RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2004; 18: 1392–1396

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1502

To the Editor-in-Chief

Sir,

Surface-activated chemical ionization

ion trap mass spectrometry in the

analysis of 21-deoxycortisol in blood

Congenital adrenal hyperplasia (CAH),

caused by a 21-hydroxylase deficit

(21-OHD), is an autosomal recessive

disorder in which deletions or muta-

tions of the CYP21 gene induce the

impairment of glucocorticoid and

mineralcorticoid synthesis.1–5 Blood

levels of adrenal hormones and precur-

sor steroids are usually measured in

order to perform CAH diagnosis.6

Over-stimulation by adrenocorticotro-

pic hormone (ACTH) is responsible

for the high concentration of circulat-

ing 17-OHP. For this reason high 17-

OHP plasma levels are used for the

diagnosis of this disorder. The excess

17-OHP is hydroxylated into 21-deoxy-

cortisol (21-DF) in the adrenal gland,

and this metabolic pathway is very

important in patients with 21-OHD.7

21-DF is a very sensitive marker

because it allows the detection of

more than 90% of heterozygous car-

riers.8–13 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.

New methods for the measurement

of 21-DF in plasma have recently been

described.14–17 In particular, a highly

sensitive method based on electro-

spray ionization (ESI) coupled to an

ion trap mass analyzer with multiple

reaction monitoring (MRM)17 has been

developed; this method has a low limit

of detection (LOD) and good linearity

range, and can be used for the quanti-

tative analysis of 21-DF.

Recently, a new high sensitivity

ionization technique named surface-

activated chemical ionization (SACI)

has been described.18 A commercially

available atmospheric pressure chemi-

cal ionization (APCI) chamber,

employed without any corona dis-

charge (no-discharge APCI), has been

modified with the insertion of a gold

surface leading to a significant

improvement in the ionization effi-

ciency. The ionization of the sample

takes place by both gas-phase and

surface-activated processes leading to

a high instrumental selectivity and

sensitivity. This fact has encouraged

us to employ it also for the analysis of

21-DF in order to verify its perfor-

mance in the analysis of this com-

pound.

In this work some results obtained

by applying the liquid chromatogra-

phy/tandem mass spectrometry multi-

ple reaction monitoring (LC/MS/

MS-MRM) approach to the analysis of

21-DF, using SACI as the ionization

source, are shown and discussed.

Standard 11-deoxycortisol (11-DF)

and 21-deoxycortisol (21-DF) were

purchased from Sigma Aldrich (Milan,

Italy). Methanol was purchased from

J.T. Baker (Deventer, Holland). Tri-

fluoroacetic acid (TFA) was purchased

from Lancaster (Eastgate, White Lund,

Morecambe, UK). Adrenocorticotropic

hormone (ACTH, Synacthen) was

purchased from Ciba-Geigy (Basel,

Switzerland). Isooctane and ethyl acet-

ate were purchased from E. Merck

(Darmstadt, Germany).

21-DF was extracted from a blood

sample of a male volunteer subject.

Plasma was obtained by blood centri-

fugation at 1550 g for 10 min at room

temperature. A 2-mL sample of plasma

was extracted twice with isooctane/

ethyl acetate (1:1, v/v). The first extrac-

tion 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 separated 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. This sec-

ond extract containing the 21-DF was

collected and dried with nitrogen. The

dried extract was re-suspended in

40 mL water/methanol (50:50), and

20 mL were analyzed. Volumes of

20 mL of standard solutions in the

concentration range 0.4–2000 ng/mL

were injected to obtain the SACI cali-

bration curves. It must be emphasized

that it is preferable to use an internal

standard (typically a deuterated com-

pound) for quantification purposes,

because SACI and ESI are both affected

by the ion suppression phenomenon

due to the biological matrix (e.g.

blood). In this paper, preliminary work

to evaluate the LOD and linearity

range, obtained by analyzing aqueous

solutions of pure standard, and the

capability of SACI to detect 21-DF

extracted from blood samples, has been

performed. However, in future work

an extensive number of quantitative

analyses will be performed and it will

be necessary to use a deuterated inter-

nal standard in order to correct the

biological matrix effect.

A Surveyor mLC (ThermoFinnigan,

Palo Alto, CA, USA) was used. The

chromatographic column was a

reverse-phase C18 (150� 1 mm, 5 mm,

300 A). A LC 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 5 min. The initial condition

was then reached over 2 min and was

maintained for 3 min to recondition

the column. The eluent flow was

100mL/min.

The SACI mass spectra were

obtained using a LCQ Deca XP ion trap

(ThermoFinnigan, San Jose, CA, USA).

The source vaporizer temperature was

in the range 150–4508C and the

entrance capillary temperature was

1508C. The ionizing surface voltage

was in the range 100–500 V. The sur-

face material was gold. The flow rate of

nebulizing gas (nitrogen) was 2.50 L/

min. The pressure of He 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.

LC-SACI chromatograms were

acquired using tandem mass spectro-

metry (MS/MS) and MRM using posi-

tive acquisition mode. For MRM the

isolation width of the precursor ion

was 3 Th and the fragment ion mass

width was also 3 Th. The collision

energy was 30% of its maximum value

(5 V peak to peak). Three microscans

were used and the microscan time in

Copyright # 2004 John Wiley & Sons, Ltd.

RCM

Letter to the Editor

MRM mode was 20 ms. In these condi-

tions, the singly charged fragment ions

of 21-DF were clearly detected. All

spectra were acquired using positive

ion mode.

The signal/noise (S/N) ratio was

calculated using the RMS algorithm.

The chromatographic data were pro-

cessed using Xcalibur qualbrowser and

Excel software.

Two isomers of deoxycortisol (21-DF

and 11-DF) are present in blood sam-

ples, but only 21-DF is used to detect

heterozygous individuals. Preliminary

results were obtained by direct infusion

of 21-DF using SACI-MS. The full scan

mass spectrum, and the MS/MS spec-

trum of the 21-DF [MþH]þ ion at m/z

347, obtained by direct infusion of a

50 ng/mL 21-DF standard solution, are

shown in Figs. 1(a) and 1(b), respec-

tively. The same behavior as that

obtained in the case of ESI and APCI

was observed.17 The two most

abundant peaks obtained by fragment-

ing the 21-DF [MþH]þ ion are atm/z311

and 293 (Fig. 1(b)), and correspond to

the loss of two and three neutral water

molecules, respectively. These peaks

are present in the fragmentation spectra

of both 21-DF and 11-DF. Thus, they are

not useful for selective discrimination

of the two steroids, but they have been

chosen for their high abundance so as to

increase the sensitivity of the MRM

method. They were monitored together

with the ions at m/z 317 and 299 during

the LC/SACI-MS/MS-MRM analysis;

these peaks were monitored in order to

increase the selectivity of the analysis.

In fact, they are present only in the

fragmentation spectrum of 11-DF

[MþH]þ ion (Scheme 1(a)), and corre-

spond to the structures proposed in

Schemes 1(b) and 1(c), respectively.

Thus the absence of these peaks in the

fragmentation spectrum confirms the

21-DF [MþH]þ ion.

21-DF is also subject to an in-source

thermal degradation phenomenon, but

in the mass spectrum shown in Fig. 1(a)

the peaks at m/z 329, 311 and 293 are

due to fragmentation of the [MþH]þ

ion of 21-DF; these peaks correspond to

loss of one, two and three neutral water

molecules, respectively. This hypoth-

esis was supported by comparing MS/

MS experiments on the ions at m/z 329,

311 and 293 (Fig. 1(a)) with MS3

experiments involving fragmentation

of the [MþH]þ molecular ion and its

fragment ions at m/z 329, 311 and 293.

In both cases the same fragmentation

pattern was observed, supporting the

interpretation that the peaks at m/z 329,

311 and 293 in the SACI mass spectrum

(Fig. 1(a)) are obtained by in-source

fragmentation of the [MþH]þ ion, but

not to the corona discharge fragmenta-

tion effect observed in previous stu-

dies,19–21 since a surface placed at zero

or low potential is used to ionize the

Figure 1. (a) Full scan mass spectrum of a 21-DF standard solution and (b) MS/MS spectrum of

21-DF [MþH]þ ion atm/z 347. The spectra were obtained by direct infusion of a 50 ng/mL standard

solution. The counts/s value of the most intense peak ([MþH]þ in the case of the mass spectrum)

is also reported.

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 1392–1396

Letter to the Editor 1393

sample in the SACI approach with no

corona discharge. Various source

vaporizer temperatures were tested

(150, 200, 250, 300, 350, 400 and 4508C)

and the best results were achieved

using 2508C.

Various surface potentials were also

used (100–500 V) and the best results

were achieved by using a low potential

(150 V). We postulate that the posi-

tively charged surface gives rise to the

adsorption and orientation of com-

pounds exhibiting a permanent dipole

moment (e.g., H2O or CH3OH

employed in the present LC/MS

experiments), thus making protons

more available for capture by the

analyte neutral molecules and thus

increasing the ionization efficiency.18

Moreover, the application of a low

surface potential makes it possible to

obtain a better focusing of the ions into

the mass analyzer. In the case of 21-DF,

the applications of 150 V surface poten-

tial gives rise to a 500% increase in

signal intensity with respect to that

achieved without applying a potential

to the floating surface.

The MRM approach17 developed

using ESI and APCI was employed in

order to verify the performance of the

SACI technique in the analysis of 21-

DF. Figures 2(a) and 2(b) show the LC/

SACI-MRM chromatograms obtained

by injecting 20 mL of a 50 ng/mL stan-

dard solution of 21-DF (1 ng injected

on-column) and a 21-DF sample

extracted from the blood of a selected

volunteer subject, respectively. In both

cases the counts/s and S/N values

were good enough to clearly detect the

analyzed molecule (2.83� 106 counts/s

with a S/N ratio of 505 for the standard

sample and 2.16� 105 counts/s with a

S/N ratio of 65 for the 21-DF sample

extracted from blood). Moreover, using

a fast chromatographic gradient of

5 min (10 min of chromatographic ana-

lysis considering the column re-equili-

bration), the 21-DF retention time

(3 min) was strongly reduced com-

pared with that achieved in the pre-

viously developed ESI approach17

using a slower chromatographic gra-

dient (10 min of chromatographic

gradient and 15 min of total chromato-

graphic analysis with a 21-DF retention

time of 7 min). It is thus possible to

reduce the time needed for each an-

alysis leading to improvements in

throughput. It must be emphasized

that, in the analysis of the sample of

21-DF extracted from blood, if the fast

gradient is used in conjunction with

ESI source, the 21-DF chromatographic

peak was detected with a lower S/N

ratio (S/N¼ 6) compared with that

achieved using SACI (S/N ratio¼ 65).

The fast LC/ESI-MRM mass chroma-

togram obtained analyzing the 21-DF

sample extracted from blood is shown

in Fig. 2(c). In this case, the decrease in

S/N ratio obtained using ESI is prob-

ably due to the matrix effect. However,

it must be emphasized that the SACI

technique is also affected by the matrix

signal suppression and enhancement

phenomenon.22 For this reason, a reli-

able quantitative analysis requires a co-

eluting internal standard or use of

matrix-matched calibration standards.

However, this experiment clearly

shows that SACI is better suited for

rapid chromatographic analysis of 21-

DF than is the ESI source. The instru-

mental LOD of the LC/SACI-MRM

approach is similar to that achieved

using the ESI source;17 in the case of ESI,

the LOD was 0.2 ng/mL injecting 20mL,

corresponding to 4 pg injected on-

column, while for SACI it was 0.25 ng/

mL injecting 20mL, corresponding to

5 pg injected on-column. The methods

usually employed for the analysis of 21-

DF (RIA and GC/MS)12,23–25 are about

4–10-fold more sensitive than the ESI

and SACI approaches described here

and previously, but they are also more

time-consuming. The linearity range

achieved for standard solutions of 21-

DF using SACI (0.4–2000 ng/mL inject-

ing 20mL, corresponding to 8–40 000 pg

injected on-column, R2¼ 0.9956) was

higher than that achieved using both

ESI (0.25–600 ng/mL injecting 20mL

corresponding to 5–12 000 pg injected

on-column, R2¼ 0.9995) and APCI

(30 – 600 ng/mL injecting 20mL corre-

sponding to 600–12 000 pg injected on-

column, R2¼ 0.9976). It must be empha-

sized that the linearity range and LOD

were obtained by analyzing aqueous

solutions of pure standard. In the case of

biological samples the matrix could

strongly increase or decrease the signal

intensity.22 Thus, under these condi-

tions, both the linearity range and the

limit of quantitation (LOQ) of the tech-

nique could be significantly different.

The data reported here suggest that

the fast LC/SACI-MRM method

employed for the analysis of 21-DF

has performance similar to the LC/

ESI-MRM method previously devel-

oped17 in terms of LOD but higher

performance in terms of linearity

range when aqueous solutions of pure

standard are analyzed. Furthermore, it

Scheme 1.

1394 Letter to the Editor

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 1392–1396

gives rise to better results in the

analysis of 21-DF compared with

APCI and it is faster than ESI. For

these reasons, it could be used in

conjunction with the LC/ESI-MRM

approach17 for the detection of hetero-

zygous individuals.

In future work other SACI instru-

mental aspects like surface rugged-

ness, surface materials, and number of

consecutive analyses that can be per-

formed without changing or cleaning

the surface, will be studied in order to

obtain the best instrumental conditions

in terms of linearity range, LOQ of the

methods and sensitivity.

AcknowledgementsThis work was supported by ARFSAG—Lombardia (Regional Association of

Figure 2. LC/SACI-MRM fast chromatograms obtained by injecting (a) 20 mL of a 21-DF

50ng/mL standard solution (1 ng injected on-column) and (b) a 21-DF sample solution extracted

from the blood of a volunteer subject, and (c) LC/ESI-MRM fast chromatogram obtained by

injecting a 21-DF sample solution extracted from the blood of a volunteer subject. The counts/s

value and S/N ratio of the 21-DF chromatographic peaks are also reported.

Letter to the Editor 1395

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 1392–1396

Families with Cortical Adrenal Hyper-plasia). The authors also thank DrsMaria Carla Proverbio and Ilaria Zam-proni for their technical support.

Simone Cristoni1*,Mariateresa Sciannamblo2,

Luigi Rossi Bernardi1, Ida Biunno3,Piermario Gerthoux4, Gianni Russo2,

Giuseppe Chiumello2 and Stefano Mora21University of Milan, Centre forBio-molecular Interdisciplinary

Studies and Industrial ApplicationsCISI, Via Fratelli Cervi 93,20090 Segrate, Milan, Italy

2Laboratory of Pediatric Endocrinol-ogy and Department of Pediatrics,

Scientific Institute H. San Raffaele, ViaOlgettina 60, 20132 Milan, Italy

3CNR-ITB, Via Fratelli Cervi 93, 20090Segrate, Milan, Italy

4University Department of LaboratoryMedicine, University of Milano-

Bicocca, Hospital of Desio,Via Mazzini 1, 20033 Desio, Milan,

Italy*Correspondence to: S. Cristoni, Universi-ta degli Studi diMilano (CISI), Via FratelliCervi 93, 20090 Segrate, Milan, Italy.E-mail: dtoycr@tin.itContract/grant sponsor: ARFSAG—Lombardia (Regional Association ofFamilies with Cortical AdrenalHyperplasia).

REFERENCES

1. Appan S, Hindmarsh PC, BrookCGD. Arch. Dis. Childhood 1989; 64:1235.

2. Bergstrand CG. Acta Pediat. Scand.1966; 55: 463.

3. Brook CGD. Clin. Endocrinol. 1990;33: 559.

4. Pang S, Wallace MA, Hofman L,Thuline HC, Dorche C, Lyon ICT,Dobbins RH, Kling S, Fufjieda K,Suwa S. Pediatrics 1988; 81:866.

5. Speiser PW, Dupont B, Rubinstein P,Piazza A, Kastelan A, New MI. Am.J. Hum. Genet. 1985; 37: 650.

6. New MI, Rapaport R, Sperling MA(eds). Pediatric Endocrinology.WB Sound: Philadelphia, 1996;281–314.

7. Finkelstein M, Shaefer JM. Physiol.Rev. 1979; 59: 353.

8. Milewicz A, Vecsei P, Korth-SchutzS, Haack D, Roseler A, Lichtwald K,Lewiska S, Mittelstaedt GV. J. SteroidBiochem. 1984; 21: 185.

9. Gueux B, Fiet J, Pham-Huu-TrungMT, Villette JM, Gourmelen M,Galons H, Brerault JL, Vexiau P,Julien R. Acta Endocrinol. (Copenha-gen) 1985; 108: 537.

10. Fiet J, Gueux B, Gourmelen M,Kutten F, Vexiau P, Coullin Ph,Pham-Huu-Trung MT, Villette JM,Raux-Demay MC, Galons H, Julien R.J. Clin. Endocrinol. Metab. 1988; 66:659.

11. Nahoul K, Adeline J, Bercovici JP. J.Steroid Biochem. 1989; 33: 1167.

12. Fiet J, Villette JM, Galons H, BoudouPh, Burthier JM, Hardy N, SolimanH, Julien R, Vexiau P, Gourmelen M,Kutten F. Ann. Clin. Biochem. 1994;31: 56.

13. Hill M, Lapcik O, Hampl R, Starka L,Putz Z. Steroids 1995; 60: 615.

14. Shimada K, Mitamura K, Higashi T.J. Chromatogr. A 2001; 935: 141.

15. Appelblad P, Irgum K. J. Chromatogr.A 2002; 955: 151.

16. Dehennin L, Nahoul K, Scholler R.J. Steroid. Biochem. 1987; 26: 337.

17. Cristoni S, Cuccato D, SciannambloM, Bernardi LR, Biunno I, GerthouxP, Russo G, Weber G, Mora S. RapidCommun. Mass Spectrom. 2004; 18: 77.

18. Cristoni S, Bernardi LR, Biunno I,Tubaro M, Guidugli F. RapidCommun. Mass Spectrom. 2003; 17:1973.

19. Cristoni S, Bernardi LR, Biunno I,Guidugli F. Rapid Commun. MassSpectrom. 2002; 16: 1686.

20. Cristoni S, Bernardi LR, Biunno I,Guidugli F. Rapid Commun. MassSpectrom. 2002; 16: 1153.

21. Cristoni S, Bernardi LR. MassSpectrom. Rev. 2003; 22: 369.

22. Mallet CR, Lu Z, Mazzeo JR. RapidCommun. Mass Spectrom. 2004; 18: 49.

23. Fiet J, Boundi A, Giton F, Villette JM,Boudou Ph, Soliman H, Morieau G,Galons H. J. Steroid Biochem. Mol.Biol. 2000; 72: 55.

24. Caulfield MP, Lynn T, Gottschalk ME,Jones KL, Taylor NF, MalunowiczEM, Shackleton CH, Reitz RE, FisherDA. J. Clin. Endocrinol.Metab. 2002; 87:3682.

25. Ichimura K, Yamanaka H, Chiba K,Shinozuka T, Shiki Y, Saito K,Kusano S, Ameniya S, Oyama K,Nozaki Y, Kato K. J. Chromatogr.1986; 374: 5.

Received 12 February 2004Revised 27 April 2004

Accepted 27 April 2004

1396 Letter to the Editor

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 1392–1396