Electrospray ionization/atmospheric pressure photoionization multimode source for low-flow liquid chromatography/mass spectrometric analysis

Analysis of several polar and non-polar compounds is performed with a newly developed dual electrospray ionization/atmospheric pressure photoionization (ESI/APPI) or ESPI source. Several variables are considered in the source, such as ESI probe heater temperature, solvent flow, dopant effects, repeller plate voltage, source geometry and photon energy (Kr vs. Ar lamp). Direct photoionization resulting in a molecular radical cation [M](*+) dominates at high temperatures (>400 degrees C) and low flow rates (<200 microL/min). Indirect photo-induced chemical ionization (PCI) involving solvent molecules becomes important at lower temperatures and higher solvent flow rates. Indirect PCI is enhanced using an Ar lamp, which yields comparable [M+H](+) signal but poorer [M](*+) signal than the Kr lamp at lower temperatures and higher flow rates. This is in support of our recent finding that the Ar lamp results in a solvent-dependent enhancement of analyte molecules via PCI. Analysis of 12 compounds in methanol under low-flow conditions (10 microL/min) demonstrates that the dual ESPI source performs favorably for most compounds versus the standard ESCI source, and significantly better than ESCI for the analysis of unstable drugs, like flurbiprofen. Several factors contributing to the benefits of the ESPI source are the shared optimal geometry for ESI and APPI sources and soft ionization of APPI versus APCI.     

Atmospheric Pressure Photoionization as a Powerful Tool for Large-Scale Lipidomic Studies

Lipidomic studies often use liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) for separation, identification, and quantification. However, due to the wide structural diversity of lipids, the most apolar part of the lipidome is often detected with low sensitivity in ESI. Atmospheric pressure (APPI) can be an alternative ionization source since normal-phase solvents are known to enhance photoionization of these classes. In this paper, we intend to show the efficiency of APPI to identify different lipid classes, with a special interest on sphingolipids. In-source APPI fragmentation appears to be an added value for the structural analysis of lipids. It provides a detailed characterization of both the polar head and the non polar moiety of most lipid classes, and it makes possible the detection of all lipids in both polarities, which is not always possible with ESI.     

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.     

Comparison of APPI, APCI and ESI for the LC-MS/MS analysis of bezafibrate, cyclophosphamide, enalapril, methotrexate and orlistat in municipal wastewater

The applicability of three different ionization techniques: atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was tested for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of five target pharmaceuticals (cyclophosphamide, methotrexate, bezafibrate, enalapril and orlistat) in wastewater samples. Performance was compared both by flow injection analysis (FIA) and on-column analysis in deionized water and wastewater samples. A column switching technique for the on-line extraction and analysis of water samples was used. For both FIA and on-column analysis, signal intensity and signal-to-noise (S/N) ratio of the target analytes in the three sources were studied. Limits of detection and matrix effects during the analysis of wastewater samples were also investigated. ESI generated significantly larger peak areas and higher S/N ratios than APCI and APPI in FIA and in on-column analysis. ESI was proved to be the most suitable ionization method as it enabled the detection of the five target compounds, whereas APCI and APPI ionized only four compounds.     

Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds

Atmospheric pressure photoionization (APPI) is a novel method of ionization in liquid chromatography/mass spectrometry (LC/MS). It was originally developed in order to broaden the range of LC/MS ionizable compounds towards less polar compounds that cannot be analyzed by electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Studies done thus far have shown that non-polar compounds that earlier were not ionizable in LC/MS can indeed be ionized by the use of APPI. However, the best ionization efficiency for low polarity samples has been achieved with low proton affinity (PA) solvents that are not suitable in reversed-phase LC (RP-LC). Here it is demonstrated that the signals for analytes with low proton affinities in acetonitrile can be increased 100-fold by using anisole as the dopant for APPI, which takes the sensitivity to the same level achieved in the analysis of high PA analytes.     

Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery

The performance of the atmospheric pressure photoionization (APPI) technique was evaluated against five sets of standards and drug-like compounds and compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI technique was first used to analyze a set of 86 drug standards with diverse structures and polarities with a 100% detection rate. More detailed studies were then performed for another three sets of both drug standards and proprietary drug candidates. All 60 test compounds in these three sets were detected by APPI with an overall higher ionization efficiency than either APCI or ESI. Most of the non-polar compounds in these three sets were not ionized by APCI or ESI. Analysis of a final set of 201 Wyeth proprietary drug candidates by APPI, APCI and ESI provided an additional comparison of the ionization techniques. The detection rates in positive ion mode were 94% for APPI, 84% for APCI, and 84% for ESI. Combining positive and negative ion mode detection, APPI detected 98% of the compounds, while APCI and ESI detected 91%, respectively. This analysis shows that APPI is a valuable tool for day-to-day usage in a pharmaceutical company setting because it is able to successfully ionize more compounds, with greater structural diversity, than the other two ionization techniques. Consequently, APPI could be considered a more universal ionization method, and therefore has great potential in high-throughput drug discovery especially for open access liquid chromatography/mass spectrometry (LC/MS) applications.     

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.     

Liquid chromatography/tandem mass spectrometric confirmatory method for determining aflatoxin M1 in cow milk: comparison between electrospray and atmospheric pressure photoionization sources

A liquid chromatography/electrospray (ESI)-tandem mass spectrometric method for the measurement of aflatoxin M1 (AFM1) in milk is described. Milk sample after protein precipitation with acetone was cleaned-up with a Carbograph-4 cartridge. Performances of the ESI source were compared with those of the atmospheric pressure photoionization source (APPI). Although a method quantification limit (MQL) of 6 ng/kg could be achieved operating with APPI source with respect to an MQL of 12 ng/kg with ESI, all the other performances being similar, then ESI was preferred as being more robust and widespread at present.     

ESI outcompetes other ion sources in LC/MS trace analysis

Choosing an appropriate ion source is a crucial step in liquid chromatography mass spectrometry (LC/MS) method development. In this paper, we compare four ion sources for LC/MS analysis of 40 pesticides in tomato and garlic matrices. We compare electrospray ionisation (ESI) source, thermally focused/heated electrospray (HESI), atmospheric pressure photoionisation (APPI) source with and without dopant, and multimode source in ESI mode, atmospheric pressure chemical ionisation (APCI) mode, and combined mode using both ESI and APCI, i.e. altogether seven different ionisation modes. The lowest limits of detection (LoDs) were obtained by ESI and HESI. Widest linear ranges were observed with the conventional ESI source without heated nebuliser gas. In comparison to HESI, ESI source was significantly less affected by matrix effect. APPI ranked second (after ESI) by not being influenced by matrix effect; therefore, it would be a good alternative to ESI if low LoDs are not required.

Graphical abstract

     

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.     

Comparison of atmospheric pressure photoionization and electrospray ionization mass spectrometry for the analysis of UV filters

A comparison was made between the electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) tandem mass spectrometric (MS/MS) responses of eleven ultraviolet (UV) filters. Four of the target compounds were favourably ionized in negative ion mode, and the other seven compounds in positive ion mode. For nine of the compounds APPI generated a similar response to that of ESI, but the APPI signal‐to‐noise (S/N) ratios were 1.3–60 times higher. The two most polar of the UV filter compounds (PBSA and BP‐4) were more efficiently ionized by ESI, offering higher signal intensities and lower detection limits. APPI was, however, less susceptible to ion suppression than ESI when real samples were injected. In order to optimize the APPI conditions different dopant solvents were examined to enhance the efficiency of the photoionization process. Among the evaluated dopants, toluene was selected as the best compromise. At a toluene flow rate of 10% of the solvent flow rates the ionization response increased by a factor of 40–50 over the use of no dopant for the compounds in positive ion mode and by more than 300 for the compounds in negative ion mode. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Comparative study of electrospray and photospray ionization sources coupled to quadrupole time-of-flight mass spectrometer for olive oil authentication

The use of fast and reliable analytical procedures for olive oil authentication is a priority demand due to its wide consumption and healthy benefits. Olive oil adulteration with other cheaper vegetable oils is a common practice that has to be detected and controlled. Rapid screening methods based on high resolution tandem mass spectrometry constitute today the option of choice due to sample handling simplicity and the elimination of the chromatographic step. The selection of the ionization source is critical and the comparison of their reliability necessary. The possibilities of the direct infusion electrospray ionization (ESI) and the recently introduced atmospheric pressure photospray ionization source (APPI), coupled to quadrupole time-of-flight (QqTOF), have been critically studied and compared to control olive oil adulteration. These techniques are very rapid (approximately 1 min per sample) and have high discrimination power to elucidate key components in the edible oils studied (olive, hazelnut, sunflower and corn). Nevertheless, both sources are complementary, being APPI more sensitive for monoacyl- and diacylglycerol fragment ions and ESI for triacylglycerols. In addition, methods reproducibility's are very high, especially for APPI source. Mixtures of olive oil with the others vegetable oils can be easily discriminated which has been tested by using principal components analysis (PCA) with both ESI-MS and APPI-MS spectra. Analogously, linear discriminant analysis (LDA) confirms methods reproducibility and detection of other oils used as adulterants, in particular hazelnut oil, which is especially difficult given its chemical similarity with olive oil.     

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.     

Determination of carotenoids by liquid chromatography/mass spectrometry: effect of several dopants

Various carotenoids were analyzed by ultra-high-pressure liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). Three different techniques to ionize the carotenoids were compared: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). For all of the carotenoids tested, it was possible to obtain characteristic transitions for their unequivocal identification using each ionization technique. APCI was shown to be a more powerful technique to ionize the carotenoids than ESI or APPI. Transitions to differentiate carotenoids that coelute were determined to distinguish antheraxanthin from astaxanthin and lutein from zeaxanthin. In addition, four dopants were evaluated to improve ionization and enhance the carotenoid signal strength in APPI. These dopants were acetone, toluene, anisole, and chlorobenzene. Carotenoids improved their response in almost all cases when a dopant was used. The use of dopants allowed the enhancement of the carotenoid signals strength up to 178-fold.     

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.     

Fourier transform ion cyclotron resonance mass spectrometry using atmospheric pressure photoionization for high-resolution analyses of corticosteroids

Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was coupled with atmospheric pressure photoionization (APPI) for the first time and used for the analysis of several corticosteroids.1 The analytes showed excellent response using APPI when compared with both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). APPI has the advantage of requiring less heat for desolvation, resulting in less thermal degradation of the analytes and higher signal-to-noise than APCI. In terms of ultimate sensitivity, APPI is more efficient than either ESI or APCI for the analysis of corticosteroids. With some compounds, the high-resolution capability of FTICRMS was necessary to obtain an accurate mass due to contributions of the M(+.) (13)C isotope in the [M+H](+) ion peak.     

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.     

Effect of eluent on the ionization process in liquid chromatography-mass spectrometry

The most widely used ionization techniques in liquid chromatography-mass spectrometry (LC-MS) are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). All three provide user friendly coupling of LC to MS. Achieving optimal LC-MS conditions is not always easy, however, owing to the complexity of ionization processes and the many parameters affecting mass spectrometric sensitivity and chromatographic performance. The selection of eluent composition requires particular attention since a solvent that is optimal for analyte ionization often does not provide acceptable retention and resolution in LC. Compromises must then be made between ionization and chromatographic separation efficiencies. The review presents an overview of studies concerning the effect of eluent composition on the ionization efficiency of ESI, APCI and APPI in LC-MS. Solvent characteristics are discussed in the light of ionization theories, and selected analytical applications are described. The aim is to provide practical background information for the development and optimization of LC-MS methods.