spectrometry would probably be detectable a t a concentration a t least one order of magnitude lower with the above-described experimental setup. Finally, it was noticed t h a t the properties manifested by the argon-hydrogenentrained air flame studied by Veillon et al. ( 2 ) , with respect to kind and degree, were not appreciably different from those manifested by the Hz/entrainedair flame in this investigation. I n view of its greater experimental simplicity, the H2/entrained-air flame may prove to be the more desirable flame. Work is currently in progress in this laboratory t o further develop the analyt-
ical usefulness of this flame in atomic fluorescence flame spectrometry. ACKNOWLEDGMENT
I t is a pleasure to acknowledge the nunieroue discussions with Janies D. Rinefordner, whose earlier work stimulated us to undertake this investigation. LITERATURE CITED
(1) Mansfield, J. AI., Winefordner, J. D., Veillon, C., ANAL. CHEM. 37, 1049
(1965).
( 2 ) Yeillon, Claude, Mansfield, J. AI.,
Parsons, AI. L., Winefordner, J. D. Ibid., 38, 204 (1966). (3) Winefordner, J. D., Staab, R. A., Ibtd., 36, 165 (1964). (4) Winefordner, J. D., Tickers, T. J., Ibid., 36, 161 (1964). D. W. ELLIS D. R . DEMERS Department of Chemistry University of New Hampshire Durham, N. H. 03824 WORKsupported in part by funds provided by the LT. S. Department of the Interior as authorized under the JT7ater Resources Research Act of 1964, Public Law 88-379 through the Water Resources Research Center of the University of Kew Hampshire.
Rapid Micromethod for Gas Chromatographic Determination of Blood Barbiturates SIR: Studies on the gas chromatographic behavior of barbiturates have been reported (2, 3, 7 , I S ) . These experiments have shown that, in general, the gas chromatography of barbiturates presents no special problems although tailing occurs with certain members of the group. Techniques designed t o reduce tailing have been described. These include the use of mixed liquid phases (19) as well as the incorporation of high molecular veight organic acids into the liquid phase ( 6 ) . Martin and Driscoll (11) minimized tailing by reducing the polarity of the barbiturate nucleus. This was accomplished by methylation with dimethyl sulfate to form the less polar 1,3-dimethyl barbituric acid derivatives. BrochmannHanssen and Svendsen (4) found that while it was not possible to separate all nienibers of a series of 21 barbiturates on a single column, all compounds could be distinguished by using 2 columns. one v-ith a nonpolar liquid phase and another with a moderately polar polyester phase. Reith, van der Heide, and Zwaal (14) too found that Xpiezon L would not adequately resolve all members of a series of 16 barbiturates. The formation of derivatives, however. permitted differentiation of unresolved pairs. Gas chromatography has been employed for the determination of barbiturates in blood ( 1 1 , 13, 14). The methods described are suitable for qualitative and quantitative analysis if adequate sample, 1 to 5 ml., is available. All, however, involve solvent extraction, filtration, and evaporation and thus are somewhat time consuming. Jain, Fontan, and Kirk ( I O ) have described a rapid micromethod for the deterniination of blood barbiturates that minimizes the preparative methodology required. Although detailed recovery studies were not reported by
these workers it appeared that recoveries of S5-9070 were obtained. In studying the metabolism of barbiturates, rapid, sensitive, and specific micromethods are required. Such methods must also be quantitatively reliable, The present paper describes such a method based on the gas chromatographic analysis of chloroforni extracts of small quantities of blood. Because of the small sample size required, the method can be used for the determination of barbiturate half-lives in single rats. I n addition the method should be useful for screening toxicological specimens for the presence of barbiturates. EXPERIMENTAL
Apparatus. A Barber - Colman Series 5000 gas chromatograph equipped with a flame ionization detector was used. The electrometer (Xodel 5040) was modified as described by Gutenmann and Lisk (8) to provide additional and 3 X ranges of 3 X IO+, ampere. The electrometer mas operated at 3 x ampere with an attenuation of 1. The column was a C-shaped borosilicate glass tube, 3.5 nim. i.d. and 5 feet long, packed with 3.4% silicone fluid DC 200 (12,500 cstks.) on Gas-Chroni Q. The column packing was prepared by the solution technique (9) using 100 ml. of a 2% (w,/v.) solution of DC 200 in toluene per 25 grams of Gas-Chrom Q. The Gas-Chrom Q was used as obtained from the supplier. The packed column was conditioned overnight a t 250" C. with the carrier gas flowing. The column temperature was adjusted to give a retention time of 7 to 9 min. and ranged from 153" to 179' C. (Table I). The detector bath and injector temperatures were 250" C. and 275" C., respectively. The nitrogen, hydrogen, and air pressures were 15, 22, and 40 p.s.i.g., respectively. Extractions were performed in a 5-ml. centrifugal separator (Kontes KO. K-
41445). An aluminum disk, punched from household aluininuni foil with a size 2 cork borer, was inserted above the septum before each use t o prevent extraction of interfering materials from the septum. Procedure. Before experimental samples were injected into the gas chromatograph 10 p1. of a 0.1% (w./v.) solution of the respective barbiturate in chloroform was injected t o saturate the column. A standard curve prepared by injecting 1 to 3 pl. of a 0.01% (w./v.) chloroform solution of the respective barbiturate, measuring the peak height and computing the number of peak height units per 0.1 pg. of barbiturate injected mas run both before and after experimental samples mere analyzed. Recovery Studies. Barbiturates, either as aqueous solutions of the sodium salts or as concentrated methanolic solutions of the free acids, were added t o heparinized rat blood obtained by aortic puncture under light ether anesthesia. Standards consisting of 10, 20, 30, 40, and 50 pg./ ml. were employed in the recovery studies. Extractions. Method A. Heparinized blood, 100 pl., was extracted for 1 niin. with 100 pl. of chloroform in the centrifugal separator using a vortex mixer. After centrifugation for 5 min., 2 t o 10 pl, of the chloroform layer, was withdrawn using a 10-pl. microsyringe and injected into the gas chromatograph. If a single barbiturate was present, as in half-life studies, a second aliquot was immediately withdrawn and injected into the gas chromatograph exactly 2 min. later and duplicate determinations were made. After the compounds were eluted the peak heights were measured and the quantity of barbiturate present in the injected aliquot computed by dividing the observed peak height by the peak height units per 0.1 pg. of the respective barbiturate as determined from the standard curve. Method B. Heparinized blood, 100 pl,, was niixed with 10 pl. of 4.OM VOL. 38, NO. 13, DECEMBER 1966
0
1945
digital computer using Fortran programs which provided values for both the horizontal and vertical intercepts, the first-order rate constant, the slope of the line, and the half-life.
1
100)
I RESULTS AND DISCUSSION
I
I
0
I
I
3
6 9 1 Time (min.)
I
2
f
5
Figure 1. Gas chromatogram of hexobarbital extracted from rat blood Peaks represent duplicate injections of the same sample
sodium dihydrogen phosphate and extracted for 1 min. with 100 pl. of chloroform. In subsequent steps, the treatment of samples according to Method B is identical with Method A. In Vivo Studies. When Method A was employed for the determination of the half-life of hexobarbital, the following procedure was used. Male Holtznian strain rats weighing 80 t o 120 grams were injected intraperitoneally with 82.5 mg./kg. of hexobarbital sodium. The volume of the solution was adjusted so that 1.0 nil. was given per 100 grams body weight. Blood samples were taken 30, 60, 90, 120, and 150 min. after the injection of the hexobarbital by severing the tip of the tail with a scapula and collecting the blood in,; disposable 100-pl. pipet ("Microcaps, Drummond Scientific Go.) previously rinsed with heparin solution. The sample was discharged into a centrifugal separator and its hexobarbital content determined eniploying Method A. The half-life was determined from a least squares analysis of a plot of the log (pg. hexobarbital/ml. blood) us. time. Actual computations were performed by a
Table
I.
Saturation of the column by injecting a relatively large quantity of barbiturate before experimental samples were analyzed was found to be essential for quantitative results. The efficacy of this step could be evaluated by preparing a standard curve both before and after experimental samples were analyzed. With a well-saturated column, excellent agreement between standard curves run both a t the beginning and a t the end of the experiment was obtained. If, on the other hand, the column was not saturated prior to the analysis of samples, the response would continue to increase throughout the experiment until saturation was reached. The results of the recovery studies are shown in Table I. With the exception of pentobarbital, the recoveries obtained by Method A increased as the pK, of the respective barbiturates increased. A possible explanation for the behavior of pentobarbital is found in data obtained by other workers which showed that the partition coefficient of pentobarbital is less than that of amobarbital between chloroform/pH 7.2, 0.17M phosphate buffer (1) and between chloroform/pH 7.4 citrate-phosphate buffer (6). I n Method B the blood was acidified before extraction so that the chloroformextractable unionized form of the drugs would predominate. This was done in an attempt to improve recoveries of pentobarbital and phenobarbital which were quite low, 70.3 and 19.5%, respectively, when Method A was employed. Preliminary experiments showed that trichloroacetic acid and dilute sulfuric acid were unsatisfactory acidifying agents because interfering materials were extracted. On the other hand, acidification by addition of 0.1 volume of 4.0M sodium dihydrogen phosphate solution to blood yielded no interfering compounds. The p H of the resulting mixture averaged 5-7. At this pH recoveries would be expected to either increase or not change depending
Recovery Studies
Column temp., "C. 153 173
Extraction procedure Barbiturate Xethod A Method B 80.6 f 3.870" (19)b 80.7 zt 4.770 (14) Amobarbital 90.6 f 1.9% (34) 75.3 f 2.970 (30) Hexobarbital 92.0 zt 2 . 4 % (10) Pentobarbital 163 70.3 f 2.S70 (14) 179 19.5 f 1,370 (13) 54.6 zt 3,870 (9) Phenobarbital 168 89.8 2.970 (23) 87.1 f 4.3% (9) Secobarbital a Mean f standard error of the mean. b Number in parentheses refers to the number of determinations.
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ANALYTICAL CHEMISTRY
0
30
60 90 120 T h o (mln.)
150
Figure 2. Blood hexobarbital levels after administration of hexobarbital or hexobarbital and SKF 525-A to rats Lower line. Hexobarbital sodium, 82.5 mg./kg. ( 3 2 0 pmoles/kg.) administered intraperitoneally. Hexobarbital half-life estimated from the plot was 35.8 min. Upper line. Hexobarbital sodium, 82.5 mg./kg. ( 3 2 0 pmoles/kg.) given 45 min. after an injection of 12.4 mg./kg. ( 3 2 pmoles/kg.) SKF 5 2 5 A. Both drugs were administered intraperitoneally. Hexobarbital half-life estimated from the plot was 98.0 min.
on the partition coefficient of the unionized barbituric acid derivative. Using Method B it was found that recoveries of pentobarbital and phenobarbital were increased while recoveries of amobarbital and secobarbital remained essentially unchanged as compared with results obtained by Method A (Table I). On the other hand, recovery of hexobarbital decreased when Method B was employed. No explanation is available for this exception. Method A was developed primarily for use in studying the in vivo metabolism of hexobarbital. Results obtained using this procedure for the determination of hexobarbital in rat blood are shown in Figure 1. It can be seen that, even when duplicate injections are made, elution of both the barbiturate peaks and the solvent is complete in about 12 min. Also to be noted in Figure 1 is the absence of spurious peaks derived from blood or solvent. It should be pointed out, however, that peaks originating from the septuni in the centrifugal separator may be observed if the aluminum disk is not securely inserted. Figure 2 shows the results of a n experiment in which Method A was employed to determine the in vivo halflife of hexobarbital alone and in the presence of an inhibitor of drug me-
tabolism, 2-diethylaminoethyl 2,2-diphenylvalerate HC1 (SKF 525-.4). Studies in which S K F 525-A, piperonyl butoxide, sesoxane, and tropital (piperonal - bis - ( 2 - [2 - 'n - butoxyethyoxy]ethyl) acetal) were employed as inhibitors of the in vivo metabolism of hesobarbital revealed no int,erfering peaks. Method 13 provides a rapid means of screening toxicological specimens for their barbiturate cont,ent. The small sample size needed permits ut,ilization of blood drawn from the finger t'ip or ear lobe. When blood containing phenobarbital, amobarbital, secobarbital, and pentobarbital was extracted according to ;\lethod B, all of t,hese compounds could be readily detected a t the 10 pg,/ml. level. Preliminary experiments indicate that the method can be used without alteration for the determination of drugs
other than barbiturates in blood. For example, Nethod A has been successfully employed for the determination of the in vivo half-life of meprobamate in rats. Experiments designed to extend the method to the analysis of other drugs are in progress. ACKNOWLEDGMENT
The author greatfully acknon-ledges the technical assistance of Victoria Blauth and Susan Shipherd. LITERATURE CITED
( I ) Bonnichsen, R., JIaehly, A. C., Frank, A,, J . Forensic Sci. 6 , 411
(1961).
(2) Braddock, L. I., JIarec, S., J . Gas Chromatog. 3, 274 (1965).
(3) Hrochmann-Hanssen, E., Svendsen, A. B., J . Pharm. Sei. 50, 804 (1961). (4)Brochmann-Hanssen, E., Svendsen, 9. B., J . Pharm. Sci. 51, 318 (1962). (5) Cieplinski, E. W., ANAL. CHEM.35, 206 11963).
(6) p b i n g , F., Scancl. J . Clin. Lab. Inwst. 7, suppl. 20, 114 (1955). ( 7 ) Gudzinowicz, B. J., Clark, S, J., J . Gas Chimnatog. 3, 147 (1965). (8) Gutenmann, W. H., Lisk, D. J., J . A g r . Food Chena. 12, 46 (1964). (9) Horning, E. C., Tan den Heuvel, W. J. A, Creech, B. G., M e t h o d s B i o c h e m . Anal. 11, 69 (1963). (10) Jain, N. C., Fontan, C. R., Kirk, P. L., AJicroch,ern. J . 8, 28 (1964). (11) Martin, H. F., Driscoll, J. L., ANAL. CHEY.38, 345 (1966). (12) Parker, K. D., Fontan, C. R., Kirk, P. L., Zbid., 35, 418 (1963). 3) Parker, K. D., Kirk, P. L., Zbid., 33, 13i8 (1961). 4) Reith, J. F., van der Heide, R. F., Zwaal, R. F. A., Pharna. Weekblad 100, 219 (1963). RI. W.ANDERS Department of Physiology Cornel1 University Ithaca, N. Y. 14850 WORKsupported by U. S. Public Health Service Grant No. GM-13527.
Determination of pK, Values of Some Substituted Nitroaliphatic Acids by Potentiometric Titration SIR: To evaluate the effect of several different functional groups on the acid strength and to obtain information on the feasibility of titrating mixtures of these acids, the pK, values of a number of aliphatic nitroacids have been determined by potentiometric titration in aqueous solution. The acids used were: 3,a-dinitro2 - hydroxybutanoic, 3.3 - dinitro2 - acetoxylbutanoic, 4,4,4 - trinitrobutanoic, 4,4,4 - trinitro - 2 - hydroxy- trinitro - 2 - acetoxylbutanoic, 4,4,4 butanoic, 4,4,4 - trinitro - 2 - acetamidobutanoic, 2 - (2,2,2 - trinitroethyl) succinic, and 4,4-dinitropentanoicS Two of these compounds. 4,4-dinitropentanoic and 4,4,4-trinitrobutanoic acids, hare been studied by Long ( 5 ) . The methods used for the determination of pH and pK, hare been described by Albert and Serjeant (2) and by Bates (2), who give extensive references to the literature. EXPERIMENTAL
Materials and Reagents. T h e compounds used were synthesized by the Applied Chemistry Division of these laboratories. All compounds were purified by several recrystallizations from suitable solvents and were dried in a n Abderhalden drying apparatus for several hours a t 65' C. Those compounds which were thought to be unstable at room temperature were analyzed immediately and kept refrigerated. Purity of the compounds was established by elemental analysis and melting point determinations.
National Bureau of Standards grade potassium hydrogen phthalate and ACS reagent grade sodium tetraborate decahydrate (Matheson Coleman and Bell) were used for standardizing the p H meter. The 0.05M solutions of these have p H values of 4.00 and 9.18, respectively, a t 25' C. Additional buffers (pHydrion) were 0.05 and used for the ranges pH 2 8 f 0.05. The distilled water used for dilution and sample preparation was boiled for 5 minutes and stored in a polyethylene container equipped with a glass tube filled with Ascarite. Carbonate-free potassium hydroxide was prepared as described by Albert and Serjeant ( I ) . Apparatus. The titration vessel consisted of a 500-ml. flask i*-ith five necks t o accommodate the following: a IO-ml. microburet, a 5-inch glass electrode (Beckman S o . 41263). a 5-inch reference sleeve-junction type calomel electrode (Beckman 41240) filled with saturated potassium chloride solution, an intake tube for nitrogen, and an outlet tube filled with Ascarite. The nitrogen was passed through a wash-bottle containing a 50% aqueous solution of potassium hydroxide to remove carbon dioxide. This bottle was connected to another bottle containing water to remoye any remaining potassium hydroxide. A magnetic stirrer was used in the solution and the temperature was maintained at 25 & 0.05' C. by a suitable water bath. Procedure. Samples of the nitroacids were weighed in 100-ml. flasks and diluted to volume with distilled viater. The sample was then placed
*
in t h e five-neck flask, stirred for sei-era1 minutes under a n atmosphere of nitrogen, and titrated with 0.1N potassium hydroxide in 10 increments, each containing approximately 0.1 meq. of KOH. After each addition, the pH was read after 1 minute. I n some cases. more than 10 increments were needed to construct a titration curve. The compounds as 0.01X solutions were titrated with 0 . 1 s potassium hydroxide so that activitv effects would bk small. The following equations were used:
pK,"
=
pH
+ log [HA] - log [A-]
pK: where, [
=
pK,"
+ 0.5 dc
(2)
(4)
] = analytical concentration,
{ H + } = hydrogen ion activity, and I , = ionic strength in moles per liter. In cases where the p H fell below 4, corrections for the hydrogen ion concentration were made in accordance with Equation 3. These corrections were hydrogen ion activities calculated from the expression ( H ) = antilog (0-pH) and employed in Equation 3. The resulting constant is a mixed ionization constant between the concentration ionization constant and the thermodynamic ionization constant. This constant is converted to the therVOL. 38, N O . 13, DECEMBER 1966
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