iqbal, Lateef Ahmad, Yasar Shah, David G. Watson

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001-10 mu g/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 mu l) was carried out with a simple one step liquid-liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 degrees C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 degrees C on Discovery HS C18 RP column (250 mm x 4.6 mm, 5 mu m) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid-liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 mu g/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases. (C) 2010 Elsevier B.V. All rights reserved.

LanguageEnglish
Pages2782-2788
Number of pages7
JournalJournal of Chromatography B
Volume878
Issue number28
DOIs
Publication statusPublished - 15 Oct 2010

Fingerprint

Plasma (human)
Thioctic Acid
Liquid-Liquid Extraction
Solid Phase Extraction
High Pressure Liquid Chromatography
Liquids
Methylene Chloride
Limit of Detection
Detectors
Plasmas
Naproxen
Solvent extraction
Medical problems
Volunteers
Buffers
Cardiovascular Diseases
Nitrogen
Phosphates
Recovery

Keywords

  • human plasma
  • solid-phase extraction
  • lipoic acid

Cite this

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title = "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",
abstract = "A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001-10 mu g/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 mu l) was carried out with a simple one step liquid-liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 degrees C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 degrees C on Discovery HS C18 RP column (250 mm x 4.6 mm, 5 mu m) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid-liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17{\%} at 0.5, 1 and 5 mu g/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97{\%}. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases. (C) 2010 Elsevier B.V. All rights reserved.",
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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. / Khan, Abad; Khan, Muhammad I.; Iqbal, Zafar; Ahmad, Lateef; Shah, Yasar; Watson, David G.

In: Journal of Chromatography B, Vol. 878, No. 28, 15.10.2010, p. 2782-2788.

Research output: Contribution to journalArticle

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T1 - 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

AU - Khan, Abad

AU - Khan, Muhammad I.

AU - Iqbal, Zafar

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AU - Shah, Yasar

AU - Watson, David G.

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AB - A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001-10 mu g/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 mu l) was carried out with a simple one step liquid-liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 degrees C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 degrees C on Discovery HS C18 RP column (250 mm x 4.6 mm, 5 mu m) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid-liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 mu g/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases. (C) 2010 Elsevier B.V. All rights reserved.

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