Reversed-phase High-performance Liquid Chromatography-mass Spectrometry 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.

Experimental hydrophobicity parameters of perfluorinated alkylated substances from reversed-phase high-performance liquid chromatography

Environmental contextPerfluorinated compounds are synthetic chemicals shown to be present in the blood of humans. To study how these contaminants get into our blood requires a good understanding of their physicochemical properties. We describe an alternative way to obtain values for how perfluorinated compounds distribute between water and fatty phases (mimicking e.g. gut content and gut wall), which is essential information for modelling and understanding the environmental fate of these chemicals. AbstractCapacity factors of perfluorinated alkylated substances were obtained from isocratic reversed-phase high-performance liquid chromatography–mass spectrometry experiments at different organic modifier strengths of the mobile phase. The resulting capacity factor v. modifier strengths plots were extrapolated to obtain capacity factors at 100% water (k0) that can serve as indicators of the hydrophobicity of the perfluorinated acids. Values of log k0 were shown to increase linearly with the number of CF2 units in the fluorinated alkyl chain.

Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography–mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture

The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile–2-propanol mobile phase. Conventional C 18 column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C 18 column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 μm C 18 particles. The separation in NARP system on C 30 column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C 18 is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.

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.

A rapid, simple, specific liquid chromatographic–electrospray mass spectrometry method for the determination of finasteride in human plasma and its application to pharmacokinetic study

A fast, accurate, sensitive, selective and reliable method using reversed-phase high-performance liquid chromatography–mass spectrometry coupling with an electrospray ionization interface was developed and validated for the determination of finasteride in human plasma. After deprotienation with acetonitrile, centrifugation, evaporation to dryness and dissolving in mobile phase, satisfactory separation was achieved on a Hypersil-Keystone C 18 reversed-phase column using a mobile phase consisting of acetonitrile–water (46:54, v/v), 0.1% acetic acid and 0.1% trifluoracetic acid. Carbamazepine (IS) was used as internal standard. This method involved the use of the [ M + H] + ions of finasteride and IS at m/ z 373 and 237 with the selective ion monitoring (SIM) mode. The calibration curve was linear in the range of 0.2–120 ng ml −1. The limit of quantification for finasteride in plasma was 0.2 ng ml −1 with good accuracy and precision. The intra-assay precision and accuracy were in the range of 2.1–11.2% and −1.3% to 8.5%, respectively. The inter-assay precision and accuracy were in the order of 3.4–12.1% and −1.5% to 11.5%, respectively. The mean sample extract recoveries of the method were higher than 85% and 74% for finasteride and internal standard (IS), respectively. The assay has been successfully used to estimate the pharmacokinetics of finasteride after oral administration of a 5 mg tablet of finasteride to 24 healthy volunteers.

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody

Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area. However, there are few reports on disulfide bond assignment of recombinant mAbs. This work describes the complete disulfide bond assignment of a recombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was used to mask free sulfhydryl groups present in the mAb. Digestion of the mAb with endoproteinase Lys-C without disulfide scrambling was achieved by denaturing the mAb in the presence of NEM in guanidine hydrochloride (GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol (DTT). Native and reduced Lys-C digests were mass analyzed by on-line reversed-phase–high-performance liquid chromatography mass spectrometry (RP–HPLC/MS). Disulfide-containing peptides were sequenced by off-line nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS) and N-terminal Edman sequencing for verifying connectivities. The recombinant IgG4 mAb was found to contain the expected disulfide linkages with the proposed method. The NEM alkylating reagent was critical in minimizing disulfide scrambling during the denaturation and digestion of the mAb. This integrated approach, combining MS and N-terminal Edman sequencing, was capable of assigning the disulfide pattern of the IgG4 mAb rapidly and completely, and should be applicable for disulfide bond assignment and structural analysis of other mAbs and large proteins with multiple disulfide bonds.

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.

Determination of phenolic derivatives of antipyrine in plasma with solid-phase extraction and high-performance liquid chromatography–atmospheric-pressure chemical ionization mass spectrometry

This manuscript describes a method to determine antipyrine and its phenolic derivatives in plasma by reversed-phase high-performance liquid chromatography–mass spectrometry (RP-HPLC–MS). The sample pretreatment consisted of a C 18 solid-phase extraction (SPE), to remove the salts and proteins. The retention behavior of antipyrine and its phenolic derivatives in the SPE procedure was estimated by the k values determined on a C 18 HPLC column at different pH values and with different buffer compositions. Recoveries of antipyrine and its phenolic products were 90% in water and 100% in plasma. Atmospheric pressure chemical ionization (APCI) was used to introduce the components into the mass spectrometer. The mass spectrometer was operated in the single ion monitoring mode (SIM mode) as well as in the selective reaction (SR) mode. The SR mode or tandem MS resulted in the best signal-to-noise ratio, with a detection limit for antipyrine of 6 pg in 20 μl. For the different phenolic antipyrines, different target ions were used and conditions were optimized for each.

Analysis of Cytochrome P450 Metabolites of Arachidonic and Linoleic Acids by Liquid Chromatography–Mass Spectrometry with Ion Trap MS2

We have used reversed phase-high performance liquid chromatography–mass spectrometry (RP-HPLC-MS) with an ion trap mass spectrometer to study the metabolism of arachidonic and linoleic acids by human recombinant cytochrome P450 (CYP) enzymes. We first recorded the MS2spectra of the carboxylate anions of epoxides, diols, ω-side chain, and bisallylic hydroxy fatty acids of arachidonic, octadeuterated arachidonic, and linoleic acids. The metabolites formed by CYP2C9 and CYP2C19 were then studied. CYP2C9 converted arachidonic and linoleic acids to epoxides/diols and monohydroxy fatty acids. Some hydroxyeicosatetraenoic acids (HETEs) were studied in detail to investigate the oxygenation mechanism. Incubation of CYP2C9 under oxygen-18 gas showed that all HETEs had incorporated oxygen-18 to the same degree. Chiral HPLC showed that CYP2C9 formed 15R-HETE (72% of the R enantiomer), 13S-HETE (90%), and 11R-HETE (57%). RP-HPLC-MS analysis revealed that CYP2C19 oxygenated arachidonic acid to 19-HETE, 14,15-epoxyeicosatrienoic acid (EET), and 8,9-EET as main metabolites. The method was sufficiently sensitive to identify arachidonic acid metabolites formed by some other isozymes. RP-HPLC-MS with MS2seems to be useful for rapid identification of fatty acid metabolites in complex mixtures formed by cytochrome P450.

High-performance liquid chromatography—continuous-flow fast atom bombardment mass spectrometry of chlorophyll derivatives

Nine chlorophyll derivatives from different spinach preparations were separated and identified using reversed-phase high-performance liquid chromatography—mass spectrometry (HPLC—MS). These pigments included chlorophylls a and b, chlorophyllides a and b, pheophorbide a, pheophytins a and b, and pyropheophytins a and b. HPLC—MS measurements were carried out using HPLC—frit-fast atom bombardment (FAB)-MS, which is a continuous-flow FAB-MS interface. The highly hydrophobic chlorophyll derivatives were eluted from a reversed-phase HPLC column using a gradient of increasing ethyl acetate concentration. Glycerol was included in the mobile phase to serve as the matrix for FAB ionization. During analysis by positive-ion HPLC—frit-FAB-MS, abundant protonated molecules, [M + H] + were detected for all nine chlorophyll derivatives. Fragment ions were observed in the mass spectra that were similar to those produced during standard probe FAB-MS. These HPLC—MS procedures were shown to be useful for the rapid separation and identification of a variety of chlorophyll derivatives from natural sources.