(W0930-02-14) HPLC Method Development for Assay of Common Topical Formulation Preservatives

Purpose: High Performance Liquid Chromatography (HPLC) assay is an integral part of topical formulation development.  Method development for analysis of various analytes in topical formulations is a dynamic and challenging process. An optimal method for analysis of an Active Pharmaceutical Ingredient (API), degradation products/impurities, and preservatives in support of R&D testing through Phase II clinical trials will include system suitability, precision, linearity, sensitivity, specificity, estimated limit of quantitation, and estimated limit of detection. Analytical characterization of preservative content is not required in early formulation development through Phase II clinical trials. However, it can be extremely helpful to begin developing methods for the analysis of preservative content prior to entering Phase III clinical trials. Common preservatives found in topical formulations are methylparaben, propylparaben, benzyl alcohol, phenoxyethanol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), benzoic acid, and sorbic acid. A method has been developed for separating each of these compounds within a standard and a gel sample by HPLC assay. This method may be further developed to accommodate specific sets of preservatives with an API and impurities.

Methods: A linear gradient method using an Agilent Zorbax Eclipse C18 column, water, and acetonitrile was initially developed to evaluate the chromatography. A diluent consisting of 50% water and 50% acetonitrile was prepared for diluting standards and samples. Propylparaben was weighed into a 25 mL flask and diluted to volume for a 100 µg/mL concentration. Each of the other seven analytes were weighed into a separate 25 mL volumetric flask to produce various concentrations that match the analytical concentration of each analyte in the sample. The propylparaben standard was aliquoted at 2.5 mL into the 25 mL flask containing the rest of the standards. The standard solution was then diluted to volume with diluent. A gel sample (also containing all eight preservatives) was prepared by weighing about 2.5 g into a 50 mL volumetric flask. The ball of the sample flask was filled halfway with diluent, and the sample was then vortexed to disperse the gel. The sample was then diluted to volume with diluent, sonicated for 5 minutes, and mixed well. The standard and sample were transferred to autosampler vials for analysis. A small amount of each reference standard was placed in individual autosampler vials and diluted with diluent up to 1.5 mL for identification of each analyte by retention time. Sorbic acid and benzoic acid co-eluted in the chromatography with the initial method. A literature review was conducted, and new mobile phases were selected. A 50 mM ammonium acetate buffer (pH = 4.5) was used as mobile phase A (MPA) and methanol was used as mobile phase B (MPB) due to its increased polarity¹. The starting conditions of the gradient were modified to increase MPB from 5% up to 30% to create a more gradual increase in the solvent wash step. The flow rate was lowered to maintain analyte retention. The final method showed good resolution of all eight compounds.

Results: Using the initial method with water as MPA and acetonitrile as MPB, six out of eight analytes had acceptable chromatography and were identified in the resulting chromatograph. Benzoic acid and sorbic acid were co-eluting. These compounds are chemically similar and therefore were retained on the column for the same amount of time¹. With the updated ammonium acetate buffer MPA and methanol MPB, all eight analytes were well-retained on the column and eluted at different times. The more gradual gradient allowed the analytes to elute earlier, and at a subsequently slower rate for each, thus increasing resolution between analytes.

Conclusion: The use of an ammonium acetate buffer facilitates the separation of benzoic and sorbic acids¹ from each other while not impacting the chromatography of the other preservatives. This buffer provides a neutral pH which enables most organic acids to ionize, thus allowing for separation². Using methanol instead of acetonitrile as MPB in the gradient also supports the ionic separation of these acids^1,2. A single method may be used to separate and quantify methylparaben, propylparaben, benzyl alcohol, phenoxyethanol, BHA, BHT, benzoic acid, and sorbic acid in one chromatograph. This method may be used as a foundation and is subject to further method development based on the formulation being analyzed.

References: 1. Abo Elhassan, Alaa Eldean & Alsayed, Mahmoud & Gomaa, Ahmed. (2017). Determination of Sorbic Acid and Benzoic Acid using Reversed- Phase High Performance Liquid Chromatography (RP-HPLC) in Different Food Commodities. Inventi Rapid: Pharm Analysis & Quality Assurance. 2017. 5.
2. Chrom Resource Center, MicroSolv Technology Corporation, 9158 Industrial Blvd. NE, Leland, NC 28451

Figure 1. Standard and sample preparation concentrations for HPLC analysis, vehicle gel formulation composition, and analytical results of vehicle gel analysis.

Figure 2. Initial development method HPLC parameters and corresponding chromatographs.

Figure 3. Final development method HPLC parameters and corresponding chromatographs.