Reversed-phase LC Research Articles

Development and application of retention time prediction models in the suspect and non-target screening of emerging contaminants

Hydrophilic interaction liquid chromatography (HILIC) and reversed phase LC (RPLC) coupled to high resolution mass spectrometry (HRMS) are widely used for the identification of suspects and unknown compounds in the environment. For the identification of unknowns, apart from mass accuracy and isotopic fitting, retention time (tR) and MS/MS spectra evaluation is required. In this context, a novel comprehensive workflow was developed to study the tR behavior of large groups of emerging contaminants using Quantitative Structure-Retention Relationships (QSRR). 682 compounds were analyzed by HILIC-HRMS in positive Electrospray Ionization mode (ESI). Moreover, an extensive dataset was built for RPLC-HRMS including 1830 and 308 compounds for positive and negative ESI, respectively. Support Vector Machines (SVM) was used to model the tR data. The applicability domains of the models were studied by Monte Carlo Sampling (MCS) methods. The MCS method was also used to calculate the acceptable error windows for the predicted tR from various LC conditions. This paper provides validated models for predicting tR in HILIC/RPLC-HRMS platforms to facilitate identification of new emerging contaminants by suspect and non-target HRMS screening, and were applied for the identification of transformation products (TPs) of emerging contaminants and biocides in wastewater and sludge.

Comprehensive Three-Dimensional LC × LC × Ion Mobility Spectrometry Separation Combined with High-Resolution MS for the Analysis of Complex Samples.

Comprehensive two-dimensional liquid chromatography (LC × LC) and ion mobility spectrometry-mass spectrometry (IMS-MS) are increasingly being used to address challenges associated with the analysis of highly complex samples. In this work, we evaluate the potential of the combination of these techniques in the form of a comprehensive three-dimensional LC × LC × IMS separation system. As application, hydrophilic interaction chromatography (HILIC) × reversed phase LC (RP-LC) × IMS-high-resolution MS (HR-MS) was used to analyze a range of phenolic compounds, including hydrolyzable and condensed tannins, flavonoids, and phenolic acids in several natural products. A protocol for the extraction and visualization of the four-dimensional data obtained using this approach was developed. We show that the combination of HILIC, RP-LC, and IMS offers excellent separation of complex phenolic samples in three dimensions. Benefits associated with the incorporation of IMS include improved MS sensitivity and mass-spectral data quality. IMS also provided separation of trimeric procyanidin isomeric species that could not be differentiated by HILIC × RP-LC or HR-MS. On the traveling wave IMS (TWIMS) system used here, both IMS separation performance and the extent of second dimension (2D) undersampling depend on the upper mass scan limit, which might present a limitation for the analysis of larger molecular ions. The performance of the LC × LC × IMS system was characterized in terms of practical peak capacity and separation power, using established theory and taking undersampling and orthogonality into account. An average increase in separation performance by a factor of 13 was found for the samples analyzed here when IMS was incorporated into the HILIC × RP-LC-MS workflow.

Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis

Method development and optimisation for comprehensive two-dimensional liquid chromatography (LC × LC) is complex, since this involves the intricate relationships between a large number of experimental parameters with the aim of achieving three conflicting goals: maximising separation performance (peak capacity), minimising analysis time and minimising dilution. This is especially true for the on-line combination of hydrophilic interaction chromatography (HILIC) and reversed phase LC (RP-LC) due to the relative elution strengths of the mobile phases used in these modes, which has severe implications for the modulation process and dilution.In this study we report a predictive kinetic optimisation tool for on-line HILIC × RP-LC which is based on theoretical relationships between the optimisation goals, the target analyte properties and chromatographic parameters (column dimensions, flow rates, mobile phases, injection volumes, etc.), allowing all chromatographic parameters to vary simultaneously within defined ranges. Experimental restrictions, such as pressure limits, flow rates, etc., are enforced to ensure all results are practically achievable on a given instrumental configuration. A Pareto-optimality approach is then used to obtain optimal sets of experimental conditions, from which the one(s) best satisfying the requirements in terms of time, dilution and/or peak capacity can be chosen. To overcome the challenges associated with mobile phase incompatibility in the coupling of HILIC and RP-LC, splitting of the first dimension HILIC flow, dilution with an aqueous make-up, or a combination of both, were investigated to establish the best approach to minimise total dilution and maximise performance. The validity of the methodology is demonstrated by deriving optimal conditions for the HILIC × RP-LC separation of procyanidins on selected columns and subsequent experimental verification of the performance for the separation of a cocoa extract.

Comprehensive Screening and Identification of Fatty Acid Esters of Hydroxy Fatty Acids in Plant Tissues by Chemical Isotope Labeling-Assisted Liquid Chromatography–Mass Spectrometry

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of lipid mediators with promising anti-diabetic and anti-inflammatory properties. Comprehensive screening and identification of FAHFAs in biological samples would be beneficial to the discovery of new FAHFAs and enable greater understanding of their biological functions. Here, we report the comprehensive screening of FAHFAs in rice and Arabidopsis thaliana by chemical isotope labeling-assisted liquid chromatography-mass spectrometry (CIL-LC-MS). Multiple reaction monitoring (MRM) was used for screening of FAHFAs. With the proposed method, we detected 49 potential FAHFA families, including 262 regioisomers, in tissues of rice and Arabidopsis thaliana, which greatly extends our knowledge of known FAHFAs. In addition, we proposed a strategy to identify FAHFA regioisomers based on their retention on a reversed-phase LC column. Using the proposed identification strategy, we identified 71 regioisomers from 11 FAHFA families based on commercial standards and characteristic chromatographic retention behaviors. The screening technique could allow for the discovery of new FAHFAs in biological samples. The new FAHFAs identified in this work will contribute to the in-depth study of the functions of FAHFAs.

A full solution for multi-component quantification-oriented quality assessment of herbal medicines, Chinese agarwood as a case

The quality of herbal medicines (HMs) is the prerequisite for their pronounced therapeutic outcomes in clinic, and multi-component (also known as quality markers, Q-markers) quantification has been widely emphasized as a viable means for quality evaluation. Because of the chemical diversity, the quality control practices are extensively dampened by four principal technical bottlenecks, including the lack of authentic compounds, large polarity span, extensive concentration range, and signal misrecognition for those potential Q-markers. An attempt to promote the potential of LC–MS/MS is made herein to cope with those obstacles and Chinese agarwood was employed as a case study. Firstly, a home-made fraction collector was introduced to automatically fragment the entire extract into a panel of fractions-of-interest. Secondly, quantitative 1H-NMR was deployed to offset the LC–MS/MS potential towards in-depth chemical profiling each fraction, and those well-defined fractions were then pooled and combined with some accessible authentic compounds to generate the pseudo-mixed standard solution. Thirdly, serial improvements were conducted for LC–MS/MS measurements. Reversed phase LC and hydrophilic interaction LC were serially coupled in respond to the large polarity window, and online parameter optimization, response tailoring, as well as RRCEC (relative response vs. collision energy curve) matching were integrated in MS/MS domain to advance the quantitative confidences. Simultaneous determination was conducted for 26 components, in total, in Chinese agarwood after method validation. In particular, authentic compound-free quantification was achieved for eight 2-(2-phenylethyl)chromone derivatives. Above all, the strategy is a promising solution to completely tackle with the technical barriers toward Q-marker quantification-oriented quality control of Chinese agarwood, as well as other HMs.

Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics.

Recent progress in top-down proteomics has driven the demand for chromatographic methods compatible with mass spectrometry (MS) that can separate intact proteins. Hydrophilic interaction liquid chromatography (HILIC) has recently shown good potential for the characterization of glycoforms of intact proteins. In the present study, we demonstrate that HILIC can separate a wide range of proteins exhibiting orthogonal selectivity with respect to reversed-phase LC (RPLC). However, the application of HILIC to the analysis of low abundance proteins (e.g., in proteomics analysis) is hampered by low volume loadability, hindering down-scaling of the method to column diameters below 2.1 mm. Moreover, HILIC-MS sensitivity is decreased due to ion suppression from the trifluoroacetic acid (TFA) often used as the ion-pair agent to improve the selectivity and efficiency in the analysis of glycoproteins. Here, we introduce a capillary-based HILIC-MS method that overcomes these problems. Our method uses RPLC trap-columns to load and inject the sample, circumventing issues of protein solubility and volume loadability in capillary columns (200 μm ID). The low flow rates and use of a dopant gas in the electrospray interface improve protein-ionization efficiencies and reduce suppression by TFA. Overall, this allows the separation and detection of small protein quantities (down to 5 ng injected on column) as indicated by the analysis of a mixture of model proteins. The potential of the new capillary HILIC-MS is demonstrated by the analysis of a complex cell lysate.

Enhancing the Possibilities of Comprehensive Two-Dimensional Liquid Chromatography through Hyphenation of Purely Aqueous Temperature-Responsive and Reversed-Phase Liquid Chromatography.

Comprehensive two-dimensional liquid chromatography (LC × LC) allows for substantial gains in theoretical peak capacity in the field of liquid chromatography. However, in practice, theoretical performance is rarely achieved due to a combination of undersampling, orthogonality, and refocusing issues prevalent in many LC × LC applications. This is intricately linked to the column dimensions, flow rates, and mobile-phase compositions used, where, in many cases, incompatible or strong solvents are introduced in the second-dimension (2D) column, leading to peak broadening and the need for more complex interfacing approaches. In this contribution, the combination of temperature-responsive (TR) and reversed-phase (RP) LC is demonstrated, which, due to the purely aqueous mobile phase used in TRLC, allows for complete and more generic refocusing of organic solutes prior to the second-dimension RP separation using a conventional 10-port valve interface. Thus far, this was only possible when combining other purely aqueous modes such as ion exchange or gel filtration chromatography with RPLC, techniques which are limited to the analysis of charged or high MW solutes, respectively. This novel TRLC × RPLC combination relaxes undersampling constraints and complete refocusing and therefore offers novel possibilities in the field of LC × LC including temperature modulation. The concept is illustrated through the TRLC × RPLC analysis of mixtures of neutral organic solutes.

A Versatile Liquid Chromatographic Method for the Simultaneous Determination of Metformin, Sitagliptin, Simvastatin, and Ezetimibe in Different Dosage Forms.

A new LC method is introduced with the concept of its versatile application to widely used drugs from different pharmacological classes. Metformin hydrochloride (MTF), sitagliptin phosphate (SIT), simvastatin (SIM) and ezetimibe (EZB) were simultaneously determined with a simple reversed-phase LC method in which a SIT-SIM binary mixture, present in a dosage form brand, was considered central for its development. Chromatographic separation was achieved with a mobile phase of acetonitrile and 0.02 M potassium dihydrogen phosphate (pH 5.2) (77 + 23, v/v) flowing through a C18 column (BDS Hypersil, 250 × 4.6 mm, 5 µm) at 1.2 mL/min at ambient temperature. UV detection was programmed to be carried out at 210 nm for EZB, SIT, and MTF, whereas SIM was detected at 240 nm. The method was validated according to International Conference on Harmonization guidelines. Linearity, accuracy, and precision were satisfactory over concentration ranges 4-40 µg/mL for EZB and SIM, 0.5-50 µg/mL for SIT, and 5-500 µg/mL for MTF. Coefficients of determination were >0.99 for the four drugs. LOQs found were 0.01 µg/mL for EZB, 0.02 µg/mL for SIT, 0.2 µg/mL for MTF, and 0.02 µg/mL for SIM. The developed method is simple, rapid, accurate, precise, and suitable for the routine QC analysis of the cited drugs in pharmaceutical products by conventional HPLC systems.

Study on the polymorphism of G-quadruplexes by reversed-phase HPLC and LC–MS

Polymorphism is inherent for G-quadruplexes (G4s), and the different structural forms are important for the participation in different biological functions of telomeres. A lot of progress has been made in the exploration of G4 polymorphism. However, quick separation and reliable assessment methods for different conformations of G4 are still very few. In this work, the polymorphism of three sequences d[(G3T)3G3], d[(G3C)3G3] and d[(G3T)4] annealed in six different solutions were investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography–mass spectrometry (LC–MS), fluorescence spectroscopy, circular dichroism spectroscopy, together with native-polyacrylamide gel electrophoresis. Different G4 conformations of these three sequences can be separated clearly by RP-HPLC, and further characterized by on-line LC–MS analysis. It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G3T)3G3] and d[(G3C)3G3] under the annealing conditions. However, flanking loop impeded d[(G3T)4] to form higher-order structures than dimer. In addition, the nature and concentration of cation, as well as the annealing solution component, all have decent influence on the stability and relative ratios of various G4 building blocks. Based on the above findings, RP-HPLC and LC–MS combined with spectroscopic techniques can be used as a facile and powerful tool for quick separation and identification of G4s in solutions, and for effective assessment of DNA sequences and annealing environments on G4 polymorphism. The established protocol provides a novel strategy for evaluating G4 polymorphism, which will facilitate studies on quadruplex structures and their biophysical properties.

Systematic evaluation of matrix effects in supercritical fluid chromatography versus liquid chromatography coupled to mass spectrometry for biological samples

Matrix effects (ME) is acknowledged as being one of the major drawbacks of quantitative bioanalytical methods, involving the use of liquid chromatography coupled to mass spectrometry (LC-MS). In the present study, the incidence of ME in SFC-MS/MS and LC-MS/MS in the positive mode electrospray ionization (ESI+) was systematically compared for the analysis of urine and plasma samples using two representative sets of 40 doping agents and 38 pharmaceutical compounds, respectively. Three different SFC stationary phase chemistries were employed, to highlight the importance of the column in terms of selectivity. Biological samples were prepared using two different sample treatments, including a non-selective sample clean-up procedure (dilute and shoot (DS) and protein precipitation (PP) for urine and plasma samples, respectively) and a selective sample preparation, namely solid phase extraction (SPE) for both matrices.The lower susceptibility to ME in SFC vs. reversed phase LC (RPLC) was verified in all the experiments performed on urine, and especially when a simple DS procedure was applied. Also, with the latter, the performance strongly varied according to the selected SFC stationary phase, whereas the results were quite similar with the three SFC columns, in the case of SPE clean-up. The same trend was observed with plasma samples. Indeed, with the PP procedure, the occurrence of ME was different on the three SFC columns, and only the 2-picolylamine stationary phase chemistry displayed lower incidence of ME compared to LC-MS/MS. On the contrary, when a SPE clean-up was carried out, the results were similar to the urine samples, with higher performance of SFC vs. LC and limited discrepancies between the three SFC columns. The type of ME observed in LC-MS/MS was generally a signal enhancement and an ion suppression for urine and plasma samples, respectively. In the case of SFC-MS/MS, the type of ME randomly varied according to the analyzed matrix, selected column and sample treatment.

Isotope-labeling derivatization with 3-nitrophenylhydrazine for LC/multiple-reaction monitoring-mass-spectrometry-based quantitation of carnitines in dried blood spots

Carnitines are diagnostic biomarkers of fatty acid oxidation defects and organic acidemias. Quantitative measurements of various carnitines in dried blood spot (DBS) have potential use in remote health applications for disease diagnosis and epidemiological surveillance. To provide an improved LC/multiple-reaction monitoring (MRM)-MS method for quantitation of carnitines in DBS, 3-nitrophenylhydrazine (3NPH) was tested as a high-efficiency chemical isotope-labeling reagent for pre-analytical derivatization of 24 routinely-analyzed species. Reaction conditions were optimized and carnitine structural isomers were separated by reversed-phase LC with positive-ion MRM/MS detection, giving on-column lower LOQs of sub- to low-femtomole levels. 13C6-3NPH was used to produce 13C6- or 13C12-labeled derivatives of the mono- and di-carboxylic carnitines in a “one-pot” reaction. These labeled analogues were used as stable isotope-labeled internal standards to compensate for possible ESI matrix effects. Combined with an optimized, two-step procedure for the extraction of carnitines from DBS, this isotope-labeling derivatizaiton - LC/MRM-MS method provided good linearity, high precision (intra-day CVs of ≤7.8% and inter-day CVs of ≤8.8%) and high accuracy (three levels of standard substances spiked in, with recoveries of 86.9%–109.7%) quantitation of carnitines in three sets of DBSs on cellulose or cotton filter paper. This method was then applied to determine the concentration changes of the analytes in the DBSs under two stability-testing regimes: 1) a one-time 4-h sunlight exposure and 2) a set of cycled temperature transitions (−20 °C for 2 days, 40 °C for 2 days, and back to −20 °C for 2 additional days). All of the carnitines showed good stabilities under the first testing condition. Under the second testing condition, free carnitine showed concentration increases of 9.3%–16.1%; acetyl carnitine, 3-OH butyryl carnitine, and malonyl carnitine showed concentration decreases of 12.2%–17.3%, 12.9%–17.1% and 10.7%–15.3%, respectively, and other 20 acyl carnitines showed concentration changes of <10% in three sets of DBSs on cellulose or cotton filter paper. These preliminary stability-testing results indicate a need to more systematically investigate the effects of various environmental conditions on the chemical stabilities of carnitines in DBS specimens if this sampling method is to be used in remote health applications.

552 - Comparison of Therapeutic Drug Levels of Adult and Pediatric Patients Undergoing Treatment for Fungal Diseases

Background: The mortality of invasive fungal infections is significant, indicating the critical need for appropriate treatment and dosing of anti-fungal drugs. Variable absorption and pharmacokinetics of triazole antifungal drugs (e.g. posaconazole and voriconazole) create a challenge for physicians to consistently achieve blood levels in the therapeutic range, particularly in patients suffering from comorbidities. The use of therapeutic drug monitoring (TDM) is critical for ensuring efficacy of voriconazole (VOR) and posaconazole (POS) treatment. Dosing in pediatric patients can prove more difficult because there have been fewer studies in these patient populations. Methods: Drug levels of POS and VOR were determined by reverse phase LC MS/MS on plasma or serum samples submitted for testing. Results were de-identified and reviewed to determine whether results fell within the therapeutic range for effective anti-fungal treatment. A large sample set (POS approximately 2500 samples and VOR approximately 5500 samples) was included to reduce the chance of sample size variability. Pediatric samples were defined as those coming from patients 17 years and younger. Results: For POS, in pediatric patients, 7.5% of samples tested had drug levels below the limit of detection of the assay (.1 mcg/mL) compared to 4% in adults; and an additional 19% of samples in pediatric and 13% in adults tested had detectable levels that were below the suggested therapeutic level of .7 mcg/mL. For VOR, the percentage of samples tested with results either above or below the therapeutic window was even higher: 6.7% of adult samples and 10.8% of pediatric samples tested had drug levels below the limit of detection of the assay (.1 mcg/mL), and an additional 21.3% of adult samples and 39.2% of pediatric sample had detectable levels that were below the suggested therapeutic range of 1.0-5.5 mcg/mL. Additionally, 13.4% of adult samples and 7.7% of pediatric samples tested had levels that were above the therapeutic range. Conclusions: From these data it is clear that an alarmingly high number of both adult and pediatric patient samples tested (24% of adult and 46% of pediatric) have antifungal drug levels below the recommended therapeutic levels. These data indicate the criticality of monitoring antifungal drug levels because of the high frequency of sub-therapeutic concentrations due to variations in metabolism, absorption or even patient compliance. Moreover, 7% of VOR results demonstrated levels above the recommended therapeutic levels, putting these patients at risk for toxicity.

High Sensitivity and Precision High-Temperature Reversed-Phase LC Analysis of Bevacizumab for Intact Bioanalysis of Therapeutic Monoclonal Antibodies

High Sensitivity and Precision High-Temperature Reversed-Phase LC Analysis of Bevacizumab for Intact Bioanalysis of Therapeutic Monoclonal Antibodies

Formation of phosphatidylethanol from endogenous phosphatidylcholines in animal tissues from pig, calf, and goat

In the presence of alcohol, phosphatidylcholine (PC) is transformed to the direct alcohol biomarker phosphatidylethanol (PEth). This reaction is catalyzed by the enzyme phospholipase D (PLD) and dependent on substrate availability. As recent methods have solely focused on the determination of PEth, information about the PC composition was generally missing. To address this issue and monitor PC (16:0/18:1 and 16:0/18:2) and PEth (16:0/18:1 and 16:0/18:2) simultaneously, a reversed phase LC–MS/MS method based on a C8 core–shell column, coupled to a Sciex 5500 QTrap instrument was developed. By application of polarity switching, at first, PC was measured in ESI positive SRM mode, while PEth was determined at a later stage in ESI negative SRM mode. The PEth method was validated for human blood samples to show its robustness and subsequently applied for the investigation of systematic in vitro PEth formation in animal tissue samples (brain, kidney, liver, and blood) from a pig, a calf, and a goat. Homogenized tissue was incubated at 37°C with varying ethanol concentrations from 1 to 7g/kg (determined by HS-GC-FID) for 5h, whereby a sample was taken every 30min. For all tissue samples, an increase in PEth was measurable. PEth concentrations formed in blood remained below the LLOQ, in agreement with literature. Data analysis of Michaelis–Menten kinetics and PC within the tissue provided a detailed insight about PEth formation, as the occurrence of PEth species can be linked to the observed PC composition. The results of this study show that PEth formation rates vary from tissue to tissue and among different species. Furthermore, new recommendations for PEth analysis are presented.

Development and Validation of Eco-Friendly Liquid Chromatographic and Spectrophotometric Methods for Simultaneous Determination of Coformulated Drugs: Phenylephrine Hydrochloride and Prednisolone Acetate.

Five simple, sensitive, and eco-friendly LC and UV spectrophotometric methods have been developed for the simultaneous determination of phenylephrine hydrochloride (PHE) and prednisolone acetate (PRD) in their combined dosage form. The first method was reversed-phase (RP) LC using methanol-water-heptane-1-sulfonic acid sodium salt (75 + 25 + 0.1, v/v/w) as a mobile phase. Separation was achieved using an XSelect HSS reversed-phase C18 analytical column (250 × 4.6 mm, 5µm). The flow rate was 1.0 mL/min and UV detection was done at 230 nm. Quantification was achieved over the concentration ranges of 5-50 µg/mL for PHE and 2-90 µg/mL for PRD. Four spectrophotometric methods were proposed, namely dual wavelength, first derivative of ratio spectra, ratio difference, and mean-centering of ratio spectra. Linearity was observed in the concentration ranges of 10-120 and 5-35 µg/mL for PHE and PRD, respectively, for the spectrophotometric methods. Green solvents were used in the proposed methods because they play a vital role in the analytical methods' influence on the environment. The suggested methods were validated regarding linearity, accuracy, and precision according to the International Conference on Harmonization guidelines, with satisfactory results. These methods could be used as harmless substitutes for routine analysis of the mentioned drugs, with no interference from excipients.

The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS

For many sample matrices, matrix effects are a troublesome phenomenon using the electrospray ionization source. The increasing use of supercritical fluid chromatography with CO2 in combination with the electrospray ionization source for MS detection is therefore raising questions: is the matrix effect behaving differently using SFC in comparison with reversed phase LC? This was investigated using urine, plasma, influent- and effluent-wastewater as sample matrices. The matrix effect was evaluated using the post-extraction addition method and through post-column infusions. Matrix effect profiles generated from the post-column infusions in combination with time of flight-MS detection provided the most valuable information for the study. The combination of both qualitative and semi-quantitative information with the ability to use HRMS-data for identifying interfering compounds from the same experiment was very useful, and has to the authors' knowledge not been used this way before. The results showed that both LC and SFC are affected by matrix effects, however differently depending on sample matrix. Generally, both suppressions and enhancements were seen, with a higher amount of enhancements for LC, where 65% of all compounds and all sample matrices were enhanced, compared to only 7% for SFC. Several interferences were tentatively identified, with phospholipids, creatinine, and metal ion clusters as examples of important interferences, with different impact depending on chromatographic technique. SFC needs a different strategy for limiting matrix interferences, owing to its almost reverse retention order compared to RPLC.

Hydrophilic interaction liquid chromatography of hydroxy aromatic carboxylic acid positional isomers

Hydrophilic interaction liquid chromatography (HILIC) has become increasingly popular as an alternative to reversed phase LC due to its ease of separating complex polar compound mixtures and the compatibility of the mobile phase with mass spectrometry (MS). Using a plain silica column (150 mm × 4.6 mm), we have shown a mixture containing three hydroxybenzoic acid isomers plus syringic and vanillic acid and three hydroxycinnamic acid isomers plus ferulic and sinapic acid can be separated using a mobile phase comprised of 90% acetonitrile (MeCN) and 10% 20 mM ammonium acetate at pH 6 in under 45 min. This method is appropriate when using UV detection at 275 nm. However, for improved MS compatibility, a buffer concentration of 10 mM is recommended but this greatly decreases the analyte retention factors. A second more nonpolar organic solvent component in the mobile phase (particularly pentane which has not been previously considered for HILIC) is found to offset this loss in retention. The optimum mobile phase is found to be 90% MeCN, 5% 10 mM ammonium acetate pH 6, and 5% pentane with resolution of eight of the ten compounds with a separation time of 30 min. Using UV detection, we have shown that detection limits range from 36 to 133 pmole and quantitation limits are spread from 94 to 376 pmole for six of the analytes. Upon testing this method on two other silica columns from different manufacturers, it is found that while resolution is similar, further optimization of the mobile phase is recommended.

Optimization of ultra-high pressure liquid chromatography – tandem mass spectrometry determination in plasma and red blood cells of four sphingolipids and their evaluation as biomarker candidates of Gaucher’s disease

While important advances have been recently achieved in the optimization of lipid classes’ separation, information on the specific determination of medium polarity lipids such as sphingolipids (SLs) in highly complex matrices remains fragmentary.In human, disorders of SL metabolism known as sphingolipidoses are a heterogeneous group of inherited disorders affecting primarily the central nervous. Early diagnosis of these conditions is of importance notably when a corrective therapy is available. The diagnosis is generally based on the determination of specific SLs in plasma and red blood cells (RBCs). For instance, glucosylceramide (GL1), glucosylsphingosine (Lyso-GL1), sphingosine (Sph), and sphingosine-1-phosphate (S1P) are proposed as relevant biomarkers for Gaucher disease (GD).Our main objective was to evaluate these biomarker candidates in a cohort of GD patients. However, most of current methods of GL1, Lyso-GL1, Sph, and S1P determination in plasma of GD patients require at least two liquid chromatographic runs. On the other hand, except for GL1 nothing is known concerning the RBC sphingolipid content. Yet, several reversed phase LC–MS methods of SLs separation and/or determination in various media with different sample preparation approaches have been proposed since 2010.Here we focused on stationary phase selection and mobile phase composition as well as on the sample preparation step to optimize and validate an UHPLC–MS/MS method for the simultaneous quantification of the four sphingolipids in both plasma and RBCs. A comparison between seven stationary phases including two RP18, two polar embedded RP18, and three HILIC phases shows that under our conditions polar embedded RP18 phases are the most appropriate for the separation of the four SLs, in terms of efficiency, peak symmetry, and separation time. In the same way, a comparison between a single step extraction with methanol and a liquid–liquid extraction with a mixture of methanol/methyl tert-butyl ether, shows that the latter mixture is the most appropriate for the extraction of SLs in terms of recovery and absence of matrix effect. After validation, this method was applied to the evaluation of the targeted SLs in a cohort of 15 known GD patients. The obtained results show that Lyso-GL1 is the only relevant biomarker in both plasma and RBCs for GD diagnosis.As the proposed method is applicable to the determination in such a highly complex matrices of four SLs with a large difference in polarity, and as the sample preparation procedure is freedom of matrix effects, this method can be easily adapted to a large diversity of samples.

Determination of Ochratoxin A in Black and White Pepper, Nutmeg, Spice Mix, Cocoa, and Drinking Chocolate by High-Performance Liquid Chromatography Coupled with Fluorescence Detection: Collaborative Study.

A method validation study for the determination of ochratoxin A in black and white pepper (Piper spp.), nutmeg (Myristica fragrans), spice mix (blend of ginger, turmeric, pepper, nutmeg, and chili), cocoa powder, and drinking chocolate was conducted according to the International Harmonized Protocol of the International Union of Pure and Applied Chemistry. The method is based on the extraction of samples with aqueous methanol, followed by a cleanup of the extract with an immunoaffinity column. The determination is carried out by reversed-phase LC coupled with a fluorescence detector. The study involved 25 participants representing a cross-section of research, private, and official control laboratories from 12 European Union (EU) Member States, together with Turkey and Macedonia. Mean recoveries ranged from 71 to 85% for spices and from 85 to 88% for cocoa and drinking chocolate. The RSDr values ranged from 5.6 to 16.7% for spices and from 4.5 to 18.7% for cocoa and drinking chocolate. The RSDR values ranged from 9.5 to 22.6% for spices and from 13.7 to 30.7% for cocoa and drinking chocolate. The resulting Horwitz ratios ranged from 0.4 to 1 for spices and from 0.6 to 1.4 for cocoa and drinking chocolate according to the Horwitz function modified by Thompson. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, and it conforms to requirements set by current EU legislation.

Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry.

Aminoacyl-tRNA synthetases-interacting multifunctional protein3 (AIMP3/p18) is involved in the macromolecular tRNA synthetase complex via its interaction with several aminoacyl-tRNA synthetases. Recent reports reveal a novel function of AIMP3 as a tumor suppressor by accelerating cellular senescence and causing defects in nuclear morphology. AIMP3 specifically mediates degradation of mature Lamin A (LmnA), a major component of the nuclear envelope matrix; however, the mechanism of how AIMP3 interacts with LmnA is unclear. Here we report solution-phase hydrogen/deuterium exchange (HDX) for AIMP3, LmnA, and AIMP3 in association with the LmnA C-terminus. Reversed-phase LC coupled with LTQ 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results in high mass accuracy and resolving power for comparing the D-uptake profiles for AIMP3, LmnA, and their complex. The results show that the AIMP3-LmnA interaction involves one of the two putative binding sites and an adjacent novel interface on AIMP3. LmnA binds AIMP3 via its extreme C-terminus. Together these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3 degradation.