BIOMEDICAL CHROMATOGRAPHY, VOL. 6,305-310 (1992)

Analysis of Chlortetracycline by High Performance Liquid Chromatography with Postcolumn Alkaline-induced Fluorescence Detection Peter D. Bryan,?* Kristina R. Hawkins,$ James T. Stewart? and Anthony C. Capomacchiaa The University of Georgia, College of Pharmacy, ?Department of Medicinal Chemistry. #Department of Pharmaceutics, Athens, Georgia 30602, USA $North Georgia College, Dahlonega, Georgia 30533, USA

A high performance liquid chromatographic method for the analysis of chlortetracycline (CTC) using postcolumn fluorescence detection has been developed. After chromatographic separation of CTC on a polystyrenedivinylbenzene copolymer column, a highly fluorescent derivative isochlortetracycline (iso-CTC) was formed postcolumn in an on-line reaction coil with the addition of 25% NaOH (wlv). Chromatographic separation was achieved on a PRP-1 column, 15 cm X 4.6 mm, with 27:73 acetonitrile:0.2% perchloric acid (vlv), at 1.0 mL/min. Fluorescence derivatization was achieved by the on-line addition of 25% NaOH (w/v), at a flow rate of 0.2 mL/ min, into the column eluant in a post-column reaction coil. The reaction coil was 9 m of teflon (1/16 in o.d., 0.3 mm i.d.) knitted into a six-sided coil. The fluorescent derivative was detected at he, 355 nm and he,,>389 nm. Using this method after a simple sample cleanup, CTC can be detected in milk at 0.04pg/mL, which is comparable to that obtained by microbiological assays. The detection method was linear between 0.02 pg/mL and 4 ,ug/mL. Because of the chromatographic separation, the method is more selective than microbiological assays and more sensitive than ultraviolet detection. With the chromatographic system described, the keto tautomeric forms of CTC and 4-epi-CTC are separated in a system which minimizes their formation on-column. In acidic aqueous organic solutions, the keto tautomer of CTC is the only product formed to any significant amount.

INTRODUCTION Chlortetracycline (CTC) is a fermentation product isolated from Streptomyces aureofaciens. CTC was the first tetracycline to be isolated and is classified as a broad spectrum antibiotic (Foye, 1989). Current United States Pharmacopeia (USP) compendia1 methods for the analysis of CTC cite microbiological assays for its quantitation (USP, 1988). Microbiological or fluorometric assays are also the most common method for the analysis of CTC from biological matrices, but this method does not discriminate between individual tetracyclines (Nouws et al., 1985; Honikel et a/., 1978; van den Bogert and Kroon, 1981). It has also been shown that CTC is unstable in the broth media used to test antimicrobial activity (Ray and Newton, 1991). Analysis of CTC has been performed by high performance liquid chromatography (HPLC) using C-18, C-8 (Sokolic et al., 1990) or polymeric (Reeuwijk and Tjaden, 1986) columns, but these methods have been applied primarily t o dosage forms or bulk drug substances. Levine et al., (1949) were the first to use an alkalineinduced fluorometric method for the detection of CTC. Blanchflower et al. (1989) have reported an HPLC method for the analysis of CTC in tissue. This work is significant because the authors formed the highly fluorescent derivative isochlortetracycline (iso-CTC) precolumn using an alkaline hydrolysis step. Fluorescence * Author to whom correspondencc should be addressed. 02h9-387919210603nS-06 $08.00

01992 by John Wiley B Sons, Ltd.

detection was used to quantitate the iso-CTC formed from CTC in the samples. A problem associated with the analysis of CTC is that it is chemically unstable and, like other tetracyclines, rapidly isomerizes in aqueous solution (pH 2-6) at the C-4 dimethylamino group to form 4-epi-tetracyclines, which have approximately 5 % of the biological activity of the parent tetracycline (Foye, 1989). The epimerization problem of tetracyclines is compounded in buffered aqueous solution. Common buffers such as citrate, phosphate and acetate increase the rate of isomerization (McCormick et al., 1957) and tautomerization. In addition to the problems of degradation products, keto-enol tautomers of CTC are rapidly formed in aqueous solutions (Naidong et al., 1990). Other degradation products limit the useful p H range of HPLC mobile phases. Below p H 1, tetracyclines form anhydrotetracyclines. In the case of CTC, above p H 7 , iso-CTC is formed. CTC and its degradation products are shown in Scheme 1. In this study, an H P I X method for the quantitation of CTC using postcolumn alkaline-induced fluorescence (PAIF) detection is reported. In this method, the sensitivity and selectivity for the analysis of CTC from whole bovine milk has been improved. Due to the increased rate of hydrolysis of bonded phase silica columns at low pH, polystyrene-divinylbenzene copolymer (PS-DVB), C-18-derivatized PS-DVB and polybutadiene-coated alumina (PBD-alumina) HPLC columns were investigated for their suitability for the HPLK analysis of CTC and its degradation products. Received 6 January 1992 Accepted 30 Marrh 1992

PETER D. BRYAN E T A L .

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EXPERIMENTAL Reagents and chemicals. CTC reference standard lot-I was obtained from the United States Pharmacopeia. 4-epiChlortetracycline (4-epi-CTC) and iso-CTC were obtained from Janssen Chimica, Beerse, Belgium. All tetracyclines and analogues were used without further purification and stored at - 4 "C. To ensure the stability of each standard, the vials were removed from the freezer and brought to ambient temperature in a vacuum desiccator. Acetonitrile (ACN) and methylene chloride (J. T. Baker Chemical Co., Phillipsburg, NJ, USA) were HPLC grade. Concentrated analytical grade perchloric acid (HCIO,), hydrochloric acid (HCI) and 50% (w/v) aqueous sodium hydroxide (NaOH) were also obtained from J . T . Baker. Distilled water was used throughout the project. Fluorescence spectrophotomelry. The conditions necessary to convert CTC to iso-CTC were determined under static conditions with a Model MFP-44A scanning fluorescence spectrophotometer (Perkin-Elmer Corp., Norwalk, CT, USA) equipped with a temperature-controlled sample turret. The sample turret temperature was controlled by a Model FE recirculating heater (Haake Instruments. Paramus, NJ, USA). The effects of temperature and percent NaOH on the fluorescence spectrum of CTC were determined by mixing 1:l 1.0 mM CTC in ACN with aqueous NaOH solutions. The sample was transferred to a 1cm quartz cuvette and placed in the temperature-controlled turret of the spectrofluorophotometer. Emission spectra from 300 nm to 700nm were obtained at a scan rate of 480ndmin using an excitation wavelength of 280 nm. The alkaline solutions used were 10, 12.5 and 25% (wlv) NaOH, and temperature was varied from 30 to 90 "C, in 10" increments. When reaction conditions were such that the maximum emission intensity occurred in less than 2 min, a chart recorder was used to determine the time to maximum fluorescence intensity. In these cases, emission at 400 nm was monitored versus time.

Chromatographic conditions. The HPLC apparatus included a Model l l 0 A pump (Beckman Instruments, Fullerton, CA, USA) and a Model 7125 injector (Rheodyne Inc., Cotati, CA, USA) equipped with a 20pL loop. HPLC columns included a PS-DVB column, 15 cm X 4.6 mm, PRP-1 (Hamilton Co., Reno, NV, USA), a C-IS-derivatked PS-DVB copolyrr.er, ACT-1, 15 cm x 4.6 mm column (Interaction Chemical Inc., Mountain View, CA, USA) or a PBD-alumina column, 15 cm X 4.6 mm (Millipore Corp., Milford, MA, USA). The column temperature was controlled with a Model TC-SO controller and a Model CI1-30 column heater (FIAtron, Milwaukee, WI, USA). Detection at 280nm was performed with a Model 2550 variable wavelength UV/Vis detector (Varian Analytical Instruments, Sunnydale, CA, USA) and data were recorded with either a Model 056-1001 chart recorder (Perkin-Elmer Corp.) or a Model 4290 integrator (Spectra Physics, San Jose, CA, USA). Mobile phases were filtered through a 0.45 pm nylon filter (Micron Separation, Inc., Westboro, MA, USA) and degassed by sonication prior to use. To determine the effects of perchloric acid on capacity and symmetry factors, the amount of perchloric acid in the aqueous portion of the mobile phase was varied from 0.01 to 0.5% (v/v). The PRP-1 column at ambient temperature was used with a mobile phase consisting of 27:73 ACNlaqueous perchloric acid, at a flow rate of 1.0 mL/min. A 20 & n L solution of CTC in 0.5% perchloric acid was injected. Standard methods were used to calculate capacity factor and asymmetry factors at half-height (Poole and Scheutte, 1986). The effects of the percent ACN in the mobile phase and column temperature on the capacity factors of CTC, 4-epiCTC and their keto tautomers were determined by varying column temperature for each mobile phase. Mobile phase concentrations were 20,25,27 and 30% ACN. The amount of perchloric acid (0.2%) in the aqueous phase remained constant. Column temperatures were varied from 25 to 70 "C in the column heater in 5" steps. The column was a PRP-1 and the flow rate was 1.0 mL/min. A CTC sample in 0.1 N HCL was allowed to degrade for 24 h before use as a qualitative standard. Postcolumn fluorescence conditions. HPLC postcolumn derivatization conditions were determined with the apparatus shown in Fig. 1. The HPLC apparatus included a Model 400 pump (Kratos, Ramsey, NJ, USA), a WISP autosampler Model 710B (Waters Associates, Milford, MA, USA) and a PS-DVB column, PRP-1, 15 cm X 4.6 mm. The postcolumn reagent was delivered from a Model 760 pump (Micromeritics, Norcross, GA, USA) and mixed with the column eluant in a mixing tee (The Lee Co., Westbrook, CT, USA). The postcolumn reactor, fabricated in house, consisted of sixsided knitted teflon tubing, 1/16 in 0.d; 0.3 mrn i.d. (Upchurch Scientific, Oak Harbor, WA, USA), similar in design to the three-dimensional coils used by Engelhardt and Neue (1982). The reaction coil was submerged in water and the temperature of the water was regulated in a doublewalled beaker by a Haake Model FE recirculating heater. Reaction products were detected by a Model 970 fluorescence

HPLC OF CHLOKTETRACYCLINE USING PAIF DETECTION

detector (Schoeffel, Westwood, NJ, USA) and data were recorded by a Model 4290 integrator. To determine the effects of postcolumn flow rate on relative fluorescence intensity, 20 pL of a 10 pg/mL solution of CTC was injected into the HPLC apparatus. The flow rates of the postcolumn reagent pump using 10% (w/v) NaOH were 0.1, 0.2, 0.5 or 1.0mUmin. A 6m reaction coil was used at 25°C and fluorescence emission was measured using I,, 355 nrn and dem>389 nm. CTC was chromatographed on the PRP-I column using 27:73 ACN:0.2% perchloric acid at 1.0 mumin. Three injections were made at each flow rate. The effects of reaction coil length and percent NaOH on the relative fluorescence intensity were determined using the same chromatographic and detection conditions described above. A postcolumn reagent flow rate of 0.2rnWmin was used. Reaction coil lengths of 3, 6 and 9m were used with NaOH concentrations of 5, 12.5 and 25% (w/v). The chromatographic and detection conditions mentioned above were also used to determine the effects of reaction coil temperature on relative fluorescence intensity for the three reaction coil lengths. A postcolumn reagent flow rate of 0.2 rnWmin was used. For the three coil lengths, the Buorescence intensity of the products formed at 25, 50 and 90°C were determined. Sample preparation. Spiked milk samples were prepared by adding 100 pL of a spiking solution containing 101.4, 10.1 or 0.80 p.g/mL CTC in 0.1 N HCI to 2 mL of milk in a 10 mL screwtop test-tube. To the blanks, 100 pL of 0.1 N HCI was added. Four replicates at each spiked level and blank were prepared. To the spiked and blank milk samples, 2.0 mL methylene chloride and 2.0 mL of aqueous 0.1 N HC1 were combined. The tubes were shaken vigorously for 30 s by hand and then centrifuged for 15 min at 5000 rpm in a Model

GLC-2B centrifuge (DuPont Instrurnents/Sorvall, Newtown,

CT,USA). The resulting supernatant was decanted and filtered through a 25 mm diameter, 0.45 pm nylon filter (Lida Manufacturing, Corp., Bensenville, TL, USA).

RESULTS AND DISCUSSION The effects of the amount of perchloric acid in the mobile phase on capacity and symmetry factors of CTC are shown in Fig. 2. As the amount of perchloric acid in the mobile phase increases, the capacity factor of CTC increases up to a 0.2% perchloric acid concentration, after which the capacity factor of CTC does not significantly increase. Also, above 0.2% perchloric acid, the peak shape of CTC is the most symmetric. This problem corresponds to the change in the equilibrium of the tautomers by increasing the rate of formation of the keto tautomer (Naidong et af.,1990). A 0.2% perchloric acid concentration was chosen as the best concentration for the mobile phase, since increasing the amount of acid above 0.2%) would only neutralize the base which is required to form the fluorescence derivative postcolumn. For chromatographic separation using UV detection, 0.2-0.5% perchloric acid was used successfully because these concentrations correspond to a p H range of 2.0-1.2, respectively. In this pH range, the rate of formation of the keto tautomer would be minimized along with the degradation products of CTC formed under strongly acidic conditions. To determine the effects of temperature and percent

307

organic modifier on the retention of the analyte on the PRP-1 column, the effects of column temperature and percent ACN in the mobile phase on the capacity factor of CTC was determined. A t 20% organic, the retention times for CTC are too long for routine analysis, except at high column temperatures, where peak shapes begin to broaden and resolution is lost. This is probably due to the increased rate of tautomerism on-column at the elevated temperatures. Above 25% ACN, the effects of temperature on retention time are minimal. At higher temperatures, resolution is also decreased due to peak broadening. The set of chromatographic conditions in which the capacity factors for 4-epi-CTC, CTC and their respective keto tautomers are between 3 and 6, are 27:73ACN:0.2% perchloric acid and ambient temperature (25 "C). Other chromatographic conditions may meet this criteria but for simplicity, the conditions at 25 "C were chosen. The studies described above were performed on the PRP-1 column because adequate retention of CTC was obtained with this column. The ACT-1 and the PBD-alumina columns were unsuitable for the separation of CTC and its degradation products because the analytes were not retained on the ACT-1 column and were irreversibly retained on the PBD-alumina column. A range of mobile phases similar to those used on the PRP-1 column were investigated for both the PBD-alumina and ACT-1 columns. The amount of organic modifier (MeOH or ACN) was varied from 0 to 100% in the case of the PBD-alumina column and from 20 to 100% (the maximum range recommended by the manufacturer) for the ACT-1 column. It has recently come to our attention that alumina columns are unstable below p H 2 and this is probably the reason for the poor results with the PBD-alumina column. In order to find chromatographic conditions to allow for the analysis of CTC, 4-epi-CTCand the more lipophilic anhydrochlortetracyclines, other columns will be investigated in future studies for this purpose. Figure 3 shows the changes in the emission spectra of 1mM CTC in ACN after addition of p H 9 NaOH (l:l), at 25 "C. Under these conditions, the maximum relative

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fluorescence intensity at 400 nm was not obtained until YO min, which is comparable to the results reported by Blanchflower et al. (1989). When 25% (w/v) NaOW was used as the derivatization reagent, maximum fluorescence intensity at 400 nm was observed at 100, 60 and 38 s for the temperatures 25,50 and 90 "C,respectively. These results show that by using NaOH solutions above pH 14 and elevated temperatures, alkaline-induced fluorescence derivatization for CTC can be achieved with the conditions obtainable in a postcolumn reaction coil. The results mentioned above were used as a basis for HPLC postcolumn derivatization. Using 10% NaOH (w/v), the maximum relative fluorescence response was obtained at a flow rate of 0.2mWmin. Also, the standard deviation of the response was minimized at this postcolumn reagent flow rate. The effects of percent NaOH and reaction coil length on relative fluorescence intensity of CTC is shown in Fig. 4. The maximum response was obtained using a 9 m reaction coil and 25% (w/v) NaOH as the postcolumn reagent at a flow rate of 0.2 mWmin. Figure 5 shows the effects of reaction coil tempera-

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Figure 5. Effects of reaction coil temperature and length on relative fluorescence intensity of CTC. Pump A-27:73 ACN:0.2% HCIO,, 1.0 mUmin; pump B-25% NaOH, 0.2 m U min; ,Iex 355 nm, 1,>389 nm.

ture and length on the relative fluorescence intensity of CTC. The maximum response was obtained using a 9 m coil at 90 "C. The postcolumn reagent was 25% NaOH (w/v) at a flow rate of 0.2mTlmin. Using the 9 m reaction coil, the fluorescence response does not significantly increase at temperatures above 50°C. The standard deviation for the fluorescence response was lower at 50°C than 90"C, and the lower temperature was chosen for the optimum conditions. The optimum conditions for PAIF detection of CTC were 25% NaOH (wh) at a flow rate of 0.2 mWmin, using a 9 m reaction coil at 50 "C. Figure 6 shows representative chromatograms from

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time (min) Figure 6. (a) Chromatogram of milk blank and (b) milk spiked with 0.04 yglmL CTC. PRP-1, 15 cm x4.6 mm; pump A--27:73 ACN:0.2% HCIO,, 1.0 mUmin; pump 8-25% NaOH, 0.2 rnlfrnin. Aex 355 nm, 1,,>389 nm.

309

HPLC OF CHLORTETRACYCLINE USING PAIF DETECTLON

Table 1. Comparisonof detection limits for CTC using microbiological assay, UV detection and PAIF alkaline induced fluorescence detection (a1

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Figure 7. (a) Chromatogram of keto-4-epi-CTC, keto-CTC, 4-epiCTC and CTC. PRP-1, 15 cm x4.6 mm. 27:73 ACN:0.2% HCIO,, 1.0 mumin, 280 nm. (b) Milk blank, 280 nm, same conditions as (a). (c) Milk spiked with 0.04 pg/mL CTC, same conditions as (a).

the analysis of CTC using PAIF detection. Figure 7 shows chromatograms using UV detection at 2SO nm. Figure 7(a) shows the separation of CTC and three degradation products formed in 0.1 N HCI after 24 h. In the above-described chromatographic system, CTC has a retention time of 9.36min and 4-epi-CTC has a retention time of 7.22 min. The other two peaks have been tentatively identified as the keto tautomers of CTC (5.55 min) and 4-epi-CTC (4.70 min). U V absorbance spectra obtained by a photodiode array detector (Waters Associates) match those obtained by Naidong er al. (1990). The C - l l a , C-12 keto-enol tautomeric forms of CTC are a particular problem because they are rapidly formed in aqueous solutions. Naidong et al. (1990) showed that the rate of formation of the keto tautomers is inversely proportional to the hydrogen ion concentration. These results were confirmed by varying the pH of aqueous organic solvents and observing the ratio of the keto tautomer formed. Using ACN/aqueous perchloric acid solvents, equilibrium was achieved in approximately 1 h. After 1 h in ACN:0.2% aqueous perchloric acid (pH 2.0), the keto-enol ratio was constant with peak-area ratios of 16:84 at 280 nm. When 100% ACN or other organic solvents were used as solvents for the standards, the chromatography was affected adversely. From these results, it follows that the stock solutions of the standards in aqueous organic solvents should be allowed to come to equilibrium

before standard curves are run. Quantitation of CTC in the sample is possible because the keto-enol ratio in the prepared samples and the standard solutions was approximately the same. Figures 6(a,b) and 7(b,c) show the chromatograms obtained from whole milk using PAIF detection and UV detection at 280 nm, respectively. Figures 6(a) and 7(b) are blank milk samples for PAIF and UV detection, respectively. Figures 6(b) and 7(c) are milk samples spiked with 100 pL of 0.808 pg/mL CTC (0.04 pg/ mL in milk sample), detection was by PAIF and UV detection, respectively. From the blank milk chromatogram (Fig. 7b), it can be seen that there was an interfering peak in the area of the chromatogram where CTC elutes, when UV detection at 280 nm was used. In the chromatogram for the milk blank using PAIF detection (Fig. Ba), the interfering peak was absent. Only when the milk was spiked at the 1.01 yg/mL level could CTC be quantitated by IJV detection at 280 nm. Even at this concentration, the precision of the measurement was compromised, with the amount of CTC quantitated at 118% of the spiked level. With PAIF detection, CTC could be detected down to 0.02pg/mL at a signal-tonoise ratio of 3 : l . In the prepared sample, 0.02 pg/mL CTC corresponds to 0.04pg/mL in the original milk sample before dilution in the sample cleanup. Table 1 shows the detection limits obtained by this method from solvents and milk, and compares the detection limits obtained by microbiological, UV detection at 280 nm and PAIF detection. Recoveries of CTC from whole milk were S l , 86 and 83% for spiked levels of 0.04, 0.50 and 5.07 pg/mL, respectively. The precision of the method was good, as shown by relative standard deviations of 5.2, 1.4 and 1.1, respectively, for the spiked samples 0.04, 0.50 and 5.07 pg/mL. The detection was linear from 0.02 to 5 pg/mL, with a correlation coefficient of 0.9997. The line for the regression equation was as follows: y = 42.892~ 0.0219. Recoveries of CTC using this method are low and can probably be improved by use of an organic modifier or protein precipitation step in the cleanup step, especially if binding to milk proteins lowers recoveries of CTC. Means of improving analyte recoveries were not exhaustively explored,

+

CONCLUSIONS An HPLC procedure for the analysis of chlortetracycline using PAIF detection has been described. The

3 10

PETER D. BRYAN E T A L

method was applied to whole bovine milk samples and for the determination Of chlortetracycline at lower levels than UV detection and is as sensitive as

microbiological assays but provides greater selectivity.

Acknowledgement P.D.B. gratefully acknowledges the financial xupport of a United States Pharmacopeia Fellowship provided by the United States Pharmacopeial Convention, Inc., Rockville, MD, USA.

REFERENCES Blanchflower, W. J., McCracken, R. J. and Rice, D. A. (1989). Analyst 114, 421. Engelhardt, H. and Neue, U. D. (1982).Chromatographia 15,403. Foye, W. 0. (1989). Principles of Medicinal Chemistry, 3rd ed. Lea and Febiger. Philadelphia. Honikel, K. O., Schmidt, U., Woltersdorf, W. and Leistner, L. (1978). J. Assoc. Off. Anal. Chem. 61, 1222. Levine, J., Garlock, E. A. Jr. and Fischbach, H. (1949). J. Am. Pharm. Assoc. 38,437. McCormick, J. R. 0..Fox, S. M., Smith, L. L., Bilter, B. A., Reichenthal, J., Origoni, V. E., Muller, W. H., Winterbottom, R. and Doerschuk, A. P. (1957). J . Am. Chem. Soc. 79,2849. Naidong, W., Roets, E., Busson, R. and Hoogmattens, J. (1990). J. Pharm. Biomed. Anal. 8, 881.

Nouws, J. F. M., Breukink, H. J., Brinkhorst, G. J., Lohuis, J., van Lith, P., Mevius, D. J. and Vree, T. B. (1985). Vet. Q. 7, 306. Poole, C. F. and Schuette. S. A. (1986). Contemporary Practices of Chromatography, 3rd ed. Elsevier Science Publishers, Amsterdam. Ray, A. and Newton, V. (1991). Antimicrob. Agents Chemo. 35, 1264. Reeuwijk, H. J. E. M. and Tjaden, U. R. (1986). J. Chromatogr. 353, 339. Sokolic, M., Filipovic, B. and Pokorny, M. (1990). J . Chromatogr. 509, 189. United States Pharrnacopeia (1988). No. XXI, Suppl. VII. The United States Pharrnacopeial Convention, Rockville, MD. van den Bogert and Kroon, A. M. (1981). J. Pharm. Sci. 70,186.