190 JOURNAL OF COSMETIC SCIENCE
Here, we demonstrate the first clinical evidence of the deposition of d31-PA into the SC of
human skin in vivo from a body wash, leave-on lotion, and a syndet bar formulation, and its
direct conversion into longer-chain FFAs and ceramides.
METHODS
STUDY DESIGN
We conducted two separate Institutional Review Board (IRB)-approved, randomized,
double-blind product application studies. The first study assessed a syndet bar formulation
and a leave-on lotion (Study 1), while the second study assessed body wash formulations
(Study 2). The data from each study were analyzed separately and conducted at Hill Top
Research, Winnipeg, Canada. Ethical approval was granted by IRB Services (Study 1:
MOD00194725, approval granted 29th January 2017 Study 2: MOD00244849, approval
granted 12th January 2018), and complied with the requirements defined in Health Canada
regulations (21 Code of Federal Regulations [CFR] parts 56 and 312.3 and US Department
of Health and Human Services [DHHS] regulations 45 CFR part 46). The IRB adheres
to good clinical practice (GCP) guidelines (e.g., International Council for Harmonisation
of Technical Requirements for Pharmaceuticals for Human Use GCP Guidelines), Health
Canada regulations, and is in compliance with 21 CFR parts 50 and 56, DHHS 45 CFR
part 46 and the Tri-Council Policy Statement for Ethical Conduct of Research Involving
Humans as appropriate to the research. Prior to inclusion, all participants gave their
informed consent to participate.
Skin dryness was graded by an expert assessor in both studies using a 0–6 scale of visual
dryness (“0 =none” to “6 =most severe”) with ½ point increments allowed. Study 1
included 12 healthy female participants aged between 35 to 50 years of age (mean age:
43.5 ± 5.1 years) with no-to-minimal dry skin on their forearms (Grade 0–1). Study 2
included 11 healthy female participants aged between 32 to 50 years of age (mean age:
41.7 ± 7.2 years) with no-to-minimal dry skin on their forearms (Grade 0–1) and moderate
dry skin on their lower legs (Grade 2–3).
Both studies included twice-daily controlled product application by trained researchers.
Applications were approximately three to four hours apart. Participants were instructed not
to use other cleansers during daily bathing or apply lotion to the test sites for the duration
of the studies.
In Study 1, after 3 days of conditioning with a mild syndet cleansing bar, one of the
following fully formulated products was applied to participants’ volar forearms twice daily
under controlled conditions:
• A mild syndet bar formulation containing FFAs and isethionate-based surfactant,
in which FFAs were partially replaced by d31-PA C16:0 (14% final). This bar
was dissolved in a 1:4 dilution with distilled water and 0.3 mL was dispensed onto
the designated pre-wet volar forearm. The solution was rubbed on the whole volar
forearm for 10 seconds, left for 30 seconds, and then rinsed off with tap water for 30
seconds.
• A leave-on lotion (0.3 mL) containing glycerine, petroleum jelly and d31-PA C16:0
(3.65% final). This was applied to the entire volar forearm and rubbed-in. Solvent
extracts and tape strips were collected at baseline and Day 28.

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