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Materials and Chemicals

Vilazodone (VIL), and Vilazodone D8 were arranged from BioOrganics Pvt. Ltd. Bangalore. Chemical structures shown in Figure 1. Acetonitrile (super gradient grade), Methanol, Methyl-t-butyl ether and Dichloromethane were obtained from RCI labscan, Thailand. Formic acid, Sodium hydrogen carbonate were used of Merck, Germany. Milli -Q-Water was used for the preparation of buffer solutions. Human frozen plasma (K2-EDTA) was received from blood bank “Jensys laboratoris”, Hyderabad, India. Analytical column, Betabasic C8 was procured from Thermo Scientific, USA.

Instrument’s, equipment’s and software’s

Quantitative analysis was performed by using triple quadrupole mass spectrometer of TSQ Quantum Ultra (Thermo scientific, USA) and Shimadzu UPLC, Japan (Prominence model) equipped with binary pump, autosampler, degasser and column oven. For working standards and buffers weighing purpose, sartorius microbalance and analytical balance were used respectively. For sample extraction purpose, refrigerated centrifuge of Thermo scientific, USA, and Multi tube vortexer used of MR scientific, India was used. Plasma samples were stored in deep freezers (Thermo electron corporation, USA) at -20+/-5°c and -70+/- 10°c. Validation data were generated by TSQ Quantum LC Quan software version 2.5.6.

Preparation of solutions

Different solutions were prepared according to defined procedure as and when required such as Diluent [Water: Methanol (60:40%v/v)], Extraction solution [Methyl-t-Butyl Ether: Dichloromethane (70:30%v/v)], Buffering agent [50mM Sodium hydrogen carbonate], Buffer for Mobile Phase [0.1%v/v Formic acid in water], Reconstitution solution [Acetonitrile: 0.1%v/v Formic acid in water (60:40%v/v)], and Rinsing solution [Methanol: Water (90:10%v/v)].

Preparation of main stock solutions and working dilutions

About 1mg of vilazodone working standard was weighed accurately and transferred in 5ml of volumetric flasks, added 2ml of methanol and sonicated to dissolve then finally diluted up to the mark with the same to obtain individual main stock concentration of about 0.200mg/mL. Further intermediate stock solution and working dilutions were prepared from main stocks by using diluent solution for spiking in plasma to obtain CC standards and QC samples. CC and QC dilutions was prepared in low light and room temperature condition. Calibration range was established from 0.300 to 300ng/mL for vilazodone.

Preparation of main stock solutions of Internal Standards and Mixed ISTD working solution

About 1mg of vilazodone D8 was weighed accurately and dissolved in 10mL of methanol to obtain main stock concentration of about 0.1mg/mL. Afterwards ISTD working solution was prepared from main stock solutions using diluent to achieve final concentration of about 1000ng/mL of Vilazodone D8.

Preparation of spiked plasma calibration curve standards and quality control samples

Calibration curve standards consisted of a set of nine non-zero concentration levels (STD-1 to STD-9) were prepared by spiking the working solutions of analyte in human K₂EDTA plasma to achieve concentration of 0.300ng/mL, 0.600ng/mL, 15.000ng/mL, 30.000ng/mL, 60.000ng/mL, 120.000ng/mL, 180.000ng/mL, 240.000ng/mL and 300.000ng/mL. In order to bracket the linearity range and for reliable quantitation, Quality Control samples were prepared at four different concentration levels as lower limit of quantification (LLOQ-0.300ng/mL), low quality control (LQC-0.750ng/mL), middle quality control (MQC-150.000ng/mL) and high-quality control (HQC-270.000ng/mL). 2%v/v of respective CC/QC dilutions was spiked in screened K₂EDTA pooled plasma to achieve the desired nominal concentration of CC/QC samples. CC/QC dilutions and spiked samples were protected from light during preparation and usage. Bulk spiked CC/QC samples was stored in an ultra-low temperature deep freezer (-70°C±10°C) until analysis.

Sample extraction procedure

Sample processing was carried out as per standard test procedure following liquid liquid extraction technique. Required frozen plasma samples were retrieved from the deep freezer and allowed to thaw at room temperature followed by adequately vortexing. Added 0.300mL of plasma sample into a prelabelled polypropylene tube and 0.015mL internal standard working solution followed by vortex to mix uniformly. 0.400mL of buffering solution was added to each tube and vortex to mix. For extraction purpose, 3mL of extraction solution was added followed by capping of tubes and vortexing for 5min using multi tube vortexer at 2500rpm. Samples were allowed to centrifuge for 5min at 4500rpm at 4°C. Supernatant organic phase was transferred into another prelabelled tube using flash freezing bath and samples were kept for drying with stream of nitrogen evaporation at 40°C. Then dried samples were reconstituted with 0.400mL of reconstitution solution and transferred to autosampler vials. Lastly, for analysis purpose, 10µL of sample was injected into UHPLC-MS/MS system.

Mass spectrometry and chromatographic conditions

Analytical separation was performed with Shimadzu prominence UPLC (Shimadzu, Japan) comprising of solvent delivery module SIL-20AT, an autoinjector SIL 20AC, a column oven CTO-20AC. Chromatography separation of analytes and internal standard was accomplished within 2.500 min using a Betabasic C8, (100*4.6mm, 5µ; Thermo Scientific, USA) column and a mobile phase consisting of Acetonitrile: 0.1% v/v formic acid in water (60:40, v/v) by binary pump at a total flow rate of 0.700 mL/min. The column and autoinjector temperature were set at 40°C and 5°C respectively. Mass spectrometer used for this work was triple quadrupole TSQ Quantum Ultra (Thermo Scientific, USA) which consisted of a heated electro spray ionization (HESI) source in positive ion mode. Multiple reaction monitoring (MRM) transitions used with dwell time set at 200 milli sec. per transition. Inert gas Nitrogen was used as the zero air for nebulizer, curtain, auxiliary, and collision gases. UPLC-MS/MS parameters are shown in table:1. Calibration standard curves were plotted by calculating the analyte to internal standard peak area ratio (y) versus analyte nominal concentrations (x). Data acquisition, processing and quantification were performed using Xcalibur version 2.2 and LC Quan version 2.7.0 software (Thermo Scientific, USA).

Method Validation

Method validation was completed according to USFDA guidelines and in compliance with principles of Good Laboratory Practice.

Carryover check

To confirm that there has been no considerable carryover of analyte from a prior injection, a carryover check experiment was conducted. At ULOQ level, this experiment was conducted. By injecting the ULOQ, blank solution, blank sample, and comparing with LLOQ samples, carryover check of the sample was performed. After each ULOQ injection, the percentage of carryover in the blank solution and blank sample was determined. Blank solution and blank sample for VIL and VIL D8 showed no carryover.

Selectivity

Twelve normal lots of K2EDTA plasma, including two lipemic and two homolytic plasma with the same anticoagulant, were used to evaluate the method's selectivity (K2EDTA). Each plasma lot was processed for blank samples and LLOQ samples in order to assess interference at the RT of the analyte and the ISTD. Fresh linearity and four sets of QC samples at LQC, MQC, and HQC levels were used for the selectivity test. The following batch-specific acceptance criteria were considered when evaluating this test: If any interfering peaks are present in the blank sample at the retention time of the analyte, their response should be less than 20% of the area of the corresponding lot LLOQ response. Any peak in the Blank sample that is present at the ISTD retention time must have the response that is less than 5% of the ISTD response average of acceptable CC/QC samples. The LLOQ samples back computed concentration should fall within 20% of the nominal value.

Matrix effect

It is carried out to evaluate the degree to which the biological matrix or any of its constituent parts affects the measurement of the analyte. To make sure that the Precision, Accuracy, and Sensitivity are not hindered by varying lots of matrix usage, matrix effect was measured in eight different lots of K2EDTA plasma, including at least one lipemic and homolytic lot plasma. At LQC and HQC, the matrix effect was assessed in triplicate. Blank samples from each lot were processed for the evaluation of the matrix effect, and after drying, the samples were reconstituted with a reconstitution solution containing the mixed analyte and ISTD to produce aqueous equivalent LQC and HQC samples.

Matrix factor (MF) for the analyte and ISTD was calculated as follows-

MF of Analyte: Mean peak analyte area in presence of Matrix samples/ Mean peak analyte area in neat aqueous sample.

MF of ISTD: Mean peak area of ISTD in presence of Matrix samples/ Mean peak area of ISTD in neat aqueous sample.

ISTD normalised M.F.- MF of Analyte/ M.F. of ISTD

MF of 1 signifies no matrix effect.

A value of less than 1 suggests ionization suppression.

A value of greater than 1 suggests ionization enhancement.

Acceptance criteria:

% CV of Matrix factor of different lots at each level for analyte and ISTD should be ≤15%.

% CV of ISTD normalised MF across different lots should be ≤15%.

Sensitivity

The sensitivity of the method that may be measured within an acceptable range of accuracy and precision is represented by the calibration curve's lowest non-zero standard (LLOQ). In this study, precision and accuracy batches were used to process six replicates of LLOQ samples. For the experiment to be considered acceptable, the LLOQ samples' accuracy must be 20% or less and their signal-to-noise ratio must be five.

Calibration curve

It shows the relationship between the analytical concentrations and the experimental response values. Four precision and accuracy batches were used to assess the method's linearity. The concentration of analyte was calculated using a weighted 1/X2 linear regression. “To meet the acceptability of calibration curve, it is desirable that coefficient of determination (r²) should be ≥0.9800. The percent nominal of LLOQ samples must be ±20% of the nominal value. The percent nominal for other than LLOQ must be ±15% of their nominal value and at least 75% of calibration curve standards including LLOQ and ULOQ must meet the above criteria” (USFDA Bioanalytical Method Validation Guidance for Industry 2018).

Precision and Accuracy

The reproducibility of a bioanalytical process is considered in terms of precision. It delivers an estimate that if the same sample is analysed more than once, the outcomes will be relatively close enough. Precision is assessed at different concentration levels during method validation utilising quality control samples such LLOQQC, LQC, MQC, and HQC. This accuracy is verified for inter-assay and intra-assay estimation [25]. The accuracy of the method is a measurement of how closely the concentration obtained during analysis resembles the actual concentration of the analyte. “Freshly spiked quality control samples i.e., LLOQC, LQC MQC and HQC and Calibration curve standard samples were processed and used for the evaluation of within batches and between batches Precision and Accuracy of the method. Percent coefficient of variation (%CV) should be within ±15 for LQC, MQC and HQC whereas for LLOQC %CV should be within ±20. Accuracy should be ±15 % for LQC, MQC and HQC whereas for LLOQC, accuracy should be within ±20%” (USFDA Bioanalytical Method Validation Guidance for Industry 2018).

Recovery

Recovery was calculated at three distinct levels at LQC, MQC, and HQC by comparing the mean peak area response obtained from extracted QC samples with identical post-spiked QC samples to represent 100% recovery. The recoveries of D8-ISTDs were assessed similarly using the respective medium QC samples as a reference.

% Recovery = Extracted peak area / Unextracted peak area*100.

It is desirable that recovery of the method should be precise and consistent. % CV of the mean recovery across different QC levels should be ≤15.

Stability

During quantitative bioanalysis by LC-MS/MS, drug stability is very important pre-analytical consideration. If appropriate preventive measures are not taken, drug instability at different stage of clinical sample handling, including collection, storage, extraction, processing and LC-MS/MS analysis, may lead to suppression or enhancement of estimation. Freshly prepared CC and QC samples at LQC and HQC levels were used for the evaluation of all matrix stability. Analyte and ISTD stock solution stability were evaluated at LLOQ and ULOQ level at room temperature. Stability experiments will be considered to be acceptable if experiment values are within ±15 for accuracy and precision.

Method Development

Optimization of quantification and separation process

Vilazodone method development was initiated with scanning and finalization followed by optimization of MS parameters using an appropriate concentration of Vilazodone and Vilazodone D8 tuning solution (~250ng/ml for both). In order to obtain optimum signal intensity and response, scanning was performed with electro spray ionization source in negative [M-H] and positive [M+H] mode polarity. However, with negative polarity mode, significantly reduced signal intensity and response were found. Therefore, in order to obtain optimum signal intensity and response positive polarity was selected to finalize the MS parameters. Abundant parent ion of VIL and VIL D8 were found at m/z [M+H⁺] 442.022 and 450.093 respectively (figure 2) while the most abundant multiple fragment ions of VIL and VIL D8 were selected at m/z 155.0 and 197.0, 157.0 and 205.0 respectively. In this study, we have noticed that single fragment transition was not producing the adequate response. Hence sum multiple fragment ions were selected to achieve the sufficient response. MS spectra are shown in figure 2. During identification of parent and product ions, skimmer offset, collision energy was optimized judiciously. The multiple reaction monitoring (MRM) mode was applied quantification of parent-product ion transitions. In order to optimize source MS condition efficiently, source parameters such as Gas, temperature and voltage were evaluated by substantially increasing or decreasing parameters which could facilitates the proper ionization, spray of mobile phase and selective quantitation. To obtain precise chromatographic decisions and symmetric peak shapes, the chromatographic factors including diverse conditions of mobile phase composition, flow rate, column selection as well as faster and shorter run times, were optimised through numerous trials. Initially different ratios of mobile phase composition consist of methanol and ammonium formate buffer with various strengths were tested. Which gives idea-based chromatography and response. Later on, in order to check the other organic solvent and buffer solution, acetonitrile and ammonium acetate buffer with different strengths were changed one by one and different trials were planned to assess the chromatographic data.  Combination of acidified ammonium formate: acetonitrile and ammonium formate: methanol was also tested to confirm the improvement upon addition of formic acid on chromatographic peak shape and response. It was observed that upon acidification of buffer, peak shape was found to be broad and response drastically reduced. However, acetonitrile gives better response and peak shape compare to methanol. Thus, above inference triggered to move for formic acid trial without ammonium formate with acetonitrile. Hence mobile phase containing formic acid in water and acetonitrile was checked with different strength of formic acid and ratios of mobile phase. Formic acid was proven to be essential for bringing the pH down in order to protonate the analytes and provide the proper response factor. With the proper ionisation and fragmentation in the mass spectrometer, the formic acid percentage was tuned to maintain this top form while remaining constant. Therefore, mobile phase consist of 0.1% formic acid in water and Acetonitrile were checked resulted better response and sharp peak shape with good precision and accuracy across the method optimization. In order to achieve sharp and symmetric peak shape with optimum response, alongside different reverse phase C₁₈ columns of varying dimension and chemistry were tested. However best reproducible chromatography in terms of response, peak shape, baseline noise, and retention time was found with beta basic C₁₈ 100*4.6mm.

Sample extraction procedure: Firstly, simple, fast, cost-effective protein precipitation technique was tested using methanol and acetonitrile as PPT agent but there was less recovery, response and suffer with huge matrix effect. Also, final extract was more turbid and not clear transparent. Therefore, moved forward for liquid-liquid extraction in which various combination of extraction solvents such as TBME, n-hexane, DCM, ethyl acetate in pure and in combination of mixture were tested. In order to make the sample unionized, pre-treatment or buffer solution was added. Sodium hydrogen carbonate, formic acid and water was used as buffering agents. Finally, mixture of extraction solvent viz. Methyl-t-Butyl Ether: Dichloromethane (70:30%v/v) and buffering agent as 50mM Sodium hydrogen carbonate was finalised for the sample extraction which gives better recovery, chromatography response with good precision and accuracy across the method optimization. Representative chromatograms are shown in figure 3.

Method Validation

Full method validation was carried out in compliance to the USFDA recommendations for the bioanalysis in biological matrix. As part of method validation, following experiments were evaluated.

Selectivity

Six lots of human K2EDTA plasma, one each lot of hemolyzed and lipemic plasma were evaluated to measure the extent to which matrix components may impact to interference of analyte or internal standard. No any interference was found at the analyte or internal standard retention time.

Sensitivity

The linearity samples and six sensitivity LLOQ samples were processed and injected. Signal-to-noise ratio, percentage coefficient of variation, and percentage difference from the nominal concentration were all examined for compliance with the acceptance criteria. All found well within the acceptance limits.

Calibration Curves

Over the prepared concentration range, the calibration curve was shown to be consistently accurate and precise. Table 2. contains the calibration curve parameters and the back-calculated concentration of the calibration standards.

Precision and accuracy

Intra-batch Precision and accuracy

A calibration curve along with four types of quality control samples (each six samples of the LLOQC, LQC, MQC and HQC) was used with four separate batches (Intra Batch 01 to 04) for the analysis and assessment of intra-batch precision and accuracy. Precision and accuracy results of each batch were compiled and mean % nominal concentration and %CV was calculated at each QC level for each P&A batch.

Inter-batch precision and accuracy

Inter-batch precision and accuracy was examined by analysing four batches consisting of calibration curve along with four types of quality control samples (each six samples of the LLOQC, LQC, MQC and HQC) on three different days. Precision and accuracy results of the batches runed on different days were compiled and global mean % Nominal concentration and global %CV were calculated at each QC level. The results obtained which are presented in table 3. were well within the acceptance limits.

Recovery

Quality control samples of low, medium and high (LQC, MQC and HQC) was processed and used for the calculation of recovery of analyte and internal standard. Six samples each of LQC, MQC and HQC were processed along with eighteen blank samples. After drying of samples, QC samples were reconstituted with reconstitution solution whereas blank samples were reconstituted with respective analyte spiking solution, internal standard and reconstitution solution to represent post extracted spiked samples with assuming 100% recovery. The obtained results were precise and consistent at each QC levels and internal standard. The results are shown in table 4.

Matrix effect

Matrix effect was tested at LQC and HQC level using six normal lots of K2EDTA plasma including single lot of lipemic and hemolysed plasma. From each lots blank samples were processed in triplicate and the final dried samples were post spiked with respective (LQC and HQC) spiking stock solution and internal standard. Along with post spike samples, Aqueous equivalent LQC and HQC samples was also prepared. The % CV obtained for matrix factors at each level, internal standard and ISTD normalized matrix factor at each level was within the acceptable limits.

Stability

The stability evaluations performed by comparing the stability samples with freshly prepared samples. Stability data is presented in Table 5. Established stabilities experiments such as Bench top stability for 08.00hrs at room temperature,  In-injector stability at 5°C in auto sampler for 79.00hrs, Freeze and thaw stability (after 4th cycle at -70±5°C), Wet extract stability at room temperature for 02.00hrs and at -20°C for 67.00hrs, Process stability at room temperature for 06.00hrs and Long term stability for 38 days at -70±5°C were found to be stable with acceptable % mean change (±15), % nominal (±15) and %CV (±15).

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