INTRODUCTION:

Azelnidipine is a novel long acting dihydropyridine calcium channel blocker having chemical name is 3-(1-diphenylmethylazetidin-3-yl)-5-isopropyl-2-amino-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-3,5 pyridine dicarboxylate (Figure1). Azelnidipine is mainly acting by blocking L and T calcium channel blocker¹. The drug has a huge potential to reduce blood pressure with a similar potency as other anti-hypertensive dihydropyridines, like amlodipine, but without increasing pulse rate². The calcium channel blockers are shown that the retard atherogenesis in animal models and to prevent the development of early lesions in human coronary arteries. They are used in the treatment of angina pectoris and hypertension³. In the previously published literature survey reveals that very few bioanalytical methods are reported for the determination of Azelnidipine.

Which includes UV-Spectroscopy⁴, HPLC^4-6, LC-MS^7,8, LC-ESI-MS^9,10 and HPLC-MS/MS methods¹¹. The present research work was to develop a single, simple, fast and suitable stability-indicating LC-ESI-MS/MS method for detection and quantification of Azelnidipine in blank human plasma. The developed method was validated as per USFDA¹² and EMA¹³ guidelines.

Figure 1: Chemical structure of Azelnidipine

MATERIALS AND METHODS:

Chemicals and Reagents:

The drug Azelnidipine’s (purity 98.7%) active pharmaceutical ingredient (API) was acquired from AKUMS Drugs and Pharmaceutical Ltd. similarly, Metoprolol was procured from Shubham Biopharma and served as Internal Standard (IS). Formic Acid (HPLC grade), DMSO (HPLC grade), Acetonitrile (HPLC grade) were procured from Merck Pvt. Ltd, Mumbai, India. Milli-Q gradient system of Millipore (Elix, Milli-Q A10 Academic, Bedford, MA, USA) was used to purify water till it reached a resistivity of 18.2 MΩ.cm The blank human plasma containing EDTA-K3 anticoagulant was collected from the Clinical Pharmacological Unit (CPU) of TAAB Biostudy Services, Kolkata, India.

Instrumentation:

Shimadzu series LC-20 AD Binary pump, CTO-10 AS VP Column oven, SIL 20 AC Autosampler, and CBM-20A Lite System Control Compartment are the various parts of the liquid chromatographic system. The quantitative determination of Azelnidipine and Metoprolol (IS) in human plasma was done using the LC-MS/MS system API 2000 with triple quadrupole tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Instruments). The Analyst 1.6.3 software (Applied Biosystems/MD SCIEX) was used to acquire, process, and quantify data.

Chromatographic conditions:

Phenomenex Kinetex C18; 50x3mm, Particle Size- 5µm column was used for chromatographic separation using Pump A: 10mM Ammonium Acetate in Water (Milli-Q) and Pump B: 0.1% Formic Acid in Acetonitrile as the mobile phase. The flow rate at 0.5mL/min was maintained. A total run time was 7.0 minutes with 10μL of injection volume. The temperature of the autosampler was maintained at 15^oC, and column oven temperature was not applicable for the said method.

Mass spectrometry:

The mass spectra of the compounds were determined using a turbo electrospray ionization (ESI) interface with multiple reaction monitoring (MRM). Positive ionization mode was used for the measurement of ions. Optimization of tuning parameters was done by injecting 100ng/mL of standard solution, which contained Azelnidipine (Analyte) and Metoprolol (IS) at 10 µL/min by using an external syringe pump that’s directly affixed to the mass spectrometer. Needle spray was done at an applied high voltage was 5.5 kV. A scan Dwell (ms) time of 200.00 msec was used to program the instrument. Azelnidipine (Analyte) and Metoprolol (IS) were measured using the MRM transition of m/z 583.200 ®167.00, m/z 268.300 ®116.100, respectively, other Mass Spectrometric Conditions are in Table-1. 400 ^oC was set as the turbo ion spray source temperature along with a voltage of 5500.00 V. The nebulizer gas (GS1) had a value of 55.00 p.s.i. Accompanied by a turbo ion spray gas (GS2) at 45 p.s.i. 30.00 and 5.00 (arbitrary scale) p.s.i. were the curtain gas (CUR) and the collision-associated dissociation (CAD) gas flow, respectively, that were maintained in the instrument.

Table 1: Mass Spectrometric Conditions:

Parameters	Azelnidipine	Metoprolol (IS)
DP (V)	89.00	25.00
FP (V)	348.00	322.00
CEP	23.10	15.23
CE (eV)	50.00	28.00
EP (V)	8.80	9.10
CXP (V)	9.00	5.00

Preparations of Standard solutions:

Preparations of solutions containing 1mg/mL of Azelnidipine and IS was prepared using DMSO, respectively. These solutions was further diluted suitably with the mobile phase to obtain a stock solution of 1 µg/mL for Azelnidipine and 15μg/mL for IS. The stock solutions prepared for the drugs was diluted to obtain seven working solutions for calibration standards. All solutions were stored at 2–8^oC. The prepared concentrations were 1.06ng/mL, 2.13ng/mL, 4.25 ng/mL, 8.5ng/mL, 17ng/mL, 34ng/mL, and 68ng/mL.

Preparations of quality control samples:

We prepared four types of quality control samples from the seven-point standard calibration solutions of Azelnidipine: LLOQ, LQC, MQC, and HQC. The LLOQ was the lowest concentration of the calibration concentration that was 1.06ng/mL. The LQC was the middle concentration of second (2.13ng/mL) and third (4.25ng/mL) concentrations of the calibration concentration that was 3.188ng/mL. The MQC was the middle concentration of fifth (17ng/mL) and sixth (34 ng/mL) concentrations of the calibration concentration of 25.50ng/mL. The HQC was the middle concentration of sixth (34ng/mL) and seventh (68ng/mL) concentrations of the calibration concentration that was 51ng/mL.

Preparations of the calibration curve:

Seven-point standard calibration solutions of Azelnidipine was prepared by spiking appropriate analyte and IS in blank human plasma to yield final concentrations. The quality control (QC) samples are LLOQ, LQC, MQC, and HQC will be prepared at three concentration levels of analyte. Calibration curves will be plotted with a peak area ratio of drug and IS on Y-axis and concentration on X-axis.

Sample preparation:

Plasma extraction was performed by protein precipitation technique (PPT). A 100μl volume of blank plasma sample was transferred to a 2mL plastic Eppendorf tube. 50μl of Azelnidipine working solution and 50μl of IS working solution (15μg/mL) was spiked into it and vortex for 1 minute. After adding 300μl of cold Acetonitrile vortexing for 10 minutes. After that, the sample was centrifuged at 12000rpm at 4^oC for 5min. Then the upper organic layer 250μl was transferred to autosampler vials then 10μl the aliquot was injected into the chromatographic system (LC-ESI-MS/MS).¹⁴

Bioanalytical Method validation:

The procedure was validated in accordance with the guidelines for selectivity, linearity, accuracy, recovery, and stability^12,13. Three validation batches were processed on three separate days. Each batch included two sets of calibration standards and five replicates of LLOQ, LQC, MQC and HQC concentrations of QC samples.

Selectivity:

For the establishment of selectivity of the method, blank plasma was collected from separate healthy human volunteers. The lower limit of quantification or LLOQ level was used for the determination of selectivity. As per the USFDA and EMA guidelines, the acceptance criteria for selectivity are that the analytes' area response should not be more than 20% in the blank than the LLOQ at retention time.^12,13

Carryover:

Blank samples were injected after HQC to analyze the possibility of interference of an injected run from an immediate previous run. If the carryover from a high concentration injection is <20% of the area of LLOQ, it qualifies under USFDA and EMA.^12,13

Sensitivity:

The sensitivity method was confirmed by injecting the spiked plasma level at LLOQ five times. The USFDA and EMA guidelines state that the analyte peak area at LLOQ has to be <5x of blank peak area. USFDA guide states that the accuracy of the injected LLOQ samples should be within ±20% and the precision should be ±20% of the coefficient of variation (CV %).^12,13

Linearity:

Regression analysis was used to evaluate linearity to create a standard calibration curve at 7 points (1.06, 2.13, 4.25, 8.5, 17, 34, and 68 ng/mL). The coefficient of determination (R²) was determined by taking the drug/IS ratio of the Y-axis and concentration on the X-axis to create the standard calibration curve.^12,13

Accuracy:

Five replications of four different concentrations (LLOQ, LQC, MQC, and HQC) were used for evaluation of the accuracy of the method. The ratio of drug to IS peak area was determined, and the line equation (y=mx+c) was used to back-calculate the concentration. The accuracy of the method was calculated from the deviation of the back-calculated value estimated from the theoretical value. Accuracy is acceptable under the USFDA guidelines if it is within ±15% of the theoretical value, which can go up to ±20% for LLOQ.^12,13

Precision:

The spiked plasma samples were injected in five replicates at levels of LLOQ, LQC, MQC, and HQC to determine the analytical procedure's precision. The %CV was calculated for the back-calculated concentrations for the repeated injections. The %CV of responses of the same day injections were determined for intraday precision calculation. The inter-day precision calculation was done by determining the %CV of the measured values of samples which were injected on different days. Under the USFDA and EMA guidelines.^12,13

Extraction Recovery:

The determination of the potentiality of the technique of sample preparation to extract Azelnidipine and IS from biological samples was done by comparison of chromatographic responses found in LQC, MQC and HQC levels of extracted samples to that of the unextracted samples of similar concentration corresponding 100% recovery.

Matrix Effect:

The effect of plasma constituents over the ionization of analyte and IS was determined by comparing the chromatographic responses of the post-extracted LQC, MQC and HQC samples with the analyte's response from neat samples at equivalent concentrations. The matrix effect was determined at the same concentration of analyte and IS as in the recovery experiment.

Limit of Detection (LOD) and Lower Limit of Quantification (LLOQ):

LOD and LLOQ were determined at signal to noise ratios with known concentrations by injecting series of dilute solutions.^15-19

Robustness:

By making deliberate minor variations in the flow rate and mobile phase composition the robustness of the method was established.^20-24

Stability:

Various stability tests like autosampler stability, freeze-thaw stability, benchtop stability, long-term stability, and short-term stability were used to estimate the drug stability in plasma. Analytes spiked blank human plasma at three quality control concentrations (HQC, MQC, and LQC). Five replicates of each were stored suitably at storage conditions of stability tests post, which it was extracted and analyzed. QC sample from the stock was freshly created just before the study to assess the stability of the analytes in the stock. In the short-term stability study, the spiked plasma sample was stored for 24 hours at - 20°C before extraction and analysis. In the case of freeze and thaw stability, the sample was stored at - 20°C and exposed to cycles of freeze and thaw by freezing at - 20°C and thawing in the laboratory's normal conditions. The samples were kept in an autosampler maintained at 15°C for 24 hours before injecting to determine the autosampler stability. The analytes spiked plasma was kept on a benchtop for 24 hours under normal laboratory conditions before analyzing to determine benchtop stability. Long-term stability study was done by storing the spiked plasma sample for 30 days at - 20°C before extraction and analysis. Freshly spiked QC sample at HQC, MQC, and MQC was used to determine the stability testing accuracy against the stability samples. If it is within ±15%, then it is acceptable under USFDA and EMA guidelines.^12,13

RESULTS AND DISCUSSIONS:

Selectivity:

Blank samples with no response and indicative of no such interference of plasma at RT similar to that of the Azelnidipine was seen in the chromatograms. Hence, the method of development meets the requirements of selectivity as the deviation in the peak area at RT in blank samples was ≤20% as compared to that of LLOQ for analytes.

Carryover:

When injected after HQC, the RT of Azelnidipine was free from any response in a blank chromatogram. As the analyte peak response was less than 20% of LLOQ, the developed method has no carryover problem.

Sensitivity:

The blanks peak area was >5x when compared with that of the analytes peak area at LLOQ. A mean accuracy of 96.18-103.71% was seen for Azelnidipine for the five LLOQ injections made. 5.88-8.53% was the precision (%CV) values for the Azelnidipine. Hence, the developed method was sensitive even at a concentration as low as 1.06ng/mL (LLOQ). Chromatographic representation of LLOQ with IS are seen in Figure 2.

Linearity:

For Azelnidipine, the calibration curve of the seven points had the R² value of 0.9975 (Figure 3). The calibration standards had a precision value ranging from 1.88% to 6.54% for Azelnidipine. Hence, the developed method linearity is present over a concentration range of 1.06 ng/mL to 68ng/mL.

Accuracy:

The back-calculated concentrations of QC samples had an accuracy range of 93.176% to 112.00% for Azelnidipine in the case of all five replicates inclusive of LLOQ. Accuracy values were within the USFDA guidelines of ±15% for HQC, MQC, and LQC and LLOQ within ±20%, as shown in the results. Table-2 and Table-3 have study results of inter-day and intra-day accuracy studies in order.

Figure 2: Chromatographic representation of LLOQ with IS of Azelnidipine

Precision:

For the Azelnidipine, the %CV of the back-calculated concentrations inclusive of the LLOQ was determined to be 1.329% to 6.037% for intra-day and for inter-day the range was between 8.20% to 9.46% in case of QC samples of three different days. Table-2 and Table-3 contain the results of intra-day and inter-day experiments, respectively, which shows that they satisfy the required criteria method's establishment of repeatability within the acceptance criteria.

Extraction Recovery:

For Azelnidipine, the QC levels mean extraction recovery was more than 98.76% - 99.12%, which clearly shows that the method of preparing the sample is well suited for drug analysis as the high extraction efficiency is seen for the analyte. The Extraction Recovery data are elaborated in Table-4.

Table 2: Inter-day-accuracy:

Quality control	Concentration (ng/mL)	Run	Mean found (ng/mL)	SD	CV (%)	Accuracy (%)
LLOQ	1.06	1	1.126	0.059	5.277	105.976
		2	0.990	0.059	5.933	93.176
		3	1.190	0.016	1.329	112.00
LQC	3.188	1	3.588	0.070	1.981	111.624
		2	2.974	0.180	6.037	93.302
		3	3.032	0.087	2.860	95.122
MQC	25.50	1	25.078	0.498	1.988	98.345
		2	22.796	0.376	1.649	89.396
		3	27.568	0.502	1.820	108.110
HQC	51	1	47.790	1.050	2.196	93.706
		2	44.786	1.062	2.371	87.816
		3	55.240	1.187	2.149	108.314

Table 3: Intraday-accuracy:

Quality control	Concentration (ng/mL)	Mean found (ng/mL)	SD	CV (%)	Accuracy (%)
LLOQ	1.06	1.102	0.098	8.85	103.72
LQC	3.188	3.188	0.294	9.24	100.02
MQC	25.50	25.147	2.062	8.20	98.62
HQC	51	49.27	4.66	9.46	96.61

Table 6: Stability Studies:

Stability	Quality control	Concentration (ng/mL)	Mean found (ng/mL)	Accuracy (%)
Freshly Thawed	LQC	3.188	3.02
	MQC	25.50	25.33
	HQC	51	50.38
Bench top (24 Hours)	LQC	3.188	2.93	97.02
	MQC	25.50	25.39	100.22
	HQC	51	51.51	102.24
Autosampler (24 Hours)	LQC	3.188	3.13	103.64
	MQC	25.50	26.29	103.81
	HQC	51	52.28	103.77
Freeze-thaw (3 Cycles)	LQC	3.188	2.90	96.03
	MQC	25.50	24.01	94.79
	HQC	51	49.39	98.03
Short term (24 Hours)	LQC	3.188	3.04	100.66
	MQC	25.50	24.07	95.03
	HQC	3.188	45.38	90.08
Long term (30 Days)	LQC	25.50	3.03	100.33
	MQC	51	23.33	92.10
	HQC	3.188	48.90	97.06

CONCLUSION:

In this present study, the bioanalytical method is developed for Azelnidipine. The method is highly specific, sensitive, and simple for detection and quantification of Azelnidipine in human plasma using LC-ESI-MS/MS. The sample preparation procedure consists of a simple protein precipitation technique using cold Acetonitrile. The method is reproducible so that the developed method is very cost-effective. The method involved a specific and straightforward sample preparation by protein precipitation technique; only 100μL human plasma needed. The method developed for the validation of the bioanalytical method was validated according to the USFDA and EMA guidelines. The developed method was found to have accepted in terms of various parameters. The overall analysis is time promising. Therefore, this study will have a significant contribution to the field of bioanalytical research, and this bioanalytical method is successfully applied in future for pharmacokinetics, bioequivalence studies and quality control laboratories.

ACKNOWLEDGEMENT:

The authors want to acknowledge M/S, TAAB Biostudy Services, Kolkata-700032, India, for providing blank human plasma samples and the necessary instrumental facilities to complete the work.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 22.07.2020 Modified on 23.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3571-3577.

DOI: 10.52711/0974-360X.2021.00618

		Extracted Blank Plasma Sample			Aqueous Sample			% OF ME
Quality Control	Concentration (ng/mL)	Mean found (ng/mL)	SD	CV (%)	Mean found (ng/mL)	SD	CV (%)	% OF ME
LQC	3.188	3.05	0.17	5.70	3.19	0.11	3.39	95.61
MQC	25.50	27.24	0.50	1.85	26.02	0.44	1.70	103.96
HQC	51	53.89	0.99	1.84	52.03	1.06	2.04	103.57

	Diluent Sample			In Plasma Sample
Quality Control	LQC	MQC	HQC	LQC	MQC	HQC
Concentration (ng/mL)	3.188	25.50	51	3.188	25.50	51
Mean found (ng/mL)	3.50	24.93	48.90	3.46	24.62	48.47
% Recovery				98.86	98.76	99.12