Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Equipment
2.3. Chromatographic Conditions
2.4. Elaboration of PLGA Microspheres
2.5. Test Sample Preparation
2.5.1. Encapsulation Efficiency Assay
2.5.2. In Vitro Release Assay
2.6. Standard Solutions Preparation
2.6.1. Encapsulation Efficiency Assay
2.6.2. In Vitro Release Assay
2.7. Validation Study
2.7.1. System Suitability Testing
2.7.2. Specificity
2.7.3. Linearity
2.7.4. Precision
2.7.5. Accuracy
2.7.6. Sensitivity
2.7.7. Robustness
2.8. Statistical Analysis
3. Results and Discussion
3.1. Validation Procedure
3.1.1. System Suitability Testing
3.1.2. Specificity
3.1.3. Linearity
3.1.4. Precision
3.1.5. Accuracy
3.1.6. Sensitivity
3.1.7. Robustness
3.2. Methods Applicability
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
Abbreviations
HPLC-UV | high performance liquid chromatography with ultraviolet detection |
UV | ultraviolet |
PLGA | poly(D,L-lactic-co-glycolic) acid |
DX | dexamethasone |
MEL | melatonin |
MSs | microspheres |
DX-MEL-MSs | dexamethasone and melatonin co-loaded PLGA microspheres |
RGC | retinal ganglion cells |
IOP | intraocular pressure |
LC/MS | liquid chromatography tandem mass spectrometry |
MeOH | methanol |
DCM | dichloromethane |
PVA | polyvinyl alcohol |
blank-MSs | unloaded microspheres |
EE | encapsulation efficiency assay |
SD | standard deviation |
PBS | phosphate buffer solution |
IVR | in vitro release assay |
SS | stock standard solution |
SS-EE | stock standard solution for encapsulation efficiency assay |
SS-IVR | stock standard solution for in vitro release assay |
WS | working solution |
WS-EE | working solution for encapsulation efficiency assay |
WS-IVR | working solution for in vitro release assay |
ICH | International Conference on Harmonization |
FDA | Food and Drug Administration |
RSD | relative standard deviation |
LOD | limit of detection |
LOQ | limit of quantification |
tR | retention time |
tW | peak width |
Rs | resolution between peaks |
IR | injection repeatability |
T | tailing factor |
N | theoretical plate number |
pS | peak symmetry |
SEM | scanning electron microscopy |
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Procedure A (for EE Determination) | Procedure B (for IVR Quantitation) | |||
---|---|---|---|---|
50 µg mL−1 | 10 µg mL−1 | |||
MEL | DX | MEL | DX | |
tR | 2.91 (0.24) | 4.69 (0.24) | 2.94 (0.13) | 4.73 (0.11) |
tW | 0.65 (1.04) | 0.63 (2.80) | 0.59 (1.54) | 0.73 (1.92) |
Rs | 3.42 (0.16) | 4.39 (0.98) | ||
IR | 0.11 | 0.21 | 0.42 | 0.45 |
T | 1.21 (0.15) | 1.20 (0.38) | 1.46 (2.09) | 1.34 (1.30) |
N | 3309.12 (1.96) | 5029.93 (1.00) | 4531.83 (2.49) | 5732.29 (2.75) |
Procedure A | Procedure B | |||
---|---|---|---|---|
MEL | DX | MEL | DX | |
Slope | 64,272.9 | 22,435.8 | 62,259.9 | 21,169.4 |
Standard error slope | 185.52 | 80.76 | 202.99 | 82.05 |
Intercept | −6243.69 | 3504.68 | −3275.71 | 916.62 |
Standard error intercept (σ) | 4878.03 | 2123.61 | 2046.99 | 827.37 |
t-Statistic slope (p) | 346.46 (0.000) * | 277.80 (0.000) * | 306.71 (0.000) * | 258.01 (0.000) * |
t-Statistic intercept (p) | −1.28 (0.2134) | 1.65 (0.113) | −1.60 (0.124) | 1.11 (0.280) |
Correlation coefficient (R) | 0.9999 | 0.9999 | 0.9999 | 0.9998 |
“Χ2” Bartlett’s test (p) | 11.89 (0.064) | 4.96 (0.549) | 6.54 (0.365) | 9.87 (0.130) |
ANOVA F-test for regression (p) | 120,032.96 (0.000) * | 77,170.01 (0.000) * | 94,070.56 (0.000) * | 66,571.46 (0.000) * |
Procedure A | Procedure B | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Conc | MEL | DX | Conc | MEL | DX | ||||||||
Day 1 | Day 2 | Day 3 | Day 1 | Day 2 | Day 3 | Day 1 | Day 2 | Day 3 | Day 1 | Day 2 | Day 3 | ||
5 | 100.78 | 99.79 | 101.70 | 101.73 | 100.67 | 102.44 | 2.5 | 100.90 | 99.11 | 100.11 | 101.63 | 101.24 | 98.46 |
100.82 | 99.52 | 101.79 | 101.96 | 102.57 | 101.32 | 101.00 | 98.82 | 99.75 | 100.88 | 101.16 | 97.26 | ||
100.32 | 100.86 | 99.09 | 98.49 | 100.86 | 102.37 | 99.95 | 101.52 | 98.89 | 99.02 | 99.90 | 99.17 | ||
100.12 | 100.18 | 99.99 | 98.79 | 100.98 | 102.33 | 100.17 | 101.37 | 98.82 | 98.14 | 100.20 | 99.50 | ||
99.83 | 100.58 | 98.07 | 102.08 | 99.35 | 99.11 | 98.80 | 99.04 | 100.74 | 100.13 | 97.47 | 101.25 | ||
99.52 | 100.31 | 97.87 | 101.88 | 99.10 | 99.54 | 98.45 | 98.67 | 100.57 | 99.41 | 97.26 | 101.94 | ||
20 | 98.87 | 100.27 | 98.28 | 98.54 | 100.03 | 98.53 | 7.5 | 100.00 | 100.86 | 99.97 | 100.26 | 101.89 | 100.06 |
98.57 | 100.32 | 98.57 | 98.68 | 99.95 | 98.71 | 100.10 | 101.00 | 99.83 | 100.17 | 101.75 | 100.18 | ||
101.57 | 101.30 | 101.63 | 101.36 | 101.61 | 101.11 | 98.49 | 99.75 | 98.25 | 98.16 | 99.86 | 98.28 | ||
101.70 | 100.75 | 101.10 | 101.70 | 101.80 | 101.09 | 98.37 | 99.79 | 98.41 | 98.38 | 99.78 | 98.38 | ||
100.44 | 99.06 | 100.62 | 100.37 | 99.15 | 100.47 | 101.31 | 98.29 | 101.00 | 101.58 | 98.38 | 102.00 | ||
100.15 | 98.90 | 100.01 | 100.59 | 99.22 | 100.16 | 100.95 | 98.22 | 101.11 | 101.95 | 99.25 | 101.69 | ||
40 | 100.58 | 98.12 | 100.64 | 101.33 | 99.87 | 101.92 | 15 | 100.80 | 99.47 | 101.05 | 99.87 | 100.04 | 101.05 |
100.65 | 98.46 | 101.06 | 101.27 | 99.70 | 101.88 | 100.60 | 99.65 | 100.37 | 99.77 | 100.92 | 101.30 | ||
100.69 | 100.91 | 100.85 | 100.99 | 100.60 | 100.50 | 98.29 | 101.26 | 100.90 | 101.62 | 101.27 | 99.22 | ||
100.72 | 101.08 | 100.95 | 101.44 | 100.90 | 100.31 | 98.25 | 101.37 | 100.80 | 101.71 | 101.68 | 99.13 | ||
98.49 | 101.19 | 98.68 | 99.59 | 101.77 | 98.36 | 101.27 | 100.92 | 99.43 | 98.82 | 99.99 | 100.02 | ||
98.67 | 101.28 | 99.00 | 99.65 | 101.82 | 98.50 | 101.33 | 101.08 | 99.30 | 97.85 | 99.23 | 100.01 |
Procedure A | Procedure B | |||
---|---|---|---|---|
MEL | DX | MEL | DX | |
Average (recovery percentages) | 100.10 | 100.54 | 99.97 | 99.99 |
RSD (%) repeatability | 1.11 | 1.26 | 1.10 | 1.38 |
RSD (%) intermediate precision | 1.09 | 1.24 | 1.08 | 1.36 |
“W” Levene’s test (p) | 2.58 (0.085) | 0.98 (0.381) | 1.35 (0.269) | 0.05 (0.950) |
ANOVA F-test inter-day (p) | 0.12 (0.889) | 0.03 (0.971) | 0.02 (0.983) | 0.05 (0.955) |
Procedure A | Procedure B | |||
---|---|---|---|---|
MEL | DX | MEL | DX | |
Average (recovery percentages) | 100.10 | 100.54 | 99.97 | 99.99 |
RSD (recovery percentages, %) | 1.09 | 1.24 | 1.08 | 1.36 |
Confidence interval recovery percentages | 99.80–100.40 | 100.20–100.88 | 99.68–100.27 | 99.62–100.36 |
“W” Levene’s test (p) | 1.02 (0.369) | 1.36 (0.265) | 0.16 (0.852) | 1.10 (0.341) |
ANOVA F-test inter-day (p) | 0.01 (0.987) | 1.44 (0.248) | 1.63 (0.206) | 0.78 (0.465) |
MEL | DX | |||||||
---|---|---|---|---|---|---|---|---|
Day | Conc | Average | SD | RSD (%) | Conc | Average | SD | RSD (%) |
1 | 0.34 | 0.35 | 0.03 | 8.09 | 0.39 | 0.40 | 0.02 | 5.77 |
0.32 | 0.37 | |||||||
0.33 | 0.42 | |||||||
0.34 | 0.43 | |||||||
0.40 | 0.39 | |||||||
0.35 | 0.42 | |||||||
2 | 0.38 | 0.35 | 0.03 | 9.07 | 0.40 | 0.41 | 0.02 | 5.19 |
0.33 | 0.42 | |||||||
0.37 | 0.39 | |||||||
0.39 | 0.43 | |||||||
0.32 | 0.44 | |||||||
0.32 | 0.39 | |||||||
3 | 0.33 | 0.34 | 0.01 | 3.66 | 0.39 | 0.41 | 0.02 | 4.62 |
0.35 | 0.42 | |||||||
0.33 | 0.41 | |||||||
0.35 | 0.43 | |||||||
0.32 | 0.38 | |||||||
0.33 | 0.40 | |||||||
Average | 0.34 | 0.41 | ||||||
SD | 0.03 | 0.02 | ||||||
RSD (%) | 7.41 | 5.22 |
Parameter | Mobile Phase (% MeOH) | Column Oven Temperature (°C) | Detection Wavelength (nm) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Value | 68 | 70 | 72 | 43 | 45 | 47 | 220.7 238.5 | 222.7 240.5 | 224.7 242.5 | ||
Procedure A | MEL | Average (recovery%) | 99.44 | 100.54 | 101.47 | 98.99 | 100.84 | 101.63 | 98.40 | 100.10 | 99.18 |
RSD (recovery%) | 0.26 | 0.44 | 0.35 | 0.55 | 0.73 | 0.63 | 0.31 | 0.42 | 0.54 | ||
tR (RSD%) | 2.97 (0.21) | 2.89 (0.21) | 2.82 (0.19) | 2.91 (0.18) | 2.89 (0.17) | 2.87 (0.23) | 2.86 (0.21) | 2.88 (0.16) | 2.88 (0.16) | ||
pS (RSD%) | 1.34 (1.11) | 1.31 (1.44) | 1.33 (1.27) | 1.32 (0.48) | 1.34 (1.54) | 1.32 (0.70) | 1.33 (1.09) | 1.35 (0.96) | 1.33 (0.68) | ||
DX | Average (recovery%) | 99.36 | 100.53 | 101.06 | 98.66 | 100.90 | 101.74 | 99.74 | 100.10 | 99.14 | |
RSD (recovery%) | 0.58 | 1.01 | 0.41 | 0.98 | 0.82 | 0.89 | 0.88 | 1.01 | 0.95 | ||
tR (RSD%) | 5.16 (0.15) | 4.68 (0.13) | 4.28 (0.10) | 4.78 (0.09) | 4.68 (0.14) | 4.58 (0.15) | 4.66 (0.09) | 4.67 (0.13) | 4.66 (0.13) | ||
pS (RSD%) | 1.38 (0.73) | 1.35 (0.69) | 1.39 (1.43) | 1.32 (0.37) | 1.37 (0.98) | 1.38 (0.42) | 1.37 (0.87) | 1.39 (0.75) | 1.37 (0.57) | ||
Procedure B | MEL | Average (recovery%) | 98.13 | 100.42 | 99.13 | 98.76 | 99.78 | 101.14 | 98.90 | 100.34 | 98.53 |
RSD (recovery%) | 0.77 | 0.79 | 0.67 | 0.48 | 0.53 | 0.55 | 0.60 | 0.35 | 0.32 | ||
tR (RSD%) | 3.01 (0.24) | 2.94 (0.22) | 2.87 (0.24) | 2.96 (0.22) | 2.94 (0.15) | 2.92 (0.21) | 2.92 (0.24) | 2.92 (0.23) | 2.92 (0.23) | ||
pS (RSD%) | 1.40 (1.90) | 1.43 (1.75) | 1.70 (1.86) | 1.49 (1.81) | 1.44 (1.78) | 1.52 (1.40) | 1.58 (1.71) | 1.52 (1.91) | 1.60 (1.77) | ||
DX | Average (recovery%) | 99.56 | 100.74 | 98.85 | 98.87 | 99.56 | 101.56 | 98.32 | 99.94 | 98.86 | |
RSD (recovery%) | 0.63 | 0.86 | 1.18 | 0.83 | 0.95 | 0.32 | 0.54 | 0.41 | 0.58 | ||
tR (RSD%) | 5.21 (0.16) | 4.75 (0.15) | 4.34 (0.17) | 4.85 (0.14) | 4.75 (0.06) | 4.64 (0.14) | 4.72 (0.18) | 4.72 (0.18) | 4.72 (0.18) | ||
pS (RSD%) | 1.40 (1.77) | 1.43 (1.73) | 1.62 (1.05) | 1.51 (1.98) | 1.41 (1.82) | 1.46 (1.76) | 1.54 (1.63) | 1.54 (1.60) | 1.56 (1.57) |
Injection Volume | |||||
---|---|---|---|---|---|
5 µL | 10 µL | 15 µL | |||
Procedure A | MEL | tR (RSD%) | 2.90 (0.17) | 2.91 (0.24) | 2.90 (0.10) |
pS (RSD%) | 1.26 (0.51) | 1.21 (0.15) | 1.15 (0.17) | ||
Area responses (mean ± RSD%) | 1,615,543.83 ± 0.11 | 3,225,811.17 ± 0.11 | 4,836,931.33 ± 0.11 | ||
p-value intercept (ANOVA) | 0.072 | ||||
p-value slope (ANOVA) | <0.001 | ||||
Correlation coefficient (R) | >0.999 | ||||
DX | tR (RSD%) | 4.68 (0.11) | 4.69 (0.24) | 4.67 (0.08) | |
pS (RSD%) | 1.23 (0.19) | 1.20 (0.38) | 1.21 (0.36) | ||
Area responses (mean ± RSD%) | 558,907.50 ± 0.13 | 1,121,940.50 ± 0.21 | 1,678,211.33 ± 0.11 | ||
p-value intercept (ANOVA) | 0.942 | ||||
p-value slope (ANOVA) | 0.002 | ||||
Correlation coefficient (R) | >0.999 | ||||
Procedure B | MEL | tR (RSD%) | 2.94 (0.31) | 2.94 (0.13) | 2.96 (0.14) |
pS (RSD%) | 1.31 (1.80) | 1.46 (1.89) | 1.40 (1.72) | ||
Area responses (mean ± RSD%) | 309,225.00 ± 0.17 | 623,427.333 ± 0.41 | 927,817.00 ± 0.42 | ||
p-value intercept (ANOVA) | 0.846 | ||||
p-value slope (ANOVA) | 0.006 | ||||
Correlation coefficient (R) | >0.999 | ||||
DX | tR (RSD%) | 4.72 (0.20) | 4.73 (0.11) | 4.75 (0.15) | |
pS (RSD%) | 1.21 (1.21) | 1.34 (1.30) | 1.34 (1.83) | ||
Area responses (mean ± RSD%) | 106,708.67 ± 0.44 | 211,243.50 ± 0.44 | 314,313.67 ± 0.29 | ||
p-value intercept (ANOVA) | 0.180 | ||||
p-value slope (ANOVA) | 0.003 | ||||
Correlation coefficient (R) | >0.999 |
Procedure A | Procedure B | |||
---|---|---|---|---|
Conc | MEL | DX | MEL | DX |
Average ± SD (n = 4) 5 min | 17.59 ± 0.18 | 76.69 ± 2.10 | 3.33 ± 0.07 | 4.38 ± 0.10 |
Average ± SD (n = 4) 10 min | 17.47 ± 0.30 | 76.69 ± 1.39 | 3.21 ± 0.11 | 4.41 ± 0.10 |
Average ± SD (n = 4) 15 min | 17.62 ± 0.39 | 77.34 ± 1.89 | 3.29 ± 0.09 | 4.41 ± 0.11 |
Average ± SD (n = 4) 20 min | 17.30 ± 0.44 | 76.30 ± 2.06 | 3.29 ± 0.08 | 4.41 ± 0.11 |
Average ± SD (n = 4) 25 min | 17.52 ± 0.41 | 76.52 ± 1.51 | 3.18 ± 0.07 | 4.43 ± 0.09 |
Average ± SD (n = 4) 30 min | 17.36 ± 0.50 | 75.08 ± 1.29 | 3.24 ± 0.06 | 4.38 ± 0.11 |
ANOVA F-test inter-group (p) | 0.43 (0.822) | 0.74 (0.603) | 1.08 (0.405) | 0.11 (0.988) |
SD (all measurements) | 0.36 | 1.69 | 0.09 | 0.09 |
RSD (all measurements) (%) | 2.06 | 2.21 | 2.65 | 2.09 |
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Brugnera, M.; Vicario-de-la-Torre, M.; Andrés-Guerrero, V.; Bravo-Osuna, I.; Molina-Martínez, I.T.; Herrero-Vanrell, R. Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release. Pharmaceutics 2022, 14, 288. https://doi.org/10.3390/pharmaceutics14020288
Brugnera M, Vicario-de-la-Torre M, Andrés-Guerrero V, Bravo-Osuna I, Molina-Martínez IT, Herrero-Vanrell R. Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release. Pharmaceutics. 2022; 14(2):288. https://doi.org/10.3390/pharmaceutics14020288
Chicago/Turabian StyleBrugnera, Marco, Marta Vicario-de-la-Torre, Vanessa Andrés-Guerrero, Irene Bravo-Osuna, Irene Teresa Molina-Martínez, and Rocío Herrero-Vanrell. 2022. "Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release" Pharmaceutics 14, no. 2: 288. https://doi.org/10.3390/pharmaceutics14020288
APA StyleBrugnera, M., Vicario-de-la-Torre, M., Andrés-Guerrero, V., Bravo-Osuna, I., Molina-Martínez, I. T., & Herrero-Vanrell, R. (2022). Validation of a Rapid and Easy-to-Apply Method to Simultaneously Quantify Co-Loaded Dexamethasone and Melatonin PLGA Microspheres by HPLC-UV: Encapsulation Efficiency and In Vitro Release. Pharmaceutics, 14(2), 288. https://doi.org/10.3390/pharmaceutics14020288