June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Research Paper

Development and validation of the simultaneous measurement of estrone and17-β estradiol in serum by LC-MS/MS for clinical laboratory applications
1,†

Mamoru Satoh2,†, Yui Miyabayashi
3

3

3

3

, Masanori Seimiya , Takahiro Kazami , Takayuki Ishige ,

3

3

4

1

Toshihiko Yoshida , Motoi Nishimura , Kazuyuki Matsushita , Makio Shozu , Fumio Nomura *1

Division of Clinical Mass Spectrometry, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japan2
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University,1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japan
3
Division of Laboratory Medicine, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japan
4
Department of Reproductive Medicine, Graduate School of Medicine, Chiba University,1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japan
Abstract Application of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical chemistry has been
increasing worldwide, especially in large institution and reference laboratories. Although immunoassays are often used formeasurement of serum estradiol (E2) when fast turnaround time is required, more sensitive and speciﬁc measurements areneeded for determination of menopausal status, estrogen deﬁciency and in the diagnosis of sex hormone related disorders.
Furthermore, simultaneous measurement of estrone (E1) and E2 is often requested particularly from gynecologic oncologists. Indeed, increased risk of endometrial cancers has been shown in subjects with high serum estrogen levels. The aimof this study is to develop and validate LC-MS/MS method for simultaneous measurement of E1 and E2 in human serum.
Serum samples were ﬁrst prepared in a 96-well supported liquid extraction plate and the eluate was derivatized by thedansyl chloride acetone solution. The derivatized samples were subjected to LC-MS/MS, and detected by selected reaction
monitoring. The lower limits of quantiﬁcation for E1 and E2 were 6.2 and 7.3 pg/mL, respectively. The 60 female sera values obtained by the LC-MS/MS method were compared with those obtained by two commercially available immunoassays.
The both of values obtained by the two immunoassays exhibited positive bias particularly at low E2 levels. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze‒thaw cycles, and the presence ofanticoagulants, had no signiﬁcant effects on these determinations. This method will aid further understanding of low-abundance estrogen, as well as the accurate determination of E1 and E2.
Key words: estrone, 17- β estradiol, dansyl chloride
Abbreviations: E1, estrone; E2, 17- β estradiol; QC, quality control; IS, internal standard; ESI, electrospray ionization; LLOQ,
lower limit of quantiﬁcation; CV, coefﬁcient of variation†

Mamoru Satoh and Yui Miyabayashi contributed equally to

Introduction

this work.
* Corresponding author

Estradiol is the most potent estrogen and plays the majorroles in the development and maintenance of female sex

Fumio Nomura

organs and also in regulation of the menstrual cycle. Since

Division of Clinical Mass Spectrometry, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japanthe ﬁrst radioimmunoassay (RIA) was reported in 1969,
serum estradiol levels have been mostly measured by

Tel: ＋81‒43‒226‒2627, Fax: ＋81‒43‒226‒2627
E-mail: fnomura@faculty.chiba-u.jp
Received April 16, 2018.

1)

immunoassays for decades . In recent years, RIAs are
increasingly replaced by non-RIA assays such as chemilu-

Accepted August 22, 2018.

minescent immunoassay (CLIA) mainly because handlingradioactive materials is a kind of nuisance hospital-based

Epub December 14, 2018.
DOI: 10.24508/mms.2019.06.001
19

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

1)clinical laboratories . For the purpose of evaluation of

The aim of this study is to develop and validate an LC/

ovarian function in which demands on assay sensitivity ismodest, immunoassays can provide relevant results. How-

MS/MS method to simultaneously measure estradiol andever, there is increasing clinical demands on highly sensitive estradiol assays for the diagnosis of variety of disor-

Also, we determined the effects of various preanalyticalders such as inborn errors of sex steroid metabolism andand the presence of anticoagulants on these determinations.

estrone in serum for application in clinical laboratories.
factors such as interferences, sample collection processes,

1)

precocious puberty . Highly sensitive assays are also
required for quantitation in men, post-menopausal women

Materials and Methods

1)and in subjects on anti-estrogen treatment . Current immu-

Calibrators and quality controls

noassays are not sensitive and accurate enough for estradiol

Mass spectrometry-grade solvents and formic acid were

measurements under these circumstances. Indeed, it wasfrom Wako Pure Chemical Industries (Osaka, Japan). Water
was from Nacalai Tesque (Kyoto, Japan). Estrone, 17- β estra-reported in a study comparing the analytical performances13
diol and 17- β estradiol- C3 were purchased from Sigma-Al13
drich (St Louis, MO, USA). Estrone- C3was purchased fromof the eight commercial estradiol immunoassays that their
coefﬁcient variation values ranged from 6.9 to 42.6% at

IsoSciences (King of Prussia, PA, USA). Estrone- C3 and 1713
β estradiol- C 3 were used as internal standards (ISs). The IS2)

13

18 pg/mL level .
The most accurate method for determination of serum
estradiol level is gas-chromatography mass spectrometry

working solution was prepared in 50% methanol and contained estrone and 17- β estradiol at each concentration of(GC-MS) . But throughput of GC-MS is not satisfactoryfor routine clinical use. In terms of sensitivity and through-10 ng/mL. The calibrators of Estrone and 17- β estradiol wereput, liquid chromatography with tandem mass spectrometry

purchased from Biocrates Life Sciences AG (Innsbruck,

3)

4‒7)

(LC/MS/MS) is increasingly used . The advantages of theuse of LC-MS/MS for steroid hormone measurement overAustria). The concentrations of the 4 Biocrates calibration

immunoassays are well recognized by one of the distin-

standards for estrone and 17- β estradiol were as follows: 30
and 20 pg/mL for calibrator level 1; 120 and 80 pg/mL for cal-guished journal in endocrinology, the Journal of Clinicalibrator level 2; 600 and 400 pg/mL for calibrator level 3; andEndocrinology and Metabolism. A position statement

1200 and 1600 pg/mL for calibrator level 4. Human Serum

released from the journal in 2013 indicated that effective

Standard Reference Material (BCR576, BCR577 andBCR578) as the quality control (QC) sample was from Sig-

January 1, 2015, manuscripts reporting sex steroid assays
8)
as important endpoint must use MS-based assays .

ma-Aldrich. The quality control value for 17- β estradiol in

Although this is a controversial subject and the journal pol-BCR576, BCR577 and BCR578 was 31.5±1.36, 187.95±

icy on this issue has been suspended for various reasons,10.88 and 365.00±19.04 pg/mL, respectively.

the position statement will deﬁnitely act as an impetus
toward accelerating use of LC-MS/MS in sex steroid mea-

Human serum samples and handling procedures

A total of 60 consecutive sera obtained from blood samples requested for estradiol measurements in Clinical labo-

surements.
Although only one analyte can be measured per one
immunoassay, LC/MS/MS permits simultaneous determina-

ratory of Chiba University Hospital were used to assesscorrelation between LC-MS/MS and chemiluminescenttions of multiple analytes by one run. Estrone, another key
estrogen, is a major form in post-menopausal females andimmunoassay (CLIA) measurements. The study protocol(No. 685) was approved the ethics committee of our insti-

simultaneous measurement of estradiol and estrone is signiﬁcant not only for better understanding of female physiol-

tute and each subject provided written informed consent

ogy, but in the ﬁeld of gynecological oncology. Circulating

prior to their enrollment in the study. Three serum samples

estrogens contribute to development of endometrial cancer
9, 10). Since increased serum estrone levand ovarian cancer

were obtained from 3 apparently healthy volunteers withvarious levels of serum E1 and E2 concentrations to vali-

11)

date the precision of this method. Venous blood samples

cancer risk, requests for simultaneous

were collected in vacutainer tubes (4S1001, Insepack. II,

measurements of estradiol and estrone are going to be

Sekisui Medical Co., Ltd., Tokyo, Japan) which contained

increased.

SiO2 as a coagulation enhancer and the samples were left at

els are reported to be positively associated with ovarian12)

and endometrial

20June 2019

Medical Mass Spectrometry Vol. 3 No. 1

room temperature for 30 min. The sera were then obtained

LC/MS/MS assay validation

by centrifugation at 1200 × g for 10 min at 4°
C. The ethics

Accuracy was estimated by measuring twenty replicatesof 17- β estradiol in BCR QC sera. Control sera of low,committee of our institute approved the study protocol, andtheir enrollment in the study.

medium and high level estrone and 17- β estradiol concentrations were used for assessing assay precision. On 20 con-Sample preparation and Dansyl chloride derivatization

secutive days, estrone and 17- β estradiol were measured
with four replicates per day (inter-day) of three level con-ume of water. IS solution (20 μL) was added to diluted

trol sera. On the day different from the 20 consecutive
days, estrone and 17- β estradiol were measured with 20

serum and vortexed for one minute. Samples were applied

replicates of the three level control sera per day (intra-day).

individually to the wells of a 96 well supported liquid

Dilution linearity was evaluated by measuring duplicates ofextraction

ﬁve dilutions of the two concentration levels of the control

each subject provided written informed consent prior to

The serum samples (100 μL) were diluted by equal vol-

(SLE)

plate

(ISOLUTE

SLE＋:

Biotage,
Uppsala, Sweden) and incubated for ﬁve minutes. The ana-

sera (medium and high) for estrone and the QC serum(BCR578 and BCR576 : BCR577＝8 : 2 (v/v)) for 17- β estra-lytes were eluted three times with 600 μL ethyl acetate : hexane (75 : 25, v/v), then the samples were evaporated to dryness under nitrogen at 45°
C using a Biotage SPE Dry 96

diol. The lower limit of quantiﬁcation (LLOQ) was determined from four replicate measurements of high level con-

(Biotage). The dry samples were dissolved in 50 μL oftrol serum for estrone and BCR578 for 17- β estradiol

sodium bicarbonate buffer (100 mM, pH 10.5) and 50 μL ofdansyl chloride solution (1.0 mg/mL in acetone), then vor-

diluted with saline as follows: 1 : 1 (not diluted), 1 : 2, 1 : 5,

texed for 1 min. The mixed samples were incubated at 60°
Clevel at which dilution linearity was conﬁrmed, S/N ratiofor 10 min, then cooled at 4°
C and a 40 μL of aliquot was
injected onto the LC-MS/MS.

was＞10 and the coefﬁcient of variance was ＜10%.

1 : 10, 1 : 20, 1 : 50 and 1 : 100. The LLOQs were set to the

Evaluation of preanalytical factors of estrone and 17β estradiol measurements

LC-MS/MS conditions
The reconstituted samples were analyzed by Advance
HPLC and EVOQ Elite triple quadrupole mass spectrome-

Effects of various preanalytical factors on the simultaneous measurement of estrone and 17- β estradiol wereter (Bruker Daltonics Inc., Fremont, CA, USA) using thefollowing conditions. The estrone and 17- β estradiol wereassessed. Except for the experiments to test the effects ofvarious clotting time, serum samples were obtained by cen-separated on a Shiseido core shell column (CAPCELL

trifugation 30 min after venipuncture. Except for the effects
of anti-coagulants or separating gel, serum samples were

CORE C18, 2.1 × 150 mm) maintained at 40°
C. The HPLC
ﬂow rate was 500 μL /min and the solvent composition of
the mobile phase was programmed to change in 10-min

collected by Insepack. II serum collection tubes (4S1001,

cycles with varying mixing ratios of solvent A (H2O con-the additive).

separating gel and silica, accelerating clotting agent used astaining 0.1% v/v HCOOH) to solvent B (CH3CN containing 0.1% v/v HCOOH): 50‒70% B 1 min, 70‒80% B 7 min,serum samples obtained from four healthy volunteers wereand 50% B 2 min. Samples were ionized using an electrospray ionization (ESI) source using the following condi-

left at 4°
C for 1, 6, 24, 72 and 168 h prior to measurement.
Freeze and thaw: To test the effects of freeze-thaw

tions: positive-ion mode; spray voltage, 5000 V; heatedcycles, serum samples were frozen at −80°
C in a deepprobe temperature, 450°
C; cone temperature, 300°
C.

freezer and then thawed at 37°
C. The samples underwent 1
to 5 freeze-thaw cycles.

Stability: To test stability of estrone and 17- β estradiol,

Selected reaction monitoring (SRM) transitions of analytes

were monitored: 504 → 171 for estrone, 507 → 171 for
13
estrone- C3, 506 → 171 for 17- β estradiol, 509 → 171 for

Effects of anti-coagulants and separating gels: To determine the effects of various anti-coagulants (EDTA, heparin,

17- β estradiol- C3. The collision energy of each analytes
13
was set at 38 V for estrone, 34 V for estrone- C3, 34 V for
13

sodium citrate), separating gels and accelerating clotting
agent (SiO2) on our LC-MS/MS based measurement of

13
17- β estradiol, 33 V for 17- β estradiol- C3. The scan time

estrone and 17- β estradiol, a total of six different blood col-was set at 50 msec for all analytes.

lection tubes were used; (I) plasma tubes (4H2011, Sekisui
21June 2019

Medical Mass Spectrometry Vol. 3 No. 1

1.7% and 0.96% for the high level samples (high), respectively. The inter-assay CVs for estrone and 17- β estradiol

Medical, sodium heparin (15 IU/mL)), (II) plasma tubes(4E4090, Sekisui Medical, dipotassium EDTA (1.5 mg/mL)), (III) plasma tubes (4E2034, Sekisui Medical, diso-

were 2.0% and 3.1% for the low, 5.5% and 5.1% for thedium EDTA (1.5 mg/mL)), (IV) serum tubes (4S1001,

medium and 1.5% and 1.1% for the high level samples,

Sekisui Medical, separating gel, silica and SiO2), (V) serum

respectively (Table 2). The results of the dilution linearity
tests for the estrone and 17- β estradiol are shown in Fig. 2a.

plain tubes (VP-P070K, Terumo, no additive) and (VI)
serum tubes (VP-AS076K, Terumo, separating gel, glass
particles and SiO2).

The correlation coefﬁcients at the all levels were more than
0.9993 and 0.9968 for estrone and 17- β estradiol, respec-

gated bilirubin, chyle and hemolyzed hemoglobin by using

surements of the medium level serum and BCR578 dilution
for estrone and 17- β estradiol, respectively. The LLOQs

the Interference Check A Plus (Sysmex Co., Hyogo, Japan)

were 6.2 and 7.3 pg/mL for estrone and 17- β estradiol,including (maximum ﬁnal concentration) 20.1 mg/dL offree bilirubin, 20.9 mg/dL of conjugated bilirubin, 1480 for-respectively (Fig. 2(b)).

mazin turbidity unit (FTU) for chyle and 520 mg/dL ofhemoglobin.

Effects of various preanalytical factors on measurement of estrone and 17-β estradiol by LC-MS/MS

Chemiluminescent immunoassay (CLIA) measurements for 17-β estradiol

the rest of the clinical samples just in case for possible
re-testing. The estrone and 17- β estradiol were found to be

Interferences: Potential interferences were tested by spiking to high level control serum with free bilirubin, conju-

tively. The LLOQ were determined by four replicate mea-

In the clinical laboratory, it is a common practice to save

The 17- β estradiol concentration of 60 female sera were
determined by the two representative commercial chemilu-

stable when left as serum samples up to 7 days at 4°
C (Fig.
3(a)).

minescent immunoassay (CLIA), ARCHITECT Estradiol II

Our results showed that repeated freeze-thaw cycles forkit (Abbott, Chiba, Japan) and ADVIA Centaur Chemilumi
E-estradiol-6 kit (Siemens, Tokyo, Japan) according to theup to 5 times had no signiﬁcant effect on the estrone and17- β estradiol serum concentrations, the CVs of the concen-

manufacturers instructions.

tration of estrone and 17- β estradiol for the 4 data points
were 1.9‒4.7% and 1.8‒3.3%, respectively (Fig. 3(b)).

Results

Previous study has shown that the gel in blood collection

Evaluation of the LC-MS/MS assays

tubes causes interference in one of the main testosterone
13)transitions . We investigated the effects of anti-coagulants

standards are shown in Fig. 1. The HPLC method provided
adequate separation of the estrogen, with 17- β estradiol

and separating gel on the estrone and 17- β estradiol assay

eluting at 5.36 min and estrone eluting at 6.20 min. Eachinternal standard was eluted at the same time as the stan-(Fig. 3(c)).

dard reference material. In addition, 17 α -estradiol was

17- β estradiol concentrations for any of the blood collection tubes. The potential interferences by hyperbilirubin-

The SRM chromatograms of estrone and 17- β estradiol

by examining a total of six different blood collection tubes
No signiﬁcant differences were noted in the estrone andeluted at 5.36 min and each transition did not interfere witheach other. The accuracy values of 17- β estradiol measure-emia, hemolysis and cylemia were tested using commer-

ment were 100.7‒100.9% (Table 1). The coefﬁcient of variation (CV) for the 17- β estradiol was lower than 4.5% at all

cially available interference test kits. No signiﬁcant effects
were noted as indicated in Fig. 3(d).

QC levels. The precision of this assay was determined byComparison of serum 17-β estradiol levels measured
by the in-house LC-MS/MS method and by two commercial CLIA kitsusing control sera containing low (21.3±1.39 and 12.5±
1.13 pg/mL), medium (52.4±2.68 and 33.2±2.10 pg/mL)
and high (124.6±2.33 and 223.0±3.03 pg/mL) level estrone
and 17- β estradiol concentrations, respectively. The
intra-assay CVs of estrone and 17- β estradiol measurements

Serum levels of E2 and E1 in 60 female subjects determined by the LC-MS/MS method were compared with

were 5.3% and 7.2% for the low level samples (low), 2.7%

those by the commercially available Abbott CLIA and Sie-and 6.4% for the medium level samples (medium) andmens CLIA.
22

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Medical Mass Spectrometry Vol. 3 No. 1

Fig. 1.

SRM chromatograms of estrone and 17- β estradiol and the ISs.

From the top panel to the bottom panel shows 504 → 171 for estrone, 507 → 171 for estrone- C3, 506 → 171 for 17- β estradiol and
13509 → 171 for 17- β estradiol- C3. The black peak is the target analyte peak. The gray peak is 17- α estradiol and can be separated
from all peaks (estrone, 17- β estradiol and ISs).
13

The ranges of the concentrations in 60 sera were 11‒872,
18‒959 and 7.6‒948.7 pg/mL as measured by the Abbott

were y (Siemens)＝1.1052x (Abbott)−0.0288 and 0.9880,
respectively. When the values obtained by the LC-MS/MS

CLIA kit, the Siemens CLIA kit and LC-MS/MS, respec-(x-axis) were compared with the values obtained by two
CLIA kits (y-axis), good correlations were obtained, but

tively. The linear regression line and correlation coefﬁcient23June 2019

Medical Mass Spectrometry Vol. 3 No. 1

y-intercepts were large (Fig. 4(a), (b)). In comparison with
the Abbott kit, the linear regression line and correlation

LC-MS/MS method and the two CLIA methods were seenin the same specimen for which the LC-MS/MS gave

coefﬁcient were y (Abbott)＝0.8597x (MS)＋15.652 and11.9 pg/mL: the values obtained by the Abbott CLIA and

0.9951, respectively. In comparison with the Siemens kit,

the Siemens CLIA were 34 and 48 pg/mL, respectively.

the linear regression line and correlation coefﬁcient werey (Siemens)＝0.959x (MS)＋15.258 and 0.9924, respecTable 2.

tively. Surprisingly, the biggest discrepancy between the
Accuracy of the LC-MS/MS assay for 17- β estradiol

Table 1.

Standard
BCR576

Expectedconc.
(pg/mL)31.5±1.36

AverageSD.
(pg/mL) (pg/mL)

CV.
(%)

Accuracy(%)

Intra-and Inter-day precisions of the LC-MS/MS
assay for estrone and 17- β estradiolConc.
(pg/mL)

CompoundEstrone17- β estradiol

Intra-day
CV (%)

Inter-day
CV (%)

Low

21.3±1.39

5.3

2.0

Medium

52.4±2.68

2.7

5.5

High

124.6±2.33

1.7

1.5

Low

12.5±1.13

7.2

3.1

30.84

1.08

4.5

100.7

BCR577 187.95±10.88 186.99

2.43

1.6

100.9

Medium

33.2±2.10

6.4

5.1

BCR578 365.00±19.04 367.56

2.56

1.3

100.8

High

223.0±3.03

0.96

1.1

Fig. 2.

Dilution linearity and LLOQ.

(a) Dilution linearity of estrone and 17- β estradiol. Dilution linearity was evaluated by measuring duplicates of 5 dilutions ofeach levels serum. (b) LLOQ of estrone and 17- β estradiol measurements. Diamonds and open circles indicate values obtained by
BCR578 dilution series and %CV at each dilution concentration, respectively. Arrowheads indicate LLOQ that meets our criteria.

24June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Fig. 3.

Effects of various preanalytical factors.

(a) Stability. (b) Freeze and thaw. (c) Effects of anti-coagulants and separating gels. (d) Interferences.

25June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Fig. 4. Linear regression analysis of the two commercial CLIA kits and in-house LC/MS/MS measurements of 17- β estradiol.

(a) The correlation between 17- β estradiol measured by CLIA and by LC/MS/MS. (b) Expanded view of low concentration in (a).
(c and d) Bland‒Altman plot of measurement value by Abbott CLIA kit and LC-MS/MS. (e and f) Bland‒Altman plot of measurement value by Siemens CLIA kit and LC-MS/MS. (d and f) Recalculated the bias with 50 pg/mL as the boundary.

LC-MS/MS-based estrogen measurements. Although PP
and LLE are simple and not costly, phospholipid, a repre-

Discussion
Here we described a simple, rapid and reproducible
LC-MS/MS method for the simultaneous assays of estrone

sentative cause of matrix effects, cannot be sufﬁciently

and 17- β estradiol considering applications into clinical lab-removed by PP , and automated preparation of microscaleoratory. We have adopted supported liquid extraction (SLE)for sample preparation and dansyl chloride as a derivatiza-

specimen is difﬁcult for PP and LLE because liquid phase14)

separation is needed. For the purpose of practical use of
LC-MS/MS, sample preparation requires to be fully auto-tion reagent.
Protein precipitation (PP), liquid‒liquid extraction

mated eventually, as is the case with currently available(LLE), solid phase extraction (SPE), or combinations ofautoanalyzers in clinical chemistry laboratories. SPE is one
candidate for automated preparation because various relat-

these methods, are generally used for sample preparation in26

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Medical Mass Spectrometry Vol. 3 No. 1

ed-products such as single cartridge and 96-well plate arepression is enough or not. For this purpose, highly sensitive

available. There is a limitation, however, because a lot ofprocesses such as washing column, equilibrating and wash-

method such as ultrasensitive gas chromatography tandem

ing after sample loading are necessary, which in turn willlead to low throughput. To overcome these problems, sev-

equivalent to the previous report, 200 to 500 μL of serum

eral sample preparation methods other than the three basic

method we described here requires only 100 μL of serum,

methods have been reported. Pucci et al. reported a method
using HybridSPE, in which PP and zirconia-coated silica

which is an advantage for pediatric use in which sample

20)

mass spectrometry has been used . Although LLOQ was
7,21)

samples had been used in the previous reports. The

volumes are limited.
Since our goal is to apply this method to real-world clinical laboratories, we determined the effects of various prean-15)

are packed for SPE to remove phospholipids . Geib et al.
reported a method using AC Extraction Plate, which was
16)
used as an immobilized sorptive material . Supported liq-

alytical factors on the LC/MS/MS measurements of serum

uid extraction (SLE) which we have used in Vitamin D
17)measurements is also another promising sample prepara-levels of E1 and E2. Indeed it was reported that analysis ofsera from blood collected in tubes containing clotting acti-tion method. With SLE, we can perform preparation byvators gave results of testosterone that were fourfold higher

using diatomaceous earth as ﬁller, and interferences can bethan that collected in plain tubes . In the present study, E1

reduced by efﬁciently removing phospholipids when theand E2 levels in samples obtained in a total of six different

13)

18)

extraction conditions were carefully optimized . In thisblood collection tubes were comparable.

study, we decided to use SLE because it may have thegreatest potential for automation of sample preparation

Serum E2 levels obtained by our LC/MS/MS method
were compared with those by the two representative immu-

because number of processing steps is small, execution

noassays available in Japan. Positive bias was noted fortime is short and also because amount of waste is small.

both immunoassays at low concentrations less than 50 pg/

Derivatization is still a key technology to improve ioniza17)
tion efﬁciency and sensitivity by ESI . The 17- β estradiol

mL. Similar bias was reported in the previous comparative
22,23). Crossstudies of immunoassays and GC-MS or LC-MS

is a well-known analyte that has low ionization efﬁciency at
ESI. For this reason, measurement of 17- β estradiol is gen-reactivity due to the limit of antibody speciﬁcity and matrix

erally derivatized mostly by dansyl chloride . Recently, 17-

Importantly, accuracies of the calibrators included in each

β estradiol measurement method without derivatization has
5‒7)
been reported using high-end model instruments . How-

immunoassay may have to be carefully evaluated as well.

ever, the sensitivities reported in these reports are compara-modest as for the purpose of evaluation of ovarian function,

ble to those obtained by the derivatization methods. Since
high-end models are expensive and require highly-trained

the conventional immunoassays can provide relevant

staff to handle, they are not suitable for routine use in clinical laboratories of standard-scale hospitals. Therefore, into be replaced by more speciﬁc and accurate LC/MS/MS

the present study we decided to use MS equipment that has

a representative journal in endocrinology . Antibodies may

been claimed to be suitable for use in clinical laboratory.

also be useful for the purpose of immunoafﬁnity extraction

The LLOQ obtained for E2 in the present study was
7.3 pg/mL and was comparable to those reported in the pre-in LC/MS/MS based measurements of steroid as werecently reported for 1,25-deihydroxyvitamin D quantiﬁca-. But the method we describe here is stilltion . This immunoafﬁnity extraction resulted in less back-not sensitive enough for some clinical situations such as

ground noise and less phospholipid effects compared to thedetermination in patients under aromatase inhibitor therapy.
Aromatase inhibitors inhibit aromatase mediated conver-conventional pretreatments .

sion of androgens, resulting in suppression of estrogen bio-

MS method for determination of serum levels of E1 and E2

synthesis. The third generation aromatase inhibitors such as

in clinical laboratories with sensitivities that can meet the
majority of clinical needs. Request of simultaneous mea-

24)

effects may be possible reasons for the discordant results .

4)vious reports

4‒7)

Under situations in which demands on assay sensitivity is

results. It is likely, however, that immunoassays are goingfor sex steroid measurements as proposed in the editorial of
5)

25)

25)

Thus, we developed a simple and reproducible LC/MS/

exemestane and letrozole are effective for the treatment of
19)
estrogen-receptor positive breast cancer . LLOQ as low assurements of E1 and E2 are going to be increased in theﬁeld of gynecological malignancy. Our LC-MS/MS method

0.1‒0.2 pg/mL is demanded to assess whether estrogen sup27

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

may be appropriate for this purpose. Worldwide, increasing

al: Simultaneous measurement of total estradiol and tes-

number of laboratories is measuring E1 and E2 by theirhome-brew LC/MS/MS methods. Signiﬁcant interlabora-

tosterone in human serum by isotope dilution liquid chro-

tory variability has been recognized even among LC-MS

Chem 409: 5943‒5954, 2017.

matography tandem mass spectrometry. Anal Bioanal

26)

E2 assay . Harmonization of the different LC/MS/MS

8) Handelsman DJ, Wartofsky L: Requirement for mass

methods will be needed to make MS-based steroid assays

spectrometry sex steroid assays in the Journal of Clinicalmore reliable and successful.

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Acknowledgments

9) Henderson BE, Feigelson HS: Hormonal carcinogenesis.
Carcinogenesis 21: 427‒433, 2000.

This research was partially supported by the Ministry of
Education, Culture, Science, Sports and Science and Tech-

10) Lukanova A, Kaaks R: Endogenous hormones and ovari-

nology (MEXT) by a Grant-in-Aid for Scientiﬁc Research(C), 2015‒2018, 15K08610 (M. Satoh).

an cancer: Epidemiology and current hypotheses. Cancer
Epidemiol Biomarkers Prev 14: 98‒107, 2005.
11) Trabert B, Brinton LA, Anderson GL, Pfeiffer RM, Falk

Conﬂict of Interest

RT, et al: Circulating estrogens and postmenopausal ovarian cancer risk in the women s health initiative observa-

All authors declare that they have no conﬂict of interest.

tional study. Cancer Epidemiol Biomarkers Prev 25: 648‒

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