Eco-friendly RP-HPLC method for determination of diazepam in coated tablet

Green RP-HPLC for diazepam determination


  • Marija Tomić Research & Development, ALKALOID AD Skopje
  • Marijana Božinovska Research & Development, ALKALOID AD Skopje
  • Nataša Anevska-Stojanovska Research & Development, ALKALOID AD Skopje
  • Jelena Lazova Research & Development, ALKALOID AD Skopje
  • Rumenka Petkovska Faculty of Pharmacy, Ss. Cyril and Methodius University in Skopje
  • Liljana Anastasova Faculty of Pharmacy, Ss. Cyril and Methodius University in Skopje
  • Natalija Nakov Faculty of Pharmacy, Ss. Cyril and Methodius University in Skopje



green/eco-friendly analytical methods, reversed-phase liquid chromatography, ethanol, diazepam, pharmaceutical analysis


A new eco-friendly RP-HPLC method for determining diazepam in coated tablets was developed and validated. The separation was achieved on a Lichrosper® 100 RP-18 (250 mm × 4 mm, 5 µm particle size) using the isocratic elution mode with a mobile phase composed of a mixture of water (H2O) and ethanol (EtOH) in a 40:60 (v/v %) ratio and a flow rate of 1.0 mL/min. The injection volume was 10 μL, and the detection wavelength was set at 254 nm. The column temperature was maintained at 35 ºC. The method was validated according to the ICH guideline, emphasizing selectivity/specificity, linearity, sensitivity, accuracy, precision, and robustness. The analytical method greenness score and Eco-scale approach were used for the method’s greenness assessment. The method was applied to determine the assay of diazepam in coated tablets and the uniformity of dosage units by content uniformity.

Applying the method proposed in this study in the pharmaceutical industry is considered environmentally sustainable. It would bring benefits in terms of a safer working environment and reduction of toxic waste formation without compromising the reliability of the analytical results.


(1) Sajid, M.; Plotka-Wasylka, J., Green analytical chemistry metrics: review, Talanta, 2022, 238 (2), 123046. DOI: 10.1016/j.talanta.2021.123046

(2) Nakov, N.; Acevska, J.; Brezovska, K.; Kavrakovski, Z.; Dimitrovska A., Green strategies towards eco-friendly HPLC methods in pharma analysis. In: High-performance liquid chromatography – recent advances and applications, IntechOpen Limited, London, 2023. DOI: 10.5772/intechopen.110035

(3) Tobiszevski, M.; Namiesnik, J., Greener organic sol-vents in analytical chemistry, Current Opinion in Green and Sustainable Chemistry, 2017, 5, 1–4. DOI: 10.1016/j.cogsc.2017.03.002

(4) Elmansi, H.; Belal, F.; Development of an eco-friendly HPLC method for the simultaneous determination of three benzodiazepines using green mobile phase, Microchemical Journal, 2019, 145, 330–336. DOI: 10.1016/j.microc.2018.10.059

(5) Scriba, G. K. E.; Sean C. Sweetman (Ed.): Martindale: The Complete Drug Reference. Chromatographia, London, 2011, 74, 647–648. DOI:

(6) World Health Organization (WHO): WHO model list of essential medicines, 22nd list, 2021. Available at: Accessed March 8, 2023.

(7) Szatkovska, P.; Koba, M.; Kośliński, J.; Wandas, J.; Bączek, T., Analytical methods for determination of benzodiazepines. A short review, Central European Journal of Chemistry, 2014, 12, 994–1007. DOI:

(8) Eldin, A. B.; Shalaby, A.; Abdallah, M. S.; Shaldam, M. A.; Abdallah, M.A., Applying green analytical chemistry (GAC) for development of stability indicating HPLC method for determination of clonazepam and its related substances in pharmaceutical formulations and calculating uncertainty, Arabian Journal of Chemistry, 2014, 12 (9), 1212–1218. DOI:

(9) El Mahjoub, A.; Staub, C., Simultaneous determination of benzodiazepines in whole blood or serum by HPLC/DAD with semi-micro column, Journal of Pharmaceutical and Biomedical Analysis, 2000, 23 (2, 3), 447–458. DOI:

(10) Gil-Agustí M.; Carda-Broch, S.; Garsía-Alvarez-Coque M.C.; Esteve-Romero, J., Use of micellar mobile phases for the chromatographic determination of clorazepate, diazepam, and diltiazem in pharmaceuticals, Journal of Chromatographic Science, 2000, 38, 521–527. DOI:

(11) Capella-Peíro, M. E.; Bose, D.; Martinavaro-Domíngues, A.; Gil-Agustí M.; Esteve-Romero, J., Direct injection micellar liquid chromatographic determination of benzodiazepines in serum, Journal of Chromatography B, 2002, 780 (2), 241–249. DOI:

(12) Bakavoli, M.; Kaykhaii, M., Quantitative determination of diazepam, nitrazepam and flunitrazepam in tablets using thin-layer chromatography-densitometry technique, Journal of Pharmaceutical and Biomedical Analysis, 2003, 31, 1185–1189. DOI:

(13) Sruthi, A.; Tejaswi, P.; Thanuja, N.; Sudheer Kumar, D.; Vivek Sagar, P., Simple RR-HPLC method for estimation of diazepam in tablet dosage form, Journal of Pharmacy Research, 2013, 6, 140–144. DOI:

(14) Djajić, N.; Krmar, J.; Rmandić, M.; Rašević, M.; Otašević, B.; Zečević, M.; Malenović, A.; Protič, A., Modified aqueous mobile phases: A way to improve retention behavior of active pharmaceutical compounds and their impurities in liquid chromatography, Journal of Chromatography Open, 2022, 2, 1000023. DOI:

(15) Kamal, A. H; El-Malla, S. F., Mixed micellar liquid chromatographic method for simultaneous determination of norfloxacin and tinidazole in pharmaceutical tablets, Microchemical Journal, 2019, 150, 104151. DOI: 10.1016/j.microc.2019.104151

(16) European Pharmacopoeia. General monographs: diazepam. 11th Ed. 2022–2023. Strasbourg France: European Directorate for the Quality of Medicines & Healthcare, 2513–2514.

(17) The United States Pharmacopoeia. The National Formulary (USP43-NP 38), 2020. Rockville, Maryland, United States Pharmacopeial Convention

(18) Yabré, M.; Ferey, L.; Touridomon Somé, I., Gaudin, K., Greening reversed-phase liquid chromatography methods using alternative solvents for pharmaceutical analysis, Molecules, 2018, 23, 1065. DOI: 10.3390/molecules23051065

(19) International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonized Tripartite Guideline: Validation of Analytical Procedures: Text and Methodology Q2(R1). ICH, 2005.

(20) AMGS calculator link

(21) Gałuszka, A.; Konieczka, P.; Migaszevski, Z. M.; Na-mieśnik, J., Analytical Eco-scale for assessing the greenness of analytical procedures, Trends in Analytical Chemistry, 2012, 37, 61–72. DOI:

(22) Mašković, M.; Jančić-Stojanović, B.; Malenović, A.; Ivanović, D.; Medenica, M., Assessment of liquid chromatographic method robustness by use of Plackett-Burman design, Acta Chromatographica, 2010, 22, 281–296. DOI: 10.1556/AChrom.22.2010.2.10



2023-11-29 — Updated on 2023-12-24


How to Cite

Tomić, M., Božinovska, M., Anevska-Stojanovska, N., Lazova, J., Petkovska, R., Anastasova, L., & Nakov, N. (2023). Eco-friendly RP-HPLC method for determination of diazepam in coated tablet: Green RP-HPLC for diazepam determination. Macedonian Journal of Chemistry and Chemical Engineering, 42(2), 153–163 . (Original work published November 29, 2023)



Analytical Chemistry