Sensitive routine liquid chromatography–tandem mass spectrometry method for serum estradiol and estrone without derivatization

Pauwels, Steven; LʼAbbate, Antonio; Jans, Ivo; Lintermans, Anneleen; Neven, Patrick; Claessens, Frank; Decallonne, Brigitte; Billen, Jaak; Vanderschueren, Dirk; Vermeersch, Pieter

doi:10.1007/s00216-013-7259-5

Cited by 57 publications

(36 citation statements)

References 25 publications

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“…Derivatization of E 2 has been widely accepted as a way to improve ionization efficiency, making it more sensitive in LC-MS/MS-based detection. Most reported methods of derivatization use dansyl chloride and found improved signal intensity by 1-2 orders of magnitude when compared to an underivatized method under positive polarity mode (23)(24)(25)(26)(27)(28). However, the derivatization step can add complexity in the sample preparation and may decrease the robustness of the overall method, which is not optimal for a routine clinical laboratory application.…”

Section: Discussionmentioning

confidence: 99%

“…With higher throughput, faster turnaround time, and minimum sample preparation, automated immunoassays are the most widely used techniques for E 2 measurements in clinical and research laboratories. However, automated immunoassays suffer from poor accuracy and precision at low physiological E 2 concentrations and are susceptible to interferences due to nonspecific antibody cross-reactivity with estrogen metabolites and other exogenous estrogens (9,(22)(23)(24). To overcome these technical limitations of immunoassays, mass spectrometry assays, especially LC-MS/MS assays, have increasingly been used in the recent years.…”

Section: Introductionmentioning

confidence: 99%

“…In a routine clinical laboratory, derivatization can increase the sample preparation time and introduce more method variations. Some attempts have been made to design methods without the need of derivatization, but those methods require an extended extraction protocol and large sample volumes (23,24,28), which are also not optimal for routine clinical practice. Our goal in this study was to develop a highsensitivity, underivatized method using micro LC-MS/MS to reliably measure E 2 concentrations in adult men, postmenopausal women, pediatric patients, and patients undergoing antiestrogen therapies.…”

Section: Introductionmentioning

confidence: 99%

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High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

Leung

Bridgman

et al. 2016

The Journal of Applied Laboratory Medicine

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Background: There are considerable demands to accurately measure estradiol (E 2 ) at low concentrations (<20 pg/mL) in postmenopausal women, men, pediatric patients, and patients receiving breast cancer treatment. Most current high-sensitivity LC-MS/MS E 2 methods require large sample volumes and involve complex sample preparations with dansyl chloride derivatization. Our study aims to develop a high-sensitivity, underivatized method using micro LC-MS/MS to reliably measure E 2 concentrations below 5 pg/mL by the use of low sample volume. Methods: A total of 290 μL of sample was mixed with internal standard (IS), E 2 -d4, and extracted with a mixture of hexane/ethyl acetate (90/10) (v/v). After extraction, sample was separated by Eksigent Ekspert™ micro LC 200 system with a flow rate of 35 μL/min in a total run time of 3.5 min and detected by SCIEX QTRAP 6500 mass spectrometer in a negative mode using transitions: 271/145 (quantifier) and 271/143 (qualifier). In this method, it was crucial to use HPLC columns with stability at a pH >10. Results:The validation study demonstrated broad linear ranges (3.0 -820.0 pg/mL) with r 2 > 0.999. Total precision was below 15% at all QC levels, and limit of quantification (LOQ) was 3.0 pg/mL. Our method showed good correlation with E 2 RIA (r 2 = 0.96, bias = −1.0 pg/mL) and modest correlation with E 2 Roche Cobas automated immunoassay (r 2 = 0.86, bias = 6.0 pg/mL). Conclusions:In conclusion, we developed and validated a routinely applicable micro LC-MS/MS method without derivatization for E 2 in blood samples with an LOQ of 3.0 pg/mL. IMPACT STATEMENTPatients under assessment of puberty delays, pubertal growth, gynecomastia, and efficiency of aromatase inhibitor (AI) treatment will benefit from the information presented here. Evidence presented on high-sensitivity LC-MS/MS assay for E 2 will allow better characterization of E 2 concentration in blood in the low measurement range. Knowledge in the field of LC-MS/MS method development for E 2 will be advanced by the information presented.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

Leung

Bridgman

et al. 2016

The Journal of Applied Laboratory Medicine

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“…Consequently, methods to evaluate such low-plasma estrogen levels in patients require a high sensitivity which makes measurement in vivo very difficult. Our research group developed a sensitive liquid chromatography-tandem mass spectrometry method for measuring low-estrogen levels (Pauwels et al 2013). The limit of quantification is 1.2 and 1.3 ng/l for estrone and E 2 respectively.…”

Section: Pharmacologymentioning

confidence: 99%

Aromatase inhibitors in the breast cancer clinic: focus on exemestane

Asten¹,

Neven²,

Lintermans³

et al. 2014

Endocrine-Related Cancer

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Breast cancer is the most prevalent type of cancer in women and responsible for significant female cancer-related mortality worldwide. In the Western world, over 80% of breast cancers are hormone-receptor positive for which endocrine therapy is administered. The main anti-estrogen treatments in use consist of selective estrogen-receptor modulators, such as tamoxifen, and third-generation aromatase inhibitors (AIs), such as exemestane, letrozole, and anastrozole. In this review, the focus will lie on exemestane, its clinical use, and its side-effect profile. Exemestane is the only third-generation steroidal AI. Its efficacy as a first-line treatment in metastatic breast cancer has been demonstrated. Therefore, exemestane could be considered a valid first-line therapeutic option, but it also can be used in second-line or further situations. Exemestane is mostly used as part of sequential adjuvant treatment following tamoxifen, but in this setting it is also active in monotherapy. Furthermore, this AI has been studied in the neoadjuvant setting as presurgical treatment, and even as chemoprevention in high-risk healthy postmenopausal women. It may reverse side effects of tamoxifen, such as endometrial changes and thromboembolic disease but may also cause some inconvenient side effects itself. Additionally, there is a lack of total crossresistance between exemestane and nonsteroidal AIs as far as their anti-tumoral efficacy is concerned; moreover the two classes of AIs display a nontotal overlapping toxicity profile. Taking together, exemestane can be considered as a useful treatment option at all stages of breast cancer.

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“…For electrospray ionization (ESI), other interesting approaches like chemical derivatization [4,5] or formation of adduct ions [6][7][8][9] have been described to increase signal. The addition of fluoride ions increases ionization for steroids and neutral drugs in negative ESI mode using a mechanism not yet completely understood [9][10][11][12]. The yield and possibility of these techniques varies with the analyte targeted, available instrumentation, and technology.…”

Section: Introductionmentioning

confidence: 96%

Possibilities and limitations of signal summing for an immunosuppressant LC-MS/MS method

Pauwels¹,

Jans

Peersman

et al. 2015

Anal Bioanal Chem

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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the method of choice for quantifying small molecules in research and clinical setting. Although there is a large toolkit to increase quantification levels for LC-MS/MS, these techniques are sometimes insufficient to attain the needed limits of quantification (LOQs) or the method becomes too impractical for routine use. We examined the possibilities and limitations of signal summing, an under-utilized, easy-to-apply practice to increase LOQs for an immunosuppressant LC-MS/MS method. The limits of signal summing for everolimus were tested by running samples of everolimus at three concentrations in triplicate programming, increasing amounts of identical transitions in a constant cycle time up to the maximum number the software permitted to sum. The increase in peak area and the signal-to-noise ratio were determined. The effect on imprecision of peak areas and response ratios was evaluated by injection of a low concentration of everolimus tenfold using respectively one and five identical transitions, retaining an identical ion counting time. We compared the imprecision, LOQ, and recovery for our routine everolimus method (using one transition for everolimus and one for d3-everolimus) and an adapted method summing three identical transitions for everolimus (and one for d3-everolimus). The increase in signal was close to the theoretically expected one with a larger experimental spread for everolimus once more than five transitions were used. There was no clear beneficial effect of summing on imprecision. The adapted everolimus method showed a lower LOQ, but comparable imprecision and recovery as the routine method. Quantification levels can be improved by signal summing. No clear effect on imprecision was observed.

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Sensitive routine liquid chromatography–tandem mass spectrometry method for serum estradiol and estrone without derivatization

Cited by 57 publications

References 25 publications

High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization

Aromatase inhibitors in the breast cancer clinic: focus on exemestane

Possibilities and limitations of signal summing for an immunosuppressant LC-MS/MS method

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