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

284 JOURNAL OF COSMETIC SCIENCE
probability of correlation between peak area and concentration. The correlation coefficient
of the calibration curve was more than 0.99, which met the validation requirements for this
parameter.16 The linear regression of calibration curves produced the equations presented in
Figure 5, where y was the peak area and x was the analyte concentration in μg/mL.
The slope of the calibration curve was different from zero according to student’s t-test as
recommended, and its high value (1691.8) indicated an appropriate response of the method
against changes in the concentrations. The calculation of residues resulted in random
data distribution with no tendency. The proportional test calculated following student’s
t-test showed that the independent terms (−16933) could be attributed to the acceptable
systematic error of the method. Overall, based on these results HPLC method is indicated
to be linear and to provide all requirements of international protocols for pharmaceutical
analytical methods.
LOD and LOQ. The LOD value of Cer-NP was 7.90 µg/mL. The LOQ value was 24.06 µg/
mL. These values were sufficient to quantify Cer-NP in the skin-simulating liposome
formulation assays such as encapsulation efficiency, in vitro performance tests, etc.
Precision. The precision of the method was facilitated to assess variability because of
random errors which cannot be controlled as those related to reagents glassware and sample
preparation. The precision results (intra- and inter-day) are presented in Table IV. Cer-NP
exhibited acceptable results (from 0.99% to 4.41%) in accordance with the literature25,26 for
intra- and inter-day analysis.
Accuracy. The accuracy of an analytical procedure expresses the closeness of agreement
between the value that is accepted either as a conventional true value or an accepted
reference value and the value found.16 The mean recovery could be within 90% to 110%
of the theoretical value for non-regulated products.27 The accuracy results of the three
analytes ranged from 97.03% to 110.21% (Table IV). Herein, it indicated that there was a
proximity between experimental and theoretical concentration values of the analytes.
Figure 4. Specificity chromatogram of Cer-NP.