and the extract was divided into two equal portions. One portion was assayed directly for heroin; detector response was linear over a concentration range.
CLIN. CHEM. 39/4,
670-675
Measurement Spectrometry Bruce Edward
of Heroin
A. Goldberger,’ J. Cone”3
A solid-phase
(1993)
and Its Metabolites D. Darwin,’
William
extraction
procedure
Terrance
was developed
for the
isolation
of heroin, 6-acetylmorphine, and morphine from blood, plasma, saliva, and urine with subsequent assay by gas chromatography/mass spectrometry. Aprotic solvents, mild elution conditions, and an enzyme inhibitor were used to ensure maximum analyte stability. Samples were extracted and the extract was divided into two equal portions. One portion was assayed directly for heroin; detector response was linear over a concentration range of 1.0 to 250 g/L. The second part of the extract was reacted with N-methyl-bis-trifluoroacetamide and assayed for the tnfluoroacetyl derivatives of 6-acetylmorphine and morphine; concentration range
detector response was linear over a of 1.0 to 500 p/L. The limit of 1.0 p.g/L for each analyte. Hydrolysis of
sensitivity was heroin to 6-acetylmorphine during extraction and analysis was 10% of the heroin was converted to 6-acetylmorphine. In addition, an elution solvent commonly used with other SPE protocols (17, 18), methylene chloride/isopropanollammonium hydroxide (80/20/2 by vol), promoted heroin hydrolysis. Consequently, we implemented the following modifications to the extraction procedure: (a) acetonitrile was substituted for methanol as the standard solution and sorbent wash solvent; (b) an acetate buffer with a pH of 674
CLINICAL
CHEMISTRY,
Vol. 39, No. 4, 1993
6.0 was used; and (c) the elution solvent was modified to eliminate isopropanol and amnionium hydroxide. In addition, sodium fluoride, an inhibitor of esterase enzyme systems, was added to the acetate buffer to prevent in vitro enzymatic hydrolysis of heroin and 6-acetylmorphune. Finally, because heroin standards were unstable in solution, we prepared fresh standards with each analytical batch. GC/MS optimization. To increase the assay response to heroin, we performed daily mass ion defect corrections for all analytes. The electron multiplier voltage was increased by 600 to 800 eV above the tune value and the mid-mass range of the mass spectrometer was optimized with modified autotuning procedures (Hewlett-Packard “UserTune”). In addition, daily GC/MS maintenance was performed, which included clipping the GC column and replacing the injector septum, liner, and gold-plated seal. Identification of heroin in biological tissues. There has been limited progress in the development of a GCIMS assay for heroin in biological specimens. Goldberger et al. (15) reported an assay for quanti1ying heroin and metabolites in hair collected from heroin users. The assay was sensitive to 0.05 pg/g of hair (with a 100-mg sample), but conditions were not optimized for heroin stability. In addition, the assay was subject to interference from biological background. The present assay was optimized for heroin stability and was free from background interference. Previously, the lack of a reliable assay for heroin led toxicologists to rely on detection of 6-acetylmorphine for evidence morphine approach
of heroin use (5, 15, 19-22). Because is a unique metabolite of heroin, also was used to distinguish heroin
6-acetyla similar use from
the use of other opium products such as codeine, morphine, and peppy seeds (19, 22). Application of the present assay provides direct identification of heroin and 6-acetylmorphine in biological specimens. Undoubtedly, the pharmacological effects of heroin are due in part to the combined effects of the active metabolites, 6-acetylmorphine and morphine. Previous pharmacodynamic evaluations of heroin have been confined to the study of heroin metabolites only; consequently, the pharmacological role of heroin is still unclear. The present assay of heroin, 6-acetylmorphine, and morphine should allow a complete evaluation of the pharmacological effects of the parent drug and its active metabolites. This
study
Research
was
funded,
in part,
by a grant
from
the
National
Council.
References 1. Sawynok
J. The therapeutic use of heroin: a review of the literature. CanJ Physiol Pharmacol 1986;64:1-6. 2. Annual emergency room data (1990), series I, no. 10-A. U.S. Department of Health and Human Services Publication No. (ADM) 91-1839. Washington, DC: DHHS, 1991. 3. Annual medical examiner data (1990), series I, no. 10-B, U.S. Department of Health and Human Services Publication No. (ADM) 91-1840, Washington, DC: DHHS, 1991. 4. Baselt RC, Cravey RH. Disposition of toxic drugs and chemicals in man. Chicago: Yearbook Medical Publishers, 1989. 5. Cone EJ, Welch P, Mitchell JM, Paul BD. Forensic drug testS
pharmacological
for opiates: I. Detection of 6-acetylmorphine in urine as an indlicator of recent heroin exposure; drug and assay considerations and detection times. J Anal Toxicol 199 1;15:1-7. 6. Way EL, Kemp JW, Young JM, Grassetti DR. The pharmacologic effects of heroin in relationship to its rate of biotransformation. J Pharmacol Exp Ther 1960;129:144-54. 7. Way EL, Young JM, Kemp JW. Metabolism of heroin and its pharmacologic implications. Bull Narc 1965;17:25-.33. 8. Yeh SY, McQuinn RL, Gorodetzky CW. Identification of diacetylmorphine metabolites in humans. J Pharm Sci 77;66:201-4. 9. Garrett ER, GOrkan T. Pharmacokinetics of morphine and its surrogates 11: methods of separation of stabilized heroin and its metabolites from hydrolyzing biological fluids and applications to protein binding and red cell partition studies. J Pharm Sci 1979; 68:26-32. 10. Elliott HW, Parker KD, Wright JA, Nomof N. Actions and metabolism of heroin administered by continuous intravenous infusion to man. Clin Pharmacol Ther 1971;12:806-14. 11. Yeh SY, McQuinn RL. GLC determination of heroin and its metabolites in human urine. J Pharm Sci 1975;64:1237-9. 12. Smith DA, Cole WJ. Rapid and sensitive gas chromatographic determination of diacetylmorphine and its metabolite monoacetylmorphine in blood using a nitrogen detector. J Chromatogr 1975; 105:377-81. 13. Umans JG, Chiu TSK, Lipman RA, Schultz MF, Shin 5, Inturi-isi CE. Determination of heroin and its metabolites by high-performance liquid chromatography. J Chromatogr 1982; 233:213-25. 14. Moore A, Bullingham R, McQuay H, Allen M, Baldwin D, Cole
CLIN.
CHEM.
39/4,
Differences for Asthma Phenotypes M.
Christine
675-679
‘Department Africa. 2Albert Bronx,
NY.
South
Africa.
Samuel
B. Reichberg,2
Claudina
of Medicine, 7 York
Einstein
June
University of the Witwaterarand Rd., Parktown, Johannesburg, 2193, South
College
Centre
Received
high-performance
liquid chromatography
for
22, 1992;
with fluorescence
detec-
tion. Anal Chim Acta 1985;170:13-20. 22. MUle SJ, Casella GA. Rendering the “poppy-seed defense” defenseless: identification of 6-monoacetylmorphine in urine by gas chromatography/mass spectrometry. Clin Chem 1988;34: 1427-30.
in Elastase-Binding Activity of a1-Protease Patients and Control Subjects with Various Gailard,’
School,
1991;15:226-31. 16. Chen BH, Taylor EH, Pappas AA. Comparison of derivatives for determination of codeine and morphine by gas chromatography/mass spectrometry. J Anal Toxicol 1990;14:12-7. 17. Huang W, Andollo W, Hearn WL. A solid phase extraction technique for the isolation and identification of opiates in urine. J Anal Toxicol 1992;16:307-10. 18. Fuller DC, Anderson WH. A simplified procedure for the determination of free codeine, free morphine, and 6-acetylmorphine in urine. J Anal Toxicol 1992;16:315-8. 19. Paul BD, Mitchell JM, Mell LD, Irving J. Gas chromatography/ electron impact mass fragmentometric determination of urinary 6-acetylmorphine, a metabolite of heroin. J Anal Toxicel 1989;13:2-7. 20. Fehn J, Megges G. Detection of 06-monoacetylmorphine in urine samples by GC/MS as evidence for heroin use. J Anal Toxicol 1985;9:134-8. 21. Derks HJGM, Van Twillert K, Zomer G. Determination of 6-acetylmorphine in urine as a specific marker for heroin abuse by
(1993)
Forty-two adult patients with asthma and 30 control subjects were investigated for elastase-binding capacities of a1-protease inhibitor and a2-macroglobulin in plasma. The binding activities of a1-protease inhibitor and a2macroglobulin in asthmatic patients with the M phenotype for the a1-protease inhibitor differed in their relationship to the values in control subjects with the same phenotype [less a1-protease inhibitor for asthmatics (35.1 ± 1.8) than for controls (42.9 ± 2.0 kU/L) (P
