Co. for providing drugs and of J. hl. Airth for statistical analysis. The valuable criticism and advice of C. C. Fulton, Food and Drug Administration, Washington, D. C., is gratefully acknowledged. LITERATURE CITED
(1) Baggesgaard-Rasmussen, H., Berger, J., Folting, K., Espersen, G., Dansk Tidsskr. Farm. 32, 81 (1958). (2) Bakre, V. J., Karaata, Z., Bartlet, J. C., Farmilo, C. G., J . Pharm. Pharmmol. 1 1 , 234 (1959). (3) Bartlet, J. C., Farmilo, C. G., Can. J. Technol. 33, 134 (1955). (4) Brandt, R., Ehrlich-Rogozinsky, S., Cheronis, N. D., Microchem. J . 5, 215
(1961). (5) Buchi, J., Huber, R., Schumacher) H., Bull. Narcotics, U . N . Dept. Social Afairs 12, No. 2, 25 (1960). (6) Eddy, ?IT. B., Fales, .H. AT., Haahti, E., Highet, P. F., Homing, E. C., May, E. L., Wildman, W. C., United Nations Document, ST/SOrl/Ser. K/114, 1961.
Fluorometric
(i) Fales, II. &I.,S I H , Bethesda 14, Nd,, private communication, 1961. (8) Farmilo, C. G., Kennett, P. hl. L., United Nations Document ST/SOA/ Ser. K/14, 1953; Ibid., 118th Meeting ACS Abstracts, p. 12B, September 1950. (9) Fischer, R., Folberth, K., ArzneimittelForsch. 5 , 66 (1955). (10) Fritz, J. S., ANAL.CHEJI.31, 1808 (1959). (11) Fulton, C. C., United Sations Document E/CN.7/117. 1948. (12) Ibid.; E/CN.7/202, 1950. (13) Ibid., ST/SOA/Ser. K/l, 1951. (14) Ibid., ST/SOA/Ser. K/34, 1954. (15) Genest, K., Farmilo, C. G., Bull. Narcotics, U N Dept. Social Aflairs 12, Yo. 1, 17 (1960). (16) Genest, K., Farmilo, C. G., J . Am. Pharm. Assoc., Sci. Ed. 48, 286 (1959). (17) Kamp, W., Pharm. Weekblad 92, 1 (1957).
L , thesis, Rutgers University, 1956. (19) Liang, Chih-Kvei, Chen, Su-Sen, J. Taiwan. Pharm. Assoc. 4, 2 (1952). (20) Lloyd, H. A,, Fales, H. M., Highet, P. F., Vandenheuvel, W. J. A,, Wildman,
(18) Klayman, D
R. C J . Am. Chene. Soc. 82,3791 ( 1NU) (21) Rliram, R , Pfeifer, S., Scz Phirnz. 27, 34 (1959). (22) Pfeifer, S., Bull. Narcotics, T .V, Dept. Socaal Afazrs 10,No. 3, 18 (1958). (23) Reifer, I., Toczko, K., Acfa Bzochtm. Polon. 3. 381 (1956). (24) Rohmisch, H., ’Pharmazie 16, 373 (1961). (25) Sakurai, H., Ann. Takamine Lab. 4, 108 (1952); C. A . 49, 3475 (1955). (26) Small, L. F., Lutz, R. E., “Chemistrv of the Oaium Alkaloids.” UD. 34,”49, 100, U: S. Treasury Deb{, Public Health Service, Washington, 1932. ~
~
RECEIVED for review March 23, 1962. Accepted August 13, 1962. Division of Analytical Chemistry, 141st Meeting, ACS, Washington, D. C., March 1962. The work reported in this paper m s undertaken as part of the international program of research on the Assay, Characteristics, Composition, and Origin of Opium pursuant to Resolutions 159 I1 C (VII), 246 F (IX), and 548 D (XT’III) of the Economic and Social Council of the United Nations.
M crodetermination of Alpha-Keto Acids
JOHN E. SPIKNER and JACK C. TOWNE Radioisotope Service, Veterans Administration Research Hospital and Department of Biochernisfry, Northwestern University Medical School, Chicago, 111.
b The quantitative conversion of submicrogram quantities of alpha-keto acids to corresponding substituied quinoxalines b y reaction with o-phenylenediamine (OPD) is described. By taking advantage of the fluorescent properties of these quinoxalines, a method has been evolved for the estimation of some alpha-keto acids. Reaction conditions, kinetics of fluorescent derivative formation, and linearity of results are described. This somewhat specific, nonreversible reaction is most useful for the determination of a single alpha-keto acid in very dilute solution. Other features include stability, reproducibility, sensitivity (range 0.05 to 0.50 pg./ml.) and the use of small reaction volumes.
T
HE METHODS customarily used for quantitation of alpha-keto acids are the quinolylhydrazone method of Robins et al. (IS) [sensitivity 4 X moles] and the similar, well established 2,4-dinitrophenylhydrazone (DNPH) procedure which is less sensitive. In the formation of the D N P H derivatives in acid media the equilibrium yield could vary with the acidity. The reaction was followed by a series of extractions wherein losses possibly occurred. Positive identification of products separated by paper or column chromatography was made difficult by the pres-
1468
ANALYTICAL CHEMISTRY
ence of s y n and anti forms of a given 2,4dinitrophenylhydrazone having different Rf values. Furthermore, homologous products were difficult to separate with a given solvent system because of chemical differences on one hand, and because of the proximity of some of their Rf values, on the other. The measurements (in each method) consisted of a spectrophotometric assay of colored products which were unstable. The D N P H method has been used by Hockenhull and coworkers ( 8 ) ,Cavallini and Frontali (b), E l Hawary and Thompson (S), and Seligson and Shapiro (15). In the similar work of Towers ( I @ , Meister (II), and Kun (9) further identification of alpha-keto acids thus isolated was made by catalytic hydrogenation of the DYPH products followed by chromatographic separation of the liberated amines. hfeister ( 1 1 ) has indicated some of the difficulties generally encountered in the D N P H procedures for alpha-keto arids. For microassay purposes the D K P H method was further limited by the reversible nature of the reaction. The details of this generally acid-dependent equilibrium are discussed by Royals (Id). Alternatively Hockenhull and Floodgate ( 7 ) described a method for the analysis of alpha-keto acids through the formation of nitroquinoxaline derivatives. Several advantages were noted in the quantitative preparation, separa-
tion, and measurement of alpha-keto acids by this means. The property of fluorescence on paper in ultraviolet light was also noted. The synthesis and the fluorescent nature of numerous substituted quinoxalines (benzopyrazines) were originally reported by Hinsberg (6) who used both 3,4diaminotoluene and o-phenylenediamine as reagents. Recent investigations in this laboratory revealed the practical application of fluorescence spectrometry to the quantitative measurement of some alpha-keto acids as their quinoxaline derivatives in acid solution. Such a derivative of pyruvic acid, 2-hydroxy-3-methylquinoxaline, is highly fluorescent and is stable in 50% sulfuric acid. Hence, as an adjunct to existing methods, a new microanalytical technique for pyruvic acid applicable to other alpha-keto acids has been developed. The procedure involves a condensation of OPD with the alpha-keto acid in acid solution. The final step is a dilution in 50% sulfuric acid followed by reading in a spectrofluorometer. EXPERIMENTAL
Apparatus. Fluorescence measurements were made with a Farrand Spectrofluoronieter having slits opened to 2 mm. and equipped with Corning filters, Nos. 9363 (primary) and 3389 (secondary), and a IP21 multiplier phototube as detector.
A 150-watt xenon arc-lamp [either Hanovia or Osram type] was the source light. A stainless steel pan 9 X 7 X 2 inches fitted with a slotted plexiglass cover (11 x 9 x 3/16 inch) and mounted on a hot plate served as an oil or water bath. The plexiglass cover was fabricated by cutting four 1-inch holes 1 6 / 8 inches apart across the cover, then milling out four '/16-inCh slots longitudinally through the holes. AS many as forty 5-ml. volumetric flasks could be inserted in the holes and suspended from the grooves in the water bath. An aluminum cover (14 X 113/8 X inch) which could support 96 samples was later made to be used with a pan 1l1/*x 91/4 x 21/2 inches deep. The latter was unaffected by heat and oil. Reagents. Solutions of reagent grade o-phenylenediaminedihydrochloride (Eastman Chemical Co.) were prepared fresh daily by dissolving 10 mg. of the salt in 100 ml. of 2N sulfuric acid. A solvent medium was prepared (2 to 3 liters) as required by carefully mixing cold, concentrated, clear, reagent grade sulfuric acid and ice-cold distilled water in equal parts by weight (50% w./w. H2S04). Alpha-Keto Acids. Solutions containing 2.5 pg./ml. of the following reagent grade alpha-keto compounds in 2N sulfuric acid or alcohol were prepared fresh when needed: sodium pyruvate, sodium glyoxylate, phenylpyruvic acid (Sigma Chemical Co.), oxalacetic acid (Mann Research Labs.), a-ketoglutaric acid (Aldrich Chemical Co.), a-ketobutyric acid (Eishell Labs), a-ketocaproic acid, a-ketoisocaproic acid, and a-ketoisovaleric acid (California Corp. Biochemical Research). These solutions were used for preparing sensitivity curves. Preparation of 2-Hydroxy-3-methylquinoxaline. An amount of 220 mg. (2 mmoles) of sodium pyruvate was dissolved in 10 ml. of 2N hydrochloric acid. A solution of 453 mg. (2.5 mmoles) of o-phenylenediaminedihydrochloride in 10 ml. of 2N hydrochloric acid was mixed with the pyruvate and allowed to stand 5 minutes at room temperature to form a white crystalline precipitate. The product was filtered with suction on a sintered glass funnel and washed twice with 10ml. portions of 2N hydrochloric acid and twice with 5-ml. portions of water. The product (210 mg., 65% yield) was air dried with suction (m.p. 230" C.). Recrystallization of the product from alcohol and vacuum drying (3 mm. a t 60" C.) gave a white product in long needles (m.p. 251" to 252" C.). By a similar procedure quinoxaline derivatives of other alpha-keto acids were prepared. The fluorescence properties of these compounds appear in Table I. Preparation of Standard Curve for Crystalline 2-Hydroxy-3-methylquinoxaline. An amount of 10.0 mg. of recrystallized, dried 2-hydroxy-3methylquinoxaline was weighed into a 10-ml. volumetric flask and diluted to the mark with 50y0 sulfuric acid. The stock solution was diluted serially to a final concentration of 0.130 pg./ml.
Fluorescence Yields of a-Keto Acid Quinoxaline Derivatives in 50% HzSO, Yield, Excitation DerivaF pa./mp mole/ fluorescence a-Keto acid m.p., C. ml. x 102 peaks, mr 338-518 2.25 199-200 Glyoxylic 365-495 9.5 197-198 a-Ketobutyric 365-498 9.15 175-178 a-Ketocamoic 360-503 a-Ketoglitaric 261-262 12.2 365-500 a-Ketoisocaproic 185-187 11.4 367-500 a-Ketoisovaleric 220 (sub) 7.74 368-498 Oxalacetic (Not isolated) 3.12 368-503 Phenylpyruvic 199-200 3.42 360-490 251-252 13.8 Pyruvic
Table 1.
Table II.
Tube
KO. 1
2 3 4 5 6 7 8 9
10
Fluorescence Data for Stability of 2-Hydroxy-3-methylquinoxoline in 50% Sulfuric Acid Sodium pyruvate, pg./ml. x 102 pa. x 0.01 1st day 2nd day 3rd day 4th day 7th day 0.52 0.32 0.40 0.27 0.228 0.27 0.75 0.95 0.55 0.54 0.56 0,456 2.1 1.1 1.7 0.674 0.84 0.80 1.2 1.1 1.0 0.99 0.913 1.0 1.4 1.6 1.4 1.3 1.35 1.14 1.9 1.5 1.75 1.7 1.7 1.35 2.1 2.0 1.9 1.9 1.9 1.59 2.1 2.3 2.2 2.1 1.82 2.1 2.7 2.35 2.3 1.92 2.3 2.35 2.28 2.6 2.6 2.8 2.6 2.9
of the derivative. The excitation and fluorescence spectral curves were drawn establishing the peaks a t 361 and 490 mp, respectively. A standard curve was prepared by plotting fluorescence against concentration. A straight line relationship was obtained. Data derived from this curve were used (denominator) in the calculations below. Sensitivity and Extent of the Reaction, A solution was prepared containing 1.15 pg./ml. of sodium pyruvate in 2N sulfuric acid (100 ml.). An amount of 1 mg. of OPD was added and the mixture was refluxed on a hot plate for 4 to 5 hours. When the reaction was complete, aliquot amounts of the mixture ranging from 0.1 ml. to 1.0 ml. were transferred from a microburet into separate 5-ml. volumetric flasks and the flasks were filled t o the mark with 50y0 sulfuric acid. These 10 samples were again diluted 1 to 10 with 50% sulfuric acid giving concentrations ranging from 0.00228 to 0.0228 pg./ml. The fluorescence of these samples was measured within 1 hour after the reaction was completJed and again on successive days. See Table 11. A linear relationship was obtained between the initial fluorescence values and concentration. Data from this curve were used in the numerator of calculations illustrated below. Repeated fluorescence measurements indicated that the lower five concentrations were stable for 2 days, while the upper concentrations were stable for 7 days. Preparation of a Sensitivity Curve for the Microreaction of Pyruvate with OPD. h solution was prepared
containing 2.28 pg./ml. of sodium pyruvate in 2N sulfuric acid. Aliquot amounts ranging from 0.1 to 1.0 ml. of the sodium pyruvate solution were placed in each of ten 5-ml. volumetric flasks, together with 1.0 ml. (100 fig.) of the OPD reagent. Two reagent blanks were prepared, and all volumes made to 3 ml. with 2N sulfuric acid. The flasks were suspended in the hot water bath and heated a t 90" to 95' C. for 3 hours. All samples were removed, cooled in ice-water and treated Rith 1.66 ml. of cold, concentrated sulfuric acid. After careful mixing (with cooling) the sample volumes were adjusted to 5.0 ml. with 50% sulfuric acid. The fluorescence of these samples was
Data for Microreaction of Pyruvate and OPD Fluorescence Pyruvate 360 (pa.t oX490 0.11, mp Tube concn., No. pg./ml. Observed Corrected Blank 1 0 0.41 Blank2 0 0.46 1 0,0456 0.79 0.36 2 0.0912 1.20 0.77 3 0.137 1.40 0.97 1.32 0.i82 4 1.75 i.62 0.228 2.05 5 1.87 2.30 0.274 6 2.27 2.70 0.319 7 2.52 2.95 0.365 8 2.82 3.25 0.410 9 3.17 3.60 0.456 10
Table 111.
VOL. 34, NO. 11, OCTOBER 1962
1469
of 10 flasks containing amounts of sodium pyruvate from 0.25 t o 2.5 pg. and 100 pg. each of OPD in 3 ml. of 0.1N sulfuric acid was prepared. An identical set of samples was prepared containing these reagents plus 2.5 pg. each of glucose. All samples were heated a t once on the water bath for 3 hours. When the reaction was complete the samples were treated as described above, and the fluorescence was measured. The data for each set were plotted giving identical linear curves as shown in Figure 2. Replication of Measurements. Triplicate samples containing 0.25 pg., 1.25 p g . , and 2.5 ug. of sodium pyruvate, oxalacetic acid, and CYketoglutaric acid were treated in a miniature reaction with OPD in 3 ml. of 2 N sulfuric acid. Samples were diluted to 5 ml. as described, then measured fluorometrically. The data are shown in Table V.
O 5u9 ITr
I
REP C TIC $1 2PTE @c PVR7,1/kTE
a'
i!YD,
A I
:-e'
c
,
-~
2
3
1
4
5 Hours
Figure 1. Reaction rates of sodium pyruvate and OPD in 2N sulfuric acid (A) and 50% sulfuric acid ( 0 )
measured and the data were recorded in Table 111. ,4 linear graph of these data was obtained by plotting fluorescence vs. concentration. Preparation of Rate Curves. The procedure for examining the rate of quinoxaline formation was to prepare 12 to 15 samples containing 2.5 pg. of sodium pyruvate and 100 pg. of OPD in 3 ml. of either 2 N or 50% sulfuric acid and to place these in the water bath a t once. At regular time intervals a sampIe was removed, cooled, and diluted to 5 ml. in 50% sulfuric acid as indicated previously, then measured fluorometrically. The fluorescence (pa.) was plotted against time (see Figure 1). The rate of reaction of pyruvate with OPD was examined in media of varying acidities. Table IV contains the data for this experiment. Fluorescence Measurement in The Presence of Carbohydrate. A set
Table IV. Rate Studies for Reaction of Pyruvate with OPD in Media of Varying Acidities Fluorescence (pa. X 0.1) Phos-
phate
Ti?,=, Sulfuric acid min. 0.1N 2N 50y0 10 0.04 0.05 0.03 15 0.07 0.14 0.09 30 0.09 0.17 0.13 45 0.10 0.19 0.15 60 0.11 0.20 0.18 75 0.12 0.22 0.21 110 0.13 0.23 0.21 135 0.13 0.21 0.23 165 0.13 0.21 0.22
1470
ANALYTICAL CHEMISTRY
buffer
PH 8.3
0.08 0.01 0 0 0 0 0 0 0
RESULTS A N D DISCUSSION
The general reaction for the preparation of quinoxalines of alpha-keto acids is expressed in Equation 1 for the formation of 2-hydroxy-3-methylquinoxaline, as originally described by Hinsberg (6).
L(
364 3 2-1 , 0
2 2 81 2 4i,
2
2
c-
v)
o
