ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
349
Simultaneous Determination of Vancomycin, Anisomycin, and Trimethoprim Lactate by High Pressure Liquid Chromatography R. L. Kirchmeier" and R. P. Upton Pfizer Inc., Quality Control, Special Testing and Analytical Development Laboratories, Groton, Connecticut 06340
A reverse phase, high pressure liquid chromatographic method for the rapid, accurate, precise, and simultaneous determination of vancomycin, anisomycin, and trimethoprim lactate has been developed, and applied to the determination of these compounds in antibiotic mixtures. No chemical methods for the determination of anisomycin have previously been reported. The methods reported in the literature for the determination of vancomycin, and/or trimethoprim lactate are imprecise, nonspecific, or time consuming, and do not allow the simuitaneous quantitation of anisomycin, vancomycin, and trimethoprim. Precision of the high pressure liquid chromatographic assay was found to be f1.8%, fl.l%, and f2.2% for vancomycin, anisomycin, and trlmethoprim lactate, respectlvely (two standard deviations relative to the average).
With the recent development of an antibiotic mixture for use as an inhibitor for the selective culture of Neisseria gonorrhoeae (1), containing vancomycin, anisomycin, and trimethoprim lactate, the development of a rapid, accurate, a n d precise chemical method of analysis for these materials in the presence of each other was desired. No previous reports of a method for t h e chemical determination of anisomycin content appear in the literature. Vancomycin has in the past been analyzed and/or quantitated by column chromatography ( 2 ) ,or T L C (3-5). Chemical methods reported for the determination of trimethoprim involve t h e use of spectrofluorimetry (6), paper chromatography ( 7 ) ,TLC (8, 9 ) , and GLC (10). Although t h e GLC method reported above for trimethoprim (10) is precise and accurate, the simultaneous determination of vancomycin and anisomycin is not possible because of their low volatilities. Simultaneous analysis of vancomycin, anisomycin, and trimethoprim by TLC or paper chromatography would not be feasible for lack of precision a n d specificity. A fluorimetric T L C procedure for trimethoprim (9) requires 24 h of development time for the fluorimetric determination of low levels of trimethoprim. Since all of t h e methods described above suffer from one or more limitations, Le., they are either nonspecific, involve long sample preparation and/or analysis tmes, or are not amenable to the simultaneous determination of vancomycin, anisomycin, and trimethoprim lactate, t h e development and application of a reverse phase high pressure liquid chromatographic procedure was undertaken. EXPERIMENTAL Apparatus and Operating Conditions. A Waters Associates Model 6000A high pressure metering pump at a flow rate of 1.0 mL/min in conjunction with a Schoeffel Model SF770 multiwavelength UV detector set at 225 nm (0.2 Absorbance Units Full Scale (AUFS)) were used for the analysis of all samples. A Valco Model CV-6-UHPa-C-20 loop injection valve (7000 psi pressure rating) fitted with a 25-pL sample loop was employed for sample injections. Spectra were recorded with a Varian Model A-25 recorder (10 mV) set at a chart speed of 10 in./h. Column. A 30 cm X 4 mm i.d. Waters Associates p Bondapak C l column ~ (a monomolecular layer of octadecyltrichlorosilane chemically bonded to totally porous p-Porasil particles having
an average diameter of 10 ym) was used at ambient temperature. Reagents and Standards. Acetonitrile distilled in glass was purchased from Burdick and Jackson. Reference standards for vancomycin (1070 Gg/mg base), anisomycin (100% 1, and trimethoprim lactate (98-100% ) were received from Elanco, Pfizer Inc., and Burroughs Wellcome, respectively, and used without further purification. Mobile Phase. Mobile phase is prepared by mixing 125 mL of acetonitrile and 875 mL of pH 6,0.05 M potassium dihydrogen phosphate buffer solution in a 1-L vacuum flask. Degassing is accomplished via house vacuum (640 Torr, approximately) with magnetic stirring for 10 min prior to use in the liquid chromatograph. Calibration Curves. Calibration curves for vancomycin, anisomycin, and trimethoprim lactate were determined on separate standard solutions (in mobile phase) containing from 0.01 to 0.07, 0.05 to 0.4, and 0.03 to 0.07 mg/mL of vancomycin, anisomycin, and trimethroprim lactate, respectively. For each solution, 25 yL was injected into the chromatographic system, and the resulting peak height was plotted vs. concentration. Sample Preparation. No sample pretreatment was required for the samples assayed in this study. Approximately 40 mg of the antibiotic mixture to be assayed (containing up to 5 mg each of vancomycin and trimethoprim and up to 30 mg of anisomycin) was accurately weighed out into a 100-mL volumetric flask, and diluted to volume with mobile phase. RESULTS AND DISCUSSION Reverse phase high pressure liquid chromatography was a logical choice for the simultaneous determination of vancomycin, anisomycin, and trimethoprim lactate. Figure 1 indicates the structures for anisomycin and trimethoprim base. Although the complete structure of vancomycin is not know, several reports of the determination of the partial structure and approximate molecular weight (1388) of vancomycin appear in t h e literature (11-13). Separation of vancomycin from anisomycin and trimethoprim was accomplished on a Sephadex G 10 column with a 0.02 M, p H 7.0 phosphate buffer mobile phase; however, trimethoprim and anisomycin were not resolved (14). In addition, approximately 4 h were required for the complete elution of vancomycin, anisomycin, and trimethoprim lactate from t h e column. The separation of vancomycin into three biologically active components via liquid chromatography on a Sephadex C-50 column has been previously reported (2). T h e average distribution ( % by peak area measured a t 280 nm) of the biologically active vancomycin components was found to be 51.5 and 73.3% (most biologically active), 20.1 and 16.8% (medium biological activity), and 15.4 and 10.1% (least biologically active) for the two vancomycin hydrochloride preparations. Under the high pressure liquid chromatographic conditions employed in this study, the chromatography of two separate preparations of vancomycin hydrochloride (Elanco Lot OAY56A and Elanco Lot 8FW78F) resulted in the elution of one major, and several minor components (by peak height measurement a t 225 nm, see Figure 2). Standard curves obtained by measuring t h e peak height of the major component, and plotting this peak height vs. concentration, agreed to within 1.2% for the two vancomycin preparations. T h e vancomycin eluate was monitored a t several wavelengths, including 280 nm; however, no significant change in t h e
0003-2700/78/0350-0349$01 .OO/O 8 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY
1978
Table I. Retention Volume vs. % Acetonitrile in Mobile Phase Mobile phase: % acetonitrile Retention volume, m L in 0.05 M, p H 6 phosphate Trime thoprim buffer Vancomycin Anisomycin lactate 20 17.5 15 OCH,
14 12.5 10
ANISOMYCIN
TRIMETHOPRIM B A S E
Flgure 1. Structure of anisomycin and trimethoprim base
218, 0.1
AUFS 225 230 235 254 280
VI
1 0
z
VI
m
3.5 4.6 6.1 19.5
6.0 10.0 13.3 16.5
6.8 12.5 17.0 21.5
34.5
46.5
...
...
Table II. Observed Peak Height vs. Detector Wavelength Detector wavelength, nm
m n
3.0
...
Observed peak height 0.04 mg/mL 0.2 mg/mL 0.05 mg/mL of vancomyof anisoof trimethoprim cin, cm mycin, cm lactate, cm 17.0 46.4 29.8 14.6 12.6 10.1 1.2 2.1
32.4 13.4 3.1 3.5 4.6
27.4 24.4 19.7 5.6 5.2
B
b
6
12
(MINI
Flgure 2. Chromatographic trace of vancomycin eluate at 225 nm
proportion of the major component was noted in either vancomycin preparation (by measurement of observed peak heights at t h e various wavelengths). In addition, a sample of vancomycin HPLC eluate was collected, lyophilized, and its bioactivity was determined vs. an unchromatographed vancomycin sample a t the same concentration in the same phosphate matrix by a previously reported biochemical procedure (15). Both samples were assayed against vancomycin hydrochloride Elanco Lot 8FW78F. No significant difference in bioactivity was found between the chromatographed and control sample of vancomycin. Thus 0.75 and 0.72 pg/mg of vancomycin hydrochloride were found in the chromatographed and control samples, respectively. T h e effect of mobile phase composition on the retention volumes of vancomycin, anisomycin, and trimethoprim lactate is given in Table I. Optimum conditions for a particular column result in the elution of all three components within approximately 20 min. Peak heights observed for vancomycin, anisomycin, and trimethoprim lactate as a function of detector wavelength are given in Table 11. The chromatographic trace of a standard mixture containing vancomycin, anisomycin, and trimethoprim lactate obtained a t 225 nm is shown in Figure 3. Calibration curves for vancomycin, anisomycin, and trimethoprim lactate were prepared on a single day. Correlation coefficients were 0.998, 0.9999, and 0.9985 for vancomycin, anisomycin, and trimethoprim lactate, respectively. The mean value of y and the y-intercept were found to be 346 and -13, 553 and +11, and 3.45 and +0.35 for vancomycin, anisomycin, and trimethoprim lactate, respectively. Repeated injections of standard solutions of vancomycin (seven injections on two separate days), anisomycin (six injections on two separate
0
6
12
IB
(MINI
Flgure 3. Chromatographic trace of a standard mixture containing 0.05 mg/mL each of vancomycin (A), and trimethoprim lactate (C), and 0.3 mg/mL of anisomycin (6)
days), and trimethoprim lactate (six injections on two separate days), gave rise to one standard deviation relative to the average peak heights of 0.6%, 0.370,and 0.4%, respectively. Thus, single point standards were used for the determination of vancomycin, anisomycin, and trimethoprim lactate by this HPLC method. Table I11 summarizes the analytical data obtained by this method on a series of 17 samples containing vancomycin, anisomycin, and trimethoprim lactate. Values are reported in terms of percent of component as determined by high pressure liquid chromatography relative to the label claim for each component. The values thus reported by HPLC tend to be 6% high for vancomycin, 2.2% low for anisomycin, and 2.0% low for trimethoprim lactate. The precision of the HPLC method as determined on four separate weighings of a single sample on two separate days was found to be f1.870, 11.17~, and &2.2% (two standard deviations relative to the average) for vancomycin, anisomycin, and trimethoprim lactate, respectively. Sample stability studies were undertaken, and indicated no trend in assay values obtained by
ANALYTICAL CHEMISTRY, VOL. 50, NO.
Table 111. Analysis of Inhibitory Antibiotic Mixture by HPLC
Sample No. 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Vancomycin content by HPLC ( % relative to label claim)
Anisomycin content by HPLC (W relative to label claim)
Trimethoprim lactate by HPLC ( % relative t o label claim)
102 106 102 102 98 99 107 109 104 104 108 106 113 107 106 109 114
99 96 98 100 96 100 99 102 98 94 98 96 99 94 97 95 101
100 99 99 101 96 101 101 102 98 93 97 95 99 94 95 95 99
HPLC with respect to temperature or pH for samples stored under various conditions for periods of several months. Samples of vancomycin, anisomycin, and trimethoprim lactate were also subjected to both acidic and basic hydrolysis (standard solutions of each material were treated with 0.1 N NaOH and 0.1 N HC1 for periods of up to 2 h). High pressure liquid chromatography of these partially decomposed materials revealed no interfering eluates. Aglucovancomycin, prepared via the acid hydrolysis of vancomycin (16),was found to elute a t the solvent front under the HPLC conditions used in this study. No other specific interferences were tested. In conclusion, the application of this reverse phase, high pressure liquid chromatographic approach for the simulta-
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neous determination of vancomycin, anisomycin, and trimethoprim lactate results in a rapid, accurate, precise method of analysis. The procedure should be applicable to determination of these materials in biological matrices subsequent to extraction of the components of interest, and application to the study of vancomycin and decomposition products obtained by various chemical means may aid in the further elucidation of the vancomycin structure. ACKNOWLEDGMENT The authors are indebted to F. Hochstein for supplying the standard materials used, and to R. Holmwood and J. Beyer for valuable aid in the development and application of this method. Appreciation is also given for the bioassay of vancomycin samples by J. Stankewich. LITERATURE CITED (1) J. Martin and J. Lewis, J . Assoc. Public Health Directors, in press. (2) G. K. Best, N. H. Best, and N. N. D u h m , Antimicrob. Agents Chemother., 115 (1968). (3) B. B. Korchagin, L. I. Serova, Z.I. Vtorova, I . I. Vagina, E. 2 . Oipinska, and S.P. Dementieva, Epidemiol.,Mikrobiol. InfeMs Boles., 8, 50 (1971). (4) I. J. McGilveray and R. D. Strickiand, J Pharm. Sci., 58, 77 (1967). (5) V. Betina, J . Chromatogr., 15, 379 (1964). (6) D. E. Ernest, B. A. Koechlin, and R. E. Weinfeld, Chemotherapy, 14, 22 (1969). (7) L. Reio, J . Chromatogr., 68, 183 (1972). (8) M. BHo and D. Bobak, Lucr. Conf. Nat. Chim. Anal., 3rd, 4, 199 (1971). (9) C. W. Sigel and M. E. Grace, J . Chromatogr., 80, 111 (1973). (10) F. L. Fricke, J . Assoc. Off. Anal. Chem., 55, 1162 (1972). (1 1) K. A. Smith, D. H. Williams, and G. A. Smith, J. Chem. Soc., Perkin Trans. 1 , 20, 2369 (1974). (12) N. K. Kochetkov and 0. S. Chizov, Biochim. Biophys. Acta, 83, 134 (1964). (13) H. Bjwndai, C. G. Hellerguist, B. Lindberg, and S. Svensson, Angew. Chem., Int. Ed. Engi., 9, 910 (1970). (14) F. A. Hochstein, Pfizer Inc., Groton, Conn. unpublished work, 1976. (15) "Code of Federal Regulations", 21, April 1, 1976 rev.. U S . Government Printing Office, Washington, D.C.. p 216. (16) F. J. Marshall, J . Med. Chem., 8, 18 (1965)
RECEIVED for review September 6,1977. Accepted November 28, 1977.
Thin-Mercury-Film Voltammetry of Inorganic Lead(I1) Complexes in Seawater Lloyd M. Petrle" and Rodger W. Baier' Graduate School of Oceanography, University of Rhode Island, Kingston, Rhode Island 0288 1
Cycllc voltammetry of M Pb(I1) model solutions proved useful In revealing that electron transfer of Pb(I1) at thin mercury film electrodes was reversible for glassy carbon electrodes at scan rates 510 mV/s and less reversible for wax-Impregnated graphite electrodes. Electron transfer was reversible for PbCI, quasi-reversible for PbS04 and PbC03, and least reversible for PbOH. Anodic stripping voltammetry (ASV) and use of *'OPb radiotracer in lo-* M Pb(I1) model solutions revealed similar trends. ASV of organic-free seawater at pH 8.5 gave peak currents less than theoretical for Pb(II), even taking Into account cell material adsorption. This was largely due to the electrochemical inactivity of PbOH. At pH 3, the peak currents were proportional to the seawater Pb(I1) concentration. Present address, Department of Chemistry, Duke University Marine Laboratory, Beaufort, N.C. 28516. 0003-2700/78/0350-035 1$01.OO/O
Because of its extensive use, its persistence in the marine environment (400 years), and its toxicity, the biogeochemical impact of lead on the marine environment needs to be more fully characterized (1-5). To more effectively predict the transport and biological effects of lead and other trace metals in the marine environment, it is necessary to determine not only their concentrations but their chemical speciation (6, 7). Because of precise and sensitive levels of detection and the capability for discrimination of discrete metal complexes, voltammetric techniques such as anodic stripping voltammetry (ASV) have been widely used to characterize metal speciation in seawater (8-10). Generally, two types of working electrodes have been used: (1) the hanging mercury drop electrode (HMDE) and (2) the thin mercury film electrode (TMFE), which consists of a liquid mercury film on an inert substrate, usually wax-coated graphite or glassy carbon. Both electrodes have distinct advantages and disadvantages with the HMDE claiming superior precision and the TMFE superior sensitivity C 1978 American Chemical Society
