A fluorometric method of analysis for tetracycline

ANALYTICAL

A

28, 222-229

BIOCHEMISTRY

Fluorometric

Method

R. G. KELLY,

(1969)

of

Analysis

L. M. PEETS,

for

Tetracycline

AND K. D. HOYT

Department of Pharmacological Research, Toxicology Research Section, LederEe Laboratories, American Cyanamid Co., Pea& Rivep, New York 10965 Received

July

11, 1968

The fluorescent properties of the tetracycline antibiotics and their derivatives have been employed for the detection of these compounds in a variety of media (l-4) as well as for their quantitative analysis (5-8). It is characteristic of tetracycline fluorescence that it is emitted by these molecules only when they are in the basic form and when certain atomic bonds are made rigid either by the formation of a metal chelate or by adsorption on a surface. Thus, for example, an area of a metal-free paper chromatogram which contains tetracycline is not fluorescent when wet, even if the pH of the solvent is alkaline. However, when sprayed with certain polyvalent cations such as calcium or magnesium, fluorescence can be seen even on a wet paper. For fluorescence emission in the absence of such cations, the tetracycline must be permitted to dry, thereby adsorbing to the paper. Commonly, a dry chromatogram is developed by exposing it to ammonia vapors and viewing the paper under long-wavelength ultraviolet light. The phenomenon of fluorescence due to the rigidity imposed by being adsorbed on a surface is also seen among certain azo dyes (9). Anhydrotetracycline, the product of acid treatment of tetracycline (lo), shares with the latter the property of enhanced fluorescence when adsorbed on paper and exposed to ammonia. This property, coupled with favorable extraction properties reported for anhydrotetracycline (11)) led to a study of the fluorescence of metal chelates of the anhydro derivative with the ultimate aim of the development of a sensitive assay procedure for tetracycline applicable to biological fluids. The analytical procedure that was developed is described here and compared with accepted analytical procedures for the determination of tetracycline (X2,13) in biological samples. 222

FLUOROMETRIC

ANALYSIS

FOR TETRACYCLINE

223

METHODS

Reagents Tetracycline hydrochloride labeled with tritium in the carbon-7 position (14). Specific radioactivity, 11.8 &mg. Microbiological activity, 1072 pg/mg. 30 % trichloroacetic acid. 5 N hydrochloric acid. 6 N sodium hydroxide. Stored in a polyethylene bottle. 1 M citrate buffer, pH 4.5. A solution of 1 M citric acid was titrated with a solution of 1 M trisodium citrate until pH 4.5 Stored in a polyethylene bottle. Reagent-grade chloroform. 0.1% AlCI,.GH,O in ethanol. 100 mg of AlCl,*GH,O was diluted to 100 ml with absolute ethanol and stirred several hours with a magnetic stirrer to effect solution. All of the distilled water employed in the preparation of reagents and in the assay procedure itself was passed through a column of a Dowex chelating resin A-l for the removal of trace metal ions. Animal Studies Five beagle dogs were given intravenous injections of tritium labeled tetracycline as shown in Table 1. Serum samples were obtained at 0, l/r, 11/, 3, 5, and 8 hr after dosing. These were divided into three aliquots, one of which was assayed microbiologicallyl; a second was assayed radiometrically as described by Takesue et al. (13) ; and the third was assayed fluorometrically as described below. Fluorometric Procedure To 0.2 ml plasma add 9.0 ml water followed by 1.0 ml 30% trichloroacetic acid. Mix thoroughly. Centrifuge and transfer 8.0 ml of the filtrate to a glass-stoppered centrifuge tube. Add 1 ml 5 N hydrochloric acid. Heat in a boiling water bath 30 min. Cool and add 1 ml 6 N NaOH. Add 1 ml pH 4.5 citrate buffer. Mix and ascertain that the pH of the resultant solution lies between 3.5 and 5.5. Add 2 ml chloroform, shake, and centrifuge. 1 We are grateful Cyanamid Company,

to Mr. A. C. Dornbush for these analyses.

of Lederle

Laboratories,

American

224

KELLY,

PEETS,

AND HOYT

Aspirate water phase and transfer 1 ml of the chloroform phase to a cIean glass-stoppered centrifuge tube. Add 1 ml 0.1% AlCl,~GH~O in absolute ethanol, mix, and permit to stand at least 1 hr (preferably overnight). Read the fluorescence of the resultant solution in an Amine0 Bowman spectrophotofluorometer using an activating wavelength of 475 rnp and a fluorescing wavelength of 550 rnp. RESULTS

AND

DISCUSSION

Figure 1 shows the spectral shift obtained when anhydrotetra cycline forms a chelate compound with aluminum under the condi. tions of the assay procedure described. Although the precise strut. ture of the metal chelate is unknown, the assignment of the lonp wavelength chromophore of tetracycline derivatives to rings B, C, and D of the tetracycline structure (14,15) would indicate the involvement of either the C-lO,C-11 peri-hydroxyl groups or the C-l&C-12 ketohydroxy systems (see Fig. 2). Using an Aminccl Bowman spectrophotofluorometer we found that the aluminum chelate was a highly fluorescent material with a maximum activating wavelength corresponding to the long-wavelength chromophore absorption peak at 475 my. The maximum wavelength of fluores cence emission was 550 rnp. The rate of development of the chelate in chloroform-ethanol (the solvent mixture to be employed in the assay procedure) way 1.2 I’

I,#’

*=---\,

\\ \, ‘,

Anhydmtetracycline 5Omcg/ml in Chloroform:

.9 -

350

400

450 Wavelength

FIG.

aluminum

1.

Absorption chelate.

spectra

500

550

in m/u

of anhydrotetracycline

and anhydrotetracycline

FLUOROMETRIC

FIG.

2.

ANALYSIS

Structure

of

FOR

225

TETRACYCLINE

anhydrotetracycline.

determined by mixing 5 ml of a 100 pg/ml solution of anhydrotetracycline hydrochloride in chloroform with 5 ml of a 0.1 “/o solution of AlCI,.GH,O in ethanol. The 475 rnp absorbance change with time was then recorded, with the results shown in Figure 3. Since at 1 hr the rate of change in absorbance was not large, it was chosen as a minimum time for fluorescence determination of analytical samples. In actual practice it is convenient to permit the development of the chelate overnight, at which time equilibrium appears to have been established. When fluorescence was plotted against concentration, the procedure as described gave a straight line over the concentration range from 0.1 to 20 pg/ml of sample. The standard curve for serum samples was not distinguishable from that derived from water samples of the same concentration except that blank serum values of from 0 to 0.1 pg/ml were obtained when the assay procedure was employed exactly as described. A blank serum value less than 0.1

3

5ml of !OOmcg /ml Anhydrotetracycline Hydrochlovide in Chlorofwm plus of 0.1% Al Cl,. 6H,O in ethanol

.6 -

51711

Lo G

t-,

.4-

i2 2 & B 6

.2 -c

J-tarf

I ioo

I 50

0

I 150

Time

FIG.

3.

Rate

of

formation

of

I 200

I 250

in Minutes

anhydrotetracycline-aluminum

chelate.

226

KELLY,

PEETS,

AND

HOYT

pg/ml could be derived from a serum sample when a larger volume of sample was employed in the assay. Table 1 shows the results obtained when samples from five dogs were assayed microbiologically, radiometrically, and fluorometrically by the procedure described. These figures were submitted to a statistical evaluation of correlation according to the method described by Snedecor (16). The correlation coefficient comparing the microbiological analyses of the sample with the fluorometric analysis was found to be +0.988.

Comparison Animal No., dose, route

1 7.5 mg/kg i.v.

of Three

Methods

Time

dose,

atft

TABLE 1 of Analysis for

Tetracycline

Concentrations. Microbiological

in Dog pg/ml

Radiometric

Serum

Serum Fluorometrie

w 1% 3 5 8 24

9.0 6.8 4.5 3.5 2.9 0.4

10.4 6.0 4.9 4.3 3.0 0.9

10.3 7.0 4.3 3.5 2.7 0.5

vi v 3 5 8 24

5.9 3.0 2.5 1.7 1.3 0.2

5.1 3.3 2.7 2.1 1.6 0.4

5.2 3.3 2.5 1.5 1.1 0.2

3 5.0 mg/kg i.v.

‘A 1?5 3 5 8 24

5.5 3.6 3.3 2.5 1.8 0.4

5.7 4.2 3.5 2.8 2.1 0.6

5.9 3.9 3.1 2.7 3.1 0.5

4 mg/kg

35 1% 3 5 8 24

2.4 1.7 1.3 1.0 0.7 0.1

2.7 1.8 1.6 1.1 0.9 0.2

2.2 2.2 1.2 1.0 0.6 0.3

?4 1% 3 5 8 24

3.1 2.1 1.7 1.3 1.0 0.3

3.7 2.3 2.1 1.7 1.4 0.4

3.2 2.2 1.9 1.3 1.2 0.3

2 5.0 mg/kg i.v.

2.5

i.v.

5 2.5 mg/kg i.v.

FLUOROMETRIC

ANALYSIS

FOR

TETRACYCLINE

227

That comparing the radiometric method with the fluorometric method was found to be f0.992. Exact correlation of two methods would be signified by a correlation coefficient of +l.OO and no correlation by a coefficient of 0. A comparison of the results obtained with microbiological and radiological techniques yielded a correlation coefficient of +0.980. Although the procedure does not appear to be useful for the analysis of demethylchlortetracycline or chlortetracycline, these compounds will produce fluorescent anhydro derivatives and therefore interfere with the analysis of tetracycline. The various 4-epitetracyclines will also interfere. Although we have not tested oxytetracycline it is reported (8) that this compound and its derivatives will also produce fluorescent substances that might interfere with the analysis. The only other interferences observed have been those due to storage of the 6 N sodium hydroxide solution in glass bottles and the use of water from which trace metals have not been removed. Presumably both of these interferences are of the same nature. That is, certain trace metals present in the reagents prevent the extraction of anhydrotetracycline into chloroform. The fluorescence yield of aluminum anhydrotetracycline is of the same order of magnitude as that observed by Kohn (6) for the calcium chelate of tetracycline and by Ibsen et al. (7) for magnesium tetracycline. We have examined the fluorescence of anhydrotetracycline in the absence of metal ion contaminants and find that the fluorescence yield of the aluminum chelate under the conditions we have described is more than 30 times greater than that of the unchelated compound at the wavelengths and conditions reported by Hayes and DuBuy (8). In addition to the high yield of fluorescence, the aluminum anhydrotetracycline complex is unique in the respect that it is excited by a relatively long wavelength of light as compared with naturally occurring biological substances. Among such compounds, only riboflavin (activation wavelength 450 rnp) shows fluorescence in this area of the spectrum. This property may prove useful in the analysis of biological samples that might ordinarily be expected to interfere with the fluorescent analyses presently in use. In 1966 and 1967 there appeared in the scientific literature several papers describing methods for the determination of tetracycline degradation products in pharmaceutical preparations (17-19). These procedures have generally employed thin-layer chromatography of small aliquots of these preparations, so that resultant eluates have been determined by low spectral absorbances. The

228

KELLY,

PEETS,

AND

HOYT

procedure described in this paper would seem to offer an advantage to such determinations, permitting elution of the various constituents by a dilute acid solution followed by detection of high sensitivity and specificity. SUMMARY

A new method of analysis for low concentrations of tetracycline in biological samples is described. The method is dependent upon the extraction of anhydrotetracycline from an acid-treated proteinfree filtrate. The anhydrotetracycline, in a chloroform extract, is then treated with ethanolic aluminum chloride to form a highly fluorescent chelate. The procedure described is compared with accepted analytical procedures for tetracycline (microbiological, radiological) in an experiment in which serum concentrations of the antibiotic were determined at various times (hours) following intravenous administration in dogs. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

Loo, T. L., TITUS, E. D., AND RALL, D. P., Science 126, 253 (1957). KELLY, R. G., AND BUYSKE, D. A., Antibiot. Chemotherapy 10, 604 (1960). RALL, D. P., Loo, T. L., LANE, M., AND KELLY, M. G., J. Natl. Cancer Inst. 19, 79 (1967). BUYSKE, D. A., EISNER, H., AND KELLY, R. G., J. Pharmacol. Exptl. Therap.130,150 (1960). LEVINE, J., GARLOCK, E. A., JR., AND FISHBACK, H., J. Am. Pharm. Assoc. (Sci. Ed.) 38, 473 (1949). KOHN, K. W., Anal. Chem. 33, 862 (1961). IBSEN, K. H., SAUNDERS, R. L., AND URIST, M. R., Anal. Biochem. 5, 505 (1963). HAYES, J. E., JR., AND DUBUY, H. G., Anal. Biochem. 7, 322 (1964). WEST, W., in “Chemical Applications of Spectroscopy,” Vol. IX of (“Technique of Organic Chemistry,” A. Weissberger, ed.), p. ‘736. Interscience, New York, 1956. BOOTH, J. H., MORTON, J., PETISI, J. H., WILKINSON, R. G., AND WILLIAMS, J. H., J. Am. CILem. Sot. 75, 4621 (1953). MISTRETTA, A. G., AND MINIERI, P. P., Antibiot. Chemotherapy 6, 13 (1956). GROVE, D. C., AND RANDALL, W. A., “Assay Methods of Antibiotics, A Laboratory Manual,” Medical Encyclopedia Inc., New York, 1955. TAKESUE, E. I., TONELLI, G., ALFANO, L., AND BUYSKE, D. A., Zntern. J. Appl. Radiation Isotopes 8, 52 (1960). STEPHENS, C. R., CONOVER, L. H., PASTERNACK, R., HOCHSTEIN, F. A., MORELAND, W. T., REGNA, P. P., PILGRIM, F. J., BRUNINGS, K. J., AND WOODWARD, R. B., J. Am. Chem. Sot. 76,3568 (1954).

FLUOROMETRIC 15.

16. 1’7. 18. 19.

ANALYSIS

FOR

TETRACYCLINE

229

MCCORMICK, J. R. D., Fox, S. M., SMITH, L. L., BITLER, B. A., REICHENTHAL, J. ORIGONI, V. E., MULLER, W. H. WINTERBOTTOM, R., AND DOERSCHUK, A. P.. J. Am. Chem. Sot. 79, 2849 (1957). Methods,” 4th ed., Chapter 7. Iowa State SNEDECOR, G. W., “Statistica College Press, Ames, Iowa, 1950. SIMMONS, D. L., KOORENGEVEL, C. K., KUBELKA, R., AND SEERS, P., J. Pharm. Sci. 55, 219 (1966). SIMMONS, D. L., Woo, H. S. L., KOORENGEVEL, C. M., AND SEERS, P., J. Pharm. Sci. 55, 1313 (1966). ASCIONE, P. P., ZAGAR, J. B., AND CHREKIAN, G. P., J. Plrrrwn. Sci. 56, 1393 (1967).