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Fluorimetric assay of ergotamine
Am2lytictr Clrinrica Acfo, 69 ( 1974) I l-17 CT:.Elscvicr Scientific Publishing Company.
FLUORIMETRIC
W. D. HOOPER. Dcprrrrnenr
ASSAY
OF
J. M. SUTHERLAND,
oj’ Mecliche.
UtIiccrsity
Amsterdam
11
- Printed in The Netherlands
ERGO-I-AMINE
M. J. EADIE
and J. H. TYRER
of Qlr~~v~.slurrtl. Ra~~tl Brishtrrw
Hospital.
Herston.
4029.
Brishrw
( Alrstrdici)
(Rcccived
14th May 1973)
Some of the potent pharmacological properties of ergot were recognized at least 2,600 years ago, and the use of ergot in obstetrics dates from mediaeval times’. In modern times the ergot alkaloids were the first adrenergic blocking agents discovered, and most aspects of their general pharmacology were elucidated by the classical studies of Dale2. According to Wolff3, the use of the vasoconstrictor property of ergot in the treatment of migraine dates from the early 1880’s. Ergotamine (Fig. 1) was the first pharmacologically active ergot alkaloid obtained in a pure crystalline form 4. It is widely used in treating migraine and migrainous neuralgia.
Fig. I. Structure
of crgotaminc.
The early calorimetric’ and spectrophotometric” assay procedures were capable of measuring only total alkaloids in the ergot. but more recently the incorporation of some type of chromatographic separation step has enabled the selective assay of specific alkaloids in either ergot extracts or pharmaceutical, preparations7.8. However, the sensitivity and selectivity of all of the methods currently available are limited by the use of spectrophotometry (u.v. or visible) for the final measurement. This paper describes studies on the fluorescence of ergotamine, and the utilization of this property in a new highly sensitive assay procedure with a potentially increased selectivity. EXPERIMENTAL
Apparatus Fluorescence measurements were made with an Aminco-Bowman spectrophotofluorimeter (Model 4-8202) with the standard slit arrangement No. 3 and reflecting mirrors. Measurements of pH were taken on an Orion Model 801 digital pH meter with a Philips combination electrode (Type CA 14/02).
12
W. D. HOOPER.
J. M. SUTHERLAND..M.
J. EADIE.
J. H. TYRER
A set of Pyrex test tubes and volumetric flasks were used exclusively for these studies, and were specially washed by soaking for 24 h in nitric acid (30’,!&, A-R.), followed by 24 h in a solution of cetyltrimethylammonium bromide (1 mg I- ’ of distilled water). with copious rinsing in distilled water after each soaking. Vacuum evaporation was performed on a Buchi Rotovapor R with a water pump.
Ergotamine tartrate (Sandoz Australia Pty. Ltd.) was used without further purification. Standard solutions from 0.001 to 5.0 jig ml -’ were prepared in 0.01 M hydrochloric acid in deionized. fluorescence-free water. by dilutions from a master standard prepared by dissolving 5.0 mp of ergotamine tartrate in 1.O 1 of hydrochloric acid (0.01 M). 2-Aminopyridine (British Drug Houses, laboratory reagent) was purified by repeated sublimation, and a IO-’ M solution in sulfuric acid (0.05 M) was prepared as a fluorescence standard”. All organic solvents were purified by fractional distillation of laboratory reagents. and their fluorescence spectra were checked to ensure absence of contamination. Glycine-sodium hydroxide buffer, pH 10.0 and I .O M with respect to glycine. was prepared. and the pH was adjusted with the use of the pH meter.
The effect of pH on the partition of crgotaminc between water and benzene was studied to ascertain the optimal pH for extraction. Several immiscible organic solvents (benzene. ether. chloroform, heptane) were tested in order to determine the least polar solvent which quantitatively removed crgotaminc from an aqueous solution ;it the optimal pH.
The effect of pH between 1 and 14 on the fiuorcsccnce intensity of aqueous solutionsofergotaminc( 1.0 jcgml-’ ) was studied. The maximal fluorescence intensity of ergotamine in water (pH 10.8) was compared with that obtained in a series of concentration (1 jig ml- ‘). organic solvents, all with the same ergotamine The stability of the fluorescent chrornophorc of ergotamine in hydrochloric acid (0.01 M) and ethanol was studied by storing solutions (cu. 2 jcg ml- ‘) in stoppered Pyrex vessels on an open laboratory bench at 20-25” for 6 months, reading the fluorescence intensity at regular intervals.
Pipette 5.0 ml of an aqueous solution containing ergotamine (standard in 0.01 M hydrochloric acid, or unknown) into a 30-m] test tube with ground-glass stopper. and add 1.0 ml of glycinc buffer and 10.0 ml of benzene. After shaking by hand for 3 min. centrifuge the tubes at 1000 g for 2 min. and pipette 8.0 ml of the benzene layer into a clean tube. Evaporate the benzene to dryness at reduced pressure, and flush the tube with a stream of dry nitrogen for l-2 min to ensure complete removal of benzene. Add 5.0 ml of ethanol, and shake the tube for 3 min.
FLUORIMETRIC
ASSAY
OF ERGOTAMINE
13
Place an aliquot of the ethanol solution in a IO-mm silica cuvette and read the fluorescence intensity with an excitation wavelength of 318 nm and an etnission wavelength of 402 nm. As crgotamine is subject to photodccomposition by ultraviolet light, the fluorescence intensity is best read at constant tune (e.g. 10 s) after the initial exposure to the exciting light. The sensitivity of the instrument was always adjusted so that a stock standard solution of 2-aminopyridine ( IO- ’ M in 0.05 M sulphuric acid) gave a predetermined fluorescence intensity. Concentrations of unknowns were determined by reference to a standard curve. The standard curve was constructed by assaying a series of 10 aqueous standards of ergotamine tartratc at concentrations between 0.1 and 5.0 pg ml- ‘, usually in triplicate. on three separate occasions. The collected data from all determinations were subjcctcd to statistical analysis. The limit of detection of the assay was established by analysing standards between 0.001 and 0.005 116 ml-‘, and comparing these with assays of deionized water blanks.
Two commercial preparations containing ergotaminc tartratc were extracted following thc’method of the United States Pharmacopeia (17th Ed., p. 236), and aliquots of the extracts were assayed following the above procedure. Replicate assays were performed twice on batches of 10 tablets, for each preparation. As one of the preparations contained caffeine citrate (100 mg/tablet), added caffeine aqueous ergotamine tartrate samples (2 /LB ml - ‘) with and without in order to detect any interference in the fluorcscencc citrate(2 mg ml- ‘) wereassayed intensity of the extracts. RESULTS
AND
DISCUSSION
Benzene was the Idast polar of the solvents tested which gave virtually quantitative extraction of ergotamine from aqueous solutions. As drug metabolites are usually more polar than the parent substancelO. and as it was hoped to apply this assay to biological fluids, benzene appeared to be the solvent of choice. offering the best compromise between completeness and selectivity of extraction. The effect of pH on the extraction of ergotamine from aqueous solutions into benzene is shown in Fig. 2. Virtually quantitative extraction was obtained in the pH range 8.5-1 1.0; the maximal percentage extraction was 98X’;, at pH 10. Since many of the solutions which one might assay would be acidic extracts (e.g. of pharmaceutical preparations). a strong buffer of appropriate pH was chosen for the pH adjustment. Adjustment by other means (e.g. titration with sodium hydroxide) is essential if solutions in acids of normality above 0.01 are to be assayed. The effect of pH on the emission spectrum of ergotamine is shown in Fig. 3. The finding of maximal fluorescence inten,sity at pH 10.8 is interesting in view of the published method for assaying lysergic acid diethylamide (containing a very similar chromophore) fIuorimetrically in acidic solution’ ‘. No LSD was available to ascertain whether it has maximal fluorescence in acid or alkali. It is also noteworthy that Vining and Taber l2 have stated that the ergot alkaloids do not fluoresce in alkaline solution. presumably because they examined only solutions of high pH
14
W. D. HOOPER.
J. M. SUTHERLAND,
M. J. EADIE.
J. H. TYRER
100
80
20
a Fig. 2. Ek~t 0r crgotaminc
of pH on extraction of ergotaminc from aqueous volume of aclucous ti3rtrutc 1.0 jig ml - ‘. Rchtivc
Fig. 3. Effect or pH on the emission of crgotuminc turlmtc 1.0 ~cg ml- ‘.
spectrum
ol’crgotaminc.
solution into bcnzcnc. phase: bcnzcnc. 3 : 5. i,,
Conccntrcltion
325 nm. i.,,,, 435 nm. Conccntrotion
(although no actual data were stated). It is probable that the most intensely fluorescent species is an anion; the pK,, for protonation of the neutral molecule’3 is 6.25 which renders it unlikely that the neutral molecule causes the peak in Fig. 3, and a deprotonation in basic solution would not be surprising. That such an ionization may occur is supported by the observation that some of the ergotamine is not extracted by benzene at pH values above 11 (Fig. 2). The excitation and emission spectra of ergotamine in water, at pH 2.1 and pH 10.8, and in ethanol are shown in Fig. 4. The original intention of measuring fluorescence intensity in water at pH 10.8 was discarded in favour of the simpler course of transferring the extracted ergotamine to alcoholic solution, with a resultant slight gain in sensitivity. However, it is important to remove all tracts of benzene completely before addition of ethanol. as further tests confirmed the capacity of hypsochromic shift” in benzene to act as a quenching agent l4 . The anticipated solution (A,, 318 nm, moving from aqueous (A,, 325 nm. i.,,, 435 nm) to ethanolic &,,, 402 nm) was observed. In fact, the fluorescence was examined in a total of 17 organic solvents and 4 mixtures of these solvents, but while the intensity in other short-chain alcohols was comparable to that in ethanol, only in propane-1,2-diol was any further significant enhancement of fluorescence obtained; this solvent was
FLUORIMETRIC
ASSAY
OF ERGO-I-AMINE
WAVELENGTH (nm)
Fig. 4. Excitation spectra (left) of crgotamine in: ( 1) water at pH 2.1 (i-.,,, 435 nm); (2) water at pH 10.8 (i.,,,, 402 nm). Emission spectra (right) of crgotaminc in: (1) water a~ pH 2.1 (j.,,, 422 nm: (3) ethanol (&, 325 nm); (2) water at 10.8 (i,, 318 nm); (3) ethanol (A,, 318 nm).
considered too oily for use in the assay. A calibration curve was obtained by processing aqueous ergotamine standards. Statistical analysis of the data showed that the points were best fitted by a straight line having the equation R = 5.201 C+O.3
13 (+=0.998)
where R is the relative fluorescence intensity, C the concentration of ergotamine tartrate, and I*~ the coefficient of determination. That such a good linear relationship was-obtained from data obtained on three separate days was taken as evidence supporting the view that daily working standardsare unnecessary. It is, ofcourse. prudent to check the calibration periodically, particularly when new batches of reagents are used. The ability to work without daily standards adds greatly to the convenience of the procedure, but is only possible as a result of the use of 2-aminopyridine as a fluorescence standard”, which enables compensation for day-to-day variations in the performance of the spectrophotofluorimeier. Reproducibility of the method was established with replicate assays of a 2.5-,ug ml- ’ standard. Eight such analyses furnished a mean relative fluorescence intensity of 13.82 units, with a standard deviation of 0.27 units. The limit of detection of the method was assessed as 0.002 r(~g ml-; a concentration of 0.001 ~16 ml --’ could not be distinguished from the water blank when a 5.0-ml sample wasassayed. and showed no peak at Acx 3 18 nm and &,,, 402 nm. Studies on the stability of the fluorophore in both 0.01 M hydrochloric acid and ethanol revealed no detectable loss of fluorescence intensity after six weeks
16
W. D. HOOPER.
J. M. SUTHERLAND.
M. J. EADIE.
J. H. TYRER
while less than lO’x, was lost after six months. These data are su,pported by previous work’ ‘. “. The results of an application of the assay arc shown in Table I which summarizes the data obtained from analyses of tablets. Both preparations contained an average of go’;:, of the labeled content (1.0 mg). Because one of the preparations contained caffeine citrate (100 mg per tablet), the possibility of interference was investigated, but none was demonstrated. TABLE
I
DETERMINATION _________ . .._ -..._
-._-_-.-_-
-
Product A Product 13 --_-.-.-.-_..-----” Mean
whc
OF ERGOTAMINE TAR-l-RATE IN PHARMACEUTICAL PREPARATIONS -_-..__._.. - _.... _... -.-.....___.- ..__._ -- _... -_ .-..---.--.---...-...--.-
r~lccrrl ‘I(, Ih~llctl
Iligl~
1’01111’111”
( “,)
_--
_-. .._.._....._.. -
x9.0 90.0
--_---
Lou I
cwrrolr
)
(‘I;,
Srtrr1tltrrtl CJ.1’ Itrhelletl
~‘(~/IfC’/? f )
.._-. . - -. ._ _..__.____-__..._ ..__....-...-__.- --93.7 03. I - _._.-.. _......_ _-.--..-.-_---.---
85.7 X6.5
__-. -.-
tkvioriou
-..-. -..-___-.-----
3.0 2.4
---.----
or 8 tlctcrminations.
The assay possesses potentially higher selectivity than previous calorimetric or spectrophotometric techniques. although related ergot alkaloids (e.0. methyser@de, i.,, 325 nm. i.,,,, 410 nm in ethanol) may still interfere if present. In this case a chromatographic separation step would become necessary’ ‘-“‘. The range of concentrations used for construction of the standard curve was chosen to span the values encountered in the present work. However, it was confirmed that the linear relationship holds down to the limit of detection of the assay (while higher concentrations exceed the capability of the spectrophotolluorimeter). SUMMARY
Studies on the fluorescence properties of ergotamine in water at various pH vnlues, and in several organic solvents are described. An assay procedure for ergotaminc. based on its intense fluorescence in ethanol, is presented. ‘Extraction of ergotamine into benzene from basic aqueous solution is followed by transfer of the extract to ethanol for fluorescence determination. The plot of fluorescence intensity us. concentration is linear up to 5 /lg ml-‘. and the assa~y has a limit of detection of 0.002 jcg ml- ‘, Reproducibility data at the 2.5-/lg ml- * level are given. .-
Une Ctude est effectuke sur les propriCtCs de fluorescence de l’ergotamine, dans l’eau, g divers pH, et dans plusieurs solvants organiques. Une mCthode est proposte, avec extraction de l’ergotamine dans le benzkne. 8.partir de solutions aqueuses basiques, et transfert de I’extrat dans I’Cthanol pour le dosage par fluorescence. La courbe de I’intensitC de la fluorescence en fonction de la concentration est IinCaire jusqu’g 5 pg ml- ’ ; la limite de dCtecticm est de 0.002 ~(8 ml- ‘. On examine la reproductibiliti: pour 2.5 116 ml-‘.
FLUORIMETRIC
ASSAY
17
OF ERGOTAMINE
ZUSAMMENFASSUNG
Untersuchungen der Fiuoreszenzeigenschaftten von Ergotamin in Wasser bei verschiedenen pH-Werten und in verschiedenen organ&hen L6sungsmitteln werden beschrieben. Ein auf der intensiven Fluoreszenz in ,&thanol beruhendes Analysenverfahren fiir Ergotamin wird vorgeschlagen. Das Ergotamin wird aus basischer wtissriger Liisung mit Bcnzol extrahicrt. anschlicssend wird der Extrakt fiir die Fluoreszenzmessung in b;thunol iiberfiihrt. Die Auftragung der Fluoreszenzintensittit gegen die Konzentration ist bis 5 118 ml -’ linear, und das Verfahren hat eine Nachweisgrenze von 0.002 /cg ml- ‘. Werte fiir die Reproduzierbarkeit bei 2.5 /lg ml-’ werden vorgelcgt. REFERENCES I L. S. Goodman and A. Gilman (Editors). 7‘/1r, f’/rtr/,,rrtrc.o/oNic,ccI Btrsis o/’ Tlwropctrrics. millnn Ltd.. London and Toronto. 4th Ed.. 1970. p. 897. 2 H. H. Dale, J. Pl~_v.sio/. (Lad.). 34 ( 1906) 163. 3 H. G. WollT. Ncoc/ccc*/rc trrul Ollrcar H~wtl hia Oxford Univ. Press. New York. 1963. 4 A. Stall, t’oh. Ntrrtrr:fbrsc/r. Gcs. B~tx*l. IO1 ( 1920) 190. 5 H. W. van Urk. I>hrrrrn. Wwkhl.. 66 ( 1929) 473. 6 A. Harmsma. /%or~n. W~d;/~/.. 65 (I 92X) I I 14. 7 V: Prochazka. F. Kuvku. M. Prucha and J. Pitru. Ccsk. Ftr~+r!~..I4 ( 1965) 154: Cl~or~. 108274 u.
9 IO II I2 13 I4 I5 I6 I7 18 I9
Collier-Mac-
Ahrr..
66 ( 1967)
Ass. 0%~. Apr. Chcm., Washington, DC.. 9th Ed.. 1960, p. 473. R. Rusnkowicz and A. C. Tcsta. J. I%_r.s. Clrot~.. 72 ( 1968) 2680. B. B. Brodic. S. Udcnfricnd and 1. E. Bacr. J. Biol. Clrcw.. IBX (1947) 299. J. Axclrod. R. 0. Brady. B. Witkop and E. V. Evarts. /lrr/r. N.Y. Ac*ot/. Sci.. 66 (1957) 435. L. C. Vining and W. A. Tahcr. Ctrrt. ./. rl/ic~ro/~io/.. 5 (1959) 441. I-i. V. Maulding and M. A. Zoglio. J. /‘/I(I~‘III. Sci.. 59 (1970) 700. R. T. Williams and J. W. Bridges. J. C/in. E’trrhol.. I7 ( 1964) 37 I. J. Trzcbinski and T. Wiecko, .4c/lr PO/. /‘/~trr.ol .. 24 ( 1967) 579: C’l~crl~.Ahsfr.. 68 ( 1968) 62652 b. E. E. Swanson. C. E. Powell. A. N. Stcvcns and E. H. Stuart. J. nn,o.. P/~trr,t~. Ass.. 21 (1932) 229. L.-N. Li and C.-C. Fang. Acrrc Phrrrrtt. Sirr.. I I ( 1964) 1x9: /Ircctl. /Ih.sfr*., I2 (1965) 2441. W. N. French and A. Wchrli. J. Plrrrrra Sci.. 54 ( 1965) I51 5. J. L. McLaughlin. J. E. Goyan ;u~d A. G. Pa~tl. J. I%ctr.r,r. Sr*i.. 53 (1964) 306.
FLUORIMETRIC
W. D. HOOPER. Dcprrrrnenr
ASSAY
OF
J. M. SUTHERLAND,
oj’ Mecliche.
UtIiccrsity
Amsterdam
11
- Printed in The Netherlands
ERGO-I-AMINE
M. J. EADIE
and J. H. TYRER
of Qlr~~v~.slurrtl. Ra~~tl Brishtrrw
Hospital.
Herston.
4029.
Brishrw
( Alrstrdici)
(Rcccived
14th May 1973)
Some of the potent pharmacological properties of ergot were recognized at least 2,600 years ago, and the use of ergot in obstetrics dates from mediaeval times’. In modern times the ergot alkaloids were the first adrenergic blocking agents discovered, and most aspects of their general pharmacology were elucidated by the classical studies of Dale2. According to Wolff3, the use of the vasoconstrictor property of ergot in the treatment of migraine dates from the early 1880’s. Ergotamine (Fig. 1) was the first pharmacologically active ergot alkaloid obtained in a pure crystalline form 4. It is widely used in treating migraine and migrainous neuralgia.
Fig. I. Structure
of crgotaminc.
The early calorimetric’ and spectrophotometric” assay procedures were capable of measuring only total alkaloids in the ergot. but more recently the incorporation of some type of chromatographic separation step has enabled the selective assay of specific alkaloids in either ergot extracts or pharmaceutical, preparations7.8. However, the sensitivity and selectivity of all of the methods currently available are limited by the use of spectrophotometry (u.v. or visible) for the final measurement. This paper describes studies on the fluorescence of ergotamine, and the utilization of this property in a new highly sensitive assay procedure with a potentially increased selectivity. EXPERIMENTAL
Apparatus Fluorescence measurements were made with an Aminco-Bowman spectrophotofluorimeter (Model 4-8202) with the standard slit arrangement No. 3 and reflecting mirrors. Measurements of pH were taken on an Orion Model 801 digital pH meter with a Philips combination electrode (Type CA 14/02).
12
W. D. HOOPER.
J. M. SUTHERLAND..M.
J. EADIE.
J. H. TYRER
A set of Pyrex test tubes and volumetric flasks were used exclusively for these studies, and were specially washed by soaking for 24 h in nitric acid (30’,!&, A-R.), followed by 24 h in a solution of cetyltrimethylammonium bromide (1 mg I- ’ of distilled water). with copious rinsing in distilled water after each soaking. Vacuum evaporation was performed on a Buchi Rotovapor R with a water pump.
Ergotamine tartrate (Sandoz Australia Pty. Ltd.) was used without further purification. Standard solutions from 0.001 to 5.0 jig ml -’ were prepared in 0.01 M hydrochloric acid in deionized. fluorescence-free water. by dilutions from a master standard prepared by dissolving 5.0 mp of ergotamine tartrate in 1.O 1 of hydrochloric acid (0.01 M). 2-Aminopyridine (British Drug Houses, laboratory reagent) was purified by repeated sublimation, and a IO-’ M solution in sulfuric acid (0.05 M) was prepared as a fluorescence standard”. All organic solvents were purified by fractional distillation of laboratory reagents. and their fluorescence spectra were checked to ensure absence of contamination. Glycine-sodium hydroxide buffer, pH 10.0 and I .O M with respect to glycine. was prepared. and the pH was adjusted with the use of the pH meter.
The effect of pH on the partition of crgotaminc between water and benzene was studied to ascertain the optimal pH for extraction. Several immiscible organic solvents (benzene. ether. chloroform, heptane) were tested in order to determine the least polar solvent which quantitatively removed crgotaminc from an aqueous solution ;it the optimal pH.
The effect of pH between 1 and 14 on the fiuorcsccnce intensity of aqueous solutionsofergotaminc( 1.0 jcgml-’ ) was studied. The maximal fluorescence intensity of ergotamine in water (pH 10.8) was compared with that obtained in a series of concentration (1 jig ml- ‘). organic solvents, all with the same ergotamine The stability of the fluorescent chrornophorc of ergotamine in hydrochloric acid (0.01 M) and ethanol was studied by storing solutions (cu. 2 jcg ml- ‘) in stoppered Pyrex vessels on an open laboratory bench at 20-25” for 6 months, reading the fluorescence intensity at regular intervals.
Pipette 5.0 ml of an aqueous solution containing ergotamine (standard in 0.01 M hydrochloric acid, or unknown) into a 30-m] test tube with ground-glass stopper. and add 1.0 ml of glycinc buffer and 10.0 ml of benzene. After shaking by hand for 3 min. centrifuge the tubes at 1000 g for 2 min. and pipette 8.0 ml of the benzene layer into a clean tube. Evaporate the benzene to dryness at reduced pressure, and flush the tube with a stream of dry nitrogen for l-2 min to ensure complete removal of benzene. Add 5.0 ml of ethanol, and shake the tube for 3 min.
FLUORIMETRIC
ASSAY
OF ERGOTAMINE
13
Place an aliquot of the ethanol solution in a IO-mm silica cuvette and read the fluorescence intensity with an excitation wavelength of 318 nm and an etnission wavelength of 402 nm. As crgotamine is subject to photodccomposition by ultraviolet light, the fluorescence intensity is best read at constant tune (e.g. 10 s) after the initial exposure to the exciting light. The sensitivity of the instrument was always adjusted so that a stock standard solution of 2-aminopyridine ( IO- ’ M in 0.05 M sulphuric acid) gave a predetermined fluorescence intensity. Concentrations of unknowns were determined by reference to a standard curve. The standard curve was constructed by assaying a series of 10 aqueous standards of ergotamine tartratc at concentrations between 0.1 and 5.0 pg ml- ‘, usually in triplicate. on three separate occasions. The collected data from all determinations were subjcctcd to statistical analysis. The limit of detection of the assay was established by analysing standards between 0.001 and 0.005 116 ml-‘, and comparing these with assays of deionized water blanks.
Two commercial preparations containing ergotaminc tartratc were extracted following thc’method of the United States Pharmacopeia (17th Ed., p. 236), and aliquots of the extracts were assayed following the above procedure. Replicate assays were performed twice on batches of 10 tablets, for each preparation. As one of the preparations contained caffeine citrate (100 mg/tablet), added caffeine aqueous ergotamine tartrate samples (2 /LB ml - ‘) with and without in order to detect any interference in the fluorcscencc citrate(2 mg ml- ‘) wereassayed intensity of the extracts. RESULTS
AND
DISCUSSION
Benzene was the Idast polar of the solvents tested which gave virtually quantitative extraction of ergotamine from aqueous solutions. As drug metabolites are usually more polar than the parent substancelO. and as it was hoped to apply this assay to biological fluids, benzene appeared to be the solvent of choice. offering the best compromise between completeness and selectivity of extraction. The effect of pH on the extraction of ergotamine from aqueous solutions into benzene is shown in Fig. 2. Virtually quantitative extraction was obtained in the pH range 8.5-1 1.0; the maximal percentage extraction was 98X’;, at pH 10. Since many of the solutions which one might assay would be acidic extracts (e.g. of pharmaceutical preparations). a strong buffer of appropriate pH was chosen for the pH adjustment. Adjustment by other means (e.g. titration with sodium hydroxide) is essential if solutions in acids of normality above 0.01 are to be assayed. The effect of pH on the emission spectrum of ergotamine is shown in Fig. 3. The finding of maximal fluorescence inten,sity at pH 10.8 is interesting in view of the published method for assaying lysergic acid diethylamide (containing a very similar chromophore) fIuorimetrically in acidic solution’ ‘. No LSD was available to ascertain whether it has maximal fluorescence in acid or alkali. It is also noteworthy that Vining and Taber l2 have stated that the ergot alkaloids do not fluoresce in alkaline solution. presumably because they examined only solutions of high pH
14
W. D. HOOPER.
J. M. SUTHERLAND,
M. J. EADIE.
J. H. TYRER
100
80
20
a Fig. 2. Ek~t 0r crgotaminc
of pH on extraction of ergotaminc from aqueous volume of aclucous ti3rtrutc 1.0 jig ml - ‘. Rchtivc
Fig. 3. Effect or pH on the emission of crgotuminc turlmtc 1.0 ~cg ml- ‘.
spectrum
ol’crgotaminc.
solution into bcnzcnc. phase: bcnzcnc. 3 : 5. i,,
Conccntrcltion
325 nm. i.,,,, 435 nm. Conccntrotion
(although no actual data were stated). It is probable that the most intensely fluorescent species is an anion; the pK,, for protonation of the neutral molecule’3 is 6.25 which renders it unlikely that the neutral molecule causes the peak in Fig. 3, and a deprotonation in basic solution would not be surprising. That such an ionization may occur is supported by the observation that some of the ergotamine is not extracted by benzene at pH values above 11 (Fig. 2). The excitation and emission spectra of ergotamine in water, at pH 2.1 and pH 10.8, and in ethanol are shown in Fig. 4. The original intention of measuring fluorescence intensity in water at pH 10.8 was discarded in favour of the simpler course of transferring the extracted ergotamine to alcoholic solution, with a resultant slight gain in sensitivity. However, it is important to remove all tracts of benzene completely before addition of ethanol. as further tests confirmed the capacity of hypsochromic shift” in benzene to act as a quenching agent l4 . The anticipated solution (A,, 318 nm, moving from aqueous (A,, 325 nm. i.,,, 435 nm) to ethanolic &,,, 402 nm) was observed. In fact, the fluorescence was examined in a total of 17 organic solvents and 4 mixtures of these solvents, but while the intensity in other short-chain alcohols was comparable to that in ethanol, only in propane-1,2-diol was any further significant enhancement of fluorescence obtained; this solvent was
FLUORIMETRIC
ASSAY
OF ERGO-I-AMINE
WAVELENGTH (nm)
Fig. 4. Excitation spectra (left) of crgotamine in: ( 1) water at pH 2.1 (i-.,,, 435 nm); (2) water at pH 10.8 (i.,,,, 402 nm). Emission spectra (right) of crgotaminc in: (1) water a~ pH 2.1 (j.,,, 422 nm: (3) ethanol (&, 325 nm); (2) water at 10.8 (i,, 318 nm); (3) ethanol (A,, 318 nm).
considered too oily for use in the assay. A calibration curve was obtained by processing aqueous ergotamine standards. Statistical analysis of the data showed that the points were best fitted by a straight line having the equation R = 5.201 C+O.3
13 (+=0.998)
where R is the relative fluorescence intensity, C the concentration of ergotamine tartrate, and I*~ the coefficient of determination. That such a good linear relationship was-obtained from data obtained on three separate days was taken as evidence supporting the view that daily working standardsare unnecessary. It is, ofcourse. prudent to check the calibration periodically, particularly when new batches of reagents are used. The ability to work without daily standards adds greatly to the convenience of the procedure, but is only possible as a result of the use of 2-aminopyridine as a fluorescence standard”, which enables compensation for day-to-day variations in the performance of the spectrophotofluorimeier. Reproducibility of the method was established with replicate assays of a 2.5-,ug ml- ’ standard. Eight such analyses furnished a mean relative fluorescence intensity of 13.82 units, with a standard deviation of 0.27 units. The limit of detection of the method was assessed as 0.002 r(~g ml-; a concentration of 0.001 ~16 ml --’ could not be distinguished from the water blank when a 5.0-ml sample wasassayed. and showed no peak at Acx 3 18 nm and &,,, 402 nm. Studies on the stability of the fluorophore in both 0.01 M hydrochloric acid and ethanol revealed no detectable loss of fluorescence intensity after six weeks
16
W. D. HOOPER.
J. M. SUTHERLAND.
M. J. EADIE.
J. H. TYRER
while less than lO’x, was lost after six months. These data are su,pported by previous work’ ‘. “. The results of an application of the assay arc shown in Table I which summarizes the data obtained from analyses of tablets. Both preparations contained an average of go’;:, of the labeled content (1.0 mg). Because one of the preparations contained caffeine citrate (100 mg per tablet), the possibility of interference was investigated, but none was demonstrated. TABLE
I
DETERMINATION _________ . .._ -..._
-._-_-.-_-
-
Product A Product 13 --_-.-.-.-_..-----” Mean
whc
OF ERGOTAMINE TAR-l-RATE IN PHARMACEUTICAL PREPARATIONS -_-..__._.. - _.... _... -.-.....___.- ..__._ -- _... -_ .-..---.--.---...-...--.-
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( “,)
_--
_-. .._.._....._.. -
x9.0 90.0
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Lou I
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)
(‘I;,
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.._-. . - -. ._ _..__.____-__..._ ..__....-...-__.- --93.7 03. I - _._.-.. _......_ _-.--..-.-_---.---
85.7 X6.5
__-. -.-
tkvioriou
-..-. -..-___-.-----
3.0 2.4
---.----
or 8 tlctcrminations.
The assay possesses potentially higher selectivity than previous calorimetric or spectrophotometric techniques. although related ergot alkaloids (e.0. methyser@de, i.,, 325 nm. i.,,,, 410 nm in ethanol) may still interfere if present. In this case a chromatographic separation step would become necessary’ ‘-“‘. The range of concentrations used for construction of the standard curve was chosen to span the values encountered in the present work. However, it was confirmed that the linear relationship holds down to the limit of detection of the assay (while higher concentrations exceed the capability of the spectrophotolluorimeter). SUMMARY
Studies on the fluorescence properties of ergotamine in water at various pH vnlues, and in several organic solvents are described. An assay procedure for ergotaminc. based on its intense fluorescence in ethanol, is presented. ‘Extraction of ergotamine into benzene from basic aqueous solution is followed by transfer of the extract to ethanol for fluorescence determination. The plot of fluorescence intensity us. concentration is linear up to 5 /lg ml-‘. and the assa~y has a limit of detection of 0.002 jcg ml- ‘, Reproducibility data at the 2.5-/lg ml- * level are given. .-
Une Ctude est effectuke sur les propriCtCs de fluorescence de l’ergotamine, dans l’eau, g divers pH, et dans plusieurs solvants organiques. Une mCthode est proposte, avec extraction de l’ergotamine dans le benzkne. 8.partir de solutions aqueuses basiques, et transfert de I’extrat dans I’Cthanol pour le dosage par fluorescence. La courbe de I’intensitC de la fluorescence en fonction de la concentration est IinCaire jusqu’g 5 pg ml- ’ ; la limite de dCtecticm est de 0.002 ~(8 ml- ‘. On examine la reproductibiliti: pour 2.5 116 ml-‘.
FLUORIMETRIC
ASSAY
17
OF ERGOTAMINE
ZUSAMMENFASSUNG
Untersuchungen der Fiuoreszenzeigenschaftten von Ergotamin in Wasser bei verschiedenen pH-Werten und in verschiedenen organ&hen L6sungsmitteln werden beschrieben. Ein auf der intensiven Fluoreszenz in ,&thanol beruhendes Analysenverfahren fiir Ergotamin wird vorgeschlagen. Das Ergotamin wird aus basischer wtissriger Liisung mit Bcnzol extrahicrt. anschlicssend wird der Extrakt fiir die Fluoreszenzmessung in b;thunol iiberfiihrt. Die Auftragung der Fluoreszenzintensittit gegen die Konzentration ist bis 5 118 ml -’ linear, und das Verfahren hat eine Nachweisgrenze von 0.002 /cg ml- ‘. Werte fiir die Reproduzierbarkeit bei 2.5 /lg ml-’ werden vorgelcgt. REFERENCES I L. S. Goodman and A. Gilman (Editors). 7‘/1r, f’/rtr/,,rrtrc.o/oNic,ccI Btrsis o/’ Tlwropctrrics. millnn Ltd.. London and Toronto. 4th Ed.. 1970. p. 897. 2 H. H. Dale, J. Pl~_v.sio/. (Lad.). 34 ( 1906) 163. 3 H. G. WollT. Ncoc/ccc*/rc trrul Ollrcar H~wtl hia Oxford Univ. Press. New York. 1963. 4 A. Stall, t’oh. Ntrrtrr:fbrsc/r. Gcs. B~tx*l. IO1 ( 1920) 190. 5 H. W. van Urk. I>hrrrrn. Wwkhl.. 66 ( 1929) 473. 6 A. Harmsma. /%or~n. W~d;/~/.. 65 (I 92X) I I 14. 7 V: Prochazka. F. Kuvku. M. Prucha and J. Pitru. Ccsk. Ftr~+r!~..I4 ( 1965) 154: Cl~or~. 108274 u.
9 IO II I2 13 I4 I5 I6 I7 18 I9
Collier-Mac-
Ahrr..
66 ( 1967)
Ass. 0%~. Apr. Chcm., Washington, DC.. 9th Ed.. 1960, p. 473. R. Rusnkowicz and A. C. Tcsta. J. I%_r.s. Clrot~.. 72 ( 1968) 2680. B. B. Brodic. S. Udcnfricnd and 1. E. Bacr. J. Biol. Clrcw.. IBX (1947) 299. J. Axclrod. R. 0. Brady. B. Witkop and E. V. Evarts. /lrr/r. N.Y. Ac*ot/. Sci.. 66 (1957) 435. L. C. Vining and W. A. Tahcr. Ctrrt. ./. rl/ic~ro/~io/.. 5 (1959) 441. I-i. V. Maulding and M. A. Zoglio. J. /‘/I(I~‘III. Sci.. 59 (1970) 700. R. T. Williams and J. W. Bridges. J. C/in. E’trrhol.. I7 ( 1964) 37 I. J. Trzcbinski and T. Wiecko, .4c/lr PO/. /‘/~trr.ol .. 24 ( 1967) 579: C’l~crl~.Ahsfr.. 68 ( 1968) 62652 b. E. E. Swanson. C. E. Powell. A. N. Stcvcns and E. H. Stuart. J. nn,o.. P/~trr,t~. Ass.. 21 (1932) 229. L.-N. Li and C.-C. Fang. Acrrc Phrrrrtt. Sirr.. I I ( 1964) 1x9: /Ircctl. /Ih.sfr*., I2 (1965) 2441. W. N. French and A. Wchrli. J. Plrrrrra Sci.. 54 ( 1965) I51 5. J. L. McLaughlin. J. E. Goyan ;u~d A. G. Pa~tl. J. I%ctr.r,r. Sr*i.. 53 (1964) 306.