A Novel RP-Chiral Separation Technique for the Quantification of Brivaracetam and its process Related Isomeric Impurities using High Performance Liquid Chromatography

 

Sandip Kumar Dey¹, Sumanta Mondal¹*, Prasenjit Mondal², Kausik Bhar¹

¹Department of Pharmaceutical Chemistry, Institute of Pharmacy,

GITAM (Deemed to be University), Visakhapatnam - 530045, A.P., India.

²Department of Pharmaceutical Technology, Brainware University,

Ramkrishnapur Road, Barasat, West Bengal, 700125, India.

 

ABSTRACT:

Reverse‐phase high‐performance liquid chromatography method has been developed for the determination of brivaracetam (BRV) with its stereoisomeric impurities (BRV-SS), (BRV-RR) and (BRV- RS). Brivaracetam with its impurities has been eluted with good resolution using the chiral column, chiralpakIG (250 × 4.6mm, 5µ) column at the detection wavelength of 210nm with the  flow rate of 0.50ml/minute. The separation was achieved with the mobile phase consisted of methanol: acetonitrile and trifluoroacetic acid in the volume ratio of 90:10:0.1. The column temperature was maintained at 30°C and detection wavelength was maintained at 210 nm. Brivaracetum (BRV), and stereoisomeric impurities, BRV-SS, BRV-RR and BRV-RS, were eluted at 13.829, 11.810, 10.448 and 9.449 min, respectively. The method showed adequate specificity, sensitivity, linearity, accuracy, precision, and robustness inline to ICH tripartite guidelines. The limit of detections were 0.2 μg/mL for BRV-SS and BRV-RR, for BRV-RS it was found 0.1μg/mL.The limit of quantification limits were 0.5μg/mL for BRV-SS, BRV-RR and for BRV-RS as well.The developed method was found to be linear over the concentration range of 0.5-20µg/mL for BRV-RS, for BRV-RR and BRV-RS it was 0.5-3µg/mLfor stereoisomeric impurities with a correlation coefficient of 0.999. The developed method was found precise (%RSD = 0.5%, 0.6% and 0.5% for BRV-SS, BRV-RR and BRV-RS,) accurate (with 96%–107% recovery) and found robust. The validation parameters of the developed method were within the limits as per the regulation of ICH Q2(R1) guidelines. Therefore, in this present method a good separation was achieved specifically for the identification of brivaracetam and its processed related stereoisomeric impurities using high performance liquid chromatography and found suitable for the quantification of stereoisomeric impurities of brivaracetam in bulk scale as well.

 

 

 

INTRODUCTION: 

Brivaracetam is a chemical analog of levetiracetam, which is a racetam derivative with anticonvulsant (antiepileptic) potential. Brivaracetam is used to treat partial-onset seizures with or without secondary generalization, in combination with other antiepileptic drugs.

 

 

It is sometimes prescribed as an alternative to the drug's analogue levetiracetam to avoid neuropsychiatric adverse effects such as mood swings, anxiety, emotional lability and depression^1,2. Brivaracetam is believed to act by binding to the ubiquitous synaptic vesicle glycoprotein 2A (SV2A), like levetiracetam. but with 20-fold greater affinity. There is some evidence that racetams including levetiracetam and brivaracetam access the luminal side of recycling synaptic vesicles during vesicular endocytosis. They may reduce excitatory neurotransmitter release and enhance synaptic depression during trains of high-frequency activity, such as is believed to occur during epileptic activity³. On 14 January 2016, the European Commission, and on 12 May 2016, the U.S. Food and Drug Administration (FDA) approved Brivaracetam under the trade name “Briviact”⁴. Even though the enantiomers have the same chemical structure, they exhibit differences in biological activities like pharmacology, toxicology, pharmacokinetics, and metabolism. Hence, the chiral analysis is outmost essential to the racemic drugs in the pharmaceutical industry and in the clinic to control or eliminate the unwanted isomer from the preparation and provide the stringent therapeutic treatment to the patient without side effects⁵. There are lots of regulatory challenges to address the regulatory queries on control and carryover by organic impurities in drug substance or drug product. These impurities are sometime genotoxic, hence the proper study of these impurities by the suitably developed analytical method is critical in drug development in today's scenario. These improved continuously with the use of new reagents, catalyst and intermediates, etc., or with changes in synthesis routes. Therefore, the control of organic or inorganic impurities in the manufacturing stages of API and its starting material plays an essential role during the process development⁶. High performance liquid chromatographic method (HPLC), even having the most common analytical methods such as optical rotation and capillary electrophoresis for the determination of isomeric purity of drug molecules, is one of the widely used analytical tools for direct chiral analysis^7-9. It can provide fast and accurate methods for well chiral separation and allows on-line detection and quantitation of isomers¹⁰. Here we have observed three isomeric impurities along with the API brivaracetam viz., (R)-2-((S)-2-oxo-4-propylpyrrolidin-1-yl) butanamide (BRV-RS) (isomer-1), (R)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl) butanamide (BRV-RR) (isomer-2), S)-2-((S)-2-oxo-4-propylpyrrolidin-1-yl) butanamide (BRV-SS) (isomer-3). There are few methods reported for quantitation of brivaracetam in pharmaceutical substances and dosage forms, The validated stability indicating UPLC method for the determination of brivaracetam in degradation impurities¹¹. Stability-indicating thin-layer chromatographic determination of brivaracetam in bulk scale and determination of the stereochemistry of diastereoisomers of brivaracetam using vibrational circular dichroism (VCD) spectroscopy were also developed^12,13. There is an obtainability of UPLC–MS/MS, assay method for identification and quantification of brivaracetam in plasma sample¹⁴. There is an availability of single UF-HPLC method¹⁵ for the determination of isomeric process impurities of brivaracetam. The disadvantages of this reported method viz., the resolution between the 1^st two impurities are less as shown in the chromatogram. There may be the opportunities to reduce the retention time because the authors utilised the costlyultra-fast liquid chromatography.Considering the above facts in mind the intensive effort has been taken to focus the present method to determine the chiral impurities of brivaracetam in higher resolution using high performance liquid chromatography, which is considerably reasonable than UPLC and to validate the developed method as per the regulation of ICH Q2(R1) guidelines¹⁶.

 

 

Fig. 1: Chemical structures of Brivaracetam and its isomeric impurities

 

MATERIALS AND METHODS:

Reagents and chemicals:

The brivaracetam standard and its three related chiral substances has been provided by the TCG lifesciences Pvt. Ltd., Salt Lake City, Kolkata, West Bengal. HPLC grade trifluoro acetic acid (99.0%) from spectrochem, methanol (99.9%) was procured form Finar Labs, Mumbai, acetonitrile (99.9%) from Merck, Mumbai. HPLC grade purified water (99.9%) was procured from Sartorious.

 

Instruments:

The analytical balance and microbalance was from Mettler Toledo XS205DU and XP6 respectively. The Ultrasonicator bath is from Bandelin DT514, Refrigerator from Samsung and vacuum pump from Divac,13500. Waters Alliance e2998 HPLC with Empower 3 software, Millford, USA, was utilised during this study.

Preparation of selective stock solutions of individual chiral impurities(BRV-RR, BRV-SS and BRV-RS):

Accurately weighted 5mg each of BRV-RR, BRV-RS and 10mg BRV SS and the individual amounts were transferred into the separate 20mL and 10mL volumetric flasks respectively. Dissolve and diluted to volume using the diluent, methanol: acetonitrile (90:10 v/v). The above three individual solutions were mentioned as stock solution-1. Then 5mL aliquot from the BRV-SS stock solution-1, 3mL aliquots from the BRV-RR and BRV-RS stock solution-1, were separately diluted to 50mL using the same diluent to obtained stock solution-2.

 

Preparation of the spiked sample solution:

About 50mg of BRV sample was weighted and transferred to the 50mL of volumetric flask. To the above flask, 5mL aliquots were spiked, each from the BRV-SS, BRV-RR and BRV-RS standard chiral impurity stock solutions-2. The volume was made up to the mark using the diluent, methanol: acetonitrile (90:10 v/v).  The obtained final concentrations were 1000ppm of BRV, 10ppm BRV-SS, 1.5ppm of BRV-RR and BRV-RS .         

 

Preparation of mobile phase, diluents:

It was prepared by mixing of HPLC grade methanol, acetonitrile and trifluoroacetic acid in the volume ratio of 90:10:0.1 (v/v/v). The prepared mobile phase was filtered using 0.45µ membrane filter and finally sonicate to degas.  Methanol: acetonitrile (90:10 v/v) was utilised as a diluent and same was used for needle wash. For seal wash water: acetonitrile (90: 10 v/v) was utilised.

 

Chromatographic procedure:

The diluent, which is considered blank, spiked sample solution was injected in six replicates and chromatograms were recorded. The chiralpakIG (250×4.6mm, 5µ) column (Diacel manufacturing plant, Shinzaike, Aboshi, Japan) has been utilised with the injection volume of 15µL. The detection was performed at 210nm with the flow rate of 0.50ml/minute. The column temperature was maintained at 30⁰C. The relative standard deviation (RSD) of NMT 5.0% from the injected six replicates of spiked standard solution for brivaracetam and its isomers (BRV-SS, BRV-RR and BRV-RS) were used to verify the specificity/system suitability criteria of the method. The resolution NLT2.0 between brivaracetam and its three isomers was set as system suitability criteria in resolution mixture.

 

Identification of impurities:

Brivaracetam crude samples were analyzed for the identification of isomeric impurities by the proposed method. Three isomeric impurities were detected in the crude sample of brivaracetam.  The m/z of detected peaks was determined by UFLC-MS technique. The observed m/z values confirmed the possible structures of isomeric impurities (Fig. No 1). The impurities (Isomer-1, Isomer-2, and Isomer-3) were synthesized and co-injected with the brivaracetam test samples to confirm its retention times. Bruker Ultrashield (400 MHz) spectrometer was used to record the 1H NMR spectra by using deuterated DMSO as a solvent and tetramethylsilane (TMS) as an internal standard. Perkin Elmer model-spectrum-100 (California, USA) instrument (KBr pellet method) was used to record the FT-IR spectra.

 

RESULTS AND DISCUSSION:

Analytical method development:

Analytical method development was performed to separate brivaracetam and its three isomeric impurities (BRV-SS, BRV-RR and BRV-RS) with short run time and excellent resolution between the separated impuritieswith a good peak shape. The resolution between isomeric impurities was critical during the method development and hence selection of proper stationary phase played a significant role during the method development. Initially several method development trials has been conducted using different stationary phases like Chiralpak-ODH, Chiralpak-ADH, Chiralpak-IA, Chiralpak-IB. The columns along with the other chromatographic conditions like column oven temperature, detection wavelength, and the ratio of organic solvent and modifier, the mobile phase composition has been tested to obtain the optimised chromatogram with satisfactory resolution between the isomeric impurities. During these trials in the search of ideal chromatogram, using aforementioned varied conditions we faced the problems with resolution between the brivaracetam and its isometric impurities. The system suitability criterion was evaluated at every time during the different trial runs of method development to ensure the strength of the developed method. The isocratic mode was preferred than the gradient mode to achieve a stable baseline. Finally the good peak shape with satisfactory resolution between the impurities were achieved using the chiralpak-IG(250×4.6mm, 5µ) column at the detection wavelength of 210 nm with the  flow rate of 0.50ml/minute at 30^oC column temperature with the mobile phase of methanol: acetonitrile and trifluoroacetic acid in the volume ratio of 90:10:0.1 (v/v/v).  It was observed that brivaracetam and its three isomers, BRV-SS, BRV-RR and BRV-RS, are well separated under the optimized conditions with a resolution greater than 2.0. The values of retention times of Brivaracetum (BRV), and stereoisomeric impurities, BRV-SS, BRV-RR and BRV-RS, were 13.829, 11.810, 10.448 and 9.449min, respectively, the chromatogram was shown in specificity chromatogram in fig. No 2.

Fig. 2: Chromatogram of Brivaracetam and its isomeric impurities(BRV-SS, BRV-RR and BRV-RS)

 

Method validation:

Specificity:

Specificity which defined is an ability of the method to measure the analyte retentions in the presence of its potential impurities.For the study of the specificity^17,18, the test sample of brivaracetam was spiked with its isometric impurities at specification level as described earlier in the preparation of spiked sample solution. Each chromatogram was recorded, and the injections was processed at the wavelength provided in the analytical method and the selectivity was demonstrated with regard to non-interference from blank (diluent) with the analytes. Peak purity was established for BRV and its specified impurities and found satisfactory.No interfering peak was observed at the retention time of BRV and its specified impurities in blank. The peak due to BRV and its isomeric impurities were well resolved from each other and other peaks.

 

LOQ Precision:

Based upon the ICH Q2 (R1) guidelines^19,20, the quantitation limit has been determined by the analysis of brivaracetam samples with known concentrations of analyte (isomeric impurities of brivaracetam i.e., BRV-SS, BRV-RR and BRV-RS) and establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision. <50 % of specification (i.e., 0.05 %) concentration was selected to determine the LOQ of the method.Further, the LOQ was verified by giving the six-replicate injection of LOQ concentration. The observed % RSD of LOQ precision was <20.0 % The signal to noise ratio of LOQ preparation was found ≥10, i.e., within the acceptance criteria.The detailresults are depicted in the Table 1 and the chromatogram was shown in fig. No 3.

 

Fig. 3: LOQ Chromatogram of Brivaracetam and its isomeric impurities(BRV-SS, BRV-RR and BRV-RS)

 

LOD verification:

The solution containing the analytes at LOD Level concentration of brivaracetam, and its impurities was prepared. 20% of specification (i.e., 0.02%) concentration was selected to determine the LOD of the method.^21,22 Further, the LOD was verified by giving the three-replicate injection of LOD concentration. Signal to noise ratio of LOD level for BRV and its specified impurities was calculated and signal to noise ratio was found ≥ 3, hence the limit of detection has been verified for this present method.The results are depicted in the Table 1.

Accuracy:

The accuracy of the developed method^23,24 has been studied by calculating the % recovery. For the study, the sample for accuracy as unspiked and spiked samples at LOQ Level, 100% and 200% level of specification limit of known impurities in triplicate has been prepared and injected into the chromatographic system. The unspiked sample of 3 preparations of BRV has been prepared in the Concentration of 1000ppm of BRV. The “accuracy – LOQ” samples in 3 preparations was made in the concentrations of 1000ppm of BRV and 0.5ppm of each of BRV-SS, BRV-RR, BRV-RS. The “accuracy-100%” solutions in 3 preparations were prepared in the concentration of 1000ppm of BRV and 10ppm of BRV-SS and 1.5ppm each of BRV-RR, BRV-RS. In the same way “accuracy-200%” solution was prepared in the concentration of 1000ppm of BRV and 20ppm of BRV-SS and 3ppm each of BRV-RR, BRV-RS. The prepared samples at these levels have been injected into the chromatographic system and obtained chromatograms are utilised to calculate the recovery of specified impurity. The percentage mean recovery was found 100% with the %RSD of 0.1 at the accuracy – LOQ” level for BRV-SS. For BRV-RR mean recovery was found 97% with the %RSD of 0.5, which are within the acceptance criteria. The details of the results at other accuracy levels for all the three impurities has been cited in the table 1. The mean recovery and %RSD values are found within the acceptance criteria. Hence the developed method, for BRV with all of its 3 isomeric impurities were found accurate.

 

Linearity:

For the study of linearity^25,26, a series of specified amount of impurities and BRV concentrations has been prepared, ranging from LOQ to 200% of specification limit. Initially several linearity stock solutions has been prepared and finally the solution with the concentration is about 100ppm each of BRV, BRV-SS and 15ppm each of BRV-RR, BRV-RS has been injected into the chromatographic system. Linearity graph of average area at each linearity level against the concentration (% w/w w.r.t. sample concentration) were prepared and determined the correlation coefficient (r), which was found more than 0.999 for BRV and its all-isomeric impurities results are cited in the table 1.

 

Method Precision:

For the study of method precision blank solution, standard solution and sample solution (6 preparations) were prepared and analyse as per the developed optimised conditions for BRV and its impurities. The prepared solutions have been injected into the chromatographic system and %RSD of the content of % area was calculated for the specified impurities results from six sample preparations. The percentage RSD was found 0.5%, 0.6% and 0.5% for BRV-SS, BRV-RR and BRV-RS, which is within the limit,that is ≤ 20%, asper the acceptance criteria of the impurity level of BRV-RR and BRV-RS (0.15%) and BRV-SS(1%).

 

Intermediate precision:

In the intermediate precision different parameters like analyst, day, column and instruments has been varied and tested in two sets. Blank solution, standard solution and sample solution (6 preparations) has been tested as per the developed analytical method. %RSD of the content of % area for the specified impurities result from six sample was calculated and found 1.8%, 6% and 10.2% for the BRV-SS, BRV-RR and BRV-RS respectively. The %RSD values are within the acceptable limit, that is ≤ 20%, as per the acceptance criteria of the impurity level of BRV-RR and BRV-RS (0.15%) and BRV-SS (1%).

 

Stability of the analytical solution:

For the stability study of the analytical solution, blank solution, standard solution, and sample solution were prepared as per the analytical method. The prepared solutions were tested initially, after 12hours, after 24 hours and after 48 hours at room temperature. The system suitability criteria were calculated as per the analytical method. The results indicated that the relative changes to the initial results was found ≤ 20% and it was found within the limit and confirms the stability of the analytical solutions for at least 48 hours at room temperature.

 

Robustness:

For the study of robustness, the blank solution, standard solution, and sample solution (duplicate preparations) were prepared as per the analytical method and injected using different chromatographic conditions by changed in flow rate (±0.1mL), changed in detection wavelength (±2nm), changed in the volume of trifluoroacetic acid (±10%) in mobile phase, change in volume of acetonitrile (±10%) in mobile phase and change in column oven temperature (±2°C). The overall %RSD with two results of same sample with the standard condition: n=8 (2+6) i.e., two data from changed condition and six data from method precision  and %RSD was found ≤ 20. Hence the developed methods for BRV with all of its 3 isomeric impurities were found robust. The detail results were cited in the Table 2.

 

CONCLUSION:

A new, specific and robust high performance liquid chromatographic method has been developed which separates the Brivaracetam and its isomeric impurities, Brivaracetam-SS, Brivaracetam-RR and Brivaracetam-RS with excellent peak shape and resolution. The developed method was further validated to ensure compliance per ICH Q2(R1) guideline, for distinctive parameters that are within the admissible limits. Therefore, the described RP-HPLC method in the present study developed, was not only suitable for the determination of isomeric purity of brivaracetam but also can be used for the quantification of stereoisomeric impurities of brivaracetam in bulk scale.

 

CONFLICTS OF INTEREST:

There are no conflicts of interest.

 

ACKNOWLEDGEMENT:

The authors are thankful to the research and development section, TCG lifesciences Pvt Ltd, Block, BN, Plot# 7, Sector V, Salt Lake electronic complex, Kolkata, 700091 for the support of this work.

 

 

Table 1: Summary of studied validation parameters

Parameters	Results of the related substances
	BRV	BRV-SS	BRV-RR	BRV-RS
	Limit of Quantitation (LOQ)
LOQ (% w.r.t.) sample concentration	0.05	0.05	0.05	0.05
	Limit of detection (LOD)
LOD (% w.r.t.) sample concentration)	0.02	0.02	0.02	0.01
	Linearity
Correlation coefficient	0.9999	0.9999	0.9999	0.9998
Y intercept	- 710.034	- 424.208	12.017	685.202
Slope	304254.896	294466.565	285944.986	288054.384
	Accuracy, % recovery (%RSD)
LOQ	-	100 (0.1)	97 (0.5)	107 (0.4)
100%	-	99 (0.1)	96 (0.6)	99 (0.0)
200%	-	99 (0.2)	98 (0.3)	99 (0.2)

 

Table 2: Robustness study results of the brivaracetam and its isomeric impurities

Parameters	Results of the related substance
	BRV-SS	BRV-RR	BRV-RS
	%RSD of Change in flow rate (± 0.1)
0.40 ml/min	0.4	1.2	1.4
0.60 mL/min	0.6	8.4	6.0
	% RSD of Change in detection wavelength (± 2 nm)
208 nm	2.7	2.6	2.4
212 nm	2.7	5.3	5.0
	% RSD of the change in the volume of acetonitrile (± 10%) in mobile phase
Methanol: ACN: TFA (91:9:0.1)	0.4	0.7	0.7
Methanol: ACN: TFA (89:11:0.1)	1.6	0.9	1.1
	%RSD of the change in the volume of TFA (±10%) in mobile phase
TFA 0.09 %	0.4	0.9	0.6
TFA 0.11 %	0.5	0.8	0.5
	%RSD of the change in column oven temperature
280C	1.6	7.2	4.1
320C	1.0	5.2	8.9

 

REFERENCES:

1.      Klein P. Diaz A.Gasalla T. Whitesides J. A review of the pharmacology and clinical efficacy of brivaracetam. Clinical  Pharmacology.2018; 10: 1-22.

2.      Malek N. Heath CA. Greene J. A review of medication adherence in people with epilepsy. Acta Neurologica Scandinavica. 2017; 135(5): 507-515.

3.      Rogawski MA. Loscher W. Rho JM. Mechanisms of Action of Antiseizure Drugs and the Ketogenic Diet. Cold Spring Harbor Perspectives in Medicine. 2016; 6(5):a022780. doi: 10.1101/cshperspect.a022780.

4.      Palleria C.Cozza G.Khengar R. Libri V. De-Sarro G. Safety Profile of the Newest Antiepileptic Drugs: A Curated Literature Review. Current PharmaceuticalResearch. 2017; 23(37):5606-5624.doi: 10.2174/1381612823666170809115429.

5.      Lien AN. Hua H. Chuong PH. Chiral drugs: an overview. International journal of Biomedical Science. 2006;2(2):85-100.PMID: 23674971; PMCID: PMC3614593.

6.      Prajapati P. Agrawal YK. Analysis and impurity identification in pharmaceuticals. Review in Analytical Chemistry. 2014; 33(2): 123-133.https://doi.org/10.1515/revac-2014-0001

7.      Zhang T. Nguyen D. Franco P, Isobe Y, Michishita T, Murakami T. Cellulose tris(3,5-dichlorophenylcarbamate) immobilised on silica: a novel chiral stationary phase for resolution of enantiomers. Journal of Pharmaceuticaland Biomedical Analysis. 2008; 46(5): 882-891.https://doi.org/10.1016/j.jpba.2007.06.008

8.      Aboul-EneinHY. High-performance liquid chromatographic enantioseparation of drugs containing multiple chiral centres on polysaccharide-type chiral stationary phases. Journal of Chromatography A. 2001; 906(1-2):185-193.doi: 10.1016/s0021-9673(00)00950-x.

9.      Purdie N. Swallows KA. Analytical applications of polarimetry, optical rotatory dispersion, and circular dichroism. Analytical Chemistry. 1989; 61(2):77A-89A.https://doi.org/10.1021/ac00177a714.

10.   Boehme W, Wagner G, Oehme U. Spectrophotometric and polarimetric detectors in liquid chromatography for the determination of enantiomer ratios in complex mixtures. Analytical Chemistry. 1982;54(4):709-711.https://doi.org/10.1021/ac00241a025

11.   Vishweshwar V.Mosesbabu J. Muralikrishna. Development and validation of stability-indicating UPLC method for the determination of brivaracetam, its related impurities and degradation products. International Journal of Pharmaceutical Science and Research. 2018; 9:2315-2327.10.13040/IJPSR.0975-8232.9(6).2315-27

12.   Shital RN. Deepali AB. Stability indicating thin-layer chromatographic determination of brivaracetam as bulk drug: application to forced degradation study. International Journal of Pharm Tech Research. 2018; 11:351-360.DOI: http://dx.doi.org/10.20902/IJPTR.2018.11406

13.   Shi Q. Abbaspour T. Sergey S. Patrick B. Wouter H. Benoit M. Stereochemistry of the brivaracetam diastereoisomers. Chirality. 2016; 28:215-225.doi: 10.1002/chir.22558

14.   Muzaffar I. Essam E. Khalid AR. UPLC-MS/MS assay for identification and quantification of brivaracetam in plasma sample: application to pharmacokinetic study in rats. Journal of Chromatography B. 2017; 1060:63-70.doi: 10.1016/j.jchromb.2017.05.039.

15.   Baksam V.Pocha SNVR. Chakka VB.Ummadi RR. Kumar P. Development of an effective novel validated stability-indicating HPLC method for the resolution of brivaracetam stereoisomeric impurities. Chirality. 2020; 32(9): 1-12.DOI: 10.1002/chir.23269

16.   Validation of Analytical Procedure, Int. Conf. Harmonization (ICH), Methodology Q2 (R1). http://www.gmp-compliance.org/guidemgr/files/Q2(R1).PDF (accessed June 2021).

17.   Tandel JN. Bhatt RV.Patel NM.Shah SK. Practical Implication of Stability Indicating Chromatographic Method for Estimation of Clomipramine Hydrochloride and its Related Substances from Capsule Dosage Form. Asian Journal of Pharmaceutical analysis. 2017; 7(2): 100-112. DOI: 10.5958/2231-5675.2017.00017.5

18.   Sireesha DM. Monika L, Bakshi V. Development and Validation of UV Spectrophotometric Method for The Simultaneous Estimation of Rosuvastatin and Ezetimibe in Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis. 2017; 7(3): 135-140.DOI: 10.5958/2231-5675.2017.00021.7

19.   Chandana OSS. Ravichandra Babu R.  Stability Indicating High Performance Liquid Chromatographic Assay for the Determination of Sildenafil Tartrate. Asian Journal of Pharmaceutical Analysis. 2017; 7(3): 196-200.DOI: 10.5958/2231-5675.2017.00031.X  

20.   Thangabalan B. Koya A.  Chaitanya G.  Sunitha N. Manohar Babu S. Stability Indicating RP–HPLC Method for the Estimation of Acamprosate in Pure and Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis. 2013, 3(4): 141-146.

21.   Kadam A S, Pimpodkar N V, Gaikwad P S. Sushila D. Chavan. Bioanalytical Method Validation. Asian Journal of Pharmaceutical Analysis. 5(4): 2015; 219-225.

22.   Pawar J, Sonawane S, Chhajed S. Kshirsagar S. Development and Validation of RP-HPLC method for simultaneous Estimation of Metformin HCl and Gliclazide. Asian Journal of Pharmaceutical Analysis. 2016; 6(3): 151-154.DOI: 10.5958/2231-5675.2016.00024.7

23.   Wagh K. Sonawane S.Chhajad S. Kshirsagar S. Development of a RP-HPLC method for separation of ezetimibe in presence of atorvastatin calcium and simvastatin and its application for quantitation of tablet dosage forms. Asian Journal of Pharmaceutical Analysis. 2017; 7(3): 169-175.DOI: 10.5958/2231-5675.2017.00027.8

24.   Edlabadkar A P. Rajput A P. RP-HPTLC Method for Determination of Garenoxacin mesylate in Bulk and in Tablet Formulation. Asian Journal of Pharmaceutical Analysis. 2018; 8(2):78-82.DOI: 10.5958/2231-5675.2018.00015.7

25.   Patil P, Patil M, Patil D D. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Piracetam and Vinpocetine. Asian Journal of Pharmaceutical Analysis. 2018; 8(2):103-108.DOI: 10.5958/2231-5675.2018.00020.0

26.   Mondal P. Kola V. A New Stability Indicating Validated RP-HPLC Method for Simultaneous Estimation of Escitalopram and Clonazepam in Bulk and Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis. 2019, 9(4), 193-198. DOI: 10.5958/2231-5675.2019.00032.2  

 

 

 

 

Accepted on 02.11.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3139-3145.