determination of ten steroid hormones in animal origin food

Analytical Methods

Simultaneous determination of ten steroid hormones in animal origin food by matrix solid-phase dispersion and liquid

chromatography–electrospray tandem mass

spectrometry

Ya-bing Fan 1,Yu-Min Yin 1,Wen-Bin Jiang,Yi-Ping Chen,Jia-Wei Yang,Jia Wu,Meng-Xia Xie ?

Analytical &Testing Center of Beijing Normal University,Beijing 100875,China

a r t i c l e i n f o Article history:

Received 19January 2013

Received in revised form 4May 2013Accepted 15June 2013

Available online 3July 2013Keywords:

Matrix solid-phase dispersion Steroid hormones UPLC–MS/MS

Complex food matrices

a b s t r a c t

An UPLC–MS/MS method for determination of ten steroid hormones in animal origin food has been devel-oped with pretreatment of the samples by matrix solid-phase dispersion (MSPD).The MSPD conditions,including the dispersing sorbents,elution solvents,ratio of sorbent to sample and the volume of the elu-tion solvent have been investigated and optimised,and the method has been evaluated and validated.The results showed that the developed method has satisfactory linearity between the MS/MS responses of the analytes and the concentration of the steroid hormones,and the limits of the detection can reach 0.01l g/kg for most of the analytes.The spiking recoveries of the steroid hormones in chicken,pork,beef and sausage samples were between 76.8%and 98.7%with RSDs lower than 10%.The results demonstrated that the developed approach has high sensitivity and repeatability,and can rapidly determinate the trace residues of steroid hormones in complex food matrices.

ó2013Elsevier Ltd.All rights reserved.

1.Introduction

Steroid hormones are endocrine-disrupting chemicals with bio-logical activity,and they have been illegally abused in animal hus-bandry for several decades due to their effects on the improvement in feed conversion ef?ciency and growth promotion.However,many evidences have indicated that the steroid hormones in ani-mal origin food have potential toxic and carcinogenic on human health,and their residues,as well as their metabolites,may be rel-evant to a variety of diseases including cancer of the breasts,ova-ries and prostate (Krieger,2008),therefore,the illicit uses of these compounds have been restricted and banned in European and many other countries.So development of sensitive and reliable analytical methods for monitoring the residuals of steroid hor-mones in foodstuffs is particularly required.

Immunoassays have been traditionally used for the determina-tion of steroid hormones (Hampl &Starka,1989),whereas they lacked the function of structural validation for the target analytes (Sokoll,Wians,&Remaley,2004)and cannot simultaneously deter-mine multi-residues in complex matrices.Gas chromatography coupled to mass spectrometry (GC–MS)has been early adopted for the quantitative determination of steroid hormone residues in

foodstuffs (Daeseleire,Vandeputte,&Van Peteghem,1998).Unfor-tunately,this technique generally requires pre-derivatization pro-cesses for the analytes (Trinh,Harden,Coleman,&Khan,2011),which would be tedious,time consuming,and moreover,not all components can be easily derivatized.High performance liquid chromatography (HPLC)combined with various mass spectromet-ric techniques,including reactive Desorption Electrospray ioniza-tion (Huang,Chen,Zhang,Cooks,&Ouyang,2007),atmospheric pressure chemical ionisation and atmospheric pressure photoioni-zation (Leinonen,Kuuranne,&Kostiainen,2002),accurate mass time-of-?ight,Fourier transform ion cyclotron resonance (Nielen,van Engelen,Zuiderent,&Ramaker,2007;Song,Li,Zeng,Liu,&Xie,2011),linear ion trap mass spectrometry (Strahm,Saudan,Sottas,Mangin,&Saugy,2007),have been tried to determine the residues of steroid hormones in complex food samples,especially,the HPLC-electrospray ionisation tandem mass spectrometry (Blasco,Van Poucke,&Van Peteghem,2007;Farke,Rattenberger,Roiger,&Meyer,2011;Guedes-Alonso,Sosa-Ferrera,&Santana-Rodriguez,2013;Penning,Lee,Jin,Gutierrez,&Blair,2010;Regal,Vazquez,Franco,Cepeda,&Fente,2009;Yang,Shao,Zhang,Wu,&Duan,2009)has been extensively utilised over the past decade due to its excellent sensitivity,high selectivity and speci?city.However,most of these approaches need multiple-steps sample pretreatment procedures,which would be more complex and time-consuming,and some techniques were more suitable for screening and identi?cation of the target analytes,while their lin-ear dynamic ranges were narrow for quantitatively determination of the steroid hormones.

0308-8146/$-see front matter ó2013Elsevier Ltd.All rights reserved.326df9b3aef8941ea76e05b0/10.1016/j.foodchem.2013.06.104

?Corresponding author.Tel.:+861058807981;fax:+861058800076.

E-mail addresses:xiemx@326df9b3aef8941ea76e05b0 ,mengxia-xie@326df9b3aef8941ea76e05b0 (M.-X.Xie).1

These authors contributed equally to this work.

Sample pretreatment procedures play a vital role for determin-ing trace residues of target analytes in complex matrices(Zhang, Liu,Xie,&Qiu,2005).Liquid–solid extraction(Shao et al.,2005) and liquid–liquid extraction(Alnouti et al.,2005)have been gener-ally employed as the preliminary step before puri?cation and enrichment of multi-residue hormones by solid phase extraction (SPE).In order to improve the ef?ciency of extraction,accelerated solvent extraction(Hooijerink,van Bennekom,&Nielen,2003), microwave-assisted extraction and supercritical?uid extraction (Tomsikova et al.,2012)were sometimes attempted.However, these procedures consumed large volumes of organic solvents, which were not environmental friendly.Some novel sample pre-treatment techniques have been investigated recently for improv-ing the selectivity and effects of puri?cation and enrichment for steroid hormones,such as solid-phase microextraction(Aufartova et al.,2011),and molecular imprinted polymers(Baggiani,Baravalle, Giovannoli,Anfossi,&Giraudi,2010;Yin et al.,2012;Zhang&Hu, 2010)as the sorbents of SPE procedures.On-line solid-phase extraction coupled to HPLC–MS/MS can also simplify the sample pretreatment procedures(Guo et al.,2013;Snow,Damon-Powell, Onanong,&Cassada,2013),which were more suitable for liquid samples.

Matrix solid-phase dispersion(MSPD)can combine the steps of homogenisation,disruption,extraction and puri?cation into one procedure(Barker,2000;Zou,Liu,Xie,Han,&Zhang,2005),and has been proven to be an effective technique for sample pretreat-ment of various biological systems ranged from solid,semi-solid to highly viscous samples.As a bene?cial alternative to other ap-proaches,MSPD has been successfully applied to the analysis of pesticides(Garcia-Rodriguez,Cela-Torrijos,Lorenzo-Ferreira,& Carro-Diaz,2012;Rallis,Sakkas,Boumba,Vougiouklakis,&Albanis, 2012),veterinary drug(Lu et al.,2012),and other pollutants(Gar-cia-Mayor,Gallego-Pico,Garcinuno,Fernandez-Hernando,&Dur-and-Alegria,2012;Pavlovic,Perisa,&Babic,2012;Zou et al., 2008)from various complex matrices.

In the present work,MSPD with ultra performance liquid chro-matography–tandem mass spectrometry(UPLC–MS/MS)was ap-plied to the extraction and determination of10steroid hormones residues in food matrices.Several sample pretreatment parame-ters,including the sorbent of MSPD,the rinsing and eluting sol-vents,and the conditions for separation and determination of the steroid hormones have been optimised.After veri?cation,the opti-mised approach has been evaluated to the simultaneous detection of the target steroids in real samples.The developed method can rapidly determine the residues of steroid hormones in complex matrices with high sensitivity and reproducibility.

2.Experimental

2.1.Chemicals and apparatus

The ten steroid hormones:Testosterone was purchased from Acros Organics(New Jersey,USA).4-Androstene-3,17-dione, Stanozolol,17a-Hydroxyprogesterone,Prednisone,Prednisolone were obtained from Dr.Ehrenstorfer GmbH(Augsburg,Germany). Megestrol acetate and Medroxyprogesterone Acetate were from TCI Chemicals(Shanghai,China).Progesterone and Hydrocortisone were provided by Aladdin Chemistry.Co.,Ltd.(Shanghai,China). The chemical structures of the above compounds are presented in Fig.S1.

Ethyl acetate,n-hexane,methylene dichloride were of HPLC-reagent grade,and provided by Sinopharm Chemical Reagent Beijing Co.,Ltd.(Beijing,China).Silica gel(40–60l m)and C18 (40–60l m)were obtained from Merck(Darmstadt,Germany), and Florisil(40–60l m)was obtained from J&K Chemical.Ltd.(Beijing,China).Doubly deionized water(DDW,18M X/cm)ob-tained from Millipore Milli-Q puri?cation system(Barnstead Corp., Boston,USA)was used throughout the experiments.HPLC grade methanol and acetonitrile were obtained from Merck(Darmstadt, Germany).Formic acid(purity>99.0%)was obtained from Waters (USA)and all the other reagents used were analytical grade.

The SPE cartridge(6mL)and sieve plates were purchased from Jiruisen(Beijing,China).Separation was carried out using a solid-phase extraction equipment,12port vacuum manifolds(Supelco, Bellefonte,PA,USA).EVA30A Nitrogen blowing instrument(Poly-tech.Co.Ltd.Beijing,China)was used for concentration.

2.2.Preparation of standard stock solutions and samples of animal origin food

Standard stock solutions of the ten compound at1.00mg/mL were prepared by dissolving inpidual target in10.0mL acetoni-trile–water(1:1,v/v)and stored at4°C in refrigerator.Working solutions(0.100ng/mL–1.00l g/mL)were obtained by subsequent dilution of the stock solution with acetonitrile–water(1:1,v/v). Tuning solutions(100ng/mL)were freshly prepared in acetoni-trile–water(1:1,v/v)containing0.1%formic acid.

The blank or negative animal origin food samples(chicken, pork,beef and sausage)used for method optimisation and develop-ment were provided by Chinese Academy of Inspection and Quar-antine.A representative portion of the samples(about200g)was homogenised and stored at4°C for analysis.

2.3.LC/MS/MS conditions

Identi?cation and quanti?cation of analytes were performed on an Acquity UPLC system equipped with a Xevo TQ MS tandem mass spectrometer(Waters Co.,USA).The Acquity UPLC BEH C18column (1.7l m,50?2.1mm)was used for the LC separation.The column oven was set to40°C,and the?ow rate of mobile phase was 0.3mL/min,and the injection volume was10l L,using full loop mode for sample injection.Acetonitrile(A)/water containing 0.1%formic acid(B)were used as mobile phase.For separation of the ten steroid hormones,a linear gradient was applied in the fol-lowing manner:the initial composition of the mobile phase was 90%B(v/v),and decreased to10%B in5min,hold in10%B for 1min,and then increase from10%to90%B in0.2min)and stabi-lized in90%B for another1min.

The mass spectrometer was operated in positive electrospray ionization using multiple reaction monitoring(MRM)mode.Nitro-gen was used as the nebulizing,desolvation and cone gas.The cap-illary voltage was held at3.7KV,the?ow of the desolvation gas was650L/h.The source temperature and desolvation gas temper-ature were set at150and350°C,respectively.Instrument opera-tion and data acquisition were processed using Waters MassLynx software.The mass transitions and collision energy used for deter-mination of the ten steroid hormones,and their quantitative ion pair(parent and daughter ions)were listed in Table1.The preci-sions of the instrument for the ten analytes were below1.0%(RSDs, n=6).

2.4.Optimisation of the matrix solid-phase dispersion procedures

The blank or negative animal tissue samples(chicken)were used for optimisation of the MSPD procedures,including the fac-tors of dispersing agents,the ratio of sorbent to sample,elution sol-vent and the volume of the elution solvent.

The spiking level of each steroid hormone was100l g/kg,and the recovery of each analyte was obtained by ratio for the peak area of the analyte to that for the corresponding standard concen-tration of the analyte.

Y.-b.Fan et al./Food Chemistry142(2014)170–177171

Florisil,C18and silica gel particles were selected as the dispers-ing agents for the MSPD procedures to observe the recoveries of the ten steroid hormones;the organic solvent methanol,acetoni-trile,ethyl acetate,dichloromethane and the mixture of acetoni-trile and ethyl acetate in the ratios of1:1to5:1(v/v)were used as eluting solvent to optimise eluting condition;the ratios of dis-persing sorbent(Florisil)to animal tissue samples ranged from 1:1to4:1(w/w)were tested to selected the ratio of sorbent to sample;5,8,10,15and20mL mixture solvents(acetonitrile and ethyl acetate,4:1v/v)were used as eluting solvent to optimise the volume of eluting solvent.The optimised MSPD procedures were as follow:

The homogenised animal tissue sample(1.0g)placed into a mortar,and then the mixture solution of the ten steroid hormones was spiked and mixed homogeneously by a pestle.After stabilized for10min,the spiked samples were blended gently with3.0g the dispersing sorbents in the mortar,and then stabilized for30min. The mixture of the sample and the dispersing sorbents was trans-ferred into a polypropylene cartridge?lled with a sieve plate at the bottom,and the column was lightly tapped and compressed.The MSPD column was washed with10mL hexane,and then eluted with10mL mixture solution of acetonitrile and ethyl acetate (4:1,v/v)at a speed of about1.0mL/min.The elution solution was collected and then evaporated to near dryness at40°C under

a gentle stream of nitrogen.The residues were dissolved with

1.00mL acetone–water solution(1:1;v/v)before UPLC–MS/MS analysis.

2.5.Method development and validation

Spiking experiments:three spiking levels(1.0,5.0and10l g/kg) were performed on chicken,pork,beef and sausage samples, respectively.The sample pretreatment processes were as described in MSPD procedures.Considering the effects of matrices on the MS/ MS responses of the analytes,control standard samples in each spiking levels were prepared.The blank animal tissue samples were treated with the MSPD procedures,and the mixture solution of the ten steroid hormones were only spiked in last step of the MSPD procedures,to the elution solution of the acetonitrile and ethyl acetate,which was evaporated to near dryness at40°C under

a gentle stream of nitrogen.The residues were then dissolved with

1.00mL acetone–water solution prior to UPLC–MS/MS analysis. The recoveries of each steroid hormone were obtained by the ratio of the peak area for the sample to that of the corresponding control sample,and each samples repeated6times.

For the matrix-forti?ed calibration curves,the blank or negative samples were pretreated with the MSPD procedures,and the spik-ing levels were ranged from0.1to200l g/kg(0.1,0.3,0.5,1.0,5.0, 20,50,100and200l g/kg),and every spiking level was repeated3 times.The calibration curves were obtained by linear regression between the peak areas of the quantitative ion pair for each steroid hormone and the concentration of the corresponding analyte, respectively.

2.6.Application of the real samples

The chicken,pork,beef and sausage samples were obtained from supermarket and each sample(200–300g)were homoge-nised and pretreated with the optimised MSPD procedures,and then analysed by UPLC–MS/MS methods.The contents of the ste-roid hormones were obtained from the matrix-forti?ed calibration curves,and every sample was repeated3times.

3.Results and discussion

3.1.LC–MS/MS conditions

For determination of steroids hormones,the mobile phases of UPLC have a signi?cant in?uence on both the separation ef?ciency and MS/MS responses of the target analytes(Blasco et al.,2007; Farke et al.,2011;Yang et al.,2009).Therefore,various kinds of mobile phases have been tried and compared,and the results showed that the mixture of acetonitrile and water with gradient elution can separate the steroids hormones better(see experimen-tal section).Owing to most of the target compounds have carbonyl groups on the molecules,positive ionisation mode was selected for the MS method,and to increase the ionisation ef?ciency,0.1% formic acid was added into the mobile phases(Farke et al.,2011; Penning et al.,2010;Regal et al.,2009).Optimisation of mass spec-trometer parameters for obtaining high sensitivity of[M+H]+were performed by introducing each tuning solution of the analyte directly into the electrospray source,and the conditions for the multiple reaction monitoring(MRM)transition,including the parent ion and the choice of corresponding daughter ion,were gen-erated automatically by the Intellistart software(see Table1). Fig.1shows the MRM chromatograms of each target compound,

Table1

The MS/MS conditions for determination of the ten steroid hormones.

Steroid hormones Parent ion(m/z)Daughter ion(m/z)Cone(v)Collision energy(eV)

4-Androstene-3,17-dione287.297.03420

109.13424 Testosterone289.297.03022

109.13022 Progesterone315.397.03422

109.13426 Stanozolol329.395.14838

121.14838 17a-Hydroxyprogesterone331.297.03620

109.13626 Prednisone359.2147.12220

171.12240 Prednisolone361.2147.12028

307.32010 Hydrocortisone363.2105.03648

121.03630 Megestrol acetate385.3267.22818

325.32816 Medroxyprogesterone Acetate387.3123.13024

327.33016

172Y.-b.Fan et al./Food Chemistry142(2014)170–177

and it can be seen that the ten steroids hormones have satisfactory MS/MS responses under the optimised conditions and can be determined within 7min.The chromatographic retention times of three components were nearly same,while they would not in?uence the determination results (Farke et al.,2011;Penning et al.,2010;Regal et al.,2009;Yang et al.,2009).3.2.MSPD procedures

Several key factors of the MSPD procedures,such as dispersing sorbent,the ratio of sorbent to sample,elution solvent and the vol-ume of the elution solvent,would affect the MSPD extraction ef?-ciencies and the recoveries of the target analytes (Barker,2000;Zhang et al.,2005;Zou et al.,2005),and so these parameters have been investigated and optimised to achieve satisfactory results of puri?cation and enrichment for the ten steroid hormones from chicken meat samples.

3.2.1.Dispersing sorbent

The effects of extraction and puri?cation for the target analytes from complex matrices have related to the properties of the dispersing sorbents (Barker,2000)in MSPD procedures,and so Florisil,C18and silica gel have been tested to select a suitable dis-persing sorbent.The concentration of each steroid hormones were 100l g/kg,and the ratio of sorbents to chicken meat were 4:1(w/w)as described in most previous reports (Barker,2000;Zhang et al.,2005;Zou et al.,2005).The rising solvent was 10mL hexane and eluting solvents were 15mL acetonitrile or ethyl acetate,a strong or middle polar eluting solvent.

In MSPD procedures,the samples were blended with the dis-persing sorbents,and then the mixtures were ?lled into a cartridge (Barker,2000;Pavlovic et al.,2012).In this case,the samples have become a part of the sorbents,and the property of the solid phase on the cartridge would be changed for some dispersing sorbents,such as the reserved phase 326df9b3aef8941ea76e05b0ually,hexane was the strongest eluting solvent for the C18sorbent in solid phase extraction (SPE)process,while it became the weakest solvent in MSPD procedure,and ordinarily utilised as rising solvent (Pavlovic et al.,2012).The average recoveries of the ten steroid hormones were all below 40%when C18was used as the dispersing sorbent of MSPD procedure and acetonitrile or ethyl acetate as eluting sol-vent,which demonstrated that C18was not a suitable sorbent for extraction and puri?cation of the selected steroid hormones in chicken

tissues.

of each target component for the ten steroid hormones (Medroxyprogesterone Acetate,Megestrol acetate,-Hydroxyprogesterone,Stanozolol,Progesterone,Testosterone and 4-Androstene-3,17-dione),and the concentration of each component were labelled in the top right corner of the chromatograms.

For the polar sorbents,silica gel and Florisil,the average recov-eries of the ten steroid hormones were about56%and67%,respec-tively,with ethyl acetate as eluting solvent,which were obviously higher than those(about45%and55%,respectively)with strong polar solvent,acetonitrile,as eluting solvent.The results showed that the recoveries and puri?cation effects of the steroid hormones with Florisil as dispersing sorbent were better than those of silica gel and C18although the recoveries were not satisfactory,which illustrated that the MSPD procedure need further optimisation. 3.2.2.Effect of eluting solvent

It was interesting to note that the average recoveries of the ste-roid hormones were higher by using the middle polar solvent ethyl acetate as eluting solvent than that by the polar solvent acetoni-trile,and so different eluting solvents with various polarities were tested for the MSPD procedures with Florisil as dispersing sorbent to observe their in?uence on the results of extraction and puri?ca-tion,and the results were shown in Fig.2.

As shown in Fig.2,the average recovery of the ten steroid hor-mones was only about20%with strong polar solvent,methanol,as the eluting solvent,and the average recovery was signi?cantly im-proved with decreasing the solvent polarity,such as acetonitrile and ethyl acetate,while the recovery would be very low with fur-ther reducing the polarity of solvent,such as dichloromethane (lower than10%,data not shown in the?gure).The results illus-trated that it was dif?cult to obtain satisfactory results for the se-lected analytes using a single eluting solvent,and then the mixture solution of acetonitrile(HCN)and ethyl acetate(EA)in the ratios of 1:1to5:1(HCN/EA,v/v)were tried.The data in Fig.2indicated that the average recovery of the ten steroid hormones has been obvi-ously improved with increasing the proportions of acetonitrile and reached about90%when HCN/EA(4:1,v/v)was used as the 3.2.3.Ratio of dispersing sorbent to sample and volume of eluting solvent

Various ratios of dispersing sorbent(Florisil)to animal tissue samples,ranged from1:1to4:1(w/w),have been investigated and the recoveries of the ten analytes were shown in Table S1. The results illustrated that the sorbents were not enough to disrupt and disperse suf?ciently the samples when the ratios were1:1and 1:2,resulting to low recoveries of the steroid hormones and many interference components in the following UPLC–MS/MS analysis. After the ratios reached to3:1and4:1,satisfactory average recov-eries of the ten analytes can be obtained(87.1%and84.7%,respec-tively),while both the average recovery of the analytes and puri?cation effects were better for the ratio of3:1,which were then selected for the following MSPD procedure.

Different volumes of mixture solutions for acetonitrile(HCN) and ethyl acetate(EA)(HCN/EA=4:1,v/v)were used as the eluting solvent to optimise MSPD conditions with the ratio of3:1for the dispersing sorbent(3g Florisil)and animal tissue(1g).The results showed that the average recoveries of the ten steroid hormones were about71%,77%and87%when the volume of the mixture sol-vent were5,8,10mL,respectively,and they would not improve with further increasing the volume of the eluting solvent to15 and20mL(the recoveries were86%and85%,respectively),which indicated that10mL mixture solvent was enough to elute the ana-lytes from the MSPD column,and larger volume of the solvent would result much interference components.

Brie?y,the optimised MSPD conditions were as following: Florisil was selected as dispersing sorbent,and the ratio of dispers-ing sorbent to sample was3:1,and the rinsing and eluting solvents were10mL hexane and10mL mixture solution of acetonitrile and ethyl acetate(4:1,v/v),respectively.

3.3.Method validation

3.3.1.The recoveries of steroid hormones at different spiking levels

Under optimised MSPD procedures and UPLC–MS/MS condi-tions,three spiking levels(1.0,5.0and10l g/kg)on blank chicken meat samples were processed and analysed,and the recovery of each steroid hormone was listed in Table2.

The data in Table2showed that the recoveries of the ten steroid hormones ranged from85.2%to95.2%with RSD lower than8.3%at the spiking levels of5.0and10.0l g/kg.Under low spiking level of 1.0l g/kg,the recoveries for most of the analytes were above80% (from81.3%to95.5%)except for Stanozolol and Hydrocortisone, which recoveries were77.8%and76.8%,respectively.The results indicated that the method can satisfy the requirement for monitor-ing the trace residues of steroid hormones in chicken meat samples (Shao et al.,2005).

The method precision was also evaluated by measuring the in-ter-day repeatability with consecutive5days at the spiking level of 1.0l g/kg,and the results showed that the RSDs of the recoveries for each steroid hormone were in the range of2.2–8.1%,illustrating that the developed approach has satisfactory repeatability.

3.3.2.Linearity and limit of detection

To observe the range of linearity for the method,the matrix-forti?ed calibration curves of the target analytes in the concentra-tion ranges of0.1–200l g/kg were performed.The MS/MS responses(peak area)versus the concentration of each steroid hormone(l g/kg)were linearly regressed,and the regression equa-tions,ranges of linearity and correlation coef?cients were summa-rised in Table3.

It can be seen from Table3that the linear ranges of4-Andro-stene-3,17-dione,Testosterone,Progesterone,Stanozolol and Pred-nisolone were from0.1to100l g/kg,and those of Megestrol acetate and Medroxyprogesterone Acetate were0.1–200l g/kg

2.The average recoveries of the ten steroid hormones under various eluting

conditions of the MSPD procedures,HCN and EA represent acetonitrile and ethyl

acetate,respectively.

174Y.-b.Fan et al./Food Chemistry142(2014)170–177

and 0.3–200l g/kg,respectively.For 17a -Hydroxyprogesterone and Prednisone,their linear ranges were 0.5–100l g/kg and 0.3–100l g/kg,inpidually,while that of Hydrocortisone was only 0.5–50l g/kg.The differences of the linear ranges for the analytes were related to their MS/MS responses (peak area)and the inter-ferences of the blank samples.It can also be noted that the corre-lation coef?cients of all the analytes were above 0.99in their linear range.

The limits of detection (LODs)for each steroid hormone of the developed approach were obtained by spiking experiments on the blank chicken meat samples,and the data were also shown in Table 3.The signal to noise ratios (S/N)were large than 3(Alnouti et al.,2005;Yang et al.,2009)when the spiking level was 0.01l g/kg for six steroid hormones (4-Androstene-3,17-dione,Testoster-one,Progesterone,Stanozolol,Prednisolone and Megestrol acetate)and 0.05l g/kg for other four components (17a -Hydroxyprogester-one,Prednisone,Hydrocortisone and Medroxyprogesterone Acetate).Similarly,the limits of quanti?cation (LOQs)were also obtained by the criterion that the signal to noise ratio was large than 10(S/N P 10)(Alnouti et al.,2005;Yang et al.,2009).The data in Table 3indicated that the ranges of linearity,LODs and LOQs of the developed method were similar or better than previous meth-od (Blasco et al.,2007;Farke et al.,2011;Nielen et al.,2007;Yang et al.,2009).Considering the enrichment factor,the LODs and LOQs of the developed method for the chicken samples were even better than those of the reported approaches for water samples (Guedes-Alonso et al.,2013;Guo et al.,2013;Snow et al.,2013).The results demonstrated that the developed approach had high sensitivity for determination of the ten steroid hormones in chicken meat samples.

3.3.3.Application to other matrices

In order to explore the suitability of the developed method to other matrices,spiking experiments on pork,beef and sausage samples were performed,and the recoveries and RSDs of each ste-roid hormone at various spiking levels (1.0,5.0and 10l g/kg)were also summarised in Table 2.

It can be seen from Table 2that the spiking recoveries of the ten analytes for the pork samples were between 79.6%and 96.9%with the RSDs in the range of 2.9–9.7%in the all spiking levels,which were similar with that of the spiking experiments on chicken meat samples.The results indicated that the developed method was suitable to determinate the residues of the steroid hormones in pork samples.

As shown in Table 2,relatively better results were obtained for spiking experiments on beef and sausage samples,especially in low spiking level (1.0l g/kg).For beef samples,the recoveries of each steroid hormone were between 82.6%and 96.8%(average recovery of the ten steroid hormones were 88.7%)at spiking level of 1.0l g/kg,which were higher than that on chicken meat and pork samples (see Table 2,their average recovery was 84.8%and 85.6%,inpiduanlly).At spiking levels of 5.0and 10.0l g/kg,the recoveries of the analytes on the beef matrix were in the range of 85.2%and 98.3%with RSDs of 1.7–8.5%,which were similar with those on chicken meat and pork samples.For sausage samples (see Table 2),the recoveries of the each steroid hormone were between 81.1%and 98.7%with RSDs lower than 9.2%at all spiking levels,and the average recovery of the ten steroid hormones at spiking le-vel of 1.0l g/kg was 88.1%,which resembled the results obtained for spiking experiment on beef samples.The results demonstrated that the MSPD procedures can effectively extract and purify the steroid hormones in beef and sausage samples.Above results illus-trated that the developed method was suitable to the pretreatment and determination of the selected analytes in various matrices.

T a b l e 2R e c o v e r i e s a n d R S D s o f t e n s t e r o i d h o r m o n e s i n f o r t i ?e d s a m p l e s w i t h d i f f e r e n t s p i k i n g l e v e l s (n =6).

S t e r o i d h o r m o n e

S p i k e d l e v e l (1.0l g /k g )

S p i k e d l e v e l (5.0l g /k g )

S p i k e d l e v e l (10.0l g /k g )

C h i c k e n

P o r k B e e f

S a u s a g e

C h i c k e n P o r k

B e e f

S a u s a g e

C h i c k e n P o r k B e e f

S a u s a g e

R (%)R S D (%)R (%)R S D (%)R (%)R S D (%)R (%)R S D (%)R (%)R S D (%)R (%)

R S D (%)R (%)

R S D (%)R (%)R S D (%)R (%)R S D (%)R (%)

R S D (%)R (%)R S D (%)R (%)R S D (%)(%)181.25.680.25.589.72.890.56.094.42.990.54.088.51.790.62.694.82.495.65.389.73.790.66.4286.48.488.36.784.87.581.13.893.84.689.66.990.85.389.74.493.53.596.96.293.94.492.75.7386.36.784.24.883.69.486.14.191.24.388.53.789.64.591.26.695.23.394.62.998.43.693.54.8477.86.687.75.889.42.581.35.885.27.487.86.888.53.890.35.685.86.086.36.489.35.788.47.2585.26.580.07.590.58.689.65.890.35.890.65.495.54.888.82.993.86.192.65.489.56.587.98.3681.77.690.25.797.66.589.21.185.88.386.56.288.77.280.66.386.96.187.58.690.62.391.45.3786.47.386.56.988.47.690.66.888.83.583.65.289.83.782.22.093.55.095.84.691.18.593.77.4876.810.279.64.982.63.588.39.288.15.789.25.885.93.786.85.692.44.994.46.591.64.894.73.1995.41.694.29.796.84.494.34.892.37.391.76.985.28.590.76.795.14.692.55.898.38.191.17.21090.14.287.77.483.3

2.689.6

3.891.1

4.090.7

4.99

5.4

6.298.6

7.892.04.690.53.795.7

6.593.35.6

1:4-A n d r o s t e n e -3,17-d i o n e ;2:T e s t o s t e r o n e ;3:P r o g e s t e r o n e ;4:S t a n o z o l o l ;5:17a -H y d r o x y p r o g e s t e r o n e ;6:P r e d n i s o n e ;7:P r e d n i s o l o n e ;8:H y d r o c o r t i s o n e ;9:M e g e s t r o l a c e t a t e ;10:M e d r o x y p r o g e s t e r o n e A c e t a t e .

Y.-b.Fan et al./Food Chemistry 142(2014)170–177175

3.4.Determination of the steroid hormones in real samples

The proposed method has been applied to analyse steroid hor-mones in chicken,pork,beef and sausage(twenty samples for each matrix),and several positive samples have been found.Fig.3 shows the MRM chromatograms for these positive samples,and re-sults indicated that the Hydrocortisone in pork,beef and sausage samples were 3.43±0.21, 3.34±0.16and 2.42±0.11l g/kg, respectively,and Progesterone has been only found in sausage sample at2.12±0.13l g/kg.

4.Summary and conclusion

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Table3

Parameters of the matrix-forti?ed calibration curves for the ten steroid hormones and their limits of detection and limits of quanti?cation.

Steroid hormones Linear equation Linear range(l g/kg)r LOD(l g/kg)LOQ(l g/kg) 4-Androstene-3,17-dione y=573.1x+2.550.1–1000.99710.010.05

Fig.3.The MRM chromatograms of Hydrocortisone in the pork(a),beef(b),sausage

(c)and progesterone in sausage(d)samples after pretreatment of MSPD

procedures.

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