Xue Xiao,Kaili He,Ya-Jun Hou,Zhangmin Xiang,Yunyun Yang

Guangdong Provincial Key Laboratory of Chemical Measurement and Emergency Test Technology,Guangdong Provincial Engineering Research Center for Ambient Mass Spectrometry,Institute of Analysis,Guangdong Academy of Sciences(China National Analytical Center,Guangzhou),Guangzhou,510070,China

Keywords:

β-blockers

Magnetic solid-phase extraction

Nanosilver-functionalized magnetic

nanoparticles

Poly(3,4-dihydroxyphenylalanine)

Fourier transform ion cyclotron resonance

mass spectrometry

A B S T R A C T

A rapid and sensitive method for analyzing trace β-blockers in complex biological samples,which involved magnetic solid-phase extraction(MSPE)coupled with Fourier transform ion cyclotron resonance mass spectrometry(FTICR-MS),was developed.Novel nanosilver-functionalized magnetic nanoparticles with an interlayer of poly(3,4-dihydroxyphenylalanine)(polyDOPA@Ag-MNPs)were synthesized and used as MSPE adsorbents to extract trace β-blockers from biological samples.After extraction,the analytes loaded on the polyDOPA@Ag-MNPs were desorbed using an organic solvent and analyzed by FTICR-MS.The method was rapid and sensitive,with a total detection procedure of less than 10 min as well as limits of detection and quantification in the ranges of 3.5-6.8 pg/mL and 11.7-22.8 pg/mL,respectively.The accuracy of the method was also desirable,with recoveries ranging from 80.9% to 91.0% following the detection of analytes in human blood samples.All the experimental results demonstrated that the developed MSPE-FTICR-MS method was suitable for the rapid and sensitive analysis of trace β-blockers in complex biological samples.

1.Introduction

β-blockers are widely used to treat various cardiovascular disorders such as hypertension,heart arrhythmias,and ischemic heart disease[1].Apart from their clinical uses and therapeutic values,βblockers are known to improve heart function by relaxing heart muscles and reducing heart rate.However,they are often misused by athletes in professional competing sports and are thus forbidden by the Medical Commission of the International Olympic Committee[1].In addition,fatal adverse drug events following the use of β-blockers have been observed in hospitalized patients in internal medicine[2].Furthermore,a series of deaths suspected to be strongly associated with the use of metoprolol[3,4]and propranolol[5,6]have been reported.The inherent relevance of β-blockers in doping,forensic science,and toxicological contexts,as well as the need for optimum dosage adjustment in clinical settings,has led researchers to develop analytical methods that can be used to accurately monitor these drugs.

To date,analytical methods including high-performance liquid chromatography(HPLC)[7,8],two-dimensional HPLC[9],and HPLC coupled with mass spectrometry(HPLC-MS)[10-17]have been developed for use in the analysis of β-blockers.However,following administration,β-blockers are present at very low concentrations in organisms.In addition,the matrices of biological samples are very complex[7-17],making it cumbersome and time-consuming to analyze these trace-molecules in biological samples.To overcome these limitations,various sample pretreatment methods such as protein precipitation(PP)[18],matrix solid-phase dispersion[19],liquid-liquid extraction (LLE)[20],solid-phase microextraction(SPE)[21,22],and magnetic solid-phase extraction(MSPE)[23,24]have been applied.Among these sample preparation methods,an obvious matrix background is still observed in the sample extracts following the use of PP,and a large volume of organic solvents is used in LLE and SPE.However,these drawbacks are avoided with MSPE,in which analytes are isolated from the sample matrix by a magnetic field,thus simplifying the sample pretreatment process[25,26].

Magnetic nanoparticles(MNPs)are a series of easily synthesized MSPE materials with large surface-to-volume ratios.However,bare MNPs have limited interaction with organic analytes[23],and chemical modifications of the particles are usually required to enhance their extraction capacity.3,4-dihydroxyphenylalanine(DOPA)is easily self-polymerized in weakly alkaline aqueous media,forming a polyDOPA coating[27].PolyDOPA shows excellent adhesive ability on various material surfaces through metal bidentate coordination and hydrogen bonding[28],and polyDOPA-coated MNPs(polyDOPA-MNPs)have been widely applied as extraction adsorbents[29].Importantly,polyDOPA-modified surfaces provide abundant active sites to immobilize molecules and grow shells through the Michael addition/Schiff base reactions[30],coordination[31],and redox[32]owing to the presence of many functional groups such as catechol,amine,carboxyl,and quinone moieties on the surface.In addition,the catechol group of DOPA can be used as a reducing agent to anchor the surface of metal nanoparticles[33].Wen et al.[34]prepared Au@polyDOPA@Ag nanoparticles to form a surface-enhanced Raman spectroscopy tag for imaging in the human lung adenocarcinoma cell line A549.A onestep in situ reduction process has been reported for the preparation of Ag@DOPA@Hg nanostructures to detect Hg2+using a colorimetric method[35].Au@DOPA nanoparticles have also been synthesized for catalytic and environmental applications[36].

Based on these reports,we developed novel nanosilverfunctionalized MNPs with an interlayer of polyDOPA(poly-DOPA@Ag-MNPs)and used them as MSPE adsorbents for the analysis of trace β-blockers(i.e.,acebutolol,metoprolol,and propranolol)in complex biological samples.The adsorption performance of polyDOPA(polyDOPA@Ag-MNPs)towards β-blockers was investigated,and a method involving MSPE coupled with Fourier transform ion cyclotron resonance mass spectrometry(FTICR-MS)for the analysis of the investigated β-blockers was developed.

2.Experimental

2.1.Materials and reagents

Ferric chloride hexahydrate(FeCl3·6H2O),sodium acetate(NaAc),diethylene glycol(DEG),ethylene glycol(EG),and acetic acid(HAc)were obtained from Tianjin Damao Chemical Reagent Factory(Tianjin,China).Sodium acrylate(Na acrylate)was purchased from Beijing Universal Century Technology Company(Beijing,China).Sodium dihydrogen phosphate dihydrate(NaH2PO4·2H2O)and disodium hydrogen phosphate dodecahydrate(Na2HPO4·12H2O)were purchased from Guangzhou Chemical Reagent Factory(Guangzhou,China).DOPA was purchased from Alfa Aesar Chemical Company(Shanghai,China).Bupivacaine hydrochloride, metoprolol tartrate salt,acebutolol hydrochloride,and propranolol hydrochloride were supplied by Shanghai Macklin Biochemical Co.,Ltd.(Shanghai,China).HPLC-grade acetonitrile and methanol were supplied by Burdick&Jackson(Muskegon,MI,USA).

2.2.Preparation of polyDOPA@Ag-MNPs

The procedure for the preparation of polyDOPA@Ag-MNPs is illustrated in Fig.1.First,carboxyl-modified MNPs(COOH-MNPs)were synthesized using a solvothermal method according to a previous study[37].Briefly,1.08 g of FeCl3·6H2O was dissolved in a mixture of 30 mL of DEG and 10 mL of EG.Next,3.0 g of NaAc and 3.0 g of Na acrylate were added,and the mixture was vigorously stirred to form a homogeneous dark yellow solution.Afterwards,the mixture was transferred to a 50 mL Teflon-lined stainless-steel autoclave and heated at 200°C for 10 h.The obtained products were washed three times with ethanol and deionized water,and dried at 60°C under a nitrogen stream.

Fig.1.Schematic illustration of the synthesis of polyDOPA@Ag-MNPs and their application as MSPE adsorbents for the analysis of trace β-blockers in biological samples using FTICRMS.DOPA:3,4-dihydroxyphenylalanine;MNPs:magnetic nanoparticles;MSPE:magnetic solid-phase extraction;FTICR:Fourier transform ion cyclotron resonance;MS:mass spectrometry;EG:ethylene glycol;DEG:diethylene glycol.

Following this,the obtained COOH-MNPs were then functionalized with polyDOPA[29].Briefly,160 mg of DOPA was dissolved in 160 mL of an aqueous solution of Tris-HCl(10 mM,pH 8.5)to prepare the DOPA solution,and 40 mg of COOH-MNPs were dispersed in the above mentioned DOPA solution and stirred for 10 h at 25°C.The obtained polyDOPA-MNPs were rinsed with water for further use.

Finally, the synthesized polyDOPA-MNPs were dispersed in 3 mL of Tris-HCl aqueous solution(pH 8.5),and200μL of AgNO3(40mM)was slowly added dropwise under sonication at 25°C,followed by the addition of 40μL DOPA(40 mM).The mixture was sonicated for another 15 min,and the final product,polyDOPA@Ag-MNPs,was obtained after washing with water three times and stored in 3 mL of H2O at 4°C.

2.3.MSPE procedure

The MSPE procedure for the extraction of β-blockers using polyDOPA@Ag-MNPs is depicted in Fig.1.First,1 mL of the liquid sample was diluted with 10-fold volumes of phosphate solution(10 mM,pH 7.0).To analyze whole blood samples,1 mL of blood was deproteinized by adding the same volume of acetonitrile,and the supernatant was collected via centrifugation at 5,000 r/min for 2 min,followed by a 10-fold dilution with phosphate solution(10 mM,pH 7.0).

PolyDOPA@Ag-MNPs(4 mg)were dispersed in the above sample solution and then mixed by sonication for 2 min.Next,polyDOPA@Ag-MNPs were separated from the solution using a magnet and washed three times with water.Subsequently,1 mL of MeOH containing 1%(V/V)HAc was utilized to desorb β-blockers from the MNPs by sonicating the mixture for 2 min,and then the eluates were filtered through a 0.22μm filter for subsequent MS analysis.

2.4.MS detection

All MS analyses were performed using a Sarix X 7T FTICR mass spectrometer(Bruker Daltonics,Bremen,Germany)in positive ion detection mode.Accurate mass measurement was acquired with a 4 M recording mode,and mass resolutions of more than 100,000 for target analytes were obtained.

3.Results and discussion

3.1.Preparation and characterization of polyDOPA@Ag-MNPs

The synthesis of polyDOPA@Ag-MNPs involved the reduction of silver cations into neutral silver atoms using polyDOPA,and the subsequent production of reactive quinones for the deposition of nanocomposites to immobilize molecules and grow shells.First,polyDOPA-MNPs were prepared according to a previous study[29].The catechol in polyDOPA films is easily oxidized to quinone,inducing a localized reduction of metal ions[35,38].Simultaneously,a polyDOPA film is spontaneously deposited outside the structure,acting as a protective layer to prevent the oxidation of the generated Ag[34].Therefore,DOPA was the reductant and stabilizer for the formation of AgNPs during the reaction process,and polyDOPA@Ag-MNPs were synthesized in situ.

The magnetic characterization of COOH-MNPs and polyDOPA@Ag-MNPs was performed using a vibrating sample magnetometer with a LakeShore7404 instrument(Lake Shore Cryotronics,Inc.,San Diego,CA,USA)at an applied field of 15 kOe and a temperature of 25°C.As shown in Fig.2A,the saturation magnetization values of COOH-MNPs and polyDOPA@Ag-MNPs were 95.5 A·m2/kg and 52.2 A·m2/kg,respectively.These results indicated that the particles exhibited an excellent magnetic response.Compared with COOH-MNPs,the decrease in the saturation magnetization value of polyDOPA@Ag-MNPs demonstrated that polyDOPA@Ag had been successfully modified on the surface of COOH-MNPs.

Next,the zeta potential and size distribution characteristics were determined using a Zetasizer Nano ZS(Malvern Instruments,Worcestershire,UK),and the zeta potentials of COOHMNPs and polyDOPA@Ag-MNPs at pH values from 4 to 9 were compared(Fig.2B).With increasing pH,the zeta potential of COOH-MNPs and polyDOPA@Ag-MNPs both decreased gradually and finally plateaued out.The zeta potential magnitude of polyDOPA@Ag-MNPs was lower than that of COOH-MNPs at each pH,indicating the successful immobilization of polyDOPA@Ag on COOH-MNPs.

The particle size and surface morphology of the COOH-MNPs and polyDOPA@Ag-MNPs were determined by transmission electron microscopy(TEM;Talos?F200x;ThermoFisher Scientific,Waltham,MA,USA).Both particles were spherical,with the diameter of approximately 300 nm.There seemed to be no significant difference between COOH-MNPs and polyDOPA@Ag-MNPs following visualization of their TEM images.However,the size distribution of the particles was different(Fig.2C).

To further prove the successful preparation of polyDOPA@Ag-MNPs,the composite of the particles was verified by energy dispersive X-ray(EDX)spectroscopy(JEM-2100HR;JEOL,Tokyo,Japan).The results obtained are shown in Fig.2D.The EDX graph of the polyDOPA@Ag-MNPs revealed that Ag was present in the particles.The weight percentage of Ag in the polyDOPA@Ag-MNPs was 4.25%.

Fig.2.(A)Vibrating sample magnetometer curves of COOH-MNPs and polyDOPA@Ag-MNPs;(B) zeta potential of COOH-MNPs and polyDOPA@Ag-MNPs at different pH values(n=3);(C)size distribution of COOH-MNPs and polyDOPA@Ag-MNPs(inset:particle size of polyDOPA@Ag-MNPs);and(D)energy dispersive X-ray spectrum of polyDOPA@Ag-MNPs.

3.2.Optimization of MSPE conditions

To achieve a good yield of β-blockers during the extraction process,factors affecting MSPE,including sample pH,adsorption time,amount of polyDOPA@Ag-MNPs,type of eluent,desorption time,and volume of eluent,were optimized using acebutolol,metoprolol,and propranolol as target analytes for examination.

The sample pH, which determines the charge of the analyte and the adsorbent,was investigated first.As shown in Fig.3A,the highest extraction efficiency was achieved when the pH of the sample solution was 7.The dissociation constant(pKa)values of acebutolol,metoprolol,and propranolol are 9.20,9.68,and 9.45,respectively[17].Therefore,these β-blockers are primarily cationic below their pKa values.Consequently,the electrostatic attraction between the analytes and the adsorbents should play a role in the adsorption process.If the electrostatic interaction plays a key role in the adsorption,theoretically, the extraction efficiencies should be higher at a pH lower than 7.0.However,in the pH range of 4.0-6.0,the extraction efficiencies were lower than those at pH 7.0,suggesting that non-specific interactions between the analytes and the adsorbents,such as π-π stacking and hydrogen bonding forces,were also at play in the adsorption process.Therefore,a sample pH of 7.0 was used in this study.

Next,the adsorption time was investigated,and 2 min was found to be the optimal adsorption time(Fig.3B).Following this,the effect of the amount of polyDOPA@Ag-MNPs on the extraction efficiency of β-blockers was investigated.As shown in Fig.3C,the extraction efficiency of polyDOPA@Ag-MNPs for each β-blocker increased with increasing amounts of the adsorbent,with a plateau attained at 4 mg.Therefore,4 mg polyDOPA@Ag-MNPs was used for further experiments.

The conditions of desorption,including the type of eluent,desorption time,and volume of eluent,were investigated.As depicted in Fig.3D,MeOH containing 1% HAc exhibited the best extraction performance.The extraction efficiencies increased with increasing eluent time until a plateau of 2.0 min(Fig.3E).Subsequently,the eluent volume associated with the optimal extraction efficiency was recorded.The extraction efficiency reached a maximum with an eluent volume of 1 mL,and further increase in the eluent volume was unfavorable(Fig.3F).

Fig.3.Effect of(A)pH,(B)adsorption time,(C)amount of polyDOPA@Ag-MNPs,(D)type of eluent,(E)desorption time,and(F)volume of eluent on the extraction efficiencies of polyDOPA@Ag-MNPs for β-blockers.

3.3.Adsorption capacity of polyDOPA@Ag-MNPs

Propranolol was selected as the target analyte to evaluate the adsorption capacity of polyDOPA@Ag-MNPs,and the adsorbed amount of the β-blocker was calculated according to Eq.(1).

Whereqe(mg/g)is the amount of propranolol adsorbed at equilibrium,c0andceare the initial and equilibrium concentrations of propranolol(μg/mL),respectively,V(mL)is the volume of solution,andm(g)is the mass of polyDOPA@Ag-MNPs used.

The Langmuir model was used to analyze the adsorption process of propranolol on polyDOPA@Ag-MNPs,and the model is expressed using Eq.(2).

whereKdrefers to the energy of adsorption andqm(mg/g)is the maximum adsorption amount of propranolol.The experiment was performed under the optimized conditions,and the obtained results are shown in Fig.4.The relationship betweence/qeandcewas expressed as an equation,ce/qe=2.57ce+0.0043,with a determination coefficient(R2)of 0.9973.The maximum adsorption capacity of polyDOPA@Ag-MNPs for propranolol was calculated as 0.39 mg/g after applying the Langmuir model.

Fig.4.Correlation of initial propranolol concentration and amount of propranolol adsorbed by polyDOPA@Ag-MNPs.

3.4.Method validation

Acebutolol,metoprolol,and propranolol were added into phosphate solution(10 mM,pH 7.0)to form a series of matrixspiked samples(with concentrations of 0.02,0.05,0.1,0.5,1,5,10,and 20 ng/mL)for construction of calibration curves.Quantitative calculations were performed using the internal standard(IS)calibration curve method.Bupivacaine was used as the IS and was added into the eluent with a concentration of 10 ng/mL.

Good linearities were obtained in the concentration range of 0.02-20 ng/mL,with correlation coefficient values(r2)＞0.9901 for analyses of the three target β-blockers(Table 1).The limits of detection(LOD)and quantification(LOQ)were from 3.5 to 6.8 pg/mL and 11.7-22.8 pg/mL,respectively,indicating desirable sensitivity of the developed MSPE-FTICR-MS method.

Table 1Reproducibility,linearity and sensitivity for β-blockers by MSPE-FTICR-MS analysis.

The repeatability experiment was performed by conducting six consecutive analyses of phosphate sample solutions(10 mM,pH 7.0)spiked with 1.0 ng/mL of each β-blocker in a single day.For reproducibility,the experiment was performed by analyzing six phosphate solution(10 mM,pH 7.0)-spiked samples(1.0 ng/mL of each β-blocker)on six different days.The obtained relative standard deviation(RSD)values of the ion intensity ratio of analyte to IS(Ianalyte/IIS)for each β-blocker are summarized in Table 1.The results indicated that our developed MSPE-FTICR-MS method had good repeatability,with RSD values ranging from 4.1% to 5.6% for six replicate analyses.In addition,our method showed desirable reproducibility,with RSD values ranging from 9.8% to 10.6% for six parallel experiments conducted on different days.

3.5.Real sample analysis

The feasibility of applying our MSPE/FTICR-MS method in the analysis of real biological samples was verified by analyzing the βblockers in spiked blood specimens from healthy human volunteers.The recovery(%)of the analyte was calculated from the measured versus added amounts according to Eq.(3).

In Eq.(3),cfoundandcspikedare the concentrations of analyte in the eluate and spiked standard solution,respectively,andV1andV0are the volumes of the eluate and spiked standard solution,respectively.To determine the recoveries of the three investigated β-blockers(i.e.,acebutolol,metoprolol,and propranolol)in the blood,three blank human plasma samples were spiked with 1 and 10 ng/mL of the mixed standard.Using the above equation,the recoveries were calculated to be in the range of 80.9%-91.0%(Table 2).

Table 2Recovery and RSD values in analysis of the spiked blood samples at two concentration levels by MSPE-FTICR-MS(n=3).

The analytical performances of the spiked blood samples(10ng/mL) with and without undergoing extraction using polyDOPA@Ag-MNPs were compared,and the results are shown in Fig.5.When the spiked blood sample was directly detected by the ESI-FTICR-MS method,the signals of the three investigated βblockers were very weak(Fig.5A).However,the signals of the investigated β-blockers in the blood sample were clearly observed using the MSPE-FTICR-MS method(Fig.5B).As is known to all,the composition of deproteinized blood is very complex and contains components such as carbohydrates,lipids,amino acids,and inorganic salts.Matrix components in biological samples often affect the sample preparation step and the ionization efficiency of MS[39].Overall,our results indicate that this method provides a novel technique for the sensitive detection of trace β-blockers in human blood samples.

Fig.5.Mass spectra of(A)spiked human blood samples(10 ng/mL of each β-blocker)without extraction and(B)spiked human blood samples after extraction with polyDOP@Ag-MNPs.

4.Conclusions

In conclusion,a polyDOPA@Ag-MNP-based MSPE coupled with FTICR-MS method was developed for the rapid analysis of trace βblockers in biological samples.The redox-active polyDOPA facilitated in situ formation of polyDOPA@Ag-MNPs,which not only effectively removed matrix interferences from biological samples,but also enhanced detection sensitivity through the enrichment of targets.By using our developed method,rapid and sensitive analysis of trace β-blockers in blood samples was successfully achieved,with an overall detection process of less than 10 min as well as LOD and LOQ in the ranges of 3.5-6.8 pg/mL and 11.7-22.8 pg/mL,respectively.Considering the excellent properties and broad application of the synthesized nanocomposites,this strategy provides a novel method for use in pharmacokinetic and pharmacodynamic studies on β-blockers.

CRediT author statement

Xue Xiao:Investigation,Writing-Original draft preparation,Methodology,Formal analysis;Kaili He:Formal analysis;Ya-Jun Hou:Validation;ZhangminXiang:Project administration;Yunyun Yang:Writing-Reviewing and Editing,Project administration,Funding acquisition.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China(Grant Nos.:21976168,22127810,and 22004113),Key Research and Development Program of Guangdong Province(Grant No.:2020B1111350002),Guangdong Basic and Applied Basic Research Foundation(Grant No.:2019A1515110420),and GDAS＇Project of Science and Technology Development(Grant No.:2021GDASYL-20210103034).