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
