Electric modeling and characterization of pulsed high‐voltage nanoelectrospray ionization sources by a miniature ion trap mass spectrometer

A better understanding of nanoelectrospray ionization (nano‐ESI) would be beneficial in further improving the performances of nano‐ESI. In this work, the pulsed high‐voltage (HV) nano‐ESI has been electrically modeled and then systematically characterized by both voltage‐current and mass spectrometry measurements. First, the equivalent resistance of a nano‐ESI source changes with respect to both emitter tip diameter and the HV applied. Increased voltage could improve both spray current and ionization efficiency of the pulsed HV nano‐ESI. Compared with conventional DC HV method, a pulsed HV has less heating effect on the capillary tip and thus allowing the application of a much higher voltage onto a nano‐ESI source. As a result, a pulsed HV nano‐ESI could further boost the ionization efficiency of nano‐ESI by employing even higher voltages than conventional DC nano‐ESI sources.     

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).     

Determination of D/L-amino acids by zero needle voltage electrospray ionisation

Ion formation may be made more efficient than in normal electrospray ionization (ESI) for certain classes of compounds, such as the polar amino acids Glu, Asn, His, Ser, Asp, Arg, Tyr and Lys, by adjusting the voltage of a normal ESI interface needle to zero voltage. For aspartic acid (Asp) the gain in signal-to-noise (S/N) ratio of the liquid chromatography/mass spectrometry (LC/MS) chromatograms obtained in the selective ion monitoring (SIM) mode (m/z 134) with zero needle potential was 40-50 times higher than detection at 4 kV. Ion formation at zero potential is likely to follow a mechanism related to sonic spray ionization. The utility of the zero needle voltage ESI was illustrated by determining the age of a human tooth by the aspartic acid epimerization method. The procedure involved separating the D- and L-aspartic acid of a tooth extract on a chiral HPLC column and detection by zero voltage ESI-MS3.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI‐MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI‐MS). A cold spray ionization providing a stable solvation‐ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug‐protein complexes with exact stoichiometries. In this paper, we apply CSI‐MS to explore the interactions of ginsenosides toward amyloid‐β‐peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI‐MS and ESI‐MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI‐MS, while dehydration products are more readily available by ESI‐MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI‐MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.     

Effect of solution acidity on cytochrome c conformations of alternating current electrospray ionization mass spectrometry

This paper reports notable observations regarding the ion charge states of thermally stable cytochrome c, generated using an alternating current (AC) electrospray ionization (ESI) device. An AC ESI sprayer entrains low-mobility ions to accumulate at the meniscus cone tip prior to the ejection of detached aerosols to produce analyte ions. Therefore, as the solvent acidity varies, protein ions entrained in the AC cone tip are found to change conformation less significantly compared with those in the direct current (DC) cone. We acquired the AC ESI mass spectra of cytochrome c at pH range from 2 to 4. Unlike the DC ESI mass spectra showing clear conformation changes due to denaturing, the AC spectra indicated that only partial denaturing occurs even at extremely acidic pH 2. More native cytochrome c in lower charge states therefore remained. Moreover, with a solvent mixture of aqueous buffer and acetonitrile (70:30), partially denatured cytochrome c was still preserved at pH 2 by using AC ESI. Completely denatured proteins are observed at pH 2 by using DC ESI.     

Electron ionization LC‐MS with supersonic molecular beams—the new concept,benefits and applications

A new type of electron ionization LC‐MS with supersonic molecular beams (EI‐LC‐MS with SMB) is described. This system and its operational methods are based on pneumatic spray formation of the LC liquid flow in a heated spray vaporization chamber, full sample thermal vaporization and subsequent electron ionization of vibrationally cold molecules in supersonic molecular beams. The vaporized sample compounds are transferred into a supersonic nozzle via a flow restrictor capillary. Consequently, while the pneumatic spray is formed and vaporized at above atmospheric pressure the supersonic nozzle backing pressure is about 0.15 Bar for the formation of supersonic molecular beams with vibrationally cold sample molecules without cluster formation with the solvent vapor. The sample compounds are ionized in a fly‐though EI ion source as vibrationally cold molecules in the SMB, resulting in ‘Cold EI’ (EI of vibrationally cold molecules) mass spectra that exhibit the standard EI fragments combined with enhanced molecular ions. We evaluated the EI‐LC‐MS with SMB system and demonstrated its effectiveness in NIST library sample identification which is complemented with the availability of enhanced molecular ions. The EI‐LC‐MS with SMB system is characterized by linear response of five orders of magnitude and uniform compound independent response including for non‐polar compounds. This feature improves sample quantitation that can be approximated without compound specific calibration. Cold EI, like EI, is free from ion suppression and/or enhancement effects (that plague ESI and/or APCI) which facilitate faster LC separation because full separation is not essential. The absence of ion suppression effects enables the exploration of fast flow injection MS‐MS as an alternative to lengthy LC‐MS analysis. These features are demonstrated in a few examples, and the analysis of the main ingredients of Cannabis on a few Cannabis flower extracts is demonstrated. Finally, the advantages of EI‐LC‐MS with SMB are listed and discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

The influence of electrospray ion source design on matrix effects

This study investigates to which extent the design of electrospray ion sources influences the susceptibility to matrix effects (MEs) in liquid chromatography–tandem mass spectrometry (LC–MS/MS). For this purpose, MEs were measured under comparable conditions (identical sample extracts, identical LC column, same chromatographic method and always positive ion mode) on four LC–MS/MS instrument platforms. The instruments were combined with five electrospray ion sources, viz. Turbo Ion Spray, Turbo V^TM Source, Standard ESI, Jet Stream ESI and Standard Z‐Spray Source. The comparison of MEs could be made at all retention times because the method of permanent postcolumn infusion was applied. The MEs ascertained for 45 pesticides showed for each electrospray ion source the same pattern, i.e. the same number of characteristic signal suppressions at equivalent retention times in the chromatogram. The Turbo Ion Spray (off‐axis geometry), Turbo V^TM Source (orthogonal geometry) and the Standard Z‐Spray Source (double orthogonal geometry) did not differ much in their susceptibility to MEs. The Jet Stream ESI (orthogonal geometry) reaches a higher sensitivity by an additional heated sheath gas, but suffers at the same time from significantly stronger signal suppressions than the comparable Standard ESI (orthogonal geometry) without sheath gas. No relation between source geometry and extent of signal suppression was found in this study. Copyright © 2012 John Wiley & Sons, Ltd.     

Optimization of electrospray interface and quadrupole ion trap mass spectrometer parameters in pesticide liquid chromatography/electrospray ionization mass spectrometry analysis

Optimization of both the ionization process and ion transportation in the mass spectrometer is of crucial importance in order to achieve high sensitivity and low detection limits and acceptable accuracy in liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) analysis. In this paper four optimization procedures of electrospray interface and quadrupole ion‐trap mass spectrometer parameters (ESI‐MS) (nebulizer gas and drying gas flow rate, end plate voltage, capillary voltage, skimmer voltage, octopoles direct current and radio frequency, trap drive and lens voltages) were studied on three pesticides – thiabendazole, aldicarb and imazalil. The results demonstrate that the methodology of optimization strongly influences the effectiveness of finding true optima of the operating parameters. Both eluent flow rate and composition during optimization have to mimic the situation during real analysis as closely as possible in order to achieve parameters giving the highest sensitivity. Therefore, post‐column addition of analyte to the mobile phase identical in composition to the one in which analyte elutes during real analysis combined with software‐based optimization was found to be the most effective and fastest method for achieving intensity maxima. The parameters most strongly affecting ion formation and transportation, hence sensitivity, were capillary voltage, direct current of the first octopole, trap drive and the second lens for all pesticides under study. In addition to sensitivity and detection limit matrix effect was considered in the optimization process. It was found that the matrix effect can be reduced but not eliminated by adjusting the ESI and MS parameters. The optimal parameters from the point of view of the matrix effect can only be found with factorial design. Parameters giving higher sensitivity tended to be more affected by matrix effect causing higher ionization suppression by co‐eluting compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Sonic spray ionization applied to liquid chromatography/mass spectrometry analysis of endocrine-disrupting chemicals in environmental water samples

A sonic spray ionization liquid chromatography/mass spectrometry (LC/SSI-MS) procedure combined with off-line solid-phase extraction was optimized for the analysis of 20 endocrine-disrupting chemicals (EDCs) in water samples. Method development included a comparison of the novel sonic spray ionization (SSI) with more traditional ion sources, i.e. pneumatically assisted electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). It was demonstrated that SSI and ESI spectra were very similar, but were more prone to the formation of solvent cluster ions as compared with APCI spectra. This phenomenon was most prominent for SSI and resulted in an increased chemical background in full-scan mass spectra. However, this chemical noise did not affect the overall sensitivity of SSI and ESI. After optimization of LC and MS parameters, the LC/SSI-MS method was validated. Recoveries ranged from 76.3 up to 113.4% for all compounds. Limits of detection (LOD) and quantitation (LOQ) were established between 3.0 and 11.5 ng/L and 9.9 and 38.0 ng/L, respectively. Within-day (n = 5) and between-day (n = 5) reproducibility were investigated at three levels and ranged from 3.3-16.5% and 7.6-19.2%, respectively. Eight-point calibration curves were established and showed linearity for all compounds (r(2) > 0.987) over a linear dynamic range of 10-10 000 ng/L.     

Systematic investigation of ion suppression and enhancement effects of fourteen stable‐isotope‐labeled internal standards by their native analogues using atmospheric‐pressure chemical ionization and electrospray ionization and the relevance for multi‐analyte liquid chromatographic/mass spectrometric procedures

In clinical and forensic toxicology, multi‐analyte procedures are very useful to quantify drugs and poisons of different classes in one run. For liquid chromatographic/tandem mass spectrometric (LC/MS/MS) multi‐analyte procedures, often only a limited number of stable‐isotope‐labeled internal standards (SIL‐ISs) are available. If an SIL‐IS is used for quantification of other analytes, it must be excluded that the co‐eluting native analyte influences its ionization. Therefore, the effect of ion suppression and enhancement of fourteen SIL‐ISs caused by their native analogues has been studied. It could be shown that the native analyte concentration influenced the extent of ion suppression and enhancement effects leading to more suppression with increasing analyte concentration especially when electrospray ionization (ESI) was used. Using atmospheric‐pressure chemical ionization (APCI), methanolic solution showed mainly enhancement effects, whereas no ion suppression and enhancement effect, with one exception, occurred when plasma extracts were used under these conditions. Such differences were not observed using ESI. With ESI, eleven SIL‐ISs showed relevant suppression effects, but only one analyte showed suppression effects when APCI was used. The presented study showed that ion suppression and enhancement tests using matrix‐based samples of different sources are essential for the selection of ISs, particularly if used for several analytes to avoid incorrect quantification. In conclusion, only SIL‐ISs should be selected for which no suppression and enhancement effects can be observed. If not enough ISs are free of ionization interferences, a different ionization technique should be considered. Copyright © 2010 John Wiley & Sons, Ltd.     

离子富集脉冲电源对提高飞行时间质谱仪灵敏度的影响

研究自制离子富集脉冲电源对飞行时间质谱仪灵敏度的提高效果。离子富集脉冲电源包括脉冲信号产生模块、放大隔离模块、开关驱动模块、高压电路模块和负载阻抗匹配模块,输出一路中压脉冲信号,两路对称高压脉冲信号。利用自制电喷雾电离源及激光解析电离源与飞行时间质谱仪联用,调试中压脉冲信号。结果显示,应用离子富集脉冲电源后仪器对不同样品的检测灵敏度可提高6~24倍。     

Sonic spray ionization interface for liquid chromatography-mass spectrometry

A sonic spray ionization (SSI) interface for liquid chromatography-mass spectrometry (LC-MS) analysis was optimized for analysis of 2-[(1R)-3-[bis(1-methylethyl)amino]-1-phenylpropyl]-4-methyl-phenol (tolterodine), used as a model drug substance, and the influence of different parameter settings was evaluated using factorial design. A comparison between SSI and electrospray ionization (ESI) was made for tolterodine, tolterodine metabolites, and a set of steroids.SSI was found to give slightly poorer repeatability and broader peaks for tolterodine compared to ESI. However, there was no significant difference in chromatographic peak shape, and the repeatability using SSI was similar to that obtained using ESI if a ratio (area of tolterodine/area of metabolite) was used. In this study, the sensitivity was higher using SSI. For the analysis of pregnanolone, less water loss was obtained using SSI, probably due to less energy being transferred to the analyte upon ionization.     

Antioxidant Pathways and One‐Electron Oxidation of Dopamine and Cysteine in Electrospray and On‐Line Electrochemistry Electrospray Ionization Mass Spectrometry

Dopamine [DA]⁺ (m/z 154), DA dimer [2DA‐H]⁺ (m/z 307) and DA quinone [DAQ]⁺ (m/z 152) are detected in positive ion mode electrospray ionization mass spectrometry (ESI MS) of dopamine in 50/1/49 (vol%) water/acetic acid/methanol. H/D exchange experiments support a covalent structure of DA dimer. Thus, ESI of DA may involve 1e^?, 1H⁺ oxidation processes followed by rapid radical dimerization. The DA quinone signal is low in ESI MS, which indicates a low efficiency of the 2e^?, 2H⁺ oxidation reaction. On‐line electrochemistry ESI MS (EC/ESI MS) with low electrochemical cell voltage floated on high ES voltage increases electrospray current and improves sensitivity for DA. The DA quinone signal increases and DA dimer signal decreases. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity of ESI and EC/ESI MS measurements. A DA quinone‐cysteine adduct [DAQ+Cys]⁺ was detected in solutions of DA with cysteine (Cys). ESI MS and EC/ESI MS indicate formation of the DA quinone‐cysteine adduct by 1e^? pathway. Oxidation pathways in ESI MS are relevant to biological reactivity of DA and Cys.     

Determination of Fusarium mycotoxins by liquid chromatography/tandem mass spectrometry coupled with immunoaffinity extraction

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T‐2 toxin (T‐2), HT‐2 toxin (HT‐2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) coupled with immunoaffinity extraction is described. A clean‐up was carried out using a DZT MS‐PREP® immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean‐up were compared. The results with the DZT MS‐PREP® IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent‐tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS‐PREP® IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03–0.33 ng·g^?1. From these studies, it is suggested that use of an IAC is effective in the clean‐up of each mycotoxin, and, when combined with LC/ESI‐MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Super‐atmospheric pressure ionization mass spectrometry and its application to ultrafast online protein digestion analysis

Ion source pressure plays a significant role in the process of ionization and the subsequent ion transmission inside a mass spectrometer. Pressurizing the ion source to a gas pressure greater than atmospheric pressure is a relatively new approach that aims to further improve the performance of atmospheric pressure ionization sources. For example, under a super‐atmospheric pressure environment, a stable electrospray can be sustained for liquid with high surface tension such as pure water, because of the suppression of electric discharge. Even for nano‐electrospray ionization (nano‐ESI), which is known to work with aqueous solution, its stability and sensitivity can also be enhanced, particularly in the negative mode when the ion source is pressurized. A brief review on the development of super‐atmospheric pressure ion sources, including high‐pressure electrospray, field desorption and superheated ESI, and the strategies to interface these ion sources to a mass spectrometer will be given. Using a recent ESI prototype with an operating temperature at 220 °C under 27 atm, we also demonstrate that it is possible to achieve an online Asp‐specific protein digestion analysis in which the whole processes of digestion, ionization and MS acquisition could be completed on the order of a few seconds. This method is fast, and the reaction can even be monitored on a near‐real‐time basis. Copyright © 2016 John Wiley & Sons, Ltd.     

Ion suppression and enhancement effects of co-eluting analytes in multi-analyte approaches: systematic investigation using ultra-high-performance liquid chromatography/mass spectrometry with atmospheric-pressure chemical ionization or electrospray ionization

In multi-analyte procedures, sufficient separation is important to avoid interferences, particularly when using liquid chromatography/mass spectrometry (LC/MS) because of possible ion suppression or enhancement. However, even using ultra-high-performance LC, baseline separation is not always possible. For development and validation of an LC/MS/MS approach for quantification of 140 antidepressants, benzodiazepines, neuroleptics, beta-blockers, oral antidiabetics, and analytes measured in the context of brain death diagnosis in plasma, the extent of ion suppression or enhancement of co-eluting analytes within and between the drug classes was investigated using atmospheric-pressure chemical ionization (APCI) or electrospray ionization (ESI). Within the drug classes, five analytes showed ion enhancement of over 25% and six analytes ion suppression of over 25% using APCI and 16 analytes ion suppression of over 25% using ESI. Between the drug classes, two analytes showed ion suppression of over 25% using APCI. Using ESI, one analyte showed ion enhancement of over 25% and five analytes ion suppression of over 25%. These effects may influence the drug quantification using calibrators made in presence of overlapping and thus interfering analytes. Ion suppression/enhancement effects induced by co-eluting drugs of different classes present in the patient sample may also lead to false measurements using class-specific calibrators made in absence of overlapping and thus interfering analytes. In conclusion, ion suppression and enhancement tests are essential during method development and validation in LC/MS/MS multi-analyte procedures, with special regards to co-eluting analytes.     

Dimerization of ionized 4‐(methyl mercapto)‐phenol during ESI,APCI and APPI mass spectrometry

A novel ion/molecule reaction was observed to occur under electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI) conditions, leading to dimerization of ionized 4‐(methyl mercapto)‐phenol followed by fast H^· loss. The reaction is particularly favored during ESI, which suggests that this ion/molecule reaction can occur both in the solution inside the ESI‐charged droplets and in the gas‐phase environment of most other atmospheric pressure ionization techniques. The dimerization reaction is inherent to the electrolytic process during ESI, whereas it is more by ion/molecule chemistry in nature during APCI and APPI. From the tandem mass spectrometry (MS/MS) data, accurate mass measurements, hydrogen/deuterium (H/D) exchange experiments and density functional theory (DFT) calculations, two methyl sulfonium ions appear to be the most likely products of this electrophilic aromatic substitution reaction. The possible occurrence of this unexpected reaction complicates mass spectral data interpretation and can be misleading in terms of structural assignment as reported herein for 4‐(methyl mercapto)‐phenol. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray ionization mass spectrometry and tandem mass spectrometry of clodronate and related bisphosphonate and phosphonate compounds

The bisphosphonate family with a P-C-P structure is a broad class of drugs, widely investigated as potential inhibitors in bone diseases and calcium metabolic disorders. In this study, the mass spectrometric (MS) behavior and fragmentation of clodronate and related bisphosphonate and phosphonate compounds was studied by using negative ion electrospray ionization (ESI) with triple quadrupole and ion trap instruments. The effect of pH on the degree of deprotonation of the polyprotic bisphosphonic and phosphonic acids in negative ion ESI-MS was investigated, and the degree of deprotonation in the ESI mass spectra and the dissociation in the liquid phase were compared. The results provide evidence that the measured ESI mass spectra do not correlate with the chemistry in the liquid phase owing to the decrease in the pH of the solvent droplets during the ion evaporation process and the charge state neutralization in the gas phase. Ion trap MS(n) provided useful information on the fragmentation study of clodronate and related bisphosphonate and phosphonate compounds, in which interesting fragmentation pathways including the direct elimination of carbon monoxide from deprotonated bisphosphonates and formation of a P-P bond were observed. Reactions between the product ions with a -PO(2) group and residual water in the ion trap or in the high-pressure region of the triple quadrupole instrument formed other unexpected fragmentation paths for all the bisphosphonates studied.     

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.