Materials

Nebivolol hydrochloride and amlodipine besylate were received as gift samples from Mylan Laboratory Pvt. Ltd., India. Analytical HPLC-grade methanol and formic acid were purchased from Merck Specialties Pvt. Ltd., Mumbai, India. Orthophosphoric acid was procured from Qualigens (Thermo Fisher Scientific). Acetonitrile and trifluoroacetic acid (TFA) were acquired from Merck. Ultra-pure HPLC-grade water was obtained from Siddhi Lab, India.

Instrumentation

The method development and validation process utilized an HPLC system comprising a 1260 Infinity II module, which included a G7111A pump, degasser, G7115A detector, and G7129A autoinjector. Analysis and separation were conducted using an Inertsil ODS-3 V column (150 mm × 4.6 mm, 5 μm). Sonication was performed with a Bio Technics India 12L300H ultrasonicator, and material weighing was carried out using an Aczet CY224 model.

Chromatographic conditions for HPLC

The analytical method was developed by preparing various solutions and optimizing the chromatographic conditions, including the composition of the mobile phase [27].

Preparation of standard stock solution (A and B)

A weighed amount of 10 mg NBH was carefully added to a 20 mL volumetric flask containing 10 mL of methanol (A). The mixture was sonicated for 10 min, and the volume was then adjusted with methanol to reach a final concentration of 500 µg/mL, resulting in a stock solution. Similarly, a separate 20 mL volumetric flask (B) was used to prepare a 500 µg/mL stock solution of AMB following the same procedure.

Working solution preparation

To prepare the working solution of NBH and AMB, 0.8 mL of each stock solution was pipetted from flasks A and B, respectively, into a 20 mL volumetric flask. The solution was then diluted with methanol to the 20 mL mark, yielding a final concentration of 20 µg/mL for both compounds.

Mobile phase preparation

The mobile phase composition was optimized by varying the ratios of acetonitrile, methanol, water, and buffer. Initially, a column with a 5 µm particle size was used for separation, and various modifiers such as chloroform, tetrahydrofuran (THF), ethanol, isopropyl alcohol (IPA), n-hexane, dichloromethane, and trifluoroacetic acid (TFA) were tested. The final mobile phase was established using a combination of acetonitrile and 0.1% TFA as the buffer, resulting in an acetonitrile–buffer ratio of 40:60 (v/v). The injection volume for all samples was set at 20 µL, and the mobile phase flow rate was maintained at 1 mL/min in isocratic mode.

Plasma sample preparation

The extraction and preparation process for plasma samples is outlined as follows [28].

Blank plasma preparation

To prepare the samples, 475 µL of human plasma was combined with 20 µL of 5% formic acid, and 1 mL of acetonitrile was added. The mixture was vortexed for 2 min and then centrifuged at 4000 rpm for 3 min. The resulting supernatant was filtered through a 0.22 µm filter, and 0.5 mL of the filtered supernatant was extracted for subsequent chromatographic analysis.

Plasma sample preparation with 5% spiking

For a 5% spiking of the sample in plasma, 475 µL of human plasma was spiked with 12.5 µL of NBH and 12.5 µL of AMB, both dissolved in methanol. The mixture was transferred to a Tarson tube and centrifuged at 4000 rpm for 10 min. The plasma was carefully separated using disposable polypropylene tips and stored in a deep freezer at − 20 ± 2 °C until further analysis.

5% spiked plasma sample extraction preparation

The protein precipitation method was used to extract the plasma samples. To prepare the samples, 500 µL of human plasma was mixed with 20 µL of 5% formic acid and the volume was adjusted to 1 mL with acetonitrile. The mixture was allowed to stand for 2 min and then thoroughly mixed. The samples were centrifuged at 4000 rpm for 3 min. The resulting supernatant, which contained the target drugs, was carefully separated. This supernatant was then dried and reconstituted with 1 mL of the mobile phase. The sample was filtered through a 0.22 µm filter, and 0.5 mL of the filtered supernatant was transferred for chromatographic analysis.

Quality control sample preparation

The quality control (QC) samples were prepared using procedures similar to those for the NBH and AMB stock solutions. To prepare the stocks, 200 mg of AMB and 250 mg of NBH were dissolved in 25 mL of methanol. From these solutions, 1 mL was pipetted and diluted with human plasma. Four concentrations of QC samples were prepared for NBH: 4.51, 13.52, 90.11, and 150.19 μg/mL, representing the LLOQ, LQC, middle-quality control (MQC), and HQC standards, respectively. Similarly, AMB QC samples were prepared at concentrations of 3.50, 10.51, 70.06, and 116.09 μg/mL for the LLOQ, LQC, MQC, and HQC standards, respectively [28].

Chromatographic conditions

In the HPLC analysis, specific chromatographic conditions were established. The analysis employed an Inertsil ODS-3 V column with dimensions of 150 mm × 4.6 mm and a particle size of 5 μm. The mobile phase was a mixture of acetonitrile and buffer in a 40:60% v/v ratio. A UV detector was used, set to a wavelength of 215 nm. The flow rate was maintained at 1.0 mL/min, and a 20 μL injection volume was utilized. The autosampler temperature was set appropriately for the analysis. To ensure the clarity of the mobile phase, it was filtered through 0.45 μm pore-size membranes and degassed. Throughout the study, the mobile phase was continuously degassed using a degasser connected to the column and was pumped in isocratic mode at a flow rate of 1 mL/min. The HPLC system received the 20 μL sample, which was then detected by the UV detector.

Method validation

Following the guidelines set by the USFDA and the EMEA, the developed and optimized method for the simultaneous determination of NBH and AMB underwent a comprehensive validation process. The validation assessed the method’s linearity, selectivity, sensitivity, accuracy, precision, recovery, and stability [29,30,31].

Linearity

Calibration curves were constructed to evaluate the linearity of NBH and AMB. The calibration ranges for NBH and AMB were established as 4.50–180.12 μg/mL and 3.50–140.06 μg/mL, respectively. Drug standard solutions were spiked into human plasma to create calibration standards. The relationship between the analyte peak area and concentration was used to generate the calibration curves [32].

Selectivity and sensitivity

The selectivity and sensitivity of the method were evaluated by analyzing six batches of human blank plasma, as well as hemolyzed and lipemic plasma samples. This assessment aimed to demonstrate the absence of chromatographic interference at the retention times of NBH and AMB. Acceptance criterion for selectivity was set at ≤ 20% of the response observed at the lower limit of quantification.

Extraction recovery

The extraction recovery of NBH and AMB was assessed as a percentage to evaluate the efficiency of the analytical procedure. This was done by comparing the response of QC samples with post-extraction spiked QC samples at the same concentration. The extraction recovery for NBH was calculated to be 97.69%, while for AMB it was 97.19%.

Accuracy and precision

Six replicates of QC samples were analyzed at four different concentration levels on three separate days to evaluate the intra-day and inter-day precision and accuracy of NBH and AMB in plasma samples. The assessment included QC samples of NBH with concentrations of 4.51 μg/mL (LLOQ), 13.52 μg/mL (LQC), 90.11 μg/mL (MQC), and 150.19 μg/mL (HQC). Similarly, QC samples of amlodipine besylate were prepared at concentrations of 3.50 μg/mL (LLOQ), 10.51 μg/mL (LQC), 70.06 μg/mL (MQC), and 116.09 μg/mL (HQC). For intra-day precision, the acceptable range was set at 85–115% of the nominal value for all concentrations, except for the LLOQ, which had a required range of 80–120%. The intra-day precision was evaluated based on the percentage relative standard deviation (% RSD), which should not exceed 15%. The LLOQ QC concentration was not to exceed a maximum of 20% RSD. For inter-day precision, the acceptable range was set at ≤ 20% of the nominal value for all concentrations except the LLOQ, where the range was ≤ 20%. The between-run precision (inter-day) criteria required that the RSD of QC samples at each concentration be within ± 2%, except for the LLOQ, which should not exceed 20% [33].

Recovery study and matrix effect

To evaluate the effectiveness of extracting the substances from plasma samples and assess the impact of the matrix, a recovery study was conducted. The substances were first added to blank plasma at standard concentrations (low, medium, and high levels) and analyzed. Solutions without the biological matrix were then prepared and spiked with the same standard concentrations. The ratio of the area of the NBH peak to the AMB peak in samples with and without the biological matrix was determined. The impact of the matrix on the quantification of NBH at the LLOQ was compared between the responses obtained with and without the matrix.

Stability studies

Stability studies of NBH and AMB in plasma samples were conducted at two concentration levels: LQC and HQC. The purpose was to determine the drug concentrations after each storage period and compare them to the initial baseline concentration. The following section details the investigations performed to assess the stability of the samples [34,35,36,37].

Bench-top stability

The bench-top stability of NBH and AMB was evaluated using low and high-concentration samples at a temperature of 25 °C. After keeping the samples at room temperature for 6 h, aliquots of the low and high-concentration plasma samples were processed and analyzed by HPLC to determine their respective drug concentrations.

Freeze–thaw stability

The freeze–thaw stability study of NBH and AMB was conducted on plasma samples with low and high concentrations over three cycles within three days. The frozen plasma samples were thawed for 2 h at ambient temperature and then refrozen for 24 h each cycle. The drug concentrations in the samples were analyzed at the end of the third cycle and compared with those at the initial (zero) cycle.

Autosampler stability

In the autosampler stability study, LQC and HQC samples obtained from the precipitate solution were reinjected 24 h after the initial injection. The percentage recovery was calculated by comparing the measured concentrations of the reinjected samples with those of freshly prepared samples.

Stability study of processed sample

The stability study of processed samples included three low-quality control (3LQC) and three high-quality control (3HQC) samples for NBH and AMB. These samples were subjected to a 6 h stability period and then analyzed by injecting them into the HPLC system.