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Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation\/emission wavelengths of 370\/463\u2009nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with \u03b3\u2010glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6\u2009ml\/min and consisting of a mixture (110:890:0.25 v\/v) of acetonitrile, ammonium acetate buffer (0.05\u2009<jats:sc>m<\/jats:sc>, pH=5.5) and hydrogen peroxide 30% (w\/w). The method was found linear over the concentration range of 25\u2013400\u2009nmol\/l. Its intra\u2010 and inter\u2010day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9\u2009nmol\/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis. Copyright \u00a9 2009 John Wiley &amp; Sons, Ltd.<\/jats:p>","DOI":"10.1002\/bdd.654","type":"journal-article","created":{"date-parts":[[2009,3,25]],"date-time":"2009-03-25T21:21:05Z","timestamp":1238016065000},"page":"138-148","source":"Crossref","is-referenced-by-count":25,"title":["An improved high\u2010performance liquid chromatography method for quantification of methotrexate polyglutamates in red blood cells of children with juvenile idiopathic arthritis"],"prefix":"10.1002","volume":"30","author":[{"given":"Milo\u0161","family":"Hroch","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jana","family":"Tukov\u00e1","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pavla","family":"Dole\u017ealov\u00e1","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jaroslav","family":"Chl\u00e1dek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2009,3,25]]},"reference":[{"key":"e_1_2_1_2_2","doi-asserted-by":"publisher","DOI":"10.1002\/1529\u20100131(199803)41:3<381::AID\u2010ART2>3.0.CO;2\u20103"},{"key":"e_1_2_1_3_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM199204163261602"},{"key":"e_1_2_1_4_2","doi-asserted-by":"publisher","DOI":"10.1002\/1529\u20100131(200008)43:8<1849::AID\u2010ANR22>3.0.CO;2\u2010F"},{"key":"e_1_2_1_5_2","doi-asserted-by":"publisher","DOI":"10.1002\/art.20288"},{"key":"e_1_2_1_6_2","doi-asserted-by":"publisher","DOI":"10.1136\/ard.60.8.729"},{"key":"e_1_2_1_7_2","doi-asserted-by":"publisher","DOI":"10.1186\/ar419"},{"key":"e_1_2_1_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0889-857X(05)70358-6"},{"key":"e_1_2_1_9_2","doi-asserted-by":"publisher","DOI":"10.1136\/ard.2005.048637"},{"key":"e_1_2_1_10_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.82.15.4881"},{"key":"e_1_2_1_11_2","doi-asserted-by":"publisher","DOI":"10.1042\/bj2360193"},{"key":"e_1_2_1_12_2","doi-asserted-by":"publisher","DOI":"10.1002\/1529\u20100131(199808)41:8<1407::AID\u2010ART9>3.0.CO;2\u2010H"},{"key":"e_1_2_1_13_2","first-page":"1830","article-title":"Methotrexate in juvenile idiopathic arthritis: Answers and questions","volume":"27","author":"Ravelli A","year":"2000","journal-title":"J Rheumatol"},{"key":"e_1_2_1_14_2","first-page":"118","article-title":"Predicting remission in juvenile rheumatoid arthritis with methotrexate treatment","volume":"20","author":"Wallace CA","year":"1993","journal-title":"J Rheumatol"},{"key":"e_1_2_1_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0889-857X(05)70359-8"},{"key":"e_1_2_1_16_2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI1554"},{"key":"e_1_2_1_17_2","doi-asserted-by":"publisher","DOI":"10.1093\/rheumatology\/keh401"},{"key":"e_1_2_1_18_2","doi-asserted-by":"publisher","DOI":"10.1093\/clinchem\/42.1.39"},{"key":"e_1_2_1_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/0021\u20109673(96)00257\u20109"},{"key":"e_1_2_1_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(86)22191-6"},{"key":"e_1_2_1_21_2","doi-asserted-by":"publisher","DOI":"10.3109\/00365518509155274"},{"key":"e_1_2_1_22_2","doi-asserted-by":"publisher","DOI":"10.1373\/49.10.1632"},{"key":"e_1_2_1_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021\u20109673(03)00776\u20103"},{"key":"e_1_2_1_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jchromb.2006.07.026"},{"key":"e_1_2_1_25_2","doi-asserted-by":"publisher","DOI":"10.1620\/tjem.215.95"},{"key":"e_1_2_1_26_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0378-4347(00)80710-0"},{"key":"e_1_2_1_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/0378-4347(87)80279-7"},{"key":"e_1_2_1_28_2","doi-asserted-by":"publisher","DOI":"10.1002\/art.20460"},{"key":"e_1_2_1_29_2","volume-title":"The Biochemistry of Folic Acid and Related Pteridines","author":"Blakley RL","year":"1969"},{"key":"e_1_2_1_30_2","unstructured":"CDER (Center for Drug Evaluation and Research) and CVM (Center for Veterinary Medicine) Guidance for Industry. 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