the plume quenching process. This result was also noted by Wiley and Veeravagu ( 5 ) . These data can be treated in several ways. Figure 3 shows the information plotted as the C / H ratio us. the per cent of methane in the product mixture and again as the per cent of acetylene. The column used did not permit separation of ethane from ethylene. Another way that these data may be treated is to consider the extent of aromaticity in these test compounds. Here we define a function A , the number of aromatic hydrogens/total number/hydrogens per molecule, this value A is shown graphically us. the per cent of methane in Figure 4. These data clearly show that the composition of the products coming from the plume-quenching process reflects the elemental composition of the sample. This happens even though a residual carbon film is left on the walls of the degradation chamber. Either the carbon is but a small fraction of the total amount of the quenching products or the percentage of carbon formed increases as the H/C ratio decreases. From visual inspection, we suspect that the second eventuality is occurring although no successful attempts to quantitate this conclusion can be reported. The data in Figure 3 show a realistic estimation of the error in these analyses. These errors result from two causes, nonreproducibility of the degradation event and from the graphical data analysis used to obtain these numbers. Experimental evidence indicating that degradation by laser radiation can be quite precise; one specific series obtained with electronic integration shows a standard deviation of less than *2.0'% (2). Therefore, we conclude that much of the error found in these values results from the method of data interpretation.
Ln Y
3
S
I
I
0.00
I
I d 20
AROHATIC!:I
Figure 4.
1
I
1
L
0.40
0.60
1 0.80
I
I
1 .oo
FUUCTIOII, A
Plasma stoichiometric analysis
YO methane yield piotted vs. the aromaticity function, defined in text. Compound identification: ( A ) paraffin, (E) durene, (C) polystyrene, (D) 1,2-diphenyIethane, (E) naphthalene, terphenyl, benzo[d,e,f]phenanthrene
These results show that this method does offer a useful route for the characterization of the type of molecule under study. Certainly the case of hydrocarbons is the most elementary. Similar estimations on compounds containing carbon, hydrogen, and oxygen must be done. These data should be, in theory, useful for an estimation of the oxygen composition. (For instance the amount of carbon monoxide might be monitored.) Such studies are now under way. We term this technique Plasma Stoichiometric Analysis and feel that it may serve as a useful technique for the routine and rapid characterization of materials. Received for review October 20, 1972. Accepted February 5 , 1973.
Use of Newer Amino Group Reagents for the Detection and Determination of Kanamycin David M. Benjamin, John J. M c C o r m a c k , and Dieter W. Gump Departments of Pharmaco'ogy and Medicine, University of Vermont College of Medicine, Buriington. Vt. 05407
Kanamycin (KM) is a polybasic aminoglycoside antibiotic which possesses clinically useful activity against a variety of pathogenic bacteria ( I ) . KM, however, is capable of producing serious adverse effects such as renal damage and auditory and vestibular dysfunction; the potential toxicity of KM, particularly in patients with preexisting renal impairment makes it highly desirable to monitor levels of this antibiotic during therapy (2). A number of empirical guides for controlling KM therapy have been used and these guides have become increasingly sophisticated ( 3 ) , but it is generally agreed that direct measurements of drug levels in serum would serve more usefully to control KM therapy. Many commonly used assays for K M in clinical laboratories have been based on inhibition of the growth of sensitive bacteria by this antibiotic; until recently, such as-
says required up to 24 hr for completion and this time factor was a definite disadvantage with such biological assays. Recently Sabath ( 4 ) and his colleagues have described a rapid microbiological method for the determination of KM but the results obtained with this method may be difficult to interpret when a patient is receiving additional antimicrobial agents concurrently with KM. The limitations of conventional microbiological assays for K M have prompted several investigations which have as their objective the development of a clinically useful method for the analysis of aminoglycoside antibiotics in biological fluids. D. H. Smith and his colleagues ( 5 ) have described very recently an elegant and rapid enzymatic assay for the aminoglycoside antibiotic gentamicin in clinical samples and this method, in principle, is applicable for the determination of kanamycin. The present report
(1) P. A . Bunn, Med. Clin. N . A m e r . . 54, 1245 (1970) (2) S. M . Finegold, A n n . N . Y.Acad. Sci., 132, 942 (1966). (3) G . E. Mawer. B R . Knowles, S. B. Lucas, R . M . Stiriand, and J. A . Tooth, Lancet, 1 , 12 (1972).
(4) L. D. Sabath. J. I . Casey: P. A. Ruch, L. L. Stumpf, and M . Finland, J . Lab. Clin. M e d . , 78, 437 (1971) (5) D. H. Smith, E. Van Otto, and A . L. Smith, New Eng. J . Med.. 286, 583 (1972) A N A L Y T I C A L C H E M I S T R Y , VOL. 45,
NO. 8,
J U L Y 1973
1531
Table I. Relation of Absorbancea to KM Concentration for the Reaction of KM with TNBS K M concn, pg/ml Absorbance, 420 n m 1.25 2.50 5.00 7.50 10.00
0.058 0.1 13 0.220 0.325 0.445
(0.055-0.059) ( 0 . 1 1 2-0.1 15) (0.21 1-0.229) (0.323-0.332) (0.443-0.446)
idase (8). We later investigated the reaction between KM and NBD chloride since this latter reagent has been described by Ghosh and Whitehouse (9) as a sensitive fluorogenic reagent for amines and amino acids; NBD chloride has a distinct advantage over another fluorigenic reagent for amines, 5-dimethylaminonaphthyl-1-sulfonylchloride (dansyl chloride) in that NBD chloride itself is not appreciably fluorescent.
'Average of three determinations (range in parentheses)
EXPERIMENTAL Table II. Apparent Absorptivitiesa of Trinitrophenylated Products Starting amino cornpd nh
Glycine KM To br amyci n
Gentamicin a
a X
7 10 9 9
0.95 2.90 3.2 2.1
f 0.02 f 0.10 f 0.14 f 0.08
Measured at the wavelength of maximum absorbance in the region Number of determinations. f standard error of the mean.
420-430 n m .
describes approaches which we have taken to develop a purely chemical assay procedure for kanamycin, which is more rapid than conventional microbiological assays for this antibiotic and which may be a useful alternative to such methods as those described by Sabath and Smith and their respective coworkers. I t should be noted that kanamycin and related aminoglycoside antibiotics can be determined by gas-liquid chromatography (6) but this approach has not been applied as yet to the estimation of aminoglycoside levels. Kanamycin is composed of a 3-glucosamine moiety and a 6-glucosamine moiety joined by glycosidic linkages to 2-deoxystreptamine; the molecule possesses four primary
OH
C H20H H
O
G
amino groups, two of which are, a t least formally, analogous to those amino groups present in simple amino sugars and two of which are analogous to the amino groups of 1,3-diaminocyclohexane. Since these amino groups are such a prominent feature of the chemical structure of kanamycin, we decided to investigate the reactivity of this antibiotic toward newer amino group reagents in an effort to establish a basis for clinical analysis of kanamycin. Our initial efforts were concentrated on a study of the reaction of KM with TNBS, a reagent introduced by Satake and his colleagues (7) for the determination of amino groups in amino acids and peptides and which has recently been employed in a spectrophotometric assay of monoamine ox(6) K . Tsuji and J. H . Robertson, Ana/. Chem.. 42, 1661 (1970). (7) K . Satake. T. Okuyama, M . Ohashi, and T. Shinoda, J , Biochem i r o k p i . 47,654 (1960)
1532
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 8, JULY 1973
Apparatus. Ultraviolet and visible spectroscopy were performed using a Perkin-Elmer Model 202 recording spectrophotometer. T h e fluorescence of spots on T L C plates was detected using a Chromato-Vue ultraviolet viewer equipped with a transilluminator high-intensity ultraviolet l a m p (long wave; 366 n m ) obtained from Ultraviolet Products, S a n Gabriel, Calif. T L C plates coated with Silica Gel G (250 p thickness) were obtained from Analtech, Inc., Newark, Del. Reagents. 2,4,6-Trinitrobenzene-l-sulfonicacid ( T N B S ) was purchased from Eastman Chemical Co., Rochester, N.Y.; 7chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) was purchased from Regis Laboratories, Chicago, Ill. Kanamycin was obtained from Bristol Laboratories, Syracuse, N.Y.; gentamicin was obtained from Schering Laboratories, Bloomfield, N.J.; a n d tobramycin was obtained from Eli Lilly Laboratories, Indianapolis, Ind. Procedures. Reaction of Kanamycin a n d Related Aminoglycosides with TNBS. T K B S (0.2 ml of a 1% solution in water) was added t o 1.6 ml of 2% sodium bicarbonate solution and then 0.2 ml of a solution of K M in 270 sodium bicarbonate was added. The reaction tubes were covered with parafilm and the color developed by heating a t 60 "C for 15 min; a blank reaction containing no K M was treated identically. The absorbance of t h e reaction mixtures was measured a t 420 n m against the reaction blank. The reactions between tobramycin and gentamicin and T N B S were carried out in an identical manner except that t h e absorbance of t h e reaction mixtures was determined at 430 n m . Reaction of K M uith NBD-Chloride. (a) Simple Aqueous Solutions. Kanamycin solutions (5-10 p1 containing 0.1-10 pg) were applied to an unactivated silica gel plate and the chromatogram was developed for a distance of 6-7 cm (approximately 20 min) using methanol-concentrated ammonia-water (3/1/1) as developing solvent. T h e plate was then sprayed with a saturated alcoholic solution of sodium acetate followed immediately by spraying with a solution of NBD chloride in absolute ethanol (2 m g j m l ) . The sprayed plate was heated in a n oven a t 120 "C for 5 min, cooled a t room temperature for 2-3 min and then rechromatographed in methanol, in the same direction as the first development, for a distance of 6-7 c m . Fluorescent spots on the plate were visualized in a Chromato-Vue using the transilluminator accessory (366 n m ) . The K M derivative appeared as a yellowgreen fluorescent spot a t R f 0.35 f 0.05. ( b ) K M in Urine. Determination of K M in urine was accomplished by applying 5 p1 of urine (containing 0-3 pg K M ) to a silica gel plate followed by chromatography, spraying. and visualization as outlined above. By visually comparing the intensity of the fluorescence of the spots corresponding to the K M derivative in unknown samples with the fluorescent intensity of K M standards it was possible to estimate the urinary concentration of K M . The time required for this analysis is 40-45 min. (c) Estimation of K M in Serum. To 1 ml of serum (containing 0-50 pg K M ) 1 ml of trichloroacetic acid (10% in water) was added; the precipitated protein was removed by centrifugation and the supernatant fluid was decanted off, transferred to a pearshaped distilling flask, and evaporated to dryness under vacuum (15-20 m m ) . The residue was dissolved in 0.1 ml of water a n d 5 p1 of this solution were applied to a silica gel plate. The plate was processed as described above and again the fluorescent K M derivative appeared a t Ri approximately 0.35.Comparison of the intensity of the K M spot in unknown samples with t h e intensity of K M standards prepared in serum permits estimation of serum K M levels in the range 0-50 pgjml. The time required t o process and analyze one sample is approximately 60 min. (8) F. Obata. A. Ushiwata, and Y . Nakarnura, i.Biochern ( T o k y o ) . 69, 349 (1971). (9) P. 6.Ghosh and M . W. Whitehouse, Biochern J.. 108, 1556 (1968).
RESULTS AND DISCUSSION We decided initially to study the reaction of KM with TNBS because it was reported (IO) that certain amino sugars react with TNBS to give products exhibiting absorption spectra, under appropriate conditions, different from those obtained for the reaction of TNBS with simple amines and amino acids; accordingly, we hoped that the amino sugar moieties in K M would react with TNBS to give a product which might have spectral characteristics different from those of the majority of amino-containing substances present in biological fluids. However, preliminary studies under a variety of experimental conditions indicated that KM reacted with T N B S to form a product which exhibited spectral characteristics (e.g., max 420 nm a t alkaline pH) which resembled closely those of the products of the reaction of simple amines and amino acids with TNBS and were unlike those observed previously for amino sugars such as glucosamine. The reaction between KM and TNBS can be used as a rapid and sensitive method for estimating the concentration of KM in samples free from other amino-containing compounds. For example, as shown in Table I, excellent linearity between absorbance and KM concentration is observed when the reaction with TXBS is carried out as outlined in the experimental section; similar results are obtained when the reaction is carried out at pH 7.0 in phosphate buffer. Using the standard system for studying the reaction of KM with TNBS ( i e . , sodium bicarbonate solution; heating a t 60 "C for 15 niin) we determined the relative absorptivity for the trinitrophenylated KM dederivative which is formed. The value for the absorptivity is presented in Table I1 along with the values obtained for the amino acid derivative glycine and aminoglycosides tobramycin and gentamicin. The molar absorptivity of KM for the TNBS reaction is approximately 3 times that of glycine, which may indicate that 3 of the 4 primary amino functions of KM are reacting to form relatively stable trinitrophenylated derivatives under the conditions employed by us; tobramycin and gentamicin appear to have three and two amino groups, respectively, which are reactive toward TNBS. It should be stressed that some caution is required in interpreting these data on relative absorptivities in view of observations which indicate that the reaction of amines with T S B S is somewhat complex ( 1 1 ) and that the absorptivities of reaction products of some amines with TKBS show considerable variations (8). While the reaction of TNBS with KM can be useful for estimating the concentration of KM in samples free of interfering amino compounds, it is not suitable for analysis of KM in complex biological fluids, especially since the absorption characteristics of the product of the reaction were not appreciably different from those which are observed for a wide variety of naturally occurring amines. In order to minimize the interference by amines present in biological samples with possible chemical determinations of KM, we decided to investigate the possibility of separating KM from such substances using thin layer chromatography. We found TLC on silica gel G using a methanol-ammonia-water system to be a satisfactory method of separating KM from many ninhydrin-positive substances present in urine; a similar TLC system (12) has been used which separates KM from other closely related aminoglycosides. In order to permit detection of small quantities of KM on TLC plates we used NBD chloride as a fluorigenic detection reagent. Addition of KM to (10) J T. Galambos and R . :Shapira. Anal. Biochem. 15,334 (1966). (11) A. R. Goldfarb, Biochemistry. 5,2570 (1966). (12) H. Maehr and C. P.Schaffner. J Chromatogr.. 30,572 (1967)
Table Ill. Comparison of KM Estimationa in Serum Samples Performed by the NBD Method and a Microbiological Methodb Sample No. 1 2 3 4
5 6 7
8 9 10 11 12 13 14 15 16 17 c 18C 19c 20c
NBD method 3.5 18 10 5 25
10 15 5
0
Microbiological method 4.2 27 7.5 1.9 29.2 12.2 15 7.2
0
30 45 40
31 33.7 43.8
10
10.1
5
5.4
Actual value 4 24 6 2 32 12 16 8
0 25 50 37.5 9 5
0
0
0
25 20 7.5 10 12.5
16 18.9 6.2 13.4 29.5
18.8
. . . .
.. .. .. ..
a All values are the average of two determinations expressed in fig/ml of serum. Method described in ref 4 . Samples of serum of patients receiving kanamycin.
a solution of NBD chloride in ethanol containing sodium acetate, a t room temperature, results in gradual appearance of an absorption maximum a t 470 nm in the reaction solution; such a peak in the 460-480 nm region is also observed with S B D derivatives of other amines (13, 14). When KM samples, spotted on silica gel plates, were sprayed first with a saturated ethanolic solution of sodium acetate and then with NBD chloride in ethanol and the plate was heated, strongly fluorescent (green-yellow) spots were observed for the NBD derivative of KM. Rechromatography in methanol of plates treated with NBD chloride resulted in a great increase in the sensitivity of the detection method. Urine samples containing KM were applied directly to TLC plates. KM in urine could easily be observed a t levels (50-600 pglml) (15) which are reached in patients treated with KM. Serum levels of KM are considerably lower than urinary levels (16) and this necessitated concentration of deproteinized serum samples prior to the chromatographic procedure. A number of unknown samples of serum containing various amounts of KM were prepared in another laboratory and submitted to us for analysis. The results obtained in determinations of KM by the NBD method coupled with TLC are shown in Table 111 which compares the present method with a rapid microbiological method. The correlation between the chemical method and the microbiological method is reasonably good, especially considering that the chemical method involves a visual estimation of the KM concentration based on comparison of the fluorescence of spots in unknown samples with appropriate standards. The last four entries in Table I11 represent estimations of KM in the serum of patients receiving KM; except for sample 20, the agreement between estimates of KM in the clinical samples is good. Attempts to elute the fluorescent KM derivative from the silica gel with a variety of solvents, for (13) D. J . Birkett. N. C. Price, G . K. Radda, and A. G. Salmon, FEBS (Fed. Eur Biochem. S o c . ) Lett.. 6, 346 (1970). (14) R . A. Kenner and A. A. Aboderin, Biochemisfry. 10,4433 (1971). (15) A. White and V . Knight, Ann. N . Y.Acad Sci., 76, 277 (1958). (16) C. M. Kunin,Ann. N . Y . Acad. Sci.. 132,811 (1966). A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 8 , J U L Y 1 9 7 3
1533
quantitative fluorimetry in solution, have been uniformly unsuccessful. At present we are endeavoring to increase the accuracy of the chemical method by direct fluorimetric scanning of TLC plates, a technique which has proved extremely useful in quantitative work (17-19). We have demonstrated the feasibility of detecting and estimating KM in samples of urine and serum by a chemical procedure; the semiquantitative nature of this procedure, based, as it is a t present, on visual comparison of fluorescent intensity with the intensity of known amounts of KM on TLC plates, is a drawback to the use of the procedure, but studies, currently in progress, of the utility of direct fluorimetric scanning of TLC plates to more accurately estimate KM levels may well result in the elimina(17) B. L. Hamman and M . M . Martin, J . Lab. Ciin. Med.. 73, 1042 (1969). (18) G . Pataki and K-T. Wang,J. Chromatogr.. 37, 499 (1968). (19) J . E. Sinsheimer, D. D. Hong, J. T. Stewart. M . L. Fink, and J. H . Burckhalter. J . Pharm. Sci.. 60, 141 (1971).
tion of this disadvantage of the present method. Preliminary studies in this laboratory have indicated that the NBD chloride procedure can be used to detect aminoglycosides such as gentamicin and tobramycin and basic peptide antibiotics such as polymyxin B, and studies are now in progress to develop assays for these agents similar to that reported here for KM.
ACKNOWLEDGMENT We are grateful to Richard Lewis for performing the microbiological assays of KM levels in serum and for kindly preparing the unknown samples of KM in pooled human serum for the analyses. Received for review June 26, 1972. Accepted January 26, 1973. A preliminary communication of this work was presented a t the Eleventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlantic City, N.J., October 1971. This work was supported in part by Grant AI 00365 (USPHS) and by a grant from Bristol Laboratories, Syracuse, N.Y.
Retention of Mercury When Freeze-Drying Biological Materials P. D. LaFleur Activation Analysis Section, Analytical Chemistry Division, National Bureau of Standards, Washington. D. C. 20234
In the past several years a number of poisonings (1-3) due to the presence of methylmercury (and/or other organomercurials) has caused widespread public concern and, concomitantly, considerable effort on the part of the scientific community to answer resulting questions of toxicology, methods of analysis, etc. ( 4 ) . Two widely used methods for the determination of mercury in biological and environmental samples are neutron activation analysis and atomic absorption spectrophotometry. Although reporting results on a dry-weight basis is not widely practiced by biological and environmental scientists, the varying degree of desiccation of a biological sample as received often makes results reported on a wetweight basis difficult to interpret; it may be preferable to report analytical results on a dry-weight basis. In the case of neutron activation analysis, it is preferable because of problems with radiolytic pressure increases in the sample container, and the desire to irradiate as much sample as possible in as small a volume as possible, to work with dried samples. Lyophilization (freeze drying) is being used more and more for drying biological samples, since temperatures may stay at, or below, room temperature during the drying process. Obviously, however, the vapor pressure of any unbound mercury, or organomercury compounds, in (1) L. T. Kurland, S. N. Faro, and H. Seidler, World Neurol.. 1 , 370 (1960). (2) T . Tsubaki, Proc. Symp.: Mercuryin Man’s Environ., 131 (1971). (3) A. Curley, V. A. Dedlak, E. F. Girling, R. E. Hawk, and W. F. Barthel, Science, 172, 65 (1971). ( 4 ) N. A. Nelson, T. C. Byerly, A. C. Kolbye, Jr., L. T. Kurland, R. E. Shapiro. S. I . Shibko, W. H . Stickel, J . E. Thompson, L. A. Van Den Berg, and A. Weissler, Environ. Res., 4, 1 (1971).
1534
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O . 8, J U L Y 1973
the sample will be greater than the pressuie in the environment of the sample during lyophilization, and losses could occur. Data indicating losses on lyophilization have been published by Pillay et al. ( 5 ) . In order to determine systematically the extent of organomercurial losses, if any, a series of experiments was designed. It was essential that mercury in the tissues of the experimental animals used be there through normal biosynthetic process, rather than in vitro spiking, since this is the case with “real” samples.
EXPERIMENTAL Some experiments were performed in 1970 at the National Bureau of Standards with gold fish which had lived in a n environment containing inorganic mercury-203 and no losses on freeze drying for those samples were observed. This agrees with the observation subsequently published by Pillay et al. (5). These experiments did not, however, assist in the evaluation of possible losses in the case of organomercurials. A series of experiments were initiated, therefore, in which inorganic mercury, methylmercuric chloride, and phenylmercuric acetate, all tagged with Z@3Hg, were fed to guinea pigs and white rats. Two guinea pigs each were fed cabbage upon which had been deposited 2@3Hg(N03)z, CH3203HgC1, and phenylmercuric acetate. Three rats each were fed standard rat pellets upon which CH32@3HgC1and phenylmercuric acetate had been deposited, and two rats were fed inorganic z03Hg on food pellets. T h e feeding regimen was followed for at least 3 weeks in all cases and the animals were then sacrificed. All the guinea pigs. one rat each from t h e CH3HgC1 and phenylmercuric acetate, and both rats being fed inorganic mercury were sacrificed within 36 hr of t h e last ingestion of tagged mercury compounds. One rat from each organomercurial regimen was sacrificed 1 2 days after the (5) K . K . S. Pillay, C. C. Thomas, Jr., J. A . Sondel. and C. H . Hyche, Anal. Chem.. 43, 1419 (1971).
