for kinetic investigations.
(3) D. L. Bloxam and W. H. Warren, Anal. Biochem., 60, 621 (1974).
LITERATURE CITED (1) S. M.Hess and S.Udenfriend, J. pharmacal. k p . Ther., 127, 175 (1959). (2) W. D. Denckla and H. K. Dewey, J. Lab. Clin. Med.,69, 160 (1967).
for review September 20! lg7& Accepted February 22, 1977.
Determination of Gentamicin Complex Components in Fermentation Broth by in-situ Fluorimetric Measurements of 4-Chloro-7-nitrobenzo-2-oxa- 1,3-diazole Derivatives Peter Kabasakalian, Sami Kalliney, and Anita W. Magatti” Development Division, Schering Corporation, Bloomfield, New Jersey 07003
A method for quantitating the individual components of gentamicin during the progress of a fermentation is described. Fluorimetric measurements are carried out in situ on the 4-chioro-7-nitrobenzo-2-oxa-l,&diaroie (NBD chloride) derivatives formed after thin-layer chromatography (TLC) of the clarified fermentation broth. This fluorimetric procedure is 800 times as sensitive as its ninhydrin analog. /-Methionine Is shown to be Involved in the methylation of gentamicin C1, to C2 and then to C1.
Gentamicin, a broad-spectrum aminoglycoside antibiotic complex produced by Micromonospora purpurea (1-3), is composed of three components, C1, Ct, and C1, (Figure 1). These differ from each other in the degree of methylation a t the 6‘ position. Although the component ratio can be followed 24 during a fermentation by microbiological estimation (4), h is required. The fast, direct densitometric method of Wilson et al. (5), using the ninhydrin chromogenic spray procedure after resolution by thin-layer chromatography (TLC), is not sensitive enough to follow directly the course of a fermentation. Since fluorescence is 10-100 times more sensitive than colorimetric procedures, fluorigenic labeling techniques were investigated. 4-Chloro-7-nitrobenzo-2-oxa-l,3-diazole (NBD chloride), which reacts with primary and secondary amines (6) while yielding a nonfluorescent hydrolysis product, was found to be the fluorigenic reagent of choice.
EXPERIMENTAL Apparatus. A Schoeffel SD 3000 double-beam densitometer equipped with a SDC 300 density computer-recorder (Schoeffel Instruments, Westwood, N.J.) was used in the reflectance (single beam) mode. It contained a high pressure xenon-mercury lamp and a miniature quartz-type monochromator set at 420 nm (excitation). The reflectance mode accessary had a built-in 420-nm sharp cut-off filter and an insertable wedge filter which was set at 530 nm (fluorescence). The inlet and exit beam slits were 1.5 and 1 mm, respectively. The settings in the density computer were positive, ratio, and numerator. Scanning and recorder speeds of 10 cm/min were used. Peak areas were measured with a Disc Integrator, Model 252A, Disc Instruments, Inc., Santa Ana, Calif. Precoated silica gel plates (0.25-mm thickness) on 20 X 20 cm glass purchased from Analtech, Inc., Newark, Del., were scored into 20 parallel lanes with a Schoeffel Scoring Device. The Chromaflex Dipping Tank was obtained from Kontes, Vineland, N. J. Reagents. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) was purchased from Aldrich Chemical Co., Milwaukee,
Wis. Gentamicin and sisomicin standards and gentamicin fermentation broths (7) were obtained from Schering Research Division. Procedure. The gentamicin fermentation broth samples were acidified to pH 2 with sulfuric acid, then centrifuged to separate mycelium and particulate matter. The pH of the aqueous solution was adjusted to 12 with sodium hydroxide and appropriate dilutions were made. TLC plates were spotted using Microcaps (Drummond Scientific Co., Broomall, Pa.) to deliver 5 pL of solution. Samples were spotted according to the “data-pair” technique of Frei et al. (8). The plates were developed according to the procedure of Wilson et al. (5), air dried, dipped in methanolic NBD chloride (0.25 mg/mL) for 2 s, heated in an oven at 120 OC for 10 min, cooled, and rechromatographed in methanol in the same direction as the first development. The plates were scanned using excitation and emission wavelengths of 420 and 530 nm, respectively.
RESULTS AND DISCUSSION Sisomicin, a broad-spectrum antibiotic produced by Micromonospora inyoensis, and a 4‘,5‘-dehydro derivative of gentamicin C1, (9), was used as the model for studying the various quantitative TLC schemes since it has only one major component. The slope of the plot of the instrumental response (integrated area) vs. the amount of sisomicin spotted (in area unitslpg sisomicin) under optimum instrumental conditions was used as the criterion of sensitivity. This value for the ninhydrin chromogenic spray procedure (5) was 0.8 area units/kg sisomicin. Spray reagents which did not require heat for fluorigenic labeling were first investigated. The fluorescamine quantitative TLC procedure of Sherma and Touchstone (10) looked promising. Although it was sensitive (27 area unitslpg sisomicin), the fluorescence decayed rapidly despite the use of a triethylamine stabilizing spray. o-Phthalaldehyde (OPT), which is soluble and stable in aqueous buffers, was reported by Benson and Hare (11)to be 5-10 times more sensitive than fluorescamine when reacted at rcmm temperature with primary amines in the presence of 2-mercaptoethanol in aqueous solution. There has been no reported use of OPT for TLC systems. The use of OPT as a fluorigenic spray for the sisomicin TLC system failed to produce any significant fluorescence even though it worked in solution. This approach was abandoned after the reason for its failure could not be ascertained. We then turned to spray reagents which required heat for fluorigenic labeling. The literature (12) indicates a preference for NBD chloride (which reacts only with primary and secondary alkylamines) over dansyl chloride (l-dimethyl-amiANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
953
Table 11. Comparison of the Sensitivity of the Ninhydrin and Best NBD Chloride Procedures for Sisomicin NBD chloride procedure Spray Dip
Ninhydrin procedure GENTAMICIN C1
R 2 R ' = CH3
GENTAMICIN Cp
R=CH3, R'=H
,
Area response Per y g sisomicin
GENTAMICIN CIO R R'; H
Figure 1. Structure of gentamicin complex
Table I. Relative Fluorescence Response Obtained When Varying the Conditions of the Separate Steps in the Analytical Procedure for Sisomicin Relative area response
Variable Redevelopment solvent and time NBD chloride reaction heating time NBD chloride dip time NBD chloride concentration in dip tank Basic spray. solution
Methanol, 45 min Ethanol, 105 min Propanol, 165 rnin 0 min 5 min 1 0 min 1 5 min
100
2s
100
3s 4s 5s 0.25 mg/mL 0.50 mg/mL 1.00 mg/mL 2.00 mg/mL Methanol-sodium acetate Ethanol-sodium acetate None
84 58 0
25 100
90 96 88 71 100
90 70 70
63 100
94
nonaphthalene-5-sulfonyl chloride) for its greater selectivity, since the latter also reacts with arylamines and phenols and yields fluorescent hydrolysis products. The usual quantitative TLC in-situ analyses of amines via NBD chloride do not use the spray technique (13-15) for fluorigenic labeling. The fluorescent derivatives are made prior to TLC; they are then separated by TLC and quantitatively analyzed by in-situ fluorimetry. The advantage of this technique is reported to be the avoidance of background irregularities due to uneven spraying. Benjamin et al. (16) reported a detection method for gentamicin, kanamycin, and tobramycin (all aminoglycosides) which involved spraying developed TLC plates with a saturated ethanolic solution of sodium acetate followed immediately by spraying with a solution of NBD chloride in absolute ethanol (2 mg/mL). The sprayed plate was heated in an oven a t 120 OC for 5 min, cooled at room temperature for 2-3 min, and then rechromatographed in methanol. The fluorescent spots on the plate were visualized a t 366 nm. The analytical procedure with quantitative readout was studied to increase the sensitivity and reduce the overall time needed for analysis (Table I): a. Since others (12-15) report excitation wavelengths of 436 to 482 nm, we scanned this region and found that 420 nm gave us the most sensitive results with our combination of light source and filters. The slope of the response line was now found to be 58 area unitslpg sisomicin. b. The redevelopment of the TLC plates to remove excess NBD chloride and clean up the background was studied using methanol, ethanol, and propanol. Methanol was found to be the best, the criteria being a reduction of time of operation and a reduction in background. 954
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
'
0
24
48
72
96
120
144
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Flgure 2. Change in gentamicin C,,, Cp, and C, fraction during normal fermentation without the addition of bmethionine at the start of the fermentation
c. The NBD chloride heating time was varied from 0 to 15 min a t 120 "C. The best results were obtained after a 10-min heating time. d. The NBD chloride application techniques of spraying and dipping (17,18) were compared. The dip technique increased the slope of the response line to 660 area units/pg sisomicin after the NBD chloride concentration of 0.25 mg/mL (which gave the best background) and a 2-s dip time were used. e. The stability of NBD chloride reagent with or without the presence of the base, sodium acetate, in methanol and ethanol was studied in the presence and absence of laboratory fluorescent light. Light was found to be detrimental when sodium acetate was present: the NBD chloride solution remained yellow in the dark, but changed to a dark green color over a 1-week period in the light. In the absence of sodium acetate, methanolic solutions colorized less than ethanolic solutions (colorless to a very pale yellow solution); the effect of light was minimal. f. Since the TLC development system included a base, ammonium hydroxide, a study was made to determine if the sodium acetate spray was necessary. It was found that a basic spray was unnecessary after the initial development in the ammonium hydroxide system before the application of NBD chloride. Using the best conditions, the area response was found to be linear up to 500 ng/spot of sisomicin, while twice the noise level represented 2 ng/spot. The results of our various improved techniques are compared with the ninhydrin procedure in Table 11. Although adjustment to pH 2 liberated gentamicin and sisomicin from the mycelia in the fermentation broths, spotting at pH 2 on the TLC plates yielded V-shaped spots not conducive to good in-situ fluorimetry. Readjusting the pH to 12 after centrifugation gave compact spots. Since these aminoglycosides in free base form adsorb carbon dioxide readily and form carbamates (19),a study of the effect of
l-methionine is involved in the methylation of CIa to Cz and then to GI. This is consistent with the work of Lee et al. (20) and Daniels et al. (21).
LITERATURE CITED (1) M. J. Weinstein, G. M. Luedemann, E. M. Oden, G. H. Wagman, J. P.
Rosselet, J. A. Marquez, C. T. Coniglio, W. Charney, H. L. Herzog, and J. Black, J. Med. Chem., 6, 463 (1983). (2) G. H. Wagman, J. A. Marquez, and M. J. Weinsteln, J. Chromatog., 34, 210 (1968). (3) D.J. Cooper, P. J. L. Daniels, M. D. Yudis, H. M. Marigliano, R. D. Guthrie, and S. T. K. Bukhari, J. Chem. Soc., 1971, 3126. (4) G. H. Wagman, E. M. Oden, and M. J. Welnsteln, Appl. Microblol., 16, 624 (1968). (5) W. L. Wllson, G. Richard, and D. W. Hughes, J. Pharm. Sci., 62, 282 i, i a m P. B. Ghosh and M. W. Whitehouse, Biochem. J., 108, 155 (1968). B. K. Lee, R. G. Condon, G. H. Wagman, K. Byrne, and C. Schaffner, J. Antlbiot., 27, 822 (1974). H. Bethke, W. Santi, and R. W. Frei, J. Chromtogr. Scl., 12, 392 (1974). H. Reimann, D. J. Cooper, A. K. Mallams, R. S.Jaret, A. Yehaskel, M. Kugelman, H. F. Vernay, and D. Schumacher, J. Org. Chem., 39, 1451 (1974). J. Sherma and J. C. Touchstone, Anal. Lett., 7 (4,279 (1974). J. R. Benson and P. E. Hare, Roc. Nat. Acad. Sci. USA, 72, 819 (1975). J. F. Lawrence and R. W. Frei, Anal.,Chem., 44, 2046 (1972). R. W. Frei and J. F. Lawrence, J. Assoc. Offic. Anal. Chem., 55, 1259 (1972). F. Van Hoof and A. Heyndrickx, Med. Fak. Landbouwet, 38 91 1 (1973). F. Van Hoof and A. Heyndrickx, Anal. Chem., 46, 286 (1974). D. M. Beniamin, J. J. McCormack. and D. W. Gumu, Anal. Chem., 45, 1531 (1973). W. Wortmann and J. C. Touchstone, in “Quantitative Thin Layer Chromatography”, J. C. Touchstone, Ed., Wiley-Interscience, New York, 1973 F: Abe and K. Sameiima, Anal. Biochem., 67, 298 (1975). R. U. Lemieux and M. A. Barton, Can. J. Chem., 49, 767 (1971). B. K. Lee, R. T. Testa, G. H. Wagman, C. M. Liu, L. McDanlel, and C. Schaffner. J. Antibiot., 26, 728 (1973). P. J. L. Daniels, A. Yehaskel, and J. €3. Morton, 18th Meeting of ICAAC, Chicago, Ill., October 1978, Paper 45.
.-. .,.
0
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120
144
- HOURS
Figure 3. Change in gentamicin Cia, C2,and C, fraction during a fermentation with the addtion of Lmethionine at the start of fermentation
carbon dioxide on the results of quantitative TLC was carried out. No effect was noticed. The variation in the gentamicin complex component area response fraction during a normal fermentation is shown in Figure 2. Gentamicin Cla, Cz, and C1 ratios remained relatively constant throughout the run. When 1-methionine was added at the start of the fermentation (Figure 3), gentamicin C2 and C1 ratios changed continuously. No detectable gentamicin Cla, the least methylated of the components, was present during most of the fermentation. Essentially, only two components were present, C2 and C1, the mono- and dimethylated derivatives of C1,. The gentamicin C1 fraction grew a t the expense of gentamicin Cz. It would appear that
RECEIVED for review January 21, 1977. Accepted March 15, 1977.
Sub-Part-per-Trillion Detection of Polycyclic Aromatic Hydrocarbons by Laser Induced Molecular Fluorescence J. H. Richardson” and M. E. Ando‘ General Chemistry Division, Lawrence Livermore Laboratory, University of California, Livermore, California 94550
Laser-induced molecular fluorescence is shown to be a sensitive and selective means for determining representative polycyclic aromatic hydrocarbons (PAH) In aqueous solutions. The limits of detection of benzene, naphthalene, anthracene, fluoranthene, and pyrene were found to be 19 ppb, 1.3 ppt (parts-per-trillion),
