Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization.

For acceptance of supercritical fluid chromatography (SFC) as a routine analysis method, the hyphenation to mass spectrometry (MS), which is typically achieved either by a splitting device or by the employment of an additional make up flow, has to be improved. Direct coupling of SFC to MS (/MS) would simplify the handling of this method. Consequently, this work focused on the direct coupling of SFC to mass spectrometry and the influence of the employed ion source on signal intensities of polar and ionic compounds in biological samples. A method for separating metabolites of the tryptophan pathway as well as other amino acids is shown. Results demonstrate that SFC is capable of separating analytes of polar and ionic nature. Modifications of the SFC system by cryostat cooling lead to higher temperature stability in the booster pump and therefore to a better reproducibility of retention times and a low dispersion nozzle inside the active back pressure regulator (ABPR) significantly improves peak shape and sensitivity when using the MS. The comparison of the ionization efficiencies using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in positive and negative ion mode shows analyte depending sensitivities. However, results indicate that APCI is better suitable for the ionization of amino acids with polar side chains, whereas ESI proved superior for the ionization of amino acids featuring hydrophobic residues. Analyte signals were suppressed with ESI when using a complex matrix such as human serum, but rather enhanced when using APCI.

Wolrab D ，Frühauf P ，Gerner C 《-》

Splitless hyphenation of SFC with MS by APCI, APPI, and ESI exemplified by steroids as model compounds.

A systematic evaluation of splitless hyphenation of supercritical fluid chromatography (SFC) with mass spectrometry (MS) was performed using different techniques for ambient pressure ionization. Interfaces commonly known from HPLC-MS/MS, i.e. electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI), were tested for their suitability in SFC-MS/MS. A triple quadrupole MS was used for data evaluation in a targeted multi-analyte design using endogenous steroids as model compounds. Individual optimization of the ionization parameters was performed in multi-dimensional design for best support of ionization in all three techniques. A post-column make-up was used to avoid analyte precipitation in the transfer capillary but also to support ionization independently from mobile phase composition. Buffer choice and concentration as well as temperature were found crucial in ESI and APCI. Best results for the multi-analyte method were obtained in both techniques using ammonium fluoride as make-up buffer. Instead of buffer solutions different organic solvents were used as dopants in APPI to support ionization. The mobile phase constituent isopropanol was already found to support ionization in APPI, however, for many analytes the addition of toluene resulted in superior results in terms of intensity. Comparing the optimized methods in terms of limit of detection (LOD), limit of quantification (LOQ), and sensitivity (slope of calibration curve) ESI was the best choice for the multiple analyte design. Only a few analytes resulted in a different optimum ionization, if focused on separately. In terms of linear dynamic range, APCI and APPI proved superior to ESI, where calibration over the whole range of concentrations (from LOD up to 5000 pg ∗ μL-1) required quadratic regression.

Parr MK ，Wüst B ，Teubel J ，Joseph JF ... -  《-》

A rapid method for the separation of vitamin D and its metabolites by ultra-high performance supercritical fluid chromatography-mass spectrometry.

In this study, a new supercritical fluid chromatography-mass spectrometry (SFC-MS) method has been developed for the separation of nine vitamin D metabolites within less than eight minutes. This is the first study of analysis of vitamin D and its metabolites carried out by SFC-MS. Six columns of orthogonal selectivity were examined, and the best separation was obtained by using a 1-aminoanthracene (1-AA) column. The number and the position of hydroxyl groups in the structure of the studied compounds as well as the number of unsaturated bonds determine the physiochemical properties and, thus the separation of vitamin D metabolites that is achieved on this column. All D2 and the D3 forms were baseline separated with resolution values>1.5. The effects of pressure, temperature, flow rate and different gradient modes were studied. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared in positive mode, both by direct infusion and after SFC separation. The results showed that the sensitivity in APCI(+) was higher than in ESI(+) using direct infusion. In contrast, the sensitivity in APCI(+) was 6-fold lower than in ESI(+) after SFC separation. The SFC-MS method was validated between 10 and 500ng/mL for all analytes with coefficient of determination (R(2))≥0.999 for all calibration curves. The limits of detection (LOD) were found to range between 0.39 and 5.98ng/mL for 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1-hydroxyvitamin D2 (1OHD2), respectively. To show its potential, the method was applied to human plasma samples from healthy individuals. Vitamin D3 (D3), 25-hydroxyvitamin D3 (25OHD3) and 24,25(OH)2D3 were determined in plasma samples and the concentrations were 6.6±3.0ng/mL, 23.8±9.2ng/mL and 5.4±2.7ng/mL, respectively.

Jumaah F ，Larsson S ，Essén S ，Cunico LP ，Holm C ，Turner C ，Sandahl M ... -  《-》

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry for the qualitative analysis of metabolites covering a large polarity range.

The applicability of ultrahigh-performance supercritical fluid chromatography coupled with mass spectrometry (UHPSFC/MS) for the qualitative analysis of metabolites with a wide polarity range (log P: -3.89-18.95) was evaluated using a representative set of 78 standards belonging to nucleosides, biogenic amines, carbohydrates, amino acids, and lipids. The effects of the gradient shape and the percentage of water (1, 2, and 5%) were investigated on the Viridis BEH column. The screening of eight stationary phases was performed for columns with different interaction sites, such as hydrogen bonding, hydrophobic, π-π, or anionic exchange type interactions. The highest number of compounds (67) of the set studied was detected on the Torus Diol column, which provided a resolution parameter of 39. The DEA column had the second best performance with 58 detected standards and the resolution parameter of 54. The overall performance of other parameters, such as selectivity, peak height, peak area, retention time stability, asymmetry factor, and mass accuracy, led to the selection of the Diol column for the final method. The comparison of additives showed that ammonium acetate gave a superior sensitivity over ammonium formate. Moreover, the influence of the ion source on the ionization efficiency was studied by employing atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The results proved the complementarity of both ionization techniques, but also the superior ionization capacity of the ESI source in the negative ion mode, for which 53% of the analytes were detected compared to only 7% for the APCI source. Finally, optimized analytical conditions were applied to the analysis of a pooled human plasma sample. 44 compounds from the preselected set were detected in human plasma using ESI-UHPSFC/MS in MSE mode considering both ionization modes.

Antonelli M ，Holčapek M ，Wolrab D 《-》

Natural compounds analysis using liquid and supercritical fluid chromatography hyphenated to mass spectrometry: Evaluation of a new design of atmospheric pressure ionization source.

The MS hyphenation performance of UniSpray®, a new design of atmospheric pressure ionization source was evaluated in both SFC and RPLC modes. Sensitivity, stability, versatility and matrix effects offered by the UniSpray were assessed in positive and negative ionization modes and systematically compared to an electrospray source (ESI) using 120 natural compounds covering an extended chemical space. In a first instance, a multivariate approach was used to screen and optimize the UniSpray source settings to maximize detection sensitivity. The position of the source capillary against the fixed charged rod was highlighted as the major parameter affecting the detection sensitivity. The sensitivity improvement in Unispray vs. ESI strongly depends on the compounds chemical class and the chromatographic mode. For a few compounds (i.e. anabasine, theanine, caproic acid, fumaric acid and protopanaxatriol), up to a 10-fold increase in sensitivity was noticed with UniSpray. The signal stability over multiple injections was also found to be equivalent between both sources with RSD values on peak intensity lower than 14% on >100 injections, in both chromatographic modes. On complex plant extract, the matrix effects occurring from the secondary metabolites were also found to be comparable between ESI and UniSpray, at least in the positive ionization mode. However, a systematic decrease of MS signal intensity was observed in SFC mode when compounds were ionized using UniSpray in the negative ion mode.

Ciclet O ，Barron D ，Bajic S ，Veuthey JL ，Guillarme D ，Grand-Guillaume Perrenoud A ... -  《-》