Reversed-phase High-performance Liquid Chromatography Mass Research Articles

Studies on potential biomarkers related to drug efficacy and toxicity of an anti-tumor drug candidate by pharmaco-metabonomics

Based on high-performance liquid chromatography-mass spectrometry (HPLC-MS) and reversed-phase high-performance liquid chromatography-mass spectrometry (RPLC-MS), the changes of drug metabolism and endogenous metabolites in normal rats and tumor model rats were studied by drug metabonomics. The changes of vital signs were used to evaluate the toxicity and pharmacological effects of the new anti-cancer candidate ST on rats, and the differences of endogenous metabolites caused by ST were analyzed by multivariate statistical method. The changes of endogenous metabolites related to ST toxicity and pharmacological effects were analyzed. Studies have shown that the correlation analysis of metabonomics based on metabonomics and changes of animal signs is helpful to screen endogenous metabolites related to drug toxicity and pharmacological effects.

Blockade of fatty acid signalling inhibits lipopolysaccharide-induced macrophage recruitment and progression of apical periodontitis.

To examine the role of palmitic acid in lipopolysaccharide (LPS)-stimulated chemotaxis of macrophages and the potential contribution of saturated fatty acid in signalling during the pathogenesis of apical periodontitis. J774, a mouse macrophage cell line, was used in the experiments. After treatment with LPS, proteolytic maturation of sterol regulatory element-binding protein-1c (SREBP-1c) and expression of fatty acid synthase (FASN) were examined by Western analysis. Levels of palmitic acid were measured by reverse phase-high performance liquid chromatography-mass spectrometry. Knockdown of SREBP-1c and FASN was accomplished by small interfering RNA technology. Secretion of CC-chemokine ligand 2 (CCL2) and cellular chemotaxis were assessed by enzyme-linked immunosorbent assay and transwell migration assay, respectively. Sulfo-N-succinimidyl oleate (SSO) treatment was used to inhibit fatty acid signalling in vitro and also in a rat model of apical periodontitis. All data were first subjected to Levene's test. In vitro data were then analysed using ANOVA followed by Tukey's multiple comparison test. Data from animal experiments were analysed by independent t-tests. The significant level was set at 0.05. LPS stimulated proteolytic maturation of SREBP-1c and FASN expression in macrophages and significantly enhanced palmitic acid synthesis (P<0.05). Knockdown of SREBP-1c attenuated LPS-enhanced FASN expression. Knockdown of FASN significantly suppressed LPS-enhanced palmitic acid synthesis (P<0.05). LPS and exogenous palmitic acid significantly enhanced CCL2 secretion and macrophage chemotaxis (all P<0.05). Inhibition of FASN expression significantly alleviated LPS-augmented CCL2 secretion (P<0.05). SSO significantly suppressed CCL2 secretion and macrophage chemotaxis augmented by LPS and palmitic acid (all P<0.05). In a rat model of induced apical periodontitis, SSO treatment significantly attenuated progression of apical periodontitis and macrophage recruitment (all P<0.05). LPS/SREBP-1c/FASN/palmitic acid signalling contributed to tissue destruction caused by bacterial infection. Modulation of lipid metabolism and signalling may be helpful for the management of apical periodontitis.

Domain-specific free thiol variant characterization of an IgG1 by reversed-phase high-performance liquid chromatography mass spectrometry

Measurement of free thiols in antibody therapeutics is important for product development and assessment of critical quality attributes. Earlier studies demonstrated fast separation of free thiol variants of IgG1 using reversed-phase high performance liquid chromatography (RP-HPLC) with diphenyl resin. Here, we report using N-tert-butylmaleimide (NtBM) alkylation followed by RP-HPLC and online mass spectrometry for rapid total and domain-specific free thiol characterization of IgG1. By increasing hydrophobicity, NtBM alkylation improves separation of free thiol variants from disulfide-linked main peak species. The unique mass shift by NtBM alkylation offers unambiguous characterization of free thiol variants by online mass spectrometry. Variant peaks separated by RP-HPLC were antibody molecules containing two NtBM-alkylated cysteines, corresponding to IgG1 containing two free thiols before alkylation. Further characterization of the collected fractions of variants by peptide mapping revealed that each variant contained unpaired cysteines located in specific IgG1 domains (CH1, CH3, VH and VL domains). Total molecular-level and domain-specific free thiol content measured by this method correlate well with orthogonal differential alkylation peptide mapping analysis, which measures free thiol level at individual cysteine residues. This method provides high throughput quantitation of total and domain-specific free thiol content in IgG1 molecules, facilitating rapid, comprehensive product and manufacturing process characterization.

Variation in anthocyanin and essential oil composition and their antioxidant potentialities during flower development of Borage (Borago officinalis L.)

The aim of this study was to examine the chemical composition and antioxidant activities of the essential oils and anthocyanin of Borago officinalis flowers. At the flowering stage, the essential oil yield in Korba (0.95 ± 0.03%) was higher than that in Beja (0.29 ± 0.03%, w/w). The essential oil composition was characterized by high proportions of (E,E)-decadienal, the main compound of monoterpene aldehydes class. The reverse phase–high-performance liquid chromatography–mass spectrometry analysis indicated that flower anthocyanins were extracted and analysed for the first time and petunidin 3,5 diglucoside (58.8% in Korba and 54.93% in Beja) was the major anthocyanin followed by delphinidin 3,5 diglucoside (36.45% in Korba and 44.45% in Beja). During the development of borage flower, anthocyanin yield increased significantly (P < 0.05) from budding to full flowering stages in the two studied regions. Antioxidant activity of anthocyanin extracts and essential oil followed the same trend as anthocyanin and essential oil yields in Korba and Beja regions. In all tests, anthocyanin extracts of borage flowers showed better antioxidant activity than essential oils. A notable variability was found among the anthocyanin and essential oil concentrations and their antioxidant activities between the two studied regions, indicating a strong influence of the degree of maturity on metabolite production.

HPLC–MS and HPLC–MS/MS analysis of seven active constituents of Xiao-Xu-Ming decoction and application to a pharmacokinetic study after oral administration to rat

Xiao-xu-ming decoction (XXMD) is a traditional Chinese medicine that has been widely used to treat theoplegia and its sequelae. This paper reports the development of three separate assays based on reversed phase high-performance liquid chromatography–mass spectrometry (HPLC–MS) and HPLC–MS/MS for the determination of seven active constituents of XXMD viz oroxylin A-7-O-glucuronide, wogonoside, liquiritigenin, cimifugin, 5-O-methylvisammiol, glycyrrhizic acid and glycyrrhetinic acid in rat plasma. All calibration curves were linear (r >0.99) with lower limits of quantitation (LLOQs)<12.4ng/mL. Intra- and inter-day precisions (as relative standard deviation) were all <10.7% with recoveries in the range of 88.7–113%. In addition, the seven analytes were shown to be stable in rat plasma samples under relevant storage conditions. The validated methods were successfully applied to a pharmacokinetic study in rat after oral administration of XXMD.

Characterization of N-acetyltryptophan degradation products in concentrated human serum albumin solutions and development of an automated high performance liquid chromatography–mass spectrometry method for their quantitation

N-acetyltryptophan (NAT) has long been used as a stabilizer in some protein solutions, such as human serum albumin, to prevent oxidative protein degradation. However, the fate of NAT has not been discussed in literature. Two NAT degradation products have been observed in concentrated albumin solutions (20% and 25%) and identified as 1-acetyl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid and 1-acetyl-3a,8a-dihydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. To monitor the levels of these two previously unidentified NAT degradation products in concentrated albumin solutions, a fully automated method, incorporating online size exclusion chromatography (SEC) trapping and reversed-phase high performance liquid chromatography–mass spectrometry (HPLC–MS) with multiple reaction monitoring (MRM) analysis, has been developed and validated for their quantitative analysis. The method does not require an internal standard. The only sample manipulation is to obtain an albumin concentration of 4% in all standards and test HPLC samples. A limit of quantitation (LOQ) as low as 20 ng/mL has been achieved for both compounds. This method can readily be adopted for the quantitative determination of other small molecules in concentrated protein solutions.

The design and use of a simple System Suitability Test Mix for generic reverse phase high performance liquid chromatography–mass spectrometry systems and the implications for automated system monitoring using global software tracking

The development of a seven-component test mixture designed for use with a generic gradient and a reversed-phase high performance liquid chromatography–mass spectrometry (RP-HPLC–MS) system is discussed. Unlike many test mixtures formulated in order to characterise column quality at neutral pH, the test mixture reported here was designed to permit an overall suitability assessment of the whole liquid chromatography–mass spectrometry (LCMS) system. The mixture is designed to test the chromatographic performance of the column as well as certain aspects of the performance of the individual instrumental components of the system. The System Suitability Test Mix can be used for low and high pH generic reverse phase LCMS analysis. Four phthalates are used: diethyl phthalate (DEP), diamyl phthalate (DAP), di-n-hexyl phthalate (DHP) and dioctyl phthalate (DOP). Three other probes are employed: 8-bromoguanosine (8-BG), amitryptyline (Ami), and 4-chlorocinnamic acid (4-CCA). We show that analysis of this test mixture can alert the user when any part of the system (instrument or column) contributes to loss of overall performance and may require remedial action and demonstrate that it can provide information that enables us to document data quality control.

Quantitative profiling of bile acids in biofluids and tissues based on accurate mass high resolution LC-FT-MS: Compound class targeting in a metabolomics workflow

We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI−). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01–1 μM range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples ( n > 250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations.

Identification of New Flavone-8-Acetic Acid Metabolites Using Mouse Microsomes and Comparison with Human Microsomes

Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3'-, 4'-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3'- and 4'-hydroxy-FAA, thus corresponding to the 3',4'-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3',4'-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3'- and 4'-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

Simple and rapid determination of adenosine in human synovial fluid with high performance liquid chromatography–mass spectrometry

A simple, fast, sensitive and selective reversed-phase high performance liquid chromatography–mass spectrometry coupling with an electrospray ionization (ESI) interface method is described for the determination of adenosine in human synovial fluid. This method involved the use of the [M + H] +ions of adenosine and 2-chloroadenosine (internal standard for the assay) at m/ z 268 and 302 in positive ion mode with selective ion monitoring (SIM). Separation was carried out on a 2.0 × 150 mm Shimadzu VP-ODS column by using an isocratic elution with a mobile phase consisting of water (94%),methanol (5%) and formic acid (1%). No interference with the components of the biological matrix was observed in the determination conditions. The calibration curve was linear in the range of 0.2–140 μg ml −1. The limits of quantification (LOQ) and detection (LOD) were 0.2 and 0.03 μg ml −1, respectively. The standard recoveries were between 93.3 and 104.0%. The method was successfully applied to determination of adenosine in some synovial fluids of patients affected by rheumatoid arthritis.

Comparison of high-performance liquid chromatography — Mass spectroscopy and capillary electrophoresis— mass spectroscopy for the analysis of phenolic compounds in diethyl ether extracts of red wines

Reversed-phase high-performance liquid chromatography-mass spectroscopy (HPLC-MS) and capillary electrophoresis-mass spectroscopy (CE-MS) have been compared for the analysis of phenolic compounds in diethyl ether extracts of red wines. MS was performed in the electrospray negative-ionization mode. Despite the much higher separation efficiency of CE compared with LC, LC-MS furnishes far superior information for elucidation of the structure of the constituents. LC-MS enabled the identification of twenty-four compounds whereas only thirteen were characterized by CE-MS.

First report of the cyanobacterial toxin cylindrospermopsin in New Zealand

Cylindrospermopsin, a hepatotoxin produced by cyanobacteria, has been unambiguously detected in cyanobacteria collected from a recreational lake in the Wellington region of New Zealand. To our knowledge this is the first report of cylindrospermopsin in New Zealand. Cylindrospermopsin and several microcystins were identified by reversed phase high-performance liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) using an atmospheric-pressure ionization source and an ionspray interface. The presence of cylindrospermopsin in New Zealand highlights the risk of direct cyanobacterial toxin exposure in recreational waters.

On-line LC-MS analysis of urinary porphyrins.

A reversed-phase high-performance liquid chromatography-mass spectrometry (LC-MS) method is described for the separation and simultaneous analysis of porphyrins related to disorders of heme biosynthesis (uro-, heptacarboxylic, hexacarboxylic, pentacarboxylic, and coproporphyrins). The method involves initial porphyrin esterification and extraction from urine. Detection and quantification is performed from the extracts by separation with a Hypersil BDS column and on-line detection by MS through coupling with an atmospheric pressure chemical ionization interface. The porphyrin esters are detected as protonated molecules [M + H]+. Their mass spectra also exhibit an [M + Na]+ fragment of lower intensity. The analytical performance of this method is compared with those of LC with UV and fluorescence detection. LC-MS used in selective [M + H]+ ion monitoring provides the lowest detection and quantitation limits. In scan mode, this LC-MS method affords, without further isolation or concentration steps, the measurement of mass spectra of unknown compounds present in the urine of patients with altered porphyrin excretion.

Biologically mediated defunctionalization of chlorophyll in the aquatic environment—I. Senescence/decay of the diatom Phaeodactylum tricornutum

To obtain further information about the origin of sedimentary tetrapyrroles such as the alkyl porphyrin desoxophylloerythroaetioporphyrin, the defunctionalization of chlorophyll a has been studied in batch cultures of the marine diatom Phaeodactylum tricornutum, which were induced to undergo senescence by the exclusion of light. Identification of pigments by combined reversed phase high-performance liquid chromatography-mass spectrometry (LC-MS), with on-line photo-diode array detection, revealed conversion of chlorophyll a to phaeophytin a and pyrophaeophorbide a. The results demonstrate that certain chlorophyll a transformation products which occur commonly in aquatic sediments can be formed in part by the action of algal enzymes in the water column. The study provides the first unambiguous identification of pyrophaeophorbide a during senescence of a non-mutant alga.