Determination of Morphine, Morphine-3-glucuronide, and Morphine-6 ...

27 downloads 757 Views 498KB Size Report
report describes an HPLC-electrospray-MS-MS method capable of detecting subnanogram concentrations of morphine (MOR) and its. 3- and 6-glucuronide ...
Journalof AnalyticalToxicology,Vol. 23, October 1999

Determination of Morphine, Morphine-3-glucuronide, and Morphine-6-glucuronidein Plasmaafter Intravenousand Intrathecal Morphine Administration Using HPLC with ElectrosprayIonization and Tandem Mass Spectrometry Matthew H. Slawson1,', Dennis J. Crouch 1, David M. Andrenyak1, Douglas E. Rollins 1, Jeffrey K. tu 2, and Peter L. Bailey2 1The Centerfor Human Toxicology, Departmentof Pharmacologyand Toxicology, Universityof Utah Health SciencesCenter, Salt Lake City, Utah 84112and 2Departmentof Anesthesiology, Universityof Utah Health SciencesCenter, Universityof Utah, Salt Lake City, Utah 84132

I Abstract High-performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization (API) massspectrometry (MS) has become a useful technique in the direct analysis of low concentrations of conjugated opiate metabolites. Previous methods using HPLC with traditional detection methods do not have the sensitivity to detect low concentrations of most conjugated drug metabolites. Methods using gas chromatography-mass spectrometry (GC-MS) require hydrolysis and derivatization of the sample followed by an indirect quantitation of conjugated metabolites. Recently, several reports have described direct analysis of opiates and their glucuronide conjugates by HPLC and API-MS. These methods report lower limits of detection than GC-MS methods and quantitation in the low nanogram-permilliliter range for the glucuronide metabolites of morphine. This report describes an HPLC-electrospray-MS-MS method capable of detecting subnanogram concentrations of morphine (MOR) and its 3- and 6-glucuronide metabolites (M3G and M6G, respectively). The assayhas a dynamic range of 250-10,000 pg/mL for M3G and M6G and 500-10,000 pg/mL for MOR. Inter- and intra-assay precision and accuracy varied by lessthan 8% for all analytes at 750-, 2500-, and 7500-pg/mL concentrations. This assaywas used for the determination of MOR, M3G, and M6G in human plasma after intravenous (IV) and intrathecal (IT) administration of MOR and its effects on the ventilatory responseto hypoxia. Peak plasma concentrations of MOR and M6G were measured 1 h after IV administration of MOR. Peak concentrations of M3G were measured 2 h after IV administration of MOR. After IT administration of MOR, peak concentrations of M3G were

* Author to whom correspondence should be addressed. Matthew H. Slawson, Center for Human Toxicology, University of Utah, 20 South 2030 East #490, Salt Lake City, UT 84112.

468

measured 8 h postdose. MOR was not detected in plasma of patients administered MOR IT. Subnanogram concentrations of M6G were measured in the plasmaof five of nine patients administered MOR IT.

Introduction The direct analysis of low concentrations of opiates and their conjugated metabolites has become possible in recent years because of technological developments in liquid chromatography (LC) and atmospheric pressure ionization mass spectrometry (API-MS). Previously, gas chromatography (GC) was used for these analyses (1-4); however, GC analysis required hydrolysis of the sample and derivatization of the analytes. Quantitation of the metabolites was estimated by comparing the hydrolyzed and nonhydrolyzed test results. Direct analysis of conjugated metabolites is possible with high-performance liquid chromatography (HPLC).Until recently, methods for the quantitation of conjugated opiate metabolites have relied on traditional HPLC detectors such as ultraviolet (5-7), fluorescence (8-13), and electrochemical (5,8,14-18). The coupling of LC with MS and API offers a much more sensitive and specific analytical technique than was previously available to directly detect and quantitate low concentrations of opiates and their glucuronide metabolites. Tandem mass spectrometry (MS-MS) affords an even greater specificitythan single-stage MS. MS-MS provides improved signal-to-noise ratios and lower limits of detection (low picogram-per-milliliter range) for many commonly detected drugs, including opiates and their conjugated metabolites.

Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.

Journal of Analytical Toxicology, Vol. 23, October 1999

Several reports have described the direct analysis of morphine (MOR)and its principal glucuronide metabolites: morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (19-22). These methods use HPLC-API-MStechniques and have limits of detection in the low nanogram-per-milliliter range for the glucuronide metabolites. The purpose of this study was to develop and validate a sensitive and specific HPLC-MS-MS method for the analysis of MOR, M3G, and M6G in plasma collected from subjects administered a single intravenous (IV)or intrathecal (IT) dose of morphine. HPLC coupled with electrospray ionization and MS-MS provided the specificity and sensitivity to detect and quantitate the low concentrations of these analytes found in the plasma samples. Therefore, we describe an analytical method capable of detecting and quantitating subnanogram concentrations of MOR, M3G, and M6G in plasma.

Materials and Methods Chemicals and reagents MOR (1 mg/mL in methanol), MOR-d3 (100 lag/mL), M3G (1 rag/mE in THF), and M3G-d3 (100 I~g/mL in THF) were obtained from Radian Corp. (Austin, TX). M6G powder was obtained from Sigma Chemical (St. Louis, MO). Clean Up| C-18 solid-phase extraction (SPE) columns were obtained from United Chemical Technologies,Inc. (Bristol, PA).HPLC-grade methanol, acetonitrile, and formic acid were obtained from Fischer Scientific (Pittsburgh, PA).Ammonium carbonate was obtained from Mallinckrodt Chemical Works (St. Louis, MO). All stock drug solutions, buffers, and HPLC mobile phase were prepared using Milli-Q grade water (Millipore,Bedford, MA). Standards and solutions Stock solutions containing MOR,M3G, and M6G (100 ng/laL) used for the preparation of the calibration curves and qualitycontrol samples were prepared in Milli-Qwater and stored at -20~ The stock solutions were used to prepare working solutions at 10, 100, and 1000 pg/laLof MOR,M3G and M6G. The working solutions were used to prepare calibration and qualitycontrol samples. Calibration curves were obtained by analyzing drug-free plasma samples fortified with MOR,M3G, and M6G at 250, 500, 750, 1000, 2500, 5000, 7500, and 10,000 pg/mL. Quality-control samples (750, 2500, and 7500 pg/mL) were prepared from solutions made with reference materials with lot numbers that were different from the reference materials used to prepare the calibration standards. Sample preparation and extraction One milliliter of calibrator, quality-control,or subject plasma was pipetted into labeled, silanized glass tubes. Internal standard (5000 pg/mL, MOR-d3and M3G-d3,50 IJL of a 100-pg/IJL stock solution) was added to each sample. Samples were vortex mixed and allowed to equilibrate for 30 min. Two milliliters ammonium carbonate (10raM pH 9) was added to each sample. The samples were again vortex mixed and then centrifuged at 3000 rpm for 10 rain.

SPE of the samples was performed using Clean Up C-18 SPE columns, a vacuum manifold device, and a vacuum source. SPE columns were prepared by sequentially passing 2 mL methanol, 2 mL water, and 2 mL 10mMammonium carbonate (pH 9) buffer through the sorbent bed. Sample supernatants were then transferred to the appropriately labeledSPE columns and allowed to pass through the column sorbent by gravity flow. The SPE columns were washed by passing 2 mL 10mM ammonium carbonate buffer (pH 9) through the column sotbent by gravity flow.The columns were thoroughly dried by applying a 15-ram Hg vacuum for 5 rain. Analytes were eluted from the column by gravity flowwith 2 mL methanol and collected into appropriately labeled tubes and evaporated to dryness under a stream of air at 35~ The residues were reconstituted with 75 IJL HPLC mobile phase (see LC-MS-MS analysis section) and transferred to 0.7-mL conical bottom autosampler vials. The vials were centrifuged at 15,000 rpm for 2 rain, and 20 I~Lof supernatant was injected onto the HPLC column.

LC-MS-MS analysis Electrospray ionization (ESI) MS-MS analysis of sample extracts was performed using a Finnigan MAT TSQ7000 (San Jose, CA) LC-MS-MS interfacedwith a Waters 626 HPLCpump and controller (Waters Corp., Milford, MA) equipped with a Leap A200S autosampler (Leap Technologies, Raleigh, NC). The HPLC mobile phase consisted of 95% water containing 0.1% formic acid and 5% acetonitrile pumped isocratically at 0.2 mL/min at ambient temperature. Chromatographic separation of analytes was achieved using a YMC ODS-AQ3 IJ, 2 x 150-ram HPLC column (YMC,Inc. Wilmington, NC). The ESI source was operated with a spray voltage of 5kV,70 psi N2 sheath gas, and 12 flow units N2 auxiliary gas. The heated capillary was maintained at 225~ Positive ion precursors for MOR (m/z 286), MOR-d3(m/z 289), M3G (rn/z462), M6G (m/z 462), and M3G-d3 (m/z 465) were selected to pass through the first quadrupole. In the second quadrupole, collision-induced dissociation (CID) was achieved using argon as the collision gas (3.5 mTorr) and offset voltages of -30 V for MOR and -35 V for the glucuronides. Product ions monitored in the third quadrupole were m/z 286 and 289 (MORand MORd3, respectively, see Discussion), m/z 286 and 289 (M3G and M3G-d3, respectively, see Discussion), and m/z 286 (M6G, see Discussion). Data were collected for the following selected reaction monitoring (SRM) transitions: m/z 286 -~ 286 (MOR), m/z 289 -~ 289 (MOR-d3),m/z 462 -~ 286 (M3Gand M6G), and m/z 465 -~ 289 (M3G-d3). The scan time was 0.2 s/scan. Quantitative analysis Quantitation of morphine and its glucumnide metabolites in plasma was achieved by calculating the peak-area ratios for the product ions of each analyte and its respective internal standards. M6G was quantitated using MOR-d3as the internal standard; MOR and M3G were ratioed to their respective internal standards isotopomers. Quadratic curve fits with 1/x weighting were used to ensure accurate quantitation over the dynamic range of the assay (250-10,000 pg/mL for M3G and M6G and 500-10,000 pg/mL for MOR). Finnigan LCQuan| (ver 1.2) 469

Journal of Analytical Toxicology,Vol. 23, O c t o b e r 1999

quantitation software was used to generate calibration curves and to calculate MOR, M3G, and M6G concentrations in analyzed samples.

Results and Discussion

Analytical method Assayprecision and accuracy were determined by analyzing Clinical protocol drug-free plasma samples (n = 4) fortified with known conHealthy, adult male volunteers were recruited by the Unicentrations of MOR, M3G, and M6G. Results are summarized versity of Utah Department of Anesthesiology to participate in in Table I. Coefficients of variation for intra-assay precision a controlled, double-blind study of the effects of IT morphine (n = 4) were determined to be less than 8% for each analyte at on the ventilatory response to hypoxia. Subjects signed in750, 2500, and 7500 pg/mL. Interassay precision was deterformed consent, and the University of Utah's Institutional Remined by comparing calculated MOR, M3G,and M6G concenview Board approved the clinical protocol. Subjects were trations from plasma samples fortified with 750, 2500, and 7500 pg/mL from three batches analyzed on three separate divided into three groups, and each subject participated in days (n = 3 for each batch analyzed). Coefficientsof variation only one group. The placebo group received placebo injecwere less than 5% for each analyte at 750, 2500, and 7500 tions intrathecally and intravenously (IV). The second group pg/mL. Accuracy was calculated by comparing the measured received 0.3 mg morphine IT and a placebo injection IV. The concentration with the target concentration and found to be third group received a placebo injection IT and 0.14 mg/kg within 9% of the target values from nine separate analyseseach morphine IV. Arterial blood was collected prior to drug adat 750, 2500, and 7500 pg/mL. ministration via vascular catheter at 1, 2, 4, 6, 8, 10, and 12 h Several analytical challenges were encountered during the after drug administration for the determination of MOR, M3G, development of this assay. Recent reports have described the and M6G concentrations. Plasma was separated from the whole analysis of MOR and its glucuronide metabolites using HPLC blood and stored frozen at-20~ until analysis. coupled to either atmospheric pressure chemical ionization (20) or ESI (19,21-23) MS. ~undsrme {%) ~,.62 These reports describe limits of quantitation greater than 1 ng/mL for the glucuronide 16 m/z2116~ ~16 metabolites. A limit of less than 1 ng/mL was 0 needed to accurately quantitate M6G in plasma 5.58 after the IT doses given in this study. The senm/z 289 -.), 289 sitivity obtained with this assay is illustrated in 0 Figure 1, which shows a plasma sample forti5.97 ~ 1 IVloq~me3-1t.luemo~de3.76mln fied with 500 pg/mL of MOR, M3G, and M6G. ~ fi-glucmvn~S.~ rain 3.76 To obtain consistent chromatographic resom/z462-) 216 0 lution of MOR, M3G, and M6G, it was neces3.75 37 ~ Mm'p~r~ 3-|kzc~'onRJe-dj sary to ensure proper wetting of the analytical ~,eeo!~,,'m.L 19 m/z465-') 2S9 column to prevent the possibilityof phase collapse of the C-18 bonded phase. This was eno i" 3.ob 3.5I~ 4.o 2.~ 2.s sured in two ways: the ODS-AQ phase in the Time (mini analytical column is hydrophilically endFigure 1. Ion chromatogramfrom an extracted plasma sample fortified with 500 pg/mL MOR capped and is specificallydesigned for use with (5.62 rain), M3G (3.76 min), and M6G (5.97 rain) and 5000 pg/mL of internal standardsMORhigh percentage of aqueous mobile phases and d3 (5.58 min) and M3G-d3 (3.75 min). the column was equilibrated daily by running

/L__

1

Table I. Precision and Accuracy for Analysis of Morphine and Metabolites in Plasma Morphine

Target concentration (pg/mL) n

Morphine-3-glucuronide Morphine-6-glucuronide Mean Mean Mean concentration % of concentration % of concentration % of (pg/mt) target % CV* (pg/mL) target % CV* (pg/mL) target % CV*

Precision Intra-assay

750 2500 7500

4 4 4

798.5 2417.1 7261.8

106.5 96.7 96.8

7.7 5.5 2.3

672.5 2429.4 7855.6

89.7 97.2 104.7

1.8 4.4 7.8

722.8 2265.0 7541.1

96.4 90.6 100.5

7.8 6.3 6.8

Interassay

750 2500 7500

9 9 9

797.4 2510.5 7466.2

106.3 100.4 99.5

4.1 3.1 3.2

734.3 2402.8 6855.9

97.9 96.1 91.4

3.0 2.5 4.6

689.0 2613.1 8125.3

91.9 104.5 108.3

4.8 3.8 1.7

*Percentcoefficientof variationfor meanconcentrationvalues(n = 4 or 9 as indicatedin the table).

470

Journal of Analytical Toxicology, Vol. 23, October 1999

Rdatlve abundance

A

(%)

285,9

Io

i

~

3.5 reTort Ar -30 V

.c.,

TO

9 i ~

Morphine m/z 2860vI+ED

"c",

Morphine m/z 286 (M+H)

zo 10 60

"8

100

120

140

16

180

2~0

m/z

220

240

260

280

I

,

I

300

B

Relative abundance

'il (%)

286.0

7O"~

-t n~': 286 (N§ Morphine~glucuronide m/z 462 (M+H) 462.1 0

P,

,'1 , , 240

;

, 260

i 280

300

320

340

360

380

400

420

440

460

m/z

Figure 2. MS-MS mass spectra of MOR (A) and MOR glucuronides (B). 3.5 mTorr of Argon was pumped into the collision cell. The offset voltage was maintained at -30 V for MOR and -35 V for M3G and M6G.

RelaUve

abundance

(%)

5.~4

7

Morphine 1535pg/mL 30~ -) 286

ff~ 5.52

3.74

0

Morphlne-6-glucuronlde5.91w.ln 3074pg~tL m~ 462-~286

5.91 3.75

2211I M~176 465 289Pg/mL -~ 0

; 0.5

~ 1.0

i 1.5

2.62.5

3.6

i 3.~

i 4.0

~ 4.5

5.6

$.5

"f 6.0

'

6.;

Tlrne (rain)

Figure 3. Ion chromatogram from an extracted patient plasma sample fortified with 5000 pg/mL internal standards MOR-d 3 (5.52 rain) and M3G-d 3 (3.75 rain). MOR (0.14 mg/kg IV) was administered 12 h prior to sample collection. Measured concentrations were 535 pg/mL (MOR, 5.54 rain), 17,722 pg/mL (M3G, 3.74 rain), and 3074 pg/mL (M6G, 5.91 min).

a gradient beginning with 100% organic solvent and ending with the initial mobile phase composition. These steps improved the interassay stability of retention times for each analyte. An additional analytical challenge encountered in the development of the assay was the CID of MOR. Several CID conditions were explored, and MOR and MOR-d3were found to fragment extensively under all conditions causing a loss of sensitivity for MOR. To overcome this, the third quadrupole was set to monitor the transmission of MOR (m/z 286 -~ 286) through the collision cell as shown in Figure 2A. This produced more baseline noise, but did not compromise the LOQ of the assay (see Figure 1). The glucuronide metabolites both showed intense CID product ions corresponding to the loss of glucuronic acid yielding unconjugated morphine (m/z 462 -~ 286) as shown in Figure 2B.

Clinical research samples All plasma samples analyzed from the subjects who receiveda placebo dose were negative for all three analytes (data not shown). After IV administration of 0.14 mg/kg of MOR, the mean plus or minus the standard error of the mean (SEM) (n = 11) peak plasma concentration was detected at 1 h for MOR and M6G (12,992.0 • 1196.3 and 23,369.0 • 6885.0 pg/mL, respectively).The mean plus or minus SEM (n = 11) peak concentration of M3G was detected at 2 h (153,795.3 + 40,692.6 pg/mL). Considerable interindividual variabilitywas observed for all three analytes as indicated by the SEM. After 12 h, M3G and M6G were still quantitatable in all subjects' plasma as illustrated in Figure 3. Mean plus or minus SEM concentrations in the 12-h sample were 21,174 • 5100.3 and 2776.4 + 508.1 pg/mL for M3G and M6G, respectively.MORwas detected in only 5 of the 11 subjects at 12 h after the IV MOR administration. Concentrations ranged from 534.8 pg/mL (Figure 3) to 1212.8 pg/mL. MOR was not detected in any subject's plasma after the 0.3-rag MOR IT dose. The mean peak M3G concentration was measured in plasma 8 h after IT administration of MOR (1576.7 • 376.6 pg/mL [mean • SEM], n ---9). M3G concentrations ranged from 290 pg/mL to 4405.6 pg/mL. M6G was detected in fiveof nine subjects administered MOR IT. The plasma concentrations of M6G in these five subjects ranged from 257.4 pg/mL to 636.2 pg/mL. The detectable plasma concentrations of M6G shown here after IT administration of MOR demonstrate the need to be able to quantitate 471

Journalof AnalyticalToxicology,Vol. 23, October 1999

subnanogram concentrations of morphine glucuronides. The ability to measure low concentrations of these analytes will allow the clinical relevance of these concentrations to be evaluated.

Conclusions This paper describes a sensitive and specific method for the analysis of subnanogram concentrations of MOR, M3G, and M6G using ESI LC-MS-MS. The assay has limits of quantitation of 250 pg/mL for M3G and M6G and 500 pg/mL for MOR. The data presented demonstrate that M3G concentrations are severalfold higher than MORand M6G after IV administration of MOR.After IT administration of MOR, only M3G is consistently detected in plasma up to 12 h postdose, with peak concentrations measured at 8 h. This method is currently being evaluated for use in the identification of subnanogram concentrations of glucuronide metabolites of MOR in other biological matrices such as urine and hair.

Acknowledgment This work was supported by the International Anesthesia Research Society through a Clinical Scholar Research Award received by Dr. Lu in 1997.

References I. M. Hoffman, J.C. Xu, C. Smith, C. Fanelli, Y. Pascal, C. Degaetano, G. Meenan, M. Lehrer, M. Lesser,and M. Citron. A pharmacodynamic study of morphine and its glucuronide metabolites after single morphine dosing in cancer patients with pain. Cancer Invest. 15:542-547 (1997). 2. E.J.Cone, R. Jufer, W.D. Darwin, and S.B. Needleman. Forensic drug testing for opiates. VII. Urinary excretion profile of intranasal (snorted) heroin. J. Anal. Toxicol. 20:379-392 (I 996). 3. E.J.Cone, P. Welch, B.D. Paul, and J.M. Mitchell. Forensic drug testing for opiates, III. Urinary excretion rates of morphine and codeine following codeine administration. J. Anal. Toxicol. 15: 161-166 (1991). 4. S.P.Gygi, F. Colon, R.B. Raftogianis, R.E. Galinsky, D.G. Wilkins, and D.E. Rollins. Dose-related distribution of codeine and its metabolites into rat hair. Drug Metab. Dispos. 24:282-287 (1996). 5. H. He, S.D. Shay, Y. Caraco, M. Wood, and A.J. Wood. Simultaneous determination of codeine and its seven metabolites in plasma and urine by high-performance liquid chromatography with ultraviolet and electrochemical detection. J. Chromatogr. B 708:185-193 (1998). 6. J. Gerostamoulos and O.H. Drummer. Solid phase extraction of morphine and its metabolites from postmortem blood. Forensic Sci. InL 77:53-63 (1996). 7. R.W. Milne, R.L. Nation, G.D. Reynolds, A.A. Somogyi, and J.T. Van Crugten. High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites: improvements to the method and application to stability studies.

472

J. Chromatogr. 565:457-464 (1991). 8. Y. Rotshteyn and B. Weingarten. A highly sensitive assay for the simultaneous determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma by highperformance liquid chromatography with electrochemical and fluorescence detection. Ther. Drug MoniL 18:179-I 88 (I 996). 9. J. Huwyler, S. Ruler, E. Kusters,and ]. Drewe. Rapid and highly automated determination of morphine and morphine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography. J. Chromatogr. B 674:57-63 (1995) 10. M. Pawula, D.A. Barrett, and P.N. Shaw. An improved extraction method for the HPLC determination of morphine and its rnetabolites in plasma. J. Pharm. Biomed. Anal. 11:401-406 (1993). 11. R. Hartley, M. Green, M. Quinn, and M.I. Levene. Analysis of morphine and its 3- and 6-glucuronides by high performance liquid chromatography with fluorimetric detection following solid phaseextraction from neonatal plasma. Biomed. Chromatogr. 7: 34-37 (I 993). 12. P.A. Glare, T.D. Walsh, and C.E. Pippenger. A simple, rapid method for the simultaneous determination of morphine and its principal metabolites in plasma using high-performance liquid chromatography and fluorometric detection. Ther. Drug Monit. 13:226-232 (1991). 13. R.R Vennand A. Michalkiewicz. Fast reliable assayfor morphine and its metabolites using high-performance liquid chromatography and native fluorescence detection. J. Chromatogr. 525: 379-388 (I 990). 14. B.A. Rashid, G.W. Aherne, M.F. Katmeh, P. Kwasowski, and D. Stevenson. Determination of morphine in urine by solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection. J. Chromatogr. A 797:245-250 (I 998). 15. J.O. Svensson, Q.Y. Yue, and J. Sawe. Determination of codeine and metabolites in plasma and urine using ion-pair high-performance liquid chromatography. J. Chromatogr. B 674:49-55 (1995). 16. M. Ohno, Y. Shiono, and M. Konishi. Simultaneous determination of dihydrocodeine and its metabolites in dog plasma by highperformance liquid chromatography with electrochemical and ultraviolet detection. J. Chromatogr. B 654:213-219 (I 994). 17. A.W. Wright, J.A. Watt, M. Kennedy, T. Cramond, and M.T. Smith. Quantitation of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in plasma and cerebrospinal fluid using solid-phase extraction and high-performance liquid chromatography with electrochemical detection [published erratum appears in Ther. Drug Monit. 20(2): 218 (1998)]. Ther. Drug Monit. 16:200-208 (1994). 18. C.P. Verwey-van Wissen, P.M. Koopman-Kimenai, and T.B. Vree. Direct determination of codeine, norcodeine, morphine and normorphine with their corresponding O-glucuronide conjugates by high-performance liquid chromatography with electrochemical detection. J. Chromatogr. 570:309-320 (1991). 19. M. Zheng, K.M. McErlane, and M.C. Ong. High-performance liquid chromatography-mass spectrometry-mass spectrometry analysis of morphine and morphine metabolites and its application to a pharmacokinetic study in male Sprague-Dawley rats. J. Pharm. Biomed. Anal. 16:971-980 (1998). 20. M.J. Bogusz, R.D. Maier, M. Erkens, and S. Driessen. Determination of morphine and its 3- and 6-glucuronides, codeine, codeine-glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography atmospheric pressure chemical ionization mass spectrometry. J. Chromatogr. B 703:115-127 (1997}. 21. 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 pressureionspray-mass spectrometry. ]. Anal. Toxicol. 21:268-277 (1997). 22. R. Pacifici, S. Pichini, I. Altieri, A. Caronna, A.R. Passa, and P. Zuccaro. High-performance liquid chromatographic-electro-

Journal of Analytical Toxicology,Vol. 23, October 1999 spray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to pharmacokinetic studies. J. Chromatogr. B 664:329-334 (1995). 23. N. Tyrefors, B. Hyllbrant, L. Ekman, M. Johansson, and B. Langstrom. Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human serum by solid-phase ex-

traction and liquid chromatography-mass spectrometry with electrospray ionisation. J. Chromatogr. A 729:279-285 (1996). Manuscript received March 22, 1999; revision received May 20, 1999.

473