Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products

Gürkan Özen; Emirhan Nemutlu

Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products

Abstract

It is difficult to analyze different concentrations of pharmaceutical active substances in dosage forms simultaneously, especially in formulations containing high amounts of excipients, on environmentally friendly principles without the need for any intervention. This study proposes a powerful analytical method for the simultaneous determination of L-ascorbic Acid and N- Acetyl Cysteine in an effervescent tablet using high performance liquid chromatography technique. In the study, it was aimed to reduce the use of toxic solvents/chemicals and waste emissions, to increase efficiency and to reduce the negative environmental consequences that may arise from them. The method was developed using a C18 (ACE-121-2546, 4.6 x 250 mm, 5 µm) column and a sodium dihydrogen phosphate buffer mobile phase. Detection wavelengths were taken as 210 and 240 nm while the flow rate was 1 mL/min. The linearity, accuracy and precision, selectivity, sensitivity, and robustness of the proposed method were validated using International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2R criteria and its green assessment was validated by AGREE and AGREEprep applications. The linear range was taken as 0.1-100 μg/mL for both compounds analyzed in the developed method. The detection limit of the method was 0.02 and 0.04 µg/mL respectively for L-ascorbic Acid and N- Acetyl Cysteine. The recovery of the method was between 98.75%- 102.20% and the accuracy and precision of the method for all compounds were 0.17% and 0.11% respectively. This new environmentally friendly method can be easily used by the chemical and pharmaceutical industries for regular analysis without any restrictions.

Keywords

References

[1] Trizna Z, Schantz SP, Hsu T. Effects of N-acetyl-L-cysteine and ascorbic acid on mutagen-induced chromosomal sensitivity in patients with head and neck cancers. Am J Surg. 1991; 162(4): 294-298. https://doi.org/10.1016/0002-9610(91)90134-Y.
[2] Yaqub H, Abdel Baky N, Attia HA, Faddah L. Hepatoprotective effect of N-acetyl cysteine and/or β-carotene on monosodium glutamate-induced toxicity in rats. Res J Med Med Sci. 2008; 3(2): 206-215.
[3] Eşrefoğlu M, Gül M, Ateş B, Batçıoğlu K, Selimoğlu MA. Antioxidative effect of melatonin, ascorbic acid and N-acetylcysteine on caerulein-induced pancreatitis and associated liver injury in rats. World J Gastroenterol. 2006; 12(2): 259-254. https://doi.org/10.3748%2Fwjg.v12.i2.259.
[4] Golonka I, Oleksy M, Junka A, Matera-Witkiewicz A, Bartoszewicz M, Musiał W. Selected physicochemical and biological properties of ethyl ascorbic acid compared to ascorbic acid. Biol Pharm Bull. 2017; 40(8):1199-1206. https://doi.org/10.1248/bpb.b16-00967.
[5] Heydari R, Elyasi NS. Ion-pair cloud-point extraction: A new method for the determination of water-soluble vitamins in plasma and urine. J Sep Sci. 2014; 37(19): 2724-2731. https://doi.org/10.1002/jssc.201400642.
[6] Müller LD. Improved extraction methods for avoiding the interference of copper in the LC determination of ascorbic acid in multivitamin-mineral tablets. J Pharm Biomed Anal. 2001; 25(5-6): 985-994. https://doi.org/10.1016/S0731-7085(01)00372-7.
[7] Sudanagunta S, Camarena-Michel A, Pennington S, Leonard J, Hoyte C, Wang GS. Comparison of two-bag versus three-bag N-acetylcysteine regimens for pediatric acetaminophen toxicity. Ann Pharmacother. 2023; 57(1): 36-43. https://doi.org/10.1177/10600280221097700.
[8] Pharoah BM, Zhang K, Khodade VS, Keceli G, McGinity C, Paolocci N, Toscano JP. Hydropersulfides (RSSH) attenuate doxorubicin-induced cardiotoxicity while boosting its anticancer action. Red Bio. 2023: 102625. https://doi.org/10.1016/j.redox.2023.102625.

[9] Stalder G, Chatte A, Alberio L, Eeckhout E. Caplacizumab for treating subacute intra-stent thrombus occurring despite efficacious double anti-platelet treatment and anticoagulation: a case report. Eur Heart J Case Rep. 2023. https://doi.org/10.1093/ehjcr/ytac497.
[10] Ebrahimi F, Zavareh S, Nasiri M. The combination of estradiol and N-acetylcysteine reduces ischemia–reperfusion injuries of mice autografted ovarian tissue. Biopreserv Biobank. 2023; 22(1): 29-37. https://doi.org/10.1089/bio.2022.0184.
[11] Zhang Y, Ding S, Li C, Wang Y, Chen Z, Wang Z. Effects of N-acetylcysteine treatment in acute respiratory distress syndrome: A meta-analysis. Exp Ther Med. 2017; 14(4): 2863-2868. https://doi.org/10.3892/etm.2017.4891.
[12] Wong KK, Lee SWH, Kua KP. N-Acetylcysteine as adjuvant therapy for COVID-19 – A perspective on the current state of the evidence. J Inflamm Res. 2021; 14: 2993-3013. https://doi.org/10.2147/jir.s306849.
[13] Foroughi MM, Beitollahi H, Tajik S, Akbari A, Hosseinzadeh R. Electrochemical determination of N-acetylcysteine and folic acid in pharmaceutical and biological samples using a modified carbon nanotube paste electrode. Int J Electrochem. 2014; 9: 8407-8421. https://doi.org/10.1016/S1452-3981(23)11056-X.
[14] Beitollahi H, Raoof J-B, Hosseinzadeh R. Fabrication of a nanostructure-based electrochemical sensor for simultaneous determination of N-acetylcysteine and acetaminophen. Talanta. 2011; 85(4): 2128-2134. https://doi.org/10.1016/j.talanta.2011.07.054.
[15] Suarez WT, Marcolino Jr LH, Fatibello-Filho O. Voltammetric determination of N-acetylcysteine using a carbon paste electrode modified with copper (II) hexacyanoferrate (III). Microchem J. 2006; 82(2):163-167. https://doi.org/10.1016/j.microc.2006.01.007.
[16] Beitollahi H, Sheikhshoaie I. Electrochemical behavior of carbon nanotube/Mn (III) salen doped carbon paste electrode and its application for sensitive determination of N-acetylcysteine in the presence of folic acid. Int J Electrochem Sci. 2012; 7(8): 7684-7697. https://doi.org/10.1016/S1452-3981(23)15815-9.
[17] Karimi-Maleh H, Hatami M, Moradi R, Khalilzadeh MA, Amiri S, Sadeghifar H. Synergic effect of Pt-Co nanoparticles and a dopamine derivative in a nanostructured electrochemical sensor for simultaneous determination of N-acetylcysteine, paracetamol and folic acid. Microchim Acta. 2016; 183: 2957-2964. https://doi.org/10.1007/s00604-016-1946-9.
[18] Shahrokhian S, Kamalzadeh Z, Bezaatpour A, Boghaei DM. Differential pulse voltammetric determination of N-acetylcysteine by the electrocatalytic oxidation at the surface of carbon nanotube-paste electrode modified with cobalt salophen complexes. Sens Actuators B Chem. 2008; 133(2): 599-606. https://doi.org/10.1016/j.snb.2008.03.034.
[19] Ogwu V, Cohen G. A simple colorimetric method for the simultaneous determination of N-acetylcysteine and cysteine. Free Radic Biol Med. 1998; 25(3): 362-364. https://doi.org/10.1016/S0891-5849(98)00024-0.
[20] Janegitz BC, Suarez WT, Fatibello-Filho O, Marcolino-Junior LH. Conductometric determination of N-acetylcysteine in pharmaceutical formulations using copper (II) sulphate as titrant. Anal Lett. 2008; 41(18): 3264-3271. https://doi.org/10.1080/00032710802507554.
[21] Rudašová M, Masár M. Precise determination of N‐acetylcysteine in pharmaceuticals by microchip electrophoresis. J Sep Sci. 2016; 39(2): 433-439. https://doi.org/10.1002/jssc.201501025.
[22] Raggi M, Cavrini V, Di Pietra A. Colorimetric determination of acetylcysteine, penicillamine, and mercaptopropionylglycine in pharmaceutical dosage forms. J Pharm Sci. 1982; 71(12): 1384-1386. https://doi.org/10.1002/jps.2600711218.
[23] Garcia-Molina F, Penalver M, Rodriguez-Lopez J, Garcia-Canovas F, Tudela J. Enzymatic method with polyphenol oxidase for the determination of cysteine and N-acetylcysteine. J Agric Food Chem. 2005; 53(16): 6183-6189. https://doi.org/10.1021/jf050197k.
[24] Alvarez-Coque MG, Hernandez MM, Camanas RV, Fernandez CM. Spectrophotometric determination of N-acetylcysteine in drug formulations with o-phthalaldehyde and isoleucine. Analyst. 1989;114(8):975-977. https://doi.org/10.1039/AN9891400975.
[25] Abu Eid M. Spectrophotometric determination of cysteine and N-acetylcysteine in pharmaceutical preparations. Microchim Acta. 1998; 129: 91-95. https://doi.org/10.1007/BF01246854.
[26] Ensafi AA, Taei M, Khayamian T, Arabzadeh A. Highly selective determination of ascorbic acid, dopamine, and uric acid by differential pulse voltammetry using poly (sulfonazo III) modified glassy carbon electrode. Sens Actuators B Chem. 2010; 147(1): 213-221. https://doi.org/10.1016/j.snb.2010.02.048.
[27] Sheng Z-H, Zheng X-Q, Xu J-Y, Bao W-J, Wang F-B, Xia X-H. Electrochemical sensor based on nitrogen doped graphene: Simultaneous determination of ascorbic acid, dopamine and uric acid. Biosens Bioelectron. 2012; 34(1): 125-131. https://doi.org/10.1016/j.bios.2012.01.030.
[28] Roe JH, Kuether CA. The determination of ascorbic acid in whole blood and urine through the 2, 4-dinitrophenylhydrazine derivavative of dehydroascorbic acid. J Biol Chem. 1943; 147: 399-407 https://doi.org/1943;147:399-407.
[29] Zannoni V, Lynch M, Goldstein S, Sato P. A rapid micromethod for the determination of ascorbic acid in plasma and tissues. Biochem Med. 1974; 11(1): 41-48. https://doi.org/10.1016/0006-2944(74)90093-3.
[30] Bajaj K, Kaur G. Spectrophotometric determination of L-ascorbic acid in vegetables and fruits. Analyst. 1981; 106(1258): 117-120. https://doi.org/10.1039/AN9810600117.
[31] Lykkesfeldt J, Loft S, Poulsen HE. Determination of ascorbic acid and dehydroascorbic acid in plasma by high-performance liquid chromatography with coulometric detection-are they reliable biomarkers of oxidative stress? Anal biochem. 1995; 229(2): 329-335. https://doi.org/10.1006/abio.1995.1421.
[32] Moghadam MR, Dadfarnia S, Shabani AMH, Shahbazikhah P. Chemometric-assisted kinetic–spectrophotometric method for simultaneous determination of ascorbic acid, uric acid, and dopamine. Anal Biochem. 2011; 410(2): 289-295. https://doi.org/10.1016/j.ab.2010.11.007.
[33] Güçlü K, Sözgen K, Tütem E, Özyürek M, Apak R. Spectrophotometric determination of ascorbic acid using copper (II)–neocuproine reagent in beverages and pharmaceuticals. Talanta. 2005; 65(5): 1226-1232. https://doi.org/10.1016/j.talanta.2004.08.048.
[34] Nojavan S, Khalilian F, Kiaie FM, Rahimi A, Arabanian A, Chalavi S. Extraction and quantitative determination of ascorbic acid during different maturity stages of Rosa canina L. fruit. J Food Compos Anal. 2008; 21(4):300-305. https://doi.org/10.1016/j.jfca.2007.11.007.
[35] Shekhovtsova TN, Muginova SV, Luchinina JA, Galimova AZ. Enzymatic methods in food analysis: Determination of ascorbic acid. Anal Chim Acta. 2006; 573: 125-132. https://doi.org/10.1016/j.aca.2006.05.015.
[36] Qi S, Zhao B, Tang H, Jiang X. Determination of ascorbic acid, dopamine, and uric acid by a novel electrochemical sensor based on pristine graphene. Electrochim Acta. 2015; 161: 395-402. https://doi.org/10.1016/j.electacta.2015.02.116.
[37] Zaporozhets O, Krushinskaya E. Determination of ascorbic acid by molecular spectroscopic techniques. J Anal Chem. 2002; 57: 286-297. https://doi.org/10.1023/A:1014938011955.
[38] Karatepe M. Simultaneous determination of ascorbic acid and free malondialdehyde in human serum by HPLC-UV. LC-GC N Am. 2004; 22(6): 362-365.
[39] Ross MA. Determination of ascorbic acid and uric acid in plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl. 1994; 657(1): 197-200. https://doi.org/10.1016/0378-4347(94)80087-1.
[40] Kall MA, Andersen C. Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid, isoascorbic acid and dehydroisoascorbic acid in food and biological samples. J Chromatogr B Biomed Sci Appl. 1999; 730(1): 101-111. https://doi.org/10.1016/S0378-4347(99)00193-0.
[41] Wu X, Diao Y, Sun C, Yang J, Wang Y, Sun S. Fluorimetric determination of ascorbic acid with o-phenylenediamine. Talanta. 2003; 59(1): 95-99. https://doi.org/10.1016/S0039-9140(02)00475-7 .
[42] Ensafi AA, Karimi-Maleh H, Mallakpour S, Hatami M. Simultaneous determination of N-acetylcysteine and acetaminophen by voltammetric method using N-(3, 4-dihydroxyphenethyl)-3, 5-dinitrobenzamide modified multiwall carbon nanotubes paste electrode. Sens Actuators B Chem. 2011; 155(2): 464-472. https://doi.org/10.1016/j.snb.2010.12.048.
[43] Kamińska A, Olejarz P, Borowczyk K, Głowacki R, Chwatko G. Simultaneous determination of total homocysteine, cysteine, glutathione, and N‐acetylcysteine in brain homogenates by HPLC. J Sep Sci. 2018; 41(16): 3241-3249. https://doi.org/10.1002/jssc.201800381.
[44] Kuśmierek K, Bald E. Determination of N-acetylcysteine and thioglycolic acid in human urine. Chromatographia. 2008; 67: 23-29.
[45] Celma C, Allue J, Prunonosa J, Peraire C, Obach R. Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A. 2000; 870(1-2): 13-22. https://doi.org/10.1016/S0021-9673(99)01078-X.
[46] Tsikas D, Sandmann J, Ikic M, Fauler J, Stichtenoth DO, Frölich JC. Analysis of cysteine and N-acetylcysteine in human plasma by high-performance liquid chromatography at the basal state and after oral administration of N-acetylcysteine. J Chromatogr B Biomed Sci Appl. 1998; 708(1-2): 55-60. https://doi.org/10.1016/S0378-4347(97)00670-1.
[47] Lewis P, Woodward A, Maddock J. Improved method for the determination of N-acetylcysteine in human plasma by high-performance liquid chromatography. J Chromatogr A. 1985; 327: 261-267. https://doi.org/10.1016/S0021-9673(01)81655-1.
[48] Mindlin RL, Butler AM. The determination of ascorbic acid in plasma; a macromethod and micromethod. J Biol Chem. 1938; 122: 673-686.
[49] Wojnowski W, Tobiszewski M, Pena-Pereira F, Psillakis E. AGREEprep–Analytical greenness metric for sample preparation TrAC Trends Anal Chem. 2022; 149: 116553. https://doi.org/10.1016/j.trac.2022.116553.
[50] Guideline IHT. Validation of analytical procedures: text and methodology. Q2 (R1). 2005;1(20):05.
[51] Pena-Pereira F, Wojnowski W, Tobiszewski M. AGREE—Analytical GREEnness metric approach and software. Anal Chem. 2020; 92(14): 10076-10082. https://doi.org/10.1021/acs.analchem.0c01887.
[52] Wojnowski W, Tobiszewski M, Pena-Pereira F, Psillakis E. AGREEprep–Analytical greenness metric for sample preparation. TrAC Trends Anal Chem. 2022: 116553. https://doi.org/10.1016/j.trac.2022.116553.
[53] Aysel KT, Merve O. Development and validation of RP-UPLC and derivative spectrophotometric methods for quantitative determination of ascorbic acid and N-acetylcysteine in effervescent tablets. Int J Life Sci Pharm Res. 2019; 9(4): 45-60. https://doi.org/10.22376/ijpbs/lpr.2019.9.4.P45-60.

Details

Primary Language

English

Subjects

Pharmaceutical Analytical Chemistry

Journal Section

Image Presentation

Authors

Gürkan Özen ^*
0000-0003-0777-6402
Türkiye

Emirhan Nemutlu
0000-0002-7337-6215
Türkiye

Publication Date

June 28, 2025

Submission Date

March 11, 2024

Acceptance Date

May 17, 2024

Published in Issue

Year 2024 Volume: 28 Number: 5

IZ

https://izlik.org/JA26HW49WA

Cite

RIS / Bibtex

APA

Özen, G., & Nemutlu, E. (2025). Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products. Journal of Research in Pharmacy, 28(5), 1777-1790. https://izlik.org/JA26HW49WA

AMA

1.Özen G, Nemutlu E. Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products. J. Res. Pharm. 2025;28(5):1777-1790. https://izlik.org/JA26HW49WA

Chicago

Özen, Gürkan, and Emirhan Nemutlu. 2025. “Green HPLC Method for Simultaneous Determination of N-Acetylcysteine and L-Ascorbic Acid in Co-Formulated Pharmaceutical Products”. Journal of Research in Pharmacy 28 (5): 1777-90. https://izlik.org/JA26HW49WA.

EndNote

Özen G, Nemutlu E (July 1, 2025) Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products. Journal of Research in Pharmacy 28 5 1777–1790.

IEEE

[1]G. Özen and E. Nemutlu, “Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products”, J. Res. Pharm., vol. 28, no. 5, pp. 1777–1790, July 2025, [Online]. Available: https://izlik.org/JA26HW49WA

ISNAD

Özen, Gürkan - Nemutlu, Emirhan. “Green HPLC Method for Simultaneous Determination of N-Acetylcysteine and L-Ascorbic Acid in Co-Formulated Pharmaceutical Products”. Journal of Research in Pharmacy 28/5 (July 1, 2025): 1777-1790. https://izlik.org/JA26HW49WA.

JAMA

1.Özen G, Nemutlu E. Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products. J. Res. Pharm. 2025;28:1777–1790.

MLA

Özen, Gürkan, and Emirhan Nemutlu. “Green HPLC Method for Simultaneous Determination of N-Acetylcysteine and L-Ascorbic Acid in Co-Formulated Pharmaceutical Products”. Journal of Research in Pharmacy, vol. 28, no. 5, July 2025, pp. 1777-90, https://izlik.org/JA26HW49WA.

Vancouver

1.Gürkan Özen, Emirhan Nemutlu. Green HPLC method for simultaneous determination of N-acetylcysteine and L-ascorbic acid in co-formulated pharmaceutical products. J. Res. Pharm. [Internet]. 2025 Jul. 1;28(5):1777-90. Available from: https://izlik.org/JA26HW49WA