Gas chromatography coupled to atmospheric pressure ionization mass spectrometry (GC-API-MS): Review

Although the coupling of GC/MS with atmospheric pressure ionization (API) has been reported in 1970s, the interest in coupling GC with atmospheric pressure ion source was expanded in the last decade. The demand of a “soft” ion source for preserving highly diagnostic molecular ion is desirable, as compared to the “hard” ionization technique such as electron ionization (EI) in traditional GC/MS, which fragments the molecule in an extensive way. These API sources include atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), atmospheric pressure laser ionization (APLI), electrospray ionization (ESI) and low temperature plasma (LTP). This review discusses the advantages and drawbacks of this analytical platform. After an introduction in atmospheric pressure ionization the review gives an overview about the history and explains the mechanisms of various atmospheric pressure ionization techniques used in combination with GC such as APCI, APPI, APLI, ESI and LTP. Also new developments made in ion source geometry, ion source miniaturization and multipurpose ion source constructions are discussed and a comparison between GC-FID, GC-EI-MS and GC-API-MS shows the advantages and drawbacks of these techniques. The review ends with an overview of applications realized with GC-API-MS.     

Atmospheric-pressure laser ionization: a novel ionization method for liquid chromatography/mass spectrometry

We report on the development of a new laser-ionization (LI) source operating at atmospheric pressure (AP) for liquid chromatography/mass spectrometry (LC/MS) applications. APLI is introduced as a powerful addition to existing AP ionization techniques, in particular atmospheric-pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photoionization (APPI). Replacing the one-step VUV approach in APPI with step-wise two-photon ionization strongly enhances the selectivity of the ionization process. Furthermore, the photon flux during an ionization event is drastically increased over that of APPI, leading to very low detection limits. In addition, the APLI mechanism generally operates primarily directly on the analyte. This allows for very efficient ionization even of non-polar compounds such as polycyclic aromatic hydrocarbons (PAHs). The APLI source was characterized with a MicroMass Q-Tof Ultima II analyzer. Both the effluent of an HPLC column containing a number of PAHs (benzo[a]pyrene, fluoranthene, anthracene, fluorene) and samples from direct syringe injection were analyzed with respect to selectivity and sensitivity of the overall system. The liquid phase was vaporized by a conventional APCI inlet (AP probe) with the corona needle removed. Ionization was performed through selective resonance-enhanced multi-photon ionization schemes using a high-repetition-rate fixed-frequency excimer laser operating at 248 nm. Detection limits well within the low-fmol regime are readily obtained for various aromatic hydrocarbons that exhibit long-lived electronic states at the energy level of the first photon. Only molecular ions are generated at the low laser fluxes employed ( approximately 1 MW/cm(2)). The design and performance of the laser-ionization source are presented along with results of the analysis of aromatic hydrocarbons.     

Atmospheric pressure photoionization. II. Dual source ionization

In this paper we describe results based on the combination of atmospheric pressure photoionization (APPI) with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The main purpose of combining more than one ionizer is to extend the range of compounds that can be simultaneously analyzed. Three modes of operation are presented; use of either ionizer, simultaneous use of two ionizers, and rapid switching between ionizers during a single chromatographic run. The dual ionizer configurations only minimally affect the performance of either ionizer relative to the standard single-ionizer sources. However, it is observed that the operation of both ionizers together does not typically give the sum signal from either source operating alone. For APCI/APPI the signal can range from less than that of either source alone to the sum of the two individual sources. For ESI/APPI, we observed large suppressions of the ESI multiply-charged signal of proteins when the APPI source was on. These behaviors are presumed to be due to the interaction of the initially formed ions by both sources and attests to the importance of ion-molecule reactions that occur during and after the primary ionization events. We give examples of compounds that are preferentially ionized by either APPI, APCI or ESI and present thermochemical arguments based on molecular structure and functionality to explain this behavior. The dual source is also shown to be able to operate in negative ion mode opening up the potential to conduct wide ranging chemical analyses.     

Atmospheric pressure laser ionization (APLI) coupled with Fourier transform ion cyclotron resonance mass spectrometry applied to petroleum samples analysis: comparison with electrospray ionization and atmospheric pressure photoionization methods

The analysis of crude oil samples remains a tough challenge due to the complexity of the matrix and the broad range of physical and chemical properties of the various individual compounds present. In this work, atmospheric pressure laser ionization (APLI) is utilized as a complementary tool to other ionization techniques for crude oil analysis. Mass spectra obtained with electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) are compared. APLI is primarily sensitive towards non‐polar aromatic hydrocarbons, which are generally present in high amounts especially in heavy crude oil samples. The ionization mechanisms of APLI vs. APPI are further investigated. The results indicate the advantages of APLI over established methods like ESI and APPI. The application of APLI in combination with Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) is thus demonstrated to be a powerful tool for the analysis of aromatic species in complex crude oil fractions. Copyright © 2011 John Wiley & Sons, Ltd.     

The distribution of ion acceptance in atmospheric pressure ion sources: Spatially resolved APLI measurements

It is demonstrated that spatially resolved mass selected analysis using atmospheric pressure laser ionization mass spectrometry (APLI MS) represents a new powerful tool for mechanistic studies of ion-molecule chemistry occurring within atmospheric pressure (AP) ion sources as well as for evaluation and optimization of ion source performance. A focused low-energy UV laser beam is positioned computer controlled orthogonally on a two-dimensional grid in the ion source enclosure. Resonance enhanced multiphoton ionization (REMPI) of selected analytes occurs only within the confined volume of the laser beam. Depending on the experimental conditions and the reactivity of the primary photo-generated ions, specific signal patterns become visible after data treatment, as visualized in, e.g., contour or pseudo-color plots. The resulting spatial dependence of sensitivity is defined in this context as the distribution of ion acceptance (DIA) of the source/analyzer combination. This approach provides a much more detailed analysis of the diverse processes occurring in AP ion sources compared with conventional bulk signal response measurements.     

Evaluation of a dual electrospray ionization/atmospheric pressure chemical ionization source at low flow rates (∼50 µL/min) for the analysis of both highly and weakly polar compounds

The atmospheric pressure ionization (API) source for a commercial mass spectrometer was modified to operate as a dual source in both the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques by simultaneously utilizing the electrospray probe and the corona discharge needle. A switching box was designed to operate in either manual or programmable modes to permit rapid switching between ionization techniques without changing sources, probes, or breaking vacuum. The source can be operated using the following ionization techniques: ESI only, APCI only, ESI/APCI simultaneously, and ESI/APCI alternatingly. The optimum operating conditions for these ionization techniques were similar to the manufacturer’s original specifications except that the APCI flow rate was lower (～50 µL/min versus 1000 µL/min) and externally heated nebulizing gas was found to be desirable. A four-component mixture, introduced by flow injection, was used to demonstrate the versatility of the dual ESI/APCI source.     

Unexpected observation of ion suppression in a liquid chromatography/atmospheric pressure chemical ionization mass spectrometric bioanalytical method

Ion suppression is a well-known phenomenon in electrospray ionization (ESI) mass spectrometry. These suppression effects have been shown to adversely affect the accuracy and precision of quantitative bioanalytical methods using ion spray. Such suppression effects have not been as well defined in atmospheric pressure chemical ionization (APCI) and there is some debate whether these effects actually occur in the ionization process using APCI. Here an example is described where clear ion suppression was observed during studies on a model compound and three metabolites using APCI liquid chromatography/tandem mass spectrometry (LC/MS/MS).     

Drug impurity profiling by capillary electrophoresis/mass spectrometry using various ionization techniques

Capillary electrophoresis/mass spectrometry (CE/MS) is predominantly carried out using electrospray ionization (ESI). Recently, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) have become available for CE/MS. With the VUV lamp turned off, the APPI source may also be used for CE/MS by thermospray ionization (TSI). In the present study the suitability of ESI, APCI, APPI and TSI for drug impurity profiling by CE/MS in the positive ion mode is evaluated. The drugs carbachol, lidocaine and proguanil and their potential impurities were used as test compounds, representing different molecular polarities. A background electrolyte of 100 mM acetic acid (pH 4.5) provided baseline separation of nearly all impurities from the respective drugs. APPI yielded both even‐ and odd‐electron ions, whereas the other ionization techniques produced even‐electron ions only. In‐source fragmentation was more pronounced with APCI and APPI than with ESI and TSI, which was most obvious for proguanil and its impurities. In general, ESI and TSI appeared the most efficient ionization techniques for impurities that are charged in solution achieving detection limits of 100 ng/mL (full‐scan mode). APPI and APCI showed a lower efficiency, but allowed ionization of low and high polarity analytes, although quaternary ammonium compounds (e.g. carbachol) could not be detected. Largely neutral compounds, such as the lidocaine impurity 2,6‐dimethylaniline, could not be detected by TSI, and yielded similar detection limits (500 ng/mL) for ESI, APPI and APCI. In many cases, impurity detection at the 0.1% (w/w) level was possible when 1 mg/mL of parent drug was injected with at least one of the CE/MS systems. Overall, the tested CE/MS systems provide complementary information as illustrated by the detection and identification of an unknown impurity in carbachol. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of eluent on the ionization efficiency of flavonoids by ion spray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization mass spectrometry.

The effect of nine different eluent compositions on the ionization efficiency of five flavonoids was studied using ion spray (IS), atmospheric pressure chemical ionization (APCI), and the novel atmospheric pressure photoionization (APPI), in positive and negative ion modes. The eluent composition had a great effect on the ionization efficiency, and the optimal ionization conditions were achieved in positive ion IS and APCI using 0.4% formic acid (pH 2.3) as a buffer, and in negative ion IS and APCI using ammonium acetate buffer adjusted to pH 4.0. For APPI work, the eluent of choice appeared to be a mixture of organic solvent and 5 mM aqueous ammonium acetate. The limits of detection (LODs) were determined in scan mode for the analytes by liquid chromatography/mass spectrometry using IS, APCI and APPI interfaces. The results show that negative ion IS with an eluent system consisting of acidic ammonium acetate buffer provides the best conditions for detection of flavonoids in mass spectrometry mode, their LODs being between 0.8 and 13 microM for an injection volume of 20 microl.     

Development of medium pressure laser ionization,MPLI. Description of the MPLI ion source

A novel pulsed valve/ion source combination capable of time-resolved sampling from atmospheric pressure has been developed for use with laser ionization time of flight mass spectrometry. The source allows ionization extremely close to the nozzle of the pulsed valve, enabling ultra-sensitive detection of a number of compounds, e.g., NO, at mixing ratios <1 pptV. Furthermore, at analyte mixing ratios in the ppbV range, the temporal resolution of the system is in the sub-second regime, allowing time-resolved monitoring of highly dynamic and complex mixtures, e.g., human breath or reacting chemical mixtures in atmospheric smog chamber experiments. Rotational temperatures of approximately 50 K have been observed for analytes seeded in the supersonic jet expansion at a distance of 1 mm downstream of the nozzle orifice. The refinement of the original ion source has drastically reduced the impact of reflected laser light and the resultant electron impact signals previously observed. The general applicability of this technique is demonstrated here by coupling the source to commercially available as well as home-built time-of-flight mass spectrometers. Finally, we discuss the MPLI technique in view of the very recently introduced atmospheric pressure laser ionization (APLI) as well as the traditional jet-REMPI approach.     

Atmospheric pressure thermospray ionization using a heated microchip nebulizer

When a standard atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) ion source is used without applying the corona discharge or photoirradiation, atmospheric pressure thermospray ionization (APTSI) of various compounds can be achieved. Although largely ignored, this phenomenon has recently gained interest as an alternative ionization technique. In this study, this technique is performed for the first time on a miniaturized scale using a microchip nebulizer. Sample ionization with the presented microchip‐APTSI (µAPTSI) is achieved by applying only heat and gas flow to a nebulizer chip, without any other methods to promote gas‐phase ionization. To evaluate the performance of the described µAPTSI setup, ionization efficiency for a set of test compounds was monitored as the microchip positioning, temperature, nebulizer gas flow rate, sample solution composition, and solvent flow rate were varied. The µAPTSI mass spectra of the test compounds were also compared to those obtained with ESI and APCI. The µAPTSI produces ESI‐like spectra with low background noise, favoring the formation of protonated or deprotonated molecules of compounds that are ionizable in solution. Multiple charging of peptides without in‐source fragmentation was also observed. Unlike ESI, however, the µAPTSI source can tolerate the presence of mobile phase additives like trifluoroacetic acid (TFA) without significant ion suppression. The µAPTSI source can be used with standard mass spectrometer ion source hardware, being a unique alternative to the present interfacing techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

Optimization of the ESI and APCI experimental variables for the LC/MS determination of s-triazines, methylcarbamates, organophosphorous, benzimidazoles, carboxamide and phenylurea compounds in orange samples

In this work, ten selected pesticides of different chemical groups, indicated to orange culture, were extracted and determined by liquid chromatography-mass spectrometry using both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) operating in the positive ion detection mode. Applying a variables selection technique verified that cone voltage, source temperature and drying-gas flow-rate are the critical variables when the ESI was used, while cone voltage was found to be the only critical variable for the MS system, operating with the APCI ionization mode. After optimization of the most important parameters through the variables selection technique, the selected ion-recording (SIR) mode, monitoring the [M + H](+) species for all the compounds, was applied for the method validation of the pesticides, in both ionization modes. In orange samples, matrix effects did not interfere with the determination of the pesticides. Pesticides quantification limits ranged from 10 to 50 microg kg(-1) for ESI and from 8.2 to 45 microg kg(-1) for APCI. Linearity was studied from LOQ upto 200 times LOQ values (r > 0.98). Recoveries obtained were in the range of 70.2-100.5% (RSDs less than 10%). In order to guarantee that the identification and confirmation of the studied pesticides in real samples were unequivocal, characteristic fragment ions of the pesticides were obtained by varying the cone voltage (in-source CID).     

New perspective on the determination of flame retardants in sewage sludge by using ultrahigh pressure liquid chromatography–tandem mass spectrometry with different ion sources

Analysis of 11 polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A bis 2,3-dibromopropylether (TBBPA-bis), tetrachlorobisphenol A (TCBPA), tetrabromobisphenol A (TBBPA) and hexabromocyclododecanes (HBCDs) was optimized by ultrahigh pressure liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) operating in negative ion (NI) mode. Electrospray ionization (ESI), atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were tested and for PBDEs APCI gave higher sensitivity than APPI while for TBBPA-bis APCI and APPI showed similar performance. ESI was the best option for TCBPA, TBBPA and HBCDs. Detection limits were between 20 and 59 fg for the compounds analyzed by ESI, 0.10 and 0.72 pg for PBDEs and 6 pg for TBBPA-bis. The matrix effect of sewage sludge extract was also tested showing negligible ion suppression for APCI and an increase of the background level of all investigated pollutants leading to a worsening of the limits of quantification by a factor between 1.2 and 3.3. The UPLC-APCI/MS/MS method for PBDEs, after pressurized liquid extraction (PLE), was validated by comparison with the concentration values from the NIST 1944 standard reference material. The advantages of the methods include low detection limits, PBDE congeners specificity using selected multiple reaction monitoring (MRM) transitions, and the absence of thermal degradation of higher PBDE congeners, especially BDE-209. The methods were applied for the determination of the above reported flame retardants in sewage sludge in order to get more information about the degradation on PBDEs (in particular BDE-209) during municipal wastewater treatments.     

Analysis of native amino acid and peptide enantiomers by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry was used for the separation and detection of amino acid and peptide enantiomers. With detection limits as low as 250 pg, 25 amino acids enantiomers were baseline resolved on a Chirobiotic T chiral stationary phase. APCI demonstrated an order of magnitude better sensitivity over electrospray ionization (ESI) for free amino acids and low molecular mass peptides at the high LC flow-rates necessary for rapid analysis. As the peptide chain length increased (peptides with M(r) > or = 300 Da), however, ESI proved to be the more ideal atmospheric pressure ionization source. A mobile phase consisting of 1% (w/w) ammonium trifluoroacetate in methanol and 0.1% (w/w) formic acid in water increased the sensitivity of the APCI method significantly. A step gradient was then used to separate simultaneously all 19 native protein amino acid enantiomers in less than 20 min using extracted ion chromatograms.     

Effect of organic mobile phase composition on signal responses for selected polyalkene additive compounds by liquid chromatography-mass spectrometry

The high performance liquid chromatography (HPLC) separation methodology employed in the study of polyalkene additive compounds by atmospheric pressure ionization mass spectrometry (API-MS) was undertaken. Both atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were examined. APPI (including dopant-assisted APPI) was found to be an inferior ionization technique to APCI in all cases. APCI ion responses were found to be highly dependent upon the organic solvent type used in the HPLC separations. Namely, employing a water/methanol gradient in place of a water/acetonitrile or a water/acetone gradient yielded improvements in analyte ion intensities between 2.3- and 52-fold for the liquid chromatography-mass spectrometry (LC-MS) experiments. Analyte and mobile phase solvent ionization energies were found to be only partially responsible, whereas mobile phase cluster formation and hydration was also implicated. Mobile phase component modification is demonstrated to be an important consideration when developing new, or modifying existing HPLC separations for use in LC-MS experiments in order to enhance analyte sensitivity for a wide variety of common polyalkene additives.     

Super‐atmospheric pressure chemical ionization mass spectrometry

Super‐atmospheric pressure chemical ionization (APCI) mass spectrometry was performed using a commercial mass spectrometer by pressurizing the ion source with compressed air up to 7 atm. Similar to typical APCI source, reactant ions in the experiment were generated with corona discharge using a needle electrode. Although a higher needle potential was necessary to initiate the corona discharge, discharge current and detected ion signal were stable at all tested pressures. A Roots booster pump with variable pumping speed was installed between the evacuation port of the mass spectrometer and the original rough pumps to maintain a same pressure in the first pumping stage of the mass spectrometer regardless of ion source pressure. Measurement of gaseous methamphetamine and research department explosive showed an increase in ion intensity with the ion source pressure until an optimum pressure at around 4–5 atm. Beyond 5 atm, the ion intensity decreased with further increase of pressure, likely due to greater ion losses inside the ion transport capillary. For benzene, it was found that besides molecular ion and protonated species, ion due to [M + 2H]⁺ which was not so common in APCI, was also observed with high ion abundance under super‐atmospheric pressure condition. Copyright © 2013 John Wiley & Sons, Ltd.     

血清脂质组学研究中多重离子化液相色谱-质谱方法的比较

系统地比较了3种常用的离子化技术电喷雾电离(ESI)、大气压化学电离(APCI)、大气压光致电离(APPI)对脂类化合物的离子化效率、检测灵敏度和覆盖范围,以探讨多重离子化液相色谱-质谱(LC-MS)方法在血清脂质组学研究中的适用性.血清样本经甲基叔丁基醚萃取后,采用Ascentiss Express C8 色谱柱(150 mm×2.1 mm, 2.7 μm)和二元线性梯度洗脱分离,流动相(A)为乙腈-水(3∶2, V/V, 含0.1%甲酸, 10 mmol/L甲酸铵),B为异丙醇-乙腈(9∶1, V/V, 含0.1%甲酸,10 mmol/L甲酸铵),分别采用ESI、APCI和APPI离子源正、负离子模式进行质谱检测.结果表明,ESI离子源对脂肪酸类、甘油脂类、甘油磷脂类化合物、鞘磷脂类化合物的离子化效率最高,对异戊烯醇脂类化合物的离子化效率与APPI离子源相当,APPI离子源对胆固醇(酯)类化合物的检测灵敏度最高,APCI离子源对各类化合物的检测灵敏度均低于ESI或APPI离子源;采用ESI和APPI离子源相结合的LC-MS脂质组学分析方法可以提高分析方法的整体灵敏度和血清中脂类信息检测的完整性.     

高效液相色谱/大气压化学电离质谱分析新型复合抗氧剂

 应用高效液相色谱 /大气压化学电离质谱技术 ,其中包括直接进样技术、同时正负离子扫描方式、可编程的源内碰撞诱导解离 (CID)技术 ,以及红外光谱分析技术 ,对一种用于润滑油的新型复合抗氧剂进行了分离分析 ,并对其中的有效组分进行了定性鉴定。该方法简便、快速、可靠。     

LC‐MS analysis of estradiol in human serum and endometrial tissue: Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

Accurate measurement of estradiol (E2) is important in clinical diagnostics and research. High sensitivity methods are critical for specimens with E2 concentrations at low picomolar levels, such as serum of men, postmenopausal women and children. Achieving the required assay performance with LC–MS is challenging due to the non‐polar structure and low proton affinity of E2. Previous studies suggest that ionization has a major role for the performance of E2 measurement, but comparisons of different ionization techniques for the analysis of clinical samples are not available. In this study, female serum and endometrium tissue samples were used to compare electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) in both polarities. APPI was found to have the most potential for E2 analysis, with a quantification limit of 1 fmol on‐column. APCI and ESI could be employed in negative polarity, although being slightly less sensitive than APPI. In the presence of biological background, ESI was found to be highly susceptible to ion suppression, while APCI and APPI were largely unaffected by the sample matrix. Irrespective of the ionization technique, background interferences were observed when using the multiple reaction monitoring transitions commonly employed for E2 (m/z 271 > 159; m/z 255 > 145). These unidentified interferences were most severe in serum samples, varied in intensity between ionization techniques and required efficient chromatographic separation in order to achieve specificity for E2. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of volatile metabolites formed by molds on barley by mass and ion mobility spectrometry

The contamination of barley by molds on the field or in storage leads to the spoilage of grain and the production of mycotoxins, which causes major economic losses in malting facilities and breweries. Therefore, on‐site detection of hidden fungus contaminations in grain storages based on the detection of volatile marker compounds is of high interest. In this work, the volatile metabolites of 10 different fungus species are identified by gas chromatography (GC) combined with two complementary mass spectrometric methods, namely, electron impact (EI) and chemical ionization at atmospheric pressure (APCI)‐mass spectrometry (MS). The APCI source utilizes soft X‐radiation, which enables the selective protonation of the volatile metabolites largely without side reactions. Nearly 80 volatile or semivolatile compounds from different substance classes, namely, alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, alkenes, terpenes, oxidized terpenes, sesquiterpenes, and oxidized sesquiterpenes, could be identified. The profiles of volatile and semivolatile metabolites of the different fungus species are characteristic of them and allow their safe differentiation. The application of the same GC parameters and APCI source allows a simple method transfer from MS to ion mobility spectrometry (IMS), which permits on‐site analyses of grain stores. Characterization of IMS yields limits of detection very similar to those of APCI‐MS. Accordingly, more than 90% of the volatile metabolites found by APCI‐MS were also detected in IMS. In addition to different fungus genera, different species of one fungus genus could also be differentiated by GC‐IMS.