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Direct Analysis of Carbonyl Compounds by Mass Spectrometry with Double Region Atmospheric Pressure Chemical Ionization Yihan Zhang, Wuduo Zhao, Dingzhong Wang, Hongtu Zhang, Guobi Chai, Qidong Zhang, Binbin Lu, Shihao Sun, and Jianxun Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05834 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 6, 2019
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Analytical Chemistry
Direct Analysis of Carbonyl Compounds by Mass Spectrometry with Double Region Atmospheric Pressure Chemical Ionization Yihan Zhang,† Wuduo Zhao,‡ Dingzhong Wang,† Hongtu Zhang,§ Guobi Chai,† Qidong Zhang,† Binbin Lu†, Shihao Sun,*,† and Jianxun Zhang*,† † Zhengzhou Tobacco Research Institute, China National Tobacco Corporation, Zhengzhou 450001, China ‡
Center for Advanced Analysis and Computational Science, Zhengzhou University, 450001, China
§ Department of Chemistry and Biochemistry, The Ohio State University, 100 W 18th Avenue, Columbus, Ohio 43210, USA ABSTRACT Direct analysis of highly reactive volatile species such as the aliphatic aldehydes as vital biomarkers remains a great challenge due to difficulties in the sample pretreatment. To address such a challenge, we herein report the development of a novel double-region atmospheric pressure chemical ionization mass spectrometry (DRAPCI-MS) method. The DRAPCI source implements a separated structural design that uses a focus electrode to divide the discharge and ionization region to reduce sample fragmentation in the ionization process. Counter-flow introduction (CFI) configuration was adopted in the DRAPCI source to reduce background noises, while ion transmission efficiency was optimized through simulating the voltage of focus electrode and the ion trajectory of the ion source. The limits of detection (LODs) of four carbonyl compounds cyclohexanone, hexanal, heptanal, and octanal by DRAPCI-MS were between 0.1 and 3 μg·m-3, approximately two to eight times lower than those by atmospheric pressure chemical ionization mass spectrometry. Additionally, the DRAPCI-MS method carried out effective in-situ analyses of the volatile components in expired milk and the exhaled breath of smokers, demonstrating the DRAPCI-MS a practical tool to analyze complex mixtures. The DRAPCI-MS method provides a rapid, sensitive, and high-throughput technique in the real-time analysis of gaseous small-molecule compounds. INTRODUCTION In the past decades, the increasing demand of in-situ analysis has accelerated the development of rapid detection technology, especially when combined with ambient-pressure mass spectrometry with soft ionization methods.1 A series of desorption ionization sources were developed and exhibited satisfactory performances in surface analyses of solid and liquid samples.1-4 However, gas sample might be absorbed or contaminated by airbags container, and not easy to concentrate, transfer and store as solid or liquid samples. Complex matrices still pose a challenge to the in-situ analysis of gas samples by soft ionization mass spectrometry techniques. 5,6 Gas analysis without sample preparation is particularly challenging for on-line mass spectrometry and demands high-throughput and sensitive instruments. Traditional methods such as solid-phase microextraction (SPME) coupled with gas chromatography mass spectrometry (GC-MS) perform well in identifying complex samples.7,8 However, these methods often involve multi-step analytical procedures and are prone to enhance risk of positive and negative artifacts.8,9 In response to the limits of these methods, ambient mass spectrometry techniques for gas detection have attracted more attention recently.10 Avoiding complex sample collection, the selected ion flow tube mass spectrometry (SIFT-MS) used a fast-flow tube reactor for the reaction between organic compounds and precursor ions generated in a microwave discharge and injected into fast helium carrier gas flow.11-13 The SIFT-MS showed great potential for high throughput and real-time analysis of volatile organic compounds (VOCs) in breaths and food samples.14,15 On the other hand, the proton transfer reaction mass spectrometry (PTR-MS) had similar chemical ionization to SIFT-MS for analyzing molecules with higher 1
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proton affinity than water. The PTR-MS has been successfully applied in on-line monitoring of VOCs down to ppt range.16-18 In another case, flowing atmospheric-pressure afterglow mass spectrometry (FAPA-MS) with He-sustained atmospheric pressure glow discharge (APGD) was developed19 and showed decrease fragmentation and potential to mitigate matrix effects.20,21 Different from the ionization mechanism of SIFT-MS, analytes were directly ionized through single- or multi-photon absorption in atmospheric pressure photoionization mass spectrometry (APPI-MS).22 To overcome the influence of the low photon flux on detection, an easily photoionizable dopant was added to act as a reagent ion.23,24 Moreover, second electrospray ionization mass spectrometry (SESI-MS) was the technology relied on the gas-phase interaction between the charged particles created by electrospray ionization source of solvent and the neutral gaseous molecules from sample.25,26 SESI coupled to high-resolution mass spectrometry had been proposed for the monitoring of metabolites in exhaled breath.27 The basic feature of these techniques is photoionization, discharge ionization, ion exchange or ion reaction with analytes, such as proton transfer for PTR and NO+ reaction for SIFT. Unfortunately, these ambient mass spectrometry methods are usually not available in standard laboratories, and the commercialization was far from the electrospray ionization mass spectrometry and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). APCI, using corona discharge at atmospheric pressure, represents a soft-ionization method where molecular ions play the dominant role in mass spectrum. APCI-MS is therefore conveniently applied in gas sample analysis and has been used in a number of areas including exhalation in medical application,28 food flavor classification,29-31 VOCs and biogenic VOCs detection,32-35 and chemical warfare agents monitor.36-39 Meanwhile, APCI has been modified to improve ionization efficiency and simplify analytical procedures. Le Quere et al. developed an APCI source with low-dead volume for on-line analyses of aroma compounds.31 Usmanov et al. developed a new analytical method for explosive detection through alternating current corona discharge APCI source which was more robust than that of the direct current corona.40 Seto et al. developed a counter-flow introduction (CFI) APCI source for the detection of chemical warfare agents in air.36-39 The LODs of the chemical warfare agents were within 1 to 8 μg·m-3 with significantly improved instrument performance. Unfortunately, the CFI-APCI combining with ion trap mass spectrometer was not in widespread use. Our group has been developing rapid detection technology for analysis of components in complex gas sample. For example, to directly introduce samples into the ionization region, the commercial APCI probe of XevoTM TQ-MS was drilled a larger hole to install heavy caliber capillary.41 In another case, acrolein and crotonaldehyde in tobacco smoke were investigated with water-assisted APCI-MS to improve the aldehyde detection.42 However, in-source ionization may result undesired fragmentation and further complicate mass spectrum, especially for highly reactive species such as aldehydes. We herein develop a novel method coupling a double-region atmospheric pressure chemical ionization (DRAPCI) source with the commercial mass spectrometer by homemade interfaces. The DRAPCI source was divided into discharge and ionization region to separate discharge and ionization processes. Additionally, a flexible capillary was installed in the modified APCI probe to directly introduce sample into the ionization region, enabling in-situ headspace sampling without pumping system. The performance of such DRAPCI-MS instrument was assessed through detecting the gas emission of food and exhaled breath of smokers. EXPERIMENTAL SECTION Sample Preparation. n-Hexanal, heptanal, cyclohexanone, and octanal were purchased from Sigma Aldrich. Distilled deionized water was acquired from Thermo Scientific GenPure (Thermo-Fisher Scientific, Stockland, Niederelbert, Germany). The 500 mg·L-1 aqueous standard stock solution was prepared and diluted to desired sample concentrations. Twenty pasteurized whole milk samples were purchased from local supermarkets in Zhengzhou, China. Ten milk samples were acquired in November 2017, while the other ten samples acquired in May 2018. All the milk samples were transferred from their original packages into 50 mL centrifuge tubes, and then stored at 4℃ until analysis. Extra virgin olive oil was purchased from the local supermarket and stored at ambient environment. Double-Region APCI Source. A novel DRAPCI source was designed to combine with commercial mass spectrometer as shown in Figure 1a. The DRAPCI source, divided by a ring-shaped focus electrode, was composed of a discharge region and an ionization region. The two regions were connected through a small hole in the focus electrode. A discharge 2
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Analytical Chemistry
needle was placed 3 mm away from the hole in the axial direction of the discharge region. Discharge occurred between the needle and the hole in focus electrode. To focus the ion in the ionization region, electric voltage was applied to focus electrode through a grounded resistor. The plane nozzle was installed between the interface of DRAPCI source and the mass spectrometer as shown in Figure 1b. Inside the plane nozzle, the sample cone connected the mass spectrometer through the cylindrical end and the DRAPCI source through the conic end. Besides, a metal interface part was designed to connect the APCI probe and the DRAPCI source. The sealing was achieved through an O-ring and optimized by adjusting the position of the APCI probe.
Figure 1. (a) Cutaway diagram of the DRAPCI source. (b) Connection between DRAPCI source and commercial instrument. (c) Schematic diagram of gas flow in DRAPCI source (not drawn to scale). Sample Injection. Samples were prepared by transferring 10 μL standard solution, 1 mL milk sample or 3mL olive oil into 20 mL glass vial (ANPEL, Shanghai, China). The samples were first incubated in vials at 25℃, and then heated at 50℃ for 3 min. The sample vapor in the glass vial was introduced into DRAPCI source through the flexible capillary at a rate of 40 mL·min-1 for 20 s. The capillary (0.53 mm i.d., 0.68 mm o.d., methyl deactivated, SGE Analytical Science, Australia) was 31 cm in length and installed in the instrument by modifying the sample tube inlet. The sample gas was forced into the chamber of DRAPCI source through the pressure difference created by the nebulizing gas according to the Venturi Effect. Pollutes from previous samples were cleanly removed from DRAPCI source by the continuous nebulizing gas. MS Measurements. Experiments were performed on a triple quadruple mass spectrometer (XevoTM TQ-MS, Waters, USA). Masslynx software was used to acquire and process MS data. The MS detection conditions were optimized in positive mode as shown in Supporting Information S-1. RESULTS AND DISCUSSION Mechanism of DRAPCI. The developed DRAPCI source is consisted of a discharge region and an ionization region as shown in Figure 1a. The ionization process of analytes proceeds in two steps: 1) formation of the reagent ions and 2) ionization of analyte molecules. The directions of reagent ion flow and sample gas flow were shown in the Figure 1c with yellow and blue arrow, respectively. Reagent ions are formed by corona discharge in the discharge region and then focused 3
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into the ionization region by the pre-set electric field. Gas sample enters the ionization region and is ionized by the reagent ions. Subsequently, ionized sample in the ionization region is drawn into the mass spectrometer. Meanwhile, the unionized gas sample carried by nebulizing gas passes through the focus electrode and then exits from the discharge region. During the ionization process, the reagent ions are introduced into the ionization region by the electric field, which is against the sample gas flow direction. The opposite direction of sample gas flow and reagent ion flow mitigates the neutral species going from discharge to ionization region and therefore reduces noise intensity. On the other hand, separating discharge from ionization process also helps to reduce analyte fragmentations by the discharge to a certain extent. Voltage is applied on the focus electrode to focus the analyst ions into the ionization region. However, the chamber of DRAPCI source is a hermetic structure, making it difficult to directly apply external voltage to the focus electrode. Application of voltage in the ionization source was achieved by applying a resistor between focusing electrode and earth, along with the corona current discharge (5 μA). In this study, N2 (purity≥99.99%) gas generated from liquid nitrogen was used as the reagent gas. Both N2+ and neutral compounds (NOx) were produced in the discharge region, and sequential reactions occurred between N2+ and H2O to form reactant ions (H3O+·H2O).42 The corresponding background mass spectrum was obtained and shown in supporting information S-2. Additionally, sampling probe and DRAPCI chamber could be cleaned by nebulizing gas, emitted from the discharged region. DRAPCI Source Simulation and Optimization. To investigate the DRAPCI source, ion transformation in the DRAPCI source was simulated by SIMION 8. A 3D hard-sphere collision model was introduced to simulate ionic motion. Ion collisions were assumed elastic, and the background gas was neutral. 20 V DC voltage was applied to the cone electrode. DC voltage of approximately 5000 V was applied to the needle electrode. Simulation structure of DRAPCI was shown in Figure 2a. Needle electrode extended to the discharge region and reagent ions were produced during discharge, which entered the ionization region to react with sample molecules. Transmission of analyte molecule ions was affected by voltage on the focus electrode, and simulation results were shown in Figure 2b. Ion transmission efficiency increased from 14.7% to 17% as the applied voltage on focus electrode increased from 1000 V to 1500 V, and then decreased as voltage continued to increase to 2750 V. Electric field lines (red line) and ion trajectory (blue and green line) were simulated at 1500 V where peak efficiency was achieved (Figure 2c). The reagent ions and sample ions were focused as passed through the focus electrode hole and plane nozzle. Figure 2d displayed the effects of resistance on ion intensity of [M+H]+ of hexanal and heptanal. Intensity of hexanal peaked when the resistance increased to 100 MΩ and gradually decayed as the resistance continued to increase. This trend was consistent with the influence of voltage on ion transmission. For these experiments, the gas flow rate was optimized at 4.0 L·min-1 to reach the maximum intensity as shown in Supporting Information S-3.
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Figure 2. (a) Simulation structure of the DRAPCI source. (b) Influence of focus electrode voltages on the ion transmission efficiency. (c) Schematic diagram of ion trajectory simulation of 1500V; Reagent ions trajectory (blue line) and sample ions trajectory (green line). (d) Effects of resistance on ion intensity of m/z 101 and m/z 115. Comparison of DRAPCI with APCI. Mass spectra of cyclohexanone, hexanal, heptanal, and octanal obtained by APCI and DRAPCI source were shown in Figure 3a. [M+H]+ was the most abundant ion in both APCI and DRAPCI sources, while more fragmented ion peak presented in the spectrum obtained by the APCI source, indicating the improvement of the double-region design. Although enhancement of the ion intensity was not significant, DRAPCI had relatively lower noise compared to that of APCI. We tentatively attribute this observation to two major aspects, the counter flow introduction configuration and the focus electrode structure. Specifically, sample gas flow might prevent part of protonated analytes entering mass analyzer, but electric field generated by the focus electrode could draw most of the protonated analytes into the plane nozzle. Moreover, ion transmission efficiency from ionization region to plane nozzle was improved. The sample gas flow was against the reagent ion flow (Figure 1c) and reduced transmission of reagent ions from discharge region to ionization region, which limited not only protonated ions but also pollutants and neutral molecules into the mass spectrometer, helped to maintain a clean background in DRAPCI source. To comfirm our assumption, signal-to-noise ratio (S/N) of DRAPCI-MS and APCI-MS was obtained with cyclohexanone, hexanal, heptanal, and octanal, respectively. The results were shown in Figure 3b. The S/N in DRAPCI source was increased between 1.5 to 3.6 times as compared with APCI source. The LODs in DRAPCI source were 2 to 8 times lower than those in APCI source (Supporting Information S-4). These results were consistent with what we expected.
Figure 3. (a) Mass spectra of cyclohexanone (1 mg·m-3), hexanal (2 mg·m-3), heptanal (2 mg·m-3), octanal (2 mg·m-3) in APCI source and DRAPCI source. (b) Signal-to-noise ratio of 40 μg·m-3 sample gases of cyclohexanone, hexanal, heptanal, and octanal in APCI source and DRAPCI source. Detection Limits and Repeatability Tests. The performance of DRAPCI was assessed with cyclohexanone, hexanal, heptanal, octanal, and the detailed experimental conditions described in Table S-1. Regression analysis indicated that the analyte peak area was linearly related to concentration (μg·m-3) with R2 values ranging from 0.976 to 0.998 for the four carbonyl compounds. The LODs of the four carbonyl compounds were in the range from 0.1 to 3 μg·m-3. Six consecutive analyses of the lowest concentration of the calibration curve were conducted and the relative standard deviations (RSDs) were in the range from 3.38% to 6.02%, showing reliably improved reading of the present system for carbonyl compounds (Supporting Information S-5). Food Analysis. Lipids are naturally present in food or added to processed food and play an important role in nutrition and flavor development. Meanwhile, lipid oxidation, contributing to undesirable off-flavor, is the major cause for quality loss in food and a big challenge to the food industry.43 Previous studies have identified the carbonyl compound hexanal as the marker generated during lipid oxidation of breast milk and bovine milk.44-45 Because of low peak intensity of [M+H]+, the peak of [M+H-H2O]+ was usually selected as the biomarker in food quality assessment.46 Due to the complex composition of milk sample, detection of [M+H]+ for hexanal is important and more desirable for the rapid detection. In this study, gas in headspace of fresh and expired milk samples were directly introduced through the flexible capillary and analyzed by DRAPCI-MS/MS. As shown in Figure 4a, the [M+H]+ peak of hexanal with m/z 101 was not obvious in the mass spectrum of fresh milk samples but present in the expired milk sample, which was identified through the hexanal 5
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MS/MS spectrum. The quantitative analysis of hexanal in headspace of different milk samples was performed by MRM mode according to the calibration curve, and results were shown in Figure 4c. Hexanal in expired milk was significantly higher than that in fresh milk (p
