wavelength will decrease the interference due to Raman ;scatter but also, unfortunately, decrease sensitivity. Most of the interference can be eliminated through the use of polarizers instead of the usual entrance and exit slits supplied with the instrument. The polarizers have proved invaluable in assaying low concentrations of phenothiazines in biological samples, because extracts that do not have a high degree of light scattering are difficult to obtain. For determination of phenothiazines in biological samples, wi: have extracted the phenothiazines wit:? heptane after the samples were made s,trongly alkaline with 10% NaOH (8). The heptane solutions have been extracted wit,h 50% acetic acid and oxidized as described above. Table IV illustrates the recovery of five phenothiazines added to different types of biological samples. Details of the procedure for biological samples will be published elsewhere. The data presented provide a start’ing point for developing a fluorometric met,hod for phenothiazines. Differences in the spect,ra of the oxidized and unoxidized phenothiazines have been emphasized because both will usually be found in most biological samples. Because of t,he differences in spectra and fluorescence intensity, fluorometric methods are most’ applicable to the determinat,ion of total phenothiazines in
Table IV.
Per Cent Recovery. of Phenothiazines Added to Biological Samples
Type of sample 1Oy0rat liver homogenate 85 105 90
Phenothiazine added, pg. Human urine Human blood Methiomeprazine, I* 103 100 Thioridazine, 1 94 94 Fluphenazine, 1 107 96 Trifluoperazine, 1 103 88 88 Chlorpromazine, 5 105 110 98 a Piumbers are per cent of phenothiazine added to sample recovered in final 50% acetic acid extract,, determined by comparison to standard made up directly in 50‘7, acetic acid. Amount of drug added to 1 ml. of indicated type of sample.
a sample and are not well suited for the differential determination of oxidized and unoxidized phenothiazines. ACKNOWLEDGMENT
The authors acknowledge the technical assistance of Vincent Estevez. LITERATURE CITED
( 1 ) Berti, T., Cima, L., Arch. Intern. Pharmacodyn. 98, 452-8 (1954). (2) Dubost, P., Pascal, S., Ann. Pharm. F ~ a n c 11,615-19 . (1953). ( 3 ) Gomori, G. “JIethodsinEnzymology,” S. P. Colowick and N. 0. Kaplan, eds , J.01. 1, pp 138-46, Academic
Press, New York, 1955. (4) Massie, S. P., Chern. Revs. 54, 797-829 (1954).
( 5 ) Mellinger, T. S., Keeler, C. E., ANAL,
CHEM.35,554-8 (1963).
( 6 ) Nadeau, G., Sobolewski, G., Can. M e d . Assoc. J . 80,826-7 (1959).
(7) Ragland, J. B., Kinross-Wright, 5’. J., Federation Proc. 2 0 , 397 (1961). (8) Ragland, J. B., Kinross-Wright, 5’. J., unpublished data, 1963. (9) Salzman, N . P., Brodie, B. B., J . Pharmacol. Exptl. Therap. 118, 46-54 (1956). (10) Udenfriend, S., “Fluorescence Assay in Biology and hiedicine,” pp. 110-14, rlcademic Press, Xew York, 1962. (11) Udenfriend, S., Iluggrrn, I). E., Vasti, B. B., Brodie, B. B., J . Pharmacol. Ezptl. Therap. 120, 26 (1957). (12) Wechsler, hl. B., Forrest, I. S., J . Seurochem. 4, 366-71 (1959). RECEIVEI) for review Piovember 26, 1963. Accept,ed March 10, 1964. Investigation supported in part by research grant hlH04578 from t,he National Institute of hIent,al Health, Public Health Service.
Determination of Submicrogram Amounts ot Selenium in BioIogicaI Materials W . H. ALLAWAY and EARLE E. CARY Agricultural Research Service, U.
S. Department of
b A method of measuring the Se content of dry and liquid biological materials is described. The sample is burned by the oxygen-flask technique, and the products of combustion are collected in 0.5N HCI. Se is separated by coprecipitation with As. The precipitate is dissolved in 1 : 1 “ 0 3 and the Se concentration measured fluorometrically after reaction with 2,3diaminonaphthalene (DAN). NH20H is used to minimize oxidation of the DAN during this reaction. Agreement with neutron activation analyses, recovery of added selenite, and svandard deviations are within 2~0.02pg. of Se. The method is adapted to II wide variety of materials without errors from interferences.
T
H E D I ~ C O V E R I that very small amounts of de will protect different animal species from certain nutritional diseases has created a need
Agriculture, Ifhaca,
N. Y.
for methods of measuring very low levels of t,his element in diets and in animal tissues. The significance of Se in nutrition has been reviewed by Scott ( I O ) , xvho points out that the critical level for deficiency of Se in poultry diets is about 0.04 p.p.m. X number of new methods for the determination of Se have appeared in response to this need ( 2 , 4, 6 , 1 1 ) . These newer methods are usually based upon measuring the fluorescence of the compound formed by the reaction of Se(1V) with an aromatic diamine. Isotopic dilution is included in some procedures ( 3 ) . Seutron activation procedures have also been used for measurement of low levels of Se in biological materials (1, 8). The method described here makes use of the oxygen-flask technique as described by Gutenmann and Lisk (6) and by Dye et aE. (4) to avoid losses of Se during the ashing of the sample. The
Se is coprecipit’ated with As according to the procedure of Cousins ( 2 ) to isolate it from interfering elements. Se(1Tr)< obtained by dissolving this precipitate in “Os, is reacted with 2,3-diaminonapthalene (DAY) and its fluoresence in Decalin is meabured after the procedures of Parker and Harvey ( 9 ) and Cukor, W-slzcyk, and Lott ( 3 ) . Oxidation of the D*iX or the Se during the formation of Se-11-W is minimized by the use of a low concentration of hydroxylamine in the reaction mixture.
EXPERIMENTAL
Reagents. Use double deionized or distilled plus deionized water. * ~ R S E N I C SOLUTION.Dissolve 0.25 gram of As203 (osidimetric standard) and 10 pellets of reagent grade S a O H in approsimately 10 ml. of water. Dilute to 200 ml. VOL. 36, NO. 7, JUNE 1964
1359
REDUCING SOLUTION.Mix two parts of concentrated HC1 and one part of 30% reagent grade hypophosphorus acid. Prepare daily as needed. STABILIZING SOLETION. 0.025M ?ja2EDTA in 2.57, hydroxylamine hydrochloride. DIAhiINONBPHTAALENE (D.kx). Dissolve 0.1 gram of 2,3-dianiinonaphthalene (hldrich Chemical Co.) in 100 ml. of 0.1-V HCI that also contains 0.5 gram of hydroxylamine hydrochloride. Extract by shaking with 10 mI. of Decalin in a separatory funnel, retaining the lower (water) phase for use and discarding the Decalin phase. This reagent should be prepared fresh daily and protected from all but yellow light during preparation and use. DECALIX-. Decahydronaphthalene
