Semiautomated Fluorometric Assay for Submicrogram Quantities of Morphine and Quinine in Human Biological Material S . J. Mu16 and P . L. Hushin N e w York State Narcotic Addiction Control Commission Testing and Research Laboratory, Brooklyn, N . Y. 11217
A simple, sensitive and specific method for the fluorometric determination of morphine and quinine extracted from biological material was developed. The fluorophore of morphine was prepared by simply treating the extract with acid, alkali, and heat. Quinine in the organic phase was re-extracted into 0.1N HzS04 and determined fluorometrically. Concentrations of 0.22 Ng/ml of morphine and 0.10 pg/ml of quinine in urine were easily detected. The application of the automated turret spectrofluorometer (ATS) to this method has allowed the laboratory to monitor large numbers of urine samples daily for morphine. The ATS method provides for effective surveillance of heroin abuse within a narcotic control, treatment, or aftercare program. METHODS CURRENTLY AVAILABLE for the determination and/or the identification of morphine in biological materials are largely based upon spectrophotometry (1-4); radioactivity (5-7) ; thin-layer chromatography ( 4 , 8-12) and gas liquid chromatography ( 4 , 13-16). None of these techniques fully meets the requirements for a rapid, reliable, simple, sensitive, and specific method to monitor human urines for morphine in a large narcotic addiction control program. In this regard fluorometry appeared most promising because of sensitivity (nanogram quantities), specificity (excitation and emission spectra), and the ease of adaptation to automated methodology (rapidity). Fluorometric studies indicated that morphine, a weakly fluorescent compound ( I 7), could be converted to a highly fluorescent fluorophore by suitable treatment with acid and alkali (18, 19). The application of fluorometry to an extract of biological material containing morphine by oxidation to pseudomorphine with potassium ferrocyanide and (1) J. M. Fujimoto, E. L. Way, and C. H. Hine, J . Lab. Clin. Med., 44, 627 (1954). (2) E . L. Way, J. W. Kemp, J. M. Young, and D. R. Grassetti, J. Pharm. Exptl. Therap., 129, 144 (1960). (3) L. A. Woods, J. Cochin, E. G. Fornefeld, and M. H. Seevers, ibid., 111, 64 (1954). (4) S. J. Mule, ANAL. CHEM., 36, 1907 (1964). (5) T. K. Adler, H. W. Elliott, and R. George, J. Pharm. Exptl. Therap., 120, 475 (1957). (6) S. J. Mule and L. A. Woods, ibid., 136,232 (1962). (7) S. Y. Yeh and L. A. Woods, J. Pharm. Sei., 59,380 (1970). (8) J. Cochin and J. W. Daly, Experientia, 18,294 (1962). (9) B. Davidow, N. Li Petri, B. Quame, B. Searle, E. Fastlich, and J. Savitzky, Amer. J . Clin. Pathol., 46, 58 (1966). (10) V. P. Dole, W. K. Kim, and I. Eglitis, J. Amer. Med. Ass., 198, 115 (1966). (11) S . J . Mule, J. Chromatogr., 39, 302 (1969). (12) A. M. Heaton and A. G. Blumberg, ibid., 41,367 (1969). 35, (13) K. D. Parker, C. R. Fontan, and P. L. Kirk, ANAL. CHEM., 346 (1963). (14) G. R. Williams and E. L. Way, Biochem. Pharmacol., 18, 1435 (19 69). (15) N. Ikekawa, K. Takayama, E. Hosoya, and T. Oka, Anal. Biochem., 28, 156 (1969). (16) F. Fish and W. D. C. Wilson, J. Chromatogr., 40,164 (1969). (17) R. Brandt, S. Ehrlich-Rogozinsky, and N. D. Cheronis, Microchem. J . , 5,215 (1961). (18) C. Fulton, J . Amer. Pharm. Ass., 26,726 (1937). (19) G. Nadeau and G. Sobolewski, Can. J . Bioclzem. Physiol., 36, 625 (1958). 708
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potassium ferricyanide under alkaline conditions has been adequately demonstrated ( 2 0 , 21) for plasma and brain tissue but not for urine. It was the specific purpose of this study to develop a fluorophore of morphine following rapid extraction primarily from human urine and other biological tissues and fluids by simply treating the extract with acid, alkali, and heat. The availability of an automated spectrophotofluorometer provided the necessary instrumentation for a rapid analysis. Quinine which has a high fluorescent quantum yield ( 2 2 ) and is extensively used in the New York City area to dilute heroin in the illicit market was also extracted from biological material and determined fluorometrically as well. This communication describes in detail the extraction of morphine and quinine from biological material, the subsequent fluorometric detection of the individual fluorophores, and the application of the automated turret spectrofluorometer (ATS) in the rapid routine monitoring of human urine samples. EXPERIMENTAL
Extraction of Morphine and Quinine from Biological Material. Two ml of sample (urine, plasma, or tissue homogenate) is transferred to a 15-ml glass stoppered centrifuge tube and the pH adjusted to 9-10 with 3.7N NHaOH. Four milliliters of chloroform :isopropanol ( 3 :1, v/v) is added to each tube and the samples are shaken by hand for 60 seconds. A two-thirds aliquot of the lower organic phase is utilized for the morphine analysis, and the remaining one third of the organic phase for the quinine analysis. FLUOROMETRIC ASSAYOF MORPHINE.The organic extract in 15-ml borosilicate glass conical centrifuge tubes is evaporated to dryness on a water bath at 85 "C under a stream of air. To ensure complete dryness, the tubes are placed in an oven at 100 "C for 10 minutes. A 0.1 ml of concd H2S04 was added to the residue in each tube and thoroughly mixed on a Vortex-Genie shaker. To each sample was added 1 ml of distilled water, mixed, and 1 ml of concd NH4OH. The mixture was shaken on the Vortex-Genie and the samples were autoclaved for 15 minutes at 120 "C under 15-18 Ib of pressure. The solutions were transferred to 7 mm X 150 mm round borosilicate glass cuvettes and fluorescence was ascertained in the automated turret spectrofluorometer (ATS) utilizing the Corning No. 4-77 filter with the Farrand 400 nm interference filter. The maximum emission wavelength was obtained by setting the monochromator drum dial at 410 nm and scanning each sample through 100 nm wavelength to 510 nm . FLUOROMETRIC ASSAYOF QUININE.Two ml of 0.1N H2S04 was added to 15-ml borosilicate glass centrifuge tubes containing the organic extract aliquot, The tubes were shaken for 30 seconds to one minute on the Vortex-Genie shaker and the upper aqueous phase transferred to the 7 mm X 150 mm borosilicate glass cuvettes. Fluorescence was determined in (20) H. Kupferberg, A. Burkhalter, and E. L. Way, J. Pharm. Exptl. Therap., 145, 247 (1964). (21) A. E. Takemori, Biochem. Pharmacol., 17, 1627 (1968). (22) R. F. Chen, Anal. Biochem., 19, 374 (1967).
A
EXCITATION
B
EMISSION
402 nm V
425nm
WAVELENGTH ( n m )
Figure 1, A . Excitation spectra of morphine fluorophore with emission monochromator at 425 nm B. Emission spectra of the morphine fluorophore with the excitation monochromatorat 392 nm. All spectra uncorrected and the Aminco-Bowman spectrofluorometer instrumental conditions as described in the Experimental Section
the automated turret spectrofluorometer (ATS) using the Corning No. 7-60 filter and scanning the emission wavelength as described for the morphine assay. Controls. In order to effectively check the method as well as the instrument, the following controls were utilized each day: two-ml urine samples (blank) without drug were extracted through the procedure and fluorescence determined on the ATS as described for both morphine and quinine; a direct standard of morphine sulfate (0.75 pg/lO p1, as free base) was evaporated to dryness and the morphine fluorophore derivative prepared as described; a direct standard of quinine sulfate (0.41 pg/ml of 0.1N HzS04, as free base); two ml of urine containing 1.5 pg of morphine (free base) and 0.82 pg of quinine (free base) was extracted and carried through the entire procedure for morphine and quinine. Recovery. To evaluate the method, 0.1 to 1.0 pg of morphine (free base) in triplicate was evaporated to dryness directly and the fluorophore derivative prepared as described. Concentrations of quinine (as free base) ranging from 0.05 to 0.50 pg were prepared in 2 ml of 0.1N H2S04. A straight line relationship between fluorescence intensity and concentration was obtained. Recoveries of 0.25 to 2.5 pg of morphine and from 0.12 to 1.2 pg of quinine from urine were 65 i 4 . 5 z and 95 i 3 . 0 z , respectively. The absence of 100% recovery was due to incomplete extraction of these drugs. Reagents. All chemicals were of reagent grade and obtained from the J. T. Baker Chemical Company. Chloroform either of spectrophotometric quality or reagent grade was found suitable. Morphine and quinine were used as the sulfate salts but all calculations were for the free base. Apparatus. The instrument used for routine assay was the Farrand automated turret spectrofluorometer (AT'S) equipped with an 85-watt high intensity mercury vapor lamp, quartz lenses, 0.2-mm slits, and a turret that holds sixteen 7 mm X 150 mm borosilicate glass cuvettes. Each sample is recorded in 20 seconds; thus the total time required for one complete turn of the turret was 5.3 minutes. With a preloaded spare turret, it is possible to scan and record 150 samples per hour. For the morphine determination, a Farrand 400 nm interference filter and a No. 4-77 Corning filter was used. For the quinine assay a No. 7-60 Corning filter was used without the interference filter.
The transmission wavelength of these filters was determined with a Perkin-Elmer VIS-UV Model 402 spectrophotometer. The Corning No. 4-77 filter provided maximum wavelengths at 400, 518, 562, and 712 nm. Combining the Corning filter No, 4-77 with the Farrand 400 nm interference filter provided a single peak with maximum transmission at 392 nm. With the Corning filter No. 7-60, peak transmission wavelengths occurred at 365 and 746 nm. Maximum wavelengths for excitation and emission were obtained by scanning the fluorophore derivative of morphine and quinine fluorescence in an Aminco-Bowman spectrophotofluorometer (No. 4-8202) with an Aminco x-y recorder (Model No. 814) attachment, This instrument was equipped with 150-watt xenon source. The excitation monochromator exit slit used was 3 mm and the emission monochromator slit was 5 mm. The meter multiplier was set at 0.3 and a sensitivity setting of 30 was used, Quartz cuvettes (10 mm) were used to obtain the fluorescence excitation and emission spectra. Setting the excitation monochromator at 392 nm provided two spectra, a scatter peak at 395 nm and an emission peak at 425 nm for the morphine (0.75 pg as free base) fluorophore. Peak excitation spectrum occurred at 402 nm when the emission monochromator was set at 425 nm. Quinine sulfate (0.82 pg/ml of 0.1N HS04, as free base) provided a peak emission spectrum at 450 nm and a minor scatter peak at 350 nm with the excitation monochromator set at 350 nm. Excitation spectra with peaks at 250 and 350 nm and minor scatter peaks at higher wavelengths were obtained when the emission monochromator was set at 450 nm. For the routine fluorometric assay of morphine with the ATS, the filters provided an excitation wavelength maximum of 392 nm and an emission wavelength maximum of 425 nm. For the quinine assay with the ATS, the filter provided a n excitation wavelength maximum of 365 nm and an emission wavelength maximum of 450 nm. The usual range switch setting for sensitivity with the ATS was 0.1 for the morphine assay and 1.0 for the quinine assay. Comments on the Procedure for Fluorometric Analysis. In our laboratory, the urine was not acid hydrolyzed prior to extraction; thus only free morphine and quinine were extracted and determined fluorometrically. Acid hydrolysis caused destruction of quinine with subsequent erratic analytic results. The quinine fluorescent assay is quite important ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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z W
BLANK
MORPHINE
QUININE
0.45 ug/rnl
0.25rg/ml
WAVELENGTH (nm)
Figure 2. Typical automated turret spectrofluorometric (ATS) spectra obtained following the extraction of a urine blank and urine containing either 0.45 pg/ml of morphine or 0.25 pg/ml of quinine Instrumental conditions: Farrand 400 nm interference filter and Corning filter No; 4-77 for the morphine assay; Corning filter No. 7-60 for the quinine assay; emission monochromator drum dial set at 410 nm and scanned over a period of 100 nm to 510 nm; 0.2mm slits at entrance and exit of the monochromator; sensitivity range switch setting at 0.1 for morphine and 1 for quinine since it is used as the primary marker for the subsequent acid hydrolysis, solvent extraction of urine at alkaline pH 9-10, and thin-layer chromatographic (TLC) confirmation of both morphine and quinine. The urine samples, however, may be hydrolyzed in 2.5N HC1 (final normality) by autoclaving for 30 minutes at 120 "C and 18 lb of pressure, adjusting the pH to 9-10 with alkali, extracting, and preparing the morphine fluorophore as described (estimation of total morphine, Le., free plus conjugated). RESULTS AND DISCUSSION Spectral Characteristics. The excitation and emission spectra (uncorrected) of the morphine fluorophore (0.75 wg as free base) at pH 8.44 k 0.31 is shown in Figure 1 . When the emission monochromator wavelength was set at 425 nm, the peak excitation spectrum occurred at 402 nm. Holding the excitation monochromator wavelength constant at 392 nm provided a peak emission spectrum at 425 nm. The routine fluorometric assay of morphine with the automated turret spectrofluorometer (ATS) equipped with filters provided excitation wavelength at 392 nm and emission wavelength at 425 nm. Effect of pH and Reagent Concentrations. The effect of pH on the fluorescence of the morphine fluorophore was determined by altering the concentration of concd H2S04 or ammonia in the reaction mixture. Maximal sensitivity was observed at the fluorometric assay pH of 8.44. An acid pH of 1.0 or alkaline pH 13-14 completely eliminated morphine fluorescence emission at 425 nm. The effect of varying the concentration of concd HzS04 on fluorescence intensity in the reaction mixture containing 1 to 5 p g of morphine was studied. Maximal fluorescence was observed when the final concentration of HzS04 was 0.55N. The optimal final concentration of NHIOH was then 3.5N. Eliminating either acid or alkali within the reaction mixture prevented the formation of the fluorophore. Optimal results were thus obtained with the reagent mixture described previously. Effect of Temperature and Time. A reduction in fluorescence intensity (20 to 85 %) at 425 nm was observed when the reaction mixture was heated in boiling water bath from 5 to 25 minutes in comparison to the fluorescence achieved with autoclaving at 120 "C with 18 lb of pressure. 710
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The fluorophine obtained with morphine (0.2 to 2 pg) appeared to be stable for a period of 48 hours under normal laboratory conditions. Specificity. To ascertain the specificity of the method for morphine, the following drugs at a level of 5 pg were extracted and carried through the entire ATS procedure as described for morphine: methadone, d-propoxyphene, codeine, cyclazocine, nalorphine, normorphine, levorphanol, ethylmorphine, dihydromorphinone, apomorphine, d-amphetamine, pentobarbital, chlorpromazine, nicotine, caffeine, and diazepam. The only drugs which yielded a fluorescence product essentially identical to morphine were nalorphine and normorphine. Neither of these drugs has an abuse potential. Nalorphine is a narcotic antagonist and normorphine is a metabolite of morphine. The other drugs did not provide a detectable fluorescent derivative. It would appear that the phenanthrene nucleus as well as a free 3-OH group is essential for the formation of the fluorophore. The nature of the morphine fluorophore as prepared by this study is unknown. However, studies (20, 21) whereby the morphine fluorescent derivative as prepared by ferriferro-cyanide oxidation indicate the compound to be pseudomophine. Although, similarities exist between the data reported with the pseudomorphine fluorophore (21) and our data, there are significant differences in spectral characteristics to indicate that pseudomorphine is not the fluorophore in this study. Urine Studies. Figure 2 shows the typical automated turret spectrofluorometer (ATS) fluorescence emission spectra obtained with negative urine (blank) and urine containing 0.45 pg/ml of morphine or 0.25 pg/ml of quinine. Correcting these concentrations for aliquots and extraction recovery provides a final total value of 390 ng for morphine and 160 ng for quinine. The minimal concentration reliably detected for morphine in urine was 0.22 pg/ml (free base) which corrected as described is equivalent to a total of 190 ng of morphine. For quinine the minimal detectable value was 0.10 pg/ml (free base) of urine that is equivalent to a corrected total value of 63 ng. Three human volunteers ingested 325 mg of quinine sulfate and urine samples were collected each morning for a period of 12 days, Quinine was detected in the urine of all the volunteers for a period of 10 to 11 days after ingestion.
It is primarily for this reason (sensitivity and time of detection) that quinine is considered a prime maker for additional thin-layer and gas chromatographic analysis of the urine samples for both morphine and quinine. Tissue Studies. Tissue homogenates (10% in 0.1N HC1, w/v) of human lung, liver, colon, kidney, and stomach (obtained at autopsy) containing 7.5 and 8.2 pg (free base) of morphine and quinine, respectively, were extracted and assayed fluorometrically as described. In all cases, morphine and quinine were quite easily detected. Comparison of Urine Samples Analyzed by the ATS and TLC Method. Three-hundred four urine samples obtained from individuals within the Narcotic Addiction Control Commission (NACC) facilities were analyzed fluorometrically (ATS) by methods described in this report and by thin-layer chromatographic (TLC) methods similar to those described previously (Zl). A comparison of the data is summarized in Table I. A good agreement existed between the number of urine samples positive for morphine by the ATS ( 1 1 . 8 x ) and the TLC (12.273 method. The comparative agreement with quinine was not as good, simply because quinine is used as the prime marker in our routine analysis and a slightly suspicious indication of this drug on a routine scan of the sample is considered positive for further confirmation by the TLC technique. The number of samples positive for morphine and negative for quinine was quite small (1.3% ATS, 1.6% TLC). The converse, however, was considerably larger as indicated by both methods of analysis (4.3 to 6.673. A higher percentage of quinine positive morphine negative urines is expected since quinine is detected in the urine following drug usage for a period of 10 to 11 days. Morphine under normal circumstances may be detected for a period of 48 to 72 hours following acute administration of the drug. It is important to note the very small percentage (1.9%) of samples negative for morphine by the ATS method and positive by the TLC method, and conversely those positive by the ATS (1.3 %) technique and not confirmed by thin-layer chromatography. Further inspection of the ATS negative morphine urine samples showed that five out of six urines were positive for quinine and therefore in the usual routine assay would be subjected to TLC confirmation analysis for quinine and morphine. In the case of quinine, 2.3% of the urine samples were negative by the ATS method and positive by the thin-layer chromatographic technique. These samples were also negative for morphine by the ATS method and therefore in the routine assay would not be analyzed by thin-layer chromatography. The percentage of samples quinine positive ATS and negative TLC (not
Table I. Comparison of 304 Urine Samples Analyzed by the ATS and TLC Method ATP TLCb Urine Urine Drug in urine samples % samples Morphine positive 36 11.8 37 12.2 Quinine positive 51 16.8 44 14.5 Morphine positive, quinine negative 4 1.3 5 1.6 Quinine positive, morphine negative 20 6.6 13 4.3 Morphine plus quinine positive 32 10.5 30 9.9 Morphine negative ATS, ... ... 6 1.9 positive TLC Morphine positive ATS, negative TLC 4 1.3 ... ... Quinine negative ATS, positive TLC ... ... 7 2.3 Quinine positive ATS, negative TLC 16 5.3 ... ... Morphine negative, quinine negative 249 81.9 255 83.9 ATS, Automated turret spectrofluorometric determination of morphine and quinine as described under the Experimental Section. b TLC, Thin-layer chromatographic determination of morphine and quinine following acid hydrolysis (2.5N HCl), solvent extraction, application of residue to silica gel plate, and detection by a series of chromogenic sprays similar to that reported by Mu16 (11).
confirmed by TLC) was 5.3%. This figure is due primarily to the inclusion of suspicious quinine positive samples as determined by the ATS as well as some destruction of quinine by acid hydrolysis prior to thin-layer chromatography. A comparison of the data by the ATS and TLC methods of analysis clearly indicates that less than 0.5 of the urine samples routinely analyzed by the ATS method for morphine and quinine might contain morphine and not be detected. Although the ATS method was primarily developed for routine urinalysis in a large narcotic addiction control program, it certainly is suitable as a simple, rapid, sensitive fluorometric technique for the analysis of morphine from biological materials in studies primarily concerned with either the disposition or metabolism of this drug. RECEIVED for review August 3, 1970. Accepted January 27, 1971. A preliminary report of these experiments was presented at the 32nd annual meeting of the Committee on Problems of Drug Dependence, National Academy of Sciences, National Research Council, February 16-1 7, 1970, in Washington, D. C.
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