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RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2004; 18: 77–82
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1284
Analysis of 21-deoxycortisol, a marker of congenital
adrenal hyperplasia, in blood by atmospheric pressure
chemical ionization and electrospray ionization using
multiple reaction monitoring
Simone Cristoni1*, Debora Cuccato2, Mariateresa Sciannamblo2, Luigi Rossi Bernardi1,Ida Biunno3, Piermario Gerthoux4, Gianni Russo2, Giovanna Weber2 and Stefano Mora2
1University of Milan, Centre for Biomolecular Interdisciplinary Studies and Industrial Applications CISI, Via Fratelli Cervi 93, 20090 Segrate,
Milan, Italy2Laboratory of Pediatric Endocrinology and Department of Pediatrics, Scientific Institute H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy3CNR-ITB, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy4University Department of Laboratory Medicine, University of Milano-Bicocca, Hospital of Desio, Via Mazzini 1, 20033 Desio, Milan, Italy
Received 12 August 2003; Revised 27 October 2003; Accepted 27 October 2003
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by 21-
hydroxylase deficit (21-OHD). Deletions or mutations of the CYP21 gene induce the impairment
of glucocorticoid and mineralcorticoid synthesis. 17-Hydroxyprogesterone (17-OHP) is the hormo-
nal marker in patients, but not in the heterozygous subjects. Excess 17-OHP is hydroxylated into 21-
deoxycortisol (21-DF), and therefore 21-DF can be used as a specific marker for diagnosis of hetero-
zygous individuals. We report an analytical method for analysis of 21-DF in blood samples using
electrospray (ESI) and atmospheric pressure chemical ionization (APCI), showing that ESI is very
sensitive for the analysis of this marker molecule. The multiple reaction monitoring (MRM)
approach was used to increase the specificity and the sensitivity of the method. Copyright #
2003 John Wiley & Sons, Ltd.
Congenital adrenal hyperplasia (CAH), caused by a 21-
hydroxylase deficit (21-OHD), is an autosomal recessive dis-
order in which deletions or mutations of the CYP21 gene
induce the impairment of glucocorticoid and mineralcorti-
coid synthesis.
The classical form of 21-OHD occurs in about 1:15 000 live
births.1–5 CAH diagnosis is usually performed by measuring
blood levels of adrenal hormones and precursor steroids.6 In
particular, 17-hydroxyprogesterone (17-OHP) is not hydro-
xylated into 11-deoxycortisol (11-DF) and cortisol synthesis is
decreased. Consequently, the secretion of adrenocorticotro-
pic hormone (ACTH), which is the promoter of the cortisol
metabolic pathway, is increased. Overstimulation by ACTH
is responsible for the high concentration of circulating 17-
OHP. For this reason, high 17-OHP plasma levels are used to
diagnose this disorder. The excess 17-OHP is hydroxylated
into 21-deoxycortisol (21-DF) in the adrenal gland, and this
metabolic pathway becomes very important in patients with
21-OHD.7 It is known that ACTH induces much higher 21-DF
plasma levels in heterozygous than in normal subjects.8–12
Several studies8–10,13–15 have demonstrated that 21-DF is a
more sensitive marker than 17-OHP because it allows the
detection of more than 90% of heterozygous carriers. Plasma
levels of 21-DF before and 60 min after ACTH stimulation is
becoming a new approach for detection of heterozygous
carriers of 21-OHD.
The methods currently used for the detection of 21-DF are
radioimmunoassay (RIA)8–10,13,14 and solid-phase time-
resolved fluoroimmunoassay (TR-FIA).16 In particular, the
RIA technique leads to high sensitivity (0.6 pg/tube)14 but on
the other hand it is labour intensive and sample loss can occur
during the extraction and preparation procedures.10 For
these reasons, the use of a mass spectrometry approach that is
highly sensitive, fast, is not labour intensive nor dangerous, is
of interest. Both gas and liquid chromatography coupled
with mass spectrometry (GC/MS and LC/MS) have been
used in recent years for the study of steroid compounds.17–19
In particular, several methods, based on coupling GC and LC
to mass spectrometry,20,21 have been developed for the
diagnosis of 21-hydroxylase deficit (21-OHD).
Liquid chromatography with tandem mass spectrometry
(LC/MS/MS) is a technique widely used to characterize and
quantify steroids.21,22 In particular, the ion sources typically
employed to analyze steroids are electrospray (ESI)23–25 and
atmospheric pressure chemical ionization (APCI).26–28 The
use of LC/MS/MS offers the advantage that there is no need
for derivatization of the samples before injection, as is
Copyright # 2003 John Wiley & Sons, Ltd.
*Correspondence to: S. Cristoni, Universita degli Studi di Milano(CISI), Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy.E-mail: [email protected]/grant sponsor: ARFSAG—Lombardia (RegionalAssociation of Families with Cortical Adrenal Hyperplasia).
required in GC/MS. Thus the detection of compounds of
interest can be achieved by directly injecting biological fluids
like urine and saliva.29,30 Kao et al.21 have developed
an approach based on the use of MS/MS for the analysis of
21-DF. They used a TurboIonSpray ionization source work-
ing at a high flow rate (1 mL/min) using a 4.6 mm i.d. column.
However, under these conditions, they can detect the 21-DF
only in serum from patients with 21-hydroxylase deficiency
(21-OHD). Furthermore, they did not use the MRM approach
that can improve the instrumental performance in terms of
specificity and sensitivity.
The aim of this study is to compare APCI and ESI
performance in the analysis of 21-DF. The 21-DF content
was extracted from the blood of four subjects, analyzed
by MRM methods, and the results thus obtained are
reported here.
EXPERIMENTAL
ChemicalsStandard 11-deoxycortisol (11-DF) and 21-deoxycortisol
(21-DF) were purchased from Sigma Aldrich (Milan, Italy).
Methanol (CH3OH) was purchased from J. T. Baker
(Deventer, Holland). Trifluoroacetic acid (TFA, CF3COOH)
was purchased from Lancaster (Eastgate, White Lund, Mor-
ecambe, UK). Adrenocorticotropic hormone (ACTH,
Synacthen) was purchased from Ciba-Geigy (Basel, Switzer-
land). Isooctane (C8H18) and ethyl acetate (CH3COOC2H5)
were purchased from E. Merck (Darmstadt, Germany).
Figure 1. Structures of (a) 21-DF and (b) 11-DF.
Figure 2. (a) LC/APCI-MS/MS analysis of a mixture containing both 21- and 11-DF, each at
a concentration of 50 ng/mL. A volume of 20 mL was injected. (b) MS/MS spectrum of
protonated 21-DF. (c) MS/MS spectrum of protonated 11-DF.
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 77–82
78 S. Cristoni et al.
Sample preparationThree heterozygous females at random stages of their men-
strual cycles and one healthy man were studied. The subjects
were relatives of CAH patients. The heterozygous state was
determined by molecular and biochemical screening.
Informed consent was obtained from each subject prior to
the study.
The ACTH stimulation test was performed by injecting
250mg of ACTH intravenously. Two blood samples were
taken both before and 60 min after ACTH stimulation.
Plasma was obtained by blood centrifugation at 1550 g for
10 min at room temperature. Plasma samples (2 mL) were
extracted twice with isooctane/ethyl acetate (1:1, v/v). The
first extraction was made with 40 mL of the solvent mixture
after vortexing for 1 min; then the pellet was frozen with
liquid nitrogen. The extract containing steroids was sepa-
rated from the frozen pellet, and the same pellet was
extracted a second time using 20 mL of the solvent mixture
following the procedure described above. Both steroid
extracts were collected and dried with nitrogen. The dried
extract was resuspended in 40 mL water/methanol (50:50),
and 20mL were analyzed. 20 mL of standard solutions in the
concentration range 0.250–600 ng/mL were injected to
obtain the APCI and ESI calibration curves.
ChromatographyA Surveyor mHPLC system (ThermoFinnigan, Palo Alto, CA,
USA) was used. The chromatographic column was a reverse-
phase C18 (250� 2.1 mm, 5mm, 300 A). A HPLC gradient was
performed using two eluents: (A) H2Oþ 0.025% TFA; (B)
CH3OHþ 0.025% TFA. A linear gradient was used, from
52% to 77% of B in 10 min. The eluent flow was 200 mL/min.
A divert valve was used in order to send the eluent to waste
for the first 4 min of HPLC analysis.
Mass spectrometryThe APCI mass spectra were obtained using a LCQXP ion trap
(ThermoFinnigan). The source temperature was 3508C and
the entrance capillary temperature was 1508C. The corona
discharge voltage was 5 kV. The flow rate of nebulizing gas
(nitrogen) was 2.00 L/min. The He pressure inside the trap
was kept constant; the pressure directly read by ion gauge
(in the absence of N2 stream) was 2.8� 10�5 Torr. The maxi-
mum injection scan time was 200 ms, 5 microscans were used,
and the automatic gain control was turned on.
The same instrument was used to obtain the ESI mass
spectra. The needle voltage was 5 kV. The entrance capillary
temperature was 2408C. The flow of nebulizing gas (nitrogen)
was 1.5 L/min. The He pressure inside the trap was kept
constant; the pressure directly read by ion gauge (in the
absence of N2 stream) was 2.8� 10�5 Torr. The maximum
injection scan time was 200 ms, 5 microscans were used, and
the automatic gain control was turned on.
HPLC/ESI and HPLC/APCI chromatograms were
acquired using tandem mass spectrometry (MS/MS) and
MRM in positive acquisition mode. The isolation width
(baseline) of the precursor ion was 3 Th. The fragment ion
mass width (baseline) was also 3 Th. The collision energy was
30% of its maximum value (5 V peak to peak). Five
microscans were used and the microscan time in MRM mode
was 20 ms. Under these conditions the instrumental resolu-
tion was effectively unit mass.
Data analysisThe signal/noise (S/N) ratio was calculated using the RMS
algorithm. The chromatographic data were processed using
Xcalibur qualbrowser and Exel software.
RESULTS AND DISCUSSION
Two isomers of deoxycortisol (21-DF and 11-DF) are present
in blood samples (Figs. 1(a) and 1(b)), but only 21-DF is used
to detect heterozygous individuals. Preliminary experiments
were performed using the APCI source. As reported in the lit-
erature,26–28 this ionization source is very efficient in the
analysis of steroids. In order to separate and quantify
the two isomers, an LC/MS/MS method was developed.
The MS/MS chromatogram, and the MS/MS spectra of the
[MþH]þ ions at m/z 347 of 21- and 11-DF isomers, are shown
in Figs. 2(a), 2(b) and 2(c), respectively (positive acquisition
mode was used). 20mL of a mixture of the 11- and 21-DF iso-
mers, each with a concentration of 50 ng/mL (1 ng injected
on-column), were injected in order to obtain the MS/MS
chromatogram. Under these conditions the signal response
of 21-DF is about 5 times higher than that of 11-DF. The
S/N ratio of the chromatographic peak of 21-DF at retention
time 7.02 min is about 100 (calculated using the RMS algo-
rithm). Under these chromatographic conditions, chromato-
graphic peaks for 21- and 11-DF are well resolved.
Scheme 1. Fragmentation pathway proposed for 11-DF.
Characterization and quantification of steroids 79
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 77–82
In the MS/MS spectrum of 11-DF (Fig. 2(c)) two peaks at
m/z 317 and 299 are observed, while in the case of 21-DF
(Fig. 2(b)) these fragments are absent. Scheme 1 shows the
fragmentation pathway proposed for 11-DF that could lead to
these fragments. The peak at m/z 317 is due to the loss of a
CH2O neutral molecule with a hydrogen rearrangement. The
peak at m/z 299 is due to a further water loss, again with
hydrogen rearrangement. The two most abundant peaks
obtained in the 21-DF fragmentation (Fig. 2(b)) are those at
m/z 311 and 293 (Scheme 2) that correspond to the loss of two
and three neutral water molecules, respectively, and are also
present in the MS/MS spectrum of 11-DF (Fig. 2(c)).
This difference in fragmentation behaviour of the two
molecules was used to perform multiple reaction monitoring
(MRM), monitoring the fragment ions atm/z 317, 311, 299 and
293. The peaks at m/z 311 and 293 are present in the
fragmentation spectra of the two isomers and thus they are
not useful for selective discrimination of the two steroids, but
they were chosen for their high abundance in the spectrum of
21-DF. As mentioned, the peaks atm/z 317 and 299 are present
only in the 11-DF fragmentation spectrum; these peaks were
monitored to increase the selectivity of the method since they
are not present in the MS/MS spectrum of 21-DF at
the retention time of 7.02 min but are clearly detected in the
MS/MS spectrum of 11-DF at the retention time of 8.31 min.
Thus, two parameters (retention time and the lack of the
peaks at m/z 317 and 299 in the MS/MS spectrum of 21-DF)
allow the reliable accurate identification of 21-DF.
The same analysis as that reported above was performed
under ESI conditions in order to determine whether the same
experimental data were reproduced also by using this
ionization source and in order to evaluate the efficiency of
this ionization approach. The same fragmentation behaviour
of the two isomers as was obtained using APCI was observed
with ESI. Moreover, increased sensitivity and linear dynamic
range were observed by using this technique. The calibration
curves for 21-DF obtained using ESI and APCI are shown in
Figs. 3(a) and 3(b), respectively. In both cases good linearity
was achieved (R2¼ 0.9995 for the ESI approach and
R2¼ 0.9976 for APCI). However, the limit of detection
obtained using the ESI source (0.20 ng/mL injecting 20mL,
corresponding to 4 pg injected on-column) was lower than
that obtained with the APCI source (1 ng/mL injecting 20mL,
corresponding to 20 pg injected on-column). The linear
dynamic range was also enhanced, from 30–600 ng/mL
(obtained under APCI conditions) to 0.25–600 ng/mL (ESI
conditions). The limit of quantitation of the developed
LC/ESI-MS/MS-MRM approach is lower than that obtained
Scheme 2. Fragmentation pathway proposed for 21-DF.
80 S. Cristoni et al.
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 77–82
previously using LC/MS/MS but without using MRM
(50 ng/mL injecting 17mL, corresponding to 850 pg injected
on-column).21 The absolute limits of quantitation of RIA
(0.75 pg/tube)14,16 and GC/MS (0.5 ng/mL, injecting 2mL,
corresponding to 1 pg injected on-column) are about 4–7
times lower than that obtained using our approach based on
the use of ESI and MRM (5 pg injected on column). However,
both RIA and GC/MS approaches require more time
consumption with respect to the LC/ESI-MS/MS-MRM
method. Furthermore, the linear range obtained (0.25–
600 ng/ml, injecting 20 mL, corresponding to 5–12 000 pg
injected on-column) is good enough if compared with
that used in the case of LC/MS/MS without using MRM
(50–250 ng/mL, injecting 17 mL, corresponding to 850–
4250 pg injected on-column)21, RIA (0.75–100 pg/tube)14,16
and GC/MS (0.5–20 ng/mL, injecting 2mL, corresponding to
1–40 pg injected on-column)20,31 approaches.
Thus, ESI was chosen in order to detect and quantify 21-DF
in eight blood samples obtained from four selected subjects.
Figures 4(a) and 4(b) show the MRM chromatograms
obtained by injecting a standard solution of 21-DF (50 ng/
mL injecting 20mL, corresponding to 1 ng injected on-
column) and a solution obtained by extracting 21-DF from a
blood sample of a selected subject. The peak corresponding to
21-DF was clearly observed in both cases.
The ESI approach was therefore used to quantitate the 21-
DF in blood samples. Table 1 summarizes the results obtained
by analyzing blood samples obtained from four selected
subjects. The samples were collected before and after ACTH
stimulation in order to distinguish between normal and
heterozygous carriers. For the healthy subject (number 2,
Table 1) no increase in the level of 21-DF was observed after
stimulation. In contrast, clearly different behaviour was
observed for heterozygous carriers (numbers 1, 3 and 4,
Table 1) in which the level of 21-DF strongly increased (about
10–15 times) after ACTH stimulation. These results fully
confirm the literature data obtained by using the radio-
immunoassay technique.8–10,13,14
Figure 3. (a) ESI calibration curve obtained using a 0.250–
600ng/mL solution concentration range of 21-DF. MRMmode
was used. (b) APCI calibration curve obtained using a 30–
600ng/mL solution concentration range of 21-DF. MRMmode
was used.
Figure 4. ESI MRM chromatograms obtained by injecting (a) 20 mL of a 21-DF
standard solution at a concentration of 50 ng/mL and (b) a plasma extract of a healthy
subject.
Table 1. Quantitative analysis of 21-DFextracted from blood
samples of four selected subjects obtained before and after
60min stimulation with ACTH
Subjects*
21-DF concentrationbefore ACTH stimulation
(ng/mL)
21-DF concentration afterACTH stimulation
(ng/mL)
1 3.1� 0.5 27.8� 3.02 2.1� 0.5 2.2� 0.53 3.5� 0.5 39.1� 2.54 1.9� 0.6 32.7� 2.8
* Heterozygous carriers are subjects # 1, 3 and 4.
Characterization and quantification of steroids 81
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 77–82
CONCLUSIONS
A method based on the use of LC/MS/MS with MRM, that
permits discrimination between 21-DF (a marker of congeni-
tal adrenal hyperplasia disease) and 11-DF, has been per-
formed using an APCI source. The data obtained using
APCI were compared with those obtained under ESI condi-
tions. The latter technique leads to a higher sensitivity and
linear dynamic range in the analysis of 21-DF, and for this rea-
son it was chosen for detecting and quantifying this com-
pound in blood samples of four selected subjects. In all
samples the 21-DF marker was readily monitored with a
10 min chromatographic analysis. Furthermore, the data
obtained clearly show that the limit of quantitation and linear
range achieved makes it possible to analyze and quantify
21-DF molecules in both healthy and heterozygous carrier
subjects; thus this method could be developed for success-
ful application in the clinic as an alternative to the labour-
intensive RIA8–10,13,14 and GC/MS21,30 approaches.
Future investigations will be focused on increasing the
sensitivity of the techniques by reducing the diameter of the
chromatographic column from 2.1 mm to 0.5 mm. The
developed methods will also be applied to the study of a
large number of blood samples in order to fully validate this
approach for the diagnosis of CAH disease.
AcknowledgementsThis work was supported by ARFSAG—Lombardia (Regio-
nal Association of Families with Cortical Adrenal Hyperpla-
sia). The authors also thank Drs Maria Carla Proverbio and
Ilaria Zamproni for their technical support.
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