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Simultaneous determination of blockers and agonists by on-fiber derivatization in selfmade solid-phase microextraction coating fiber Wei Liu, Zhimin Yan, Xiayang Huang, Jinfeng Chen, Minghua Lu, Lan Zhang, Guonan Chen
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Received date: 10 April 2014 Revised date: 19 July 2014 Accepted date: 21 July 2014 Cite this article as: Wei Liu, Zhimin Yan, Xiayang Huang, Jinfeng Chen, Minghua Lu, Lan Zhang, Guonan Chen, Simultaneous determination of blockers and agonists by on-fiber derivatization in self-made solid-phase microextraction coating fiber, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.07.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination of blockers and agonists by on-fiber derivatization in self-made solid-phase microextraction coating fiber
Wei Liu1, 2, Zhimin Yan1, Xiayang Huang1, Jinfeng Chen1,3, Minghua Lu1, 2, Lan Zhang1, 2*, Guonan Chen1* 1. Ministry of Education Key Laboratory of Analysis and Detection for Food Safety (Fuzhou University) 2. Test Center of Fuzhou University, Fuzhou University, Fuzhou, Fujian, 350002, China 3. Longyan Entry-Exit Inspection and Quarantine Bureau, Longyan, Fujian, 364000, China
Corresponding author: Lan Zhang Postal address: Department of Chemistry, Fuzhou University, Fuzhou 350002, Fujian, China Fax: 86-591-87893207 Tel: 86-591-87892448 E-mail: [email protected]
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Abstract: An environmentally friendly and sensitive method for determination of blockers and agonists was described in this paper. The method is based on a homemade sol-gel solid-phase microextraction (SPME) coating with simultaneous on-fiber derivatization and subsequent gas chromatography mass spectrometry (GC/MS) analysis. The influences of the main factors on the type and thickness of the homemade fiber coatings, conditions of the derivatization, extraction and desorption of SPME were investigated in detail. The proposed procedure showed limits of detection lower than 0.2 ng mL-1. The linearity was in the 0.5-150 ng mL-1 for clenbuterol and 1.0-100 ng mL-1 for metoprolol and propranolol. The variation in measurements (repeatability) was below 8.7% (n = 6) and the degree of difference between (reproducibility) was below 11.4% (n = 3). In the application, spiked human saliva samples and real human saliva samples were analyzed, it was found that saliva would affect the detection of propranolol when it was a very low content. The established method can be feasible in practical application and helpful for agonists and blockers preliminary screening during the competition.
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Keywords: blockers, agonists, on-fiber derivatization, solid-phase microextraction, gas chromatography-mass spectrometry
1. Introduction Clenbuterol is one of the most widely used 2-adrenoceptor agonists, while metoprolol and propranolol are the most common -antagonists (-blockers). They both work on the body’s -adrenergic receptors with contrary effects and they are structural related amino alcohols. So they can be manipulated and analyzed together with similar detection procedures. The mainstream methods for determination of blockers and agonists include GC-MS [1, 2], liquid chromatography [3, 4], Capillary electrophoresis mass spectrometry (CE-MS)[5, 6] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [7-9]. The involved biological samples include hair [10], whole blood [11], serum [12], plasma [13, 14] and urine [5]. In the Medical Commission of the International Olympic Committee, the 2-agonists and -blockers have been forbidden. And the official detection requirement is concentration of 0.5 g mL−1 in urine by GC-MS after derivatization. In fact, no matter what kind of biological sample analysis, the pre-treatment and concentration is essential. And the new sample preparation method solid-phase microextraction (SPME) integrated sampling, extraction, concentration and sample introduction into one step, it has been considered as a very powerful sample preparation tool[15]. While introducing derivatization in SPME-GC/MS can further
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expand the application areas of SPME-GC/MS and bring lower limits of detection (LODs), there have been some researches and several reviews on this topic [16-20]. In brief, achieve derivatization operation during SPME-GC/MS process have three modes: in the gas chromatography injector port [21], in the sample matrix [22-24] and on the fiber coating after SPME sampling [25, 26]. The injection-derivatization method is the inchoate derivatization approach of SPME-GC/MS which might lead to incomplete derivatization reaction and possible damage to the capillary column because of the excessive derivative agent. Derivatization reaction in the sample matrix with SPME sampling simultaneously is the simplest mode but with some limitations. While the derivatization occurs on the SPME fiber coating, the fiber is not only a separation/extraction device but also being turned into a reaction vessel. It makes the whole process simple and fast. Until now, GC-MS is the major detection technique for beta2-agonists and beta-antagonists. And analysis of 2-agonists and -blockers in saliva is a great choice because of its noninvasive, easy to collect and less confidential in comparison with blood and urine. In this paper, homemade sol-gel SPME coatings were prepared and contrasted for on-fiber derivatization and SPME operation; an entire GC-MS determination method for blockers and agonists was trying to establish and the application in positive saliva samples were realized so as to the unlimited and sensitive preliminary screening of clenbuterol, metoprolol and propranolol in competitions.
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2. Experimental Procedures 2.1. Chemicals and Reagents Silylating reagents of N-methy-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was obtained from Sigma (SIGMA, St. Louis, MO, USA). Clenbuterol, metoprolol and propranolol (Fig. 1) were from national institute for the control of pharmaceutical and biological products (Beijing, China). Methyl methacrylate (MMA), benzoyl peroxide (BPO), tetramethyl orthosilicate (TMOS), trifluoroacetic acid (TFA) and polyethylene glycol 20 000 (PEG 20 000) were from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China). Hydroxy-terminated silicone oil (OH-TSO) and poly methyl hydrogen siloxane (PMHS) were purchased from Santai Organic Silica Material Factory (Zhejiang, China). Divinylbenzene (DVB) was obtained from Shengzhong Fine Chemical Co.Ltd. (Shanghai, China). Position for Fig. 1 A stock standard mixture containing clenbuterol, metoprolol and propranolol (1.00 mg mL-1, respectively) was prepared in methanol and stored at 4 . Working standard solutions were prepared by diluting with methanol and renewed every week. Spiked water samples were prepared weekly by adding the appropriate volume of the working standards solution to the 20 mL water sample. Milli-Q ultrapure water (Millipore, Bedford, USA) was used throughout the experiments and the resistivity was about 18 M·cm (25
).
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2.2. Instrumentation and operating conditions All the gas chromatographic experiments were performed on an Agilent 6890N-5973I GC-MSD (Hewlett-Packard, Palo Alto, CA, USA). A HP-5MS GC column (0.25 m film thickness, 0.25 mm×30 m, Agilent Technologies) was used for separation. The SPME device for manual extraction was consisted in a homemade holder assembly and several replaceable homemade fiber assemblies, which were prepared as described in reference [27]. The thickness of the homemade SPME fiber coating was monitored by environmental scanning electron microscope (XL30 ESEM, Philips). The injector temperature was maintained at 270
. Splitless injection was
employed and the injection volume was 1.0 L. The oven temperature was set at 80 for 1 min, and then increased to 260 heated at 15
min-1 to 300
at 25
min-1 and held for 1 min, and then
!
. Carrier gas was helium (Purity >99.999 ) and the
flow rate was set at 1.2 mL min-1. The electron impact (EI) ion source, quadrupole mass analyzer, and the interface temperature were maintained at 230, 150 and 300 , respectively. Electron impact ionization (70 eV) was utilized. m/z range was 50-550, and solvent delay was 7 min. Positive mode was used for detection. Selected ion monitoring (SIM) was employed for quantitative measurement. For clenbuterol, the qualitative ions were 57, 73, 86 and 262 (m/z), and the quantitative ion was 86 (m/z); for metoprolol, the qualitative ions were 72, 101, 223 and 324 (m/z), and the
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quantitative ion was 72 (m/z); for propranolol, the qualitative ions were 72, 115, 144 and 215 (m/z), and the quantitative ion was 72 (m/z).
2.3. Preparation of the homemade SPME fiber coatings Before sol-gel coating, commercial silica fiber (Yongnian Optical Fiber Factory, Yongnian, Heibei Province, China) was immersed in acetone for 15 min to remove the polyimide on its surface, then rinsed with water and dried. After that, a rehydroxylation process was performed to maximize the number of silanol groups on the silica surface before the preparation of fiber coatings. At firsr, the fiber was immersed in 1.00 mol L-1 sodium hydroxide solution for 60 min to increase the number of silanol groups on fiber wall, then rinsed with water and dried. Finally, the fiber was immersed in 0.10 mol L-1 hydrochloric acid for 20 min to reestablishment of equilibrium, then rinsed with water again and dried. The procedure was repeated for 3 times. Then,
four
different
carbowax-divinylbenzene
fibers
coatings
(CW-DVB),
with
carbowax
different (CW),
polarities
hydroxyl
--
silicone
oil-polymethylhydrosiloxane (OH-TSO-PMHS) and polyacrylate (PA) -- were prepared according to the procedures in our previous work [27]. Briefly, for the preparation of OH/TSO-PMHS fiber coating, a volume of 600 L of TMOS (precursor), 440 L of OH-TSO (coating polymer), 60 L of PMHS (deactivation reagent), and 400 L of TFA (acid catalyst) were thoroughly shaken and degasification for 5 min by supersonic purifier. The mixture was then centrifuged at
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4000 r min-1 for 5 min. The precipitate was removed and the top clear sol solution was used for fiber coating. For the preparation of CW fiber coating, 400 L of TMOS, 200 L of OH-TSO, 50 L of PMHS, 300 L of 99% TFA, 200 mg of PEG-20,000 and 300 L of acetone were mixed in a 2 mL tube. For CW/DVB fiber coating, 400 L of TMOS, 200 L of OH-TSO, 50 L of PMHS, 300 L of TFA, 200 L of DVB, 200 mg of PEG-20,000 were mixed, and for PA fiber coating, 300 L of TMOS, 220 L of OH-TSO, 30 L of PMHS and 200 L of 99% TFA were mixed. When fibers were dipped vertically into a freshly prepared sol solution, it was held inside the sol solution for 20 min with sol–gel coating forming on the bare outer surface of the fiber end. For each fiber, this coating process was repeated for 3 times. Then it was placed in a desiccator at room temperature for 12 h. 2.4. SPME and derivatization procedure The SPME fibers were conditioned on GC injection port at 250;
for 2 h before
use. A 20 mL of measured solutions was placed in a 25 mL glass vial with a silicon-septum cap and spiked with analytes, and mixed with 4.5 g of NaCl adjusted pH to 11.00 with 1.0 mol L-1 NaOH. The vial was capped, and placed on a stirrer and heated to 30;
with 650 r min-1 for 30 min. After extraction, the fiber was exposed in
the headspace of a 2.0 mL sample vial containing 3.0 L MSTFA at 40;
for 10 min.
Finally, the fiber was pulled out from the vial and immediately inserted into the GC injection port for thermal desorption with 270; and 4 min.
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2.5. Sample Preparation Saliva samples were collected from three healthy male volunteers. All saliva samples were collected before or 2 h after eating 50.00 mg metoprolol. To obtain the mixed saliva samples, each volunteer was told not to swallow the saliva but to store it 6 min in their mouths when sampling. Then, saliva was collected in Eppendorf tubes and stored at −20
immediately. Every saliva sample was taken out of 200 L in
serum bottle, added 4.50 g NaCl for SPME extraction and diluted to 20.00 mL by deionized water then adjusted to pH 11.00 with 1.00 mol L-1 NaOH solution.
3. Results and discussion Because clenbuterol, metoprolol and propranolol are polar materials and cannot respond in GC/MS without derivatization, they need to be derivatized to achieve good recoveries and precision. In this paper, based on the merits of on-fiber derivatization method introduced above, the typical derivatizing reagent for hydroxyl compounds (the silylating reagent of MSTFA with -agonists and -blockers) was chosen to be loaded on the fiber after analytes extraction[28, 29]. In optimization experiments, every measuring datum was repeated three times, and an average was applied.
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3.1 Selection of SPME fiber Fiber coating is one of the key factors in the extraction efficiency to analytes. One objective of this study was to select the suitable SPME fiber coating for the extraction and derivatization of blockers and agonists from aqueous samples. In this experiment, three types of homemade fiber coating were selected for investigation as strong polar coatings CW and CW-DVB, moderate polar coating PA and weak polar coating OH-TSO/PMHS. As the rule of “like dissolves like”, it could be seen from Fig.2 that CW-DVB fiber exhibited the best effect to all three analytes. Position for Fig. 2 Fiber thickness, one of critical factors in the determination of the adsorption capacity of SPME fiber coating, is related to the number of coating times. Coating fiber of the CW-DVB column was executed from 2 to 5 layers, and the thickness of them were about 10, 21, 32 and 39 m, mesured by ESEM. It was found that the adsorption amount of analytes reached maximum when 32 m fiber coating was used.
3.2 Optimization of derivatization reactions The derivatization reaction was affected by various parameters, including the reaction temperature, the derivatization time and the volume of the reagent. The derivatization conditions were optimized to achieve both high derivatization yield and high extraction efficiency. First of all, the effect of the reaction temperatures was tested at 25 , 30 , 35 , 40
, 45
and 50
with 3 L MSTFA to the analytes, respectively. When the
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temperature was lower than 40 , the extraction yields was positively correlated with the temperature; but when the temperature was higher than 40 , the correlation was reversed because of the side reactions of derivatization (Fig. 3). So, the derivatization temperature was chosen at 40 . Position for Fig. 3 In order to examine the effect of the reaction time on the derivatization efficiency, experiments were performed at 5, 8, 10, 15 and 20 min by using MSTFA at 40 . As shown in Fig. 4, the highest peak area for the derivatives of compounds was at 10 min and with no significant change until 20 min. As a result, subsequent experiments were carried out using a 10 min derivatization time. Position for Fig. 4 Although the amount of derivatization reagents should be enough for the derivative reaction, immoderate excess of derivatization reagents is actually disadvantageous to the fiber coating lifetime. In this experiment, the effect of 1-5 L of derivatization reagent MSTFA was compared with the same amount of analytes at 40
for 10 min. It can be seen from the Fig.5 that the peak areas of three TMS
derivatives increased as the volume of MSTFA increased to a maximum of 3 L. As a consequence, 3 L of reagent was used to ensure that enough derivatizing reagent was present during the extraction process. Position for Fig. 5
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3.3 Optimization of SPME extraction conditions Many factors, including extraction temperature, extraction time, stirring speed, pH of the solution and concentration of NaCl, can influence SPME extraction efficiency. These parameters were carefully optimized in this SPME study. The effect of extraction temperature to the target compounds was examined from 20 to 60;
and the results were illustrated in Fig.6. Temperature affects both the
kinetics and the thermodynamics of the extraction process of analytes. Higher temperature can accelerate the mass transference; at the same time, partition coefficients of the analytes between the fiber and the solution decrease with the increase of temperature. In conclusion, the effect of various temperatures on the response of metoprolol was insignificant, whereas the effects on propranolol and clenbuterol reached maximum at 30; ; thus 30;
was selected as the optimum value.
Position for Fig. 6 The duration of SPME extraction was also studied. SPME is an equilibrium-based technique, and the best extraction efficiency usually reached after equilibrium. To find the optimum extraction time, the samples were stirred at 30; with 650 r min-1 for 5 to 60 min. It was found that the responses of metoprolol and clenbuterol increased with increasing extraction time until 30 min and propranolol reached maximum at 45 min. Taking into account of the effect of extraction time to all three analytes, 30 min was selected as the extraction time. The optimization of stirring speed was performed from 500 to 1100 r min-1. The
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results indicated that increasing stirring speed to 650 r min-1 could markedly enhance the extraction amounts of compounds, especially propranolol. High stirring speed can accelerate the diffusion from the bulk of the sample solution to the coating surface and reduce the equilibrium time, while the frangible silicon dioxide fiber could be damaged with excessively high stirring speed. So, 650 r min-1 was selected. One of the most important parameters in the extraction process is the pH. pH values influenced not only the dissociation of functional groups in analytes, but also the surface properties of the fiber. Generally, in order to increase the extraction efficiency of the target compounds in sample solution, appropriate pH should be chosen to keep these compounds as molecular form and prevent them from dissociating during the SPME process. The effect of the pH of the sample solution was examined by varying pH from 8.00 to 12.00 (adjusted by 1.0 mol/L HCl and 1.0 mol/L NaOH). At pH 11.00, all three analytes achieved maximum responses. Increasing the ionic strength of the sample solution can reduce the solubility of analytes in water due to the formation of hydration spheres around the ionic salt molecules. However, the additional ions brought competitive adsorption with analytes and changed the surface properties of the fiber, which were unfavorable for the extraction. For investigating the effect of salt addition, various concentrations of NaCl in sample solutions (0, 0.075, 0.150, 0.225, 0.300 and 0.350 g mL-1 ) were tested. Results showed that 0.225 g mL-1 NaCl yielded the most extracted amounts to all three target compounds; hence NaCl concentration of 0.225 g mL-1 was chosen for next experiments.
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3.4 Optimization of desorption conditions After SPME extraction and derivatization, the target compounds were desorpted using the GC injection port. With respect to the desorption conditions, both the desorption temperature and the desorption time were optimized. For removing the extracted analytes from the fiber coating to the vaporizing chamber, the temperature of injection port, namely the desorption temperature, was optimized from 240 to 290
(Fig. 7). Optimal desorption temperatures were decided
by the capabilities of the instrument, the properties of analytes and the specifications of the SPME fiber. The SPME fiber used in this method could endure 290
with
gradually degradation and the inappropriate temperature would result in the coating bleed of the fiber and could be disadvantageous to the chromatographic column. Considering precision and recovery, 270
was chosen as suitable desorption
temperature. Position for Fig. 7 Then, for achieving the total desorption of analytes with no memory effects, desorption time was optimized and the optimal time for extracting maximal amounts of all compounds was 4 min. To sum up, the optimal conditions of extraction, derivatization and desorption were as follows: in 20 mL spiked water solution, adjusting pH to 11.0, including 0.225 g mL-1 NaCl, using 32 m homemade CW-DVB fiber coating extracted
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analytes for 30 min with 650 r min-1 stirring rate in 30; ; then derivatization for 40; and 10 min with 3.0 L MSTFA; desorption was performed in 270;
for 4 min.
After choosing related influence factors, the typical total ion current (TIC) chromatograms and the extracted ion current (EIC) chromatograms of three analytes in blank water solution and spiked water solutions (2.0 ng mL-1 and 100 ng mL-1) were given as Fig. 8a and Fig.8b. It could be seen from Fig.8a that, derivatization introduced some impurities to the SPME fiber in the TIC mode; but it didn’t affect the detection of extracted ions of clenbuterol (ion 86), metoprolol (ion 72) and propranolol (ion 72) (see Fig. 8b). It demonstrated that the water matrix and SPME fiber did not affect the detection of clenbuterol, metoprolol and propranolol. Position for Fig. 8 3.5 Method performance Calibration solutions with spiked deionized water samples were adsorbed, derivatizated, desorpted, analyzed with the optimized conditions and detected with GC-MS. The regression curves, correlation coefficients (R2), linear ranges and limits of detection (LODs) of three targets were shown in Table 1. The linearity of this method was investigated over a concentration range of 0.5-150 ng mL-1 for clenbuterol and 1.0-100 ng mL-1 for metoprolol and propranolol. The linear relationship between peak areas and concentrations were observed with R2 greater than 0.9933. This allowed the quantification of the compounds by the method of external standardization. The LODs for the analytes were estimated at S/N = 3 under GC/MS conditions (SIM mode) by considering the lowest concentration of each of the
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analytes. The LODs were lower than 0.2 ng mL-1. Repeatability, expressed as the relative standard deviation of the analysis of seven samples spiked at 100 ng mL-1, was below 8.7% (n = 6). Reproducibility between days, expressed as the relative standard deviation of three samples spiked at 100 ng mL-1 and analyzed on three different days, was below 11.4% (n = 3).
3.6 Method application 3.6.1 Spiked and real human saliva sample analysis Each of the 200 L blank saliva sample obtained from healthy people was added 4.50 g NaCl and diluted with deionized water to 20.00 mL. Then they were adjusted by 1.00 mol L-1 NaOH to pH 11.00. After that, the diluted saliva solutions were spiked with analytes to 1.0, 5.0 and 50 ng mL-1, respectively. Under the optimized conditions, the blank and spiked target compound solutions were detected three times, and the recoveries and precisions were given in Table 2. As shown in the table, the recoveries of the target compounds were between 105.5% and 120.3%,, and the precisions were lower than 13.1%; so this method could be used for lightly determination of trace clenbuterol, metoprolol and propranolol in real saliva positive samples without more tedious additional disposals such as deproteinization.
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3.6.2 Application to positive human saliva sample analysis A male volunteer was treated with one pill of metoprolol tablet (~50 mg metoprolol) and the saliva was collected after 2 hours. After pre-treatment with description in 2.3.6, the proposed method was applied to the detection of metoprolol in collected positive saliva samples under the optimal conditions. The EIC chromatograms (ion 72) of metoprolol positive saliva samples and blank saliva samples using optimized parameters were shown in Fig.9. Position for Fig. 9 It could be seen that there was an obvious chromatographic peak in positive saliva sample chromatogram at metoprolol’s peak time which was significantly different from the blank sample chromatogram. And unfortunately, a small peak of blank saliva sample was found appearing at the position of propranolol in the EIC chromatogram for ion 72. It would affect the detection of extracted ion of propranolol when propranolol was a very low content.
4. Conclusion A selective analytical method based on SPME followed by on-fiber derivatization with MSTFA coupled to GC-MS was used to determine clenbuterol, metoprolol and propranolol. The important parameters involved in the method such as appropriate polarity and thickness of fiber coating material with consistent extraction, derivatization and desorption parameters were evaluated. This method is timesaving
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and easy to perform as the total analytical time is less than 1 hour; it is environmental friendly and avoids using toxic organic solvents and excess derivatization reagents. This method is fully validated and shown to be suitable for the simultaneous confirmation and quantitation of blockers and agonists with good LODs lower than 0.2 ng mL-1. The repeatability was below 8.7% (n = 6) and the reproducibility was lower than 11.4% (n=3). This simple and sensitive method has been applied to the measurement of clenbuterol, metoprolol and propranolol in human saliva. It showed that in saliva samples the performance of the method is still descent in presence of enzymes and proteins. The established method can offer a preliminary screening means for clenbuterol, metoprololand and propranolol. Since on the one side, large competitions have huge amount of samples need to screening and on the other side, confirmation of abuse drugs still using urine samples. This method was feasible in practical application and helpful for agonists and blockers control during the competition. It can provide an alternative option to extend the scope of doping monitoring and biological monitoring of blockers and agonists.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21275029), National Basic Research Program of China (No.2010CB732403), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1116) and the Nature Sciences Funding of Fujian Province (2010J05021).
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matrices, Anal. Chim. Acta 631 (2009) 47-53. [28] W. Liu, L. Zhang, Z. Wei, S. Chen, G. Chen, Analysis of beta-agonists and beta-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry, J. Chromatogr. A 1216 (2009) 5340-5346. [29] C. Brunelli, C. Bicchi, A. Di Stilo, A. Salomone, M. Vincenti, High-speed gas chromatography in doping control: fast-GC and fast-GC/MS determination of beta-adrenoceptor ligands and diuretics, J. Sep. Sci. 29 (2006) 2765-2771.
23
Highlights
It was the first time to use SPME and on-fiber derivatization method to extract blockers and agonists. This method is sensitive. Compared with other detecting methods, the detection limits are lower than ng mL-1 level. Compared with other literatures about the analytes, the pretreatment in this method is very simple, green and time-saving. The entire process including extration, desorption, separation and determination can complete in one hour.
24
Table 1 The regression equations, linearity, correlation coefficients, LODs, repeatability and reproducibility of the methoda Analytes
Equationb
R2
Linear
LOD
Repeatabilit
Reproducibilit
range
c
yd
ye
(ng
(ng
mL-1)
mL-1)
n=6)
0.2
7.2
10.6
0.5
8.7
11.4
0.5
8.3
10.9
Clenbutero
y=
0.997
0.5-15
l
298100x+10555
7
0
Metoprolol
y=
0.993
1.0 -
117237x+16144
3
100
Propranolo
y=334490x+3915
0.998
1.0 -
l
6
5
100
a b c
(%RSD,
(%RSD, n=3)
Conditions as in Fig.8. S/N=3 The parameters, x and y, refer to the concentration of the target compound (ng mL-1) and the
corresponding peak area. d
100 ng mL-1
e
100 ng mL-1
25
Table 2 Table 2 Extraction recoveries of spiked saliva samples by SPME with on-fiber derivatizationa Recoveries (%RSD, n=3) Analytes
1.0
5.0
50
(ng mL-1)
(ng mL-1)
(ng mL-1)
109.7
112.7
106.1
(7.9)
(7.3)
(8.6)
111.4
105.5
107.7
(9.0)
(8.2)
(7.4)
120.3
116.8
114.5
(13.1)
(11.7)
(12.0)
Clenbuterol
Metoprolol
Propranolol a
Conditions as in Fig.8.
26
Figure captions Fig. 1 Structures of the studied molecules
Fig. 2 Selection of SPME fiber coatings. SPME conditions: extraction temperature, 30
; extraction time, 30 min; stirring rate,
650 r min-1; pH, 11.00; NaCl concentration, 0.225 g mL-1; MSTFA, 3 L; derivatization temperature, 40 270
; derivatization time, 10 min; desorption temperature,
; desorption time, 4 min.
The concentration of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 3 Selection of derivatization temperature. CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 4 Selection of derivatization time CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 5 The volume of MSTFA CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 6 Selection of extraction temperature CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively. 27
Fig. 7 Selection of the desorption temperature CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 8 The chromatograms of analystes with on-fiber derivatization after SPME (a) TIC chromatogram; (b) EIC chromatogram
CW-DVB fiber coating, SPME conditions are as Fig.2.
Fig. 9 The EIC chromatograms of blank and positive metoprolol saliva samples SPME conditions are as Fig.8.
Graphical abstract
28
Fig. 1a
Fig.1b
Fig.1c
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8a
Fig. 8b
Fig. 9
Simultaneous determination of blockers and agonists by on-fiber derivatization in selfmade solid-phase microextraction coating fiber Wei Liu, Zhimin Yan, Xiayang Huang, Jinfeng Chen, Minghua Lu, Lan Zhang, Guonan Chen
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(14)00632-8 http://dx.doi.org/10.1016/j.talanta.2014.07.064 TAL14973
To appear in:
Talanta
Received date: 10 April 2014 Revised date: 19 July 2014 Accepted date: 21 July 2014 Cite this article as: Wei Liu, Zhimin Yan, Xiayang Huang, Jinfeng Chen, Minghua Lu, Lan Zhang, Guonan Chen, Simultaneous determination of blockers and agonists by on-fiber derivatization in self-made solid-phase microextraction coating fiber, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.07.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination of blockers and agonists by on-fiber derivatization in self-made solid-phase microextraction coating fiber
Wei Liu1, 2, Zhimin Yan1, Xiayang Huang1, Jinfeng Chen1,3, Minghua Lu1, 2, Lan Zhang1, 2*, Guonan Chen1* 1. Ministry of Education Key Laboratory of Analysis and Detection for Food Safety (Fuzhou University) 2. Test Center of Fuzhou University, Fuzhou University, Fuzhou, Fujian, 350002, China 3. Longyan Entry-Exit Inspection and Quarantine Bureau, Longyan, Fujian, 364000, China
Corresponding author: Lan Zhang Postal address: Department of Chemistry, Fuzhou University, Fuzhou 350002, Fujian, China Fax: 86-591-87893207 Tel: 86-591-87892448 E-mail: [email protected]
1
Abstract: An environmentally friendly and sensitive method for determination of blockers and agonists was described in this paper. The method is based on a homemade sol-gel solid-phase microextraction (SPME) coating with simultaneous on-fiber derivatization and subsequent gas chromatography mass spectrometry (GC/MS) analysis. The influences of the main factors on the type and thickness of the homemade fiber coatings, conditions of the derivatization, extraction and desorption of SPME were investigated in detail. The proposed procedure showed limits of detection lower than 0.2 ng mL-1. The linearity was in the 0.5-150 ng mL-1 for clenbuterol and 1.0-100 ng mL-1 for metoprolol and propranolol. The variation in measurements (repeatability) was below 8.7% (n = 6) and the degree of difference between (reproducibility) was below 11.4% (n = 3). In the application, spiked human saliva samples and real human saliva samples were analyzed, it was found that saliva would affect the detection of propranolol when it was a very low content. The established method can be feasible in practical application and helpful for agonists and blockers preliminary screening during the competition.
2
Keywords: blockers, agonists, on-fiber derivatization, solid-phase microextraction, gas chromatography-mass spectrometry
1. Introduction Clenbuterol is one of the most widely used 2-adrenoceptor agonists, while metoprolol and propranolol are the most common -antagonists (-blockers). They both work on the body’s -adrenergic receptors with contrary effects and they are structural related amino alcohols. So they can be manipulated and analyzed together with similar detection procedures. The mainstream methods for determination of blockers and agonists include GC-MS [1, 2], liquid chromatography [3, 4], Capillary electrophoresis mass spectrometry (CE-MS)[5, 6] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [7-9]. The involved biological samples include hair [10], whole blood [11], serum [12], plasma [13, 14] and urine [5]. In the Medical Commission of the International Olympic Committee, the 2-agonists and -blockers have been forbidden. And the official detection requirement is concentration of 0.5 g mL−1 in urine by GC-MS after derivatization. In fact, no matter what kind of biological sample analysis, the pre-treatment and concentration is essential. And the new sample preparation method solid-phase microextraction (SPME) integrated sampling, extraction, concentration and sample introduction into one step, it has been considered as a very powerful sample preparation tool[15]. While introducing derivatization in SPME-GC/MS can further
3
expand the application areas of SPME-GC/MS and bring lower limits of detection (LODs), there have been some researches and several reviews on this topic [16-20]. In brief, achieve derivatization operation during SPME-GC/MS process have three modes: in the gas chromatography injector port [21], in the sample matrix [22-24] and on the fiber coating after SPME sampling [25, 26]. The injection-derivatization method is the inchoate derivatization approach of SPME-GC/MS which might lead to incomplete derivatization reaction and possible damage to the capillary column because of the excessive derivative agent. Derivatization reaction in the sample matrix with SPME sampling simultaneously is the simplest mode but with some limitations. While the derivatization occurs on the SPME fiber coating, the fiber is not only a separation/extraction device but also being turned into a reaction vessel. It makes the whole process simple and fast. Until now, GC-MS is the major detection technique for beta2-agonists and beta-antagonists. And analysis of 2-agonists and -blockers in saliva is a great choice because of its noninvasive, easy to collect and less confidential in comparison with blood and urine. In this paper, homemade sol-gel SPME coatings were prepared and contrasted for on-fiber derivatization and SPME operation; an entire GC-MS determination method for blockers and agonists was trying to establish and the application in positive saliva samples were realized so as to the unlimited and sensitive preliminary screening of clenbuterol, metoprolol and propranolol in competitions.
4
2. Experimental Procedures 2.1. Chemicals and Reagents Silylating reagents of N-methy-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was obtained from Sigma (SIGMA, St. Louis, MO, USA). Clenbuterol, metoprolol and propranolol (Fig. 1) were from national institute for the control of pharmaceutical and biological products (Beijing, China). Methyl methacrylate (MMA), benzoyl peroxide (BPO), tetramethyl orthosilicate (TMOS), trifluoroacetic acid (TFA) and polyethylene glycol 20 000 (PEG 20 000) were from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China). Hydroxy-terminated silicone oil (OH-TSO) and poly methyl hydrogen siloxane (PMHS) were purchased from Santai Organic Silica Material Factory (Zhejiang, China). Divinylbenzene (DVB) was obtained from Shengzhong Fine Chemical Co.Ltd. (Shanghai, China). Position for Fig. 1 A stock standard mixture containing clenbuterol, metoprolol and propranolol (1.00 mg mL-1, respectively) was prepared in methanol and stored at 4 . Working standard solutions were prepared by diluting with methanol and renewed every week. Spiked water samples were prepared weekly by adding the appropriate volume of the working standards solution to the 20 mL water sample. Milli-Q ultrapure water (Millipore, Bedford, USA) was used throughout the experiments and the resistivity was about 18 M·cm (25
).
5
2.2. Instrumentation and operating conditions All the gas chromatographic experiments were performed on an Agilent 6890N-5973I GC-MSD (Hewlett-Packard, Palo Alto, CA, USA). A HP-5MS GC column (0.25 m film thickness, 0.25 mm×30 m, Agilent Technologies) was used for separation. The SPME device for manual extraction was consisted in a homemade holder assembly and several replaceable homemade fiber assemblies, which were prepared as described in reference [27]. The thickness of the homemade SPME fiber coating was monitored by environmental scanning electron microscope (XL30 ESEM, Philips). The injector temperature was maintained at 270
. Splitless injection was
employed and the injection volume was 1.0 L. The oven temperature was set at 80 for 1 min, and then increased to 260 heated at 15
min-1 to 300
at 25
min-1 and held for 1 min, and then
!
. Carrier gas was helium (Purity >99.999 ) and the
flow rate was set at 1.2 mL min-1. The electron impact (EI) ion source, quadrupole mass analyzer, and the interface temperature were maintained at 230, 150 and 300 , respectively. Electron impact ionization (70 eV) was utilized. m/z range was 50-550, and solvent delay was 7 min. Positive mode was used for detection. Selected ion monitoring (SIM) was employed for quantitative measurement. For clenbuterol, the qualitative ions were 57, 73, 86 and 262 (m/z), and the quantitative ion was 86 (m/z); for metoprolol, the qualitative ions were 72, 101, 223 and 324 (m/z), and the
6
quantitative ion was 72 (m/z); for propranolol, the qualitative ions were 72, 115, 144 and 215 (m/z), and the quantitative ion was 72 (m/z).
2.3. Preparation of the homemade SPME fiber coatings Before sol-gel coating, commercial silica fiber (Yongnian Optical Fiber Factory, Yongnian, Heibei Province, China) was immersed in acetone for 15 min to remove the polyimide on its surface, then rinsed with water and dried. After that, a rehydroxylation process was performed to maximize the number of silanol groups on the silica surface before the preparation of fiber coatings. At firsr, the fiber was immersed in 1.00 mol L-1 sodium hydroxide solution for 60 min to increase the number of silanol groups on fiber wall, then rinsed with water and dried. Finally, the fiber was immersed in 0.10 mol L-1 hydrochloric acid for 20 min to reestablishment of equilibrium, then rinsed with water again and dried. The procedure was repeated for 3 times. Then,
four
different
carbowax-divinylbenzene
fibers
coatings
(CW-DVB),
with
carbowax
different (CW),
polarities
hydroxyl
--
silicone
oil-polymethylhydrosiloxane (OH-TSO-PMHS) and polyacrylate (PA) -- were prepared according to the procedures in our previous work [27]. Briefly, for the preparation of OH/TSO-PMHS fiber coating, a volume of 600 L of TMOS (precursor), 440 L of OH-TSO (coating polymer), 60 L of PMHS (deactivation reagent), and 400 L of TFA (acid catalyst) were thoroughly shaken and degasification for 5 min by supersonic purifier. The mixture was then centrifuged at
7
4000 r min-1 for 5 min. The precipitate was removed and the top clear sol solution was used for fiber coating. For the preparation of CW fiber coating, 400 L of TMOS, 200 L of OH-TSO, 50 L of PMHS, 300 L of 99% TFA, 200 mg of PEG-20,000 and 300 L of acetone were mixed in a 2 mL tube. For CW/DVB fiber coating, 400 L of TMOS, 200 L of OH-TSO, 50 L of PMHS, 300 L of TFA, 200 L of DVB, 200 mg of PEG-20,000 were mixed, and for PA fiber coating, 300 L of TMOS, 220 L of OH-TSO, 30 L of PMHS and 200 L of 99% TFA were mixed. When fibers were dipped vertically into a freshly prepared sol solution, it was held inside the sol solution for 20 min with sol–gel coating forming on the bare outer surface of the fiber end. For each fiber, this coating process was repeated for 3 times. Then it was placed in a desiccator at room temperature for 12 h. 2.4. SPME and derivatization procedure The SPME fibers were conditioned on GC injection port at 250;
for 2 h before
use. A 20 mL of measured solutions was placed in a 25 mL glass vial with a silicon-septum cap and spiked with analytes, and mixed with 4.5 g of NaCl adjusted pH to 11.00 with 1.0 mol L-1 NaOH. The vial was capped, and placed on a stirrer and heated to 30;
with 650 r min-1 for 30 min. After extraction, the fiber was exposed in
the headspace of a 2.0 mL sample vial containing 3.0 L MSTFA at 40;
for 10 min.
Finally, the fiber was pulled out from the vial and immediately inserted into the GC injection port for thermal desorption with 270; and 4 min.
8
2.5. Sample Preparation Saliva samples were collected from three healthy male volunteers. All saliva samples were collected before or 2 h after eating 50.00 mg metoprolol. To obtain the mixed saliva samples, each volunteer was told not to swallow the saliva but to store it 6 min in their mouths when sampling. Then, saliva was collected in Eppendorf tubes and stored at −20
immediately. Every saliva sample was taken out of 200 L in
serum bottle, added 4.50 g NaCl for SPME extraction and diluted to 20.00 mL by deionized water then adjusted to pH 11.00 with 1.00 mol L-1 NaOH solution.
3. Results and discussion Because clenbuterol, metoprolol and propranolol are polar materials and cannot respond in GC/MS without derivatization, they need to be derivatized to achieve good recoveries and precision. In this paper, based on the merits of on-fiber derivatization method introduced above, the typical derivatizing reagent for hydroxyl compounds (the silylating reagent of MSTFA with -agonists and -blockers) was chosen to be loaded on the fiber after analytes extraction[28, 29]. In optimization experiments, every measuring datum was repeated three times, and an average was applied.
9
3.1 Selection of SPME fiber Fiber coating is one of the key factors in the extraction efficiency to analytes. One objective of this study was to select the suitable SPME fiber coating for the extraction and derivatization of blockers and agonists from aqueous samples. In this experiment, three types of homemade fiber coating were selected for investigation as strong polar coatings CW and CW-DVB, moderate polar coating PA and weak polar coating OH-TSO/PMHS. As the rule of “like dissolves like”, it could be seen from Fig.2 that CW-DVB fiber exhibited the best effect to all three analytes. Position for Fig. 2 Fiber thickness, one of critical factors in the determination of the adsorption capacity of SPME fiber coating, is related to the number of coating times. Coating fiber of the CW-DVB column was executed from 2 to 5 layers, and the thickness of them were about 10, 21, 32 and 39 m, mesured by ESEM. It was found that the adsorption amount of analytes reached maximum when 32 m fiber coating was used.
3.2 Optimization of derivatization reactions The derivatization reaction was affected by various parameters, including the reaction temperature, the derivatization time and the volume of the reagent. The derivatization conditions were optimized to achieve both high derivatization yield and high extraction efficiency. First of all, the effect of the reaction temperatures was tested at 25 , 30 , 35 , 40
, 45
and 50
with 3 L MSTFA to the analytes, respectively. When the
10
temperature was lower than 40 , the extraction yields was positively correlated with the temperature; but when the temperature was higher than 40 , the correlation was reversed because of the side reactions of derivatization (Fig. 3). So, the derivatization temperature was chosen at 40 . Position for Fig. 3 In order to examine the effect of the reaction time on the derivatization efficiency, experiments were performed at 5, 8, 10, 15 and 20 min by using MSTFA at 40 . As shown in Fig. 4, the highest peak area for the derivatives of compounds was at 10 min and with no significant change until 20 min. As a result, subsequent experiments were carried out using a 10 min derivatization time. Position for Fig. 4 Although the amount of derivatization reagents should be enough for the derivative reaction, immoderate excess of derivatization reagents is actually disadvantageous to the fiber coating lifetime. In this experiment, the effect of 1-5 L of derivatization reagent MSTFA was compared with the same amount of analytes at 40
for 10 min. It can be seen from the Fig.5 that the peak areas of three TMS
derivatives increased as the volume of MSTFA increased to a maximum of 3 L. As a consequence, 3 L of reagent was used to ensure that enough derivatizing reagent was present during the extraction process. Position for Fig. 5
11
3.3 Optimization of SPME extraction conditions Many factors, including extraction temperature, extraction time, stirring speed, pH of the solution and concentration of NaCl, can influence SPME extraction efficiency. These parameters were carefully optimized in this SPME study. The effect of extraction temperature to the target compounds was examined from 20 to 60;
and the results were illustrated in Fig.6. Temperature affects both the
kinetics and the thermodynamics of the extraction process of analytes. Higher temperature can accelerate the mass transference; at the same time, partition coefficients of the analytes between the fiber and the solution decrease with the increase of temperature. In conclusion, the effect of various temperatures on the response of metoprolol was insignificant, whereas the effects on propranolol and clenbuterol reached maximum at 30; ; thus 30;
was selected as the optimum value.
Position for Fig. 6 The duration of SPME extraction was also studied. SPME is an equilibrium-based technique, and the best extraction efficiency usually reached after equilibrium. To find the optimum extraction time, the samples were stirred at 30; with 650 r min-1 for 5 to 60 min. It was found that the responses of metoprolol and clenbuterol increased with increasing extraction time until 30 min and propranolol reached maximum at 45 min. Taking into account of the effect of extraction time to all three analytes, 30 min was selected as the extraction time. The optimization of stirring speed was performed from 500 to 1100 r min-1. The
12
results indicated that increasing stirring speed to 650 r min-1 could markedly enhance the extraction amounts of compounds, especially propranolol. High stirring speed can accelerate the diffusion from the bulk of the sample solution to the coating surface and reduce the equilibrium time, while the frangible silicon dioxide fiber could be damaged with excessively high stirring speed. So, 650 r min-1 was selected. One of the most important parameters in the extraction process is the pH. pH values influenced not only the dissociation of functional groups in analytes, but also the surface properties of the fiber. Generally, in order to increase the extraction efficiency of the target compounds in sample solution, appropriate pH should be chosen to keep these compounds as molecular form and prevent them from dissociating during the SPME process. The effect of the pH of the sample solution was examined by varying pH from 8.00 to 12.00 (adjusted by 1.0 mol/L HCl and 1.0 mol/L NaOH). At pH 11.00, all three analytes achieved maximum responses. Increasing the ionic strength of the sample solution can reduce the solubility of analytes in water due to the formation of hydration spheres around the ionic salt molecules. However, the additional ions brought competitive adsorption with analytes and changed the surface properties of the fiber, which were unfavorable for the extraction. For investigating the effect of salt addition, various concentrations of NaCl in sample solutions (0, 0.075, 0.150, 0.225, 0.300 and 0.350 g mL-1 ) were tested. Results showed that 0.225 g mL-1 NaCl yielded the most extracted amounts to all three target compounds; hence NaCl concentration of 0.225 g mL-1 was chosen for next experiments.
13
3.4 Optimization of desorption conditions After SPME extraction and derivatization, the target compounds were desorpted using the GC injection port. With respect to the desorption conditions, both the desorption temperature and the desorption time were optimized. For removing the extracted analytes from the fiber coating to the vaporizing chamber, the temperature of injection port, namely the desorption temperature, was optimized from 240 to 290
(Fig. 7). Optimal desorption temperatures were decided
by the capabilities of the instrument, the properties of analytes and the specifications of the SPME fiber. The SPME fiber used in this method could endure 290
with
gradually degradation and the inappropriate temperature would result in the coating bleed of the fiber and could be disadvantageous to the chromatographic column. Considering precision and recovery, 270
was chosen as suitable desorption
temperature. Position for Fig. 7 Then, for achieving the total desorption of analytes with no memory effects, desorption time was optimized and the optimal time for extracting maximal amounts of all compounds was 4 min. To sum up, the optimal conditions of extraction, derivatization and desorption were as follows: in 20 mL spiked water solution, adjusting pH to 11.0, including 0.225 g mL-1 NaCl, using 32 m homemade CW-DVB fiber coating extracted
14
analytes for 30 min with 650 r min-1 stirring rate in 30; ; then derivatization for 40; and 10 min with 3.0 L MSTFA; desorption was performed in 270;
for 4 min.
After choosing related influence factors, the typical total ion current (TIC) chromatograms and the extracted ion current (EIC) chromatograms of three analytes in blank water solution and spiked water solutions (2.0 ng mL-1 and 100 ng mL-1) were given as Fig. 8a and Fig.8b. It could be seen from Fig.8a that, derivatization introduced some impurities to the SPME fiber in the TIC mode; but it didn’t affect the detection of extracted ions of clenbuterol (ion 86), metoprolol (ion 72) and propranolol (ion 72) (see Fig. 8b). It demonstrated that the water matrix and SPME fiber did not affect the detection of clenbuterol, metoprolol and propranolol. Position for Fig. 8 3.5 Method performance Calibration solutions with spiked deionized water samples were adsorbed, derivatizated, desorpted, analyzed with the optimized conditions and detected with GC-MS. The regression curves, correlation coefficients (R2), linear ranges and limits of detection (LODs) of three targets were shown in Table 1. The linearity of this method was investigated over a concentration range of 0.5-150 ng mL-1 for clenbuterol and 1.0-100 ng mL-1 for metoprolol and propranolol. The linear relationship between peak areas and concentrations were observed with R2 greater than 0.9933. This allowed the quantification of the compounds by the method of external standardization. The LODs for the analytes were estimated at S/N = 3 under GC/MS conditions (SIM mode) by considering the lowest concentration of each of the
15
analytes. The LODs were lower than 0.2 ng mL-1. Repeatability, expressed as the relative standard deviation of the analysis of seven samples spiked at 100 ng mL-1, was below 8.7% (n = 6). Reproducibility between days, expressed as the relative standard deviation of three samples spiked at 100 ng mL-1 and analyzed on three different days, was below 11.4% (n = 3).
3.6 Method application 3.6.1 Spiked and real human saliva sample analysis Each of the 200 L blank saliva sample obtained from healthy people was added 4.50 g NaCl and diluted with deionized water to 20.00 mL. Then they were adjusted by 1.00 mol L-1 NaOH to pH 11.00. After that, the diluted saliva solutions were spiked with analytes to 1.0, 5.0 and 50 ng mL-1, respectively. Under the optimized conditions, the blank and spiked target compound solutions were detected three times, and the recoveries and precisions were given in Table 2. As shown in the table, the recoveries of the target compounds were between 105.5% and 120.3%,, and the precisions were lower than 13.1%; so this method could be used for lightly determination of trace clenbuterol, metoprolol and propranolol in real saliva positive samples without more tedious additional disposals such as deproteinization.
16
3.6.2 Application to positive human saliva sample analysis A male volunteer was treated with one pill of metoprolol tablet (~50 mg metoprolol) and the saliva was collected after 2 hours. After pre-treatment with description in 2.3.6, the proposed method was applied to the detection of metoprolol in collected positive saliva samples under the optimal conditions. The EIC chromatograms (ion 72) of metoprolol positive saliva samples and blank saliva samples using optimized parameters were shown in Fig.9. Position for Fig. 9 It could be seen that there was an obvious chromatographic peak in positive saliva sample chromatogram at metoprolol’s peak time which was significantly different from the blank sample chromatogram. And unfortunately, a small peak of blank saliva sample was found appearing at the position of propranolol in the EIC chromatogram for ion 72. It would affect the detection of extracted ion of propranolol when propranolol was a very low content.
4. Conclusion A selective analytical method based on SPME followed by on-fiber derivatization with MSTFA coupled to GC-MS was used to determine clenbuterol, metoprolol and propranolol. The important parameters involved in the method such as appropriate polarity and thickness of fiber coating material with consistent extraction, derivatization and desorption parameters were evaluated. This method is timesaving
17
and easy to perform as the total analytical time is less than 1 hour; it is environmental friendly and avoids using toxic organic solvents and excess derivatization reagents. This method is fully validated and shown to be suitable for the simultaneous confirmation and quantitation of blockers and agonists with good LODs lower than 0.2 ng mL-1. The repeatability was below 8.7% (n = 6) and the reproducibility was lower than 11.4% (n=3). This simple and sensitive method has been applied to the measurement of clenbuterol, metoprolol and propranolol in human saliva. It showed that in saliva samples the performance of the method is still descent in presence of enzymes and proteins. The established method can offer a preliminary screening means for clenbuterol, metoprololand and propranolol. Since on the one side, large competitions have huge amount of samples need to screening and on the other side, confirmation of abuse drugs still using urine samples. This method was feasible in practical application and helpful for agonists and blockers control during the competition. It can provide an alternative option to extend the scope of doping monitoring and biological monitoring of blockers and agonists.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21275029), National Basic Research Program of China (No.2010CB732403), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1116) and the Nature Sciences Funding of Fujian Province (2010J05021).
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Highlights
It was the first time to use SPME and on-fiber derivatization method to extract blockers and agonists. This method is sensitive. Compared with other detecting methods, the detection limits are lower than ng mL-1 level. Compared with other literatures about the analytes, the pretreatment in this method is very simple, green and time-saving. The entire process including extration, desorption, separation and determination can complete in one hour.
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Table 1 The regression equations, linearity, correlation coefficients, LODs, repeatability and reproducibility of the methoda Analytes
Equationb
R2
Linear
LOD
Repeatabilit
Reproducibilit
range
c
yd
ye
(ng
(ng
mL-1)
mL-1)
n=6)
0.2
7.2
10.6
0.5
8.7
11.4
0.5
8.3
10.9
Clenbutero
y=
0.997
0.5-15
l
298100x+10555
7
0
Metoprolol
y=
0.993
1.0 -
117237x+16144
3
100
Propranolo
y=334490x+3915
0.998
1.0 -
l
6
5
100
a b c
(%RSD,
(%RSD, n=3)
Conditions as in Fig.8. S/N=3 The parameters, x and y, refer to the concentration of the target compound (ng mL-1) and the
corresponding peak area. d
100 ng mL-1
e
100 ng mL-1
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Table 2 Table 2 Extraction recoveries of spiked saliva samples by SPME with on-fiber derivatizationa Recoveries (%RSD, n=3) Analytes
1.0
5.0
50
(ng mL-1)
(ng mL-1)
(ng mL-1)
109.7
112.7
106.1
(7.9)
(7.3)
(8.6)
111.4
105.5
107.7
(9.0)
(8.2)
(7.4)
120.3
116.8
114.5
(13.1)
(11.7)
(12.0)
Clenbuterol
Metoprolol
Propranolol a
Conditions as in Fig.8.
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Figure captions Fig. 1 Structures of the studied molecules
Fig. 2 Selection of SPME fiber coatings. SPME conditions: extraction temperature, 30
; extraction time, 30 min; stirring rate,
650 r min-1; pH, 11.00; NaCl concentration, 0.225 g mL-1; MSTFA, 3 L; derivatization temperature, 40 270
; derivatization time, 10 min; desorption temperature,
; desorption time, 4 min.
The concentration of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 3 Selection of derivatization temperature. CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 4 Selection of derivatization time CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 5 The volume of MSTFA CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 6 Selection of extraction temperature CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively. 27
Fig. 7 Selection of the desorption temperature CW-DVB fiber coating, other SPME conditions are as Fig.2. The concentrations of clenbuterol, metoprolol and propranolol were 2.0 ng mL-1, respectively.
Fig. 8 The chromatograms of analystes with on-fiber derivatization after SPME (a) TIC chromatogram; (b) EIC chromatogram
CW-DVB fiber coating, SPME conditions are as Fig.2.
Fig. 9 The EIC chromatograms of blank and positive metoprolol saliva samples SPME conditions are as Fig.8.
Graphical abstract
28
Fig. 1a
Fig.1b
Fig.1c
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8a
Fig. 8b
Fig. 9
