pH-Induced Shifts in Ultraviolet Maxima for Determination of Isoniazid and Acetyl Isoniazid in Mixtures SIR: For rapid measurement, differentiation, and characterization of isoniazid (1") and acetyl isoniazid (acetylI N H ) in mixtures where sensitivity below 1.0 y per ml. is not required, pH-induced spectral shifts in ultraviolet maxima can be a valuable analytical tool. Goldman (2) noted the reversible shift of I N H from 265 mp in acid to 300 mp a t pH 13.0, and felt that this absorption change depended upon the presence of a free ring nitrogen and an unsubstituted hydrazid group. Howeveb the major metabolite of INH-namely, acetyl-INH-was not investigated. Maher et al. (3) reviewed and extended Goldman's work, but found that both free isoniazid and the acetyl derivative shifted absorption maxima to 300 mp a t pH 13.0, and thoughithat this would exclude the use of ultraviolet absorption for the determination of I S H and acetyl-INH in body fluids. However, the behavior of acetyl-INH below the pK. of I N H (pH 7.0 to 10.0) was not investigated. Although acetyl-ISH and free I K H exhibit maxima a t 265 mp in acid and a t approximately 300 mp in alkaline solutions of pH 12.0, the two compounds actually make this pH-engendered tautomeric shift to 300 mp a t different pH 1.0
,
I
4
0.0 240
280
280
WAVE L E N Q T H ,
Figure 1.
720
ethylene dichloride-isobutyl alcohol (1:9:10), and were then removed from the triplet by extraction with 0.1N sulfuric acid. Using this extraction procedure 96 =!= 2% of added I S H and acetyl-INH was recovered from whole blood, serum, and urine. By recording ultraviolet absorbance readings of the acid extract and by recording further absorbance readings (after the extract had been made alknline to pH 10.0 by addition of solid sodium carbonate and to pH 13.0 by dropwise addition of saturated sodium hydroxide) , values for acetylIT\", free INH, and total I N H could be computed These characteristic ultraviolet absorption patterns have also been used by this laboratory in determining I N H and acetyl-INK after separation on a strongly cationic ion exchange column (1) (Dowex 50-X8 200 to 400 mesh, H + form). After separation on the ion exchange resin, pH-induced shift permitted the exact characterization of the compounds, and for quantitative improvement the mathematical subtraction of background. I n the process of developing the cationic exchange technique, this ultraviolet method was an invaluable aid as a rapid screening technique to facilitate the
values, which serves to differentiate the two. This fact has not been reported previously in the literature. As shown in Figure 1, acetyl-INH (20 y per ml.) shows complete shift in maxima to 300 mp a t pH 10.0. At this pH (Figure 2), I N H (20 y per ml.) does not exhibit any shift in its absorption peak from 265 mp; it does not exhibit any tendency to shift its absorption peak until pH 11.0, and the complete shift to 300 mp is not noted until a pH of 12.0 is obtained. This distinctive difference in a b s o r p tion maxima due to pH points the way to rapid analytical methods for the individual determination of these two compounds in mixtures or for definite characterization after chemical separation. The ratio of absorbance t o concentration for both ionic forms of acetyl-ISH and I N H was found to be linear for concentrations from 1 to 20 y per ml., which is satisfactory for most analytical purposes. These ultraviolet absorption differences have been used for the determination of I;?JH and acetyl-IT\" after extraction of the compounds from 2 to 3 ml. of whole blood, serum, or urine saturated with ammonium sulfate. The compounds mere extracted into an organic phase composed of heptane-
300
my
Spectral shift, acetyl-INH
ANALYTICAL CHEMISTRY
320
340
240
I
I
1
I
I
1
260 WAVE LENQTH , mp
Figure 2.
Spectral shift, INH
1
1
1
best choice of resin and elution conditions. The spectral shift technique is also applicable for studies on the pharmacology of I S H where it may be important to know the true value of acetyl-INH as distinguished from free I S H and other metabolites of I N H such as isonicotinic acid. Isonicotinic acid, for esample, does not exhibit a n ultraviolet spectral shift in alkaline solution, and thus does not interfere with acetyl-IXH measurenirnts. Characterization and quantitative determination of I K H and its mctabolites have also been made by the ultraviolet method of pH-engendered spectral shifts after paper chromatographic separation. I X H and acetyl-INH were separated using descending chromatography on
acid-washed Whatman S o . 1 filter paper which had been saturated with 0.2M phosphate buffer (pH 6.0) and dried. The solvent system used was 78y0n-butyl alcohol, 20% 2,4-lutidineJ and 2% salicylaldehyde saturated with p H 6.0 phosphate buffer. The discrete spots were detected by spraying the developed paper with 0.lX hydrochloric acid saturated with salicylaldehyde, and exposing the papers t o ammonia. The papers were then examined under an ultraviolet light source. Some R, values obtained with this technique were: I N H , 0.82; acetyl-IN", 0.60; m-sulfobenzaldehyde isonicotinoyl hydrazone, 0.26. Before eluting the chromatograph spots for ultraviolet spectrophotometric quantitation, the papers were thoroughly dried by heat, thus driving off the salicylaldehyde so that it did not interfere. The marked spots containing 5 t o 20 y of the chromatographed compound were then eluted
into 4 ml. of 0.ln' sulfuric acid placed in 1-cm. quartz cuvettes, and absorbance was determined in the range 220 t o 340 mp using a Beckman DK-2 ratio recording spectrophotometer. LITERATURE CITED
(1) Belles, Q. C., Littleman, M. L., A m
Rev. Respiratory Diseases, in press. ( 2 ) Goldman, D. S., Science 120, 315 (1954). (3) Maher, J. R., Whitney, J. M., Chambers, J. s., Stanonis, D. J., Am. Rev. Tuberc. Pulmonary Diseases 76, 852 (1957). Q.C. BELLES M. L. LITTLEMAN
Biochemistry Laboratory Leahi Hospital Honolulu 16, Hawaii
RECEIVED for review Februar 11, 1960. -4ccepted March 24, 1960. k o r k supported in p a r t by a Ford Foundation Grant.
Analysis of Lithium Metal and Lithium(1) Mixtures SIR: I n studies concerned Rith correlation of physical properties of Li" dispersed in mineral oil as a function of volume concentration, it was necessary to evaluate Li" concentration in Li"Li- mixtures by a n independent method. A procedure is based on the controlled hydrolysis of the metal in a n osygen atmosphere, catalytic combination of the evolved hydrogen with the carrier gas, and titration of the hydrolysis residue. A schematic representation of the reaction train is given in Figure 1, and the pertinent features of the hydrolysis chamber arc given in Figure 2. Water is distilled into the reaction chamber, one drop at a time, to avoid prolonged bursts of hydrogen. During the reaction, the lithium hydroside which forms tends to shield the unreacted metal. Cpon dilution of the hydroxide with the continued dropwise addition of water, the reaction resumes. Irregularities in the hydrogen evolution are minimized by the 2-liter mixing chamber. To ensure quantitative combination of the evolved hydrogen, approximately a 10 to 1 osygen to hydrogen ratio is maintained during the course of the controlled hydrolysis. The moist effluent is dried by a series of three 9-inch phosphorus pentoxide towers prior to passage through the catalytic chamber, which consists of a quartz tube, 500 X 10 mm., tightly packed with 1-mil platinum wire. The hot zone, maintained a t 450" C., is approximately 9 inches long. Water generated in the combustion tube is subsequently trapped in a series of four 50-cc. phosphorus pentoside weighing bottles. Two mercury check valves,
the second filled with concentrated sulfuric acid, are used to prevent backdiffusion of moisture into the system, and a dibutyl phthalate-filled U-tube flowmeter monitors the flow rate. A chemical analysis of the lithium in
mineral oil dispel sions was required for correlation nith nuclear magnetic resonance (NMR) measurements ( 1 ) . Because the dispersed lithium was partially osidizcd and because unsaturated fatty acids vere present as stabilizers,
Kp- DISTILLATION REACTION CHAMBER
2- LITER MIXING CHAMBER ~
i ~ P 05 z DRYING TOWERS
FREEZEOUT TRAP
OW ICE TREHLOROETtWLENE
U-TUBE FLOWMETER
Figure 1.
Schematic representation of analysis train
Table I. Data for System Blank Time, Weighing Bottle Increase, Mg.
Hours
Oxygen purifier 16.00 in 15.75 16.50 15.75 15.75 22.75 16.00 21.75 22.75 22.75
Oxygen purifier 16.25 out 15.75
15.50 15.50 15.50
1 1.3 3.9 0.1 2.3 1.9 2.4 1.7 -0.4 1.3 0.1
2 1.0
3 0.5 0.7 0.8 -0.3 -0.3 0.4 0.3 0.0 0.2 -0.3 -0.3 0.6 0.3 -1.9 -2.0 1.5 0.9 -1.2 -0.6
66.3 0.4 69.5 -0.4 60.3 0.2 64.5 1.2 60.6 1.2
4 0.1 0.9 -0.5 1.3 -0.2 -0.1 0.1 -1.0 0.3 -0.3
1 1.6 5.0 0.1
3.0
2.4 2.2 2.2 -0.4 1.2 0.1 Av. 1 . 7 * 1.2 -0.3 -0.1 81.6 0.7 0.5 88.2 0.0 0.7 77.8 1.1 1.4 83.2 1.7 1.4 78.2 Av. 8 1 . 8 i 3.1
Mg./20 2 1.2 0.8 -0.4 0.6 0.0 -0.2 0.8 -1.8 1.4 -1.0
0.1 i 0.8 0.5 -0.5 0.2 1.6 1.6 0.7 i 0.7
Hours 3 0.6 1.0 -0.4 0.4 0.2 -0.2 0.4 -1.8
4 0.1 1.2 -0.6 2.4 -0.2 -0.1 0.2 -1.0 0.8 0.2 -0.6 -0.2 0.4 0.2 f * 0.6 0.6 -0.4 -0.1 0.8 0.6 0.0 0.8 1.4 1.8 2.2 1.8 0.8 1.0 * * 0.8 0.7
VOL. 32, NO. 6, MAY 1960
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