Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

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Abstract

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C8 columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm2 and of SA from 0.1 to 5 μg/cm2 in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm2) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.

Introduction

The use of topically administered acetylsalicylic acid (ASA) in various solvents has been recently proposed as treatment for pain relief in skin infections 1, 2. To determine whether the analgesic effect is related to the concentrations of the drug in the skin or in plasma, i.e. if the analgesic effect was due to a local or systemic mechanism [3], a method to measure the concentrations of ASA in the skin had to be developed. To date there is only one study in which ASA has been measured in skin but, as is the case with other drugs, a radiolabeled compound was used [4].

A number of methods have been described for the extraction of antinflammatory drugs ASA and SA from plasma 5, 6involving the use of liquid–liquid extraction with organic solvents. The use of solid adsorbents as an alternative method of extraction is becoming increasingly popular 7, 8. These adsorbents offer the advantage of avoiding emulsion formation and reducing the volume of solvent required for an efficient extraction. Solid-phase extraction methods are also simple and far less time consuming than liquid–liquid extractions.

This study describes an HPLC method to determine the concentrations of ASA and SA in human stratum corneum using the stripping method and direct injection as well as in plasma using solid-phase extraction.

Section snippets

Chemicals and reagents

ASA, SA, Piroxicam (PIR, tape internal standard, I.S.) and Phenobarbital (PHE, plasma I.S.) were acquired from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were HPLC-grade and the other reagents and solvents were of analytical grade (Merck, Darmstadt, Germany).

Tape strippings were performed using 5 cm of 3M adhesive tape (Scotch Magic, 3M) in a ribbon of 2-cm width.

Isolute liquid–solid extraction columns, packed with 100 mg of C8-bonded 40 μm silica with average pore size of 60 Å and

Determination of ASA and SA in the tape samples

Whereas no interfering peaks near the retention times of ASA, SA and I.S. were present in the chromatograms of blank or basal (time 0) plasma samples, in the blank of tape samples there was a peak interfering with phenobarbital (the I.S. used for plasma) and therefore a different I.S. (piroxicam) had to be used. Representative chromatograms are shown in Fig. 1 (tape sample).

Conclusions

The use of topically administered drugs is becoming a widespread system to overcome toleration problems with certain drugs. Thus, noninvasive methods to measure the concentration of drugs at the site of administration are important, particularly with nonradioactive techniques.

This method using noninvasive tape-stripping sampling and HPLC chromatography appears to be very suitable for the accurate determination of aspirin and its main metabolite, salicylic acid, within the human stratum corneum.

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