Negative-ion Atmospheric Pressure Chemical Ionization Research Articles

Fragmentation studies of neutral per- and polyfluoroalkyl substances by atmospheric pressure ionization-multiple-stage mass spectrometry.

The establishment of fragmentation pathways has a great interest in the identification of new or unknown related compounds present in complex samples. On that way, tentative fragmentation pathways for the ions generated by atmospheric pressure ionization of neutral per- and polyfluorinated alkyl substances (PFASs) have been proposed in this work. Electrospray (ESI), atmospheric pressure chemical ionization (APCI) and photoionization (APPI) were evaluated using mobile phases and source conditions that enhance the ionization efficiency of ions generated. A hybrid mass spectrometer consisting of a linear ion trap and an Orbitrap was used to combine the information of both multiple-stage mass spectrometry (MSn) and mass accuracy measurements to characterize and establish the genealogical relationship between the product ions observed. The ionization mechanisms to generate ions such as [M-H]-, [M]-•, and [M+O2]-• or the in-source collision-induced dissociation (CID) fragment ions in each API source are discussed in this study. In general, fluorotelomer olefins (FTOs) ionized in negative-ion APCI and APPI generated the molecular ion, while fluorotelomer alcohols (FTOHs) also provided the deprotonated molecule. Besides, fluorooctane sulfonamides (FOSAs) and sulfonamido-ethanols (FOSEs) led to the deprotonated molecule and in-source CID fragment ions, respectively. The fragmentation pathways from these precursor ions mainly involved initial α,β-eliminations of HF units and successive losses of CF2 units coming from the perfluorinated alkyl chain. Moreover, FTOHs and FOSEs showed a high tendency to generate adduct ions under negative-ion ESI and APPI conditions. The fragmentation study of these adduct ions has demonstrated a strong interaction with the attached moiety. Graphical abstract.

A comparison of electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization for the liquid chromatography/tandem mass spectrometric analysis of bisphenols. Application to bisphenols in thermal paper receipts and U.S. currency notes.

Human health concerns have arisen for common bisphenols (BPA and BPS) as well as new structural analogs. While native sulfone bisphenols and sulfone-derivatized bisphenols are amenable to electrospray ionization (ESI), alternative ionization methods such as atmospheric pressure chemical ionization (APCI) and APPI were investigated to analyze non-derivatized, non-sulfone-containing bisphenols. Ionization of bisphenols using negative ion APPI was compared to negative ion ESI and negative ion APCI for their relative abilities to produce [M-H]- precursor ions for subsequent collision-induced dissociation (CID) in a triple quadrupole mass analyzer. Multiple reaction monitoring (MRM) transitions were optimized using the most sensitive ion transitions and coupled with high-performance liquid chromatographic (HPLC) separation to detect and quantify BPA, BPB, BPF, BPZ, BPAP, BPS, and BPSIP. APPI provides a more universal sensitivity over ESI toward the classes of bisphenols studied with detection limits of 20-50 pg on-column. The LC/APPI-MS/MS method was used to examine the levels of these seven bisphenols in thermal paper receipts and in U.S. paper currency. In both receipts and currency, BPA and BPS were the dominant bisphenols found in the paper. The measurement of BPA and bisphenol analogs in thermal paper receipts and transfer of bisphenols from receipts to currency notes is of increasing importance to assess human dermal exposure routes to this class of compounds. Equivalent sensitivity between sulfone- and non-sulfone-containing bisphenols is achievable through the use of alternate ionization sources such as APCI and APPI that circumvents tedious and time-consuming derivatization procedures to render analytical sensitivity by ESI.

Application of orbitrap mass spectrometry for analysis of model bio-oil compounds and fast pyrolysis bio-oils from different biomass sources

Pyrolysis bio-oils have great potential for the future use as biofuels and source of oxygenated chemicals. To optimize a pyrolysis process, detailed knowledge about the chemical composition of bio-oils is necessary. In recent years, high-resolution mass spectrometry (HRMS) has successfully been used to the characterization of pyrolysis bio-oils from lignocellulosic biomass. This method enabled to detect thousands of semivolatile and nonvolatile, high-molecular-weight bio-oil compounds and provided partial information about their structure. In this work, we used high-resolution orbitrap mass spectrometry to characterize semivolatile and nonvolatile, high-molecular-weight compounds of four bio-oils obtained from the ablative flash pyrolysis of different biomass sources. Before the analyses of these bio-oils, we analyzed model bio-oil compounds and commercially available bio-oil from fast pyrolysis of wood using positive-ion and negative-ion electrospray (ESI) and positive-ion and negative-ion atmospheric pressure chemical ionization (APCI) orbitrap mass spectrometry and compared the results. Based on this comparison, a combination of negative-ion ESI and APCI was found to be well suited for the characterization of pyrolysis bio-oils; these techniques were thus used for the study of bio-oils from different biomass sources and the obtained results were compared. In the studied bio-oils, mostly compounds with 1–8 oxygen atoms per molecule were detected and their degree of unsaturation (DBE) was about 1–10 (negative-ion ESI) and 1–17 (negative-ion APCI), respectively. Among the studied bio-oils, the differences were observed mostly in abundances of their major compounds (compound classes). The analyses of model bio-oil compounds brought valuable information about their behavior during the HRMS characterization of bio-oils. The presented results could help to improve the understanding of bio-oil composition and HRMS characterization of bio-oils and facilitate their further utilization.

Monitoring hydrogen sulfide in simulated breath of anesthetized subjects

Human breath can be a complex mixture of trace gases in humid air, with the addition of various anesthetizing agents it becomes even more complex and can present a challenge for the accurate quantification of components. The purpose of this paper was to investigate the quantification of H2S as a function of several potential matrix/interference compounds using negative-ion atmospheric pressure chemical ionization with mass spectrometric detection. Analysis was performed by humidifying zero-air and adding various compounds that would be encountered in a perioperative situation. Nitrous oxide, carbon dioxide and to a lesser extent humidity have significant effects on the instrument response to hydrogen sulfide concentration, while halogenated species were found to have little if any complicating effect. It was found that H2S can be accurately quantified by using either [SO3]− or [SO2]−/[SO3]−, both of which eliminate the response variability due to interferences.

High-throughput determination of barbiturates in human plasma using on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry

A high-throughput method was developed for determinations of eight barbiturates (barbital, allobarbital, phenobarbital, cyclobarbital, amobarbital, secobarbital, thiopental, and thiamylal) in human plasma by on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry (MS–MS). Plasma samples (100 μl) spiked with the eight barbiturates and 5-(4-methylphenyl)-5-phenylhydantoin (internal standard) were diluted with 300 μl of 13.3 mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced onto the analytical column (Phenomenex Onyx monolithic C18 column, 100 mm × 4.6 mm i.d.) by column switching. Quantification was performed by multiple reaction monitoring with negative-ion atmospheric pressure chemical ionization. Good peak separation and peak shapes of the eight drugs were achieved within an analysis time of 3 min, including the extraction time. All drugs spiked into plasma showed recoveries of 80–93 %. The regression equations for the eight drugs showed excellent linearities in the range of 10–5000 ng/ml of plasma, and the limits of detection ranged from 1.0 to 10 ng/ml. The lower and upper limits of quantitation were 10–50 ng/ml and 5000 ng/ml, respectively. Intraday and interday coefficients of variation for all the drugs were not >9.1 %. The accuracies of quantitation were 92.0–108 %. The method was successfully applied to determination of the level of amobarbital in human plasma after its oral administration to a volunteer.

Atmospheric pressure chemical ionization tandem mass spectrometry of carotenoids

Carotenoids are natural pigments synthesized by plants and photosynthetic microorganisms, some of which, like β-carotene, are precursors of vitamin A, and others such as lutein and lycopene might function in the prevention of age-related macular degeneration and prostate cancer, respectively. Mass spectrometry provides high sensitivity and selectivity for the identification and quantitative analysis of carotenoids in biological samples, and previous studies have described how atmospheric pressure chemical ionization (APCI) offers distinct advantages over electrospray and fast atom bombardment for the analysis of specific carotenoids. Since APCI product ion tandem mass spectra have been reported for only a few carotenoids, a detailed investigation of twelve carotenes and xanthophylls was carried out using both positive ion and negative ion APCI tandem mass spectrometry with collision-induced dissociation. Using protonated molecules as precursor ions in positive ion mode and radical anions in negative ion mode, characteristic fragment ions were identified that may be used to distinguish between carotenoids.

Ionization Mechanisms Related to Negative Ion APPI, APCI, and DART

A recent report found that negative ion atmospheric pressure photoionization (Ni-APPI) and direct analysis in real time (Ni-DART) ionize compounds by electron capture, dissociative electron capture, proton abstraction, and anion adduction. The authors of this report suggested that the common ionization of Ni-APPI and Ni-DART demonstrated that these techniques ionize a wider array of compounds than negative ion atmospheric pressure chemical ionization (Ni-APCI). Here we show that Ni-APCI, using the atmospheric sample analysis probe (ASAP) technique, in the absence of solvent vapors, ionizes the same and similar compounds by the same reported mechanisms. These results are supported by previous publications, which show that each mechanism is active for Ni-APCI. This work demonstrates that irrespective of the initial method of ionization, at atmospheric pressure, similar ion/electron-molecule chemistries prevail.

Ionization Mechanism of Negative Ion-Direct Analysis in Real Time: A Comparative Study with Negative Ion-Atmospheric Pressure Photoionization

The ionization mechanism of negative ion-direct analysis in real time (NI-DART) has been investigated using over 42 compounds, including fullerenes, perfluorocarbons (PFC), organic explosives, phenols, pentafluorobenzyl (PFB) derivatized phenols, anilines, and carboxylic acids, which were previously studied by negative ion-atmospheric pressure photoionization (NI-APPI). NI-DART generated ionization products similar to NI-APPI, which led to four ionization mechanisms, including electron capture (EC), dissociative EC, proton transfer, and anion attachment. These four ionization mechanisms make both NI-DART and NI-APPI capable of ionizing a wider range of compounds than negative ion-atmospheric pressure chemical ionization (APCI) or negative ion-electrospray ionization (ESI). As the operation of NI-DART is much easier than that of NI-APPI and the gas-phase ion chemistry of NI-DART is more easily manipulated than that of NI-APPI, NI-DART can be therefore used to study in detail the ionization mechanism of LC/NI-APPI-MS, which would be a powerful methodology for the quantification of low-polarity compounds. Herein, one such application has been further demonstrated in the detection and identification of background ions from LC solvents and APPI dopants, including water, acetonitrile, chloroform, methylene chloride, methanol, 2-propanol, hexanes, heptane, cyclohexane, acetone, tetrahydrofuran (THF), 1,4-dioxane, toluene, and anisole. Possible reaction pathways leading to the formation of these background ions were further inferred. One of the conclusions from these experiments is that THF and 1,4-dioxane are inappropriate to be used as solvents and/or dopants for LC/NI-APPI-MS due to their high reactivity with source basic ions, leading to many reactant ions in the background.

Negative Ion-Atmospheric Pressure Photoionization: Electron Capture, Dissociative Electron Capture, Proton Transfer, and Anion Attachment

To better guide the development of liquid chromatography/electron capture-atmospheric pressure photoionization-mass spectrometry (LC/EC-APPI-MS) in analysis of low polarity compounds, the ionization mechanism of 19 compounds was studied using dopant assisted negative ion-APPI. Four ionization mechanisms, i.e., EC, dissociative EC, proton transfer, and anion attachment, were identified as being responsible for the ionization of the studied compounds. The mechanisms were found to sometimes compete with each other, resulting in multiple ionization products from the same molecule. However, dissociative EC and proton transfer could also combine to generate the same [M - H](-) ions. Experimental evidence suggests that O(2)(-*), which was directly observed in the APPI source, plays a key role in the formation of [M - H](-) ions by way of proton transfer. Introduction of anions more basic than O(2)(-*), i.e., C(6)H(5)CH(2)(-), into the APPI source, via addition of di-tert-butyl peroxide in the solvent and/or dopant, i.e., toluene, enhanced the deprotonation ability of negative ion-APPI. Although the use of halogenated solvents could hinder efficient EC, dissociative EC, and proton transfer of negative ion-APPI due to their EC ability, the subsequently generated halide anions promoted halide attachment to compounds that otherwise could not be efficiently ionized. With the four available ionization mechanisms, it becomes obvious that negative ion-APPI is capable of ionizing a wider range of compounds than negative ion chemical ionization (NICI), negative ion-atmospheric pressure chemical ionization (negative ion-APCI) or negative ion-electrospray ionization (negative ion-ESI).

Fragmentation study of hexanitrostilbene by ion trap multiple mass spectrometry and analysis by liquid chromatography/mass spectrometry

The fragmentation pathways of three explosive compounds with similar structures, hexanitrostilbene (HNS), cyclotrimethylene trinitramine (RDX), and 2,4,6-trinitrotoluene (TNT), have been investigated by multiple mass spectrometry (MSn, n = 1, 2, 3) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The electron capture mechanism for these compounds in negative ion APCI and ESI mode differs from the usual negative ion mechanism, deprotonation or addition of other species. This was shown for HNS and TNT, which both gave a [M]- anion but not a [M-H]- ion in APCI, and the [M]- anion of HNS was observed in ESI. The quantitative analysis of HNS was performed by liquid chromatography (LC)/ESI-MS, and the results obtained by the internal standard (ISTD) method were compared with those from the external standard (ESTD) method, demonstrating that both quantitation approaches are useful, with good sensitivity, reproducibility and linearity, and ESTD is preferable in routine applications.

Quantitative analyses of β‐carotene and retinol in serum and feces in support of clinical bioavailability studies

Among more than 50 provitamin carotenoids, beta-carotene is the most metabolically active source of retinol. Despite diets rich in fruits and vegetables containing beta-carotene, vitamin A deficiency is the leading cause of blindness and childhood mortality in developing countries. In addition, the uncertainty of beta-carotene bioconversion into vitamin A suggests that new data are needed to update the nutritional guidelines in developed countries. Previously, we reported the development of a carotene/retinol plateau isotopic enrichment method (CarRet PIE) for the determination of beta-carotene bioavailability and bioconversion into retinol, which utilizes positive ion atmospheric pressure chemical ionization (APCI) liquid chromatography/mass spectrometry (LC/MS). While seeking to validate the CarRet PIE using a mass balance approach requiring fecal measurements of beta-carotene and retinol, interference was encountered that required substantial modifications of the LC/MS assay. Here we report a new LC/MS assay that is based on the detection of molecular anions of beta-carotene using negative ion APCI with a reversed-phase C30 column for HPLC separation. Sample preparation required saponification to eliminate interfering triglycerides. The limit of detection (LOD) of beta-carotene was 0.25 pmol calculated on the basis of an injection of 20 microL of 0.0125 microM beta-carotene, and the limit of quantitation (LOQ) was 1.0 pmol based on the injection of 20 microL of 0.050 microM beta-carotene. The linear range was 1.1 to 2179 pmol on-column. The wide linear range and low LOD and LOQ of this assay facilitated the sensitive and selective quantitative analysis of beta-carotene in both serum and fecal samples in support of an on-going clinical investigation of beta-carotene bioavailability and bioconversion into vitamin A.

Applications of HPLC-MS in compound Ilex pubescens extract study

In this paper, high performance liquid chromatography (HPLC) along with mass spectrometry (MS) and HPLC along with a diode array detector (DAD) was used to study the compound Ilex pubescens extract. Two ionization techniques: electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were used in this work. The liquid chromatograms obtained by DAD, total ion chromatograms (TIC) from positive-and negative-ion ESI-MS and the positive-and negative-ion APCI-MS were compared. The liquid chromatograms obtained by TIC from ESI-MS provided more information on chromatographic peaks than those obtained by DAD or TIC from APCI-MS. It is suggested that the fingerprints of the compound Ilex pubescens extract should be provided by the liquid chromatograms obtained by DAD together with TIC from the negative-ion ESI-MS. The molecular weights of the nine main components in an HPLC-DAD chromatogram were determined by the corresponding positive-and negative-ion ESI and the positive-and negative-ion APCI mass spectra information. In the liquid chromatogram obtained by TIC from the negative-ion ESI-MS, the molecular weights of 23 main components were determined based on the corresponding positive-and negative-ion ESI mass spectra information.

Flavonoids in Leguminosae: analysis of extracts of T. pratense L., T. dubium L., T. repens L., and L. corniculatus L. leaves using liquid chromatography with UV, mass spectrometric and fluorescence detection.

Reversed-phase LC on C-18 bonded silica with a methanol-ammonium formate gradient was used to determine the main flavonoids in leaves of four species of the Leguminosae family. The detection modes were diode-array UV absorbance, fluorescence, and (tandem) mass spectrometry. LC-UV was used for a general screening, sub-classification, and the calculation of total flavonoid contents. LC-FLU was included to identify isoflavones on the basis of their native fluorescence. Most structural information regarding aglycons, sugar moieties, and acidic groups was derived from LC-MS in both the full-scan and extracted-ion mode, using negative-ion atmospheric pressure chemical ionization. MS/MS did not provide much additional information, because the same fragments were observed as in full-scan MS. In T. pratense and T. repens, the main constituents were flavonoid glucoside-(di)malonates, while T. dubium and L. corniculatus mainly contained flavonoid (di)glycosides. Satellite sets comprising an aglycon, the glucoside and glucoside-malonates or -acetates, were abundantly present only in T. pratense. Generally speaking, the main aglycons and sugars in the four plant species are surprisingly different. In addition, while the results for T. pratense are similar to those reported in the literature, there is little agreement in the case of the other species. Finally, total flavonoid contents ranged from 50-65 mg/g for L. corniculatus and T. dubium, to 15 mg/g for T. pratense and only 1 mg/g for T. repens.

Atmospheric pressure chemical ionization mass spectrometry and in‐source fragmentation of lutein esters

Negative-ion atmospheric pressure chemical ionization (APCI) mass spectrometry and in-source collisionally induced dissociation (CID) were employed to obtain structural information of lutein esters from marigold extract. Both molecular ions and structurally significant fragments corresponding to the loss of fatty acids were observed in high abundance in the current study. Six lutein diesters including lauroylmyristoyl-lutein (LML), dimyristoyl-lutein (dML), myristoylpalmitoyl-lutein (MPL), dipalmitoyl-lutein (dPL), palmitoylstearoyl-lutein (PSL) and distearoyl-lutein (dSL) were characterized in a marigold flower extract. Breakdown curves (plots of relative ion abundance vs. internal energy) of three lutein diesters were established by monitoring the relative ion abundance of molecular and fragment ions at different cone voltages during negative-ion APCI-LC/MS analysis.

Identification of acylated xanthone glycosides by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry in positive and negative modes from the lichen Umbilicaria proboscidea

The xanthoside composition of the crude extract of Umbilicaria proboscidea (L.) Schrader was characterized using LC–UV diode array detection and LC–atmospheric pressure chemical ionization (APCI) MS methods. The presence of acylated xanthone- O-glucosides was determined by both positive and negative ion LC–APCI-MS methods. Based on UV and MS spectral data and NMR spectroscopy, a total of 14 compounds (6- O-acylated umbilicaxanthosides A and B) were identified in U. proboscidea for the first time. In order to further develop the applicability of LC–MS techniques in phytochemical characterization, the effect of different ionization energy on fragmentation was studied using APCI. The optimal ionization conditions were achieved in positive ion APCI by using ammonium acetate buffer and in negative ion APCI by using formic acid (pH 4).

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.

Simultaneous determination of all-trans, 9-cis, 13-cis retinoic acid and retinol in rat prostate using liquid chromatography-mass spectrometry.

Since retinoic acid (RA) and RA receptors are key developmental regulators during organogenesis, they might participate in the abnormal development of the prostate caused by early estrogen exposure. In order to test this assumption, a sensitive analytical method that can differentiate 9-cis, 13-cis, and all-trans RA in small tissue samples ( approximately 8 mg) is required. Since retinol is the metabolic precursor to RA, simultaneous quantification of retinol would also provide valuable information. Here, we report a liquid chromatography-mass spectrometry method for simultaneous determination of retinol and 9-cis, 13-cis, and all-trans RA in rat prostate. Mass spectrometric signal responses for RA were compared using positive ion atmospheric-pressure chemical ionization (APCI) and electrospray, as well as positive ion and negative ion APCI. Positive ion APCI was selected for all subsequent analysis for its better sensitivity, and to provide simultaneous determination of retinol and RA. Ventral prostate tissue samples were homogenized and extracted following simple protein precipitation without derivatization. Baseline separation of 9-cis, 13-cis, and all-trans RA standards was obtained by using a non-porous silica C18 column. Selected ion monitoring of the ions m/z 301 and m/z 269 was carried out for mass spectrometric quantitative analysis. The ion of m/z 301 corresponded to the protonated molecule of RA, whereas the ion of m/z 269 corresponded to loss of water or acetic acid from the protonated molecule of retinol or the internal standard retinyl acetate respectively. The method has a linear response over a concentration range of at least three orders of magnitude. The limit of quantitation was determined to be 702 fmol all-trans RA injected on-column. The method showed excellent intra- and inter-assay reproducibility and good recovery, and is suitable for analyzing RA and retinol in small tissue samples (approximately 8 mg).

Multi-residue analysis of avermectins in swine liver by immunoaffinity extraction and liquid chromatography–mass spectrometry

A multi-residue LC–MS method was developed to determine avermectin residues in swine liver. Abamectin and ivermectin (22,23-dihydroabamectin) were extracted, and cleaned up by immunoaffinty columns with immobilized anti-avermectin polyclonal antibodies. The cleaned samples were separated by high-performance liquid chromatography (HPLC) with a C 8 column and determined by negative-ion atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) using selective ion monitoring (SIM) of [M-H] −. Recoveries of abamectin and ivermectin from fortified samples at 5–100 μg kg −1 levels ranged from 74 to 94% and from 65 to 87%, respectively. The limits of detection were 5 μg of abamectin or ivermectin in 1 kg sample.

Naphthenic Acids in Crude Oils Characterized by Mass Spectrometry

The presence of naphthenic acids in crude oils is of concern in the petroleum industry due to their corrosivity to refinery units. It is desirable to determine the ring type and carbon number distributions because the corrosivity of naphthenic acids is dependent on the sizes and structures. The characterization of naphthenic acids is also of interest to geochemical studies, particularly migration and biodegradation, and to refinery wastewater treatment for environmental compliance. We have evaluated chemical ionization, liquid secondary ion mass spectrometry (fast ion bombardment), atmospheric pressure chemical ionization (APCI), and electrospray ionization in both positive and negative ion modes for the determination of molecular distribution of acids without derivatization. Negative-ion APCI using acetonitrile as a mobile phase yields the cleanest spectra with good sensitivity among the ionization techniques evaluated. The selectivity of negative-ion APCI for naphthenic acids has also been demonstrated by comparing results for a whole crude oil with those for the isolated acid fraction. APCI also holds a great potential for on-line liquid chromatography−mass spectrometric (LC/MS) to separate acids by high-performance liquid chromatography (HPLC) followed by mass spectrometric characterization of acids.

Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography–mass spectrometry with negative ion atmospheric pressure chemical ionisation

A new, rapid and sensitive method has been developed for the determination of nivalenol (NIV) and deoxynivalenol (DON) by using HPLC in combination with an atmospheric pressure chemical ionization (APCI)-interface and a single quadrupole mass spectrometer. Different LC and MS parameters have been optimized prior to this in order to obtain better results and sensitivity. The effect of nebulizing temperature on the sensitivity and fragmentation of NIV and DON in an APCI interface was investigated. Also, the influence of the cone voltage on the fragmentation pattern was studied, which was shown to have a tremendous effect. Furthermore, the effect of modifiers such as ammonium acetate, acetic acid and ammonia on the ionisation yield of the above substances have been investigated. The extraction was carried out using acetonitrile–water. A two step purification was then applied on two different Mycosep clean up columns. We have used a modified, rapid and isocratic HPLC method combined with a negative ion APCI–MS for the separation and quantitative determination of NIV and DON in wheat extract. An RP C18 column was used for the separation of selected compounds in wheat extract with water–acetonitrile–methanol (82:9:9, v/v/v) at a flow-rate of 1 ml/min without a split. Calibration curves show good linearity and reproducibility. The detection limit and precision were determined for NIV and DON. Both compounds could be detected down to μg/kg level in wheat using selected ion monitoring of the [M–H]− ions and the main fragments.