Analysis of docosanol using GC/MS: Method development, validation, and application to ex vivo human skin permeation studies

Authors: SHANKARA, VIJAY - University Of Mississippi; Wang, Mei; AJJARAPUA, SRINIVAS - University Of Mississippi; KOLIMI, PRAVEEN - University Of Mississippi; AVULA, BHARATHI - University Of Mississippi; MURTHY, REENA - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi; NARAISIMHA, MURTHY - University Of Mississippi

Submitted to: Journal of Pharmaceutical and Biomedical Analysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2021
Publication Date: 8/29/2021
Citation: Shankara, V., Wang, M., Ajjarapua, S., Kolimi, P., Avula, B., Murthy, R., Khan, I., Naraisimha, M. 2021. Analysis of docosanol using GC/MS: Method development, validation, and application to ex vivo human skin permeation studies. Journal of Pharmaceutical and Biomedical Analysis. https://doi.org/10.1016/j.jpha.2021.08.004.
DOI: https://doi.org/10.1016/j.jpha.2021.08.004

Interpretive Summary: Docosanol is used in the treatment of recurrent oral-facial herpes simplex labialis (HSL) also known as cold sores or fever blisters. HSL is caused by herpes simplex virus-1 (HSV-1), primarily transmitted through contact with an infected person’s oral or genital lesion and secretions. During the period of 2015–2016, the prevalence of HSV-1 infection was 47.8% and 63.6% of US and global population, respectively. Docosanol acts against HSV-1 by inhibiting viral entry into host cells and prevents the development of drug-resistant mutant viral strains. Topical docosanol is the only FDA approved over the counter alternative to topical penciclovir and acyclovir for treatment of HSL. Various generic docosanol products are under development stages and bioequivalence studies are required to assess these products. In order to demonstrate in vitro bioequivalence of generic docosanol cream, the tested products generally require qualitative, quantitative, physicochemical, microstructural, and in vitro release characteristics similar to reference listed drug. Attempts have been made to establish a nonclinical bioequivalence study, such as in vitro release and ex vivo permeation testing as biowaivers to obtain a market approval of these topical products. Docosanol is a 22-carbon saturated fatty alcohol which are devoid of chromophore or fluorophore. To date, there are no sensitive and specific analytical method for quantification of docosanol in creams, in vitro, ex vivo, or clinical samples. The present study is designed to develop and validate a gas chromatography/selected ion monitoring-mass spectrometry (GC/SIM-MS) method for the quantification of docosanol in ex vivo study samples. It was demonstrated that the validated GC/SIM-MS method was sensitive, specific, and suitable for quantification of docosanol as a quality control tool. This method can be used in routine analysis as a cost-effective alternative to other advanced techniques.

Technical Abstract: Docosanol is the only over the counter topical product approved by USFDA for the treatment of recurrent oral-facial herpes simplex labialis. The validated analytical methods for docosanol are required to demonstrate bioequivalence of docosanol topical products. A gas chromatography/selected ion monitoring mode-mass spectrometry (GC/SIM-MS) method was developed and validated for the determination of docosanol in the studied biological samples. Docosanol and isopropyl palmitate (internal standard) were separated on a high-polarity GC capillary column with (88% Cyanopropy)aryl-polysiloxane as the stationary phase. The ions of m/z 83 and 256 were selected to monitor docosanol and isopropyl palmitate, respectively, and the total run time was 20 min. The GC/SIM-MS method was validated in accordance with USFDA guidelines and the results of study met the acceptance criteria. The calibration curve was linear over a concentration range of 100–10000 ng/mL (R2 >0.994). The recoveries for the docosanol from receptor fluid and skin homogenate were >93.2% and >95.8%, respectively. The validated method was successfully applied to analyze ex vivo human cadaver skin permeation study samples. The amount of docosanol penetrated skin on application of creams of Abreva tube and Abreva pump at 48h was 21.5 ± 7.01 and 24.0 ± 6.95 ng/mg of human cadaver skin, respectively. It was concluded that the validated GC/SIM-MS was sensitive, specific, and suitable for quantification of docosanol as a quality control tool. This method can be used in routine analysis as a cost-effective alternative to other techniques.