CLIN.CHEM.37/7, 1281-1283 (1991) CLINICAL CHEMISTRY, Vol. 37, No. 7, 1991 1281 Isocratic Reversed-Phase HPLC Method to Measure Pyrimethamine Extracted from Plasma of Infants Treated for Toxoplasmosis T. H. Zytkovicz,’ Julia Salter,’ Laura Hennigan,’ Ralph Timperi,’ James Maguire,2 and Rodney Hoff’ An isocratic HPLC method for measuring pyrimethamine extracted from infant plasma is reported. The method is an improvement over previously published methods by requiring lower volumes of plasma (100 L) and having increased sensitivity to pyrimethamine at 210 nm. The procedure, which entails a basic organic extraction and subsequent HPLC chromatography of the reconstituted extract, can detect 1.4 ng and quantify 4.0 ng of py- rimethamine per 4O-L injection, with two analyses per 100-FL sample. Analytical recovery of pyrimethamine added to plasma at 10, 50, and 125 ng/100 L averaged 80%, 92%, and 101%, respectively (n = 20). Within- and between-day Cvs were <7%. Studies of various plasma samples from adults and infants (n = 15) revealed no interference from other plasma peaks with the analyte of interest. AdditIonal Keyphrases: infection pediatric chemistry acquiredimmunodeficiencysyndrome Toxoplasmosis, a common protozoal infection, can be life-threatening in infants who are infected congenitally (1) and in immunodeflcient persons, especially those with acquired immunodeficiency syndrome (AIDS) (2). The preferred treatment for toxoplasmosis involves py- rimethamine, a basic, lipophilic, protein-bound drug with a half-life in adults of 23 to 175 h (3-7). What concentration of pyrimethamine in blood is needed for effective treatment of congenital toxoplasmo- sis has not been thoroughly established. To monitor the blood concentrations of pyrimethamine in infants treated as part of a statewide screening program, we developed a sensitive analytical method for py- rimetharnine that could be performed with the small volumes of plasma that are obtainable from newborn infants. A method to measure pyrimethamine in plasma would help define the appropriate treatment of congen- ital toxoplasmosis. Pyrimethamine has been assayed by microbiological methods (8), gas-liquid chromatography (9-11), thin- layer chromatography (12), spectrophotometry (13) and HPLC (3-5,14-16). Here we describe a modified HPLC method for quantifying pyrimethamine in blood sam- ples from pediatric patients. The main advantages of the HPLC method described here are (a) greater sensitivity ‘Massachusetts Department of Public Health, State Laboratory Institute, and the Theobald Smith Research Institute, 305 South St., Jamaica Plain, MA 02130. 2Bngham and Womens Hospital, Division of Infectious Dis- eases, 75 Francis St., Boston, MA 02115. Received August 24, 1990; accepted April 17, 1991. to pyrimethamine and (b) requirement for small vol- umes of plasma, which also allows duplicate analyses on the same sample. The analysis is done with a standard isocratic reversed-phase HPLC system with a 210-nm detector and a recorder/integrator. Materials and Methods Solutions. A 50 mgfL stock solution of pyrimethamine (Sigma Chemical Co., St. Louis, MO) was prepared in acetonitrile (J. T. Baker, Phillipsburg, NJ). Working standards of pyrimethamine (100, 250, 500, 937.5, and 1250 tg/L, corresponding to 4, 10, 20, 37.5, and 50 ng of pyrimethamine per 40-FL injection) were prepared from the stock solution by dilutions with the mobile phase. The mobile phase, sodium phosphate buffer (0.1 mol/L, pH 2.5)/acetonitrile (79/21, by vol), contained tetrabutyl- ammonium hydroxide (Fisher Scientific, Fair Lawn, NJ), 0.5 mmolJL. Before use, we filtered the mobile phase through a 0.45-ian pore-size Nylon 66 filter under reduced pressure. Instrumentation. The HPLC system consisted of a Model 2150 HPLC pump (LKB, Piscataway, NJ), a 250 x 4.6 mm (i.d.) 5-sm particle size Ultrasphere C18 Altex column (Alitech Assoc., Deerfield, IL), a Model 7126 sample injector (Rheodyne, Cotati, CA) fitted with a 100-pL loop, a Model 166 Programmable ultraviolet detector (Beckman Instruments, San Ramon, CA) oper- ating at 210 nm and 0.01 absorbance unit full-scale, and a Model PC-8300 controller (NEC, Wood Dale, IL). The detector was connected to a Model LCI-100 computing integrator (Perkin-Elmer, Norwalk, CT) operating in the peak-height mode. Extraction of pyrimethamine from plasma. To a 15-mL silanized screw-top glass centrifuge tube containing 100 p.L of heat-inactivated plasma (at 56#{176}C for 30 mm) (17), add 650 L of distilled water and 250 p.L of 2 mol/L sodium hydroxide reagent. Add 1 rnL of acetonitrile to the sample, mix briefly, then add 5 mL of methylene chloride (Fisher Scientific). Vortex-mix for 1 mm, then centrifuge at 450 x g for 10 mm and remove the lower (organic) phase into a 15-mL silanized glass centrifuge tube. Evaporate the solvent at 37 #{176}C under a stream of nitrogen. Re-extract the mixture as above, and combine the organic phase with the dried first extract, and again evaporate the solvent. If the extracted sample is not analyzed immediately, store it no longer than four days at 4#{176}C until analysis. Reconstitution and analysis. To reconstitute the dried organic extract, add 21 L of acetonitrile and vortex- mix, then add 79 j.tL of 0.1 mol/L sodium phosphate buffer (pH 2.5) and again vortex-mix. Analyze two 40-L aliquots from each sample by HPLC with a flow