Quantitative Determination of Bulk Molecular Concentrations of β


Rapid quantitative determination of bulk molecular concentration in solid samples without sample pretreatment is demonstrated using the internal extra...
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Rapid quantitative determination of bulk molecular concentrations of #-agonists in pork tissue samples by direct internal extractive electrospray ionization mass spectrometry Jiaquan Xu, Shengrui Xu, Yipo Xiao, Konstantin Chingin, Haiyan Lu, Runhan Yan, and Huanwen Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00517 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Analytical Chemistry

Rapid quantitative determination of bulk molecular concentrations of β-agonists in pork tissue samples by direct internal extractive electrospray ionization mass spectrometry Jiaquan Xu, Shengrui Xu, Yipo Xiao, Konstantin Chingin, Haiyan Lu, Runhan Yan and Huanwen Chen* Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang, P. R. China. *Email: [email protected]; Fax: +86-791-83896370 ABSTRACT: Rapid quantitative determination of bulk molecular concentration in solid samples without sample pretreatment is demonstrated by the internal extractive electrospray ionization mass spectrometry (iEESI-MS) analysis of six β-agonists, including salbutamol (Sal), clenbuterol (Cle), ractopamine (Rac), terbutaline (Ter), tulobuterol (Tul), brombuterol (Bro), in pork tissue samples. Single sample analysis only required 1 min. The linear range of detection was about 0.01-1000 µg/kg (R2>0.9994). The limit-of-detection (LOD) varied from 0.002 µg/kg for Sal to 0.006 µg/kg for Tul. Relative standard deviation (RSD) of quantitation was in the range 6.5-11.3%. The analytical results were validated by gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography mass spectrometry (LC-MS), showing the accuracy rates of 92-105%. The current study extends the power of ambient MS as a method for the quantification of molecules at the surface of solid samples (e.g. in µg/cm2 units) toward the quantification of molecules in bulk sample volume (i.e. in µg/kg units), which commonly required in food safety control, biomedical analysis, public security and many other disciplines.

Owing to the minimal sample pretreatment, ambient ionization techniques such as desorption electrospray ionization (DESI),1 direct analysis in real time (DART),2 desorption atmospheric pressure chemical ionization (DAPCI),3 lowtemperature plasma probe (LTP),4 electrospray-assisted laserdesorption/ionization (ELDI),5 extractive electrospray ionization (EESI),6 etc. increase the simplicity7-11 and throughput12-17 of mass spectrometry (MS) analysis. Efforts are now exerted to improve the quantitation power of ambient MS analysis.18 Fast quantification of analytes on the sample surface in µg/cm2 units has been achieved for many types of solid samples.1-5, 1920 However, quality assessment protocols typically require bulk analyte concentration (e.g. in µg/L units).21-24 For the real-world solid samples, the surface concentration can significantly differ from the bulk-phase concentration25 and thus cannot be reliably used to represent the bulk concentration of the given analytes. Gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LCMS) are widely employed as the standard methods for the determination of bulk molecular concentration in solid samples,26-31 but these methods require multiple-step sample pretreatment and long analysis time.32-34 So far, bulk molecular concentration in ambient MS can only be derived for solution samples.6,21 The rapid quantitative molecular analysis of bulk solid samples would therefore greatly strengthen the application of ambient MS analysis to the real-world problems. Internal extractive electrospray ionization mass spectrometry (iEESI-MS) detects analytes inside a bulk sample without sample pretreatment.25 In iEESI, a charged solution such as methanol/water (1:1) is directly infused through the volume of a bulk sample. The solution is supplied through the capillary inserted into the sample. Driven by the high electric field between the inserted capillary and the ion entrance of the mass spectrometer, the solution diffuses toward the ion entrance and forms an ionic plume at the edge of the sample, which gives rise to gas-phase ions similar to that in ESI. Up to now, iEESIMS has mainly been applied for the qualitative characteriza-

tion of various biological samples (tissues, fruits, vegetables, etc.),35-39 motivating further development of iEESI-MS for the accurate quantitative analysis of analytes in biological bulk sample. Herein, a disposable iEESI device consisting of a sample chamber, seal cage, sample holder and adapter was developed and applied for the direct quantitation of analytes inside bulk samples (Figure 1a-b). As a illegal feed additives, β-agonists were always used to promote animal growth rates and increase muscle leanness by inducing redistribution of fat in the muscle tissues of mammals,40,41 which were harmful to human health.42 Therefore, as a model demonstration, six types of βagonists such as salbutamol (Sal), clenbuterol (Cle), ractopamine (Rac), terbutaline (Ter), tulobuterol (Tul), brombuterol (Bro) in pork tissue samples were successfully quantified by iEESI-MS with the analysis time of 1 min per sample, linear response range of 0.01-1000 µg/kg (R2>0.9994) and a lowest LOD of 0.002 µg/kg. The analytical results were validated using GC-MS and LC-MS, with the accuracy of 92-105%. The work offers a new strategy for direct quantitation of analytes in a bulk sample using MS without tedious sample pretreatment.

EXPERIMENTAL SECTION Equipment, materials and reagents. The iEESI-MS experiments were carried out using a homemade disposable iEESI source installed onto a linear trap quadruple (LTQ) mass spectrometer (Thermo Scientific, San Jose, U.S.A.). Being improved from the iEESI source described in previous work 19, the disposable iEESI was composed by four easy-to-made parts including a sample chamber, a seal cage, a sample fixer, and an adapter with capillary (Figure 1a). The fused silica capillary (0.10 mm i.d., 0.15 mm o.d., Agilent Technologies Co., Ltd., U.S.A.) was used to inject the extraction solution into the tissue samples. Methanol (HPLC grade) was purchased from ROE Scientific Inc. (Newark, U.S.A). The ul-

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Qualitative and quantitative analysis. A standard pork sample contained known β-agonist was prepared by spiking known amount β-agonist according to previously reports25,40 and national standards30,31. Firstly, β-agonist-free pork tissue samples which were confirmed by GC-MS in the reference laboratory of Sport Science Institute of Jiangxi Province were used as reference blank samples.31 Secondly, blank pork tissue strips (2 g each, sized as 50 × 20 × 2 mm) were immersed into the series of β-agonists standard solutions (50 mL each) with different concentration levels for 10 h inside an air-lightly sealed beaker with gently shaking/stirring for 3 min after every 30 min intervals at the room temperature, allowing the βagonists evenly distributed in the tissue strips. After incubation, the pork strips were flushed using water for 3 times once they were taken out from the β-agonists standard solution. The flushed tissue samples were placed on a glass for 5 min to dry and then the tissue strip was cut into small pieces (1.5 mg each, sized as 1 × 1 × 2 mm), which was then loaded in the iEESIMS chamber for direct analysis without further treatment. The eluent plus the solution left inside the beaker were quantified to obtain the left-over β-agonists using the characteristic fragments of each β-agonist obtained by ESI-MS.43 The total amount of the β-agonists diffused into the pork tissue strips (Q) were calculated as follows: Q=Qb-Qa, where Qb was the total amounts of the β-agonists added into the solution before the tissue strip was added; Qa was the total amounts of the β- agonists remained inside the solution after the tissue strip was incubated for 10 h. For better comparison, a big piece of pork (lean tissue) was cut into small piece and mixed first, and then divided into three part for iEESI-MS, GC-MS (5 g) and LC-MS (2 g) analysis. Note that due to the fixed small volume of the sampling chamber of iEESI device, the big pieces of lean pork were analyzed by following the procedure of iEESI-MS analysis described elsewhere25,35.

trapure water used for the experiments was produced by Millipore ultrapure water system. Standards: Cle, Sal, Rac, Ter, Tul, Bro with purity of 99.5% ±0.1% were purchased from Laboratorien Berlin-Adlershof GmbH company (Berlin, Germany). Standard stock solutions: 10 mg/L standard stock solutions were prepared by dissolving the standards in distilled water respectively, followed by storing at 255 K. Standard solution: Standard solutions with a serials concentration of 0.01, 0.1, 1, 10, 100, 1000 µg/L were prepared by diluting the standard stock solution. As noted by earlier studies, β-agonists preferably accumulate in lean tissue. Thus only lean meat samples are necessary to be analyzed for the purpose. The lean pork samples were provided by the Sports Science Institute of Jiangxi Province. The protein and fat of the lean pork samples were about 23.5% and 3.6%, respectively. Only those samples contained no βagonists and passed the quality control by national standards of China31 were employed as the blank samples for further experiments. iEESI-MS analysis. The sampling ionization procedure is schematically illustrated in Figure 1b. The iEESI sampler was designed to allow accurate sampling of a tiny amount (1.5 mg) of tissue sample. As shown in Figure 1b, for iEESI-MS analysis, the pork tissue sample was directly loaded in the sampler chamber by pressing the sampler on the tissue with a single punch, requiring no sample pretreatment. Due to the defined capacity of the sample chamber, the amount of sample loaded in the chamber was defined. Then, the sample chamber filled with the pork tissue was assembled with the other three components to form an iEESI source, which was equipped on the MS instrument for direct tissue sample analysis (Figure 1c). The distance between the tip of the iEESI and the mass spectrometer inlet was 4.0 mm. Biased by a high voltage (+5 kV), the mixture of methanol/water (V:V=1:1) was slowly infused at 1 µL/min as the extraction reagent into the 3-dimensional volume of the pork by a syringe pump. The analytes were extracted by the infused solvent and carried forward by the electric field gradient inside the bulk volume of the pork tissue. A stable electrospray plume was generated from sample chamber tip, in front of the MS inlet (Figure 1d), producing the analytes ions in the open-air for mass analysis. The LTQ mass spectrometer was set to run in the positive ion detection mode. The temperature of the heated capillary of the LTQ MS instrument was 150 °C. The tube lens voltage was +100 V and the capillary voltage was +10 V. Because the molecular weight of six β-agonists is below 400, the m/z range was set between 50 and 400 during the β-agonists analysis. Collision induced dissociation (CID) experiments were performed with an isolation mass-to-charge ratio window width of 1.5 Da and normalized collision energy (CE) of 16-25%. All the mass spectra were recorded about 7.0 min with background subtracted. The MS1 and the MS2 spectra were collected alternately with about 0.5 min intervals controlled by software (Thermo Xcalibur), unless labeled elsewhere. The MS2 spectra of Sal, Cle, Bro, Ter, Tul, Rac were recorded successively. The intensity averaged for about 30 s of the characteristic fragment signal of a given β-agonist was used for quantification. The rest parameters were set as the default values of the LTQ instrument, and no further optimization was performed.

Figure 1 Schematic illustration of iEESI-MS for direct quantitative analysis of pork tissue sample using a disposable iEESI source. (a) The disposable iEESI device and its components; (b) Analytical process of pork tissue sampling using iEESI-MS. Ca. 1.5 mg pork tissue was loaded into the sample chamber with a punch requiring no sample pretreatment. The distance between the tip of the iEESI and the mass spectrometer inlet was 4.0 mm. Biased by a high voltage (+5 kV), the mixture of methanol/water (V:V=1:1) was infused at 1 µL/min as the extraction reagent; (c) A photo of the interface between iEESI device and MS; (d) A photo of a stable plume from the tip of sample chamber marked by a red arrow.

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Analytical Chemistry the apex of the sample chamber (Figure 1 c, d). The air-tight sealed design of the configuration prevented material loss during iEESI process, resulting in the improved sensitivity and repeatability of analysis. Qualitative performance of iEESI-MS. The typical total ion chromatography (TIC, Figure 2a) and extracted ion chromatography (EIC, Figure 2b-e and Figure S-1a-d) for pork analysis showed that the iEESI-MS signals of targeted analytes lasted more than 3 min for MS1 and 0.5 min for MS2 of each β-agonist, allowing the sufficiently stable signals and enough time for qualitative and quantitative analysis. More data about TIC and EIC about Sal, Cle, Bro, Ter, Tul and Rac were showed in Figure 2 d-e and Figure S-1e-h. All the β-agonists including Sal (MW 239), Cle (MW 276), Rac (MW 301), Ter (MW 225), Tul (MW 227) and Bro (MW 366) were detected as protonated molecules in the iEESI-MS spectra (Figure S-2a), which were in agreement with the previous data obtained using LC-MS44-46. Tandem mass spectrometry experiments were carried out to confirm the molecular ions of six β-agonists. The corresponding MS2 data (Figure S-2b-g, Table S-4) were matched using authentic β-agonist compounds, showing the CID fragmentation patterns which were very similar to the reported data in the literature44,48 and identical to those detected from the spiked pork tissue samples. For each kind of β-agonists, the strongest fragment ions, marked with red star (Figure S-2b-g), was selected as the signal ions for quantitative measurements.

Thus, the standard concentration of each pork tissue sample (C) was available via the formula below in the unit of bulkphase concentration (µg/kg): C=Q/m, where Q was the total amounts of the β-agonists diffused into the pork tissue strips; m was the mass of each blank port tissue strip weighted before the sample was soaked into the β-agonists solution. The values of Q and C of prepared standard pork samples were displayed in Table S-3. Once the series of standard port tissue samples were prepared, iEESI-MS analysis was performed to obtain the characteristic fragment of β-agonists in the MS/MS spectra. Finally, a linear correlation curve between the intensity levels of the characteristic fragment (Y) and the concentration levels of β-agonists in each pork tissue sample (C) was obtained in the range of 0.01-1000 µg/kg with the logarithm scales. Such a linear curve was then used as the working calibration curve for real sample analysis. For direct infusion ESI-MS experiments, the β-agonists in pork tissue (1.5 mg) were pressed out from the tissue into a 1.5 mL vial and then diluted by 100 µL CH3OH/H2O (V:V=1:1) solution, which was then directly infused at a flow rate of 1 µL/min for ESI-MS analysis. The mass analyzer was LTQ, the ESI high voltage was set at +5 kV (i.e., the same as that for iEESI), and nitrogen sheath gas was 0.8 MPa for the ESI experiments. To validate the accuracy of the iEESI-MS results, conventional GC-MS and LC-MS, which was operated as that required in a national standard method30,31 for detection of βagonists in bulk-phase concentration (detailed in SI, Table S-1 and Table S-2), were performed as the reference method.

RESULTS AND DISCUSSION Development of the disposable iEESI device. To obtain better quantitation performance, it is necessary to accurately control the total volume of the bulk sample which is sampled during the iEESI process. For precise sampling and minimizing potential contamination, a new disposable iEESI device (Figure 1a) was developed based on the earlier configuration introduced in our previous work.25 The new configuration consists of four major parts, sample chamber for sample loading; sample holder; sealing cage; adapter with capillary (Figure 1a). The material used for preparation of sample chamber was stainless steel, while the material used for preparation of seal cage, sample holder and adapter was polyetheretherketone. The sealing cage was used for air-tight sealing and mounting the sample chamber to the adapter. A tissue sample was loaded through a 2 mm hole by pressing the sample chamber with sharpened edges against the bulk tissue sample (Figure 1b). The sampling device allowed high sampling reproducibility with RSD (n=6) values no more than 3% for 6 duplicated sampling. Although the sampling part could be used to load the same amount of tissue for more than 20 times, it was recommended for single use only since the part was disposable and thus the potential contamination could be avoided. After the sample was loaded, the sample chamber was assembled with the other parts to form an integrated iEESI source, accompanying with insertion of the capillary into the sample for injecting extraction reagent into the sample. The capillary was tightly fixed on the adapter to prevent deviations in the depth of capillary penetration into the sample. A distance of 2 mm between the tip of capillary and the front edge of sample was precisely controlled by a fixture. Ionic spray was generated through a tiny hole (7.0 µm, i.d.) at

Figure 2 Typical TIC and EIC of 6 β-agonists in pork tissue samples analysed by iEESI-MS. (a) TIC of pork sample, (b) EIC of Sal, (c) EIC of Cle, (d) TIC of Sal in CID analysis, (e) TIC of Cle in CID analysis. The duration of 0.5-1.1 min, 1.7-2.3 min, 2.8-3.4 min, 4.0-4.6 min, 5.25-5.9 min, 6.5-7.1 min were used for CID analysis of Sal, Cle, Bro, Ter, Tul and Rac, respectively.

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Table 1 Analytical results of iEESI-MS, LC-MS and GC-MS for the same pork tissue samples purchased from local market Sample

1

2

3

4

5

Sal

LC-MS GC-MS iEESI-MS Acc L Acc G LC-MS GC-MS iEESI-MS Acc L Acc G LC-MS GC-MS iEESI-MS Acc L Acc G LC-MS GC-MS iEESI-MS Acc L Acc G LC-MS GC-MS iEESI-MS Acc L Acc G

Content (µg/kg)

RSD (%)

0.073 0.069

10.1 8.3

Cle Acc (%)

Content (µg/kg)

RSD (%)

-

-

94.5 4.761 5.067 4.674

8.5 10.8 10.1

0.195 0.183

8.2 7.8 8.5

7.6 11.3

0.751 0.712 0.738

10.7 9.1 6.8

-

2.237 2.537 2.312

9.1 10.5 8.8

Acc (%)

-

5.852 5.580 5.402

8.5 9.8 8.3

Ter Content (µg/kg)

RSD (%)

0.198 0.184

8.7 10.3

-

9.7 10.3 7.6 9.2 10.9 9.8

94.3 103.8 0.495 0.486 0.465

98.5 104.9

-

98.3 0.968 0.879 0.913

8.1 8.3 7.3

RSD (%)

-

7.9 9.2 6.5

92.1 104.1 1.111 1.044 1.095

Content (µg/kg)

93.8 -

97.8 92.6 3.095 2.740 2.850

Acc (%)

-

98.1 92.3 11.854 12.527 11.594

Bro

0.040 0.037

-

RSD (%)

-

-

-

-

9.8 10.7

-

-

-

Acc (%)

Content (µg/kg)

RSD (%)

7.458 7.813 7.134

8.1 7.5 7.9

-

9.6 8.7 11.9

-

102.6 97.6 27.030 28.574 26.080

6.7 7.4 8.6

0.094 -

11.2

Acc (%)

95.6 91.3 7.854 7.312 7.505

-

92.5 -

-

93.9 95.6

Content (µg/kg)

-

92.3 96.9

-

Acc (%)

Rac

92.9 -

103.3 91.1 -

Tur

96.5 91.3 40.302 40.728 37.253

4.9 7.9 7.4 92.4 91.5

-

21.063 23.504 21.563 -

7.8 6.8 9.3 102.4 91.7

* Acc L represent accuracy of iEESI-MS based on LC-MS and Acc G represent accuracy of iEESI-MS based on GC-MS. Acc L = Result obtained by iEESI-MS/Result obtained by LC-MS. Acc G = Result obtained by iEESI-MS/Result obtained by GC-MS. (n=3 for RSD)

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Quantitative performance of iEESI-MS. Five measurements were carried out in parallel to evaluate the precision (i.e, RSD values) of the calibration. As the results, the RSDs of 5 measurements were less than 9% for all the data points employed to make the calibration curve. This finding indicated that the iEESI-MS was of high precision, and all the samples prepared in this study were highly uniform. Figure 3 presents six calibration curves obtained according to the experimental procedure, with the calibration equations such as (Ysal)= 0.317X+1.807, (Ycle)= 0.229X+1.408 for Sal and Cel, respectively. A wide linear response range of 0.01-1000 µg/kg in the logarithmic scales with R2 > 0.9994 was obtained for the six βagonists tested. The LOD of β-agonists in pork tissue defined by a signal-to-noise of 3 were 0.002 µg/kg (Sal), 0.005 µg/kg (Cle), 0.003 µg/kg (Rac), 0.003 µg/kg (Ter), 0.006 µg/kg (Tul) and 0.002 µg/kg (Bro). The direct extraction of β-agonists from three dimension in pork sample by charged extraction agent could greatly improve the analytical volume of the sample per unit volume of extraction agent, resulted in increasing the concentration of β-agonists per unit extraction agent. This may be the main reason for low detection limit of iEESI-MS. Detailed iEESI-MS data to make the calibration curves were provided in Table S-5. Nice recoveries of β-agonists were also obtained by iEESI-MS with a range of 91.2-102.7% (Figure S6). For performance reference, direct infusion ESI-MS was employed to obtain the LOD values of 6 β-agonists spiked into the pork tissue samples. The experimental results showed that the LOD values of direct infusion ESI-MS varied in the range of 0.26-0.75 µg/kg (detailed in Table S-7), which were all higher than those obtained by iEESI-MS. Note that only a tiny amount of solvent (less than 10 µL) was used to extract the tissue sample (1.5 mg) in the iEESI process, making the analyte levels in the electrospray solution were relatively higher than those for the direct infusion ESI process. Alternatively, the inferior performance could also be attributed to other factors such as insufficient squeezing/collection of the analytes, low ionization efficiency and chemical interference in the direct infusion ESI case. For instance, all signals of 6 β-agonists were detectable by iEESI-MS using the pork tissues subjected to sever squeezing, confirming that the β-agonists were not all collected for ESI in the direct infusion experiments.

Figure 3. The calibration curves of six β-agonists obtained by iEESI-MS. The error bars represent standard deviations of 4 replicates (n=4).

Method validation by GC-MS and LC-MS. Figure 4 shows the mass spectral data recorded by iEESI-MS from pork tissue samples contaminated with 6 β-agonists at varied levels for the LOD determination. The experimental results demonstrated that the LOD values obtained using iEESI-MS were significantly lower than those obtained by conventional methods recommended by the national standards of China, probably due to the factors such as highly sensitive detection by LTQ-MS instrument and high ionization efficiency of iEESI. To demonstrate the accuracy of iEESI-MS for practical analysis of unknown samples, large pieces of pork tissue samples purchased from local markets were analyzed by the iEESI-MS. The iEESI-MS analytical results were validated following the GC-MS or LC-MS procedures (detailed in SI) as regulated by the national standards of China30,31. For iEESIMS analysis, the pork tissue sample (e.g., 1.5 mg, sized as 1 × 1 × 2 mm) was directly loaded in the sampler with a single punch, requiring no sample pretreatment. Characteristic fragment ions of β-agonists were detected for quantitation according to the calibration curve (Figure 3), and the results were shown in Table 1. Note that large amounts of pork samples (e.g., 5-10 g for GC-MS; 2-5 g for LC-MS) were consumed for those containing β-agonists at sub-ppb levels. However, increasing the sample consumption was not always effective, especially for the samples containing β-agonists at tens ppt levels, probably the tiny amounts analytes were impossibly to be effectively concentrated for GC/LC-MS detection according to the standard operation procedure. For such samples, a novel sample pretreatment method must be innovated prior to method validation using GC/LC-MS. However, this has not been attempted due to the scope of the current study. Given the results obtained by GC-MS and LC-MS as the true values, the accuracy of the iEESI-MS corresponding to GC-MS (AccG) and LC-MS (AccL) was calculated, respectively. As shown in Table 1, the iEESI-MS accuracy of 92-105% was obtained for direct detection of β-agonists in the bulk tissue samples with β-agonists at 0.2-40 µg/kg. For iEESI-MS it took less than 1 min for a single sample run. On the other hand, timeconsuming pretreatments such as mashing, ultrasonic processing, centrifuging, extracting, concentrating, filtering, purifying, etc. were demanded to make the β-agonists solutions ready for GC-MS or LC-MS analysis. The concentrations of βagonists were then calculated according to the linear equation of GC-MS (Table S-8) or LC-MS (Table S-9). The whole process of GC-MS analysis took the skillful technician more than

LOD values of 0.1-0.7 µg/kg (detailed in Table S-8) were obtained by GC-MS with 5.0 g sample. Alternatively, the LOD values of 0.01-0.08 µg/kg (detailed in Table S-9) were obtained by LC-MS with 2.0 g sample. The LODs between different methods could be affected by many factors. The MS instruments were one of the major factors contributing significantly to the LOD values in our case. In this work, GC-MS was performed on a commercial instrument (Agilent 7000c) equipped a triple quadrupole mass analyzer and EI source with SIM scanning mode, while a most sensitive mass analyzer of LTQ were used to obtained LC-MS (ESI source) and iEESIMS (iEESI source) data with MRM and SIM scanning model, respectively. Apparently, the remarkable enhancement of the sensitivity achieved in this work was impossibly contributed only by the iEESI technique. Besides of the derivatization for GC-MS detection, multi-step pretreatments including homogenate, enzymolysis, centrifugation, extraction (isopropanol/ethyl acetate, V/V=4:6), purification, concentration etc. were required for both GC-MS and LC-MS to obtain the analytical solutions of β-agonists, such a tedious sample pretreatment process could cause low sensitivity in analytes detection due to the inefficient extraction and analytes losses.

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a half day in this study. Promisingly, iEESI-MS presents an alternative choice for the quantitative analysis of trace analytes such as β-agonists in bulk pork tissue samples, with significantly improved analysis speed and the bulk molecular concentrations.

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Supporting Information Experimental description of GC-MS for analysis of six βagonists; Experimental description of LC-MS for analysis of six β-agonists; The parameters of prepared standard pork samples; EIC and TIC (CID fragments) analysis of four βagonists in pork sample by iEESI-MS; iEESI-MS spectra of pork sample with β-agonists; Characteristic mass spectra peak of six β-agonists; Calibration curve data of six kinds of βagonists obtained by iEESI-MS; Recoveries obtained for iEESI-MS of 6 β-agonists; Calibration curve data of six kinds of β-agonists obtained by direct infusion ESI-MS; Calibration curve data of 6 kinds of β-agonists obtained by GC-MS; Calibration curve data of 6 kinds of β-agonists obtained by LC-MS.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes The authors declare no competing financial interest and no conflicts of interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21225522), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (No. IRT13054), Jiangxi Province Program for the support of Science and Technology in Universities (No. KJLD13051), the China Academy of Metrology Science and Technology (No. 40AKYKF1601).

REFERENCES (1) (2)

Figure 4. Mass spectra of β-agonists in pork tissue samples recorded by iEESI-MS/MS using CH3OH/H2O (V/V=1:1) as the extraction solvent for LOD determination. (a) Full scan mass spectrum; (b) MS/MS spectrum of Sal at 0.002 µg/kg; (c) MS/MS spectrum of Cle at 0.005 µg/kg; (d) MS/MS spectrum of Bro at 0.003 µg/kg; (e) MS/MS spectrum of Ter at 0.003 µg/kg; (f) MS/MS spectrum of Tul at 0.006 µg/kg; (g) MS/MS spectrum of Rac at 0.002 µg/kg. The fragments marked by red star were used for quantitation of the corresponding β-agonist.

(3) (4) (5)

(6)

CONCLUSION

(7)

The distinctive feature of the presented ambient iEESI-MS approach is that it allows precise molecular quantification of molecular analytes in the bulk volume of solid samples rather than on a sample surface. Bulk-phase concentration was directly obtained without sample pretreatment using the disposable iEESI source dedicated for quantitative analysis. Six βagonists in pork tissue samples were successfully quantitatively determined by iEESI-MS with 1 min per sample, linear response range of 0.01-1000 µg/kg (R2>0.9994) and LOD of 2 ng/kg and accuracy 92-105%. The unique capability of iEESIMS for the rapid and accurate quantification of trace analytes in bulk-phase concentration units (e.g., ng/kg) may promote the application of ambient mass spectrometry in multiple disciplines (e.g., for the food safety, biomedical, public security) in the near future.

(8) (9) (10) (11) (12) (13) (14) (15) (16) (17)

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