title: Measurement of salicylic acid, 2,3-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid in human serum by hplc with electrochemical detection: Method development and validation
Creators: name: Campbell, Graham Ross
Publisher: name: University of Glasgow
Year: 2000
Subjects: subject: Analytical chemistry.

description: Free radicals, the highly reactive, unstable molecular species implicated in the development of many diseases, are introduced and the analytical methods available for their estimation reviewed. One approach, the measurement of the aspirin metabolites salicylic acid (SA) and 2,3- and 2,5- dihydroxybenzoic acid (DHBA) is fully examined. The improvement and validation of a method for the routine measurement of SA in-patients receiving prophylactic doses of aspirin resulted in an assay with the following characteristics:- 0.5ml serum, acidified to pH 2.5, and applied to a HAX(TM) (Jones Chromatography) mixed phase SPE column preparation in succession with methanol and pH 2.5 acetate buffer. After further buffer washes, the SA was eluted with methanol containing 3%NH3 (v/v). The dried extract was reconstituted in 500). mul HCl (0.05M). HPLC separation, free from interference, was achieved on a 250 x 4.6 mm C18 column of 5mu particle size, at a temperature of 30°C, using a mobile phase of 30niM citrate buffer pH 4.75 containing 28.6% (v/v) methanol flowing at l.0 ml/min. The electrochemical detector (Antec Ltd) oxidising potential was set at 1.1 OV. Mean SA extraction efficiencies were 85% over a 0.1 - 50) muM linear range. The internal standard, 4- methylsalicylic acid (4-MeSA), was selected from a number of candidates, based on chromatographic, voltammetric and extraction characteristics. Intra-assay precision was 7.1, 6.2 and 4.6% (CV, n = 9), at 1.5, 15 and 35muM respectively; inter-assay precision was 16.9, 8.5 and 6.6% (CV, n = 9) at 1.5, 15 and 35muM respectively. SA was stable for 13 days in water, hydrochloric acid and serum at pH 7.3 - 7.5 and pH 2.5 at -30°C, 4°C and room temperature. Development of an assay for 2,3-DHBA and 2,5-DHBA was only partially completed as described below:- 2,3-DHBA and 2,5-DHBA were also extracted by SPE as described above. Chromatography took place on a similar C18 column at 30°C at a flow rate of 1.0ml/min. Successful resolution of analytes, IS and unknown extracted peaks was obtained using 30mM citrate buffer containing no methanol, by modification of the pH to 5.3. However, a gradient of increasing methanol concentration was added to elute the polar compounds and shorten run times. An optimum oxidation potential of 0.75V was required for the detection of 2,3- and 2,5-DHBA making the selection of a new IS, 3,4-DHBA, with a similar voltammogram, necessary. Preliminary extraction efficiencies for 2,3- and 2,5-DHBA at a concentration of 100nM were found to be 79% and 73% respectively. 2,3- and 2,5-DHBA were found to be stable for up to 2.5 hours in aqueous solution at room temperature. Both analytes are therefore less stable than SA. Precision data are required to complete the validation of this assay. Clinical application of the SA method was carried out using blood from 8 diabetic patients known to be taking low doses of aspirin. SA was detected in all specimens, with levels of SA ranging from 0.23 - 10.51muM after administration of low-doses of aspirin from 75 - 300mg/day. Blank serum samples from subjects known not to have taken aspirin were analysed for SA (n = 6) and 2,3 and 2,5-DHBA (n = 11) by the methods described above. Unexpectedly, SA was detected in all samples in the range 15 - 75pmol/ml. Similarly, peaks with the same retention time as 2,3- and 2,5-DHBA were found in all samples. The presence of all three analytes was confirmed by voltammetric comparison with standard solutions. The origin of these analytes in samples from control subjects is known, but since SA, 2,3- and 2,5-DHBA have been detected in a number of different foodstuffs, 62,65 a dietary source should be considered. With little further development, the methods described above are sufficiently sensitive, robust and reliable for use in measuring the extent of free radical involvement in various disease states by assaying relative levels of these analytes following low-dose aspirin administration.