Differentiation of estriol glucuronide isomers by chemical derivatization and electrospray tandem mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 1429–1440

) DOI: 10.1002/rcm.2463
Published online in Wiley InterScience (www.interscience.wiley.com
Differentiation of estriol glucuronide isomers by

chemical derivatization and electrospray tandem

mass spectrometry

Matilda Lampinen-Salomonsson1, Ulf Bondesson1,2, Carl Petersson3

and Mikael Hedeland2*1Division of Analytical Pharmaceutical Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23 Uppsala, Sweden2Department of Chemistry, National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden3DMPK & BAC, AstraZeneca R&D Sodertalje, SE-151 85 Sodertalje, Sweden

Received 23 December 2005; Revised 27 February 2006; Accepted 28 February 2006

*CorrespoNationalE-mail: M

This paper describes a way of differentiating between the three isomers of estriol glucuronide by the

use of chemical derivatization and liquid chromatography/electrospray tandem mass spectrometry

(MS/MS). In their native form, these isomers gave rise to almost identical product ion spectra,

involving the neutral loss of 176Da (i.e. monodehydrated glucuronic acid), whichmade it impossible

to determine the position of conjugation by MS/MS alone. In order to change the fragmentation

pathways, positive charges were introduced into the analytes by chemical derivatization. The

following reagents were tested: 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylamino-

propyl)carbodiimide and 2-picolylamine. Interestingly, derivatization using a combination of all

three reagents gave a selective fragmentation pattern that could differentiate between the isomers

estriol-16-glucuronide and estriol-17-glucuronide. Estriol-3-glucuronide, which lacks a free phenolic

group, could be differentiated through a different type of reaction product when exposed to 2-chloro-

1-methylpyridinium iodide. Furthermore, in order to assist structural assignment of the fragments,

their accurate masses were determined using a hybrid quadrupole time-of-flight mass spectrometer

and fragmentation pathways were elucidated by the use of MS3 on an ion trap mass spectrometer.

Copyright # 2006 John Wiley & Sons, Ltd.

Estriol (3,16a,17b-trihydroxy-1,3,5(10)-estratriene), the

natural female estrogen hormone, is formed via 16a

hydroxylation of estrone in the ovaries and through 16a-

hydroxydehydroepiandrosterone in the placenta of pregnant

women.1 Estrogens are primarily excreted in the urine after

conjugation, i.e. as sulfates and glucuronides.2 The isomeric

forms of estriol glucuronides are estriol 3b-D-glucuronide

(E3G), estriol 16a-(b-D-glucuronide) (E16G) and estriol-17b-

(b-D-glucuronide) (E17G)2,3 (Fig. 1).

The use of tandemmass spectrometry for the identification

of drugs and their metabolites is a well-known approach.

However, the limitation of this technique alone becomes

evident in the fragmentation pattern of conjugates, such as

glucuronides. The neutral loss of 176Da, i.e. monodehy-

drated glucuronic acid, at low collision energies is a general

feature in this context. This gives a problem with the

determination of the position of glucuronidation.4,5 Nuclear

magnetic resonance (NMR) spectroscopy is another tech-

nique for structural determination,6 but it requires larger

amounts of sample material than mass spectrometry, and

this can be difficult to accomplish in bioanalytical appli-

cations.4,5 Determining the exact position of glucuronidation

ndence to: M. Hedeland, Department of Chemistry,Veterinary Institute (SVA), SE-751 89Uppsala, [email protected]

is important from a biochemical and pharmacological point

of view, as different isomers may have different toxicological

or pharmacological activities. One example is morphine-6-

glucuronide, which is more pharmacologically active than

morphine itself, and morphine-3-glucuronide, which has

been claimed to antagonize the analgesic effect.1 Regulatory

authorities are becoming increasingly more stringent regard-

ing exposure to metabolites.7 Metabolic studies require

synthetic standards and, in order to synthesize these, exact

knowledge of the structures are required.

Chemical derivatization in combination with liquid

chromatography coupled to tandem mass spectrometry

(LC/MS/MS) for the enhancement of detection sensitivity

is a well-established technique.8–13 Introduction of an easily

ionizable function or a constantly charged group effectively

increases the sensitivity of detection of steroids in electro-

spray ionization mass spectrometry (ESI-MS),14 and the

fragmentation pattern can also be affected due to the ‘charge

remote fragmentation’ phenomenon.15 Leavens et al.8 used 2-

chloro-1-methylpyridinium iodide (CMP) in combination

with triethylamine (TEA) for the activation of carboxylic

acids before introducing tris(trimethoxyphenyl)phosphonium

(TMPP), an ionic species, for sensitivity enhancement in


Figure 1. Structures of estriol and its metabolites estriol-3-

glucuronide, estriol-16-glucuronide and estriol-17-glucuronide.

1430 M. Lampinen-Salomonsson et al.

LC/ESI-MS/MS. Quirke et al.13 improved the detection

sensitivity of steroids by using the reagent 2-fluoro-1-

methylpyridium p-toluenesulfonate (FMP).13 Many com-

pounds used for these applications, e.g. 1-ethyl-3-(3-dimethy-

laminopropyl)carbodiimide (EDC)16,17 andCMP,18,19 arewell-

known activating reagents for subsequent fluorescence

tagging.20

There have been a few reports on the use of derivatization

for the structural evaluation of conjugated metabolites. Cui

and Harvison21 used a reaction with 3-pyridylcarbinol in

order to determine the site of glucuronidation for N-(3,5-

dichlorophenyl)succinimide. Selective acetylation with

acetic anhydride was proven to be successful by Schaefer

et al.22 for discriminating between different possible

isomers of the glucuronides of carvedilol, and Kondo

et al.23 described the structures of two conjugated

metabolites of candesartan cilexetil by methylation with

diazomethane.

The aim of the present study was to investigate if mass

spectral differentiation between the three isomers of estriol

glucuronide could be imposed by chemical derivatization.

The collision-induced dissociation (CID) spectra of estriol, its

metabolites and their different derivatives have been

compared and evaluated. The occurrence of selective

fragmentation as well as differences in reactivity was

studied. Furthermore, in order to facilitate the structural

assignment of the product ions, data from different mass

spectrometers, such as triple quadrupole, quadrupole time-

of-flight and ion trap, have been used in combination.

EXPERIMENTAL

Chemicals and materialsThe model compounds p-nitrophenyl b-D-glucuronide

(NPG), estriol (3,16a,17b-trihydroxy-1,3,5(10)-estratriene)

and its metabolites estriol 3b-D-glucuronide sodium salt

(E3G), estriol 16a-(b-D-glucuronide) (E16G) and estriol-17b-

(b-D-glucuronide) (E17G) were all purchased from Sigma-

Aldrich (Steinheim, Germany). The chemical derivatization

reagents 2-chloro-1-methylpyridinium iodide (CMP), 2-

picolylamine (PA) and 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide hydrochloride (EDC) were also purchased

from Sigma-Aldrich. Triethylamine (TEA) and polyethyle-

neglycol (PEG) 200, 400 and 600 were obtained from


Fluka Chemika (Buchs, Switzerland). The internal calibrant-

clemastine fumarate was purchased from Novartis

Sweden AB (Taby, Sweden), 4-acetaminophen was obtained

from Sigma-Aldrich and codeine phosphate hemihydrate

was a gift from AstraZeneca (Sodertalje, Sweden). The water

was purified using a Milli-Q water purification system

(Millipore, Bedford, MA, USA). All other chemicals were of

analytical reagent grade or better and used without further

purification.

Liquid chromatographyA Jasco liquid chromatography system (Jasco, Tokyo, Japan)

with two PU-1585 pumps, a DG-1580-53 degasser, a HG-

1580-32 dynamic mixer and a Rheodyne 7725i (Rheodyne,

Cotati, CA, USA) injector or an Agilent 1100 series system

(Agilent Technologies, Waldbronn, Germany) with a degas-

ser, a binary pump, a well-plate autosampler and a column

conditioner set at 258C was used. For chromatographic

separation a Synergi Polar reversed-phase (RP) column

(Phenomenex, Torrance, CA, USA) with a particle diameter

of 4mm and dimensions of 2.0mm� 150mm (i.d.� length)

or a Zorbax Eclipse XDB-C18 column (Agilent Technologies)

with a particle diameter of 3.5mm and dimensions of

2.1mm� 100mm (i.d.� length) was used. Both were

equipped with a C18 precolumn (Phenomenex) with

dimensions 2.0mm i.d.� 4mm. The mobile phase compo-

sition methanol/0.1% acetic acid in Milli-Q water was varied

for the different columns but mainly the combination was

50:50 v/v and 40:60 v/v for the Synergi Polar RP column and

the Zorbax Eclipse XDB-C18 column, respectively. The flow

rate was 0.2mL/min and the injection volume was 20mL.

Mass spectrometryThree different electrospraymass spectrometers were used, a

triple quadrupole mass spectrometer (TSQ 7000), an ion trap

(LCQ) (both ThermoFinnigan, San Jose, CA, USA) and a

hybrid quadrupole time-of-flight mass spectrometer (Q-TOF

I) (Micromass, Waters, Manchester, UK).

TSQ 7000The software for controlling the TSQ 7000 instrument and for

data evaluation, processing and integration of peaks was

Xcalibur 1.2 (ThermoFinnigan). For mass calibration of the

instrument in both positive and negative ionmode, a 100mM

NaI solution in isopropanol/water (50:50, v/v) was used. In

negative ion mode, a standard solution (30mM) of one of the

model compounds (E17G) was used for tuning. Due to the

low response for themodel compounds in positive ionmode,

tuning was done with the NaI solution (Na5I4þm/z 622.5667).

The tuning was performed by direct infusion with a syringe

pump (Harvard Apparatus pump II, Holliston, MA, USA).

The syringe pump flow (10mL/min) was mixed in a

connecting T with the LC flow before entering the mass

spectrometer (total flow rate 0.2mL/min). The ESI source

parameters were capillary temperature 2508C, sheath gas 56

psi, and spray voltage 4.5 kV. The collision gas was argon at

1.15mTorr and the collision energy was varied between 5 and

75V. The multiple-ion detector was set at 1400V for MS1

analysis and 1700V for product ion analysis. The instrument

mode was varied between negative/positive MS1 in scan


DOI: 10.1002/rcm

Differentiation of estriol glucuronide isomers 1431

mode, negative product ion mode and positive product ion

mode.

LCQThe ion trap mass spectrometer used was an LCQ

(ThermoFinnigan). The software for instrument control

and data acquisition was Xcalibur v.1.3 (ThermoFinnigan).

The LCQ was tuned with constant infusion of metoprolol

(3.7mM) from a syringe pump (10mL/min) mixed in a

connecting T with the LC flow (total flow rate 0.2mL/min).

The ESI source parameters were spray voltage 6.0 kV,

capillary temperature 2508C, capillary voltage 45V, tube

lens offset 5.0, sheath gas flow 80, and auxiliary gas flow 10

(the two latter in arbitrary units). The instrument was used in

MS1 scan mode, MS2 scan mode and MS3 scan mode.

Q-TOFFor accurate mass determinations, a Q-TOF I instrument,

upgraded with a 3.6 GHz time-to-digital converter card, was

used (Micromass, Manchester, UK). The controller for this

instrument was a PC with MassLynx v. 3.4 software. This

program was also used for data acquisition and processing.

The calibration was performed by direct infusion to the mass

spectrometer with a syringe pump (Harvard Apparatus

pump II). The mass calibration for negative ion analysis was

done with NaI (100mM) in isopropanol/water (50:50, v/v)

and for positive ion analysis with PEG 200/400/600 (1 nM) in

ammonium formate (2mM) in water and acetonitrile (50:50,

v/v). The source block temperature and desolvation

temperature were 120 and 3508C, respectively, and the

nebulizer and desolvation gas flows were 15 and 350 L/h.

The instrument parameter settings in negative mode were

capillary voltage 3000V, cone 40V, extractor 0V, and MCP

2700V and in positive mode capillary voltage 3500V, cone

25V, extractor 2V, and MCP 2700V.

As internal calibrants in negative mode, I�m/z 126.9045, 4-

acetaminophen [M–H]�m/z 150.0555 and NaI2�m/z 276.7987

were used. In positive mode 4-acetaminophen [MþH]þm/z

152.0711, codeine [MþH]þm/z 300.1599, clemastine

[MþH]þm/z 344.1781, and PEG 200/400/600 m/z 371.2281

were used. The internalmass calibrants were directly infused

through a connecting T and mixed with the LC flow post-

column. The acquisition was done using product ion scan

mode with separate acquisition channels for the internal

calibrants and the analyte. The collision energy for the

internal calibrants was 4 eV and for the analyte the collision

energy was varied. As the data from the analyte and internal

calibrant ended up in two different spectra when running

MS/MS, the acquisition channels were added with the

command ‘combine functions’. This was done in order to

obtain the analyte and the internal calibrant peaks in the

same spectrum, a prerequisite for lock mass recalibration.

The spectra were combined, smoothed and centered

according to the following software settings. The TOF

constants, resolution and number of pushes correction factor

(Npmultiplier) were set to zero. The procedure was repeated

once with the TOF constants set to 5000 and 0.7, resolution

and Np multiplier, respectively, to check that no dead time

distortion was present (the m/z values should be the same in

both centered spectra).24 To achieve an acceptable calibration


for accurate mass measurements (mean residual <3mTh) a

polynomial fit of 5 was used according to the recommen-

dations of the manufacturer.

Possible empirical formulae for the fragments were

calculated using the software SoftShell MS Calculator v.

1.3 (SoftShell International Ltd., Grand Junction, CO, USA).

The maximum tolerated m/z deviation used was 10 mTh.

Constraints regarding atom types and number of atoms were

set taking the composition of the precursor ion, MS3 data and

the nitrogen rule into account.

Derivatization method developmentNPA (0.06mM) was used as a model compound to test the

derivatization conditions.The tested concentrations of TEA,

CMP, EDC and PA were evaluated by comparison of the

chromatographic peak areas of the derivatives as a relative

measure of the reaction yield. An overview of the intended

reactions is given in Fig. 2.

The conditions for the reaction involving activation with

CMP and substitution with PAwere studied as follows. First,

the CMP concentration in the reaction tubes was held

constant at 23mM and the PA concentration was varied in

the interval 10–60mM. Secondly, the PA concentration was

held constant at 23mM and the CMP concentration was

varied between 3 and 33mM. The TEA concentration was

also varied in the interval 3–100mMwith constant CMP and

PA concentrations (3 and 23mM, respectively).

The conditions for the reaction involving activation with

EDC and substitution with PAwere also evaluated. The EDC

concentration was varied between 5 and 35mM at constant

PA concentration (22.5mM). The PA concentration was also

varied between 7.5 and 45mM at constant EDC concen-

tration (35mM). The reaction conditions selected for the

further experiments and the procedures are given separately

for the different reactions in the sections below.

Derivatization with CMPStandard solutions of the model compounds (NPG, estriol,

E3G, E16G and E17G) were prepared in acetonitrile. To an

aliquot ofmodel compound solution (200mL, 0.03mM), CMP

(70mM) and TEA (140mM) in acetonitrile (200mL) and pure

acetonitrile (200mL) were added. Thus, the final concen-

trations of the compounds in the reaction tubes were

0.01:23:47mM model compound/CMP/TEA. The samples

were placed in an ultrasonic bath for 30min and then

evaporated at 658C to dryness under a flow of nitrogen. The

residues were dissolved in acetic acid (0.1M) in Milli-Q

water (200mL) and vortexed.

Activation with CMP and substitution with PAThe (CMP)-PA derivatization (the parentheses symbolize

that the activator was exchanged by substitution of PA) was

performed as follows. To an aliquot of model compound

solution (200mL, 0.03mM), CMP (10mM) and TEA (20mM)

in acetonitrile (200mL) and PA (70mM) in acetonitrile

(200mL) were added. Thus, the final concentrations of the

compounds in the reaction tubes were 0.01:3:7:23mMmodel

compound/CMP/TEA/PA. The samples were placed in an

ultrasonic bath for 30min and then evaporated at 658C to

dryness under a flow of nitrogen. The residues were


DOI: 10.1002/rcm

Figure 2. Reaction schemes for (A) activation with CMP and PA substitution; (B) activation

with EDC and PA substitution; and (C) CMP binding to the free phenolate for E16G and E17G

(for explanation of R2 and R3, see Fig. 1).


dissolved in acetic acid (0.1M) in Milli-Q water (200mL) and

vortexed.

Derivatization with EDCTo an aliquot of model compound in acetonitrile (100mL,

0.06mM), acetonitrile (100mL) was added. The sample was

vortexed and the activator EDC (40mM) in acetonitrile

(200mL) was added together with concentrated TEA (20mL).

Thus, the final concentrations of the compounds in the

reaction tubeswere thus 0.014:19:342mMmodel compound/

EDC/TEA. The samples were placed in an ultrasonic bath for

30min and then evaporated at 658C under a flow of nitrogen

to dryness. The residues were dissolved in acetic acid (0.1 M)

in Milli-Q water (200mL) and vortexed.

Activation with EDC and substitution with PATo an aliquot of model compound in acetonitrile (100mL,

0.06mM), PA (100mM) in acetonitrile (100mL) was added.

The sample was vortexed and the activator EDC (40mM) in

acetonitrile (200mL) was added together with concentrated

TEA (20mL). Thus, the final concentrations of the compounds

in the reaction tubes were 0.014:19:342:24mM model

compound/EDC/TEA/PA. The samples were placed in

an ultrasonic bath for 30min and then evaporated at 658Cunder a flow of nitrogen to dryness. The residues were

dissolved in acetic acid (0.1M) in Milli-Q water (200mL) and

vortexed.

Derivatization with both EDC and CMPTo an aliquot of model compound in acetonitrile (100mL,

0.06mM), acetonitrile (100mL) was added. The sample was

vortexed and the activator EDC (40mM) in acetonitrile

(200mL) was added together with concentrated TEA (20mL).


Thus, the final concentrations of the compounds in the

reaction tubes were 0.014:19:342mM model compound/

EDC/TEA. The sampleswere placed in an ultrasonic bath for

30min, where later CMP (70mM) and TEA (140mM) in

acetonitrile (200mL) were added. The final concentrations of

the compounds in the tubes were 0.01:13:277:23mM model

compound/EDC/TEA/CMP. The samples were placed in

the ultrasonic bath for another 30min. They were then

evaporated at 658C to dryness under a flow of nitrogen and

finally redissolved in acetic acid (0.1M) in Milli-Q water

(200mL) and vortexed before injection into the chromato-

graphic system (for the reaction scheme, see Fig. 3).

Activation with EDC, substitution with PA andreaction with CMPTo an aliquot of model compound in acetonitrile (100mL,

0.06mM), PA (100mM) in acetonitrile (100mL) was added.

The sample was vortexed and the activator EDC (40mM) in

acetonitrile (200mL) was added together with concentrated

TEA (20mL). Thus, the concentrations of the compounds in

the reaction tubes for the first reaction were

0.014:19:342:24mM model compound/EDC/TEA/PA. The

samples were placed in an ultrasonic bath for 30min,

whereafter CMP (70mM) and TEA (140mM) in acetonitrile

(200mL) were added. The final concentrations of the

compounds in the tubes were 0.01:13:277:16:23mM model

compound/EDC/TEA/PA/CMP. The samples were placed

in the ultrasonic bath for another 30min. They were then

evaporated at 658C to dryness under a flow of nitrogen and

finally redissolved in acetic acid (0.1 M) in Milli-Q water

(200mL) and vortexed before injection into the chromato-

graphic system.


DOI: 10.1002/rcm

Figure 3. Derivatization reaction scheme for the double reaction of E16G. The same

procedure was applied for E17G.


Figure 4. Mass spectra from (A) the negative product ion

mode analysis with precursor ion m/z 287 [M–H]� of estriol

and with a collision energy of 40 eV and (B) the positive

product ion mode analysis with precursor ion m/z 289

[MþH]þ of estriol and with a collision energy of 20 eV

(Q-TOF instrument).

RESULTS AND DISCUSSION

Two different mass spectrometers, the TSQ 7000 and the

Q-TOF, were used for the evaluation of the fragmentation of

underivatized estriol and estriol glucuronides in both

negative and positive product ion mode. These initial

experiments were performed in order to obtain reference

data for comparison with the fragmentation of the deriva-

tives, as well as to demonstrate the difficulty in establishing

the position of glucuronidation by mass spectrometry alone.

Fragmentation of estriolEstriol fragmentation was investigated and used as a

reference for the further structural evaluation of the product

ions of the glucuronides and their derivatives. The TSQ

instrument could not detect estriol at the investigated

concentrations (120mM). The Q-TOF instrument on the

other hand gave a signal for this compound in both negative

and positive ion mode with precursors m/z 287 for [M–H]�

andm/z 289 for [MþH]þ, respectively, but the signal intensity

in positive ion mode was low.

The product ion spectrum of estriol in negative mode is

shown in Fig. 4(A). Structures for the ions atm/z 143, 145 and

171 have previously been proposed by Croley et al.25 The

corresponding positive product ion spectrum of estriol

agreed well with previous studies26,27 (Fig. 4(B)). These

fragments were subjected to accurate mass measurement,

using the Q-TOF. Suggested formulae and structures of the

ions together withm/z differences compared with theoretical

values are given in Tables 1 and 2. Different collision energies

were used in some of the accurate mass determinations in

order to regulate the relative abundance, and thereby the

mass accuracy, of different product ions.


Fragmentation of estriol glucuronidesStandards of the metabolites E3G, E16G and E17G (30mM)

were chromatographed and analyzed in both negative and

positive product ion mode with precursor ions m/z 463 [M–

H]� and m/z 465 [MþH]þ, respectively. Fragmentation

patterns for the metabolites were compared at different

collision energies (15, 35, 55, and 75 V with the TSQ


DOI: 10.1002/rcm

Table 1. Accurate mass determination and proposed formulae of estriol ions in negative ion ESI. As internal mass calibrants,

I�m/z 126.9045, 4-acetaminophen [M–H]�m/z 150.0555 and NaI�2m/z 276.7987 were used. Proposed structures for negatively

charged estriol ions are also shown

MS determined (m/z) Internal calibrant ions (m/z) Proposed fragments Theoretical mass (m/z) Mass difference (mTh)

287.1645 276.7987 C18H24O3 [M–H]� 287.164716 0.2

171.0798 150.0555 CH2

O

C12H11O25

171.080990 1.2

169.0607 150.0555 CH2

O

C12H9O

169.065339 4.6

145.0661 150.0555

O

C10H9O25

145.065339 0.8

143.0517 150.0555

O

C10H7O25

143.049689 2.0

119.0483 126.9045 CH2

O

C8H7O

119.049689 1.8


instrument and 15, 25, 35, 45, 55 and 65 eV with the Q-TOF

instrument).

No significant differences in fragmentation could be

observed between the three estriol glucuronides using any

of the instruments in the negative ion mode (Q-TOF spectra

are shown in Fig. 5). The accurate masses of the negative ESI

product ions from E16G were also determined using the Q-

TOF (Table 3). Almost all the proposed theoreticalm/z values

of the ions were in good agreement with the determined

ones. The deviations were between 2.3 and 4.2mTh, except

for m/z 113. The chemical structures of some of the product

ions of E16G are proposed in Table 3. The m/z 175 ion

originated from the glucuronic acid,m/z 113 corresponds to a

loss of CO2 and water from m/z 175,28 and m/z 85 to a loss of

CO fromm/z 113.4,28 Thus, the estriol glucuronides produced

mainly the aglycone and fragments from the glucuronic acid

in negative ion mode. One exception was m/z 171, which

resulted from fission of the steroid skeleton.

The conjugated metabolites were also analyzed in positive

product ionmode (Q-TOF spectra are shown in Fig. 6). Many

of the product ions were formed from the aglycone, as they

were also observed in the positive product ion spectrum of

estriol (cf. Fig. 4(B)). E3G also had product ions atm/z 313, 355

and 411, which were not observed for E16G and E17G, but

the signal intensity was very low, making these data

uncertain. The accurate masses of the positive product ions


were also determined, and the differences between the

determined m/z values and the theoretical ones for the

suggested ions were lower than the corresponding results

from negative mode (Table 4).

From these analyses, all three conjugated estriol metab-

olites could not be differentiated from each other according

to their fragmentation patterns. Furthermore, E16G and

E17G could not be separated from each other chromato-

graphically (Fig. 7).

The present case was thus a further example of the general

phenomenon with structural evaluation of glucuronides

using MS/MS, i.e. the loss of 176Da at low collision

energies.5,21 This makes the determination of the position

of glucuronidation impossible. In order to determine

whether this problem could be solved by chemical

derivatization, different reaction procedures were tested.

The hypothesis was that introduction of a positive charge

into the steroid glucuronides would alter their fragmentation

pattern due to the possibility of a charge remote mechan-

ism.15 This would also increase their detection sensitivity in

positive ion ESI.

Choice of derivatization conditionsThe limited availability of model compounds is a general

problem when developing methods for phase II metabolites.

NPA was therefore used as a model compound for


DOI: 10.1002/rcm

Table 2. Accurate mass determination and proposed formulae of estriol product ions in positive ion ESI with collision energy

10 eV and 20 eV. As internal mass calibrants, 4-acetaminophen [MþH]þm/z 152.0711 and codeine [MþH]þm/z 300.1599 were

used. Proposed structures for positively charged estriol product ions are also shown

MS determined(m/z) 10 eV

MS determined(m/z) 20 eV

Internal calibrantions (m/z)

Proposedfragments

Theoretical(m/z)

Mass difference(mTh) 10 eV


289.1791 289.1824 300.1599 C18H25O3 [MþH]þ 289.180369 1.3 2.0

271.1702 — 300.1599 C18H23O2 [M–H2OþH]þ 271.169805 0.4 —

253.1600 253.1603 300.1599CH

+

HO

CH3

C18H21O[M – 2 H2O+H]+

253.159240 0.8 1.0

159.0835 159.0784 152.0711

OH

CH2

+

C11H11O

159.080990 2.5 2.6

157.0632 157.0643 152.0711

OH

CH2

+

C11H9O

157.065339 2.1 1.0

133.1065 133.0647 152.0711

OH

CH2

+

C9H9O27

133.065339 41.2 0.6

107.0615 107.0556 152.0711C

+

CH3OH

C7H7O

107.049689 11.8 5.9

Figure 5. Mass spectra from the negative product ion mode

with precursor ionm/z 463 [M–H]� for E3G, E16G and E17G,

and with a collision energy of 35 eV (Q-TOF instrument).



development of the reaction conditions, due to its availability

and low cost. No full optimization of reaction conditions was

performed, as the aim of this study primarily was qualitative.

Two different activation methods for the carboxylic acid

were tested, reactions with CMP or EDC (Fig. 2(A) and 2(B)).

Both reagents form an ester with the model compound for

further nucleophilic substitution by PA. TEAwas added as a

catalyst for the activation. The carboxylic acid activation

method with CMP was slightly modified from an earlier

method developed by Leavens et al.8

Themethod development data for the activationwith CMP

and substitution with PA showed that there was a slight

increase in the amount of PA derivative formed at low CMP

concentration in the mixture at constant PA concentration

(results not shown). A variation in the PA concentration with

constant CMP concentration gave on the other hand no

significantly increased amount of the PA derivative (results

not shown). The variation in TEA concentration did not have

a significant effect on the reaction yield, and a concentration

in the lower part of the tested region was selected. The

chosen concentrations of the reagents in the reaction tubes for


DOI: 10.1002/rcm

Table 3. Accurate mass determination of the estriol-16-glucuronide ions in negative ion ESI. As internal calibrants, I�m/z

126.9045, 4-acetaminophen [M–H]�m/z 150.0555 and NaI�2m/z 276.7987 were used

Mass determined(m/z)


Proposedfragments

Theoreticalmass (m/z)

Mass difference(mTh)

287.1670 276.7987 CH3

O

OH

OH

C18H23O3

287.164716 2.3

175.0266 150.0555

O

OH

OHOH

O

O

C6H7O64, 28

175.024263 2.3

171.0784 150.0555 CH2

O

C12H11O

171.080990 2.6

117.0146 126.9045 C4H5O4 117.018783 4.2

113.0143 126.9045 O

-O O m/z 113C5H5O3

28

113.023869 9.6

Nd O

-O m/z 85C5H8O

4

nd¼ accurate mass not determined, ion only acquired on TSQ 7000 with unit mass resolution.

Figure 6. Mass spectra from the positive product ion mode

analysis with precursor ion m/z 465 [MþH]þ for E3G, E16G

and E17G, and with a collision energy of 10 eV (Q-TOF

instrument).


further experiments were 3mM CMP (containing 7mM

TEA) and 23mM PA. When CMP was used as the sole

reagent, its concentration was changed to 23mM, as this

concentration was found to increase the amount of CMP

derivative formed.


No significant effect on the product yield was obtained

from the variation of the reagent concentration for the

activation with EDC and substitution of PA (result not

shown). The chosen concentrations for the future derivatiza-

tions were 19mM EDC and 24mM PA (concentrations in the

reaction tubes).

Derivatization with CMPCMP was first tested as a sole reagent in the absence of a

nucleophile for further substitution.10 The quaternary

products (denoted EXG-CMP) were observed in positive

product ion mode with m/z of 556 Mþ for E16G-CMP and

E17G-CMP. However, for E3G-CMP, only traces were found.

The products were further studied by CID. The collision

energy was set at 15, 35 or 55V. The major product ion from

E16G-CMP and E17G-CMP was m/z 380, which corre-

sponded to the loss of 176Da, i.e. the monodehydrated

glucuronic acid (Fig. 8). This observation led to the suspicion

that the CMP was coupled to the phenol in the 3-position for

E16G and E17G instead of to the intended carboxylic acid (cf.

Figs. 2(A) and 2(C)). This was probably due to the fact that

the 3-phenolate was a stronger nucleophile than the

carboxylate. Leavens et al.8 also reported a low recovery

when coupling CMP to glucuronic acid. As CMP apparently

could bind in two different positions for E16G and E17G, i.e.


DOI: 10.1002/rcm

Table 4. Accurate mass determination of the estriol-16-glucuronide product ions in positive ion ESI with collision energy 10 or

20 eV. As internal calibrants, 4-acetaminophen [MþH]þm/z 152.0711, codeine [MþH]þm/z 300.1599 and clemastine [MþH]þm/z

344.1781 were used

Mass determined(m/z) 10 eV

Mass determined(m/z) 20 eV


Proposedfragments




465.2119 465.3241 344.1781 C24H33O9 [MþH]þ 465.212457 0.5 111.7447.2005 — 344.1781 C24H31O8 [M–H2OþH]þ 447.201893 1.4 —429.1891 429.1898 344.1781 C24H29O7 [M–2H2OþH]þ 429.191328 2.2 1.5411.1931 — 344.1781 C24H27O6 [M–3H2OþH]þ 411.180763 12.4 —289.1821 289.1803 300.1599 C18H25O3 [AglyconeþH]þ 289.180369 1.7 0.07271.1704 271.1698 300.1599 C18H23O2 [Aglycone–H2OþH]þ 271.169805 0.6 0.005253.1595 253.1585 300.1599 C18H21O [Aglycone–2H2OþH]þ 253.159240 0.3 0.7159.0268 159.0278 152.0711

OH

CH2

+

C11H11O

159.080990 54.2 53.2

Figure 7. E3G, E16G and E17G 15mM injected separately

and analyzed with MS scan (m/z 50–750) (TSQ instrument).

Extracted ion chromatogram for m/z 463.3 is shown.

Figure 8. Mass spectrum from positive product ion analysis

of precursor ion m/z 556 (Mþ of E17G-CMP) with a collision

energy of 35V (TSQ instrument).


of precursor ion m/z 380 (Mþ of estriol-CMP) with a collision

energy of 45 eV (TOF instrument).


to the phenol in the 3-position and/or to the carboxylic acid

on the glucuronide, these reactions could thus theoretically

result in a bis-CMP product, but no such peaks were

observed. Furthermore, no significant differences were


found between the product ion spectra for E16G-CMP and

E17G-CMP. E3G-CMP, with the glucuronic acid attached to

the 3-phenolic position, gave no product ion at m/z 380.

Instead a product ion at m/z 110 was found, which

corresponded to 2-hydroxy-1-methylpyridinium. The

absence of a neutral loss of 176Da indicated that CMP

had bound to the glucuronic acid on this compound.

To confirm the hypothesis that CMP could bind to the 3-

phenol, the derivatization was also performed on estriol. An

ion at m/z 380 was then observed in MS1, corresponding to

Mþ of an estriol-CMP product. This ion was fragmented in

positive product ion mode with collision energies up to

65 eV, using the Q-TOF. The m/z 380 ion was very stable;

however, some product ions could be observed, such as m/z

110, which corresponded to 2-hydroxy-1-methylpyridinium

(Fig. 9). These results strengthened the hypothesis that CMP

could react in the 3-phenolic position. This derivative had a

higher response in positive ionMS1 than estriol itself, and the

accurate masses of the ions from the product ion analysis

(precursor ion m/z 380) were determined (Table 5).

Although no tuning could be performed with the

derivatives, both E16G-CMP and E17G-CMP had a higher

response in positive ion mode than the E16G and E17G in

either positive or negative ion mode. In conclusion, the CMP

reaction was very useful as an aid to the use of mass

spectrometry to discriminate phenols from alcohols, in


DOI: 10.1002/rcm

Table 5. Accurate mass determination of estriol-CMP product ions in positive ion ESI with collision energy 45 eV. Internal

calibration substance, clemastine [MþH]þm/z 344.1781

Mass determined (m/z) Internal calibrant ions (m/z) Proposed fragments Theoretical mass (m/z) Mass difference (mTh)

380.2222 344.1781 See Fig. 9 for suggested structure. 380.222569 0.4C24H30NO3

362.2125 344.1781 C24H28NO2 [M–H2O]þ 362.212004 5.0348.1966 344.1781 C23H26NO2 [M–CH4O]þ 348.196354 0.25


of precursor ion m/z 555 ([MþH]þ of E17G-(EDC)-PA) with a

collision energy of 35 V (TSQ instrument).


addition to the increased detection sensitivity in positive ion

ESI.

Activation with CMP and substitution with PACMP activation followed by derivatization with PA (Fig. 2(A))

should theoretically give a protonated molecule at m/z 555.

This derivative was found for E3G but not for E16G and

E17G. An explanation for this is that CMP reacted quickly

with the phenolic group in the 3-position for the two latter

metabolites creating a stable derivative, and that no

activation of the carboxylate occurred. Other possible

derivatives could be those with CMP attached to the 3-

position and with PA bound to the glucuronic acid, but no

indications of this kind of product were found. The E3G

carboxylic acid group was, on the other hand, activated by

CMP, followed by substitution with PA. Interestingly, the

E3G-(CMP)-PA derivative gave a more intense signal than

E3G-CMP. It also gave a higher response than E3G itself in

the positive ion scan mode.

Derivatization with EDCAs CMP bound to the phenolic position, a more selective

reagent than CMPwas needed. EDC, a well-known carboxylic

acid activator,16,17,20 was therefore tested (Fig. 2(B)).

EDC appeared to give a stable product ([MþH]þm/z 620.6)

with all three metabolites, and the positive product ionmode

gave ions at m/z 129, 261 and 549. The m/z 380 ion (loss of

176Da) which appeared with CMP bound to the phenol in

E16G and E17G could not be found, indicating that the

glucuronic acids had been derivatized by EDC. The

responses for E16G-EDC and E17G-EDC were somewhat

lower than for E16G-CMP and E17G-CMP, respectively. This

could be explained by the fact that the CMPderivatives had a

constantly positively charged pyridinium group, or by

differences in reaction yield.

Activation with EDC and substitution with PAEDC activation followed by PA substitution should give the

same product for E3G as (CMP)-PA derivatization. As

expected, a comparison of the product ionmass spectra of the

E3G-(CMP)-PA and E3G-(EDC)-PA derivatives showed no

significant differences. As mentioned above, for E16G and

E17G no (CMP)-PA-derivative could be found, but, when

using EDC as an activator of the carboxylic acid, PA could

bind to all three isomers. However, there were no significant

differences in their fragmentation patterns. As an example,

the product ion spectrum of E17G-(EDC)-PA together with

suggested structures of the ions are shown in Fig. 10. It is

notable that the fragmentation was significantly different

than for estriol and the native estriol glucuronides. Most

product ions from this derivative seemed to contain the PA


and glucuronyl residues. This was probably a result of the

pyridine nitrogen carrying the positive charge.

Derivatization with both EDC and CMPTo enhance the possibility of obtaining significant differences

in fragmentation between E16G and E17G, a double reaction

was tested (see the reaction scheme in Fig. 3). First, EDC

derivatization was performed, and this was followed by

reaction with CMP. The results from these experiments

showed that CMP and EDC had both bound to E16G and

E17G, forming a product with m/z 711.6 for Mþ and 356.4 for

[MþH]2þ in the spectrum recorded with the TSQ 7000 mass

spectrometer. The derivatives were analyzed in positive

product ion mode with precursor ions m/z 711.6 and 356.4,

respectively. The collision energy was varied from 5 to 55V

and the product ion from the doubly charged molecules

agreed with the fragmentation of the singly charged

molecules. No significant fragmentation differences were

found between E16G-EDC-CMP and E17G-EDC-CMP

(results not shown).

Activation with EDC, substitution with PA andreaction with CMPThe EDC-CMP derivatization, described above, was

extended by substitution of EDC with PA for E16G and

E17G (Fig. 3). Both a singly charged derivative (m/z 646.8Mþ)

and a doubly charged derivative (m/z 323.7 [MþH]2þ) were

formed with identical product ion spectra for the respective

isomer. Interestingly, however, the fragmentation patterns

differed for the E16G and E17G derivatives on all three

instruments. The data obtained from the Q-TOF and the ion

trap are shown in Figs. 11 and 12, respectively. The major

product ion from the E16G-(EDC)-PA-CMP derivative was

m/z 306, which could not be found for E17G-(EDC)-PA-CMP.


DOI: 10.1002/rcm

Figure 11. Mass spectra from product ion analysis of pre-

cursor ion m/z 646 (Mþ) with a collision energy of 55 eV (Q-

TOF instrument) for the E16G-(EDC)-PA-CMP and E17G-

(EDC)-PA-CMP derivatives.

Figure 12. Mass spectra from product ion analysis of pre-

cursor ion m/z 646 (Mþ) with a collision energy of 55% (LCQ

ion trap) for the E16G-(EDC)-PA-CMP and E17G-(EDC)-PA-

CMP derivatives.


Furthermore, m/z 362 was significantly more abundant for

E16G-(EDC)-PA-CMP, whereas the opposite situation

occurred for m/z 348. The product ion spectra differed

somewhat between the Q-TOF and the ion trap in the higher

mass region, probably due to somewhat different fragmenta-

tion mechanisms, but the occurrence of the above-mentioned

characteristic product ions was the same for both instrument

types. This important finding thus made mass spectrometric

differentiation between these two isomers possible.

In order to aid elucidation of the structures of the

characteristic product ions, accurate mass measurements

and MS3 experiments were performed on the Q-TOF and the

ion trap, respectively. The ion at m/z 380 probably resulted


from the loss of monodehydrated glucuronic acid and PA

from the derivative, yielding estriol-CMP (Table 6, cf. Fig. 9).

The formation of the characteristic ion at m/z 306 indicated

a neutral loss of 74 Da fromm/z 380. However, as expected,

MS3 experiments performed on the ion trap did not

demonstrate that this ion was formed from m/z 380.

Assuming that m/z 306 was an even-electron ion, it should

contain an odd number of nitrogen atoms according to the

nitrogen rule. Taking the proposed structure of the E16G

derivative into account, it would be most probable that m/z

306 contained only one and not three nitrogen atoms, as the

mass difference from the precursor ion was 340Da.

Calculations of possible elemental compositions based

on the determined accurate mass, assuming that the

remaining nitrogen was on the pyridinium ring, did not

yield any realistic formula other than C21H24NO, using a

tolerated m/z difference of �10mTh. A further notable

observation was that this characteristic ion was not formed

from the other derivatives of E16G or E17G with a CMP

group in the 3-position (cf. Fig. 8). This implies that the

remote pyridine of the PA groupwas somehow involved in

the formation of m/z 306.

The m/z 362 ion indicated a loss of water (18Da) from m/z

380 (Table 6). MS3 experiments performed on the ion trap

confirmed thatm/z 362 was a secondary fragment of m/z 380.

Using this information as a constraint, there were no other

probable elemental compositions than the expected

C24H28NO2, using a tolerated m/z difference of �10mTh

from the determined accurate mass. However, there must be

alternative mechanisms for its formation, as the ion was

significantly more abundant for the E16G derivative.

The E17G-(EDC)-PA-CMP derivative gave two product

ions, m/z 346 and 348, which were less abundant for the

E16G-(EDC)-PA-CMP derivative (Table 7 and Fig. 11). MS3

experiments showed that m/z 348 was a secondary product

ion from m/z 380. Calculations of the accurate mass data,

taking this fact into account, gave C23H26NO2 as the only

realistic formula, indicating a neutral loss of CH4O from m/z

380. This hypothesis was strengthened by the fact that this

ion also was formed from estriol-CMP (cf. Fig. 9 and Table 5),

with a determined accurate mass that even more closely

matched a loss of CH4O.

The ion at m/z 346 was more difficult to explain, as the

measurement difference was very high when assuming a net

loss of CH6O (34Da) from m/z 380 (Table 7). Furthermore,

there were no indications from the MS3 experiments that this

ion was formed from m/z 380. Assuming that m/z 346 was an

even-electron ion, it should contain an odd number of

nitrogen atoms. As discussed above for m/z 306, the only

realistic possibility is thus that it only has the pyridinium

nitrogen remaining. However, a calculation of different

possible empirical formulae within am/z error of 10 mTh did

not give rise to any composition that could yield a realistic

structure. One remaining possibility is that m/z 346 was an

odd-electron ion.

CONCLUSIONS

Chemical derivatization has been successfully utilized in

order to enable structural discrimination between isomeric


DOI: 10.1002/rcm

Table 6. Accurate mass determination of the E16G-(EDC)-PA-CMP product ions in positive ion ESI with collision energy 55 eV.

Internal calibration substance, clemastine [MþH]þm/z 344.1781



Proposedfragments



380.2279 344.1781 See Fig. 9 for suggested structure. C24H30NO3 380.222569 5.3

362.2202 344.1781 C24H28NO2 [M–266–H2O]þ 362.212004 8.2

306.1944 344.1781

N+

CH3

CH3

O

C21H24NO

306.185789 8.6

Table 7. Accurate mass determination of E17G-(EDC)-PA-CMP product ions in positive ion ESI with collision energy 55 eV.

Internal calibration substance, clemastine [MþH]þm/z 344.1781



Proposedfragments

Theoretical mass(m/z)


380.2289 344.1781 See Fig. 9 for suggested structure. C24H30NO3 380.222569 6.3348.2032 344.1781 C23H26NO2 [M–266–CH4O]þ 348.196354 6.8346.2238 344.1781 (C23H24NO2)

� 346.180704 43.1

�This is not a suggested fragment, only the atomic composition of the theoretical m/z used for the calculation of the m/z difference.


glucuronides by mass spectrometry. Estriol-16-glucuronide

(E16G) and estriol-17-glucuronide (E17G) could be differ-

entiated from each other by collision-induced dissociation

(MS/MS) only after a certain covalent modification. A

characteristic base peak product ion for the E16G derivative

was found at m/z 306, which could not be observed for the

E17G derivative. Furthermore, estriol-3-glucuronide (E3G)

could be selectively detected by a different type of reaction

product, due to the absence of a free phenol function.

AcknowledgementsThe authors are grateful to Professor Dr. Wilfried Niessen

(hyphenMassSpec, The Netherlands) for fruitful discussions

on fragmentation pathways.

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DOI: 10.1002/rcm

Documents

Differentiation of estriol glucuronide isomers by chemical derivatization and electrospray tandem mass spectrometry