Determination of exemestane and 17-hydroxyexemestane by high-performance liquid chromatography coupled with tandem mass spectrometry and high-resolution mass spectrometry

An approach was developed for determining and confirming the presence of exemestane and its metabolite 17-hydroxyexemestane in urine. It is based on the application of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/MS) and atmospheric pressure chemical ionization high-resolution mass spectrometry (HPLC-HRMS). To detect hydroxyexemestane, the analysis of the hydrolyzed fraction of urine is preferable. The recovery rates of exemestane and 17-hydroxyexemestane were 83 and 91%, respectively. The detection limits were 1 ng/mL for HPLC-MS/MS and 2.5 ng/mL for HPLC-HRMS. In spite of a considerable effect of ionization suppression, the sensitivity and selectivity of the determination are affected by the selection of the optimal detection conditions in HPLC-MS/MS and by the high accuracy of mass determination in mass spectrometry with orbitrap detection, enabling resolution at a level of 5 ppm. The procedures can be used for screening and confirmatory analysis.     

Analysis of brain lipids by directly coupled matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance thin-layer chromatography

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization MS technique providing only minor fragmentation of the analyte. Therefore, the method is basically suitable for mixture analysis, although the ion yields strongly depend on the basicity/acidity of the analyte in relation to the applied matrix. Accordingly, less sensitively detectable compounds may be suppressed by more sensitively detectable compounds. Thus, separation of the mixture into the individual compounds is normally indispensable. This paper demonstrates the capabilities and limitations of a direct, simple, and inexpensive MALDI-high-performance thin-layer chromatography (HPTLC) coupling for the analysis of a crude lipid extract from porcine brain. Brain lipids were chosen because they represent a rather complex mixture and are of currently significant research interest. It was found that normal-phase HPTLC-separated lipids can be easily characterized by direct MALDI-TOF-MS analysis with sufficient resolution to allow the assignment of virtually all lipid classes, even rather minor species such as phosphorylated phosphoinositides or complex glycolipids as gangliosides. Advantages and disadvantages of this approach are discussed.     

Identification of aprotinin degradation products by the use of high-performance capillary electrophoresis, high-pressure liquid chromatography and mass spectrometry.

A preparation of bovine aprotinin, bovine pancreatic trypsin inhibitor, was subjected to high-performance capillary electrophoresis (HPCE) analysis and the purity was calculated to be approximately 80%. The two dominating contaminants were integrated to approximately 7% each as compared to the intact molecule. Characterization by high-pressure liquid chromatographic (HPLC) and mass spectrometric analysis was carried out on digests of the reduced and alkylated molecules. The contaminants were identified as truncated aprotinin, missing one and two amino acids, respectively, at the C-terminus. No such structures were identified in similar amounts in preparations of recombinant aprotinin by HPLC or HPCE.     

Determination of vitamin B5 in human urine by high-performance liquid chromatography coupled with mass spectrometry

In the present work, we have developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the identification and quantification of vitamin B5 in human urine. Urine was spiked with vitamin B5 internal standard, hopantenic acid (HOPA), and then diluted with the LC mobile phase prior to its analysis by LC/MS. The quantification was performed in single ion monitoring mode. The calibration curve was linear (r2 = 0.999) between 0.25 to 10 microg/mL. With a limit of detection of 0.1 microg/mL the method was sensitive enough to determine low levels of vitamin B5 in urine. The overall quantitative efficiency of the method was evaluated by spiking urine samples with four different concentrations of vitamin B5; the intra-assay coefficient of variation was below 5% and the recoveries were between 96 to 108%. The results of the present study show that the proposed method is selective and sensitive enough for the quantification of vitamin B5 in urine.     

Use of high-performance liquid chromatography coupled with high-resolution mass spectrometry for the identification and quantitative determination of tetrodotoxin in pharmaceuticals

A procedure is developed for the identification and determination of tetrodotoxin in pharmaceuticals by liquid chromatography coupled with high resolution mass spectrometry (HPLC?HRMS). The components of a pharmaceutical sample were separated in the gradient elution mode on a HILIC hydrophilic sorbent and detected by HRMS with electrospray ionization in the mode of registration of selected ion transitions. The calibration curve is linear in the range 0.75?1.25 μg/mL with the correlation coefficient > 0.9995.     

Identification of heat-induced degradation products from purified betanin, phyllocactin and hylocerenin by high-performance liquid chromatography/electrospray ionization mass spectrometry

Betanin, phyllocactin (malonylbetanin) and hylocerenin (3-hydroxy-3-methylglutarylbetanin) were isolated from purple pitaya (Hylocereus polyrhizus [Weber] Britton and Rose) juice, and their degradation products generated by heating at 85 degrees C were subsequently monitored by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Thermal degradation of phyllocactin and hylocerenin in purified solution excluding the alleged protective effects by the juice matrix is reported for the first time. Betanin was predominantly degraded by hydrolytic cleavage, while decarboxylation and dehydrogenation were of minor relevance. In contrast, hylocerenin showed a strong tendency to decarboxylation and dehydrogenation, hydrolytic cleavage of the aldimine bond occurring secondarily. Phyllocactin degradation was most complex because of additional decarboxylation of the malonic acid moiety as well as generation and subsequent degradation of betanin due to phyllocactin demalonylation. Upon prolonged heating, all betacyanins under observation formed degradation products characterized by an additional double bond at C2-C3. Hydrolytic cleavage of the aldimine bond of phyllocactin and hylocerenin yielded previously unknown acylated cyclo-dopa derivatives traceable by positive ionization, while application of ESI(-) facilitated the detection of a glycosylated aminopropanal derivative and dopamine, which have never been described before as betanin degradation products.     

Arsenic speciation by coupling high-performance liquid chromatography with inductively coupled plasma mass spectrometry

Summary An ICP-MS detector in combination with HPLC has been evaluated for the analysis of six arsenic compounds. The influence of the presence of an organic modifier in the mobile phase on arsenic response and the quality parameters of the analysis are discussed. Detection limits for arsenic species under study range from 10 to 30 pg. The determination of arsenic compounds in solutions simulating fish or sediment extracts has been used to demonstrate the applicability of this technique.     

Screening hydrolysis products of sulfur mustard agents by high-performance liquid chromatography with inductively coupled plasma mass spectrometry detection

Sulfur mustard (HD), bis(2-chloroethyl)sulfide, is one of a class of mustard agents which are chemical warfare agents. The main chemical warfare hydrolysis degradation products of sulfur mustards are: thiodiglycol, bis(2-hydroxyethylthio)methane, 1,2-bis(2-hydroxyethylthio)ethane, 1,3-bis(2-hydroxyethylthio)propane, and 1,4-bis(2-hydroxyethylthio)butane. The aim of this study is to identify these five hydrolysis degradation products utilizing reversed-phase high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (ICP-MS) for element-specific sulfur detection using a collision/reaction cell and electrospray ionization mass spectrometry to confirm the identification. To date, this is the first study utilizing ICP-MS with ³²S element-specific detection for the analysis of vesicant chemical warfare agent degradation products.     

Analysis of aspartyl peptide degradation products by high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

A reversed-phase HPLC method for the analysis of degradation products of the model aspartyl tripeptides Phe-Asp-GlyNH2 and Gly-Asp-PheNH2 after incubation at pH 2 and 10 was developed. Most of the compounds could be separated with a gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. Resolution of the isomeric pairs L-Phe-alpha-L-Asp-GlyNH2/L-Phe-beta-L-Asp-GlyNH2 and L-Phe-alpha-D-Asp-GlyOH/L-Phe-beta-D-Asp-GlyOH was achieved with a gradient of acetonitrile in phosphate buffer, pH 5.0. Under acidic conditions the major degradation pathway was cleavage of the peptide backbone amide bonds yielding dipeptides and amino acids, C-terminal deamidation as well as formation of succidinimyl peptides. At alkaline pH both deamidation of the C-terminal amide as well as isomerization and concomitant enantiomerization of Asp were observed. The peaks were identified both by reference substances and by online electrospray mass spectrometry. The results were compared to a previous developed capillary electrophoresis method. Diastereomeric pairs ofpeptides that could not be separated by capillary electrophoresis were resolved by HPLC while the separation of corresponding pairs of alpha- and beta-Asp peptides was not always achieved by HPLC in contrast to capillary electrophoresis illustrating that both techniques can be complimentary in peptide analysis.     

Identification of 19 phthalic acid esters in dairy products by gas chromatography with mass spectrometry

A detection method for 19 kinds of phthalic acid ester compounds analyzed by n‐hexane/ether/acetonitrile 1:7:8 v/v/v mixed solvent extraction, quick, easy, cheap, effective, rugged, and safe purification and internal standard method of quantitative gas chromatography with mass spectrometry was established. This method can effectively remove interfering materials, such as lipids, fatty acids, and pigments, from dairy products. The 19 kinds of phthalic acid ester compounds were within a 0.025–0.2 mg/kg range, the recovery rate was 65.2–125.7%, relative standard deviation was 7.9–15.4% (n = 6), and the limit of detection was 0.005–0.02 mg/kg. Concentrations of the 19 kinds of phthalic acid ester compounds ranged between 0.01 and 0.12 mg/kg in ten dairy materials and 20 dairy products. The established method is simple, rapid, accurate, and highly sensitive.     

Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.     

Characterization of atenolol transformation products on light-activated TiO2 surface by high-performance liquid chromatography/high-resolution mass spectrometry

We have studied the photocatalytic transformation of atenolol, 4-[2-hydroxy-3-[(1-methyl)amino]propoxyl]benzeneacetamide, a cardioselective beta-blocking agent used to treat cardiac arrhythmias and hypertension, under simulated solar irradiation using titanium dioxide as photocatalyst. The investigation involved monitoring drug decomposition, identifying intermediate compounds, assessing mineralization, and evaluating toxicity. High-performance liquid chromatography (HPLC) coupled to high-resolution mass spectrometry (HRMS) via an electrospray ionization (ESI) interface was a powerful tool for the identification and measurement of the degradation products; 23 main species were identified. Intermediates were characterized through their chromatographic behavior and evolution kinetics, coupled with accurate mass information. Through the full analysis of MS and MS(n) spectra and a comparison with parent drug fragmentation pathways, the diverse isomers were characterized. Neither atenolol nor the intermediates formed exhibit acute toxicity. All intermediates are easily degraded and no compound identified could withstand 2 h irradiation. Photomineralization of the substrate in terms of carbon mineralization and nitrogen release was rapid and, within 4 h of irradiation, organic nitrogen and carbon were completely mineralized.     

Synthesis of 1-aryl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acids and esters

Ethyl 1-aryl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylates 2 were prepared by the condensation of arylhydrazines with ethyl 3-ethoxy-2-(trifluoroacetyl)-2-propenoate (1a) at low temperature. The corresponding acids were also synthesized. X-ray diffraction analysis of an amide derivative 4 verified the position of the trifluoromethyl group on the pyrazole ring.     

Analysis of carbohydrates in plants by high-performance anion-exchange chromatography coupled with electrospray mass spectrometry

A mass spectrometer was coupled to high-performance anion-exchange chromatography (HPAEC) with the help of electrochemical neutralization of the eluent and post-column addition of lithium chloride for carbohydrate analysis. Parallel selective channels (single ion monitoring) were used to decrease the detection limits and separate unresolved peaks. The mass specific detection allowed the simultaneous analysis of a wide range of sugar alcohols, mono-, di- and oligosaccharides. Carbohydrates extracted from leaves of poplar submitted to drought stress were analyzed using pulsed amperometric detection (PAD), then mass spectrometry. It allowed the confirmation of peak attribution and the identification of salicin as a major compound in the extracts. Different responses to water deficit and re-hydration were obtained for several carbohydrates, suggesting different roles in osmoprotection processes.     

Rapid quantification and characterization of soyasaponins by high-performance liquid chromatography coupled with electrospray mass spectrometry

A method using high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) in the negative mode is presented for the quantification and characterization of different soyasaponins using six authentic soyasaponin standards. This method was successfully applied to the rapid separation of diverse soyasaponins, more than 50, including soyasaponins A in different degrees of acetylation, and soyasaponins B in both their 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated and non-conjugated forms in different samples in one single run for only 30 min. Standard calibration curve was linear over the concentration range of 0.010-1.0 mg/L for each soyasaponin. Within-day and day-to-day relative standard deviations were less than 9.2 and 13.1%, respectively.