(3) zyxwvutsrqponm D. T. Salt, B. Arret, and J. Wilner, zyxwvutsrqp J. Pharm. Sci., zyxwvut 62, 2040 zyxwvut (4) R. W. Squires and S. E. Hartsell, Appl. Microbiol., 3,40 (1955). zyxwvutsr (5) D. H. Howard, J. RacterioL, 71,625 (1956). (6) zyxwvutsrq S. W. Hwang, "Freezing and Drying of Microorganisms," Uni- (7) B. J. h y e t and J. F. Keane, Jr., Riodynarnica, 139,119 (1952). ACKNOWLEDGMENTS The authors thank J. Felix, M. Emery, and F. Mamaril, Quality Control Department, Hoffmann-La Roche, for providing technical consultation and for reviewing this manuscript. In addition, they thank C. Bernhart for her technical consultation and R. Russomanno of the same Quality Control Department for his interest and encouragement. (1973). versity Park Press, Baltimore, Md., 1969, p. 169. Thiamine Whole Blood Pharmacokinetics in Rats Using Both a Specific 35S-Thiamine Liquid Scintillation Assay and the Thiochrome Fluorescence Assay J. D. PIPKIN and V. J. STELLAX Received June 16,1977,from the Department zyxwvutsrq of Pharmaceutical Chemistry, Uniuersity of Kansas, Lawrence, KS 66045. publication September 30, 1977. Accepted for Abstract zyxwvutsrqp 0 The study of factors altering the CNS and GI absorption of thiamine In rats required the development of a specific assay for thi- amine from 10O-pl samples of blood and plasma and small quantities of tissue. The specific thiochrome fluorescence assay for thiamine was modified to handle microsamples and to use "S-thiamine. This sensitive and specific radioassay using "S-thiamine gave pharmacokinetic pa- rameters for 4-mg/kg iv doses of thiamine in rats equivalent to those using the less sensitive thiochrome fluorescence assay. The new assay, because of its lower limit of detection, allowed the study of the time profile of thiamine after a 1-mg/kg iv dose in rats. Such a time profile could not have been followed using the standard thiochrome fluorescence assay. Keyphrases Thiamine-radiochemical analysis in blood, compared to fluorometric analysis, pharmacokinetics in rats Radiochemistry- analysis, thiamine in blood, compared to fluorometric analysis 0 Phar- macokinetics-thiamine in rats 0 Vitamins-thiamine, radiochemical analysis in blood, compared to fluorometric analysis, pharmacokinetics in rat The thiochrome (11, Scheme I) fluorescence assay for thiamine (I) in biological fluids was described previously (1-12). Specific assays for thiamine with either 14C- or "S-thiamine utilized electrophoric separations (12) or t,he ion-exchange system as in the thiochrome fluorescence assay (1).The study (1) using ion exchange did not, how- ever, determine assay specificity in an in vivo system. The present paper reports the modification of the spe- cific thiochrome fluorescence assay for thiamine in whole blood to its use with microsamples and "S-thiamine and demonstrates its use in following the pharmacokinetics of intravenously administered thiamine at low doses to rats. Quantitative studies of factors altering the central nervous system (CNS) and GI absorption of thiamine (vitamin B,) have been limited. Thiamine, being a very water-soluble compound with a quaternary nitrogen, is poorly absorbed into the CNS (13) and poorly absorbed from the GI tract (14-17). Thiamine passes through those barriers because it is actively transported (13-17). How- ever, as with other active transport systems, thiamine absorption is both saturable and/or easily inhibited (13-17). These absorption characteristics have been im- plicated in Wernicke's encephalopathy (14-18) in alco- holics, in the terminal children's disorder Leigh's disease (l9-27), and in polioencephalomalacia in feedlot cattle (28, 29). A study of thiamine pharmacokinetics in whole blood as a preliminary to the study of the effects of lipid-soluble thiamine prodrugs (30) in altering thiamine distribution required the development of a specific and sensitive assay for free thiamine (nonphosphorylated) and total thiamine (thiamine plus its mono-, di-, and triphosphate esters) in 100-~1 samples of whole blood and plasma and small quantities of tissue, e.g., brain tissue. EXPERIMENTAL Reagents and Materials-All reagents were analytical grade, and all aqueous solutions were prepared using glass-distilled deionized water. The primary source for reagent preparation and storage was the review of Mickelsen and Yamamoto (6). Reagent preparation and storage also The 35S-thiamine was obtained commercially1as two different lots of varying specific activity. Radiochemical purity was determined by paper chromatography and TLC, and chemical purity was checked by IR spectrophotometry. Thiamine hydrochloride was obtained commercially2 with its purity confirmed by bioassay. CH 3 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR dkd I [H,cJ~yJtcH,cH,o,l - - c'- KEZT *HC'l I were discussed elsewhere (1-12). JQr2XCH> CH,CH,OH H ,C I1 * = '-Slabel Scheme I AmershadSearle Corp. Arlington Heights, Ill., lotA05006, specific activity 176 mCi/mmole and lot E06046, specific activity 233 mCi/mrnole. 2 Sigma Chemical Co., St. Louis, Mo. 0 18 / Journal of Pharmaceutical Sciences