Simultaneous Determination of Naturally Occurring Estrogens and

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Simultaneous determination of naturally occurring estrogens and mycoestrogens in milk by UHPLC-MS/MS analysis Anna Laura Capriotti, Chiara Cavaliere, Susy Piovesana, Serena Stampachiacchiere, Roberto Samperi, Salvatore Ventura, and Aldo Laganà J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Sep 2015 Downloaded from http://pubs.acs.org on September 28, 2015

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Journal of Agricultural and Food Chemistry

Simultaneous determination of naturally occurring estrogens and mycoestrogens in milk by UHPLC-MS/MS analysis

Anna Laura Capriotti, Chiara Cavaliere*, Susy Piovesana, Serena Stampachiacchiere, Roberto Samperi, Salvatore Ventura, Aldo Laganà

Dipartimento di Chimica, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy

*Corresponding author: Chiara Cavaliere Dipartimento di Chimica, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy Phone: +39 06 4991 3834. E-mail: [email protected]

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Abstract 1

A simple, fast and reproducible method for the simultaneous determination of natural estrogens and

2

mycoestrogens (resorcylic acid lactones) in milk by ultra-high performance liquid chromatography

3

combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC/ESI–

4

MS/MS) is described. The extraction was carried out by solid phase extraction (SPE) using

5

graphitized carbon black as solid sorbent. The use of carbon black allowed to avoid any type of

6

sample pretreatment and the extraction was performed simply by diluting milk samples in water.

7

Correlation coefficient values were obtained in the range between 0.9991 to 1, with good recoveries

8

(67-107% at the lowest spiked level), repeatability (4.8%-16.8%) and reproducibility (3.2%-16.3%).

9

Moreover, a very low matrix effect was observed for both estrogens and mycoestrogens. With

10

respect to a previous method based on SPE with OASIS MAX cartridges, the one here described

11

allowed to detect all the analytes under investigation, at the lowest tested concentration level,

12

including free estrogens (in particular estriol).

13

Finally, the developed UHPLC/ESI–MS/MS method was applied to the analysis of some whole

14

milk samples from different lactating animals (cow, goat and donkey), as well as UHT cow milk

15

and powder milk samples.

16 17

Keywords

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Estrogens;

mycoestrogens;

zearalenone;

milk;

liquid chromatography-mass


spectrometry

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19

INTRODUCTION

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Milk is one of the most consumed and important food in human diet. Cow milk is the most

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widespread one, however, various types of milk, characterized by different composition and

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nutritional values, are commercially available.

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One of the most known problems affecting milk production concerns the possible contamination

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with natural estrogens and other endocrine disruptor compounds (EDCs) of both natural and

25

synthetic origin. According to their origin, there are three main sources of EDCs in milk and its

26

derivatives. The first source of EDCs is provided by the animals themselves, and constitutes the so

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called “natural contamination” of milk, which is the most challenging to control. Commercial milk

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can be potentially contaminated with natural hormones, such as estrone (E1), estradiol (E2) and

29

estriol (E3). In fact, due to the continuous demand and consumption of milk, about 75% of

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commercial milk is produced from pregnant cows.1,2 It is known that during gestation state the

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concentration of natural estrogens is relatively high, therefore relevant quantities of estrogens can

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occur in raw milk1 and then in ready-to-drink milk, leading to increased incidence of health

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problems, especially reproductive apparatus disorders.

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The second source of EDCs in milk is constituted by substances of synthetic/industrial origin, such

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as dioxins, alkylphenols and phthalates that can reach milk and milk products during industrial

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processes, like packaging and storage.3 Because of their structure, they may mimic the estrogen

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activity and therefore interfere with the hormonal system. Although the latest industrial processes

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are well able to control and avoid such contamination, EDCs still represent a very important issue.

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For this reason in the literature several works have been published, regarding methodologies for the

40

screening of these compounds in milk and milk based products.4,5

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Finally, the third source of EDCs is provided by animal feed, which could be potentially

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contaminated. In this regard, mycotoxins are the most important animal feed contaminants, and in

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particular, mycoestrogens are mycotoxins displaying the same effects of estrogens at health level

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due to their chemical structures. Zearalenone (ZEA) and its metabolites are the most known 3 ACS Paragon Plus Environment


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compounds belonging to mycoestrogen group. ZEA is one of the most widely distributed

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mycotoxins produced by the Fusarium species, in particular F. graminearum and F. culmorum. The

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fungi that produce ZEA most frequently contaminate corn, but can also colonize barley, oats, wheat,

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sorghum, millet, and rice.6 Crops are then used as feed for animals and in this way mycotoxins

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might enter the human food chain via animal products, including milk and derivative products.

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Studies on cows consuming feed containing relatively high levels of mycotoxins have shown the

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mycotoxins and their metabolites can transfer to milk7,8 but, despite these studies, the presence of

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mycoestrogen residues in milk has been largely neglected, until now.

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One of the ZEA metabolite, namely α-zearalanol (α-ZAL) is principally used as illicit anabolic

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substance for animal growth. During its metabolism in animals, α-ZAL is then oxidized to

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zearalanone (ZAN) and is further reduced to β-zearalanol (β-ZAL) which is its primary metabolite

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in bovine. Therefore, all of these analytes could be found in animals that had consumed feed

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naturally contaminated with mycoestrogens9,10 or treated with the growth promoter α-ZAL.

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Moreover α-ZAL and its metabolites are also related to the metabolism of ZEA in animals and can

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occur from its metabolic pathway. In all cases, all of these compounds could reach milk with the

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same effects of free estrogens, therefore their presence should be monitored too. To reach this goal

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an analytical methodology should be able to simultaneously detect all these compounds, natural

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estrogens and mycoestrogens, at trace level, despite the lack of maximum residue levels in milk and

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other dairy products. Due to the complexity of milk, the analysis of estrogens and related

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compounds is sometimes challenging, and a lot of steps during sample preparation are required.

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Delipidation and deprotenization are commonly employed procedures in milk analysis, but they are

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time consuming and can lead to analyte loss. At the same time it would be difficult to employ the

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conventional commercial solid phase extraction (SPE) cartridges without any sample pretreatments.

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Also the newest and fast extraction methodologies, like QuEChERS, are rarely used for this type of

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analytes in milk.


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To overcome these challenges, in this work we developed and validated an analytical methodology

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for fast, simple and simultaneous determination of mycoestrogens and natural estrogens in different

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commercial milk samples, without any sample pretreatment. The developed method, based on ultra

73

high performance liquid chromatography coupled to tandem mass spectrometry via electrospray

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source (UHPLC/ESI-MS/MS), is able to reach very good recoveries, negligible or very low matrix

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effect and lower limits of detection and quantification than previously published methods, without

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any selectivity loss.

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MATERIALS AND METHODS

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Chemicals and reagents

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Acetonitrile (ACN), Methanol (MeOH), CH2Cl2, NH3 (30%), HCOOH (> 98%), and HCl were

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supplied by Sigma-Aldrich (Milan, Italy). All reagents were of analytical reagent grade, solvents

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were LC-MS grade. Ultrapure water (resistivity 18.2 MΩ cm-1) was obtained using an Arium water

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purification system (Sartorious, Florence, Italy).

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Pure standards of all selected compounds, i.e., E1, 17β-estradiol (β-E2), 17α-estradiol (α-E2), E3,

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ZEA, α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), ZAN, α-ZAL, β-ZAL, were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). The commercially available deuterated standards 17β-

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estradiol-16,16,17-d3 (β-E2-d3) and zearalenone-d6 (ZEA-d6) were purchased as pure standards

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from Wellington Laboratories (Toronto, Ontario, Canada) and were used as internal standards (ISs).

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Full names and abbreviations of the selected compounds are reported in Figure 1. Individual stock

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standard solutions of the analytes were prepared in MeOH at 1 mg mL−1 level. Composite working

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standard solutions were prepared by suitable dilution of stocks and used for recovery experiments

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and renewed weekly.

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Extraction and clean-up apparatus



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A model ST ultrasonic bath at a frequency of 50±3 kHz from Stimin (Milan, Italy) and an ALC

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(Milan, Italy) multispeed refrigerated centrifuge PK131R were used. Polypropylene tubes,

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polyethylene frits, and the vacuum manifold were from Supelco (Bellefonte, PA, USA). All

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samples were extracted and cleaned by SPE cartridges packed with Carbograph-4 supplied by

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LARA (Rome, Italy). Carbograph-4 is a graphitized carbon black (GCB) similar to Carboprep 200

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(Restek) and ENVI-carb X (Supelco).11,12 Acrodisc 13 mm syringe filters with 0.2 µm GHP

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membrane (used for the filtration of samples prior to the injection into the chromatographic system)

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were from Pall (Pall Corp., MI, USA).

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Sample collection and preparation

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Different types of cow whole milk, UHT pasteurised whole milk (3.6 g fat; 3.30 g protein per 100

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mL), goat milk (3,9 g fat; 3,2 g protein per 100 mL), donkey milk (1,21 g fat; 1,74 g protein per 100

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mL) and powder milk (0.1 g fat and 3.3 g protein per 100 mL of reconstituted milk) were purchased

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from local markets. The method was developed on whole milk, considered as the worst case in

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terms of interferents content (such as proteins and fats).

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Fortified samples were prepared by spiking 10 mL of milk with suitable amounts of composite

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working standard solutions and defined concentration of ISs (2 µg/L for β-E2-d3 and 1.5 µg/L for

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ZEA-d6), diluted to 250 mL with ultrapure water, and passed through the Carbograph-4 cartridges

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at about 20 mL min−1. Cartridges were prepared by placing 500 mg of the adsorbent inside 6 mL

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polypropylene tubes between two polyethylene frits. Before processing the samples, these

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cartridges were attached to a vacuum manifold apparatus and washed sequentially with 10 mL of

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CH2Cl2/MeOH (80/20, v/v), 5 mL of MeOH, 20 mL of 10 mmol L−1 HCl solution, and 10 mL of

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ultrapure water. Then, the cartridge was washed sequentially with 50 mL of ultrapure water, 10 mL

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of MeOH acidified with 50 mmol L−1 HCOOH, and 5 mL of MeOH. Acidified MeOH was

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necessary to elute some compounds that could interfere with the analysis of free estrogens,

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especially E3.13 Analytes were then eluted with 15 mL of CH2Cl2/MeOH (80/20, v/v). The eluate 6 ACS Paragon Plus Environment

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was evaporated to dryness in a water bath at 37 °C under a gentle stream of nitrogen. The residue

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was dissolved in 500 µL of water/MeOH (50/50, v/v) and then forced through a 0.2 µm GHP

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membrane syringe filter. Five microliters of each final solution was analyzed by UHPLC/ESI-MS/

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MS.

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UHPLC/ESI-MS/MS analysis

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Liquid chromatography was performed by using an Ultimate 3000 LC system (Thermo Fisher

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Scientific, Bremen, Germany) that consisted of a binary four-line pump, equipped with a degasser, a

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thermostatted microwell-plate autosampler, and a thermostatted column oven.

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A 5 µL aliquot of sample was injected on a Hypersil Gold (50×2.1 mm i.d., 1.9 µm particle size)

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equipped with a Security guard Hypersil Gold (4×2.1 mm i.d., 5 µm particle size), both from

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Thermo Fisher Scientific. The column was maintained in the thermostatted column compartment at

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40° C. Mobile phases were water (A), and ACN (B). For separation of the analytes, gradient elution

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was performed by linearly increasing the amount of B in A at flow rate of 300 µL min-1. The

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starting mobile phase composition was 15 % B. After an isocratic step at 15 % B for 1 min, B was

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linearly increased to 50% within 7 min, then brought to 99% in 2 min, and held constant for 5 min.

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Finally, the B content was lowered to 15% and the column allowed to re-equilibrated for 5 min, for

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a total cycle time of 20 min.

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A freshly prepared solution of NH3, 80 mmol L−1 in water/MeOH (50/50, v/v), was added to the LC

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column effluent at a flow rate of 50 µL min−1 by means of a Perkin-Elmer series 200 micropumps

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(Norwalk, CT, USA). MS/MS detection was performed with a TSQ VantageTM triple-stage

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quadrupole mass spectrometer (Thermo Fisher Scientific) connected with the LC system via a

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heated ESI source, operated in negative ionization mode. Spray voltages of 2.6 kV were applied,

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whereas declustering voltage was maintained to 0 V. The vaporizer temperature was set to 280°C

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and the capillary (ion transfer tube) temperature to 220°C. Sheath gas pressure, ion sweep gas

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pressure, and auxiliary gas pressure were set to 40, 0, and 20 (arbitrary units), respectively. Mass 7 ACS Paragon Plus Environment


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calibrations and resolution adjustments on the resolving lens and quadrupoles were automatically

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performed using the manufacturer’s solution introduced via infusion pump at 5 µL min−1 flow rate.

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In order to optimize the tuning parameters for each compound, 1 ng µL−1 standard solutions in

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water/MeOH (50/50, v/v) were infused at 10 µL min−1. The negative precursor ions were selected

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by the first quadrupole and fragmented in the collision cell with the appropriate collision energy.

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From the MS/MS full-scan spectra, suitable transitions were selected for acquisition in selected

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reaction monitoring (SRM) mode. The whole LC-MS system was managed by Xcalibur software

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(v.2.1, Thermo Fisher Scientific).

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Method Performance

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The method developed was evaluated according to recovery, matrix effect, linearity, precision

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(intra- and inter-day precision), limits of detection (LODs) and limits of quantifications (LOQs)

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under the optimized conditions.

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Recovery

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The relative recoveries of the analytes were calculated by comparing the peak area ratio relative to

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that of the ISs obtained by spiking milk samples containing no detectable analyte quantities with the

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composite working standard solutions and ISs at four different concentration levels (5, 0.5, 0.2 and

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0.05 µg/L) before and after the extraction procedure in triplicates.

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Matrix Effect

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Signal suppression (or enhancement) on ESI-MS/MS response due to matrix components, i.e.

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matrix effect (ME), was assessed by comparing the response of each analyte in matrix with respect

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their response in solvent via the formula: [1-(Amatrix/Astandard)]·100 where Amatrix and Astandard are the

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relative peak area with respect to the ISs area of the analyte in matrix and in solvent, respectively. It

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was assessed during recovery experiments for each considered concentration. 8 ACS Paragon Plus Environment

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Linearity

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For linearity evaluation two sets of calibration lines, named “standard” and “matrix-matched”,

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respectively, were constructed. The standard calibration curve was constructed by diluting

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appropriate volumes of the working standard solution in water/MeOH (50/50, v/v), and adding the

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same amount of ISs at all times. Each point of matrix-matched calibration curve was prepared

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following the procedure applied for recovery experiments as described in the previous section. Also

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in this case the same amount of IS was added at all times. Both standard and matrix-matched

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solutions were prepared at six concentration levels, ranging from 5 µg/L to 0.02 µg/L.

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Precision

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Precision of the method was assessed by the repeatability and reproducibility studies and expressed

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as recoveries and the relative standard deviation (RSD, %). The intra-day precision (RSDr) was

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expressed as the standard deviation of the recovery values (n=6) of the spiked samples at three

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levels measured during the same day. The inter-day precision (RSDR) was determined by analysing

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the spiked samples (n=3) for six different days at the same levels.

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LODs and LOQs

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Method sensitivity was evaluated by LODs and LOQs.

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The LODs and LOQs can be either instrumental (ILODs and ILOQs) or relative to the applied

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methodology (MLODs and MLOQs). To calculate LODs the standard deviation of the response (σ)

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was divided by the slope of the calibration curve (S), via the formula: LOD = 3 σ/S using the

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standard calibration curve for ILOD and matrix-matched calibration curve for MLOD.

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In the same way, ILOQ and MLOQ were estimated according to the formula: LOQs = 10 σ/S. These

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values were calculated employing data generated from regression statistic performed using standard

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and matrix-matched calibration points in the range of the lower concentrations (0.02 µg/L- 0.2 9 ACS Paragon Plus Environment


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µg/L). The sum of the ion currents of the SRM transitions was considered to determine LOQs,

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while the less intense transition was considered to evaluate LODs.

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RESULTS AND DISCUSSION

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LC-MS/MS optimization

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The ESI-MS/MS fragmentation profile of all considered analytes was investigated by infusion

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experiments of single standards of approximately 1 ng/µL, prepared in water/MeOH (50/50, v/v).

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Mycoestrogens and free estrogens were studied in negative ionization mode, giving in all cases [M-

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H]- as precursor ion. The list of precursor and fragment ions is shown in Table 1 together with their

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optimized S-Lens and collision energies (CE).

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After that UHPLC conditions were also investigated in order to find out the best mobile phases to

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separate the analytes using C18 stationary phase.

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It has just been reported14 that neutral mobile phases, such as water/ACN or water/MeOH, showed

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great results in term of free estrogens separation, therefore preliminary experiments were carried out

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testing these solvents. ACN resulted the most appropriate organic solvents for chromatographic

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elution of all analytes in terms of peak separation and S/N ratio, especially because ACN allowed to

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well separate ZAN, α-ZOL and β-ZOL, which coeluted using MeOH as organic mobile phase.

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In addition, to achieve more stable and reproducible signals and enhance the ionization of free

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estrogens, post column addition of NH3 was introduced, because of the incompatibility of high pH

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mobile phases with the silica-bonded C18 stationary phase. The addition of the basic modifier can

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significantly improve the deprotonation of the very weakly acid free estrogens; this resulted in a

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significant increase in the intensity and stability of the response of ESI-MS system.

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Concentration of 80 mmol/L of NH3 in water/MeOH (50/50, v/v) mixture was chosen due to the

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volatility of the phase. The best conditions in term of peak area reproducibility were achieved at a

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flow rate of 50 µL min-1.


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The mass-chromatograms of free estrogens and mycoestrogens are shown in Figure 2a and Figure

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2b, respectively. All the examined compounds were analysed in one chromatographic run. Each

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peak is relative to the sum of the selected transitions in an analytes-free milk sample spiked at the

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lowest level considered for recovery experiments (0.05 µg/L) using the composite standard working

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solution.

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Sample preparation

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Solid phase extraction with GCB

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Milk analysis is very often challenging because a lot of interfering compounds, such as proteins and

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fats, are present. They can suppress analyte signals, especially for compounds that can be present at

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trace level. For this reason deproteinization and/or defatting are often employed before extraction.

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This could lead to analyte loss, reduced reproducibility of the method, and long and time consuming

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procedures. The use of Carbograph-4, thanks to its sorbent qualities, can avoid these limitations. It

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is essentially a non-specific and non-porous sorbent with a surface area of about 200 m2 g-1. The

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surface framework of this type of sorbents used in SPE was shown to be contaminated by oxygen

239

complexes. These groups are able to interact so strongly with sufficiently acidic compounds that

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conventional solvent systems are not able to desorb them.15,16 Because of the presence of positively

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charged chemical heterogeneities on their surface, GCB can be considered to be both reversed-

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phase sorbent and anion exchanger, showing great versatility. Therefore it exhibits the advantage of

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being able to simultaneously extract neutral, basic and acidic compounds.

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In our recent work we used Carbograph-4 for the determination of free and conjugated estrogens in

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milk.17 In this case backflush elution mode was necessary, due to the excessive retention of the

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conjugated into the sorbent.

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Here we chose to directly focus on the free estrogens and mycoestrogens, present in milk and

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potentially dangerous for human health, therefore the methodology was adapted and optimized for

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this purpose. With the optimized procedure it was possible to elute all the analytes with 11 ACS Paragon Plus Environment


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CH2Cl2/MeOH after a simple dilution of milk in water. Recovery experiments were performed at

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four concentration levels and the results are shown in Table 2. The employed methodology

252

provided very good recoveries with negligible matrix effect also at the lowest concentration level,

253

demonstrating the effectiveness of this SPE for a very complex matrix, such as whole milk.

254 255

Comparison with other extraction methods

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Searching the literature for works dealing with endocrine disruptors in milk provides several

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publications, but only very few methods addressed the determination of resorcyclic acid lactones

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and endogenous estrogenic compounds in milk. In particular most works were especially dedicated

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to the presence of free estrogens in milk and dairy products. On the other hand methods for the

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determination of ZEA and its metabolites were developed, but they comprised other mycotoxin

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compounds as well. In this context only two works described methods for their analysis in milk as

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estrogenic compounds together with mycoestrogens, by LC-MS/MS18,19 and in only one work free

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estrogens were also considered as well, but analyzed with other techniques.20

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In their work Xia et al.18 analysed the six resorcyclic acid lactones with SPE using the Oasis MAX

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cartridges in whole milk samples. This polymeric sorbent has similar properties to GCB sorbent

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because it contains quaternary amine groups on the surface, therefore it can act as reversed-phase

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sorbent and anion exchanger. For this reason we decided to verify if the cited procedure could work

268

also for simultaneous extraction of estrogens and mycoestrogens in comparison to our optimized

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procedure.

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For this purpose recovery experiments were carried out using Oasis MAX cartridges at two

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concentration levels (1 µg/L and 0.05 µg/L) following the procedure reported Xia et al.18 Results

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demonstrated that at high spiking levels recoveries range between 70 to 90%, except for E3 (only

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13%, data not shown). This can be explained probably because being E3 the most polar analyte, it

274

could be lost during cartridge washing with ACN. At lower spiking levels, which is the scenario in

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milk samples, this methodology was unable to recovery free estrogens, and also mycoestrogens 12 ACS Paragon Plus Environment

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recoveries decreased significantly. This result was attributed to the low concentration levels, in

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which case the sample pretreatment (deproteinization step) can become a crucial and limiting step;

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in fact, the unavoidable analyte loss and the inefficiency of the commercial sorbent to retain and

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elute the analytes significantly affected sample recoveries. On the other hand with the optimized

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method using GCB as sorbent it was possible to reach lower MDLs and MQLs, with negligible or

281

very low matrix effect, which did not influence analyte signals also at low concentration,

282

demonstrating the effectiveness of the clean up for all the considered compounds.

283 284

Method Performance

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The developed method showed good linearity for all the analytes investigated also in the range of

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the low concentrations with correlation coefficients ranging between 0.9991 to 1, as shown in Table

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3. In the majority of the works previously published LOD and LOQ values were calculated using

288

the signal to noise ratio of the transitions of the analyzed compounds. However when operating in

289

MS/MS mode with modern triple quadrupole mass spectrometers, the baseline noise is very often

290

quite totally absent, therefore the extrapolated LOD and LOQ values are notably underestimated

291

and not realistic.

292

For this reason the detection and quantification limits were assessed considering the standard

293

deviation of the response and the slope, i.e.: LOD = 3 σ/S, where σ is the standard deviation of the

294

response and S the slope of the calibration curve. For the estimate of σ, the standard deviation of y-

295

intercept of the regression line was used. In the same way, LOQ was estimated as 10 σ/S. For LOQ

296

measurements, for each analyte the sum of the two transitions was considered, whereas for LOD

297

measurements only the response of the second most intense SRM transition was considered. To

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assess the reliability of the results we used the standard deviation and the slope of the linear

299

regression generated from the lower calibration points (between 0.02 µg/L and 0.2 µg/L, nearer to

300

LODs and LOQs values). Then, to verify the obtained values for the developed method, blank milk

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samples were spiked at MDL and MQL concentrations. Moreover, as reported in the Commission 13 ACS Paragon Plus Environment


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Decision 2002/657/EC21, we decided that the presence of a compound is confirmed if the relative

303

intensities of their selected transitions correspond to those in the calibration standard, under certain

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conditions set, within set tolerances. For this reason the relative intensities of the selected transition

305

for all the analytes were checked and compared in each point of the standard and matrix matched

306

calibration curve for their real detection. Finally the calculated values were confirmed by spiking a

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blank sample with the found concentrations. As shown in Table 3, with this method it is possible to

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reach good detection and quantification limits, which are lower with respect to the other published

309

methods. This result is crucial for this type of analysis, especially because natural contamination of

310

estrogenic compounds is often present at trace levels.

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Analysis of milk samples

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To demonstrate its applicability, the developed method was employed to analyse different whole

314

milk samples: sixteen cow milk samples, two UHT cow milk samples, three powder cow milk

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samples, four goat milk samples, and five donkey milk samples. As for the method development,

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we chose whole milk type considered as the worst case in term of complexity and because it is also

317

known that free estrogens could be found in larger amounts in samples richer in fats. Results

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obtained in sample analysis indicated that in four of the sixteen samples of cow milk E1 was found

319

at concentrations between its MLOD and MLOQ value, and in one of them also ZEA was found

320

between its MLOD and MLOQ value. The other samples did not show any contamination.

321 322

REFERENCES

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(1)

Malekinejad, H.; Scherpenisse, P.; Bergwerff A. A. Naturally occurring estrogens in

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processed milk and in raw milk (from gestated cows). J. Agric. Food. Chem. 2006, 54, 9785-

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9791.


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(2)

Johnson, A. C.; Williams, R. J.; Matthiessen, P. The potential steroid hormone contribution of

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estrogen-mimicking compounds in food. J. Chromatogr. A 2013, 1313, 62-77.

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Zollner, P.; Jodlbauer, J.; Kleinova, M.; Kahlbacher, H.; Kuhn, T.; Hochsteiner, W.; Lindner,

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chromatography/mass spectrometry. Mass Spectr. Rev. 2012, 31, 466-503 15 ACS Paragon Plus Environment

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with


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C.;

Foglia,

P.;

Pastorini,

E.;

Samperi,

R.;

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A.

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chromatography/tandem mass spectrometric confirmatory method for determining aflatoxin

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M1 in cow milk: Comparison between electrospray and atmospheric pressure photoionization

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sources. J. Chromatogr. A 2006, 1101, 69-78.

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assay based on hot water extraction and liquid chromatography–tandem mass spectrometry

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for monitoring quinolone residues in bovine milk. Food. Chem. 2008, 108, 354-360.

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spectrometry applied to the analysis of natural and synthetic steroids in environmental waters.

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Anal. Lett. 2001, 34, 913-926.

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free estrogens and their conjugates in sewage and river waters by solid-phase extraction then

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liquid chromatography-electrospray-tandem mass spectrometry. Chromatographia 2002, 56,

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from Soil and Natural Waters Using Soil Column Extraction and Off-Line Solid-Phase

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Extraction Followed by Liquid Chromatography with UV Detection or Liquid

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Chromatography/Electrospray Mass Spectroscopy. Anal. Chem. 1998, 70, 121-130.

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Laganà A. Ultra-high-performance liquid chromatography-tandem mass spectrometry for the

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analysis of free and conjugated natural estrogens in cow milk without deconjugation. Anal.

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(18) Xia, X.;Li, X.; Ding, S.; Zhang, S.; Jiang, H.; Li, J.; Shen, J. Ultra-high-pressure liquid

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chromatography–tandem mass spectrometry for the analysis of six resorcylic acid lactones in

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S. Evaluation of the combination of a dispersive liquid–liquid microextraction method with

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micellar electrokinetic chromatography coupled to mass spectrometry for the determination of

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estrogenic compounds in milk and yogurt. Electrophoresis 2015, 36, 615-625.

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36.

389



390

FIGURE CAPTIONS

391 392

FIGURE 1 Chemical Structure, names and abbreviation of the free estrogens and resorcyclic acid

393

lactones analyzed.

394 395

FIGURE 2a Selected ion chromatograms of MRM of the investigated free estrogens. Each peak is

396

relative to the sum of the selected transitions for each estrogen in a milk sample spiked at 0.05 µg/L

397

with the composite standard working solution.

398 399

FIGURE 2b Selected ion chromatograms of MRM of the investigated resorcyclic acid lactones.

400

Each peak is relative to the sum of the selected transitions for each estrogen in a milk sample spiked

401

at 0.05 µg/L with the composite standard working solution.


Page 18 of 25

Page 19 of 25


Table 1. Full name, abbreviation, retention time, precursor and product ions and MS parameters of the analysed compounds.

Compound Abbreviation

Retention time Precursor ion (min) [M-H](m/z)

Product ion (m/z)

CE

S-Lens

Estriol (E3)

4.87

287.1

143.0 145.1

50 40

160

β-zearalanol (β-ZAL)

6.71

321.0

277.2 161.2

23 32

126

β-zearalenol (β-ZOL)

6.80

319.0

275.1 160.0

20 28

150

β-Estradiol-d3 (β-E2-d3)

7.28

274.0

143.0 145.1

60 60

181

α-zearalanol (α-ZAL)

7.29

321.0

277.2 161.2

23 32

126

β-estradiol (β-E2)

7.31

271.1

145.1 183.2

40 40

159

α-zearalenol (α-ZOL)

7.44

319.0

275.1 160.0

23 36

149

α-estradiol (α-E2)

7.56

271.1

145.1 183.2

40 40

159

Estrone (E1)

7.84

269.1

145.1 159.1

40 32

128

Zearalanone (ZAN)

8.12

319.0

275.2 205.1

22 25

127

Zearalenone-d6 (ZEA-d6)

8.12

323.0

175.1 131.1

26 33

125

Zearalenone (ZEA)

8.17

317.0

175.1 131.1

25 33

150



Page 20 of 25

Table 2. Relative recoveries (n=3 for each level) and matrix effect of free estrogens and resorcyclic acid lactones in milk samples spiked at four different concentration levels. 5 µg/L

0.5 µg/L

0.2 µg/L

0.05 µg/L

R% (RSD)

ME%

R% (RSD)

ME%

R% (RSD)

ME%

R% (RSD)

ME%

E3

93.0 (2.5)

2

81.6 (3.4)

-8

94.6 (4.0)

6

107.2 (6.4)

-2

β-ZAL

92.4 (1.4)

6

101.7 (2.0)

2

97.6 (3.3)

4

100.7 (5.2)

5

β-ZOL

85.3 (3.5)

12

90.3 (4.1)

1

100.8 (7.6)

-6

106.4 (9.0)

-4

α-ZAL

91.6 (4.0)

10

95.6 (9.6)

-1

94.4 (2.6)

2

101.1 (3.4)

2

β-E2

90.3 (2.6)

9

83.8 (6.7)

0

89.1 (7.8)

9

71.9 (5.4)

0

α-ZOL

96.8 (5.3)

5

98.2 (3.0)

8

110.2 (6.8)

-1

99.6 (4.6)

-3

α-E2

85.7 (7.1)

6

70.8 (8.2)

7

68.9 (8.1)

8

67.4 (10.2)

7

E1

91.4 (3.7)

4

82.0 (2.3)

7

100.9 (5.0)

2

96.8 (8.1)

8

ZAN

95.2 (2.5)

2

92.1 3(3.5)

0

102.3 (4.3)

10

98.0 (3.0)

6

ZEA

90.0 (2.0)

3

84.9 (4.0)

4

97.9 (3.9)

-9

99.6 (2.3)

-2

Analyte


Page 21 of 25


Table 3. Validation parameters of the proposed LC/ESI-MS/MS method for the selected compounds in milk.

Analyte

R2

R (%); RSDra (%) 0.5 µg/L

R (%); RSDRb (%) 0.5 µg/L

R (%); RSDra (%) 0.2 µg/L

R (%); RSDra (%) 0.05 µg/L

R (%); RSDRb (%) 0.2 µg/L

R (%); RSDRb (%) 0.05 µg/L

ILOD (pg inj)

ILOQ (pg inj)

MLOD (µg/L)

MLOQ (µg/L)

E3

0.9999

80.0; 6.4

77.8; 7.9

91.0; 8.5

96.0; 13.2

93.5; 13.4

90.0: 10.0

2.5

3.6

0.06

0.09

β-ZAL

0.9998

95.0; 7.0

92.3; 5.6

84.3; 22.6

78.7; 10.7

90.8; 31.1

94.8: 11.4

1.0

3.5

0.06

0.2

β-ZOL

0.9992

88.4; 6.6

83.3; 8.8

79.5; 15.2

91.0; 16.8

87.9; 18.8

93.2: 14.7

3.5

6.0

0.1

0.1

α-ZAL

0.9999

93.7; 5.3

90.1; 6.1

92.0; 13.6

89.1; 7.7

92.6; 15.7

101.0: 3.2

2.5

4.5

0.06

0.09

β-E2

0.9999

81.3; 10.2

82.8; 15.0

95.6; 7.4

90.3; 11.1

95.0; 6.8

92.0: 16.3

2.0

6.5

0.02

0.06

α-ZOL

0.9999

96.0; 3.5

94.5; 5.0

100.9; 4.8

95.8; 5.2

98.2; 5.7

97.4: 6.8

1.5

3.0

0.03

0.07

α-E2

0.9998

75.1; 9.4

88.0; 10.0

75.8; 9.3

85.7; 14.8

90.3; 14.2

92.6: 12.4

4.0

4.0

0.01

0.02

E1

0.9991

84.1; 6.7

86.0; 9.4

87.0; 8.6

94.1; 8.0

97.6; 5.1

91.0: 7.8

3.5

3.5

0.03

0.06

ZAN

1.0000

95.2; 3.3

93.3; 5.9

93.4; 7.8

88.0; 9.1

85.1; 11.4

95.5: 9.7

0.28

1.5

0.004

0.01

ZEA

1.0000

82.4; 5.1

80.8; 4.7

85.7; 6.8

91.6; 4.8

93.9; 7.2

89.4: 3.9

1.0

3.0

0.02

0.02

a) Intra-day precision n=6 within the same day b) n=6 in 6 different days; n=3 within each day

21

ACS Paragon Plus Environment


FIGURE 1

22

ACS Paragon Plus Environment

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FIGURE 2 a

b




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TOC Graphic


Simultaneous Determination of Naturally Occurring Estrogens and

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