Mixed-Mode Chromatography: Studies on Hybrid Retention Mechanisms of Some Antihypertensive Drugs
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Instrumentation
2.3. Sample Preparation
2.4. Method Performance
3. Results and Discussion
3.1. HPLC Method Development
3.1.1. Setting the Wavelength for DAD Detection
3.1.2. Compound Speciation over pH
3.2. Design of Experiments
− 0.02893 pH × TEA − 0.3929 pH × Flow + 0.01425 TEA × Flow
3.3. Optimized Chromatographic Method
3.4. Study of the Mechanism of Chromatographic Retention
3.4.1. Influence of Mobile Phase Composition on Chromatographic Separation
3.4.2. Effect of Temperature on Chromatographic Separation
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Compound | Acidity Constants * as pKa | Species Structure at pH 6.50/ Charge |
---|---|---|
Indapamide | 0.0000 | |
Atenolol | +0.9983 | |
Metoprolol | +0.9983 | |
Propranolol | +0.9983 | |
Bisoprolol | +0.9983 |
Run Order | Factor Levels | Response | ||
---|---|---|---|---|
pH | TEA Conc., mM | Flow Rate, mL/min | Asymmetry of IDP | |
1 | 6.5 | 20 | 2 | 1.14 |
2 | 7.2 | 0 | 2 | 1.30 |
3 | 6.5 | 0 | 2 | 0.76 |
4 | 6.5 | 20 | 1 | 0.88 |
5 | 6.5 | 0 | 1 | 0.73 |
6 | 7.2 | 20 | 1 | 1.29 |
7 | 7.2 | 20 | 2 | 1.33 |
8 | 7.2 | 0 | 1 | 1.60 |
Term | Effect | Coef | SE Coef | T-Value | p-Value |
---|---|---|---|---|---|
Constant | 1.1288 | 82.09 | 0.008 | ||
pH | 0.5025 | 0.2513 | 0.0138 | 18.27 | 0.035 |
TEA | 0.0625 | 0.0313 | 0.0138 | 2.27 | 0.264 |
Flow | 0.0075 | 0.0038 | 0.0138 | 0.27 | 0.830 |
pH × TEA | –0.2025 | –0.1012 | 0.0138 | –7.36 | 0.086 |
pH × Flow | –0.1375 | –0.0687 | 0.0138 | –5.00 | 0.126 |
TEA × Flow | 0.1425 | 0.0712 | 0.0138 | 5.18 | 0.121 |
Compound | Retention Time (min) | Asymmetry Factor | Resolution |
---|---|---|---|
ATN | 3.23 | 1.24 | 0 |
MTP | 6.45 | 1.37 | 7.46 |
IDP | 11.03 | 1.21 | 5.62 |
BSP | 13.46 | 1.42 | 2.32 |
PPL | 22.10 | 1.47 | 5.89 |
Compound | Slope | Intercept | Correlation Coefficient, R | LOD (µg/L) | LOQ (µg/L) |
---|---|---|---|---|---|
ATN | 4,013,278 | −10,894.63 | 0.9997 | 0.028 | 0.084 |
MTP | 2,749,484 | 29,446.29 | 0.9997 | 0.027 | 0.082 |
IDP | 3,636,080 | −4049.268 | 0.9996 | 0.032 | 0.099 |
BSP | 1,732,658 | 29,179.05 | 0.9999 | 0.012 | 0.037 |
PPL | 9,803,196 | 11,9691.9 | 0.9995 | 0.035 | 0.11 |
Compound | Synthetic Sample (0.4 μg/L) | River Water Sample | ||||
---|---|---|---|---|---|---|
Intra-Day | Inter-Day | Before Spiking *, μg/L | After Spiking **, μg/L | |||
0.32 | 0.40 | 0.48 | ||||
RSD % | Recovery ± RSD % | |||||
ATN | 0.95 | 1.24 | <LOD | 97.51 ± 3.77 | 99.51 ± 2.70 | 101.4 ± 1.90 |
MTP | 1.02 | 1.56 | <LOD | 103.3 ± 3.85 | 105.0 ± 6.76 | 103.7 ± 4.26 |
IDP | 1.46 | 0.98 | <LOD | 101.0 ± 4.29 | 97.97 ± 2.71 | 99.63 ± 2.62 |
BSP | 1.35 | 1.61 | <LOD | 97.39 ± 5.41 | 97.11 ± 4.46 | 92.73 ± 4.68 |
PPL | 1.74 | 1.87 | <LOD | 95.91 ± 6.34 | 94.22 ± 4.05 | 96.37 ± 5.31 |
ATN | MTP | IDP | BSP | PPL | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Parameter | Variation | Rt (min) | R | Rt (min) | R | Rt (min) | R | Rt (min) | R | Rt (min) | R |
Wavelength, nm | 222 | 3.26 | 0.00 | 6.39 | 7.55 | 11.16 | 5.57 | 13.56 | 2.27 | 22.51 | 5.78 |
226 | 3.23 | 0.00 | 6.45 | 7.45 | 11.03 | 5.62 | 13.46 | 2.31 | 22.10 | 5.89 | |
230 | 3.24 | 0.00 | 6.55 | 7.57 | 11.21 | 5.59 | 13.68 | 2.32 | 22.47 | 6.60 | |
Mobile phase pH | 7.10 | 3.24 | 0.00 | 6.42 | 7.54 | 11.06 | 5.61 | 13.34 | 2.27 | 22.29 | 5.80 |
7.20 | 3.23 | 0.00 | 6.45 | 7.45 | 11.03 | 5.62 | 13.46 | 2.31 | 22.10 | 5.89 | |
7.30 | 3.19 | 0.00 | 6.38 | 7.56 | 10.92 | 5.65 | 13.44 | 2.33 | 22.20 | 5.93 | |
Temperature, °C | 23 | 3.25 | 0.00 | 6.53 | 7.48 | 10.89 | 5.69 | 13.60 | 2.28 | 21.78 | 5.91 |
25 | 3.23 | 0.00 | 6.45 | 7.45 | 11.03 | 5.62 | 13.46 | 2.31 | 22.10 | 5.89 | |
27 | 3.22 | 0.00 | 6.50 | 7.40 | 11.12 | 5.71 | 13.70 | 2.30 | 22.25 | 5.97 |
K | log k | %MeOH | Equation | R | |
---|---|---|---|---|---|
Intercept | Slope | ||||
ATN | |||||
2.05 | 0.312 | 20 | 0.425 | −0.0063 | 0.9421 |
1.78 | 0.249 | 25 | |||
1.69 | 0.228 | 30 | |||
1.63 | 0.213 | 35 | |||
MTP | |||||
11.0 | 1.04 | 20 | 0.145 | −0.0329 | 0.9762 |
6.02 | 0.780 | 25 | |||
4.37 | 0.640 | 30 | |||
3.46 | 0.539 | 35 | |||
IDP | |||||
28.2 | 1.45 | 20 | 0.104 | −0.0503 | 0.9946 |
13.1 | 1.12 | 25 | |||
8.19 | 0.913 | 30 | |||
4.79 | 0.680 | 35 | |||
BSP | |||||
41.4 | 1.62 | 20 | 0.172 | −0.0528 | 0.9868 |
17.6 | 1.24 | 25 | |||
10.2 | 1.01 | 30 | |||
6.56 | 0.817 | 35 | |||
PPL | |||||
51.8 | 1.71 | 20 | 0.0445 | −0.0446 | 0.9987 |
28.9 | 1.46 | 25 | |||
17.4 | 1.24 | 30 | |||
10.9 | 1.04 | 35 |
Temperature (K) | 1/T | k | ln k | Equation | ΔH (kJ/mol) | ΔS J/(mol·K) | ||
---|---|---|---|---|---|---|---|---|
Intercept | Slope | R | ||||||
ATN | ||||||||
298.15 | 0.0033540 | 1.692 | 0.52567 | −0.7967 | 393.9 | 0.9986 | −3.27 | −6.62 |
308.15 | 0.0032451 | 1.617 | 0.48039 | |||||
313.15 | 0.0031933 | 1.583 | 0.45951 | |||||
318.15 | 0.0031432 | 1.558 | 0.44359 | |||||
MTP | ||||||||
298.15 | 0.0033540 | 4.375 | 1.4759 | −0.5783 | 611.9 | 0.9987 | −5.08 | −4.80 |
308.15 | 0.0032451 | 4.075 | 1.4049 | |||||
313.15 | 0.0031933 | 3.951 | 1.3737 | |||||
318.15 | 0.0031432 | 3.854 | 1.3480 | |||||
IDP | ||||||||
298.15 | 0.0033540 | 8.092 | 2.0908 | −5.305 | 2208 | 0.9970 | −18.3 | −44.1 |
308.15 | 0.0032451 | 6.552 | 1.8794 | |||||
313.15 | 0.0031933 | 5.775 | 1.7535 | |||||
318.15 | 0.0031432 | 5.059 | 1.6210 | |||||
BSP | ||||||||
298.15 | 0.0033540 | 10.25 | 2.3272 | +0.5865 | 518.1 | 0.9992 | −4.30 | +4.81 |
308.15 | 0.0032451 | 9.609 | 2.2626 | |||||
313.15 | 0.0031933 | 9.367 | 2.2371 | |||||
318.15 | 0.0031432 | 9.208 | 2.2201 | |||||
PPL | ||||||||
298.15 | 0.0033540 | 17.37 | 2.8550 | −2.091 | 1475 | 0.9997 | −12.3 | −17.2 |
308.15 | 0.0032451 | 14.89 | 2.7008 | |||||
313.15 | 0.0031933 | 13.71 | 2.6180 | |||||
318.15 | 0.0031432 | 12.74 | 2.5449 |
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Badea, I.A.; Mihăilă, A.; Popa, D.E.; Tencaliec, A.M.; Buleandră, M. Mixed-Mode Chromatography: Studies on Hybrid Retention Mechanisms of Some Antihypertensive Drugs. Separations 2025, 12, 136. https://doi.org/10.3390/separations12060136
Badea IA, Mihăilă A, Popa DE, Tencaliec AM, Buleandră M. Mixed-Mode Chromatography: Studies on Hybrid Retention Mechanisms of Some Antihypertensive Drugs. Separations. 2025; 12(6):136. https://doi.org/10.3390/separations12060136
Chicago/Turabian StyleBadea, Irinel Adriana, Andrei Mihăilă, Dana Elena Popa, Anca Monica Tencaliec, and Mihaela Buleandră. 2025. "Mixed-Mode Chromatography: Studies on Hybrid Retention Mechanisms of Some Antihypertensive Drugs" Separations 12, no. 6: 136. https://doi.org/10.3390/separations12060136
APA StyleBadea, I. A., Mihăilă, A., Popa, D. E., Tencaliec, A. M., & Buleandră, M. (2025). Mixed-Mode Chromatography: Studies on Hybrid Retention Mechanisms of Some Antihypertensive Drugs. Separations, 12(6), 136. https://doi.org/10.3390/separations12060136