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

measured 8 h postdose. MOR was not detected in plasma of

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-per-
milliliter 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.
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 be-
cause 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 chro-
matography (HPLC).Until recently, methods for the quantita-
tion 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.

468 Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.

Journal of Analytical Toxicology, Vol. 23, October 1999
Several reports have described the direct analysis of mor-
phine (MOR)and its principal glucuronide metabolites: mor-
phine-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 sen-
sitive and specific HPLC-MS-MS method for the analysis of
MOR, M3G, and M6G in plasma collected from subjects ad-
ministered 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 quality-
control samples were prepared in Milli-Qwater and stored at
-20~ The stock solutions were used to prepare working so-
lutions at 10, 100, and 1000 pg/laLof MOR,M3G and M6G. The
working solutions were used to prepare calibration and quality-
control 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 stan-
dard (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 sot-
bent by gravity flow.The columns were thoroughly dried by ap-
plying a 15-ram Hg vacuum for 5 rain. Analytes were eluted
from the column by gravity flowwith 2 mL methanol and col-
lected into appropriately labeled tubes and evaporated to dry-
ness 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 au-
tosampler 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 ex-
tracts 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 sepa-
ration 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 pre-
cursors 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, colli-
sion-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 MOR-
d3, 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 re-
action 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 stan-
dards. M6G was quantitated using MOR-d3as the internal stan-
dard; 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.
Clinical protocol
Healthy, adult male volunteers were recruited by the Uni-
versity of Utah Department of Anesthesiology to participate in
a controlled, double-blind study of the effects of IT morphine
on the ventilatory response to hypoxia. Subjects signed in-
formed consent, and the University of Utah's Institutional Re-
view Board approved the clinical protocol. Subjects were
divided into three groups, and each subject participated in
only one group. The placebo group received placebo injec-
tions intrathecally and intravenously (IV). The second group
received 0.3 mg morphine IT and a placebo injection IV. The
third group received a placebo injection IT and 0.14 mg/kg
morphine IV. Arterial blood was collected prior to drug ad-
ministration via vascular catheter at 1, 2, 4, 6, 8, 10, and 12 h
after drug administration for the determination of MOR, M3G,
and M6G concentrations. Plasma was separated from the whole
blood and stored frozen at-20~ until analysis.
~undsrme
{%)
16
Results and Discussion
Analytical method
Assayprecision and accuracy were determined by analyzing
drug-free plasma samples (n = 4) fortified with known con-
centrations of MOR, M3G, and M6G. Results are summarized
in Table I. Coefficients of variation for intra-assay precision
(n = 4) were determined to be less than 8% for each analyte at
750, 2500, and 7500 pg/mL. Interassay precision was deter-
mined by comparing calculated MOR, M3G,and M6G concen-
trations from plasma samples fortified with 750, 2500, and
7500 pg/mL from three batches analyzed on three separate
days (n = 3 for each batch analyzed). Coefficientsof variation
were less than 5% for each analyte at 750, 2500, and 7500
pg/mL. Accuracy was calculated by comparing the measured
concentration with the target concentration and found to be
within 9% of the target values from nine separate analyseseach
at 750, 2500, and 7500 pg/mL.
Several analytical challenges were encountered during the
development of this assay. Recent reports have described the
analysis of MOR and its glucuronide metabolites using HPLC
coupled to either atmospheric pressure chem-
ical ionization (20) or ESI (19,21-23) MS.
~,.62 These reports describe limits of quantitation

0
m/z2116~ ~16

5.58
/L__ greater than 1 ng/mL for the glucuronide
metabolites. A limit of less than 1 ng/mL was
needed to accurately quantitate M6G in plasma
after the IT doses given in this study. The sen-
m/z 289 -.), 289
sitivity obtained with this assay is illustrated in
0 5.97 Figure 1, which shows a plasma sample forti-
~ 1 IVloq~me3-1t.luemo~de3.76mln
~ fi-glucmvn~S.~ rain 3.76
fied with 500 pg/mL of MOR, M3G, and M6G.
m/z462-) 216 To obtain consistent chromatographic reso-
0 3.75 lution of MOR, M3G, and M6G, it was neces-
37 ~ Mm'p~r~ 3-|kzc~'onRJe-dj
sary to ensure proper wetting of the analytical

o
1 ~,eeo!~,,'m.L
19 m/z465-') 2S9

i" 3.ob 3.5I~ 4.o

column to prevent the possibilityof phase col-
lapse of the C-18 bonded phase. This was en-
2.~ 2.s
Time (mini sured in two ways: the ODS-AQ phase in the
Figure 1. Ion chromatogramfrom an extracted plasma sample fortified with 500 pg/mL MOR analytical column is hydrophilically end-
(5.62 rain), M3G (3.76 min), and M6G (5.97 rain) and 5000 pg/mL of internal standardsMOR- capped and is specificallydesigned for use with
d3 (5.58 min) and M3G-d3 (3.75 min). high percentage of aqueous mobile phases and
the column was equilibrated daily by running

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

Morphine Morphine-3-glucuronide Morphine-6-glucuronide

Target Mean Mean Mean
concentration concentration % of concentration % of concentration % of
(pg/mL) n (pg/mt) target % CV* (pg/mL) target % CV* (pg/mL) target % CV*

Precision Intra-assay 750 4 798.5 106.5 7.7 672.5 89.7 1.8 722.8 96.4 7.8
2500 4 2417.1 96.7 5.5 2429.4 97.2 4.4 2265.0 90.6 6.3
7500 4 7261.8 96.8 2.3 7855.6 104.7 7.8 7541.1 100.5 6.8

Interassay 750 9 797.4 106.3 4.1 734.3 97.9 3.0 689.0 91.9 4.8
2500 9 2510.5 100.4 3.1 2402.8 96.1 2.5 2613.1 104.5 3.8
7500 9 7466.2 99.5 3.2 6855.9 91.4 4.6 8125.3 108.3 1.7

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

470
Journal of Analytical Toxicology, Vol. 23, October 1999

a gradient beginning with 100% organic sol-

A vent and ending with the initial mobile phase
Rdatlve composition. These steps improved the in-
abundance
(%)
285,9 terassay stability of retention times for each
analyte.
An additional analytical challenge encoun-
Io
i ~
.c., 3.5 reTort Ar
9 i ~ "c", tered in the development of the assay was the
TO CID of MOR. Several CID conditions were ex-
-30 V
plored, and MOR and MOR-d3were found to
Morphine Morphine fragment extensively under all conditions
m/z 2860vI+ED m/z 286 (M+H)
causing a loss of sensitivity for MOR. To over-
come this, the third quadrupole was set to
zo monitor the transmission of MOR (m/z 286 -~
10 286) through the collision cell as shown in
I , I
Figure 2A. This produced more baseline noise,
60 "8 100 120 140 16 180 2~0 220 240 260 280 300
m/z but did not compromise the LOQ of the assay
(see Figure 1). The glucuronide metabolites
both showed intense CID product ions corre-
B sponding to the loss of glucuronic acid yielding
Relative
abundance unconjugated morphine (m/z 462 -~ 286) as
shown in Figure 2B.

'il
(%) 286.0

Clinical research samples

7O"~ All plasma samples analyzed from the sub-
jects who receiveda placebo dose were negative
for all three analytes (data not shown). After IV
-t
administration of 0.14 mg/kg of MOR, the
n~': 286 (N§ mean plus or minus the standard error of the
Morphine~glucuronide mean (SEM) (n = 11) peak plasma concentra-
m/z 462 (M+H)
tion was detected at 1 h for MOR and M6G
462.1
(12,992.0 • 1196.3 and 23,369.0 • 6885.0
0 P, ,'1 , ,
240
;
260
, i
280 300 320 340 360 380 400 420 440 460 pg/mL, respectively).The mean plus or minus
m/z
SEM (n = 11) peak concentration of M3G was
Figure 2. MS-MS mass spectra of MOR (A) and MOR glucuronides (B). 3.5 mTorr of Argon was detected at 2 h (153,795.3 + 40,692.6 pg/mL).
pumped into the collision cell. The offset voltage was maintained at -30 V for MOR and -35 V Considerable interindividual variabilitywas ob-
for M3G and M6G. served for all three analytes as indicated by the
SEM. After 12 h, M3G and M6G were still quan-
titatable in all subjects' plasma as illustrated in
RelaUve
abundance Figure 3. Mean plus or minus SEM concentra-
(%) 5.~4
tions in the 12-h sample were 21,174 • 5100.3
7 Morphine
1535pg/mL ff~ and 2776.4 + 508.1 pg/mL for M3G and M6G,
30~ -) 286
respectively.MORwas detected in only 5 of the
5.52 11 subjects at 12 h after the IV MOR adminis-
tration. Concentrations ranged from 534.8
pg/mL (Figure 3) to 1212.8 pg/mL.
3.74 MOR was not detected in any subject's
Morphlne-6-glucuronlde5.91w.ln plasma after the 0.3-rag MOR IT dose. The
3074pg~tL 5.91
0 m~ 462-~286 mean peak M3G concentration was measured
3.75 in plasma 8 h after IT administration of MOR
2211I M~176
465
289Pg/mL
-~ (1576.7 • 376.6 pg/mL [mean • SEM], n ---9).
M3G concentrations ranged from 290 pg/mL to
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.;
4405.6 pg/mL. M6G was detected in fiveof nine
Tlrne (rain) subjects administered MOR IT. The plasma
Figure 3. Ion chromatogram from an extracted patient plasma sample fortified with 5000 pg/mL
concentrations of M6G in these five subjects
internal standards MOR-d 3 (5.52 rain) and M3G-d 3 (3.75 rain). MOR (0.14 mg/kg IV) was ad- ranged from 257.4 pg/mL to 636.2 pg/mL. The
ministered 12 h prior to sample collection. Measured concentrations were 535 pg/mL (MOR, detectable plasma concentrations of M6G
5.54 rain), 17,722 pg/mL (M3G, 3.74 rain), and 3074 pg/mL (M6G, 5.91 min). shown here after IT administration of MOR
demonstrate the need to be able to quantitate

471

subnanogram concentrations of morphine glucuronides. The
ability to measure low concentrations of these analytes will
allow the clinical relevance of these concentrations to be eval-
uated.
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 quantita-
tion 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 consis-
tently detected in plasma up to 12 h postdose, with peak con-
centrations measured at 8 h.
This method is currently being evaluated for use in the iden-
tification 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.
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473