Online 2D-LC for Complex N-Glycan Analysis from Biopharmaceuticals.

EPO has a complex glycosylation pattern with differently branched and charged glycans. A combination of hydrophilic interaction chromatography (HILIC) with weak anion exchange chromatography (WAX) enables highly orthogonal separation. Comprehensive 2D-LC analysis with HILIC in the first and WAX in the second dimension provides high resolution 2D chromatography together with simultaneous charge profiling. Meanwhile, multiple heart-cutting 2D-LC analysis combining WAX and HILIC separation provides a flexible alternative whereby the user can select multiple peaks to be analyzed in the second dimension and, moreover, run longer gradients in the second dimension.

Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism.

Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.

Non-destructive plasticizer screening using a direct inlet probe-atmospheric pressure chemical ionization source and ion trap mass spectrometry.

RATIONALE: In recent years, several ambient ionization techniques, where solid and/or liquid samples are brought directly into the ion source without any sample preparation and chromatographic separation, have been introduced for mass spectrometric (MS) analyses. Using the direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI)-MS and DIP-APCI-MS(n) methods presented here, a non-destructive screening analysis for plasticizers directly from plastic articles can be performed. METHODS: The DIP-APCI ion source developed in our laboratory uses a temperature-programmed push rod to introduce solid or liquid samples into a homemade APCI ion source. The DIP-APCI ion source was coupled to an ion trap (IT) mass spectrometer and selected source parameters were optimized. To enable a screening analysis for plasticizers, standards substances of several phthalates and other plasticizers were analyzed and their fragmentation behavior during collision-induced dissociation (CID) was studied. RESULTS: Using DIP-APCI-ITMS, plasticizers can be detected directly from plastic articles and identification is possible through MS(n) experiments. For example, the isomeric phthalates di(2-ethylhexyl) phthalate and di-n-octyl phthalate can be differentiated according to their fragmentation behavior. CONCLUSIONS: There are several advantages of the DIP-APCI source in comparison to many other ambient desorption ion sources: (i) well-defined gas phase matrix, (ii) precisely adjustable reagent gases (e.g. O2 for negative APCI), (iii) well-defined probe temperature, and (iv) fully automated operation.

Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection.

There has been evidence linking elevated polyamines (PAs) and acetylpolamines (AcPAs) level and cancer. So the simultaneous analysis of these compounds has become important task for cancer diagnosis and antitumor drug monitoring. A simple, fast and inexpensive CZE-LIF method has been developed for the determination of cadaverine (CAD), putrescine (PUT), spermine (SPM), spermidine (SPD), acetylspermine (ASPM), and acetylspermidine (ASPD) in human urine using 4-chloro-7-nitro-2,1,3-benzooxadiazole as a fluorescent reagent. Labeling reaction conditions were systematically investigated and were found to be 20 mM borate buffer at pH 7.4, labeling reaction time, and temperature were 10 min and 70°C, respectively. Under these optimized conditions the four PAs, two AcPAs and the internal standard were separated in 6 min. An Exactive-MS with an ESI source was used for identification of the bis-derivative of the ASPM. The method was validated in term of linearity, LODs, repeatability, intra- and interday assays, recovery, and selectivity. The LODs for CAD, PUT, SPM, SPD, ASPM, and ASPD were found to be 7.6, 10.0, 9.0, 8.8,7.8, and 3.3 nM, respectively. The method was successfully applied for the analysis of PAs and AcPAs in healthy human urine samples.

Quantification of coumarin in cinnamon and woodruff beverages using DIP-APCI-MS and LC-MS.

The use of the direct inlet probe-atmospheric-pressure chemical ionization (DIP-APCI) ion source developed in our laboratory coupled to a high resolution Q-TOF MS for the quantitative analysis of coumarin in different cinnamon samples was demonstrated in this study. Extraction of coumarin from various cinnamon samples was followed by DIP-APCI-mass spectrometry (MS) and liquid chromatography (LC)-MS analysis. For quantification, an external calibration with and without the use of stable isotope-labeled coumarin as internal standard was compared. The results obtained by DIP-APCI-MS and LC-MS were in good agreement. Even without the use of an internal standard satisfying linearity (R(2) > 0.997), recovery (94-104% for spiking levels between 100 and 5,000 mg/kg) and intra- and interday repeatability (2.2-13.8%RSD) was demonstrated using DIP-APCI-MS. To reduce the number of samples requiring quantitative analysis, the possibility of semi-quantitative screening of coumarin directly from powdered cinnamon using DIP-APCI-MS was shown. The analysis of woodruff-flavored beverages and cinnamon-flavored chewing gum by DIP-APCI-MS resulted in the formation of an artifact interfering with coumarin detection. As with other ambient ionization methods, special attention has to be paid to possible spectral interferences due to isobaric substances present in the sample matrix or formed from matrix components after ionization. The temperature-programmed vaporization in DIP-APCI-MS combined with the use of stable isotope-labeled coumarin as internal standard helped in recognizing this interference.

Development, optimization, and use of an APCI source with temperature-controlled vaporization of solid and liquid samples.

A trend is observed in mass spectrometry, in which solid samples without prior dissolution and chromatographic separation are brought directly into the ion source and are ionized, e.g., by corona discharge (Atmospheric Solids Analysis Probe) or plasma (Direct Analysis in Real Time). The Direct Inlet Probe-atmospheric-pressure chemical ionization (APCI) ion source presented here, which was coupled to a high-resolution quadrupole time-of-flight-mass spectrometer, differs from most of the other ion sources in having temperature-programmed heating of the sample. The resulting possibility to reduce ion suppression and ion-molecule reactions in the ion source was shown by the separation of two fatty acid methyl esters as a result of their boiling point difference. Using caffeine as sample, certain source parameters such as the auxiliary gas flow, the drying gas flow, and the position of the probe tip in the ion source were optimized. The ability to perform quantitative analyses was shown by the linear concentration response (R(2) = 0.9984) observed when analyzing different caffeine concentrations. An extract of a Chinese medicinal herb was used to examine the reproducibility (relative standard deviations of the most abundant m/z signals were ≤8.1 %). It was also possible to distinguish milled samples of Radix Angelicae sinensis and Radix Angelicae gigas from each other and to identify the coumarins they contain without sample preparation. Supplying synthetic air instead of nitrogen to the ion source makes APCI in the negative mode possible as well; this was proven by the analysis of n-nonyl-ß-D-maltoside.

High-performance liquid chromatography-mass spectrometry profiling of phenolic compounds for evaluation of olive oil bitterness and pungency.

Bitterness and pungency are important parameters for olive oil quality. Therefore, two instrumental methods for evaluation of these taste attributes were developed. The first one is based on the photometric measurement of total phenolic compounds content, whereas the second one is based on the semiquantitative evaluation of hydrophilic compounds by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Evaluation of total phenolic compounds content was performed by a modified method for the determination of the K(225) value using a more specific detection based on the pH value dependency of absorbance coefficients of phenols at λ = 274 nm. The latter method was not suitable for correct prediction, because no significant correlation between bitterness/pungency and total phenolic compounds content could be found. For the second method, areas of 25 peaks detected in 54 olive oil samples by a HPLC-MS profiling method were correlated with the bitterness and pungency by partial least-squares regression. Six compounds (oleuropein aglycon, ligstroside aglycon, decarboxymethyl oleuropein aglycon, decarboxymethyl ligstroside aglycon, elenolic acid, and elenolic acid methyl ester) show high correlations to bitterness and pungency. The computed model using these six compounds was able to predict bitterness and pungency of olive oil in the error margin of the sensory evaluation (±0.5) for most of the samples.