282 JOURNAL OF COSMETIC SCIENCE
variations in the chromatographic conditions and lipids’ representative chromatogram of
the developed HPLC method are presented in Table II and Figure 3, respectively.
During the study, the major problem was peak overlapping, which is derived from the
physicochemical similarity of the phosphatidylcholine and Cer-NP.19 Namely, cholesterol
has saturated and unsaturated cyclic hydrocarbons. However, phosphatidylcholine and
Cer-NP similarly have a long fatty acid chain and polar head group, which state the
movement through the column.20 Furthermore, the steric hindrance derived from branching
and cycling indicates partitioning to the pore of column material hence, cholesterol might
have distinctness because of its cyclic hydrocarbons.12 Additionally, the functional groups
on the backbone affect the affinity of the analyte with the stationary phase.12 Herein, the
polarity of the head group on phosphatidylcholine could arise from its phosphate ions while
Cer-NP’s polarity was based on its hydroxyl groups.
On the other hand, the solubility of phosphatidylcholine, Cer-NP, and cholesterol have
differences based on their lipophilicities. Cer-NP has solubility problems in various solvents
and aqueous media.21 Even though cholesterol has a significantly lower lipophilic character
(log P of 8.7) rather than the other compounds (phosphatidylcholine: 12.9, Cer-NP: 12.4),
the chemical structure specified the retention time. Considering the chemical structure
similarity of phosphatidylcholine and Cer-NP, their retention times are expected to be very
similar. Hence, various compositions of mobile phase, flow rates, and injection volumes
were tested to prevent the overlapping of phosphatidylcholine, Cer-NP, and cholesterol
peaks, which could improve the retention times of the peaks of these compounds. The
retention times of lipid materials were studied in different conditions. In addition, tailing
factors (T
f )and the number of theoretical plates (N) of the Cer-NP peak were presented in
Table II. To improve peak resolution, the serial mobile phase composition (trials 1–5) was
tested with the mobile phase consisting of the mixture of methanol: acetonitrile from 60:40
(v/v) to 100:00 (v/v). These subsequent modifications in the mobile phase composition with
a reduction of its polarity caused an increase in lipid retention times. Trial 5 resulted in
low resolution (2.32) with the phosphatidylcholine peak still in conflict with the Cer-NP
peak. Thereby, the flow rate was fixed to be 0.8 mL/min in trials 12–14, and, then 0.5 mL/
min in trials 15–17. Also, the injection volume was decreased from 100 µL to 10 µL, to get
sharper peaks (trials: 12–17). The suitable method was repeated with a C8 column with
Figure 3. Representative HPLC chromatogram of the lipid components in skin-simulating liposome
formulation.

283 CERAMIDE-NP IN SKIN-SIMULATING LIPOSOME FORMULATIONS
the same column dimension and pore diameter to show the stationary phase effect. The
phosphatidylcholine showed an approximate retention time of 11.648, when Cer-NP and
cholesterol were detected at 4.58 and 5.18, individually.
Based on the tailing factor and the number of theoretical plate parameters, the method
in which the mixture of methanol: acetonitrile (60:40, v/v) as mobile phase with
0.5 mL/min flow rate and 10 µL of injection volume showed adequate peak separation
for the lipid components. In this final method shown in Figure 3, the retention times of
phosphatidylcholine, Cer-NP, and cholesterol were 10.7 minutes, 13.5 minutes, and 16.0
minutes, respectively.
The system suitability parameters are determined based on USP guidelines. The resolution
of phosphatidylcholine and Cer-NP was 4.14, which had been a problem in the other
trials. The tailing factor of Cer-NP was less than 1.5 as defined by USP.22,23 The number
of theoretical plates was 5913.479 in agreement with the limit of the FDA’s established
parameter of N 2.000.24 The acceptance criteria of system suitability on USP and Trial
17 values are listed in Table III.
VALIDATION OF METHOD
Specificity. The specificity is a defining parameter that has been the ability of the method to
quantify the analyte of interest in the presence of interferences.16 Herein, it was proposed to
determine whether a contamination peak derived from the manufacturing process formed
in the Cer-NP retention times during the preparation of liposome formulation. In the set
condition analysis, no contamination peak was detected in the Cer-NP retention times.
Moreover, the peak area and retention time of Cer-NP from the liposome formulation
showed no significant difference in comparison with the standard solution of Cer-NP,
confirming the method selectivity (p ≤ 0.05). The specificity chromatogram was shown
in Figure 4.
Linearity. The stock solution was prepared at a lipid concentration of skin-simulating
liposome formulation. The linearity of the method was evaluated at five concentration points
by diluting the standard stock solution to get solutions over the range of 80 µg/mL and
480 µg/mL for each of all lipids according to ICH Q2 (R1).16 The results were plotted graph
concentration versus an area to evaluate the correlation coefficient, which has shown a high
Table III
The Acceptance Criteria of System Suitability and Method Values
Acceptance criteria Method values Equation Suitability
Number of theoretical plate 2000 5913.479 N =16 (tx/wx) Suitable
Selectivity factor 1 1.42 (Phosphatidylcholine and
Cer-NP)
1.21 (Cer-NP and Cholesterol)
α =(ty − tm)/
(tx − tm)
Suitable
Resolution 1.5 4.14 (Phosphatidylcholine and
Cer-NP)
R =(ty − tx)/​
0.5(wy +wx)
Suitable
Tailing factor 2 1.19 Tf =w0.05/2f Suitable
Capacity factor 0.5 – 20 2,64 K =(tx − tm)/tm Suitable
RSD 2 1,13 =SD × 100/mean Suitable