Journal of Analytical Toxicology, Vol. 33, September 2009 Determination of Heroin and Its Main Metabolites in Small Sample Volumes of Whole Blood and Brain Tissue by Reversed-Phase Liquid Chromatography– Tandem Mass Spectrometry Ritva Karinen*, Jannike Mørch Andersen, Åse Ripel, Inger Hasvold, Anita Braute Hopen, Jørg Mørland, and Asbjørg S. Christophersen Division of Forensic Toxicology and Drug Abuse, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, 0403 Oslo, Norway Abstract A high-performance liquid chromatography–tandem mass spectrometry (LC–MS–MS) method has been developed for the quantitative analysis of heroin and its major metabolites 6-acetylmorphine, morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood and brain tissue, using 0.1-mL samples. We evaluated this method for analysis of heroin and its metabolites in samples from heroin treated mice. Ice-cold acidic buffer containing sodium fluoride was immediately added to blood and brain homogenate samples. Sample preparation was achieved by protein precipitation on ice-bath, using a mixture of ice-cold acetonitrile and methanol. The supernatant was evaporated to dryness, reconstituted with mobile phase, and injected into the chromatographic system. Separation was performed on a Xterra® C18 column with gradient elution. The MS analysis was performed in positive ion mode, and multiple reaction monitoring (MRM) was used for drug quantification. The limits of quantification for the different opiates varied from 0.0007 to 0.02 mg/L in blood and from 0.002 to 0.06 µg/g in brain tissue. Day-to-day relative standard deviation ranged from 3.1 to 14.5%, and within-day variation ranged from 2.1 to 11.4%. The recoveries were between 80 and 111%. The stability of heroin was tested, and the study showed that heroin is more stable in brain tissue than in blood. Introduction Heroin is a potent narcotic analgesic with substantial potential for abuse. The half-life of heroin is extremely short [approximately 2–5 min (1)]. The compound is rapidly deacetylated to the specific metabolite 6-acetylmorphine (6-AM) both spontaneously (2) and by enzymatic hydrolysis and further to morphine. In humans, morphine is metabolized to morphine3-glucuronide (M3G) and, to a lesser extent, morphine-6glucuronide (M6G) and normorphine (3). In rodents, morphine is preferentially conjugated to M3G (4). * Author to whom correspondence should be addressed. E-mail: [email protected]. Heroin is known to be unstable in both aqueous and organic solutions (2). It has been documented that the rate of deacetylation of heroin to 6-AM in aqueous solution is dependent on pH and temperature, increasing with higher pH and temperature. The breakdown of the drug is significantly inhibited in aqueous solution at pH 4 and at a temperature of 4°C. 6-AM is also unstable in aqueous solutions, but its degradation to morphine is limited under acidic conditions (2). Different techniques such as high-performance liquid chromatography (HPLC) with ultraviolet (UV), diode-array (DAD), and electrochemical (EC) detection (1,4,5) and gas chromatography with nitrogen-phosphorus detection (GC–NPD), mass spectrometry (MS), and tandem MS have been used for analyzing heroin (6–9). LC–MS and LC–MS–MS have been more commonly used recently for simultaneous analysis of heroin and its metabolites in a biological matrix because of their high selectivity and sensitivity (10–12). Many of these methods include a pretreatment of the samples with liquid– liquid or solid-phase extraction and/or derivatization, which is time consuming. In addition, they require relatively large sample volumes (0.25–2.0 mL), which is a disadvantage when the sample size is limited, as in experiments using small animals like mice that are often used in research on drugs of abuse. The aim of our study was, therefore, to develop a sensitive LC–MS–MS method for the determination of heroin, 6-AM, morphine, M3G, and M6G in small samples of blood and brain tissue from mice combined with a sample treatment procedure to minimize the degradation of heroin and 6-AM in biological samples. Experimental Chemicals Methanol (HPLC grade) and acetonitrile (for UV HPLC) were purchased from LAB-SCAN (Dublin, Ireland). AnalaR® ammo- Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. 345 Journal of Analytical Toxicology, Vol. 33, September 2009 methanol, except the solutions for heroin and the morphine glucuronides, which were prepared in acetonitrile and water, respectively. All stock solutions were stored at –20°C. Stock solutions for calibration samples and quality control (QC) samples were prepared independently. Working standard solutions for all compounds, except heroin, were prepared in a 5 mM ammonium formate buffer (pH 3.1). Heroin was prepared separately in the same buffer. Because it was difficult to obtain sufficient amounts of blank blood from mice without sacrificing a large number of animals, human whole blood was used to make blood standards. Spiked blood standards and mice brain tissue homogenate standards were prepared at six concentration levels (one replicate of each level) from the working standard solutions at the concentration range shown in Table I. Stock solutions for the deuterated analytes were prepared in methanol except for the morphine glucuronides, which were prepared in water. An internal standard working solution of each compound was prepared in water (0.5 µM). QC and calibration samples were freshly prepared for each analysis. nium formate was from BDH Laboratory Supplies (Poole, England); heparin sodium was from SIGMA (Sigma-Aldrich Norway AS, Oslo, Norway); and formic acid and sodium fluoride (NaF) were from Merck (VWR International AS, Oslo, Norway). Heroin, M6G, M3G, and 6-AM were supplied by Lipomed (Lipomed, Cambridge, MA). Morphine was from NMD (NMD Grossisthandel AS, Oslo, Norway). M6G-d3, M3G-d3 (0.1 mg/mL in methanol), morphine-d 6 , and 6-AM-d 6 (1 mg/mL in methanol) were supplied by Cerilliant® (Austin, TX). Deionized water was obtained from a Milli-Q UF Plus water purification system (Millipore, Bedford, MA). Human whole blood, used for the preparation of calibration standards and quality control samples, was supplied by the blood bank at Ullevål University Hospital (Oslo, Norway). Blood and brain tissue samples from mice The Norwegian Review Committee for the Use of Animal Subjects approved the experimental protocol of this study. Each mouse (C59BL/6J-Bom) was given an injection (15 µmol/kg, sc) of heroin dissolved in 0.9% NaCl. The injections were given in total volumes of 0.1 mL/10 g mouse. Five minutes after injection the mice were CO2 anesthetized. Blood sampling (500 µL) was performed by heart puncture with a syringe containing 80 µL ice-cold NaF (final concentration 4 mg/mL) dissolved in heparin (100 IE/mL). The blood was transferred to a microcentrifuge tube, and aliquots of 100 µL were taken out into plastic tubes and diluted (1:1, v/v) in ice-cold ammonium formate buffer (5 mM, pH 3.1) containing NaF (final concentration 4 mg/mL). The blood samples were immediately frozen in liquid N2 and stored at –20°C until analysis. After blood sampling, the brain (except cerebellum) was quickly removed, washed in ice-cold ammonium formate buffer containing NaF, blotted on a filter paper, and homogenized with an Ultra Turrax T8 homogenizer (IKA, Jake and Kunkle, Germany) in ice-cold ammonium formate buffer (5 mM, pH 3.1) to a final concentration of 0.33 g tissue/mL homogenate. Thereafter, a 100-µL aliquot of the brain tissue homogenate was mixed with 100 µL of ice-cold formate buffer in a plastic tube. Samples were immediately frozen in liquid N2 and stored at –20°C. The brain homogenate was thawed and frozen once before analysis to break intact cells. Preparation of samples One-hundred microliters of working standard/control solutions was added to an aliquot of 100 µL human whole blood or mice brain tissue homogenate in a plastic tube. Fifty microliters of the internal standard mixture (0.5 µM in water) was added to all samples, followed by immediate agitation on a Multitube vortex mixer. Five-hundred microliters of ice-cold acetonitrile/methanol (85:15) was added to each tube, followed by shaking on a Whirlmixer. The tubes were capped and placed in the deep-freezer at –20°C for a minimum of 10 min, then centrifuged at 4500 rpm (3900 × g) at 4°C for 10 min. The organic phase was transferred to a glass tube and evaporated to dryness at 40°C under a gentle stream of nitrogen. The dry residue was then reconstituted with 100 µL of cold mobile phase (3% acetonitrile/97% 5 mM ammonium formate buffer pH 3.1, v/v), centrifuged, and transferred to auto sampler vials. LC–MS–MS LC was performed using an integrated system from Waters with a 2695 Separations Module. Chromatographic separation was performed at 35°C on a Xterra® MS C18 column (2.1 mm × 150 mm, 3.5-µm particle size) using gradient elution with a mobile phase consisting of 5 mM ammonium formate buffer pH 3.1 (A) and acetonitrile (B). The flow rate was 0.2 mL/min. Preparation of standard solutions Stock standard solutions for all compounds were prepared in Table I. Molecular Weight, LOD, LOQ, Dynamic Range, and Coefficient of Determination (r2) with %CV in Blood (Human and Mouse) and Brain Tissue Samples LOD LOQ Substance Molecular Weight Blood (mg/L) Brain tissue (µg/g) M3G M6G Morphine 6-AM Heroin 461.5 461.5 285.3 327.4 369.4 0.0065 0.00060 0.00049 0.00033 0.00096 0.020 0.0018 0.0015 0.0010 0.0029 346 Blood (mg/L) 0.019 0.0014 0.0012 0.00065 0.0025 Dynamic Range Brain tissue (µg/g) 0.059 0.004 0.0036 0.0020 0.0077 Blood (mg/L) 0.02–11.5 0.002–1.15 0.003–1.43 0.0007–0.16 0.002–0.89 Brain tissue (µg/g) Mean r2 (n = 10) 0.07–35.0 0.007–3.5 0.009–4.3 0.002–0.5 0.006–2.7 0.99973 0.99947 0.99949 0.99541 0.99878 %CV 0.03 0.10 0.06 0.05 0.17 Journal of Analytical Toxicology, Vol. 33, September 2009 A gradient was carried out starting from 3% B, increased to (the same as those used in within-day and between-day varia60% over the next 8 min, and maintained for 2 min, before retion determinations, see Table III) for all compounds. turning to its initial conditions within 0.1 min and equiliStability study of working solutions and sample extracts brating for 5.9 min. Total run time was 16 min. Injection The stability of the working solutions of all compounds at volume was 10 µL. All compounds were eluted within 12 min. three concentration levels was tested by analyzing stored soMS detection was performed on a Quattro Premier XE triplelutions against freshly prepared solutions. The stability of quadrupole MS. Ionization was achieved using electrospray in sample extracts was examined by reanalysis of some QC samthe positive mode (ESI+) and multiple reaction monitoring (MRM). The source block temperature was 120°C, and the capples either after storage in the LC autosampler at 10°C for up illary voltage was 2 kV. The desolvation gas (nitrogen) was to 14 days or after storage in the freezer at –20°C for up to one heated to 400°C, and the flow was set to 1097 L/h. The cone gas month. (nitrogen) was delivered at a flow rate of 48 L/h, and the colliStability study of heroin in authentic samples from mice sion gas (argon) pressure was maintained at 2 psi. Data acquiTwenty mice, given heroin sc, were used to examine the stasition, peak integration, and calculation were interfaced to a bility of heroin in blood and brain tissue homogenate. After computer workstation running MassLynx 4.0 SCN509 software. The masses monitored, along with Table II. LC–MS–MS Method the respective cone voltage, collision energy, and retention times for all analytes, Rt MRM 1* MRM 2 Cone Voltage Collision Energy are listed in Table II. Substance Method validation Quantitative results were obtained by integrating the peak height of the specific MRM chromatogram in reference to the integrated height of the internal standard. A second order calibration curve (y = ax2 + bx + c) was used for quantification because of the wide concentration range (six-point calibration). Origin was excluded and a weighing factor 1/x was used. Limits of detection (LOD) and quantification (LOQ) were determined as a mean of background noise + 3 standard deviations (SD) and + 10 SD, respectively. LOD was confirmed from blank blood samples, spiked with decreasing concentrations of the analytes, where the ion ratio was acceptable (within ± 20% from the mean ion ratio of standards and controls). The ion ratio was calculated as the peak height of the quantification transition divided by the peak height of the qualifier transition. Imprecision and inaccuracy of the method at the LOQ was determined by analyzing blood samples spiked near LOQ (n = 4, three replicates). Within-day and between-day variations were determined by analysis of spiked human whole blood and/or brain homogenate samples at three different concentration levels for all compounds. Extraction recovery and matrix effect were studied using the method developed by Matuszewski et al. (13). For this study, four mouse brain homogenates and mouse blood samples and three human blood samples, two replicates of each, were spiked at three concentration levels (min) (m/z) (m/z) (V) (eV) M3G M6G Morphine 6-AM Heroin† 2.7 4.1 4.6 8.1 9.6 462.0 > 286.0 462.0 > 286.0 286.0 > 201.0 328.0 > 211.0 370.0 > 268.0 462.0 > 268.0 462.0 > 268.0 286.0 > 152.0 328.0 > 165.0 370.0 > 165.0 45 45 45 45 50 30/30 30/30 20/40 25/40 30/40 M3G-d3 M6G-d3 Morphine-d6 6-AM-d6 2.7 4.1 4.8 8.1 465.0 > 289.0 465.0 > 289.0 292.0 > 201.0 334.0 > 211.0 50 50 45 45 30 30 25 25 * First transition (MRM 1) was used for quantification. † 6-AM-d was used for internal standard. 6 Table III. Within-Day Precision (%CV), Between-Day Precision (%CV) and Bias (%) of the QC Samples (n = 10) Concentration Blood (mg/L) Brain tissue (µg/g) Within-Day Precision (%CV) (Blood) M3G 0.032 0.32 3.2 0.098 0.98 9.79 2.9 3.3 5.1 10.2 6.1 4.9 6.0 8.8 3.6 M6G 0.0032 0.032 0.32 0.0098 0.098 0.98 3.7 2.4 4.1 7.0 3.8 3.1 4.4 8.2 4.7 Morphine 0.0043 0.043 0.43 0.013 0.13 1.30 5.7 2.1 11.4 5.5 6.1 4.3 1.1 3.0 -0.4 6-AM 0.0010 0.010 0.10 0.0030 0.030 0.30 4.6 4.0 5.9 10.5 14.5 8.9 0.4 –7.1 –15.0 Heroin 0.0052 0.016 0.16 0.016 0.047 0.47 5.7 4.7 10.2 11.3 6.9 12.8 1.9 0.5 8.9 Substance Between-Day Precision (%CV) (Blood and brain tissue) Bias (%) 347 Journal of Analytical Toxicology, Vol. 33, September 2009 sampling, the samples were stored at –20°C. Aliquots of 100 µL were analyzed 3 h, 1 day, and 1 week after the experiment was performed. Statistical analysis was performed using paired samples T-test. A value of p < 0.05 was considered statistically significant. LC–MS–MS instrument without any further clean-up of the samples. HPLC separation and MS–MS methods A sufficient separation of all the compounds was achieved within 12 min. Gradient elution ensured a satisfactory separation to allow time-programmed MS detection, that is, detection Results and Discussion windows according to the retention time of different compounds. Sample preparation LC–MS–MS was carried out using the electrospray ionisation Sample preparation was achieved by simple precipitation of technique (ESI) in positive mode and multiple reaction monmacromolecules by addition of ice-cold acetonitrile/methanol itoring (MRM). The MS–MS detector was programmed to demixture to 100 µL whole blood or brain tissue homogenate. tect two ion transitions of the compounds within a time frame The recoveries of all the different compounds were found to where the compounds were known to elute. For the correbe between 78 and 111% (Table IV, mean of the three consponding deuterated analogues, only one transition was moncentration levels tested). The recoveries of the deuterated initored. ternal standard analogues were of similar values. The superIn LC–MS–MS time segment 1, the following compounds natant, after centrifugation, was injected directly into the were detected: morphine, M3G, and M6G. 6-AM and heroin were detected in MS–MS time segment 2. Chromatograms of blank, calibrator, and Table IV. Recovery (%), Matrix Effect (%), and Corrected Matrix Effect (%) in an authentic sample are shown in Figure 1. Blood (Human and Mouse) and Brain Tissue (Mouse)* Substance Recovery (%) Matrix Effect (%) Corrected Matrix Effect (%) 97 102 104 M3G Brain samples Blood samples (mice) Blood samples (human) 93 80 87 99 113 92 M3G-d3 Brain samples Blood samples (mice) Blood samples (human) 92 78 84 102 111 88 M6G Brain samples Blood samples (mice) Blood samples (human) 107 88 87 331 424 529 M6G-d3 Brain samples Blood samples (mice) Blood samples (human) 107 88 84 334 417 545 Morphine Brain samples Blood samples (mice) Blood samples (human) 87 100 104 108 125 161 Morphine-d6 Brain samples Blood samples (mice) Blood samples (human) 101 98 101 84 121 144 6-AM Brain samples Blood samples (mice) Blood samples (human) 111 108 110 105 120 144 6-AM-d6 Brain samples Blood samples (mice) Blood samples (human) 112 107 107 105 120 124 Heroin Brain samples Blood samples (mice) Blood samples (human) 106 97 –† 124 124 –† * Mean calculated from three concentration levels. † Not carried out. 348 99 102 99 128 103 111 100 101 108 124 105 Validation A six-point calibration curve was set up for the concentration ranges listed in Table I. The calibrations curves were found to be reproducible in the concentration ranges listed and with correlation coefficients greater than 0.995 for all the analytes. Between-day and within-day precisions for all compounds were determined at three concentration levels. The results from the between-day variation, within-day precision, and bias are shown in Table III (n = 10). Day-to-day variations were in the range 3.1–14.5%, and intraday variations at the same concentration levels were between 2.1 and 11.4%. The method was found to be highly reproducible for all compounds. Whole blood samples from humans and mice, and brain tissue homogenates from mice were used for determination of LOD and LOQ. The imprecision and inaccuracy at the LOQ was within ± 11.7% from the nominal value with a %CV within ± 0.3% for all compounds. There was no significant difference in background noise between human blood, mouse blood, and brain tissue homogenate samples. LOD, LOQ, dynamic range, and coefficient of determination (r2) with %CV are shown in Table I. LOQ for all compounds were found to be satisfactory for research purposes. The results from the matrix effect experiment are shown in Table IV. The table shows that the analytes, except M6G and M6G-d6, had no or only minor ion en- Journal of Analytical Toxicology, Vol. 33, September 2009 hancement/suppression. M6G and its deuterated analogue had very large ion enhancement. When the results were corrected against their deuterated analogues (response used in calculations) the matrix effects were eliminated. However, some ion enhancement was observed for morphine and heroin (internal standard for heroin was 6-AM-d6) in brain samples. Stability of working solutions and sample extracts Working solutions were found to be stable for at least three months when stored at 4°C. No significant difference was found for any compound in sample extracts after storage in the auto sampler at 10°C for up to 14 days or after freezing for up to one month. The results from these studies are not shown. The 6-AM concentration in spiked heroin standard extracts was also examined (n = 5). The results are shown in Figure 2. Blood and brain extracts were found to contain approximately 3–11% and 0.5–9% 6-AM, respectively. Heroin dissolved in physiological saline, used for injections, was stable for at least 10 h (results not shown). Measured value (µM) Figure 1. MRM chromatograms of extracted blank blood sample from human (A), QC sample from human (B), and blood sample from mouse taken 5 min after injection of heroin (15 µmol/kg, sc) (C). Theoretical value (µM) Figure 2. Mean concentration of heroin and 6-AM measured in extracted standards for brain and blood spiked with heroin (n = 5). Stability of heroin in authentic samples The stability of heroin in blood and brain tissue homogenate samples stored at –20°C from 20 mice injected with heroin is shown in Figure 3. There was no difference in the heroin concentration in brain tissue homogenates analyzed 3 h, 1 day, and 1 week after sampling, indicating high stability. However, in blood the heroin concentration fell significantly from the first measurement at 3 h to day 1 and further from day 1 to 1 week. Low pH, low temperature and NaF were used to minimize 349 Heroin (µg/g) Heroin (µg/g) Journal of Analytical Toxicology, Vol. 33, September 2009 Time Time p < 0.05 from first measurement; # p < 0.05 from second measurement (paired samples T-test) Figure 3. Concentration of heroin in brain tissue homogenate (n = 20) and blood (n = 19) samples from mice injected with heroin in vivo (15 µmol/kg, sc) and analyzed after storage at –20°C for 3 h, 1 day, and 1 week. The results are mean + SD. deacetylation of heroin after sampling. This treatment was not effective in stabilizing heroin in blood. This result supports an earlier report of different esterases in the two tissues (1). Conclusions A reversed-phase HPLC–MS–MS method with high precision, selectivity, and sensitivity for analysis of heroin and the major metabolites 6-AM, morphine, M6G, and M3G in small samples of whole blood and brain tissue homogenate has been developed and validated. Sample preparation was a simple protein precipitation, which was timesaving. The method was found to be highly reproducible. Use of sodium fluoride combined with immediate freezing, followed by sample preparation at low temperature, and low pH was found to stabilize heroin in the brain tissue. For blood samples, this treatment was less effective. We, therefore, recommend that blood samples are extracted as soon as possible after collection because of the instability of heroin. Acknowledgments The authors would like to thank Elisabeth Leere Øiestad for valuable comments and for critical reading of the manuscript. References 1. A.Y. Salmon, Z. Goren, Y. Avissar, and H. Soreq. Human erythrocyte but not brain acetylcholinesterase hydrolyses heroin to morphine. Clin. Exp. Pharmacol. Physiol. 26: 596–600 (1999). 2. D.A. Barrett, A.L. Dyssegaard, and P.N. Shaw. The effect of temperature and pH on the deacetylation of diamorphine in aqueous solution and in human plasma. J. Pharm. Pharmacol. 44: 606–608 (1992). 3. R.E. Aderjan and G. Skopp. Formation and clearance of active and inactive metabolites of opiates in humans. Ther. Drug Monit. 20: 561–569 (1998). 350 4. M. Handal, M. Grung, S. Skurtveit, A. Ripel, and J. Mørland. Pharmacokinetic differences of morphine and morphineglucuronides are reflected in locomotor activity. Pharmacol. Biochem. Behav. 73: 883–892 (2002). 5. D. Bourquin, T. Lehmann, R. Hämmig, M. Bührer, and R. Brenneisen. High-performance liquid chromatographic monitoring of intravenously administered diacetylmorphine and morphine and their metabolites in human plasma. J. Chromatogr. Biomed. Sci. Appl. 694: 233–238 (1997). 6. B.A. Goldberger, W.D. Darwin, T.M. Grant, A.C. Allen, Y.H. Caplan, and E.J. Cone. Measurement of heroin and its metabolites by isotope-dilution electron-impact mass spectrometry. Clin. Chem. 39: 670–675 (1993). 7. J.G. Guillot, M. Lefebvre, and J.P. Weber. Determination of heroin, 6-acetylmorphine, and morphine in biological fluids using their propionyl derivatives with ion trap GC–MS. J. Anal. Toxicol. 21: 127–133 (1997). 8. P. Kintz, P. Mangin, A.A.J. Lugnier, and A.J. Chaumont. Simultaneous identification and quantification of several opiates and derivatives by capillary gas chromatography and nitrogen selective detection. Int. J. Legal Med. 103: 57–62 (1989). 9. A. Polettini, A. Groppi, and M. Montagna. Rapid and highly selective GC/MS/MS detection of heroin and its metabolites in hair. Forensic Sci. Int. 63: 217–225 (1993). 10. S.G. Phillips and K.R. Allen. Acetylcodeine as a marker of illicit heroin abuse in oral fluid samples. J. Anal. Toxicol. 30: 370–374 (2006). 11. E.J. Rook, M.J.X. Hillebrand, H. Rosing, J.M. van Ree, and J.H. Beijnen. The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by highperformance liquid chromatography coupled with tandem mass spectrometry. J. Chromatogr. B 824: 213–221 (2005). 12. P. Zuccaro, R. Ricciarello, S. Pichini, R. Pacifici, I. Altieri, M. Pellegrini, and G. D’Ascenzo. Simultaneous determination of heroin 6-monoacetylmorphine, morphine, and its glucuronides by liquid chromatography–atmospheric pressure ionspray-mass spectrometry. J. Anal. Toxicol. 21: 268–277 (1997). 13. B.K. Matuszewski, M.L. Constanzer, and C.M. Chavez-Eng. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal. Chem. 75: 3019–3030 (2003). Manuscript received December 16, 2008; revision received May 6, 2009.
© Copyright 2024