Quantitation of hepcidin in human urine by liquid chromatography–mass spectrometry

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Analytical Biochemistry 384 (2009) 245–253

0003-2697/$ - see front matter Crown Copyright © 2008 Published by Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2008.09.045

Contents lists available at ScienceDirect

Analytical Biochemistry

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Hep ci din, a pep tide hor mone that is pro duced pre dom i nantly

by hepa to cytes, has a major role in main tain ing iron homeo sta sis

in mam mals. It acts as a neg a tive reg u la tor of iron entry into the

sys temic cir cu la tion from mac ro phages and duo de nal en tero cytes,

and it pro vides the principle to avoid repetition mech a nism for

con trol ling plasma iron lev els [1].

Hep ci din was orig i nally iden ti fied by two groups as a liver-

induced anti mi cro bial pep tide (LEAP-1) [2] and as a uri nary anti-

mi cro bial pep tide (HAMP) [3]. Hep ci din is syn the sized in the liver

as the 60-res i due pro-hep ci din but is largely pres ent in serum

and urine in the form of the 25-res i due pep tide, which con tains

8 cys teine res i dues, con strained by four disul fide bridges. The

orig i nally pro posed disul fide con nec tiv i ties 1-4, 2-8, 3-7, and 5-6

[3], based on the cys teine knot anti mi cro bial pep tides, were later

revised [4] as a result of detailed struc tural stud ies to 1-8, 2-7,

3-6, and 4-5. This struc ture results in a dis torted b-sheet with a

hair pin loop, sta bi lized by disul fide pair ing and hydro gen bonds,

giv ing rise to an amphi pathic struc ture that includes an unusual

vic i nal disul fide.

Hep ci din func tions by bind ing to the cel lu lar iron export

pro tein, fer ro por tin, expressed by mac ro phages, pla cen tal syncy-

tio troph o blasts, hepa to cytes, and duo de nal en tero cytes. This bind-

ing results in inter nal i za tion of the hep ci din–fer ro por tin com plex

and subsequent deg ra da tion of fer ro por tin that pre vents cel lu lar

iron release [5]. Phys i o log i cal con cen tra tions of hep ci din result

in nor mal trans fer rin sat u ra tion lev els and phys i o log i cal rates of

eryth ro poi e sis through the reg u lated entry of iron into plasma.

Over ex pres sion of hep ci din leads to rapid inter nal i za tion and deg-

ra da tion of fer ro por tin, result ing in mac ro phage iron reten tion,

with decreased plasma iron lev els, as seen in the ane mia of chronic

dis ease. In con trast, hep ci din defi ciency leads to increased activ ity

of fer ro por tin, result ing in increased iron entry into the blood, high

trans fer rin sat u ra tion, with excess iron depo si tion in the liver and

other tis sues, as seen in hered i tary he mo chro ma to sis. Lev els of

plasma hep ci din are reg u lated by inflam ma tory stim uli, includ ing

cyto kines (espe cially IL-6), plasma iron, ane mia, and hypoxia [6,7].

More recently, results from mouse mod els indi cate that other cyto-

kines, includ ing IL-1, as well as sol u ble he mo juv elin and growth

fac tors such as mem bers of the bone mor pho ge netic pro tein fam-

ily, BMP-2, -4, and -9, also reg u late hep ci din syn the sis [8–10]. To

fully under stand these many inter ac tions, a reli able method for

hep ci din quan ti ta tion is essen tial.

Quantitation of hepcidin in human urine by liquid chromatography–mass

spectrometry

Sukhvinder S. Bansal a,*, John M. Halket a, Adrian Bomford b,c, Robert J. Simpson b, Nisha Vasavda d, Swee Lay Thein d,e, Robert C. Hider a

a Phar ma ceu ti cal Sci ences Divi sion, King’s Col lege Lon don, 150 Stam ford Street, Water loo, Lon don SE1 9NH, UKb Nutri tional Sci ences Divi sion, King’s Col lege Lon don, Water loo, Lon don SE1 9NH, UKc Insti tute of Liver Stud ies, King’s Col lege Hos pi tal, Lon don SE5 9RS, UKd Divi sion of Gene and Cell Based Ther apy, James Black Cen ter, King’s Col lege Lon don School of Med i cine, Lon don SE5 9NU, UKe Depart ment of Hema to log i cal Med i cine, King’s Col lege Hos pi tal, Lon don SE5 9RS, UK

a r t i c l e i n f o a b s t r a c t

Article history:

Received 16 June 2008

Available online 5 October 2008

Hep ci din is a pep tide hor mone that func tions as a key reg u la tor of mam ma lian iron metab o lism. Serum and

urine lev els are increased in inflam ma tion and sup pressed in he mo chro ma to sis, and they may have diag nos-

tic impor tance. This study describes the devel op ment and val i da tion of an ana lyt i cal method for the quan ti ta-

tive deter mi na tion of the con cen tra tion of hep ci din in clin i cal sam ples. A sta ble, iso to pi cally labeled inter nal

stan dard, [15N,13C2]Gly12,20-hep ci din, was syn the sized and a stan dard quan tity was added to urine sam ples.

Extrac tion was per formed using weak cat ion exchange mag netic nano par ti cles. An ion trap mass spec trom-

e ter was used to quan tify hep ci din in the sam ples. The hep ci din assay was val i dated, and good recov ery of

hep ci din was obtained. The assay is accu rate and pre cise. Uri nary hep ci din lev els of 3 to 9 nmol/mmol cre at-

i nine¡1 were found in healthy con trols, with reduced lev els in he mo chro ma to sis (P < 0.00006) and ele vated

lev els in inflam ma tion (P < 0.00035). In sickle cell dis ease, a wide range was found, with the mean value not

dif fer ing sig nifi cantly from con trols (P < 0.26). In sum mary, a val i dated method has been devel oped for the

quan ti ta tion of hep ci din using a sta ble, iso to pi cally labeled inter nal stan dard and applied to deter mine the

con cen tra tions of hep ci din in the low nano mo lar range in urine sam ples from patients and con trols.

Crown Copyright © 2008 Published by Elsevier Inc. All rights reserved.

Key words:

LC–MS/MS

Sta ble iso tope

Pep tide quan ti ta tion

Hep ci din

Iron metab o lism

* Cor re spond ing author.

E-mail address: suk hi.ban [email protected] (S.S. Bansal).


246 Quantitation of hepcidin in human urine by LC–MS / S.S. Ban sal et al. / Anal. Biochem. 384 (2009) 245–253

To date, the rela tion ship between hep ci din con cen tra tions and

other reg u la tory and trans port pro teins involved in iron metab o lism

and asso ci ated dis ease con di tions have been deter mined largely by

meth ods based on dot blot ting [7,11] or an enzyme-linked immu no-

sor bent assay (ELISA)1 for pro-hep ci din [12]. Sur face-enhanced laser

desorp tion/ion i za tion time-of-flight mass spec trom e try (SEL DI–TOF

MS) has also been used to deter mine hep ci din lev els [13–15]. To cor-

rect for dif cul ties in quan ti ta tion, des-Asp hep ci din has been used as

an inter nal stan dard [16]. Recently, a quan ti ta tive approach employ-

ing liquid chro ma tog ra phy tan dem mass spec trom e try (LC–MS/MS)

with cal ci to nin gene-related pep tide (CGRP) as the inter nal stan-

dard [17] has been devel oped. The suit abil ity of CGRP as an inter-

nal stan dard for hep ci din quan ti ta tion is not ideal given that CGRP

has a larger mass and dif fer ent hydro pho bic ity, pI, and net charge.

In the cur rent work, we describe the syn the sis and appli ca tion of

[15N,13C2]Gly12,20-hep ci din, a com pound that is phys i co chem i cally

iden ti cal to hep ci din, for the quan ti ta tive mea sure ments of hep ci din

by MS. Fol low ing sat is fac tory val i da tion of the assay, the method was

adapted for mea sure ment of hep ci din in urine sam ples from dif fer-

ent clin i cal sit u a tions. Preliminary results on serum and urine anal y-

sis have been reported pre vi ously [18,19].

Mate ri als and meth ods

Mate ri als

All sol vents were HPLC grade and were obtained from Fisher

Sci en tific UK (Leices ter shire, UK). Fmoc pro tected amino acids

and pep tide syn the sis reagents were pur chased from Ba chem

(Mersey side, UK). Gly cine (U-13C2 98%, 15N 98%) was obtained

from Cam bridge Iso tope Lab o ra to ries (Ando ver, MA, USA). Mag-

netic nano par ti cles were obtained from Bru ker Dal ton ics (Bre men,

Ger many). Hep ci din was obtained from Pep tides Insti tute (Osaka,

Japan). Matrix-assisted laser desorp tion/ion i za tion (MALDI)–TOF

MS was car ried out on a Bru ker Auto flex (Bru ker Dal ton ics). LC–MS/

MS anal y ses were car ried out on an LCQ Deca XP with a Sur veyor

LC sys tem (ThermoFisher Scientific, San Jose, CA, USA).

Prep a ra tion of hep ci din inter nal stan dard

Lin ear [15N,13C2]Gly12,20-hep ci din (DTHFPICIFCCGCCHRSKCG

MCCKT) was syn the sized on FmocThr(tBu)-Trt-PEG-PS flow resin

(0.14 mmol/g¡1). Fmoc depro tec tion was achieved with 20% piper i-

dine in dimethylform am ide (by vol ume), and acyl a tion cycles were

car ried out using 4 equiv a lents of the Fmoc amino acid deriv a tive

pre ac ti vat ed with N-[1H-ben zotriazol-1-yl)(dimeth yl amino)meth-

y lene]-N-methyl me than a min i um hexa fluoro phos phate N-oxide

(HBTU) and diiso pro pyl eth yl amine in a molar ratio (1:0.98:1.5 by

vol ume) in dimethylform am ide. Fmoc [15N,13C2]gly cine was incor-

po rated at posi tions 12 and 20 dur ing the syn the sis. After com-

ple tion of the syn the sis, the lin ear pep tide was cleaved from the

resin with tri flu o ro ace tic acid (TFA)/water/eth ane di thiol/phe-

nol/thio ani sole (8.75:0.5:0.25:0.5:0.5). The pep tide was reduced

in dithi o thre i tol and puri fied by preparative reverse-phase high-

per for mance liquid chro ma tog ra phy (RP–HPLC) using a Waters

LC Prep sys tem (Mil ford, MA, USA) apply ing a water/ace to ni trile

1 Abbre vi a tions used: ELISA, enzyme-linked immu no sor bent assay; SEL DI–TOF

MS, sur face-enhanced laser desorp tion/ion i za tion time-of-flight mass spec trom-

e try; LC–MS/MS, liquid chro ma tog ra phy tan dem mass spec trom e try; CGRP, cal ci-

to nin gene-related pep tide; MALDI, matrix-assisted laser desorp tion/ion i za tion;

HBTU, N-[1H-ben zotriazol-1-yl)(dimeth yl amino)meth y lene]-N-methyl me than a-

min i um hexa fluoro phos phate N-oxide; TFA, tri flu o ro ace tic acid; RP–HPLC, reverse-

phase high-per for mance liquid chro ma tog ra phy; SCA, sickle cell ane mia; Hb,

hemo glo bin; SI, serum iron; SF, serum fer ri tin; WCX, weak cat ion exchange; ESI,

elec tro spray ion i za tion; SRM, selected reac tion mon i tor ing; LLOQ, lower limit of

quan ti ta tion; CID, col li sion-induced dis so ci a tion.

gra di ent con tain ing 0.1% TFA. The cor rect molec u lar mass of the

iso lated lin ear pep tide (m/z 2803.09 for [M+H]+) was ver i fied by

MALDI–TOF MS using a-cyano-4-hydroxy cin namic acid in ace to-

ni trile and 0.1% TFA as the matrix. The intra mo lec u lar fold ing of

the reduced pep tide was per formed in degassed redox buf fers

con tain ing ammo nium bicar bon ate (100 mM), gua ni dine hydro-

chlo ride (2 M), reduced glu ta thi one (1 mM), and oxi dized glu ta thi-

one (0.1 mM) at a 5-lM con cen tra tion for 60 h. The prod uct was

puri fied by preparative RP–HPLC as described above, and the cor-

rect molec u lar mass (m/z 2794.01 for [M+H]+) was deter mined by

MALDI–TOF MS. The purity of the pep tide was greater than 97%

as assessed by RP–HPLC and MS. The pep tide con tent was deter-

mined using amino acid anal y sis with ortho-phth al de hyde deriv-

a ti za tion [20]. Amino acid anal y sis of the hydro ly sate was car ried

out on a Zor bax Eclipse HPLC col umn (150 £ 3 mm i.d., 3.5 lm, Ag i-

lent Tech nol o gies, Santa Clara, CA, USA) on an 1100 HPLC sys tem

(Ag i lent Tech nol o gies). The elu ent was mon i tored using exci ta tion

at 340 nm and emis sion at 450 nm on the fluo res cence detec tor.

Finally, [15N,13C2]Gly12,20-hep ci din was used to pre pare stock

solu tions at 1780 nmol/L in 0.1% aque ous for mic acid/ace to ni trile

(4:1, v/v) for the subsequent anal y sis.

Sam ple col lec tion and stor age

Urine was col lected from healthy sub jects and from patients

with hered i tary he mo chro ma to sis, sys temic infec tion and sickle cell

ane mia (SCA, Hb SS). The study was approved by the King’s Col lege

Hos pi tal research eth ics com mit tee, and all par tic i pants pro vided

informed writ ten con sent. A total of 12 healthy sub jects (age range

24–50 years, 6 males and 6 females) par tic i pated in the study. The

hemo glo bin (Hb), serum iron (SI), and serum fer ri tin (SF) lev els in all

of these sub jects were within the nor mal ranges for the lab o ra to ries

in this insti tu tion (for males: Hb 15.1 ± 1.0 g/dl, SI 15.3 ± 2.3 lmol/L, SF

175 ± 38 lg/L; for females: Hb 12.9 ± 0.7 g/dl, SI 14.3 ± 4.4 lmol/L, SF

39.3 ± 17.2 lg/L [all val ues means ± SD]). The 12 patients with hered-

i tary he mo chro ma to sis (age range 32–65 years, all males) attended

the clin i cal ser vice of the Insti tute of Liver Stud ies, King’s Col lege

Hos pi tal. All were homo zy gous for C282Y muta tion in the he mo chro-

ma to sis gene, HFE, and at the time of study had been fully treated

by phle bot omy with SF lev els of approx i mately 50 lg/L¡1. At least 3

months had elapsed between the time of the most recent ther a peu-

tic phle bot omy and the time the sam ples were taken. The 6 patients

(age range 45–61 years, 4 males and 2 females) with sys temic bac te-

rial infec tion and inflam ma tion were receiv ing intra ve nous anti bi ot-

ics and had nor mal renal func tion as judged by cre at i nine clear ance

within the nor mal range.

Sam ples from 15 patients with SCA (age range 32–65 years, 8

males and 7 females) were col lected dur ing steady-state clin i cal

con di tions at rou tine fol low-up in spe cial ist clin ics from Feb ru-

ary to April 2007. Urine sam ples from all patient groups and the

healthy sub jects were col lected between 15.00 and 17.00 h.

In all patients and con trols, mid stream urine sam ples were

col lected into plain ster ile tubes. Part of the urine was used for

cre at i nine deter mi na tion using the Jaf fe reac tion [21], and the

remain der was stored at ¡80 °C until fur ther anal y sis. The sam-

ples were thawed and ana lyzed within 8 weeks of col lec tion. The

hep ci din lev els were nor mal ized to cre at i nine con cen tra tion and

pre sented as nmol/mmol cre at i nine¡1 to cor rect for dif fer ences in

urine con cen tra tion.

Hep ci din assay

The assay was based on the quan ti ta tive detec tion of the

hep ci din sig nal by MS rel a tive to the heavy iso tope-labeled

inter nal stan dard that is added at a known con cen tra tion. Hep-

ci din and [15N,13C2]Gly12,20-hep ci din are of iden ti cal amino acid


Quantitation of hepcidin in human urine by LC–MS / S.S. Ban sal et al. / Anal. Biochem. 384 (2009) 245–253 247

com po si tion and exhibit iden ti cal prop er ties as judged by reten-

tion times on LC and MS frag men ta tion with the excep tion of the

mass dif fer ence. Because the sig nal inten sity is lin ear for both hep-

ci din and [15N,13C2]Gly12,20-hep ci din, the con cen tra tion can be

cal cu lated directly from the ratio of the sig nal responses.

Sam ple prep a ra tion

Weak cat ion exchange (WCX) mag netic nano par ti cles were ac-

ti vated by wash ing with ammo nium ace tate (25 mM, pH 6.8). Sam-

ples were allowed to thaw at ambi ent tem per a ture and then were

vor texed. Urine (200 ll) was spiked with 40 ll of [15N,13C2]Gly12,20-

hep ci din solu tion (178 mol·L¡1), made up to 1 ml with 25 mM

ammo nium ace tate in a poly pro pyl ene tube, and vor tex-mixed

(final pH 6.8). A 10-ll ali quot of WCX mag netic nano par ti cles

(2.5 lg of pro tein bind ing capac ity) was added to the sam ple and

gently agi tated for 30 min. Unbound pro teins were removed by

cap tur ing the nano par ti cles onto a mag netic sep a ra tor, the super-

na tant was dis carded, and the nano par ti cles were washed with

water (5 £ 500 ll). Finally, the bound cat ionic pep tides were eluted

from the mag netic nano par ti cles with 20 ll of 0.1% aque ous for-

mic acid/ace to ni trile (4:1, v/v) for 5 min, the spent nano par ti cles

were cap tured on the mag netic sep a ra tor, and the super na tant was

trans ferred into a mi cro vial for LC–MS/MS anal y sis.

LC and MS oper at ing con di tions

Chro mato graphic sep a ra tion was car ried out on a Sur veyor

sys tem com posed of a deg as ser, a pump, an auto sam pler, and a

col umn heater (ThermoFisher Scientific). Liquid chro ma tog ra phy

was per formed on a Poro shell C18 col umn (75 £ 1 mm i.d., 5 lm,

300 Å, Ag i lent Tech nol o gies) oper ated at a col umn tem per a ture

of 35 °C. A flow rate of 200 ll/min¡1 was employed. The mobile

phase used for the sep a ra tion of hep ci din was com posed of water

con tain ing 0.1% for mic acid (sol vent A) and ace to ni trile (sol vent

B). The gra di ent applied was 20% B, ris ing to 90% B in 3.5 min fol-

lowed by wash ing and re equil i bra tion steps. The sam ple (3 ll) was

injected via a 20-ll sam ple loop. The ana lyt i cal col umn was inter-

faced with an LCQ Deca XP using elec tro spray ion i za tion (ESI). The

mass spec trom e ter was oper ated in the positive mode with the

heated cap il lary set at 260 °C and 2.5 kV applied to the elec tro spray

nee dle. The sheath gas flow was set to 40 units, and the aux il iary

gas flow was set to 10. The data were acquired in a full-scan mode

(m/z 200–1400) and in MS/MS selected reac tion mon i tor ing (SRM)

mode using auto matic gain con trol with 1 mi cro scan, a max i mum

ion time of 50 ms, and MS/MS col li sion energy of 25 eV.

The mass spec trom e ter param e ters were opti mized by infus-

ing a solu tion of hep ci din (1780 nmol/L) to obtain the max i mum

response of the tri ply charged ion at m/z 931 ([M+3H]3+). The tri-

ply charged pre cur sor ion pro duced two major frag ments: (y21)2+

and (y19)2+. The [15N,13C2]Gly12,20-hep ci din pre cur sor ion also

pro duced the cor re spond ing (y21)2+ and (y19)2+ frag ments. The iso-

la tion widths for the pre cur sor masses and scan widths for the

prod uct ion were set to 2 Da. Data anal y sis was per formed using

Xcal i bur soft ware (ver sion 2.06, ThermoFisher Scientific) from the

raw spec tral data. Peak inte gra tion param e ters used were as fol-

lows: smooth ing points, 7: base line win dow, 40; area noise fac tor

8, and peak noise fac tor, 30.

Quan ti ta tion of hep ci din

Urine (200 ml) was stripped with char coal (200 mg) for 2 h.

A 200-ll ali quot of this stripped urine was prepared using WCX

mag netic nano par ti cles as described above and then ana lyzed by

LC MS/MS to show that the hep ci din had been com pletely removed

from the sam ple.

Lin e ar ity

In the hep ci din recov ery exper i ment, a series of hep ci-

din solu tions in 200 ll of char coal-stripped urine were used

to pre pare cal i brants over the range of 0, 0.9, 1.8, 4.5, 9.0, 17.9,

35.9, 53.8, 89.6, and 179.2 nmol/L. After the addi tion of 40 ll

of [15N,13C2]Gly12,20-hep ci din (178 nmol/L), the sam ples were

prepared using the mag netic nano par ti cle pro ce dure described

above and ana lyzed by LC–MS/MS, and then the stan dard curves

were gen er ated.

Hep ci din recov ery

For the recov ery exper i ment, the con trol sam ples were not

spiked with [15N,13C2]Gly12,20-hep ci din but were eluted from the

mag netic nano par ti cles with 20 ll of 0.1% aque ous for mic acid/ace-

to ni trile (4:1, v/v) con tain ing 7.2 pmol of [15N,13C2]Gly12,20-hep ci-

din and were ana lyzed by LC–MS/MS.

In the precision and accu racy exper i ments, char coal-stripped

sam ples were enriched with hep ci din, seri ally diluted, and ana-

lyzed by LC–MS/MS. Precision and accu racy exper i ments were

per formed in char coal-stripped urine (n = 6) enriched with 1.8, 9.0,

71.7, and 179 nmol/L hep ci din. Finally, four clin i cal sam ples were

ana lyzed in trip li cate on 3 con sec u tive days to obtain intra- and

in ter day repro duc ibil ity.

Results

Hep ci din and [15N,13C2]Gly12,20-hep ci din coe lut ed on HPLC

but exhib ited a mass dif fer ence of 6 Da in the MALDI–TOF MS

(Fig. 1) and in ESI–MS. The net pep tide con tent was deter mined

to be 84% using amino acid anal y sis, and a 1780 nmol/L solu tion

in 0.1% aque ous for mic acid/ace to ni trile (4:1, v/v) was prepared for

subsequent anal y sis. The syn the sized [15N,13C2]Gly12,20-hep ci din

was estab lished to be bio log i cally active in an in vitro fer ro por tin

inter nal i za tion assay [1].

0 5 10 15 20 25 30 35 40 45

0.00

0.01

0.02

0.03

0.04

0.05

2788 2792 2796 28000

500

1000

1500

2000

2500

3000

3500

Rel

ativ

e Ab

unda

nce

Abso

rban

ce (2

20 n

m)

Time (min)

m/z

Fig. 1. Chro mato graphic anal y sis of hep ci din and [15N,13C2]Gly12,20-hep ci din.

Shown is an HPLC chro mato gram of a mix ture of hep ci din and [15N,13C2]Gly12,20-

hep ci din (0.1 mg/ml). Sta tion ary phase: Vy dac 218TP54 col umn; mobile phase A:

0.1% TFA; mobile phase B: 90% ace to ni trile; gra di ent: lin ear 1% B/min; flow rate:

1 ml/min; detec tion: 220 nm. Inset: MALDI–TOF MS of the same sam ple using a-

cyano-4-hydroxy cin namic acid as the matrix.



WCX mag netic nano par ti cles were used because no pre treat ment

of the sam ple, includ ing fil tra tion or cen tri fu ga tion, was required,

thereby min i miz ing losses. Frac tion ation with the mag netic nano-

par ti cles is very rapid and ef cient. The basic pep tides, includ ing

hep ci din, were cap tured from the urine and con cen trated 10-fold.

We used a Poro shell 300SB-C18 col umn with 300-Å par ti cle

size, which is suit able for pep tide anal y sis. The Poro shell sta tion-

ary phase is a unique mi crop ar tic u late, super fi cially porous C18

col umn pack ing that is espe cially use ful for car ry ing out ultra-fast

gra di ent sep a ra tions and can tol er ate rel a tively high flow rates

(2 ml/min¡1 for a 2.1-mm i.d. col umn). Gra di ent elu tion from 20

to 90% ace to ni trile within 3.5 min was used. Using the Poro shell

col umn, the mean reten tion time for 300 injec tions was 2.78 min

with a coef cient of var i ance of 1.50%.

Mass spec trom e try

The mass spec tro met ric param e ters were opti mized by direct

infu sion of a 1780 nmol/L solu tion of hep ci din in the positive mode

and showed the dou bly, tri ply, and qua dru ply charged molec u lar

ions with m/z 1395.4 ([M+2H]2+), m/z 930.8 ([M+3H]3+), and m/z

698.2 ([M+4H]4+) (Fig. 2A). A sim i lar series of ions were observed

for [15N,13C2]Gly12,20-hep ci din at m/z 1397.9 ([M+2H]2+), m/z 932.6

([M+3H]3+), and m/z 699.9 ([M+4H]4+), respec tively (Fig. 3A). The most

abun dant ion observed for both hep ci din and [15N,13C2]Gly12,20-

hep ci din was the qua dru ply charged molec u lar ion. This pre cur sor

ion was able to absorb frag men ta tion ener gies; how ever, the prod uct

ions were many, yield ing no prod uct ion of suf cient inten sity. Frag-

men ta tion of the pre cur sor molec u lar ion at m/z 930.8 ([M+3H]3+)

resulted in the ef cient loss of 501.2 Da (b4)+ to yield (y21)2+ due to

cleav age of the N-ter mi nal tet ra pep tide at the pro line res i due. A

less abun dant prod uct with a loss of 353 Da (b3)+ from (y22)2+ due

to cleav age on the N-ter mi nal tri pep tide was also observed in the

full-scan spec tra (Fig. 2B). Sim i lar frag men ta tion was also observed

for [15N,13C2]Gly12,20-hep ci din (Fig. 3B). Hep ci din con tains three

res i dues with high gas phase basicity (two lysines and one argi-

nine). In the qua dru ply charged ion, all three sites are expected to

be pro ton ated, leav ing the addi tional mobile pro ton to engage in

200 400 600 800 1000 1200 1400

0

1x106

2x106

3x106

4x106

200 400 600 800 1000 1200 1400

0

1x106

2x106

3x106

4x106

5x106

y2+

22Rel

ativ

e A

bund

ance

m/z

y2+

21

DTHFPICIFCCGCCHRSKCGMCCKT

b4b

3

b3

b4

y21

y22

MS/MS of precursor ion at m/z 930.3

[M+5H]5+

[M+4H]4+

[M+2H]2+Rel

ativ

e A

bund

ance

m/z

[M+3H]3+

Full scale MS of hepcidinA

B

Fig. 2. MS of hep ci din. (A) Direct infu sion of 1780 nmol·L¡1 solu tion of hep ci din gave molec u lar ions at m/z 1395.4 ([M+2H]2+), m/z 930.8 ([M+3H]3+), and m/z 698.2 ([M+4H]4+).

(B) Frag men ta tion of pre cur sor ion at m/z 930.8 resulted in the ef cient loss of b4 to yield (y21)2+. A loss of b3 to yield (y22)2+ was also observed.



charge-directed frag men ta tion. In con trast, the tri ply charged ion will

be pro ton ated at the three basic sites; how ever, there are no mobile

pro tons avail able for charge-med i ated frag men ta tion, thereby rais-

ing the acti va tion energy for frag men ta tion path ways [22,23]. Using

the frag men ta tion of the tri ply charged ion results in a tre men dous

increase in sen si tiv ity for SRM and is an ideal tran si tion for mon i tor-

ing hep ci din in an LC–MS/MS assay.

Assay val i da tion

Matrix effects

Because urine con tains endog e nous hep ci din, it was dif cult to

ana lyze blank sam ples. How ever, urine was suc cess fully stripped

of hep ci din as deter mined by LC–MS/MS and then was spiked with

stan dard amounts of syn thetic hep ci din for val i da tion. In these

exper i ments, sam ples from he mo chro ma to sis patients were used

because they were found to con tain low con cen tra tions of hep ci-

din. Finally, some 300 sam ples from patients with a wide range

of clin i cal con di tions, includ ing healthy con trols, were ana lyzed to

show that there is no inter fer ence from the matrix.

Lin e ar ity

A non weight ed lin ear regres sion line was fit ted to the con cen-

tra tion ratio cov er ing the range of 0 to 179 nmol/L of hep ci din. The

data are expressed as the ratio of the con cen tra tion of hep ci din/

con cen tra tion of the inter nal stan dard against the peak area ratio

of hep ci din/inter nal stan dard (n = 6) (Fig. 4). The lin e ar ity of hep-

ci din was mea sured with an inter cept not sig nifi cantly dif fer ent

from zero (R2 = 0.996, y = 0.956x + 0.0978) (Fig. 4). The lower limit

of quan ti ta tion (LLOQ) was deter mined as the level of the least

con cen trated cal i bra tion stan dard with a sig nal/noise ratio greater

than 3:1 (Fig. 5).

Hep ci din recov ery

The recov er ies of hep ci din after mag netic nano par ti cle frac tion-

ation and LC–MS/MS were deter mined to be 81, 79, and 78% (n = 6)

200 400 600 800 1000 1200 1400

0

1x106

2x106

3x106

4x106

200 400 600 800 1000 1200 1400

0

1x106

2x106

3x106

4x106

5x106

y2+

22

Rel

ativ

e A

bund

ance

m/z

y2+

21

DTHFPICIFCCGCCHRSKCGMCCKT

Full scale MS of [15N,13C2]Gly12,20-hepcidin

b3

b4

b3

b4

y21

y22

[M+2H]2+

[M+3H]3+

[M+4H]4+

Rel

ativ

e A

bund

ance

m/z

[M+5H]5+

MS/MS of the precursor ion an m/z 932.8

A

B

Fig. 3. MS of [15N,13C2]Gly12,20-hep ci din. (A) Direct infu sion of 1780 nmol·L solu tion of [15N,13C2]Gly12,20-hep ci din gave molec u lar ions at m/z 1397.3 ([M+2H]2+), m/z 932.8 ([M+3H]3+),

and m/z 699.8 ([M+4H]4+). (B) Frag men ta tion of pre cur sor ion at m/z 932.8 resulted in the ef cient loss of b4 to yield (y21)2+. A loss of b3 to yield (y22)2+ was also observed.



for the addi tion of 179, 17.9, and 1.8 nmol L¡1 hep ci din, respec tively.

The sam ple prep a ra tion does not involve any solu tion trans fers,

although the mag netic nano par ti cle pro cess ing will result in some

losses. Dur ing elu tion of hep ci din from the nano par ti cles, aque ous

for mic acid and ace to ni trile were used to main tain the pep tide in

solu tion. The recov ery is both repro duc ible and inde pen dent of the

amount of hep ci din added.

Par al lel ism (dilu tion recov ery)

Char coal-stripped urine was spiked with hep ci din and seri ally

diluted to give con cen tra tion of hep ci din/[15N,13C2]Gly12,20-hep-

ci din ratios of 4:1, 2:1, 1:1, 0.5:1, 0.25:1, and 0.125:1. The peak area

of hep ci din/[15N,13C2]Gly12,20-hep ci din against the con cen tra tion

of hep ci din/[15N,13C2]Gly12,20-hep ci din gave a lin ear rela tion ship

(R2 = 0.991, y = 0.977x ¡ 0.0155) (Fig. 4).

Accu racy and precision

To eval u ate the repro duc ibil ity of the hep ci din mea sure ments,

six inde pen dent mea sure ments were made on four dif fer ent con-

cen tra tions of spiked hep ci din. The mea sured con cen tra tions,

per cent age rel a tive errors (accu racy), and coef cients of var i a tion

(precision) are pre sented in Table 1. We found that the mea sure-

ments were highly repro duc ible, with the coef cient of var i a tion

below 10%, except for sam ples that con tained very low or very high

con cen tra tions of hep ci din. In addi tion, the accu racy of the mea-

sure ments was within 10% of the expected val ues.

Hav ing estab lished the repro duc ibil ity of the hep ci din deter-

mi na tions in stripped urine, we eval u ated the repro duc ibil ity and

precision of the mea sure ments in clin i cal sam ples. Four sam ples

from patients with a wide range of hep ci din con cen tra tions were

ana lyzed in trip li cate on 3 con sec u tive days. The mea sured con cen-

tra tions, coef cients of var i a tion, and total mean precision from 3

days are pre sented in Table 2. Over all, the total coef cient of var-

i a tion for the 3 days was below 10%. Typ i cal chro mato grams from

the LC–MS/MS assay are shown in Fig. 6 based on the frag men ta-

tion of the tri ply charged molec u lar ion with a loss of b4 to yield

(y21)2+. The LLOQ of the assay on the LCQ Deca XP was 1 nmol/L,

equiv a lent to 8 pg on col umn.

To eval u ate the val i dated assay, hep ci din con cen tra tions in 50

urine sam ples from healthy vol un teers and patients with hered-

i tary he mo chro ma to sis, sys temic infec tion, or SCA were deter-

mined. (Fig. 7).

Dis cus sion

This is the first report of the quan ti ta tion of hep ci din in clin i-

cal urine sam ples using an ion trap mass spec trom e ter.2 The ana-

lyte and inter nal stan dard are of iden ti cal amino acid com po si-

tion and exhibit iden ti cal chem i cal prop er ties, but they dif fer in

their masses, with 2787.023 and 2793.042 Da for hep ci din and

[15N,13C2]Gly12,20-hep ci din, respec tively. Because the sig nal inten-

sity is lin ear for both the ana lyte and the inter nal stan dard, the

con cen tra tion of hep ci din can be cal cu lated directly from the ratio

of the sig nal responses. The sam ples were frac tion ated with out

any preliminary fil tra tion or cen tri fu ga tion using mag netic nano-

par ti cles. The extrac tion pro ce dure is rapid, ef cient, and highly

repro duc ible with good recov ery.

We ini tially attempted to carry out the hep ci din anal y sis on

a tri ple quad ru pole mass spec trom e ter using col li sion-induced

dis so ci a tion (CID), but it was not pos si ble to gen er ate an abun-

dant ion for use in SRM. Hep ci din was able to absorb substantial

frag men ta tion ener gies in the tri ple quad ru pole before any frag-

men ta tion was observed. Once frag men ta tion was achieved, the

prod uct ions were many and had low and var i able inten si ties

over all, and it proved to be dif cult to gen er ate a sin gle prod-

uct. Mur phy and cowork ers reported the anal y sis of hep ci din

using MS–MS and observed the ion at m/z 698.4 frag ment ing to

m/z 644.1 for the qua dru ply charged pre cur sor ion to yield the

prod uct ion (y23)4+ of very low abun dance [17]. More recently,

hep ci din was ana lyzed by the tran si tion of the quin tu ply charged

ion m/z 558.7 frag ment ing to m/z 120.1 (im mo ni um ion of Phe),

which lacks spec i fic ity [25].

In the cur rent work, we suc cess fully used the ion trap with SRM

based on the m/z 930 to (y21)2+ tran si tion that was spe cific for hep-

ci din and the m/z 933 to (y21)2+ tran si tion for [15N,13C2]Gly12,20-

2 At the time when this man u script was under review, a com mu ni ca tion describ-

ing a dif fer ent LC–MS/MS method for the quan ti ta tion of hep ci din in serum was

pub lished [24].

0 1 2 3 4 50

1

2

3

4

5

Pea

k A

rea

(Hep

cidi

n/ [15

N,13

C2]

Gly

12,

20-H

epci

din)

Ratio of concentration (Hepcidin/ [15N,13C2]Gly 12,20-Hepcidin)

Fig. 4. Cal i bra tion and serial dilu tion plot for hep ci din. j, Rela tion ship between the

peak area of hep ci din/[15N,13C2]Gly12,20-hep ci din (7.19 pmol) and the con cen tra-

tion of hep ci din/[15N,13C2]Gly12,20-hep ci din (7.19 pmol). s, Serial dilu tion of char-

coal-stripped sam ple spiked with hep ci din: frag men ta tion of pre cur sor ion at m/z

930.8 with the ef cient loss of b4 to yield (y21)2+, 4:1 to 0.125:1.

Table 1

Accu racy and precision of LC–MS/MS anal y sis of hep ci din spiked into char coal-

stripped urine at dif fer ent con cen tra tions.

Sta tis tics Hep ci din con cen tra tion

1.8 nmol/L 9.0 nmol/L 71.7 nmol/L 179 nmol/L

Mean 1.9 9.4 77.1 170.7

Accu racy (% RE) 5.6 4.4 7.5 –4.6

Precision (% CV) 11.4 9.9 8.7 12.5

Note. Shown are the mean con cen tra tion, accu racy (per cent age rel a tive error, %RE),

and precision (per cent age coef cient of var i a tion, %CV).

Table 2

Intra- and in ter day val i da tion sta tis tics for four clin i cal sam ples ana lyzed in trip li-

cate over 3 days.

Day Sta tis tics Val i da tion sam ple con cen tra tion of hep ci din

(nmol/L)

1 Mean (nmol/L) 193.6 107.9 43.7 9.07

Precision (% CV) 8.59 6.22 4.54 8.17

n 6 6 6 6

2 Mean (nmol/L) 201.5 114.4 44.1 6.68

Precision (% CV) 8.14 4.18 4.28 10.36

n 6 6 6 6

3 Mean (nmol/L) 186.8 114.4 44.10 9.96

Precision (% CV) 7.02 5.68 8.42 11.45

n 6 6 5 6

All Mean (nmol/L) 193.3 112.2 44.10 9.39

Precision (% CV) 8.07 4.96 4.32 10.08

n 18 18 17 18



hep ci din (Fig. 5). Cen tral to the accu rate quan ti ta tion of hep ci din is

the use of an appro pri ate inter nal stan dard given that the meth ods

reported to date lack spec i fic ity (dot blot) and/or require extrac-

tion or quan ti ta tion tech niques that give rise to poten tially var i-

able recov ery of hep ci din.

In this arti cle, we reported a novel assay for hep ci din that in

val i da tion stud ies is highly repro duc ible and dem on strates high

rates of recov ery (80%) from urine. We car ried out hep ci din anal-

y sis in char coal-stripped urine and obtained good accu racy and

good within- and between-day precision. The method has a limit

of quan ti ta tion of 1 nmol L¡1, which can in prin ci ple be low ered by

the use of larger vol umes of urine and more mag netic nano par ti-

cles.

We found the mean hep ci din con cen tra tion for the 12 con trol

urine sam ples to be 5.2 ± 1.82 nmol/mmol cre at i nine¡1. Ne meth

and Ganz [26] reported the con cen tra tions in the urine of con trol

sub jects to be in the range of 3 to 30 nmol/L, and we found a sim i lar

range of 3 to 9 nmol/mmol cre at i nine¡1 with one out lier. There is

clearly con sid er able var i a tion in uri nary hep ci din in healthy sub-

jects that may be related to diur nal var i a tion [14].

The mean urine hep ci din con cen tra tion in 12 hered i tary he mo-

chro ma to sis patients was found to be 0.55 ± 0.52 nmol/mmol cre-

at i nine¡1 with three out li ers between 3.4 and 4.5 nmol/mmol

cre at i nine¡1. This find ing con firms pre vi ous reports that uri nary

hep ci din lev els in he mo chro ma to sis patients under main te nance

phle bot omy are sup pressed [7,14], con sis tent with the idea that

deple tion of iron stores may fur ther exac er bate the HFE-related

defect in hep ci din syn the sis [27]. Con fir ma tion that serum hep ci-

din is innately low in HFE-related he mo chro ma to sis was recently

reported using the SEL DI tech nique [28].

Six sam ples from patients with sys temic infec tion and inflam-

ma tion dem on strated a marked increase in hep ci din con cen tra-

tions of between 10 and 50 nmol/mmol cre at i nine¡1 with a mean

of 26.5 ± 14.5 nmol/mmol cre at i nine¡1. Increased lev els of hep-

ci din in infec tion are thought to be related to increased lev els

of the cyto kine IL-6 [11], a likely inducer of hep ci din syn the sis.

Although IL-6 is also known to be increased in sickle cell ane-

mia [11,29] and this cyto kine is a known inducer of hep ci din, the

range of val ues observed may be related to the com plex inter play

among inflam ma tion, iron bur den, and increased eryth ro poi e-

sis due to hemo ly sis. Indeed, Kear ney and cowork ers [30] found

that hep ci din con cen tra tion was sup pressed rel a tive to the iron

bur den in such patients. Of the 15 patients with sickle cell ane-

mia inves ti gated in the cur rent study, the major ity had uri nary

hep ci din lev els of less than 15 nmol/mmol cre at i nine¡1 with two

excep tions at 19.7 and 65.5 nmol/mmol cre at i nine¡1. The dif fer-

ence may be related to the dif culty in dif fer en ti at ing mild pain-

ful cri ses when IL-6 lev els are increased from steady state. Some

of the steady-state sam ples may well have been col lected in the

res o lu tion phase of mild pain ful cri ses that had been man aged

at home.

In this report, [15N,13C2]Gly12,20-hep ci din was used as an

inter nal stan dard for the deter mi na tion of hep ci din con cen tra-

tion in urine sam ples with sen si tiv ity down to 1 nmol/L. Using

urine from nor mal sub jects and patients with hered i tary he mo-

chro ma to sis, sys temic infec tion, and SCA, an ef cient and repro-

duc ible frac tion ation pro ce dure for the sen si tive and selec tive

deter mi na tion of hep ci din was devel oped. The frac tion ation pro-

ce dure is ame na ble for high-through put anal y sis for both frac-

tion ation and MS.

RT: 0.00 - 10.00 SM: 9G

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RT: 1.521.523AA: 52383BP: 1146.2

RT: 9.789.778AA: 19787BP: 1144.2

RT: 2.102.098AA: 14987BP: 1144.3

RT: 3.293.291AA: 13157BP: 1144.7

RT: 0.830.830AA: 8066BP: 1144.0

2.101144.3 3.81

1143.8 5.261146.3

RT: 2.732.732AA: 1329892BP: 1148.9

RT: 9.669.662AA: 40398BP: 1148.8

RT: 0.450.455AA: 21374BP: 1147.2

RT: 3.113.112AA: 17463BP: 1147.4

RT: 5.985.982AA: 11972BP: 1147.1

NL: 2.22E4TIC F: + c ESI SRM ms2 931.00 [1143.50-1146.50] MS H12944_080224011303

NL: 1.27E5TIC F: + c ESI SRM ms2 933.00 [1146.60-1150.00] MS ICIS H12944_080224011303

Fig. 5. Lower lim its of quan ti ta tion. Char coal-stripped urine (200 ll) was spiked with 10 ll of hep ci din (18 nmol/L) and 40 ll of [15N,13C2]Gly12,20-hep ci din (178 nmol/L). The

sam ple was puri fied using WCX mag netic nano par ti cles and eluted with 20 ll ace to ni trile/0.1% for mic acid (4:1), and 3 ll was injected onto a Poro shell col umn and ana lyzed

by frag men ta tion of pre cur sor ions at m/z 930.8 and 932.8 with the ef cient loss of b4 to yield (y21)2+ for hep ci din and [15N,13C2]Gly12,20-hep ci din, respec tively. Shown are

the upper trace peak area for hep ci din and the lower trace peak area for the inter nal stan dard.



Acknowl edg ments

The authors thank Andrew Kic man and David Cowan (Drug

Con trol Cen ter, King’s Col lege Lon don) for their con struc tive

advice and care ful read ing of the paper, and we thank Ki shor Raja

(King’s Col lege Hos pi tal) for the cre at i nine mea sure ments and

Andrew McKie for functional assay of hepcidin. We also thank

the reviewer for high light ing the mobile pro ton model. This

work was sup ported by the Guy’s and St. Thomas’ Trust R031154

(S.S.B., A.B., and R.J.S.), Med i cal Research Coun cil (MRC UK) grant

G00001249 (ID56744, S.L.T.), and an MRC UK Grant G9001437

(ID 52073, R.C.H. and R.J.S.).

Ref er ences

[1] T. Ganz, Hep ci din: A pep tide hor mone at the inter face of innate immu nity and iron metab o lism, Curr. Top. Micro biol. Immu nol. 306 (2006) 183–198.

[2] A. Kra use, S. Nei tz, H.J. Mag ert, A. Schulz, W.G. Forss mann, P. Schulz-Knap pe, K. Ad er mann, LEAP-1, a novel highly disul fide-bonded human pep tide, exhib its anti mi cro bial activ ity, FEBS Lett. 480 (2000) 147–150.

[3] C.H. Park, E.V. Va lore, A.J. War ing, T. Ganz, Hep ci din, a uri nary anti mi cro bial pep tide syn the sized in the liver, J. Biol. Chem. 276 (2001) 7806–7810.

[4] H.N. Hunter, D.B. Ful ton, T. Ganz, H.J. Vo gel, The solu tion struc ture of human hep ci-din, a pep tide hor mone with anti mi cro bial activ ity that is involved in iron uptake and hered i tary he mo chro ma to sis, J. Biol. Chem. 277 (2002) 37597–37603.

[5] E. Ne meth, M.S. Tut tle, J. Po wel son, M.B. Vau ghn, A. Dono van, D.M. Ward, T. Ganz, J. Kap lan, Hep ci din reg u lates cel lu lar iron efflux by bind ing to fer ro por-tin and induc ing its inter nal i za tion, Sci ence 306 (2004) 2090–2093.

[6] G. Nic olas, C. Chau vet, L. Vi atte, J.L. Da nan, X. Bi gard, I. Dev aux, C. Beau mont, A. Kahn, S. Va u lont, The gene encod ing the iron reg u la tory pep tide hep ci din is reg u-lated by ane mia, hypoxia, and inflam ma tion, J. Clin. Invest. 110 (2002) 1037–1044.

[7] E. Ne meth, E.V. Va lore, M. Ter rito, G. Schil ler, A. Lich ten stein, T. Ganz, Hep ci din, a puta tive medi a tor of ane mia of inflam ma tion, is a type II acute-phase pro-tein, Blood 101 (2003) 2461–2463.

[8] R.H. Wang, C. Li, X. Xu, Y. Zheng, C. Xiao, P. Zer fas, S. Coo per man, M. Eck-haus, T. Roua ult, L. Mish ra, C.X. Deng, A role of SMAD4 in iron metab o lism through the positive reg u la tion of hep ci din expres sion, Cell Metab. 2 (2005) 399–409.

[9] J. Tru ksa, H. Peng, P. Lee, E. Beu tler, Bone mor pho ge netic pro teins 2, 4, and 9 stim-u late murine hep ci din 1 expres sion inde pen dently of Hfe, trans fer rin recep tor 2 (Tfr2), and IL-6, Proc. Natl. Acad. Sci. USA 103 (2006) 10289–10293.

[10] J.L. Ba bitt, F.W. Hu ang, D.M. Wright ing, Y. Xia, Y. Si dis, T.A. Sa mad, J.A. Cam pa-gna, R.T. Chung, A.L. Sch ney er, C.J. Woolf, N.C. Andrews, H.Y. Lin, Bone mor pho-ge netic pro tein sig nal ing by he mo juv elin reg u lates hep ci din expres sion, Nat. Genet. 38 (2006) 531–539.

-10

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Hep

cidi

n (n

mol

.mm

ol c

reat

inin

e-1)

DiagnosisHH CS SCA IA

Fig. 7. Hep ci din con cen tra tions of clin i cal sam ples. Shown are urine hep ci din con-

cen tra tions deter mined by LC–MS/MS. HH, hered i tary he mo chro ma to sis; CS, con-

trol sam ples; SCA, sickle cell ane mia; IA, infec tion.

RT: 0.00 - 9.99 SM: 7G

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RT: 3.093.085AA: 34895BP: 1145.7

RT: 9.429.423AA: 33729BP: 1144.2

RT: 1.301.296AA: 19297BP: 1145.6

RT: 1.891.892AA: 17147BP: 1146.3

RT: 3.823.821AA: 6670BP: 1145.2

RT: 2.782.780AA: 1035914BP: 1147.6

RT: 3.193.191AA: 63228BP: 1149.5

RT: 0.770.774AA: 28938BP: 1149.4

RT: 2.372.367AA: 40509BP: 1149.0

RT: 4.624.616AA: 25432BP: 1148.5

RT: 9.169.158AA: 19630BP: 1149.2

RT: 8.358.351AA: 30093BP: 1148.0

RT: 6.876.871AA: 15553BP: 1148.5

RT: 6.176.172AA: 8921BP: 1147.6

NL: 1.28E5TIC F: + c ESI SRM ms2 931.00 [1143.50-1146.50]MS ICISH12999_080225050500

NL: 1.32E5TIC F: + c ESI SRM ms2 933.00 [1146.60-1150.00]MS ICISH12999_080225050500

Fig. 6. Anal y sis of a typ i cal urine sam ple. A urine sam ple was spiked with 40 ll of [15N,13C2]Gly12,20-hep ci din (178 nmol/L), puri fied using WCX mag netic nano par ti cles, and

eluted with 20 ll ace to ni trile/0.1% for mic acid (4:1), and 3 ll was injected onto a Poro shell col umn and ana lyzed by the frag men ta tion of pre cur sor ions at m/z 930.8 and

932.8 with the ef cient loss of b4 to yield (y21)2+ for hep ci din and [15N,13C2]Gly12,20-hep ci din, respec tively. Shown are the upper trace peak area for hep ci din and the lower

trace peak area for the inter nal stan dard.



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