AJAC  Vol.2 No.4 , August 2011
Rapid HPLC Method for Determination of Parachloroaniline in Chlorhexidine Antiseptic Agent in Mouthrinses, Ophthalmic and Skin Solution
ABSTRACT
We described a simple and rapid method to quantify simultaneously chlorhexidine (CHD) and its major metabolite, para Chloroaniline (pCA) by HPLC with UV detection without the additional need of mobile-phase amine modifiers or ion-pairing reagents, with good resolution between pCA and CHD, symmetry peak of the compound and short run time. HPLC-UV analyses were performed using a Dionex® Summit liquid chromatograph (Dionex Corp, Sunnyvale, CA, USA). Chromatographic separations were carried out on a Luna® 150 mm×3 mm i.d. column packed with 3 µm CN (cyano) particles (Phenomenex®), guarded by an on-line filter. Mobile phase consist of methanol:water with sodium chloride with 0.02% of formic acid (55:45). Wavelengths for pCA and for CHD are 238 and 255 nm respectively. Influence of methanol and of sodium chloride content in the eluant has been studied. Linearity of CHD is very good, from 0.5 up to 21.2 µg/l while linearity of pCA is in the range of 0.05 to 10 µg/l with correlation coefficients above 0.999. Resolution between the components is above 4, asymmetry is about 1.3 and 1.7 for pCA and CHD respectively and the run time is less than 5 minutes. This method has been applied to CHD solution of different medical devices. No interference has been reported, and the analysis of direct injection of solution, without any treatment is achieved in less than five minutes.In conclusion, we present a validated method for dosage of CHD and its major impurity pCA, known to be carcinogen, available into medical products or medicinal device for in-vitro diagnostic.

Cite this paper
nullA. Nicolay, E. Wolff, M. Vergnes, J. Kaloustian and H. Portugal, "Rapid HPLC Method for Determination of Parachloroaniline in Chlorhexidine Antiseptic Agent in Mouthrinses, Ophthalmic and Skin Solution," American Journal of Analytical Chemistry, Vol. 2 No. 4, 2011, pp. 422-428. doi: 10.4236/ajac.2011.24051.
References
[1]   D. S. Paulson, “Efficacy Evaluation of a 4% Chlorhexidine Gluconate as a Full-Body Shower Wash,” American Journal of Infection Control, Vol. 21, No. 4, 1993, pp. 205-209. doi:10.1016/0196-6553(93)90033-Z

[2]   J. M. Albandar, J. Rise, P. Gjermo and J. R. Johansen, “Radiographic Quantification of Alveolar Bone Level Changes. A 2-Year Longitudinal Study in Man,” Journal of Clinical Periodontology, Vol. 13, No. 3, 1986, pp. 195-200. doi:10.1111/j.1600-051X.1986.tb01459.x

[3]   P. Gjermo, “Chlorhexidine in Dental Practice,” Journal of Clinical Periodontology, Vol. 1, No. 3, 1974, pp. 143- 152. doi:10.1111/j.1600-051X.1974.tb01250.x

[4]   S. S. Block, “Disinfection, Sterilization and Preservation,” Lea and Febiger, Philadelphia, 1991, p. 274.

[5]   G. W. Denton, “Chlorhexidine,” 5th Edition, Lippincott Williams & Williams, Philadelphia, 2001, p. 32.

[6]   F. A. Másquio Fiorentino, M. A. Corrêa and H. R. Nunes Salgado, “Analytical Methods for the Determination of Chlorhexidine: A Review,” Critical Reviews in Analytical Chemistry, Vol. 40, No. 2, 2010, pp. 89 -101. doi:10.1080/10408340903232020

[7]   V. R. Hebert, J. R. Middleton, E. Tomaszewska and L. K. Fox, “Methodology for Quantifying Residues of Chlor-hexidine in Raw Dairy Milk,” Journal of Agricultural and Food Chemistry, Vol. 51, No. 3, 2003, pp. 567-570. doi:10.1021/jf020915s

[8]   H. Tsuchiya, T. Miyazaki and S. Ohmoto, “High-Per- formance Liquid Chromatographic Analysis of Chlorhe- xidine in Saliva after Mouthrinsing,” Caries Research, Vol. 33, No. 2, 1999, pp. 156-163. doi:10.1159/000016510

[9]   T. Pesonen, J. Holmalahti and J. Pohjola, “Determination of Chlorhexidine in Saliva Using High-Performance Liq-uid Chromatography,” Journal of Chromatography, Vol. 665, No. 1, 1995, pp. 222-225.

[10]   L. K. Revelle, W. H. Doub, R. T. Wilson, M. H. Harris and A. M. Rutter, “Identification and Isolation of Chlor-hexidine Digluconate Impurities,” Pharmaceutical Re-search, Vol. 10, No. 12, 1993, pp. 1777-1784. doi:10.1023/A:1018986501194

[11]   Y. Ha and A. P. Cheung, “New Stability-Indicating High Performance Liquid Chromatography Assay and Proposed Hydrolytic Pathways of Chlorhexidine,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 14, No. 8-10, 1996, pp. 1327-1334. doi:10.1016/S0731-7085(96)01763-3

[12]   J. de Vries, J. Ruben and J. Arends, “Determination of Chlorhexidine in Saliva and in Aqueous Solutions,” Ca-ries Research, Vol. 25, No. 6, 1991, pp. 410-414. doi:10.1159/000261403

[13]   J. E. Jensen and F. Christensen, “A Study of the Elimina-tion of Chlorhexidine from the Oral Cavity Using a New Spectrophotometric Method,” Journal of Periodontal Research, Vol. 6, No. 4, 1971, pp. 306-311. doi:10.1111/j.1600-0765.1971.tb00622.x

[14]   K. Kudo, N. Ikeda, A. Kiyoshima, Y. Hino, N. Nishida and N. Inoue, “Toxicological Analysis of Chlorhexidine in Human Serum Using HPLC on a Polymer-Coated ODS Column,” Journal of Analytical Toxicology, Vol. 26, No. 2, 2002, pp. 119-122.

[15]   L. R. Brougham, H. Y. Cheng and K. A. Pittman, “Sensi-tive High-Performance Liquid Chromatographic Method for the Determination of Chlorhexidine in Human Serum and Urine,” Journal of Chromatography, Vol. 383, No. 2, 1986, pp. 365-373.

[16]   H. Below, N. Lehan and A. Kramer, “HPLC Determina-tion of the Antiseptic Agent Chlorhexidine and Its De-gradation Products 4-Chloroaniline and 1-Chloro-4-Ni- trobenzene in Serum and Urine,” Microchimica Acta, Vol. 146, No. 2, 2004, pp. 129-135.

[17]   Y. W. Lam, D. C. Chan, S. Y. Rodriguez, J. H. Lintakoon and T. H. Lam, “Sensitive High-Performance Liquid Chr- omatographic Assay for the Determination of Chlorhex-idine in Saliva,” Journal of Chromatography, Vol. 612, No. 1, 1993, pp. 166-171.

[18]   P. Wainwright and M. Cooke, “Direct Determination of Chlorhexidine in Urine by High-Performance Liquid Chr- omatography,” The Analyst, Vol. 111, No. 11, 1986, pp. 1343-1344. doi:10.1039/an9861101343

[19]   H. Matsushima, K. Sugimoto, K. Shibata and N. Sakurai, “Determination by Mass Fragmentography of the Chlor-hexidine in Biological Samples,” Japanese Journal of Hygiene, Vol. 37, No. 5, 1982, pp. 762-767.

[20]   V. G. Alder, D. Burman, R. A. Simpson, J. Fysh and W. A. Gillespie, “Comparison of Hexachlorophane and Chl- orhexidine Powders in Prevention of Neonatal Infection,” Archives of Disease in Childhood, Vol. 55, No. 4, 1980, pp. 277-280. doi:10.1136/adc.55.4.277

[21]   E. Read, “A Note of Hibitane Assay with Final Iodina-tion,” Methodological Surveys in Biochemistry, Chichester Ellis, Horwwod, 1978.

[22]   W. K. Gavlick and P. K. Davis, “Gas Chromatographic Determination of p-Chloroaniline in a Chlorhexidine Digluconate-Containing Alcohol Foam Surgical Scrub Product,” Journal of AOAC International, Vol. 77, No. 3, 1994, pp. 583-586.

[23]   A. Ono, “Gas-Liquid Chromatographic Separation of Toluidine, Chloroaniline and Dichloroaniline Isomers on Various Stationary Phases Including Heteroaromatic Compounds,” Analyst, Vol. 107, No. 1275, 1982, pp. 600-605.

[24]   Y. Zhu, Y. Yang and Q. Qi, “Determination of Chlor-hexidine Acetate in Disinfectant by High Performance Liquid Chromatography,” Wei Sheng Yan Jiu, Vol. 32, No. 1, 2003, pp. 51-87.

[25]   L. Havlíková, L. Matysová, L. Nováková, R. Hájková and P. Solich, “HPLC Determination of Chlorhexidine Gluconate and p-Chloroaniline in Topical Ointment,” Vol. 43, No. 3, 2007, pp. 1169-1173.

[26]   European Medicines Agency, “Guideline on the Limits of Genotoxic Impurities,” EMEA/CHMP/QWP/251344/2006, 2006, pp. 1-8.

[27]   A. P. Cheung, R. Mavar, C. Carlson and W. K. Chiang, “Problems Affecting the Liquid Chromatographic Quan-titation of Chlorhexidine Digluconate in Ophthalmic So-lutions,” Journal of Pharmaceutical and Biomedical Ana- lysis, Vol. 9, No. 1, 1991, pp. 41-45. doi:10.1016/0731-7085(91)80235-2

[28]   L. E. Stevens, J. R. Durrwachter and D. O. Helton, “Ana- lysis of Chlorhexidine Sorption in Soft Contact Lenses by Catalytic Oxidation of [14C]Chlorhexidine and by Liquid Chromatography,” Journal of Pharmaceutical Sciences, Vol. 75, No. 1, 1986, pp. 83-86. doi:10.1002/jps.2600750120

[29]   L. Zhao, L. Zhu and H. K. Lee, “Analysis of Aromatic Amines in Water Samples by Liquid-Liquid-Liquid Mi-croextraction with Hollow Fibers and High-Performance Liquid Chromatography,” Journal of Chromatography, Vol. 963, No. 1-2, 2002, pp. 239-248. doi:10.1016/S0021-9673(02)00544-7

[30]   F. M. Musteata and J. Pawliszyn, “Assay of Stability, Free and Total Concentration of Chlorhexidine in Saliva by Solid Phase Microextraction,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 37, No. 5, 2005, pp. 1015-1024. doi:10.1016/j.jpba.2004.09.055

[31]   ICH Harmonised Tripartite Guideline, “Validation of Analytical Procedures: Methodology: ICH Topic Q2B,” CPMP/ICH/281/95, 1996, pp. 1-15.

[32]   J. Vial and A. Jardy, “Experimental Comparison of the Different Approaches to Estimate LOD and LOQ of an HPLC Method,” Analytical Chemistry, Vol. 71 No. 14, 1999, pp. 2672-2677.

[33]   K. Usui, T. Hishinuma, H. Yamaguchi, N. Tachiiri and J. Goto, “Determination of Chlorhexidine (CHD) and Non-ylphenolethoxylates (NPEOn) Using LC-ESI-MS Method and Application to Hemolyzed Blood,” Journal of Chro-matography, Vol. 831, No. 1-2, 2006, pp. 105-109.

 
 
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