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Development and Validation of Bioanalytical Method for Determination of Nebivolol and Valsartan in Human Plasma by Using RP-HPLC

R. N. Kachave
R. N. Kachave
 and 
A. G. Mundhe
A. G. Mundhe
   | Mar 12, 2022
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Article Category: Original Paper
Published Online: Mar 12, 2022
Page range: 49 - 58
Received: Apr 14, 2018
Accepted: Jun 01, 2018
DOI: https://doi.org/10.2478/afpuc-2021-0019
Keywords
© 2021 R. N. Kachave et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1a

Structure of Nebivolol.
Structure of Nebivolol.

Figure 1b

Structure of Valsartan.
Structure of Valsartan.

Figure 2

Chromatogram of Optimized Chromatographic Method.
Chromatogram of Optimized Chromatographic Method.

Figure 3

Extracted standard blank sample.
Extracted standard blank sample.

Figure 4a

Calibration Curve of Nebivolol.
Calibration Curve of Nebivolol.

Figure 4b

Calibration Curve of Valsartan.
Calibration Curve of Valsartan.

j.afpuc-2021-0019.tab.010

ABBREVIATIONS FORMS
NL Nebivolol
VL Valsartan
RP Reverse phase
HPLC High-performance liquid chromatography
BA Bioavailability
BE Bioequivalence
API Active pharmaceutical ingredient
UPLC Ultra-performance liquid chromatography
UV Ultraviolet
QC Quality control
RT Retention time
USP United States Pharmacopeia
CV Coefficient of variation
IS Internal standard
SD Standard deviation

Matrix samples’ stability of nebivolol and valsartan at −70°C ± 5°C for 37 days.

Parameters Nebivolol Valsartan
HQC (comparison sample) HQC (stability sample) LQC (comparison sample) LQC (stability sample) HQC (comparison sample) HQC (stability sample) LQC (comparison sample) LQC (stability sample)
n 6 6 6 6 6 6 6 6
Mean 7.9880 8.0145 1.4953 1.4902 6386.5000 6401.5000 1194.5000 1188.6667
SD 0.02458 0.05858 0.02273 0.01646 22.29574 21.37054 17.14351 14.17980
% CV 0.31 0.73 1.52 1.10 0.35 0.33 1.44 1.19
% Mean accuracy 99.85 100.18 99.69 99.34 99.79 100.02 99.54 99.06
% Mean stability 100.33 99.65 100.23 99.51

Observation of optimised method chromatogram.

System suitability parameters Atorvastatin Nebivolol Valsartan
RT 2.597 3.189 4.374
Area 97,579 5671 11,302
USP plate count 9792.8 9135.0 8011.3
USP tailing 1.4 1.4 1.1
USP resolution - 4.6 6.9
Area ratio - 0.05686 0.3545
SD - 0.0316 1.00
% CV - 0.0512 1.18

Matrix factor evaluation of nebivolol and valsartan.

Parameters Nebivolol Valsartan
HQC LQC HQC LQC
n 18 18 18 18
Mean 7.9592 1.4937 6400.5556 1190.4444
SD 0.07785 0.01768 44.02035 30.91016
% CV 0.98 1.18 0.69 2.60
% Mean accuracy 99.49 99.58 100.01 99.20
No. of QC failed 0 0 0 0

Intra-day precision and accuracy of nebivolol and valsartan.

Parameters Nebivolol Valsartan
HQC MQC LQC LLOQ QC HQC MQC LQC LLOQ QC
n 6 6 6 6 6 6 6 6
Mean 7.9513 4.9662 1.4943 0.4985 6403.1667 4017.5000 1196.000 396.5000
SD 0.05211 0.10071 0.01707 0.01320 10.60974 107.61924 11.6666 8.06846
% CV 0.66 2.03 1.14 2.65 0.17 2.68 0.97 2.03
% Mean accuracy 99.39 99.32 99.62 99.70 100.05 100.44 100.44 99.13

Observation table for linearity of nebivolol and valsartan.

Nebivolol Valsartan
Final conc. in ng/mL AUC Area ratio Final conc. in ng/mL AUC Area ratio
0.5 570 0.006 400 3415 0.0350
1 1235 0.013 800 6724 0.0689
1.5 1785 0.018 1200 10,160 0.1041
4 4643 0.048 3200 26,678 0.2731
5 5880 0.060 4000 34,216 0.3506
6 6902 0.071 4800 41,162 0.4217
8 9176 0.094 6400 52,728 0.5402
10 11,175 0.115 8000 67,420 0.6910
Parameters Nebivolol Valsartan
Correlation coefficient 0.998 0.9994
Regression equation y = 1140× 8.4073×
Linearity range 0.5–10 ng/mL 400–8000 ng/mL

Recovery study of nebivolol and valsartan.

Parameters Nebivolol Valsartan
Unextracted (HQC) Extracted (HQC) Unextracted (MQC) Extracted (MQC) Unextracted (LQC) Extracted (LQC) Unextracted (HQC) Extracted (HQC) Unextracted (MQC) Extracted (MQC) Unextracted (LQC) Extracted (LQC)
n 6 6 6 6 6 6 6 6 6 6 6 6
Mean 9445 9253 5965 5782 1886 1857 54,658 53,310 35,376 34,761 10,665 10,507
SD 59.17 70.20 53.57 66.05 12.32 31.02 657.59 400.50 416.36 210.54 143.61 70.53
% CV 0.62 0.75 0.89 1.14 0.65 1.67 1.20 0.75 1.17 0.88 1.34 0.67
% Mean recovery 97.96 96.93 98.46 97.54 98.26 98.52
Overall % mean recovery 97.784 98.112

Inter-day precision and accuracy of nebivolol and valsartan.

Parameters Nebivolol Valsartan
HQC MQC LQC LLOQ QC HQC MQC LQC LLOQ QC
n 6 6 6 6 6 6 6 6
Mean 7.9238 4.9362 1.4907 0.4977 6394.3333 3999.0000 1194.8333 393.5000
SD 0.08962 0.08039 0.01770 0.01799 15.83246 70.11419 11.72035 6.05805
% CV 1.13 1.63 1.19 3.61 0.25 1.75 0.98 1.54
% Mean accuracy 99.05 98.72 99.38 99.53 99.91 99.98 99.57 98.38

Long-term stock solution stability of nebivolol and valsartan.

Parameters Nebivolol Valsartan
HQC LQC HQC LQC
n 6 6 6 6
Mean 7.9938 1.4927 6391.6667 1193.5000
SD 0.01950 0.02082 21.42584 20.08731
% CV 0.24 1.39 0.34 1.68
% Mean accuracy 99.92 99.51 99.87 99.46

Matrix samples’ stability of nebivolol and valsartan at −20°C ± 5°C for 37 days.

Parameters Nebivolol Valsartan
HQC (comparison sample) HQC (stability sample) LQC (comparison sample) LQC (stability sample) HQC (comparison sample) HQC (stability sample) LQC (comparison sample) LQC (stability sample)
n 6 6 6 6 6 6 6 6
Mean 7.9930 7.8872 1.4872 1.4898 6394.5000 6391.1667 1193.1667 1188.5000
SD 0.02481 0.15394 0.02729 0.01393 23.57753 18.50856 20.48821 18.98157
% CV 0.31 1.95 1.83 0.94 0.37 0.29 1.72 1.60
% Mean accuracy 99.91 98.59 99.14 99.32 99.91 99.86 99.43 99.04
% Mean stability 98.68 100.18 99.95 99.61

Batch-to-batch precision and accuracy of nebivolol and valsartan.

Parameters Nebivolol Valsartan
HQC MQC LQC LLOQ QC HQC MQC LQC LLOQ QC
n 18 18 18 18 18 18 18 18
Mean 7.9577 4.9657 1.4917 0.4981 6398.5000 3993.7222 1194.17 396.1667
SD 0.07478 0.08347 0.01596 0.01470 11.92254 79.93071 11.1596 7.10634
% CV 0.94 1.68 1.07 2.95 0.19 2.00 0.93 1.79
% Mean accuracy 99.47 99.31 99.44 99.61 99.98 99.84 99.44 99.04
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