Skin lipid analysis - HPTLC - Farwanah et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 HPTLC (AMD, Camag) 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Recovery 7 Internal Standard 8 Reference

Abstract

Farwanah et al. presented an improved method for the seperation and subsequent quantification of major stratum corneum lipids, including particularly the seven known ceramide classes in the same run. The method was successfully applied to skin surface extracts obtained in vivo.

Analysed Matrices

skin lipid extracts

Analytes

• extracted lipids:

Cholesterol-3-sulfate; Ceramides; Cholesterol; Free fatty acids; Triglycerides; Sterol esters

• 7 extracted ceramides:

Cer EOS; Cer NS; Cer NP; Cer EOH; Cer AS; Cer AP; Cer AH

Sample Preparation

• skin lipid extracts from six test persons (in vivo, caucasian males, age 28 +/- 4 years)
• filling a cylindrical glass beaker with 4-cm I.D. (contact area: 12,56 cm2) with 8 ml n-hexane/EtOH (2:1)for surface extraction
• tightly pressing the open side to skin area at the inner forearm to prevent lateral leakage
• extraction time: 5 min
• evaporation of extraction mixture (50 °C) and drying of residue under a stream of argon
• dissolving the dryed lipids in 500 μl CHCl3/MeOH (1:1)

Analytical Method

HPTLC (AMD, Camag)

application of samples

• washing of HPTLC plates (three times) with CHCl3/MeOH (65:35) before use
• automatic sample application with Automatic TLC Sampler 4 (Camag): dosage speed = 100 μl/ s
• 15 samples on each plate: 6 lanes for skin lipid samples (each 6 μl) and 9 lanes for reference lipids (from blank to 4 μg for each standard lipid)
• start line: 8 mm from the bottom
• band lenght: 8 mm
• distance between lanes: 4 mm

development of plates

• automatically using a AMD-2 apparatus (Camag)
• 17 step gradient of decreasing polarity
• after drying, the plates were dipped into a aqueous solution of 10 % CuSO4, 8 % H3PO4, and 5 % MeOH for 20 s
• drying of plates in a drying oven (150 °C, 20 min)

densitometry

• scanning of developed and visualised plates from 7 cm to front by TLC Scanner 3 (Camag)
• reflectance mode; wavelength of 546 nm
• slit dimensions: 4 * 0,1 mm
• scan speed: 20 mm/s
• data resolution: 25 μm per step
• integration and quantification based on peak areas by CATS software (Camag)

Method Validation

Accuracy

Precision

LOD

LOQ

Recovery

Internal Standard

Cer NP

Reference

• H. Farwanah, R. Neubert, S. Zellmer, K. Raith Journal of Chromatography B 2002, 780, 443-450.

Institute of Pharmaceutics and Biopharmaceutics; Martin Luther University; W.- Langenbeck-Str. 4, 06120 Halle (S.); Germany.

Tel.: 49-345-552-5215; Fax: 49-345-552-7292; e-mail: raith@pharmazie.uni-halle.de (K. Raith)