Analysis of multiplex endogenous estrogen

Analytica Chimica Acta 711 (2012) 60–68
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Analytica Chimica
Acta
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a c
a
Analysis of multiplex endogenous estrogen metabolites in human urine using ultra-fast liquid chromatography–tandem mass spectrometry:A case study for breast cancer
Jiang Huang a ,Jianghao Sun a ,Yanhua Chen a ,Yongmei Song b ,Lijia Dong b ,Qinmin Zhan b ,Ruiping Zhang a ,∗,Zeper Abliz a ,∗
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China b
State Key Laboratory of Molecular Oncology,Cancer Institute and Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China
a r t i c l e
i n f o
Article history:
Received 7August 2011
Received in revised form 12October 2011Accepted 27October 2011
Available online 7 November 2011
Keywords:
Estrogen metabolite Breast cancer Urine
Ultra-fast liquid chromatography–tandem mass spectrometry Metabolic profiling
a b s t r a c t
A rapid,sensitive,specific and accurate analytical method of ultra-fast liquid chromatography combined with tandem mass spectrometry (UFLC–MS/MS)was established for simultaneous quantitative analysis of 16distinct endogenous estrogens and their metabolites (EMs)in postmenopausal female urine.The quantitative method utilized a hydrolysis/extraction/derivatization step and a UFLC system to achieve separation in 16min.The lower limit of quantitation for each estrogen metabolite was 2pg mL −1with the percent recovery of a known added amount of estrogen at 93.2–109.3%.The intra-batch accuracy and precision for all analytes were 87.5–107.7%and 0.6–11.7%,respectively,while inter-batch accuracy and precision were 87.0–105.8%and 1.2–10.2%,ing this developed and validated method,the comprehensive metabolic profiling of 16EMs in urine samples of 86postmenopausal female breast cancer patients and 36healthy controls was investigated by systematic statistical analysis.As a result,the circulating levels of 6EMs were found to be different by a comparison of patients and healthy controls.The parent estrogens,estrone (E1)and 17␤-estradiol (E2),as well as 2-hydroxyestradiol (2-OHE2)and 4-hydroxyestradiol (4-OHE2)were produced in higher abundance,whereas 16␣-hydroxyestrone (16␣-OHE1)and 2-methoxyestradiol (2-MeOE2)were decreased in the breast cancer group.2-OHE2and 4-OHE2in particular showed significant elevation in patients,which are consistent with the carcinogenic mechanism hypothesis that catechol estrogens can react with DNA via quinones,resulting in mutations to induce breast cancer.Thus,2,4-hydroxylation may be the dominant metabolic pathway for parent estrogens rather than 16␣-hydroxylation.The lower level of 2-MeOE2in the breast cancer group was believed to correlate with its protective effect against tumor formation.This study could provide valuable information on the association of the EM metabolic pathway with carcinogenesis as well as identify potential biomarkers for estrogen-induced breast cancer risk.
© 2011 Elsevier B.V. All rights reserved.
1.Introduction
Exposure to endogenous estrogens and their metabolites (EMs)has been believed to be associated with the development and growth of breast cancer [1]for more than a century,ever since it was reported that oophorectomy resulted in the remission of breast cancer in women [2].While the general importance of estro-gen metabolites as risk factors is clear,the mechanisms for their carcinogenic effects have not been firmly established and certain aspects remain controversial.Two types of general mechanisms are
∗Corresponding authors.Tel.:+861063165218;fax:+861063165218.
E-mail addresses:rpzhang@ (R.Zhang),zeper@ (Z.Abliz).
usually considered for how estrogens contribute to breast cancer.The first is that elevated levels of some estrogens directly or indi-rectly increase the rate of genotoxic cell proliferation by stimulating estrogen receptor-mediated transcription and thereby increase the number of errors occurring during DNA replication [3–6].An alter-native hypothesis is that the generation of quinone derivatives of some specific estrogen metabolites,such as the reactive catechol estrogens,can react with DNA to form predominantly stable and depurinating adducts [7–9],resulting in oncogenic mutations to induce breast cancer [10].
The absolute levels and patterns of EMs in the body may play a critical role in the etiology of breast cancer.Estrone (E1)and 17␤-estradiol (E2),which are the parent estrogens of the metabolic pathways,mainly induce tumors by stimulation of aberrant breast
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J.Huang et al./Analytica Chimica Acta711 (2012) 60–6861
epithelial cell proliferation or formation of genotoxic catechol estrogen-quinones[4,9].Meanwhile,higher concentrations of2-hydroxyestradiol(2-OHE2)and4-hydroxyestradiol(4-OHE2)were found in cancer cases and interpreted as a higher potential risk for quinone formation to react with DNA through inappropriate alternations in the regulation of gene expression[3,4,11].
When it comes to the role of16␣-hydroxyestrone(16␣-OHE1)in cancer,there is no universal agreement.It was reported that women who metabolize a larger proportion of endogenous estrogen through the16␣-hydroxylation pathway may be highly correlated with the growth of tumors[12–14].On the other hand, in several studies,the extent of16␣-OHE1was not linked to higher risk of breast cancer[15–17].Furthermore,substantial evi-dence has demonstrated that the formation of catechol estrogens attribute more to DNA damage than16␣-hydroxylation of estro-gens[18].Moreover,the roles of2-hydroxyestrone(2-OHE1)and 4-hydroxyestrone(4-OHE1)in cancer development,which were assumed to be“good estrogen”[19,20]through the inhibition of cell proliferation or potential genotoxic factors for breast car-cinogenesis,remain unclear[12,13].Therefore,it is important to appropriately profile estrogens and their metabolites to provide valuable information in elucidating the mechanism of hormone carcinogenesis and for further application in thefields of early diagnosis,screening,prevention and treatment of breast cancer.
To the best of our knowledge,a number of methods have already been applied and developed to provide specific descriptive and quantitative information concerning each EM,including radio immunoassay(RIA)[21,22],enzyme immunoassay(EIA)[23,24], high performance liquid chromatography with electrochemical detection(HPLC-ECD)[25,26],gas chromatography–mass spec-trometry(GC–MS)[27,28]and HPLC–MS2[29–31].Owing to its high sensitivity,specificity,accuracy,and reproducibility,multi-ple reaction monitoring(MRM)has been developed as the method of choice for quantitative and qualitative determination of EMs in human serum[32,33]and of estrogen sulfates in human urine [34,35].
A total of15EMs in urine[30]and serum[31]from pre-menopausal and post-menopausal women were analyzed by HPLC–MS/MS in75min to gain insight into the possible mechanisms for estrogen-related breast carcinogenesis.Recently, simultaneous quantitative analysis of12EMs in35min was also reported[36].However,the long chromatographic run was inappropriate for large-scale studies.Because of their trace concen-trations,similar structures and complex sample matrices,analysis of these compounds is quite challenging in terms of sensitivity and selectivity.Ultra performance liquid chromatography(UPLC)[37], ultra-fast liquid chromatography(UFLC)and rapid resolution liquid chromatography(RRLC)[38,39]provide high throughput,excel-lent chromatography resolution and high sensitivity,which have made them potential methods for the entire profiling of estrogen metabolites.
In this study,we present a UFLC–MS/MS method that requires only simple hydrolysis,extraction and derivatization and only 0.5mL of urine,yet is capable of accurately and precisely measur-ing16endogenous estrogens and their metabolites in urine within 16min.Due to the low volatility and polar nature of estrogen,rapid derivatization by dansyl chloride[29,30]was applied for ultra trace analysis and greatly improved the MS detection sensitivity as much as1000-fold[40,41].
It is known that a90%decrease in estrogen excretion in post-menopausal women due to gradual atrophy of ovaries[42,43], leading to a more stable EM profile in postmenopausal women on the cumulative lifetime[1].On the contrary,in premenopausal women,the total exposure to estrogen is strongly affected by a variety of ing the developed UFLC–MS/MS method,we assayed the concentration levels of16EMs in86postmenopausal female urine samples to explore perturbations in the metabolic pathway of EMs and to discover potential biomarkers for estrogen-induced breast cancer initiation.
2.Experimental
2.1.Chemicals
Sixteen estrogens and estrogen metabolite(EM)standards are shown in Fig.1.Among these,E1,E2,16␣-OHE1,4-OHE1,2-OHE2,4-OHE2,estriol(E3)and16-epiestriol(16-epiE3) were obtained from Sigma–Aldrich(St.Louis,MO),and2-OHE1,17-epiestriol(17-epiE3),16-ketoestradiol(16-ketoE2), 2-methoxyestradiol(2-MeOE2),4-methoxyestradiol(4-MeOE2), 2-methoxyestrone(2-MeOE1),4-methoxyestrone(4-MeOE1),and 2-hydroxyestrone-3-methyl ether(3-MeOE1)were obtained from Steraloids,Inc.(Newport,RI).␤-Glucuronidase/sulfatase from Helix pomatia(Type H-2)was obtained from Sigma–Aldrich.Dansyl chlo-ride(reagent grade)was obtained from Alfa Aesar(MA,USA).The internal standard(IS),tanshinone IIA,was obtained from National Institutes for Food and Drug Control(Beijing,China).Acetonitrile (HPLC grade)and formic acid were obtained from Merck(Darm-stadt,Germany),methanol(HPLC grade)was obtained from J&K Chemca,and ammonium acetate(HPLC grade)was obtained from Fluka(Netherlands).Acetone was obtained from Beijing Chemical Works and sodium bicarbonate and l-ascorbic acid were obtained from Sinopharm Chemical Reagent Co.,Ltd.
2.2.Sample collection
The breast cancer group(BC group)consisted of86post-menopausal females with a diagnosis of primary breast cancer confirmed at the Cancer Institute of the Chinese Academy of Medi-cal Sciences(Beijing,China)who were enrolled in this study.All of these subjects were newly diagnosed and had not been on estro-gen containing treatments during the sampling period.Thirty-six healthy Chinese female volunteers were selected for the control group(NC group),individually matched by age.The average ages for the BC and NC groups were59.0±7.1and59.5±7.9years old, respectively.Other criteria for exclusion among subjects included currently pregnant or lactating as well as taking oral contracep-tives or exogenous hormones[44].Each subject was involved under Informed Consent according to the Guidelines of the Hospital Ethics Committee and approved by corresponding regulatory agen-cies.Morning spot urine samples were collected with appropriate instruction and immediately stored at−80◦C prior to analysis.Cre-atinine analysis was carried out by the Inspection Department of the Cancer Institute and Hospital of the Chinese Academy of Med-ical Sciences using an enzymatic procedure with0.5mL of urine.
2.3.Preparation of standard solutions and QC samples
Stock solutions of EMs were each prepared at100␮g mL−1by dissolving1mg of each compound in methanol containing0.1% (w/v)l-ascorbic acid to afinal volume of10mL.Working standard solutions in the presence of each estrogen at1␮g mL−1were freshly prepared by mixture of a50␮L aliquot of each stock solution with acetonitrile/water(60:40,v/v)containing0.1%(w/v)l-ascorbic acid [32].Estrogen-free urine with no detectable level of any EMs was prepared by vortexing human urine with activated charcoal and was employed for the preparation of calibration standards and quality control(QC)samples.Serial dilution of EM working stan-dard solutions spiked into0.5mL estrogen-free urine generated calibration standards in the range from2pg mL−1to20ng mL−1. These calibration samples were prepared with10␮L of tanshi-none IIA(50ng mL−1in methanol)as an internal standard and were
62J.Huang et al./Analytica Chimica Acta 711 (2012) 60–68
E1
E2
16α-OHE1
16-ketoE2
E3
2-MeOE1
2-OHE24-OHE2
3-MeOE12-OMeE24-
OMeE2
Tan shinone A
(IS)
H 3H 3CO
H 3OCH 3
Fig.1.Chemical structures of 16endogenous estrogens,their metabolites and the internal standard (IS).
assayed in duplicate.QC samples were prepared under the above-mentioned procedure at three levels (5pg mL −1(QC L),0.2ng mL −1
(QC M),and 5ng mL −1(QC H))of each EM.All solutions were stored at −20◦C until use.
2.4.Sample preparation
Sample preparation,including hydrolysis,extraction and derivatization,was adapted from a method previously described [30].Since endogenous EMs are mainly excreted as glucuronide and sulfate conjugates in urine with a small amount of free estrogen at pg mL −1levels,enzymatic hydrolysis with ␤-glucuronidase/sulfatase was applied to measure the total urinary EMs.All urine samples were first thawed at 4◦C and centrifuged at 10,000×g at 4◦C for 10min.Next,a 0.5mL aliquot of super-natant was collected,followed by the addition of 0.5mL of freshly prepared enzymatic hydrolysis buffer containing 5␮L of ␤-glucuronidase/sulfatase,2.5mg l -ascorbic acid,and 0.5mL of 0.15M sodium acetate buffer (pH 4.6)in the presence of 10␮L tan-shinone IIA in methanol (50ng mL −1).These reactions were then incubated for 16h at 37◦C.
For cleanup and trace level enrichment,a solid-phase extrac-tion (SPE)approach was selected.After hydrolysis,samples were loaded onto a SPE cartridge (GracePure,C 18-max,100mg/1mL),which was activated,purified and preconditioned with 3mL
acetonitrile and 3mL purified water,and eluted with 2mL ace-tonitrile.The extracted fraction was evaporated to dryness under a stream of nitrogen at 60◦C.The residue was re-dissolved in 100␮L of sodium bicarbonate buffer (0.1M,pH 9)and 100␮L of dansyl chloride in acetone (1mg mL −1).After vortex mixing for 45s,sam-ples were incubated at 70◦C for 9min to form the dansyl derivatives for UFLC–ESI-MS/MS analysis.Calibration standards,QC samples and urine samples were all prepared in the same way.
2.5.UFLC–MS/MS analysis
UFLC–MS/MS analysis was carried out on a Shimadzu UFLC-XR system (Shimadzu Corp.,Kyoto,Japan)coupled to a 5500QTRAP system (Applied Biosystems/MDS Sciex,Toronto,Canada).An Extend-C 18reversed-phase column (1.8␮m,100mm ×2.1mm,Agilent,USA)with an online filter was used for chromatographic separation,and the column was maintained at 50◦C.The compart-ment of the autosampler was set to 4◦C.A 10␮L pretreated sample was injected onto the column and eluted with a linear gradient at a flow rate of 300␮L min −1,where solvent A was 0.1%(v/v)formic acid in water and solvent B was acetonitrile,both adjusted with ammonium acetate (0.2mM,pH 6.80).The gradient started with 60%B and linearly increased to 90%B over 7min,then was held for 3min followed by re-conditioning to the initial conditions over
J.Huang et al./Analytica Chimica Acta711 (2012) 60–6863 Table1
MRM conditions for the derivatized EMs and the internal standard.
Analyte Precursor ion
(m/z)Product ion
(m/z)
DP(V)CE(eV)
E1504.2171.17045
E2506.2171.17048
16␣-OHE1520.2171.17545
16-ketoE2520.2171.17545
E3522.2171.17545
16-epiE3522.2171.17545
17-epiE3522.2171.17545
2-MeOE1534.2171.17545
3-MeOE1534.2171.17545
4-MeOE1534.2171.17545
2-MeOE2536.2171.17545
4-MeOE2536.2171.17545
2-OHE1753.3170.18550
4-OHE1753.3170.18550
2-OHE2755.3170.18550
4-OHE2755.3170.18550 Tanshinone IIA295.2277.212028
0.5min and re-equilibration with60%B for5.5min prior to the next injection.
For MS analysis,a5500QTRAP system equipped with a Turbo ion spray source was employed.The dansyl derivatives of the estrogen metabolites were detected by positive ESI UFLC–MS/MS in multi-ple reaction monitoring(MRM)mode.The compound-dependent MRM conditions for EM-dansyl,including declustering potential (DP)and collision energy(CE),are summarized in Table1.Other source-dependent parameters were optimized as follows:ion spray voltage at5.5kV;vaporizer temperature at650◦C;and nebulizing gas at65psi,drying gas at50psi,curtain gas at20psi and colli-sion gas at5psi.Nitrogen gas was used as both the nebulizing and drying gas.Data acquisition and processing were performed with Analyst1.5software.
3.Results and discussion
3.1.Method development
Optimization of MS detection was performed by direct infusion of standard solutions with IS at a concentration of20ng mL−1and aflow rate of10␮L min−1.The tetracyclic compound tanshinone IIA was proved to be an appropriate internal standard.It has con-sistent extraction recovery and similar retention behavior to the dansylated EMs.The transition ion of tanshinone IIA,295.2/277.2 was tracked,corresponding to the loss of a water molecule.The transition ions of the dansylated EMs were selected based on the highest intensities of the ions obtained in the ESI-MS/MS spec-tra.12estrogens involving one phenolic hydroxyl group react with one dansyl chloride,which generated m/z171fragments[31]with higher intensities.Other four estrogens involving two phenolic hydroxyl groups,such as2-OHE1,4-OHE1,2-OHE2and4-OHE2, react with two dansyl chloride,which produced m/z170ions with higher intensities.It could be speculated that the different distri-bution of charge induced homolytic and heterolytic cleavage of the dansyl moiety to produce m/z171radical ion and m/z170ion, respectively.
To obtain both reproducible separation and high sensitivity for EMs and IS within a reasonable separation time,UFLC conditions were optimized.As a result,a mobile phase consisting of ace-tonitrile and0.1%(v/v)formic acid containing0.2mM ammonium acetate was applied to improve the separation.A representative MRM chromatographic profile by UFLC–MS/MS is shown in Fig.2. It should be noted that interferences were observed even though SPE had been used for extensive cleanup and MRM was used as a selective detection method.Table2
Typical calibration curves obtained by UFLC–MS/MS.
Analyte Equations Correlation
coefficient(R2)
LOQ
(pg mL−1)
E1y=13.1x+0.7840.99972
E2y=7.67x+0.6930.99942
16␣-OHE1y=7.19x+0.5430.99982
16-ketoE2y=8.02x+0.8330.99922
E3y=10.8x+0.2790.99982
16-epiE3y=10.3x+0.06250.99992
17-epiE3y=16.7x+0.1260.99962
2-MeOE1y=2.86x+0.0600.99982
3-MeOE1y=4.83x+0.04490.99902
4-MeOE1y=2.64x+0.0590.99982
2-MeOE2y=2.48x+0.05590.99952
4-MeOE2y=4.7x+0.0450.99962
2/4-OHE1y=11.1x+0.01430.99962
2-OHE2y=2.46x+0.03690.99942
4-OHE2y=3.4x+0.06490.99852
All16EMs were fully baseline-resolved except for2-OHE1and 4-OHE1,whose very similar structures led to extremely close reten-tion times:9.95min(2-OHE1)and9.93min(4-OHE1).Changing gradient parameters had little effect on the retention behavior of 2-OHE1and4-OHE1.Although all15EMs were baseline-resolved as previously reported[29–31],75min was required for each run. Since its application in epidemiology was taken into consideration, the successful simultaneous quantification of16dansylated deriva-tives of EMs within16min was considered a major improvement. However,2-OHE1and4-OHE1had to be considered as one sig-nal in this method(expressed as2/4-OHE1).Therefore,the current method is more competitive for the rapid metabolic profiling of EMs in a large number of urine samples.
3.2.Validation of the developed method
3.2.1.Linearity and carryover
Estrogen-free urine spiked with IS and EM standards at9 concentration levels were treated with the above-mentioned extraction and derivatization in duplicate to establish calibration curves.Calibration curves for each EM were constructed from the ratios of the peak area of the dansylated EMs to that of the IS against the analyte concentrations andfitted by linear regression with1/X weighting.Satisfactory linearity was observed over a wide linear range from2pg mL−1to20ng mL−1for each dansylated EMs, and the linear correlation coefficients were all better than0.99. The lower limit of quantification(LLOQ)was defined as the lowest concentration on the calibration curve that could be reliably and reproducibly measured with accuracy and precision of less than 20%(relative standard deviation,RSD)and a signal-to-noise ratio greater than10.
The LLOQ of all16EMs was2pg mL−1for this method.Charcoal-stripped urine from six different healthy females was employed as a blank control to estimate carryover with no detection of dansylated EMs and IS.This suggested that any chromatographic interference from carryover around the retention times of EMs and IS was neg-ligible,which improved the LLOQ.The calibration curves(Table2) were also used to measure specific analytes in the real urine sam-ples accurately with adequate sensitivity.
3.2.2.Precision and accuracy
The precision and accuracy of the method were assessed by performing replicate analysis of samples at four concentration lev-els of QC and LLOQ for each EM.Accuracy was determined as the percentage recovery of a known amount of EMs spiked into estrogen-free urine;intra-batch and inter-batch precisions were both measured by the percentage relative standard deviation(RSD).
64J.Huang et al./Analytica Chimica Acta 711 (2012) 60–68
50%100 %50%
100%50%100
%50%
100 %50%100
%50%
100 %50%
100
%a
b
50%100
%1412108
6
4
Time ( min)
16
50%
100%50%100
%50%
100%50%
100
%50%100%50%100%50%100%50%100%4
6
8
10
12
14Time( min)
E1
E2
E3
16-epi E3
17-epi E3
3-M
eOE14-M eOE1
2-M
eOE12/4-OHE1
4-OHE2
2-OHE22-M eOE2
4-M
eOE216-ketoE2
16α-OH E1
16
Fig.2.(a)MRM profiles of derivatized estrogens and their metabolites by UFLC–MS/MS in estrogen-free human urine sample.(b)MRM profiles of derivatized estrogens and
their metabolites by UFLC–MS/MS in estrogen-free urine spiked with EM standards.
Intra-batch precision was based on replicates of six analyses,and inter-batch precision was measured on the basis of two repli-cates in each of four batches.The upper limits of the intra-batch and inter-batch samples were considered acceptable when less than 15%,with the exception of the LLOQ,which was fixed at 20%according to the FDA guidelines [46].Tables 3and 4summa-rize the results:for the three levels,5pg mL −1(QC L),0.2ng mL −1(QC M),and 5ng mL −1(QC H),the intra-batch accuracies were 87.5–101.1%,92.5–107.7%,and 90.8–106.8%;the intra-batch
precisions were 2.3–11.7%,1.5–11.2%,and 0.6–9.5%;the inter-batch accuracies were 87.0–101.5%,89.8–105.8%,and 89.1–104.9%;and the inter-batch precisions were 2.1–10.2%,2.3–6.7%,and 1.2–4.9%,respectively.
For the LLOQ,the accuracy was ranged from 89.0%to 98.1%,and the precision was ranged from 0.6%to 6.4%.These results demonstrated that the accuracy and precision were within gen-erally acceptable limits and that the quantification of the current method was reproducible and accurate.
J.Huang et al./Analytica Chimica Acta711 (2012) 60–6865 Table3
Accuracy and intra-batch precision for measurement of dansylated EMs in urine(n=6).
Analyte QC low QC medium QC high
Accuracy(%)a Precision(%)b Accuracy(%)a Precision(%)b Accuracy(%)a Precision(%)b
E187.5 3.3101.2 1.595.60.6
E294.1 3.9103.0 4.098.4 5.1
16␣-OHE191.8 5.395.411.299.29.5
16-ketoE297.4 6.096.2 4.199.4 1.4
E392.8999.4 2.895.9 2.9
16-epiE390.37.996.4 4.4105.9 3.3
17-epiE399.1 4.792.5 4.5106.8 1.7
2-MeOE194.78.2101.67.591.0 1.9
3-MeOE1101.1 2.3105.8 2.090.8 1.0
4-MeOE196.1 6.7105.1 6.298.4 1.9
2-MeOE292.0 5.596.0 6.091.6 1.2
4-MeOE294.68.396.1391.3 3.8
2/4-OHE192.511.7105.8 2.094.0 3.7
2-OHE294.5 6.5107.77.094.1 1.4
4-OHE291.6 4.993.77.694.8 1.2
a Accuracy was measured as the percentage recovery of a known amount of EMs spiked into estrogen-free urine.
b Precision was measured by the percentage relative standard deviation(RSD).
3.2.3.Matrix effect and recovery
Matrix suppression or enhancement was evaluated by compar-ing the peak area of dansylated EMs spiked into estrogen-free urine with that of EM standards prepared in methanol.The two sets were treated identically in the hydrolysis,extraction and derivatization procedure described above.No significant MS response change was observed within the retention time window of the16EM derivatives.For three concentrations of QC level,the ratios of peak response were within the following acceptable ranges:85.4–92.7% for QC L,85.7–94.1%for QC M and85.2–90.3%for QC H.Addition-ally,the relative standard deviations(RSDs)were all less than12.5%. The results indicated that a negligible matrix effect occurred when using this method.
The relative recoveries of EMs were investigated through mea-sured concentrations of three replicates of the three QC level samples,which were calculated from the biological calibration curves.The recoveries of each EM after the above sample prepa-ration were obtained by dividing the average measured value of spiked EMs by the nominal value that was added.As a result,the relative recoveries ranged from95.8%to109.3%for QC L,from 93.2%to105.8%for QC M,and from94.7%to108.5%for QC H.The relative standard deviations(RSDs)were0.6–10.2%,1.2–7.8%,and 0.8–10.9%.3.2.4.Stability
Sample stability was evaluated by analysis of the QC samples at three levels for intra-batch stability and inter-batch stability and determined by the percentage relative standard deviation(RSD). The analytes were considered stable if the value deviated less than 15%from the expected concentration of freshly prepared samples. The results were as follows:2.2–7.7%at QC L level,0.8–7.9%at QC M level,and1.2–3.0%at QC H level for intra-batch stability;and 2.1–10.2%,2.3–6.7%,and1.2–4.9%at three levels for inter-batch stability.Since all urine samples were run over four days,the dan-sylated products were considered to be stable during this analysis period.
3.3.Metabolic profiling of urinary EMs by UFLC–MS/MS
The developed method was applied to the metabolic profiling of EMs in real clinical samples;86breast cancer patients and36 healthy controls were recruited within this study(see Section2.2). Briefly,0.5mL aliquots from each urine sample were hydrolyzed, extracted,derivatized,and analyzed to determine target deriva-tized EM concentrations.The identification of target EMs was achieved by comparing the chromatographic retention times and MS/MS spectra of the samples with those of reference standards.
Table4
Accuracy and inter-batch precision for measurement of dansylated EMs in urine(n=8).
Analyte QC low QC medium QC high
Accuracy(%)a Precision(%)b Accuracy(%)a Precision(%)b Accuracy(%)a Precision(%)b
E191.1 4.497.7 3.790.1 4.9
E288.2 3.3100.5 5.199.4 4.1
16␣-OHE190.210.098.2 5.4103.0 3.6
16-ketoE296.0 5.495.8 3.699.3 1.6
E387.0 3.8100.1 3.996.5 2.2
16-epiE390.3 6.994.3 6.4104.9 3.2
17-epiE391.7 6.289.8 3.8104.6 3.0
2-MeOE195.5 6.6100.2 4.789.1 3.0
3-MeOE1101.5 2.1104.0 3.690.4 1.2
4-MeOE194.8 5.9105.8 5.698.7 1.9
2-MeOE289.5 6.097.0 5.490.8 1.8
4-MeOE290.98.295.5 2.390.3 4.2
2/4-OHE191.410.2103.1 4.694.0 3.2
2-OHE293.8 5.7103.6 5.393.7 1.4
4-OHE290.4 5.192.9 6.793.7 3.2
a Accuracy was measured as the percentage recovery of a known amount of EMs spiked into estrogen-free urine.
b Precision was measured by the percentage relative standard deviation(RSD).
66J.Huang et al./Analytica Chimica Acta 711 (2012) 60–68
30%
100
50%
3.0%2.0%
Time( min)
Fig.3.MRM profiles of derivatized estrogens and their metabolites by UFLC–MS/MS in postmenopausal urine samples.
Fig.3shows typical MRM chromatographic profiles of derivatized
EMs from UFLC–MS/MS on urine samples from postmenopausal women.The isotopic peak interferences for closely eluted EMs were investigated.Except the isotopic peak interference of 16␣-OHE1on 16epiE3,the case was different from other compounds due to con-siderable chromatographic separation with each other.After the overlapping of isotopic peak was corrected,the absolute levels of 16individual dansylated EMs were then adjusted in accordance with creatinine (5000␮mol L −1)and evaluated by statistical anal-ysis (Table 5).
Fig.4shows the creatinine-normalized data presented as the mean ±SD.The statistical significance of the difference in these
Table 5
Urinary endogenous EMs excretion in postmenopausal healthy female (H-post)and breast cancer patients (BC-post).Data were calculated in accordance with creati-nine concentration of 5000␮mol L −1in every urine sample and shown as mean and standard error.
Analyte
Mean ±SD (g ␮mol −1of creatinine)
p value
H-post
BC-post
E10.030±0.0220.047±0.0540.037E2
0.027±0.0310.047±0.0450.05516␣-OHE10.106±0.1290.056±0.0530.04116-ketoE20.149±0.1760.136±0.1370.779E3
0.041±0.0350.024±0.0190.08016-epiE30.033±0.0280.023±0.0260.32917-epiE30.231±0.1460.241±0.2540.9222-MeOE10.208±0.1480.173±0.1690.2213-MeOE10.032±0.0370.027±0.0210.3674-MeOE1NA
NA
NA 2-MeOE20.019±0.0220.006±0.0060.0384-MeOE20.004±0.0060.004±0.0030.4802/4-OHE10.007±0.0060.009±0.0090.3722-OHE20.018±0.0140.036±0.0400.0374-OHE2
0.032±0.034
0.063±0.052
0.048
analytes between the breast cancer group and healthy controls was carried out by a paired t -test.All p -values are from two-sided tests.Overall,the levels for E1,E2,2-OHE2,4-OHE2,16␣-OHE1and 2-MeOE2were significantly different between the two groups.The breast cancer patients excreted greater amounts of E1,E2,2-OHE2and 4-OHE2,but lesser amounts of 16␣-OHE1and 2-MeOE2com-pared with healthy females.Due to the lack of detection in both groups,4-MEOE1was excluded for statistical analysis.Other EMs were found at levels not deemed to be significant.These interest-ing results suggested that the six discrepant EMs might be highly associated with breast cancer risk.
The parent estrogens (E1and E2)were at higher levels in breast cancer patients.It has been previously determined that E1is the main circulating unconjugated estrogen in postmenopausal women [13]and is readily converted into E2at the C17posi-tion,which is a reversible process [47].E2is thought to be a key estrogen in a dual mechanism where E2can act either as a hor-mone to stimulate aberrant epithelial cell proliferation or as the precursor to the formation of genotoxic hydroxylated metabo-lites,such as catechol estrogens [4,9].Therefore,increased levels of E1and E2synergistically lead to the appearance of spontaneous mutations that subsequently increase breast cancer risk [11].In addition,humans excrete great amount of estrogen metabolites from the parent estrogens through three hydroxylation pathways [45,48].The 2-hydroxylation pathway includes 2-OHE1,2-OHE2,2-MeOE1,2-MeOE2and 3-MeOE1;the 4-hydroxylation pathway includes 4-OHE1,4-OHE2,4-MeOE1and 4-MeOE2;and the 16␣-hydroxylation pathway includes 16␣-OHE1,E3,16-epiE3,17-epiE3and 16-ketoE2.
As expected,2-OHE2and 4-OHE2,indirectly formed from the parent EMs by the catechol hydroxylation pathway,were present in elevated amounts in females with breast cancer.This observation was in accordance with the main mechanism of carcinogene-sis [3,8].These two compounds are primarily metabolized to。