Ultra-high Performance Liquid Chromatography Separation Research Articles

A discovery biotransformation strategy: combining in silico tools with high-resolution mass spectrometry and software-assisted data analysis for high-throughput metabolism

Understanding compound metabolism in early drug discovery aids medicinal chemistry in designing molecules with improved safety and ADME properties. While advancements in metabolite prediction brings increased confidence, structural decisions require experimental data. In vitro metabolism studies using liquid chromatography and high-resolution mass spectrometry (LC–MS) are generally resource intensive and performed on very few compounds, limiting the chemical space that can be examined. Here, we describe a novel metabolism strategy increasing compound throughput using residual in vitro clearance samples conducted at drug concentrations of 0.5 µM. Analysis by robust ultra high-performance liquid chromatography separation and accurate-mass MS detection ensures major metabolites are identified from a single injection. In silico prediction (parent cLogD) tailors chromatographic conditions, with data-dependent tandem mass spectroscopy targeting predicted metabolites. Software-assisted data mining, structure elucidation and automatic reporting are used. Confidence in the globally aligned workflow is demonstrated with 16 marketed drugs. The approach is now implemented routinely across our laboratories. To date, the success rate for identification of at least one major metabolite is 85%. The utility of these data has been demonstrated across multiple projects, allowing earlier medicinal chemistry decisions to increase efficiency and impact of the design–make–test cycle thus improving the translatability of early in vitro metabolism data.

Structural characterization of human milk oligosaccharides using ultrahigh performance liquid chromatography-helium charge transfer dissociation mass spectrometry.

The combination of helium charge transfer dissociation mass spectrometry (He-CTD-MS) with ultrahigh performance liquid chromatography (UHPLC) is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). The research focuses on the identification of the monosaccharide sequence, the branching patterns, the sialylation/fucosylation arrangements, and the differentiation of isomeric oligosaccharides in the mixture. Initial studies first optimized the conditions for the UHPLC separation and the He-CTD-MS conditions. Results demonstrate that He-CTD is compatible with UHPLC timescales and provides unambiguous glycosidic and cross-ring cleavages from both the reducing and the nonreducing ends, which is not typically possible using collision-induced dissociation. He-CTD produces informative fragments, including 0,3An and 0,4An ions, which have been observed with electron transfer dissociation, electron detachment dissociation, and ultraviolet photodissociation (UVPD) and are crucial for differentiating the α-2,3- versus α-2,6-linked sialic acid (Neu5Ac) residues present among sialyllacto-N-tetraose HMOs. In addition to the linkage positions, He-CTD is able to differentiate structural isomers for both sialyllacto-N-tetraoses and lacto-N-fucopentaoses structures by providing unique, unambiguous cross-ring cleavages of types 0,2An, 0,2Xn, and 1,5An while preserving most of the labile Neu5Ac and fucose groups.

The utility of silica hydride-based stationary phases for dual-mode ultra high performance liquid chromatography separation of synthetic cathinone positional isomers.

Emerging drugs usually mimic the effects of traditional drugs, but are not always controlled due to the chemically altered structures. Positional isomers of emerging drugs are difficult to analyze because they challenge the separation and detection techniques commonly employed by forensic laboratories. The utility of silica hydride stationary phases for the ultra-high performance liquid chromatography separation of synthetic cathinone positional isomers was studied in this manuscript. SiH phases are operable under both reversed phase and aqueous normal phase chromatographic conditions without the need to change solvent reservoirs. The separation of eight positional isomers of the synthetic cathinone, pentedrone, was investigated using five silica hydride phases, and compared to a classical dual column reversed phase, hydrophilic interaction chromatography system, and to a bimodal pentaflurophenyl column. Significant selectivity differences were observed using either a combination of a classical reversed phase C18 and NP Silica columns or the various bimodal columns. The silica hydride silica-C column, which contains no derivatized ligands attached to the silica hydride backbone, not only gave the most orthogonal separation of the bimodal columns, but provided a unique separation of all eight positional isomers (resolution ≥ 1) using the combination of reverse phase and aqueous normal phase chromatographic conditions.

Comprehensive Comparison of Clinically Relevant Grain Proteins in Modern and Traditional Bread Wheat Cultivars.

Bread wheat (Triticum aestivum L.) is one of the most valuable cereal crops for human consumption. Its grain storage proteins define bread quality, though they may cause food intolerances or allergies in susceptible individuals. Herein, we discovered a diversity of grain proteins in three Ukrainian wheat cultivars: Sotnytsia, Panna (both modern selection), and Ukrainka (landrace). Firstly, proteins were isolated with a detergent-containing buffer that allowed extraction of various groups of storage proteins (glutenins, gliadins, globulins, and albumins); secondly, the proteome was profiled by the two-dimensional gel electrophoresis. Using multi-enzymatic digestion, we identified 49 differentially accumulated proteins. Parallel ultrahigh-performance liquid chromatography separation followed by direct mass spectrometry quantification complemented the results. Principal component analysis confirmed that differences among genotypes were a major source of variation. Non-gluten fraction better discriminated bread wheat cultivars. Various accumulation of clinically relevant plant proteins highlighted one of the modern genotypes as a promising donor for the breeding of hypoallergenic cereals.

Data-Dependent Acquisition and Database-Driven Efficient Peak Annotation for the Comprehensive Profiling and Characterization of the Multicomponents from Compound Xueshuantong Capsule by UHPLC/IM-QTOF-MS.

The state of the art ion mobility quadrupole time of flight (IM-QTOF) mass spectrometer coupled with ultra-high performance liquid chromatography (UHPLC) can offer four-dimensional information supporting the comprehensive multicomponent characterization of traditional Chinese medicine (TCM). Compound Xueshuantong Capsule (CXC) is a four-component Chinese patent medicine prescribed to treat ophthalmic disease and angina. However, research systematically elucidating its chemical composition is not available. An approach was established by integrating reversed-phase UHPLC separation, IM-QTOF-MS operating in both the negative and positive electrospray ionization modes, and a “Component Knockout” strategy. An in-house ginsenoside library and the incorporated TCM library of UNIFITM drove automated peak annotation. With the aid of 85 reference compounds, we could separate and characterize 230 components from CXC, including 155 ginsenosides, six astragalosides, 16 phenolic acids, 16 tanshinones, 13 flavonoids, six iridoids, ten phenylpropanoid, and eight others. Major components of CXC were from the monarch drug, Notoginseng Radix et Rhizoma. This study first clarifies the chemical complexity of CXC and the results obtained can assist to unveil the bioactive components and improve its quality control.

PO-267 PHGDH and PSAT confer metabolic vulnerability to IDH2-driven reprogramming in breast cancer

IntroductionReprogrammed energy metabolism is one among the distinguished cancer hallmarks that unravels the dysregulated bioenergetics of cancer cells. Isocitrate dehydrogenase 2 (IDH2), a mitochondrial metabolic enzyme has been reported to be overexpressed in several cancers, yet the mechanisms of wildtype IDH2-induced transformation are still obscure. We previously identified IDH2, as a novel poor prognosis marker of breast cancer progression. In agreement, transcriptomics (TCGA) and proteomics data of breast cancer clinical samples showed similar expression changes of increased wildtype IDH2 from luminal, to triple-negative and to HER-2 positive, which correlated with tumour aggressiveness.Material and methodsWe performed intracellular metabolite tracing with heavy labelled glucose (13C6) and glutamine (13C5) followed by ultra-high performance liquid chromatography separation and mass spectrometric analysis on the Q-Exactive Plus instrument. Genome-scale metabolic modelling analysis was performed to identify the IDH2-dependent metabolic genes using Minimization of Metabolic Adjustment (MOMA) algorithm.Results and discussionsOverexpression of IDH2 increased serine biosynthesis, increased glycolytic rate and oxidative stress resistance. Genome-scale metabolic modelling of breast cancer tumour data identified dependence on serine biosynthesis as one of the metabolic vulnerability of IDH2 overexpressing cells. CRISPR-Cas9 mediated knockout of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT) specifically inhibited IDH2 overexpressing cells, even in the presence of exogenous serine and glycine, and reduced the glycolysis rate and glucose uptake. Furthermore, loss of serine synthesis pathway hampered the flux from glycolysis, into the TCA cycle. Moreover the metabolic perturbations of synthetic dosage lethal partners, PHGDH and PSAT sensitised the IDH2 overexpressing cells to oxidative stress.ConclusionThis study reveals WT-IDH2 as a critical metabolic regulator in breast cancer that supports the pro-tumorigenic Warburg effect. These findings may contribute to translate the metabolic dependency to improve targeted therapeutics.

Sensitivity enhancement by chromatographic peak concentration with ultra-high performance liquid chromatography–nuclear magnetic resonance spectroscopy for minor impurity analysis

High performance liquid chromatography can be coupled with nuclear magnetic resonance (NMR) spectroscopy to give a powerful analytical method known as liquid chromatography-nuclear magnetic resonance (LC-NMR) spectroscopy, which can be used to determine the chemical structures of the components of complex mixtures. However, intrinsic limitations in the sensitivity of NMR spectroscopy have restricted the scope of this procedure, and resolving these limitations remains a critical problem for analysis. In this study, we coupled ultra-high performance liquid chromatography (UHPLC) with NMR to give a simple and versatile analytical method with higher sensitivity than conventional LC-NMR. UHPLC separation enabled the concentration of individual peaks to give a volume similar to that of the NMR flow cell, thereby maximizing the sensitivity to the theoretical upper limit. The UHPLC concentration of compound peaks present at typical impurity levels (5.0–13.1 nmol) in a mixture led to at most three-fold increase in the signal-to-noise ratio compared with LC-NMR. Furthermore, we demonstrated the use of UHPLC-NMR for obtaining structural information of a minor impurity in a reaction mixture in actual laboratory-scale development of a synthetic process. Using UHPLC-NMR, the experimental run times for chromatography and NMR were greatly reduced compared with LC-NMR. UHPLC-NMR successfully overcomes the difficulties associated with analyses of minor components in a complex mixture by LC-NMR, which are problematic even when an ultra-high field magnet and cryogenic probe are used.

UHPLC-PDA Assay for Simultaneous Determination of Vitamin D3 and Menaquinone-7 in Pharmaceutical Solid Dosage Formulation.

A newly developed method based on ultrahigh performance liquid chromatography (UHPLC) was optimized for the simultaneous determination of vitamin D3 and menaquinone-7 (MK-7) in tablet formulation in the present study. UHPLC separation of vitamin D3 and MK-7 was performed with ACE Excel 2 C18-PFP column (2 μm, 2.1 × 100 mm) at 0.6 mL min−1 flow rate, whereas the mobile phase consisted of methanol/water (19 : 1, v/v, phase A) and isopropyl alcohol (99.9%, phase B) containing 0.5% triethylamine. Isocratic separation of both the analytes was performed at 40°C by pumping the mobile phases A and B in the ratio of 50 : 50 (v/v, pH, 6.0). Both analytes were detected at a wavelength of 265 nm and the injection volume was 1.0 μL. The overall runtime per sample was 4.5 min with retention time of 1.26 and 3.64 min for vitamin D3 and MK-7, respectively. The calibration curve was linear from 5.0 to 100 μg mL−1 for vitamin D3 and MK-7 with a coefficient of determination (R2) ≥ 0.9981, while repeatability and reproducibility (expressed as relative standard deviation) were lower than 1.46 and 2.21%, respectively. The proposed HPLC method was demonstrated to be simple and rapid for the determination of vitamin D3 and MK-7 in tablets.

Development and Validation of a UHPLC UV Method for the In-Process Control of Bosentan Monohydrate Synthesis.

Bosentan monohydrate (4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]benzene-1-sulfonamide monohydrate) is a dual endothelin receptor antagonist (ERA) applied in the treatment of pulmonary arterial hypertension. To achieve effective process control of the bosentan monohydrate synthesis, it was necessary to develop a selective and not highly time-consuming method for ultra-high performance liquid chromatography (UHPLC). The method is characterized by adequate sensitivity, reproducibility and selectivity for the determination of bosentan monohydrate and related compounds from all synthetic stages. The UHPLC separation was carried out by reversed phase chromatography on the Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with a mobile phase composed of solvent A (0.1 %, v/v, acetic acid in water) and solvent B (methanol), in the gradient mode at the flow rate of 0.4 mL min−1. Limits of detection and quantification for the compounds were ≤0.1 µg mL−1 and 0.3 µg mL−1, respectively. The linearity for all related compounds was investigated as in the range for the active pharmaceutical ingredient (API) and as in the range for the in-process control. The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.

Optimisation by response surface methodology of microextraction by packed sorbent of non steroidal anti-inflammatory drugs and ultra-high performance liquid chromatography analysis of dialyzed samples

A procedure based on microextraction by packed sorbent (MEPS) followed by ultra-high performance liquid chromatography (UHPLC) with photodiode array (PDA) detection has been developed for the analysis of seven selected non steroidal anti-inflammatory drugs (NSAIDs) in human dialysates. The influence on MEPS efficiency of pH of the sample, pH of the washing solvent and methanol content in the hydro-alcoholic elution mixture has been investigated by response surface methodology based on a Box-Behnken design of experiments. Among the above factors, pH of sample is the variable that mostly influences MEPS recovery. UHPLC separation of the NSAIDs was completed within less than 4min under isocratic elution conditions on a Fortis SpeedCore C18 column (150×4.6mm I.D., 2.6μm) using acetonitrile-phosphate buffer as the mobile phase. Calibration curves of the NSAIDs were linear over the concentration range 0.025–15μg/mL, with correlation coefficients≥0.998. Intra- and inter-assay relative standard deviations were <8% and recovery values ranged from 94% to 100% for the quality control samples. The results reveal that the developed MEPS/PDA-UHPLC method exhibits a good accuracy and precision and is well suited for the rapid analysis of human dialysate from patients treated with the selected NSAIDs.

Micellar and sub-micellar ultra-high performance liquid chromatography of hydroxybenzoic acid and phthalic acid positional isomers

Micellar liquid chromatography (MLC) has been used primarily for the separation of neutral analytes of varying polarities, most commonly phenols and polyaromatic hydrocarbons, but does not seem to have been used to study aromatic hydroxy acids in detail. We have studied the separation of hydroxybenzoic acid mixtures, including monohydroxybenzoic and dihydroxybenzoic acid positional isomers by MLC. Sodium dodecylsulfate (SDS) is investigated as the modifying surfactant on a C18 ultra-high performance liquid chromatography (UHPLC) column (100×2.1mm, 1.8μm). The addition of only SDS (no organic solvent) to the mobile phase reduced the influence of hydrophobic interactions while improving the retention times, resolution, and peak shapes, even at concentrations below the critical micellization concentration (CMC). The UHPLC separation of 7 hydroxybenzoic acids, including 6 dihydroxybenzoic acid positional isomers and one trihydroxybenzoic acid, is achieved with high efficiency using 0.1% SDS in 1.84mM sulfuric acid (pH 2.43) mobile phase, in less than 6min with a flow rate of 0.3mLmin−1, and in less than four min with a flow rate of 0.7mLmin−1. Six monohydroxybenzoic acid isomers are also effectively separated by MLC, using a 0.5% SDS mobile phase modifier, in less than 20min with a flow rate of 0.3mLmin−1, and in less than 14min with a flow rate of 0.7mLmin−1. The 3 phthalic acid isomers could be separated using a similar mobile phase and flow rates in less than 6 and 4min. Solute-micelle equilibrium constants and partition coefficients are calculated for 6 monohydroxybenzoic acids based on a plot of MLC retention factor vs. mobile phase micelle concentration. All aromatic acid isomers studied can be classified as binding solutes in the MLC retention mechanism. Less effective separations are observed with shorter chain surfactants, leading to higher retention times and poor peak shapes. It is concluded that increasing chain length led to more efficient MLC separations, and SDS is the preferred modifying surfactant for the examined separation.

A validated simultaneous quantification method for vonoprazan (TAK-438F) and its 4 metabolites in human plasma by the liquid chromatography-tandem mass spectrometry.

Vonoprazan fumarate (TAK-438) is a potassium-competitive acid blocker which was approved in Japan for a treatment of acid-related diseases. In this study a simple and validated bioanalytical method, which can simultaneously determine vonoprazan (TAK-438F) and its four metabolites (M-I, M-II, M-III and M-IV-Sul) in human plasma, was developed. The method is based on protein precipitation and subsequent ultra-high performance liquid chromatography separation followed by tandem mass spectrometry detection. The mass spectrometric parameters for detection of TAK-438F, M-I, M-III and M-IV-Sul were modified from their optimum values in order to achieve a simultaneous quantification while retaining enough sensitivity and wide dynamic ranges for all the target analytes. The validity and robustness of the method was verified through a validation study as per the regulatory guidance on bioanalytical method validation. The calibration ranges are 0.1-100 ng/mL for TAK-438F and M-III, and 1-1000 ng/mL for M-I, M-II and M-IV-Sul using the 100 μL of human plasma. The total run time per sample is 5 min. The working solution for M-III was recommended to be prepared separately, especially for the long-term use, in order to avoid the instability of M-III in the mixed working solutions, which could cause the high consumption of reference standards. The established method was applied to clinical pharmacokinetic studies and concentrations of all the analytes in human plasma were successfully determined with high reproducibility ensured by incurred sample reanalysis, indicating the suitableness of the established method.

Screening approach by ultra-high performance liquid chromatography-tandem mass spectrometry for the blood quantification of thirty-four toxic principles of plant origin. Application to forensic toxicology.

Plant poisonings have left their mark on history and still cause many deaths, whether intentional or accidental. The means to show toxicological evidence of such poisonings should be implemented with great care. This article presents a technique for measuring thirty-nine toxic principles of plant origin in the blood, covering a large amount of toxins from local or exotic plants: α-lobeline, α-solanine, aconitine, ajmaline, atropine, brucine, cephalomannine, colchicine, convallatoxin, cymarine, cytisine, digitoxin, digoxin, emetine, gelsemine, ibogaine, jervine, kavain, lanatoside C, lupanine, mitragynine, neriifolin, oleandrin, ouabain, paclitaxel, physostigmine, pilocarpine, podophyllotoxin, proscillaridin A, reserpine, retrorsine, ricinine, scopolamine, senecionine, sparteine, strophanthidin, strychnine, veratridine and yohimbine. Analysis was carried out using an original ultra-high performance liquid chromatography separation coupled with tandem mass spectrometry detection. Extraction was a standard solid phase extraction performed on Oasis(®) HLB cartridge. Thirty-four of the thirty-nine compounds were put through a validation procedure. The assay was linear in the calibration curve range from 0.5 or 5 μg/L to 1000 μg/L according to the compounds. The method is sensitive (LOD from 0.1 to 1.6 μg/L). The within-day precision of the assay was less than 22.5% at the LLOQ, and the between-day precision was less than 21.5% for 10 μg/L for all the compounds included. The assay accuracy was in the range of 87.4 to 119.8% for the LLOQ. The extraction recovery and matrix effect ranged from 30 to 106% and from -30 to 14%, respectively. It has proven useful and effective in several difficult forensic cases.

Determination of Acequinocyl and Hydroxyacequinocyl Residues in Food by Ultra-High Performance Liquid Chromatography Separation and Tandem Mass Spectrometric Detection

A sensitive method for the quantification of acequinocyl and hydroxyacequinocyl in foodstuff (beef, chicken muscle and liver, fish, peach, cucumber, Chinese cabbage and broad bean) was developed by ultra-high performance liquid chromatography and tandem mass spectrometry. Acequinocyl and hydroxyacequinocyl were found to be stable in the presence of formic acid at low temperature (−18 °C) in the dark. The target compounds in solid food samples were successively extracted by acetonitrile containing 0.5 % (v/v) formic acid, and then cleaned up by using Florisil columns, and then evaporated to near dryness under a nitrogen stream at 40 °C, and finally dissolved in acetonitrile containing 0.5 % (v/v) formic acid. No matrix effect was observed from any foodstuff. Linear calibration curves with correlation coefficients of 0.9996 for acequinocyl and 0.9998 for hydroxyacequinocyl over the same range 2 to 100 μg L−1 were obtained. The intra-day and inter-day relative standard deviations were both superior to 3 % (N = 10) for 5 μg L−1. The method detection limits were 1.4 μg kg−1 for acequinocyl and 1.3 μg kg−1 for hydroxyacequinocyl. The method quantification limits were 4.6 and 4.3 μg kg−1, respectively, which were at least five times lower than their maximum residue limits for food. Neither acequinocyl nor hydroxyacequinocyl was detected in any foodstuff. The recoveries at spiked levels of 5, 10, and 50 μg kg−1 varied in the ranges 81–100 % for acequinocyl and 77–103 % for hydroxyacequinocyl in the foodstuffs, validating the accuracy of the proposed method.

New Trend in the LC Separation Analysis of Pharmaceuticals – High Performance Separation by Ultra High-performance Liquid Chromatography (UHPLC) with Core-shell Particle C18 Columns –

This article presents a mini-review of the recent results in the ultra high-performance liquid chromatography (UHPLC) separation of pharmaceuticals by our group. High performance UHPLC separation employing core-shell particle C18 columns was demonstrated. High performance (high theoretical plate number of approximately 20000/10 cm, low theoretical plate height of 5 μm) was obtained without any specific devices in the conventional HPLC apparatus, only through changing detector sampling times and the inner diameter of the connecting tube. High theoretical plate numbers with low column back pressure obtained by the core-shell particle columns enabled fast separation of the analytes. Methanol, which gives high column pressure drops in the reversed-phase mode HPLC compared with acetonitrile, can be used without any trouble. One analysis of the purity testing of diltiazem hydrochloride was performed within 100 s. One analysis in the photostability testing of mecobalamin (vitamin B12 analogue) was successful within 180 s.

Determination of N-nitrosamines in rubber products by solid phase extraction purification and ultra high performance liquid chromatography-tandem mass spectrometry

A method using C18 solid phase extraction (SPE) for purification and ultra high performance liquid chromatography (UHPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for detection was developed to simultaneously determine 13 N-nitrosamines in rubber products. The analytes were extracted with methanol (60 degrees C, 30 min) with the aid of ultrasonic technique and then purified by a C18 SPE cartridge. The analytes were separated on a C18 chromatographic column and qualitatively and quantitatively detected by a mass spectrometer with positive ESI at multiple reaction monitoring (MRM) mode. The operating parameters for UHPLC separation and ESI-MS/MS detection were also optimized. Under optimum operating conditions, the relative standard deviations (RSDs, n = 7) were less than 10% at spiked level of 50 microg/kg for all analytes except N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) which were spiked at 500 microg/kg. The recoveries spiked in real from 70.7% to 117.0%. The limits of detection (LODs, 10 times of standard deviation) were in the range of 0.5 - 500 microg/kg. The method has been successfully applied to the simultaneous determination of the 13 N-nitrosamines in rubber products.

Structural characterization of silicone polymers using compositional ultra-high performance liquid chromatography separation, electrospray ionization, and high resolution/accurate mass

In this article, the development of an on-line ultra-high performance liquid chromatography (UHPLC)-high resolution/accurate mass (HR/AM) methodology for timely characterization of a broad range of silicone polymers is described. Electrospray ionization was optimized for silicone polymers to enable the direct coupling of the separation with mass spectrometry. Reverse-phase UHPLC was optimized to provide a high resolution separation of oligomers by molar mass and by end-group composition. With the high resolution compositional separation, multiple pseudo-molecular ion species were indexed with retention time and assisted in rapid identification of oligomer species. Relative retention times of different oligomer end-group species separated based on polarity provided additional information to aid data interpretation. These additional pieces of information were combined with accurate mass to provide a high information content analysis in a relatively short time frame. This methodology was applied to polymer end-group characterization, as well as comparative analysis examples.

Quantitative Analysis of Gangliosides in Bovine Milk and Colostrum-Based Dairy Products by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry

Milk gangliosides have gained considerable attention because they participate in diverse biological processes, including neural development, pathogen binding, and activation of the immune system. Herein, we present a quantitative measurement of the gangliosides present in bovine milk and other dairy products and byproducts. Ultrahigh performance liquid chromatography separation was used for high-throughput analysis and achieved a short running time without sacrificing chromatographic resolution. Dynamic multiple reaction monitoring was conducted for 12 transitions for GM3 and 12 transitions for GD3. Transitions to sialic acid fragments (m/z 290.1) were chosen for the quantitation. There was a considerable amount of gangliosides in day 2 milk (GM3, 0.98 mg/L; GD3, 15.2 mg/L) which dramatically decreased at day 15 and day 90. GM3 and GD3 were also analyzed in pooled colostrum, colostrum cream, colostrum butter, and colostrum buttermilk. The separation and analytical approaches here proposed could be integrated into the dairy industry processing adding value to side-streams.

Comparative analysis of nucleosides and nucleobases from different sections of Elaphuri Davidiani Cornu and Cervi Cornu by UHPLC–MS/MS

A rapid and sensitive hydrophilic interaction liquid chromatography method was developed for the identification and quantitative determination of 17 nucleosides and nucleobases by using ultra-high performance liquid chromatography separation system coupled to triple quadrupole mass spectrometer. Sufficient separation of the target compounds was achieved on an Acquity BEH Amide column (2.1mm×100mm, 1.7μm) in a single run of 9min. The LOD and LOQ were in the range of 0.017–50.00ngml−1 and 0.056–146.00ngml−1, respectively. Then, the method was applied for the analysis of nucleosides and nucleobases in Elaphuri Davidiani Cornu and Cervi Cornu. Multivariate statistical analysis was also used to investigate the differences in the nucleosides and nucleobases profiles among the samples. The present study also provided helpful information for quality control of Elaphuri Davidiani Cornu and Cervi Cornu.

Comprehensive Analysis of Oil Sands Processed Water by Direct-Infusion Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry with and without Offline UHPLC Sample Prefractionation

Oil sands processed water (OSPW) is the main byproduct of the large-scale bitumen extraction activity in the Athabasca oil sands region (Alberta, Canada). We have investigated the acid-extractable fraction (AEF) of OSPW by extraction-only (EO) direct infusion (DI) negative-ion mode electrospray ionization (ESI) on a 12T-Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS), as well as by offline ultrahigh performance liquid chromatography (UHPLC) followed by DI-FTICR-MS. A preliminary offline UHPLC separation into 8 fractions using a reversed-phase C4 column led to approximately twice as many detected peaks and identified compounds (973 peaks versus 2231 peaks, of which 856 and 1734 peaks, respectively, could be assigned to chemical formulas based on accurate mass measurements). Conversion of these masses to the Kendrick mass scale allowed the straightforward recognition of homologues. Naphthenic (CnH2n+zO2) and oxy-naphthenic (CnH2n+zOx) acids represented the largest group of molecules with assigned formulas (64%), followed by sulfur-containing compounds (23%) and nitrogen-containing compounds (8%). Pooling of corresponding fractions from two consecutive offline UHPLC runs prior to MS analysis resulted in ~50% more assignments than a single injection, resulting in 3-fold increase of identifications compared to EO-DI-FTICR-MS using the same volume of starting material. Liquid-liquid extraction followed by offline UHPLC fractionation thus holds enormous potential for a more comprehensive profiling of OSPW, which may provide a deeper understanding of its chemical nature and environmental impact.