Comprehensive quantification of ceramide species in human stratum corneum

Abstract

One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantification of the overall CER species by improving our previously reported profiling technique using normal-phase liquid chromatography-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm x 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. This method opens lipidomics approaches for CERs in the SC.

Fig. 1.

Multi-SIM chromatograms of CER species in human SC using NPLC-ESI-MS. (a) inner forearm SC. (b) cheek SC. Sample: crude lipids obtained from a 35-year old male using a piece of tape 5 mm × 10 mm. NPLC conditions: column, Inertsil SIL 100A-3 (1.5 mm i.d. × 150 mm); column temperature, 40 C; mobile phase, (A) n-hexane/2-propanol/formic acid, 95:5:0.1, by vol, (B) n-hexane/2-propanol/50 mM ammonium formate aqueous solution, 25:65:10, by vol; flow rate, 0.1 ml/min; gradient elution, see Materials and Methods; injection volume, 10 μl; postcolumn addition, 2.5 mM ammonium formate containing 2-pro panol/methanol (50:50, by vol) at a flow rate of 0.1 ml/min. ESI-MS conditions for simultaneous SIM measurement of 182 ions: polarity, positive; heater temperature of nitrogen gas, 300 C; flow of heated dry nitrogen gas, 8.0 l/min; nebulizer gas pressure, 20 psi; capillary voltage, 3500 V; fragmenter voltage, 150 V; cycle time; 1.15 s/cycle; dwell time for each ion; 7 ms.

Fig. 2.

Correlation between total carbons of 12 authentic CER[NS] and their relative responses in the inter-day analyses. Relative responses are presented as means ± SD. R, correlation coefficient.

Fig. 3.

Average levels of CER classes in three different regions each of the forearm and cheek SC of a 35-year-old male. Levels are presented as means ± SD.

Fig. 4.

Average levels of CER[NS] species in three different regions each of the forearm and cheek SC of a 35-year-old male. Levels are presented as means ± SD.