Determination of Tetracyclines by Extraction of Fluorescent Complexes Application to Biological Materials W. KOHN
KURT
Downloaded via NORTHERN KENTUCKY UNIV on July 17, 2018 at 11:02:37 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
National Cancer Institute, National Institutes of Health, Bethesda, Md.
extractable form Tetracyclines mixed complexes with calcium and barbiturates. Preliminary studies on the nature of these complexes are described. Procedures are developed, based on this phenomenon, which combine the use of fluorometry and complexometric extraction to yield a highly sensitive and specific method for the tetracycline antibiotics (with the exception of oxytetracycline). Convenient procedures are devised for determinations in body fluids and soft tissues over the usual pharmacologic range of drug concentration. Reasonably good agreement with bioassay was obtained. on the binding of in tumors and bone (7) and in experimental gastric ulcers (3) have emphasized the need for a suitable chemical method for the estimation of these antibiotics in tissues. Such a method might avoid some of the problems of microbiological assays, and could be of value in studies of the pharmacology and mechanism of action of these
reports
Recent tetracyclines
drugs. Various physical and chemical properties of tetracyclines have been used for determinations in pharmaceuticals [see references given by Chiccarelli et al. (2) ] but none has found general applicability to biological materials. The methods to be described are based on the solvent extraction of highly fluorescent tetracycline complexes. The extractability of a chlortetracycline calcium chelate has been noted (6), but this extraction fails at low concentrations of the antibiotic. This deficiency does not occur in the case of the complexes formed in the present method. Some evidence relating to the nature of these complexes will be presented. The methods have been used in this laboratory in a pharmacologic study in dogs (6), and some of the data relevant to the evaluation of the methods will be included. APPARATUS
Fluorescence intensities were measured by means of a Farrand Model A
862
·
analytical chemistry
fluorometer equipped with a General Electric AH3 mercury vapor lamp and an RCA 1P21 multiplier phototube. Two 405- µ interference filters were inserted in the incident light path and a 530- µ interference filter plus a Corning No. 3384 yellow glass filter were placed in the path of the emitted light. The smallest (5 mm.) light aperture was used. Fluorescence spectra were obtained with an Aminco-Bowman spectrophotofluorometer using Vie-inch defining slits. The instrument was calibrated with a mercury vapor lamp, but no corrections were made for nonlinearity of multiplier phototube (RCA 1P21) response. Absorbance measurements were performed using a Beckmann DU spectrophotometer. Tissues were ground with a stainless steel tissue press, consisting of a 2-cm. diameter cylinder fitted with a piston. The tissue is placed inside the cylinder and is forced through a stainless steel screen having 0.5-mm. diameter holes. REAGENTS
Sodium barbital, National Formulary (Fisher Scientific Co.) was used in the procedures without further purification. A 0.9M aqueous solution is sufficiently stable for 2 to 4 weeks at room temperature. Pentobarbital was prepared from the U.S.P. sodium salt by neutralization of an aqueous solution with HC1, and wTas recrystallized from heptane-ethyl acetate mixtures. Reagent grade ethyl acetate was refluorescent imdistilled to remove
purities. Tetracyclines. Tetracycline (TO), 7 6 chlortetracycline demethyl (DMCT), and 7-chlortetracycline (chlor-TC) were supplied by Lederle Laboratories, 5-hydroxy tetracycline (oxy-TC) by Chas. Pfizer & Co., Inc., and Ar-pyrrolidinomethyltetracycline (PMT) by Farbwerke Hoechst AG. All of these were in the form of hydrochlorides. Tetracycline has the following structure: -
-
-
PROCEDURES
Extraction of Tetracyclines from Tissues. Approximately 0.5-gram samples of fresh or frozen tissue were ground with a tissue press and exweighed. Tetracyclines were tracted by shaking with O.LV HC1 (19 ml. per gram of tissue) at 5° C. for 1 hour followed by centrifugation at 1000 g for 30 minutes. Measurement of Fluorescence. All fluorescence intensity readings were referred to primary fluorescence standards prepared as follows: 2.00 ml. of 50 µ aqueous tetracycline hydrochloride was mixed with 0.40 ml. of 0 9M sodium barbital and diluted to 50 ml. with a solution of 3 X 10~43/ CaCl2 and 8 X 10~33/ barbital (U.S.P.) in methanol. The fluorescence of these standards deteriorated less than 1 % per week at room temperature in the dark. There was no detectable change in fluorescence reading after 20 minutes’ continuous exposure of the standard solution to the fluorometer source (using the largest aperture). Readings were made by setting the standard to 90 on the galvanometer scale (or, for more intense samples, to 45 or 30 and multiplying sample readings by 2 or 3, respectively). Secondary standards of different tetracycline concentrations were sometimes convenient.
Procedure I. (This one-step procedure may be used for samples not containing protein or phosphates.) An aliquot of sample is measured into a glass-stoppered centrifuge tube, and diluted with water to a total volume of 2.5 ml. To this are added 2.0 ml. of a solution of 0.44Ar trichloroacetic acid and 0.043/ calcium chloride, 3.00 ml. of ethyl acetate, and 3.0 ml. of 0.93/ sodium barbital. The tube is shaken vigorously for about 2 minutes and the phases are allowed to separate. (Barbital precipitates, but then dissolves in the ethyl acetate phase.) The fluorescence of the upper layer is measured. Procedure II. (For phosphate-free samples which may contain protein.) An aliquot of sample is diluted with water to make a total volume of 5.5 ml. Proteins are precipitated with 1.00 ml. of a solution of 1.8Ar trichloroacetic acid and 0.163/ calcium chloride, and centrifuged. A 4.00-ml. portion of supernatant is pipetted into a glass-stoppered centrifuge tube containing 3.00 ml. of ethyl acetate and 4.0 ml. of 0.93/ sodium
barbital. The remainder of the procedure is the same as in 1. Procedure III. (For samples containing phosphates, with or without
protein.) 1. An aliquot of sample is diluted with water to make 4.00 ml. Proteins are precipitated with 1.00 ml. of a solution of ,9/V trichloracetic acid and 0.004.1/ KH2PO4. (For the case of tissue extracts in 0.1 " IIC1, the concentration of trichloroacetic acid is reduced to 1.5N so as to maintain a total of 1.9 meq. of acid.) 2. Four milliliters of supernatant are mixed with 2.00 ml. of a solution of 0.06.1/ Pb(N03)2 and 2,0.1/ sodium acetate. (In the absence of protein, 2.50 ml. of the latter solution can be added directly to step 1.) Ten to 30 minutes are allowed for completion of precipitation before centrifuging. 3. Four milliliters of supernatant are mixed with 1.00 ml. of 0.32.1/ KI03 and centrifuged after standing 30 to 60 minutes. 4. Four milliliters of the supernatant are added to a glass-stoppered centrifuge tube containing 3.00 ml. of ethyl acetate, 3.0 ml. of 0.9.1/ sodium barbital, and 3.0 ml. of 0.1.1/ 1
CaCl2. The remainder of the procedure is the same as in I.
Distribution Studies. Five-milliliter volumes of aqueous and organic phases were equilibrated for 2 to 6 hours at 21.5° ± 1.0° C. Tetradetercycline concentrations were mined in both phases by procedure I, using appropriate standards. Extraction was also followed by visual observation of fluorescence excited with a long wave ultraviolet mercury vapor lamp, or by absorbance measurement (385 µ) of the organic phase. RESULTS
AND DISCUSSION
Tetracyclines form chelates with divalent metal ions (1, 4, 8). The present work was begun with an attempt
to use the strong fluorescence of the Ca+2 chelate for determinations in tissues. The method was insensitive, however, because of high background fluorescence. Extraction of the fluorescent species into organic solvents, was, therefore, attempted. Several solvents were found to extract an intensely fluorescent complex at pH 9 from highly dilute (2 µ ) solutions of tetracycline (TC) in barbital buffer containing CaCl2 (20 ml/). (The barbital buffer consisted of a mixture of 3.0 mmoles of sodium barbital and 1.0 mmole of hydrochloric or trichloracetic acid in 10 ml. of aqueous solution. The resulting barbital precipitate dissolved in the organic phase.) Extraction succeeded with 1-butanol, isoamyl alcohol, ethyl acetate, isoamyl acetate, and chloroform but not with ethyl ether, benzene, or hexane. Extraction, when it occurred, was immediate. Ethyl acetate was selected for the procedures because it demonstrated most clearly the specificity among tetracyclines to be described.
Nature of Extractable Complexes. Omission of Ca+2 from the above system prevents extraction. Addition of as little as 0.1 µ ß of Ca+i (to 3 ml. of organic solvent mixed with 10 ml. of aqueous solution) produced a marked increase in absorbance (385 µ) of the ethyl acetate phase when a large excess of TC (0.5 Mmole) wras present. (Ethylenedinitrilo) tetraacetate (EDTA) reverses both the extraction and the fluorescence induced by Ca+2. The distribution ratio in the case of highly dilute TC solutions, was 20 in the presence of Ca+2 and 0.2 in its absence. Replacement of Ca+2 with Mg+2 permits partial extraction into 1-butanol, but not into ethyl acetate. Both solvents extract a somewhat less fluorescent Zn+2 complex. Mn+!, Fe+2, Co+2, Ni+2, Cu+2, Al+S, and Fe+3 abolish both extraction and fluorescence. Omission of barbital likewise prevents extraction (when this is attempted from highly dilute TC solutions): there was no extraction from tris (hydroxymethyl) aminomethane (TRIS) or ammoniaHC1 buffers at pH 9. Extraction can be induced, however, by addition of barbital or another barbiturate. These observations suggest the formation of an uncharged mixed complex of tetracycline and barbiturate ligands bridged by a calcium ion. The following observations support this picture. /Vj/V-Dimethylbarbital, which has no displaceable hydrogen, is completely inert as a replacement for barbital, while /V-methylphenobarbital, which retains an ionizable group, is nearly as active as phenobarbetal. Several other compounds capable of forming organic anions were found to induce extraction, although to a lesser degree than do the barbiturates. These included p-hydroxypropiophenone, nand sulbutyl-p-hydroxybenzoate, fathiazole. Another class of compounds capable of inducing extraction are the linear ßdiketones. Acetylacetone, hexane-2,4dione, and ethyl acetoacetate were found to have the same order of effectiveness as the barbiturates, although the fluorescence produced was less intense. On the other hand, hexane-2,5-dione (a 7-diketone) and 5,5-dimethyicyclohexane-l,3dione (a cyclic /3-diketone) were completely inert. The /3-diketones are noted for their ability to dissociate a proton, and, in the case of the linear compounds, to form metal chelates. The cyclic compounds cannot chelate. This evidence, therefore, favors the calcium ion as the site of attachment of the /3-diketone to the extracted complex, and indicates the possibility that the calcium ion may bind simultaneously to TC and to a second ligand. Since the probable chelating group of TC is an enolized /3-diketone (4), TC itself should be able to serve as the -
Extraction should then at high TC concentrations even in the absence of barbiturate or other secondary ligand, and should vary with the square of the TC concentration in the aqueous phase. This was verified in distribution studies with an aqueous phase of TRIS-HC1 buffer (pH 8.9, ,0.5M) and 8 mM CaCl2, and with isoamyl acetate, ethyl acetate, isoamyl alcohol, or mixtures thereof as solvents. The log-log plot of TC concentration in the organic phase vs. its concentration in the aqueous phase was in each case linear over a 10-fold variation of aqueous TC concentration. The slopes fell in the range 2.1 ± 0.2. Similar experiments were performed with 0.1/1/ pentobarbital or 0.1/1/ acetylacetone in isoamyl acetate as solvent. The slopes were now 1.25 and 1.16, respectively. These results indicate that barbiturates and linear /3-diketones cause a change in the major extracted species from a complex containing two molecules of TC to forms having only a single TC molecule. All of these complexes must contain Ca+2, since extraction does not occur in the absence of this ion. All the evidence at hand is compatible with the formation and extraction of mixed complexes of two ligands linked through a calcium ion. There may, however, be additional stabilization through hydrogen bonding between the two ligands. One such possibility is shown below, for the case of the complex with barbital. second ligand. occur
*
The fluorescence spectra of the extracted calcium-barbital complexes of several tetracyclines are shown in Figure 1. Properties of the Extraction System. The reagent quantities in the procedures were chosen so as to give a final pH of 8.9 to 9.0 in the aqueous phase of the extraction step. This falls in the range for optimal fluorescence (Figure 2). There is a slow fall in pH (approximately 0.3 unit in 24 hours) due to the alkaline hydrolysis of -ethyl acetate. Nevertheless, the fluorescence does not diminish more than 6% after 24 hours at room temperature (in the dark). The readings were found VOL. 33, NO. 7, JUNE 1961
·
863
in
t
-
ico
---DMCT
TC
_---OXY-TC
.....
Table II. Effects of Potential Interfering Substances on Readings Procedure I with 4.0 MMmoles of
CHLOR-TC
Tetracycline Per
Cent Compound Added
ReadMmole ing
None
kh2po< Sodium oxalate
Figure 1. Apparent activation and fluorescence spectra of complexes of tetracyclines Tetracyclines diluted to 2.0 µ in ethyl acetate phase, prepared as in procedure I
Sodium citrate Sodium urate
Glycine
0.001 0.002 0.005 0.04 0.08 0.04 0.08
0.04 0.08 0.2 0.4
0.001 0.01 EDTA 0.02 0.001 FeCl, +0.02 mmole EDTA 0.01
FeCl,
to be independent of the amount of CaCl2 used (over the range, 0.01 to 0.2 mmole). The fluorescence intensity is proportional to the amount of tetracycline in the system, except for slight deviations below 0.3 µ ß and above 10 mamóles. The lower limits for measurement by procedure I are approximately 0,3 mamóle (0.15 Mg·) for TC and PMT and 0.2 mamóle (0.1 Mg.) for DMCT. The method is somewhat less sensitive for chlor-TC and much less sensitive for oxy-TC. This is due to the lower fluoof the complexes formed by rescence these antibiotics (Figure 1), and, in the case of oxy-TC, also to incomplete extraction into ethyl acetate (Table I). The system has considerable selectivity for those tetracycline derivatives with high antibiotic potency (Table I). The upper five compounds in the table are potent antibiotics. All of these, except oxy-TC, show nearly complete extraction and form highly fluorescent complexes. The derivatives below oxy-TC in the table have much less
Table
I.
Fluorescence Reading and Per Cent Extraction
10 MMmoles of Test Compound
in Procedure I
Per Cent Read- Extrac-
Test Compound
DMCT PMT TC
chlor-TC oxy-TC 4-epi-DMCT 5a-epi-TC desdimethylaminoTC 4-epi-TC anhydro-TC 12a-deoxy-TC
Riboflavin
864
·
ing 310 230 210 150 70 65 40 15 10 9 6 8
ANALYTICAL CHEMISTRY
tion >95
90 90
>95 50 30 70 25 10
>90 35
