The quantitative determination of the total free fatty acids (FFAs) is an important analytical task because FFAs exhibit important physiological effects and are also relevant in many other fields, for instance, in food research. Our aim was to investigate whether a commercially available enzymatic test kit developed for the determination of FFAs in human serum is also suitable to determine different physiological and nonphysiological FFAs and to which extent the impact on the sensitivities (i.e., the accuracy by which a given FFA can be determined) differ. It will be shown that the chain length as well as the double bond content has a significant impact on the sensitivity by which a given FFA can be determined. For instance, palmitic acid (16:0) is determined with an approximately 20 times higher sensitivity in comparison to docosahexaenoic acid (22:6n-3). All data were obtained by measuring the concentrations of the FFAs by gas chromatography, and selected FFAs were also determined in a complex matrix of human serum. It is concluded that this kit is not useful if major alterations of the FFA composition of a complex mixture are expected because the individual FFAs are not detected with the same sensitivities: the concentrations of polyunsaturated FFA determined by this kit are wrong. Figure
The used enzymatic kit detects different free fatty acids with significantly different sensitivities: the number of carbon atoms and the number of double bonds massively contribute to these differences相似文献
Here we describe an integrative protocol for metabolite extraction and the measurement of three cellular constituents, chlorophyll
a, total protein, and glycogen from the same small volume of cyanobacterial cultures that can be used as alternative sample
amount parameters for data adjustment in comparative metabolome studies. We conducted recovery experiments to assess the robustness
and reproducibility of the measurements obtained for the cellular constituents. Also, we have chosen three profile-intrinsic
parameters derived from gas chromatography-mass spectrometry (GC/MS) data in order to test their utility for spectral data
adjustment. To demonstrate the relevance of these six parameters, we analyzed three cyanobacteria with greatly different morphologies,
comprising a unicellular, a filamentous, and a filamentous biofilm-forming strain. Comparative analysis of GC/MS data from
cultures grown under standardized conditions indicated that adjustment of the corresponding metabolite profiles by any of
the measured cellular constituents or chosen intrinsic parameters led to similar results with respect to sample cohesion and
strain separation. Twenty-one metabolites significantly enriched for the carbohydrate and amine superclasses are mainly responsible
for strain separation, with a majority of the remaining metabolites contributing to sample group cohesion. Therefore, we conclude
that any of the parameters tested in this study can be used for spectral data adjustment of cyanobacterial strains grown under
controlled conditions. However, their use for the differentiation between different stresses or physiological states within
a strain remains to be shown. Interestingly, both the adjustment approaches and statistical tests applied effected the detection
of metabolic differences and their patterns among the analyzed strains. 相似文献
Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method which can be applied to virtually all natural products including oligosaccharides. Unfortunately, however, the unequivocal identification of a TLC spot is normally difficult. Fortunately, this problem can be minimized when mass spectrometry (MS) such as matrix-assisted laser desorption and ionization time-of-flight is used to identify the TLC spots. This work is dedicated to the TLC/MS analysis of oligosaccharides derived from native chondroitin sulfate and hyaluronan. We will show that the thickness of the silica gel layer (200 versus 100 µm) has a tremendous influence on the quality of the mass spectra: a reduced silica gel thickness enhances the spectral quality and, in particular, improves the achievable signal-to-noise ratio. Additionally, unwanted formylation of the GAG oligosaccharides (which occurs due to the high moiety of formic acid in the mobile phase) can also be minimized if MS-grade HPTLC plates are used.
A HPLC and a HPTLC-densitometric method were developed for the quantification of prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol the major chromone glucosides in the roots of Saposhnikovia divaricata. The validation of both methods resulted in comparable parameters regarding stability, specificity, linearity, robustness, precision and recovery, whereas complementary advantages were obtained concerning LOD and LOQ. The HPTLC-based densitometry revealed a lower LOD (1.11 versus 4.37 μg mL−1 in HPLC) and LOQ (3.36 versus 13.24 μg mL−1 in HPLC) for prim-O-glucosylcimifugin, whereas the HPLC resulted in a lower LOD (1.00 versus 4.10 μg mL−1 in HPTLC-densitometry) and LOQ (3.04 versus 12.46 μg mL−1 in HPTLC-densitometry) for 4′-O-β-d-glucosyl-5-O-methylvisamminol. Both methods revealed nearly matching contents of the chromones after analysis of different commercially available batches of Saposhnikoviae divaricatae radix with a total content for both chromone glycosides in the range from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC and between 0.34 ± 0.011 and 0.61 ± 0.009 % determined by HPTLC. The plant material cultivated in Germany showed a very similar content and ratio of both chromone glucosides in comparison to the standard batches originating from China.
MALDI-TOF MS is traditionally used for “proteomics”, but is also a useful tool for lipid analysis. Depending on the applied matrix, however, some lipid classes are more sensitively detected than other ones and this may even lead to suppression effects if complex mixtures are analyzed. Therefore, a previous separation into the individual lipid classes is necessary. Using artificial lipid mixtures or easily available tissue extracts, it has been already shown that HPTLC-(High Performance Thin-Layer Chromatography)-separated lipids can be conveniently analyzed by MALDI-TOF MS directly on the TLC plate. Here we present an initial TLC-MALDI study of the lipid composition of ovine mesenchymal stem cells. Due to the complex composition of these cells, data are also compared to lipids extracted from human erythrocytes. It will be shown that even very minor lipid classes can be easily detected and with much higher sensitivity than by common staining protocols. Additionally, MS images of the developed TLC plates will be shown and potential applications, new methods of data analysis as well as problems discussed.
Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is "proteomics," there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that "clean" spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract. 相似文献
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization MS technique providing only minor fragmentation of the analyte. Therefore, the method is basically suitable for mixture analysis, although the ion yields strongly depend on the basicity/acidity of the analyte in relation to the applied matrix. Accordingly, less sensitively detectable compounds may be suppressed by more sensitively detectable compounds. Thus, separation of the mixture into the individual compounds is normally indispensable. This paper demonstrates the capabilities and limitations of a direct, simple, and inexpensive MALDI-high-performance thin-layer chromatography (HPTLC) coupling for the analysis of a crude lipid extract from porcine brain. Brain lipids were chosen because they represent a rather complex mixture and are of currently significant research interest. It was found that normal-phase HPTLC-separated lipids can be easily characterized by direct MALDI-TOF-MS analysis with sufficient resolution to allow the assignment of virtually all lipid classes, even rather minor species such as phosphorylated phosphoinositides or complex glycolipids as gangliosides. Advantages and disadvantages of this approach are discussed. 相似文献
A HPLC and a HPTLC-densitometric method were developed for the quantification of prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol the major chromone glucosides in the roots of Saposhnikovia divaricata. The validation of both methods resulted in comparable parameters regarding stability, specificity, linearity, robustness, precision and recovery, whereas complementary advantages were obtained concerning LOD and LOQ. The HPTLC-based densitometry revealed a lower LOD (1.11 versus 4.37 μg mL?1 in HPLC) and LOQ (3.36 versus 13.24 μg mL?1 in HPLC) for prim-O-glucosylcimifugin, whereas the HPLC resulted in a lower LOD (1.00 versus 4.10 μg mL?1 in HPTLC-densitometry) and LOQ (3.04 versus 12.46 μg mL?1 in HPTLC-densitometry) for 4′-O-β-d-glucosyl-5-O-methylvisamminol. Both methods revealed nearly matching contents of the chromones after analysis of different commercially available batches of Saposhnikoviae divaricatae radix with a total content for both chromone glycosides in the range from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC and between 0.34 ± 0.011 and 0.61 ± 0.009 % determined by HPTLC. The plant material cultivated in Germany showed a very similar content and ratio of both chromone glucosides in comparison to the standard batches originating from China. 相似文献