INTRODUCTION:

Residual solvents are undesirable compounds (solvents) that are produced or employed during the production of pharmaceutical formulations, excipients, or drugs and that don't seem to be eliminated by reasonable means in the final product.^1,2In nature, these solvents could be dangerous. As a result, residual solvent analysis becomes an essential tool for the routine administration of prescription medications. The ICH^3,4specifies the proper limits for certain chemicals. Pharmaceutical materials are manufactured, purified, and processed using a lot of solvents. Since these solvents have no medicinal function but may have unfavorable effects on consumers, any residues must be eliminated as much as possible. The concentration of these solvent residues shouldn't go beyond the upper bounds listed in the ICH guidelines.^5,6The boundaries that are to be used must match those nominatives. This offers methods for analyzing residual solvents, while other legitimate approaches could also show adherence to the specified bounds. The limitations outlined below do not extend to excipients, pharmaceutical compounds, or dietary constituents other than those specified in the specific monographs. However residual solvent levels given in pharmaceuticals, excipients, and related food additives are also used to show compliance as a crucial component of the management plan, which lessens or eliminates the need for examination inside the product. Unless category one solvents are used in the production of coating materials, colorants, Flavors, capsules, and acquisition inks, residual solvents are often not needed.^7,8Vinegar contains acetic acid, which has been utilized by humans for thousands of years. Vinegar was used as a medication to cure wounds and chronic coughs in ancient Greece.⁹ The ICH lists the proper limits for various chemicals. Pharmaceutical materials are manufactured, purified, and processed using a lot of solvents. Since these solvents have no medicinal function but may have unfavorable effects on consumers, any residues must be eliminated as much as possible. The concentration of these solvent residues shouldn't be more than the upper bounds listed in the ICH recommendations.¹⁰ Because vinegar has antibacterial and preservative properties, it is also being utilized to safeguard food. Acetic acid has been connected to several healthiness rewards in recent years, with antihypertensive and antihyperglycemic effects.^11-13 Certain varieties of vinegar have anticancer properties as well. Human leukemia cells undergo apoptosis when visible to naturally fermented sugar cane vinegar (kibizu) from Amami Ohshima Island.¹⁴Moreover, the vinegar derived from the rice-shochu post-distillation slurry inhibits the formation of tumours and increases the longevity of mice with solid tumours.¹⁵ A little over five percent of vinegar is made up of acetic acid, which has been demonstrated to have anticancer properties. We found in a prior work that 0.5 vol% acetic acid caused cell death, especially in cancer cells.¹⁶ On the other hand, after topical therapy, 60% Volume of acetic acid results in cellular necrosis and ulcers.¹⁷ These findings suggested that the chemical's concentration affects the cytotoxic properties of acetic acid. Still unclear is the exact mechanism by which acetic acid causes cell death.

The monocarboxylic transporter (MCT), a membrane transporter that carries acetic acid and other monocarboxylic acids into cells, is responsible for incorporating acetic acid into the tricarboxylic acid cycle (TCA) as a substrate of acetyl-CoA.¹⁸ The acetic acid may cause oxidative stress in cancer cells, which would lead to apoptosis. The TCA cycle makes reactive oxygen species (ROS), with superoxide radicals, which cause cancer cells to undergo apoptosis.^19-20 Since acetic acid is an open-chain hydrocarbon and cannot be seen with a UV detector, it cannot be examined using UPLC or HPLC, making it a very difficult task to estimate the amount of acetic acid in API. Scientists attempted to analyze the residual solvent using the GC-HS Technique but discovered that the acetic acid's response was very low and could not be identified or detected. As a result, they felt the need for derivatization and began attempting it using a variety of reagents, including N, Obis, and trimethylsilyl trifluoroacetamide (BSTFA), which is frequently used in this reaction ²¹. For a particular silylating reagent, the sequence of ease of derivatization of different functional groups is as follows: alcohol > phenol > carboxylic acid > amine > amide. Steric hindrance will similarly affect reactivity towards a certain silylating reagent inside this sequence.²² Alcohols' ease of reactivity is therefore arranged as follows: primary > secondary > tertiary for alcohols, and primary > secondary for amines. The amount of acetic acid in the antidiabetic medication Empagliflozin was ascertained by derivatizing the medication with BSTFA (N,Obis(trimethylsilyl trifluoroacetamide).²³

Chemicals and Reagents:

Samples of Empagliflozin were obtained from a Pharmaceutical Company, Analytical grade Dimethylformamide was obtained from J.T Baker, Acetic acid standard was procured from Honeywell Fluka Company, BSTFA (N,Obis (trimethylsilyl tri fluoroacetamide) procured from Honey well Fluka.

METHOD:

Instruments:

Gas Chromatography - Turbomatrix Headspace Clarus-690 Perkin Elmer.

Column: DB-624, 30m x 0.53mm x 3.0um or equivalent to the oven Temperature:

Rate in ⁰C	Temperature in ⁰C	RT in (min)
0.0	50	2
10	220	2

Injector: 200⁰C, Detector- 240⁰C, Carrier gas- Nitrogen, Nitrogen Flow- 2.0ml/minute, Attenuation-4, Range-1, Split ratio-15:1, Hydrogen Flow-40ml/minute, Air Flow- 400 ml/minute, Run time- 21minute.

Head Space: Vial of the Oven Temperature-90°C, Needle Temperature- 100⁰C, Transfer line temperature-110°C/minute, Vial Thermostating time- 20minutes, Pressurization time: 3min, Withdrawal time- 0.2min, GC-Cycle time- 30min, PPC-20 psi, Injection time- 0.1 minutes, Injection Volume - 0.2ml. Observed Relative retention time concerning Dimethyl Formamide (DMF) are given below:

Component	Retention time (min)	RRT
Acetic acid	About 9.040	0.76
N,N Dimethyl Formamide	About 11.676	1.00

Preparation of Solutions:

Diluent:

Prepare the Standard Stock Solution I: Weigh precisely 500 mg of standard acetic acid (5000 ppm) in a 100 ml standard volumetric flask mix, then dilute it to the mark with diluent.

Preparation of Standard Stock Solution:

Acetic acid concentration (250ppm) is the first step in the preparation of the standard stock solution. Next, pipette 5ml of Standard Stock Solution I into a standard 100ml volumetric flask mix and dilute with diluent to up to the mark.

Preparation of Blank Vials:

Fill three clean, dry HS vials with 2.0ml of diluent and 0.4ml of BSTFA. Tightly seal the vials.

Preparation of Test Solution Vials:

Weigh accurately about 100mg of test sample in a clean and dry HS-Vial. Add 2ml of diluent and 0.4ml of BSTFA in it. Seal the vial properly.

RESULTS:

Method Development:

To establish the technique, a solution of empagliflozin in diluent was created using acetic acid and dimethylformamide at 500ppm and 50000ppm, respectively. Many stationary phases are utilized to create a robust and appropriate GC-HS method for the separation process. The preliminary column screening used several column types with various stationary phases, such as the DB-5 (30m x 0.53mm x 5um). These columns gave selectivity between the peaks of acetic acid and dimethylformamide using a mobile phase as nitrogen and a FID detector, however, the peaks are wide. We kept looking for the ideal oven program that would provide the highest resolution and acetic acid and dimethylformamide selectivity. On Column DB-624 (30 m x 0.53mm x 3um), good separation was obtained using the oven program and other GC-Parameters listed in the method.

Note: Since only solvents are identified and quantified in the GC-HS Method, we won't see the API peak (Empagliflozin).

Chromatographic Conditions:

More technique optimization and acetic acid quantification were done on the GC-HS DB-624,30m x 0.53mm x 3um column as a result of the improved chromatographic findings achieved on this column. Based on information gathered from technique development and optimization efforts, the GC Column DB-624, 30m x 0.53mm x 3um, with nitrogen as the mobile phase and 200°C injector and 240°C detector temperatures, was designed. 0.2 minutes of injection time, 0.2 milliliters of volume, 50°C initial column temperature, and ambient sample temperature are used to calculate the flow rate. Dimethylformamide and acetic acid were separated under these circumstances, with the peak of the former eluting after the acid.

Acetic acid had a relative retention time of 0.76 and dimethylformamide had a mean retention time of 9.0 minutes and 11.6 minutes in the optimised technique. The following figure depicts the structures of acetic acid, dimethylformamide, and empagliflozin.

Figure 1: Structure of Empagliflozin

DISCUSSION:

Method Validation:

It offers direction on the choice and assessment of several analytical technique validation tests. A glossary of words is provided in this guideline to help bridge the gaps that are frequently found between different compendia and papers from the regulatory bodies of ICH member states.

Specificity:

The capacity to evaluate the analyte without a doubt in the presence of potentially present components is known as specificity. These might typically contain matrices, degradants, and contaminants. ²⁴

Table 1: Preparation of Stock Solution (Standard)

Standard	Wt. (mg)	Dil. to ml	Volume taken in ml	Dil. to ml	Conc. (ppm)
Acetic acid	253.15	100	---	----	5053.38
Standard Solution in ml
Acetic acid	----	---	2.5	50	252.67

Table 2: Relative Retention Time

Component	Retention time (min)	RRT
Acetic acid	About 9.040	0.76
N,N-dimethyl Formamide	About 11.676	1.00

Precision:

The accuracy of an analytical method expresses the degree of agreement between a set of measurements performed from many samplings of the same homogeneous sample under specific conditions. Acetic acid's system and method precision were assessed at the 250 ppm specification level in relation to a 50000 ppm analyte concentration. The percentage RSD of the method and system precision for acetic acid was found to be 1.80% and 0.69%, respectively, demonstrating an acceptable level of method precision. ²⁵

Table 3: System Precision

Preparation of Standard Stock Solution
Standard	Wt. (mg)	Diluted in ml	Volume taken in ml	Dil. to ml		Conc. (ppm)
Acetic acid	255.82	50	---	----		5106.68
Standard Solution in ml
Acetic acid	----	---	2.5	50	255.33

Table 4: Six Replicate of System Precision Standard Solution

Acetic acid
Injections	Area
1	230648
2	237544
3	241328
4	239907
5	241506
6	241839
Mean	238795
SD	4294.34
RSD	1.80

Table 5: Method Precision

Preparation of Standard Stock Solution--I
Standard	Wt. (mg)	Dil. to ml	Volume taken in ml	Dil. to ml	Conc. (ppm)
Acetic acid	255.82	50	---	----	5106.68
Standard Solution- I ml
Acetic acid	----	---	2.5	50	255.33

Table 6: Method Precision (Acetic acid)

Solution	Test Splike wt. in mg	Amount Added in ppm	Area in test Spike	Amount found in ppm	Amount Recovered in ppm	Avg. of Recovered ppm	SD	% RSD
100% Spike	100.35	5088.87	239664	5107.39	5107.39	5105.72	35.33	0.69
	100.56	5078.24	238825	5078.88	5078.88
	100.29	5091.91	240361	5125.30	5125.30
	100.53	5079.76	240909	5124.73	5124.73
	100.45	5083.80	243463	5183.18	5183.18
	100.34	5089.37	241474	5146.47	5146.47

Table 7: Intermediate Precision (Acetic acid)

Solution	100% Spike
Test splike wt. in mg	100.98	100.38	100.51	100.06	100.56	101.06
Amount added in ppm	5038.14	5070.78	5061.70	5054.46	5059.18	5034.15
Area in test Spike	480	493	500	534	518	489
Amount found in ppm	4708.88	4863.61	4923.83	5282.30	5098.55	4789.30
Amount Recovered in ppm	4704.88	4863.61	4923.83	5282.30	5098.55	4789.30
Avg of Recovered ppm	4943.75
SD	194.21
% RSD	3.93

GC-MS Method:

Column: Agilent Technologies DB-FFAP 30mx 0.530mm, 1.0micron Instrument: PerkinElmer, GC 2014, Detector: FID, Carrier gas: Nitrogen Temp programming: Initial 50°C hold for one minute, 5^oC Ramp/minutes, 100°C hold for one minute, 25^oC Ramp up to 250°C hold for two minutes, run time 20 minutes, the purity by GC-FID of the sample was 99.95%.

Peak #	Time[min]	*Area[uV sec]**	Height [UV]	Area %
1	1.639	162.63	71.81	0.01
2	3.401	1379830.27	138642.12	99.95
3	9.787	534.13	136.07	0.04
		1380527	138850.00	100.00

Figure 2: Spectra of GC-FID

GC-MS Spectrum:

Column: Agilent Technologies, Elite -5MS, 30 m X 0.25 mm, 1.0-micron Instrument: Perkin Elmer, Carrier gas: Helium Source Temp.: 230°C, Transfer line: 250°C Inlet Temp.: 180°C, Diluent: Methanol Source energy: 70eV. The Identification by GC-MS: Conform to molecular of acetic acid.

Figure 3: Spectra of GC-MS

Figure 4: IR Spectra of Acetic acid

Figure 5: NMR Spectra of Acetic acid

Test	Specification	Result
Description	Colourless Liquid	Colourless Liquid
Clarity	Clear	Clear
Assay (GC-FID)	≥ 99.50%	99.95%
Water (by KF)	≤ 0.5%	0.4078%
¹H-NMR	Confirm Structure	Conforms
GC-MS	Confirm Molecule	Conforms
IR	Confirm Structure	Conforms

Limit of Detection and Limit of Quantitation:

The detection limit of a certain analytical technique is the lowest concentration of analyte in a sample that can be recognized but may not always be quantified as an exact quantity. The quantitation limit of a certain analytical procedure is the lowest concentration of analyte in a sample that can be quantitatively determined with suitable precision and accuracy.²⁶

Table 8: Results of Precision at LOD and LOQ

Acetic acid
Precision at LOD	Precision at LOQ
21217	71861
21902	71834
22002	73033
21391	72540
21514	73967
21419	74604
21574	72973	Mean
309.62	1129	SD
1.44	1.55	% RSD

Linearity:

An analytical procedure's linearity is its capacity to produce test findings that, within a certain range, are directly proportional to the analyte concentration in the sample.²⁷

Table 9: Linearity Level Solutions

Linearity Levels	Volume of Standard Stock Solution-I in ml	Diluted to ml
I- (LOQ%)	0.75	50
II-(50%)	1.25	50
III- (80%)	2.00	50
IV- (100%)	2.5	50
V- (120%)	3.0	50
VI- (150%)	3.75	50

Table 10: Results of Linearity:

Linearity Levels	Conc in ppm	Area-1	Area-2	Area-3	Avg Area
I- (LOQ)	76.31	71602	72388	72865	72285
II-(50%)	127.19	115876	116810	114987	115891
III-(80%)	203.50	188095	191645	192431	190724
IV-(100%)	254.38	248736	247977	242523	246412
V-(120%)	305.25	298147	282045	289620	289937
VI-(150%)	381.56	352814	362448	347953	354405

Table 11: Result Summary of Standard Solution (Accuracy)

Levels	Test wt Spike mg	Amt added in ppm	Area in Test Spike	Amt Found in ppm	Amt Recovered in ppm	% Recovery	Mean% Recovery	SD	RSD
I- LOQ	100.67	1521.81	70027	1487.57	1487.57	96.31	94.95	1.19	1.25
	100.59	1523.02	68684	1460.21	1460.21	94.44
	100.60	1522.87	68441	1454.90	1454.90	94.10
II- 50%	100.52	2540.13	115892	2465.55	2465.55	96.20	96.78	1.22	1.26
	100.60	2538.11	118261	2513.95	2513.95	98.18
	100.49	2540.89	115617	2460.44	2460.44	95.97
III- 100%	100.35	5088.87	239664	5107.39	5107.39	99.93	99.91	0.32	0.32
	100.56	5078.24	238825	5078.88	5078.88	99.58
	100.29	5091.91	240361	5125.30	5125.30	100.22
IV- 150%	100.56	7617.36	359684	7649.08	7649.08	100.13	100.08	0.27	0.27
	100.35	7633.30	358483	7639.49	7639.49	99.79
	100.51	7621.15	360379	7667.67	7667.67	100.32

Accuracy:

The degree of agreement between the value obtained and the value recognized as either a conventional true value or an approved reference value is what determines an analytical procedure's accuracy.²⁸

Figure 6: Linearity of Acetic Acid

CONCLUSION:

It confirms that during the process of the drug, the unknown impurity was identified and it was confirmed by IR, NMR, and GC-MS techniques. The assay of GC-FID specification was ≥99.50 but we got the 99.95%. Residual solvents are undesirable compounds (solvents) produced or employed during the production of pharmaceutical formulations, excipients, or drugs, that don't seem to be eliminated by reasonable means in the final product. Method validation was carried out using GC-Column DB-624 (30m x 0.53mm x 3um) due to better separation in GC-HS mode. The validated method was demonstrated to be specific, accurate, precise, and sensitive. The developed and validated method can be implemented for the determination and quantitation of Acetic acid in Empagliflozin bulk drug. An unknown impurity was identified during bulk drug analysis. It was isolated and subsequently characterized using various analytical techniques, such as IR, NMR, GC, and GC-MS. Acetic acid, a genotoxic residual solvent, was identified as an unknown impurity and needs to be quantified and validated for routine analytical work.

CONFLICT OF INTEREST:

No conflicts of interest regarding this investigation.

ETHICS APPROVAL:

Research Related to the Ph.D. degree in Chemistry and The topic approval was granted by the concerned university, the University of Mumbai, Mumbai.

FUNDING:

No funding was received to conduct this research study.

AUTHORS CONTRIBUTIONS:

All authors contributed equally to the study.

ACKNOWLEDGEMENT:

The authors wish to express their sincere gratitude to Supriya Life Sciences, Mumbai, for providing the samples for this research work.

ABBREVIATIONS:

LOD: Limit of Detection, LOQ: Limit of Quantification, IR: Infrared, GC-MS: Gas Chromatography-Mass spectroscopy, NMR: Nuclear magnetic resonance, GC-HS: Gas Chromatography-Headspace, ICH: International Council for Harmonisation, Avg.: Average, MCT: monocarboxylic transporter, TCA: Tricarboxylic acid cycle, Conc.: Concentration, ROS: Reactive oxygen species, UV: Ultra Violet, Dil: Dilute, UPLC: Ultra-performance liquid chromatography, HPLC: High-Performance Liquid Chromatography, BSTFA: N,Obis (trimethylsilyl tri fluoroacetamide), GC-FID: Gas-Chromatography Flame ionization detector, SD: Standard Deviation. RSD: Relative Standard Deviation, API: Active Pharmaceutical Ingredient, wt.: Weight.

SUMMARY:

During the process of the Empagliflozin drug, the unknown impurity was identified, this impurity was confirmed by GC-FID, GC-MS, IR, and NMR. The unknown impurity was identified this impurity was acetic acid.

REFERENCES:

1. Patel R. James B. ElShaer A. Pharmaceutical Excipients and Drug Metabolism: A Mini-Review. Inter J Mol Sci. 2020; 21(21): 8224. Doi:10.3390/ijms21218224.

2. Dave J. Tirgar P. Patel B. A Pharmacological Analysis of Sodium-Glucose Cotransporter-2 Inhibitors for the Treatment of Diabetes and the Complications Associated. Res J Pharmacy Tech. 2024; 17(8): 3625-3622. doi: 10.52711/0974-360X.2024.00566

3. Gupta A. Goel R. Jain S. Saini V. A Review on Impact of ICH and its Harmonization on Human Health Care and Pharmaceuticals. J Pharm Res Clin Prac. 2014; 4(2):1-10.

4. ICH the need to harmonize [Online]. [cited 2014 May 02]; Available from: URL:http://www.ich.org/about/history.html..

5. ICH Harmonized Guideline, Analytical Procedure Development Q14 Draft version Endorsed on 24 March 2022.

6. ICH Harmonized Guideline, https://www.ich.org/page/quality%E2% 80% 93 guidelines.

7. Shrivastava AK. Verma S. Awasthi H. HPTLC Phytochemical Profiling and Simultaneous Quantification of Quercetin and Gallic in Prosopis juliflora. Res J Pharmacy Tech. 2024; 17(8): 3801-6. doi: 10.52711/0974-360X.2024.00590

8. Sojitra C. Tehare A. Dholakia C. Sudhakar P. Agarwal S. Singh KK. Development and validation of residual solvent determination by headspace gas chromatography in Imatinib Mesylate API. Appl Sci. 2019; 1: 233. doi: 10.1007/s42452-019-0233-x

9. Pikoulis EA. John CBP. Tsigris C. Pikoulis N. Leppäniemi AK. Emmanouil P. Trauma Management in Ancient Greece: Value of Surgical Principles through the Years, world J Surgery. 2004; 28(4): 425-430. doi:10.1007/s00268-003-6931-x.

10. Zhunussova M. Tursynova S. Abdullabekova R. Murzalieva G. Karieva Y. Akhmetova S. Development of the composition and research of soft dosage forms with carbon dioxide extract from Scabiosa ochroleuca L. Res J Pharmacy Tech. 2022; 15(8): 3434-2. doi: 10.52711/0974-360X.2022.00575.

11. Carbonari D. Chiarella P. Mansi A. Pigini D. Iavicoli S. Tranfo G. Biomarkers of susceptibility following benzene exposure: Influence of genetic polymorphisms on benzene metabolism and health effects. Bio mark Med. 2016; 10: 145–163. doi: 10.2217/bmm.15.106.

12. Sharma S. Arora V. Simultaneous Spectrophotometric Estimation of Curcumin and Quercetin in mixture. Res J Pharmacy Tech. 2022; 15(8): 3502-6. doi: 10.52711/0974-360X.2022.00587.

13. ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). 2016. Impurities: Guidelines for Residual Solvents Q3C(R6). Geneva, Switzerland: ICH.

14. Inoue C. Tomomi K. Yukiko M. Bungo S. Katsuya F. Kuniyoshi S. Masao S. Katakura Y. Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells. Cytotechnology.2015; 67: 721–725. doi: 10.1007/s10616-015-9849-x.

15. Hanan RHM. Tulbah FSA. El‑Ghor AA. Eissa SM. Suppression of tumor growth and apoptosis induction by pomegranate seed nano‑emulsion in mice bearing solid Ehrlich carcinoma cells. Sci Reports. 2023; 13: 5525. doi:10.1038/s41598-023-32488-6.

16. Abid A. Dekmouche M. Bechki L.Bireche K. Belkhalfa H. Messaoudi A. Belfar ML. Bioactive Composition Analysis using HPLC-UV Profile and Evaluation of Antioxidant activities of different extracts from Aerial parts of Atractylis aristata batt. Res J Pharmacy Tech. 2022; 15(8): 3370-6. doi: 10.52711/0974-360X.2022.00564

17. Hui Y. Lu Y. Xiao-Feng Z. Ling L. Rong-Ping Z. Zhong-Kun R. Xu L. Antichronic Gastric Ulcer Effect of Zinc-Baicalin Complex on the Acetic Acid-Induced Chronic Gastric Ulcer Rat Model, Gastroenterology Res Prac. 2018; 1275486: 1-9. doi:10.1155/2018/1275486.

18. Arnold PK. Lydia WSF. Regulation and function of the mammalian tricarboxylic acid cycle. J Biol Chem. 2023; 299(2): 102838, doi: 10.1016/j.jbc.2022.102838.

19. Amal M. Abou AA. MereyHA. El Kalla RA. El Gendy AE. Validated Spectrophotometric Methods for Simultaneous Determination of Sulphadoxine and trimethoprim in a Veterinary Pharmaceutical Dosage Form. Res J Pharmcy Tech. 2020; 13(11): 5151-5157. doi: 10.5958/0974-360X.2020.00901.4

20. Panieri E. Santoro MM. ROS homeostasis and metabolism: a dangerous liaison in cancer cells. Cell Death Disea. 2016; 7: e2253. doi:10.1038/cddis.2016.105.

21. Zhu K. Binghe G. Michael K. Markus M. Luong J. Matthias P. Elimination of N, O-bis (trimethylsilyl)trifluoroacetamide interference by base treatment in derivatization gas chromatography-mass spectrometry determination of parts per billion of alcohols in food additive. J Chromatography A. 2017; 1490: 74-79.doi:10.1016/j.chroma.2017. 02.025.

22. Soudi AT. Hussein OG. Elzanfaly ES. Zaazaa HE. Mohamed A. Stability Indicating TLC–Densitometric Method for Determination of Alcaftadine in Presence of its Degradation Products and Dosage form Preservatives. Res J Pharmcy Tech. 2020; 13(11): 5171-5176. doi: 10.5958/0974-360X.2020.00904.X

23. Begou O. Weber K. Beckmann B. Tsikas D. GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine. Molecules. 2021: 26(11): 3206. doi:10.3390/molecules26113206.

24. Tanuja A. Ganapathy S. Bio-analytical Method Development and Validation for Simultaneous Determination of Bictegravir, Emtricitabine, and Tenofovir Alafenamide Fumarate in Human Plasma by LC-MS/MS. Ind J Pharm Edu Res. 2022; 56(4): 1190-1204. doi: 10.5530/ijper.56.4.201.

25. Lakhmapure SB. Kothari S. Lokhande MV. Validation of gas chromatography (GC) method for residual solvent in brompheniramine maleate (API), Inter J Pharm Sci. Res. 2020; 11(10): 5039-5052. doi: 10.13040/IJPSR.0975-8232.11(10).5039-52.

26. Maurya CP. Lokhande MV. Characterization and validation of impurities related to pharmaceutical bulk drug (API) by using some analytical techniques. Inter J Pharm Sci Res. 2017; 8(8): 3325-3340. doi: 10.13040/IJPSR.0975-8232.8(8).3325-40.

27. Savitha K. Ravichandran S. Method Development and Validation for Simulataneous Estimation of Biotin and Folic Acid in Bulk and Tablet dosage form by RP-HPLC. Res J Pharmcy Tech. 2020; 13(11): 5289-5292. doi: 10.5958/0974-360X.2020.00925.7.

28. Nazira S. Yaser B., Mohamad MS. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Aspirin and Rivaroxaban in Synthetic Mixture. Res J Pharmacy Tech. 2020; 13(11): 5459-5465. doi: 10.5958/0974-360X.2020.00953.1

Received on 27.06.2024 Revised on 19.11.2024

Accepted on 08.02.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2670-2676.

DOI: 10.52711/0974-360X.2025.00384

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.