Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS

Research Article

Synthesis and bioanalytical evaluation ofmorphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma usingLC-MS/MS

The aim of this work was to synthesize morphine-3-O-sulfate and morphine-6-O-sulfate

for use as reference substances, and to determine the sulfate conjugates as possible heroin

and morphine metabolites in plasma and urine by a validated LC-MS/MS method.

Morphine-6-O-sulfate and morphine-3-O-sulfate were prepared as dihydrates from

morphine hydrochloride, in overall yields of 41 and 39% with product purities of 499.5%

and 498%, respectively. For bioanalysis, the chromatographic system consisted of a

reversed-phase column and gradient elution. The tandem mass spectrometer was oper-

ated in the positive electrospray mode using selected reaction monitoring, of transition

m/z 366.15 to 286.40. The measuring range was 5–500 ng/mL for morphine-3-O-sulfate

and 4.5–454 ng/mL for morphine-6-O-sulfate in plasma. In urine, the measuring range

was 50–5000 ng/mL for morphine-3-O-sulfate and 45.4–4544 ng/mL for morphine-6-O-

sulfate. The intra-assay and total imprecision (coefficient of variation) was below 11% for

both analytes in urine and plasma. Quantifiable levels of morphine-3-O-sulfate in

authentic urine and plasma samples were found. Only one authentic urine sample

contained a detectable level of morphine-6-O-sulfate, while no detectable morphine-6-O-

sulfate was found in plasma samples.

Keywords: Human / LC-MS/MS / Morphine-3-O-sulfate / Morphine-6-O-sulfate /SynthesisDOI 10.1002/jssc.201100739

1 Introduction

Morphine is a natural product with historical use as

therapeutic and abused drug. The two glucuronidated

metabolites morphine-3-O- (M3G) and morphine-6-O-

glucuronide (M6G) have received considerable attention

ever since methods for their determination in body fluids

became available in the 1980 s [1–3]. M3G is the main

metabolite excreted in urine, while M6G is pharmacologi-

cally active and may accumulate during long-term treatment

[4, 5].

The corresponding metabolites morphine-3-O-sulfate

(M3S) and morphine-6-O sulfate (M6S) have received much

less attention and are assumed to be less abundant than the

glucuronides. Morphine ethereal sulfate was reported as a

morphine metabolite in human adults and in cats but this

was based on tentative identification [6–8]. Choonara et al.

detected M3S as a urinary morphine metabolite in children

using HPLC [9]. However, no sulfate metabolites were

detected from heroin in urine [10], or as morphine meta-

bolite in children in plasma [9].

Since glucuronidation capacity is low at birth the sulfate

metabolites are of interest when studying morphine meta-

bolism in children [9]. Further reasons for interest in sulfate

conjugates of morphine is the polymorphism in glucu-

ronidation enzymes [11] making sulfation potentially more

important in some individuals, and the potential pharma-

cologic activity of morphine-O-sulfate conjugates [12].

A fundamental obstacle for this work was the lack of

commercially available M3S and M6S reference substances.

The available synthetic routes to morphine-6-O-sulfate

(M6S, Scheme 1, 3) and morphine-3-O-sulfate (M3S,

Scheme 2, 6) rely on manipulation of 3-acetylmorphine

[13–15] or 6-acetylmorphine [13], respectively. However,

Maria Andersson1

Tomasz Janosik2

Hamid Shirani2

Johnny Slatt3

Andreas Fischer3

Olof Beck4

1Department of LaboratoryMedicine, Division of ClinicalPharmacology, KarolinskaUniversity Hospital, Stockholm,Sweden

2Department of Biosciences andNutrition, Karolinska Institute,Novum, Huddinge, Sweden

3Division of Inorganic Chemistry,Royal Institute of Technology,Stockholm, Sweden

4Department of Medicine,Division of ClinicalPharmacology, KarolinskaUniversity Hospital, Stockholm,Sweden

Received August 19, 2011Revised October 31, 2011Accepted November 7, 2011

Abbreviations: M-d3, morphine-d3; M3G, morphine-3-O-glucuronide; M6G, morphine-6-O-glucuronide; M3S,morphine-3-O-sulfate; M6S, morphine-6-O-sulfate; SRM,selecting reaction monitoring

Correspondence: Maria Andersson, Division of Clinical Pharma-cology C1:68, Huddinge Karolinska University Hospital, 141 86Stockholm, SwedenE-mail: [email protected]: 146-8-58581050

& 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2012, 35, 367–375 367

these approaches (which all rely on the Welsh acetylation of

morphine) [16] gave, in our hands, products of insufficient

purity, as was revealed by product characterization with

LC-MS/MS. Therefore alternative synthetic procedures were

developed and are included in this report.

Previous methods used for M3S and M6S analysis

comprise TLC, GC and HPLC [8, 9], while LC-MS and

LC-MS/MS are commonly used in more recent work on

other morphine metabolites. In this study we aimed at

developing an LC-MS/MS method for determination and

quantification of M3S and M6S in human urine and

plasma. The purpose is to apply the method to the study

of morphine pain treatment of neonates (www.neoopioid.

eu/) and for study of abnormal metabolic patterns of heroin

addicts [17].

2 Material and methods

2.1 Chemicals

Morphine-d3 (M-d3) was obtained from Cerilliant (Round

Rock, TX, USA). Morphine HCl was from Apoteket AB

(Stockholm, Sweden). Formic acid (pro analysis quality) and

acetonitrile (LiChrosolv isocratic grade for Liquid Chroma-

tography) were from Merck KGaA (Darmstadt, Germany).

Methanol (HiPerSolv CHROMANORM for HPLC-gradient

grade) was purchased from VWR (Radnor, PA, USA).

Dimethyl sulfoxide was from J. T. Baker (Philipsburg,

Netherlands). Ultra-pure water was produced in-house by a

Milli-Q Millipore Water system. All reagents and solvents

for synthesis originated from commercial sources, and were

used as received, except pyridine, which was stored over

sodium hydroxide pellets. Column chromatography was

performed using silica gel (particle size 40–63 mm).

2.2 Synthesis

2.2.1 Morphine-6-O-sulfate (3, Scheme 1)

Morphine hydrochloride (1) (3.22 g, 10 mmol) was

suspended at 0–51C (ice-water bath) under vigorous stirring

in a mixture of CH2Cl2 (50 mL) and saturated aqueous

NaHCO3 (50 mL). Acetic anhydride (2.83 mL, 30 mmol) was

then added over ca. 5 min at 0–51C. The cooling bath was

removed, and the mixture was allowed to warm to room

temperature over 1 h. The layers were separated, and the

aqueous phase was extracted with CH2Cl2 (3� 20 mL). The

combined organic extracts were washed with brine, dried

(MgSO4), and evaporated in vacuum, leaving morphine-3-O-

acetate as a colorless glass. This material was dissolved

immediately in anhydrous pyridine (25 mL), and sulfur

trioxide–pyridine complex (3.50 g, 22 mmol) was added in

one portion. The resulting mixture was stirred at 55–601C

for 4 h. After cooling, most of the solvent was removed

in vacuum, and the foamy tan residue was treated

with ice-cold water (30 mL) and CHCl3 (20 mL). The

organic layer was removed using a pipette, and the solid

material which precipitated in the aqueous layer was

collected by filtration, washed with several portions of

cold water, and dried in vacuum, providing the intermediate

2 as a pinkish solid (2.54 g). The material above was

added in one portion to a 5% solution of NaOH in

methanol (50 mL). The mixture was stirred at room

temperature for 1 h and 45 min. After concentration of the

mixture at reduced pressure to ca. 20% of the original

volume, 5% aqueous solution of acetic acid was added,

adjusting the pH to ca. 5–6. Upon standing at room

temperature for 24 h, crystals separated from the

solution. This material was collected by filtration, washed

with water, and recrystallized twice from water (with

filtration while hot), to give M6S (3) as a dihydrate (1.66 g,

41% over three steps) as colorless needles. The 1H-NMR

data were in good agreement with those published

previously [18]. The purity of the material produced using

this procedure was typically 499.5% as determined by

LC-MS/MS. The structure was further confirmed by X-ray

diffraction (Fig. 1A), which also revealed that 3 crystallizes

as a dihydrate. Crystal data: C17H21NO8S, orthorhombic,

Scheme 1. Reagents and conditions: (a) Ac2O, NaHCO3 (aq),CH2Cl2, 0–51C to room temperature, 1 h; (b) pyridine �SO3,pyridine, 55–601C, 4 h; (c) NaOH, MeOH, room temperature,1 h, 41% from 1.

HO

HO

O

HNMe

HCl

a,b

AcO

TBSO

O

HNMe

c

HO

TBSO

O

HNMe

d

O3SO

HO

O

HN

H

Me

41

5 6

Scheme 2. Reagents and conditions: (a) Ac2O, NaHCO3 (aq),CH2Cl2, 0–51C to rt, 1 h; (b) TBSCl, imidazole, DMF, roomtemperature, 72 h; (c) Na2CO3, MeOH, 48 h, 69% from 1; (d)pyridine �SO3, pyridine, 55–601C, 4 h, 56% from 5.

J. Sep. Sci. 2012, 35, 367–375368 M. Andersson et al.


P212121, a 5 9.799(4), b 5 11.688(11), c 5 15.50(4) A,

V 5 1775(5) A3, Z 5 4, 3443 reflections, 250 L.S. parameters,

R1 5 0.070, wR2 5 0.147, Flack parameter x 5 0.18(19). A

CIF file with the crystallographic data is available as

Supporting Information.

2.2.2 Morphine-3-O-sulfate (6, Scheme 2)

Morphine-3-O-acetate was prepared as above on half the

scale, starting from morphine hydrochloride (1) (1.61 g,

5 mmol) and acetic anhydride (1.42 mL, 15 mmol). After

work-up as above, the crude product was dissolved in dry

DMF, followed by addition of tert-butyldimethylsilyl chloride

(TBSCl) (1.51 g, 10 mmol), and imidazole (1.36 g, 20 mmol).

The resulting mixture was stirred at room temperature for

72 h, whereupon it was partitioned between CHCl3 (50 mL)

and water (100 mL). The layers were separated, and the

aqueous phase was extracted with CHCl3 (3� 25 mL). The

combined organics were dried over MgSO4, and concen-

trated in vacuum, leaving crude 4 as a colorless viscous oil.

The product 4 was dissolved in methanol (100 mL), and

Na2CO3 (3.20 g, 30 mmol) was added. The resulting mixture

was stirred at room temperature for 36 h. Removal of the

solvent in vacuum gave a solid residue, which was treated

with water (100 mL), followed by addition of 10% aqueous

acetic acid until pH 6. The mixture was extracted with

CHCl3 (4� 25 mL). The combined organic extracts were

washed with water, dried over MgSO4, and concentrated in

vacuum to give a solid cream-colored residue. This was

subjected to flash column chromatography, initially using

CH2Cl2 as the eluent, followed by 5% methanol in CH2Cl2containing 0.5% triethylamine, to provide the TBS-protected

intermediate 5 (1.38 g, 69% over 3 steps) as a white solid

([M1H]1 m/z 400). During some runs, a di-silylated deri-

vative (identified by LC-MS (ESI); m/z 514 [M1H]1),

originating from unreacted morphine from the first step,

was present as an impurity even after chromatography

(elutes just before the product). This can be easily removed

by trituration of 5 with Et2O, leading to high purity material.

A mixture of 5 (195 mg, 0.49 mmol) and sulfur trioxide-

pyridine complex (400 mg, 2.51 mmol) in pyridine (10 mL)

was heated at 55–601C for 4 h. After cooling, the mixture was

evaporated in vacuum at 30–401C until completely dry. Water

was added to the residue (50 mL), followed by saturated

aqueous NaHCO3, adjusting the pH to 6. This mixture was

heated until a clear solution was obtained. After filtration while

warm, the solution was left standing at room temperature for

18 h, resulting in crystallization of the product as colorless

needles. The crystals were collected by filtration, washed with

several portions of water. This material was recrystallized from

water, to provide M3S (6) as a dihydrate (110 mg, 56%) as

colorless needles. MS (ESI) m/z 364 [M–H]�. The 1H-NMR

data were in good agreement with those published previously

[18]. The purity of the product obtained using this procedure

was typically 498% as determined by LC-MS/MS. The overall

yield from morphine hydrochloride (1) was 39%. The single

crystal X-ray analysis (Fig. 1B) demonstrated that 6 was

obtained as a dihydrate. Crystal data: C17H21NO8S, ortho-

rhombic, P212121, a 5 6.9041(8), b 5 13.564(2), c 5 18.867(2) A,

V 5 1766.8(4) A3, Z 5 4, 3225 reflections, 248 L.S. parameters,

R1 5 0.054, wR2 5 0.149, S 5 1.06, Flack parameter

x 5 –0.09(16). A CIF file with the crystallographic data is

available as Supporting Information.

2.3 Patient samples

Patient samples were de-coded surplus samples obtained

from the routine flow of clinical samples sent to the

laboratory for analysis.

2.4 Sample preparation

2.4.1 Urine

Urine samples were prepared for analysis by dilution

with ultra-pure water containing internal standard M-d3

(340 ng/mL). A 25-mL aliquot of urine was added to 100 mL

of internal standard solution in an autosampler vial and

mixed for about 10 s.

Figure 1. The crystal structures of (A) morphine-6-O-sulfate(M6S, 3) dihydrate (B) morphine-3-O-sulfate (M3S, 6) dihydrate.In the case of 6, the water molecules are omitted, as the locationof their hydrogen atoms could not be determined.

J. Sep. Sci. 2012, 35, 367–375 Liquid Chromatography 369


2.4.2 Plasma

Whole blood was sampled in heparinized vacuum tubes, the

samples were centrifuged at 3000� g for 10 min and the

plasma was collected. The plasma samples were then

prepared by acetonitrile precipitation. A 50-mL aliquot of

plasma and 100 mL of acetonitrile containing the internal

standard M-d3 (500 ng/mL) were mixed in Eppendorf tubes

for about 10 s, and centrifuged for 10 min at 16 000� g. The

resulting supernatant was transferred to a glass-tube and

evaporated to dryness under nitrogen at 401C. Finally, the

residue was reconstituted with 30 mL of 0.1% aqueous

formic acid and transferred into an autosampler vial.

2.5 Analysis

The instrument used was an ACQUITY UPLC system

connected to a Quattro Premier XE tandem mass spectro-

meter (Waters, Milford, MA, USA). All data were collected

and analyzed with the MassLynx 4.1 software. The analytical

column used was an ACQUITY UPLC HSS T3,

2.1� 100 mm, 1.8 mm particle size, kept at 601C. For the

analysis of plasma extracts, the analytical column was

combined with an ACQUITY HSS T3 VanGuard Pre-

column, 2.1� 5 mm, 1.8 mm particle size. The injection

volume was 2 mL for the urine extracts (using partial loop

with needle overfill) and 3 mL for the plasma extracts (full

loop). The injector loop volume was 5 mL.

A gradient elution was used using mobile phase A: 0.1%

formic acid and mobile phase B: methanol. The gradient for

the urine extracts started at 9.5% mobile phase B for

0.50 min, increased to 30% B for 1.60 min, then further

increased to 95% B for 1.40 min and was held at 95% B for

0.40 min. The column was then equilibrated for 1.60 min at

9.5% B. The mobile phase flow was 0.20 mL/min. The

gradient for the plasma extracts was modified to be held at

100% B for 2.00 min and the column was equilibrated for

1.00 min at 9.5% B. This made the run time for plasma

extracts 1 min longer. Total run times were 5.5 min for

urine and 6.5 min for plasma samples.

The tandem mass spectrometer was operated in the

positive electrospray mode using selecting reaction moni-

toring (SRM). The capillary voltage was 1 kV, extractor

voltage 4 V and the RF lens was set to 0 V. The source

temperature was 1301C and the desolvation temperature

was 3501C. The cone gas flow rate, N2, was 50 L/h, desol-

vation gas, N2, flow rate was 900 L/h and the collision gas,

Ar2, flow rate was 0.30 mL/min. Table 1 shows the indivi-

dual settings for each analyte. One transition was monitored

for quantification, while recording in negative mode was

used for qualification because of lack of a second prominent

transition ion. The analysis of M, M3G and M6G was

performed separately according to an earlier reported

procedure [19].

2.6 Standards and quality controls

Calibration curves were constructed from peak area ratios

between SRM transitions for M3S and M6S and the internal

standard, M-d3. The weighting factor used for the linear

regression calculation was 1/x. Standards were prepared in

both blank urine and blank plasma from stock solutions in

dimethyl sulfoxide. Seven concentration levels were used for

plasma ranging from 5 to 500 ng/mL for M3S and 4.5 to

454 ng/mL for M6S and 8 concentration levels were used for

in urine ranging from 50 to 5000 ng/mL for M3S and 45.4 to

4544 ng/mL for M6S. Quality controls were prepared in the

same manner at three levels, 15, 50 and 300 ng/mL for M3S

in plasma and 13.6, 45.4 and 272 ng/mL for M6S in plasma.

The three concentration levels prepared in urine were 150,

500 and 3000 ng/mL for M3S and 136, 454 and 2720 ng/mL

for M6S. The calibrators and quality controls were stored at

�201C.

2.7 Validation

The limit of detection (LOD) was estimated by using spiked

blank plasma and urine. The lower limit of quantification

(LLOQ) was estimated based on the total CVo20% criterion

and accuracy 80–120%. Accuracy and precision were studied

by analyzing a replicate of five of each quality control level

during five days (n 5 25). The recovery of analytes during

plasma protein precipitation was calculated according to:

recoveryð%Þ ¼ 100� peak area of plasma spiked before precipitation

peak area of spiked blank plasma extract

Ion suppression was studied using two experimental

designs. The first experiment was post column infusion of a

solution containing both analytes (10 mg/mL) at a rate of

10 mL/min. Extracts of three different blank patient urine

and three different blank patient plasma samples were

thereafter injected while monitoring the SRM transition

366.15 to 286.40. In the second experiment, M3S and M6S

Table 1. MS/MS acquisition parameters used for the SRM mode and retention time

Analyte Precursor ion (m/z) Product ion (m/z) Dwell time (s) Cone voltage (V) Collision energy (eV) Retention time (min)

M3S 366.15 286.40 0.150 40 24 1.80

M6S 366.15 286.40 0.150 40 24 2.55

M-d3 289.35 201.35 0.050 45 25 2.17



were added to 12 patient plasma samples at the level of 50

and 45.4 ng/mL, respectively, and 15 patient urine samples

at the level of 1000 and 909 ng/mL, respectively. The area

response of analytes was then compared with the area

response of analytes from a reference solution prepared at

the same concentration levels in mobile phase A. The matrix

effect was then calculated according to:

matrix effectð%Þ ¼ 100� peak area of spiked plasma extract

peak area of spiked mobile phase A� 100

The stability in plasma and urine was studied for 24 h in

room temperature and at 41C. The stability at �201C was

studied for 16 days for plasma and 2.5 month for urine.

Stability of extracts on the autosampler was studied for 12 h

at 121C. Stock solution stability was studied for 5 months in

dimethyl sulfoxide at �201C. The three-cycle freeze and

thaw stability was also studied.

3 Results

3.1 Synthesis of M3S and M6S

Upon screening of various conditions of the preparation of

3-acetylmorphine, we noted that treatment of morphine

hydrochloride (1) with acetic anhydride in a two-phase

mixture consisting of saturated aqueous NaHCO3 and

CH2Cl2 at 0–51C resulted in efficient transformation into

the desired product, without co-formation of detrimental

levels of side-products. The resulting 3-acetylmorphine was

directly subjected to sulfatation [13, 14], providing the

known intermediate 2 (Scheme 1). Cleavage of the acetyl

group of 2 was performed using NaOH in methanol [13],

leading to morphine-6-O-sulfate (3). The high product

purity (499.5%) and overall yield (41%) were very

consistent between different runs. Recrystallization of this

product from water gave 3 as a dihydrate, which was

revealed by X-ray crystallography (Fig. 1). In this context, it

should also be noted that the crystal structure of the

intermediate 2 has previously been reported [20].

M3S (6) has previously been prepared from 6-ace-

tylmorphine following a similar path involving installation

of the sulfate moiety, followed by deacetylation [13].

However, since 6-acetylmorphine is not readily available as

synthetic starting material, a different protecting strategy

was used, where morphine hydrochloride (1) was initially

converted into 3-acetylmorphine, which was immediately

treated with TBSCl in DMF in the presence of imidazole,

providing the silyl ether 4 (Scheme 2). Subsequent deace-

tylation of 4 with sodium carbonate in methanol produced

the 6-O-silylated derivative 5 in 69% overall yield from 1.

The corresponding tert-butyldiphenylsilyl-protected deriva-

tive has previously been used as an intermediate towards 3-

amino-3-desoxy derivatives, and was prepared by a selective

desilylation of 3,6-bis-tert-butyldiphenylsilyl ether at the

phenolic hydroxyl group [21]. Exposure of 5 to sulfur triox-

ide-pyridine complex as in the sequence above was followed

by removal of the pyridine in vacuo. Upon addition of water,

the acidic medium caused the final desilylation, to provide

morphine-3-O-sulfate (M3S, 6) as a dihydrate in 56% yield.

The overall yield from morphine hydrochloride (1) was 39%.

The purity of the material obtained using this protocol was

typically 498%. The identity of 6 was finally rigorously

proven by a single crystal X-ray study, which showed that 6was isolated as a dihydrate.

3.2 Method development

The SRM transitions as well as cone voltage and the

collision energy were optimized by direct infusion of each

analyte including the internal standard, M-d3. A compound

solution of M3S and M6S was infused at a concentration of

10 mg/mL and at a rate of 15 mL/min. Only one useful SRM

transition was found that could be used for both M3S and

M6S. However, a second SRM transition was obtained for

identification by data recording in negative mode.

Two analytical columns were evaluated during the

method development of the LC system. The ACQUITY

UPLC HSS T3, 2.1� 100 mm, 1.8 mm and the ACQUITY

BEH C18 1.0� 100 mm, 1.7 mm. The HSS T3 column is

designed to separate and retain polar compounds in the

reversed-phase mode. There was good separation between

M3S and M6S on both columns but, in addition, separation

between M, M3G and M6G was best on the HSS T3.

Acetonitrile and methanol were both evaluated as mobile

phase B during development of the chromatographic

system. Methanol gave the best separation. The best choice

of internal standard would have been isotopic-labeled M3S

and M6S but these were not available. Instead, M3G-d3,

M6G-d3 and M-d3 were tested for M3S and M6S. M-d3 was

selected because it gave the best calibration curves, i.e.

correlation coefficients for both M3S and M6S.

Figure 2A shows a chromatogram from a plasma cali-

brator containing 100 ng/mL M3S and 91 ng/mL M6S. M3S

and M6S are well separated from each other. The chroma-

togram also shows were M3G, M and M6G elutes in the

chromatographic system. M3S, M and M6G are barely

separated at the baseline. However, this is not necessary

because they are monitored by different transitions in the

mass spectrometer. There is no contribution at the M3S and

M6S channel from the M3G, M6G or M. In the M channel,

there is a small contribution from M3S and M6S transition

and the M3G and M6G transition due to in-source frag-

mentation. A chromatogram obtained from analysis of a

urine calibrator is shown in Fig. 2B. The capacity factor (k0)for the first eluting compound M3G is 0.38, k0 for M3S is

0.62 and k0 for M6S is 1.3.

3.3 Validation

A linear relationship between peak area ratio of analyte to

internal standard and the analyte concentrations were



observed in the range of 5–500 ng/mL for M3S and

4.5–454 ng/mL for M6S in plasma (r 5 0.999, n 5 3). In

urine, the linearity was observed in the range of

50–5000 ng/mL for M3S and 45.4–4544 ng/mL for M6S

(r 5 0.999, n 5 3). LOD (s/n 5 3) was estimated to 6 ng/mL

for M3S and 4 ng/mL for M6S in urine. In plasma

LOD (s/n 5 3) was estimated to 0.6 ng/mL for M3S and

0.3 ng/mL for M6S. The LLOQ was according to the

criterion CV o20% and accuracy 80–120% for the lowest

concentration levels in the calibration curves. Thus, the

LLOQ for M3S in plasma was 5 ng/mL (CV 5 7.4%, n 5 5),

for M6S in plasma 4.54 ng/mL (CV 5 14.8%, n 5 5), for

M3S in urine 50 ng/mL (CV 5 7.0%, n 5 5) and for M6S in

urine 45.4 ng/mL (CV 5 3.7%, n 5 5). The difference of

LLOQ between matrices was due to the dilution factor. A

summary of intra-assay and total imprecision and accuracy

data are shown in Table 2, a and b for both urine and

plasma. All the CV values were o11% and the accuracy was

within 98–111% from their nominal value for all analytes in

both urine and plasma.

The average recovery of M3S during plasma protein

precipitation (n 5 3) was 97.2% at the concentration level of

50 ng/mL, and the average recovery of M6S was 96.7%

(n 5 3) at the concentration level of 45.4 ng/mL. The first

study of matrix effect on ionization was an infusion

experiment with injection of blank extracts of both urine

and plasma. The SRM signal was markedly reduced for both

M3S and M6S following injection of blank urine and plasma

extracts after elution of the void volume. Both M3S and M6S

elutes before the recovery of the signal. The second ion

suppression experiment M3S and M6S were spiked in 12

different plasma samples and 15 different urine samples

and in mobile phase A. The matrix effect data are

summarized in Table 3.

Inte

nsity

(cp

s)

0

500000

1.35 1.8 2.25 2.7Time (min)

M3S

M6S

M-d3

M

M3G

M6G

Inte

nsity

(cp

s)

0

45000

2 2.5 31.5

Time (min)

m/z 289.35 201.35

m/z 366.15 286.40

m/z 289.35 201.35

m/z 366.15 286.40

M-d3

M3S

M6S

A

B

Figure 2. Chromatograms obtained from the LC-MS/MS analysis(A) a plasma calibrator containing 100 ng/mL M3S and 91 ng/mLM6S. Peaks for M3G, M and M6G are also shown in thechromatogram at the concentration level of 100 ng/mL for eachanalyte; (B) a urine calibrator containing 500 ng/mL M3S and454 ng/mL M6S.

Table 2. Intra-assay, total imprecision and accuracy data for M3S and M6S in (a) urine and (b) plasma

M3S M6SConc. added (ng/mL)

n Accuracy% CV%

Conc. added (ng/mL)

n Accuracy% CV%

(a) Urine

Intra-assay Intra-assay

150 25 98 5.4 136 25 105 3.9

500 25 98 6.9 454 25 107 6.4

3000 25 101 5.2 2720 25 111 4.9

Total imprecision Total imprecision

150 25 98 5.4 136 25 105 8.3

500 25 98 9.7 454 25 107 10.1

3000 25 101 7.1 2720 25 111 8.5

(b) Plasma

Intra-assay Intra-assay

15 25 99 8.3 13.6 25 117 10.5

50 25 96 5.2 45.4 25 107 6.6

300 25 98 5.5 272 25 110 6.5

Total imprecision Total imprecision

15 25 99 8.3 13.6 25 117 10.5

50 25 96 5.2 45.4 25 107 10.9

300 25 98 5.5 272 25 110 10.7

The experiment was performed with five replicates for five days.



The matrix effect was greater from plasma extracts than

from urine extracts. The data presented in Table 3 was

obtained from 12 different plasma and 15 different urine

specimens and documents the variability between indivi-

duals.

The stability study was performed on triplicate samples at

the level of 50 ng/mL for M3S and 45.4 ng/mL for M6S in

plasma. Freshly prepared calibrators were used as the refer-

ence. In plasma, M3S and M6S were stable (97–106%) at all

studied storage conditions. In urine, M3S and M6S were stable

(107–116%) at all studied storage conditions. Autosampler

stability of M3S and M6S was documented in urine extracts

(98–101%) and in plasma extracts (90–92%) for 12 h at 121C.

Stock solutions of both M3S and M6S were found to be stable

(101–102%) for 5 months in dimethyl sulfoxide at �201C

using freshly prepared stock solutions as reference.

3.4 Application

Nine authentic plasma samples were analyzed of which

eight samples contained quantifiable levels of M3S ranging

from 5.8 to 12.9 ng/mL (Table 4). No detectable levels of

M6S in any of the plasma patient samples could be found. A

representative chromatogram from one plasma patient

sample is shown in Fig. 3A. This sample contained

9.6 ng/mL M3S but no detectable M6S. This patient sample

also contained M3G, M and M6G, see Table 4.

A number of 18 patient urine samples were analyzed

(Table 4) selected on the basis of the presence of the heroin-

specific metabolite, 6-acetylmorphine (6-AM). All these

samples contained detectable levels of M3S, 13 samples were

above the quantification level ranging from 69 to 1500 ng/mL.

Only one urine patient sample contained a detectable level of

M6S together with 101 ng/mL M3S (Fig. 3B). Identification

was confirmed by monitoring the SRM transition 364.2 to

364.2 in negative mode (dwell time 0.100 s, cone voltage to

40 V and collision energy 10 eV). This sample also

contained high levels of M, M3G, M6G and 6-AM. Table 4 also

shows the ratio between M3S and M3G. The ratio between

M3S and M3G appears to be higher in plasma as compared

with urine.

Table 3. Results from matrix effect experiment

Urine n 5 15 Plasma n 5 12

M3S M6S M3S M6S

Matrix effect% �4.4710.4 14.577.6 �47.875.8 �37.474.8

Table 4. De-coded surplus samples collected at random from the routine flow of clinical samples sent to the laboratory for analysis

Patient sample M3S (ng/mL) M6S (ng/mL) Morphine (ng/mL) M3G (ng/mL) M3S/M3G %

Plasma 1 12.9 a) 5.0 2200 0.59

Plasma 2 6.25 a) a) a) No ratio

Plasma 3 a) a) a) 230 No ratio

Plasma 4 6.5 a) a) 510 1.3

Plasma 5 5.8 a) a) 3.0 193

Plasma 6 8.2 a) 50 60 13.7

Plasma 7 7.3 a) 60 80 9.1

Plasma 8 9.6 a) 60 140 7.0

Plasma 9 8.2 a) 20 100 8.2

Urine 1 101 b) 15 780 78 900 0.13

Urine 2 b) a) 890 12 100 No ratio

Urine 3 81 a) 3870 33 800 0.24

Urine 4 b) a) 780 11 200 0.17

Urine 5 74 a) 2560 38 600 0.19

Urine 6 155 a) 10 210 71 100 0.22

Urine 7 1500 a) 57 960 230 800 0.65


Urine 9 69 a) 1560 26 600 0.26

Urine 10 93 a) 3530 29 600 0.31

Urine 11 493 a) 18 530 108 700 0.45

Urine 12 94 a) 2670 44 300 0.21

Urine 13 73 a) 7320 54 100 0.13

Urine 14 499 a) 38 760 199 100 0.25


Urine 16 460 a) 24 800 111 500 0.41

Urine 17 b) a) 610 6400 No ratio

Urine 18 83 a) 3530 29 600 0.28

(a) Not detectable (M3S o 0.6 ng/mL and M6S o 0.3 ng/mL in plasma and M3S o 6 ng/mL and M6S o 4 ng/mL in urine).

(b) Detectable.



4 Discussion

4.1 Synthesis of M3S and M6S

With M6S (3) as the first target, the conditions described by

Welsh for the acetylation of morphine were used (acetic

anhydride in the presence of saturated aqueous NaHCO3)

[16]. The material obtained from these experiments was

thereafter exposed to the sulfur trioxide–pyridine complex,

followed by cleavage of the acetyl group, in line with

previous syntheses [13, 14]. However, we found that the

quality of the resulting M6S (3) was not satisfactory, as it

was contaminated with morphine residues. We attributed

this issue to the fact that, in our hands, the Welsh

acetylation procedure gave 3-acetylmorphine containing

the side-product heroin, as well as some unreacted

morphine, as judged from TLC and LC-MS analyses. In

addition, it was noted that 3-acetylmorphine is rather labile,

and even carefully purified samples underwent degradation

within days into mixtures containing morphine and heroin

upon cold storage in the dark. This implied that

3-acetylmorphine must be used immediately after prepara-

tion, thereby avoiding problems with impurities and side-

reactions in subsequent steps. Very similar approaches to

those mentioned above have been employed very recently

for synthesis of both M6S (3) and M3S (6) [18], but

unfortunately, neither the product yields, nor the purities

were accounted for. Consequently, as highly pure samples

of M6S (3) and M3S (6) were required for reference

purposes, it was decided that modified synthetic approaches

had to be employed.

4.2 Bioanalytical aspect

A validated bioanalytical method for accurate determination of

M3S and M6S in plasma and urine was successfully

developed. One further improvement could be to include

stable isotope labeled analogues of M3S and M6S as internal

standards to better compensate the matrix effect, but these

substances are not yet available. We considered it important to

obtain separation between morphine and metabolites in both

the chromatographic system as well as in the mass spectro-

meter. This is mainly due to the production of morphine from

the conjugates by in-source fragmentation. The availability of

M3S and M6S as a reference substance is of value in the

validation of possible interference in any analytical method for

morphine determination in biological samples.

Our study confirms that the morphine sulfate conju-

gates are less abundant than the glucuronides. We have for

the first time been able to demonstrate the presence of M3S

in urine and plasma as morphine metabolites using highly

selective mass spectrometry technique. In future work, the

upper limit of the measuring range may be lowered fivefold

for both plasma and urine. In the work by Choonara et al.,

M3S could be detected using HPLC UV in urine, but not in

plasma [9]. This could be explained by a more than

25-fold increased sensitivity in our method. Regarding M6S,

a signal indicating its presence was observed in only one

urine sample. Therefore, it still remains to more convin-

cingly demonstrate M6S as a morphine metabolite in

humans.

This project was supported by the European Community’sSeventh Framework Programme under grant agreement no.223767 and through the regional agreement on medical trainingand clinical research (ALF) between Stockholm County Counciland Karolinska Institutet, GA 20090490.

The authors have declared no conflict of interest.

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0

450000

1.5 2 2.5 3Time (min)

Inte

nsity

(cp

s)

M-d3

M3S

m/z 289.35 201.35

1.50

42000

2 2.5 3

Inte

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(cp

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Time (min)

M-d3

M3S

M6S

m/z 366.15 286.40

m/z 289.35 201.35

m/z 366.15 286.40

A

B

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Documents

Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS