Complementary approach for analysis of phospholipids by liquid chromatography hyphenated to elemental and molecular mass spectrometry.

Phospholipids are one of the most important lipid categories with multiple functions in biological systems. Their analysis can contribute to a better understanding of metabolomic and kinetic processes in living cells. Comprehensive methods based on liquid chromatography coupled to mass spectrometry are available for phospholipid identification and quantification. However, quantification of phospholipids using electrospray ionization-mass spectrometry with internal standards is still challenging due to several reasons. In particular, the detector response of phospholipid species differs with variation of the head group as well as the fatty acid chain length and double bond number. Inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) provides an alternative approach for their absolute quantification with universal detector response for phosphorus independent of its chemical form and proportional to its quantity. Therefore, a quantification method based on compound-independent calibration using hydrophilic interaction liquid chromatography (HILIC) coupled to ICP-MS/MS was developed. An inverse gradient system was implemented for constant mobile phase composition after HILIC separation, which provides steady plasma ionization conditions. Isobaric phosphorus interferences were decreased by using the oxygen reaction mode of the triple quadrupole based ICP-MS/MS instrument. Complementary molecular information was obtained by ESI-high-resolution MS and MS/MS. The applicability of this approach was demonstrated in a proof of concept by complementary analysis of a total lipid extract of baker's yeast.

Elemental Bioimaging by Means of Fast Scanning Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry.

One of the most common setups for elemental bioimaging, the hyphenation of a laser ablation (LA) system and an inductively coupled plasma mass spectrometer (ICP-MS), was expanded by adding full scan mass spectrometric information as another dimension of information. While most studies deal with the analysis of typically not more than up to 10 isotopes per scan cycle, a fast scanning quadrupole mass analyzer was utilized to record the full mass spectrum of interest in this work. Mass-to-charge ratios from 6 to 250 were observed within one cycle. Besides the x- and y-position on the ablated sample and the intensity, the m/z-ratio served as fourth variable for each pixel of the obtained data, closing thereby the gap between "inorganic" and "organic" mass spectrometric imaging techniques. The benefits of this approach include an improved control of interferences, the discovery of unexpected elemental distributions, the possibility to plot isotopic ratios, and to integrate the intensities of a certain number of mass channels recorded for each isotope, thus virtually increasing sensitivity. The respective data are presented for dried droplets as well as embedded animal and human tissue slices. Limits of detection were calculated and found to be in accordance with counting statistics. A dedicated software macro was developed for data manipulation prior to common evaluation and image creation.

Rapid cell mode switching and dual laser ablation inductively coupled plasma mass spectrometry for elemental bioimaging.

RATIONALE: Two different approaches to improve the limits of detection (LODs) in elemental bioimaging have been developed. They both consider the fact that for the widely applied quadrupole-based instruments, metals in the mass range <100 u are analyzed with the best figures of merit in the kinetic energy discrimination (KED) mode; much better LODs are achieved for some metalloids and nonmetals by the introduction of more reactive gases, e.g., oxygen, into the collision/reaction cell (CRC). METHODS: While the first approach simultaneously utilizes two inductively coupled plasma mass spectrometry (ICP-MS) detectors hyphenated to one laser ablation (LA) system, the second is based on a single ICP-MS instrument with fast cell mode switching (CMS) of the CRC between individual line scans. RESULTS: Both methods were evaluated concerning their respective improvements by the analysis of rat brain samples. The utilization of two detectors showed improved LODs compared with conventional KED-only analysis in dependency on the gas flow splitting ratio, e.g., for sulfur by about 3.5 orders of magnitude. CMS provided even better results with a further improvement by a factor of 1.6. CONCLUSIONS: As a CRC with a small inner volume was used, fast cell gas switches at the end of every line prevented issues related to the reproducibility of the laser ablation stage for the CMS approach. Linear interpolation was found to be a valuable tool without affecting the spatial resolution of the images. In addition, a software macro is presented, which facilitates data evaluation.

On-line species-unspecific isotope dilution analysis in the picomolar range reveals the time- and species-depending mercury uptake in human astrocytes.

In order to reveal the time-depending mercury species uptake by human astrocytes, a novel approach for total mercury analysis is presented, which uses an accelerated sample introduction system combined on-line with an inductively coupled plasma mass spectrometer equipped with a collision/reaction cell. Human astrocyte samples were incubated with inorganic mercury (HgCl2), methylmercury chloride (MeHgCl), and thimerosal. After 1-h incubation with Hg(2+), cellular concentrations of 3 µM were obtained, whereas for organic species, concentrations of 14-18 µM could be found. After 24 h, a cellular accumulation factor of 0.3 was observed for the cells incubated with Hg(2+), whereas the organic species both showed values of about 5. Due to the obtained steady-state signals, reliable results with relative standard deviations of well below 5 % and limits of detection in the concentration range of 1 ng L(-1) were obtained using external calibration and species-unspecific isotope dilution analysis approaches. The results were further validated using atomic fluorescence spectrometry.