Int J App Pharm, Vol 14, Issue 6, 2022, 167-177Original Article

SIMULTANEOUS DETERMINATION OF FLUPENTIXOL AND NORTRIPTYLINE HCl USING RP-HPLC WITH PDA DETECTOR

SUGANDHA KUMAR P.a, KANTIPUDI RAMBABU*

*aDepartment of Chemistry, RVR and JC College of Engineering, Guntur 522019, A. P., India
Email: [email protected]

Received: 11 Aug 2022, Revised and Accepted: 09 Sep 2022


ABSTRACT

Objective: In the current investigation, to separated and validate the cancer healing drugs (Nortriptyline HCl and Flupentixol) through the HPLC (e-2695) instrument containing a PDA detector.

Methods: A simple, selective, validated and well-defined stability that shows isocratic RP-HPLC methodology for the quantitative determination of Nortriptyline HCl and Flupentixol. The chromatographic strategy utilized Agilent eclipse XDB column of dimensions 250x4.6 mm, 5 micron, using isocratic elution with a mobile phase of Methanol and 0.1% orthophosphoric acid (40:60). A flow rate of 1 ml/min and a detector wavelength of 250 nm utilizing the PDA detector were given in the instrumental settings. Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines.

Results: LOD and LOQ concentrations for Flupentixol were 0.015 µg/ml, 0.05 µg/ml and for Nortriptyline HCl were 0.3 µg/ml, 1.0 µg/ml. The calibration charts plotted were linear with a regression coefficient of R2>0.999. Recovery, specificity, linearity, accuracy, robustness, ruggedness were determined as a part of method validation and the results were found to be within the acceptable range.

Conclusion: The proposed method to be fast, simple, feasible and affordable in assay condition. During stability tests, it can be used for routine analysis of production samples and to verify the quality of drug samples during stability studies.

Keywords: Nortriptyline HCl, Flupentixol, RP-HPLC, Development, Validation


INTRODUCTION

Flupentixol (INN), also known as flupenthixol (former BAN), marketed under brand names such as Depixol and Fluanxol is a typical antipsychotic [1, 2] drug of the thioxanthene class. Flupentixol's main use is as a long-acting injection given once in every two or three weeks to individuals with schizophrenia [3, 4] who have poor compliance with medication and suffer frequent relapses of illness, though it is also commonly given as a tablet. There is little formal evidence to support its use for this indication but it has been in use for over fifty years [5]. Flupentixol is also used in low doses as an antidepressant [6-8]. There is tentative evidence that it reduces the rate of deliberate self-harm among those who repeatedly self-harm [9].

Nortriptyline, sold under the brand name Pamelor, among others, is a medication used to treat depression. This medicine is used for: neuropathic pain [10, 11], attention deficit hyperactivity disorder (ADHD) [12, 13], smoking cessation [14] and anxiety [15]. As with many antidepressants [16, 17], its use for young people with depression and other psychiatric disorders [18] may be limited due to increased suicidality in the 18-24 population initiating treatment. Nortriptyline is a less preferred treatment for ADHD and stopping smoking. It is taken by mouth. Common side effects include dry mouth, constipation, blurry vision, sleepiness, low blood pressure with standing, and weakness. Serious side effects may include seizures [19], an increased risk of suicide in those less than 25 y of age, urinary retention [20], glaucoma [21], mania [22], and a number of heart issues. Nortriptyline may cause problems if taken during pregnancy. Use during breastfeeding appears to be relatively safe. It is a tricyclic antidepressant (TCA) [23, 24] and is believed to work by altering levels of serotonin [25] and norepinephrine. This medication is in capsule or liquid and is taken by the mouth one to four times a day, with or without food. Usually, people are started on a low dose and it is gradually increased. A level between 50 and 150 ng/ml of nortriptyline in the blood generally corresponds with an antidepressant effect [26]. Both drug structures are shown in fig. 1. The aim of the study is to separate the pharma ingredients Nortriptyline HCl and Flupentixol by using RP-HPLC.

A B

Fig. 1: Structure of (A) Nortriptyline HCl and (B) Flupentixol

Till today there are no HPLC method was reported in the literature. Hence we developed a method for the simultaneous quantification of Nortriptyline HCl and Flupentixol. The developed HPLC method was utilized for the estimation of the combined drugs by in vitro method.

MATERIALS AND METHODS

Chemicals

Methanol, HPLC-grade orthophosphoric acid, water were purchased from Merck India Ltd, Mumbai, India. APIs of Nortriptyline HCl and Flupentixol standards were procured from Glenmark, Mumbai.

The instrumentation

Waters alliance liquid chromatography (model 2695) [27, 28] was monitored with empower 2.0 data handling system and a detector of photodiode array (model 2998) [29, 30] was used for this study.

Method optimization

To optimize the chromatographic conditions, different ratios of phosphate buffer and the acetonitrile in the mobile phase with isocratic and gradient mode was tested. However, the mobile phase composition was modified at each trial to enhance the resolution and also to achieve acceptable retention times. Finally, 0.1% OPA buffer and methanol with isocractic elution was selected because it results in a greater response of active pharmacy ingredients. During the optimization of the method, various stationary phases such as C8, C18 phenyl and amino, Symmetry C18 columns were tested. From these trials the peak shapes were relatively good with a agilent eclipse XDB column of 250 x 4.6 mm, 5 µ with a PDA detector. The mobile phase flow rate has been done at 250 nm in order to obtain enough sensitivity. By using the above conditions we get retention times of Nortriptyline HCl and Flupentixol were about 2.717 and 4.123 min with a tailing factor of 1.04 and 1.15. The number of theoretical plates for Nortriptyline HCl and Flupentixol were 3812, 4499, which indicate the column’s successful output. The proposed approach suggests that it is extremely precise. According to ICH guidelines, the method established was validated.

Validation procedure

According to ICH Q2 (R1) guidelines [31, 32], analytical parameters were validated [33, 34] such as system appropriateness, precision, specificity, accuracy, linearity, robustness, LOD, LOQ, forced deterioration, and stability.

Preparation of buffer

In 1 Lt of HPLC Water, 1 ml of orthophosphoric acid was dissolved and filtered through 0.45 µ filter paper.

Chromatographic conditions

The HPLC analysis was performed on a reverse phase HPLC system with isocratic elution mode using a mobile phase of methanol and 0.1% OPA and agilent eclipse XDB column (250x4.6 mm, 3.5 μ) column with a flow rate of 1 ml/min.

Diluent: Mobile phase was used as diluent.

Preparation of the standard stock solution

Standard stock solution of Nortriptyline HCl (100 mg) and Flupentixol (5 mg) were prepared in 100 ml volumetric flask. It was filtered through a 0.45μ syringe filter. Standard stock solution concentrations of Nortriptyline HCl (1000μg/ml) and Flupentixol (50μg/ml) were obtained.

Preparation of the sample stock solution

Five Flupentixol and Nortriptyline HCl tablets were accurately weighed and triturated to get a fine powder. A 100 mg Nortriptyline HCl and 5 mg Flupentixol equivalent weight tablet powder was transferred into a 100 ml volumetric flask and dissolved in diluent. The solution was ultra-sonicated for 10 min and made the volume with diluent. The tablet sample stock solution was then filtered through 0.45 micron syringe filter and utilized for preparing sample solution for the assay.

RESULTS AND DISCUSSION

The main analytical challenge during development of a new method was to separate active Pharma ingredients. In order to provide good performance, the chromatographic conditions were optimized.

System suitability

In System suitability [35-38], injecting standard solution and reported USP tailing and plate count values are tabulated in table 1.

Table 1: Results of system suitability

System suitability parameter Acceptance criteria Nortriptyline HCl Flupentixol
Mean Std dev Mean Std dev
USP Plate Count NLT 2000 3845 10.264 4485 9.457
USP Tailing NMT 2.0 1.06 0.482 1.12 0.647
USP Resolution NLT 2.0 7.65 2.248
Retention time NLT 2.0 2.726 1.254 4.134 1.149

mean±SD (n=6)

Fig. 2: Chromatogram of system suitability

Specificity

In this test method, placebo, sample and standard solutions were analyzed individually to examine the interference [39, 40].

The below fig. shows that the active ingredients were well separated from blank and their excipients and there was no interference of placebo with the principal peak. Hence the method is specific.

Fig. 3: Chromatogram of blank

Linearity

The area of the linearity peak versus different concentrations has been evaluated for Nortriptyline HCl, Flupentixol as 10, 25, 50, 100, 125, 150 percent, respectively [41-43]. Linearity was performed in the range of 5-75µg/ml of Nortriptyline HCl and 2-30µg/ml of Flupentixol. The correlation coefficients achieved greater than 0.999 for all.

Table 2: Linearity of nortriptyline HCl and flupentixol

S. No. Conc µg/ml Nortriptyline HCl area count Conc. µg/ml Flupentixol area count
1 25.00 495068 1.25 161527
2 50.00 988745 2.50 325949
3 75.00 1475496 3.75 478598
4 100.00 1956477 5.00 651899
5 125.00 2414459 6.25 818873
6 150.00 2965454 7.50 958745
Correl coef 0.99985 0.99979
Slope 19575.54 129053.63
intercept 2648.86 1133.32

(n=3)

A

B

Fig. 4: Calibration plots of (A) Nortriptyline HCl (B) Flupentixol

Accuracy

In this method, Accuracy was conducted in triplicate by analyzing active pharma ingredient sample solution at three kinds of concentration levels of 50, 100 and 150% of each at a specified limit. Percentage recoveries were measured and found to be within the limit. The accuracy and reliability of the developed method were established. The results are given in table 3.

Table 3: Results of accuracy

S. No. % Level Nortriptyline HCl Flupentixol
Mean % recovery Std dev Mean % recovery Std dev
1 50 99.8 0.633 100.2 0.29
2 100 98.9 0.27 98.8 0.591
3 150 99.2 1.062 100.3 0.178

(n=3)

Precision

In the method precision study prepare six different samples in the concentration of Nortriptyline HCl (100 ppm) and Flupentixol (5 ppm) are injected into HPLC system.

Intraday precision

Six replicates of a sample solution containing Nortriptyline HCl (100μg/ml) and Flupentixol (5μg/ml) were analysed on the same day. Peak areas were calculated, which were used to calculate mean, SD and %RSD values. These results are given below table 4 [44, 45].

Inter-day precision

Also called Intermediate precision. In this, six replicates of a sample solution containing Nortriptyline HCl (100μg/ml) and Flupentixol (5μg/ml) were analysed on a different day. Peak areas were calculated which were used to calculate mean, SD and %RSD values [46]. The present method was found to be precise as the RSD values were less than 2% and also the percentage assay values were close to be 100%.

LOD and LOQ

The LOD concentrations for Nortriptyline HCl are 0.3 µg/ml and s/n values is 3 and Flupentixol 0.015 µg/ml and s/n value 3. The LOQ concentration for Nortriptyline HCl 1.0 µg/ml and its s/n value is 10 and Flupentixol 0.05 µg/ml and s/n value is 10. The method is validated as per the ICH guidelines [47, 48].

Robustness

The conditions of the experiment were designed to test the robustness of the established system intentionally altered, such as flow rate, mobile phase in organic percentage in all these varied conditions [49-51]. Robustness results for Nortriptyline HCl and Flupentixol were found to be within the limit and results are tabulated in table 7.

Table 4: Precision results of nortriptyline HCl and flupentixol

S. No. % Assay
Nortriptyline HCl Flupentixol
M. P. I. P. M. P. I. P.
1 100.1 100.1 99.8 99.9
2 100.2 100.0 100.0 99.8
3 100.1 100.0 100.1 100.2
4 100.0 100.1 100.2 100.1
5 100.0 100.1 100.7 100.5
6 100.2 100.1 100.1 100.7
Mean (n=6)±SD 100.1±0.084 100.1±0.051 100.1±0.316 100.2±0.336
%RSD (n=6) 0.10 0.05 0.32 0.34

Fig. 5: Chromatogram of method precision

Table 5: LOD and LOQ for nortriptyline HCl and flupentixol

Nortriptyline HCl Flupentixol
LOD LOQ LOD LOQ
Concentration s/n Concentration s/n concentration s/n Concentration s/n
0.3 µg/ml 3 1.0 µg/ml 10 0.015 µg/ml 3 0.05 µg/ml 10

Table 6: Robustness data of nortriptyline HCl and flupentixol

Parameter name Nortriptyline HCl % assay Flupentixol % assay
Mean Std dev Mean Std dev
Flow minus (0.8 ml/min 99.9 0.04 99.9 0.006
Flow plus (1.2 ml/min) 100.1 0.049 99.8 0.124
Organic minus (36:64) 99.0 0.032 99.5 0.072
Organic plus (44:56) 100.1 0.026 99.8 0.107

RSD-Relative standard deviation; (n=3)

Degradation studies

The Flupentixol and Nortriptyline HCl sample was subjected into various forced degradation conditions to effect partial degradation of the drug. Studies of forced degradation [52, 53] have carried out to find out that the method is suitable for products of degradation [54, 55]. In addition, the studies provide details about the conditions during which the drug is unstable, in order that the measures are often taken during formulation to avoid potential instabilities.

Acid degradation

Acid degradation was done at 1N HCl and degradation was formed 13.3% for Nortriptyline HCl and 13.4% for Flupentixol.

Alkali degradation

Alkali degradation was done at 1N NaOH and degradation was formed 13.7% for Nortriptyline HCl and 13.9% for Flupentixol.

Peroxide degradation

Peroxide degradation was done at 20% hydrogen peroxide and degradation was formed 14.4% Nortriptyline HCl and 15.1% for Flupentixol.

Reduction degradation

In reduction degradation, 4.6% Nortriptyline HCl and 8.7% Flupentixol degradation were observed.

Thermal degradation

In thermal degradation the standard was degraded to 5% of Nortriptyline HCl and 4.2% of Flupentixol.

Hydrolysis degradation

In hydrolysis degradation, the standard was degraded to 4.4% of Nortriptyline HCl and 2.9% of Flupentixol.

All degradation results are tabulated in table 9.

Table 9: Forced degradation results of nortriptyline HCl and flupentixol

Degradation condition Nortriptyline HCl % deg Flupentixol % deg
Mean Std dev Mean Std dev
Control degradation 0.1 0.026 0.2 0.074
Acid degradation 13.3 0.452 13.4 0.245
Alkali degradation 13.7 0.247 13.9 0.361
Peroxide degradation 14.4 0.196 15.1 0.289
Reduction degradation 4.6 0.153 8.7 0.524
Thermal degradation 5 0.187 4.2 0.521
Hydrolysis degradation 4.4 0.241 2.9 0.183

(n=3)

CONCLUSION

We present in this article simple, selective, validated and well-defined stability that shows isocratic RP-HPLC methodology for the quantitative determination of Nortriptyline HCl and Flupentixol. All the products of degradation formed during the stress conditions and active pharma ingredients are well separated and peaks were well resolved from each other and separate with an appropriate retention time, indicating that the proposed method to be fast, simple, feasible and affordable in assay condition. Therefore the developed method during stability tests, it can be used for routine analysis of production samples and to verify the quality of drug samples during stability studies.

ACKNOWLEDGEMENT

The authors are grateful to the R. V. RandJ. C. college of engineering for providing them with the resources they needed to perform this study.

AUTHORS CONTRIBUTIONS

All the authors have contributed equally

CONFLICTS OF INTERESTS

Declared none

REFERENCES

  1. Lally J, MacCabe JH. Antipsychotic medication in schizophrenia: a review. Br Med Bull. 2015;114(1):169-79. doi: 10.1093/ bmb/ldv017, PMID 25957394.

  2. Budman CL. The role of atypical antipsychotics for the treatment of Tourette’s syndrome: an overview. Drugs. 2014;74(11):1177-93. doi: 10.1007/s40265-014-0254-0, PMID 25034359.

  3. Laursen TM, Nordentoft M, Mortensen PB. Excess early mortality in schizophrenia. Annu Rev Clin Psychol. 2014;10:425-48. doi: 10.1146/annurev-clinpsy-032813-153657, PMID 24313570.

  4. Agarwal P, Sarris CE, Herschman Y, Agarwal N, Mammis A. Schizophrenia and neurosurgery: A dark past with hope of a brighter future. J Clin Neurosci. 2016;34:53-8. doi: 10.1016/j.jocn.2016.08.009. PMID 27634495.

  5. Shen X, Xia J, Adams CE. Flupenthixol versus placebo for schizophrenia. Cochrane Database Syst Rev. 2012;11:CD009777. doi: 10.1002/14651858.CD009777.pub2. PMID 23152280.

  6. Johnson DA. A double-blind comparison of flupenthixol, nortriptyline and diazepam in neurotic depression. Acta Psychiatr Scand. 1979;59(1):1-8. doi: 10.1111/j.1600-0447.1979.tb06940.x. PMID 369298.

  7. Young JP, Hughes WC, Lader MH. A controlled comparison of flupenthixol and amitriptyline in depressed outpatients. Br Med J. 1976;1(6018):1116-8. doi: 10.1136/bmj.1.6018.1116, PMID 773506.

  8. Tam W, Young JP, John G, Lader MH. A controlled comparison of flupenthixol decanoate injections and oral amitriptyline in depressed out-patients. Br J Psychiatry. 1982;140:287-91. doi: 10.1192/bjp.140.3.287, PMID 7093597.

  9. Hawton K, Witt KG, Taylor Salisbury TL, Arensman E. Pharmacological interventions for self-harm in adults (PDF). Cochrane Database Syst Rev. 2015;7:CD011777:CD011777. doi: 10.1002/14651858.

  10. Kaur J, Ghosh S, Sahani AK, Sinha JK. Mental imagery training for treatment of central neuropathic pain: a narrative review. Acta Neurol Belg. 2019;119(2):175-86. doi: 10.1007/s13760-019-01139-x, PMID 30989503.

  11. Al-Quliti KW. Update on neuropathic pain treatment for trigeminal neuralgia. The pharmacological and surgical options. Neurosciences (Riyadh). 2015;20(2):107-14. doi: 10.17712/nsj.2015.2.20140501, PMID 25864062.

  12. Foreman DM. Attention deficit hyperactivity disorder: legal and ethical aspects. Arch Dis Child. 2006;91(2):192-4. doi: 10.1136/adc.2004.064576, PMID 16428370.

  13. Faraone SV, Larsson H. Genetics of attention deficit hyperactivity disorder. Mol Psychiatry. 2019;24(4):562-75. doi: 10.1038/s41380-018-0070-0, PMID 29892054.

  14. Kalkhoran S, Benowitz NL, Rigotti NA. Prevention and treatment of tobacco use: JACC Health Promotion series. J Am Coll Cardiol. 2018;72(9):1030-45. doi: 10.1016/j.jacc.2018. 06.036. PMID 30139432.

  15. Robinson OJ, Pike AC, Cornwell B, Grillon C. The translational neural circuitry of anxiety. J Neurol Neurosurg Psychiatry. 2019;90(12):1353-60. doi: 10.1136/jnnp-2019-321400, PMID 31256001.

  16. Healy D, Le Noury J, Mangin D. Enduring sexual dysfunction after treatment with antidepressants, 5α-reductase inhibitors and isotretinoin: 300 cases. Int J Risk Saf Med. 2018;29(3-4):125-34. doi: 10.3233/JRS-180744, PMID 29733030.

  17. Rochester MP, Kane AM, Linnebur SA, Fixen DR. Evaluating the risk of QTc prolongation associated with antidepressant use in older adults: a review of the evidence. Ther Adv Drug Saf. 2018;9(6):297-308. doi: 10.1177/2042098618772979, PMID 29854391.

  18. Stein DJ. What is a mental disorder? A perspective from cognitive-affective science. Can J Psychiatry. 2013;58(12):656-62. doi: 10.1177/070674371305801202, PMID 24331284.

  19. Sharma AN, Hoffman RJ. Toxin-related seizures. Emerg Med Clin North Am. 2011;29(1):125-39. doi: 10.1016/j.emc.2010.08.011, PMID 21109109.

  20. Kowalik U, Plante MK. Urinary retention in surgical patients. Surg Clin North Am. 2016;96(3):453-67. doi: 10.1016/j.suc.2016.02.004. PMID 27261788.

  21. Mi XS, Yuan TF, So KF. The current research status of normal tension glaucoma. Clin Interv Aging. 2014;9:1563-71. doi: 10.2147/CIA.S67263. PMID 25258525.

  22. Berrios GE. Of mania: introduction. Hist Psychiatry. 2004;15(57 Pt 1):105-24. doi: 10.1177/0957154X04041829, PMID 15104084.

  23. Gillman PK. Tricyclic antidepressant pharmacology and therapeutic drug interactions updated. Br J Pharmacol. 2007;151(6):737-48. doi: 10.1038/sj.bjp.0707253. PMID 17471183.

  24. Glazener C, Evans J, Glazener PR, Cathryn MA, editors. Tricyclic and related drugs for nocturnal enuresis in children. Cochrane Database Syst Rev. 2016:pub2:CD002117:CD002117. doi: 10.1002/14651858.

  25. Srinivasan S, Sadegh L, Elle IC. Serotonin regulates C. elegans fat and feeding through independent molecular mechanisms. Cell Metab. 2008;7(6):533-44. doi: 10.1016/j.cmet.2008.04.012, PMID 18522834.

  26. Orsulak PJ. Therapeutic monitoring of antidepressant drugs: guidelines updated. Ther Drug Monit. 1989;11(5):497-507. doi: 10.1097/00007691-198909000-00002, PMID 2683251.

  27. Subrahmanyam Talari AV, prathyusha Ks, New A. Validated RP-HPLC method for cisplatin and topotecan in API and vaccine form and its stress studies. Int J Res Pharm Sci. 2021;12:808-14.

  28. Syed Rafi, Kantipudi Rambabu. Stability indicating validated HPLC method for the determination of aceclofenac and misoprostol in bulk and pharmaceutical formulation. IJRPS 2020;11(4):7848-53. doi: 10.26452/ijrps.v11i4.4669.

  29. Bhavani P, Prasada Rao K, Mohan S. Novel validated reversed-phase high-performance liquid chromatography method for determination of glucosamine, diacerein, and methyl sulfonyl methane in micro-sample rat plasma and its application to pharmacokinetic and dissolution studies. Asian J Pharm Clin Res. 2020;13:50-63. doi: 10.22159/ajpcr.2020.v13i12.36547.

  30. Shivani CP, Maheshwari DG. Development and validation of UV spectrometric and HPLC method for estimation of escitalopram oxalate and flupentixol dihydrochloride in the combined dosage form. AJPTI. 2016;4:59-70.

  31. Naresh Kumar DS, Patel D. Stability indicating chromatographic method development and validation for the simultaneous estimation of escitalopram oxalate and flupentixol in its pharmaceutical dosage form by HPLC. WJPR. 2017;6:549-66.

  32. Supriya T, Naresh D, Vijaya Kumar G, Haneer MA. Stability indicating RP-HPLC method development and validation for simultaneous estimation of escitalopram and flupentixol pure and marketed formulation. Asian J Pharm Res. 2018;8:4-10.

  33. Malathi S, Devakumar D. Development and validation of RP-HPLC method for the estimation of escitalopram oxalate and flupentixol dihydrochloride in combined dosage form and plasma. Int J Pharm Pharm Sci. 2021;13:61-6.

  34. Naykode MD, Bhagwat DA, Jadhav SD, More HN. Analytical and bioanalytical method for quantification of pure azilsartan, not its salts by RP-HPLC. Res J Pharm Technol. 2017;10(3):708-14. doi: 10.5958/0974-360X.2017.00133.0.

  35. Singh M, Charde M, Shukla R, Rita MC. Determination of calcipotriene in calcipotriene cream 0.05% w/w by RP-HPLC method development and validation. Res J Pharm Technol. 2011;4:1219-23.

  36. Eluru A, Surendra Babu K. A study of method development, validation and forced degradation for simultaneous quantification of povidone-iodine and ornidazole in bulk and pharmaceutical dosage form by using RP-HPLC. IJPSR. 2021;12:1217-22.

  37. Senthil Rajan D, Muruganathan G, Shivkumar K, Ganesh T. Development and validation of HPLC method for simultaneous quantification of vasicine, glycyrrhizin and piperine in poly herbal cough syrup. Int J Curr Pharm Res. 2020;12:15-9.

  38. Malak Y, Al-Bathish AA, gazy MK, El-Jamal. RP-HPLC and chemometric methods for the determination of two antidiabetic mixtures; metformin hydrochloride-canagliflozin and metformin hydrochloride-gliclazide in their pharmaceutical formulation. Int J Pharm Pharm Sci. 2020;12:83-94.

  39. Gadhvi MP, Bhandari A, Suhagia BN, Desai UH. Development and validation of RP-HPLC method for simultaneous estimation of atazanavir and ritonavir in their combined tablet dosage form. Res J Pharm Technol. 2013;6:200-3.

  40. Swati K, Abhishek P, Sushank S, Bothiraja C, Atmaram P. High-performance liquid chromatography for the simultaneous estimation of cefoperazone and sulbactam in rat plasma and its importance in therapeutic drug monitoring. Int J Pharm Pharm Sci. 2020;12:92-7.

  41. Gomathy S, Narenderan ST, Meyyanathan SN, Gowramma B. Development and validation of hplc method for the simultaneous estimation of apigenin and luteolin in commercial formulation. Crit Rev. 2020;7:4785-90.

  42. Vijayakumari M, Reddy Ch B. Stability indicating validated hplc method for the determination of zanubrutinib in bulk and pharmaceutical dosage form. Asian J Pharm Clin Res. 2020;13:159-62.

  43. Raziq A. Syed Umer Jan. Relative comparison of stability and degradation of methylcobalamin tablets of different brands at different storage settings. Int J Appl Pharm. 2021;13:171-5.

  44. Rajakumari R, Sreenivasa Rao S. Stress degradation studies and development of a validated RP-HPLC method for determination of tiagabine in the presence of its degradation products. Int J Pharm Pharm Sci. 2016;8:230-6.

  45. Charu Pandya P, Sadhana Rajput J. Development and validation of stability indicating method RP-HPLC method of acotiamide. Int J Pharm Pharm Sci. 2018;10:1-8.

  46. Athavia BA, Dedania ZR, Dedania RR, Swamy SMV, Prajapati CB. Stability indicating HPLC method for determination of vilazodone hydrochloride. Int J Curr Pharm Sci 2017;9(4). doi: 10.22159/ijcpr.2017v9i4.20975.

  47. Unade TT, Krishnamanjari Pawar A. A new stability indicating UPLC method for the determination of two anti-diabetic drugs in combination: applications to bulk and tablet formulation. Int J Appl Pharm. 2022;14:192-9.

  48. Manoranjani M. A study of method development, validation and forced degradation for simultaneous quantification of cisplatin and fluorouracil in bulk and pharmaceutical dosage form by RP-HPLC. J Pharm Sci and Res. 2021;13: 155-61.

  49. Prasada Rao PTSRK. A study of method development and validation of pemetrexed and cisplatin using RP-HPLC. J Pharm Sci and Res. 2021;13:143-8.

  50. Satya Dev S. TNVSS. A new selective method development and validation of cabozantinib and Nivolumab using HPLC. J Pharm Sci and Res. 2021;13:188-92.

  51. Manoranjani M. Assay method development and validation of cilnidipine and ramipril, characterization of its degradants by using LC-MS/MS. Int J Appl Pharm. 2022;14:276-85. doi: 10.22159/ijap.2022v14i2.43570.

  52. Prasada Rao PTSRK. HPLC method development and validation of lercanidipine HCl and aatenolol, characterization of its degradants by using LC-MS/MS. Int J Appl Pharm. 2022;14:125-34.

  53. Satya Dev S. TNVSS, Chintalapudi Ramakrishna. A new related substances method development and validation of two anti-cancer drugs by using an effective liquid chromatographic method. Int J Appl Pharm. 2022;14:116-24.

  54. David Raju M. Simultaneous method development and validation of choline salicylate and tannic acid using RP-HPLC in bulk and pharmaceutical dosage form. Int J Appl Pharm.. 2022;14:227-35.

  55. David Raju M. Development and validation of HPLC method for the determination of lasmiditan drug in bulk and tablet dosage form. J Pharm Sci and Res. 2021;13:170-3.