Bioanalytical Method Development and Validation of Brexpiprazole in Human Plasma By RP-HPLC Method

Brexpiprazole is a BCS class II type called (7-{4-[4- (1-benzothiophen-4-yl) piperazin-1-yl] butoxy} quinoline2(1H)-one is a new medication in psychiatric 1,2 and also in the treatment of depression which has a high affinity for monoamine neurotransmitters like serotonin, dopamine, and noradrenaline receptors, For other major depressive disorder like Alzheimer’s disease, schizophrenia 3,4 , neurobehavioral disorders 5 , combat disorder, bipolar disorder treatment, adjunctive treatment it is mostly used. It has more potency than other antipsychotic drugs with little aqueous solubility and substantial intestinal permeability. 6,7 Brexpiprazole was originally approved in the United States in July 2015 for use as an adjunctive action for major depressive disorder (MDD) and schizophrenia. 9 Antipsychotics are first and second-generation drugs that are mainly active as D2 receptor antagonists. 10,11 Brexpiprazole acts as a partial agonist of the serotonin 5-HT1A receptor and the dopamine D2 and D3 receptors. Partial agonists have both blocking properties and stimulating properties at the receptor they bind to. The ratio of blocking activity to stimulating activity determines a portion of its clinical effects. . 12 brexpiprazole is a substrate of CYP2D6 and CYP3A4, like its predecessor aripiprazole. A simple, accurate, and precise method for the quantification of brexpiprazole in human plasma was developed by using the RP-HPLC (Reverse phase-High performance Liquid Chromatographic) technology to construct an internal standard of oriconazole.

MATERIALS AND METHODS

MATERIALS:

1. API:
2. Jai Ram Biosciences generously provided a sample of brexpiprazole API.
3. Human plasma:

Table No. 1: Human plasma

4. Chemicals:

3.  Instruments:

Method Development:

Diluent: In order to ensure that the medications would dissolve properly, we used a diluent consisting of 0.1% orthophosphoric acid and 50% acetonitrile.

How to make a stock solution of brexpiprazole (100 µg/ml):

Make a solution of 10 mg of Brexpiprazole in 100 ml of volumetric flask to achieve a concentration of 100 µg/ml...

Preparation of Brexpiprazole Spiking Solutions (0.010 µg/ml to 0.4µg/ml):

Brexpiprazole was generated from the stock solution at the following concentrations: 0.1 µg/ml, 0.2 µg/ml, 0.3 µg/ml, 1.6 µg/ml, 2.00 µg/ml, 2.4 µg/ml, 3.2 µg/ml, and 4.0 µg/ml.  The eight 10-milliliter volumetric flasks were thereafter filled to the top with diluent. Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with analyte dilutions from working stocks at concentrations of 10, 20, 30, 160, 200, 240, 320, plus 400 ng/ml, respectively.

Preparation of internal standard Solution (oriconazole):

Stock-1: Put 5 mg of Oriconazole into 100 mL of volumetric flask and dilute it until the volume reaches 50 mg per mL.

Stock-2: Dilute the solution to a concentration of 5µg/ml by adding 1 millilitre to a 10-milliliter volumetric flask from the previous solution..

Final concentration: To achieve an ISD concentration of 1µg/ml, 0.5 ml of the solution from the preceding stage should be mixed with blank plasma using working stock dilutions of the analyte.

Extraction procedure

Put 750µl of plasma, 500µl of internal standard, 250µl of Brexpiprazole from both spiking solutions, and 1 ml of acetonitrile into a centrifuging tube.  Stir for another 15 seconds.   Next, vertex for 2 minutes after spinning for 5 minutes at 3200 rpm.   After the centrifugation process, gently add 10 µ L of the filtered sample to the HPLC directly.

There was a 15-second mixing period between 250µl of Brexpiprazole, 500µl of internal standard, and 750µl of plasma.   acetonitrile and a chemical blender for two minutes. Node set Drain by spinning the mixture at 3200 rpm for 5 minutes.   Collecting a specimen from the liquid above  Using a 0.45µ filter constructed of polyvinylidene difluoride or polyvinylidene fluoride, squeeze the sample out.   Put 20 µ L into the HPLC.

Trial (optimized method):

Chromatographic conditions

Phase of mobility: 0.01 nanoohms A 60:40 mixture of acetonitrile and potassium dihydrogen phosphate at a pH of 4.8, with a flow rate of one millilitre per minute. Applying a Phenomenex c18 column (150 x 4.6 mm, 5 ?).   Detector beam width: 215 nanometres. Column temperature: 300 °C   Two thousand three hundred microlitres is the prescribed injection volume.

Six minutes, tops

Resolved: 0.01%   This is a 2:5 solution of potassium dihydrogen phosphate in acetonitrile.

Table No: 10 and Figure No: 4 show the trial's outcomes.

Method Validation:

System Suitability:

All indicators for system appropriateness were adequate and within the permitted range, as per ICH requirements. During the system compatibility test, the percentage CVs for retention time (RT) and area ratio (analyte area/IS area) were in the range of 0.06 to 0.08. This is because the auto-sampler was fed a cocktail of extracted and unextracted samples for analysis.   The results of this experiment did not show any evidence of carryover..

Matrix factor evaluation:

The matrix impact is a key component in assessing pharmacokinetic studies.   The internal standard normalised matrix factor, which was close to 1, varied from 0.90 to 0.99, indicating that plasma samples do not show any ionisation suppression or augmentation.

Quality control samples:

1. The chromatography observed during the period of Brexpiprazole was good, as shown in Figures 5-9, which present example chromatograms of the standard zero, QC-LLOQ, QC-MQC, and QC-HQC samples, respectively.
2. Selectivity/Specificity:

By examining the retention time of Brexpiprazole and the internal standard for interference with the endogenous plasma components, we validated the approach and demonstrated its selectivity.   The mass detection of the extracted blank plasma demonstrated good drug and internal standard selectivity in six sets of K2EDTA blank plasma that were evaluated for selectivity.   No interferences were detected that would have altered the retention periods of the analytes or the internal standard.   Below are chromatograms illustrating the internal standard sample made up of pooled plasma, as well as the standard blank prepared using a representative sample.   This result is shown in Figures 10 and 12...

3. Linearity:

The calibration curve for Brexpiprazole was found to be linear from 10 to 400 ng/mL.   The coefficient of correlation (r2) was more than 0.999 in every case.   It appears that the results are linear because the peak area ratios are strongly related for all analyte concentrations.

4. The third accuracy and precision batch acquired a typical calibration curve, as shown in Figure 25.   In Table 14, Figure 13–21, we can see the combined results of the three calibration curves' concentrations that were back-calculated..

Section 4: Accuracy & Precision:

5. We were able to evaluate the precision and accuracy both within and between days by analysing six replicates at five different QC levels (LLOQ, LQC, MQC, and HQC).   Using six replicate analyses, the procedure's precision and accuracy were evaluated at four distinct concentrations: 10ng/ml (LLOQ), 30ng/ml (LQC), 200ng/ml (MQC), and 320ng/ml (HQC).   When evaluating the accuracy of plasma samples, we found that intraday accuracy ranged from 99.91% to 103.000% and interday accuracy ranged from 100.07% to 102.23%, both of which are excellent.   Intraday, the precision of the analyte ranged from 0.36 percent to 2.07 percent, and interday, the precision of the plasma sample ranged from 0.4 percent to 2.09 percent.   For a rundown of the findings, see Tables 15 and 16, as well as Figures 22–27..
6. Recovery: Extracting blank plasma spiked with standards containing the same area with a known dose of Brexpiprazole allowed us to estimate recovery, as did measuring the peak areas of prepared plasma samples.   It was found that the overall mean recovery rate with Brexpiprazole was 98.12%.
7. The results for Brexpiprazole at three distinct QC concentration levels are summarised in Table No. 17.
8. A recovery rate of 99.00% was found for Oriconazole overall.   Table 18 provides a synopsis of the Oriconazole recovery results.  12.  Main Ideas:
9.  How long does the stock solution of Brexpiprazole remain effective?   On a bench top, six distinct LQC and HQC samples were evaluated for stability, with concentrations of 30 and 320 ng/ml, respectively.
10. The experiment was conducted in a controlled atmosphere for 9 hours on the benchtop.   In terms of mean stability, 100.78% was calculated for LQC and 101.18% for HQC.   Table 19 showed this result.Matrix samples' stability at room temperature for 37 days and at -80 ± 5°C.
11. A 37-day period of refrigeration at -28°C and -80°C proved that the Brexpiprazole stock solution maintained its long-term stability at a concentration of LQC-HQC.   At 28 ± 5°C, the average stability of Brexpiprazole was found to be 100.28%, 100.05% & 101.88%, 101.86, and at the same temperature, 99.90%, 100.44% & 101.85%, 101.36—respectively.
12. The long-term stability of Brexpiprazole is shown in Tables 20, 21.

RESULTS AND DISCUSSIONS

Method Development:

The following conditions were considered when developing the method for determining Brexpiprazole: drug solubility; PKA value; and compliance with current ICH standards.

Trial 4 (optimized method):

Fig No. 1: Chromatogram of trial 4

Observation: Both peaks exhibited excellent peak shape, retention time, and tailing-free elution.

Method Validation:

System suitability of Brexpiprazole

Auto sampler carryover of Brexpiprazole:

Matrix factor evaluation:

Table No. 7: Reviewing matrix factors in the absence of a matrix

Quality control samples

Standard zero sample:

Fig. No. 2: Chromatogram of a calibration standard

Fig. No. 3: The QC-LLOQ sample chromatogram

Brexpiprazole QC-LQC

Fig. No. 4: Brexpiprazole chromatogram from a QC-LQC sample

Fig. No. 5: Sample chromatogram for QC-MQC

Fig. No. 6: The Brexpiprazole QC-HQC sample chromatogram

Fig. No. 7: Obtaining a blank, standard sample

Fig. No. 8: Oriconazole Chromatogram (Internal standard)

Fig. No. 9: Brexpiprazole pure medication chromatogram)

Linearity of brexpiprazole

Accuracy (Brexpiprazole intra-day runs):

Fig No. 10: Intraday precision 1

Fig No. 12: Intraday precision 3

5. Recovery:

Recovery of brexpiprazole

Recovery - Internal standard

6. Stabilities:

Long term stock solution stability

Matrix samples stability at -80 ± 5?C for 37days