this is the most sensitive procedure for t h e determination of formaldehyde. It has a wide versatility in qualitative and quantitative methods. Other d i p h a t i c aldehydes react just as readily as formaldehyde with the reagent. The reagent in t h e procedure has to be pure. Otherwise, the presence of impurities such as 3methyl-2-benzothiazolone azine in t h e reagent can result in a final turbid solution. However, purification is readily accomplished (10). Chromotropic
Acid
Procedure.
This procedure is highly selective for formaldehyde; however, formaldehyde-releasing compounds, such as piperonylic acid, anisyl alcohol, and dextrose, also react. J Acid Procedures A and B. These procedures appear t o be highly selective for formaldehyde. Formaldehyde-releasing compounds also react. Any compound t h a t gives a color in sulfuric acid would interfere in t h e procedure. Very large amounts of
aliphatic aldehydes interfere. Although these procedures are not so highly selective as t h e C and D procedures, they are somewhat more sensitive because of their low dilution factor. J Acid Procedures C and D. These procedures are highly selective for formaldehyde. Compounds containing combined formaldehyde will also react. These procedures are about twice as sensitive as the chromotropic acid method for equivalent dilution factors. Phenyl J Acid Procedure. This procedure is also highly selective for formaldehyde. Compounds containing combined formaldehyde can react. The sensitivity of this procedure is approximately two and one-half times t h a t of t h e chromotropic acid procedure. LITERATURE CITED
(1) Beroza, M., ANAL. CHEY. 26, 1970 (1954).
( 2 ) Boos, R. N., Zbid., 20, 964 (1948). (3) Bricker, C. E., Johnson, H. R., Zbid., 17, 400 (1945). (4) Bricker, C. E., Vail, W. -4., Zbid., 22, 720 (1950). (51 ~, Eenriwe. E.. 2. Anal. Chem. 110. 22 (lU937).’ ’ (6) Grant, W.M., ANAL.CHEM.20, 267 (1948). ( 7 ) Kamel, M., Wizinger, R., Helv. Chim. Acta 43, 594 (1960). (8) Kramm. D. E.. Kolb, C. L.. ANAL. CHEM.27. 1076 (1955’1. ( 9 ) Sawicki,’E., Hauser, T. R., Ibid., 32, 1434 (1960). (10) Sawicki, E., Hauser, T. R., Stanley, T. R., Elbert, W.C., Zbid., 33,93 (1961). ( 1 1 ) Sanicki, E., Stanlev, T. W..Mikro‘ chim. Acta’1960, 510. (12’1 Tanenbaum. M.. Bricker. C. E.. LNAL.CHEY. 23,354 (1951). ’ (13) West, P. W., Sen, B., 2. Anal. Chem. 153, 477 (1956). (14) Yoe, J. A., Reid, L. C., IND.ENO. CHEM.,ANAL.ED. 13, 238 (1941). _
I
\ -
RECEIVEDfor review January 25, 1962. Accepted June 19, 1962. Division of Water and Waste Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
Simultaneous Determination of Morphine, Codeine, and Porphyroxine in Opium by Infrared and Visible Spectrometry
’
KLAUS GENEST and CHARLES G. FARMILO Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Ont., Canada Morphine and codeine in opium can be rapidly and accurately determined simultaneously by infrared spectrometry in carbon tetrachloride after quantitative instantaneous acetylation, The minor alkaloid porphyroxine can b e estimated in the presence of other opium alkaloids b y a specific color reaction. Results for morphine and codeine compare favorably with those obtained by a liquid-liquid extraction method. Absolute values for porphyroxine in opium are given for the first time. The method is recommended for use in determination of the origin of opium for the UN opium research program and has 9570 confidence limits for morphine, codeine, and porphyroxine of f0.26, 3~0.28, and j~0.0083, respectively.
A
for the determination of morphine and codeine in opium require separation of the two alkaloids. Many methods are based on the classical liquid-liquid extraction or include chromatographic techniques (14, 17, 19, 22, 23, 25). LL K N O W S METHODS
1464
ANALYTICAL CHEMISTRY
The final determination is made by gravimetric, titrimetric, or spectrophotometric methods. Quantitative paper chromatography by strip densitometry (16, $1) and after elution (24) was used. A gas chromatographic procedure, which improves the separation step considerably is not yet quantitative (6, 7 , 20). .inother method based on the observation that morphine and codeine show different fluorescence spectra after treatment with sulfuric acid rvas not applied t o opium (4). Porphyrovine was analyzed separately in samples of opium by another method (8, 11). Earlier, from our laboratory, a method for the simultaneous infrared analysis of narcotine, thebaine, and papaverine in opium was published (2). Riorphine cannot be analyzed directly by infrared spectrometry because of its poor solubility in suitable solvents; diacetylmorphine, hoyever, is soluble in carbon tetrachloride. The catalyzed quantitative transformation of morphine and codeine by acetic anhydride rapidly yields diacetylmorphine and acetylcodeine at room temperature which can be assayed simultaneously by
infrared spectrometry. The object of this paper is to describe a n infrared method for the simultaneous determination of morphine and codeine as acetyl derivatives and a spectrophotometric method for porphyroxine in the presence of other opium alkaloids in opium. EXPERIMENTAL
Apparatus. A Perkin-Elmer Model 221 infrared spectrophotometer and a Beckman D K 2 recording spectro-
photometer were used. Reference Standards and Reagents.
Diacetylmorphine (121.8 t o 243.8 mg.) and acetylcodeine (11.4 to 57.0 mg.) were dissolved in anhydrous carbon tetrachloride and made u p to 25 ml. in volumetric flasks which were protected from light. Porphyroxine (0.26 mg.) was dissolved in methanol and, after treatment with 2 N hydrochloric acid (for details of procedure see Porphyroxine), diluted to 10 ml. with methanol. MECKE’SREAGENT.Selenious acid (0.5 gram) was disrolved in concen1 Third part of the series “The Determination of the Origin of Opium” ( 2 , 9).
trated sulfuric acid (100 ml.). The reagent produces a green-blue color on contact with acetylcodeine and a blue color with diacetylmorphine. PHOSPHORIC ACID (85%). Thebaine gives an orange-yellow color. ACETYLATION REAGEKT.Cold acetic anhydride (50 ml.) was mixed with cold perchloric acid (70 to 72%, 2.35 ml.) while cooling, and the solution was stored in the refrigerator. The reagent is yellowish and stable for about 5 days. Caution: There is no hazard under the conditions given in the above procedure; however, solutions containing perchloric acid should not be heated and should be diy~osedof promptly after use. PAPER CHROMATOGRAPHY. Ascending method on (NH4)2S04-impregnated paper and isobutanol-acetic acid-water (100: 10:24) as described earlier (15) was used. Preparation of Opium Extract. Opium particles (1 gram), which were powdered, sieved (20 to 40 mesh) and dried for 2 hours a t 103' C. to 105' C. are triturated in a mortar with 2 nil. of water until a smooth paste is obtained. Five grams of alumina (arid, Woelm) is gradually added and mixed thoroughly with the opium triturate. 9 20 x 10 cm. column is half filled with water and charged with 5 grams of dry alumina. The adsorbent is allowed to settle, air bubbles are removed with the aid of a glass rod, and the water is allowed to run out until a 3-cm. layer remains above the alumina. The opium-alumina triturate and remaining traces in the mortar are wiped out with a wet pad of cotton wool and placed on top of the column. The elution is made with water a t a rate of about one drop per second. The first 5 ml. of effluent water are recycled to the reservoir. A total of 40 ml. of eluate is collected. Removal of Secondary Alkaloids. The column eluate is transferred to a separatory funnel and 10 ml. of acetic acid is added. A second separatory funnel containing 10 ml. of Fater and a cotton pledget in its stem is prepared. Portions of chloroform (15 ml. each) are used t o extract the secondary alkaloids from the first separatory funnel. The chloroform layers are washed. The Fater in the second separatory funnel holds back traces of morphine and codeine which are transferred by the chloroform. The evtraction is continued until two drops of the extract give a negative reaction with sirupy phosphoric acid indicating absence of thebaine. The chloroform extracts are collected and the solvent is evaporated on a water bath. The residue is set aside for porphyroxine analysis. Preparation of the Extract for Infrared Analysis. A liquid-liquid extractor is charged with chloroform: Isopropyl ( 3 : l ) and 5 ml. of concentrated ammonia. After the chloroform extraction the aqueous phases are put into the extractor, made strongly ammoniacal (a total of about 15 ml. of ammonia is needed), and a pinch of sodium bisulfite is added. The morphine-codeine group is then extracted,
for about 2 hours, until a negative Mecke's test is obtained. The extract is evaporated to dryness on the water bath and dissolved in 10 ml. of methanol in a volumetric flask. Two 5-ml. aliquots are removed for porphyroxine analysis. The remaining methanol solution is again evaporated to dryness, then dissolved in 5 ml. of acetic acid. Acetylation reagent (2 ml.) is added and the solution is left a t room temperature for 10 minutes. Then 50 ml. of water is added and the solution is carefully neutralized with 11 grams of sodium bicarbonate and transferred to a separatory funnel. A second separatory funnel containing 10 ml. of water is added to the setup. Both stems of the funnels should contain a pad of cotton wool. The extraction of the acetylated alkaloid is made with 15-ml. portions of carbon tetrachloride until a negative Mecke's test with 2 drops of extract is obtained. The collected extracts are transferred into an opaque vessel and concentrated under reduced pressure on a water bath a t 38" C. to about 20 ml. The carbon tetrachloride solution is transferred to a 25-ml. volumetric flask filled up to the mark and its infrared spectrum is measured (PerkinElmer 221) under the conditions: frequency range 1115-1020 em.-', prism NaCl, slit 130, speed 50 cm.-'/min., source 0.3 amp., filter out position, attenuator speed 11, automatic gain control off, automatic suppression 6, gain 4.5, cells 1.03-mm. path length, pen position s e t a t 0.1 with sample and reference in beams a t 1085 scale 4 cm. = 100 cm.-', compensation CC1,. The percentages of morphine and codeine are calculated according to the formulas Jf%
=
oA*mX6 f
C% = (24.2 Ai
- 21.6 Az) X f
where
AZ=
A1038
- A1026
and A1
-4105s -
-41087
f = 1.11
to correct for the removal of aliquot for porphyroxine analysis. If porphyroxine is not analyzed, this factor is left out. Porphyroxine. The chloroform residue of secondary alkaloids is dissolved in 10 ml. of methanol in a volumetric flask. A 0.5-ml. aliquot of this solution and a 0.5-ml. aliquot of the methanolic solution of the morphine-codeine group prior to acetylation are combined in a 3-ml. volumetric flask which is filled with methanol. This solution serves as a blank. Another 0.5-ml. aliquot from each of the two methanolic solutions is dried (in dishes) on the boiling water bath and the residues are transferred with 0.5-, 0.3-, and 0.2-ml. portions of 2N hydrochloric acid to a 3-ml. volumetric flask immersed for 3 minutes in a water bath a t 80" C. After cooling, the solutions are made up to volume with methanol and the spectrum is obtained
from 700 to 380 mw. The content of porphyroxine is calculated from the absorbance in a 1-cm. cell a t the 525mp maximum by the formula Porphyroxine yo =
A521
X 0.147
RESULTS AND DISCUSSION
Purification Procedure. The chromatography of opiates from opium by water on the alumina column is based on experiences of earlier authors (5, 9). Morphine, codeine, and porphyroxine are completely eluted in the first 40 mi. of aqueous eluate, because paper chromatography testing of further eluate showed only narcotine. The secondary alkaloids papaverine and narcotine are removed easily by chloroform, whereas thebaine is more difficult to extract. The color test with phosphoric acid if done properly will assure the final removal of thebaine without running the risk of losing codeine by too many chloroform extractions. Acetylation. Acetylation of t h e morphine-codeine residue by the HC104catalyzed reaction (IO) is an improvement over other methods for these compounds ( 1 ) because it is instantaneous and complete. KO morphine or codeine can be detected by paper chromatography a t 1 minute after addition of fresh acetylation reagent. Seutralization of the acetylation mixture releases the acetyl derivatives for extraction in carbon tetrachloride. Ammonia was not suitable for this step because of heat development and lack of p H control, which causes some hydrolysis of the diacetylmorphine to 6-acetylmorphine and sometimes to 3-acetylmorphine. Although the former has nearly the same analytical infrared properties, it is difficult to extract with carbon tetrachloride from a neutral solution. Sodium bicarbonate is a suitable neutralizer: It does not generate heat and, if it is added in small batches, losses by frothing can be avoided. The final p H is between 7.9 and 8.2, under which conditions no hydrolysis products were found by paper chromatography. The carbon tetrachloride extract has to be protected from light. Prolonged exposure to diffuse daylight or fluorescent light causes precipitates of hydrochlorides of the acetylated alkaloids. Infrared Method. The infrared spectra from 1850 to 850 cm.-' of diacetylmorphine, acetylcodeine, and a mixture of the two are shown in Figure 1. The most suitable peak for morphine analysis is a t 1038 cm.-l for which the validity of Beer's law was checked for concentrations corresponding to the usual range of 10 to 20% morphine in opium. Acetylcodeine has no absorbance from 1038 to 1025 cm.-', and the morphine concentration can be calculated directly from the A2 value. At the analytical peak chosen for the VOL. 34, NO. 1 1 , OCTOBER 1962
1465
WAVELENGTH ,MICRONS
6
0 01
I
8
7
I
I
I
I
9
IO
!
I
12
11
1
in carbon tetrachloride is given in Figure 2. Tracings of the analytical region for three opiums of various origins can be seen in Figure 3. It shows a n opium high in morphine and low in codeine (Yugoslavia), one which is low in morphine and high in codeine (China), and one a-ith intermediate values for both alkaloids (Iran). Quantitative results by the infrared method for six opiums compared n-ith those obtained by a classical chemical method are listed in Table 11. The results indicate good agreement betneen the infrared and chemical methods. Interferences. The alkaloids thebaine, papaverine, and narcotine interfere in t h e morphine-codeine determination and must be removed. Thebaine is transformed by acetylation t o phenolic end products, which are soluble in carbon tetrachloride, while the pair papaverine-narcotine is naturally sufficiently soluble to occur in the final eutract. Because i t was not known whether the small amounts of minor phenolic alkaloids-e.g., porphyroxine-which were detected by paper chromatography would interfere with the morphinecodeine assay, it mas necessary to prepare some fractions especially for the interference studies. An Indian Esport opium mas processed using Fulton's liquid-liquid extraction procedure (14) to obtain three fractions containing, respectively, minor phenolic alkaloids, a n unknown base, and narceine. These opium isolates, and the known (26) crystalline alkaloids. cryptopine, cotarnine, and laudanine \?ere acetylated, extracted with carbon tetrachloride, and their solution spectra obtained. They showed no absorption from 1025 to 1038, or 1058 to 108'7 em.-' The morphine-codeine solutions from opium extracts were colored yellow, but otherwise gave practically identical spectra from 850 to 1850 cm.-' Compared with solution spectra of pure diacetylmorphineacetylocodeine. Earlier con-
I3 14 15 1 , I I
,
I
01 0 2
03 0 4 0 5 06
0 8 I O
01 W
$ 0 2
a 03 0 4 051
I
" I
I
01
0 2
03 04 0 5 0 6
0 8 I O
I2
1800
1700
1600
1500 1400 1300
1200
1100
900
1000
800
700
W A V E N U M B E R , CM-'
Figure 1. Infrared spectra A.
Diacetylmorphine ( 1 82.5 mg.) Acetylcodeine (57 mg.) Mixture of diacetylmorphine ( 1 4 6 mg.] and acetylcodeine (34.2 mg.) in CCla (25 ml.)
6. C.
Table I.
Absorbances a t Analytical Maxima of Infrared Spectra Pure Diacetylmorphine and Acetylcodeine
Conc. mg./25 Compounds Diacetylmorphine Acetylcodeine
ml.
Absorbances at frequencies, em.-'
cch
121.8 57
A1058
A1087
Ai
0.323 0.307
0.048 0.100
0.275 0.207
analysis of codeine (1058 cm.-'), on the other hand, diacetylmorphine absorbs as well. Beer's law was found valid for concentration ranges corresponding to 1 to 5% codeine in opium. The codeine content is calculated by a two-component formula. Typical absorbance data for the two acetylated compounds are given in Table I. Other possible analytical peaks are discussed under "Interferences." Processing of various amounts of pure acetylated compounds and their mixtures through the purification and acetylation steps showed no losses as checked by paper chromatography and infrared spectrometry. Opium Analysis. T h e spectrum of a typical acetylated opium extract 1466
0
ANALYTICAL CHEMISTRY
Al03&
0.372 0.197
A~OZE AO
0.068 0.197
0.304 0.000
WAVELENGTH ,MICRONS 8
7
6
9
IO
II
12
13
14 15
01 W
g a m E
0 2
03
0
fn 0 4
2
0 5 0 6 0 8 I O
1
I800
1700
1600
1500 1400
!
1300
I
1200
I
1100
1000
900
800
WAVENUMBER, CM-'
Figure 2.
Infrared spectrum of Yugoslavian opium One gram of opium per 2 5 ml. of CCI4
(UN38)extract
700
sideration had been given to using the infrared spectral regions 1175 t o 1215, 1270 to 1300, and 1755 to 1790 cm.-' for the simultaneous assay of morphine and codeine; however, i t was found t h a t the minor phenolic fractions do absorb there, too. The diacetylmorphine and acetylcodeine maxima at 1283, 1195, and 1776 cm.-'are therefore unsuitable for analytical purposes. It has been shown conclusively that the alkaloids thebaine, papaverine, narcotine, crjytopine, cotarnine, laudanine, an unknon-n base, and the minor phenolic opium extracts will interfere in this assay. Suitable precautions, either by chemical means or by careful selection of infrared bands for assay, must he taken to prevent the interferences. Porphyroxine. The minor phenolic opium alkaloid porphyrosine is converted 11:treatment with diluted mineral acid into a red colored compound, porphyr hydrochloride (11). Earlier met'hods for porphyrosine are based on measurement of the red color obtained when an ether estract from a lime-opium niisture was acidified with mineral acid. Because no pure porphyroxine was available, the values reported eo far were only relative (mostly given in Lovibond units) ( 8 ) . Klayman ( I S ) \vas successful in isolating pure porphyrosine from opium and reported its empirical formula ultraviolet and infrared spectra. H e also showed t h a t porphyr hydrochloride consists of a t least three components, one colorless and two colored. The molar absorptivity of porphyr hydrochloride based on the amount of porphyroxine given by Klayman (18) was used. The validity of Beer's law was established earlier (8). Thus i t is possible to give absolute values for porphyrosine in opium (Table 11). There is a more than tenfold spread in concentration of porphyroxine between opiums of various origins. The content in Indian opium is higher than assumed by earlier authors (11, 18). About one third of the porphyroxine amount of Indian opium accompanies the secondary alkaloids. If the porphyroxine analysis in the morphine-codeine group is done after the acetylation step, lower values are found. Precision. The 95% confidence limits were used to express t h e precision of the chemical and t h e infrared methods. For the chemical method t h e d a t a are based o n t h e Indian Export opium sample Ex 3398. I n addition to values given in Table I1 other d a t a as outlined earlier (4) where used for the calculation. For morhpine the 95% confidence limit &'as found to be k1.83 and for codeine *0.71, both for 16 degrees of freedom. The precision of the infrared method was calculated from data given in Table
Table It.
Comparative Results
of Analysis of Opium Samples b y Infrared and Chemical Methods
Content of alkaloid, yo (calculated to anhydrous opium)
Opium LN 111, Iran
L S IV, Turkey
Codeine IR Chemical method methods 2.29 2.09 2.37 3.03 3.17
UN 38,Yugoslavia
1.26 1.48
UN 37B,China
5.16 5.04
U N 35,India
3.40 3.04
Indian Export 3398
3.18 2.97 3.37
Morphine IR Chemical method methods
~
2.87" 2.26b 2 . 23c 3.56" 2.5gb 2.72s 1.67" 1 .26b 1.32" 6.630d 5.50b 5.SOc
11.85 11.78 11.88 12.45 12.60
11.13" 11 .OOb 10,97' 12.25" 11,l o b 11.05e 20.33" 17 5Gb 19.60" 12.08" 10,90* 10.9oc
18.89 19.35 11.28 11.31
3.45. 3.42b 3.38c 3.3OC
13.75 13.55
Porghyroxine, spectrophotometric
method 0 024 0 024 0 110 0.113 0,059 0 054 0.060 0.059 0.060 0.054 0.206 0 213
12.94" 12.00b 12.82s 12.41"
12.75 12.75 12.70
0,304 0.290 0.317 0.292 0.315O
Fulton's values, by K/34 method (14). Fulton's values by earlier methods (12,IS). Values from this laboratory by K/34 method. This value is reported t o contain "unknown base" (14). Value analyzed by K/14 method (8).
WAVELENGTH, MICRONS
9
IO
IO
9
9
IO
00
Figure 3. Infrared spectrum of analytical region of three opium extracts
I
;0 2 z
a A.
Yugoslavian E. Chinese C. Iranian One gram of opium per 25 ml. of CC14
03 0
