A liquid chromatographic method for determining the tetracycline antibiotics was automated. The technique employed the use of a valve arrangement through which twenty to thirty samples in solution were sequentially applied to a single column for chromatography.
Each chromatogram consisted of a continuous flow separation of components followed by a spectral determination of the column eluate at a rate of twelve samples per hour. The method was applicable to crystalline tetracycline antibiotics and their various pharmaceutical dosage forms. P. P. ASCIONE, J. B. ZAGAR, G. I'. CNREKIAN
method is not expected to be a substitute for one providing separate analysis for de-' composition products and closely related compounds, the features of ease of column preparation, rapidity of analysis and repetitive use of the same column makes it a technique highly desirable for the determination of tetracycline purity. The advantages of specificity, accuracy, speed and precision normally inherent to automated chromatography were realized.
MATERIAL AND METWODS
Apparatus
The apparatus used in the automated CC includes the following components: (a) Cheminert Metering Pump Model CMP-3, automatic-sample-injection valve Model SVA-8031, three-way valve Model CAV-3031, Cheminert Column Model LC-s-13, polytetrafiuoroethylene tubing 0.031 in. and 0.063 in. bore, pneumatic actuators Model PA-875, solenoid air valves and flanging tool (Chromatronix Inc., Berkeley, Calif;) ; (b) multiple-program cycling timer Model 540 (Conrac Corp., Old Saybrook, Conn.) ; (c) Sampler II, proportioning pump, assorted tubing and glass fittings (Technicon Corp., Tarrytown, N.Y.); (d) spectrophotometer DBG, equipped with 4 mm rectangular flow cell (Beckman Instruments, Inc., Fullerton, Calif *) ; (e) recorder, modified with a linear retransmitting slidewire (Bristol Co., Waterbury, Conn.); (f) digital integrator Model 3370A (Hewlett-Packard, Avondale, Pa.) .
Reagents EDTA, 'pH 6.0. Dissolve 37.2 g of ethylenediaminetetraacetic acid d&odium salt in cu. 800 m.l of distilled water. Adjust the pH to 6.0 with ammonium hydroxide and dilute to I 1 with distilled water.
Polyethylene glycol 400 (PEG 400), 20 % in glycerine. To So ml of glycerine, add sufficient PEG 400 to make IOO ml. Mix well.
Buffer. To 95 ml of EDTA pH 6.0, add sufficient 20 Y. PEG 400 in glycerine to make IOO ml. Shake well.
Calcium lactate-morpholine solution (CLM). Dissolve 0.30 g of calcium lactate in 450 ml of methanol, Add 4 ml of morpholine, swirl and dilute to 500 ml with methanol.. Working reference standard. Prepare a solution containing 0.6 mg/m.l of reference standard by dissolving 60 mg of powder in IO m.l of methanol and diluting ,to IOO m.l with chloroform.
Methanol, chloroform, benzene and morpholine, reagent grade, Acid-washed diatomaceous earth (marketed as Celite@ 545 by Johns-Manville Co.). Preparation has been described previously12.
Preparation of colman sufiport
Mix 100 g of acid-washed diatomaceous earth with 30 ml of buffer, in a plastic bag or in a glass jar. Roll or mix until the support is completely homogenous.
Co&nn firejbavation
1 Place inlet assembly into the LC-J/-x3 column and secure the collar to the column. Weight 8.0 f 0.1 g of co1um.n support and add to the column in four 2.0 g