Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high...

AnalyticalMethods

PAPER

aUnidad de Lıpidos y Riesgo Cardiovasc

Molecular, Hospital Universitario Miguel Se

Aragon, Zaragoza, SpainbI3A, Departamento de Quımica Analıtica, EsUniversidad de Zaragoza, Campus Rio Ebr

Spain. E-mail: [email protected]; Tel: +34cUnitat de Lıpids, Servei d'Endocrinolog

Biomediques August Pi i Sunyer, Hospital

Obesidad y Nutricion (CIBERobn), Instituto

SpaindProteomics Unit, Instituto de Investigacion

de Ciencias de la Salud (I+CS), SpaineDepartamento de Bioquımica y BiologıaZaragoza, Zaragoza, Spain

Cite this: Anal. Methods, 2013, 5, 2249

Received 13th November 2012Accepted 22nd February 2013

DOI: 10.1039/c3ay26395a

www.rsc.org/methods

This journal is ª The Royal Society of

Simultaneous determination of oxysterols, phytosterolsand cholesterol precursors by high performance liquidchromatography tandem mass spectrometry in humanserum

Lucıa Baila-Rueda,ab Ana Cenarro,a Montserrat Cofan,c Irene Orera,d Silvia Barcelo-Batllori,d Miguel Pocovı,e Emilio Ros,c Fernando Civeira,a Cristina Nerınb

and Celia Dome~no*b

Afast and sensitivehighperformance liquidchromatographywithatmosphericpressure chemical ionization

tandem mass spectrometry (HPLC-APCI-MS/MS) method to identify and quantify oxysterols, phytosterols

and non-cholesterol sterols at the pico-molar concentration level in human serum in only one run was

developed in this study. This method allows the simultaneous separation and quantitation of individual

phytosterols, cholesterol precursors and oxidized derivatives of cholesterol without a derivatization step

in a single run, thus providing a more confident quantitation of sterols in serum. After saponification,

solid-phase extraction (SPE) used as a clean-up step and HPLC separation, detection by MS was developed

using APCI and multiple ion monitoring modes. This method employs reversed-phase C18 SPE cartridges

and serum calibrators, as well as isotopically labelled cholesterol as an internal standard added before

sample processing. The time consumed for a single sample is reduced from the 4 hours of conventional

sterol analysis to 1 hour including the chromatographic run time. The method has been evaluated by

analyzing a certified cholesterol sample as well as by comparison to other two methods used as reference,

based on GC and enzymatic reaction, respectively. Serum from 14 individuals was successfully analyzed.

Detection limits for oxysterols, phytosterols and non-cholesterol sterols, all determined in a single run in

small serum volumes, were between 0.47 and 1.69 pM. Intra-day precision was <7% for all sterols in in-

house-made lipoprotein-deficient serum. The limits of detection showed high sensitivity with very good

intraday- and interday-precision for all sterols. The recoveries of the phytosterols, cholesterol precursors

and cholestanol ranged from 77% to 92%. These analytical parameters provide a reliable and

reproducible method for the identification and quantitation of human sterols.

1 Introduction

Sterols are lipid molecules that occur naturally in the animal andplant kingdom. The most abundant animal sterol is cholesterol,and the most frequently occurring plant sterols, also named

ular, and Laboratorio de Investigacion

rvet, Instituto Investigacion Sanitaria de

cuela de Ingenierıa y Arquitectura (EINA),

o, Marıa de Luna, 3, 50018, Zaragoza,

976765100

ia i Nutricio, Institut d'Investigacions

Clınic and CIBER Fisiopatologıa de la

de Salud Carlos III (ISCIII), Barcelona,

Sanitaria Aragon (IIS), Instituto Aragones

Molecular y Celular, Universidad de

Chemistry 2013

phytosterols, are b-sitosterol, sitostanol, campesterol and stig-masterol. All sterolshave asimilar chemical structure, a tetracycliccyclopentaphenanthrenewith an iso-octyl side-chain at carbon 17and a hydroxyl group at carbon 3. The difference between distinctsterols lies in the addition of an extra methyl, ethyl or hydroxylgroup to the sidechain.Themolecular structurecontainsadoublebond and, when hydrolyzed, saturated plant stanols are formedfrom the unsaturated molecules. Other important human sterolsare the precursors of cholesterol synthesis, such as desmosterol,lanosterol and lathosterol. These precursors are found in muchsmaller amounts than cholesterol in human serum. Humansterols also include oxidized cholesterols: 24-hydroxycholesterol,27-hydroxycholestrol and 7a-hydroxycholestenone, which areformed during the hepatic conversion of cholesterol into bileacids,1 and cholestanol, a cholesterol metabolite which isproduced directly from cholesterol oxidation.

The concentration of serum cholesterol depends on acomplex and highly regulated mechanism involving cholesterol

Anal. Methods, 2013, 5, 2249–2257 | 2249

Analytical Methods Paper

absorption, synthesis, metabolism and intestinal elimination.Phytosterols, phytostanols and cholesterol share the sameintestinal absorption and secretionmechanisms;2 hence, serumphytosterols have been extensively used as surrogate markers ofintestinal cholesterol absorption and dietary plant sterolconsumption. Serum levels of cholesterol precursors are relatedto liver cholesterol synthesis3,4 and oxysterols are markers ofcholesterol metabolism in a specic tissue or organ.5,6 Thequantitatively most abundant oxysterol in the circulation is 27-hydroxycholesterol. Due to the association between non-cholesterol sterols and cholesterol absorption, synthesis andelimination, these compounds have been proposed as a usefultool to explore the mechanisms of the variation in serumcholesterol levels, including familial forms of hyperlipidemia.7

In fact, increased levels of plasma sterols other than cholesterolcould serve as markers for abnormalities in lipid metabolismassociated with clinical disease. Premature atherosclerosis andxanthomatosis occur in two rare lipid storage diseases, cere-brotendinous xanthomatosis (CTX) and sitosterolemia. In CTX,cholestanol is present in all tissues. In sitosterolemia, dietarycampesterol and sitosterol accumulate in plasma and red bloodcells. Plasma accumulation of oxo-sterols is associated withinhibition of bile acid synthesis and other abnormalities inplasma lipid metabolism. Inhibition of cholesterol biosynthesisis associated with appearance in plasma of precursor sterols.8

Non-cholesterol sterols are difficult to analyze in their nativestate by modern instrumental analytical techniques due to theirchemical structure and low concentrations. The presence ofmuch higher concentrations of cholesterol in serum representsadditional difficulties in the simultaneous analysis of non-cholesterols. Non-cholesterol sterols have been historicallyanalyzed by gas chromatography (GC).9 GC offers high temporalresolution, allowing for the separation of sterols of similarcomposition such as positional isomers and diastereomers.Other analyses by GC rely upon derivatization of sterols on amicrober to reduce their polarity and simultaneously increasethe volatility of derivatives, which are further vaporized in theinjection port of the GC with a ame ionization detector (FID).10

However, derivatization adds additional steps to sample prepa-ration, increases instrumentmaintenance requirements and canincrease the complexity of the resulting chromatogram. A GC-MSmethod has been applied to the determination of sterols aerderivatization as pentauorobenzyl esters in human plasma.11

GC with FID,12,13 high-performance liquid chromatography(HPLC) with ultraviolet (UV) detection,14,15 HPLC with refractiveindex (RI) detection16 and ultra high performance liquid chro-matography (UHPLC)-atmospheric pressure chemical ioniza-tion (APCI)-MS for phytosterols17 are the most frequently usedmethods for the analyses of sterols. A rapid development of arange of analytical platforms, including GC, HPLC, UPLC, andCE coupled to MS spectroscopy, enables separation, detection,characterization and quantitation of such metabolites andrelated metabolic pathways.18 Liquid chromatography (LC) hasalso been applied in combination with uorescence detection(FD)19 or evaporative light-scattering detection (ELSD),20 butthese techniques need additional steps for analyses. A rapid andsensitive method involving atmospheric pressure thermal

2250 | Anal. Methods, 2013, 5, 2249–2257

desorption chemical ionization mass spectrometry (APTDCI-MS) is able to ionize sterols for qualitative and quantitativeanalysis directly from dried plasma/blood spots.21 However,most of the techniques are focused on some of the sterols andnone of the published papers deal with all sterols and theirprecursors in a single run. The possibility of having simulta-neously all the data concerning oxysterols, phytosterols andcholesterol precursors in a single run together with the absenceof matrix effects, due to the clean-up step, which is the objectiveof the present study, has the advantage of permitting themonitoring of many samples from different patients in a shortertime. The aim of our study was to develop a reproducible andsensitive HPLC-APCI-MS/MSmethod to identify and quantify allsterols, including oxysterols in only one run, at low concentra-tions in human serum without a derivatization step. One keypoint in this study is the clean-up procedure, using reversed-phase C18 cartridges, to eliminate the matrix interferences.Without the previous clean-up step it is very difficult to analyzethe minor compounds, as they can be easily overlapped by thebackground noise produced by the matrix. The use of serumcalibrators and the addition of an isotopically labelled internalstandard before sample processing facilitate the analysis.

The proposed method is compared with the enzymaticprocedure22 which is used to determine cholesterol in humanserum samples. This comparison conrmed that the newprocedure is a robust and useful tool for the determination ofnon-cholesterol sterols.

2 Materials and methods2.1 Non-cholesterol sterol standards

All sterol stock solutions were prepared in isopropanol. Ninecalibration levels were prepared by mixing and diluting thesterol stock solutions in isopropanol containing 10.5 mM ofinternal standard ([2H6] cholesterol-26,26,26,27,27,27) to nalconcentrations between 0.26 and 25.8 mM for cholestanol; 0.24and 24.3 mM for stigmasterol; 0.24 and 24.0 mM for sitostanol;0.24 and 24.1 mM for b-sitosterol; 0.23 and 23.5 mM for lano-sterol; 0.26 and 26.0 mM for desmosterol; 0.25 and 25.0 mM forcampesterol; and 0.25 and 24.9 mM for 24-hydroxycholesteroland 27-hydroxycholesterol. All sterol standards were obtainedfrom Sigma-Aldrich, Spain.

2.2 Subjects

Fourteen patients with primary hyperlipoproteinemia who wereoverweight and had not undergone lipid-lowering treatmentwere included in this study. All patients gave written informedconsent to a protocol approved by a medical ethics committee(Comite Etico de Investigacion Clınica de Aragon ECRR/00034,Spain). Serum samples from these patients were used in thisstudy.

2.3 Sample preparation

Fasting blood samples were obtained for the measurement ofnon-cholesterol sterols. Aer centrifugation at 3000 r.p.m. for20 min, serum was stored in polypropylene tubes at �80 �C

This journal is ª The Royal Society of Chemistry 2013

Paper Analytical Methods

until analysis. For the hydrolysis of non-cholesterol sterols, theserum samples were rst subjected to saponication. Serum(100 ml) was transferred to a screw-capped vial, and 6 ml ofdeuterium-labelled internal standard (7.9 mM) was added. Aerthe addition of 1 ml of freshly prepared 0.71 M ethanolicsodium hydroxide, alkaline hydrolysis was performed for 20min at 60 �C in an ultrasound bath.23 Hydrolysis reagents wereprepared immediately prior to use. Thereaer, 500 ml ofdeionized water was added. The sample was extracted twicewith 3 ml of hexane. The extract was dried at 30 �C under astream of nitrogen and reconstituted in 200 ml methanol. Thisextract was loaded onto the SPE cartridge. The C18 cartridgesused for the SPE extraction (100 mg, Discovery DSC-18, Supelco,Spain) were preconditioned with 400 ml of methanol and gravityeluted. The non-cholesterol sterols were desorbed with 1.4 ml of2-propanol by gravity and 40 microliters of the nal mixture wasinjected into the HPLC-MS/MS system.

2.4 Cholesterol determination by the enzymatic method

To determine the cholesterol concentration, three patients and acommercial serum sample were studied. These samples weretreated as previously described in Section 2.3 but they werediluted 1 : 100 for the HPLC-MS/MS analyses because serumcholesterol concentrations are 100 times higher than those ofnon-cholesterol sterols. The enzymatic method to determine thecholesterol concentration was carried out on a “Synchron LX20System” (Beckman Coulter, Fullerton, CA). A minimum samplevolume of 0.6 ml was needed for the Multi-Analyte Panel. Asample volumeof 3ml and300ml reagentwereused for individualtests. The samplewas run as part of aMulti-Analyte BiochemistryPanel. Sensitivity for cholesterol determination is 5mg dl�1 with95%condence and the analytical range is 5–750mgdl�1. In thisprocedure cholesterol esters in a sample are hydrolysed bycholesterol esterase (CHE). The free cholesterol produced isoxidised by cholesterol oxidase (CHO) to cholestene-3-one withthe simultaneous production of hydrogen peroxide (H2O2),which oxidatively couples with 4-aminoantipyrine and phenol inthe presence of peroxidase (POD) to yield a chromophore. Thered quinoneimine dye formed can be measured spectrophoto-metrically at 540/600 nm as an increase in absorbance.

2.5 Tandem mass spectrometry detection

The HPLC is coupled to a 4000 QTrap triple quadrupole massspectrometer (Applied Biosystems, Foster City, CA) through aTurboV� source equipped with either an ESI (TurboIonSpray)probe or an APCI (Atmospheric Pressure Chemical Ionization)probe. Both probes can be inserted into the source housingthrough the same port. These interchangeable probes have anorthogonal orientation to the ion inlet orice.

The source with the ESI probe is operated in positive modewith a spray voltage of 5500 V, a curtain gas of 10 psi (nitrogen),and ion Source Gas 1 and 2 set to 20 psi (both nitrogen). Gas 2 ismaintained at 500 �C. The source with the APCI probe is oper-ated in the positive mode with a curtain gas of 10 psi (nitrogen),a nebulizer current of 3 mA, an ion Source Gas 1 of 20 �C(nitrogen) and a heated temperature of 500 �C.


The settings optimized in the quadrupoles allowed ions ofwider mass distribution to pass through quadrupole 1 into thecollision cell, where ions were fragmented and resolved byquadrupole 3. The signal–noise values of the transitions wereobtained with Analyst 1.5 soware (Applied Biosystems, FosterCity,CA).Non-cholesterol sterol quantitationwasperformedwithMultiQuant 1.2 soware (Applied Biosystems, Foster City, CA).

2.6 High performance liquid chromatography separation

Sterols were separated using reversed-phase C18 HPLC. A 40 mlaliquot of the puried extract (100% 2-propanol) was loadedonto a RP-HPLC column (Zorbax Eclipse Plus C18 2.1� 150 mm,3.5 mm particle; Agilent, Spain) equipped with a guard column(C18, 4 � 2.5 mm). Aer this step, sterols were separated bygradient elution. The gradient program used for the APCIionization was 80% solvent B (100%methanol) at the beginningfor 1 minute, and thereaer ramped to 100% solvent B over a 4min period and held for 20 min. Then, within a minute solventB was at 80% and it was held for 9 minutes (solvent A: 100%HPLC grade water. Solvent B: 100% LC-MS grade methanol,Sigma-Aldrich, Spain). For testing the ESI ionization thegradient program with 100% solvent B was used (85% meth-anol, 5 mM ammonium acetate) at the beginning for 2 min, andthereaer ramped to solvent A (100% methanol, 5 mM ammo-nium acetate) over a 13 min period and held for 10 min. Thecolumn was maintained at 30� using a column oven. The owrate was 200 ml min�1 for both ionizations modes.

2.7 Gas-chromatography (GC)-FID method

Serum non-cholesterol sterol concentrations were also analyzedby gas chromatography, using a modication of a methodpreviously described by Heinemann et al.9 Briey, epi-coprostanol (0.0052 mol) was added to serum (100 ml) as theinternal standard. Aer alkaline hydrolysis, extraction andderivatization to trimethylsilyl ethers, the sterols were quanti-ed by gas chromatography on a 30 m nonpolar capillarycolumn (TRB-Esterol; Teknokroma, Barcelona, Spain) with aPerkin-Elmer GC Autosystem (Perkin-Elmer, Norwalk, CT) and aFID detector. In each run, lanosterol, cholestanol, campesterol,b-sitosterol, and stigmasterol were quantied.

Calculation of sterol concentrations was performed usingepicoprostanol as internal standard by one-point calibration.The area of the corresponding sterol peak in the chromatogramis divided by the area of epicoprostanol and multiplied by theamount of epicoprostanol added to the sample and by theresponse factor. To validate this calculation method we usedstandard curves for each sterol with epicoprostanol as internalstandard.

2.8 Statistical analysis

Analyses were performed using SPSS (version 15.0, SPSS Inc.,Chicago, IL). All data are presented as means and standarddeviations (SD). Normal distribution was assessed with theKolmogorov–Smirnov test. Correlation coefficients between GCand HPLC-MS/MS data were determined using a bivariatecorrelation test.

Anal. Methods, 2013, 5, 2249–2257 | 2251


2.9 Analytical features

To test the matrix effects, the recovery experiments were per-formed using in-house-made lipoprotein-decient serum(LPDS). LPDS controls containing 7.7 mM of cholestanol, 7.3 mMof stigmasterol, 7.2 mM of sitostanol, 7.2 mM of b-sitosterol, 7.8mM of lanosterol, 7.8 mM of desmosterol, 7.5 mM of campesterol,7.5 mM of 24-hydroxycholesterol and 27-hydroxycholesterol, and7.9 mM of internal standard were used to calculate accuracy.Within- and between-day precisions were determined using anin-house-made LPDS control. Five replicates of the same samplespiked at two different concentration levels (approx. 7–15 mM foreach sterol) were analyzed to calculate RSD values. Limits ofdetection (LOD) and limits of quantitation (LOQ) were deter-mined by injecting serial dilutions of the lowest calibration limitof the linear range. For theLOD, the concentration giving a signalto noise (S/N) ratio of 3 in 50% of the analyzed replicates wasused. For LOQ the S/N ratio applied was 10.

3 Results and discussion3.1 Separation of individual cholesterol related compounds

A rapid HPLC-MS/MSmethod for the simultaneous quantitation offree cholestanol, stigmasterol, sitostanol, b-sitosterol, lanosterol,desmosterol, campesterol, 24-hydroxycholesterol, and 27-hydrox-ycholesterol in serum samples is described in this work. In partic-ular, these sterols have been chosen for the study because they arethemost representative sterols of cholesterolmetabolism, see Fig. 1.

3.2 Optimization of detection parameters

Electrospray ionization (ESI) is a broadly applicable method forpolar compounds in a wide range of molecular weights. In

Fig. 1 MRM ion chromatogram derived from the analysis of a mixture of commonintensities and retention times are represented.

2252 | Anal. Methods, 2013, 5, 2249–2257

addition, this ionization source is generally exchangeable in thesame mass spectrometer with an APCI source that is applicableto less polar and low molecular weight compounds. Thus, ESIand the complementary use of APCI have recently become thestandard ionization methods for HPLC-MS. Since sterols areless polar and relatively low molecular weight compounds withhigh gas phase ionization potentials, we selected APCI for thisanalysis by HPLC-MS/MS. However, the presence of the hydroxylgroup in the sterol molecules led us to compare the ESI sourceto APCI for their detection.24 Table 1 shows the results of theMRM transitions of the two ionization sources by infusingindividual sterol standards at the same concentration. Con-cerning the MRM transitions, different behaviour of thecompounds was observed in ESI. Most of the compounds werepreionized but stigmasterol showed an adduct with NH4

+

present in the mobile phase. Cholestanol and sitostanol lost thehydroxyl group (17 units). In all cases the MRM transitions werereproducible and unequivocally related to each individualcompound. An APCI source was chosen for the study aerobserving signals one order of magnitude better than thosefrom the ESI detection. MRM pairs were chosen by infusingindividual sterols into the MS and generating product ions MS/MS from which these values were selected and optimized.Individual transitions, optimal declustering potentials, colli-sion energies and retention times are given in Table 2. The ionsmonitored resulted in the form [M + 1 � 18]+, corresponding tothe loss of the hydroxyl group of the sterols as a water molecule,which is common in chemical ionization of alcohols.25

Some sterols such as 24-hydroxycholesterol and desmosterolshowed the same MRM transitions, but this was not a problembecause they were separated in the HPLC. Fragmentation of

sterols. Corresponding peak colours are given in the right top of the figure. Peak


Table 1 MRM transitions and intensity of the two ionization sourcesa

Common name

ESI APCI

MRM transitions Intensity (cps) MRM transitions Intensity (cps)

Cholestanol 371.3/129.0 15 � 103 371.3/81.0 22 � 104

371.3/67.0 29 � 103 371.3/95.1 23 � 104

Stigmasterol 429.1/73.0 15 � 103 395.4/55.1 6 � 104

429.1/340.9 22 � 103 395.3/91.1 7 � 104

Sitostanol 399.3/57.0 1 � 103 399.4/81.1 13 � 104

399.3/69.1 1 � 103 399.4/95.1 13 � 104

b-Sitosterol 415.7/73.0 8 � 103 397.4/81.1 31 � 104

415.7/326.9 6 � 103 397.4/147.1 45 � 104

Desmosterol 385.3/73.0 3 � 103 367.4/91.4 21 � 104

385.3/295.0 4 � 103 367.4/95.1 23 � 104

Campesterol 401.9/283.3 2 � 103 383.4 117 � 104

382.5/323.3 3 � 103 383.4/81.2 13 � 104

Lanosterol 427.9/91.0 7 � 104 409.4/95.0 9 � 104

427.9/119.1 40 � 104 409.4/191.1 17 � 104

24-Hydroxycholesterol 367.3/59.3 28 � 104 367.4/81.1 3 � 104

367.3/105.2 14 � 104 367.4/91.1 3 � 104

27-Hydroxycholesterol 367.3/114.9 7 � 103 385.4/67.0 17 � 104

367.3/91.1 8 � 103 385.4/81.1 16 � 104

a Concentration of each compound: 7.7 mM of cholestanol, 7.3 mM of stigmasterol, 7.2 mM of sitostanol and b-sitosterol, 7.8 mM of lanosterol anddesmosterol, 7.5 mM of campesterol, 24-hydroxycholesterol and 27-hydroxycholesterol. ESI, electrospray ionization; APCI, atmospheric pressurechemical ionization; MRM, multiple reaction monitoring.

Table 2 Molecular weight and instrumental parameters of studied sterols. Selected ion monitoring and multiple reaction monitoring signal to noise and intensitya

Common name MW RT (min) DP (V) CE (V) Dwell time (ms)

SIM MRM

Ion S/N Transition S/N

Cholestanol 388.7 21.3 81 57 100 371.3 2977 371.3/81.0 48481 47 100 371.3/95.1 505

Stigmasterol 412.7 21.5 71 83 100 395.3 500 395.4/55.1 87971 93 100 395.4/91.1 718

Sitostanol 416.7 24.3 81 49 100 399.4 1072 399.4/81.1 208081 55 100 399.4/95.1 7920

b-Sitosterol 414.7 22.9 51 35 100 397.3 771 397.4/81.1 111051 65 100 397.4/147.1 1383

Desmosterol 384.6 18.2 76 93 100 367.3 264 367.4/91.1 154976 49 100 367.4/95.1 1492

Campesterol 400.7 21.5 81 57 100 383.3 3262 383.4 326281 47 100 383.4/81.1 723

Lanosterol 426.7 21.0 61 19 100 409.4 1824 409.4/95.0 20761 47 100 409.4/191.1 655

24-Hydroxycholesterol 402.7 12.2 61 83 100 367.3 343 367.4/81.1 158361 57 100 367.4/91.0 1599

27-Hydroxycholesterol 402.7 12.3 56 55 100 385.3 390 385.4/67.0 106756 83 100 385.4/81.1 1107

Cholesterol 6D 392.7 20.0 51 35 100 375.3 202 375.4/81.2 51085 53 100 375.4/147.1 547

a MW,molecular weight; MRM,multiple reactionmonitoring; CE, collision energy; DP, declustering potential; RT, retention time. S/N, signal–noisevalue. Sterol concentrations: 25.8 mM for cholestanol, 24.3 mM for stigmasterol, 24.0 mM for sitostanol, 0.15 mM for sitosterol, 2.6 mM fordesmosterol, 12.5 mM for campesterol, 11.7 mM for lanosterol, 12.4 mM for 24-hydroxycholesterol and 27-hydroxycholesterol and 2.6 mM fordeuterated cholesterol.


sterols is very complex because of their polycyclic structure.However, the signal-to-noise ratios of MRM transitions, whichinvolve fragmentation of sterols, were better in most cases thanthose of selected ion monitoring (SIM), which did not involvefragmentation26 (Table 2). However, when injecting a lower


concentration of each compound the background noise alsodecreased and the ratio S/N was much better than thatexpressed in Table 2. A crucial point in the development of themethod was the chromatographic separation of 24-hydrox-ycholesterol and desmosterol, because both analytes have the

Anal. Methods, 2013, 5, 2249–2257 | 2253

Table

3Precisiondata,

calib

rationcu

rves,linea

rdyn

amicrange,

detectionlim

itan

dquan

titationlim

itofthedifferen

tsterolsa

Analyte

Intra-da

yprecision(RSD

%)

Inter-day

precision(RSD

%)

Calibration

curve

Lineardy

nam

icrange

(mM)

Detection

limit(pM)

Qua

ntitation

limit(pM)

Con

centration

adde

d(mM)

Con

centration

foun

d(mM)

Total

recovery%

Cholestanol

3.5

8.3

y¼

0.43

618x

+0.00

560,

R2¼

0.98

90.26

–25.9

0.95

4.05

7.7

6.8

88Stigmasterol

3.7

8.5

y¼

0.26

326x

+0.00

465,

R2¼

0.99

30.24

–24.3

1.12

4.05

7.2

5.7

79Sitostan

ol2.5

10.7

y¼

0.38

388x

+0.00

345,

R2¼

0.99

50.24

–24.2

0.77

4.05

7.2

6.2

87b-Sitosterol

3.0

7.5

y¼

0.98

095x

+0.01

511,

R2¼

0.99

20.24

–24.2

1.06

4.05

7.2

6.7

92Desmosterol

4.1

3.8

y¼

0.60

079x

+0.00

760,

R2¼

0.99

10.26

–26.4

0.86

4.05

7.9

6.9

88Cam

pesterol

2.3

10.5

y¼

1.15

511x

+0.02

386,

R2¼

0.98

60.30

–23.6

0.58

4.05

7.5

6.1

81La

nosterol

4.7

3.8

y¼

1.22

438x

+0.00

996,

R2¼

0.99

40.23

–23.4

0.47

4.05

7.1

5.4

7724

-Hyd

roxych

olesterol

7.4

12.5

y¼

0.42

320x

+0.00

539,

R2¼

0.98

90.30

–24.8

1.49

4.05

7.5

4.9

6627

-Hyd

roxych

olesterol

7.0

16.8

y¼

1.05

979x

+0.00

157,

R2¼

0.98

60.25

–24.9

1.69

4.05

7.4

4.4

59

aRSD

,relativestan

dard

deviation.


same molecular mass and behave identically through the ioni-zation process. Sterols eluted from the column between 10 and25 min and in general more hydrophilic oxysterols, such ashydroxycholesterols, eluted before cholesterol. The latesteluting sterol was sitostanol, the most hydrophobic sterol. Allother detected sterols eluted between these two extremes.Widths were generally less than 30 seconds at the full-width,half maximum setting, which gave the best resolution. Allsterols had at least two MRM pairs for a single compound. Thisadditional MRM pair added a higher condence for compoundidentication based on relative intensity. An MRM ion chro-matogram of a mixture of sterols is shown in Fig. 1. As can beseen, most sterols are separated in the chromatographiccolumn and the use of specic mass fragments also facilitatesthe unequivocal identication and quantitation of others, whenthe separation is not possible. This way, their individualquantitative determination is now possible.

3.3 Clean-up step by SPE

Chromatographic columns can be easily blocked by the lipidscontained in serum. Lembcke et al. in 2004 proposed to quan-tify free and steried phytosterols in human serum by directinjection of the sample aer the saponication and cyclohexaneextraction into the HPLC-APCI-MS/MS. However, without theSPE clean-up, the matrix interferences were very high whenanalyzing many samples, although it was not a problem for onesingle analysis.27 To apply a clean-up step extends the life of thecolumn and that of the rest of the instruments. In the presentprocedure, a C18 SPE cartridge, previously optimized bystudying the performance of several stationary phases, has beenused. A critical issue in this step is the washing solvent andelution time of the cartridge for each sample. The efficiency ofthe whole process was also evaluated. The use of a solid phaseextraction (SPE) produced a cleaner extract than without theSPE. The procedure without the SPE yielded values similar tothose obtained by the addition of the SPE method but theanalytical system was dirty aer a few injections and long serialanalysis was not possible due to the matrix interference. Withthis additional step the extract was clean enough to injectseveral samples online into the HPLC-MS/MS. Under theseconditions the matrix effect was negligible. This is an importantachievement, as in these conditions the simultaneous analysisof oxysterols, cholesterol precursors and phytosterols can beperformed and many samples can be sequentially analysed.

The recoveries of non-cholesterol sterols using this proce-dure were studied with and without the SPE step, but includingin all cases the hydrolysis step. For this purpose de-lipiedserum spiked with the non-cholesterol sterols was used as wellas the [2H6]-cholesterol as internal standard. All sterols wereanalyzed. Recoveries of sterols were in the range of 77–92% andcalculated accuracy was about 85%, as can be seen in Table 3.

3.4 Analytical features

The intra-day and inter-day variations were lower than 11% forall sterols in LPDS, except for 24-hydroxycholesterol and 27-hydroxycholesterol, for which the inter-day variation was 12%

2254 | Anal. Methods, 2013, 5, 2249–2257 This journal is ª The Royal Society of Chemistry 2013

910

11RSD

%

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

2.18

0.24

5.24

0.31

2.81

0.24

3.5

7.4

2.16

0.10

3.68

0.15

3.05

0.06

3.7

6.7

2.19

0.65

4.07

1.57

2.26

0.66

3.0

22.65

0.69

4.76

1.35

2.72

0.73

4.1

3.1

1.73

0.80

3.25

1.85

2.37

0.96

2.3

1.9

0.15

0.05

0.13

0.08

0.15

0.06

4.7

14.9

0.12

—0.26

—0.12

—2.5

—0.45

—0.21

—0.29

—7.4

—0.08

—0.17

—0.08

—7.0

—


and 17%, respectively. Calibration curves for all sterols showeda linear range of two to three orders of magnitude with corre-lation coefficients around 0.99, except for cholestanol (0.989),campesterol (0.986), 24-hydroxycholesterol (0.989), and 27-hydroxycholesterol (0.986). All quantitation limits were belowtheminimum concentration reported in serum according to theliterature (Table 3).

Because of the high sensitivity of the MS/MS detection, thelaborious derivatization procedures required for GC, such aspreparation of volatile and thermostable derivatives, can beomitted. The time consumed in the procedure for a singlesample could be reduced from 4 h to 1 h. This complex samplepre-treatment represents the limiting step in GC analysis.11,28

MS represents a sensitive and selective detection technique thatis efficient for the analysis of biological samples. Coupling ofHPLC with MS offers the advantage that sterols can be analyzedin the extracts as hydrolyzed sterols.27

Table

4Individual

values

ofserum

concentrationsofnon-cholesterolsterols(10�

2mM)determined

byHPLC-M

S/MSan

dGC-FID

in11

subjectsa

10�2mM

12

34

56

78

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

HPL

C-

MS/MS

GC

Cholestanol

3.66

0.26

6.27

0.31

2.05

0.21

2.78

0.26

8.52

0.35

5.22

0.21

3.73

0.29

5.57

0.26

Stigmasterol

1.90

0.09

1.06

0.12

3.48

0.09

0.23

0.07

3.91

0.08

3.99

0.16

3.33

0.08

4.25

0.12

b-Sitosterol

8.80

0.91

5.44

0.49

14.63

0.62

1.53

0.49

19.96

3.13

4.69

1.57

3.54

0.97

4.10

1.11

Desmosterol

4.21

0.87

4.64

0.79

4.93

1.30

3.23

0.67

6.84

1.46

5.75

1.30

4.26

0.89

5.63

1.19

Cam

pesterol

2.38

1.15

1.96

0.81

1.83

0.87

1.22

0.59

6.04

2.81

3.14

1.68

2.24

0.94

3.14

1.42

Lanosterol

0.18

0.06

0.24

0.09

0.37

0.13

0.12

0.04

0.58

0.16

0.26

0.13

0.23

0.08

0.22

0.10

Sitostan

ol0.09

—0.03

—0.12

—0.01

—0.19

—0.31

—0.22

—0.27

—24

-Hyd

roxych

olesterol0.14

—0.17

—0.08

—0.05

—0.17

—0.17

—0.21

—0.18

—27

-Hyd

roxych

olesterol0.09

—0.18

—0.08

—0.07

—0.14

—0.18

—0.11

—0.18

—

aGC,G

asch

romatog

raph

y;HPL

C-M

S/MS,

highpe

rforman

celiqu

idch

romatog

raph

ymasssp

ectrom

etry.

3.5 Evaluation of the method

3.5.1 Comparison between GC-FID and HPLC-MS/MSmethods in 11 patients. Serum sterols from 11 patients wereanalyzed by GC-FID and HPLC-MS/MS to validate this newtechnique. Table 4 shows the individual values of serumconcentrations of non-cholesterol sterols (10�2 mM) deter-mined by HPLC-MS/MS and GC-FID. The non-cholesterol sterolconcentrations obtained by HPLC-MS/MS are in the rangesaccording to the literature reported.8,29 HPLC-MS/MS measure-ments obtained were higher than GC-FID values. The highervalues obtained by using the HPLC-MS/MSmethod are probablydue to the fact that, unlike GC-FID, this method does notrequire the derivatization step, and it is well known that therecovery in the derivatization reaction applied to the GC-FIDmethod is limited.9 In the GC-FID method, high temperatureduring GC-FID separation may have caused degradation of therelatively thermolabile steroid structure of the sterols. Accord-ing to the review of Busch and King,30 labeled cholesterolshould be used to identify the likely analytical artifacts duringthe analysis. In this paper labeled cholesterol has been used asan internal standard in all studies, so that the values obtainedby the HPLC method are not overestimated. Unfortunatelyother labeled sterols were not available, unless specicsynthesis was performed, but this task was outside the scope ofthis work. In contrast, the GC-MS method did not use thelabeled cholesterol standard. We cannot be sure about theunderestimation of the quantitative values obtained by GC-MS.At any rate, the GC-FID method involves a derivatization stepand has less sensitivity and probably leads to a higher error forsmall concentrations of sterols than HPLC-MS/MS.

The developed extraction method lasts for 45 minutes, whilethe conventional one (GC-FID), currently applied for choles-terol, takes more than 3 hours. Although this method is wellestablished, it has the disadvantage of being laborious and isnot suited to detecting changes in cholesterol synthesis occur-ring within the time scale of less than a few days.8 Moreover, themethod requires that the subjects be in a steady state withregard to cholesterol metabolism. For these reasons, alternative

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 2249–2257 | 2255

Table 5 Serum concentration of cholesterol (mM) obtained by using the enzymatic method and the HPLC-MS/MS method in three subjects and in a standardreferencea

12 13 14Certied serumsample RSD%

HPLC-MS/MS Enzymatic





Cholesterol (mM) 3.81 6.42 4.27 6.42 5.00 8.34 3.55 3.96 3.5 2.5

a HPLC-MS/MS, high performance liquid chromatography mass spectrometry.


methods have been sought to monitor whole body cholesterolsynthesis in which the non-cholesterol sterols play a role. TheHPLC-MS/MS developed here is able to analyze all the non-cholesterol sterols in a single run.

Concerning the concentration of non-cholesterol sterolsfound in serum, it is worth emphasizing that these levels canbe affected by several factors such as the intake of plantsterols in the general population. Since these factors mayhave a considerable effect on the levels of plant sterols withinthe blood, it is essential that they are controlled in studiesseeking to determine the relationship between circulatingplant sterol levels and cholesterolemia risk.29 Future work onthis issue can be carried out by applying the proceduredeveloped here for the individual analysis of non-cholesterolsterols.

3.5.2 Comparison between the enzymatic and the HPLC-MS/MS methods. As a certied serum sample for all sterols isnot available, a sample of serum with a certied concentrationof cholesterol (3.96 � 2.5 mM) was also analyzed by using theHPLC-MS/MS method proposed here and by the enzymaticmethod. Three replicates were analyzed and the concentrationfound by HPLC-MS/MS was 3.55 � 3.5 mM. There were nostatistically signicant differences between the values providedby the twomethods. Data in Table 5 show the values obtained inboth methods.

Cholesterol was also analyzed in the serum from threedifferent subjects with a well standardized enzymatic methodand the HPLC-MS/MS method proposed here. Table 5 providesthe values obtained by the twomethods. The data show a highervalue obtained by using the enzymatic method than by HPLC-MS/MS in the serum from the patients. These differences, notobserved when working with the certied cholesterol sample,could be attributed to the presence of other sterols in the realserum samples. The quantitation by LC-MS/MS is based on thecharacteristic MS fragments, thus it is not possible to over-estimate the concentration found. These results emphasize theimportance of a method that can provide the individualconcentration of each sterol to avoid overestimation and wrongdiagnosis.

4 Conclusions

The new HPLC-MS/MS analytical method reported hereinallows a simultaneous, reproducible, precise and sensitivedetermination of non-cholesterol sterols, including oxysterols,

2256 | Anal. Methods, 2013, 5, 2249–2257

phytosterols and cholesterol precursors, in a single run, usingsmall serum volumes without extensive sample pre-treatment.Moreover, because this new method could be easily automated,it is feasible to run many clinical samples in a short time. Thismethod could be especially suited for large scale clinical studiesto monitor sterols in different situations, such as the assess-ment of cholesterol homeostasis, adherence to dietary phytos-terol intake, or the evaluation of the lipid lowering effects ofdrugs. Furthermore, non-cholesterol sterols in serum seem tobe a useful tool for the differential diagnosis of genetic hyper-cholesterolemias. The combination of the SPE clean-up stepand the powerful HPLC-MS/MS provides the required tool for aclear and fast diagnosis that could prevent the risk of heartattack and the serious diseases derived fromhypercholesterolemia.

Abbreviations

GC
Gas chromatography FD Fluorescence detection ELSD Evaporative light-scattering detection APCI Atmospheric pressure chemical ionization LPDS Lipoprotein-decient serum IUPAC International Union of Pure and Applied Chemistry LDLR Low density lipoprotein receptor APOE Apolipoprotein E SPE Solid phase extraction RP-HPLC
Reversed phase high-performance liquidchromatography

MRM
Multiple reaction monitoring SIM Selected ion monitoring.
Acknowledgements

This research has been supported by Project PI037/09 andGrupo T-10 (Grupo Universitario de Investigacion Analıtica,GUIA) from Gobierno de Aragon and PS09/00673, PI09/00665and PI10/00387 from Fondo de Investigacion Sanitaria (FIS),Instituto de Salud Carlos III (ISCIII), Spain Grupo Consolidadode Investigacion-Gobierno de Aragon. Centro de InvestigacionBiomedica en Red Fisiopatologıa de la obesidad y nutricion(CIBERobn) is an initiative of Instituto de Salud Carlos III,Spain.



References

1 I. Bjorkhem, M. Boberg and E. Leitersdorf, in Inborn Errorsin Bile Acid Biosynthesis and Storage of Sterols other thanCholesterol, The Metabolic and Molecular Basis of InheritedDisease, New York, 1995, pp. 2961–2981.

2 E. Ros, Atherosclerosis, 2000, 151, 357–379.3 R. S. Tilvis and T. A. Miettinen, Am. J. Clin. Nutr., 1986, 43,92–97.

4 T. A. Miettienen, R. S. Tilvis and Y. A. Kesaniemi, Am. J.Epidemiol., 1990, 131, 20–31.

5 C. Galman, I. Arvidsson, B. Angelin and M. Rudling, J. LipidRes., 2003, 44, 859–866.

6 L. Iuliano, F. Micheletta, S. Natoli, S. G. Corradini,M. Iappelli, W. Elisei, L. Giovannelli, F. Violi andU. Diczfalusy, Anal. Biochem., 2003, 312, 217–223.

7 A. L. Garcıa-Otın, M. Cofan, M. Junyent, D. Recalde,A. Cenarro, M. Pocovı, E. Ros and F. Civeira, J. Clin.Endocrinol. Metab., 2007, 92, 3667–3673.

8 A. Kuksis, J. Chromatogr., A, 2001, 935, 203–236.9 T. Heinemann, G. Axtmann and K. Bergmann, Eur. J. Clin.Invest., 1993, 23, 827–831.

10 C. Dome~no, B. Ruiz and C. Nerin, Anal. Bioanal. Chem., 2005,381, 1576–1583.

11 A. Sevanian, R. Seraglia, P. Traldi, P. Rossato, F. Ursini andH. Hodis, Free Radical Biol. Med., 1994, 17, 397–409.

12 I. Bjorkhem, T. Miettinen, E. Reihner, S. Ewerthv, B. Angelinand K. Einarsson, J. Lipid Res., 1987, 28, 1137–1143.

13 F. Kardani, A. Daneshfar and R. Saharai, Anal. Chim. Acta,2011, 28, 232–237.

14 P. B. Hylemon, E. J. Studer, W. M. Pandak, D. M. Heuman,Z. R. Vlahcevic and Y. L. Chiang, Anal. Biochem., 1989, 182,212–216.


15 B. Ruan, N. Gerst, G. T. Emmons, J. Shey andG. J. Schroepfer, Jr, J. Lipid Res., 1997, 38, 2615–2626.

16 T. Saldanha, A. C. Sawaya, M. N. Eberlin and N. Bragagnolo,J. Agric. Food Chem., 2006, 54, 4107–4113.

17 B. Y. Lu, Y. Zhang, X. Q. Wu and H. Shi, Anal. Chim. Acta,2007, 588, 50–63.

18 A. H. Zhang, H. Sun, P. Wang, Y. Han and X. J. Wang, Analyst,2012, 137, 293–300.

19 Y. T. Lin, S. S. Wu and H. L. Wu, J. Chromatogr., A, 2007,1156, 280–287.

20 P. Breinholder, L. Mosca and W. Lindner, J. Chromatogr., B:Anal. Technol. Biomed. Life Sci., 2002, 777, 67–82.

21 G. Paglia, O. D'Apolito, M. Gelzo, A. Dello Russo andG. Corso, Analyst, 2010, 135, 789–796.

22 Current status of blood cholesterol measurement in clinicallaboratories in the United States: a report from theLaboratory Standard Panel of the National CholesterolEducation Program. Clin. Chem.1988, 34, 193–201.

23 M. Orozco-Solano, J. Ruiz-Jimenez and M. Luque de Castro,J. Chromatogr., A, 2010, 1227–1235.

24 J. G. McDonald, M. B. Thompson, C. E. McCrum andW. D. Russell, Methods Enzymol., 2007, 432, 145–170.

25 M. E. Rose, Interpretation of Mass Spectra, in RapidCommunications in Mass Spectrometry, University ScienceBooks, Mill Valley, 4th edn, 1994.

26 M. Pelillo, G. Galletti and G. Lercker, Rapid Commun. MassSpectrom., 2000, 14, 1275–1279.

27 J. Lembcke, U. Ceglarek, G. M. Fiedler, S. Baumann,A. Leichtle and J. Thiery, J. Lipid Res., 2005, 46, 21–26.

28 S. Dzeletovic, O. Breuer, E. Lund and E. Diczfalusy, Anal.Biochem., 1995, 225, 73–80.

29 Y. Chan, K. A. Varady, Y. Lin, E. Trautwein, R. P. Mensink,J. Plat and P. Jones, Nutr. Rev., 2006, 64, 385–402.

30 T. P. Busch and A. J. King, Anal. Biochem., 2009, 388, 1–14.

Anal. Methods, 2013, 5, 2249–2257 | 2257

Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high...

Documents

Transcript of Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high...