ACKNOWLEDGMENT The authors gratefully acknowledge the assistance of Kent J. Eisentraut for providing additional details and advice concerning the base dissolution of B e 0 and of Michael L. Taylor for advice on the construction of the Sephadex-base column for removing H(tfa). Received for review July 20, 1973. Accepted November 12, 1973. The research reported in this paper was conducted in part by personnel of the Aerospace Medical Research Laboratory, Aerospace Medical Division, Air Force Sys-
tems Command, Wright-Patterson Air Force Base, Ohio, and in part by personnel of Monsanto Research Corporation, Dayton, Ohio, under Contract No. F33615-71-C-1794. This work was supported in part by the National Institute of Occupational Safety and Health, Division of Toxicology and Pathology, Cincinnati, Ohio. The experiments reported herein were conducted according t o the “Guide for the Care and Use of Laboratory Animals,” DHEW 73-23. This is the same material as in AMRL-TR-72-72. Further reproduction is authorized to satisfy the needs of the 1J.S. Government.
High Pressure Liquid Chromatographic Determination of Tetracyclines Kiyoshi Tsuji, J. H. Robertson, and W. F. Beyer Control Analytical Research and Development, The U p j o h n C o m p a n y , Kalamazoo, Mich. 4900 1
A high pressure liquid chromatographic method for the determination of tetracycline is described. The method requires no derivatization or gradient elution, and it can separate tetracycline (TC), anhydrotetracycline (ATC), 4-epi-tetracycline (ETC), 4-epi-anhydrotetracycline (EATC), chlorotetracycline (CTC), and doxycycline in less than 3 0 minutes. A 1-meter column packed with ZIPAX, HCP was used with a mobile phase of 1 3 % methanal in 0 . 0 2 M sodium phosphate, dibasic and 0 . 0 1 M phosphoric acid at pH 2.5 at a flow rate of 0.85 ml/min (1,000 psi). The relative standard deviation of the method i s 1.02% and the method is sensitive to approximately 10 nanograms TC per sample injected.
Many analytical methodologies have been reported for the determination of tetracycline (1-12), the latest being the gas liquid chromatographic (GLC) method of Tsuji and Robertson (13). The GLC method, however, requires derivatization of tetracyclines with trimethylsilyl reagents and the derivatization process, under certain conditions, forms degradation compounds of TC. The sensitivity for ATC, ETC, and EATC by the thin-layer chromatographic P. P. Ascione. J. E . Zagar. and G . P. Chrekian, J. Pharm. Sci.. 5 6 , 1393 (1967) I . C. Kiykhuis and M . R Brommet,J Pharm. S o . . 59, 558 (1970) A . A Fernandez, V . T . Noceda. and E. S. Carrera, J . Pharm. S o . . 5 8 , 443 (1969). P. B. Lloyd and C. C. Cornlord. J. Chromatogr. 5 3 , 403 (1970). Y . Nishimoto, E Tsuchida. and S Toyoshima, J. Pharm. SOC. (Jap.),87, 516 (1967). D. L. Simmons, R . J. Ranz, H . S. L. Woo, and P. Picotte, J. Chromatogr.. 43, 141 (1969). A . Sina, M . K . Youssef. A. A. Kassem, and I . A. Attia, J Pharm. SCi.. 60, 1544 (1971). B. W . Griffiths. R . Brunet. and L. Greenberg, Can. J. Pharm. Sci.. 5, 101 (1970). P. P Ascione and G . P. Chreklan, J . Pharm Scr . 59, 1480 (1970). P. P. Ascione, J. 8.Zagar. and G. P. Chrekian, J. Pharm. Sci.. 56, 1396 (1967). W . W. Fike and N . W . Brake, J. Pharm. Sci.. 61, 615 (1972). B. G. Kelly, J Pharm. Sci.. 53, 1551 (1964) K . Tsuji and J. H.Robertson, Anal. Chem . 45, 2136 (1973)
method is low ( I ) , and the diatomaceous earth column “classical” liquid chromatographic method (DECLC) is time consuming (12). The application of high-pressure liquid chromatography (HPLC) to the analysis of TC is preferred over other analytical methods because of its high speed and superior sensitivity.
EXPERIMENTAL Apparatus. A Laboratory Data Control (LDC. Riviera Beach, Fla.) modular liquid chromatograph equipped with a 280-nm UV monitor (Model 1285), a Milton Roy Minipump (LDC), and a pulse dampener (Model ’709)was used. An empty duPont stainless steel column, 2.1 X 1000 mm, was treated as follows. The column was first rinsed with tetrahydrofuran followed by vigorous scraping of the inside of the tubing with a cotton string pre-soaked with tetrahydrofuran to remove loose metal particles. Chloroform was then drawn through the column by vacuum and the column was dried by a stream of dry nitrogen. The column was then conditioned by soaking overnight in 0.1M ethylenediaminetetraacetic acid (EDTA) solution. The column was rinsed with double-distilled water followed by absolute methanol and dried. Stainless steel frits of 2- and 10-pm pore size were degassed by vacuum and soaked in 0.1MEDTA overnight. The 10-pm frit thus treated was fitted into the inlet end of the column, and a hex nut (duPont, No. 820349) with stainless steel front and back lock ferrules and cap (duPont, KO. 201724) was attached to the column. Zipax-hydrocarbon polymer (HCP, duPont) was dry packed into the open end of the column by adding a small amount of HCP at a time and lightly tapping on the floor. After the column was tightly packed, an EDTA pre-treated 2-pm pore size frit was inserted into the outlet end ofthe column. The column t h s packed was attached to a duPont injector port and to the 280-nm UV monitor. The column was then conditioned by first pumping 0.1M EDTA solution for approximately 4 hours prior to the mobile phase. The theoretical plates of the column thus prepared were 900 per meter for the TC peak. Reagents. Mobile Phase. A. 13% Methanol and 0.001M EDTA in 0.01M phosphoric acid and 0.02M dibasic sodium phosphate at pH 2.5 or B. 3% Acetonitrile in 0.001M EDTA and 0.01M phosphoric acid and 0.02M dibasic sodium phosphate at p H 2.5 were used. Depending upon the performance of the column, either -4or B mobile phase may be used to obtain the maximum separation of tetracyclines. Internal Standard Solution. Approximately 70 ml of anhyANALYTICAL C H E M I S T R Y , VOL. 46, NO. 4, APRIL 1974
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Figure 1. High-pressure liquid chromatography of tetracyclines using a 1-meter HCP column at 0.45 ml/min (500 psi) at room temperature. Mobile phase: 13% methanol and 0.001M EDTA in
phosphoric acid and 0.02M dibasic sodium phosphate,
0.02M
pH 2.5 ( 1 ) injection, ( 2 ) 4-epf-tetracycline, (3) tetracycline, ( 4 ) chlorotetracycline, (5) impurity in doxycycline, (6) doxycycline, ( 7 ) internal standard (prednisolone),(8) 4-epi-anhydrotetracycline, (9) anhydrotetracycline
drous methanol was added to a 250-ml graduated cylinder containing a) 250 mg prednisolone, or b) 125 mg of 4-(N-butylamino)benzoic acid (Aldrich Chemical Co., Milwaukee, Wis.). stoppered and shaken to dissolve. The mobile phase was then added to the volume. The internal standard solution ( a ) was used with mobile phase A and (b) for B. Preparation of TC Standard. Tetracycline hydrochloride U.S.P. Reference Standard, Issue H was dried at 60 "C under
