Liquid chromatographic assay for common sunscreen agents: application to in vivo assessment of skin penetration and systemic absorption in human volunteers

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Abstract

The purpose of the present study was to develop a reverse-phase high-performance liquid chromatographic (HPLC) assay for quantifying four common sunscreen agents, namely 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 2-ethylhexylsalicylate (octylsalicylate) and salicylic acid 3,3,5-trimethcyclohexyl ester (homosalate) in a range of biological matrices. This assay was further applied to study the skin penetration and systemic absorption of sunscreen filters after topical application to human volunteers. Separation was achieved utilizing a Symmetry C18 column with methanol–water as the mobile phase. The assay permits analysis of the sunscreen agents in biological fluids, including bovine serum albumin (BSA) solution, plasma and urine, and in human epidermis. The assay was linear (r2>0.99) with minimum detectable limits of 0.8 ng for oxybenzone, 0.3 ng for octylmethoxycinnamate, and 2 ng for homosalate and octylsalicylate. The inter- and intra-day variation for the four sunscreens was less than 3% at the upper end of the linear range and less than 6% at the lower end. Recoveries of sunscreens from plasma, 4% (w/v) BSA solution and epidermal membranes were within the range of 91–104%. Recoveries from urine of the four sunscreens, and oxybenzone with its metabolites were more than 86%. Up to approximately 1% of the applied dose of oxybenzone and its metabolites was detected in the urine. Appreciable amounts were also detected in the stratum corneum through tape stripping. The HPLC assay and extraction procedures developed are sensitive, simple, rapid, accurate and reproducible. Results from the preliminary clinical study demonstrate significant penetration of all sunscreen agents into the skin, and oxybenzone and metabolites across the skin.

Introduction

The use of sunscreens has increased with the awareness of the detrimental effects of sun exposure on human skin such as erythema, skin aging and cancers. Sunscreen products are formulated to provide a specific sun protection factor (SPF) and to absorb a broad spectrum of ultraviolet radiation (UVR). In addition to traditional sunscreen products, sunscreen chemicals are also incorporated into a wide range of everyday hair and skin products and may therefore be used without the wearer making a conscious decision to apply a sunscreen.

The actives used in topical formulations are generally classified as either chemical or physical sunscreens. Physical sunscreens comprise of particles that act by scattering, reflecting, or absorbing the passage of radiation. Chemical sunscreens act by absorbing incident UVR and then dissipating it as longer wavelength energy, thereby protecting the skin from potentially damaging UVR. The efficiency of sunscreens is estimated by the sun protection factor, which depends on the content of UV filters in the formulation. The necessity to provide high SPF and screening efficiency against both UV-A (320–400 nm) and UV-B (290–320 nm) wavelengths has led to the development of sunscreen preparations containing many different sunscreen chemical combinations. Benzophenones, dibenzoylmethanes and anthranilates are the most common UV-A filters, whereas the UV-B filters include p-aminobenzoic acid (PABA) derivatives, salicylates, cinnamates, digalloyl triolate, lawsone, acrylates and benzimidazole derivatives. Of the approved sunscreen chemicals, oxybenzone (OX, benzophenone-3), octylmethoxycinnamate (OMC), butylmethoxydibenzoylmethane (BDM), octylsalicylate (OS) and homosalate (HS) are some of the most common active ingredients used in sunscreen formulations.

Recent studies have provided evidence that some sunscreens are absorbed systemically following topical application to the skin [1], [2], [3]. These studies involved determination in skin layers only or measurement of urinary excretion of absorbed sunscreens and their metabolites. Neither provided a full pharmacokinetic analysis, as only a single measure of absorption or excretion was assessed. It would be advantageous to quantify penetration within the skin tissue and systemic distribution of sunscreen agents following topical application. This would aid in the determination of the exposure of viable tissues to sunscreen chemicals, provide a better understanding of the potential for toxicity both locally and systemically, and facilitate design of novel formulations to target the outer skin layers.

In addition to in vivo studies, skin penetration of chemicals and drugs is frequently investigated using in vitro techniques. The in vitro technique utilizes diffusion cells, which consist of a receptor and donor phase separated by a synthetic or skin membrane. Where lipophilic solutes are investigated, as is the case for many sunscreens, bovine serum albumin (BSA) or other solubility modifiers are used as receptor fluids to provide adequate solubility and ensure sink conditions [4], [5]. A suitable extraction procedure and high-performance liquid chromatographic (HPLC) assay is required to facilitate these studies.

Many of the HPLC assays published for sunscreen agents are designed for product evaluation and determination of concentrations in cosmetic formulations [6], [7], [8]. Few assays for evaluation of sunscreens in biological samples have been reported [5], [9], [10], [11]. A reliable analytical method for the quantitative determination of the common sunscreen chemicals in biological fluids will facilitate the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data.

The aim of this study was to develop simple, rapid and reliable operating procedures for quantification of sunscreen chemicals in a range of biological matrices. Butylmethoxydibenzoylmethane, octylmethoxycinnamate, octyldimethyl PABA, octylsalicylate, oxybenzone and homosalate are the most common sunscreen agents. Most of them present similar retention times in previously published assays and are therefore difficult to resolve. HS is especially problematic because it presents two peaks corresponding to two isomeric forms [6]. This paper provides a reproducible and accurate assay, by which four of the most common sunscreen agents (Fig. 1), including HS, can be resolved simultaneously. Using this assay procedure, a preliminary investigation of the penetration into the skin tissues, plasma and excretion in the urine of four common sunscreens, as active ingredients in a commercially available sunscreen product, was studied. This paper also provides procedures for the extraction of sunscreens from tape strips, skin tissue and biological matrices including plasma, urine and bovine serum albumin.

Section snippets

Materials and methods

OX, OS and BSA were purchased from Sigma–Aldrich (USA). OMC and HS were gifts from BASF Corporation (NJ, USA) and EM Industries (Germany), respectively. Coppertone sunblock lotion (Schering-Plough Health Care Products Inc.) was the commercially available sunscreen product used for the study. HPLC grade methanol was from Fisher Scientific (USA). De-ionized water (Milli-Q, Waters Inc., USA) was used and all other chemicals used were analytical reagent grade.

HPLC instrumentation and conditions

An Alliance liquid chromatographic

Chromatography and resolution

HPLC chromatograms of the four sunscreen agents after sample preparation from an extract of 4% (w/v) BSA, plasma and tape strips are shown in Fig. 2. Many of the HPLC assays published are designed for product evaluation and determination of concentrations in cosmetic formulations [6], [7], [8], [19]. The assay method previously published for evaluation of sunscreens in biological samples is useful but offers certain limitations in terms of sensitivity, especially with octylsalicylate [5].

Acknowledgements

The authors wish to acknowledge the financial support of the Canada Foundation for Innovation and the Manitoba Infrastructure Fund and Hill Top Research Inc. VS acknowledges the support of a University of Manitoba Graduate Fellowship and Leslie F. Buggey Graduate Fellowship. Technical support from Dr. Frank Burczynski (UM) is highly appreciated.

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