Automated analysis for thiourea and its derivatives in biological fluids

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SHORT

Automated

CON~IUSICATIOKP

Analysis for Thiourea and in Biological Fluids1

Its Derivatives

The method of Williams et (11. (1) for the analysis of thiourea and its clcrivntivcs has been adapted to the Technicon AutoAnalyzer. These compounds react with Grotc’s reagent (2) to produce a blue color. The automated procedure for thiouren concentrations of 0.1 to 15 111111 is diagramiiicd in Fig. 1. Grote ‘s reagent is prepared as follows (2) : 1.0. TUBE SIZE

CONSTANT TEMPERATURE DIALYZER 38°C

BATH

DISCARD c Plus 0.5ml/L

40 ft COIL 1.6 m m I.D.

I~I(:. 1. Ma.nifold

nsseml)ly

and

flow

Techmcon

Brij

diagram.

5.0 gm of sodium nitroferricyanide and 5.0 gm of hydrosylnmine hydrochloride are dissolved in 100 ml of mater in a beaker; 10 gm of NaI-ICO,, is added and the beaker covered. When gas ceases to evolve, 1.0 ml of bromine is added and the beaker covered again. After gas evolution is complete, this solution is filtered, diluted to 1 liter and placed in a dark bottle. The diluted reagent was used for no longer than 1 week. If a precipitate should form in Grotc’s reagent, irregular (“noisy”) records will result. This difficulty can be removed by pIacing a small filter, prepared by lightly packing glass wool into :L piccc of ‘I’ygon tubing, in the line leaving the reagent bottle. Even if the records are not irregular, t,he precipitate may accumulate in t,hc heating bath. It ‘Supported by a grant Health Service, National

(AM-06700) to Dr. Institute of Arthritis

Nathan Lifson and Metabolic

from the Disease.

U. S. Public

YHORT

COblMUKIC.~TIOSS

359

can be removed by flushing the bath coils with water at, the end of the day’s mnlyses. Standards made up in water, 0.9% NaCl, or 0.03 A’ HCl yielded the same results. For the above-mentioned range of concentrations, optical density was a linear funct,ion of concentration. The maximum absorption peak was found to be 600 m/1. In this study the r:lte of wi~pling was rout#inely 20/‘hr, but lilnited espcricnw indicntccl th:ltS :i rate of 4O/hr nould provide equally atcccptable values. The manifold assembly in Fig. 1 can of course l)e moclificd. For PSample, for thiourea concentrations of 1OkjO ItiX, tlw sample tube was changed to a tube of 0.020” diameter; an(1 a tube of 0.110” diameter pumping water was joined to the tube between the clialyzcr and the bath. Fluids analyzed wtisfactorily for thiourcn includctl c:lninc blood, plasma, and serum, and :ko Ringer’s fluid and SaCl solution!: placed in loops of dog small intestine and removed several minutes later. The standard deviation for a series of replicate values was less th:ui 1% of the mwn value. For the above solutions the blank value was the same as watc’r and recovery of the coml~ountl was 96-102s. Tlic dcflertion was only l-2$ lers for :L given :miount of coml~~ound per unit volume of saline, pl:~sn~a, or blood than for water, and hcncc up t.o 205G nonsolvent volume in the uamplc had only n sm:tll effect’. There was no interference from glucose (27 mX), fructose (27 mill), sylox (7 mM), or urea (50 niN), with the concentrations in parentheses the highest tested. Results for mcthylthiourcn (I), trimethylthiourea (II), and methylrliethyltl~iourea (III ) , were similar to those for t~hiourca.’ With t,hc standard membrane (Ttschnicon rlwignntion) the optical clensity at a given conccntrntion for iI) was 0.98 of t’hat for thiourea; for (II) and (III) the corresponding values were 0.73 an11 0.56, rcspectivcly. According to analvses of the effluents from the tlialyzer, the donor and recipient, streams came to approximately 40, 35, 26, anti 237; of diffusion equilibrium for thiourea, (Ii, (II), and (III), rwpectiwly. n’hen thiourea was n~ltled to urine obtained either from normal humans or dogs, the values obtained UX’I’C ul) to 27% low. Crentinine, glycine, arginine, and :rlimine in concentratioiw of O.Sc/r in thiourea solutions lowered the thiourea values 4, 27, 11, and 7%, respectively. The urines studied gave :L positive blank value ; tile amino acids did not. \\:e have found that all the above remarks apply if a 6G0 mj~, filter is used instead of the designated one (600 1111~ 1, but the sensitivity is 60% ‘?Thiourra and its methyl derivative were obtained commercially. The trimethyl and methyl diethyl derivatives were synthesized from methyl isothiocpanate and the appropriate amine. The puritp of the lntt,er two was confirmed by melting point, mensurcmrnts and elem+xt:u-y analysis.

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SHORT

less. This offers analyzed withollt

I wish

to thank

COMMTJKICATIONS

a may to expand the range of concentrations any other modifications.

Dr.

Snthan

Lifbon

lor

his many

helpful

1. \VILI.I.\MS, R. H., JAXDONE~, IS. J., AND ICAY, G. -I., J. Lab. (1944). 2. GHOTE, I. W., J. b’iol. C~ICU~. 93, 25-30 (1931).

suggestions.

Clir~.

Med.

JULIENE

An Improved

Column

for Gas-Liquid

of Substituted

Cholanic

to be

A.

29, 329-336

GERFAST

Chromatography Acids

The +(al)arnt,ion of bile acids by gas-liquid chromatography (GIL 1 constitutes a significant advance in the analytical methodology of the substituted cholanic acids (1). A variety of st’ationnrp phases, selective :md nonsclcctirc, have been used to rharactcrize the gas-chromatog~a~~hic behavior of compounds in the cholanic acid series (2). Studies contluctcd in this laboratory (3-5)) in agrcceinent wit’11 published obeervations (6 ) , hart revealed that the best separations were obtainable with a fluoronlkyl ~~oly~ilosanc 1~11:~ (PP-1265) ,I which csliibit,s selective retcntion c+fcct:: on cpiincric hyrlrosyl and ketonin functional groups, and clistinguishce positional isoumkm in Ftcroirls. Ho~.c~‘cr, the merits of this phase have lmm seriously offset by the tailing of peaks as well as by the great difficulty mcounteml in obktining reproducible columns. Since our studier on mixed-phase columm have shown that it ir posail)lc to obtain appropriate mistum of polar ant1 nonpolar phases which would provide spcific rctcntion effects sought for in any system (7-10)) it was tlecnml rlcairnhle to forniulatc a siinilar column for bile acicls. During the courx~ of two years, rcveral combinations of FS-1265 with neopcntylglycol succinate polyester (NGS) and SE30 (relatively ’ Dow

Corning

Corporation.

Midland,

Michigan

; viscosity,

10,000

centistokes