Bioanalytical RP-HPLC method development and validation for estimation of azelnidipine and Olmesartan medoxomil in human plasma

An accurate, rapid and simple reversed-phase high performance liquid chromatography (RP-HPLC) bioanalytical method was developed for estimation of Azelnidipine (AZP) and Olmesartan medoxomil (OLM)in human plasma. The AZP, OLM and internal standard (IS) were extracted by liquid-liquid extraction technique. Chromatographic separation was accomplished using BDS Hypersil C18, 250 mm X 4.6 mm, 5µ analytical column. The mobile phase consisted of Acetonitrile: Water, pH adjusted with ortho-phosphoric acid in the ratio 60:40 with flow rate of 1ml/min. Detection and quantification were performed by UV detector at 256 nm. The retention times were found to be 10.05±0.02, 4.3±0.02 and 6.9±0.02 for AZP, OLM and IS respectively. The linearity range was found to be 0.5 to 12 µg /ml and 1 to 15 µg /ml for AZP and OLM respectively.The method was validated as per CDER guideline, was found to be suitable for analysis in biological fluid.

OLM is converted to Olmesartan after hydrolysis and it binds to the AT1receptor in vascular smooth muscle and competes with angiotensin II . This prevents vasoconstriction and decreases aldosterone production, which prevents aldosteronestimulated retention of sodium and excretion of potassium. Thus, OLM is used to treat the hypertension and it relaxes blood vessels to allow the flow of blood more easily [4,5] . and is marketed in Japan as Rezaltas® by Daiichi Sankyo Company [6] . This combination of azelnidipine, a dihydropyridine calcium channel antagonist and Olmesartan medoxomil, an orally active angiotensin receptor antagonist, is proving to be better choice than other combinations used to treat hypertension [7,8] . The clinical studies on this combination revealed that it helps to reduce the possible side effects and control the heart rate with minimum or no changes in normal heart rate [9,10] . Hence, we targeted this novel combination for the analytical study. Chromatographic (RP-HPLC) method [11] and rapid reversed phase Ultra-Fast liquid chromatography (UFLC) method for simultaneous determination of AZP and OLM in pharmaceutical dosage form has been reported with greater precision and accuracy [12] . Ramesh et al., (2017) developed novel, rapid, specific and sensitive liquid chromatography tandem mass spectrometry (LCMS/MS) method for the simultaneous determination of AZP and OLM in human plasma.
Prabhakar et al., (2017) developed RP-HPLC method for AZP with retention time of 6.130 min in mobile phase methanol: water containing 0.1% glacial acetic acid (75:25) [14] . Modi et al., (2016) [15] and Gore et al., (2016) [16]  in water and acetonitrile [18] . Chavda et al., published review on analytical methods for estimation of AZP and telmisartan [19] , while recently developed HPLC methods for AZP and telmisartan have also been reported [20,21] .  The liquid-liquid extraction was carried out in following manner: • Pooled human plasma frozen at below -25ºC were thawed at RT followed by vertexing to confirm the homogeneity. • The spiked samples were kept for 1 hr to reach equilibrium concentration.
• The tubes were vortexed for 2 minutes and ethyl acetate: ACN: 0.5% formic acid in the ratio 5:2.5:2.5 were added.
• It was then vortexed for 1 minute and centrifuged for 10 min at 5000 rpm. The supernatant was collected and evaporated on hotplate.
• Then it was reconstituted with 1ml mobile phase and was transferred into pre-labelled auto sampler vials and injected to HPLC system with optimized chromatographic conditions.

Calibration standards (CS) and quality control (QC) samples preparation
As per CDER guidelines, the calibration curve must contain at least six dilutions, which are known as calibration standards (CS).
Quality control standards (QC) has critical role to measure the performance of optimized method and these should be selected in such a way that it should cover the whole range of calibration line.

Selection of LLOQ
Initially, 100 µg/ml of both drugs, AZP and OLM were analysed in combination using optimized conditions and peak areas and peak heights were observed. Accordingly, the dilution for LLOQ was prepared so that it can be detected with a minimum height of 2 to 3 units. Thus, 0.5 µg/ml AZP and 1 µg/ml OLM were selected as LLOQ.

Linearity
Similarly, other dilutions were analysed by making serial dilutions and the dilutions having peak area values in linear fashion were selected for calibration curve. Preparation of calibration curve was done by plotting peak area ratio on y-axis and respective concentrations in µg/ml on x-axis. The slope, y-intercept, linear equation and regression coefficient (r 2 ) were calculated for each drug.

Selectivity
Selectivity for the developed bioanalytical assay is normally performed by analyzing blank samples of the blood plasma from multiple sources. Thus, 6 normal blank plasma lots from different sources were processed as per the developed method. It is also mentioned in the guidelines that the IS should be analyzed to avoid interference. Thus, 6 different zero calibrators were also processed as per method.

Sensitivity
As per the CDER guidelines, method sensitivity is better determined by LLOQ thus it becomes necessary to perform it during method development. Thus, LLOQ was spiked in plasma and six different LLOQ dilutions were processed. The CV for the area of six LLOQ samples was calculated.

Carry Over
Carry over between two runs was studied by injecting blank plasma initially, followed by ULOQ and then two blank runs back to back.

Accuracy and Precision (A and P)
A and P determines the readiness of developed method for validation as well as its application to marketed products or biological samples. It was performed by preparing fresh dilutions of all QCs in triplicate and analysing them at specific intervals.

Within Run
The QCs were analysed in triplicate for three times on same day at the interval of 2 hours and recovered concentrations were

Bench Top
Spiked plasma samples of AZP and OLM were kept on bench for 4, 6 and 24 hrs. LQC and HQC were prepared in triplicate and processed after 4, 6 and 24 hrs and injected immediately after the processing was completed. The percentage recovery was calculated.

Long Term
Spiked plasma samples were kept at intended temperature for 10, 20 and 30 days. LQC and HQC were prepared and processed after 10, 20 and 30 days and injected immediately after processing completed. The percentage recovery was calculated.

RESULTS AND DISCUSSION Solvent and IS Selection
In accordance with the trials for solubility and stability of analytes in various solvents, methanol was found to be very ACN is hydrogen bond donor, has a higher dipole moment, low UV cut-off, higher boiling point and lower viscosity. All the peaks were eluted with proper separation with good peak shape.
.Water with adjusted pH is always a good solvent for RP-HPLC and water with specific pH monitors the elution properties of the analytes by maintaining the ionization characteristics of drugs.
Depending upon the observations of chromatographic runs, the combination of ACN: water pH adjusted with OPA (60:40) was finalized as mobile phase for the bioanalytical method of AZP and OLM. AZP and OLM were showing well separated peaks along with proper peak shapes, sufficient number of theoretical plates, good symmetry and good resolution.

Chromatographic Conditions
There were some fixed chromatographic conditions depending upon the availability of instrument type at analytical lab.
These include the parameters like type of column was BDS hypersil 0.5% formic acid in the ratio 5:2.5:2.5 proved to be the best extracting solvents with good peak shape and better recovery in the range of 85 -91%.

Validation
The validation of developed bioanalytical method was performed as per CDER guidelines.

Calibration Curve
The linearity was observed in the range 0.5, 1.5, 3, 6, 9 and 12 µg/ml for AZP and 1, 3, 6, 9, 12 and 15 µg/ml for OLM (Table 2). The calibration graphs were obtained with regression equations, y = 0.1146x + 0.5213 and y = 0.1028x + 0.4342 having r2 values 0.9972 and 0.9986 for AZP and OLM respectively (Fig 4 and 5).  Selectivity Selectivity has to be performed to check the interference of biological fluids or matrices in the chromatograms of analytes. For this purpose, normal blank human plasma collected from 6 different sources was combined and analysed under the developed chromatographic conditions. As per blank chromatogram there was no intervention of plasma at the Rts of analytes. Also the analysis of IS was conducted to find any interferences with the analyte. 6 different zero calibrators were analysed and it was found that there is no any interference of IS at the retention times of analytes.

Sensitivity
The detection ability, to observe all analytes at all concentrations including a very small quantity of analyte is tested in the validation parameter called sensitivity. It can be defined as the lowest concentration of analyte that can be measured with tolerable precision and accuracy (i.e., LLOQ).
Thus, sensitivity was determined by analyzing 6 replicates of LLOQs of AZP and OLM along with IS (Table 6). It was observed that AZP at 0.5 µg/ml and OLM at 1 µg/ml were detectable with the height of 3 and 2 units respectively. The RSD of peak areas was 3.75, 3.88 and 2.27 for AZP, OLM and IS respectively, which shows the sensitivity of the method.
Similarly, sensitivity of method was tested by observing system suitability parameters of the chromatographic runs.
Acceptance limit of asymmetry factor is 1, theoretical plates should be more than 1500 and resolution should be more than 2. It was observed that the values of asymmetry factor, theoretical plates and resolution of AZP, OLM and IS (Table 3) were within acceptance limit indicating that the method is selective.

Accuracy and Precision (A and P)
QCs were obtained according to calibration curves plotted for both analytes. LLOQ, LQC, MQC and HQCs of both analytes were prepared freshly and analysed for A & P parameters. As per the guidelines, acceptance criteria for accuracy is that in each validation run, the analysed QCs should be ±15% of nominal concentrations, whereas at LLOQ, they should be ± 20% of nominal concentrations. Table 4 shows the recovered concentrations, percentage accuracy, SD and RSD or CV (%) of 6 replicates of QCs of AZP and OLM analysed for three times on same day at the interval of 2 hours.

Between Run
The values of recovered concentrations, percentage accuracy, SD and RSD or CV (%) of QCs of AZP and OLM resp. analysed in triplicate for one time on three consecutive days are listed in Table 4. It was found that, percentage accuracy was within the percentage assay limit of the analytes and all the CV (%) values were within acceptance criteria as per the guidelines.

Recovery
Extraction of respective QC concentrations of AZP and OLM in combination was done, analysed and percentage recovery was computed. The recovery was within assay limit of respective drugsas shown in Table 5.