1. bookVolume 68 (2021): Edizione 2 (December 2021)
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Development and Validation of Bioanalytical Method for Determination of Nebivolol and Valsartan in Human Plasma by Using RP-HPLC

Pubblicato online: 12 Mar 2022
Volume & Edizione: Volume 68 (2021) - Edizione 2 (December 2021)
Pagine: 49 - 58
Ricevuto: 14 Apr 2018
Accettato: 01 Jun 2018
Dettagli della rivista
License
Formato
Rivista
eISSN
2453-6725
Prima pubblicazione
25 Nov 2011
Frequenza di pubblicazione
2 volte all'anno
Lingue
Inglese
INTRODUCTION

Nebivolol (NL) is chemically described as 1-(6-fluoro-3, 4-dihydro-2H-1-benzopyran-2-yl)-2-{[2-(6-fluoro-3, 4-dihydro-2H-1-benzopyran-2-yl)-2-ydroxyethyl]amino} ethan-1-ol (Figure 1a). Its molecular formula is C22H25F2NO4 and its molecular weight is 405.435 g/mol. Valsartan (VL) is chemically described as (2S)-3-methyl-2-[N-({4-[2-(2H-1, 2, 3, 4-tetrazol-5-yl) phenyl] phenyl}methyl) pentanamido] butanoic acid (Figure 1b). Its empirical formula is C24H29N5O3 and its molecular weight is 435.52 g/mol [1,2,3,4,5].

Figure 1a

Structure of Nebivolol.

Figure 1b

Structure of Valsartan.

VL belongs to the angiotensin II receptor blocker (ARB) family of drugs, which also includes telmisartan, candesartan, losartan, olmesartan and irbesartan. ARBs selectively bind to angiotensin receptor-1 (AT1) and prevent the protein angiotensin II from binding and exerting its hypertensive effects, which include vasoconstriction, stimulation and synthesis of aldosterone and ADH, cardiac stimulation and renal reabsorption of sodium, among others. Overall, the physiological effects of VL lead to reduced blood pressure, lower aldosterone levels, reduced cardiac activity and increased excretion of sodium. NL is a racemic mixture of two enantiomers, wherein one is a β-adrenergic antagonist and the other acts as a cardiac stimulant without β-adrenergic activity. Treatment with NL leads to a greater decrease in systolic and diastolic blood pressure than atenolol, propranolol or pindolol. NL and other β-blockers are generally not first-line therapies as many patients are first treated with thiazide diuretics. They are indicated for hypertension to lower the blood pressure and reduce the risk of fatal and non-fatal cardiovascular events, primarily strokes and myocardial infarction. NL is a competitive and selective β1-receptor antagonist, has little or no effect on β2 receptors at doses <10 mg, lacks intrinsic sympathomimetic and membrane-stabilising activity at therapeutically relevant concentrations and reduces systemic vascular resistance. VL blocks the binding of angiotensin II to type 1 angiotensin receptors, causing a reduction in blood pressure, and blocks vasoconstrictor and aldosterone-secreting effects of angiotensin II. Byvalson (VL and NL tablet) 80 and 5 mg is a fixed-dose combination for the treatment of hypertension [1,2,3,4,5,6,7,8].

The literature survey presents different methods available for the detection of NL and VL separately and also in a combined form. Ultra-performance liquid chromatography (UPLC) method for detection of both drugs [9], an isocratic (high-performance liquid chromatography [HPLC]) method [10], an ion-pair HPLC method [11], RP-HPLC method for the estimation of both drugs [12], ultraviolet (UV) simultaneous estimation of NL and VL [13] – these all are the methods available for the detection of NL and VL.

This study aims to develop a rapid analytical technique for the estimation of NL and VL in human plasma. The proposed method is a rapid, precise, selective and sensitive RP-HPLC method that has short and simple extraction procedures that consume small amounts of solvent and biological fluid for extraction and time. This method is used in the bioavailability/bioequivalence (BA/BE) study for analysis of NL and VL in biological samples like blood, plasma and urine.

MATERIALS AND METHODS

NL and VL active pharmaceutical ingredients (APIs) were received as a gift sample from Spectrum Pharma Research solutions. HPLC-grade acetonitrile and methanol were procured from Rankem, Avantor Performance Material India Limited. Plasma used for the study was received from Deccan pathological lab, Hyderabad. Analytical grade phosphate buffer, potassium dihydrogen phosphate and orthophosphoric acid were also procured from Rankem, Avantor Performance Material India Limited.

Chromatographic conditions

Chromatographic separation was performed at 30°C of column temperature with the mobile phase consisting of 0.01 N potassium dihydrogen phosphate pH 3.0:acetonitrile in the ratio of 60:40. Separation of NL and VL was achieved on Symmetry C18 (150 × 4.6 mm, 5 μm) at a flow rate of 1.0 mL/min; the injection volume was 50 μL and detector wavelength was 280 nm.

Preparation of standard stock solution
Diluents

Based upon the solubility of the drugs, diluent was selected; 0.01 N potassium dihydrogen phosphate and acetonitrile were taken in the ratio of 50:50.

Preparation of NL stock solution (5000 ng/mL)

Take 5 mg of NL in a 100-mL volumetric flask and add diluent to give a volume of 50,000 ng/mL. Take 1 mL of the prepared solution and dilute to 10 mL with diluent to produce 5000 ng/mL.

Preparation of NL spiking solutions

From the above-mentioned NL stock solution, 0.01, 0.02, 0.03, 0.08, 0.10, 0.12, 0.16 and 0.20 mL was pipetted and transferred to eight individual 10-mL volumetric flasks and the volume was made up to the mark with diluent to produce 5, 10, 15, 40, 50, 60, 80 and 100 ng/mL. Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 0.5, 1, 1.5, 4, 5, 6, 8 and 10 ng/mL.

Preparation of VL stock solution (400 μg/mL)

Take 40 mg of VL in a 100-mL volumetric flask and add diluent to give a volume of 400,000 ng/mL.

Preparation of VL spiking solutions

From the above-mentioned VL stock solution, 0.1, 0.2, 0.3, 0.8, 1.0, 1.2, 1.6 and 2.0 mL was pipetted and transferred to eight individual 10-mL volumetric flasks, and the volume was made up to the mark with diluent to produce 4.0, 8.0, 12, 32, 40, 48, 64 and 80 μg/mL. Calibration standards and QC samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 400, 800, 1200, 3200, 4000, 4800, 6400 and 8000 ng/mL.

Preparation of atorvastatin (internal standard [IS]) spiking solutions

Fifty milligrams of atorvastatin was weighed, dissolved and diluted to 100 mL by diluent (500 μg/mL). From this stock solution, 2 mL was pipetted, diluted to 100 mL with diluent and used as a spiking solution (10 μg/mL).

EXTRACTION PROCEDURE
Protein precipitation method
Preparation of calibration standard and QC samples

Take 750 μL of plasma and add 500 μL of IS, 250 μL of NL and 250 μL of VL from the spiking solutions into a centrifuging tube; add 1 mL of acetonitrile. Mix by cyclomixer for 15 sec. Then vertex for 2 min and finally centrifuge for 5 min at 3200 rpm. After centrifugation, collect the supernatant, filter it and directly inject 50 μL into HPLC.

Preparation of patient's samples

Take 1250 μL of the patient's sample and add 500 μL of IS into a centrifuging tube; add 1 mL of acetonitrile. Mix by cyclomixer for 15 sec. Then vertex for 2 min and finally centrifuge for 5 min at 3200 rpm. After centrifugation, collect the supernatant, filter it and directly inject 50 μL into HPLC.

Method validation

Method validation for an analytical method is a process used to verify/confirm whether the newly developed method is suitable for its intended purpose as per the ICH guidelines [14,15,16,17,18,19]

Parameters involved in bioanalytical validation are as follows:

System suitability test

Selectivity

Matrix factor evaluation

Precision and accuracy

Linearity

Recovery

Stability

System suitability test

System suitability test was performed before each batch sample analysis to ensure the reproducibility of the chromatographic system. This test was performed by running six injections of the diluted drug and IS in the linear region of the calibration curve and measuring the percentage of relative standard deviation (% RSD).

Selectivity/specificity

To establish the selectivity of the method, possible interferences at the retention time (RT) of NL, VL and IS due to endogenous plasma components were checked during validation. Selectivity was performed by testing six lots of K2EDTA blank plasma, and the analyte detection of extracted plasma has good selectivity of both drugs and IS.

Matrix factor evaluation

The combined effect of all components of the sample other than the analyte on the measurement of quantity. Three blank specimens from each of not less than six batches of the matrix under screening were extracted. For the matrix effect, LQC and HQC spiking dilutions were spiked, and IS dilutions were also spiked in the previously extracted blank specimens.

Precision and accuracy

The intra-day and inter-day accuracy and precision were assessed by analysing six replicates at four different QC levels like LLOQ, LQC, MQC and HQC. Accuracy and precision method performance was evaluated by six replicate analyses for NL at four concentration levels, that is, 0.5 ng/mL (LLOQ), 1.5 ng/mL (LQC), 5 ng/mL (MQC) and 8 ng/mL (HQC), and for VL at 400 ng/mL (LLOQ), 1200 ng/mL (LQC), 4000 ng/mL (MQC) and 6400 ng/mL (HQC). The intra-day and inter-day accuracies of plasma samples were assessed and excellent mean % accuracies were calculated. The precision of the method was expressed in terms of % RSD, and accuracy was expressed as a percentage of theoretical concentration (observed concentration × 100/theoretical concentration).

Linearity

The calibration curve in a bioanalytical method is a linear relationship between concentration (independent variable) and response (dependent variable) using a least-squares method. This relationship is built to predict the unknown concentrations of the analyte in a complicated matrix. The linearity sample should consist of a blank sample, a zero sample and six to eight non-zero samples covering the expected range, including LLOQ.

Recovery

Recovery was determined by comparing the peak areas obtained from prepared plasma samples with those extracted from blank plasma spiked with the same amount of NL and VL standards at three QC concentration levels.

Stabilities

Long-term stock solution stability for NL: In bench-top stability, six replicates of LQC and HQC samples (1.5 and 8 ng/mL) were analysed after 9 h of storage at room temperature in the laboratory bench. The % mean stability was calculated.

Long-term stock solution stability for VL: In bench-top stability, six replicates of LQC and HQC samples (1200 and 6400 ng/mL) were analysed after 9 h of storage at room temperature in the laboratory bench. The % mean stability was calculated.

Matrix samples’ stability at −20°C ± 5°C and −70°C ± 5°C after for 37 days of storage

Long-term stock solution stabilities for NL and VL were determined at LQC and HQC concentration levels after a storage period of 37 days at −20°C and −70°C in the deep freezer. The % means stabilities of NL and VL were calculated.

RESULTS
Optimisation of chromatographic method

Chromatographic separation of NL and VL by RP-HPLC method was performed by various mobile phase trials. The ideal separation of NL and VL was obtained by using a mobile phase consisting of 0.01 N potassium dihydrogen phosphate (pH 3.0):acetonitrile in the ratio of 60:40. A representative chromatogram of the optimised chromatographic method is shown in Figure 2.

Figure 2

Chromatogram of Optimized Chromatographic Method.

System suitability test

All the system suitability parameters were within the range and satisfactory as per the ICH guidelines. The % coefficient of variation (CV) for system suitability test was in the range of 1.0 ± 0.0316 for RT of NL, 1.18 ± 0.0512 for RT of VL and 0.23% for the area ratio (analyte area/IS area) of atorvastatin. Full results of the system suitability test are shown in Table 1.

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
Selectivity

Evaluation of selectivity was performed by testing six batches of K2EDTA blank plasma. The analysis of extracted blank plasma showed that the developed method has good selectivity for both drugs and IS. Representative chromatograms are shown in Figure 3 of standard blank and blank with IS sample using pooled plasma.

Figure 3

Extracted standard blank sample.

Matrix factor evaluation

The matrix effect plays a vital role in the assessment of pharmacokinetic studies. It was expressed as an IS-normalised matrix factor and it varied from 0.90 to 0.99, which was close to 1, which indicates that there was no analytical signal suppression or enhancement in plasma samples. Results are shown in Table 2.

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
Precision and accuracy

The intra-day and inter-day accuracies of plasma samples were assessed and excellent mean % accuracies for NL and VL were obtained with range varying from 99.32% to 99.70% and from 99.13% to 100.44% for intra-day accuracy and from 99.31% to 99.61% and from 99.84% to 99.98% for inter-day accuracy, respectively. The precision (% CV) of the analytes in the plasma samples was calculated and found to be 0.66%–2.65% and 0.17%–2.68% for intra-day accuracy and 0.94%–2.95% and 0.19%–2.00% for inter-day accuracy, respectively. Results are shown in Tables 3–5.

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

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

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
Linearity

Calibration was found to be linear over the concentration range of 0.5–10 ng/mL for NL and 400–8000 ng/mL for VL. The coefficient of determination (r2) value was found to be consistently greater than 0.999 in all the cases shown in Figure 4a and b. Linearity of results shows an excellent correlation between the peak area ratios for each concentration of analytes; results are shown in Table 6.

Figure 4a

Calibration Curve of Nebivolol.

Figure 4b

Calibration Curve of Valsartan.

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

The overall % mean recoveries for NL and VL were found to be 97.96% and 98.11%, respectively. The recoveries were obtained for NL and VL at three QC concentration levels. The overall % mean recovery for atorvastatin (IS) was found to be 99.00%. The results are shown in Table 7.

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
Stabilities
Long-term stock solution stability for NL

The % mean stability of six replicates of LQC and HQC samples (1.5 and 8 ng/mL) was analysed after 9 h of storage at room temperature on the laboratory bench. These were calculated and found to be 99.51% for LQC and 99.92% for HQC. The result is shown in Table 8.

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
Long-term stock solution stability for VL

The % mean stability of six replicates of LQC and HQC samples (1200 and 6400 ng/mL) was analysed after 9 h of storage at room temperature on the laboratory bench. These were calculated and found to be 99.51% for LQC and 99.92% for HQC. The result is shown in Table 8.

Matrix samples’ stability at −20°C ± 5°C and −70°C ± 5°C after 37 days of storage

The % mean stability of NL was found to be 100.18% (LQC) and 98.68% (HQC) at 20°C ± 5°C and 99.65% (LQC) and 100.33% (HQC) at 70°C ± 5°C. The % mean stability of VL was found to be 99.61% (LQC) and 99.95% (HQC) at 20°C ± 5°C and 99.51% (LQC) and 100.23% (HQC) at 70°C ± 5°C. The results are shown in Tables 9a and b.

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

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
DISCUSSION

After studying literature about NL and VL, the method development was started according to hydrophobic and hydrophilic interaction of the drugs. The absorbance spectra of both drugs were measured, overlayed and the wavelength of optimal sensitivity of determination was chosen (280 nm). The IS was selected based on the presence of chemical properties similar to the compounds of interest. Atorvastatin was chosen as the IS. After selecting the wavelength, the next step was the selection of the extraction procedure by observing the % recovery. The protein precipitation method was selected as the extraction procedure for better recovery. After selection of the extraction method, the method development process started with a flow rate of 1.0 mL/min at a column temperature 30°C, mobile phase 0.1% orthophosphoric acid (pH 2.2):acetonitrile (50:50), column Agilent C18 (150 mm × 4.6 mm, 5 μm) and injection volume 50 μL. In these parameters, VL was not eluted. So, further trial was carried out. In the second trial, the mobile phase and its ratio were changed to 0.01 N potassium dihydrogen phosphate (pH 3.0):acetonitrile (55:45) and other chromatographic conditions were the same as first trial. In the second trial, the resolution between atorvastatin and NL was unacceptable. So, third trial was carried out using 0.1% orthophosphoric acid (pH 2.2):acetonitrile (60:40) mobile phase and Symmetry C18 (150 mm × 4.6 mm, 5 μm) column. VL was not eluted in this trial. In the fourth trial, mobile phase 0.01 N potassium dihydrogen phosphate (pH 3.0):acetonitrile (70:30) and column Agilent C18 (150 mm × 4.6 mm, 5 μm) were used. In this trial, NL United States Pharmacopeia (USP) plate count was low and RTs were unnecessarily high. So, in the next trial, 0.01 N potassium dihydrogen phosphate (pH 3.0):acetonitrile (60:40) was used as the mobile phase and Symmetry C18 (150 × 4.6 mm, 5 μm) was used as the column. All peaks eluted with good peak shape and RT. The tailing tests were also passed. So, this method is used as an optimised method for the separation of NL and VL from human plasma. After optimisation was completed, the method was evaluated for various validation parameters as per the ICH M10 guideline.

CONCLUSION

A simple, accurate and precise method was developed for the estimation of NL and VL in human plasma using atorvastatin as the IS. RTs of NL and VL were found to be 3.146 and 4.346 min, respectively. The % CV of NL and VL was found to be 1.0% and 0.58%, respectively. Per cent recoveries were obtained as 98.28% and 98.11%, respectively. The linearity concentrations were in the range of 0.5–10 ng/mL for NL and 400–8000 ng/mL for VL. The lower limits of quantification were 0.5 ng/mL for NL and 400 ng/mL for VL, which reached the levels of both drugs possibly found in human plasma. Further, the reported method was validated as per the ICH guidelines and found to be well within the acceptable range. The proposed method is simple, rapid, accurate, precise and appropriate for pharmacokinetic studies and therapeutic drug monitoring in clinical laboratories.

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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