Introduction: Acetylsalicylic acid (ASA, aspirin) ( Figure ) has been being used as an analgesic-antipyretic for a long time; it is contained in many of over-the-counter drugs. Although ASA is relatively safe, various poisoning symptoms, such as lowered consciousness levels, hypotension, pulmonary edema and convulsion, were reported upon ingestion of a large amount of this drug [1]. For analysis of ASA, methods by HPLC [2–19], GC [20–23], GC/MS [24] and capillary electrophoresis [25–27] were reported; among these methods, HPLC is most popular. . | Acetylsalicylic a cid by Einosuke Tanaka Introduction Acetylsalicylic acid ASA aspirin Figure has been being used as an analgesic-antipyretic for a long time it is contained in many of over-the-counter drugs. Although ASA is relatively safe various poisoning symptoms such as lowered consciousness levels hypotension pulmonary edema and convulsion were reported upon ingestion of a large amount of this drug 1 . For analysis of ASA methods by HPLC 2-19 GC 20-23 GC MS 24 and capillary electrophoresis 25-27 were reported among these methods HPLC is most popular. In this chapter the methods for ASA analysis by HPLC 9 16 and GC 23 are presented. O Figure COOH OOCCH3 Structure of acetylsalicylic acid ASA . HPLC analysis of ASA and its metabolites in plasma 16 Reagents and their preparation ASA salicylic acid gentisic acid and salicyluric acid can be purchased from Sigma St. Louis MO USA . ASA is dissolved in acetonitrile to prepare 1 mg mL solution. 2-Methylbenzoic acid MBA internal standard IS Bayer Leverkusen Germany and other manufacturers is dissolved in purified water to prepare 100 pg mL solution. For constructing each calibration curve methanolic solutions of ASA and its metabolites at various concentrations in the range of pg mL are prepared. HPLC conditions Column a reversed phase columna b Novapak 150 x mm . particle diameter 4 pm Waters Eschborn Germany . Mobile phase purified water 85 phosphoric acid acetonitrile 740 mL 900 pL 180 mL v v pH about . Detection wavelength 237 nm flow rate 1 mL min column oven temperature 30 C. Springer-Verlag Berlin Heidelberg 2005 344 Acetylsalicylic acid Procedure i. A volume of plasmac and 200 pL of MBAd solution are placed in a volume microtube and mixed well for 1-2 s. ii. The pH of the mixture is adjusted to about . iii. A volume of acetonitrile is added to the above mixture. iv. It is vortex-mixed well at 4 C for 15 min and centrifugal at 10 500 g for 1 min. v. The
