Comparison of universal detectors for high-temperature micro liquid chromatography

The fragmentation behavior of taxoids was studied using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources with multi-stage tandem mass spectrometry. In the positive ion mode taxoids gave prominent [M+Na]+ and [M+K]+ ions with the ESI source, and [M+NH4]+ or [M+H]+ ions with the APCI source. The MS/MS fragmentations of ions produced by APCI and ESI sources were very similar. For both sources, the presence of cinnamoyl or benzoyl groups could be characterized by initial losses of 148 or 122 u, respectively, from molecular adduct ions. However, the elimination of cinnamic acid was relatively difficult for the molecular adduct ions formed by APCI, and was comparable in importance to the loss of acetic acid. The other fragments involved losses of CH2CO, CO, and H2O. The 5/7/6 type taxoids underwent characteristic losses of 58 or 118 u from ions produced by both APCI and ESI sources. The fragmentation behavior was remarkably influenced by substitution locations. The elimination of the C-10 benzoyl group was usually the first fragmentation step, while that of the C-2 benzoyl group was relatively difficult. The acetoxyl group at C-7 was more active than those at C-2, C-9, and C-10, which in turn were more active than that at C-4. These fragmentation rules could facilitate the rapid screening and structural characterization of taxoids in plant extracts by high-performance liquid chromatography/mass spectrometry (HPLC/MS).

Direct coupling of supercritical fluid extraction to a gas phase atmospheric pressure chemical ionisation source ion trap mass spectrometer for fast extraction and analysis of food components

An evaluation of over 75 pesticides by high-performance liquid chromatography/mass spectrometry (HPLC/MS) clearly shows that different classes of pesticides are more sensitive using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). For example, neutral and basic pesticides (phenylureas, triazines) are more sensitive using APCI (especially positive ion). While cationic and anionic herbicides (bipyridylium ions, sulfonic acids) are more sensitive using ESI (especially negative ion). These data are expressed graphically in a figure called an ionization-continuum diagram, which shows that protonation in the gas phase (proton affinity) and polarity in solution, expressed as proton addition or subtraction (pKa), is useful in selecting APCI or ESI. Furthermore, sodium adduct formation commonly occurs using positive ion ESI but not using positive ion APCI, which reflects the different mechanisms of ionization and strengthens the usefulness of the ionization-continuum diagram. The data also show that the concept of "wrong-way around" ESI (the sensitivity of acidic pesticides in an acidic mobile phase) is a useful modification of simple PKa theory for mobile-phase selection. Finally, this finding is used to enhance the chromatographic separation of oxanilic and sulfonic acid herbicides while maintaining good sensitivity in LC/MS using ESI negative.

Atmospheric pressure chemical ionization (APCI) sources operated with point to plane DC discharges ('Coronas') frequently suffer from point electrode degradation and potentially lead to oxidation and/or fragmentation of the generated analyte ions. It is postulated that these adverse effects are caused by the interaction of these ions with the discharge chemistry as well as en route to the mass analyzer region. The corona discharge metal point electrode is replaced by the conically shaped liquid effluent evolving from a fused-silica capillary, which is analogous but not identical to the Taylor cone formation in electrospray ionization. The liquid consisting of either pure water or water containing 0.1 %V formic acid is fed via a nano-flow delivery stage at typical flow rates between 1-800 μL/h. The liquid flow is continuously replenishing the surface of the point electrode. The source is directly coupled to the inlet capillary of appropriate mass spectrometers, e.g., the Bruker Daltonics and Agilent varieties. The actively pumped liquid flow is supplying a constant amount of the reagent gas (H2O) to the corona region in the 20 ppmV to 30 %V range, leading to controlled, very stable operation of the source. The typical light emission observed for corona discharges is in very close proximity to the aqueous surface. Analyte protonation is the dominating ionization pathway. The degree of primary analyte fragmentation is extremely low. We have developed a novel atmospheric pressure chemical ionization source designed for the hyphenation of nano-flow liquid chromatography and gas chromatography with atmospheric pressure ionization mass spectrometry. The proposed reaction mechanism including the electrochemistry occurring in the source along with formation of protonated analyte molecules via collision-induced dissociation (CID) is in full accord with the experimental results. The system exhibits an extremely stable performance over prolonged operation times, sole generation of protonated molecules, and low degree of analyte ion fragmentation.

The capabilities of a recently launched atmospheric pressure chemical ionization (APCI) source for mass spectrometry (MS) coupled to gas chromatography (GC) have been tested in order to evaluate its potential in pesticide residue analysis in fruits and vegetables. Twenty-five pesticides were selected due to their high fragmentation under electron ionization (EI), making that the molecular ion (M+) is practically absent in their spectra. The fragmentation of these pesticides under APCI conditions was studied, with the result that M+ was not only present but also highly abundant for most compounds, with noticeable differences in the fragmentation patterns in comparison with EI. Moreover, the addition of water as modifier was tested to promote the formation of protonated molecules ([M+H]+). Under these conditions, [M+H]+ became the base peak of the spectrum for the majority of compounds, thus leading to an increase of sensitivity in the subsequent GC–MS/MS method developed using triple quadrupole analyzer (QqQ). Highly satisfactory sensitivity and precision, in terms of repeatability, were reached and linearity was satisfactory in the range 0.01–100ng/mL. The developed methodology was applied to apple, orange, tomato and carrot QuEChERS fortified extracts in order to evaluate the matrix effects. In summary, the soft and reproducible ionization in the APCI source has greatly favored the formation of [M+H]+ oppositely to EI where abundant fragmentation occurs and where the molecular ions have low abundance or are even absent in the mass spectrum. In this way, the use of APCI has facilitated the development of tandem MS methods based on the selection of abundant [M+H]+ as precursor ion.

Determination of bisphenol analogues in food-contact plastics using diode array detector, charged aerosol detector and evaporative light-scattering detector

Evaporative light scattering detector(ELSD) and charged aerosol detector(CAD) methods were established in this study for the content determination of four kinds of sugars in Zhusheyong Yiqi Fumai(YQFM), and the factors affecting the accuracy of CAD methods were discussed. HPLC-ELSD chromatographic separation was performed on a Shodex Asahipak NH2 P-50 column with acetonitrile-water(75∶25)as the mobile phase, with a flow rate of 0.8 mL·min~(-1), drift tube temperature of 80 ℃. The analysis by HPLC-CAD was performed on the same column with acetonitrile-water as mobile phase for gradient elution, with a flow rate of 0.8 mL·min~(-1), a neb temperature of 45 ℃, and power function(PF) of 1.3. The samples of YQFM were detected by ELSD and CAD respectively. It was found that YQFM was composed of fructose, glucose, sucrose and maltose. The linear relationship of the two methods was good, and the recoveries, reproducibility and stability of these four kinds of sugars measured by the two methods satisfied the requirements of methodology. Both CAD and ELSD detectors were accurate and reliable in detecting saccharides components in YQFM. In addition, it was revealed in this study for the first time that the PF parameter of CAD had an important influence on the accuracy of sugar determination and acted as the key parameter of CAD method. It was also found that for CAD, a non-linear detector, there was no significant difference between the results of linear regression and logarithmic regression.

The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208 degrees C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay.

Indoles are common building blocks of new pharmacologically active chemical entities in drug discovery and development. Due to their poor ionization in electrospray ionization mass spectrometry, atmospheric pressure chemical ionization (APCI) is the method of choice for LC-MS analysis of simple indoles. Three types of ions, including [M − 1]+, , and [M + H]+, can be observed in APCI but the relative intensities of these ions may vary depending upon the structural properties of the indoles and the mass spectral source conditions. We report in this paper the observation of [M − 1]+ions for methylindoles in an Agilent multimode ion source and the investigation into their formation. By means of tandem mass spectrometric experiments performed on a Thermo Fisher Scientific LTQ ion trap mass spectrometer equipped with an APCI source, it was found that [M − 1]+ions can be generated from ions upon-collision induced dissociation. This suggests that the [M − 1]+ions might be the in-source fragmentation product of ions. It was proposed that both [M − 1]+and ions are probably generated through a charge transfer mechanism while [M + H]+ions are the product of proton transfer. The basicity of the analytes might play an important role in dictating which ionization mechanism is operative. For 3-methylindole, the charge transfer process appears to be more dominant than for 2-methylindole since the former is less basic. As expected, substituting electron withdrawing groups on 3-methylindole, such as fluorine, promotes charge transfer and vice versa. Therefore, it is expected that formation of the [M − 1]+ions is more pronounced for less basic methylindoles.

Characterization of ribostamycin and its impurities using a nano-quantity analyte detector: Systematic comparison of performance among three different aerosol detectors

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.

The effects of the chaotropic agent perchloric acid on high temperature liquid chromatography (HTLC) with water as mobile phase have been investigated over a temperature range from 130°C to 200°C. Aniline, N-methylaniline, 3-ethylaniline and N,N-dimethylaniline were used as analytes. Similar to the effects of increasing the organic content of the mobile phase in regular reverse phase HPLC, the increase of column temperature in HTLC results in the decrease of capacity factors for all the aniline test compounds. By adding low concentrations of perchloric acid in the mobile phase, the capacity factors of the anilines also decrease which is equivalent to the increase of column temperature. Comparisons of HClO4 gradient and temperature program for the separation of the four anilines in HTLC are presented.

A new ionization method, named surface-activated chemical ionization (SACI), was employed for the analysis of five amphetamines (3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDE), amphetamine and methamphetamine) by ion trap mass spectrometry. The results so obtained have been compared with those achieved by using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) using the same instrument, clearly showing that SACI is the most sensitive of the three. The limit of detection and linearity range for SACI were compared with those obtained using APCI and ESI, showing that the new SACI approach provides the best results for both criteria. SACI was used to analyze MDA, MDMA MDE, amphetamine and methamphetamine in four urine samples, and the quantitation results are compared with those achieved using ESI.

Liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in the positive and negative ion modes was used for the characterization of a block copolymer consisting of methoxy poly(ethylene oxide) (mPEO), an epsilon-caprolactone (CL) segment and linoleic acid (LA), used as surfactant in water-based latex paints. Chromatographic separation was obtained based on the number of CL units in the polymer species and the presence of an mPEO and/or LA tail. Different ionization methods were found to be complementary and only their combination allowed the qualitative profiling of the chemical composition. The LC/MS method has proven valuable for following the reaction in time, as well as for comparison of different polymeric surfactants.

Because most lipid extracts are a mixture of saturated and unsaturated molecules, the most successful strategies for the quantitative analysis of lipids have involved the use of so-called "mass" or universal detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC "mass" detector, a charged aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents, charging the aerosol particles, and measuring the current from the charged aerosol flux. In the present study, the CAD was evaluated with several normal phase and reverse phase HPLC methods commonly used for the quantitative analysis of lipid classes and lipid molecular species. The CAD detected common lipids such as triacylglycerols, diacylglycerols, glycolipids, phospholipids, and sterols. Lower molecular weight lipids such as free FA had smaller peak areas (50-80% lower). FAME were not detected by the CAD, probably because they were completely evaporated and did not form aerosol particles. The minimum limits of detection of the CAD with lipids varied with different mobile phase solvents. Using solvent systems that were predominantly hexane, the minimum limits of detection of triacylglycerols, cholesterol esters, and free sterols were about 1 ng per injection and the mass-to-peak area ratio was nearly linear from the range of about 1 ng to about 20 mg per injection. Three other solvents commonly used for HPLC lipid analysis (methanol, isopropanol, and acetonitrile) caused higher levels of background noise and higher minimum limits of detection. These experiments indicate that the CAD has the potential to become a valuable tool for the quantitative HPLC analysis of lipids. Long-term studies are needed to evaluate full instrument performance.