3.2.1 Method validation
The purpose of this analytical procedure is to quantify the isomer impurities of the pidotimod and oral solution. For this reason, method validation was performed according to the corresponding China pharmacopeia guidelines. Typical validation characteristics such as specificity, LOD and LOQ, linearity, accuracy, precision, as well as robustness were evaluated.
i) Linearity, LOD and LOQ
The linearity of the method was generated by regression analysis with seven standard solutions having a concentration range of 0.20–3.5 µg·mL− 1 (equal to 0.02%-0.3% of sample resolution) of S,R and S,S, and 0.70–3.5 µg·mL− 1 (equal to 0.06%-0.3% of sample resolution) of R,S and R,R, respectively. The correlation coefficient curves showed good linearity (r2 > 0.9992) within the test ranges. The LOD and LOQ were very low at about 0.12 ng·mL− 1 and 0.4ng·mL− 1, respectively.
ii) Specificity, accuracy, and precision
Specificity assays are necessary to unequivocally identify the analytes from background noise, and the technique of QDa itself always provides a high degree of specificity. No interference from the formulation excipients or blank solvent was observed at the retention times of the isomers. The system suitability solution showed clear baseline separation of all the isomers, and the resolution was above 1.5. Thus, the analytical method is considered specific. The accuracy of the spiked samples, which had three replicates, varied from 92–109% of the true value. The precision test was carried out by injecting 2 µL of the standard mixture (1.77 µg·mL− 1) for five replicates. The relative standard deviations (RSDs) of S,R, S,S, R,S, and R,R were 1.7%, 2.1%, 1.6%, and 1.5%, respectively. The obtained results are given in Tables 1 and 2.
Table 1
QDa method validation parameters of pidotimod isomers.
Validation parameters
|
(S,R)-
|
(S,S)-
|
(R,S)-
|
(R,R)-
|
Precision (n = 5, RSD)
|
1.7%
|
2.1%
|
1.6%
|
1.5%
|
LOD-LOQ
|
|
|
|
|
LOD (ng)
|
0.12
|
0.13
|
0.41
|
0.42
|
LOQ (ng)
|
0.40
|
0.42
|
1.38
|
1.41
|
Linearity (m/ɀ245)
|
|
|
|
|
Calibration range (µg·mL− 1)
|
0.20–3.34
|
0.21–3.50
|
0.69–3.44
|
0.71–3.54
|
Calibration points
|
7
|
7
|
6
|
6
|
Correlation coefficient(r)
|
0.9998
|
0.9997
|
0.9998
|
0.9996
|
Regression equation
|
y = 303339x -6543.1
|
y = 382843x -17107
|
y = 316393x -2754.3
|
y = 354403x -22501
|
Table 2
QDa method accuracy results.
Standard concentration
(µg/ml)
|
Recovered concentration (µg/ml)
|
Accuracy (%), n = 3
|
S,R
|
S,S
|
R,R
|
S,R
|
S,S
|
R,R
|
0.6688
1.0032
1.3376
|
0.6241 ± 0.0022
0.9329 ± 0.0052
1.2513 ± 0.0369
|
0.7113 ± 0.0113
1.0626 ± 0.0424
1.3760 ± 0.0511
|
0.7803 ± 0.0288
1.1067 ± 0.0026
1.4670 ± 0.0516
|
93.4
(RSD = 0.4%)
92.8
(RSD = 0.6%)
93.0
(RSD = 3.2%)
|
102.0
(RSD = 1.8%)
105.1
(RSD = 4.0%)
97.9
(RSD = 3.8%)
|
108.4
(RSD = 1.8%)
104.3
(RSD = 0.2%)
103.5
(RSD = 3.5%)
|
iii) Stability and Robustness
The sample solution was stable within 48 h. Robustness indicates the reliability of an analytical method when a small deliberate change is made to the method parameters. To that end, we altered the flow rate from 0.8 to 1.0 ml·min − 1, the column temperature from 30 ℃ to 40 ℃, and the mobile solvent composition by ± 2%. In this experiment some of the system suitability parameters such as k’, tailing factor, and USP plate count were measured. The obtained results indicate that there was no significant impact on system suitability. Hence, the method was considered robust.