A Bioanalytical Method for Eliglustat Quantification in Rat Plasma

A Bioanalytical Method for Eliglustat Quantification in Rat Plasma Abstract A simple and sensitive bioanalytical HPLC–UV method has been developed and validated for quantification of eliglustat in rat plasma. The liquid–liquid extraction method was found to be more efficient compared to protein precipitation technique. Chromatographic separation of eliglustat was achieved using Kromasil C18 column with a mobile phase consisting of a mixture of methanol and ammonium acetate (pH 3.2) in a ratio of 60:40. Detection wavelength was set at 282 nm. The developed method was specific, accurate, precise with good recovery and stability profile. The calibration curve constructed over a range of 0.3–10 μg/mL was linear (R2 > 0.997). Accuracy in intra and inter-day assay were found to be 96.27–107.35% and 96.80–106.57%, respectively. The corresponding precision (%CV) values were within 4.31–10.90% and 4.82–9.97%, respectively. Till date, no method is available for bioanalysis of eliglustat in any type of biological matrix. This is the first time to report a bioanalytical method for this molecule. The developed bioanalytical method was applied to quantitate eliglustat in the plasma samples of a single dose oral pharmacokinetic study in Sprague Dawley rat. Introduction Bioanalysis plays a key role in drug discovery, pharmacokinetic and toxicokinetic study (1). Nowadays, bioanalysis is an integrated component of the drug development process. Drug discovery and its further development into a new drug substance are the two crucial stages in the drug product lifecycle. The human body comprises of series of complex systems to affect the action and fate of drugs (2–4). Therefore, not only what drug affects the body but also what the body does to the drug is equally important. Determination of pharmacokinetic properties of drugs and their metabolites in the body can be beneficial to correlate their variable effects amongst different species. It is thus helpful to adjust the dose of a drug (5). Developing a quantification method of drugs in biological matrices is challenging because of the presence of various exogenous and endogenous compounds. The extraction of the analyte from such matrices prior to their analysis need to be optimized (6–9). Different bioanalytical techniques have been reported for the pre-treatment of the samples for achieving appropriate accuracy, precision, selectivity and sensitivity (7). Gaucher disease is a hereditary disease caused by the accumulation of glucocerebroside in the cells and other organs. The disease is characterized by enlargement of liver and lungs, and decreament in the white blood cell count resulting in anemia (10). Due to the lack of enzyme glucocerebrosidase in the patients suffering from this disease, glucocerebroside accumulates inside the cells. Eliglustat inhibits the enzyme glucosylceramide synthase and prevents the generation of glucocerebroside and thereby, ameliorate Gaucher disease (11, 12). Chemically, eliglustat is N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)octanamide (2R,3R)-2,3-dihydroxysuccinate. The objective of this study was to develop and validate a reverse phase-high-performance liquid chromatography (RP-HPLC) method for the estimation of eliglustat in rat plasma and evaluation of its applicability in a pharmacokinetic study. There is no bioanalytical method available till date for quantitation of eliglustat in rat plasma or any other type of biological matrices. This is the first time to report an accurate and precise bioanalytical method for quantitation of eliglustat in rat plasma. Materials and Methods Chemicals and reagents Eliglustat (Figure 1A) (purity > 98%) was received as a gift sample from Torrent Pharmaceuticals, India and haloperidol (Figure 1B) (purity > 99%) was purchased from Sigma Aldrich. Dichloromethane, methanol (HPLC grade), acetonitrile (HPLC grade), ethyl acetate and tertiary butyl methyl ether (TBME) were purchased from Fisher Scientific. Ammonium acetate and trichloroacetic acid were also procured from Sigma Aldrich. Milli-Q grade water generated from a Milli-Q gradient system of Millipore was used for the analysis. Figure 1. View largeDownload slide Molecular structure of (A) Eliglustat and (B) Haloperidol. Figure 1. View largeDownload slide Molecular structure of (A) Eliglustat and (B) Haloperidol. Method development The UV spectra of both eliglustat and haloperidol (internal standard (IS)) were recorded in the range of 200–400 nm in a UV–Visible spectrophotometer (UV–Vis 1800, Shimadzu). Initial method development trials for HPLC analysis were carried out using ammonium acetate buffer of 10 mM concentration of different pH. The eliglustat concentration for method development trials was 10 μg/mL. Initially, extraction of eliglustat from rat plasma was carried out with protein precipitation technique using methanol in acidic and neutral conditions. In acidic medium, trichloroacetic acid (TCA) (2%) was used as an acidifying agent. In either of the protein precipitation technique (neutral or acidic condition), the recovery was not optimum. Therefore, we moved to liquid–liquid extraction (LLE) to extract eliglustat from rat plasma. In LLE, four different solvents namely dichloromethane, ethyl acetate, TBME and diethyl ether were used for optimizing the extraction process. All the LLEs were carried out in the acidic, basic and neutral medium. Instrumentation The Agilent 1200 infinity series HPLC instrument coupled with UV detector with open lab software was used for the development and validation of the method for estimation of eliglustat in rat plasma. Sample extraction procedure A volume of 10 μL of eliglustat (100 μg/mL) was taken in a plastic RIA (radioimmunoassay) tube, evaporated in nitrogen evaporator till dryness, added 80 μL of blank rat plasma and vortex mixed. Thereafter, 10 μL of 100 μg/mL of IS solution was added and vortex mixed. The extraction solvent TBME was added to it (2 mL) and vortexed. The tubes were centrifuged for 10 min at 5,000 rpm at 4°C and kept in −80°C for 7 min. The organic layer was separated and evaporated in nitrogen evaporator till dryness. The dried extract was then reconstituted with 300 μL of mobile phase and finally, 5 μL was injected into the HPLC system. Preparation of stock solution and buffer Individual stock solutions of 100 μg/mL were prepared for eliglustat and haloperidol in methanol. The buffer solution (10 mM) was prepared by dissolving ammonium acetate in Milli-Q water. It was then filtered through a 0.2 μm Millipore filter and the pH was adjusted to 3.2 with diluted acetic acid. Bioanalytical method validation The developed bioanalytical method was validated according to the USFDA bioanalytical method validation guideline (13, 14). System suitability System suitability was performed by taking mid-quality control (MQC) sample. Six MQC samples were prepared separately and injected. Deviations in the area and retention time were evaluated. Specificity Specificity was determined at the lower limit of quantitation (LLOQ) level. Blank plasma samples from six different sources were collected and percent interference was checked at LLOQ level of 0.3 μg/mL. For each plasma samples, six determinants were evaluated (13, 15). Calibration curve Calibration curves were constructed using six concentrations levels of 0.3, 1, 2, 5, 8 and 10 μg/mL. The acceptance criteria for the linearity study was set to get the regression coefficient (R2) of the curve greater than 0.99. Accuracy, precision and recovery Accuracy and precision were evaluated at three quality control (QC) concentrations namely low-QC (LQC), MQC and high-QC (HQC) level. The concentrations of LQC, MQC and HQC level were 0.9, 4 and 8 μg/mL, respectively. Six determinations at each concentration were injected for determining the accuracy and precision of the method. Acceptance criteria were set to ensure that at least 67% of samples should be within 85–115% of nominal concentration and the %CV of the six replicate injections should be <15%. On the other hand, as per USFDA guideline, the %CV at LLOQ should be ±20% (13, 16). Recovery of the analyte was determined by comparing the response found in the extracted sample to that of the unextracted sample. Stability Two QC levels were selected (LQC and HQC) to conduct the stability experiments. Benchtop, autosampler, freeze–thaw and long-term stability were checked at different time intervals and storage conditions. The acceptance criteria for all the stability studies were set to ensure that at least 67% of QC samples should be in the limits of 85–115% and at an individual QC level, there should be more than 50% pass samples (13, 16). Benchtop stability The spiked QC samples were stored at room temperature for a period of 4 h and analyzed thereafter for determining benchtop stability of eliglustat. Freshly spiked QC samples were used for comparison of the concentration of the analyte with the samples kept on the benchtop for the stated time. Autosampler stability The processed QC samples were stored for 12 h inside the autosampler and analyzed for autosampler stability. Freshly spiked QC samples were used for comparison of the concentration of the analyte with the samples stored inside the autosampler (13, 17). Freeze–thaw stability Three freeze and thaw cycles were performed by storing the samples at −80°C and thawing at room temperature. Freshly spiked QC samples were used for comparison of the concentration of eliglustat with the freeze–thaw samples (13). Long-term stability The QC samples were stored at −80°C for 30 days to check the long-term stability of eliglustat in rat plasma. Freshly spiked QC samples were used for comparison of concentration of eliglustat with the long-term stability samples (13). Pharmacokinetic study Study design Pharmacokinetic study of eliglustat was performed in male Sprague Dawley rats. Total number of animals used in the study was 16. The average weight of rats was 220–250 g. Animals were dosed according to their body weight. The dose of eliglustat was 160 mg/kg which was administered through oral route. Blood samples were collected at different time points after dosing. The study protocol was approved by the Institutional Ethical Committee before commencing the experiment (approval no# NIPERA/IAEC/2018/21). The samples were analyzed using the validated HPLC method. Blood sampling Approximately 0.4 mL of blood sample was collected at zero hours (pre-dose) and then at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h post-dose from each animal (Lee et al., 2008). Samples were collected through retro-orbital route and transferred into the 1.5 mL microcentrifuge tubes. Plasma was separated by centrifugation at 7,000 rpm for 10 min at 4°C and stored at −80°C until analysis (18, 19). Data analysis The plasma concentration versus time points curve was plotted to determine the maximum plasma concentration (Cmax), time to reach maximum concentration (tmax) and other pharmacokinetic parameters. The area under the curve (AUC) was calculated taking the sum of the area from 0 to 12 h following linear trapezoidal rule. The three points on straight line of the curve after Cmax, i.e., in the elimination phase were taken to determine elimination rate constant (kel). The relationship of 0.693/kel was used to determine elimination half-life (t1/2) (20, 21). Results HPLC method development Eliglustat showed to have good retention in a C18 Kromosil column (250 mm × 4.6 mm, 5μm). At the 50:50 (v/v) ratio of methanol and ammonium acetate buffer, eliglustat got eluted at the retention time of 21 min. After adjustment of the mobile phase ratio to 80:20 of methanol and ammonium acetate, the retention time of eliglustat was found to be 3 min. However, at the mobile phase ratio of 60:40, the retention time was 8.9 min. The optimum wavelength at which both eliglustat and haloperidol showed good absorbance was selected for detection of the analytes in HPLC analysis. Finalized HPLC method for eliglustat quantification in rat plasma consisted of a mobile phase having 60:40 ratio of methanol and ammonium acetate buffer with pH 3.2. The pKa of eliglustat is 8.17. At pH 3.2, eliglustat remains unionized and showed to have a better chromatographic profile in terms of peak shape and repeatability. The retention time of eliglustat and IS was ~8.9 and 5.6 min, respectively. The flow rate, injection volume and detection wavelength was 1 mL/min, 5 μL and 282 nm, respectively. Total chromatographic run time was 12 min. In protein precipitation method of sample extraction, recovery of eliglustat and IS were found to be 61 and 89%, respectively. However, in LLE, recovery of eliglustat and IS were comparatively higher which were found to be 83 and 95%, respectively. Hence, LLE offered better extraction for the analyte as well as IS. Highest recovery for eliglustat was obtained in neutral condition with TBME as an extracting solvent. Method validation System suitability System suitability test was performed using six replicates of MQC at a single concentration level. This test was performed to check the instrument performance before each analysis. In this test, we checked the %CV of area and retention time of six replicates. System suitability was performed at the start of the method validation and on each day before the use of the instrument. The CV for retention time and area of system suitability testing was found to be below 0.59 and 0.37%, respectively. Specificity There was no interfering peak observed at the retention time of drug and IS in the blank samples. Therefore, the developed method can be considered to be highly specific for quantification of eliglustat in rat plasma. Representative chromatograms of blank plasma sample and rat plasma spiked with eliglustat have been shown in Figure 2A and B, respectively. Figure 2. View largeDownload slide A representative chromatogram of (A) blank and (B) rat plasma sample spiked with analyte and IS. Figure 2. View largeDownload slide A representative chromatogram of (A) blank and (B) rat plasma sample spiked with analyte and IS. Linearity The developed bioanalytical method was linear over the calibration range of 0.3–10 μg/mL. The average regression coefficient (R2) was found to be more than 0.99 indicating a good correlation between peak area ratios (drug/IS) and concentration of eliglustat. Linearity results have been summarized in Table I. Table I Accuracy and Precision of Linearity Standards CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 *CS, calibration standard. Table I Accuracy and Precision of Linearity Standards CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 *CS, calibration standard. Accuracy and precision According to the USFDA guideline, the %CV of the accuracy of QC samples should not be more than 15% except at LLOQ at which it should not be more than 20%. Accuracy in intra and inter-day assay were found to be 96.27–107.35% and 96.80–106.57%, respectively. The corresponding precision (%CV) values were within 4.31–10.90% and 4.82–9.97%, respectively. The intra-day and inter-day accuracy-precision results have been summarized in Table II. Table II. Intra- and Inter-day Accuracy and Precision Data of the Analyte Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 %Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Table II. Intra- and Inter-day Accuracy and Precision Data of the Analyte Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 %Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Extraction recovery USFDA guideline recommends to carry out extraction recovery at three concentrations. However, we have determined the recovery at two concentration levels (LQC and HQC) which was expected to reflect the recovery of the analyte from the samples of the entire calibration range. Extraction recovery for eliglustat at LQC and HQC levels was found to be 91.79 and 96.12%, respectively. Hence, the developed LLE procedure can be considered to be efficient enough to extract the eliglustat from rat plasma samples. Stability In benchtop stability study, accuracy was found to be 98.97 and 97.51% for HQC and LQC samples, respectively. In autosampler stability study, the accuracy for HQC and LQC were 98.66 and 94.87%, respectively. The accuracy in freeze–thaw stability study for HQC and LQC were 101.69 and 100.88%, respectively. Finally, in long-term stability study, the accuracy was found to be 101.40 and 104.86% for HQC and LQC, respectively. The stability data for validation of the method has been summarized in Table III. Table III. Stability Data of the Analytes in Rat Plasma Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 % Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Table III. Stability Data of the Analytes in Rat Plasma Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 % Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Pharmacokinetic study The developed bioanalytical method was employed to analyze the plasma samples of a pharmacokinetic study of eliglustat in the rat. The Cmax of eliglustat was 1.58 μg/mL at 3 h (tmax). The kel and t1/2 were found to be 0.13/h and 5.23 h, respectively. The area under curve up to 12 h (AUC0–12) was 11.51 μg h/mL and AUC to infinite time (AUC0–α) was 15.06 μg h/mL. Profiles of the mean plasma concentration of eliglustat over time have been shown in Figure 3. Figure 3. View largeDownload slide Pharmacokinetic profile of eliglustat in rat. Figure 3. View largeDownload slide Pharmacokinetic profile of eliglustat in rat. Discussion Comparison of the extraction techniques revealed that liquid–liquid method had a maximum recovery for eliglustat compared to protein precipitation technique. Therefore, the LLE procedure was selected as a suitable sample preparation technique for this bioanalytical method. The results in the system suitability study were found to be satisfactory before each of the analysis performed. The developed bioanalytical method was selective and specific and able to differentiate as well as quantify the eliglustat in the presence of other matrix components. The linearity experiments revealed that the method is suitable for accurately quantify eliglustat in the linearity range of 0.3–10 μg/mL. The accuracy and precision of the method evaluated at three QC levels were found to be well in accordance with the USFDA bioanalytical method validation guideline. The higher value of %CV in HQC was due to more variation of observed concentration in HQC samples compared to MQCs and LQCs. However, at the LLOQ level, %CV was found to be 12.62 (data not shown). So, there was no direct relationship between concentration and %CV found in this method. The variations were random irrespective of concentration and were within the acceptance limit specified in USFDA guideline for bioanalytical method validation. Stability of eliglustat in bench top (4 h), autosampler (12 h), freeze–thaw (three cycles) and long-term (30 days) stability study was within the acceptable limit. The applicability of the method has been established by analyzing the plasma samples of a single dose oral pharmacokinetic study of eliglustat in the rat. All the pharmacokinetic parameters (Cmax, tmax, t1/2, AUC0–t, AUC0–α) were determined after quantifying eliglustat in rat plasma samples using the method developed for it. Thus, the method can be considered to be useful for the quantification of eliglustat in studies where the rat plasma sample is involved. Conclusion A selective and sensitive bioanalytical RP-HPLC method has been developed for quantitation of eliglustat in rat plasma. The bioanalytical sample preparation technique optimized for achieving the best recovery from the plasma samples. LLE method with TBME as extraction solvent showed to have maximum extraction recovery. The developed method was validated according to USFDA bioanalytical method validation guideline. All the validation parameters including accuracy, precision and stability studies were found to be within the acceptance limit as specified in USFDA guideline. The method has been successfully applied to quantitate eliglustat in the plasma samples of a pharmacokinetic study in the rat. However, the applicability of the developed method has been demonstrated through a short pharmacokinetic study using a dose that is not equivalent to that of human patients treated with eliglustat. Therefore, a full pharmacokinetic study under real conditions needs to be established in future. The novelty of this method can be justified with the unavailability of any previously reported bioanalytical method for quantification of eliglustat in rat plasma. This method thus can be considered to have a significant contribution in the field of bioanalysis. Ethical declaration The protocol for animal experiments was approved by the Institutional Animal Ethics Committee (IAEC) prior to initiation of the experiment. The approval number is NIPERA/IAEC/2018/21. Acknowledgments The author would like to acknowledge NIPER-AHMEDABAD for providing financial help and necessary laboratory facilities for this research work. Authors would also like to thank Mr Prakash Niguram for his help in arranging eliglustat for this study. References 1 Pandey , S. , Pandey , P. , Tiwari , G. , Tiwari , R. ; Bioanalysis in drug discovery and development ; Pharmaceutical Methods , ( 2010 ); 1 ( 1 ): 14 – 24 . Google Scholar Crossref Search ADS PubMed 2 Selinger , K. , Fung , E.N. , Bryan , P. 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

A Bioanalytical Method for Eliglustat Quantification in Rat Plasma

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Oxford University Press
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© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmz033
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Abstract

Abstract A simple and sensitive bioanalytical HPLC–UV method has been developed and validated for quantification of eliglustat in rat plasma. The liquid–liquid extraction method was found to be more efficient compared to protein precipitation technique. Chromatographic separation of eliglustat was achieved using Kromasil C18 column with a mobile phase consisting of a mixture of methanol and ammonium acetate (pH 3.2) in a ratio of 60:40. Detection wavelength was set at 282 nm. The developed method was specific, accurate, precise with good recovery and stability profile. The calibration curve constructed over a range of 0.3–10 μg/mL was linear (R2 > 0.997). Accuracy in intra and inter-day assay were found to be 96.27–107.35% and 96.80–106.57%, respectively. The corresponding precision (%CV) values were within 4.31–10.90% and 4.82–9.97%, respectively. Till date, no method is available for bioanalysis of eliglustat in any type of biological matrix. This is the first time to report a bioanalytical method for this molecule. The developed bioanalytical method was applied to quantitate eliglustat in the plasma samples of a single dose oral pharmacokinetic study in Sprague Dawley rat. Introduction Bioanalysis plays a key role in drug discovery, pharmacokinetic and toxicokinetic study (1). Nowadays, bioanalysis is an integrated component of the drug development process. Drug discovery and its further development into a new drug substance are the two crucial stages in the drug product lifecycle. The human body comprises of series of complex systems to affect the action and fate of drugs (2–4). Therefore, not only what drug affects the body but also what the body does to the drug is equally important. Determination of pharmacokinetic properties of drugs and their metabolites in the body can be beneficial to correlate their variable effects amongst different species. It is thus helpful to adjust the dose of a drug (5). Developing a quantification method of drugs in biological matrices is challenging because of the presence of various exogenous and endogenous compounds. The extraction of the analyte from such matrices prior to their analysis need to be optimized (6–9). Different bioanalytical techniques have been reported for the pre-treatment of the samples for achieving appropriate accuracy, precision, selectivity and sensitivity (7). Gaucher disease is a hereditary disease caused by the accumulation of glucocerebroside in the cells and other organs. The disease is characterized by enlargement of liver and lungs, and decreament in the white blood cell count resulting in anemia (10). Due to the lack of enzyme glucocerebrosidase in the patients suffering from this disease, glucocerebroside accumulates inside the cells. Eliglustat inhibits the enzyme glucosylceramide synthase and prevents the generation of glucocerebroside and thereby, ameliorate Gaucher disease (11, 12). Chemically, eliglustat is N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)octanamide (2R,3R)-2,3-dihydroxysuccinate. The objective of this study was to develop and validate a reverse phase-high-performance liquid chromatography (RP-HPLC) method for the estimation of eliglustat in rat plasma and evaluation of its applicability in a pharmacokinetic study. There is no bioanalytical method available till date for quantitation of eliglustat in rat plasma or any other type of biological matrices. This is the first time to report an accurate and precise bioanalytical method for quantitation of eliglustat in rat plasma. Materials and Methods Chemicals and reagents Eliglustat (Figure 1A) (purity > 98%) was received as a gift sample from Torrent Pharmaceuticals, India and haloperidol (Figure 1B) (purity > 99%) was purchased from Sigma Aldrich. Dichloromethane, methanol (HPLC grade), acetonitrile (HPLC grade), ethyl acetate and tertiary butyl methyl ether (TBME) were purchased from Fisher Scientific. Ammonium acetate and trichloroacetic acid were also procured from Sigma Aldrich. Milli-Q grade water generated from a Milli-Q gradient system of Millipore was used for the analysis. Figure 1. View largeDownload slide Molecular structure of (A) Eliglustat and (B) Haloperidol. Figure 1. View largeDownload slide Molecular structure of (A) Eliglustat and (B) Haloperidol. Method development The UV spectra of both eliglustat and haloperidol (internal standard (IS)) were recorded in the range of 200–400 nm in a UV–Visible spectrophotometer (UV–Vis 1800, Shimadzu). Initial method development trials for HPLC analysis were carried out using ammonium acetate buffer of 10 mM concentration of different pH. The eliglustat concentration for method development trials was 10 μg/mL. Initially, extraction of eliglustat from rat plasma was carried out with protein precipitation technique using methanol in acidic and neutral conditions. In acidic medium, trichloroacetic acid (TCA) (2%) was used as an acidifying agent. In either of the protein precipitation technique (neutral or acidic condition), the recovery was not optimum. Therefore, we moved to liquid–liquid extraction (LLE) to extract eliglustat from rat plasma. In LLE, four different solvents namely dichloromethane, ethyl acetate, TBME and diethyl ether were used for optimizing the extraction process. All the LLEs were carried out in the acidic, basic and neutral medium. Instrumentation The Agilent 1200 infinity series HPLC instrument coupled with UV detector with open lab software was used for the development and validation of the method for estimation of eliglustat in rat plasma. Sample extraction procedure A volume of 10 μL of eliglustat (100 μg/mL) was taken in a plastic RIA (radioimmunoassay) tube, evaporated in nitrogen evaporator till dryness, added 80 μL of blank rat plasma and vortex mixed. Thereafter, 10 μL of 100 μg/mL of IS solution was added and vortex mixed. The extraction solvent TBME was added to it (2 mL) and vortexed. The tubes were centrifuged for 10 min at 5,000 rpm at 4°C and kept in −80°C for 7 min. The organic layer was separated and evaporated in nitrogen evaporator till dryness. The dried extract was then reconstituted with 300 μL of mobile phase and finally, 5 μL was injected into the HPLC system. Preparation of stock solution and buffer Individual stock solutions of 100 μg/mL were prepared for eliglustat and haloperidol in methanol. The buffer solution (10 mM) was prepared by dissolving ammonium acetate in Milli-Q water. It was then filtered through a 0.2 μm Millipore filter and the pH was adjusted to 3.2 with diluted acetic acid. Bioanalytical method validation The developed bioanalytical method was validated according to the USFDA bioanalytical method validation guideline (13, 14). System suitability System suitability was performed by taking mid-quality control (MQC) sample. Six MQC samples were prepared separately and injected. Deviations in the area and retention time were evaluated. Specificity Specificity was determined at the lower limit of quantitation (LLOQ) level. Blank plasma samples from six different sources were collected and percent interference was checked at LLOQ level of 0.3 μg/mL. For each plasma samples, six determinants were evaluated (13, 15). Calibration curve Calibration curves were constructed using six concentrations levels of 0.3, 1, 2, 5, 8 and 10 μg/mL. The acceptance criteria for the linearity study was set to get the regression coefficient (R2) of the curve greater than 0.99. Accuracy, precision and recovery Accuracy and precision were evaluated at three quality control (QC) concentrations namely low-QC (LQC), MQC and high-QC (HQC) level. The concentrations of LQC, MQC and HQC level were 0.9, 4 and 8 μg/mL, respectively. Six determinations at each concentration were injected for determining the accuracy and precision of the method. Acceptance criteria were set to ensure that at least 67% of samples should be within 85–115% of nominal concentration and the %CV of the six replicate injections should be <15%. On the other hand, as per USFDA guideline, the %CV at LLOQ should be ±20% (13, 16). Recovery of the analyte was determined by comparing the response found in the extracted sample to that of the unextracted sample. Stability Two QC levels were selected (LQC and HQC) to conduct the stability experiments. Benchtop, autosampler, freeze–thaw and long-term stability were checked at different time intervals and storage conditions. The acceptance criteria for all the stability studies were set to ensure that at least 67% of QC samples should be in the limits of 85–115% and at an individual QC level, there should be more than 50% pass samples (13, 16). Benchtop stability The spiked QC samples were stored at room temperature for a period of 4 h and analyzed thereafter for determining benchtop stability of eliglustat. Freshly spiked QC samples were used for comparison of the concentration of the analyte with the samples kept on the benchtop for the stated time. Autosampler stability The processed QC samples were stored for 12 h inside the autosampler and analyzed for autosampler stability. Freshly spiked QC samples were used for comparison of the concentration of the analyte with the samples stored inside the autosampler (13, 17). Freeze–thaw stability Three freeze and thaw cycles were performed by storing the samples at −80°C and thawing at room temperature. Freshly spiked QC samples were used for comparison of the concentration of eliglustat with the freeze–thaw samples (13). Long-term stability The QC samples were stored at −80°C for 30 days to check the long-term stability of eliglustat in rat plasma. Freshly spiked QC samples were used for comparison of concentration of eliglustat with the long-term stability samples (13). Pharmacokinetic study Study design Pharmacokinetic study of eliglustat was performed in male Sprague Dawley rats. Total number of animals used in the study was 16. The average weight of rats was 220–250 g. Animals were dosed according to their body weight. The dose of eliglustat was 160 mg/kg which was administered through oral route. Blood samples were collected at different time points after dosing. The study protocol was approved by the Institutional Ethical Committee before commencing the experiment (approval no# NIPERA/IAEC/2018/21). The samples were analyzed using the validated HPLC method. Blood sampling Approximately 0.4 mL of blood sample was collected at zero hours (pre-dose) and then at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h post-dose from each animal (Lee et al., 2008). Samples were collected through retro-orbital route and transferred into the 1.5 mL microcentrifuge tubes. Plasma was separated by centrifugation at 7,000 rpm for 10 min at 4°C and stored at −80°C until analysis (18, 19). Data analysis The plasma concentration versus time points curve was plotted to determine the maximum plasma concentration (Cmax), time to reach maximum concentration (tmax) and other pharmacokinetic parameters. The area under the curve (AUC) was calculated taking the sum of the area from 0 to 12 h following linear trapezoidal rule. The three points on straight line of the curve after Cmax, i.e., in the elimination phase were taken to determine elimination rate constant (kel). The relationship of 0.693/kel was used to determine elimination half-life (t1/2) (20, 21). Results HPLC method development Eliglustat showed to have good retention in a C18 Kromosil column (250 mm × 4.6 mm, 5μm). At the 50:50 (v/v) ratio of methanol and ammonium acetate buffer, eliglustat got eluted at the retention time of 21 min. After adjustment of the mobile phase ratio to 80:20 of methanol and ammonium acetate, the retention time of eliglustat was found to be 3 min. However, at the mobile phase ratio of 60:40, the retention time was 8.9 min. The optimum wavelength at which both eliglustat and haloperidol showed good absorbance was selected for detection of the analytes in HPLC analysis. Finalized HPLC method for eliglustat quantification in rat plasma consisted of a mobile phase having 60:40 ratio of methanol and ammonium acetate buffer with pH 3.2. The pKa of eliglustat is 8.17. At pH 3.2, eliglustat remains unionized and showed to have a better chromatographic profile in terms of peak shape and repeatability. The retention time of eliglustat and IS was ~8.9 and 5.6 min, respectively. The flow rate, injection volume and detection wavelength was 1 mL/min, 5 μL and 282 nm, respectively. Total chromatographic run time was 12 min. In protein precipitation method of sample extraction, recovery of eliglustat and IS were found to be 61 and 89%, respectively. However, in LLE, recovery of eliglustat and IS were comparatively higher which were found to be 83 and 95%, respectively. Hence, LLE offered better extraction for the analyte as well as IS. Highest recovery for eliglustat was obtained in neutral condition with TBME as an extracting solvent. Method validation System suitability System suitability test was performed using six replicates of MQC at a single concentration level. This test was performed to check the instrument performance before each analysis. In this test, we checked the %CV of area and retention time of six replicates. System suitability was performed at the start of the method validation and on each day before the use of the instrument. The CV for retention time and area of system suitability testing was found to be below 0.59 and 0.37%, respectively. Specificity There was no interfering peak observed at the retention time of drug and IS in the blank samples. Therefore, the developed method can be considered to be highly specific for quantification of eliglustat in rat plasma. Representative chromatograms of blank plasma sample and rat plasma spiked with eliglustat have been shown in Figure 2A and B, respectively. Figure 2. View largeDownload slide A representative chromatogram of (A) blank and (B) rat plasma sample spiked with analyte and IS. Figure 2. View largeDownload slide A representative chromatogram of (A) blank and (B) rat plasma sample spiked with analyte and IS. Linearity The developed bioanalytical method was linear over the calibration range of 0.3–10 μg/mL. The average regression coefficient (R2) was found to be more than 0.99 indicating a good correlation between peak area ratios (drug/IS) and concentration of eliglustat. Linearity results have been summarized in Table I. Table I Accuracy and Precision of Linearity Standards CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 *CS, calibration standard. Table I Accuracy and Precision of Linearity Standards CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 CS*1 CS2 CS3 CS4 CS5 CS6 Nominal conc. 0.3 1 2 5 8 10 Run1 0.33 0.75 2.13 4.83 8.24 9.80 Run2 0.35 0.88 2.12 4.96 8.03 10.26 Run3 0.34 0.99 1.96 5.01 7.96 9.96 N 3.00 3.00 3.00 3.00 3.00 3.00 Mean 0.34 0.87 2.07 4.93 8.07 10.00 SD 0.01 0.12 0.10 0.09 0.15 0.23 CV% 4.02 13.66 4.61 1.90 1.83 2.33 % Accuracy 112.57 86.98 103.30 98.66 100.92 100.04 % Deviation 12.57 −13.02 3.30 −1.34 0.92 0.04 *CS, calibration standard. Accuracy and precision According to the USFDA guideline, the %CV of the accuracy of QC samples should not be more than 15% except at LLOQ at which it should not be more than 20%. Accuracy in intra and inter-day assay were found to be 96.27–107.35% and 96.80–106.57%, respectively. The corresponding precision (%CV) values were within 4.31–10.90% and 4.82–9.97%, respectively. The intra-day and inter-day accuracy-precision results have been summarized in Table II. Table II. Intra- and Inter-day Accuracy and Precision Data of the Analyte Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 %Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Table II. Intra- and Inter-day Accuracy and Precision Data of the Analyte Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Intra-day accuracy and precision data Quality control Run Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 1 0.95 0.04 4.31 105.61 2 0.96 0.05 5.10 106.74 3 0.97 0.06 5.71 107.35 MQC 1 3.93 0.29 7.37 98.22 2 3.92 0.37 9.36 100.57 3 3.91 0.34 8.78 97.63 HQC 1 7.74 0.84 10.90 96.75 2 7.70 0.78 10.17 96.27 3 7.79 0.83 10.71 97.36 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 Inter-day accuracy and precision data LQC 0.96 0.05 4.82 106.57 MQC 3.92 0.31 8.03 98.70 HQC 7.74 0.77 9.97 96.80 %Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Extraction recovery USFDA guideline recommends to carry out extraction recovery at three concentrations. However, we have determined the recovery at two concentration levels (LQC and HQC) which was expected to reflect the recovery of the analyte from the samples of the entire calibration range. Extraction recovery for eliglustat at LQC and HQC levels was found to be 91.79 and 96.12%, respectively. Hence, the developed LLE procedure can be considered to be efficient enough to extract the eliglustat from rat plasma samples. Stability In benchtop stability study, accuracy was found to be 98.97 and 97.51% for HQC and LQC samples, respectively. In autosampler stability study, the accuracy for HQC and LQC were 98.66 and 94.87%, respectively. The accuracy in freeze–thaw stability study for HQC and LQC were 101.69 and 100.88%, respectively. Finally, in long-term stability study, the accuracy was found to be 101.40 and 104.86% for HQC and LQC, respectively. The stability data for validation of the method has been summarized in Table III. Table III. Stability Data of the Analytes in Rat Plasma Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 % Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Table III. Stability Data of the Analytes in Rat Plasma Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 Quality control Stability Measured concentration of eliglustat (μg/mL) Mean SD % CV % Accuracy LQC 12 h—autosampler 0.90 0.08 8.38 93.34 12 h—benchtop 0.94 0.04 4.76 97.52 3rd freeze/thaw 0.91 0.06 6.33 100.88 30 day at −20°C 0.94 0.05 5.39 104.87 HQC 12 h—autosampler 7.69 0.81 10.53 98.67 12 h—benchtop 7.64 0.78 10.22 98.97 3rd freeze/thaw 7.65 0.75 9.85 101.69 30 Days at −20°C 7.59 0.87 11.43 101.40 % Accuracy: (Mean assayed concentration – nominal concentration)/(nominal concentration) × 100. Pharmacokinetic study The developed bioanalytical method was employed to analyze the plasma samples of a pharmacokinetic study of eliglustat in the rat. The Cmax of eliglustat was 1.58 μg/mL at 3 h (tmax). The kel and t1/2 were found to be 0.13/h and 5.23 h, respectively. The area under curve up to 12 h (AUC0–12) was 11.51 μg h/mL and AUC to infinite time (AUC0–α) was 15.06 μg h/mL. Profiles of the mean plasma concentration of eliglustat over time have been shown in Figure 3. Figure 3. View largeDownload slide Pharmacokinetic profile of eliglustat in rat. Figure 3. View largeDownload slide Pharmacokinetic profile of eliglustat in rat. Discussion Comparison of the extraction techniques revealed that liquid–liquid method had a maximum recovery for eliglustat compared to protein precipitation technique. Therefore, the LLE procedure was selected as a suitable sample preparation technique for this bioanalytical method. The results in the system suitability study were found to be satisfactory before each of the analysis performed. The developed bioanalytical method was selective and specific and able to differentiate as well as quantify the eliglustat in the presence of other matrix components. The linearity experiments revealed that the method is suitable for accurately quantify eliglustat in the linearity range of 0.3–10 μg/mL. The accuracy and precision of the method evaluated at three QC levels were found to be well in accordance with the USFDA bioanalytical method validation guideline. The higher value of %CV in HQC was due to more variation of observed concentration in HQC samples compared to MQCs and LQCs. However, at the LLOQ level, %CV was found to be 12.62 (data not shown). So, there was no direct relationship between concentration and %CV found in this method. The variations were random irrespective of concentration and were within the acceptance limit specified in USFDA guideline for bioanalytical method validation. Stability of eliglustat in bench top (4 h), autosampler (12 h), freeze–thaw (three cycles) and long-term (30 days) stability study was within the acceptable limit. The applicability of the method has been established by analyzing the plasma samples of a single dose oral pharmacokinetic study of eliglustat in the rat. All the pharmacokinetic parameters (Cmax, tmax, t1/2, AUC0–t, AUC0–α) were determined after quantifying eliglustat in rat plasma samples using the method developed for it. Thus, the method can be considered to be useful for the quantification of eliglustat in studies where the rat plasma sample is involved. Conclusion A selective and sensitive bioanalytical RP-HPLC method has been developed for quantitation of eliglustat in rat plasma. The bioanalytical sample preparation technique optimized for achieving the best recovery from the plasma samples. LLE method with TBME as extraction solvent showed to have maximum extraction recovery. The developed method was validated according to USFDA bioanalytical method validation guideline. All the validation parameters including accuracy, precision and stability studies were found to be within the acceptance limit as specified in USFDA guideline. The method has been successfully applied to quantitate eliglustat in the plasma samples of a pharmacokinetic study in the rat. However, the applicability of the developed method has been demonstrated through a short pharmacokinetic study using a dose that is not equivalent to that of human patients treated with eliglustat. Therefore, a full pharmacokinetic study under real conditions needs to be established in future. The novelty of this method can be justified with the unavailability of any previously reported bioanalytical method for quantification of eliglustat in rat plasma. This method thus can be considered to have a significant contribution in the field of bioanalysis. Ethical declaration The protocol for animal experiments was approved by the Institutional Animal Ethics Committee (IAEC) prior to initiation of the experiment. The approval number is NIPERA/IAEC/2018/21. Acknowledgments The author would like to acknowledge NIPER-AHMEDABAD for providing financial help and necessary laboratory facilities for this research work. Authors would also like to thank Mr Prakash Niguram for his help in arranging eliglustat for this study. References 1 Pandey , S. , Pandey , P. , Tiwari , G. , Tiwari , R. ; Bioanalysis in drug discovery and development ; Pharmaceutical Methods , ( 2010 ); 1 ( 1 ): 14 – 24 . Google Scholar Crossref Search ADS PubMed 2 Selinger , K. , Fung , E.N. , Bryan , P. 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Journal of Chromatographic ScienceOxford University Press

Published: Nov 9, 21

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