HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYTICAL METHOD VALIDATION FOR GLUTARALDEHYDE AND BENZALKONIUM CHLORIDE IN DISINFECTANTS
Objective: The aim of this study was to produce a selective, accurate, and faster high-performance liquid chromatography (HPLC) analytical method
for benzalkonium chloride and glutaraldehyde in disinfectants using ultraviolet (UV)-visible detection.
Methods: Glutaraldehyde has no chromophore, so it was first derivatized using 2,4 dinitro phenylhydrazine. Acetonitrile:water (75:25) was used as
the mobile phase for glutaraldehyde and acetonitrile-acetate pH 4 (75:25) for benzalkonium chloride, both at a flow rate of 1.2 mL/min. The optimized
assay was validated with respect to accuracy, precision, linearity, selectivity, limit of quantitation (LOQ), and limit of detection (LOD).
Results: The method was linear for benzalkonium chloride, with correlation coefficient of 0.9995, LOD of 14.55 ppm, and LOQ of 48.51 ppm. The
correlation coefficient for glutaraldehyde was 0.9995, with LOD of 0.49 ppm and LOQ of 1.64 ppm. Accuracy was between 98% and 102%, and
precision was below 2% for both the tests.
Conclusion: The HPLC analytical method for benzalkonium chloride and glutaraldehyde in disinfectants using UV-visible detection in this research
was successful to produce a selective, accurate, and faster method.
2. Koyama K, Shimazu T. Benzalkonium Chloride. Berlin Heidelberg:
Springe-Verlag; 2005. p. 407-13.
3. Directorate General of Livestock Product Development Indonesian
Ministry of Agriculture. Indonesian Veterinary Drug Pharmacopoeia.
Jakarta: Indonesian Ministry of Agriculture; 2008.
4. Labranche LP, Dumont SN, Levesque S, Carrier A. Rapid determination
of total benzalkonium chloride content in ophthalmic formulation.
J Pharm Biomed Anal 2007;43:989-93.
5. Menet MC, Gueylard D, Fievet MH, Thuillier A. Fast specific separation
and sensitive quantification of bactericidal and sporicidal aldehydes by
high-performance liquid chromatography: Example of glutaraldehyde
determination. J Chromatogr Sep Tech 1997;692:79-86.
6. Harmita. Physicochemistry Analysis. Depok: Faculty of Pharmacy,
Universitas Indonesia; 2012.
7. Rediatning W, Kartini N. Analysis of amino acids with high performance
liquid chromatography derivatized prakolom and pascakolom. Proc
8. Rao N, Gawde KD. Method development and force degradation
studies for simultaneous estimation of salbutamol sulfate, etofylline
and bromhexine hydrochloride in pharmaceutical dosage form using
reversed-phase high-performance liquid chromatography method.
Asian J Pharm Clin Res 2018;11:378-82.
9. Shaikh S, Jain V. A novel reverse-phase high-performance liquid
chromatographic method for simultaneous estimation of ellagic acid,
quercetin, and piperine in ayurvedic formulations. Asian J Pharm Clin
10. Ramesha B, Reddy KR, Unni KM, Ankolekar V, Anuradha P,
Amith KM. A validated UHPLC method for the determination of
atorvastatin acetonide tert-butyl ester and 4-fluoro-alpha-(2-methyl-1-
oxopropyl)-gamma-oxo-n, beta-diphenylbenzene butaneamide. Asian J
Pharm Clin Res 2012;5:115-22.
11. ICH. ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and
Methodology. International Conference on Harmonization, 1994; 1996.
12. Thompson M, Ellison SL, Wood R. Harmonized guidelines for singlelaboratory
validation of methods of analysis (IUPAC Technical Report).
Pure Appl Chem 2002;74:835-55.
13. Bretnall AE, Clarke GS. Validation of analytical test methods. In: Ahuja
S, Scypinski S, editors. Handbook of Modern Pharmaceutical Analysis,
Separation Science and Technology Series. 2nd ed. Waltham, MA, USA:
Academic Press (Elsevier); 2011. p. 9-457.
14. Anderson J, Berthod A, Pine V, Stalcup AM. Analytical Separation
Science. Harlow, UK: Prentice-Hall; 2016.