Table
II
The
Chromatographic
Conditions
of
Trials
Mobile
Phase
System,
Flow
Rate
and
Injection
Volume,
and
Results
the
Retention
Times,
Tailing
Factors,
and
the
Number
of
Theoretical
Plates
of
Cer-NP
Chromatograms
HPLC
conditions
Retention
time
(min)
Tailing factor
Number
of theoretical plates
Suitability
Mobile
phase
system (MeOH:ACN)
Flow
rate
Stationary phase
Injection volume
Phosphatidylcholine
Cer-NP
Cholesterol
Cer-NP
Cer-NP
Peak
quality
Trial
1
10:0
1
C18
100
5.7
6.3
8.3
1.162
320.7
Peak
depletion
Trial
2
9:1
1
C18
100
4.9
6.7
8.6
1.336
2569.6
Peak
depletion
Trial
3
8:2
1
C18
100
6.7
7.3
9.1
1.169
5969.9
Overlapping
Trial
4
7:3
1
C18
100
7.1
8.2
9.9
2.693
1257.1
Overlapping
Trial
5
6:4
1
C18
100
7.3
8.9
10.4
1.383
4759.4
Unqualified
Trial
6
9:1
0.8
C18
80
6.6
ND
8.4
ND
ND
Peak
depletion
Trial
7
9:1
0.8
C18
50
6.4
ND
8.2
ND
ND
Peak
depletion
Trial
8
9:1
0.8
C18
10
6.4
ND
8.1
ND
ND
Peak
depletion
Trial
9
9:1
0.5
C18
80
10.3
ND
13.2
ND
ND
Overlapping
Trial
10
9:1
0.5
C18
50
10.3
ND
13.1
ND
ND
Overlapping
Trial
11
9:1
0.5
C18
10
10.4
ND
13.2
ND
ND
Overlapping
Trial
12
6:4
0.8
C18
80
6.9
8.4
10.3
1.303
3594.0
Suitable
Trial
13
6:4
0.8
C18
50
6.6
8.1
9.9
ND
ND
Unqualified
Trial
14
6:4
0.8
C18
10
6.9
8.9
10.3
1.258
5983.8
Suitable
Trial
15
6:4
0.5
C18
80
10.5
13.1
15.9
ND
ND
Unqualified
Trial
16
6:4
0.5
C18
50
10.5
13.0
15.8
0.851
3792.8
Suitable
Trial
17
6:4
0.5
C18
10
10.7
13.5
15.8
1.194
5913.4
Suitable
Trial
17’
6:4
0.5
C8
10
11.6
ND
ND
1.067
ND
Peak
depletion
Note:
ND
=
Not
determined.
280 JOURNAL OF COSMETIC SCIENCE

281 CERAMIDE-NP IN SKIN-SIMULATING LIPOSOME FORMULATIONS
proportionality tests were evaluated based on the residual variance using the student t-test
(p =0.05). The residues were calculated based on the difference between theoretical and
experimental values, which were estimated from the calibration curve.18
LOD and LOQ. The LOD and LOQ values of the method were calculated from the
calibration curves according to the following equations based on the standart deviation of
the response and slope:17
LOD SD
S =× 3 3 .
LOQ SD
S =× 10
SD is the mean standard deviation of y-axis interception values of calibration curves
S is the mean angular coefficient of calibration curves.
Precision. The precision of the method was evaluated for two levels: repeatability (intra-
assay) and intermediate (inter-assay) precision. The intra-assay precision was performed
with liposome analytes at 80%, 100%, and 120% (320, 400, 480 µg/mL) using three
replicates within a day which qualified the ICH Q2(R1) specifications.16 The inter-assay
precision was performed on three different days at analyte concentrations of (320, 400, and
480 µg/mL) in three replicates using refresh samples prepared by the same analysis. The
results of precision were calculated as the coefficient of variation (CV) in each level for each
analyte concentration, using the following equation:
CV deviation of the peak areas for each analyte/ =(standard
a average of the peak areas for each analyte) × 100
Accuracy. The accuracy of the method was carried out at 80%, 100%, and 120% of
liposome analytes (320, 400, 480 µg/mL) of Cer-NP based on ICH Q2(R1).16 The liposome
formulation including a 600 µg/mL concentration of analytes was prepared with the
appropriate dilutions. The experiments were performed in three replicates. The results of
accuracy were calculated based on the peak areas of Cer-NP in terms of recovery (R), as
described in the following equation:
R measured concentration in the liposomes/
measured concent
=(
tration in the solutions) × 100
RESULTS AND DISCUSSION
METHOD DEVELOPMENT AND OPTIMIZATION OF CHROMATOGRAPHIC CONDITIONS
Three compounds (phosphatidylcholine, Cer-NP, and cholesterol) exhibited the maximum
absorption at 210 nm hence the wavelength was standardized in all studies. The main