159 CHAPTER-4 A validated LC method for the determination of chiral purity of (S)-2azido-3-methylbutanoic acid: a key raw material of Valganciclovir hydrochloride
160 4.1 Introduction (S)-2-azido-3-methylbutanoic acid [Fig.4.1.F1] is a key starting raw material of valganciclovir hydrochloride. (S)-2-azido-3-methylbutanoic acid, a single enantiomer is very critical as it affects the overall chiral purity of valganciclovir hydrochloride (Valcyte, manufactured by Roche)[1]bulk drug. Valganciclovir hydrochloride is an antiviral drug used to treat cytomegalovirus infections. Valganciclovir is L-valyl ester of ganciclovir that exits as a mixture of two diastereomers [2], after oral administration [3], it is rapidly converted to ganciclovir by intestinal and hepatic esterases. Fig. 4.1.F1: Chemical structure of (S)-2-azido-3-methylbutanoic acid 2-azido-3-methylbutanoic acid has S- and R-enatiomeric forms [Fig. 4.1.F2] and (R)-2-azido-3-methylbutanoic acid could be present as a chiral impurity in (S)-2-azido-3-methylbutanoic acid. A control and accurate quantification of undesired enantiomers in pharmaceuticals is essential [4] in this connection and LC is generally opted for this purpose.
161 Fig. 4.1.F2: Chemical structure of (S) and (R)-2-azido-3methylbutanoic acid. (S)-2-azido-3methylbutanoic acid (R)-2-azido-3methylbutanoic acid Since, there is a chance of formation of (R)-2-azido-3-methylbutanoic acid during synthesis of (S)-2-azido-3-methylbutanoic acid. The separation of (R)-2-azido-3-methylbutanoic acid from (S)-2-azido-3methylbutanoic acid followed by its determination is one of the major concern as it affects the overall chiral purity of valganciclovir hydrochloride. The synthetic scheme of valganciclovir hydrochloride was presented in Fig.4.1.F3. The determination of the stereo isomeric composition of pharmaceuticals is rapidly becoming one of the key issues in the development of new drugs. Among the methods currently used to achieve chiral separation of enantiomers, high resolution liquid chromatographic systems based on chiral stationary phases, CSPs (direct methods) are more rapid and suitable for the resolution of racemic mixtures of pharmacologically active chemical entities [5-7]. Several CSPs are now available to allow the direct separation of enantiomers. Amylose CSPs are one of these normally employed phases used for the separation
162 of enantiomers and determination of chiral purity. Enantiomeric inclusion in chiral cavities, which might be multiple, and competitive in amylase and cellulose based chiral stationary phases seems to be responsible for the chiral discrimination. Fig. 4.1.F3: Synthetic scheme of valganciclovir hydrochloride Reviewed the literature for analytical methods for the separation of 2azido-3-methylbutanoic acid enantiomers. Till date, there is no reported validated chiral HPLC method in the literature for the enantiomeric separation of 2-azido-3-methylbutanoic acid. Therefore, it is very essential to develop a simple and suitable analytical method for the enantiomeric measurement of 2-azido-3-methylbutanoic acid. Our purpose was to develop a simple, sensitive, precise and reliable method for enantiomeric purity determination of (S)-2-azido-3-methylbutanoic acid and quantification of its opposite enantiomer of (R)-2-azido-3-
163 methylbutanoic acid. The developed liquid chromatographic method for the enantiomeric separation of 2-azido-3-methylbutanoic acid using an amylose based chiral stationary phase, chiralpak-ia was validated as per International Conference of Harmonization (ICH) guidelines [8-9] in terms of precision, linearity, accuracy, specificity and robustness. 4.2 Experimental 4.2.1 Materials: Samples of S and R-enantiomers of 2-azido-3-methylbutanoic acid confirmed by spectral characterization and SOR (specific optical rotation) were obtained from Process Research Department of Dr. Reddy s Laboratories Ltd, Hyderabad, India. HPLC-grade n-hexane was procured from Tedia company, Inc., Canada. Ethanol and IPA were purchased from Ranbaxy Fine Chemicals, New Delhi, India. Analytical Reagent grade tri-fluoroacetic acid (TFA) was purchased from Fluka. 4.2.2 Equipment: Waters make An Alliance HPLC (Alliance 2695 Model, Waters Corporation, Milford, USA) equipped with in-built auto sampler and 2487 dual absorbance detector was used for the analysis. The output signal was monitored and processed using Waters Millenium software. Photo diode array detection was also used for determining peak purity. The chiral columns used in method development were chiralcel OD, chiralpak AD, chiralcel OJ, chiralpak IA, chiralpak IB and chiralpak IC [10]. All are Daicel make (Daicel Chemical Industries, Japan) with 5µm
164 particle size in (250 x 4.6) mm dimension. The other columns used crownpak (Daicel Chemical Industries, Japan), chiral AGP (Advanced Separation Technologies Inc., New Jersey) and chirobiotic (Chiron Technologies, Inc., New Jersey) are 5 µm particle size in (150 x 4.6) mm dimension. 4.2.3 Chromatographic conditions: The chromatographic conditions were optimized using a chiralpak IA column. The mobile phase, a mixture of n-hexane, ethanol, isopropyl alcohol and TFA in the ratio of 98:1.5:0.5:0.1 ml with a flow rate of 1.0 ml min-1 was employed. The column temperature was maintained at 25 C and the detection was monitored at a wavelength of 215 nm. The injection volume was 10 µl. 4.2.4 Standard preparation: The stock solutions of S and R-enantiomers of 2-azido-3-methylbutanoic acid were prepared individually by dissolving an appropriate amount of the substances in diluent of mobile phase. Working solutions were prepared in mobile phase. The target analyte concentration was fixed as 2.0 mg ml-1. This solution was diluted further to get the required concentrations for the method validation [11]. 4.3 Results and discussion 4.3.1 Optimization of chromatographic conditions: The selection of an appropriate chiral stationary phase (CSP) is an important consideration in separating the enantiomers in method
165 development. To date, different types of CSPs include such as proteins, Pirkle-type phases, cyclodextrins, polysaccharide, ligand exchange and antibiotics, etc., have been effectively applied for enantiomeric separation of different pharmaceutical compounds [12-14]. Initial experiment was carried out for the separation of S and Renantiomers of 2-azido-3-methylbutanoic acid on crownpak CR (+)150 x 4.6 mm, 5 microns (daicel) column. This column contains a chiral crown ether as a chiral selector which is coated onto a 5μm silica support. This column is a reference column for achieving amino acid separations. The separation was tried in reverse phase with the mobile phases consisting of buffer 0.01 M (perchloric acid) and methanol in the ratio of 90:10. The partial resolution was observed between enantiomers, but it was not satisfactory. The retention times of S and R-enantiomers were 2.5 and 2.9 min respectively [Fig. 4.3.F1]. Fig. 4.3.F1: Trial chromatogram on crownpak CR (+) column X-axis: Retention time in min and Y-axis: Peak response in mau The next trail, chiral AGP column 150 x 4.6 mm, 5 microns was used, in which the chiral selector α1-acid glycoprotein (AGP) is immobilized onto spherical silica particles. It separates amines, acids and non-protolytes. The separation was tried in reverse phase with the
166 mobile phases consisting of buffer (0.01 M potassium dihydrogen sulphate whose ph was adjusted to 4.0) and methanol in the ratio of 50:50. A single peak was observed at a retention time of 6.2 min indicating that the separation was not achieved for 2-azido-3methylbutanoic acid enantiomers [Fig4.3.F2]. Fig. 4.3.F2: Trial chromatogram on chiral AGP column 0.60 4 0.2 6 0.40 U A 0.20 0.00 0.00 2.00 4.00 6.00 8.00 Minutes 10.00 12.00 14.00 X-axis: Retention time in min and Y-axis: Peak response in AU In the further trail using chirobiotic T column 150 x 4.6 mm, 5 microns (Chromtech) in which covalently bonding macrocyclic glycopeptides are bonded to high pure silica gel. This column is ideal for underivatized Amino Acids, N-derivatized Amino Acids, Carboxylic acids, Phenols and small Peptides. The separation was tried in reverse phase with the mobile phase consisting of buffer (0.02 M potassium dihydrogen sulphate, the ph was adjusted to 4.0) and methanol in the ratio of 85:15. A single peak was observed at a retention time of 5.5 minutes indicating that the separation was not achieved for 2-azido-3methylbutanoic acid enantiomers [Fig. 4.3.F3].
167 Fig. 4.3.F3: Trial chromatogram on chirobiotic T column. X-axis: Retention time in min and Y-axis: Peak response in AU Literature survey reveals that among the available CSP s, the carbamate-derivatized amylose (Chiralpak AD-H) and cellulose (Chiralcel OD-H) stationary phases are the most popular phases because of their selectivity and versatility [15-17]. Hence, the experiments were carried out by using chiralpak AD-H and chiralcel OD-H columns. When chiral Pak AD-H column (250 x 4.6 mm, 5 micron) was used with a mixture of n-hexane, isopropanol, triethylamine (80:20:0.1 v/v/v) as mobile phase at 1.0 ml min-1 flow rate. A single peak was observed at a retention time of 6.0 min indicating that the separation was not achieved for 2-azido-3-methylbutanoic acid enantiomers [Fig. 4.3.F4].
168 Fig. 4.3.F4: Trial chromatogram on chiralpak AD-H column 0.60 0.40 U A 0.20 0.00 0.00 2.00 4.00 6 6.9 5 re m o it n a n e S & R 6.00 8.00 Minutes 10.00 12.00 14.00 X-axis: Retention time in min and Y-axis: Peak response in AU When chiralcel OD-H column (250 x 4.6 mm, 5 micron) was used with the same chromatographic conditions, a single peak was observed at a retention time of 4.8 min indicating that the separation was not achieved for 2-azido-3-methylbutanoic acid enantiomers [Fig. 4.3.F5]. Fig. 4.3.F5: Trial chromatogram on chiralpak OD-H column 0.30 U A 0.20 0.10 0.00 0.00 4 6 7. 4 rs e m o it n a n e S & R 5.00 10.00 Minutes 15.00 20.00 25.00 X-axis: Retention time in min and Y-axis: Peak response in AU The next trails were carried out by using the CHIRALPAK IA, IB and IC columns which offer enhanced compound solubility, greater column stability and novel column selectivity, all of which improve column
169 lifetime. Using the immobilized column allows a greater freedom of solvent choice than for coated columns. This immobilization confers two major advantages. One is that the CSP can no longer be changed or destroyed by use of a forbidden solvent-there are no forbidden organic solvents with the new columns. The other advantage is that with this total freedom of choice of solvent. Unlike coated columns, this immobilized column has universal solvent compatibility. Polar organic solvents, typically EtOH, MeOH and ACN are widely used as mobile phases for chiral separation on the polysaccharide-derived CSPs. The use of solvents of this kind has several advantages such as the simplicity in mobile phase preparation, fast separations, compatibility with LC-MS and the possibility to enhance the sample solubility in the mobile phase. A basic additive (most commonly DEA) in the mobile phase is in general beneficial for separation of chiral compounds of basic nature. When chiralpak IC column (250 x 4.6 mm, 5 micron) was used with a mixture of n-hexane, ethanoll, trifluoroacetic acid (96:4.0:0.1 v/v/v) as mobile phase at 1.0 ml min -1 flow rate. A single peak was observed at 5.6 min indicating that the separation was not achieved for 2-azido3-methylbutanoic acid enantiomers [Fig. 4.3.F6].
170 Fig. 4.3.F6: Trial chromatogram on chiralpak-ic column 0.80 4 4 6. 5 r e m o it n a n e S & R 0.60 U A 0.40 0.20 0.00 0.00 5.00 10.00 Minutes 15.00 20.00 X-axis: Retention time in min and Y-axis: Peak response in AU In the next trail the chiralpak-ib column (250 x 4.6 mm, 5 micron) was used with a mixture of n-hexane, ethanol, trifluoroacetic acid (96:4.0:0.1 v/v/v) as mobile phase at 1.0 ml min -1 flow rate. A single peak was observed at 6.3 min indicating that the separation was not achieved for 2-azido-3-methylbutanoic acid enantiomers [Fig. 4.3.F7]. Fig. 4.3.F7: Trial chromatogram on chiralpak-ib column 0.30 1 2 3. 6 r e m o it n a n -e S & R 0.20 U A 0.10 0.00 0.00 5.00 10.00 15.00 Minutes 20.00 25.00 30.00 X-axis: Retention time in min and Y-axis: Peak response in AU
171 In the further trail chiralpak-ia column (250 x 4.6 mm, 5 micron) was used with a mixture of n-hexane, ethanol, trifluoroacetic acid (98:2.0:0.1 v/v/v) as mobile phase at 1.0 ml min-1 flow rate. The separation was achieved between S and R-enantiomers of 2-azido-3methylbutanoic acid [Fig. 4.3.F8]. But resolution has to be improved between S and R-enantiomers. The chiral selector contained in Chiralpak-IA [Fig. 4.3.F8] is Amylose tris (3,5-dimethylphenylcarbamate) immobilized on 5µm silica-gel. Fig. 4.3.F8: The structure of CPS of Chiralpak IA Now, to improve the resolution between S and R-enantiomers of 2-azido-3-methylbutanoic acid, isopropyl alcohol was added in the mobile phase. Base to base separation was achieved between S and R enantiomers of 2-azido-3-methylbutanoic acid [Fig. 4.3.F9] with retention times of 7.5 and 8.1 respectively. The resolution is more than 2 between two enantiomers.
172 Fig. 4.3.F9: Typical final trial chromatogram on chiralpak IA column X-axis: Retention time in min and Y-axis: Peak response in AU Finally, good resolution achieved for the 2-azido-3-methylbutanoic acid enantiomers on chiralpak-ia column consisting of mobile phase n-hexane, ethanol, isopropyl alcohol and trifluoroacetic acid in the ratio of 98:1.5:0.5:0.1 (v/v/v/v). The resolution between S and Renantiomers of 2-azido-3-methylbutanoic acid was found to be 2.5 [Fig. 4.3.F9]. The elution was monitored at 215 nm and the flow rate was maintained at 1.0 ml min-1. 4.3.2 Diluent selection: Based on the solubility nature of 2-azido-3-methylbutanoic acid enantiomers the diluent finalized as mobile phase. 4.3.3 Optimized chromatographic conditions: Based on the method development experiments and solution stability studies the below chromatographic conditions were finalized for the determination of R-2-azido-3-methylbutanoic acid content in S-2-azido-
173 3-methylbutanoic acid. Chromatographic conditions are tabulated in Table 4.3.T1. Table 4.3.T1: Chromatographic conditions Column Chiralpak-IA, 250mm x 4.6mm x 5.0 m. Mobile phase A mixture of n-hexane, ethanol, isopropyl alcohol and trifluoroacetic acid in the ratio of 98: 1.5: 0.5: 0.1 (v/v/v/v) Flow rate 1.0 ml min-1 Column temperature Ambient (25 C + 2 C). Wavelength 215 nm Injection volume 10 l Diluent Mobile phase Concentration 2.0 mg ml-1 4.3.4 Method specificity: Forced degradation studies were also performed on (S)-2-azido-3methylbutanoic acid sample to provide an indication of the stability indicating property and specificity of the proposed method. The stress conditions employed for degradation study include acid hydrolysis (0.1 N ethanolic HCl/24 h reflux), base hydrolysis (0.1 N ethanolic NaOH/24 h reflux), and peroxide degradation (3% w/v H 2O2/3 h reflux). Peak purity of stressed sample of (S)-2-azido-3-methylbutanoic acid was checked by using photo diode array detector. Purity angle was found less than purity threshold in all stress samples and no interference was found from the blank which demonstrate the analyte peak homogeneity.
174 4.4 Method validation The developed HPLC method was taken up for validation. The analytical method validation was carried out in accordance with ICH guidelines. 4.4.1 System Suitability Test: The purpose of the system suitability test is to ensure that the complete testing system (including instrument, reagents, columns, analysts) is suitable Chromatography for General the intended Chapter states application. system The suitability USP as: "System suitability tests are an integral part of liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such." Weighed each 5 mg of S- 2-azido-3-methylbutanoic acid and R-2azido-3-methylbutanoic acid into a 10 ml volumetric flask, dissolved and diluted to volume with diluent. This solution injected into the HPLC system. The corresponding system suitability chromatogram and data are shown in Fig. 4.4.F1.
175 Fig. 4.4.F1: Representative chromatogram of system suitability X-axis: Retention time in min and Y-axis: Peak response in AU Acceptance criteria: The resolution between the S-2-azido-3- methylbutanoic acid and R-2-azido-3-methylbutanoic acid should be greater than 2.0. The observed resolution between both the enantiomers is 2.6. This indicates that the system meets the required system suitability criteria. 4.4.2 Precision: The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. The precision of method was checked by injecting six individual preparations of (S)-2-azido-3-methylbutanoic acid (2.0 mg ml-1) spiked with 0.5% of (R)-2-azido-3-methylbutanoic acid with respect to (S)-2-azido-3-methylbutanoic acid analyte concentration.
176 The % RSD of (R)-2-azido-3-methylbutanoic acid peak tabulated in Table 4.4.T1 and the typical chromatogram presented in Fig. 4.4.F2. Table 4.4.T1: Precision results S.No R- 2-azido-3-methylbutanoic acid peak area Prep-1 19768 Prep-2 19622 Prep-3 20062 Prep-4 19614 Prep-5 20265 Prep-6 20591 Average 19987 %RSD 1.96 Acceptance criteria: The % RSD should not be more than 2.0 %. The %RSD value obtained from the six injections is well within the acceptance criteria, which indicates that the method is precise at specification level limit [0.2 % level]. Fig. 4.4.F2: Reference chromatogram of 0.2% precision X-axis: Retention time in min and Y-axis: Peak response in AU
177 4.4.3 Limit of detection (LOD) and limit of quantification (LOQ): LOD and LOQ established for R-2-azido-3-methylbutanoic acid peak based on signal to noise ratio method. 4.4.3.1 Limit of detection (LOD): The limit of detection of an analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. Prepared a series of dilutions of R-2-azido-3-methylbutanoic acid impurity in different concentrations, until to get the signal to noise ratio between 2 and 3. Injected each dilution once. The LOD values were represented in Table 4.4.T2. Table 4.4.T2: LOD value for R- 2-azido-3-methylbutanoic acid S.No Impurity Name S/N Ratio Limit of detection ( in µg ml-1) Limit of detection ( wrt test concentration) 2.6 0.16 0.0075% R-2-azido-31 methylbutanoic acid Acceptance criteria: The S/N ratio should be between 2 and 3. 4.4.3.2 Limit of quantification (LOQ): The Limit of quantification (LOQ) of an analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Prepared a series of dilutions of (R)-2-azido-3-methylbutanoic acid impurity in different
178 concentrations, until to get the signal to noise ratio between 9.5 and 10.4. Injected each dilution once. The limit of quantification results were represented in Table 4.4.T3. Table-4.4.T3: LOQ value of R- 2-azido-3-methylbutanoic acid S.No Impurity Name S/N Ratio Limit of Quantification ( in µg ml-1) Limit of Quantification ( wrt test concentration) 10.6 0.5 0.025% R-2-azido-31 methylbutanoic acid Acceptance criteria: The S/N ratio should be between 9.5 and 10.4. Fig. 4.4.F3: Representative chromatogram of LOQ. X-axis: Retention time in min and Y-axis: Peak response in AU 4.4.4 Precision at limit of quantification level: Prepared six individual solutions containing (R)-2-azido-3- methylbutanoic acid at the limit of quantification level. Injected each solution once and calculated the % RSD for the area of (R)-2-azido-3methylbutanoic acid peak. The precision at limit of quantification for (R)-2-azido-3-methylbutanoic acid peak was less than 10.0%,
179 confirming good precision of the method at LOQ. The results were tabulated in Table-4.6 and the typical LOQ chromatogram shown in Fig. 4.4.T5. Table 4.4.T4: Precision at LOQ level S.No Preparation R- 2-azido-3-methylbutanoic acid peak area 1 2 3 4 5 6 Preparation-1 2010 Preparation-2 1965 Preparation-1 2115 Preparation-2 1895 Preparation-1 1901 Preparation-2 2185 %RSD 5.83 Acceptance criteria: The % RSD should not be more than 10. The % RSD value is well within the acceptance criteria, which indicates that the method is precise at LOQ level limit [0.025 % level]. 4.4.5 Accuracy at limit of quantification level: The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness.
180 Prepared three different solutions containing R-2-azido-3- methylbutanoic acid at the limit of quantification level and injected each solution once. Prepared the test solution for three times from the same homogeneous sample. Prepared three different sample solutions containing R-2-azido-3methylbutanoic acid at the limit of quantification level and injected each solution once, calculated % Recovery for the impurity. The results were tabulated in Table 4.4.T5. S.No Table 4.4.T5: Accuracy at LOQ level Impurity Name % Recovery R-2-azido-3-methylbutanoic 1 85 acid Acceptance criteria: The percentage recovery should not be less than 70.0 and should not be more than 130.0 4.4.6 Linearity The linearity of an analytical procedure is its ability to obtain test results, which are directly proportional to the concentration of analyte in the test sample. Linearity experiments were carried out by preparing the (S)-2-azido-3-methylbutanoic acid sample solutions containing (R)-2- azido-3-methylbutanoic acid from LOQ to 200% (i.e. LOQ, 25%, 50%, 75%, 100% and 200%) with respect to their specification limit (0.50%).
181 Calibration curve was drawn by plotting area of the (R)-2-azido-3methylbutanoic acid peak on the Y-axis and concentration on the X- axis. The linearity results were tabulated in Table 4.4.T6. The slope and Y-Intercept of (R)-2-azido-3-methylbutanoic acid peak in the linearity study was found to be 4060 and 275.6 respectively. Calibration curve obtained by least square regression analysis between average peak area and the concentration showed (Fig. 4.4F4) linear relationship with a regression coefficient of 0.998. The best fit linear equation obtained was Y =4060 Con 275.6 for R-vildagliptin peak. The typical linearity chromatograms were shown in Fig. 4.4.F5 to F.4.F10. Table-4.4.T6: (R)-2-azido-3-methylbutanoic acid linearity results Concentration (µg ml-1) (R)- 2-azido-3-methylbutanoic acid area 0.5 2382 1.5 6002 2.5 9205 5.0 19961 7.5 29298 10.0 41128 Correlation Coefficient(r) 0.998 Slope 4060.35 Intercept -275.60
182 Fig. 4.4.F4: Linearity plot for (R)-2-azido-3-methylbutanoic acid X-axis: Concentration in µg ml-1 and Y-axis: Peak area mau 4.4.6.1 LOQ level linearity R-2-azido-3-methylbutanoic acid impurity spiked at LOQ level to (S)-2azido-3-methylbutanoic acid. Representative chromatogram was shown in Fig. 4.4.F5. Fig. 4.4.F5: Representative chromatogram of LOQ linearity. X-axis: Retention time in min and Y-axis: Peak response in AU
183 4.4.6.2 Linearity at 25% level: R-2-azido-3-methylbutanoic acid impurity spiked at 25 % level [0.125 %] level to (S)-2-azido-3-methylbutanoic acid. Representative chromatogram was shown in Fig. 4.4.F6. Fig. 4.4.F6: Representative chromatogram of 25% linearity X-axis: Retention time in min and Y-axis: Peak response in AU 4.4.6.3 Linearity at 50% level: R- 2-azido-3-methylbutanoic acid impurity spiked at 50 % level [0.25%] to (S)-2-azido-3-methylbutanoic acid. Representative chromatogram was shown in Fig. 4.4.F7. Fig. 4.4.F7: Representative chromatogram of 50% linearity. X-axis: Retention time in min and Y-axis: Peak response in AU
184 4.4.6.4 Linearity at 100% level: R- 2-azido-3-methylbutanoic acid [0.50%] to impurity spiked at 100% level (S)-2-azido-3-methylbutanoic acid. Representative chromatogram was shown in Fig. 4.4.F8. Fig. 4.4.F8: Representative chromatogram of 100% linearity. X-axis: Retention time in min and Y-axis: Peak response in AU 4.4.6.5 Linearity at 150% level: R- 2-azido-3-methylbutanoic acid [0.75%] to impurity spiked at 150% level (S)-2-azido-3-methylbutanoic acid. Representative chromatogram was shown in Fig. 4.4.F9. Fig. 4.4.F9: Representative chromatogram of 150% linearity. X-axis: Retention time in min and Y-axis: Peak response in AU
185 4.4.6.6 Linearity at 200% level: R- 2-azido-3-methylbutanoic acid impurity spiked at 200% level [1.0%] to (S)-2-azido-3-methylbutanoic acid. Representative chromatogram was shown in Fig. 4.4.F10. Fig. 4.4.F10: Representative chromatogram of 200% linearity X-axis: Retention time in min and Y-axis: Peak response in AU 4.4.7 Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy of the method established at 50%, 100% and 150% of the impurities specification limit (0.50%). Test solution prepared in triplicate (n=3) with (R)- 2-azido-3-methylbutanoic acid at 0.25%, 0.5%, and 0.75% level w.r.t. analyte concentration (i.e. 2.0 mg/ml). Each solution was injected once into HPLC. Mean % recovery of impurities calculated in the test solution using the area of (R)-2-azido-
186 3-methylbutanoic acid standard at 0.50% level with respect to analyte. The recovery results were tabulated in Table 4.4.T7. Table 4.4.T7: Accuracy results S.No. Recovery levels % of Recovery 1 50% 102.8 2 100% 100.9 3 150% 101.9 Acceptance criteria: The % recovery should not be less than 85 and should not be more than 115. The above data reveals that the method is accurate. 4.4.8 Solution stability: The solution stability of 2-azido-3-methylbutanoic acid was carried out by leaving both unspiked and spiked sample solutions at temperature 25 C on a laboratory bench for 48 h. The content of Renantiomer was determined for every 6 h interval sample solution in tightly capped volumetric flask at room temperature for two days. significant change was observed in the content of No (R)-2-azido-3- methylbutanoic acid during solution stability experiments up to 24 hours. Hence concluded as (S)-2-azido-3-methylbutanoic acid sample solutions are stable for at least 24 hours in the developed method. The results were summarized in Table 4.4.T8.
187 Table 4.4.T8: Solution stability results, Batch No: 001 Time interval % of R- 2-azido-3-methylbutanoic acid Initial 6 Hrs ND ND 12 Hrs ND 18 Hrs ND 24 Hrs ND 4.4.9 Robustness study: To determine the robustness of the developed method, experimental conditions were deliberately altered and the resolution between both the isomers was evaluated. In each of the deliberately altered chromatographic condition (flow rate 0.8 ml min -1 and 1.2 ml min-1, column temperature 23 C and 27 C) the resolution between both the isomers greater than 2.0, illustrating the robustness of the method. The effect of change in percent ratio of additive was also studied. Table 4.4.T9: Results of robustness study Parameter Temperature (± 5 0C of set temperature) Variation Resolution between R-enantiomer and Senantiomer Flow rate (± 0.2 ml min-1of the set flow) 200C 300C 0.8 ml min-1 1.2 ml min-1 2.3 2.2 2.4 2.2
188 4.4.10 Batch analysis data: Using the above validated method, (S)-2-azido-3-methylbutanoic acid samples were analyzed as per the finalized conditions and the data is presented in Table 4.4.T10. Table 4.4.T10: (S)-2-azido-3-methylbutanoic acid batch analysis data S.No. Batch number % of R- 2-azido-3methylbutanoic acid 1 AMB001 Not detected 2 AMB002 Not detected 3 AMB005 Not detected The above results reveal that the R- 2-azido-3-methylbutanoic acid sample is not detected in the above three batches. 4.5 Summary and Conclusion A new isocratic chiral HPLC method was developed for the separation of two enantiomers of 2-azido-3-methylbutanoic acid. Chiralpak IA column has shown excellent selectivity for 2-azido-3methylbutanoic acid enantiomers and developed method is quite simple, sensitive, and reproducible. The limit of quantitation is 0.5 µg ml-1. The method is validated as per ICH guidelines and found to be linear, precise, accurate, specific, roboust and rugged for the determination of (R) - 2-azido-3-methylbutanoic acid in (S) -2-azido-3methylbutanoic acid. The method can be used for the determination of enantiomeric purity of (S)-2-azido-3-methylbutanoic acid.
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