ANALYTICAL CHEMISTRY
1604
Sample 1 2 3 4
5 6
7 8 9
10
Table IV. Reproducibility of Method % Desoxycholic Acid Average 90.5-90.0-90.4 91.5-94,1-89.4 90.0-92.7-91.5 98.6-98.8-99.5 95,1-96.2-94.3 98.7-97.0-98.7 81.2-80.4-80.0 84.1-83.9-84.1 96.1-95.9-96.6 92.1-94.2-92.0
90.3 f0.3 9 1 . 7 12 . 2 9 1 . 4 10 . 9 98.9 1 0 . 3 9 5 , 2 =t0 . 7 98.1 + 0 . 8 80.5 1 0 . 4 84.0 1 0 . 1 96.2 * 0 . 3 9 2 . 8 11 . 0
Table V. Desoxycholic Acid in Typical Biles Sample
DISCUSSION OF RESULTS
The results given in Table IV indicate the reproducibility of this method when applied to commercially produced desoxycholic acid, and Table V presents assays of typical biles collected from various countries. LITERATURE CITED
(1) Abe, Yoshimi, J . Biochem. ( J a p a n ) , 25, 181-9 (1937). (2) deHaen, P., J . Am. P h a r m . Assoc., 33, 161 (1944). (3) Gregroy, R., and Pascoe, T. A , J . Biol. Chem., 83,35-42 (1929). (4) Kajiro, K., and Shimada, T., 2. physiol. Chem., 254, 57-60 (1938). (5) Kawaguti, S., J . Biochem. ( J a p a n ) , 28, 445-9 (1938). (6) Sair, W., Biochem. J., 11,ll-13 (1917).
Desoxycholic Acid Found, % 10.3-10.9-10.6 12.0-12.4-11.9 12.5-12.1-12.3 10.8-10.9-1 1 . 3 9.5-9.9 14.2-14.8 12.7-12.8 10.9-10.6-11.0 0.5- 0 . 5 4.8- 4 . 6 9.6- 9 . 6 12.3-11.8-12.2 1.4- 1.5 1.1- 1 . 1
Average, % 10.6 12.1 12.3 11.0 9.7 14.5 12.8 10.9 0.5 4.7
9.6 12.1 1.5 1.1
(7) Pregl, F., and Buchtala, H., 2. physiol. Chem., 74, 198-211 (1911). (8) Reinhold, J. G., and Wilson, D. W.,J . Biol. Chem., 96, 637-46 (1932). (9) Scherrer, I., Helv. Chim. A c t a , 22, 1329-40 (1939). (10) Shimada, T., J . Biochem. ( J a p a n ) , 28, 149-60 (1938). (11) Ibid., 29,41-50 (1939). (12) TTebb, T. J., ASAL. CHEM.,20, 100-3 (1948). RECEIVED for review April 16, 1952. Accepted July 31, 1952. Presented a t the Meeting-in-3Iiniature, of the North Jersey Section, AXERICASCHEMICAL SOCIETY, January 28,1952.
Determination of Barbiturates Ultraviolet Spectrophotometric Method with Diflerentiation of Several Barbiturates LEO R. GOLDBAUM Department of Biochemistry, Army Medical Service Graduate School, Washington, D. This study was made to perfect a n ultraviolet absorption procedure for the determination and differentiation of barbiturates in biological materials. A method was developed which is based on the change in the absorption spectra of barbiturates i n strong alkali and i n solution a t pH 10.5. When the optical densities in pH 10.5 solution are subtracted from those in strong alkali, differences appear that are highly characteristic of all barbiturates except the n-methyl and thio derivatives. By comparingthe differences a t various wave lengths with t h a t a t 260 mp, ratios are obtained t h a t can differentiate among many of the commonly used barbitu-
R
ECESTLY a number of ultraviolet spectrophotometric
procedures have been developed for the determination of barbiturates (f-4), but none has been able to combine specificity, accuracy, sensitivity, and simplicity, nor have any attempted a differentiation of the various barbiturates. Such a procedure is needed in toxicological investigations of barbiturate poisonings and in pharmacological studies of these sedatives. This report described a simple, rapid, ultraviolet spectrophotometric procedure for the specific identification and quantitative determination of micro quantities of barbiturates and for the differentiation of many barbiturates. EXPERIMENTAL
This method is based on the observation that substituted barbiturates have an absorption band in strong alkali characteristic
C.
rates. Absorbing substances appearing in the extracts of drug-free biological materials as well as other drugs show no significant differences a t these pH’s and do not interfere in this procedure. The difference i n optical density a t 260 mp is related to the barbiturate concentration. The method can determine barbiturate concentrations as low as 0.1 mg. per 100 ml. of blood and 0.3 mg. per 100 grams of tissue. This rapid, simple, highly specific procedure is invaluable in the clinical diagnosis of barbiturate poisoning, in toxicologicalinvestigation of barbiturate deaths, and in pharmacological investigation of barbiturate metabolism.
of one resonance form, while in solution of approximately pH 10 there is a different absorption band characteristic of another resonance form. Thus, the absorption spectra of barbiturates in strong alkali show characteristic maxima a t 255 mp and minima a t about 235 mp. I n a buffer solution a t pH 9.8 to 10.5 there occur higher maxima a t 240 mp with no minima. When the optical densities of the buffered solution a t the various n-ave lengths are subtracted from the optical densities of the strong alkali, differences result which provide a highly specific identification of barbiturates. The differences are greatest a t 260 mp, decreasing through zero a t about 280 mp (isosbestic point) to a maximum negative a t around 240 or 235 mp, and increasing through zero a t about 227 mp (isosbestic point) to positive differences a t the lower wave lengths. The differences a t 260 mp for alkaline solutions containing 2 to 30 micrograms of bar-
V O L U M E 2 4 , NO. 10, O C T O B E R 1 9 5 2 biturates per ml. follow Beer's law (Figure 1) and are used for a quantitative measure of barbiturate concentrations. Figure 2 illustrates the absorption spectra of a barbiturate (amobarbital) in 0.45 S sodium hydroxide and in solution of pH 10.5, together with the characteristic differences that appear when the optical densities of the pH 10.5 solution are subtracted from the optical densities of the 0.43 S sodium hydroxide.
BARBITAL
90.
0 AMOBARBITAL
60 W v)
50 K LL
40
30 a
Y
*
20
0
5 10 MICROGRAMS
20 of
30
BARBITURATE
Figure 1. Relation between Optical Density Differences at 260 mp and Barbiturate Concentration Ordinate reuresents averaee differences obtained b y subtraciing optical d-ensity of barbiturate standards in 0.45 S NaOH from those a t pH 10.5 a n d abscissa, barbiturate concentrations in micrograms per rnl. of 0.45 S S a O H
1605 butallylonal has a positive ratio of $0.96, phenobarbital +0.43, and secobarbital +0.21, while pentobarbital and amobarbital have negative ratios, -0.15 and -0.33, respectively. Those optical densities a t 270, 260, 252, 249, 247, 240, 235, 232, and 228 mfi are sufficient to identify and differentiate barbiturates. Some of the other barbiturates investigated have rlosely related ratios according to the substitutions on the 5 position of the malonylurea ring. Those barbiturates that have a secondary alkyl radical in addition to an ethyl group-for example, probarbital (ethyl, isopropyl)-show ratios similar to pentobarbital (ethyl, 1-methglbutyl). Those having a primary alkyl radical in addition to an ethyl group-for example, barbital (diethyl)are similar to amobarbital (ethyl, isoamyl). Aprobarbital (allyl, isopropyl) is similar to secobarbital (allyl, 1-methyl butyl). An attempt is being made to differentiate these closely related barbiturates by their optical density differences a t other pH's, and by their rate of decomposition in alkali. Determination of Barbiturates in Biological Materials. The presence of absorbing substances normally found in extracts of biological materials, as well as other absorbing drugs, is a major difficulty in ultraviolet spectrophotometric procedures because these substances interfere with the specificity, accuracy, and sensitivity of any method. To eliminate these interfering substances in the determination of barbiturates, other investigators (1, 3, 4) have limited the size of the sample and used complicated extraction procedures, xhich resulted in loss of sensitivity, low recoveries, and lengthy procedures. They also n ere unable to eliminate interference by other drugs. The determination of differences in the optical densities in pH 10.5 solution and in 0.45 S sodium hydroxide furnishes a simple means of identifying, estimating, and differentiating barbiturates in biological materials. There occurs no interference from normally absorbing substances which appear in the alkaline extracts of blood, urine, or tissue, as these substances do not
0.80.
DBerentiation of Barbiturates. The optical density difference of amobarbital in alkali and in pH 10.5 solution (Figure 2) is characteristic of all barbiturates except the n-methyl and thio derivatives, which have considerably different absorption spectra. Houever, when these optical density differences obtained for a number of barbiturates are closely examined, many of them can be differentiated. Significant variations in the relative optical density differences occur a t various wave lengths. These become apparent when the optical density differences a t a given wave length are compared with that a t 260 mp. The ratios of some representative barbiturates obtained by dividing the differences a t a given wave length by the difference a t 260 mp are shown in Table I. Considerable differences in the ratios a t various wave lengths are found with these barbiturates. For example, a t 270 mp butallylonal has the highest ratio, +0.81, follom-ed by secobarbital +0.69, pentobarbital +0.61, phenobarbital f0.59, and amobarbital +0.52. At 228 mp
Table I.
> w
n
W A V E L E N G T H IN
dicate differences between optical densities i n p H 10.5 and in 0.45 A' NaOH. Wavelength,mp270 260 252 249 247 240 235 232 228 Differences +0.22 f 0 . 4 3 +0.24 fO.05 -0.09 - 0 43 -0.46 -0.38 - 0 14
Differentiation of Several Representative Barbiturates by Their Difference Ratios
Optical density difference a t given wave length ' Optical'denbity difference a t 260 mp Wave Lengths, mp 270 260 252 249 247 240 Ratios ----.---.--"I_ Amnharhital /i.n..ny], ethy1)a fO.52 1.00 +0.56 +0.12 -0.21 -1.00 Pentobarbital (1-methyl butyl, ethy1)a f0.61 1 00 f0.40 -0.05 -0.38 -1.02 Secobarbital (1-methylbutyl allyl)Q +0.69 1.00 -I-0.30 -0.13 -0.39 -1.04 Butallylonal (sec-butyl B-bromoallyl)a fO.81 1.00 +0.07 -0.63 -0.35 -1.03 +0.59 1.00 +0.36 Phenobarbital (ohenyl: ethyl)" -0.09 -0.36 -0.96 Cyclopal @-cyclopenten-l-yl, allyla) +O 80 1 00 fO.11 -0.34 -0.63 -1.10 a Substituents in 5 position of malonylurea nucleus.
(
j.L__.
Mu
Figure 2. Ultraviolet Absorption Spectra of Amobarbital (Isoamyl, Ethyl, Barbituric Acid) 0. I n 0.45 N NaOH. X . I n borate buffer, ,pH 10.5. Vertical solid linea in-
)
235
232
228
-1.09 -1.00 -0.89 -0.72 -0.84 -0.94
-0.88 -0.80 -0.63 +0.20 -0.48 -0.50
-0.33 -0.15 +0.21 +0.96
fO.43 f0.71
A N A L Y T I C A L CHEMISTRY
1606
Table 11. Optical Density Differences from Extracts of Drug-Free Blood, Urine, and Liver Wave Length, mfi 305
Extract from 10 mi. of blood O.D. of 0.45 N NaOH 0.125 O.D. of p H 10.5a 0.130 Differences -0.005 Extract from 3 gram@of liver O.D. of 0.90 N N a O H 0.150 O.D. of p H 10.5a 0.145 Differences $0.005 Extract from 5 ml. of urine O.D. of 0.45 N NaOH 0.255 O.D. of p H 10.5a 0.250 Differences $0.005 a
2 70
260
252
249
247
240
235
232
228
0.150 0.150
0.165 0.160
0.215 0.205
0.250 0.245
0.275 0.265
0.340 0.330
0.365 0.350
0.380 0.370
0.470 0,445
SO.005
0.000 0.340 0.340
$0.010
+0.005
+0.010
+0.010
iO.015
$0.020
$0.025
0.390 0.375 S0.015
0.450 0.420 $0,030
0.510 0.480 i0.030
0.550 0.520 +0,030
0.650 0.63 +o,020
0.450 0.450 $0.000
0.580 0.560 $0.020
0.640 0.620 $0,020
0.680 0.660 $0.020
0.780 0.750
0.365 0.360
0.365 0.360
S0.005
10.005
0.375 0.370 $0.005
0.550 0.550
0.420 0.425
+O.OOO
-0.005
0.400 0.400 $0.000
0.425 0.420 $0.005
+O.OOO
$0.030
Optical density readings corrected for dilution by buffer.
Table 111. Optical Density Differences from Blood Extract [5 ml. of blood containing 100 micrograms of added amobarbital (isoamyl, ethyl barbituric acid)]
Wave Lengths, mp O.D. of 0.45 N NaOH extract O.D. of p H 10.5a
305
270
260
252
249
247
240
235
232
228
0.085 0.100
0.39 0.18
0.67 0.27
0.78 0.55
0.77 0.71
0.75 0.83
0.67 1.08
0.62 1.05
0.64 0.97
081 0.92
$0.52 4-0.52
100 1.00
+0.58 SO.56
O.D. differences O.D. difference a t 260 mp Ratios of standard amobarbital (Table I) Ratio =
-0.15 -0.12
-0.20 -0.21
-1.02 -1.00
- 1 08 -1.08
-0.85
-0.28
-0.88
-0.33
Optical density readings corrected for dilution by buffer.
significantly change their absorption in strong alkali and in pH 10.5 solution (Table 11). For the extraction of barbiturates from blood, urine, or tissues, the procedure used is simple and rapid and affords good recovery of added barbiturates (3). Reagents. BORATEBUFFER. Dissolve 12.369 grams of boric acid and 14.911 grams of potassium chloride in water and dilute to 200 ml. to prepare a 1 M solution of these salts. After standing a t room temperature for 24 hours, filter off any undissolved salts. SODIUMHYDROXIDE SOLUTIONS.Prepare an approximately 0.45 N sodium hydroxide from saturated sodium hydroxide. Using a pH meter, adjust the normality until pH 10.5 iB obtained when 2 parts of alkali are added to 1 part of borate buffer. Similarly prepare an approximately 0.90 N sodium hydroxide from saturated sodium hydroxide. Adjust the normality until pH 10.5 is obtained when 2 parts of alkali are added to 2 parts of borate buffer. SOLVENTS.Wash a reagent grade chloroform or ethylene dichloride successively with approximately 1 iV sodium hydroxide and twice with water. For every liter of solvent, use 100 ml. of wash solution. Waeh only the volume needed for daily use. On standing, the chloroform tends to decompose. Procedure for Blood. Oxalated blood or plasma is extracted with chloroform in a separatory funnel or glass-stoppered bottle. For samples of 1 to 5 ml., 50 ml. of chloroform are used; for larger samples (5 to 10 ml.) 75 ml. of chloroform are used. A clear aliquot (usually 40 or 60 ml., depending on the amount of chloroform) is obtained by running the chloroform through a fast filter paper (Whatman 41) and then is extracted wlth 4 ml. of approximately 0.45 N sodium hydroxide in a dry separatory funnel. The chloroform layer is discarded while the alkaline solution is run into a small test tube and centrifuged. Three milliliters of the clear alkaline extract are transferred to the 1-cm. quartz cuvette of a Beckman DU quartz photoelectric spectrophotometer. Distilled water in place of the blood sample is run through the extraction procedure and its alkaline extract is transferred to a cuvette to be used as the reference solution. Optical density readings are made a t nave lengths 305, 270, 260, 252, 247, 250, 235, 232, and 228 mp. The optical density a t 305 mp is an indication of tke amount of absorbing substances other than barbiturates. The differences a t this wave length should be insignificant. Then 2 ml. of the alkaline extracts of blood and the reference solution are ipetted into a tube containing 1 ml. of 1 Af boric acid potassium choride buffer to yield a pH below 10.6 and above 10.2. The buffered solutions are transferred to d r r cuvettes
and again the optical densities are determined a t the above m ave lengths. These optical densities are corrected for dilution with the buffer by multiplying by 1.5, and then subtracted from the optical densities of the alkaline extract.
If barbiturates are present, the characteristic spectral differences appear; if absent, the differences of normally present absorbing substances are insignificant, especially a t 260 mp where a quantitative estimation is made (Table 11). I n the analysis of numerous barbiturate-free blood samples, the difference a t 260 m9 amounts to less than 0.02 n-ith blood samples as large as 10 ml. The error introduced by this difference is approximately 1 microgram per ml. of blood nhen 5 ml. of blood are analyzed. Procedure for Urine. One to 5 nil. of urine (pH below 7.0) are extracted with 50 ml. of chloroform. To remove interfering substances, the chloroform layer is separated and returned to R clean separatory funnel and shaken with 5 ml. of 1 M phos hate buffer pH 7.4. A clear aliquot is obtained by running the clloroform through a Whatman 41 filter paper. The procedure is continued as described under blood. Procedure for Tissue. Tissues are prepared for extraction by homogenizing with distilled water in an all-glass tissue homogenizer (Tenbrook). Chloroform is the solvent of choice for all tissues except brain, nhere ethylene dichloride is used. A weighed sample of tissue may be homogenized and made up to a specified volume, using an aliquot for analyses; or a small sample may be homogenized and quantitatively transferred into a separatory funnel containing the solvent. For the extraction of 5 ml. of homogenate, containing 1- or 2-gram samples, 75 ml. of solvent are used. The analysis is continued as 'described for blood, except that the aliquot of clear filtered solvent is extracted with approximately 0.9 N sodium hydroxide. The normality of the alkali is increased to eliminate the turbidity that sometimes occurs with weaker alkali (2). The adjustment of pH is made b s the addition of 2 ml. of the alkaline extract to 2 ml. of the 1 M boric acid-potassium chloride buffer to yield a pH between 10.5 and 10.2. The optical densities of the buffer solution are multiplied by 2, then subtracted from those of the strong alkali. Calculations. A quantitative estimation of barbiturate concentration is made by comparing the optical density difference a t 260 mp in pH 10.5 and in 0.45 T sodium hydroxide of the unknown extract with the optical density difference of an extract obtained n-hen a known quantity of barbiturate is added
V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 to a sample and carried through the procedure. Table I11 illustrates the optical densities from an alkaline estract of 5 ml. of blood containing 100 micrograms of amobarbital, Thr difference in optical densities a t 260 mp equivalent to 20 micrograms per ml. of alkaline extract is 0.40. The ratios obtained by dividing the optical density difference a t a given wave length by the difference a t 260 mp are similar to those obtained from a standard alkaline solution of amobarbital. Table IV lists the optical density differences a t 260 mp obtained when some rcpresentative Iiarbiturates (100 micrograms) are added to blood. F h e n these optical density differences (Table IV) equivalent to 20 micrograms of barbiturates per ml. of alkaline extract are compared with those obtained with standards (Figure I), approsimately 95% of those barbiturates studied, except for barbital, is recovered in the estraction procedure. The following formula c:m be used for the calculation of the barbiturate concentration in Iliological materials: hficrograms of barbiturate per ml. or gram of sample = a X b X c X d e X f X g
where a
=
b = c = d =
e =
f
=
g =
difference of unknown extract in alkali and in pH 10.5 a t 260 mp volume of chloroform used to extract sample volume of alkali used to extract chloroform aliquot micrograms of barbiturate per ml. of knowi alkaline extract difference of known extract in alkali and in pH 10.5 at 260 mp chloroform aliquot amount of sample DISCUSSION
Sensitivity and Specscity. In some of the other ultraviolet spectrophotomet’ric procedures, the presence of barbiturate is determined by the characteristic absorption band a t pH 10 (Figure 2) and barbiturates are estimated by the difference in absorption a t 240 mp in alkali and in an acid pH. I n the presence of large amounts of other absorbing substances, even high concentrations of barbiturates may show no characteristic maxima.
Table IV.
Differences in Optical Densities of Extracts
(At 260 mp in 0.45 N 5 a O H and pH 10.5 obtained from 5-ml. blood samples containing 100 micrograms of added representative barbiturates) Av. Difference at 260 mp Equivalent to 20 y of Barbiturate per MI. of Alkaline Barbiturate Extract Amobarbital 0.40 3~ 0 . 0 2 a Barbital 0.48 =t0.02 Butallylonal 0.27 f 0 . 0 2 Pentobarbital 0.39 f 0.02 Phenobarbital 0.36 zt 0.02 Seconal 0.36 =t0 02 a Standard error of mean.
In such cases reliance is placed only on the difference in the optical densities a t 240 mp in pH 10 and in acid. Thus, no specific identification of barbiturates can be made. In the present procedure, barbiturates are identified by the appearance of the evt r e n d y characteristic optical density differences which are a composite of two different absorption bands. Thebe differences occur even when small concentrations of barbiturates are present with large amounts of interfering substances. A barbiturate is indicated only when a mayimum positive difference appears a t 260 mp, which decreases to a negative difference at around 250 mp and a maximum negative difference a t 240 nip. Then 3 quantitative estimation can be made from the difference at 260 mp. Considering a difference of 0.04 a t 260 mp as significant, the concentration of barbiturates needed to give this difference is about 2 micrograms per ml. of alkali. From the above formula, when a 10-ml. sample of blood is analyzed, from which is obtained a chloroform aliquot equivalent to ‘/iof the sample. the differ-
1607 ence, 0.04, inclicates the presence of approxirnstely 1.0 microgram of barbiturate per ml. of blood. I n the deterniination of barbiturates in tissue, the sensitivity is not as great, as there is a larger amount of normally absorbing substances, thus limiting the size of the sample. When a 3-gram tissue sample is analyzed from which an aliquot is obtained equivalent to 4 / s of the sample, the difference, 0.04, indicates the presence of approximately 3.3 micrograms of barbiturate per gram of tissue. The blood levels that occur after therapeutic doses of the commonly used barbiturate. range from 1 to 10 micrograms per ml., and after toxic doses range from 15 to 100 micrograms per nil. The corresponding tissue levels are higher. The sample used for analysis should be such that the alkaline extract should contain about 25 micrograms of barbiturates per ml. With these concentration
