Porous Graphitic Carbon Column Research Articles

Correction: Detection of lard adulteration in 3 kinds of vegetable oils by liquid chromatography–mass spectrometry with porous graphite carbon column

Correction: Detection of lard adulteration in 3 kinds of vegetable oils by liquid chromatography–mass spectrometry with porous graphite carbon column

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

IntroductionUntargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose.ObjectivesThe current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices.MethodsSeparation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others.ResultsAll three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column.ConclusionThe results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix.Graphical abstract

Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn.

Despite their many important physiological functions, past work on the diverse sequences of human milk oligosaccharides (HMOs) has been focused mainly on the highly abundant HMOs with a relatively low degree of polymerization (DP) due to the lack of efficient methods for separation/purification and high-sensitivity sequencing of large-sized HMOs with DP ≥ 10. Here we established an ultrahigh-temperature preparative HPLC based on a porous graphitized carbon column at up to 145 °C to overcome the anomeric α/β splitting problem and developed further the negative-ion ESI-CID-MS/MS into multistage MSn using a combined product-ion scanning of singly charged molecular ion and doubly charged fragment ion of the branching Gal and adjacent GlcNAc residues. The separation and sequencing method allows efficient separation of a neutral fraction with DP ≥ 10 into 70 components, among which 17 isomeric difucosylated nona- and decasaccharides were further purified and sequenced. As a result, novel branched difucosyl heptaose and octaose backbones were unambiguously identified in addition to the conventional linear and branched octaose backbones. The novel structures of difucosylated DF-novo-heptaose, DF-novo-LNO I, and DF-novo-LNnO I were corroborated by NMR. The various fucose-containing Lewis epitopes identified on different backbones were confirmed by oligosaccharide microarray analysis.

Detection of lard adulteration in 3 kinds of vegetable oils by liquid chromatography-mass spectrometry with porous graphite carbon column.

Liquid chromatography‒mass spectrometry employing porous graphite carbon columns and an n-octane-isopropanol mobile phase was utilized for the separation of triacylglycerols (TAGs) in various edible oils, aiming to identify lard adulteration in soybean, corn, and sunflower seed oils. Experiments were conducted using a Hypercarb column (2.1mm × 100mm, 5µm) and an n-octane-isopropanol (70:30, V/V) mobile phase at a flow rate of 0.25mL· min-1 and a column temperature of 60°C. Detection was achieved through atmospheric pressure chemical ionization-mass spectrometry. Analysis of diverse edible oil samples revealed that oils of the same type shared similar TAG compositions, while different types exhibited distinct TAG profiles. Distinct variations in triglyceride composition were observed across different edible oils. Based on liquid chromatography‒mass spectrometry analysis, the characteristic component 1-stearic acid-2-palmitic acid-3-oleic acid glyceride (SPO), which may also include PSO, was identified in lard through principal component analysis and orthogonal partial least squares discriminant analysis. This component served as a marker for detecting as low as 0.1% lard adulteration in soybean, corn, and sunflower seed oils. The technique offers a precise and effective approach for the identification of lard adulteration in these edible oils.

Ultrahigh-Performance Supercritical Fluid Chromatography-Multimodal Ionization-Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts.

Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

Development of a column switching for direct online enrichment and separation of polar and nonpolar analytes from aqueous matrices

Trace substances in surface waters may threaten health and pose a risk for the aquatic environment. Moreover, separation and detection by instrumental analysis is challenging due to the low concentration and the wide range of polarities. Separation of polar and nonpolar analytes can be achieved by using stationary phases with different selectivity. Lower limits of detection of trace substances can be obtained by offline enrichment on solid phase materials. However, these practices require substantial effort and are time consuming and costly. Therefore, in this study, a column switching was developed to enrich and separate both polar and nonpolar analytes by an on-column large volume injection of aqueous samples. The column switching can significantly reduce the effort and time for analyzing trace substances without compromising on separation and detection. A reversed phase (RP) column is used to trap the nonpolar analytes. The polar analytes are enriched on a porous graphitized carbon column (PGC) coupled serially behind the RP column. A novel valve switching system is implemented to enable elution of the nonpolar analytes from the RP column and, subsequently, elution of polar analytes from the PGC column and separation on a hydrophilic interaction liquid chromatography (HILIC) column. To enable separation of polar analytes dissolved in an aqueous matrix by HILIC, the water plug that is flushed from the PGC column is diluted by dosing organic solvent directly upstream of the HILIC column. The developed method was tested by applying target analysis and non-target screening, highlighting the advantage to effectively separate and detect both polar and nonpolar compounds in a single chromatographic run. In the target analysis, the analytes, with a logD at pH 3 ranging from -2.8 to + 4.5, could be enriched and separated. Besides the 965 features in the RP phase, 572 features from real wastewater were observed in the HILIC phase which would otherwise elute in the void time in conventional one-dimensional RP methods.

Stable Isotope Dilution LC/ESI-SRM/MS Analysis for Highly Polar Bioactive Dipeptides Found in Fermented Brown Rice Product using a Porous Graphitic Carbon Column

In this study, a validated analytical method based on liquid chromatography/electrospray ionization-selected reaction monitoring/mass spectrometry (LC/ESI-SRM/MS) was developed for dipeptides in a functional food, fermented brown rice and rice bran with Aspergillus oryzae (FBRA). The aim of this study was to screen and quantify dipeptides in the water-soluble fraction of FBRA. There are few studies focusing on dipeptides in FBRA, even though FBRA is expected to contain bioactive peptides: FBRA or its aqueous extract has shown variety of biological activities. An anti-hypertensive dipeptide was found in similar rice bran products digested by thermolysin. Dipeptides are the smallest class of peptides, and many show a variety of biological activities. Some bioactive peptides are widely found in fermented foods. However, because dipeptides are generally too polar to be retained on versatile LC columns, such as octadecylsilyl columns, dipeptides have been often overlooked and not quantified because of the analytical difficulties. The LC was performed using a porous graphitic carbon column with the mobile phases of 0.1% formic acid/0.1% trifluoroacetic acid/1% tetrahydrofuran in water and acetonitrile. The stable isotope-labeled internal standards and SRM for dipeptides made it possible to develop a validated method. Five dipeptides were identified and quantified in FBRA in much higher concentrations than related materials (malted brown rice, rice bran, brown rice, and white rice) as follows: Ile-Arg, 82.1 µg/g; Ala-Phe, 27.8 µg/g; Ala-Tyr, 31.6 µg/g; Val-Phe, 46.3 µg/g; and Ile-Tyr, 49.9 µg/g. This method was simple and robust and would be applicable for other functional foods.Graphical

Application of a porous graphitic carbon column to carbon and nitrogen isotope analysis of underivatized individual amino acids using high-performance liquid chromatography coupled with elemental analyzer/isotope ratio mass spectrometry.

Isolation of underivatized amino acids (AAs) using high-performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ13 C) and nitrogen isotope (δ15 N) analyses of AAs because of the high analytical precision and for performing dual-isotope analysis. However, some AAs in natural samples, especially small, hydrophilic AAs, are not suitably separated using reversed-phase columns (e.g., C18) and ion-exchange columns (e.g., Primesep A). We developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ13 C and δ15 N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ13 C and δ15 N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel (Scomber australasicus). Chromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ13 C and δ15 N values of AA standards remained unchanged during the whole experimental procedure. The δ13 C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ13 C analysis. The good separation performance of hydrophilic AAs and the reliability of δ13 C and δ15 N analyses of individual AAs using the porous graphite column offer a significant advantage over conventional settings. We suggest that, in the future, the HPLC × EA/IRMS method can be used for reliable δ13 C and δ15 N analyses of AAs in natural samples.

Evaluation of PGC Stationary Phases Under High-Temperature LC Conditions for the Analysis of Parabens in Food Samples

Porous graphitic carbon (PGC) columns for liquid chromatography (LC) represent an alternative to octadecyl‑bonded silica columns for the separation of both polar and nonpolar molecules. This is accomplished by exploiting the polarizability of the stationary phase interacting with the functional groups of the analytes. However, the elution of nonpolar compounds requires a high percentage of organic solvent, losing the intrinsic advantage of reversed‑phase aqueous separations. In this article, we aimed to exploit an additional advantage of such columns, viz. the resistance at high temperatures. Superheated water was employed as the mobile phase, taking advantage of the decrease in water dielectric constant by increasing the temperature. In this context, our goal was to minimize the percentage of organic solvent utilizing high temperatures (up to 250 °C) to achieve fast and “green” separations. The new developed high-temperature LC instrument was applied to the analysis of parabens in food samples.

In-depth characterization of 1,4-butanediol diglycidyl ether substituted hyaluronic acid hydrogels

BDDE substituted HA hydrogels remain the most commonly used HA product in the biomedical field. The physical and biochemical properties of the hydrogels are dependent on the degree of modification and substitution patterns/positions, thus, characterizing their fine structure is of great importance for quality assurance. In this study, we developed novel LC-MS methods for accurate determination of MoD as well as in-depth characterization of the linkage network. Fragments resulted from enzymatic depolymerization were resolved by a porous graphitic carbon column followed by online tandem-MS for determining the modification site/residue. With high-resolution separation, two types of previously unknown structures were detected in the cross-linked fragments of 2-B-2 and 4-B-2. Based on the feature of resistance to NaBH4 reduction, these structures contain a GlcNAc residue modified at OH1. This special sugar unit likely derived from reducing end of the native polysaccharide could be a signature to discriminate subtle batch to batch variations.

Selective determination of formaldehyde by high-performance liquid chromatography with porous graphitic carbon column using N,N′-bis(9-anthrylmethyl)propane-1,3-diamine as derivatizing reagent

Aromatic compounds containing two secondary amino groups were designed and prepared as new derivatizing reagents for aldehydes. One of them, N,N'-bis(9-anthrylmethyl)propane-1,3-diamine (APD), could achieve selective determination of formaldehyde (FA) on a porous graphitic carbon (PGC) column using xylenes, chlorobenzene, and 1-chloronaphthalene as mobile phases by high-performance liquid chromatography (HPLC). The APD-FA derivative was eluted from the PGC column, while the other APD-aldehyde derivatives remained on the column during the HPLC measurements. This specific elution was not observed using mobile phases such as acetonitrile, 1,4-dioxane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, chloroform, benzene, toluene, benzyl alcohol, 2-ethyl-1-hexanol, and pyridine. The APD-FA derivative had a six-membered ring of two tertiary amines identified using 1H NMR spectroscopy. When the π-π interaction of the solvent molecule of the mobile phase with PGC overcame that between the APD-FA derivative and PGC, the APD-FA derivative could be eluted from the column. The best resolution between the peak of the APD-FA derivative and that of free APD was observed when using o-xylene. The optimum derivatization and the HPLC conditions for selective HPLC determination of FA were to conduct the derivatization of FA by heating in an aqueous phase with APD in o-xylene at 100°C. In this method, FA could be derivatized with APD at a mildly neutral pH of 6.7, unlike the low pH required for the derivatization of aldehydes with 2,4-dinitrophenylhydrazine (DNPH), which is commonly used for the derivatization of aldehydes. The detection and quantification limits of FA were 0.8 and 3.5ngmL-1 in this HPLC method with fluorescent detection, respectively. This selective HPLC method could be applied to the determination of FA in various water samples. It was found that only APD among the derivatizing reagents containing two secondary diamines was useful for the selective determination of FA.

Analysis of the solvent strength parameter (linear solvent strength model) for isocratic separations in reversed-phase liquid chromatography

The solvent strength parameter (slope) and intercept (log kw) of the linear solvent strength model are determined for methanol-water (17 columns), acetonitrile-water (15 columns), acetone-water (7 columns), tetrahydrofuran-water (6 columns), and 2-propanol-water (4 columns) for varied compounds. It is shown that the linear region of the plots of the retention factor (log k) against the volume fraction of organic solvent (ϕ) is largely a system property independent of solute type for compounds with accessible retention factors. Solvent strength parameters for methanol (3.12 ± 0.12), acetonitrile (2.78 ± 0.18), acetone (2.71 ± 0.11), and tetrahydrofuran (2.95 ± 0.24) are assigned for siloxane-bonded silica and porous graphitic carbon columns as an average of the compound-specific experimental values. Statistical analysis indicates that the compound-specific solvent strength parameters are column dependent and limited as a general solvent property. Evaluation of the column-specific solvent strength parameters provides further evidence that the average solvent strength parameter is column dependent. The column-specific solvent strength values are compared with the hypothetical water-organic solvent distribution systems using the system constants of the solvation parameter models as variables confirming the column dependence of the solvent strength parameter. The column-specific solvent strength parameters can be predicted by the solvation parameter model with a typical deviation of about 0.12 over a range of 1.69 to 6.33 for the experimentally determined values. The intercept of the linear solvent strength model is shown to be both column and organic solvent dependent. Compound column-specific values are adequately described by the solvation parameter model. Log kw cannot be recommended as a descriptor of solute properties since it has no clear connection to a real distribution system.

Characterization of Humulus lupulus glycosides with porous graphitic carbon and sequential high performance liquid chromatography quadrupole time-of-flight mass spectrometry and high performance liquid chromatography fractionation

Monoterpenes contribute to the characteristic aroma of several hop varieties and may occur as nonvolatile glycosides. Upon hydrolysis, the volatile terpenes are released from the glycoside precursors. Little is known, however, about the glycoside composition of hops. Seven pentose-hexose monoterpene alcohol glycosides from dried Humulus lupulus L. cv. Citra cones were isolated using high performance liquid chromatography separation and fractionation on a reverse phase phenyl-hexyl column. Further evaluation of each isolated fraction through HPLC qTOF MS with porous graphitic carbon (PGC) showed that the seven isolated monoterpenyl glycoside fractions could be further resolved into 20 isomers. Isolation on phenyl-hexyl followed by separation on PGC was needed to distinguish each isomer present. Additionally, the hop cones were screened for potential aroma glycosides. Using the PGC column combined with a database of over 900 potential glycosides, the identification of 21 additional monoterpene-polyol, norisoprenoid, volatile phenol, and aliphatic alcohol glycosides is reported.

Risk assessment for nitrosated pharmaceuticals: A future perspective in drug development.

Since June 2018, thousands of drug products from around the world had to be recalled due to the unexpected presence of nitrosamines (NAs). Starting with the pharmaceutical group of sartans, antidiabetic drugs, antihistamines, and antibiotics also became the subject of investigation. The occurrence of NAs has shown that pharmaceutical companies and regulatory agencies did not focus on these substances in the past during drug development. In this study, we incorporated a nitrosation assay procedure into high-resolution supercritical fluid chromatography (SFC)-mass spectrometry screening to test the potential of direct nitrosation of active pharmaceutical ingredients (APIs). The forced degradation study was performed with a four-fold molar excess of sodium nitrite, relative to the drug substance, at pH 3-4 for 4 h at 37°C. Chromatographic separation was performed on a porous graphitic carbon column by SFC. The mass analysis then focused on direct N-nitrosation or N-nitroso compounds (NOCs) formed after dealkylation. Substances (n = 67) from various pharmaceutical classes were evaluated and 49.3% of them formed NOCs, of which 21.2% have not yet been reported in the literature. In addition, for two APIs, which are known to form an unidentified NOC, the structure could be identified. A few substances also showed multiple NOCs and even N,N'-dinitroso-species. As NAs are carcinogens, they have to be eliminated or at least limited to prevent cancer in patients, who rely on these drugs. This study contributes a procedure that can be implemented in preapproval drug development and postapproval risk assessment to prevent unexpected findings in the future.

UPLC-ESI-MS analysis of salts using a porous graphitic carbon column

A method for the identification and quantification of salts relevant in the analysis of explosives was developed for a porous graphitic carbon (PGC) UPLC/HPLC column combined with ESI-MS detection. The presented method covers a wide range of energetic salts and provide a valuable analytical tool for security, forensic as well as environmental monitoring. Both anionic and cationic species can be identified and quantified in a single chromatographic run. Afterward, analytes can be indubitably identified by LC-MS equipped with an atmospheric pressure ionisation (API) interface. Since the retention mechanism of PGC is due to combination of different interactions, a tunable retention can be obtained by adjustments of the composition of the mobile phase. The use of LC-MS in combination with PGC is an improved alternative to the existing analytical methods based on silica derivatised reversed phase (RP) columns. A high percentage of ammonia in the mobile phase allowed elution of the most highly retained analytes. The effects of different solvents and their combinations and mobile phase additives were evaluated. The developed method improves the energetic salts detection in terms of sensitivity and selectivity. The effectiveness of the method is demonstrated by the analysis of a reference mixture of anions and cations.

Improving temperature gradient interaction chromatography of polyolefins by simultaneous use of different stationary phases

Temperature gradient interaction chromatography (TGIC) at high temperatures is a powerful method for the chemical composition separation of polyolefins. TGIC is a two-step process where the sample is crystallized on the stationary phase at low temperature followed by the elution of the sample components using a temperature gradient towards high temperatures. For TGIC typically a porous graphitic carbon (PGC) stationary phase is used. The separation mechanism is based on crystallization and adsorption/desorption phenomena and it has been shown that co-crystallization and co-adsorption may affect the separation.The present study reports on the simultaneous use of a non-adsorptive and an adsorptive stationary phase (column) in series to utilize both crystallization and adsorption for improved separation in TGIC. A silica column is used as the non-adsorptive support to allow for the crystallization of the polyolefin sample in the absence of an adsorptive force followed by the typical PGC column for adsorption/desorption. Accordingly, the loci of crystallization and adsorption/desorption are well separated from each other and can be adjusted independently.This novel column setup allows the sample to be introduced slowly onto the second (adsorptive) column eliminating possible co-adsorption and poor selectivity. Low molar mass polyethylene comprising of oligomers with approximately C30C130 was used to illustrate the importance of a non-adsorptive column for improved separation. Utilizing a non-adsorptive silica column allows for higher dynamic flow rates during crystallization, which improves separation. Shorter adsorptive columns are found to be more efficient in this experimental protocol as compared to standard TGIC experiments. Smaller PGC column sizes result in reduced longitudinal and Eddy diffusion and, hence, higher resolution of low and high molar mass polyolefins.

Enhancement of teprenone isomer separation by subcritical fluid chromatography using porous graphitic carbon column

Teprenone is a therapeutic anti-ulcer agent developed in Japan. As described in the Japanese Pharmacopoeia (JP) 17th Edition, gas chromatography/hydrogen flame ionization detection (GC/FID) and high-performance liquid chromatography/ultraviolet detection (HPLC/UV) have been used for the assay of active pharmaceutical ingredients (APIs) and teprenone capsules, respectively. The critical aspect of the assay is a separation of the structural isomers (mono-cis and all-trans) of teprenone. Herein, we propose an improved quantitative method for the quality control of teprenone in APIs and capsules via subcritical fluid chromatography/photo diode array detection (SubFC/PDA) using a porous graphitic carbon column. SubFC conditions, i.e., type and content of the organic modifier in the mobile phase, column temperature, injection volume, and flow rate, were optimized. The developed SubFC/PDA method was validated according to ICH guidelines Q2(R1) in terms of accuracy, precision (repeatability and intermediate precision), specificity, linearity, quantification range, robustness, and stability. Comparison of SubFC/PDA method with the GC/FID or HPLC/UV method (described in JP) revealed that the SubFC/PDA method gave better resolution and run time than the JP methods. The developed SubFC/PDA method is expected to be useful for pharmaceutical analysis or quality control of teprenone isomers.

In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans.

Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.

Optimized method for analysis of ethanolamines, hydrolysis products of nitrogen mustards, from urine samples using LC-MS/MS

Highly polar ethanolamines (EAs), excreted in urine, are hydrolysis products of nitrogen mustards (NMs), which are prohibited by the Chemical Weapons Convention (CWC). The methods established for biological matrices are essential for verification analysis of the CWC related chemicals. This paper describes a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed for qualitative and quantitative analysis of EAs, N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA) and triethanolamine (TEAOH) from urine samples. After optimization of sample preparation and chromatographic conditions, the method was fully validated. Silica solid-phase extraction (SPE) cartridges and a porous graphite carbon (PGC) column were selected for validation studies. The method is linear from 5 to 500, 0.5 to 250, and 0.5 to 500 ng/mL for TEAOH, EDEA, and MDEA, respectively. It is also precise and accurate. A minimum sample amount of 0.5 mL urine was used. The limit of quantification using this approach was 0.4, 5.5, and 6.3 ng/mL for MDEA, EDEA and TEAOH, respectively. The combination of the PGC column and high pH eluents in analysis retained and separated the studied EAs. Retention times were 2.11, 2.56 and 2.98 min for MDEA, EDEA and TEAOH, respectively. The method is applicable for verification analysis of the CWC.

Total Retention Liquid Chromatography-Mass Spectrometry to Achieve Maximum Protein Sequence Coverage.

Peptide identification by liquid chromatography-mass spectrometry (LC-MS) requires retention and elution of peptides from the LC column. Although medium and hydrophobic peptides are readily retained by the C18 columns that are commonly used in proteomics, short and hydrophilic peptides are not retained nor measured by MS due to their elution in the void volume after sample injection. These nonretained peptides can possess important post-translational modifications, such as glycosylation or phosphorylation. We describe a total retention LC-MS method that employs a reverse phase C18 column and porous graphitic carbon (PGC) column to retain both hydrophobic and hydrophilic peptides for LC-MS analysis. Our setup uses a single valve with a trapping column and two LC pumps run at low microliter/minute flow rates to deliver separate gradients to parallel capillary C18 and PGC columns. Our capillary LC system balances the need for high sensitivity with ease of implementation as compared to other 2D LC systems that use nanocolumns with multiple trapping columns and multiport valves. We demonstrate the utility of the method identifying hydrophilic peptides that went undetected when only a C18 nanocolumn was used. These missed hydrophilic peptides include tripeptides and N-glycosylated species.