Amplifier Circuit. The amplifier circuit, shown in Figure 1, is basically similar to the differential amplifier described previously (7). I n order to achieve fast response, the input stage consists of two 143 A F E T amplifiers (Analog Devices, Cambridge, Mass.) specially selected to have bias currents of less than 5 PA. The overall circuit response time is improved and ringing eliminated by including a small trimming capacitor in the negative feedback loop of the differential amplifier stage. To allow small potential changes t o be observed at high sensitivity-e.g., on an oscilloscope-an offset of up to 100 mV is summed with the output of the differential stage. Greater offsets can be obtained by reducing the value of the 500 KO input resistor t o the output amplifier. The offset is derived from a high stability f 5 V supply, a 805-V5 voltage regulator (Beckman Instruments, Fullerton, Calif.) driven by the amplifier power supply. Amplifier Response. The gain-frequency response of the circuit, Figure 2, shows the bandwidth to be greater than 100 KHz. The common mode rejection ratio, adjusted to better than 10j:l at dc shows a decrease with increasing frequency at approximately 20 db/decade. The response of the amplifier t o a step input, Figure 3, shows the response time t o be
about 2 psec for a small signal. This is approximately 1000 times faster than for the previously described unit (7). Measurement of the amplifier input current is difficult because it is so small but the following method gave an estimate. An ac oscillator with a n output of 10 mV was connected in series with a screened lo9 O resistor across the amplifier input. The amplifier output was then measured o n a n oscilloscope as a function of frequency. Difference between the input and output voltage was equated t o a n IR drop in the lo9O resistor, thus allowing the input current t o be calculated. Figure 4 shows that a t low frequencies, the current is about 1 pA while at high frequencies it is about 10 PA. The properties of this amplifier should allow the experimental study of membrane electrode potential relaxation processes. I n addition, interesting possibilities exist for the use of membrane electrodes to follow fast reactions in solution by stopped-flow techniques. RECEIVED for review June 26, 1970. Accepted August 31, 1970. This work was supported by a grant from the National Science Foundation.
Gas-Liquid Chromatographic Determination of Amino-Glycoside Antibiotics: Kanamycin and Paromomycin Kiyoshi Tsuji and John H. Robertson Conlrol Analytical Research and Decelopment, The Upjohn Company, Kalamazoo, Mich. 49001
KANAMYCIN ( I ) , paromomycin ( 2 ) , and neomycin ( 3 ) are water soluble, basic antibiotics which are markedly similar in chemical, physical, and biological properties. Commercial preparations of these amino glycoside antibiotics contain various isomers (Figures 1 , 2 , 3). They may be differentiated by paper, thin layer, and column chromatography ( 4 , 5 ) and quantitated by microbiological assay methods (6). The gas chromatographic assay method for neomycin (7) is adapted in this paper for the separation and determination of kanamycin and paromomycin and their isomers. EXPERIMENTAL
Apparatus. F & M Model 400 with flame ionization detector was used. Gas flow rates: hydrogen 40 ml/min, air 600 ml/min, and carrier gas (helium) 70 ml/min. Chart speed 0.25 inch/min. Oven temperature: 290°C for paromomycin and 300 “C for kanamycin. (1) T. Takeuchi, T. Hikiji, K. Nitta, S. Yamazaki, S. Abe, H. Takayama, and H. Umezawa, J . Antibiot. Ser A, 10, 107 (1957). (2) J. W. Davisson, I. A. Solomons, and T. M. Lees, Antibiot. Chemother. ( Washiitgton, D.C.) 2,460 (1952). (3) E. A. Swart, D. Hutchison, and S. A. Waksman, Arch. Biochem., 24, 92 (1949). (4) R. T. Schillings and C. P. Schaffner, “Antimicrobial Agents
and Chemotherapy,” Braun-Brumfield, Inc., Ann Arbor, Mich. 1961, p 274. ( 5 ) E. Roets and H. Vanderhaeghe, Pharm. Tijdschr. Belg., 44, 57 (1967). (6) Code of Federal Regulations, Title 21-Food and Drugs, U. S. Government Printing Office, Washington, D. C., 1968. (7) K. Tsuji and J. H. Robertson, ANAL.CHEM., 41, 1332 (1969).
Column. Glass column, 3 mm X 1830 mm (6 ft) packed with (1) 0 . 7 5 x OV-1 o n Gas Chrom Q, 100/120 mesh (Applied Science Laboratories, Inc., State College, Pa.) for paromomycin and (2) 3 z OV-1 o n Gas Chrom Q, 100/120 mesh (Applied Science Laboratories) for kanamycin were used. The columns were non-flow conditioned at 330 “C for 1 hour, followed by a n injection of approximately 50 p1 of Silyl-8 (Pierce Chemical Co., Rockford, Ill.) with flow at 200 and 250 “C. Silylated samples of the antibiotics were injected a t 300 “C. The columns thus prepared had 210 and 250 theoretical plates per foot for silylated paromomycin I and kanamycin A, respectively. Internal Standard-Silylation Reagent. For paromomycin determinations, a solution containing 3 mg of trilaurin (Supelco, Inc., Bellefonte, Pa.) and 70 p1 N-trimethylsilyldiethylamine (Pierce Chemical) per ml of Tri-Si1 Z (Pierce Chemical) was prepared. F o r kanamycin determinations, a solution containing 8 mg of trilaurin and 25 pl of N-trimethylsilyldiethylamine per ml of Tri-Si1 Z was prepared. Reference Standard. A water solution containing 10.0 mg of paromomycin sulfate or kanamycin sulfate reference standard per milliliter was prepared. One-milliliter aliquots were pipetted in duplicate into 2-cm3 serum vials (No. 621133174, Kimble Glass, Div. of Owens-Illinois, Toledo, Ohio) and freeze dried. The vials were capped using red rubber closures (No. V-35, West Company, Phoenixville, Pa.) and aluminum retainers (No. 13-FA-003, West Co.). Sample. Samples were prepared in the same way as the reference standard. Silylation Procedures. One milliliter of the internal standard-silylation reagent was added to the sample and the reference standard vials using a tuberculin syringe. The
ANALYTICAL CHEMISTRY, VOL. 42,
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H O m H ZN
/
0 KANAMYCIN A KANAMYCIN B
KANAMYCIN C
R NHz R:= NH2
Rz=OH
R1ZOH
RZZNH2
-
R,= NH,
4 Min
Figure 1. Structure of kanamycins C)4zOH_
PAROMA M IN E
0-
PAROMOMYCIN PAROMOMYCIN
I 11
R1"H
R t -CH,NH2
R,:CH,NH,
RZ:H
Figure 2. Structure of paromomycins - OH
. \
Figure 4. Composite chromatograms indicating the separation of paromomycins I and I1 as well as neomycins I3 and C isothermally at 290 "C.
CH2NH2
\
Neobiosamine
(1) paromomycin I, (2) neomycin B, (3) paromomycin 11, (4) neomycin c
. ..
\
\
'.
N
0Neomycin B
R1=H
R2=CH2NH2
R~=NHz
Neomycin C
R1 =CH2NH2
R2=H
R3 =NH2
Neomycin LPB
R1 =H
R2=CH2NH2
R3=NHCOCH3
Neomycin LPc
R1 =CH2NH2
R2=H
R3=NHCOCH3
4 Min
Figure 3. Structure of neomycins vials were heated in a 75 "C oil bath for 45 minutes, swirling occasionally.
I 2
i'
RESULTS AND DISCUSSION
1,
Separation of Paromomycins I and 11. The merit of this gas chromatographic determination of paromomycin is its ability to separate and quantitate paromomycins I and 11 with greater facility than other methods. A typical chromatogram, which shows the separation of paromomycins I and I1 and neomycins B and C when present in the same sample (8), is shown in Figure 4. Note that paromomycins I and I1 differ only in their stereochemistry at Csin the paromose moiety (Figure 2). The CH2NH2group at the Cs position of paromomycin I is axial and that of paromomycin I1 is equatorial.
Figure 5. Composite chromatograms indicating the separation of kanamycins isothermally at 300 "C
(8) K. L. Rinehart, W. T. Shier, E. J. Hessler, H. K. Jahnke, J. H. Robertson, and K . Tsuji, J. Anribiot., in press.
(1) aminoglucosyl deoxystreptamine, (2) kanamycin B, (3) kanamycin A, and (4) kanamycin C
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The order of elution of the silylated paromomycins I and I1 is analogous to silylated neomycin (7). It may be speculated, therefore, that the equatorial trimethylsilylamine group at the C 6position is exposed to the outer environment to a greater extent than the corresponding axial group of paromomycin I. As a result, paromomycin I1 is more soluble in the liquid support phase than paromomycin I. This coupled with the expected lower volatility of the equatorial form (9) would explain why paromomycin I1 is retained longer on the OV-1 column than paromomycin I. Separation of Kanamycins. Figure 5 shows a chromatogram of the kanamycins. The compounds were identified by the LKB 9000 gas chromatograph-mass spectrometer in order of elution as (1) aminoglucosyl deoxystreptamine, (2) kanamycin B, (3) kanamycin A. The fourth peak is assumed to be kanamycin C ; however, mass spectrometric analysis of this peak gave inconclusive data. The order of elution between kanamycins A and B is analogous to paromomycin I and neomycin B (Figure 4) and can be attributed to their structural differences which are at the C2 position of the glucosamine moiety (OH for kanamycin A us. NH2 for kanamycin B) (Figure 1). Characterization of Derivatives. Mass spectrometry was used to characterize silylated derivatives of paromomycin and kanamycin. Because silylated amines are readily hydrolyzed, drying and transfer of the derivatives into a mass spectrometer is extremely difficult. T o minimize hydrolysis, a sample was introduced into the chromatographic inlet system of an LKB 9000 gas chromatograph-mass spectrometer. Although the molecular ion of totally silylated paromomycin (1551) and kanamycin (1276) are beyond the optimum capability of the
LKB 9000, mass in excess of 1200 were obtained. The mass spectrum of paromomycin I exhibited strong intensities at 376, 449 or 450, and 726 mje indicating the presence of silylated deoxystreptamine, paromose and/or glucosamine, and paromobiosamine. These data indicate that all active hydrogens on both the hydroxy and amine groups in paromomycin are completely silylated. Active hydrogens on kanamycin were also silylated, since the mass spectrum of kanamycin A exhibited strong intensities at 376 and 451 mje indicating the presence of silylated deoxystreptamine and kanosamine. The mass spectrum of silylated paromamine gave the molecular ion of 899 mje. Quantitative Determination. PAROMOMYCIN. The precision of the gas chromatographic method was determined by assaying 8 separate preparations of paromomycin sulfate powder. The relative standard deviation of the quantitative determination of paromomycin I is 1.7% in a sample containing 10.7% paromomycin 11. The precision of the method was determined KANAMYCIN. by assaying 7 separate preparations of kanamycin sulfate powder. The relative standard deviation for the determination of kanamycin A is less than 1 %. ACKNOWLEDGMENT
The supply of paromomycin sulfate from Parke, Davis & Company and kanamycins A and B from Bristol Laboratories is acknowledged. E. J. Hessler is acknowledged for the supply of paromamine, paromomycins I and 11. P. B. Bowman and M. L. Knuth are acknowledged for the mass spectrometric analysis. RECEIVED for review June 18, 1970. Accepted August 24, 1970.
(9) W. J. A. Vander Heuvel, J. Chromatogr., 27,85 (1967).
Voltammetric Determination of Europium(ll1) Using the Lanthanum Hexaboride Electrode D. J. Curran and K . S . Fletcher IIP Department of Chemistry, Uniuersity of Massachusetts, Amherst, Mass. 01002
RECENTLY we described lanthanum hexaboride as an electrode for electrochemical studies( I ) and employed this material for the electrochemical generation of lanthanum(II1) for use in titrations ( 2 , 3) and as a polarized indicator electrode for acid-base titrations ( 4 ) . The present note describes the application of this electrode as a voltammetric indicating electrode for the determination of europium(II1). Few electrochemical studies of the europium system have been reported. Laitinen and Taebel (5) discuss a polarographic investigation using 0.1N NH4C1supporting electrolyte Present address, Research Center, The Foxboro Company, Foxboro, Mass. 02035. (1) (2) (3) (4) (5)
D. J. Curran and K. S. Fletcher 111, ANAL.CHEM., 40,78 (1968). Ibid., p 1809. Ibid., 41, 267 (1969). Ibid., 40, 1804 (1968). H. A. Laitinen and W. A. Taebel, ibid., 13,825 (1941).
and report the half-wave potential as -0.671 V us. SCE and the ratio of the average diffusion current to concentration, id/C, as 2.88 kA-l./mole. Using this value of id/C and the capillary characteristics given by these workers, the diffusion coefficient is calculated as 5.87 X 10-6 cm*/sec from the Ilkovic Equation. Gierst and Cornelissen (6, 7) also investigated this system polarographically using several supporting electrolytes. These workers report the half-wave potential to be -0.60 V us. SCE and the mean value (for several supporting electrolytes) of the diffusion coefficient to be 7.1 X 10-6 cm2/sec. Anderson and Macero (8) determined the formal potential of the system (Eo’= -0.60 V us. SCE in (6) L. Gierst and P. Cornelissen, Collect. Czech. Chem. Commun., 25, 3004 (1960). (7) L. Gierst, in “Transactions of the Symposium on Electrode Processes,” E. Yeager, Ed., John Wiley and Sons, New York, 1961, pp 109-144. (8) L. B. Anderson and D. J. Macero, J. Phys. Chem., 1942 (1963).
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