Simultaneous Determination of Heroin, 6-Monoacetylmorphine ...

Journal of Analytical Toxicology,Vol. 21, July/August1997

SimultaneousDetermination of Heroin, 6-Monoacetylmorphine, Morphine, and its Glucuronides by Liquid ChromatographyAtmospheric Pressureionspray-MassSpectrometry R Zuccaro 1, R. Ricciarello 2, S. Pichini 1, R. Pacifici 1, I. Altieri 1, M. Pellegrini 1, and G. D'Ascenzo 2 IClinical Biochemistry Department, Istituto Superiore di SanitY, Roma, Italy and 2Chemistry Department, University "La Sapienza'; Roma, Italy

[ Abslract ] A new analytical technique has been developed for the simultaneous

determination of heroin, 6-monoacetylmorphine, morphine, morphine-6- and 3-g|ucuronides, and codeine in serum using liquid

chromatography coupled with ionspray mass spectrometry. The analytes and the internal standard, nalorphine, were subjected to solid-phase extraction (SPE) using ethyl SPE columns before chromatography. The chromatographic separation of the analytes was achieved using a normal phase column and a water-methanol-acetonitrile-formic acid mobile phase at a flow rate of 230 IJL/min. The mass spectrometer was operated in selected-ion monitoring mode. Under these conditions, the limit of quantitation was 0.5 ng/mL for heroin, 4 ng/mt for 6-monoacetylmorphine, 4 ng/ml_ for morphine, 1 ng/mL for morphine-3-glucuronide, 4 ng/mL for morphine-6-glucuronide, and 4 ng/mL for codeine. Serum levels of heroin metabolites were determined in C57BL/6 inbred mice after a dose of 20 mg/kg heroin administered subcutaneously. 6-Monoacetylmorphine showed a peak concentration of 0.93 pg/mL serum at 3 rain, whereas morphine and morphine-3-glucuronide achieved their peak concentrations of 9.6 and 2.9 pg/mL serum at 10 and 20 rain, respectively. Finally, the absence of morphine-6-glucuronide and codeine excluded the possibility of their formation from morphine in this animal model.

Introduction Heroin (3,6-o-diacetylmorphine, diamorphine, DAM) is the opioid most commonly sold on the illicit drug market. Heroin has an extremely short half-life (approximately 5 min) and is rapidly metabolized by deacetylation to 6-monoacetylmorphine (6-MAM)and further to morphine. Morphine is metabolized by conjugation to morphine-3-glucuronide (M3G)and morphine-6glucuronide (M6G) and is present in blood and urine for prolonged periods of time (Figure 1) (1). Although a number of methods exist to determine heroin, 6-MAM, and morphine (2,3) or morphine and its glucuronides (4-8), only one analytical assay for simultaneous determination of heroin, 6-MAM,and morphine and its glucuronides has 268

been reported in the literature recently because of the differences in the physicochemical characteristics of the various analytes (9). Liquid chromatography (LC)appears to be the technique that can separate both the Zipophilicand the hydrophflic anaZytes without any pretreatment (10). However,it remains difficult to find a detection method among the detectors commonly used with LC (ultraviolet, amperometric, coulometric, fluorimetric, etc.) that gives maximum sensitivity for all the substances. In the past few years, considerable research has been carried out on high-performance liquid chromatography-mass spectrometry (HPLC-MS) coupling. Indeed, MS, used as a detector, offers the characteristics of universality and selectivity, even if HPLC-MS can suffer from a lackof sensitivity. SeveralHPLC-MS interfaces were developed to solve this problem, and the atmospheric pressure ionspray interface, which allows the direct passage from atmospheric pressure to the sorgent vacuum, proved useful to achieve sensitivities similar to those of gas chromatography-mass spectrometry (GC-MS) (11). The present paper describes a method for the simultaneous determination of heroin, 6-MAM,morphine and its glucuronides, and codeine using LC coupled with atmospheric pressure ionspray-MS and its application on serum samples from herointreated C57BL/6 inbred mice. Furthermore, the pharmacokinetics of all the metabolites of heroin in this simple animal model were investigated for the first time.

Materials and Methods Chemicals Heroin free base, morphine-HC1, codeine-HCl, M3G, nalorphine-HCl, and 6-MAMwere purchased from Salars (Como, Italy). M6G was obtained from Sigma (Poole, U.K). Methanol and acetonitrile were supplied by Farmitalia-Carlo Erba. All other reagents were of analytical grade from Farmitalia-Carlo Erba. Ethy] solid-phase extraction columns (1-mL volume, 100 mg sorbent) were from J.T. Baker (Milan, Italy).

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JournalofAnalyticalToxicology,Vol.21,July/August1997

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Journal of Analytical Toxicology, Vol. 21, July/August 1997

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Journal of Analytical Toxicology, Vol. 21, July/August1997

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Table I. Intraday and Interday Retention Time Reproducibility of Analytes in Selected-Ion Monitoring

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Ion

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* Abbreviations:6-MAM,6-monoacetylmorphine;M3G,morphine-3-glucuronide; M6G,morphine-6-glucuronide.

Standards preparation

Solutions of stock reference standards (1 mg/mL, 10 pg/mL, and 1 pg/mL ) were prepared in methanol and stored below 0~ Dilutions were freshly made daily for each analysis. A methanol-diluted mixture of all the analytes was used to investigate intraday and interday retention time reproducibility. Serum standards were prepared daily by adding known amounts of stock standards to drug-free serum; these standards were used to create HPLC-MS calibration curves as a control and to determine analytical recoveries and intraday and interday variabilities. The two latter parameters were de-

termined from analyses of fivespiked serum samples performed for up to six days. Instrumentation and conditions Chromatography was performed using a model 140B dual-

syringe solvent-delivery pump (Applied Biosystems, Norwalk, CT) equipped with a model 8125 Rheodyne valve (Rheodyne, Berkeley,CA).Chromatographic separation was achieved using a Supelcosil LC-Si (25 crn x 2.l-ram i.d., 5-pm particle size, Supelco, Bellefonte, PA). The column was pretreated for 24 h with water-methanol (50:50, v/v) at a flow rate of 230 pL/min. The mobile phase consisted of methanol-acetonitrilewater-formic acid (59.8:5.2:34.65:0.35, v/v/v/v). Separation was achieved by isocratic solvent elution at a flow rate of 230 pI,/min split to 46 pL/min before the atmospheric pressure ionization source of the MS. An API I MS (Perkin-Elmer, Norwalk, CT) was used for signal detection. The API system consisted of a single quadrupole MS with an articulated ionspray inlet designed for the introduction and ionization of the sample in a flowing liquid stream. The sample flow was introduced through a silica thread 100 r in length and 100 pm in diameter. The MS was operated with a capillary tip voltage of 5000 V. Sample molecules were ionized by reagent ions created by the corona discharge between the needle tip and the interface plate, which was set at 650 V. The orifice for separating atmosphere and vacuum was maintained at 50 V for M3G and M6G, and at 70 V for nalorphine, morphine, heroin, and codeine. Vacuum pressure inside the quadrupole chamber was 1.8 x 10-s torr. Source temperature 273


voltage to apply, the behavior (molecular peak and possible fragmentation) of all the analytes as this parameter varied was investigated. The mass spectra of individual analytes at various orifice voltages (50, 70, 90 V) obtained with a dwell time of 1 ms and a 0.1 ainu are shown in Figures 2-8. The soft ionization due to instrumental assembly did not allow extensive fragmentation; hence, only mass-to-charge ratios corresponding to molecular ions and one or two fragments were obtained. Under our working conditions, the mass spectrum of heroin displays a molecular peak corresponding to the adduct [M+H]§ (m/z 370) and a very weak signal of molecular ion adducted with sodium (Figure 2). Similarly, the mass spectrum of 6-MAMdisplayeda molecular peak corresponding to the adduct [M+H]+ (m/z 328) and a very weak signal of molecular ion adducted with sodium (Figure 3). The mass spectrum of the morphine displays a molecular peak [M+H]§ m/z 286 accompanied by two other peaks at rn/z 163 and 215. These are found also in the spectra of nalorphine, codeine, and heroin, which refer, respectively, to the methanol clusters (Figure 4). The peak at m/z 279 common to all the spectra is due to the formation of clusters. A signal of [M+H]§ m/z 462 was obtained for morphine-6glucuronide, and m/z 484.0 was obtained for the sodium adduct

was set at 60~ The ions were generated using nitrogen gas. These parameters were checked daily and modified slightly when needed to achieve maximum sensitivity. Signal optimization was achieved by using a polypropylene-glycolsolution as described in the operating handbook. The mass spectra were obtained using an infusion pump at a flow rate of 10 IJL/min in positive ion mode [M+H]§ The analyte standards were dissolved in methanol without the addition of formic acid because heroin is a chemically unstable compound that is hydrolyzed at low pH values. It was therefore deemed inappropriate to use acid solvents for standard solution preservation. The soft ionization obtained using the ionspray source yielded a strong molecular peak signal and a drastically reduced fragmentation, as can be seen in the mass spectra (Figures 2-8). The mobile phase flow rate has no particular effect on detector response (10, 30, 50 IJL/min). Therefore, a flow rate of 10 IJL/min was used to obtain the spectra. In order to optimize the signal versus tip voltage, the standards (1 ng/1JL)were injected in triplicate in flow-injected analysis mode using a water-methanol (50:50, v/v) mobile phase at a flow rate of 10 IJL/min. The tip voltage value that yielded maximum sensitivity was found to be 5000 V. In order to select which orifice .

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Time (min) Time (min) Figure 9. Selected-ion monitoring chromatograms of methanolic standard mixture of all the analytes under investigation (2 pg/mL) (A) and extract of serum samples from mice 10 rain after the treatment with 20 mglkg heroin (B). 6-Monoacetylmorphine (0.2 iJglmL), morphine (2.9 pg/mL), morphine-3-glucuronide (3.4 IJglmL), and nalorphine (internet standard 2 IJglmL).

274

Journal of Analytical Toxicology, Vol, 21, July/August 1997

of [M+Na]§ (Figure 5). For M3G, a mass spectrum with a molecular peak ofm/z 462 was also obtained for the hydrogen adduct and a peak of m/z 484 for the sodium adduct (Figure 6). The fragmentations of these two morphine metabolites do not differ appreciably. In the case of both M3G and M6G, a fragment with m/z 286 that is due to the loss of a glucoronide fragment is produced by increasing the orifice voltage. This fragment had the same mass-to-charge ratio as the molecular peak of morphine. Codeine displays a typical peak at rn/z 300. There are three additional characteristic signals: the sodium adduct (m/z 322) and the two solvent peaks (Figure 7). The mass spectrum of nalorphine displays a molecular peak [M+H]§ at m/z 312. Also present are peaks at m/z 215, m/z 163, and rn/z 288, which were probably due to impurities in the standard (Figure 8). The chromatographic separation was achieved with a silicabased column and a mobile phase commonly used for reversedphase chromatography. For this reason, the column had to be initially pretreated with water-methanol (50:50 v/v) for 24 h to saturate silica hydrophilic sites. The water-methanol mixture was also used at the end of each day to store the column (30 rain at a flow rate of 230 pL/min). Furthermore, in order to assess the stability of the stationary phase in the column, intraday and interday retention time reproducibilities of the analytes were determined. For the purpose of investigating intraday retention time reproducibility, 15 injections (each of 2 lag/mL standard mixture) were made in the course of the day. The mean and standard deviation of the retention times were then calculated. Interday retention time reproducibility

Table II. Analytical Recoveries of All the Analytes from Serum Samples and the Intraday and Interday Coefficients of Variation (CV)* Concentration

Recovery (mean • S.D.)

CV (%)

Intraday Interday

(ng/mL)

(%)

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72.0 + 3.5 74.8 + 2.7

4.8 3.6

5.3 4.0

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99.5 + 1.0 99.8 + 0.9

1.0 1.0

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99.0 • 1.7 99.2 • 1.1

1.7 1.1

2.2 1.6

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77.2 • 4.8 79.6 _+.2.9

6.2 3,6

6.7 4.0

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43.8 _+2.5 44.6 • 1.8

5.6 4.0

6.2 4.5

Codeine 10 500

99.3 • 1.1 99.6 • 0.9

1.1 0.9

1.6 1.2

* n=15.

was evaluated by injecting the standard mixture (2 pg/mL) in triplicate for a period of one week (Table I). The results obtained showed a good stability in the stationary phase in the column.

Extraction of biological samples A 1-mL aliquot of serum with 200 pL of nalorphine (1 pg/mL aqueous solution) added was extracted using ethyl solid-phase extraction columns. The columns were conditioned with two column volumes of methanol, one column volume of water, and two column volumes of 0.001M ammonium hydrogen carbonate buffer (pH 9.3). Serum samples were loaded onto the column, washed with one column volume of 0.001M buffer, and eluted with one column volume of methanol. The eluate was evaporated to dryness under nitrogen at room temperature and redissolved in 100 IaL of mobile phase, and a 5-1JLaliquot was injected onto the HPLC column. Pharmacokinetic studies in mice C57BL/6 Inbred mice (Charles River, Calco, Como, Italy), 8--10 weeks of age, 18-20 g in weight, were treated with heroin free base at a dose of 20 mg/kg administered subcutaneously. Heroin free base (approximately20 rag) was dissolvedin a minimum volume (100-200 IJL)of saline mixed with KH2PO40.5M and diluted with saline. The pH was adjusted to 6.4 with 1M Na2HPO4, and saline was added to make a final volume of 10 mL. Blood samples were collected at 0, 0.5, 1, 1.5, 3, 5, 10, 20, 40, 60, and 120 rain and 6, 12, and 24 h after administration. Five mice were used for each test. Serum obtained by centrifugation was stored at -20~ until analysis.

Results and Discussion Figure 9 shows selected-ion monitoring (SIM) chromatograms of a methanolic standard mixture of all the analytes under investigation and an extract of serum sample from heroin-treated mice. The analytical recoveries of all the analytes from serum samples and the intraday and interday coefficients of variation are shown in Table II. It must be said that solid-phase extraction with ethyl columns gave the best results in terms of analytical recoveries of both lipophilic and hydrophilic compounds without the use of nonvolatile salts of buffers, which could not

Table III. Quantitation Limit and Linearity of the Method

Compound Heroin 6-Monoacetylmorphine Morphine Morphine-3-glucuronide Morphine-6-glucuronide Codeine

Quantitation limit (ng/mL) 0.5 4.0 4.0 1.0 4.0 4.0

Correlation Linearity coefficients y = 3.2x + 1 y = 1.7x + 5 y = 1.3x- 7 y = 1.0x- 0.3 y = 0.Tx- 0.7 y = 1.6x- 3.3

0.999 0.998 0.996 0.998 0.998 0.997

* • = amount of the analytes (ng/mL); y = peak area (counts per second).

275


be injected onto the MS. The quantitation limit (signal-tonoise ratio of 3) and the linearity of the method are given in Table III. The calibration curves were linear between the quantitation limit and 10 IJg/mL for each analyte with correlation coefficients always higher than 0.99. The procedure described previously was used to study the pharmacokineticsof heroin metabolites in C57BL/6mice treated with a 20-mg/kg subcutaneous dose (Figure 10). Using this dosage and this route of administration, heroin was not found in serum samples from mice, not evenjust after the administration. This result fairly agrees with the observation of Umans and Inturrisi (12). Conversely,6-MAMshowed a peak concentration of 0.93 pg/mL serum at 3 rain, rapidly declined to 0.06 pg/mL within 2 h, and then disappeared. Morphine and M3G achieved their peak concentrations of 9.6 and 2.9 ]Jg/mLserum at 10 and 20 rain and were detectable up to 2 and 6 h, respectively (Figure 9). Morphine-6-glucumnide was not detected in serum samples at this heroin dosage or at a double dosage (data not shown). Indeed, M6G was not detected in 24 h urine of mice treated with 10 mg/kg morphine, although in vitro studies demonstrated the capability of mice liver toward the glucuronidation of the 6-hydroxylgroup of morphine (13). Finally, 1.2

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Conclusion Heroin metabolism in human and animal models has been investigated by several authors (15-17). Nonetheless, at the moment, only one report that determines all heroin metabolites in one patient who was intravenously treated with 200 mg heroin exists (9), but detection limits and analysis times were not stated. The difficultiesencountered in the chromatographic separation of parent compound, intermediate metabolites, and the glucuronides were due to the different polarities. Previous reports describe chromatographic separations of morphine from its two glucoronides using salt concentrations that are incompatiblewith mass spectrometry. The use of a column with a silica stationary phase (conditioned for 24 h as described previously) proved extremely effective.By virtue of the polarity of the stationary phase, the compounds being tested can be retained. Columns such as C2, C4, C8,and C1scould not be used to separate these compounds simultaneously and completely unless salts were used. If this is done, they can no longer be used for chromatographic analysis coupled with mass spectrometry. A new method was proposed for the quantitative determination of heroin, 6-MAM, morphine, M3G, M6G, and codeine using nalorphine as the internal standard. The assay also permitted a complete evaluationof heroin metabolism at low doses because of its high sensitivity, especially in the case of heroin (0.5 ng/mL) itself, which was usually I 24.c present in traces in biological fluids. Solid6.0 phase extraction made the methodology rapid and reliable, and the analysis of a biological sample (extraction and chromatographic run) could be concluded in approximately 40 rain. A simple animal model was used to investigate the complete metabolism of heroin, which was administered subcutaneously at a known dosage. The analytical technique proposed can also be applied to the determination of heroin and its metabolites in biological samples (serum, hair, and urine) and should allow an evaluation of the pharmacokinetics of the parent drug and its active metabolites for clinicotoxicological investigations. 24.0 6.o

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7.

8.

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