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Determination-of-phenylethanolamine-A

Journal of Chromatography B,900 (2012) 94–99

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Journal of Chromatography

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o m

Short communication

Determination of phenylethanolamine A in animal hair,tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS

Ming-Xia Zhang a ,Cun Li a ,b ,Yin-Liang Wu c ,?

Hebei University of Engineering,Handan 056038,PR China b

Tianjin Agricultural University,Tianjin 300380,PR China c

Ningbo Academy of Agricultural Sciences,Ningbo 315040,PR China

a r t i c l e

i n f o

Article history:

Received 6March 2012Accepted 24May 2012

Available online 2 June 2012

Keywords:

Phenylethanolamine A QuEChERS Hair

Animal tissues Animal feeds LC–MS/MS

a b s t r a c t

A simple,sensitive and reliable analytical method was developed for the determination of a new beta-agonist phenylethanolamine A in animal hair,tissues and animal feeds by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS)with QuEChERS.Samples were extracted with acetonitrile/water (80:20,v/v).The extract was puri?ed through QuEChERS method,then was dried with nitrogen and residues were redissolved in mobile phase for hair sample or directly diluted with 0.1%formic acid in water for other samples,and analyzed by LC–MS/MS on a Waters Acquity BEH C 18column with 0.1%formic acid in water/methanol as mobile phase with gradient elution.The samples were quanti?ed using phenylethanolamine A-D 3as internal standards.The pro-posed method was validated according to the European Commission Decision 2002/657/EC determining speci?city,decision limit (CC ?),detection capability (CC ?),recovery,precision,linearity,robustness and stability.The CC ?values ranged from 0.10to 0.26?g/kg.The CC ?values ranged from 0.20to 0.37?g/kg.The mean recoveries of 95.4–108.9%with intra-day CVs of 2.2–5.6%and inter-day CVs of 3.1–6.2%were obtained.The method is demonstrated to be suitable for the determination of phenylethanolamine A in animal hair,tissues and animal feeds.The total time required for the analysis of one sample except animal hair sample,including sample preparation,was about 25min.

1.Introduction

Beta-agonists are originally used in the therapeutic treatment of asthma and preterm labor in humans [1].However,these com-pounds are also misused as growth promoters in livestock by diverting nutrients from fat deposition to the production of muscle tissues in animals [2].This misuse had caused some severe acci-dental poisonings in humans [3,4].Therefore,all beta-agonists are banned as feed additives for growth promotion in animals in China and EU [5,6].With the crackdown of banned beta-agonists,such as clenbuterol,salbutamol and ractopamine,a new beta-agonist named phenylethanolamine A has been illegally used in livestock in China (Fig.1)[7].In order to control the use of phenylethanolamine A,speci?c and sensitive methods for identifying and quantifying of phenylethanolamine A in animal hair,tissues and animal feeds are required.

Although gas chromatography–mass spectrometry (GC–MS)[8–11]and liquid chromatography–mass spectrometry (LC–MS)

?Corresponding author.Tel.:+8657487928060;fax:+8657487928062.E-mail address:wupaddy?eld@https://www.wendangku.net/doc/cc13408151.html, (Y.-L.Wu).

[12–17]methods have been developed to identify beta-agonists in biological samples,there are little literatures on identi?cation of phenylethanolamine A in animal tissues and feeds [7,18].A LC–MS/MS method had been recently developed by Sun and Yan in swine muscle [7].However,the pre-treatment procedure of the method was very complicated and time-consuming because the combination of enzymatic hydrolysis,liquid–liquid extraction and solid phase extraction (SPE)were used to cleanup.Therefore,rapid,speci?c and sensitive methods for the identi?cation and quanti?-cation of phenylethanolamine A in hair,animal tissues and feeds are required.

The QuEChERS method was previously used by Anastassiades et al.for the determination of pesticides in fruit and vegetable samples with primary and secondary amine (PSA)as the base sor-bent [19].The method has already received worldwide acceptance because of the simplicity and high throughput enabled a labora-tory to process signi?cantly a large number of samples in a given time.Moreover,the method had recently been used for the deter-mination of veterinary drug residue and mycotoxins [20,21].Due to high ef?ciency of QuEChERS,it is necessary to develop QuECh-ERS method for phenylethanolamine A in animal hair,tissues and feeds.

M.-X.Zhang et al./J.Chromatogr.B900 (2012) 94–99

Fig.1.Mass spectrum of phenylethanolamine A at30eV and its structure.

Here we developed a simple and reliable con?rmatory LC–MS/MS analytical method for analyzing phenylethanolamine A in animal hair,tissues and feeds with QuEChERS cleanup procedure. Validation parameters tested were speci?city,CC?,CC?,recovery, precision,linearity,robustness and stability.

2.Materials and methods

2.1.Materials and reagents

Methanol,acetonitrile and formic acid were LC grade.Hexane was analytical grade.Phenylethanolamine A was purchased from Dr.Ehrenstorfer(Augsburg,Germany).Phenylethanolamine A-D3 was purchased from WITEGA Laboratorien Berlin-Adlershof GmbH (Berlin,Germany).Sodium chloride,anhydrous magnesium sul-phate,hydrochloric acid and sodium hydroxide were obtained from Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,PR China).PSA sorbent was purchased from Agilent Technologies Inc.(Santa Clara, CA,USA).The water was puri?ed with a Milli-Q reverse osmosis system(Millipore,Milford,MA,USA).

2.2.Standard solutions

Stock solutions of phenylethanolamine A and phenylethanolamine A-D3(100?g/mL)were prepared in methanol.Three fortifying standard solutions(25,37.5and 50ng/mL)were prepared by diluting stock standard solution with acetonitrile.Two internal working standard solutions of phenylethanolamine A-D3(50and500?g/L)were prepared in acetonitrile.Six working standard solutions(0.025–2.5?g/L)of phenylethanolamine A were prepared by diluting stock solu-tion with0.1%formic acid in water/methanol(80:20,v/v).Two individual working standard solutions(1000?g/L)for MS–MS optimization were prepared by diluting stock solutions with0.1% formic acid in water/methanol(80:20,v/v).

2.3.Chromatographic conditions

A Waters Acquity UPLC instrument(Milford,MA,USA)was used in the present experiment.Separation was carried out on an Acquity BEH C18column(100mm×2.1mm,1.7?m)maintained at30?C. The mobile phase consisted of solvent A(0.1%formic acid in water) and solvent B(methanol).Initial gradient conditions were set to 20%

B and held for0.5min before incorporating a linear gradi-ent increasing to80%B at2.5min and held for1.0min.At3.6min the gradient was programmed to initial conditions to reequilibrate the column for1.4min(total run time5min).The?ow rate was 0.30mL/min.The injection volume was10?L in full loop injection mode.2.4.Mass spectrometry conditions

Detection was carried out by a Waters Xevo TM TQ triple-quadrupole MS?tted with electrospray ionization(ESI)probe operated in the positive ion mode.The following parameters were optimal:capillary voltage,3000V;ion source temperature,150?C; desolvation gas temperature,500?C;desolvation gas?ow rate, 1000L/h.Detection was carried out in multiple reaction moni-toring(MRM)mode.Argon was used as the collision gas,and the collision cell pressure was4mbar.The selected MRM tran-sitions for phenylethanolamine A were m/z345.3>117.8and 345.3>150.0with a dwell time of0.25s.The transition chosen for quanti?cation was345.3>150.0.The selected MRM transition for phenylethanolamine A-D3was348.3>153.1with a dwell time of 0.25s.The cone voltage was20V.As for the collision energy,it was30eV for m/z345.3>117.8,22eV for m/z345.3>150.0and m/z 348.3>153.1.

2.5.Sample preparation

2.5.1.Animal tissues and feeds

A5g of homogenous representative sample was weighed in a 50mL plastic centrifuge tube and100?L of internal standard solu-tion at500?g/L was added.Then vortexed for15s and allowed to stand at room temperature for30min.After addition of25mL of acetonitrile/water(80:20,v/v),the sample was homogenized by a high-speed blender(Ultra-Tyrrax T25;IKA,Germany)for1min. After addition of2.0g of NaCl and10mL of hexane(5mL of water were added for feed samples),the mixture was shaken vigorously for1min.To separate aqueous and organic phase,the sample was centrifuged at5000rpm for3min.An aliquot of middle organic phase(2mL)was transferred to a5mL centrifuge tube with150mg anhydrous MgSO4and50mg PSA.After shaking vigorously on vor-tex for1min,the tube was centrifuged at10,000rpm for2min.The puri?ed extract was diluted with0.1%formic acid in water in4:6 (0.40mL puri?ed extract mixed with0.60mL of0.1%formic acid in water)ratio.Prior to?nal instrumental analysis,sample solution was passed through the0.20?m?lter.

2.5.2.Animal hair

Hair(500mg),which had been cut into small pieces shorter than 1mm with scissors,was incubated overnight at45?C in5mL0.1M HCl after addition of100?L of internal standard solution at50?g/L. After cooling,the mixture was adjusted pH to7.0with0.5M NaOH. Then,20mL of acetonitrile/water(80:20,v/v)was added.The sam-ple was homogenized by a high-speed blender(Ultra-Tyrrax T25; IKA,Germany)for1min.The following cleanup steps were identi-cal to Section2.5.1.After puri?cation,the puri?ed extract(1.5mL) was evaporated to dryness in a water bath at40?C under nitrogen and reconstituted in500?L of0.1%formic acid in water/methanol (80:20,v/v).Prior to?nal instrumental analysis,sample solution was passed through the0.20?m?lter.

2.6.Method validation

The evaluation of the suitability of the method for the deter-mination of phenylethanolamine A in swine hair,swine tissues, chicken mixed feed and swine mixed feed was carried out according to the European Commission Decision2002/657/EC[22].

To verify the absence of interfering substances around the reten-tion time of phenylethanolamine A,20blank samples for each kind of sample were analyzed.

The linearity of the method was determined by assaying each calibration standard at six concentration levels(0.025,0.05,0.1,

0.25,1.0and2.5?g/L)over the linear range of phenylethanolamine

A.The concentration of internal standard was1.0?g/L.Linear

96M.-X.Zhang et al./J.Chromatogr.B900 (2012) 94–99

regression analysis of calibration standard data was performed by plotting the peak area ratio of quantitative ion pair of phenylethanolamine A to internal standard.

The CC?was established by analyzing20blank materials per matrix to be able to calculate the signal to noise ratio at the time window in which the analyte is expected.Three times the signal to noise ratio(qualitative ion pair)was used as CC?.The CC?was established by analyzing21blank materials per matrix forti?ed with phenylethanolamine A at0.50?g/kg.

Recovery of phenylethanolamine A was measured in blank sam-ples that were forti?ed at0.50?g/kg,0.75?g/kg and1.0?g/kg.The forti?ed samples were analyzed and the recoveries were calculated by comparing the measured concentrations to the forti?ed concen-trations.The repeatability was measured on the21forti?ed blank samples(n=7replicates per concentration level and analyzed in three independent analytical runs)for each kind of sample and expressed by coef?cient of variation(inter-day CVs).The within-laboratory reproducibility was measured on the21forti?ed blank samples(n=7replicates at0.50?g/kg forti?cation level and ana-lyzed at three occasions with three different operators)for each kind of sample and expressed by coef?cient of variation(CV R).

Robustness?rst was established by introducing changes in vol-ume of extract solution(15,20,25and30mL)for forti?ed liver samples at0.50?g/kg.Then,robustness was evaluated by intro-ducing small changes in the chromatographic system,like?ow rate(0.25,0.30and0.35mL/min),column temperature(25,30,35 and40?C)and the concentration of formic acid in solvent A(0.05, 0.10and0.15%).Robustness of chromatographic system change was assessed by injecting the same matrix(liver)forti?ed standard solution(2.0?g/L)six times under each above chromatographic condition.

The stability was determined in two different ways:(a)in solvent(stock solutions)and(b)in matrix(forti?ed level was 0.50?g/kg,each kind of matrix was forti?ed15samples).

3.Results and discussion

3.1.LC–MS/MS optimization

Working solutions of1000?g/L were infused to opti-mize the MS–MS parameters of phenylethanolamine A and phenylethanolamine A-D3and to select the appropriate diagnostic ions.The infusion process was carried out with the same chro-matographic conditions as those used during analysis.The ESI+was selected due to its sensitivity,ruggedness and easy handling and maintenance.

Because beta-agonists belong to group A of Annex I,Council Directive96/23/EC[23],a minimum of four identi?cation points are required,which were obtained by monitoring one parent ion (1point)and two transitions(each1.5points).The selected transi-tions for phenylethanolamine A and the optimal MS–MS conditions are described in Section2.4.

After optimization of MS parameters,mobile phase compo-sition had been investigated.We had found the sensitivity of phenylethanolamine-A can be signi?cantly enhanced by adding small amounts of formic acid in mobile phase.There was no sig-ni?cant difference between two mobile phases(0.1%formic acid in water/acetonitrile and0.1%formic acid in water/methanol)in sensitivity.In order to protect column,the gradient elution sys-tem with0.1%formic acid in water/methanol had been chosen to analyze phenylethanolamine-A.

3.2.Sample preparation

To develop a simple pretreatment procedure,we decided to enhance the selectivity of extraction by means of QuEChERS employing partitioning of acetonitrile/water mixture induce by addition of inorganic salts.While the analytes are transferred into an organic phase,some more polar matrix impurities are left in aqueous layer.Moreover,hexane was added to remove fat.

After centrifugation,the middle phase of the extract was directly diluted with0.1%formic acid in water and injected to LC–MS/MS without PSA puri?cation for animal tissues and feeds.However, there were about20–35%and25–45%ion suppression at2:8and 4:6dilution ratio,respectively.To decrease the matrix effect,the matrix effects(signal suppression/enhancement,SSE)were eval-uated by comparing of external matrix matched calibration slope with the external solvent calibration slope in range0.025–2.5?g/L at4:6dilution ratio after PSA puri?cation for animal tissues and feeds and3-fold concentration factor after PSA puri?cation for animal hair(Fig.2).The results revealed that the matrix effect of phenylethanolamine A was decreased with the increase of the amount of PSA from0mg to50mg and no signi?cant difference between50mg and100mg.So,50mg of PSA was used in the present study.

A suitable deconjugation step is required for simultaneous determination of beta-agonists in animal tissues and urine.How-ever,the main form is free state in animal tissues and urine for aniline-type beta-agonist including phenylethanolamine A[24]

. Fig.2.The effect of different PSA amounts on the matrix effect at4:6dilution ratio after PSA puri?cation.

M.-X.Zhang et al./J.Chromatogr.B900 (2012) 94–9997

Fig.3.The MRM chromatogram of phenylethanolamine(0.05?g/L,S2and S3)and phenylethanolamine-D3(1.0?g/L,S1)in standard solution and the MRM chromatograms of blank and forti?ed samples at the0.50?g/kg level for phenylethanolamine in muscle(a),liver(b),kidney(c),fat(d),hair(e),chicken mixed feed(f)and swine mixed feed (g).

98M.-X.Zhang et al./J.Chromatogr.B900 (2012) 94–99 Table1

Mean recoveries of phenylethanolamine A from animal hair,tissues and animal feeds by LC–MS/MS.

Type of sample Forti?ed

concentration

(?g/kg)Intra-day mean

recovery and CVs

(%,n=7)

Inter-day mean

recovery and CVs

(%,n=21)

CV R(%)

Muscle0.5098.3(4.3)101.3(5.5)96.7(4.2)98.8(4.9) 5.1

0.7598.7(2.2)99.2(2.5)97.9(4.4)98.6(3.1)–

1.099.7(

2.7)99.0(

3.2)101.7(

4.3)100.1(3.5)–Liver0.509

5.4(5.0)97.5(3.0)103.7(5.5)98.9(5.7) 5.8

0.7596.0(3.9)103.1(3.5)96.2(2.3)98.4(4.7)–

1.097.6(4.3)103.3(4.4)97.6(

2.9)99.5(4.7)–Kidney0.50104.4(4.5)102.0(

3.9)99.7(

4.9)102.0(4.6)7.1

0.7597.0(3.8)103.1(5.4)99.0(4.7)99.7(5.2)–

1.098.4(3.5)97.2(4.6)99.3(3.3)98.3(4.2)–Fat0.50104.2(4.7)97.4(3.6)10

2.1(4.1)101.2(5.3) 5.1

0.7597.5(3.4)105.4(5.0)98.9(3.2)100.6(6.2)–

1.0101.3(4.5)97.8(3.1)98.4(3.7)99.2(4.3)–Hair0.5096.4(4.9)10

2.4(

3.3)99.1(

4.1)99.3(4.6) 6.3

0.75100.5(4.2)96.5(4.0)96.8(3.6)97.9(5.0)–

1.097.8(4.0)100.7(3.2)98.5(

2.9)99.0(4.8)–

Chicken feed 0.5096.3(4.7)95.8(3.8)99.8(4.1)97.3(5.1) 6.8

0.75106.2(3.8)97.2(3.3)98.6(4.0)100.7(5.3)–

1.098.5(4.2)96.6(3.8)98.1(3.5)97.7(4.4)–

Swine feed 0.5098.8(5.1)103.6(3.2)108.9(4.7)103.8(6.2) 6.6

0.7597.4(3.9)105.1(4.4)96.4(4.2)99.6(5.8)–

1.095.9(4.0)99.4(5.6)97.0(3.5)97.4(5.2)–

Hooijerink et al.had found the conjugated rate was about only5% for clenbuterol in urine samples[25].Moreover,a positive muscle sample from Henan Institute of Veterinary Drug Control was ana-lyzed using the method and another method developed by Sun and Yan with enzymatic hydrolysis[7].The result was5.89±0.17?g/kg (n=3)for our method with external quanti?cation and higher than the concentration(5.52±0.33(n=3))analyzed by the another method.So,enzymatic hydrolysis had not been used in the present study.

For the determination of?2-agonists residue in biological sample,the pretreatment of developed methods usually include buffer solution extraction,enzymatic hydrolysis and SPE procedure [11–13,16].Consequently,at least2–3h(sometimes over18h) must be required for determination of one https://www.wendangku.net/doc/cc13408151.html,pared with the conventional pretreatment procedures,the established QuECh-ERS pretreatment procedure was very simple and economic.It can meet the requirements of rapid increase in the number of samples.

3.3.Method validation

3.3.1.Linearity

The calibration graph was obtained by plotting the peak area of quantitative ion pair of phenylethanolamine A to internal standard at0.025–2.5?g/L.The linear equation was Y=1.3827X+0.0108 with the correlation coef?cient(R2)of0.9993.The maximum indi-vidual residual deviate was5.8%at all of standard https://www.wendangku.net/doc/cc13408151.html,ing the curve,recoveries can be calculated at each forti?cation level.The MRM chromatograms of standard solution are shown in Fig.3. 3.3.2.Speci?city

The speci?city was evaluated by analyzing20blank samples for each kind of sample.Fig.3indicates that there were no interfer-ing peaks from endogenous compounds at the retention time of phenylethanolamine A.3.3.3.Recovery and precision

Based on the level recommended by the EU-CRL for other aniline-type beta-agonists(26),a level of0.50?g/kg of phenylethanolamine A was chosen as the target analytical level. And,three different forti?ed concentrations(0.50,0.75and 1.0?g/kg)had been chosen according to the European Commis-sion Decision2002/657/EC[22].The results are summarized in Table1.The mean recoveries,intra-day CVs,inter-day CVs and within-reproducibility(CV R)varied from95.4to108.9%,from2.2 to5.6%,from3.1to6.2%and from5.1to7.1%,respectively.

3.3.

https://www.wendangku.net/doc/cc13408151.html,?and CCˇ

According to the concept of the European Commission Decision 2002/657/EC,three times of the signal to noise ratio(qualitative ion pair)was used for CC?.The CC?was established by analyzing21 blank materials per matrix forti?ed with phenylethanolamine A at 0.50?g/kg(Fig.3).The value of the decision limit(CC?)plus1.64 times the standard deviation of the within-laboratory reproducibil-ity of the measured content equals the detection capability(CC?). Results are presented in Table2.The results of the CC?ranged from 0.10to0.26?g/kg.The CC?values ranged from0.20to0.37?g/kg. Additionally,limit of detection(LOD)was0.13?g/kg after a10-fold dilution for swine muscle sample.The LOD is better than the pre-viously published LC–MS/MS method for phenylethanolamine A in swine muscle(0.20?g/kg)even if a10-fold concentration had been used[7].

3.3.5.Robustness

After robustness experiment on volume change of extract solu-tion for forti?ed liver sample at0.50?g/kg,it was found that the mean recoveries(n=7)were106.2,97.5,103.7,and104.1%for15, 20,25,30mL of extract solution,respectively.There were not signif-icant differences for these recoveries.The possible reason was the use of isotope compound as internal standard for quanti?cation in the present study.

Table2

CC?and CC?(?g/kg)obtained for phenylethanolamine A in animal hair,tissues and animal feeds by LC–MS/MS.

Matrix Muscle Liver Kidney Fat Hair Chicken mixed feed Swine mixed feed CC?0.130.120.100.120.110.230.26

CC?0.210.220.220.200.210.340.37

M.-X.Zhang et al./J.Chromatogr.B900 (2012) 94–9999

The Waters Xevo TM TQ MS is a robust platform for quantitative LC–MS/https://www.wendangku.net/doc/cc13408151.html,pared with conventional LC–MS/MS,much more MRM transitions can be acquired with higher sensitivity in a sin-gle analysis,especially combined with ultra high performance LC systems.

The analytical results of the matrix forti?ed standard solu-tion(2.0?g/L)were quanti?ed with an external standard solution (2.0?g/L)analyzed in the same chromatographic condition.The concentrations of the matrix standard solution were1.90–2.01?g/L for phenylethanolamine A.The CVs ranged from0.52%to 1.89%.These results demonstrate that changes of chromato-graphic conditions did not signi?cantly in?uence the analytical results.

3.3.6.Stability

The stock standard solutions in methanol were stored for at least one month at?20?C.The stock solutions were analyzed every week by UPLC and the instrumental responses were compared with the peak areas obtained at the moment of solution prepa-ration(t=0).The acceptance criterion was a response comprised between95%and105%of the initial one[26].Forti?ed samples at 0.50?g/kg stored at?20?C were analyzed after3,7and14days. It was found that the recoveries of phenylethanolamine A had no obvious change.

3.4.Applications of the method

Forty samples(twenty swine muscle and twenty swine liver sample)commercially available from the local market were ana-lyzed for phenylethanolamine A using the above method.No residue was found in these samples.

4.Conclusion

In the present study,a fast and sensitive method was developed for the determination of phenylethanolamine A in seven kinds of matrixes(hair,muscle,liver,kidney,fat,chicken mixed feed and swine mixed feed)by LC–MS/MS with QuEChERS.This method was validated with forti?ed blank samples and satisfactory recoveries were obtained.The CC?and CC?were found to be suf?ciently low to determine the residue of phenylethanolamine A in animal hair, tissues and animal feeds.

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