application of hyphenated mass spectrometric techniques to the determination of corticosteroid...

Application of Hyphenated MassSpectrometric Techniques to theDetermination of Corticosteroid Residuesin Biological Matrices

J.-P. Antignac &, F. Monteau, J. Negriolli, F. Andre, B. Le Bizec

Laboratoire d’Etude des Residus et Contaminants dans les Aliments (LABERCA), Ecole Nationale Veterinaire de Nantes (ENVN),Route de Gachet, BP 50707, 44307 Nantes Cx 3, France; E-Mail: [email protected]

In Honour of Professor Edward Houghton: A Celebration of 30 Years in Racing Chemistry

Received: 7 October 2003 / Revised: 3 November 2003 / Accepted: 18 November 2003Online publication: 6 February 2004

Abstract

Fifty years after the discovery of natural corticosteroid hormones and their anti-inflammatoryproperties many synthetic derivatives of these molecules are now available. Most are widely usedin human and veterinary medicine, legally but under regulated conditions. These compounds canalso be used as growth promoters in animal breeding, although such use is illegal in Europe.Consequently, analytical methods have been developed to monitor use of corticosteroids in cattle.This paper, based on the authors experience and the main relevant literature, describes thedifferent mass spectrometric approaches used for measurement of corticosteroid residues (parentdrug, metabolites, and esters) in biological matrices (urine, meat, hair), including gas chroma-tography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry(LC–MS2). The respective advantages of liquid chromatography and gas chromatography, inconjunction with different derivatisation reactions, are discussed. The behavior of corticosteroidswith different ionization techniques is also discussed. Application to monitoring corticosteroidmisuse and to investigation of pharmacokinetics and metabolism in bovine species is describedand new data are presented relating to elimination and hair fixation kinetics for free and esterforms and the nature and proportions of corticosteroid phase I and phase II metabolites. Finally,this work reviews ten years experience of the use of a variety of mass spectrometric techniques foranalysis of corticosteroids in animals produced as food.

Keywords

Column liquid chromatography – mass spectrometryGas chromatography – mass spectrometryCorticosteroidsResidues in biological matrices

Introduction

Fifty years after the discovery of natural

corticosteroid hormones and their anti-

inflammatory properties, many synthetic

derivatives of these molecules are now

available. In human and veterinary medi-

cine their legal use is strictly regulated,

with withdrawal periods between treat-

ment and slaughter, andmaximum residue

levels in edible material for some of the

compounds. Low concentrations of

glucocorticosteroids are also known to

promote weight gain, to reduce feed con-

version ratio, and to have a synergetic

effect with other molecules, for example b-agonists or anabolic steroids [1–3]. The

compounds have therefore been used as

growth promoters in cattle, a practice not

allowed in Europe. For many years a

variety of analytical methods have been

proposed for identification of corticoste-

roid residues in edible tissue or urine

samples, all based on detection of the

parent drug. During the 1980s and early

1990s themain detectionmethods used for

corticosteroids were radioimmunology

[4, 5], fluorimetry [6, 7], or liquid chroma-

tography with UV detection [8–12]. These

methods had the advantages of rapidity,

automation, and high-throughput in rou-

tine screening analysis but sometimes

lacked sensitivity and/or specificity.

Nowadays, mass spectrometry (MS) cou-

pled to gas (GC) or liquid (LC) chroma-

tography is the method of choice for

unambiguous identification of these com-

pounds at trace levels in biological matri-

ces. GC–MS with electron-impact (EI)

ionization was the first mass spectrometric

technique to be applied to these com-

pounds [13–23]. Carbon isotope-ratio

mass spectrometry (GC–C-IRMS) has

also been used to distinguish natural

endogenous corticosteroids from their

exogenous analogues [24]. The further

development of LC–MS [25–28] and LC–

MSn [29–38] with atmospheric-pressure

ionization (API) techniques has been

responsible for spectacular improvements

of method performance for these moder-

ately polar molecules.

DOI: 10.1365/s10337-003-0179-3

2004, 59, S13–S22

� 2004 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

Review Chromatographia Supplement Vol. 59, 2004 S13

If this context, the first purpose of this

paper is to review and discuss the advan-

tages and disadvantages of these different

techniques for analysis of corticosteroid

residues, including parent drugs, esterified

forms, and metabolites, that could be re-

garded as innovative improvements in the

field. Different aspects of the analysis, for

example chromatographic separation

(comparison of GC with LC), ionization

technique (comparison of EI with API),

mode of acquisition (comparison of MS

with MSn), and sample preparation have

been considered.

In drug-residue analysis, and for other

fundamental or practical purposes also,

knowledge of the main metabolic path-

ways of the target analyte should be of

major interest. The parent drug can be

subject to biotransformations basically

divided into phase I and phase II metab-

olism. Phase Imetabolism usually includes

oxidation and/or reduction reactions (for

instance hydroxylation and/or hydroge-

nation). Phase II metabolism basically in-

volves conjugation reactions with polar

groups such as glucuronide or sulfate,

leading to a highly hydrophilic product,

which facilitates elimination in the urine or

feces. Very few studies have been devoted

to the nature and relative proportions of

the different phase I and phase II metab-

olites of corticosteroids in cattle; the liter-

ature refers to studies on horses [39–41],

humans [42–47], and rodents [48] only. In

this context, the second purpose of this

paper was to describe some applications of

mass spectrometric techniques dedicated

to investigation of the pharmacokinetics

and metabolism of corticosteroids in cat-

tle. The applications and complementarity

of the different mass spectrometric tech-

niques for detection of phase I metabolites

and determination of the relative propor-

tions of phase II metabolites is discussed.

Experimental

Reagents and Chemicals

Methanol, cyclohexane, diethyl ether,

ethyl acetate, glacial acetic acid and 1 M

HCl were analytical or HPLC grade from

Solvents Documentation Syntheses (SDS,

Peypin, France), who also supplied C18

and SiOH SPE cartridges (2 g and 1 g

stationary phase, respectively). Sodium

acetate and sodium carbonate were pur-

chased from Merck (Darmstadt, Ger-

many). The enzyme forphase IImetabolite

deconjugation was a purified Helix poma-

tia preparation from Sigma (St Louis,

MO, USA). The derivatization reagents

N-methyl - N-(trimethylsilyl)trifluoroace-

tamide (MSTFA) and trimethyliodosilane

(TMIS) were purchased from Fluka

(Buchs, Switzerland). Dithiothreitol

(DTE) was fromAldrich (Milwaukee,WI,

USA). Standard reference corticosteroids

were purchased from Sigma. Standard

solutions (1 mg mL)1) were prepared in

methanol. Working solutions were pre-

pared by successive tenfold dilution with

methanol and stored in the dark at)20 �C.

Sample Preparation

The analytical procedures used for

extraction and purification of corticos-

teroids from urine, edible tissue, and hair

have been described elsewhere [49–51].

Briefly, the initial sample preparation

step was enzymatic hydrolysis for urine

(Helix pomatia, 50 �C, 4 h) and liquid–

liquid extraction for edible tissue (meth-

anol–2 M acetate buffer, 45:55 v/v). For

hair samples, acid hydrolysis (methanol–

1 M HCl, 40:60 v/v, 50 �C, 4 h) of un-

treated hair was used for measurement of

the parent drug (i.e. total dexametha-

sone) whereas liquid extraction of pul-

verized hair (methanol, ultrasonic bath,

30 min) was used for the measurement of

the ester forms of the drug. Extracts were

purified by reversed-phase SPE of C18

with subsequent alkaline liquid–liquid

clean-up (Na2CO3 10%), eventually fol-

lowed by normal-phase SiOH SPE.

Gas Chromatography

Gas chromatography was performed with

an HP6890 chromatograph (Agilent

Technologies, Palo-Alto, USA). Com-

pounds were separated on a 30 m · 0.25-

mm i.d. column coated with a 0.25-lmfilm of OV-1 (Ohio-Valley). The column

oven temperature was maintained at

150 �C for 3 min after injection then

programmed at 5 �min)1 to 270 �C which

was maintained for 10 min. Injector and

transfer line temperatures were 250 and

280 �C, respectively. Helium (N55) was

used as carrier gas at a flow rate of

1 mL min)1. the different derivatisation

reactions tested were classical silylation

with MSTFA–TMIS–DTE (1000:5:5,

v/v/w, 50 �C, 12 h), formation of oxime

derivatives by use of methoxylamine

(MOX 8% in pyridine, 70 �C, 30 min),

and formation of boronic esters (meth-

ylboronic acid in ethyl acetate, room

temperature, 15 min).

Liquid Chromatography

HPLC was performed with an Alliance

2690 HPLC pump with automatic injector

(Waters, Milford, MA, USA). Reversed-

phase liquid chromatography was per-

formed on a 50 mm · 2 mm i.d., 5-lmparticle, Nucleosil C18AB octadecyl-graf-

ted silica column (Macherey–Nagel,

Duren, Germany) with guard column

(10 mm · 2 mm i.d., 5-lm particle, Nu-

cleosil C18AB). The mobile phase was a

gradient prepared from methanol (A) and

0.5% (v/v) acetic acid in water (B). Mobile

phase composition (A:B; v/v) was:

0–10 min, 40:60; 10–20 min, 90:10; and

20–30 min, 40:60. The flow rate was

220 lL min)1 and the volume injectedwas

10 lL.

Mass Spectrometry

GC–MS was performed with an

HP5989A mass spectrometer (Agilent

Technologies, Palo-Alto, CA, USA) with

electron-impact (EI) ionization, or

chemical ionization with methane as re-

agent gas, in positive-ion (PCI) or nega-

tive-ion (NCI) mode. Source and

quadrupole temperatures were 280 and

100 �C, respectively.LC–MS and LC–MS2 experiments

were performed with a QuattroLC triple-

quadrupole analyzer (Micromass, Man-

chester, UK) in positive or negative

electrospray ionization mode. Nitrogen

was used as nebulization and desolvation

gas, at flow rates of 90 and 600 L h)1,

respectively. Source and desolvation

temperatures were 130 and 400 �C,respectively. Potentials applied to the

capillary (from 3.0 to 4.0 kV) and cone

(from 15 to 35 V) were optimized for

each molecule, as was the energy applied

in the collision cell (from 5 to 30 V).

Results and Discussion

Comparison of GC and LC forSeparation of Corticosteroids

Typical GC and LC ion chromatograms

obtained for different corticosteroids,

S14 Chromatographia Supplement Vol. 59, 2004 Review

corticosteroid phase I and II metabolites,

and corticosteroid esters are presented in

Fig 1. Because of the moderate polarity

of corticosteroids, as a result of hydroxyl

and keto groups on the sterane skeleton,

their gas-chromatographic analysis re-

quires preliminary derivatization to in-

crease their volatility and avoid

adsorption phenomena leading to tailing

peaks and natural water loss. Most cur-

rently used techniques employ chemical

oxidation, then negative chemical ioni-

zation or classical silylation, then positive

electron-impact ionization [15]. Typical

ion chromatograms obtained from

bovine urine samples by use of these two

techniques are shown in Fig 1. If chemi-

cal ionization is used (Fig. 1a), isomeric

by-products and interfering compounds

can complicate interpretation. A better

chromatographic profile is obtained after

silylation (Fig. 1b) but sensitivity is low-

er, because of the formation of multiple

by-products (a mixture of polysilylated

compounds). Finally, GC is preferable to

LC when used with simple MS, because

of the better resolution of complex bio-

logical extracts and better isomer differ-

entiation. Indeed, the poor separation

sometimes obtained in liquid chroma-

tography combined with a single mass-

filtering stage can be insufficient for

unambiguous interpretation. Despite the

suitability and efficiency of GC–NCI-MS

its field of application is mainly limited to

screening purposes—because different

compounds can lead to the same deriva-

tive, complementary analysis is usually

needed for unambiguous analyte identi-

fication.

Liquid chromatography does not re-

quire a derivatization step and enables

direct measurement of corticosteroids.

Although improved sensitivity is clearly

apparent from the profile obtained from

liquid chromatography–negative electro-

spray tandem mass spectrometry

(Fig. 1c), most of the analytes of interest

are not well separated. With regard to

mass differences between the native un-

derivatized target molecules, however,

the specificity of the extracted ion chro-

matograms is satisfactory. This technique

is, moreover, particularly suitable for di-

rect measurement of polar metabolites, as

shown in Figs. 1d and 1e, which depict

typical diagnostic ion chromatograms for

four conjugated phase-II corticosteroid

metabolites and six hydroxylated phase-I

corticosteroid metabolites, respectively.

Other authors [38] have, moreover, re-

ported the possibility of separating the

two isomers dexamethasone and beta-

methasone under isocratic conditions

(acetonitrile–water, 90:10 (v/v) containing

0.3% formic acid) with a specific column

(100 mm · 2.1 mm i.d., 5-lm particle,

Hypercarb; Thermo-Hypersil, Runcorn,

UK), a separation that was successfully

reproduced in our laboratory (Fig. 1f).

Disadvantages of LC include limitation

to reversed-phase separations, because of

solvent compatibility requirements with

API interfaces and, sometimes, ion-sup-

pression phenomena when target analytes

are co-eluted with the highly polar unre-

tained fraction of the extract. LC–MS

and LC–MS2 are also highly suited to

direct separation and measurement of

corticosteroid esters, as shown in Fig. 1g.

Nevertheless, with the development of

new interfaces (e.g. photoionization) ex-

pected to enable extended choice of mo-

bile phases, and introduction of more

specific stationary phases, liquid chro-

matography might be the ultimate choice

for analysis of corticosteroids and corti-

costeroid metabolites.

Comparison of EI, CI, and ESIfor Ionization ofCorticosteroids

Typical mass spectra obtained for dexa-

methasone by use of a variety of ioniza-

tion techniques are presented in Fig 2.

Silylation with N-methyl-N-trimethylsi-

lyl-trifluoroacetamide–trimethyliodosi-

lane–dithiothreitol (MSTFA–TMIS–

DTE, 1000:5:5, v/v/w) for 12 h at 50 �Cled to a penta-TMS derivative (Fig. 2a).

The weak intensity of the molecular ion

and the huge fragmentation of the

derivative led to poor sensitivity. Such

derivatization then EI+ ionization can

be used for screening purposes on the

basis of full-scan or SIM acquisition and

use of library databases. The main limi-

tations of this technique are the derivat-

isation time and the difficulty of

extending it to a large number of corti-

costeroids—indeed, derivatization effi-

ciency and global sensitivity decrease

perceptibly as the number of hydroxyl

and ketone groups increases, leading to

very poor signals for the most polar

corticosteroids (e.g. triamcinolone and

prednisolone).

To avoid the multiple reaction prod-

ucts obtained from derivatization by si-

lylation, some authors have proposed

protecting these functional groups by

oxime formation before silylation [23,

40]. Methoxylamine (MOX), the reagent

most often used, enables replacement of

C=O functionality by C=N–OCH3

groups. The EI+ mass spectrum ob-

tained after reaction of dexamethasone

with MOX (8% in pyridine, 30 min,

70 �C) then classical silylation with

MSTFA–TMIS (1000:5, v/v) is shown in

Fig. 2b. Although the sensitivity achieved

by use of this technique is slightly inferior

to that of silylation alone, its advantage is

to produce four diagnostic ions with high

masses. Houghton et al. [40] also re-

ported the strong influence of C16 sub-

stitution and isomerism on methoxime

formation at position C20; this can be

used to distinguish betamethasone

(mono-MO-tris-TMS derivative) from

dexamethasone (bis-MO-tris-TMS deriv-

ative) and to improve the detectability of

the corticosteroids in the NCI mode.

Another means of derivatisation is the

preparation of boronic esters. This tech-

nique, which has been widely applied to

a–c diol steroids, entails reaction with

methylboronic acid in ethyl acetate for

15 min at ambient temperature then

classical silylation. A typical EI mass

spectrum obtained from the tri-TMS

methylboronic derivative of dexametha-

sone is shown in Fig. 2c. The stability of

the derivative is evident; an intense

molecular ion can be observed for a–cdiol compounds. Its main disadvantage,

however, is, once again, insufficient sen-

sitivity for analysis of trace levels of res-

idues in complex biological matrices.

A last chemical reaction enabling GC–

MS analysis of corticosteroids is elimi-

nation of the polar C17 side-chain by

chemical oxidation and subsequent oxi-

dation of residual hydroxyl groups to

ketone functionality. This technique was

the most widely used before development

of LC–MSn systems [5, 14, 15, 18–20, 40].

Among the three possible oxidative re-

agents, pyridium chlorochromate (PCC)

is probably the most powerful, leading to

the C17 keto product with complete oxi-

dation of the C11 and C6 hydroxyl

groups. One peculiarity of this oxidative

reaction is the formation of two reaction

products from C16 substitution (the a and

b isomers). In positive chemical-ioniza-

tion (PCI) mode the oxidation products

of dexamethasone are characterized by a

relatively intense pseudomolecular ion

[M + H]+ and a moderately intense

[M ) HF + H]+ ion (Fig. 2d). In the


Fig. 1. GC and LC ion chromatograms obtained from corticosteroids (phase I and phase II metabolites and esters). (FLU ¼ fludrocortisone,DEX ¼ dexamethasone, BET ¼ betamethasone, FLM ¼ flumethasone, BCL ¼ beclomethasone, PRN ¼ prednisone, PRL ¼ prednisolone,MPRL ¼ methylprednisolone, CRN ¼ cortisone, CRL ¼ cortisol, TRI ¼ triamcinolone, FLC ¼ fluocinolone acetonide, CLB ¼ clobetasol, TH ¼tetrahydro-, DH ¼ dihydro-, H ¼ hydroxy-, g ¼ glucuronide, s ¼ sulfate, a ¼ acetate, h ¼ hemisuccinate, da ¼ diacetate, p ¼ propionate, pv ¼pivalate, dp ¼ dipropionate)


Fig. 2. Typical mass spectra obtained from dexamethasone by use of different derivatization, ionization, and signal-acquisition techniques


negative chemical-ionization (NCI) mode

two diagnostic ions are detected,

[M ) HF]) and [M ) HF ) CH3]), with

sensitivity approximately four times

higher than for PCI or EI (Fig. 2e). Fi-

nally, chemical oxidation coupled with

NCI seems to be the method of choice for

the detection of corticosteroids by GC–

MS, because of advantages in terms of

sensitivity. Nevertheless, the formation of

isomeric products, difficult application to

all corticosteroids, and the need for

unambiguous identification resulted in

less interest in this technique than in more

recent developments in LC–MSn, at least

for confirmatory purposes.

Electrospray (ESI) and atmospheric

pressure chemical ionization (APCI) are

clearly very suitable for corticosteroids.

Under slightly acidic conditions these

relatively polar compounds give an in-

tense pseudomolecular ion [M + H]+ in

ESI+ and an adduct with the conjugated

base of the organic acid used

[M + base]) in ESI) [29, 52]. These

diagnostic ions can be selected as pre-

cursor ions for further fragmentation in

MS2. In ESI+ (Fig. 2f), numerous but

not very specific fragment ions can be

observed; these correspond to losses of

water molecules and/or halogen atoms

and other minor cleavages within the B

and C rings. In ESI) (Fig. 2g) fragmen-

tation is reduced to two ions, the

pseudomolecular ion [M ) H]) and the

fragment corresponding to cleavage of

the side chain with loss of formaldehyde

[M ) CH2O ) H]). This fragmentation

pathway seems to be extremely efficient

for measurement of many corticosteroids

at trace residue levels in biological

matrices. If additional diagnostic ions are

required a high cone potential inducing

in-source fragmentation of the

[M + base]) ion can be used to select the

resulting [M ) CH2O ) H]) fragment as

precursor; this ion can be further frag-

mented in the collision cell (Fig. 2h). Fi-

nally, atmospheric-pressure ionization in

the negative-ion mode is now the tech-

nique of choice for ionization and frag-

mentation of corticosteroids, because

sensitivity and specificity are better

than for all other ionization techniques.

Also, if chromatographic separation of

isomers such as dexamethasone and

dexamethasone is unsatisfactory, the rel-

ative intensities of diagnostic ions pro-

duced by electrospray ionization can be

used to distinguish these two isomers,

by use of a conventional model [53] of

a multidimensional statistical approach

[54].

Fig. 3. LC–ESI())-MS2 diagnostic ion chromatograms obtained from spiked urine and meat samples


Application to MonitoringCorticosteroid Misuse

LC–ESI())-MS2 diagnostic ion chroma-

tograms obtained from spiked urine and

meat samples are shown in Fig 3. These

profiles were obtained during validation

of the methods in accordance with

European criteria requirements fixed by

the EC/2002/657 decision [49, 55]. The

very clean chromatograms obtained were

indicative of the specificity of the triple-

quadrupole system operating in negative

electrospray ionization mode and the

MRM acquisition technique. The result-

ing sensitivity, with detection capability

(CCb) lower than 0.1 ppb for most of the

15 corticosteroids investigated, enables

very efficient monitoring of these mole-

cules. Such performance is linked to the

ionization efficiency of API sources and

to specificity of MS2. Ion chromatograms

from single and tandem mass spectro-

metric analysis of fluocinolone acetonide

in the same spiked liver sample are com-

pared in Fig 4; the sensitivity and speci-

ficity of MS2 are clearly superior.

Selection of appropriate diagnostic

ions is the key to success. To illustrate this

point six different ion chromatograms

from analysis of beclomethasone in a

spiked liver sample are presented in Fig 5;

each trace is that of a characteristic ion

determined during method development.

It is obvious here that characteristic does

not mean systematically diagnostic. In-

deed, monitoring of the [M + acetate])

ion (m/z ¼ 467) as precursor without

fragmentation is clearly a bad choice,

because of high noise levels in all the time

windows. The transition [M + acet-

ate]) ! [M ) H]) (i.e. 467 ! 407) is no

better, because of the significant proba-

bility that interfering compounds with the

same molecular weight will form the ad-

duct and fragment in the same way. The

transition [M + acetate]) ![M ) CH2O ) H]) (i.e. 467 ! 377) is

clearly more specific and sensitive and

subsequent fragmentation of the specific

ion [M ) CH2O ) H]) enables powerful,

unambiguous identification. Finally, all

potential characteristic ions must be sys-

tematically tested on real extracts before

being declared diagnostic and finally re-

tained in the programmed method.

After having considered these instru-

mental factors, much of the specificity

and final performance of the method is

highly dependent on sample pretreat-

ment. In our work improved sample

purification was applied—enzymatic

hydrolysis of the conjugated phase II

metabolites before reversed-phase C18

SPE then liquid–liquid clean-up with

sodium carbonate and normal-phase

SiOH SPE [49]. Even if use of LC–MS is

expected to enable reduced sample

preparation, we assume such a process

remains necessary to avoid potential

ion suppression and quantification diffi-

culties, because of competition phenom-

enon between analyte and matrix

interferences, and to maintain the long-

term stability and repeatability of the

system.

Application to Kinetic andMetabolic Investigations

This methodology has been used to

determine the kinetics of elimination of

corticosteroids from cattle after intra-

muscular administration. Concentrations

measured in urine and hair from a cow

successively treated with three different

corticosteroids are presented in Table 1.

The results show that the steroids were

clearly identified in the urine, that the

maximum rate of excretion was on the

day after injection, and that elimination

was rapid irrespective of the type of ester

administered (i.e. the concentration was

Fig. 4. Comparison of single and tandem mass spectrometry for analysis of fluocinolone acetonide.LC–ESI())-MS2 traces obtained from a spiked liver extract (1 lg kg)1, ppb)


below the detection limit after only 3–6

days). The second observation was the

important disparity in the maximum ob-

served concentrations—values varied

from 1.6 to 45.4 lg L)1 (ppb). Even if the

doses administered were not exactly the

same, this phenomenon must be investi-

gated to determine the dependence of the

transfer and metabolism of these com-

pounds on compound structure and the

type of ester injected. Our results were,

however, in accordance with those from

other studies [4, 15].

Our results showed that the esterified

and free forms of hemisuccinate hydro-

philic esters were both detectable in hair

for approximately 2 weeks, demonstrat-

ing the suitability of this matrix, with

urine, for medium-term monitoring pur-

poses, as has already been mentioned by

other authors [56]. No residues of the

esterified or free forms of acetate or di-

propionate lipophilic esters were detected

in hair samples. We have no explanation

for this and additional experiments are in

progress to investigate the phenomenon.

It is, however, assumed that the physi-

cochemical properties of the ester

administered probably affect transfer and

biotransformation of these hormonal

residues.

This study has confirmed the

suitability of LC–MS2 for kinetic study of

a variety of biological matrices—the

technique enables efficient monitoring

and direct identification of the target

analytes.

Our second application was collection

of new data about the nature and relative

proportions of the main phase I and II

metabolites of corticosteroids in bovine

urine. Application of our LC–MS2

method to optimization of the separation

Table 1. Measured concentrations (ng g)1, ppb) of dexamethasone (DEX), methylprednisolone (MPRL), and beclomethasone (BCL) in urine and hairfrom an adult cow successively treated (intramuscular injection) with 40 mg DEX acetate, 120 mg MPRL hemisuccinate, and 80 mg BCL dipropionate

Urine samplesDay D0 D1 D2 D3 D6 D7 D8 D9

DEX –* 45.4 25.6 5.4 0.2 0.2 – –MPRL – 1.6 0.3 – – – – –BCL – 14.5 5.8 2.5 – – – –

Hair samplesDays D0 D1 D3 D6 D10 D15 D19 D22DEX – – – – – – – –DEX ester – – – – – – – –MPRL – 17.6 87.2 27.6 24.2 1.3 2.3 –MPRL ester – 449.7 1699.9 477.9 184.2 8.1 4.3 –BCL – – – – – – – –BCL ester – – – – – – – –

*Not detected

Fig. 5. Six different LC–ESI())-MS2 ion chromatograms obtained from analysis of beclometha-sone in a spiked liver sample (1 lg kg)1, ppb)


and specific hydrolysis of conjugated

phase II metabolites, and final results

indicative of differences between endoge-

nous and exogenous corticosteroids, has

been described elsewhere [57]. Houghton

et al. [39, 40] have studied the phase I

metabolism of dexamethasone in the

horse, but very few data were available

for cattle. It was therefore decided to

adapt this methodology for evaluation of

the metabolite profile of corticosteroids

in urine samples, taking into account

biotransformations reported in the liter-

ature, including hydroxylation,

oxidation, and reduction [16, 17, 42–48].

The theoretical masses of different oxi-

dized and reduced analytes were moni-

tored. A reference urine sample collected

before treatment and a urine sample col-

lected one day after administration of

40 mg dexamethasone acetate were com-

pared. The ion chromatograms obtained

are shown in Fig 6. The most important

quantitative results were obtained for

dexamethasone. Two hydroxylated

metabolites are readily apparent (m/z 466

and 470) as are one reduced (m/z 448)

and five oxidized (m/z 452 and 454)

metabolites. Complete structural identi-

fication of these compounds is not readily

achievable by LC–ESI-MS2, however.

Indeed, the very soft ionization process

inherent to API interfaces is a limitation

of this technique in terms of structure

elucidation. Other ionization techniques,

for example APCI, APPI, or EI, in

association with complementary tech-

niques like GC–MS2 or GC–HRMS,

might usefully give additional structural

information.

Conclusion

This paper summarizes ten years of

experience in the field of corticosteroid

analysis using mass spectrometry. The

first GC–MS approaches were devoted to

investigation of the possibilities of gas

chromatographic separations, to explo-

ration of the advantages of different

derivatization reactions, and to study of

corticosteroid behavior on application of

electron impact and chemical ionization

to find the best diagnostic ions for

screening and confirmatory analysis.

Current methods are, however, based on

liquid chromatography coupled with

tandem mass spectrometry, mainly be-

cause LC separation and atmospheric

pressure ionization are highly suited to

analysis of moderately polar corticoster-

oids—LC–MS2 systems are currently the

ideal analytical tool for many of the lab-

oratories monitoring corticosteroids. To

achieve the best sensitivity and specificity

for a limited number of known analytes,

use of these systems to focus on charac-

teristic ions is the best choice. Improve-

ment of our knowledge of corticosteroid

metabolism in breeding animals is a major

challenge and combined use of LC–API-

MSn and GC–EI-MSn for detection and

identification of metabolites is certainly a

key to success. LC-based systems enable

very rapid and efficient comparative

analysis which reveals the presence of

potential analytes of interest whereas GC-

Fig. 6. LC–ESI())-MS2 ion chromatograms corresponding to theoretical masses of different potential phase I metabolites of dexamethasone,monitored in a blank urine sample collected before treatment and in a urine sample collected one day after administration of 40 mg dexamethasoneacetate. (FLU ¼ fludrocortisone, internal standard; DEX ¼ dexamethasone)


based systems furnish more structural

data, enabling unambiguous identifica-

tion. The distinction between natural

(cortisol, cortisone) and suspected

endogenous (prednisolone, prednisone)

corticosteroids and their exogenous ho-

mologs, on the basis of carbon-isotope-

ratio mass spectrometry or metabolic

profiling, is also an emerging future

challenge.

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