Determination of Chloral Hydrate, Trichloroacetic Acid, Trichloroethanol, and Urochloralic Acid in the Presence of Each Other and in Tissue Homogenates Bernard E. Cabanal and Peter K. Gessner2 Pharmacology Departmenf, School of Medicine, State Uniwrsity of New York at Buffalo, Buffalo, N . Y .
STUDIES OF THE KINETICS of chloral hydrate metabolism in mice required sensitive analytical procedures for the estimation of chloral hydrate and its metabolites: trichloroacetic acid, trichloroethanol, and urochloralic acid in tissue homogenates and in the presence of each other. Although a number of methods are available for the estimation of chloral hydrate, trichloroacetic acid, and trichloroethanol in mixtures thereof, most require prior quantitative separation of trichloroethanol from the other components of the mixture ( I , 2). Friedman and Cooper (3) have published a method for the determination of chloral hydrate, trichloracetic acid, and trichloroethanol in the presence of each other. The method involves subjecting two aliquots of the same sample to the Fujiwara reaction ( 4 ) , one of the aliquots having been previously heated with alkali so as to destroy chloral hydrate. These authors took advantage of the fact that all three compounds form chromophores with absorption maxima at 370 m p but only trichloroethanol forms a chromophore possessing a second absorption maximum a t 440 mM. The simplicity of the method makes it rather attractive. However, these same authors comment that absorption of chloral hydrate and trichloroethanol is not strictly additive. Probably for this reason the method was not used in later studies by Mackay and Cooper (5)and has failed t o find acceptance as evidenced by the continued development and use by investigators in this area of more complex methods involving prior separation of trichloroethanol (6-8). We have reinvestigated this procedure and have confirmed that, if trichloroethanol is present in the mixture being assayed, absorbance measurements a t 370 mp fail to exhibit the principle of additivity. We find, furthermore, that the chromophore formed by either chloral hydrate or trichloroacetic acid (Chromophore I) absorbs significantly at 440 mp. AccordPresent address, Pharmacology Department, Bristol Laboratories, Syracuse. N. Y . Please address correspondence to this author.
(1) T. C. Butler. J . Pliurmuco/.~92,49 (1948). (2) E. K. Marshall, Jr., and A. H. Owens, Jr., BUN. Joliris Hopkbis Hosp., 95, 1 (1954). (3) P. J. Friedman and J. R. Cooper, ANAL. CHEM., 30, 1674 (1958). (4) K. Fujiwara, Srrzber: Abliuridl. Natirrforsch. Ges. Rostock, 6 , 33 (1914). (5) F. J. Mackay and J. R. Cooper, J . Pliurmucol., 135, 271 (1962). (6) L. Buchel, Arch. Sci. P/iysio/., 23, 115 (19640). (7) Zbid.. p. 225 (1964b). (8) E. R. Garrett and H. J. Lambert. J . Pl1ur.m. Sci.: 55, 812 (1966).
0.8
1
0.7
0.5 1
O'
460
450 500 550 Wavelength (millimicrons)
600
Figure 1. Absorption spectra of ( A ) Chromophore I and ( B ) Chromophore I1 ingly we have developed a n alternative procedure. This procedure takes advantage of the fact that a t 540 mp, the wavelength corresponding to a second absorption maximum of Chromophore I, there is no significant absorption of the chromophore formed by trichloroethanol (Chromophore 11). This is the same maximum as that originally used in the determination of chloral hydrate and trichloroacetic acid by Butler ( I ) and Marshall and Owens ( 2 ) . The procedure also takes advantage of the fact that although a t 440 m p (the wavelength corresponding to the second absorption maximum of Chromophore 11) there is significant absorption by Chromophore I (Figure l), the principle of additivity is adhered to. Furthermore, in this procedure, by subjecting an aliquot to hydrolysis prior to the Fujiwara reaction, it is possible t o determine urochloralic acid by difference. Finally by use of appropriate simultaneous equations, this procedure allows estimation of all four compounds in a sample containing a mixture thereof. EXPERIMENTAL
Reagents. Chloral hydrate, U.S.P., mp. 51-52' C, trichloroacetic acid, pyridine, and 5-sulfosalicylic acid, all of reagent grade, were purchased from the Baker Chemical Co. Pyridine was purchased in 1-pint bottles, and kept in the dark. A fresh bottle of pyridine was opened a t least every two weeks, high blanks resulting from bottles kept for longer periods of time. 2,2,2-Trichloroethanol (technical grade) was purchased from the Aldrich Chemical Co. and further VOL. 39,
NO. 12, OCTOBER 1967
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Comparison of Absorbance by Chromophores I and I1 When Formed Separately and When Formed in Same Solution Comoosition of solution Concenand wavelength tration, ~ _ _ Absorbance _ 440 mp 540 mp 370 mr Chromophore Compound rg/ml Mean f s.e. Mean f s.e. Mean i see. I Chloral hydrate 25 0.600 i 0.004 0.022 + 0.002 0.258 i 0.002 I1 Trichloroethanol 25 0.179 i 0.003 0.210 i 0.002 0.005 i 0.001 Chloral hydrate 25 0.637 i 0.002 I and I1 0.231 i 0.003 0.262 f 0.003 Trichloroethanol 25
Table I.
purified by fractional distillation a t 20 mm Hg, followed by recrystallization from heptane a t low temperatures. The recrystallized trichloroethanol had a melting point of 15-16" C. Apparatus. The UV absorption spectra were determined using a Beckman DB-G Spectrophotometer. The absorbance measurements of the chromophore were made using a Beckman DU Spectrophotometer. Melting point determinations were taken on a Kofler hot stage microscope apparatus (A. H. Thomas Co.). Assay Procedures. Procedure A can be used to determine either chloral hydrate or trichloroacetic acid in the range of 6-60 pg and trichloroethanol in the range 5-80 pg. If both chloral hydrate and trichloroacetic acid are present, procedure A will determine the sum of these, necessitating the determination of trichloroacetic acid separately by procedure B. Procedure C is used to determine urochloralic acid. Procedure A. To a 1.0-ml test sample in a test tube is added 5.0 ml of pyridine. The contents of the tube following this, and indeed every other addition, are thoroughly mixed by use of a Vortex mixer. The tube is placed in an ice bath for 3 min, 2.0 ml of 10M potassium hydroxide is added, the tube is transferred to a boiling water bath for 5 min and then returned to the ice bath for a further 5 min. A 3.0-ml aliquot of the pyridine layer is now transferred to a second test tube which is also kept in the ice bath. Finally, immediately prior to transfer to a cuvette, 0.5 ml of water is added to the pyridine for clarification. The absorbance of the sample is determined a t 440 and 540 mp. Among the steps of Procedure A, the placing of samples in a n ice bath for a period of 3 min following addition of the pyridine, but prior to the addition of the alkali, allowed less water to be used in the final clarification procedure, and increased the sensitivity of the assay. Procedure B. To a 1.0-ml test sample in a test tube is added 2.0 ml of 10M potassium hydroxide. The test tube is placed in a boiling water bath for exactly 2 min, transferred to a n ice bath for 5 minutes, 5.0 ml of pyridine is added, the tube placed in a boiling water bath for 5 minutes and returned t o an ice bath for a further 5 minutes. A 3.0ml aliquot of the pyridine layer is now transferred to a second test tube which is also kept in the ice bath. Clarification of the pyridine is achieved as in procedure A and the absorbance is determined at 540 mp. Procedure C. To a small flask containing 3.0 ml of the test sample is added 3.0 ml of 12N hydrochloric acid. The flask is then stoppered, incubated at 80" C for 16 hours, cooled, and a 1.0-ml aliquot of the hydrolyzate is assayed by procedure A, 1 6 M rather than 10M potassium hydroxide being used. The potassium chloride which is formed can lead t o the formation of a gel-like substance in the pyridine layer. This gel can be readily disrupted by centrifugation a t 500 g for 5 minutes. Preparation of Tissue Extracts. Mice are killed by cervical dislocation and are immersed immediately in liquid nitrogen for 2 minutes. The mice thus frozen are coarsely pulverized 1450
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ANALYTICAL CHEMISTRY
by use of a stainless steel cylinder and two closely fitting stainless steel pistons and are homogenized in a Waring Blendor for 2 minutes with 7 volumes of precooled (0" C) 8 % sulfosalicylic acid. The homogenate is transferred to a second Waring Blendor equipped with a Willem Polytron Assembly (Will Scientific, Inc.), homogenized for a further two minutes, and allowed to stand, with occasional shaking, a t 0" C for 45 minutes to allow complete protein precipitation. Thereafter, sizeable portions of the homogenate are centrifuged at 500 g for 5 minutes and the supernate filtered through 2 layers of Whatman No. 2 filter paper. The filtered supernates are then assayed. Recoveries from Tissues. When determining the tissue content of these compounds, all calculations should be based on contemporaneously run recoveries of known amounts (30-60 pg) of the compounds from the sulfosalicylic acid containing tissue homogenates. Specifically recoveries should be run of trichloroethanol by procedures A and C , of trichloroacetic acid by procedure B and of either trichloroacetic acid or chloral hydrate by procedure A, the recoveries of these two compounds by procedure A not being significantly different (see Table 11). For trichloroacetic acid and trichloroethanol, compounds having relatively long half lifes in vivo, we find recoveries obtained by addition of the compound to the sulfosalicylic acid containing tissue homogenate to be identical to those obtained by injecting the compound to mice 20 seconds prior to their being killed. Statistical Procedures. The least squares regression coefficient of absorbance on the concentration of the trichloro compound, as determined by a given procedure, was calculated using a Fortran IV computer program. This same program was used to determine the significance of the differences between regression coefficients as described by Batson (9). The significance of differences between means was determined using Students T test. RESULTS AND DISCUSSION
When subjected to the Fujiwara reaction, chloral hydrate and trichloroacetic acid give rise to the same chromophore (Chromophore I) with absorption maxima a t 370 and 540 mp. Trichloroethanol, when subjected to the same reaction forms a chromophore (Chromophore 11) with absorption maxima a t 370 and 440 mp. The non-additivity at 370 m p of the absorption of Chromophore I and Chromophore I1 when these are formed in the same solution (3) was confirmed. Assays were run in quintuplicate, using procedure A, on solutions containing 25 pg/ml chloral hydrate, 25 pg/ml trichlorethanol and a mixture containing 25 pg/ml of each chloral hydrate and trichloroethanol. The sum of absorptions of Chromophore I and Chromophore I1 when these are formed separately (Table I) is significantly (9) H. C. Batson, "An Introduction to Statistics in the Medical Sciences," Burgess Publishing Co., Minneapolis, 1963, p. 60.
Table II. Regression Coefficients of Absorbance on Concentration of Trichloro Compound Being Assayed Regression Coefficients with %e. Whole body Recoveries Trichloro Standard solutions, homogenates, from whcle body compound Procedure A, mp absorbance/pg/ml absorbance/pg/ml homogenates P Chloral hydrate A 540 0.0116 + 0.0002 0.0106 =t0.0003 91.4 0.05) from each other as would be expected, given the same chromophore is formed in each case. Therefore, using the convention, T C E ~ ~ O=A contribution by the trichloroethanol in the mixture t o the absorbance a t 440 mp following procedure A, and the abbreviations CH, TCA, and UCH for chloral hydrate, trichloroacetic acid, and urochloralic acid, respectively, we can write (P
fi = TCAMOA/TCAS~OB
(7)
Substituting from 7 into 2,
fi
~ ~ O . B
(8)
- X540B . fi
(9)
T C A B ~ O=AX and substituting 8 into 3 CHUOA=
X540A
Further substituting 1 into 8 and 9 we can write: T C ~ O=Af i . fi
. XSIOB
(10)
and CHUOA= f i ( X 5 4 0 ~- XSIOB. fi)
(11)
substituting 10 and 11 into 5 TCEUOA=
X440A
-fi
.
XSIOA
(12)
and finally since the ratios T C A ~ ~ O A / T C AC~H~ O ~ ~CO, A / C H ~ ~ O C , and T C E ~ ~ O A / T C Edo ~ J not O C differ significantly (P < 0.01) from unity we can subtract Equation 5 from 6 to give UCH44oc
= XGOC
- X44o~
(13)
Thus the amounts of trichloroacetic acid, chloral hydrate, trichloroethanol, and urochloralic acid in a mixture can be calculated by use of Equations 2, 9, 12, and 13, respectively. We find the regression of absorbance on the concentration of the trichloro compound to be linear for both standard solutions and tissue homogenates in the concentration ranges studied-Le., 5-60 pg/ml for chloral hydrate and trichloroacetic acid and 5-80 pg/ml for trichloroethanol. The results are summarized in Table 11. In each case recovery from tissue was significantly lower than from standard solution. Several other factors are worth noting. The regression coefficients for chloral hydrate and trichloroacetic acid for procedure A are almost identical for recovery from standard solutions and from tissues which reinforces the concept that the same fi = CH~~OA/CH = ~C~HO~A~ O C / C H =~ ~ O C chromophore is involved. Procedure B leads to a 10.4% TCA~*OA/TC&~ =OTAC A ~ . ~ O C / T C A(1) ~ ~ O C decrease for the recovery of trichloroacetic acid from standard solutions as compared to Procedure A. This difference Further, using the convention, is significant (P < 0.05) and can be attributed to a partial x440.4 = absorbance of an unknown solution at 440 mp decomposition of the trichloroacetic acid by the treatment following procedure A, with hot alkali. Procedure B is seen to lead to an even we can write: greater decrease, relative to Procedure A, when recoveries from tissues are considered. This latter decrease (39.9x) is X ~ ~ O = BT C A S ~ O B (2) not only significant in itself ( P < 0.001) but is also significantly XMOA= TCASIOA C H ~ ~ O A (3) (P< 0.001) greater than the observed difference in recoveries from standard solutions and can be attributed to an interx540C = TCAs4oc CHstoc (4) ference with chromophore formation or quenching of the chromophore by the alkali treated tissue supernatant. The XMOA= TCAUOA -t- CHMOA T C E ~ ~ ~ A ( 5 ) recovery from tissues of trichloroethanol or trichloroacetic xuoc = T C A ~ ~ O C CHt40c TCE44oc UCH44oc (6) acid following i.p. injection to live animals (84.2 and 87.3% and respectively) is entirely comparable with the recoveries of
+ +
+
+
+
VOL. 39, NO. 12, OCTOBER 1967
1451
these compounds following their addition to the sulfosalicylic acid homogenate (84.2 and 89.0%, respectively ; Table 11). Urochloralic acid, the glucuronic acid conjugate of trichloroethanol, yields trichloroethanol on hydrolysis. We have found that when mice are injected with 500 mg/kg of trichloroethanol, killed between 1 and six hours later and the trichloroethanol is estimated after subjecting tissue supernatants to hydrolysis, as described in procedure C, 97.3 i 0.7 of the injected trichloroethanol can be accounted for. If the trichloroethanol content of tissue is determined by procedure A, that is without prior hydrolysis, trichloroethanol is found to have a biological half life of 3.5 hours. We conclude, therefore, that the difference in recoveries of trichloroethanol by procedures A and C is due to a metabolite which on hydrolysis yields trichloroethanol. The only known metabolite which will do this is urochloralic acid.
procedure used. When whole body homogenates are being assayed, it is, therefore, essential to take into account tissue recovery values. Similar consideration would presumably apply to other biological materials. Furthermore, the chromophore formation is affected by minor variations in the temperatures and time intervals of the assay procedure. Accordingly calculations should always be based on contemporaneously run recoveries of known amounts of the compounds from tissue homogenates. Since the recoveries from tissue homogenates of trichloroacetic acid and chloral hydrate using procedure A are not significantly different, the necessity to run both is obviated. The considerable decrease in sensitivity which occurs for tissue recoveries of trichloroacetic acid using procedure B should be particularly stressed. Failure to take this fact into account when analyzing mixtures containing both chloral hydrate and trichloracetic acid leads to an erroneously high value for the former and low values for the latter.
CONCLUSIONS
The procedures described herein afford a sensitive, accurate, and reproducible method for the estimation of chloral hydrate and its metabolites, trichloroethanol, trichloroacetic acid, and urochloralic acid, in whole body homogenates without necessitating prior extraction or separation procedures. Other compounds possessing trihalogens substituted carbon atoms can undergo the Fujiwara reactions ( 4 ) and would be expected, if present, to interfere with the assay. When tissue recoveries are performed, there always occurs a significant loss of sensitivity, although the magnitude of this effect differs with the chromophore being formed and the
ACKNOWLEDGhIENT
We thank the Computing Center of the State University of New York at Buffalo for the use of its equipment. This Computing Center is partially supported by National Institute of Health Grant FR-00126 and National Science Foundation Grant GP-5675. RECEIVED for review May 15, 1967. Accepted June 26, 1967. Investigation supported by a grant from the Licensed Beverage Industries and by U. S. Public Health Grants 5T1 GM 107 and R01 MH 12542.
Gas Chromatographic Determination of Normal Paraffins iln Kerosine H. S . Knight Shell Decelopment Cornpanj*,Emerycille, Calif. A SUBTRACTION METHOD for the determination of normal paraffins in kerosine and gas oil was developed by Whitham ( I ) . The sample was analyzed twice by gas chromatography, once with and once without a Molecular Sieve 5A column for selectively sorbing the normal paraffins (normals). By comparison of the two independent curves, Whitham was able to calculate the normals content. This method is not very sensitive. Eggertsen and Groennings ( 2 ) developed a more elaborate gas chromatographic method in which the normals were released from the sieve by heating. Only one sample was needed and the normals and non-normals areas were observed independently, which resulted in improved sensitivity. The method, limited to gasoline, employed a specialized apparatus for programming the sieve and analytical columns independently and oxidizing the effluent to COz and water prior to detection. By modifying the apparatus and the temperature programs Blytas and Peterson were able to extend the method to the kerosine range (3).
Ponnamperuma and Pering ( 4 ) and Brunnock (5) reported on methods for recovering the normal paraffins from the sieve by destroying the sieve lattice with H F and extracting the released normals, The need for specialized gas chromatographic apparatus is thus avoided, particularly if the sorption step is also independent of the G C equipment. The sorption of the normals external to the G C is usually carried out as described by Brunnock. The sample is diluted with a non-normal solvent and an excess of molecular sieve is added. The mixture is then refluxed until the normals are all sorbed. This takes many hours. In the present work the kerosine is added directly to an excess of sieve and the free-flowing mixture is warmed to 100"120" C for half an hour. This completes the sorption step. The sieve is washed free of non-normals and is then destroyed with dilute H F and the normals are extracted with isooctane. The more complicated recovery procedure used by Brunnock for higher boiling normals is not necessary for kerosine. H F is hazardous and the usual handling precautions must
(1) B. T. Whitham, Nature, 182, 391 (1958). (2) F. T. Eggertsen and S. Groennings, ANAL.CHEM., 33, 1147 (1961).
(3) G. C . Blytas and D. L. Peterson, ANAL. CHEM.,39, 1434 (1967). (4) C. Ponnamperuma and K. Pering, Nafure, 209, 979 (1966).
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ANALYTICAL CHEMISTRY
