4.94.9 © Springer-Verlag Berlin Heidelberg 2005 II.4.9 Salicylic acid By Einosuke Tanaka Introduction Salicylic acid (> Figure 9.1) is being used as a keratolytic agent for treatment of corns or ver- rucae; the acid is included in adhesive plasters, ointments and liquid medicines as their ingre- dient for removing the stratum corneum. Since such products containing salicylic acid are easily obtainable, salicylic acid poisoning ( salicylism) cases are relatively many especially for infants not older than 5 years. Its poisoning symptoms were reported to be headache, vertigo, tinnitus, bradyacusia, amblyopia and sweating [1]. For analysis of salicylic acid, HPLC methods were reported [2–7]. In this chapter, a method for HPLC analysis of salicylic acid in serum specimens is presented [3]. Structure of salicylic acid. ⊡ Figure 9.1 Reagents and their preparation • 8-Chlorotheophylline (internal standard, IS, Sigma, St. Louis, MO, USA) is dissolved in ethanol to prepare 150 µg/mL solution. • Salicylic acid (Sigma) is dissolved in methanol to prepare 5–500 µg/mL solutions. • A 2.7-g aliquot of sodium acetate is dissolved in 1 L water, followed by addition of 15 mL acetic acid to prepare acetate bu er solution (pH 3.6). HPLC conditions Column: a reversed phase column a , µBondapak C18 Radial-Pak (100 × 5 mm i.d., particle di- ameter 10 µm, Waters, Milford, MA, USA). Mobile phase: acetate bu er solution (pH 3.6)/methanol (72:28, v/v). Detection wavelength b : 280 nm; ow rate: 1 mL/min: column (oven) temperature: room tem- perature. 392 Salicylic acid Procedure i. A 100-µL volume of serum, 20 µL IS solution and 200 µL of 1 M hydrochloric acid solu- tion are placed in a glass centrifuge tube with a ground-in stopper and mixed well. ii. A 2-mL volume of dichloromethane/isopropanol (9:1, v/v) is added to the above mixture. iii. A er mixing or shaking for 5 min, it is centrifuged at 2,500 g for 2 min. iv. e organic layer (lower phase) is transferred to a clear test tube. v. e organic extract is evaporated to dryness under a gentle stream of air (or nitrogen) with warming at 50 °C. vi. e residue is dissolved in 100 µL of the mobile phase to be used as test solution. vii. A 20-µL aliquot of it is injected into HPLC. viii. For constructing calibration curves, various concentrations of salicylic acid and a xed amount of IS are processed according to the same procedure. Assessment and some comments on the method > Figure 9.2 shows HPLC chromatograms for the authentic standard salicylic acid and for the extract of human serum, into which salicylic acid and IS had been spiked. In this method, the linearity of salicylic acid was found in the range of 20–500 µg/mL; the detection limit was 3 µg/mL. Since salicylic acid is also a main metabolite of acetylsalicylic acid, the present method can be used for the metabolite analysis for acetylsalicylic acid (see Chapter 4 of general drugs). Conversely, the HPLC conditions described in Chapter 4 can be also used for analysis of sali- cylic acid. Methyl salicylate, being used as a topical analgesic-antiphlogistic drug, is also me- tabolized into salicylic acid; therefore, the present method can be used for analysis in methyl salicylate poisoning. Blood therapeutic, toxic and fatal concentrations [8] Blood therapeutic concentrations: 20–250 µg/mL; toxic concentrations: 150–500 µg/mL; fatal concentrations: not lower than 500 µg/mL. Poisoning case [9] Twenty-year-old and 21-year-old brothers were su ering from ichthyosis and were by them- selves treating their corni ed trichophytia (hyperkeratomycosis) with a drug. It was a gel drug consisting of propylene glycol, 20% ethanol, 20% water and 6% salicylic acid. For one bandage, about 100 g of the gel drug was applied; they continued such treatment for several years. Just a er each treatment, they complained of noisy and continuing tinnitus. e concentration of salicylic acid in serum sampled from the elder brother was 366 µg/mL, which was at a toxic level. e toxic dose of salicylic acid is about 50 mg/kg. e concentration was estimated to reach its maximum about 12 h a er topical application the gel. e body weight of above pa- tient (elder brother) was 62 kg; about 60% (3.6 g in 100 g gel) of the total amount of the drug 393 applied was considered to be absorbed percutaneously. If this assumption is correct, his dose per kg is calculated to be 58 mg/kg, which exceeds the above toxic dose (50 mg/kg). Notes a) In many reports, reversed phase octadecyl (C 18 ) chemical-bonded silica gel columns with 10–15 cm lengths and 2.5–5 µm particle sizes are being used. b) e wavelength to be used is somewhat di erent in di erent methods; the optimum wave- length should be checked for each detector and for each mobile phase. HPLC chromatograms for the authentic salicylic acid and other compounds (A) and for an extract of human serum, into which salicylic acid and IS had been spiked (B) [3]. 1: acetaminophen (100 µg/mL, retention time 3 min); 2: theophylline (20 µg/mL, 4.6 min); 3: salicylic acid (200 µg/mL, 6.2 min); 4: IS ( 8-chlorotheophylline) (8.7 min). ⊡ Figure 9.2 Salicylic acid 394 Salicylic acid References 1) Cooper P (1989) Handbook of Drug Poisonings, 2nd edn. Ishiyaku Publishers Inc., Tokyo, pp 611–612 (in Japa- nese) 2) Rumble RH, Roberts MS, Wanwimolruk S (1981) Determination of aspirin and its major metabolites in plasma by high-performance liquid chromatography without solvent extraction. J Chromatogr 225;252–260 3) Kinberger B, Holmen A (1982) Simultaneous determination of acetaminophen, theophylline and salicylate in serum by high-performance liquid chromatography. J Chromatogr 229:492–497 4) Kruk RJ, Adams MA, Philp RB (1984) Rapid and sensitive determination of acetylsalicylic acid and its meta bolites using reversed-phase high-performance liquid chromatography. J Chromatogr 310:343–352 5) Brandon RA, Eadie MJ, Smith MT (1985) A sensitive liquid chromatographic assay for plasma aspirin and sali- cylate concentrations after low doses of aspirin. Ther Drug Monit 7:216–221 6) Chubb SA, Campbell RS, Price CP (1986) Rapid method of measuring salicylate in serum by high-performance liquid chromatography. J Chromatogr 380:163–169 7) Odou P, Barthelemy C, Robert H (2001) Influence of seven beverages on salicylate disposition in humans. J Clin Pharm Ther 26:187–193 8) Stead AH, Moffat AC (1983) A collection of therapeutic, toxic and fatal blood drug concentrations in man. Hum Toxicol 2:437–464 9) Soyka PA, Soyka LF (1980) Absorption of salicylic acid. JAMA 244:660–661 . used for analysis of sali- cylic acid. Methyl salicylate, being used as a topical analgesic-antiphlogistic drug, is also me- tabolized into salicylic acid;. Brandon RA, Eadie MJ, Smith MT (1985) A sensitive liquid chromatographic assay for plasma aspirin and sali- cylate concentrations after low doses of aspirin.