191 IN VIVO ELONGATION OF TOPICALLY APPLIED FATTY ACIDS
In Study 2, after 5 days conditioning with a soap bar the body wash formulations were
applied to marked 5 cm × 5 cm test sites (three on each forearm and leg) twice daily
under controlled conditions. The cleansers were fully formulated body wash formulations
containing FFAs and glycinate-based surfactants, in which FFAs were replaced with
different concentrations of d31-PA C16:0 (0.05%, 0.25%, 0.75% and 2%). Masslinn towels
were prepared with 0.15 mL of body wash before washing the pre-wetted test sites for 10
seconds. After 90 seconds, the test sites were rinsed under running water for 15 seconds.22
Tape strips were collected at baseline and Day 28.
LIPID COMPOSITION ANALYSIS
In Study 1, solvent extracts were collected from designated solvent collection sites by three
consecutive applications of 3 mL 80:20 methanol/ethyl acetate to a 3 cm diameter skin
circle, enclosed with a glass ring. The solvent extracts were further re-extracted using a
modified Bligh et al. method.23 Twenty sequential tape strips were collected. Extraction of
the lipids from tape strips was done with 10 mL per tape of 80:20 methanol/ethyl acetate
on a roller at room temperature for three hours.
The resulting extracts were subjected to solid-phase extraction (SPE) on amino-propyl
columns to remove interfering matrix material and partition the samples into ceramide and
FFA fractions. Following SPE column conditioning with organic solvents to remove residual
lipids, ceramide fraction was eluted with 66:33 chloroform: isopropanol FFA fraction
was eluted with methanol +2% acetic acid. Tape strips collected from the body wash
application sites in Study 2 followed the same methods of lipid extraction and fractionation
using SPE amino-propyl columns.
Obtained lipid fractions were detected and semi-quantitated by liquid chromatography
tandem mass spectrometry (LC/MS/MS) using area under the curve values. Deuterated
FFA analysis and deuterated sphingosine (a surrogate marker for ceramides) analyzes were
performed on an ACQUITY UPLC I-Class connected to a Xevo TQ-S mass-spectrometer.
For FFA analysis, samples were separated on a reversed-phase Halo C18 2.1 × 150 mm,
particle size 2.7 µm analytical column, applying mobile phase A (75:25 methanol/water
with 0.1% ammonium acetate) and mobile phase B (99:1 isopropanol/methanol with 0.1%
ammonium acetate) in a gradient elution method. MS/MS measurements were performed
using negative electrospray ionization mode (ESI), utilizing multiple-reaction monitoring
(MRM) to selectively detect and semi-quantitate the following deuterated FFA molecules:
palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0),
lignoceric acid (C24:0) and cerotic acid (C26:0).
For sphingosine analysis, samples were separated on a reversed-phase Phenomenex Kinetex
EVO C18, 2.1 × 150 mm, particle size 1.7 µm analytical column, applying mobile phase
A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) in
a gradient elution method. MS/MS measurements were performed using positive ESI,
utilizing MRM to selectively detect and semi-quantitate deuterated sphingosine released
by acid hydrolysis of the ceramide fraction. MRM methodology is a gold standard in mass
spectrometry, based on the measurement of peak areas for specific parent-to-daughter ion
transitions. This approach allows for reduction of noise and increased accuracy of detection.
Acid hydrolysis of the ceramides was performed using a modification of the Naoi et al.
method.24 Briefly, the ceramide fraction from the SPE column separation was split

192 JOURNAL OF COSMETIC SCIENCE
equally into two aliquots after dissolving in chloroform: methanol (2:1) the solvent was
evaporated at 50°C under a stream of nitrogen gas. The two ceramide aliquots for each
sample were dissolved in methanol. The hydrolysis aliquot was acidified with concentrated
HCl (12.1 mL). The samples were incubated at 60°C for 20 hours, dried and submitted
for analysis.
RESULTS
FFA ELONGATION FROM LEAVE-ON FORMULATION
After four weeks application of the leave-on formulation, elongation of d31-PA was detected
in solvent extracts from all participants. Higher levels of three elongated FFAs (C18:0,
C20:0, and C22:0) were observed versus baseline samples. A consistent trend in the levels
of elongated FFAs was observed across the study population: at Day 28, we detected higher
levels of C22:0 than C20:0 and C18:0, and higher levels of C20:0 than C18:0. Although
all participants showed detectable levels of elongated FFAs, there was variability in the
extent of FFA elongation between participants (Figure 1a). LC/MS/MS analysis of tape
strips showed elongation up to C26:0 in a subset of three participants (Figure 1b).
FFA ELONGATION FROM SYNDET BAR FORMULATION
After four weeks application of the syndet bar formulation, deuterated elongated FFAs were
detected in solvent extract samples from 9 out of 11 participants. Similar to the leave-on
lotion, a trend was observed across participants. Higher levels of C20:0 were detected at
Day 28 than C18:0 in all nine participants, and higher levels of C22:0 than C20:0 were
detected in five of the nine participants (Figure 2a). Elongation up to C26:0 was not detected
in any of the solvent extract samples. However, LC/MS/MS analysis of tape strips showed
formation of C24:0 and C26:0 in a subset of three participants. MRM peak area assessment
revealed that C24:0 was the most prevalent FFA in all tape strip extracts (Figure 2b).
FFA ELONGATED FROM BODY WASH FORMULATIONS
After four weeks application of the body wash formulations to the arm and leg, detectable
levels of elongated FFAs (C20:0 to C24:0) were found in tape strip samples for all body
wash concentrations (Figure 3). Detectable levels of these elongated FFAs were found in all
tape strip samples following application of the 2% d31-PA to the arm (Figure 3a) and leg
(Figure 3b).
DETECTION OF DEUTERATED SPHINGOSINE
Solvent extracts and skin tape strip extracts were analyzed for the presence of deuterated
sphingosine following leave-on lotion, syndet bar and bodywash application. Detectable
levels of d29 sphingosine were observed (Figure 4) these levels increased after acid hydrolysis
for the leave-on lotion (Figure 4a), syndet bar (Figure 4b) and the different concentrations of
body wash application (Figure 4c). This indicates that the d31-PA supplied by the topical