VdaPP) = -b,,/2b,2
(17)
where bzl and bzz are obtained from Equation 16 a s bn = Fi / S,
If a similar procedure is followed for a Gaussian function, y = f(u) = a exp[-(u - V ~ ) ~ P ' ~ I (21) in which @' = W W H M , the correction factor becomes
(18) We now expand t as defined in Equation 9 in a Taylor's series about U R = 0, t
=
€0
+ u*&)
+
'..
(19)
VR'0
Since-c" = -uR(app) by Equation 9: Equations 16, 17, and 18 show that t o vanishes for the symmetrical Lorentzian function. By tedious differentiation of t (as given by Equation 9, we get finally .^
J The same coordinate transformations have been used and @ is again the value of p' in units of A .
ACKNOWLEDGMENT Assistance in the early stages of this work from P. M. Fast and D. L. Nordlund is greatly appreciated.
1
(20)
Substitution of this expression into Equation 9 leads to the desired results as given in Equations 10 and 11.
Received for review October 24, 1972. Accepted February 5, 1973. We are grateful to the National Science Foundation for support of this work by grants GP-15127 and GJ32213, and to the University of Kansas Computation Center for computer time on the HW-635. One of us ( U J ) appreciates the support from an NDEA traineeship. Laboratory computer equipment was provided by NSF grant GJ 332.
Screening for Heroin-A Comparison bf Current Methods David Sohn Department of Pathology, New York Medical College Center for Chronic Disease, Welfare Island, New York, N. Y . 1001 7
Julius Simon, Moheeb A. Hanna, Guirguis V . Ghali, Ramadan A. Tolba, and Vahe Melkonian Laboratory for Chromatography, Bayside, N. Y. 11364
Heroin (diacetylmorphine) is excreted as morphine primarily in urine. Two-thirds or more of urinary morphine is glucuronide conjugated. Quinine is a major diluent of heroin. A study is made of 3009 samples positive for quinine among some 50,000 urine samples examined during the first half of 1972. They were analyzed by thin layer chromatography (TLC), spectrophotofluorometry (SPF), and automated morphine analyzer (AMA). Yields of morphine-positive samples were 19.9, 24.3, and 27.0%, respectively, by TLC, AMA, and SPF. Additional studies including efficacy of hydrolysis; gas-liquid chromatography (GLC): and Hemagglutination Inhibition ( H I ) immunoassay are presented. Results indicate the need for hydrolysis in techniques sensitive only to unconjugated morphine: that new columns, with shorter retention times for morphine, may permit GLC to be used for initial screening: and that H I which is also sensitive to conjugated morphine may represent a superior method of screening. Limits of sensitivity were on the order of 0.5, 0.2, 0.1, 0.1, and 0.03 Fg/ml, respectively, for TLC, SPF, AMA, GLC, and HI.
Addiction to heroin, or diacetylmorphine, is a major medical-social problem. In the body, heroin is rapidly deacetylated to morphine. In the human, urine is the major route of excretion. Morphine is excreted either free or conjugated, primarily to glucuronide. 1498
ANALYTICAL CHEMISTRY, VOL. 4 5 , NO. 8, JULY 1973
Studies by Way and Adler ( I ) and others, in man, indicate that over 50% of the total dose is excreted in the urine within the first 8 hr and roughly 90% during the first 24 hr following injection. Free morphine represents between 5 and 20% of the total amount excreted in urine, primarily between 3 and 8 hr after parenteral administration. Measurable urinary free morphine persists for 48 hr or longer. Following the same dose of injected morphine, there are great individual variations in the ratios of free and conjugated morphine a t any given time as well as in the interval during which free morphine is present at detectable levels. The overall sensitivity of any method of analysis depends on whether it is free or total morphine that is identified. The time interval between administration of the drug and sampling is therefore an extremely critical determinant of overall sensitivity. In practice, among a large group of specimens containing morphine, administration of the drug may have taken place from several hours to several days prior to sampling. Therefore, this is a study of distribution within a population and many samples must be studied if different methods of analysis are to be compared. In the basic study presented here, a total of some 50,000 urine samples were reviewed. Current methods of bioanalysis detect either free morphine only or total morphine. Since total morphine levels (1) E. L. Way and T. K . Adler. "The Biological Disposition of Morphine and its Surrogates," W.H.O.. Geneva, 1962.
in urine samples may be 1 pg/ml or less, extraction techniques with organic solvents are frequently employed to concentrate the morphine. In techniques utilizing a partition between urine and organic solvent, conjugated morphine remains in the aqueous phase and only free morphine enters the organic phase. To raise overall sensitivity, hydrolysis of the urine s a p p l e breaks the glucuronide bond and so may increase the free morphine entering the organic phase. Quinine, one of the most common diluents of heroin, is readily detected by many of the same methods of analysis used to detect morphine. After intake of “street” heroin, quinine may be detected in urine for several days after detectable morphine has disappeared. The most common methods of analysis in current use include thin layer chromatography (TLC), gas-liquid chromatography (GLC), spectrophotofluorometry (SPF), automated morphine analysis employing fluorometric techniques (AMA), and most recently, immunologic methods either by radioimmunoassay or by hemagglutination inhibition (HI). One other available immunoassay method-the free radical assay technique-is neither widely used, being used by only 6 of the 155 participating laboratories in the most recent Toxicology Proficiency Testing Program of the Department of Health, Education, and Welfare ( 2 ) , nor is it specific, false positives were reported in Vietnam with Ornade, Tigan, paregoric, and codeine ( 3 ) .
MATERIALS AND METHODS Experience with TLC, GLC, SPF, AMA, and HI will be discussed. Approximately 50,000 urinalyses for heroin (as morphine) were performed during February through May 1972. Since “street” .heroin is commonly diluted with quinine, and since virtually all specimens positive for morphine contained quinine, to study heroin detection, urine samples positive for quinine were selected for statistical analysis. In this interval, 3603 samples contained quinine. During part of this period, technical problems precluded parallel comparisons of some samples and 3009 urine samples positive for quinine were retained. Each of these samples was analyzed by TLC without hydrolysis, by SPF, and by AMA (Table I). TLC, using glass plates precoated with silica gel G, was performed by the authors’ ( 4 ) modification of Davidow’s procedure. SPF determinations employed a Farrand automated turret spectrofluorometer (Farrand Optical Corporation, M t . Vernon, N.Y. 10550) with excitation monochrometer set a t 392 nm to yield a n emission peak for morphine a t 425 nm. The fluorophore was prepared from a n unhydrolyzed extract of urine following the procedure of Mu16 and Husbin ( 5 ) .Prior acid hydrolysis caused quinine destruction and yielded erratic analytic results. The fluorophore derived by this procedure is not pseudomorphine. In Mulb’s work, the minimum concentration reliably detected for morphine in urine was 0.22 pg/ml. Our sensitivities appeared to be in the same range. AMA determinations utilized the Technicon automated morphine analyzer (Technicon Instrument Corp.; Tarrytown, N.Y. 10591). This automated photofluorometric analyzer has claimed sensitivity of 0.2 pg/ml. The fluorophore is pseudomorphine (6). Analysis followed the procedure of the manufacturer employing reagents supplied by the manufacturer. The manufacturer has indicated that a hydrolysis cartridge not now on the market will be available. GLC studies used a Bendix Toxichron or a Bendix Life Sciences gas chromatograph Series 2500 (Bendix Corporation, Ronceverte, W.Va. 24970). The columns were respectively 3 ft X 2 ( 2 ) D. Bayse and D. S. Lewis, “Drug Abuse, Survey I I ; Amphetamine-
Barbiturate-Methadone--Morphine,” Department
(3) (4) (5)
(6)
of
Health, Educa-
tion, and Welfare, Atlanta, June 1972. S. L. Baker, Jr., Arner. J. Pub/. Health, 62, 857 (1972). D. Sohn and J . Simon, Clin. Chem.. 18, 405 (1972). S. J . Muleand P. L. H u s h i n , Anal. Chern., 43, 708 (1971). M . Sansur, A. Buccafuri, and S. Morgenstern, J. Ass. Offic. Anal. Chem., 55,880
(1972)
Table I. Specimens Positive for Morphine among 3009 Specimens Containing Quinine Analyzed from February through June 1972 Total positive (SPF and/or Method TLCa SPF AMA AMA) N. u mber 598 815 731 91 2 Per cent
19.9
27.0
24.3
30.1
a Not hydrolyzed.
Table II. TLC Analysis of 156 Hydrolyzed Urine Samples Containing Conjugated Morphine at Various Concentrations (May 1972)a %of samples in which Morphine concn, pg/ml morphine was detected 0.25 0.50 1 .oo
0 (4)* 87.5 ( 1 6 ) 100.0 ( 9 ) 100.0 ( 4 ) 100.0 ( 3 )
2.00 3.00
a N o morphine was detectable by TLC or A M A before hydrolysis. bTotal number of samples containing morphine at a given concentration (36 of 156 samples contained morphine).
mm i.d. and 6 f t X 4-mm i.d. glass columns packed with 3% OV-7 on 100/120 Gas Chrom Q (Bendix Corporation). The operating conditions were as follows: temperatures, column 240 (260) “C, detector 260 (280) “C, injection port 250 (270) “C; hydrogen flow, 40 (40) ml/min; nitrogen flow, 45 (60) ml/min; air flow, 300 (600) ml/min (4).(Figures in parentheses refer to the 6-ft column.) Immunoassay was by the hemagglutination-inhibition method of Adler (7) using material supplied by him. This antiserum reacts with morphine and with codeine, dihydromorphinone, pethidine, dextromethorphan, and, occasionally, propoxyphene. Sensitivity is approximately 0.03 pg/ml.
RESULTS Among 3009 samples containing quinine, 19.9% were positive for morphine by TLC, 27.0% by SPF, 24.3% by AMA, and 30.1% by either SPF, AMA, or both (Table I). Specimens positive for morphine by TLC were also positive either by S P F or by AMA. Statistical analysis showed significant differences between positive findings by TLC and by S P F with the probability of less than five chances in a thousand. The differences between analysis by AMA and TLC or by AMA and S P F were not statistically significant. TLC sensitivity is meaningful only when measured in terms of total morphine. To evaluate overall TLC sensitivity, urine specimens negative for morphine by TLC prior to hydrolysis were studied. After hydrolysis, concentrations of 0.25 pg/ml were not detectable. But, 87.5% of those with concentrations of 0.5 pg/ml and all of those with higher concentrations were detected (Table 11). Hydrolysis followed the method described by Mule (8). A representative group of 160 urine samples from methadone patients was examined by TLC before and after hydrolysis. Among 42 quinine-containing samples, 62% were positive for morphine before and 78.5% after hydrolysis (Table III). Comparison of TLC, before and after hydrolysis, with AMA and with HI was made in a group of 89 urine specimens positive for quinine. Before hydrolysis, 7.9% of the specimens were positive for morphine, and 25.8% were (7) F. L . Adler and C. Liu, J. lmrnunol., 106, 1684 (1971). (8) S. J. Mul6, Routine Identification of Drugs of Abuse in Human Urine. New York State NACC Testing and Research Laboratory. New York, N.Y., 1971.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, J U L Y 1973
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Table Ill. 160 Urine Samples Analyzed by TLC before and after Hydrolysis (May 1972)a Samples positive for morphineb
Number of total
Before hydroiysis
After hydrolysis
~ncrease
26 62.0
33 78.5
7 16.5
a Hydrolysis by autoclaving: 1 hr at 225 OF, 1 ml concd HCl/lO mi urine. of 42 samples containing quinine.
Table I V . Comparisop of 89 Urine Samples Containing Quinine Analyzed by TLC, before and after Hydrolysis, by AMA, and by HI TLC
TLC-Ha
AMAb
AMAC
HI
Total positive
No. of
positives (Positives/no. containing
7
23
22
38
59
62
7.9
25.8
24.7
42.7
66.3
69.6
quinine) X
100
a After hydrolysis. AMA analysis utilizing a four-line difference as positive (manufacturer’s criteria). AMA analysis at limit of sensitivity (see text). ~
Table V. Morphine Detection: Comparison of AMA and GLC of 70 Urine Specimens Containing Quinine (July-AUguSt 1972) AMA/Hla GLC/Hlb
Positives
65% (13/20)c 83.3% (25/30)c By HI-38 Of 70 (53%) Total ( A M A and/or GLC and/or H I ) 42 of 70 (60%)
AMA/HI ratio of those positive by AMA divided by number positive by HI for specimens analyzed by AMA and by HI *GLC/Hi Ratio of specimens positive by GLC divided by number positive by HI for Specimens analyzed by GLC and by HI Actual numbers of specimens
positive after hydrolysis. There were 24.7% positive by AMA. HI found 66.3% positive. When the AMA charts were reviewed after the HI results were obtained, additional samples were found for a total of 42.7% which did not meet the criteria suggested by the manufacturer (four-line difference or more between the curves generated by the test sample and by its blank) but which showed a definite difference between the two curves recorded by the instrument. This will be called the “limits of sensitivity” in the discussion (Table IV). To compare GLC to AMA and to HI, 70 urine specimens containing quinine were all analyzed by HI and also by GLC and/or AMA. Of the samples, 38 were simultaneously analyzed by all three methods. To compare these methods, ratios AMA/HI and GLC/ HI, representing, respectively, as numerator the number of specimens positive by AMA or by GLC and, respectively, as denominator the total number of specimens positive by HI, were analyzed both by AMA and HI or GLC and HI. The ratios permit an evaluation of GLC and AMA in terms of HI, the procedure common to both. AMA/HI was 65% while GLC/HI was 83.3%. Specimens positive by HI represented 53% of those analyzed (Table V).
DISCUSSION In this study, SPF provided greater yields of morphinepositive specimens than either TLC, without hydrolysis, or AMA (Table I). SPF is a simple method permitting analyses of 500 or more samples per instrument day. 1500
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
Among several hundred compounds analyzed, the only fluorescence products essentially identical to morphine are nalorphine (a morphine antagonist) and normorphine (a morphine metabolite) ( 5 ) . I n the study by Mu16 and Hushin of 51 urine samples containing quinine among 304 analyzed, there was no statistical difference between TLC and SPF ( 5 ) .In our 3009 urines containing quinine, there was a striking contrast (19.9% positive by TLC, 21.0% positive by SPF). SPF gave greater yields than AMA, though not to a statistically significant degree. The AMA instrument costs about five times as much as the SPF but requires no preparation of the sample. I t can analyze some 40 specimens an hour. Preparation for SPF requires an extraction and formation of a fluorophore by autoclaving. One hundred or more samples per hour can be analyzed with it. The differences in yields must be considered in terms of experience with each instrument. The SPF is a well-seasoned instrument. The one in our laboratory had been in operation for well over two years when the present study began. It had been employed in tens of thousands of analyses. The AMA was first available commercially in November of 1971. The one utilized in this study was installed in December 1971. In the period during which the present study was undertaken (February to May 1972), operational problems related both to the instrument and to its components occurred with this instrument. One major operational problem yielded completely unanticipated results. In the AMA procedure ( 6 ) , undiluted, raw urine samples are aspirated by the sampler. Morphine is extracted at pH 9.4 with a mixture of organic solvents. Following two separations, the organic phase is treated with dilute NaOH. After final separation, the aqueous phase is retained, split into two equal fractions, and mixed with buffer of p H 9.4. Potassium ferricyanide solution is added to the “sample” stream, water t o the “blank” stream. “Free” (unconjugated) morphine is converted by the potassium ferricyanide to its highly fluorescent dimer pseudomorphine. The resulting solutions are measured simultaneously in separate fluorometers a t 440 f 20 nm. The fluorescence of the sample and of its background are each recorded in phase on the same chart with a two-pen recorder. Drug-free urine is used to obtain “blank” recordings for both channels. Recorder output is adjusted to render both peak heights identical. Identification of morphine depends on the difference between the increased fluorescence of the pseudomorphine dimer (red pen) and that of the blank (green pen). Standardization employs a high-morphine standard of 2.0 pg/ml adjusted to yield a full-scale deflection, a low-morphine standard of 0.2 pg/ml, and one or more standards of intermediate concentration. The low standard will generally produce a deflection difference of between four or five scale lines, or 5.7% of full-scale deflection on the recording paper provided with the instrument. In practice, the minimum level of detection routinely employed is 0.2 pg/ml for 4- to 5-line differences in deflections. A high blank may be present in the urine of patients receiving drugs other than morphine, particularly phenothiazines, the background fluorescence may be highly variable, and false-positive results would be obtained in certain cases if these were not corrected by the blank (6). Elevated background fluorescence appears related to nonspecific factors including aging of the urine with bacterial contamination and/or suspended crystals coming out of solution or cooling to room temperature. Hydrolysis of urine samples without subsequent “clean up” resulted in extremely high blanks that made analysis impossible
Table VI. Sample and Background Fluorescence and Differences in Fluorescence for Various Specimens Analyzed on AMA
Fluorescencea Sample
Sample
Standard 1 Standard 1 ‘ Drug-free samples for blank ( 6 ) c
117 119
7 9
6 70 26 12 29 121 125 9 96 35 50 70 34 64 63 77 50 40 37 107
5 6 7 8
Standard 2 Standard 2‘ Drug-free samples for blank ( 5 ) c 5’ 6’ 7‘ 8’ 41 43 42 40 57 59 60 61
Background
Fluorescenceb
Differenced
Sample
Background
110 110
125 125
8 8
6 61 19 9 24 9 10
0 9P 7P 3 5P 112 115
9 83 35 12 34 132 135
9 85 21 8 32 9 9
9 72 29 39 64 32 39 47 67 42 36 29 114
0 24P 6P 11P 6P 2 25P 16P 1OP 8P 4P 8P -7
8 67 26 31 55 15 38 33 51 62 35 26 118
8 95 32 40 70 17 19 27 64 74 38 33 118
Differenced 117 117
0 -2 14P 4P 2 123 126 0
- 28 -6 -9 -15 -2 19P 6P -13 -12 -3 -7 0
Measured in scale lines, red-pen fluorometer used for “samples.” Measured in scale lines, green-pen fluorometer used for “samples.” parentheses refers to total number of blanks analyzed. P means that a value of four or more scale units would be read as positive.
a
even following dilution with water or with fresh drug-free urine of low-background fluorescence, procedures which will frequently work well with other urines with high blanks. In some samples of drug-free urine with high-background fluorescence, sample fluorescence exceeded that of the background by the 4- to 5-line difference considered as positive by the critera employed with the instrument. This occurred despite the zeroing of the instrument with urine of low-background fluorescence. The disparity between sample and blank may be due to changes in the relative sensitivities of each of the fluorometers. If the “sample” fluorometer’s sensitivity became even slightly greater than that for the “blank” fluorometer, though each remained linear, for drug-free urine of low blank the curves might be readily superimposed on one another. However, when urines with high-background fluorescence are analyzed. the unequal sensitivities may result in increasing separation of the two curves yielding false-positive results. These may be due to changes in the light sources, the optics, or the electronic circuitry due to aging of components or dust accumulations. If the AMA is the single instrument used for the detection of morphine, spurious identifications might arise despite due care on the part of the operator (following the instructions of the manufacturer) who would be unaware that these false positives were occurring. I t emphasizes the need for any drug identification to be confirmed by separate independent procedures (9). To evaluate these changes, an experimental protocol was followed. The AMA was standardized with known morphine standards, the two recorders adjusted so that drug-free urine had coincident curves on each fluorometer (this step was repeated 5 to 6 times), and urine samples with varying background fluorescence were analyzed. The (9) 6 S Finkle, Anal Chern., 44 (9), 18A (1972)
Number in
final segments of tubing, each terminating in one of the two fluorometers, were then interchanged and the entire procedure was repeated. Results of 16 typical samples are presented in Table VI. Negative differences ( i e . , “blank” curve greater than “sample” curve) are considered negative for the presence of morphine. Substitution of reagents employed with fresh carefully checked reagents did not alter the results. The conclusion to be drawn from these results is that the two fluorometer-pen combinations do not match. To prevent the reporting of false-positive results morphinefree samples with high-background fluorescence should be interspersed with test specimens and also that the fluorometer-pen combination and/or primary filters be interchanged on a regular basis and specimens be reanalyzed and any discrepancies noted. The time of sampling, as has been mentioned, represents an extremely critical determinant of overall yield and, in turn, sensitivity. For the sensitivities developed in Table 11, no morphine a t all was detectable either with TLC before hydrolysis or with AMA. In samples taken more than 24 hr after parenteral administration, this would be the usual situation. I t is postulated that the samples presented in Tables I and I11 are random temporal distributions of free and conjugated morphine as seen in any large group of heroin users. In addition, it is assumed that the samples presented in each of the tables come from the same population. If this is so, then it could be inferred that hydrolysis of the urine samples in the original group of 3009 quinine-containing specimens (and 598 positive for morphine before hydrolysis) would give an increased yield on TLC of specimens positive for morphine comparable to the increased yield following hydrolysis of the 160 samples presented in Table 111. This increment of 16.5% would give overall yields directly comparable and statistically indistinguishANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
1501
able from those of SPF or AMA. This highlights the fact that a relatively insensitive technique like TLC (in comparison to SPF or AMA) may have much higher overall yields when the 75 to 95% of conjugated morphine is hydrolyzed before analysis than would be the yields of more sensitive and efficient instrumental techniques like SPF or AMA which depend on the detection, when hydrolysis is not employed, of the 5 to 25% of free morphine present. I t should also be noted from Table I that the overall total yield for both S P F and for AMA was 30.1% indicating that in significant numbers of specimens only one of these techniques was positive in this series. The difference between TLC and SPF in Table I should not be attributed to inefficient extraction since the results with TLC were statistically within the range of the AMA yield. Extraction would therefore appear to have been adequate. The 89 specimens containing quinine analyzed by TLC, before and after hydrolysis, by AMA, and by HI are compared in Table IV. Though these results are not statistically significant, the yield with HI alone is 66.3% compared with 24.7% with AMA (Table IV). Adler (7) indicates that sensitivities with HI are on the of the minimum order of 0.001 Fg/ml or are about concentration detectable with SPF or AMA. The samples analyzed by HI were predominantly from methadone maintenance program patients. The presence of methadone in these urines and other “blank” uiines from these patients did not interfere with the HI reaction in those samples studied thus far. If HI indeed measures total morphine, hydrolysis would be unnecessary and this technique would be independent of the time elapsed following parenteral administration in terms of the varying ratios of free and conjugated morphine. Review of the AMA recordings with the knowledge of the HI results suggested an additional 16 unhydrolyzed specimens which might be considered positive by AMA. Future sharpening of criteria for identification and introduction of hydrolysis may significantly increase yields with AMA (within the limits of the instrument as indicated above). Comparisons of AMA and GLC were made utilizing the relative yields of each as compared with those with HI as described above (Table V). In the limited group of 70 specimens, of which 38 were positive by HI, and 42 were positive by one or more of the three methods, the relative yield for AMA/HI was 65% while for GLC/HI it was 83.3%. Nonhydrolyzed urine samples were employed. The contrast between AMA and GLC reflects the difference in sensitivities of AMA and GLC, which is on the order of 0.2 Wg/ml with AMA, while GLC is routinely sensitive to nanogram and even picogram quantities of morphine, depending on the detectors employed. We have used GLC primarily for confirmation of compounds such as cocaine, methadone, amphetamine, barbiturates, nonbarbiturate
1502
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
sedatives, and phenothiazines. The relatively long retention times for morphine associated with many liquid phases limited GLC to confirmation rather than initial screening. Experiences with a newly available OV-7 column (Bendix Corporation, Process Instrument Division, Ronceverte, W.Va. 24970) have demonstrated retention times of the order of 4 min with good differentiation of diverse compounds. These columns may permit initial screening by GLC. In this context, we had had more than 2 years of experience with GLC when this study was begun. A major conclusion to be drawn from this progress report is the importance of hydrolysis in studies of urinary excretion of drugs where both free and conjugated compounds are excreted. In these studies of excreted morphine, yields by TLC following hydrolysis appeared to compare favorably with those employing other methods. The sensitivity of a given technique or instrument cannot be equated with anticipated yields of positive specimens unless the drugs to be analyzed are converted to a form (free morphine in this study) which is amenable to analysis. Where hydrolysis was not performed, AMA, a fully automated procedure, requiring no initial sample preparation, compared favorably to other methods within the limitations indicated. GLC, a sensitive technique, had been limited in screening applications primarily to confirmation. Developments in liquid phases may permit shorter retention times and its use in morphine screening. Immunoassay techniques are sensitive to both free and conjugated morphine. In these limited studies (Table IV and V) yields with HI exceeded those with other techniques. These data suggest that where morphine excretion is the single parameter of interest, immunologic techniques, requiring little or no instrumentation, may attain or surpass the instrumental techniques in specificity, sensitivity, high volume potential, and economy. Further experience with these techniques, which must be subjected to confirmation by more specific and sensitive methods such as mass spectroscopy, is necessary to prove the ultimate usefulness of immunoassay methods.
ACKNOWLEDGMENT Material for hemagglutination-inhibition studies was supplied through the courtesy of F. L. Adler of the Public Health Research Institute of the City of New York, New York, N.Y. 10016: Received for review September 11, 1972. Accepted February 1, 1973. This paper was presented a t the 24th National Meeting of the American Association of Clinical Chemists, Cincinnati, Ohio, August 25, 1972.
