eliminate the possibility of human errors and also facilitate manual analytical processes much more effectively.
J. Ramirez-Munoz, Anal. Chim. Acta, 71,321-331 (1974). T. Higuchi and E. Brochmann-Hanssen, "Pharmaceutical Analysis," Interscience, New York, N.Y., 1961. V. Das Gupta and D. E. Cadwallader, J. fharm. Sci., 57, 112 (1968). J. Y. Park, paper presented at the Pitisburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 6, 1974, Cleveland, Ohio. (9) J. Y. Park, submitted to J. Pharm. Sci., "Determinations of vitamin A and vitamin E in vitamin tablets or capsules by discrete sample automatic analysis." (10) Rolf Strobecker and Heinz M. Henning, "Vitamin Assay Tested Methods," Verlag Chemie, Weinkeim, Germany, 1966, pp 65-97.
ACKNOWLEDGMENT The author thanks Juan Ramirez-Muiioz and W. F. U1rich for productive discussion and suggestions.
LITERATURE CITED (1) H L. Reynolds, H. P. Eiduson, J. R. Weatherwax, and D. D. Dechert, Anal. Chern., 44, (13), 22A-34A (1972). (2) U S. Pharmacopeia XVIIi, U S . Pharmacopeia Convention, Inc., Bethesda, Md, 1970. (3) National Formulary XIII, American Pharmaceutical Association, Washington, D.C., 1970. (4) D. G. Rohrbaugh and J. Ramirez-Munoz, Anal. Chim. Acta, 71, 31 1-320 (1974).
RECEIVEDfor review June 3, 1974. Accepted October 25, 1974. Presented in part a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1974.
Systematic Identification of Unknown Drugs in Powder Form by Means of Ultraviolet Spectrophotometry in Forensic Toxicology Mark Martens, Frank Martens, Patrick Maenhout, and Aubin Heyndrickx Department of Toxicology, State University of Ghent, Hospitaalstraat 13, 8-9000 Ghent, Belgium
Since rapid and safe determination of unknown pharmaceutical powders has become a need, several laboratories have designed identification systems by means of punched card systems. It is our aim to attempt identification by using only the electronic properties of the unknown molecule. Using buffers of pH 2, 7, and 12 and solvents of different polarity as ethanol and n-heptane, it is possible to build up a pharmaceutical Keydex punched card system, which offers the possibility of determining unknown powders by 125 UV criteria. By optical coincidence of the punched cards, carrying the UV information, one molecule can be selected out of hundreds. Afterwards, additional GLC and TLC analyses may affirm Its identity.
The increasing amount of drugs, being seized by the police daily, has made a fast, accurate, and simple method indispensable for the determination of narcotics and psychoanaleptic pharmaceuticals. Usually these samples are delivered as crystalline or amorphous powders. In such cases, it is a nearly impossible task to identify unambiguously the unknown powder by means of a common spot test and a single UV spectrum, taking into consideration the enormous variety of pharmaceuticals. Lowell W. Bradford and James W. Brackett ( I ) built up an identification system based on the comparison of UV spectra and the computation of the specific absorptivities. By taking advantage of the electronic properties of a series of chemical compounds in alkaline and acid solutions, further differentiation of unknown substances is described. But many questions may arise about the system: To what extent do the unknown molecules change their electronic behavior as a function of the polarity of the solvent used? What is the reproducibility of the UV spectra of pharmaceutical compounds extracted by a water-solvent equilibration system, when putrefied human forensic samples are considered? To this last question, one can only answer that UV spectrophotometry is an inadequate tool in the analysis of biological samples. TLC, GLC, and GC-MS are undoubtedly 458
the only means to obtain plausible solutions to this particular problem. Furthermore, if a classification has to be set up, one must consider the possibility of extending the system with new compounds, without disturbing the uniformity. Charles McArdle (2) designed an efficient identification system for tablets by means of a punched card system. He chose color, size, marking, and taste as identification criteria for tablets. Later on, the Anti-poison Center of Nancy, France, developed a more complete system, also based on marginal perforation. This laboratory switched over to the optical coincidence punched card system recently ( 3 ) . At the Metropolitan Police Laboratories in London, optical coincidence systems have been in use for a long time. Not only the morphology of tablets is registered, but also the electronic spectra of the active substances in alkaline and acid media ( 4 ) . On the basis of the systems mentioned, we set up a method to determine unknown powders by using the electronic properties of the compound only. The Frank-Condon principle states that during the electronic transition, atoms do not move. Electrons, including those of the solvent molecules, may reorganize. Most transitions result in an excited state, more polar than the ground state; the dipole-dipole interactions with solvent molecules will, therefore, lower the energy of the excited state more than that of the ground state. Thus, it is usually observed that ethanol solutions give longer wavelength maxima than n- heptane solutions do. The weak transition of the oxygen lone pair in ketones, the n T * transition shows a solvent effect in the opposite direction. The solvent effect is now due to the lesser extent to which solvents can hydrogen-bond to the carbonyl group in the excited state. In case of potentially tautomeric molecules, the change in the absorption maxima with the change of the p H is due sometimes to a change in the chromophore as a result of the tautomerism and sometimes to simple protonation or deprotonation. Therefore it was necessary not only to consider acid-base shifts in aqueous media but also the change of the curve shape ii; solvents of different polarity. Furthermore, the information, obtained from the interpretation of the UV
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
-.
uasJIIIL.u 111 a
neyuex p"L'b-,'"u ta,u
aysL"L1,.
l l U J 1s "e"IJeu
in s u c h a way that the system is able to store the UV properties of 10,000 pharmaceuticals in powder form. Our a i m was to design a fast identification system which could be used in police laboratories with modest equipment and where untrained analysts can run the system after a brief learning period.
Figure 2. iransterring organic layer in me quam cuvene of the microseparator
OY
means
EXPERIMENTAL Apparatus. A UV spectrophotometer Pye Unieam SP 800 and
.... L.1 carus ...L L . . J . . . >r.n.. pmrc1,t.u nryusx WLCDSe ~
m. . . .
7,-
l.lnLoa ""=
I."IIIl npI-III"
I, "*c A ' 0 0 ' 1 1 , , ,
~~~~~~
we1e ~~~~~
used. The first two figures of the Keydex serial numher are read on the ordinate while the two last figures are read on the ahcissa. One punched card has room for ten thousand compounds with Keydex serial numbers going from 0000 to 9999 (Figure 1). The mieraseparatar, especially designed t o separate the n- heptane layer from the aqueous solution without any mutual contamination, consists of a calibrated Pyrex glass tuhe and a Teflon plunger on which a capillary tube is fixed. By pushing the plunger downwards, the upper organic layer can he transferred to another tuhe via the capillary; in this way two immiscible layers can he completely separated (Figure 2). Reagents. Buffer pH 2, mixture of 25 ml 0.2M KCI and 6.5 ml 0.2M HCI; buffer pH 7, mixture of 50 ml 0.1M KHzP04 and 29.1 ml 0.1M NaOH buffer pH 12, mixture of 50 ml0.05M NazHP04 and 26.9 ml 0.1M N a O H a saturated K&Os solution: analytical reagent grade ethanol and analytical reagent grade n- heptane were used. All drugs (analytical grade), Considered as being narcotic drugs, are involved. The list includes psychoanaleptics and m r eotics as well and is completed by chemicals with similar UV properties to facilitate a more accurate differentiation of the unknown. Procedure. Subdivision of the Punched Card System. The punched card system is divided in two major groups. The first group consists of a numher of punched cards to characterize the identity of the unknown compound, considering an extinction maximum in a certain medium in a certain wavelength zone. Therefore, the UV spectra recorder sheets are subdivided in 18 zones (Figure 3). These zones are 200-215 nm, 215-225 nm, 225230 nm, 230-235 nm, 235-240 nm, 240-245 nm, 245-250 nm, 250260 nm, 26W270 nm, 270-275 nm, 275-280 nm, 280-285 nm, 285290 nm, 290-295 nm, 295-300 nm, 300-325 nm, 325-350 nm, 350400 nm, and 4 0 M 5 0 nm. For the zone (20W215 nm), no punched card has been made hecause of its analytical uselessness. Each spectrum zone consists of 5 punched cards, according t o the five solvents used: pH 2, pH 7, pH 12, ethanol, and n- heptane. Far example: 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, pH 12; 260-270 nm, ethanol; and 260-270 nm, n-heptane. The seeand series of punched cards carries the information abaut the number of peaks in the wavelength zone hetween 240 and 300 nm. For each of the five media used, there are 7 punched cards to register the numher of peaks. In this way 35 punched
cards complete the system. An example for one medium is: 240300 nm, pH 2 , 0 peaks; 240-300 nm, pH 2 , 1 peak; 240-300 nm, pH 2 , 2 peaks; 240-300 nm, pH 2 , 3 peaks; 240-300 nm, pH 2 , 4 peaks; 240300 pH 2, 5 peaks; and 240-300 nm, pH 2, >5 peaks (more than 5 peaks). In total the system contains 125 classification possibilities for one compound based an UV interpretation only.. Arrangements for Programming UV spectra. When Must an Extinction Maximum Be Considered as a "Peak"? Whether or not a peak may he considered depends on the angle a (Figure 4). As long as the angle a between the tangent through the hending point in the curve and the parallel to the abscissa of the spectrum is zero, a peak is programmed. When a is significantly greater than zero, no peak is programmed. In doubtful eases, a peak is always programmed. These definitions are valuahle only when the unknown solute is diluted in such a way that the mast important extinction maxima show an absorbance reading hetween 0.5 and 1.5. Extinction Maxima in a Defined Spectrum Zone. When the extinction maximum is clearly and completely situated in a certain defined spectrum zone, this zone is programmed. On the other hand, when a peak coincides exactly with the border of two spectrum zones, both adjacent zones are programmed. To ascertain repraducihle operation of the UV apparatus during a long period of time, it has to he calibrated regularly hy means of a holmium filter. Recording the Spectra. A few hundred milligrams of powder is divided in four test tubes, one of which is a carefully calibrated
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
459
.PEAK-
o(=o
I
i
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Figure 6. U V spectra of unknown powder. example 1
Figure 1
Figure 7. UV spectra of unknown powder, example 2
Figure 5.
UV
spectr;3 of etnyia
.._. .... -....e tube, used for mic roseparati-.. buffer pH 2 in the cdibrated test tube, 3 ml of buffer pH 7, pH 12, ""A 1 I. ,.C..+L"",., *.," ...L in every other test tube. The powders are thoroughly mixed with these solvents on a whirling mixer for 2 minutes and centrifueed afterwards. The clear aliouot is earefullv . Doured . into a quartz cuvette. If the UV curve goes outscale, the aliquot is diluted as described above. No reference cuvette is used. The rest of the aliquot of the pH 2 solution in the calibrated test tube is alkalinized with l ml of saturated K&03 solution and an equal volume of n-heptane is added. The aqueous phase is extracted by the organic layer by means of a whirling mixer. After separation of both layers by centrifugation, the n-heptane layer is transferred into the cuvette by means of the microseparator. This UV spectrum, in apolar medium, must be recorded with a n-heptane reference cuvette. For all UV recordings, the same sheet is used. The overall picture of the combined UV curves can almost be considered as a fingerprint o f t l_l
I
.,
I
RESUL'I ple concerns ethylamphetamine; the spectra are recorded as described under "Procedure" (Figure 5). We check systematically every UV zone from 215 nm up to 400 nm and select those punched cards out of the system, which tell us in which UV zone and under which medium conditions a peak is present. Next punched cards are then successively put on the light box: 240-245 nm, ethanol; 245-250 nm, pH 2; 245-250 nm, pH 7; 245-250 nm, ethanol; 245-250 nm, nheptane; 250-260 nm, p H 2; 250-260 nm, pH 7; 250-260 nm, pH 12; 250-260 nm, ethanol; 250-260 nm, n- heptane; 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, pH 12; 260-270 nm, ethanol; and 260-270 nm, n- heptane. 460
For counting the number of peaks in zone 240 nm-300 nm, we select next cards out of the svstem and add them to the firs t series on the light box: 240-300 nm, pH 2,4 peaks; 240-301 3 nm, pH 7, 4 peaks; 240-300 nm, pH 12, 3 peaks; 240-301 0 nm, ethanol, 5 peaks; 240-300 nm, ethanol, >5 peaks; i8nd 240-300 nm, n-heptane, >5 peaks. .~.. .. Through this series of Keydex cards a hole is drilled in the quadrangle, of which the position on the punched card is indicated hy the Keydex serial number. This Keydex serial number is the code number of Ethylamphetamine. If any doubt may arise about the definition of a peak in the spectrum of ethylamphetamine, one can solve the problem as follows: the spectrum in ethanol clearly shows five maxima and one "doubtful Deak" in zone 240-300 nm (Figure 5). T o avoid loss of infoimation, card "240-300 nm ethanolI I ,5 peaks" is predicted to have a hole at the appropriate Coor din ates. -~ Example of Identification of Unknown Powders. The recording of the UV spectrais done in exactly the same way as mentioned above. Example 1 is shown in Figure 6. Next cards are taken from the Keydex system: 270-275 nm, pH 2; 270-275 nm, pH 7; 270-275 nm, ethanol; 270-275 nm, n-heptane; 275-280 nm, pH 2; 275-280 nm, pH 7; 275-280 nm, pH 12; 275-280 nm, ethanol; 275-280 nm, nheptane; 280-285 nm, pH 2; 280-285 nm, pH 7; 280-285 nm, pH 12; 280-285 nm, ethanol; 280-285 nm, n- heptane; 285-290 nm, pH 2; 285-290 nm, pH 7; 285-290 nm, pH 12; 285-290 nm, ethanol; 285-290 nm, n- heptane; 290-295 nm, ethanol; and 290-295 nm, n- heptane. The number of peaks in the zone 240-300 nm are 240300 nm, p H 2 , 2 peaks; 240-300 nm, pH 2 , 3 peaks; 240-300 nm, pH 7,2 peaks; 240-300 nm, pH 7 , 3 peaks; 240-300 nm, pH 12, 1 peak; 240-300 nm, pH 12, 2 peaks; 240-300 nm, ethanol, 2 peaks; 240-300 nm, ethanol, 3 peaks; and 240300 nm n- heptane, 4 peaks. ~~
~
~
~
~
~
.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
~
By optical coincidence, two Keydex serial numbers are selected, namely 0080 and 0086, which correspond respectively with diethyltryptamine and dimethyltryptamine. Both substances can easily be differentiated by TLC and GLC. Example 2 is shown in Figure 7. Next cards are taken from the Keydex system: 235-240 nm, pH 7; 235-240 nm, pH 12; 240-245 nm, pH 12; 245-250 nm, n-heptane; 250-260 nm, pH 2; 250-260 nm, pH 7; 250-260 nm, ethanol; 250-260 nm, n-heptane; 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, ethanol; and 260-270 nm, n- heptane. The number of peaks in the zone 240-300 nm are 240300 nm, pH 2 , 3 peaks; 240-300 nm, p H 7 , 2 peaks; 240-300 nm, pH 12, 1 peak; 240-300 nm, ethanol, 3 peaks; 240-300 nm, n- heptane, >5 peaks. By optical coincidence, 1 Keydex serial number becomes visible, namely 0015; pentobarbital-amphetamine, sold on the Belgian market as Pentoadiparthrol. This can further be affirmed by TLC and GLC.
mixtures are programmed, like the amphetamine-barbiturate complexes for instance. The relative extinctions of the different peaks of one spectrum are not considered in the Keydex system; of course this would complete the total of UV characteristics. The one-year experience we now have, shows that this kind of UV characteristics need not be programmed. When one Keydex analysis points out four serial numbers, we can still compare the overall shape of the spectra of the selected numbers with the spectrum of the unknown. To reduce the influence of concentration on the shape of U.V.-curves, a log absorbance plot us. wavelength may be introduced ( I ) . At our department, parallel to this method, a similar system has been worked out for the morphological identification of pharmaceutical preparations with special attention to tablets. The morphological section combined with the UV spectra programming offers reliable and rapid determination possibilities.
ACKNOWLEDGMENT We acknowledge Oscar Van den Broecke for his excellent cooperation in building up the system. We thank the Firm Van Ermenghem, Leuven, Belgium, for turning the idea of the microseparator into a practical form.
CONCLUSIONS The examples prove that this kind of Keydex system means progress in the rapid and safe identification as far as narcotic and psychoanaleptic drugs are concerned. Examples are shown where complete recording of a spectrum was possible. Sometimes drugs do not dissolve in a given medium or do not go over to the n-heptane phase; these “negative” spectra are programmed as well since solubility and partition are determining for the drug itself and therefore offer supplementary information. On the other hand, the question of mixed powders may arise; of course the spectra of the present compounds overlap and cannot be identified by the presented system, unless some often-encountered
LITERATURE CITED (1) L. W. Bradford and Brackett J. W., Mikrochim. Acta, 1958/3, 353-381. (2) C. McArdle, The General Hospital, Birmingham, U.K., personal communication, 1973. (3) J. F. Lorentz, Centre Hospitalier de Nancy, Nancy, France, personal communication, Symposium on tablet identification, July 1973, Nancy, France. (4)J. V. Jackson, Forensic Toxicology Department, Metropolitan Police Laboratories, London, U.K., personal communication, 1972.
RECEIVEDfor review April 29, 1974. Accepted September 12, 1974.
Rapid, Sensitive Spectrophotometric Method for the Determination of Ascorbic Acid Mohamed A. Eldawy,’ A. S. Tawfik, and S. R. Elshabouri Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Assiut, Assiut, A. R. Egypt
A spectrophotometric procedure based on the interaction of dimethoxydiquinone with ascorbic acid was developed. A buffered ascorbic acid solution was reacted with the reagent and the resulting color was measured at 510 nm. Absorbance vs. concentration was linear up to 80 Fg/ml. Replicate analyses showed good agreement, and an average recovery of 99 f 0.6% was obtained for analysis of synthetic mixtures. Other reducing substances, preservatives, stabilizers, minerals, vitamins, and hormones, likely to be present along with ascorbic acid do not interfere with precision of the method or the development of the color. The method is applicable to single as well as multicomponent formulations. Assay results on various commercial samples were reported.
To whom all correspondence should be addressed.
Numerous methods for, and excellent reviews on, the analysis of ascorbic acid are available in the literature ( I 3 ) . The official methods adopted for the analysis of this vitamin depend upon its reducing properties ( 4 , 5 ) . These methods whether iodometric, 2,6-dichlorophenol-indophenol, or the ceric sulfate, in spite of many modifications, still reflect analytical procedures which suffer from lack of specificity. Other reducing substances are oxidized by iodine which is the reagent adopted by U.S.P. XVIII ( 4 ) ,for the analysis of this vitamin. The standard dichlorophenolindophenol procedure used for the official assay of ascorbic acid injection is not a specific assay for the title vitamin (2). Among other substances, sulfohydryl compounds, thiosulfate, riboflavin, and ferrous compounds do interfere with the determination by this method (2). The oxidation of ascorbic acid by ceric sulfate does not correspond to any definite oxidation stage and is largely influenced by time of contact of the reactants, the relative ratio of acidity of the medium and the temperature (6).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
461
J. Ramirez-Munoz, Anal. Chim. Acta, 71,321-331 (1974). T. Higuchi and E. Brochmann-Hanssen, "Pharmaceutical Analysis," Interscience, New York, N.Y., 1961. V. Das Gupta and D. E. Cadwallader, J. fharm. Sci., 57, 112 (1968). J. Y. Park, paper presented at the Pitisburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 6, 1974, Cleveland, Ohio. (9) J. Y. Park, submitted to J. Pharm. Sci., "Determinations of vitamin A and vitamin E in vitamin tablets or capsules by discrete sample automatic analysis." (10) Rolf Strobecker and Heinz M. Henning, "Vitamin Assay Tested Methods," Verlag Chemie, Weinkeim, Germany, 1966, pp 65-97.
ACKNOWLEDGMENT The author thanks Juan Ramirez-Muiioz and W. F. U1rich for productive discussion and suggestions.
LITERATURE CITED (1) H L. Reynolds, H. P. Eiduson, J. R. Weatherwax, and D. D. Dechert, Anal. Chern., 44, (13), 22A-34A (1972). (2) U S. Pharmacopeia XVIIi, U S . Pharmacopeia Convention, Inc., Bethesda, Md, 1970. (3) National Formulary XIII, American Pharmaceutical Association, Washington, D.C., 1970. (4) D. G. Rohrbaugh and J. Ramirez-Munoz, Anal. Chim. Acta, 71, 31 1-320 (1974).
RECEIVEDfor review June 3, 1974. Accepted October 25, 1974. Presented in part a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1974.
Systematic Identification of Unknown Drugs in Powder Form by Means of Ultraviolet Spectrophotometry in Forensic Toxicology Mark Martens, Frank Martens, Patrick Maenhout, and Aubin Heyndrickx Department of Toxicology, State University of Ghent, Hospitaalstraat 13, 8-9000 Ghent, Belgium
Since rapid and safe determination of unknown pharmaceutical powders has become a need, several laboratories have designed identification systems by means of punched card systems. It is our aim to attempt identification by using only the electronic properties of the unknown molecule. Using buffers of pH 2, 7, and 12 and solvents of different polarity as ethanol and n-heptane, it is possible to build up a pharmaceutical Keydex punched card system, which offers the possibility of determining unknown powders by 125 UV criteria. By optical coincidence of the punched cards, carrying the UV information, one molecule can be selected out of hundreds. Afterwards, additional GLC and TLC analyses may affirm Its identity.
The increasing amount of drugs, being seized by the police daily, has made a fast, accurate, and simple method indispensable for the determination of narcotics and psychoanaleptic pharmaceuticals. Usually these samples are delivered as crystalline or amorphous powders. In such cases, it is a nearly impossible task to identify unambiguously the unknown powder by means of a common spot test and a single UV spectrum, taking into consideration the enormous variety of pharmaceuticals. Lowell W. Bradford and James W. Brackett ( I ) built up an identification system based on the comparison of UV spectra and the computation of the specific absorptivities. By taking advantage of the electronic properties of a series of chemical compounds in alkaline and acid solutions, further differentiation of unknown substances is described. But many questions may arise about the system: To what extent do the unknown molecules change their electronic behavior as a function of the polarity of the solvent used? What is the reproducibility of the UV spectra of pharmaceutical compounds extracted by a water-solvent equilibration system, when putrefied human forensic samples are considered? To this last question, one can only answer that UV spectrophotometry is an inadequate tool in the analysis of biological samples. TLC, GLC, and GC-MS are undoubtedly 458
the only means to obtain plausible solutions to this particular problem. Furthermore, if a classification has to be set up, one must consider the possibility of extending the system with new compounds, without disturbing the uniformity. Charles McArdle (2) designed an efficient identification system for tablets by means of a punched card system. He chose color, size, marking, and taste as identification criteria for tablets. Later on, the Anti-poison Center of Nancy, France, developed a more complete system, also based on marginal perforation. This laboratory switched over to the optical coincidence punched card system recently ( 3 ) . At the Metropolitan Police Laboratories in London, optical coincidence systems have been in use for a long time. Not only the morphology of tablets is registered, but also the electronic spectra of the active substances in alkaline and acid media ( 4 ) . On the basis of the systems mentioned, we set up a method to determine unknown powders by using the electronic properties of the compound only. The Frank-Condon principle states that during the electronic transition, atoms do not move. Electrons, including those of the solvent molecules, may reorganize. Most transitions result in an excited state, more polar than the ground state; the dipole-dipole interactions with solvent molecules will, therefore, lower the energy of the excited state more than that of the ground state. Thus, it is usually observed that ethanol solutions give longer wavelength maxima than n- heptane solutions do. The weak transition of the oxygen lone pair in ketones, the n T * transition shows a solvent effect in the opposite direction. The solvent effect is now due to the lesser extent to which solvents can hydrogen-bond to the carbonyl group in the excited state. In case of potentially tautomeric molecules, the change in the absorption maxima with the change of the p H is due sometimes to a change in the chromophore as a result of the tautomerism and sometimes to simple protonation or deprotonation. Therefore it was necessary not only to consider acid-base shifts in aqueous media but also the change of the curve shape ii; solvents of different polarity. Furthermore, the information, obtained from the interpretation of the UV
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
-.
uasJIIIL.u 111 a
neyuex p"L'b-,'"u ta,u
aysL"L1,.
l l U J 1s "e"IJeu
in s u c h a way that the system is able to store the UV properties of 10,000 pharmaceuticals in powder form. Our a i m was to design a fast identification system which could be used in police laboratories with modest equipment and where untrained analysts can run the system after a brief learning period.
Figure 2. iransterring organic layer in me quam cuvene of the microseparator
OY
means
EXPERIMENTAL Apparatus. A UV spectrophotometer Pye Unieam SP 800 and
.... L.1 carus ...L L . . J . . . >r.n.. pmrc1,t.u nryusx WLCDSe ~
m. . . .
7,-
l.lnLoa ""=
I."IIIl npI-III"
I, "*c A ' 0 0 ' 1 1 , , ,
~~~~~~
we1e ~~~~~
used. The first two figures of the Keydex serial numher are read on the ordinate while the two last figures are read on the ahcissa. One punched card has room for ten thousand compounds with Keydex serial numbers going from 0000 to 9999 (Figure 1). The mieraseparatar, especially designed t o separate the n- heptane layer from the aqueous solution without any mutual contamination, consists of a calibrated Pyrex glass tuhe and a Teflon plunger on which a capillary tube is fixed. By pushing the plunger downwards, the upper organic layer can he transferred to another tuhe via the capillary; in this way two immiscible layers can he completely separated (Figure 2). Reagents. Buffer pH 2, mixture of 25 ml 0.2M KCI and 6.5 ml 0.2M HCI; buffer pH 7, mixture of 50 ml 0.1M KHzP04 and 29.1 ml 0.1M NaOH buffer pH 12, mixture of 50 ml0.05M NazHP04 and 26.9 ml 0.1M N a O H a saturated K&Os solution: analytical reagent grade ethanol and analytical reagent grade n- heptane were used. All drugs (analytical grade), Considered as being narcotic drugs, are involved. The list includes psychoanaleptics and m r eotics as well and is completed by chemicals with similar UV properties to facilitate a more accurate differentiation of the unknown. Procedure. Subdivision of the Punched Card System. The punched card system is divided in two major groups. The first group consists of a numher of punched cards to characterize the identity of the unknown compound, considering an extinction maximum in a certain medium in a certain wavelength zone. Therefore, the UV spectra recorder sheets are subdivided in 18 zones (Figure 3). These zones are 200-215 nm, 215-225 nm, 225230 nm, 230-235 nm, 235-240 nm, 240-245 nm, 245-250 nm, 250260 nm, 26W270 nm, 270-275 nm, 275-280 nm, 280-285 nm, 285290 nm, 290-295 nm, 295-300 nm, 300-325 nm, 325-350 nm, 350400 nm, and 4 0 M 5 0 nm. For the zone (20W215 nm), no punched card has been made hecause of its analytical uselessness. Each spectrum zone consists of 5 punched cards, according t o the five solvents used: pH 2, pH 7, pH 12, ethanol, and n- heptane. Far example: 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, pH 12; 260-270 nm, ethanol; and 260-270 nm, n-heptane. The seeand series of punched cards carries the information abaut the number of peaks in the wavelength zone hetween 240 and 300 nm. For each of the five media used, there are 7 punched cards to register the numher of peaks. In this way 35 punched
cards complete the system. An example for one medium is: 240300 nm, pH 2 , 0 peaks; 240-300 nm, pH 2 , 1 peak; 240-300 nm, pH 2 , 2 peaks; 240-300 nm, pH 2 , 3 peaks; 240-300 nm, pH 2 , 4 peaks; 240300 pH 2, 5 peaks; and 240-300 nm, pH 2, >5 peaks (more than 5 peaks). In total the system contains 125 classification possibilities for one compound based an UV interpretation only.. Arrangements for Programming UV spectra. When Must an Extinction Maximum Be Considered as a "Peak"? Whether or not a peak may he considered depends on the angle a (Figure 4). As long as the angle a between the tangent through the hending point in the curve and the parallel to the abscissa of the spectrum is zero, a peak is programmed. When a is significantly greater than zero, no peak is programmed. In doubtful eases, a peak is always programmed. These definitions are valuahle only when the unknown solute is diluted in such a way that the mast important extinction maxima show an absorbance reading hetween 0.5 and 1.5. Extinction Maxima in a Defined Spectrum Zone. When the extinction maximum is clearly and completely situated in a certain defined spectrum zone, this zone is programmed. On the other hand, when a peak coincides exactly with the border of two spectrum zones, both adjacent zones are programmed. To ascertain repraducihle operation of the UV apparatus during a long period of time, it has to he calibrated regularly hy means of a holmium filter. Recording the Spectra. A few hundred milligrams of powder is divided in four test tubes, one of which is a carefully calibrated
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
459
.PEAK-
o(=o
I
i
_-
IF-
Figure 6. U V spectra of unknown powder. example 1
Figure 1
Figure 7. UV spectra of unknown powder, example 2
Figure 5.
UV
spectr;3 of etnyia
.._. .... -....e tube, used for mic roseparati-.. buffer pH 2 in the cdibrated test tube, 3 ml of buffer pH 7, pH 12, ""A 1 I. ,.C..+L"",., *.," ...L in every other test tube. The powders are thoroughly mixed with these solvents on a whirling mixer for 2 minutes and centrifueed afterwards. The clear aliouot is earefullv . Doured . into a quartz cuvette. If the UV curve goes outscale, the aliquot is diluted as described above. No reference cuvette is used. The rest of the aliquot of the pH 2 solution in the calibrated test tube is alkalinized with l ml of saturated K&03 solution and an equal volume of n-heptane is added. The aqueous phase is extracted by the organic layer by means of a whirling mixer. After separation of both layers by centrifugation, the n-heptane layer is transferred into the cuvette by means of the microseparator. This UV spectrum, in apolar medium, must be recorded with a n-heptane reference cuvette. For all UV recordings, the same sheet is used. The overall picture of the combined UV curves can almost be considered as a fingerprint o f t l_l
I
.,
I
RESUL'I ple concerns ethylamphetamine; the spectra are recorded as described under "Procedure" (Figure 5). We check systematically every UV zone from 215 nm up to 400 nm and select those punched cards out of the system, which tell us in which UV zone and under which medium conditions a peak is present. Next punched cards are then successively put on the light box: 240-245 nm, ethanol; 245-250 nm, pH 2; 245-250 nm, pH 7; 245-250 nm, ethanol; 245-250 nm, nheptane; 250-260 nm, p H 2; 250-260 nm, pH 7; 250-260 nm, pH 12; 250-260 nm, ethanol; 250-260 nm, n- heptane; 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, pH 12; 260-270 nm, ethanol; and 260-270 nm, n- heptane. 460
For counting the number of peaks in zone 240 nm-300 nm, we select next cards out of the svstem and add them to the firs t series on the light box: 240-300 nm, pH 2,4 peaks; 240-301 3 nm, pH 7, 4 peaks; 240-300 nm, pH 12, 3 peaks; 240-301 0 nm, ethanol, 5 peaks; 240-300 nm, ethanol, >5 peaks; i8nd 240-300 nm, n-heptane, >5 peaks. .~.. .. Through this series of Keydex cards a hole is drilled in the quadrangle, of which the position on the punched card is indicated hy the Keydex serial number. This Keydex serial number is the code number of Ethylamphetamine. If any doubt may arise about the definition of a peak in the spectrum of ethylamphetamine, one can solve the problem as follows: the spectrum in ethanol clearly shows five maxima and one "doubtful Deak" in zone 240-300 nm (Figure 5). T o avoid loss of infoimation, card "240-300 nm ethanolI I ,5 peaks" is predicted to have a hole at the appropriate Coor din ates. -~ Example of Identification of Unknown Powders. The recording of the UV spectrais done in exactly the same way as mentioned above. Example 1 is shown in Figure 6. Next cards are taken from the Keydex system: 270-275 nm, pH 2; 270-275 nm, pH 7; 270-275 nm, ethanol; 270-275 nm, n-heptane; 275-280 nm, pH 2; 275-280 nm, pH 7; 275-280 nm, pH 12; 275-280 nm, ethanol; 275-280 nm, nheptane; 280-285 nm, pH 2; 280-285 nm, pH 7; 280-285 nm, pH 12; 280-285 nm, ethanol; 280-285 nm, n- heptane; 285-290 nm, pH 2; 285-290 nm, pH 7; 285-290 nm, pH 12; 285-290 nm, ethanol; 285-290 nm, n- heptane; 290-295 nm, ethanol; and 290-295 nm, n- heptane. The number of peaks in the zone 240-300 nm are 240300 nm, p H 2 , 2 peaks; 240-300 nm, pH 2 , 3 peaks; 240-300 nm, pH 7,2 peaks; 240-300 nm, pH 7 , 3 peaks; 240-300 nm, pH 12, 1 peak; 240-300 nm, pH 12, 2 peaks; 240-300 nm, ethanol, 2 peaks; 240-300 nm, ethanol, 3 peaks; and 240300 nm n- heptane, 4 peaks. ~~
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By optical coincidence, two Keydex serial numbers are selected, namely 0080 and 0086, which correspond respectively with diethyltryptamine and dimethyltryptamine. Both substances can easily be differentiated by TLC and GLC. Example 2 is shown in Figure 7. Next cards are taken from the Keydex system: 235-240 nm, pH 7; 235-240 nm, pH 12; 240-245 nm, pH 12; 245-250 nm, n-heptane; 250-260 nm, pH 2; 250-260 nm, pH 7; 250-260 nm, ethanol; 250-260 nm, n-heptane; 260-270 nm, pH 2; 260-270 nm, pH 7; 260-270 nm, ethanol; and 260-270 nm, n- heptane. The number of peaks in the zone 240-300 nm are 240300 nm, pH 2 , 3 peaks; 240-300 nm, p H 7 , 2 peaks; 240-300 nm, pH 12, 1 peak; 240-300 nm, ethanol, 3 peaks; 240-300 nm, n- heptane, >5 peaks. By optical coincidence, 1 Keydex serial number becomes visible, namely 0015; pentobarbital-amphetamine, sold on the Belgian market as Pentoadiparthrol. This can further be affirmed by TLC and GLC.
mixtures are programmed, like the amphetamine-barbiturate complexes for instance. The relative extinctions of the different peaks of one spectrum are not considered in the Keydex system; of course this would complete the total of UV characteristics. The one-year experience we now have, shows that this kind of UV characteristics need not be programmed. When one Keydex analysis points out four serial numbers, we can still compare the overall shape of the spectra of the selected numbers with the spectrum of the unknown. To reduce the influence of concentration on the shape of U.V.-curves, a log absorbance plot us. wavelength may be introduced ( I ) . At our department, parallel to this method, a similar system has been worked out for the morphological identification of pharmaceutical preparations with special attention to tablets. The morphological section combined with the UV spectra programming offers reliable and rapid determination possibilities.
ACKNOWLEDGMENT We acknowledge Oscar Van den Broecke for his excellent cooperation in building up the system. We thank the Firm Van Ermenghem, Leuven, Belgium, for turning the idea of the microseparator into a practical form.
CONCLUSIONS The examples prove that this kind of Keydex system means progress in the rapid and safe identification as far as narcotic and psychoanaleptic drugs are concerned. Examples are shown where complete recording of a spectrum was possible. Sometimes drugs do not dissolve in a given medium or do not go over to the n-heptane phase; these “negative” spectra are programmed as well since solubility and partition are determining for the drug itself and therefore offer supplementary information. On the other hand, the question of mixed powders may arise; of course the spectra of the present compounds overlap and cannot be identified by the presented system, unless some often-encountered
LITERATURE CITED (1) L. W. Bradford and Brackett J. W., Mikrochim. Acta, 1958/3, 353-381. (2) C. McArdle, The General Hospital, Birmingham, U.K., personal communication, 1973. (3) J. F. Lorentz, Centre Hospitalier de Nancy, Nancy, France, personal communication, Symposium on tablet identification, July 1973, Nancy, France. (4)J. V. Jackson, Forensic Toxicology Department, Metropolitan Police Laboratories, London, U.K., personal communication, 1972.
RECEIVEDfor review April 29, 1974. Accepted September 12, 1974.
Rapid, Sensitive Spectrophotometric Method for the Determination of Ascorbic Acid Mohamed A. Eldawy,’ A. S. Tawfik, and S. R. Elshabouri Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Assiut, Assiut, A. R. Egypt
A spectrophotometric procedure based on the interaction of dimethoxydiquinone with ascorbic acid was developed. A buffered ascorbic acid solution was reacted with the reagent and the resulting color was measured at 510 nm. Absorbance vs. concentration was linear up to 80 Fg/ml. Replicate analyses showed good agreement, and an average recovery of 99 f 0.6% was obtained for analysis of synthetic mixtures. Other reducing substances, preservatives, stabilizers, minerals, vitamins, and hormones, likely to be present along with ascorbic acid do not interfere with precision of the method or the development of the color. The method is applicable to single as well as multicomponent formulations. Assay results on various commercial samples were reported.
To whom all correspondence should be addressed.
Numerous methods for, and excellent reviews on, the analysis of ascorbic acid are available in the literature ( I 3 ) . The official methods adopted for the analysis of this vitamin depend upon its reducing properties ( 4 , 5 ) . These methods whether iodometric, 2,6-dichlorophenol-indophenol, or the ceric sulfate, in spite of many modifications, still reflect analytical procedures which suffer from lack of specificity. Other reducing substances are oxidized by iodine which is the reagent adopted by U.S.P. XVIII ( 4 ) ,for the analysis of this vitamin. The standard dichlorophenolindophenol procedure used for the official assay of ascorbic acid injection is not a specific assay for the title vitamin (2). Among other substances, sulfohydryl compounds, thiosulfate, riboflavin, and ferrous compounds do interfere with the determination by this method (2). The oxidation of ascorbic acid by ceric sulfate does not correspond to any definite oxidation stage and is largely influenced by time of contact of the reactants, the relative ratio of acidity of the medium and the temperature (6).
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