Interaction Liquid Chromatography-tandem Mass Spectrometry Research Articles

A Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Quantitative Method for Determination of Baricitinib in Plasma, and Its Application in a Pharmacokinetic Study in Rats.

Baricitinib, is a selective and reversible Janus kinase inhibitor, is commonly used to treat adult patients with moderately to severely active rheumatoid arthritis (RA). A fast, reproducible and sensitive method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of baricitinib in rat plasma has been developed. Irbersartan was used as the internal standard (IS). Baracitinib and IS were extracted from plasma by liquid–liquid extraction using a mixture of n-hexane and dichloromethane (1:1) as extracting agent. Chromatographic separation was performed using Acquity UPLC HILIC BEH 1.7 µm 2.1 × 50 mm column with the mobile phase consisting of 0.1% formic acid in acetonitrile and 20 mM ammonium acetate (pH 3) (97:3). The electrospray ionization in the positive-mode was used for sample ionization in the multiple reaction monitoring mode. Baricitinib and the IS were quantified using precursor-to-production transitions of m/z 372.15 > 251.24 and 429.69 > 207.35 for baricitinib and IS, respectively. The method was validated according to the recent FDA and EMA guidelines for bioanalytical method validation. The lower limit of quantification was 0.2 ng/mL, whereas the intra-day and inter-day accuracies of quality control (QCs) samples were ranged between 85.31% to 89.97% and 87.50% to 88.33%, respectively. Linearity, recovery, precision, and stability parameters were found to be within the acceptable range. The method was applied successfully applied in pilot pharmacokinetic studies.

Simultaneously quantitative analysis of peptides and chemical components in Cervus and Cucumis polypeptide injection (Songmeile®) using reversed phase liquid chromatography-hydrophilic interaction liquid chromatography–tandem mass spectrometry

Co-occurrences of peptides and chemical components are usually observed in complicated matrices. Notably, those traditional Chinese medicine prescriptions (TCMPs) contain both plant and animal ingredients. It is still challenging to simultaneously monitor peptides and chemical components attributing to their different liquid chromatographic (LC) and mass spectrometric (MS) behaviors. Herein, efforts were made to pursue an eligible approach enabling simultaneous determination of peptides and chemical components in a TCMP namely Cervus and Cucumis polypeptide injection (CCPI, Songmeile®) that is prepared from the acid hydrolytic peptide-enriched extract of Sika deer (Cervus nippon Temminck) bone and the aqueous extract of muskmelon (Cucumis melo L.) seeds. Reversed phase liquid chromatography and hydrophilic interaction liquid chromatography were serially connected (RPLC-HILIC) to achieve comprehensive retention and separation. Sensitive detection was accomplished with selected reaction monitoring (SRM) mode, and multiply charged and singly charged ion transitions were defined for peptides and chemical components, respectively. Inter-batch variations of CCPI were evaluated in an authentic compound-independent manner. In particular, online energy-resolved MS was proposed to gain optimal parameters for five targeted peptides after that CCPI peptidome was profiled using nanoLC-LTQ Orbitrap Velos Pro MS. A so-called universal metabolome standard (UMS) sample was built for calibration curve construction and subsequently applied to acquire the quasi-contents of all 31 analytes, including five peptides and 26 chemical components, in ten batches of CCPI (CCPI1–CCPI10). The quantitative dataset revealed mild fluctuation for the quasi-content profiles of analytes-of-interest within different batches. More importantly, RPLC-HILIC–SRM is a promising method to fully address the demands of simultaneous measurement of peptides and chemical components in complicated matrices, and it might be a robust analytical tool for in-depth quality evaluation of CCPI as well as other TCMPs.

Halomethanesulfonic Acids-A New Class of Polar Disinfection Byproducts: Standard Synthesis, Occurrence, and Indirect Assessment of Mitigation Options.

Halomethanesulfonic acids (HMSAs) are recently discovered polar disinfection byproducts without commercially available reference materials. To allow for their accurate quantification, we successfully synthesized standards for the four presumably most prevalent HMSA congeners: chloromethanesulfonic acid, bromomethanesulfonic acid, dichloromethanesulfonic acid, and bromochloromethanesulfonic acid. After structure confirmation and quantification with high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, we integrated them into a multilayer solid phase extraction and hydrophilic interaction liquid chromatography-tandem mass spectrometry method dedicated to the analysis of polar water contaminants. With this method we monitored HMSAs in drinking water production plants from four European countries and tap water samples taken in six countries. HMSAs were detected in the low μg/L range after the chlorination step during drinking water production, all tap waters samples, and two surface waters used for drinking water production. Concentrations in tap water samples ranged from 0.07 μg/L to 11.5 μg/L while the HMSA concentrations in surface waters were in the range of 100 ng/L. We utilized the HMSA formation potential to indirectly assess the behavior of hitherto unknown HMSA precursors, consequently identifying ozonation, filtration through activated carbon, and reverse osmosis as efficient removal tools for HMSA precursors, thus limiting their formation during subsequent water disinfection.

Occurrence of Tetrodotoxin in Bivalves and Gastropods from Harvesting Areas and Other Natural Spaces in Spain.

Tetrodotoxin (TTX) is a potent neurotoxin that is receiving increasing interest in the European Union because it has been found in different fishery products (fish, bivalves and gastropods) captured in European waters. Since available information is scarce, further analytical data regarding the incidence of this toxin in European fishery products is needed in order to perform an appropriate risk assessment devoted to protecting consumers’ health. Hence, samples of bivalves and gastropods were collected at different points of the Spanish coast and analyzed by high-performance hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) to evaluate the presence of TTX. None of the analyzed samples showed TTX above an internal threshold of 10 µg/kg or even showed a peak under it. Our results on TTX occurrence obtained in bivalve molluscs and gastropods did not show, at least in the studied areas, a risk for public health. However, taking into account previous positive results obtained by other research groups, and since we did not detect TTX in our samples, a more completed study increasing sampling frequency is needed to ensure proper risk evaluation towards the food safety of these products.

Analysis of degradation products of nitrogen mustards via hydrophilic interaction liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for qualitative and quantitative analysis of ethanolamines (EAs), which are nitrogen mustard degradation products. With this method, the retention times of the highly hydrophilic EAs on the HILIC column were sufficient (retention times: methyl diethanolamine, 12.2 min; ethyl diethanolamine, 11.2 min; and triethanolamine, 9.5 min) and the EAs were analyzed more efficiently than with reported HILIC-MS/MS methods. The detection limits of methyl diethanolamine and ethyl diethanolamine in serum and urine using this approach were 15–20 ng/mL. The suitability of the method for real samples was evaluated via recovery tests involving urine and serum, and the method was validated. The MS/MS fragmentation of EAs was discussed based on density functional theory calculations.

Investigation of free and conjugated seleno-amino acids in wheat bran by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

An analytical method for determining seleno-methionine, methyl-seleno-cysteine, and seleno-cystine in wheat bran was developed and validated. Four different extraction procedures were evaluated to simultaneously extract endogenous free and conjugated seleno-amino acids in wheat bran in order to select the best extraction protocol in terms of seleno amino acid quantitation. The extracted samples were subjected to a clean-up by a reversed phase/strong cation exchange solid-phase extraction and analyzed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. The optimized extraction protocol was employed to validate the methodology. Process efficiency ranged from 58 to 112% and trueness from 73 to 98%. Limit of detection and limit of quantification were lower than 1ng/g. Four wheat bran samples were analyzed for both total Se and single seleno-amino acids determination. The results showed that Se- seleno-methyl-lselenocysteine was the major seleno-amino acid in wheat bran while seleno-methionine and seleno-cysteine were both minor species.

Quantification of clitidine in caps and stems of poisonous mushroom Paralepistopsis acromelalga by hydrophilic interaction liquid chromatography–tandem mass spectrometry

A simple and high-throughput analytical method for determining clitidine in Paralepistopsis acromelalga using hydrophilic interaction liquid chromatography–tandem mass spectrometry (LC–MS/MS) was established. To determine clitidine in the mushrooms, a simple procedure including dilution with methanol solution and filtering by cartridge was employed just before quantification by LC–MS/MS for high-throughput analysis. In this report, concentrations of clitidine in mushrooms were determined by the standard addition method. The present established method was successfully applied to the analysis of fruit bodies of P. acromelalga, which were obtained from five different locations in Japan. Results on concentrations of clitidine in each stem and cap of P. acromelalga specimens tested showed that their concentrations were quite different, not only between stems and caps, but also among locations and strains; the concentrations of clitidine in stems and caps ranged from 1.41 to 9.30 mg/g and 3.17 to 14.4 mg/g, respectively. This is the first report to present a detailed quantitative analysis of clitidine by MS and the distribution of clitidine in stems and caps of P. acromelalga. This analytical method for clitidine was thought to be useful in P. acromelalga poisoning cases to identify the causative toxic mushroom.

Hydrophilic interaction liquid chromatography–tandem mass spectrometric approach for simultaneous determination of safingol and D-erythro-sphinganine in human plasma

A simple and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for the simultaneous determination of C18-L-threo-sphinganine (safingol, an anti-neoplastic in phase I trials) and its diastereomer, C18-D-erythro-sphinganine (sphinganine), in human plasma. Sample pretreatment involved a protein precipitation with methanol using 25 μL aliquots of plasma. Chromatographic separation of the diastereomers and C17-D-erythro-sphinganine, an internal standard, was achieved on a Xbridge HILIC (3.5 μm, 100 × 2.1 mm) using isocratic elution with the mobile phase of 2 mM ammonium bicarbonate in water (pH 8.3) and acetonitrile at a flow rate of 0.3 mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were linear over the concentration range of 0.2–100 ng/mL with a lower limit of quantification of 0.2 ng/mL. The relative standard deviation of intra-day and inter-day precision was below 8.27%, and the accuracy ranged from 92.23 to 110.06%. The extraction recoveries were found to be higher than 93.22% and IS-normalized matrix effect was higher than 90.92%. The analytes were stable for the durations of the stability studies. The validated method was successfully applied to the analyses of pharmacokinetic samples from patients treated with safingol and all-trans-N-(4-hydroxyphenyl)retinamide; (fenretinide, 4-HPR) in a current phase I clinical trial (SPOC-2010-002, ClinicalTrials.gov Identifier: NCT01553071).

Multi-year assessment of paralytic shellfish toxins in hard clam species along the coastline of Jiangsu Province, China

Paralytic shellfish toxins (PSTs) are notorious neurotoxins that threaten public health and food safety worldwide. Although PST monitoring programs have recently been established throughout China, the profiles and variation of PSTs in important commercial clams (e.g., Mactra veneriformis, Ruditapes philippinarum, and Meretrix meretrix) along the Jiangsu Province coastline remain largely unexplored. In this study, a validated hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method was used to examine PST profiles and levels in 540 clam samples from natural production areas along Jiangsu Province coastline during 2014–2016. Although the PST levels (≤6.38 μg saxitotoxin equivalents (eq)/kg) were consistently below European Union regulatory limits (≤800 μg saxitotoxin eq/kg) during this time period, saxitotoxin, decarbamoylsaxitotoxin, and gonyautoxins 1 and 4 were detected, and nearly 40% of the samples were saxitotoxin-positive. The PST levels also varied significantly by seasons, with peak values observed in May during 2014–2016. This is the first systematic report of PSTs in clams from Jiangsu Province, and additional research and protective measures are needed to ensure the safety of clams harvested in this area.

Sensitive analysis of trehalose-6-phosphate and related sugar phosphates in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography–tandem mass spectrometry

Trehalose-6-phosphate (T6P) is an important signaling metabolite that is involved in many physiological processes. However, the mechanism of the biological functions of T6P is not fully understood. Quantification of T6P in plants will be beneficial to elucidate the mechanism. However, it is still a challenge to chromatographically separate and sensitively detect T6P and related sugar phosphates. In the current study, we developed a method for effective separation and sensitive detection of glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), sucrose-6-phosphate (S6P) and T6P in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry (ChD-HILIC-MS/MS). With this method, two pairs of isomers (G1P/G6P and S6P/T6P) could be well separated on a HILIC column and sensitively detected by MS with limits of detection (LODs) ranging from 0.1 to 0.6 ng mL−1. The developed method was successfully applied to the detection of endogenous G1P, G6P, S6P and T6P in small amounts of plant tissues, such as 1 mg fresh weight of Oryza sativa shoot.

Quantitative Profiling of Endogenous Metabolites Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry (HILIC-MS/MS).

Dynamic modeling of metabolic reaction networks requires absolute quantification of intracellular and extracellular metabolite concentrations with high precision and accuracy. This chapter presents a robust HILIC-ESI-MS/MS procedure for targeted quantitative profiling of more than 50 polar key metabolites in multicomponent endogenous extracts. Without using ion-pairing-agents or prior derivatization protocols, organic acids, amino acids, sugar phosphates, coenzymes, and nucleotides are measured on a triple quadrupole platform in positive and negative electrospray ionization modes with preoptimized MRM transitions. Robust polymer-based zwitterionic stationary phases (ZIC®-pHILIC) support alkaline mobile phase conditions (pH9.2) for enhancing retention and chromatographic performance of polar analytes in bicratic elution mode without unfavourable column bleed. The quality of the method was extensively validated and demonstrated by absolute metabolite quantification in endogenous Escherichia coli extracts by comparative use of standard-based external calibration, isotope dilution, and standard addition as quantification strategies. In sum, alkaline ZIC®-pHILIC chromatography emerged as an efficient approach providing high selectivity and sensitivity for comprehensive metabolic studies.

Investigation of free seleno-amino acids in extra-virgin olive oil by mixed mode solid phase extraction cleanup and enantioselective hydrophilic interaction liquid chromatography-tandem mass spectrometry.

An analytical method for determining seleno-methionine (SeMet), methyl-seleno-cysteine and seleno-cystine in extra-virgin olive oil (EVOO) was developed and validated. EVOO sample (15 g) was diluted with hexane, extracted with methanol/water 80:20 (v/v), and cleaned up by a reversed phase/strong cation exchange solid phase extraction. Analysis was performed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. Process efficiency ranged between 49 and 97% and trueness between 87 and 126%, with intermediate precision, expressed as standard deviation, lower than 10%. Method detection limits (MDLs) and method quantification limits (MQLs) were lower than 1 μg kg-1. Thirty-two EVOO samples from different Italian regions were analyzed for both total Se and single seleno-amino acids determination. Only l-SeMet was found at level MQL (0.2 μg kg-1)-1.42 μg kg-1 in ten samples, while total Se was in the range of MDL-9.1 μg kg-1. Concentration of l-SeMet (5-6% of total Se) and total Se correlated very well to each other (R2 = 0.995).

High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS) was developed for the simultaneous determination of 28 amphetamine-type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid-liquid extraction (LLE) at pH9.5 using methyl tert-butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization. All stimulants were eluted within 7minutes and baseline separation was achieved for isomeric and isobaric compounds using HILIC chromatography. Extraction efficiency was greater than 80% and matrix effect was acceptable for most stimulants. The limit of detection (LOD) was in the range of 10-50pg/mL and the lower limit of quantification (LLOQ) was in the range of 50-100pg/mL. Quadratic regression was employed for quantification and the dynamic range of quantification was 50-10000pg/mL. Confirmatory analysis criteria were established using product ion ratios and retention time. The limit of confirmation (LOC) was in the range of 20-100pg/mL. Stability study results indicated that some stimulants were unstable in equine plasma at room temperature and 4°C. However, all the stimulants studied were stable at -20°C and-80°C for the 6month study period.

Novel zwitterionic HILIC stationary phase for the determination of ethyl glucuronide in human hair by LC-MS/MS

Some recent studies have described a shift from traditional reversed-phase to more hydrophilic LC chemistry for EtG determination in hair (hEtG). The reason relies on the poor retention of C8– and C18-based columns for polar compounds, even in presence of great amount of aqueous phase. This work presents the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-LC-MS/MS) method based on a novel zwitterionic stationary phase for the analysis of hEtG. The linearity was assessed in the range of 5–100 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1.4 pg/mg and 4.5 pg/mg in hair, respectively. Suitable diagnostic sensitivity was achieved without the introduction of a sample purification step, or a post column solvent addition. The method was successfully applied to real hair samples after full validation. This method, based on a separation at neutral conditions, confirmed the optimum retention and thus selectivity for weak acids in zwitterionic HILIC columns.

Multi-residue methodology for the determination of 16 coccidiostats in animal tissues and eggs by hydrophilic interaction liquid chromatography – Tandem mass spectrometry

A simple, sensitive and efficient confirmatory method was developed and validated for the determination of 16 coccidiostats in animal tissues and eggs using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS). The sample preparation consisted of a solid-liquid extraction with ACN and dispersive SPE cleanup with MgSO4 and C18. Analysis was realized in an Acquity BEH HILIC silica column, in SRM mode. Both positive and negative ionization was performed, using polarity switching. Isocratic elution was used with a mobile phase of ACN: aqueous ammonium formate 1 mM with 0.1% formic acid (80:20, v/v). Method validation was performed in eggs, poultry, bovine, ovine, porcine and rabbit tissue and exceptionally low LODs were achieved, varying from 0.004 μg kg−1 (decoquinate in porcine tissue) to 0.560 μg kg−1 (halofuginone in eggs). The developed methodology was applied in 82 muscle and egg samples through the Greek National Residue Control Plan for coccidiostats.

Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP7) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP7 and its precursor inositol hexakisphosphate (InsP6) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP6 and InsP7 peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP6 and InsP7 on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP6 and InsP7 can be quantitated from 20–500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP6 in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP7 is induced by NaF treatment and ectopic expression of InsP6K2, a primary kinase for InsP7 synthesis. Furthermore, this analysis reveals the abundance of InsP6 (50.46 ± 18.57 pmol/106 cells) and scarcity of InsP7 in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP6 and InsP7 in mouse and human samples, and we expect that the method will contribute to further understanding of InsP7 functions in mammalian pathobiology.

Phospholipidome of endothelial cells shows a different adaptation response upon oxidative, glycative and lipoxidative stress

Endothelial dysfunction has been widely associated with oxidative stress, glucotoxicity and lipotoxicity and underlies the development of cardiovascular diseases (CVDs), atherosclerosis and diabetes. In such pathological conditions, lipids are emerging as mediators of signalling pathways evoking key cellular responses as expression of proinflammatory genes, proliferation and apoptosis. Hence, the assessment of lipid profiles in endothelial cells (EC) can provide valuable information on the molecular alterations underlying CVDs, atherosclerosis and diabetes. We performed a lipidomic approach based on hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) for the analysis of the phospholipidome of bovine aortic EC (BAEC) exposed to oxidative (H2O2), glycative (glucose), or lipoxidative (4-hydroxynonenal, HNE) stress. The phospholipid (PL) profile was evaluated for the classes PC, PE, PS, PG, PI, SM, LPC and CL. H2O2 induced a more acute adaptation of the PL profile than glucose or HNE. Unsaturated PL molecular species were up-regulated after 24 h incubation with H2O2, while an opposite trend was observed in glucose- and HNE-treated cells. This study compared, for the first time, the adaptation of the phospholipidome of BAEC upon different induced biochemical stresses. Although further biological studies will be necessary, our results unveil specific lipid signatures in response to characteristic types of stress.

Ubiquity of the neurotoxin β-N-methylamino-L-alanine and its isomers confirmed by two different mass spectrometric methods in diverse marine mollusks

In recent years, the neurotoxin β-N-methylamino-L-alanine (BMAA) has been reported in some marine mollusk species. To further discover BMAA in marine animals, a total of 59 samples belonging to 3 phyla, 22 families, and 43 species, were collected from Dalian, Rongcheng, and Zhoushan cities, China, in April 2017. All samples were quantified by a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) analysis of underivatized extract, and ten samples were also analyzed by a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using a precolumn AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate)-derivatization method. Results indicated that 48 mollusk samples contained BMAA with concentrations above the limit of detection (0.31 μg g−1 wet weight), and the isomers of BMAA, β-amino-N-methylalanine (BAMA) and 2,4-diaminobutyric acid (DAB) were universally present in most samples. However, N-(2-aminoethyl) glycine (AEG) was not found in any sample. Comparison of both analytical methods showed that BMAA and BAMA were not completely separated by the HILIC column although they still could be identified by specific transitions. In contrast the C18 column provided good separation for the AQC-derivatives of BMAA and all of its isomers. Development of analytical methods and stable isotope tracing of BMAA should be carried out in the future.

A novel malic acid-enhanced method for the analysis of 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-methylcytidine and 5-hydroxymethylcytidine in human urine using hydrophilic interaction liquid chromatography-tandem mass spectrometry

5-Methyl-2′-deoxycytidine (5-mdC), 5-hydroxymethyl-2′-deoxycytidine (5-hmdC), 5-methylcytidine (5-mrC) and 5-hydroxymethylcytidine (5-hmrC) are epigenetic marks of DNA and RNA, and aberrant levels of these modified nucleosides were found to be associated with various cancers. Urine is a preferred source of biological fluid for biomarker discovery because the sample collection process is not invasive to patients. Herein, we developed a novel malic acid-enhanced hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for sensitive and simultaneous quantification of the modified cytosine nucleosides in human urine. Malic acid markedly increased the detection sensitivities of all four cytosine nucleosides, with the limits of detection (LODs) for 5-mdC, 5-hmdC, 5-mrC and 5-hmrC being 0.025, 0.025, 0.025 and 0.050 fmol, respectively. By using this method, we demonstrated, for the first time, the presence of 5-hmrC in human urine, and we successfully quantified 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine samples collected from 90 patients with colorectal cancer (CRC) and 90 healthy controls. We found that the levels of 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine were all substantially decreased in CRC patients, suggesting that these modified nucleosides might have great potential to be noninvasive biomarkers for early detection and prognosis of CRC. Together, we established a novel and sensitive method for detecting 5-methylated and 5-hydroxymethylated cytosine nucleosides in human urine and the results from this study may stimulate future investigations about the regulatory roles of these cytosine derivatives in the initiation and development of CRC.

A novel strategy for isolation and determination of sugars and sugar alcohols from conifers

The ultrasound-assisted extraction method for isolation of 17 sugars and sugar alcohols from conifers with a subsequent hydrophilic interaction liquid chromatography-tandem mass spectrometry method for their determination is proposed. The optimization of extraction parameters was carried out using Taguchi – L9 (34) orthogonal array experimental design for the following parameters—a methanol concentration in the extraction solution, an extraction time, a type of plant sample and an extraction temperature. The optimal ultrasound-assisted extraction conditions were—MeOH concentration – 30% (water – 70%), extraction time – 30 min, type of plant sample – II (grinded leaves 2–4 mm long), extraction temperature – 60 °C. Pure water and acetonitrile were used as eluents in gradient elution mode to separate the analytes. Direct determination of multiple sugars and sugar alcohols was carried out using a mass spectrometric detector operated in a multiple reaction monitoring mode, providing detection limits in the range between 0.1 and 20 ng/mL and good analytical characteristics of the method without derivatization. The developed approach was validated by multiple successive extraction method applied to test its performance on a series of 10 samples, i.e. 2 samples per each of 5 genera: Abies, Larix, Picea, Pinus (Pinaceae) and Juniperus (Cupressaceae), widely distributed in the boreal conifer forests of Eurasia. The novel strategy can be used for profiling of sugars and sugar alcohols in a wide range of plant species.