torted the recovery obtained (101.97%) was unsatisfactorily high. This can be attributed to the reaction of carbon dioxide with the strongly basic titrant. It may be concluded that TMU is a useful solvent for the titration of very weak to strong acids and for bases of moderate to high basicities. The ability to differentiate this broad range of substances, availability at moderate cost, and ease of handling should lead to further uses of tetramethylurea as a nonaqueous titration medium.
their tendency to form adducts with solute species, as evidenced by the steeply sloping plateaus of the titration curves obtained, it may be that such associations, perhaps in the form of a 1 :3 adduct, are responsible for the additional inflection observed. It also may be that the water present in the original perchloric acid sample contributes to the formation of different acidic species. The possibility that the added methanol from the titrant could act as a source of potential protons was discounted in view of the titration behavior exhibited by the other substances. Also due to the very weak basicity of TMU, it would not be expected that the methanol acidity would be greatly enhanced with respect to its aqueous acidity. However, it may be possible that methanol does act as a complicating component in the perchloric acid system. Further studies will be made in an attempt to explain this behavior. The effect of atmospheric contaminants was studied by titrating benzoic acid without a protective atmosphere. Although the shape of the resulting titration curve was not dis-
ACKNOWLEDGMENT
The authors thank the General Electric Company of Evendale, Ohio, for providing the facilities for this research and the Ott Chemical Company for providing an initial sample of tetramethylurea. RECEIVED for review March 17, 1969. Accepted May 12, 1969. __
Gas-Liquid Chromatographic Determination of Neomycins B and C Kiyoshi Tsuji and John H. Robertson Control Analytical Research and Development, The Upjohn Company, Kalamazoo, Mich. 49001 THETERM neomycin usually refers to mixtures of two stereoisomers, neomycins B and C, and their degradation product neamine or neomycin A. The determination of neomycin is made difficult by the close structural similarity between neomycin B and neomycin C, Figure 1. Biological responses of the neomycins are not constant and frequently are influenced by chemical and physical factors ( I ) . Several chemical methods have been reported to separate and quantitatively determine neomycins B and C. Kaiser ( 2 ) prepared * 4C-labeled hexa-N-acetyl derivatives of neomycins B and C and separated them by paper chromatography. The derivatives were eluted and quantitated with a liquid scintillation counter. Brodasky (3) reported the use of thin layer (carbon) chromatography for the separation of neomycins B and C. The use of column chromatography for the separation of neomycins B and C has also been reported (4-7). These methods are time consuming and are not suitable for a laboratory in which a large number of neomycin products are quantitated routinely. To our knowledge, this paper is the first to describe the separation and quantitation of neomycins B and C by gas chromatography. EXPERIMENTAL
Apparatus. F & M Model 400 with flame ionization detector was used with gas flow rates of hydrogen 20 ml/min, air 550 ml/min, and carrier gas (helium) 40 ml/min, chart (1) W. T. Sokolski, C. G. Chidester, and D. G. Kaiser, J. Phnrm. Sci., 53, 726 (1964). (2) D. G. Kaiser, ANAL.CHEM., 35,552 (1963). (3) T. F. Brodasky, ibid., 35, 343 (1963). (4) G. Nomine and L. Penasse, U. S. Patent 3,062,807 (1962). ( 5 ) H. Maeher and C. P. Schaffner, ANAL.CHEM.,36, 104 (1964). (6) S. Inoue and H. Ogawa, J . Chroniatogr., 13, 536 (1964). (7) A. E. Kaptionak, E. Biernacka, and H. J. Pazdera, Technicon Symposia, Mediad Press, New York, N. Y . 1965, p 27.
1332
ANALYTICAL CHEMISTRY
O0OH”
-HO
“2
OH
-0
“2
0
CH,OH
2,6-diamineglucose
deoayslreptamine
D-ribose
neamine
neosamine
B
neobiosomine B
4;
NEOMYCIN B
H OO
o
G
H
*
0
“2
OH
0
“2
CH,OH
2,6-diomineglucose deoxystreptomine
Y
D-ribose
neosamine
c
j -
neamine
neobiosomine C
NEOMYCIN C Figure 1. Structure of neomycins B and C
speed of 0.25 inch/min, and an oven temperature of 290 “C. Column. A glass column, 3 mm X 1830 mm (6 ft.) and packed with 0.75% OV-1 on GAS-CHROM Q, 100-120 mesh (Applied Science Laboratory, Inc., State College, Pa.) was used. The column was non-flow conditioned at 330 “C followed by the injection of Silyl-8 (Pierce Chemical Co., Rockford, Ill.) and silylated neomycin. The column thus prepared had 300 theoretical plates per foot for silylated neomycin B. Internal Standard Solution. Prepare a pyridine solution containing approximately 3 mg/ml of trilaurin (Supelco, Inc., St. Bellefonte, Pa.). TRI-SIL Z (Pierce Chemical) may be substituted for pyridine for better stability of silylated neomycin.
Table I. Precision of Gas Chromatographic Assay of Neomycin in Neomycin Sulfate Powder Total neomycin (rglmg)= Neomycin C (neomycins Neomycin B B & C) (I.cg/mg) (dmg)
Average Standard deviation Relative standard deviation 0
pg of
692.0 703.6 702.7 695.6 686.9 700.0
579.2 599.0 599.5 588.9 581.2 592.7
112.8 104.6 103.2 106.7 105.7 107.3
696.8
590.1
106.7
6.55
8.65
3.33
0.94%
1.47%
3.12%
neomycin base per mg of neomycin sulfate powder.
Reference Standard. Use USP lot I neomycin Reference Standard at 767 pg neomycin base per mg anhydrous neomycin sulfate. Weigh approximately 6 mg neomycin powder into a one-dram vial and dissolve with one ml water. Place this vial in an appropriate apparatus and freeze-dry. The resulting freeze dried sample dissolves more readily during silylation. Sample. Prepare in identical way to the reference standard. Silylation Procedure. Add 1.O ml of the internal standard solution and 80 p1 of N-trimethylsilyldiethylamine(Pierce Chemical) to each vial containing the freeze dried sample. Heat the vial in a 75 "C oil bath for 40 minutes. Silylated neomycins thus prepared are extremely sensitive to moisture and better stability may be obtained when the sample is prepared in a 1.5-ml serum vial with a 13-mm natural red rubber closure. Calculation: [R1/R2]X [ Wr/ Ws] X F
=
pg neomycin per mg of sample.
Where: of the neomycin sample peak R1 = Area _ _ _ _ - ~ Area of the internal standard peak of the. neomycin standard._ peak R1 = Area -. Area of the internal standard peak Wr: Weight of neomycin reference standard in mg.
Ws: Weight of neomycin sample in mg. F:
Assigned value of neomycin reference standard expressed in pg of anhydrous neomycin base per mg of reference standard. RESULTS
The merit of the gas chromatographic assay method of neomycin is its capability to separate and quantitate neomycins B and C with greater facility than other methods. Under the conditions of the experiment, the response of one lot of neomycin C was 98.5%, while another lot was 100.2x when compared to the USP lot I Reference Standard. Therefore, the response of neomycin C is approximately equal to that of neomycin B. A typical chromatogram indicating the separation and response of neomycins B and C is shown in Figure 2.
Figure 2. Isothermal separations of neomycins B and C a t 290 "C
Quantitative Determination of Neomycins B and C. The precision of the assay method was determined by assaying six separate preparations of neomycin powder against the USP lot I Reference Standard. Table I indicates that the relative standard deviation is about 1% when expressed as total neomycin (neomycins B and C). Larger relative standard deviations were noted for the individual response; 1.5% for neomycin B and 3.12% for neomycin C. These larger errors are probably due to the incomplete separation of the neomycin isomers. Comparison of Neomycin Reference Standards. The neomycin contents of the FDA Working Standard and the International Reference Standard were determined against the USP lot I Reference Standard, Table XI. The result for the FDA Working Standard agrees well with the assigned value, however, the agreement was poor with the International Reference Standard. This discrepancy could be due to the high neomycin C content in the International Reference Standard (27.2%) which would have given a lower microbiological response in relationship to neomycin B content. The neomycin C results of the USP lot I and the FDA standards agree well with values reported in literature (2, 8). The presence of neamine was detected in the FDA and the International Standards at a level of approximately 3 and 5 respectively. Gas Chromatography of Neamine, Neobiosamine in the Presence of Neomycins B and C. A mixture of silylated neamine, neobiosarnine, and neomycins B and C were chromatographed using temperature programming (Figure 3). Neamine and neobiosamine were clearly separated from each other and from neomycins B and C. A shoulder on
x,
VOL. 41, NO. 10, AUGUST 1969
e
1333
Neomycin reference standard USP lot I FDA
International
Assigned drug content (pg/mg)
Table 11. Comparison of Neomycin Reference Standards Assay value Total neomycin Neomycin C Neamine Values reported in literature (Pg/mg) (neomycins B & C) (72) (7.3 Neomycin C Neamine (xj
767 700 680
(z)
767@ 715 635
