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Spectrophotofluorometric method for the microdetection and estimation of morphine and codeine
MICROCHEMICAL
VOL.
JOURNAL
Spectrophotofluorometric Microdetection
Method and Estimation
Morphine RICHARD NICHOLAS
V, PAGES
215-223
(1961)
for the of
and Codeine
BRANDT, SARAH EHRLICH-ROGOZINSKY * and D. CHERONIS, Department OJ Chemistry, Brooklyn College, Brooklyn, New York
I. Introduction II complete review of the chemical methods for the microdetermination and estimation of morphine and related compounds will be pubThe present work deals lished in a later paper from this laboratory. with the microdetection and estimation of morphine and codeine by means of their fluorescence. Early experiments on the fluorescence of alkaloids were reported He found that morphine fluoresced about 50 years ago by Heller.’ with a weak blue and codeine with a weak gray color. Andant later studied the fluorescence of alkaloids under irradiation from a mercury vapor lamp using a Wood’s filter and the apparatus developed by Bayle and Fabre. 3 He reported4,5 that codeine fluoresced bot’h in the visible and in the ultraviolet, while morphine fluoresced only in the ultraviolet. This was confirmed later by Beguiristans who found that morphine hydrochloride fluoresced at a maximum of 4715 A., while Kosyakova7 report’ed that morphine does not fluoresce. Radley and Grant8 in a work on fluorescence analysis base their statement that morphine and codeine fluoresce on the cited reports. Bowman and collaborators9 who developed the modern spectrofluorometer published data on the fluorescence of an appreciable number of organic compounds. Morphine is reported to fluoresce with arlivation spectra at 270-290 rnp and fluorescent spectra at 365 rnp * On leave from the WeizmannInstitute of Science, Rehovoth, 215
Israel.
216
BRANDT,
EHRLICH-ROGOZINSKY,
AND
CHERONIS
using the precursor of the present Aminco instrument with the II’ 28 photomultiplier tube. In previous work by one of the authors in this laboratory, it was observed that solutions of many alkaloids including morphine and codeine fluoresce in aqueous solutions (made by adding a solution of the alkaloid in acetone to water at pH of 4-6), when irradiated by an ultraviolet hand lamp (long wave type 3660 A., Ultra-Violet Product,s, Inc.). It was determined that morphine and codeine could be detected visually at a lower limit of 40 pg./ml. In the present work, it was found that solutions of morphine in 0.1 N sulfuric acid show a fluorescence peak at 350 rnp and a main activation peak at 285 rnp. A second activation peak was observed at 245 rncc. Varying the pH from 1 to 3 gave no change in emission intensity. The intensity of the emission at pH 7 decreases and at pH 10-12, the fluorescence is negligible. Codeine, the methyl ether of morphine, gives the same fluorescence and activation peaks as morphine with similar intensity in O.lN sulphuric acid. However, at pH’s of 10-12, the intensity of the fluorescence of codeine solutions is unchanged. This difference bctween morphine and codeine at high pH’s qflords a method of distinguishing and determining the two allcaloids in the presence of each other. Quantitative studies showed the optimal range of determination is bet,ween 0.1 and 50 wg./ml. The lower limit of detection is 10 nanograms/ml. At concentrations above 100 Mg./ml. quenching is quite noticeable.
II. Experimental Apparatus and Reagents (A) Aminco-Bowman Spectrophotofluorometer with l-ml. (minimum volume) quart’z cuvets, II’ 21 and 1P 28 photomultiplier tubes, Mosely X-Y recorder, photomultiplier microphotometer, and various light apertures. Quinine in O.lN sulfuric acid was used to check the correct functioning of the instrument.lO No corrections for intensity of emission were used.‘1 (B) Recording Beckman BK Spectrophotometer. Morphine, codeine, and quinine alkaloids (New York Quinine Co.) of U.S.P. grade were used at first,. A purified grade was made by recrystallization and also by vacuum fractional sublimation of the MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
2
MORPHINE
AND
CODEINE
DETECTION
217
Chloroform and acetone, “Spectra” grade (Fisher U.S.P. product. Scientific Co.). Sulfuric acid and sodium hydroxide used were of reagent grade. Water was prepared by triple distillation using an efficient fractionating column. All glassware and cuvets were cleaned by boiling with concentrated nitric acid and rinsed with triple distilled water.
III. Procedure A fresh stock solution of 100 pg./ml. of morphine and of codeine was prepared in (a) 0.1, (b) 0.01, and (c) O.OOlN sulfuric acid. These solutions were diluted to various concentrations. Stock solutions of the alkaloids in sodium hydroxide at concentrations of 0.1, 0.01, and O.OOlN were also prepared. For morphine solution at neutral pH, a few drops of acetone were used as a solvent followed by dilution with water. Codeine was dissolved directly in water. Blank values were determined for all solvents. Values in per cent transmission were read from the microphotometer at the peak activation and peak fluorescence monochromator settings. The product of the meter multiplier and the per cent transmission is the relative per cent transmission. The meter multiplier setting is a measure of resistor values substituted in the microphotometer as indicated on a scale from 1 to 0.001 with the 0.001 setting being the most sensitive.lO Activation and fluorescence spectra are determined in the usual manner.g
IV. Results Experiments with bot,h morphine and codeine dissolved in acet,one gave negligible fluorescence beyond the blank value. In chloroform, a solut,ion of 10 pg./ml. of codeine gave only a slight fluorescence (0.01 relative y0 T, slit, no. 3, 1P 21 photomultiplier tube), while the same concentrat’ion of morphine gave no noticeable fluorescence at t,his instrument set’ting. Figure 1 shows the activation curve for a solution of morphine in O.lN sulfuric acid at a concentration of 10 pg./ml., while Figure 2 shows the fluorescence peak (at 350 1~1~)for the 285 rnp activation at various concentrat,ions. Apparemly two activation peaks are obt>aiued: t,he first at 24.5 rnp and the second and more int,ense at 285 rnp. When activat’ed at 245 mp, morphine also gives a fluorescent peak at
BRANDT,
EHRLICH-ROGO%INSI
ANI)
CHERONIS
* i 200
i
\ m+
400
l
\ 200 mr
\ 400
Fig. 1 (left). Morphine (10 pg./ml.) in O.lN sulfuric acid. Activation spcctra peaks at 245 and 285 rnp at the peak fluorescence of 350 m$. Fig. 2 (center). Morphine (various concentrations): (.A) 100 pg./ml., (U) 50 pg./ml., (C) 20 pg./ml, (D) 10 Pg./ml., in O.lN sulfuric acid. Fluoresccncc spectrum (peak at 350 mp) at the activation peak of 285 mp. Fig. 3 (right). Codeine (10 Ng./ml.) in O.LV sulfuric acid. Activation spectrum (peaks at 245 and 285 mp); fluorescence spectrum (peak at 350 mp).
350 rnp, which is less intense (not illustrated here). I;or codeine, the same activation and fluorescent peaks are observed as shown in Figure 3, but the ratio of the 243 rnp peak to the 285 rnp differs from that of morphine. The same peaks are obtained with both the 1P 21 and II’ 28 photomultiplier tubes, but, the relative per cent transmission is higher with the 11’ 28 t.ube. Figure 4 shows the linear relation of the log concentration versus log relative per cent’ t’ransmission for morphine (blank values have been subtracted) at the same slit widths and different photomultiplier tubes. Figure 5 shows a linear relationship for codeine using different slits and different phot’omultiplier tubes. This proportionality is shown in the range of 0.1 pg./ml. to 50 pg./ml. MICROCHEhlIC.4L
.TOURNAL,
VOL.
V,
ISSUE
2
MOltI’HINE
I
451)
CODEINE
DETECTION
I 2 0 LOG CONC. l’ig. 4. Quantitative estimation of morphine. Log relative y0 transmission log concentration. All values at peak fluorescence of 350 rn$ and using #S. Using the lP28 photomultiplier tube: (X) the 285 rnp activation, (0) 215 mg activation. TJsing the 1P 21 photomultiplier tube: (M) the 285 mr Cvation, (A) the 245 mp activation. -2
219
-I
vs slit the ac-
when using either photomult’iplier tube. Quantities lower than 0.1 pg./ml. show irregularities in their determinations. However, such solutions still exhibit their characteristic activation and fluorescence peaks. Figure 6 shows the spectra of a mixture of .5 Mg./ml. each of morphine and codeine in 0. IN sulfuric acid. This mixt,ure has a relat,ivc per cent transmission similar to that of 10 pg./ml. of morphine or WJdeine. l’igure 6 also shows the spectra of t!he same mixture in O.lN sodium hydroxide. The intensity of the emission is decreased to a value similar to that of 3 pg./ml. of codeine (see Table I). This permits the est,imation of codeine at higher pH and also the estimation of morphine hy the decrease in emission int’ensity.
220
BRANDT,
EHRLICH-ROGOZINSKY,
AND
CHERONIS
Fig. 5. Quantitative estimation of codeine. Log relative y0 transmission vs. log concentration. Using the 1P 28 photomultiplier tube and slit #5: (X) the 285 rnp activation, (0) the 245 rnp activation. Using the 1P 21 photomultiplier t.ube and slit 83: (m) the 285 mp activation, (A) the 245 mp activation.
Typical
Results of a Mixture
Compound Codeine Morphine Misture: codeinemornhine
Weight, w/ml.
TABLE I of Codeine and Morphine Slit 5) Found at activation 245 rnN, 285 mr, PR. rg.
at, Bask pH’s. ( 11’ 28 and
Error at activation, % 245 mp 285 rnp
13.0 11.8
14.1 0.0
12.6 0.0
8.5
:3. I
6.5 5 .!I
6.8
6.0
-1 6
1.5
MICROCHEMICAL
.JOURNAL.
VOL.
V, ISSUE
2
MORPHINE
AND CODEINE
DETECTION
221
Coherent scatter has been deleted from the graphic representation of the spectra, shown in Figures l-3 and G and 7. V. Discussion ‘The peak observed at’ 245 rnp was thought t,o br a peculiarity of the light source of the type indicated by Duggan et al.,” for t)hc activation Wit,h pyridoxine, it was observed that as the spectra of pyridoxine. concentration increased, the second activation peak formed. In t,he case of codeine and morphine, the opposit,e occurred. The 245 rnp peak decreases in the typical manner of concentration quenching (see Fig. 7). Purification of morphine by four crystallizations and also by fractional vacuum sublimation did not change the activation peaks. The previous report8ed13absorpt,ion peak of morphine and codeine is
Fig. 6 (left). Morphine and codeine at pH 1 and pH 12 (5 pg./ml.). Activation spectra (peaks at 245 and 285 rnp) at the peak fluorescence of 350 m+ Abscissa: Wavelength in nw. Ordinate: Arbitrary intensity. Fig. 7 (right). Morphine (various concentrations): (A) 100 pg./ml., (B) 50 pg./ml., and (C) 20 pg./ml. in O.lN sulfuric acid. Activation spectra (peaks at 285 and 245 rnp) at the fluorescence peak of 350 rnp. Abscissa: Wavelength in rnp. Ordinate: Arbitrary intensity.
222
BRANDT,
EHRLICH-ROGOZINSKY,
AND CHERONIS
285 mp. The absorption spectra of morphine at various concentrations (see Fig. 8) in dilute sulfuric acid shows a peak at 285 rnE.rand also intense absorption below 250 rnp. A similar absorption is shown wit,h codeine (not illustrat,ed).
Fig. 8. Morphine (various concentrations): (A) 100 pg./ml., (B) 50 pg./ml., and (C) 25 pg./ml. in O.lN sulfuric acid. Absorption spectra with Beckman 1)U Spectrophotometer. (Peak at 283 mp.) Abscissa: Wavelength in rnp. Ordinate: Transmittance, ‘%.
Williams14 reported that the fluorescence of phenol depends upon the pH. He found maximum fluorescence at pH 1 and minimum at pH 13. Anisole, the methyl ether of phenol, fluoresces equally at pH 1,7,10, and 14 with approximately the same intensity as phenol. The The ability to form quinphenoxide ion is apparently nonfluorescent. oidal resonance forms most likely accounts for this fluorescence. Similarly, in the present study of morphine, the formulas shown indicate the formation of the anion, which would not be able to assume a quinoidal structure due to the oxygen bridge at carbon 4 and thus the MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
2
MORPHINE
AND
CODEINE
‘VO-/(Ii)\ ~pcIr:, _ o,l
DETECTIOS
223
HO-~, (_-l----l ,/
‘2.-N-CH,
t “\ HO-
\
\/j 0
-o-
,
\/ 0
~
,
nonfluorescent species is formed. Codeine most likely does not form this anion and also does not show any emission or spect,ral change. The application of this method to the d&e&on and estimation of ot,her alkaloids is under invcst~igntion.
Summary A spectrofluoromet,ric method for the detection and estimation of morphine and codeine in the presence of each other at the microgram and submicrogram range is described. The detection and estimation of codeine in the presence of morphine is based on t,he fact bhat at’ high pH, the fluorescence of codeine remains unchanged while t)hat, of morphine is quenched.
References 1. Heller, R., 2. Phys. Chem Biol., 2,39T (1916). 2. Andant, A., Corn@. rend., 189,98 (1929). 3. Bayle, E., and R. Fabre, J. pharm. chintz., 1,248 (1025). 4. Andant, A., Bull. sci. pharmacol., 37,28 (1930). 5. Andant, A., Bull. xi. pharmacol, 37,89 (1930). 6. Beguiristan, J. M. B., Chem. Zentr., I, 2800 (1942). 7. Kosyakova, I. A., Zhur. Anal. Khim., 2,27 (1947). 8. Radley, J. A., and J. Grant, Fluorescence Analysis in Ultraaiolet Light, Chapman and Hall Ltd., London, 1954. 9. Bowman, R. L., P. A. Caulfield, and S. Udenfriend, Science, 122, 32 (1955). 10. Instruction and Service Manual No. 768, -4merican Instrument Co., Inr., Silver Spring, Md. 11. White, E. C., M. Ho., and E. Q. Weimer, Anal. Chem., 32,438 (1960). 12. Duggan, I>. E., R. 1,. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Riophys., 68,l (1957). 13. Bradford, L. W., and W. B. Bracket, Report of Systematic Ultra Violet Spectrophotometric Data of Dangerous Drugs, Poisons, and Narcotics, Lahoratoq of Criminalistics, Office of D. A., County of Santa Clara, San Jose, Calif. 14. Williams, R. T., J. Royal Inst. Chenl., 86,611 (1959).
VOL.
JOURNAL
Spectrophotofluorometric Microdetection
Method and Estimation
Morphine RICHARD NICHOLAS
V, PAGES
215-223
(1961)
for the of
and Codeine
BRANDT, SARAH EHRLICH-ROGOZINSKY * and D. CHERONIS, Department OJ Chemistry, Brooklyn College, Brooklyn, New York
I. Introduction II complete review of the chemical methods for the microdetermination and estimation of morphine and related compounds will be pubThe present work deals lished in a later paper from this laboratory. with the microdetection and estimation of morphine and codeine by means of their fluorescence. Early experiments on the fluorescence of alkaloids were reported He found that morphine fluoresced about 50 years ago by Heller.’ with a weak blue and codeine with a weak gray color. Andant later studied the fluorescence of alkaloids under irradiation from a mercury vapor lamp using a Wood’s filter and the apparatus developed by Bayle and Fabre. 3 He reported4,5 that codeine fluoresced bot’h in the visible and in the ultraviolet, while morphine fluoresced only in the ultraviolet. This was confirmed later by Beguiristans who found that morphine hydrochloride fluoresced at a maximum of 4715 A., while Kosyakova7 report’ed that morphine does not fluoresce. Radley and Grant8 in a work on fluorescence analysis base their statement that morphine and codeine fluoresce on the cited reports. Bowman and collaborators9 who developed the modern spectrofluorometer published data on the fluorescence of an appreciable number of organic compounds. Morphine is reported to fluoresce with arlivation spectra at 270-290 rnp and fluorescent spectra at 365 rnp * On leave from the WeizmannInstitute of Science, Rehovoth, 215
Israel.
216
BRANDT,
EHRLICH-ROGOZINSKY,
AND
CHERONIS
using the precursor of the present Aminco instrument with the II’ 28 photomultiplier tube. In previous work by one of the authors in this laboratory, it was observed that solutions of many alkaloids including morphine and codeine fluoresce in aqueous solutions (made by adding a solution of the alkaloid in acetone to water at pH of 4-6), when irradiated by an ultraviolet hand lamp (long wave type 3660 A., Ultra-Violet Product,s, Inc.). It was determined that morphine and codeine could be detected visually at a lower limit of 40 pg./ml. In the present work, it was found that solutions of morphine in 0.1 N sulfuric acid show a fluorescence peak at 350 rnp and a main activation peak at 285 rnp. A second activation peak was observed at 245 rncc. Varying the pH from 1 to 3 gave no change in emission intensity. The intensity of the emission at pH 7 decreases and at pH 10-12, the fluorescence is negligible. Codeine, the methyl ether of morphine, gives the same fluorescence and activation peaks as morphine with similar intensity in O.lN sulphuric acid. However, at pH’s of 10-12, the intensity of the fluorescence of codeine solutions is unchanged. This difference bctween morphine and codeine at high pH’s qflords a method of distinguishing and determining the two allcaloids in the presence of each other. Quantitative studies showed the optimal range of determination is bet,ween 0.1 and 50 wg./ml. The lower limit of detection is 10 nanograms/ml. At concentrations above 100 Mg./ml. quenching is quite noticeable.
II. Experimental Apparatus and Reagents (A) Aminco-Bowman Spectrophotofluorometer with l-ml. (minimum volume) quart’z cuvets, II’ 21 and 1P 28 photomultiplier tubes, Mosely X-Y recorder, photomultiplier microphotometer, and various light apertures. Quinine in O.lN sulfuric acid was used to check the correct functioning of the instrument.lO No corrections for intensity of emission were used.‘1 (B) Recording Beckman BK Spectrophotometer. Morphine, codeine, and quinine alkaloids (New York Quinine Co.) of U.S.P. grade were used at first,. A purified grade was made by recrystallization and also by vacuum fractional sublimation of the MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
2
MORPHINE
AND
CODEINE
DETECTION
217
Chloroform and acetone, “Spectra” grade (Fisher U.S.P. product. Scientific Co.). Sulfuric acid and sodium hydroxide used were of reagent grade. Water was prepared by triple distillation using an efficient fractionating column. All glassware and cuvets were cleaned by boiling with concentrated nitric acid and rinsed with triple distilled water.
III. Procedure A fresh stock solution of 100 pg./ml. of morphine and of codeine was prepared in (a) 0.1, (b) 0.01, and (c) O.OOlN sulfuric acid. These solutions were diluted to various concentrations. Stock solutions of the alkaloids in sodium hydroxide at concentrations of 0.1, 0.01, and O.OOlN were also prepared. For morphine solution at neutral pH, a few drops of acetone were used as a solvent followed by dilution with water. Codeine was dissolved directly in water. Blank values were determined for all solvents. Values in per cent transmission were read from the microphotometer at the peak activation and peak fluorescence monochromator settings. The product of the meter multiplier and the per cent transmission is the relative per cent transmission. The meter multiplier setting is a measure of resistor values substituted in the microphotometer as indicated on a scale from 1 to 0.001 with the 0.001 setting being the most sensitive.lO Activation and fluorescence spectra are determined in the usual manner.g
IV. Results Experiments with bot,h morphine and codeine dissolved in acet,one gave negligible fluorescence beyond the blank value. In chloroform, a solut,ion of 10 pg./ml. of codeine gave only a slight fluorescence (0.01 relative y0 T, slit, no. 3, 1P 21 photomultiplier tube), while the same concentrat’ion of morphine gave no noticeable fluorescence at t,his instrument set’ting. Figure 1 shows the activation curve for a solution of morphine in O.lN sulfuric acid at a concentration of 10 pg./ml., while Figure 2 shows the fluorescence peak (at 350 1~1~)for the 285 rnp activation at various concentrat,ions. Apparemly two activation peaks are obt>aiued: t,he first at 24.5 rnp and the second and more int,ense at 285 rnp. When activat’ed at 245 mp, morphine also gives a fluorescent peak at
BRANDT,
EHRLICH-ROGO%INSI
ANI)
CHERONIS
* i 200
i
\ m+
400
l
\ 200 mr
\ 400
Fig. 1 (left). Morphine (10 pg./ml.) in O.lN sulfuric acid. Activation spcctra peaks at 245 and 285 rnp at the peak fluorescence of 350 m$. Fig. 2 (center). Morphine (various concentrations): (.A) 100 pg./ml., (U) 50 pg./ml., (C) 20 pg./ml, (D) 10 Pg./ml., in O.lN sulfuric acid. Fluoresccncc spectrum (peak at 350 mp) at the activation peak of 285 mp. Fig. 3 (right). Codeine (10 Ng./ml.) in O.LV sulfuric acid. Activation spectrum (peaks at 245 and 285 mp); fluorescence spectrum (peak at 350 mp).
350 rnp, which is less intense (not illustrated here). I;or codeine, the same activation and fluorescent peaks are observed as shown in Figure 3, but the ratio of the 243 rnp peak to the 285 rnp differs from that of morphine. The same peaks are obtained with both the 1P 21 and II’ 28 photomultiplier tubes, but, the relative per cent transmission is higher with the 11’ 28 t.ube. Figure 4 shows the linear relation of the log concentration versus log relative per cent’ t’ransmission for morphine (blank values have been subtracted) at the same slit widths and different photomultiplier tubes. Figure 5 shows a linear relationship for codeine using different slits and different phot’omultiplier tubes. This proportionality is shown in the range of 0.1 pg./ml. to 50 pg./ml. MICROCHEhlIC.4L
.TOURNAL,
VOL.
V,
ISSUE
2
MOltI’HINE
I
451)
CODEINE
DETECTION
I 2 0 LOG CONC. l’ig. 4. Quantitative estimation of morphine. Log relative y0 transmission log concentration. All values at peak fluorescence of 350 rn$ and using #S. Using the lP28 photomultiplier tube: (X) the 285 rnp activation, (0) 215 mg activation. TJsing the 1P 21 photomultiplier tube: (M) the 285 mr Cvation, (A) the 245 mp activation. -2
219
-I
vs slit the ac-
when using either photomult’iplier tube. Quantities lower than 0.1 pg./ml. show irregularities in their determinations. However, such solutions still exhibit their characteristic activation and fluorescence peaks. Figure 6 shows the spectra of a mixture of .5 Mg./ml. each of morphine and codeine in 0. IN sulfuric acid. This mixt,ure has a relat,ivc per cent transmission similar to that of 10 pg./ml. of morphine or WJdeine. l’igure 6 also shows the spectra of t!he same mixture in O.lN sodium hydroxide. The intensity of the emission is decreased to a value similar to that of 3 pg./ml. of codeine (see Table I). This permits the est,imation of codeine at higher pH and also the estimation of morphine hy the decrease in emission int’ensity.
220
BRANDT,
EHRLICH-ROGOZINSKY,
AND
CHERONIS
Fig. 5. Quantitative estimation of codeine. Log relative y0 transmission vs. log concentration. Using the 1P 28 photomultiplier tube and slit #5: (X) the 285 rnp activation, (0) the 245 rnp activation. Using the 1P 21 photomultiplier t.ube and slit 83: (m) the 285 mp activation, (A) the 245 mp activation.
Typical
Results of a Mixture
Compound Codeine Morphine Misture: codeinemornhine
Weight, w/ml.
TABLE I of Codeine and Morphine Slit 5) Found at activation 245 rnN, 285 mr, PR. rg.
at, Bask pH’s. ( 11’ 28 and
Error at activation, % 245 mp 285 rnp
13.0 11.8
14.1 0.0
12.6 0.0
8.5
:3. I
6.5 5 .!I
6.8
6.0
-1 6
1.5
MICROCHEMICAL
.JOURNAL.
VOL.
V, ISSUE
2
MORPHINE
AND CODEINE
DETECTION
221
Coherent scatter has been deleted from the graphic representation of the spectra, shown in Figures l-3 and G and 7. V. Discussion ‘The peak observed at’ 245 rnp was thought t,o br a peculiarity of the light source of the type indicated by Duggan et al.,” for t)hc activation Wit,h pyridoxine, it was observed that as the spectra of pyridoxine. concentration increased, the second activation peak formed. In t,he case of codeine and morphine, the opposit,e occurred. The 245 rnp peak decreases in the typical manner of concentration quenching (see Fig. 7). Purification of morphine by four crystallizations and also by fractional vacuum sublimation did not change the activation peaks. The previous report8ed13absorpt,ion peak of morphine and codeine is
Fig. 6 (left). Morphine and codeine at pH 1 and pH 12 (5 pg./ml.). Activation spectra (peaks at 245 and 285 rnp) at the peak fluorescence of 350 m+ Abscissa: Wavelength in nw. Ordinate: Arbitrary intensity. Fig. 7 (right). Morphine (various concentrations): (A) 100 pg./ml., (B) 50 pg./ml., and (C) 20 pg./ml. in O.lN sulfuric acid. Activation spectra (peaks at 285 and 245 rnp) at the fluorescence peak of 350 rnp. Abscissa: Wavelength in rnp. Ordinate: Arbitrary intensity.
222
BRANDT,
EHRLICH-ROGOZINSKY,
AND CHERONIS
285 mp. The absorption spectra of morphine at various concentrations (see Fig. 8) in dilute sulfuric acid shows a peak at 285 rnE.rand also intense absorption below 250 rnp. A similar absorption is shown wit,h codeine (not illustrat,ed).
Fig. 8. Morphine (various concentrations): (A) 100 pg./ml., (B) 50 pg./ml., and (C) 25 pg./ml. in O.lN sulfuric acid. Absorption spectra with Beckman 1)U Spectrophotometer. (Peak at 283 mp.) Abscissa: Wavelength in rnp. Ordinate: Transmittance, ‘%.
Williams14 reported that the fluorescence of phenol depends upon the pH. He found maximum fluorescence at pH 1 and minimum at pH 13. Anisole, the methyl ether of phenol, fluoresces equally at pH 1,7,10, and 14 with approximately the same intensity as phenol. The The ability to form quinphenoxide ion is apparently nonfluorescent. oidal resonance forms most likely accounts for this fluorescence. Similarly, in the present study of morphine, the formulas shown indicate the formation of the anion, which would not be able to assume a quinoidal structure due to the oxygen bridge at carbon 4 and thus the MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
2
MORPHINE
AND
CODEINE
‘VO-/(Ii)\ ~pcIr:, _ o,l
DETECTIOS
223
HO-~, (_-l----l ,/
‘2.-N-CH,
t “\ HO-
\
\/j 0
-o-
,
\/ 0
~
,
nonfluorescent species is formed. Codeine most likely does not form this anion and also does not show any emission or spect,ral change. The application of this method to the d&e&on and estimation of ot,her alkaloids is under invcst~igntion.
Summary A spectrofluoromet,ric method for the detection and estimation of morphine and codeine in the presence of each other at the microgram and submicrogram range is described. The detection and estimation of codeine in the presence of morphine is based on t,he fact bhat at’ high pH, the fluorescence of codeine remains unchanged while t)hat, of morphine is quenched.
References 1. Heller, R., 2. Phys. Chem Biol., 2,39T (1916). 2. Andant, A., Corn@. rend., 189,98 (1929). 3. Bayle, E., and R. Fabre, J. pharm. chintz., 1,248 (1025). 4. Andant, A., Bull. sci. pharmacol., 37,28 (1930). 5. Andant, A., Bull. xi. pharmacol, 37,89 (1930). 6. Beguiristan, J. M. B., Chem. Zentr., I, 2800 (1942). 7. Kosyakova, I. A., Zhur. Anal. Khim., 2,27 (1947). 8. Radley, J. A., and J. Grant, Fluorescence Analysis in Ultraaiolet Light, Chapman and Hall Ltd., London, 1954. 9. Bowman, R. L., P. A. Caulfield, and S. Udenfriend, Science, 122, 32 (1955). 10. Instruction and Service Manual No. 768, -4merican Instrument Co., Inr., Silver Spring, Md. 11. White, E. C., M. Ho., and E. Q. Weimer, Anal. Chem., 32,438 (1960). 12. Duggan, I>. E., R. 1,. Bowman, B. B. Brodie, S. Udenfriend, Arch. Biochem. Riophys., 68,l (1957). 13. Bradford, L. W., and W. B. Bracket, Report of Systematic Ultra Violet Spectrophotometric Data of Dangerous Drugs, Poisons, and Narcotics, Lahoratoq of Criminalistics, Office of D. A., County of Santa Clara, San Jose, Calif. 14. Williams, R. T., J. Royal Inst. Chenl., 86,611 (1959).