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Determination of geochemically important sterols and triterpenols in sediments using UHPLC-MS/MS Giovana Anceski Bataglion, Eduardo C. Meurer, Ana Cecília Rizatti de AlbergariaBarbosa, Márcia Caruso Bícego, Rolf Roland Weber, and Marcos Nogueira Eberlin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01517 • Publication Date (Web): 01 Jul 2015 Downloaded from http://pubs.acs.org on July 9, 2015

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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

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Determination of geochemically important sterols

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and triterpenols in sediments using UHPLC−MS/MS

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Giovana Anceski Bataglion,*,† Eduardo Meurer,† Ana Cecília Rizzatti de Albergaria-Barbosa,§

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Márcia Caruso Bícego,‡ Rolf Roland Weber,‡ Marcos Nogueira Eberlin†

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(Unicamp), 13083-970, Campinas, SP, Brazil

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§

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40170-020, Salvador, BA, Brazil

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ThoMSon Mass Spectrometry Laboratory, Chemistry Institute, University of Campinas

Deparment of Oceanography, Geoscience Institute, Federal University of Bahia (UFBA),

Marine Organic Chemistry Laboratory, Oceanography Institute, University of São Paulo (USP),

05508-120, São Paulo, SP, Brazil

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ABSTRACT

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A fast, sensitive and selective ultra-high performance liquid chromatography tandem

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mass spectrometry (UHPLC–MS/MS) method that is able to quantify geochemical biomarkers in

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sediment is described. A pool of ten sterols, which can be used as biomarkers of autochthonous

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(cholesterol, cholestanol, brassicasterol, ergosterol), allochthonous (stigmasterol, β-sitosterol,

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campesterol and stigmastanol) and anthropogenic (coprostanol and epicoprostanol) organic

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matter (OM) and three triterpenols, lupeol and α- and β-amyrin, were chosen as the analytes. The

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method showed excellent analytical parameters, and compared with the traditional GC–MS

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methods that are commonly applied for the analysis of sterols, this method requires no sample

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clean-up or derivatization and presents improved values for the LOD and LOQ. UHPLC can

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separate the diastereoisomers, epicoprostanol, coprostanol and cholestanol, and the isomers,

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lupeol, α- and β-amyrin. The method was successfully applied for the quantification of the

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biomarkers, and thus, it was applied to assess the OM sources and the impacts of anthropogenic

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activities in sediments from different environments, such as Antarctica and other Brazilian

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systems (Continental Shelf, São Sebastião Channel and Santos Estuary). Unique profiles of the

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biomarkers were observed for the contrasting environments, and β-amyrin and cholesterol were

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more predominant in the Santos Estuary and Antarctica samples, respectively. The sterol ratios

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indicated a higher level of sewage contamination in the Santos Estuary.

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Analytical Chemistry

INTRODUCTION

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The organic matter (OM) composition in sediments is complex and includes compounds

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that are synthesized by aquatic organisms (autochthonous) and compounds that are introduced

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through the terrestrial input from the surrounding watershed via stream/river inflows

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(allochthonous).1 Classes of lipid biomarkers or specific lipids that represent different sources of

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OM have been widely used as tracers or proxies to identify their sources to the sedimentary

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record over a range of temporal and spatial scales.2-6 Some classes that are typically used as

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geochemical biomarkers are alkanes, hopanes,7-9 fatty acids, alcohols, triterpenols and sterols.10-

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Sterols are commonly used in the assessment of OM sources in aquatic environments

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when typical distributions are found in phytoplankton, terrestrial vascular plants and sewage

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matter.13-15 Cholesterol is widely distributed in many organisms; however, in aquatic sediments,

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its presence is primarily associated with algae because many species present it as the

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predominant sterol.16-18 Phytosterols, such as stigmasterol and β-sitosterol, are produced

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primarily by plants.19-21 Triterpenols (α-amyrin, β-amyrin and lupeol) are considered highly

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specific biomarkers for higher plants, and their abundant presence in sediments confirms the

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widespread input of terrestrial OM.11,19

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Fecal sterols, such as coprostanol and epicoprostanol, have been shown to be reliable

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forensic biomarkers for assessing the sewage contamination in sediments.22,23 Coprostanol

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constitutes approximately 60 % of the total sterols in human feces, while only very small

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amounts have been found in anaerobic sediments that have not been previously contaminated by

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human fecal materials and in feces from other animals.24-26 Considering that the concentrations

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of coprostanol alone may not indicate sewage contamination, a more robust and reliable

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assessment includes investigating the ratios of sterols, such as coprostanol/cholesterol,

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coprostanol/cholestanol and (coprostanol + cholestanol)/cholestanol.27-29

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To fully understand the geochemical and environmental significance of sterols,

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demanding methodologies are usually required for their detection and quantification. Sterol

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analysis in sediment samples is generally performed by gas chromatography coupled to mass

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spectrometry (GC–MS), which require a clean-up step using alumina and silica columns, where

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sterols are separated from other classes of compounds. The fraction containing the sterols is

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usually derivatized to increase the volatility and to decrease LOD, but this step prolongs the

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analysis and is the major potential source of bias and imprecision.30-33

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For the assessment of a few phytosterols and cholesterol in human serum and edible oils,

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novel analytical platforms that are based on liquid chromatography tandem mass spectrometry

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(LC–MS/MS), particularly using the multiple reaction monitoring (MRM) mode, have been

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developed.34-36 However, when using this approach, there is no comprehensive method to

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analyze the wide variety of important biomarkers in organic geochemistry.

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Therefore, the main purpose of the current study was to develop a simple, sensitive, fast

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and reliable UHPLC–MS/MS method for the analysis of major sterols and triterpenols in

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sediment samples. We have chosen 13 compounds of interest, including phyto/zooplankton,

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vascular plants and sewage discharge sterols (Table S1, Supporting Information).

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MATERIALS AND METHODS

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Standard Chemicals. (3β)-Cholest-5-en-3-ol (cholesterol; ≥ 99 % purity), (3β,5α)-Cholestan-3-

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ol (cholestanol; > 95 % purity), (3β,5β)-Cholestan-3-ol (coprostanol; > 95 % purity), (3α,5β)-

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Cholestan-3-ol (epicoprostanol; > 95 % purity), (3β,22E)-Ergosta-5,7,22-trien-3-ol (ergosterol; >

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95 % purity), (3β,22E)-Ergosta-5,22-dien-3-ol (brassicasterol; > 95 % purity), (3β,24R)-Ergost-

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5-en-3-ol (campesterol; > 65 % purity), (3β)-Stigmast-5-en-3-ol (β-sitosterol; > 98 % purity),

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(3β,22E)-Stigmasta-5,22-dien-3-ol (stigmasterol; > 95 % purity), (3β,5α)-Stigmastan-3-ol

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(stigmastanol; > 95 % purity) and (3β)-Lup-20(29)-en-3-ol (lupeol; ≥ 94 % purity) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). The α-amyrin ((3β)-Urs-12-en-3-ol) and

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β-amyrin ((3β)-Olean-12-en-3-ol) were isolated and used only to determine their retention times

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and MRM transitions.

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Based on the structural similarities between all of the sterols and their retention times,

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cholesterol-d6 was used as the internal standard (IS) for quantification of all of the compounds.

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HPLC grade ethanol, methanol, n-hexane and dichloromethane were obtained from J. T. Baker

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(Mexico City, D.F., Mexico), and the water was purified by a Milli-Q gradient system

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(Millipore, Milford, MA, U.S.A.).

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Study Area and Sampling. To test the performance of the developed method, sediment samples

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from contrasting environments, such as from Antarctica, the Brazilian Continental Shelf, the São

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Sebastião Channel and the Santos Estuary, were chosen.

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The Santos Estuary is located on the coast of the São Paulo state, Brazil. The area is

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heavily urban, and it holds the largest commercial port in the country. It is also near one of the

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important industrial complexes on the Brazilian coast.37,38 The São Sebastião Channel represents

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a marine passage between the continent and the São Sebastião Island, which is also an important

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tourism area on the coast of the São Paulo state, Brazil.3,39 The southeastern Brazilian

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Continental Shelf corresponds to an arc-shaped sector extending from 23S to 28S, where two

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main sectors may be identified: the north and south São Sebastião Island.40,41 The study area in

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Antarctica is the Admiralty Bay, which is situated on King George Island. The embayment has

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three large inlets: Martel, Mackelar and Ezcurra, where several research stations are hosted.15,22

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Eight surface sediment samples were collected using a Van-Veen in these 4 different

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environments (Table S2, Supporting Information). The samples were frozen at -18 °C and

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freeze-dried at -50 °C under a vacuum pressure of 270 mbar using a Thermo Savant ModulyoD

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freeze dryer.

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Extraction of Biomarkers from the Sediment Samples. The freeze-dried sediment samples

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were macerated, and approximately 20 g were spiked with IS solution to obtain a final

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concentration of 500 ng mL-1, and they were then extracted with 50 mL of n-hexane and

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dichloromethane (1:1, v:v). The extraction was performed using a microwave system, MarsX

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(CEM, Mathews, USA), operating at 1600 W with pressure and temperature gradients reaching

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200 psi and 85 °C in 5 min, respectively. The system was kept isothermal for 15 min, and then it

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returned to the initial pressure and temperature conditions. The organic extracts were evaporated

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to 1 mL, and an aliquot of 50 µL was diluted to exactly 1000 µL of methanol.

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UHPLC–MS/MS Method Validation. Analysis of the sterols and triterpenols in the

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sedimentary OM extracts was conducted using an UHPLC–MS/MS 8040 system (Shimadzu,

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Kyoto, Japan) that consisted of a triple quadrupole mass spectrometer equipped with an

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atmospheric pressure chemical ionization (APCI) source.

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The chromatographic separation was performed on a Shim-pack XR-ODS III 2.2 µm, 2.0

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mm i.d., 150 mm column (Shimadzu, Kyoto, Japan) using the gradient elution at 30 °C as

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follows: 0-2 min (90 % B), 2-8 min (100 % B), 8-9 min (90 % B) at a flow rate of 0.6 mL min-1.

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Solvent A was water, and solvent B was methanol. The autosampler temperature was maintained

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at 10 °C, and the injection volume was 10 µL.

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The precursor ions were determined in full scan experiments, and the [M + H − H2O]+

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species were chosen as the precursor ions instead of the traditional ones [M + H]+. The product

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ions were chosen, and the analyses were then conducted by MRM using two transitions for each

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standard compound and 20 msec of dwell time. The settings of the mass spectrometer were

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optimized for each transition (Table S3, Supporting Information). The APCI source parameters

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were as following: corona current, 4.0 µA; heat block temperature, 300 °C; desolvation line

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temperature, 250 °C; collision induced dissociation gas pressure (Ar), 224 kPa and no drying gas

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flow was used. The data were acquired by Labsolution software version 5.53 SP2.

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The calibration curves were generated in concentrations of 20, 50, 75, 100, 200, 400, 600,

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800 and 1000 ng mL-1 that contained an equal amount of IS (500 ng mL-1), and they were run in

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triplicate (intraday and interday (3)). These concentrations represent 0.2, 0.5, 0.75, 1, 2, 4, 6, 8

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and 10 ng on-column of each analyte. Both α-amyrin and β-amyrin were quantified using the

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calibration curve that was obtained for lupeol. The limit of detection (LOD) and limit of

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quantification (LOQ) of each analyte was defined according to: LOD = 3 s/S and LOQ = 10 s/S,

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respectively, where s is the standard deviation of the regression line equation, and S is the

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calibration curve slope. The intraday and interday accuracy and precision were determined using

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quality control (QC) samples prepared by spiking 50, 400 and 800 ng mL-1 of each sterol in

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sodium sulfate, and then, they were extracted using the same method as described for the

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sediment samples.

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Accuracy values that were in the range of 80−120 % and coefficients of variation (% CV)

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that were less than 20 % were considered acceptable. The extraction recoveries were assessed in

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the sediment samples by spiking them with IS prior to extraction to obtain a final concentration

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of 500 ng mL-1. The extraction recovery was calculated by the calibration curve that was

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obtained for IS, and it was considered acceptable if it was greater than 80 %.

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The solid phase extraction (SPE) was tested using Bond Elut C18 (Varian) and Oasis

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HLB (Waters) as the cartridges, but the results were similar as for those obtained by analyzing

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the extracts without treatment. Therefore, the diluted extracts were analyzed directly by

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UHPLC–MS/MS, and the elution was discarded during the first four min.

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RESULTS AND DISCUSSION

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UHPLC–MS/MS Method Validation

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The specificity of the UHPLC–MS/MS method was assessed by analyzing a mixture of

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all of the analytes in the same chromatographic run. Figure 1 shows the individual

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chromatograms corresponding to two transitions (quantification and confirmation) for each

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analyte plus the retention time, which are used as reliable identification because sterols have

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similar fragmentation patterns and the sediment sample represents a complex matrix.42-44

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Analytical Chemistry

350000 epicoprostanol/coprostanol/ 350000 cholestanol 300000 300000 371>81 250000 250000 371>95 200000 200000

500000 cholesterol 500000 369>95 400000 400000 369>135 300000 300000

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100000 1:378,90>83,00(+) 100000 ergosterol 379>69 75000 379>83 75000

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20000000 20000000 lupeol/β β-amyrin/ 17500000 17500000 α-amyrin 15000000 15000000 409>95 12500000 12500000 409>137 10000000 10000000 7500000 7500000 5000000 5000000 2500000 2500000 00 1,0 2,0 3,0 4,0 1,0 2,0 3,0 4,0

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Figure 1. Representative UHPLC–MS/MS chromatograms for all of the analytes in a standard solution mixture showing the quantification and confirmation transitions.

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Because the structures are somewhat similar and the fragmentation patterns rely on

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cleavages of the steroid skeleton, common product ions were selected for different compounds.

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However, the different precursor ions ensure proper MRM selectivity even for coeluting

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analytes. All three C27∆0 stanols (epicoprostanol, coprostanol and cholestanol) and all the

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isomeric triterpenols (lupeol, β-amyrin and α-amyrin) were properly separated.

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Although it was not the objective of this work, this method could be adapted to the

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purification of the sterol fraction present in organic extracts. This purification may be useful in

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diversified studies related to sterols. We tested the separation of sterols between 0.2 and 10 ng

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on-column of each analyte for analytical finality, but a higher total dry mass could be tested for

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other purposes.

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The calibration curves were linear over the concentration range used with determination

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coefficients (r2) that were greater than 0.998. The LODs were between 1.2 and 3.0 ng g-1, and the

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LOQs were between 3.6 and 9.0 ng g-1. Table 1 summarizes the analytical parameters (linearity,

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LOD and LOQ) of the method for all of the analytes.

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Table 1. Analytical parameters of the method (linearity, LOD and LOQ) for all of the compounds Retention LOD LOQ Compound r2 Slope time (min.) (ng g-1) (ng g-1) cholesterol 6.4 0.999 4384 ± 48 2.2 6.8 epicoprostanol 5.8 0.999 3042 ± 25 1.4 4.1 coprostanol 6.6 0.999 4622 ± 52 1.3 4.0 cholestanol 7.0 0.999 2614 ± 29 1.7 5.1 ergosterol 5.5 0.999 1935 ± 21 2.7 6.8 brassicasterol 6.1 0.998 3099 ± 45 1.6 4.8 campesterol 7.0 0.999 6133 ± 28 1.3 4.1 stigmasterol 6.8 0.999 682 ± 3 3.0 9.0 β-sitosterol 7.5 0.999 2366 ± 11 1.2 3.6 stigmastanol 8.2 0.999 1924 ± 19 2.0 6.1 lupeol 6.1 0.999 13988 ± 165 1.6 4.7

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Some previous studies that were based on the LC–MS/MS approach reported

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quantifications of a few sterols that are studied here only in human serum and edible oil samples.

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All of these studies used only one transition for each compound.34-36 Table 2 shows a comparison

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of the LOD and LOQ for sterols that were obtained with the different techniques, and the

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UHPLC–MS/MS method described herein presents similar or better LODs and LOQs with

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improved specificity, while monitoring a total of 13 of the most important sterols and triterpenols

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in geochemical and environmental analysis.

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Table 2. Comparison of the LODs and LOQs for sterols obtained using different techniques LOQ LOD Technique Sample Details Reference (ng mL-1) (ng mL-1) LC-MS/MS edible oil 2-25 10-100 Direct analysis 35 (APCI) 4 min.; 6 analytes GC-MS (EI)

sediment

50-250

Derivatization 60 min.; 11 analytes

30

LC-MS/MS (APPI)

human serum

0.25-0.68

Direct analysis 6 min.; 4 analytes

34

UHPLC-MS/MS (APCI)

sediment

1.2-3.0

3.6-9.0

Direct analysis 10 min.; 13 analytes

This work

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The accuracy and precision that were measured with the intraday and interday analyses

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were also quite satisfactory (Figure 2). The intraday accuracy and precision ranged from 90.7 to

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116.7 % and from 0.3 to 10.2 %, respectively, whereas the interday accuracy and precision

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ranged from 94.8 to 110.2 % and from 0.8 % to 6.9 %, respectively. The method recovery was

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assessed by spiking an IS that had similar characteristics to the analytes in the native samples.

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After extraction, the IS concentration was comparable to the theoretical value with recovery

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values ranging from 82.6 to 107.1 and a CV between 1.9 and 5.8 %. Thus, reliable quantification

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was demonstrated for the 13 sterols and triterpenols that were tested.

214 A)

100 80

Intraday 1 Intraday 2 Intraday 3

120 Accuracy (%)

Accuracy (%)

B)

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100 80 60 8 6 4 2 0 1

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Compound

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Figure 2. Intraday and interday accuracy and precision for the QC samples at three levels: A) 50

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ng mL-1, B) 400 ng mL-1 and C) 800 ng mL-1. Ergosterol (1), epicoprostanol (2), coprostanol (3),

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cholestanol (4), cholesterol (5), campesterol (6), stigmasterol (7), β-sitosterol (8), stigmastanol

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(9), brassicasterol (10) and lupeol (11).

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According to the analytical parameters shown, a rapid, precise and sensitive

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UHPLC−MS/MS method that can perform the simultaneous determination of a total of 13

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representative sterols and triterpenols in sediments was developed and validated.

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Field-Testing Application

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The method was then successfully applied to quantify sterols and triterpenols in sediment

225

samples from environments that were subjected to different OM sources. Table 3 lists the total

226

and individual concentrations of the sterols and triterpenols in the surface sediments from

227

Antarctica, the Brazilian Continental Shelf, the São Sebastião Channel and the Santos Estuary.

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Table 3. Concentration (ng g-1 dry weight) of sterols and triterpenols in sediment samples Concentration (ng g-1 dry weight) Compounds Ergosterol Epicoprostanol Coprostanol

Santos Estuary 2 n.d.

Antarctica 1

Antarctica 2 n.d.

São Sebastião Channel 1 n.d.

São Sebastião Channel 2 n.d.

Brazilian Continental Shelf 1 n.d.

Brazilian Continental Shelf 2 n.d.

n.d.

n.d.

8.1 ± 0.2

3.4 ± 0.5

n.d.

3.5 ± 0.4

5.1 ± 0.6

n.d.

n.d.

21.7 ± 2.0

48.2 ± 2.4

12.3 ± 1.3

n.d.

10.4 ± 0.9

6.9 ± 0.3

n.d.

n.d.

Cholestanol

63.4 ± 6.0

85.4 ± 6.8

28.2 ± 6.1

n.d.

72.2 ± 6.0

96.6 ± 7.9

15.48 ± 3.4

41.7 ± 7.5

Cholesterol

92.2 ± 17.7

167.2 ± 30.5

76.8 ± 12.9

n.d.

57.8 ± 7.9

68.7 ± 8.2

28.83 ± 6.0

135.71 ± 11.3

Campesterol

34.5 ± 3.2

45.3 ± 1.3

5.1 ± 0.3

n.d.

29.2 ± 1.6

18.9 ± 0.9

4.38 ± 0.4

28.02 ± 0.6

Stigmasterol

182.8 ± 9.3

89.4 ± 1.6

n.d.

n.d.

46.4 ± 0.6

35.2 ± 0.3

18.99 ± 1.3

71.5 ± 4.0

β-sitosterol

510.9 ± 23.3

173.5 ± 4.9

25.2 ± 2.8

n.d.

78.7 ± 3.2

66.3 ± 1.4

42.09 ± 2.3

139.27 ± 1.5

Stigmastanol

255.1 ± 15.8

76.3 ± 5.7

13.6 ± 0.9

n.d.

52.4 ± 3.3

57.8 ± 3.9

11.92 ± 1.0

61.45 ± 0.5

Brassicasterol

23.4 ± 4.1

47.2 ± 5.1

13.3 ± 1.4

n.d.

40.1 ± 1.8

36.7 ± 1.1

25.71 ± 2.0

n.d.

Lupeol

415.7 ± 36.2

70.5 ± 4.1

n.d.

n.d.

3.1 ± 0.1

2.4 ± 0.2

n.d.

2.81 ± 0.3

β-amyrin

1682.0 ± 119.9

516.9 ± 36.7

14.6 ± 0.9

n.d.

40.8 ± 1.8

25.1 ± 0.8

6.06 ± 0.2

22.23 ± 0.9

α-amyrin

103.7 ± 7.64

11.6 ± 1.6

n.d.

n.d.

5.2 ± 0.4

3.3 ± 0.3

n.d.

n.d.

− − − −

439.8 ± 28.0

423.0 ± 25.9

153. 46 ± 16.6

502.69 ± 26.6

0.13

0.07

0.18

0.10

0.14

0.07

− − −

− − −

Total

229

Santos Estuary 1 n.d.

3385.4 ± 245.1

1339.6 ± 100.9

192.5 ± 27.1

Ratio I

0.25

0.36

0.30

Ratio II

0.24

0.29

0.16

Ratio III

0.34

0.56

0.43

Ratio I: coprostanol/(coprostanol+cholestanol); Ratio II: coprostanol/cholesterol; Ratio III: coprostanol/cholestanol

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Higher total concentrations (3385.4 and 1339.3 ng g-1 dry weight) of sterols and

231

triterpenols were observed for the sediment samples from the Santos Estuary. These results are

232

consistent with a semi-closed area that is dominated by the Atlantic rain forest, mangrove forest

233

and saltmarsh, which provide large amounts of OM to the estuarine environment.38 Somewhat

234

lower total concentrations (439.8 and 423.0 ng g-1 dry weight) were observed for samples from

235

the São Sebastião Channel, which is a marine passage between the continent and the São

236

Sebastião Island39 and is thus more exposed to hydrodynamism than an estuary. Samples from

237

the Brazilian Continental Shelf presented total concentrations of 153.5 and 502.7 ng g-1 dry

238

weight. This difference is consistent with the site of these samples. Brazilian Continental Shelf 1

239

was collected farther from the continent and deeper (200 m − 308 m depth) than Brazilian

240

Continental Shelf 2 (100 m − 121 m depth). A concentration of 192.4 ng g-1 dry weight was

241

observed for sample Antarctica 1, which represents the Martel Inlet in the northern part of

242

Admiralty Bay. No sterols and triterpenols could be detected in sample Antarctica 2, which was

243

collected close to the Comandante Ferraz Brazilian Antarctic station.

244 245

Figure 3 shows the differences in the biomarker profiles between the samples from environments with distinct inputs of OM.

246 247 248 249 250 251 252

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Antarctica 1

Brazilian Shelf Brazilian Shelf

Relative Percentage (%)

Relative Percentage (%)

30 30

20

10

0 1

2

3

4

5

6

7

8

25 20 15 10 5 0

9 10 11 12

1

2

3

4

5

Compound

25

6

7

8

9 10 11 12

Compound

50

São Sebastião Channel 1 São Sebastião Channel 2

20

Relative Percentage (%)

Relative Percentage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15 10 5 0

Santos Estuary 1 Santos Estuary 2

40 30 20 10 0

1

2

3

4

5

6

7

8

9 10 11 12

1

2

3

Compound

4

5

6

7

8

9 10 11 12

Compound

253

Figure 3. Relative distribution of sterols and triterpenols in sediment samples from Antarctica,

254

the Brazilian Continental Shelf, the São Sebastião Channel and the Santos Estuary.

255

Epicoprostanol (1), coprostanol (2), cholesterol (3), cholestanol (4), brassicasterol (5),

256

campesterol (6), stigmasterol (7), β-sitosterol (8), stigmastanol (9), lupeol (10), β-amyrin (11), α-

257

amyrin (12).

258 259

The Santos Estuary presents higher a contribution of the triterpenol β-amyrin than all of

260

the sterols, indicating a predominance of OM from higher plants.11,19 Indeed, the Santos Estuary

261

is characterized by the intense input of OM from the surrounding vegetation – the Atlantic rain

262

forest, the mangrove forest and the saltmarsh.38,45 However, samples from the São Sebastião

263

Channel presented lower contributions of triterpenols and higher contributions of β-sitosterol,

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cholesterol and cholestanol. These findings are consistent with a coastal environment, which

265

receives OM from terrestrial plants and also has a high diversity of aquatic organisms.46,47 The

266

sterol profiles of the Brazilian Continental Shelf samples are quite similar to those obtained for

267

the São Sebastião Channel with lower contributions of triterpenols and similar contributions of

268

cholesterol and β-sitosterol. These results are consistent considering that the Brazilian

269

Continental Shelf samples were collected near the São Sebastião region. However, Antarctica 1

270

shows a much higher contribution of cholesterol, which agrees with the strong predominance of

271

autochthonous sources of OM in that glacier region because the vegetation is not well developed

272

and it is a remote environment.48

273

The differences in the biomarker profiles among the samples from contrasting

274

environments can also be visualized by principal component analysis (PCA) using the sterols and

275

triterpenols proportions to remove any concentration effects (Figure S1, Supporting

276

Information). A discrimination of the Santos Estuary samples was revealed by the principal

277

contribution of the β-amyrin proportion for the F1 axis, whereas a discrimination of the

278

Antarctica sample was revealed by the principal contribution of the cholesterol proportion for the

279

F2 axis. Factor 1 (F1) and factor 2 (F2) contributed to 73.4 and 14.3 %, respectively, for the

280

discrimination of the samples.

281

In addition to evaluating the profiles of sterols and triterpenols in the sediment from the

282

contrasting environments, we were also interested in demonstrating the method applicability for

283

assessing the levels of contamination by sewage discharge.

284

The highest concentration of coprostanol was found for the Santos Estuary 2 (48.2 ng g-1

285

dry weight), which is located on the estuary bank near a solid waste disposal that is used by the

286

Baixada Santista cities and is close to small towns. Santos Estuary 1, Antarctica 1 and São

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Sebastião Channel 1 and 2 presented coprostanol concentrations of 21.7, 12.3, 10.4, and 6.9 ng g-

288

1

289

present for Santos Estuary 1. Although the Antarctica region is considered one of the best-

290

preserved environments in the world, we could quantify both coprostanol and epicoprostanol in

291

one of the samples. Indeed, previous studies have shown detectable concentrations of sewage

292

markers in surface samples around the scientific stations.15,22

dry weight, respectively. Epicoprostanol was also detected for these samples, but it was not

293

There is no consensus with respect to the level of coprostanol in the sediments that is

294

indicative of sewage contamination; however, the threshold concentration in natural pristine

295

coastal sediments is usually < 10 ng g-1.27 Thus, samples from Santos were found to have

296

coprostanol concentrations that were considerably higher than the threshold value.

297

Because of the lack of a reliable quantitative criterion involving the presence of

298

coprostanol exclusively from domestic sewage, the ratios of some sterols, such as coprostanol

299

(coprostanol + cholestanol), coprostanol/cholesterol and coprostanol/cholestanol, have been

300

reported as useful indicators to evaluate the level of sewage contamination in sediments.27-29

301

Based on these ratios, sewage influence is most evident in the sediment samples from the Santos

302

Estuary (Table 1), which presented coprostanol/cholesterol values that were > 0.2 and

303

coprostanol/cholestanol values that were > 0.3. These are values that indicate that coprostanol

304

has fecal origin.28,29 This result was expected because Santos Estuary is a highly populated area

305

with solid disposal, intensive shipping and waste discharge from industries.37,38

306

However, the majority of the other samples displayed low values for these ratios,

307

suggesting an insignificant contribution from domestic sewage discharge. Regardless, permanent

308

human activities in these regions require monitoring programs to evaluate the continuing trends

309

and prevent the increase of anthropogenic impacts from sewage discharge.

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Analytical Chemistry

CONCLUSION

311

We developed a fast, sensitive and selective UHPLC–MS/MS method to quantify 13

312

geochemical biomarkers in sediments. We evaluated the sensitivity, the intraday and interday

313

accuracy and the precision and recovery, and we obtained satisfactory results. The new method

314

offers considerable performance compared with traditional GC–MS methods that have been

315

commonly applied for the analysis of sterols. This method exhibits an improved LOD and LOQ

316

and requires no sample clean-up or derivatization. Thus, the developed method presents a

317

reduction in both sample preparation time and analysis time. We tested the validated method for

318

the quantification of sterols and triterpenols in sediments from different environments for which

319

unique profiles of biomarkers were obtained, revealing their contrasting characteristics. The

320

results show the potential of the method to monitor levels of anthropogenic contamination from

321

sewage discharge in sediment samples and to assess changes in the OM cycle related to

322

environmental processes.

323

ASSOCIATED CONTENT

324

Supporting Information

325

Table S1. Description, main sources and chemical structures of the sterols and triterpenols

326

Table S2. Location of the collection sites

327

Table S3. Mass spectrometry parameters for the MRM transitions: Q1 voltage, Q3 voltage and

328

collision energy (CE)

329

Figure S1. Principal component analysis score and loading plots using the sterols and triterpenols

330

proportions.

331

This information is available free of charge via the Internet at http://pubs.acs.org/.

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332

AUTHOR INFORMATION

333

Corresponding Author

334

*Phone: 55-19-3521-3049; fax: 55-19-3521-3073; e-mail: [email protected]

335

Author Contributions

336

The manuscript was written through contributions of all authors. All authors have approved the

337

final version of the manuscript.

338

Funding Sources

339

This study was supported by FAPESP (scholarship 2012/21395-0).

340

Notes

341

The authors declare no competing financial interests.

342

ACKNOWLEDGMENT

343

The authors would like to thank FAPESP (scholarship 2012/21395-0) for sponsoring this study.

344

ABBREVIATIONS

345

UHPLC-MS/MS, liquid chromatography tandem mass spectrometry; OM, organic matter; MRM,

346

multiple reaction monitoring; APCI, atmospheric pressure chemical ionization; LOD, limit of

347

detection; LOQ, limit of quantification; CV, coefficient of variation.

348

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349

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MS).

A fast, sensitive, and selective ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method that is able to quantify ge...
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