suggestion seems very unlikely. A more reasonable explanation for their observations, in accord with our proposals, would be the condensation of 2-iminoindandione (VI) with 2-hydroxyindandione (111), according to our Equation 14. The source of 2-hydroxyindandione in their solutions was probably from hydrolysis of 2-aminoindandione (IV) according to Equation 12. This suggestion is supported by the fact that Zelensky et al. claim to have isolated 2-hydroxyindandione from their solutions.
ACKNOWLEDGMENT Special thanks are extended to M. F. Marcus for his invaluable assistance with the electrochemical experiments. Received for review January 9, 1973. Accepted March 29, 1973. These studies were supported by grants from the Marquette University Committee on Research. The support of an NDEA Title IV Fellowship (PJL) is also gratefully acknowledged.
Spectrofluorometric Method for the Determination of Hydroxylated Anthracene Derivatives and Its Application to the Assay of Senna Derivatives in Biological Tissues A. C. Lane Department of Pharmacology, Reckitt a n d Colman, Pharmaceutical Division, Dansom Lane, Hull HU8 7DS, England
A new rapid and sensitive fluorometric method is described for the assay of sennosides and related hydroxylated anthracene derivatives. A fluorescent complex is formed in aqueous sodium tetraborate in the presence of the reducing agent sodium dithionite. Fluorescence intensity is measured at 510 nm with excitation at 410 nm. Optimal conditions for the development of the complex have been found. Limits of detection are between 2 and 10 ng/ml. The assay of ten test samples from a solution of sennoside A gave a relative standard deviation of &3.35% and, when compared with a standard spectrophotometric assay procedure, a relative error of -1.65%. The application of the method to the assay of anthracene derivatives in biological tissues is also described.
Currently accepted methods for the analysis of senna (I-3), cascara ( 4 4 , and aloes (6) are based on extraction followed by absorption spectrometry. There are also biological assays based on the cathartic action of sennosides (7, 8 ) , aloes (91, and anthraquinones (10). All these methods are cumbeysome and require relatively large amounts of drug. The ability of hydroxylated anthraquinones to form fluorescent chelates with boron compounds in concentrated sulfuric acid has been used in the detection of boron (11). Also hydroxylated anthraquinones have been detected in biological tissues, after an extraction procedure, by their fluorescence in an aqueous borate solution (12). "European Pharmacopoea," 1971, p 358. "British Pharmacopoea," Addendum, 1971 (1968),p 91. Analyst (London), 90, 582 (1965). Analyst (London). 93, 749 (1968). J. W . Fairbairn. and S. Simic, J. Pharm. Pharmacol., 16, 450, ( 1964).
Analyst (London), 92, 593 (1 967). T. C. Lou, J. Pharm. Pharmacol.. I , 673 (1949). R. T Brittain, P. F. D'Arcy, and J . J . Grimshaw, J. Pharm. Pharmacol., 14, 715 (1962). J. C. Munch, "Bioassays," Williams and Wilkins. Baltimore, Md., 1931, p 771.
Viehover, J. Amer. Pharm. A s s . , 25, 1 , 112 (1936). Radley. Analyst (London), 69, 47 (1944). R . Joachimovits, Monatsschr. Geburfsh. Gynaek., 83, 42 (1929).
A.
J. A .
A method was required for the determination of small amounts of drug to facilitate a study of the absorption and distribution of anthraquinones in animals. It was decided to try to develop a fluorescence assay based on the observation (13) that anthraquinones form a highly fluorescent complex in borate solution by reduction with sodium dit hionite. This report describes a simple, rapid, and sensitive method for the fluorometric assay of hydroxylated anthracene derivatives and its application to their assay in biological tissues.
EXPERIMENTAL A p p a r a t u s . A double monochromator spectrofluorometer (Aminco-Bowman, American Instrument Company, Silver Spring, Md.) fitted with a xenon l a m p a n d a n RCA IP28 photomultiplier was used. Fused quartz cells (10 X 10 X 48 m m ) were employed. T h e slit width was 2 or 3 m m (Aminco slit arrangement No. 3) in both the excitation a n d analyzing monochromators. Except where otherwise indicated, the excitation monochromator was set a t 410 n m and the emission monochromator a t 510 nm. Reagents. Primary Fluorescence Standard. Chrysazin or 1,8dihydroxyanthraquinone (1,8D) was chosen as t h e primary fluorescence standard because it could be obtained cheaply a n d with a high degree of purity. Also stock solutions of 1,8D in absolute ethanol were stable for a n indefinite period. T h e crystalline 1,8D was obtained from R. N . Emanuel Limited, Wembley, England, and recrystallized from ethyl acetate ( m p 193 "C). Secondary Fluorescence Standards. Whenever possible secondary fluorescence standards were dissolved in 2% sodium tetraborate, otherwise they were dissolved in absolute ethanol. Because of their instability in alkaline solution, the secondary standards were prepared immediately before each assay. Borate Solution. A 2% solution of sodium tetraborate (prepared in glass-distilled water) having a p H of 9.2 was used for all solutions except for lengthy procedures involving solid tissues. For t h e latter, a 2% solution containing 1% 2-mercaptoethanol (antioxid a n t ) was used. Reducing Agent. A 1.6570 sodium dithionite solution was prepared fresh for each assay. Calibration G r a p h for 1,s-Dihydroxyanthraquinone.S a m ples (0.075, 0.15, 0.75, 1.50, 7.50, a n d 15.0 pg) of 1,8D dissolved in 50 pI of absolute ethanol were each added t o borate solution (10 (13) C. A. Friedman, and H . A. Ryan, unpublished work. Westminster Laboratories, 1960.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
1911
Assay of Samples of Non-biological Origin (Procedure 1). An accurately weighed amount of about 1 mg was dissolved in either borate or ethanol. A suitable aliquot containing between 0.1 and 10 fig of assayable material was then diluted to 10 ml with borate solution and the initial fluorescence intensity was recorded. Reducing reagent (1 ml) was added and the tube contents were mixed. T h e contents of the tube were heated for 15 min in a boiling water bath. T h e tube was cooled a n d the relative intensity of fluorescence was measured. Assay of Blood Samples (Procedure 2). Blood samples were drawn into heparinized syringes. A 0.10-mi sample of plasma obtained after centrifugation of t h e blood a t 2500 X g for 10 min was diluted with 1.9 ml of borate and the initial fluorescence was measured. Reducing reagent (0.2 ml) was added a n d the mixture was then heated in a boiling water bath for 15 min. T h e tube was cooled and the fluorescence intensity was recorded. Assay of Urine and Bile Samples (Procedure 3). Samples of bile (0.02 ml) and urine (0.10 ml) were diluted with borate to give a total volume of 10 ml. T h e procedure was then as described for Procedure 1. Assay of Tissues from the Gastrointestinal Tract (Procedure 4). Samples of stomach, small intestine, and colon were freed of adhering matter by flushing them with water. If the gastrointestinal tract contents were to be analyzed, then the flushing was performed with borate solution containing 1% w/v 2-mercaptoethan01 a s a n antioxidant. After blotting the tissue dry about 10 g was accurately weighed and homogenized by grinding it with acidwashed sand in two volumes of borate containing 1% 2-mercaptoethanol. T h e mixture was centrifuged a t 20,000 x g for I5 min and a portion of the clear supernatant was assayed. Genera!ly 0.10-ml samples of extract diluted to 10 ml with borate were suitable for assay. T h e procedure was then as for Procedure 1. Gastrointestinal contents were homogenized in ten volumes of borate solution containing 1% w/v 2-mercaptoethanol by using a glass homogenizer. The homogenate was then centrifuged a t 20,000 X g for 15 min. T h e clear supernatant (0.10 m!) was then diluted to 10 ml with borate a n d assayed as described for Procedure 1.
1
.-* L
W
C Q
L
C Q 0
C Q U W
Q L
0
-a 5 4
0.0
Wavelengt h(nm)
Figure I. Fluorescence excitation and emission spectra ( 0 ) 0.75 pg/rnl sennoside A worked through Procedure 1 , and ( 0 )a reagent blank worked through Procedure 1 . Spectra are uncorrected
11
*
.-
1
W
C 0
-
1
C
0 V C
m V (Io
m
L
0
a
LL
13
5
a
11
PH
Figure 2. Effect of pH on the development of fluorescence pg/ml 1.8D worked through Procedure 1
of 1
ml) in a test tube. Reducing agent (1 ml) was added and the contents were mixed. T h e tubes were then heated in a boiling water bath for 15 min; then, after cooling, the final fluorescence intensities were recorded. T h e calibration graph was rectilinear and passed through the origin after correction for the reagent blank fluorescence. 1912
RESULTS AND DISCUSSION Sennosides, cascarosides, and other hydroxylated anthracene derivat,ives form highly fluorescent complexes in aqueous borate solution with sodium dithionite. The nature of this complex is not certain, but it is probably a boron chelate of a substituted dianthranol. The fluorophore is not extractable into organic solvents. The conditions for the optimal development of the fluorophore and its stabilization have been investigated. Spectral Characteristics. The fluorescence excitation and emission spectrum for 1,8D (0.75 pgjml) and a reagent blank are shown in Figure 1. The two spectra are similar with excitation maxima a t 370, 410, and 440 nm and a single emission peak a t 510 nm. The spectrum of the blank is mainly due to the contribution of borate ions. For the purposes of routine assay, the solutions were excited a t 410 nm since this wavelength offers the best aiscrimination between blank and test solutions. Effect of pH on Fluorophore Development. 1,8D (1 pg/ml) was taken through Procedure 1. Solutions of borate below pH 9.2 were obtained by mixing a 2% solution of boric acid with a 2% solution of sodium tetraborate to give the required pH. Borate solutions above pH 9.2 were obtained by the addition of 3N potassium hydroxide. The results are shown in Figure 2 . The fluorescence intensities showed a fairly wide pH optimum between pH 6.6 and 9.6. Below pH 5.4 the fluorophore was unstable and rapidly decayed. For routine purposes, a 2% aqueous solution of borate was used since most of the substances assayed were more soluble in alkaline solution. Effect of Borate Concentration on the Development of Fluorescence. The effect of borate concentration on 10 pg of 1,8D taken through Procedure 1 is shown in Figure 3. The effect of borate concentration was marked over the range 0-2mM when a plateau was reached. For practical
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
I - -
- -
0.1
I 0
1
2
. 10
. 30
.
.
50
Conc.Borate (mM1
-
- - -
-1-
- -
-
-
0.9 v 2
0.5
- - -
6
i 10
C 0 n c . D it hioni t e ( m M 1
. Figure 4. Effect of dithionite concentration on the development
of fluorescence of
1 pg/ml 1,BD worked through
Procedure 1
Figure 3. Effect of borate concentration on the development of fluorescence of 1 r g / m l 1,BD worked through Procedure 1
purposes, it was convenient to make up a 2% w/v solution (52.5mM) of sodium tetraborate since this solution was near the saturation point a t room temperature. Effect of Dithionite Concentration on the Development of Fluorescence. The effect of dithionite concentration was investigated over the range 0.01 to 9.5mM by taking 1,8D (10 r g ) through Procedure 1. The results are shown in Figure 4. Below 0.07mA4, the fluorophore was unstable and rapidly deteriorated a t room temperature. Above 0.95mM dithionite, there was some loss in fluorescence intensity presumably due to the presence of a pale yellow color giving rise to an inner filter effect. For routine purposes, it was decided to use 1.65% (0.71mM dithionite. The Stability of the Fluorophore. When the optimal amounts of reagents already described were used and 10 fig of rhein or 1,8D was taken through Procedure 1, the fluorophore was stable (no loss of fluorescence) for a t least 150 min a t room temperature (22 "C). Time Taken for Development of Fluorescence. Some substances ( e . g . , 1,8D and rhein) fluoresced in the presence of cold reducing reagent, whereas others ( e . g . , sennosides A and B) needed a short period of heating in a boiling water bath for fluorescence to develop fully. There was no suggestion from the analyzed samples of bile, urine, or plasma from animals which had been given 1,8D, rhein, rheinanthrone, or sennosides A or B that conjugation or other metabolic transformations caused any change in the time taken for fluorescence to develop. The time curve for the development of fluorescence of samples collected from anaesthetized pigs uia cannulae in the gall and urinary bladders 3 hr after the intravenous administration (3 mg/kg) of either sennoside A or 1,8D is shown in Figure 5 . There is no evidence that the assay measures substances other than the unaltered (parent) compounds in biological tissues. Calibration Curves in the Presence of Biological Material. Pure samples of a range of hydroxylated anthraqui-
.
. 0
12
6
18
T i m e (min.1
Figure 5. Effect of heating in a boiling water bath on fluores-
cence development
(e---.)
0.3 pg/rnl pure sennoside A w o r k e d through Procedure 1. (0--0) 100 @I pig urine containing sennoside A rnetabolite(s) worked through Procedure 3, ( A - - A ) 0.25 r g / m l pure 1,8D worked t h r o u g h Procedure 1 , (A--A) 10 @I pig bile containing 1,8D rnetabolite(s) worked through Procedure 3
nones were taken through Procedure 1 to give a standard curve for each substance. After subtraction of the reagent blank which was obtained by omitting the drug, the calibration graphs were rectilinear and passed through the origin. Limits of sensitivity (fluorescence intensity greater than twice background) were 2, 3, 3, 5, and 11 ng/ml for rheinanthrone, sennoside A and B, 1,8D, and rhein, respectively. The graphs were rectilinear up to at least 20
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 1 1 , SEPTEMBER 1973
1913
~~~
~~
~
~~
Table I. Relative Fluorescence Intensities of Various Hydroxylated Anthracene Derivatives Assayed Separately and as Mixtures by Procedure I Borate
Heated
1. Rhein (0.66 pg/ml)
Substance(s) assayed
0.019
3.80
2. Rheinanthrone (0.035 pg/ml) 3. Sennoside A (0.66pg/ml) 1f 2 1f 3
1.48 0.036 1.53 0.057
1.50 18.3
24-3
1.54 1.54
l f 2 + 3
5.4 21.65 19.35
23.5
Table I I. Relative Fluorescence Intensities of Various Hydroxylated Anthracene Derivatives at a Concentration of 1 pg/ml Assayed by Procedure 1 Borate Compound
Sennoside A Sennoside B Sennidin A Sennidin B Cascaroside A 4- B Barbaloin Aloe emodin Rhein Rhein-8-glucoside Rheinanthrone 1-Hydroxyanthraquinone 1,8-Dihydroxyanthraquinone
only
0.004 0.008 0.042 0.100 0.059
2.3
Cold reducing reagent
0.206 0.52 0.282 0.525 0.35 3.35 12.7
Heated with reducing reagent
27.6 25.8 25.4 24.0 0.99
5.54
4.34 12.7 5.70
0.029 43.8
0.282 43.2
5.16 43.8
0.005
4.7
4.7
0.001
17.4
17.4
0.008 0.022
CONCLUSIONS
pg/ml. When the drug standard curves were repeated in the presence of 20 pl bile, 100 pl of urine, or 100 11 plasma, after subtraction of the appropriate tissue blank there was no significant difference from the control. The Accuracy and Precision of the Assay. The assay of ten samples from a homogeneous solution containing sennoside A gave a relative standard deviation of 13.35%. An independent assay of the same solution of sennoside A by the accepted method ( 2 ) gave a figure of 1.21 mg/ml, compared with the fluorometric assay figure of 1.19 mg/ ml. The Simultaneous Assay of Rhein, Rheinanthrone, and Sennoside A. To ascertain if drug mixtures would behave anomalously, three components of a mixture were assayed separately and combined as for Procedure 1. The results are summarized in Table I, and each result is the average of two readings. I t was concluded that the combined drug assays gave a true cumulative figure.
1914
The Comparative Fluorescence of Sennosides and Related Anthracene Derivatives. Representative examples of various classes of hydroxylated anthracene derivatives were worked through Procedure 1. The results are tabulated in Table 11, and each result is the average of two readings. Only rheinanthrone fluoresced to its full extent in borate only. Some substances, e.g., 1-hydroxyanthraquinone and rhein, gave maximal fluorescence on addition of the cold reducing reagent whereas the sennosides and rhein8-glucoside required a period of heating in a boiling water bath to reach maximum fluorescence intensity. It was apparent that neither rhein nor rhein-8-glucoside fluoresced to the same extent as rheinanthrone on a mole for mole basis. Presumably under the assay conditions, dithionite was not able to reduce rhein t o rheinanthrone. The Effect of Organic Compounds and Inorganic Ions on the Assay. A large number of colorless organic compounds including sugars, acids, and lipids commonly found in blood, bile, and urine, failed to interfere with the fluorescence assay. Nevertheless, fluorescence was reduced by an inner filter effect and was related to the color of bile and urine. For the same reason, clear plasma samples were preferred to those which were hemolyzed. There was no interference from chloride, bromide, bicarbonate, sulfate, or phosphate anions or sodium, potassium, ammonium, aluminum, calcium, zinc, or magnesium cations a t lOmM concentration. In the presence of 1mM stannous and ferrous ions, there was interference due to the formation of a precipitate.
Hydroxylated anthracene derivatives can be assayed by this spectrofluorometric method, with an approximate 100-fold increase in sensitivity over conventional spectrophotometric procedures. The method is suitable for the detection of low levels of hydroxylated anthracene derivatives in biological tissues and is simpler than the involved extraction procedures which are required for the spectrophotometric methods.
ACKNOWLEDGMENT
C. Young is thanked for supplying samples of cascaroside A and B, barbaloin, aloe emodin, and rhein-8-glucoside and for discussions on the analytical procedure. I thank also I. R. MacFarlane for supplying biological samples for fluorometric analysis and H. E. Dobbs for his help and encouragement. Received for review January 29, 1973. Accepted April 5 , 1973.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
