Evaluation of an Isotope Dilution HPLC Tandem Mass Spectrometry

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Article

Evaluation of an isotope dilution HPLC tandem mass spectrometry candidate reference measurement procedure for total 17-#estradiol in human serum Julianne Cook Botelho, Ashley Ribera, Hans C Cooper, and Hubert W. Vesper Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03220 • Publication Date (Web): 16 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Analytical Chemistry

Evaluation of an isotope dilution HPLC tandem mass spectrometry candidate reference measurement procedure for total 17-β β estradiol in human serum Julianne Cook Botelho1, Ashley Ribera1, Hans C Cooper1, and Hubert W Vesper1*

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Health, Division of Laboratory Sciences, Clinical Chemistry Branch, 4770 Buford Hwy

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NE, Atlanta, Georgia, USA

Centers For Disease Control And Prevention, National Center For Environmental

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*Send correspondence to this author: Hubert W Vesper, Centers for Disease Control and

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Prevention, 4770 Buford HWY NE, MS-F25, Atlanta, Georgia 30341, Fax (770) 488-

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7030, E-mail: [email protected]

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Disclaimer: The findings and conclusions in this report are those of the author(s) and do

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not necessarily represent the views of the Centers for Disease Control and Prevention

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Key words: Reference measurement procedure, total estradiol, hormone standardization

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1 ACS Paragon Plus Environment


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Abstract

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The inaccuracy of 17-β estradiol (E2) measurements affects its use as a biomarker in

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patient care and research. Clinical and research communities called for accurate and

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standardized E2 measurements. Reference Measurement Procedures (RMPs), part of the

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CDC Hormone Standardization Program (HoSt), are essential in addressing this need and

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ensuring that methods are accurate and comparable across testing systems, laboratories,

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and over time.

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A candidate RMP (cRMP) was developed for the measurement of total E2 in serum using

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liquid chromatography tandem mass spectrometry (LC-MS/MS) without derivatization.

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The cRMP meets suggested performance criteria for accuracy and precision through the

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use of isotope dilution, calibrator bracketing, and gravimetric measurements. The cRMP

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demonstrated high agreement with certified reference materials (no significant bias to

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BCR576, 577, and 578) and established RMPs (slope 1.00, 95% CI 1.00 to 1.01; intercept

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0.02, 95%CI -0.01 to 0.06). The cRMP is highly precise with intra-assay, inter-assay, and

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total percent CVs of 2.7%, 1.3%, and 2.4%, respectively. A higher specificity was

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achieved by measuring E2 without derivatization, compared to methods using

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derivatization agents.

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The

cRMP can serve as a higher-order standard for establishing measurement

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traceability and provides an accuracy base against which routine methods can be

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compared in HoSt.

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1.0 Introduction

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inhibitor therapy)1, assess fertility2, monitor sex steroid metabolism disorders3, identify

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E2 secreting tumors4, and assess fracture risk5. Several research studies and meta-

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analyses found that elevated E2 levels in postmenopausal women increase the risk for

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breast cancer with relative risk ratios similar to those observed for cholesterol levels and

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cardiovascular disease risk6,7. Additional studies have reported associations between E2

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and the risk of endometrial cancer8,9, cardiovascular disease10, and cognitive function11.

Estradiol (E2) measurements are used to monitor antiestrogen therapy (e.g. aromatase

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E2 measurements are reported to be inaccurate and highly variable, especially at the low

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concentrations observed in men, postmenopausal women, and women on aromatase

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inhibitor therapy.1,12–14 It has been suggested that the source of high variability at low

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concentrations is due to lack of method specificity1,12,13,15, which can impact the

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diagnosis and treatment assessments of patients. For example, it has been suggested that

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in post-menopausal women on aromatase inhibitor therapy, inaccurate measurements can

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lead to incorrect assessments of treatment efficacy.15-17

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A recent report from the clinical and research community and a position statement by The

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Endocrine Society recognized the problem with inaccurate E2 measurements and the

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need to improve steroid hormones measurements.1,12 To make improvements, clinical

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laboratories, proficiency testing (PT) providers, and assay manufacturers need access to

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highly accurate and precise reference measurement procedures (RMPs) to assess the

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trueness of analytical methods used in patient care and research. To address these needs,


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the CDC established the Hormone Standardization Program (HoSt).18,19 One key

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component of this program is an RMP for E2.

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RMPs are analytically involved, laborious, require larger sample volumes, and as a result

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are not practical for routine laboratories to adopt. Only three RMPs for E2 in serum are

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recognized by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) as

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meeting criteria for higher-order reference method standards according to ISO 15193.20,21

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Two of the methods use GC/MS22,23 and one uses LC-MS/MS methodology24. The new

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proposed candidate RMP (cRMP) meets the accuracy and precision recommendations for

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measuring E2 with sufficient sensitivity for assigning target values in serum of men and

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women (pre- and postmenopausal) and is comparable to established RMPs. In contrast to

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the published LC-MS/MS RMP, this method uses optimized chromatography without

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requiring derivatization to improve specificity. In addition, no operational RMP exists in

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the United States to address the needs of the clinical laboratories, PT providers, and assay

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manufactures. The cRMP presented here is suitable to meet these needs.

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2.0 Materials and Methods

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Certified primary reference standard for 17-β estradiol NMIJ CRM 6004-a lot 009 from

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National Metrology Institute of Japan (Ibaraki, Japan) with a purity of 98.4% was used as

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calibrators. [2H5]-estradiol lot B0369 (Steraloids, Newport, RI), with an isotopic purity of

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99.0%, was used as an internal standard (IS). HPLC-grade methanol and hexane, as well

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as reagent-grade ammonium carbonate and sodium acetate were obtained from Fisher

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Scientific (Suwanee, GA). Anhydrous ethanol was purchased from Sigma-Aldrich (St.



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Louis, MO). Ammonium fluoride was obtained from ACROS (Pittsburgh, PA). Steroids

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analyzed for interference testing (Table 1) were obtained from Steraloids (Newport, RI)

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and Cerilliant (Round Rock, TX).

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materials (BCR 576, BCR 577, and BCR 578) were purchased from the Institute for

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Reference Materials and Measurements (IRMM, Geel, Belgium). Fresh-frozen, single-

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donor serum materials were obtained from Solomon Park Research Laboratories

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(Kirkland, WA). Other serum materials for QC preparation and matrix evaluations were

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obtained from BioreclamationIVT (Westbury, NY).

Three levels of serum-based certified reference

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2.1 Calibrator Preparation

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All volumetric steps for the calibrator and sample preparation were controlled by

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gravimetric measurements. Mass fractions (mg/g) were calculated for all calibrator stock

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solutions and calibrators. For the purpose of this manuscript, mass fractions (mg/g) were

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converted into volume concentrations using the specific density determined with a DMA

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500 density meter (Anton Paar, Ashland,VA). A primary stock solution (PSS) was

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prepared by dissolving 300 mg of E2 in 100 mL anhydrous ethanol, and further diluting

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was achieved through addition of 200 µL of this solution in 100 mL anhydrous ethanol

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(Stock B). Stock C was created by diluting 100 µL of Stock B solution in 100 mL

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anhydrous ethanol. Three working standard solutions were prepared by diluting 5.0 mL,

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1.0 mL and 100 µL of Stock C in 100-mL anhydrous ethanol: WS1 (1.10 nM [300

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pg/mL]), WS2 (220 pM [60.0 pg/mL]) and WS3 (22.0 pM [6.00 pg/mL])., Internal

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Standard (IS) PSS and working standard solutions ISWS1, ISWS2, and ISWS3 were

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prepared using [2H5]-estradiol in the same manner as unlabeled E2 to obtain similar final


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concentrations.

WS1 and ISWS1 (High) solutions were used to quantitate values

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typically reported in premenopausal women (>257 pM [70.0 pg/mL]), WS2 and ISWS2

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(Medium) for men and some postmenopausal women (36.7-257 pM [10.0-70.0 pg/mL]),

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and WS3 and ISWS3 (Low) for postmenopausal women (< 36.7 pM [10.0 pg/mL]).

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Similar to procedures previously described, five point calibration curves were prepared

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by adding varying amounts of ISWS solution to WS solution to obtain mass ratios of

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unlabeled to labeled E2 of 0.5, 0.8, 1.0, 1.2, and 1.5.25,26 The procedure was applied to

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establish calibration curves for a concentration range of High 440-734 pM (120-200

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pg/mL), Medium 88.1-147 pM (24.0-40.0 pg/mL) and Low 8.81-14.7 pM (2.40-4.00

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pg/mL). Calibration curves were prepared in triplicate.

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2.2 Sample Preparation

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Determination of E2 concentration in a sample was performed using bracketing

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calibration as described previously.25,26 In brief, the amount of ISWS solution was

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adjusted to achieve an approximate mass ratio of one to the endogenous E2. For that, a

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routine method measured the approximate concentration of E2 in the sample. The result

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from this measurement was used to determine the amount of ISWS solution needed to

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achieve the ratio of one. The ISWS solution was transferred to a glass vial, and the

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solvent was evaporated under vacuum prior to addition of serum to avoid protein

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precipitation. For analysis of High concentration samples, 0.5 mL of serum was used; for

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Medium concentration samples 1.0 mL was used; and for Low concentration samples

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3.0-5.0 mL was used, depending upon the approximate concentration of the sample. The



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samples and IS were allowed to equilibrate at room temperature for 30 min while gently

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shaking as previously described to improve accuracy and reproducibility 22.

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E2 was released from binding proteins with the addition of sodium acetate (0.5 M, pH

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5.5) at a sample-to-buffer ratio of 1:2 (v/v) and equilibrated at room temperature (20oC)

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for 2 hours. Liquid/liquid extraction (LLE) was performed in duplicate using 2.5 mL of

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an ethyl acetate: hexane (3:2, v/v) solution. The two organic extracts were combined,

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evaporated to dryness under vacuum, and reconstituted in 0.5 mL of ammonium

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carbonate (0.2 M, pH 8.2). To remove polar lipids, the solution was extracted using 3 mL

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of ethyl acetate: hexane (3:2, v/v). The organic layers were dried under vacuum and

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reconstituted with 120 µL of a water:methanol mixture (80:20, v/v) for LC-MS/MS

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analysis. Three quality control (QC) materials from unaltered, pooled serum (QClow,

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QCmedium, and QChigh) and three IRMM certified reference materials (BCR576,

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BCR577, and BCR578)) were prepared by reconstitution according to the certificates of

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analysis and analyzed in duplicate in each run.

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2.3 LC-MS/MS Conditions

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Two injections for each preparation were performed on an AB/Sciex API 5500 tandem

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mass spectrometer (Foster City, CA) equipped with a Shimadzu LC-10AD/SIL-30AC

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Nexera system (Columbia, MD). A Phenyl/Hexyl Kinetex, 50 × 4.6 mm, 2.6 µm

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analytical column with a guard column (Phenomenex, Torrance, CA) heated to 40oC was

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used with 0.2 mM ammonium fluoride in water (Buffer A) and methanol only (Buffer B).

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The HPLC separation was achieved with a 9 minute gradient from 48% to 62% Buffer B


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at a flow rate of 750 µL/min. The mass spectrometer was operated with electrospray

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ionization (ESI) in the negative ion mode with voltage set to -4,000 V, collision energy

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set to -50 eV, and Turbo Ion Spray Source temperature set at 650 oC. Selected reaction

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monitoring (SRM) was used with the following transitions: m/z 271 → 145 (quantitation

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ion - QI) and m/z 271 → 183 (confirmation ion- CI) for E2 and with 276 → 147 (QI) and

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m/z 276 → 187 (CI) for the IS.

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2.4 Data Analysis

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Analyst 1.6.2 software (AB/Sciex, Framingham, MA) was used to integrate selected

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peaks. Quantitation was performed with the peak area count ratio of the E2 QI over the

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IS QI. The mean area count ratios from the two injections were used for data analysis.

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To convert the mass concentration to the commonly used volume concentration, the

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density of the serum samples was used. For value assignment, duplicate preparations of

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serum were made in two independent runs (n=4).

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measurements was used to assign an E2 concentration to the sample. Statistical

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evaluations for Deming regression and difference plots were performed with Analyse–It

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Software, Ltd Version 2.26 (Leeds, UK) using t-test, α=0.05.

The mean value from four

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2.5 Method Validation

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Accuracy was evaluated through a comparison with a JCTLM-recognized RMP at the

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University of Ghent (JCTLM code-NRMeth 10)23 and IRMM certified reference

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materials. The mean percent bias of the cRMP to the JCTLM-recognized RMP was

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determined following the CLSI document EP 9 A2 protocol comparing 40 single-donor



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serum samples.27 The cRMP was compared to the JCTLM-recognized RMP values using

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a Deming regression analysis. In addition, IRMM certified reference materials were

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analyzed, and the mean percent difference from the certified value was determined and

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evaluated for significance as described in NIST Special Publication 82928.

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Imprecision was evaluated by analyzing 3 serum pools in duplicate over 5 days. CLSI EP

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10-A3 was used to calculate the intra-assay, inter-assay, and total percent coefficients of

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variation (CVs).29 Standard Uncertainty and Expanded Uncertainty of the cRMP

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following ISO Guide to the Expression of Uncertainty in Measurement 2008 was also

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evaluated.30 The estimated variance of Type A uncertainty was calculated from repeated

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measurements and Type B uncertainty was calculated using uncertainties in the purity of

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the reference compound, inaccuracy in the weighing of each component, and the

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measurement of the serum density.

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combined to determine the Standard Uncertainty and multiplied by a coverage factor,

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k=2, to determine the Expanded Uncertainty using QClow, QCMedium, and QCHigh.

Calculated Type A and B uncertainties were

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Twenty-nine structural analogs and other steroid hormones with relative molecular

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masses close to E2 and the IS were tested as potential interferences (Table 1). The

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absence of a peak with the mass transitions of E2 (m/z 271 → 145) or the IS (m/z 276 →

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187) at the retention time of E2 at 11.3 minutes confirmed that the structural analogs did

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not interfere with the quantitation of E2. In addition, the QI/CI ratio of E2 was monitored

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in the 40 single-donor samples and compared to the QI/CI ratio of the calibrators. A


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difference of less than +20% was used to confirm that no interfering compounds were

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present.31

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The effect of different sample matrices was determined following procedures previously

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described.25 Four sample matrices (0.9% saline solution, charcoal stripped serum, male

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serum, and female serum) and one set of neat samples in ethanol were evaluated. A 7-

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point calibration curve ranging from 147- 848 pM (40-231 pg/mL) was prepared in each

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matrix and spiked with 551 pM (150 pg/dL) IS solution. The calibrators in the matrices

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were subjected to the sample preparation described above. The MS response (area count

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ratios of analyte over IS) was compared in all four matrices to the MS response of the

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neat samples prepared in ethanol (matrix-free). The sample matrix effect (ME) was

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calculated with the following equation: ME %= B/A × 100, where B is the area count

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ratios of E2 to IS obtained from samples in matrix, and A is the area count ratios in

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matrix-free samples.

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3.0 Results and Discussion

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The trueness and imprecision of an RMP needs to be smaller than that of a routine

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measurement procedures to ensure target values assigned have a sufficiently low

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uncertainty so that, any uncertainty added by a routine method can then still lead to

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clinically meaningful results. RMPs should have a limit that is half the limit for a routine

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method for imprecision and a limit that is one third the maximum bias.32,33 The CDC

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HoSt Program for E2 certifies analytical performance criteria for a routine E2 assay with

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a difference of +/-12.5% at concentrations 73.4pmol/L (20.0 pg/mL) from the true value and includes a suggested

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imprecision of 11.3%.15 The described cRMP meets the suggested performance criteria

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for E2 with a bias within ±2.8% and imprecision of 100%). The serum samples had a ME% of 96.2% and 92.2%,

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indicating slight ion suppression from the matrix, as expected with increased serum

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volume requirements for samples with lower concentrations of E2 such as male samples.

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However, our evaluations have shown no significant effect of this matrix effects on the

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accuracy and precision of our method in both male and female samples.

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Sample preparation of the cRMP plays a key role in ensuring that no interferences and no

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significant matrix effects exist. This is achieved in part through the selected conditions

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used during the isolation of the analyte from the matrix. The first LLE isolated the lipids

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from polar compounds. A solution of ethyl acetate:hexane at 3:2, v:v, was selected to

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maximize isolation of the analyte from the matrix. The addition of a basic solution

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(ammonium carbonate buffer, pH 8.2) to the sample extract and the second LLE allowed

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for the removal of acidic impurities, such as fatty acids and phospholipids. These lipids

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are a source of ion suppression and matrix effects, particularly in LC MS/MS analyses.37

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A higher pH for the ammonium carbonate buffer would increase the removal of

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impurities25; however, the pH of 8.2 was selected to be two pH units below the pKA of

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E2 (10.5) to avoid deprotonation and subsequent decrease in recovery of E240.

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The cRMP has been automated using the Hamilton Starlet (Reno, NV) equipped with an

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analytical balance. The enhancement to the method doubled the sample throughput

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compared to the manual sample preparation which helps to meet the increasing demand

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for target value assignments.

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4.0 Conclusion

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The described ID LC-MS/MS cRMP for measuring total 17-β estradiol with sufficient

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sensitivity for assigning target values to sera from men, and pre- and postmenopausal

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women, has higher specificity at similar accuracy and precision performance compared to

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existing RMPs. High specificity is needed to ensure accurate target value assignment,

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especially in samples with low E2 concentrations, where small amounts of interfering



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compounds can have a high impact on the measurement result. The cRMP is suitable to

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address the current unmet needs of clinical laboratories, assay manufacturers, and PT

3

providers by providing value assignments to serum for calibration and verification of

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method performance as part of the CDC HoSt Program.

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Acknowledgements

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The authors would like to acknowledge support from the Division of Laboratory Science

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at the CDC, the CDC Foundation, Drs. Linda Thienpont and Kathleen Van Uytfanghe

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from the University of Ghent, CDC Hormone Laboratory members Otoe Sugahara, Jacob

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Farris, Lumi Duke, Paul Kim, Brandon Laughlin, Chelsea Harrod, Susan Ma, Krista

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Poynter, Christopher Ghattas, Dr. Uliana Danilenko, and CDC PBL/LRL. In addition, the

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authors would like to acknowledge The Endocrine Society, AACC, and the Partnership

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for the Accurate Testing of Hormones (PATH) for their support.

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Figure 1- Chromatograms of neat and serum sample for E2 (retention time- 11.3 minutes) A: Neat – E2 Concentration of 20.3 pM (5.52 pg/mL) B: Native Serum Sample- E2 Concentration of 26.5 pM (7.21 pg/mL) resolved from additional interference peaks 100

A

50

B

50

0

0 6

5 6 7

100 Relative Intensity (%)

Relative Intensity (%)

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8

9

10

11

12

13

14

6

Time (min)

7

8

9

10

11

12

Time (min)


13

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Table 1: List of steroid hormones used for interference analysis including the molecular weight, concentration used during testing, and source of material used

MW (g/mol)

Steroid

Concentration Vendor nM (ng/mL)

4, 16-Androstadien-3β-ol

272.43

0.41 (0.11)

5, 16-Androstadien-3β-ol

272.43

0.41 (0.11)

Androstenediol

290.44

0.38 (0.11)

Androstenedione

290.44

0.38 (0.11)

16, (5α)-Androsten-3-one

272.43

0.41 (0.11)

2, (5α)-Androsten-17-one

272.43

0.41 (0.11)

Androsterone

290.4

0.38 (0.11)

5α-Cholestane

372.67

0.30 (0.11)

6-Dehydronandrolone

272.38

0.41 (0.11)

Dehydroepiandrosterone (DHEA)

288.42

0.39 (0.11)

Dehydroepiandrosterone sulfate (DHEAS)

368.49

0.30 (0.11)

1-Dehydrotestosterone

286.41

0.39 (0.11)

3-Deoxydehydroepiandrosterone (3-deoxy DHEA)

272.43

0.41 (0.11)

17-Desoxyestradiol

256.4

0.43 (0.11)

5a-Dihydrotestosterone (DHT)

290.44

0.38 (0.11)

Epiandrosterone

290.44

0.38 (0.11)

17α-Estradiol (epiestradiol)

272.38

0.41 (0.11)

Epitestosterone

288.42

0.39 (0.11)

1,3,5(10)-Estratrien-16β, 17β-epoxy-3-ol

272.38

0.41 (0.11)

5(10)-Estren-3, 17-dione

272.4

0.41 (0.11)

Estriol

288.38

0.39 (0.11)

Estrone

270.37

0.41 (0.11)

17α-Ethynylestradiol

296.4

0.37 (0.11)

Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) LGC Standards (Manchester, NH) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Cerilliant (Round Rock, TX) Cerilliant (Round Rock, TX) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Cerilliant (Round Rock, TX) Sigma-Aldrich (St. Louis, MO) Steraloids, Inc. (Newport, RI) Cerilliant (Round Rock, TX) Steraloids, Inc. (Newport, RI) Steraloids, Inc. (Newport, RI) Cerilliant (Round Rock, TX) Cerilliant (Round Rock, TX) Cerilliant (Round


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Rock, TX) Etiocholanolone

290.4

0.38 (0.11)

2-Methoxyestradiol

302.41

0.37 (0.11)

17α-Methyl-5α-androstane-3α,17β-diol

306.49

0.36 (0.11)

17-α Methyltestosterone

302.46

0.37 (0.11)

19-Norethindrone

298.42

0.37 (0.11)

D(−)-Norgestrel

312.45

0.36 (0.11)

Cerilliant (Round Rock, TX) Steraloids, Inc. (Newport, RI) Cerilliant (Round Rock, TX) Cerilliant (Round Rock, TX) Steraloids, Inc. (Newport, RI) Sigma-Aldrich (St. Louis, MO)

1 2



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1 2 3

Page 22 of 30

Table 2: Method comparison to a recognized RMP using 40 single-donor samples and Deming regression analysis.

Sample Group

n

Range

Slope

Intercept

pM

(95% CI)

(95% CI)

(pg/mL)

[SE, p]

[SE, p]

1.00

0.02

(0.997 to 1.008)

(-0.02 to 0.05)

[0.003, 0.363]

[0.018, 0.311]

1.00

1.38

(0.978 to 1.011)

(-1.17 to 3.93)

[0.007, 0.499]

[1.170, 0.260]

0.99

0.38

(0.962 to 1.010)

(-0.06 to 0.83)

[0.011, 0.234]

[0.206, 0.0850]

2.61 – 26.5

0.99

0.03

(0.71 -7.21)

(0.979 to 1.008)

(-0.02 to 0.08)

[0.006. 0.3654]

[0.021, 0.1973]

2.61 – 1,050 All

40 (0.71 –285)

High 277 – 1,050 (>257 pM)*

14 (75.4 – 285)

Medium 43.7 – 203 (36.7-257 pM)+

15 (11.9 – 55.3)

Low (

Evaluation of an Isotope Dilution HPLC Tandem Mass Spectrometry

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