Measurement of ethylenethiourea using thermospray liquid chromatography-mass spectrometry.

Reliable measurement of ethylenethiourea (ETU) is important because ETU is a potent thyroid carcinogen. A method for the separation and identification of ethylenethiourea (ETU) by applying reversed-phase high-pressure liquid chromatography (HPLC) followed by thermospray (TSP) mass spectrometry (MS) detection is described. Single ion recording detection applying HPLC-MS of ETU appeared to be highly selective and equally sensitive as an HPLC method applying UV detection reported in our earlier study (1). The detection limit for ETU was 100 pg per injection.     

Biotransformation of amlodipine. Identification and synthesis of metabolites found in rat, dog and human urine/confirmation of structures by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry

Metabolism of the dihydropyridine calcium antagonist (R,S)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbony l-5- methoxycarbonyl- 6 -methyl- 1,4-dihydropyridine (amlodipine) has been studied in animals and man using 14C-labelled drug. The metabolite patterns are complex; 18 metabolites have been isolated from rat, dog and human urine. Based on chromatographic and mass-spectral evidence, structures have been proposed for the main metabolites and confirmed by synthesis of unambiguous reference compounds. Comparison of all reference compounds and isolated metabolites was made by gas chromatography-mass spectrometry pressure liquid chromatography on-line thermospray-mass spectrometry of underivatised compounds directly in urine. The metabolites are largely pyridine derivatives. The methods used in structure designation are presented, along with the proposed route of metabolism, which indicates that the metabolic pattern for amlodipine in man has features in common with those of both rat and dog.     

Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry.

Soybean products containing isoflavones are widely consumed in Western and Asian diets for putative health benefits, but adverse effects are also possible. The conjugated forms of isoflavones present in a soy nutritional supplement (predominately acetyl glucosides) and in blood from two human volunteers after consuming the supplement (7- and 4'-glucuronides and sulfates) were identified using liquid chromatography coupled with electrospray/tandem mass spectrometry. Circulating conjugates of genistein and daidzein were quantified using selective enzymatic hydrolysis and deuterated internal standards for liquid chromatography-electrospray/mass spectrometry. The levels of isoflavone glucuronides were much greater than the corresponding sulfates or aglycones. The substrate activities of genistein and daidzein were evaluated with recombinant human UDP glucuronosyl transferase (UGT) and sulfotransferase (SULT) by using enzyme kinetics. The SULTs 1A1*2, 1E, and 2A1 catalyzed formation of a single genistein sulfate; however, SULTs 1A2*1 and 1A3 had no observed activity. None of the SULTs showed activity with daidzein. Although several UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) catalyzed 7- and 4'-glucuronidation of genistein or daidzein, the UGT 1A10 isoform, which is found in human colon but not liver, was found to be specific for genistein. Glucuronidation of only genistein was observed in human colon microsomes, although nearly equal activity was observed for daidzein in human liver and kidney microsomes. These findings suggest a prominent role for glucuronidation of genistein in the intestine concomitant with absorption, although hepatic glucuronidation of absorbed genistein and daidzein aglycones is also likely.     

Investigation of the toxin profile of Greek mussels Mytilus galloprovincialis by liquid chromatography-mass spectrometry.

Samples of Mytilus galloprovincialis were harvested from five different locations in Thermaikos gulf, Greece after harmful algae bloom. All of the mussel samples were found positive by mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins. Liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and yessotoxins (YTXs). In order to investigate the presence of okadaic acid esters, alkaline hydrolysis was performed for all the samples, and LC-MS analyses were carried out on the samples before and after hydrolysis. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analyses were also carried out to investigate the presence of domoic acid and paralytic shellfish poisoning (PSP) toxins at trace levels. All of the samples were found to be contaminated only with okadaic acid at levels 0.10-0.20 microg/g.     

Determination of roxithromycin in rat lung tissue by liquid chromatography-mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method for the determination of roxithromycin in rat lung tissue is described. Liquid-liquid extraction was adopted for sample preparation with recoveries from 72.5 to 76.9% at levels of 0.1, 5.0 and 20.0 microg/ml. Chromatographic separation was performed on a C18 column using a mixture of methanol, water and formic acid (80:20:1, v/v/v) as mobile phase delivered at a flow rate of 0.5 ml/min. Positive selected ion monitoring (SIM) mode was used for the quantification of roxithromycin at m/z 837.7 and clarithromycin (internal standard) at m/z 748.7. The linearity was obtained over the concentration range of 0.05-20.0 microg/ml and the lower limit of quantification was 0.05 microg/ml. For each QC level of roxithromycin, the intra- and inter-day precisions relative standard deviation (R.S.D.) were less than 4.1 and 7.5%, respectively, and accuracy (RE) was +/-10.0%. The proposed LC-MS method has been successfully used for the determination of roxithromycin in rat lung tissue after oral administration of roxithromycin formulations to 44 SD rats. The present study demonstrates that the concentration of roxithromycin in rat lung tissues can be significantly increased by ambroxol when they are formulated in combination.     

Quantitative determination of daumone in rat plasma by liquid chromatography-mass spectrometry

Daumone, 6-(3,5-dihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-heptanoic acid is a pheromone secreted by Caenorhabditis elegans, and has been known as a pivotal regulator of chemosensory processes in development and ageing. A quantification method using mass spectrometry was developed for the determination of daumone in rat plasma. After simple protein precipitation with acetonitrile including an internal standard, the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations after a single intravenous administration of daumone in rats.     

Investigation of the temperature stability of premarin intravenous using liquid chromatography-mass spectrometry

Stability of Premarin^®Intravenous was investigated in dry and reconstituted forms by monitoring major components in samples for a period of six months, using liquid chromatography–mass spectrometry. The components, largely comprising a series of estrogen and steroid hormone sulfates, were considered to be fairly stable (variation ≤ 10%) for dry samples stored at room temperature and at 38 °C (100 °F) during the experimental time frame. However, significant variation, especially after 2 months of storage, was observed in reconstituted solutions. This variation was significantly larger for samples stored at elevated vs. room temperature. It was interesting to note that the concentration of equilenin sulfate increased over time, whereas that of other major components were seen to fluctuate and decrease. This phenomenon was partially explained by the conversion of equilin compounds into their corresponding equilenin forms, a phenomenon which was further investigated through a storage study with pure standard solutions and by tandem mass spectrometry.     

Characterization of antisense oligonucleotide-peptide conjugates with negative ionization electrospray mass spectrometry and liquid chromatography-mass spectrometry

Covalent post-synthesis or solid-phase conjugation of peptides to oligonucleotides has been reported as a possible method of delivering antisense oligonucleotides into cells. While synthesis strategies for preparing these conjugates have been widely addressed, few detailed reports on their structural characterization have been published. This paper discusses the negative ion electrospray ionization mass spectrometric (ESI-MS) and liquid chromatography-mass spectrometric (LC-MS) analysis of various peptide-oligonucleotide conjugates ranging from small T(6)-nucleopeptides to large peptide-oligonucleotide phosphorothioate conjugates and ribozyme-peptide hybrids (3-13 kDa). Molecular weight determination with mass errors of 0.1-3.1 amu were conducted, employing on-line IP-RP-HPLC and high m/z range mode to facilitate the analysis of large compounds and difficult modifications.     

High-performance liquid chromatography-mass spectrometry in the clinical laboratory

The development of HPLC-atmospheric pressure ionization-mass spectrometry (HPLC-MS) has presented clinical laboratories with a powerful analytic tool. The aim of this paper is to provide an overview of the current status of HPLC-MS in the clinical laboratory; to discuss the challenges to mass spectrometry in this setting; and to present some of the latest developments in instrumentation and illustrate their potential application. Currently, the major clinical applications for HPLC-MS are neonatal screening for metabolic disorders, therapeutic drug monitoring of immunosuppressant and HIV/AIDS drugs, and toxicological investigations. The major barrier to the uptake of this technology in the clinical laboratory is the initial capital outlay for instrumentation. A secondary reason is the lack of suitably trained scientists. The challenges that clinical HPLC-MS face are (I) ease of use and automation, (2) interpatient variability in relation to matrix effects, (3) availability of suitable internal standards, and (4) harmonization of methods to meet regulatory requirements. The development of the triple quadrupole linear ion trap mass analyzer allows the quantification power of a triple quadrupole mass analyzer to be combined with the scanning ability of an ion trap.This hybrid instrument allows different permutations of scan combinations. The combination of selected reactant monitoring and MS3 is an attractive combination for quantification. The ion source, atmospheric pressure photoionization, has recently been developed and is well suited to nonpolar analytes, although its role is yet to be established. This ion source complements other interfaces used in HPLC-MS. Both of these advances in instrumentation add to the potential applications of HPLC-MS. How HPLC-MS goes forward into the clinical laboratory is dependent on clinical scientists, instrument manufacturers, and regulatory authorities.     

Quantitative determination of nitrendipine in human plasma using high-performance liquid chromatography-mass spectrometry

A sensitive and highly selective liquid chromatography-mass spectrometry (LCMS) method was developed to determine nitrendipine (4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3, 5-pyridinedicarboxylic acid ethyl methyl ester, CAS 39562-70-4) in human plasma. The analyte and the internal standard nimodipine (CAS 66085-59-4) were extracted from plasma samples by n-hexane-isopropanol (95:5, v/v), and analyzed on a commercially available column Interfaced with a mass spectrometer. Positive atmospheric pressure chemical ionization (APCI) was empolyed as the ionization source. The samples were detected by the use of selected ion monitoring (SIM) mode. The mobile phase consisted of methanol-water (75:25, v/v). The method has a limit of detection (LOD) of 0.1 ng/ml. The linear calibration curves were obtained in the concentra tions range of 0.3-40 ng/ml (r2 > or = .99). The intra- and inter-day batch precisions were lower than 10% in terms of relative standard deviation (R. S. D.), and the accuracy ranged from 85 to 110% in terms of percent accuracy. The overall extraction recoveries were determined to be about 75% on average. This validated method was successfully applied to the evaluation of the pharmacokinetic profiles of nitrendipine tablets administered to 8 Chinese healthy volunteers.     

Characterization of in vitro and in vivo metabolism of leelamine using liquid chromatography-tandem mass spectrometry

1. Leelamine is a diterpene compound found in the bark of pine trees and has garnered considerable interest owing to its potent anticancer properties. The aim of the present study was to investigate the metabolic profile of leelamine in human liver microsomes (HLMs) and mice using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

2. We found that leelamine undergoes only Phase I metabolism, which generates one metabolite that is mono-hydroxylated at the C9 carbon of the octahydrophenanthrene ring (M1) both in vitro and in vivo. The structure and metabolic pathway of M1 were determined from the MSⁿ fragmentation obtained by collision-induced dissociation using LC-MS/MS in HLMs.

3. Cytochrome p450 (CYP) 2D6 was found to be the dominant CYP enzyme involved in the biotransformation of leelamine to its hydroxylated metabolite, whereas CYP2C19, CYP1A1, and CYP3A4 contributed to some extent.

4. Moreover, we identified only one metabolite M1, in the urine, but none in the feces. In conclusion, leelamine was metabolized to a mono-hydroxyl metabolite by CYP2D6 and mainly excreted in the urine.

     

High-speed simultaneous determination of nine antiepileptic drugs using liquid chromatography-mass spectrometry

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important and widely practiced. However, simultaneous AED assays usually concentrate only on old or new AEDs. A new simultaneous assay was developed to monitor both older and newer AEDs in the same sample. This assay measures zonisamide (ZNS), lamotrigine (LTG), topiramate (TPM), phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), carbamazepine-10,11-diol (CBZ-Diol), 10-hydroxycarbamazepine (MHD), and carbamazepine-10,11-epoxide (CBZ-E). Sample pretreatment consisted of a single solid-phase extraction (SPE) for all AEDs in a 100-microL plasma sample. HPLC separation was achieved on a Shimdazu Shimpack XR-ODS (4.6 id x 50 mm, 2.2-mum) column with a gradient mobile phase of acetate buffer, methanol, acetonitrile, and tetrahydrofuran. Four internal standards were used. Detection was achieved by atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in selected-ion monitoring (SIM) mode with constant polarity switching. High recovery (88%-96%) was obtained for all compounds by SPE. Linearity was observed throughout an 80-fold concentration range, with correlation coefficient (r) values higher than 0.99 for all AEDs. For the standards, the accuracy ranged from 89.3% to 111.8%. The within-run coefficient of variation (CVw) value was < or =9.7%, the between-run coefficient of variation (CVb) was < or =16.2%, and the total variability (CVt) was < or =16.8%. For the quality controls (QCs), accuracy ranged from 89.3% to 108.8%, CVw was < or =9.6%, CVb was < or =14.1%, and CVt was < or =15.1%. The correlation r values for comparison of this assay with existing validated assays in our laboratory (GC-MS or LC-MS) were 0.95, 0.91, 0.87, 0.95, and 0.95 for PHT, LTG, CBZ, CBZ-E, and CBZ-Diol, respectively.     

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.     

Structural elucidation of hydroxylated metabolites of the isoflavan equol by gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry.

Equol has, as have other isoflavonoids, recently gained considerable interest due to its possible health effects. However, detailed studies on the metabolism of equol are scarce. Therefore, we investigated the phase I metabolism of equol using liver microsomes from Aroclor-treated male Wistar rats as well as from a male human. The identification of the metabolites formed was elucidated using high performance liquid chromatography (HPLC) with diode array detection, HPLC/atmospheric pressure ionization electrospray mass spectrometry, and gas chromatography-mass spectrometry, as well as reference compounds. (+/-)-Equol was converted to 11 metabolites by the liver microsomes from Aroclor-pretreated rats comprising three aromatic monohydroxylated and four aliphatic monohydroxylated as well as four dihydroxylated products. The main metabolite was identified as 3'-hydroxy-equol. Using human liver microsomes, equol was converted to six metabolites with 3'-hydroxy- and 6-hydroxy-equol as main products. Furthermore, the aliphatic hydroxylated metabolite 4-hydroxyequol, which was recently detected in human urine after soy consumption, was formed. On the basis of these findings, it is suggested that phase I metabolism of equol is part of a complex biotransformation of the soy isoflavone daidzein in humans in vivo.     

Characterization of in vitro metabolites of the aryl hydrocarbon receptor ligand 6-formylindolo[3,2-b]carbazole by liquid chromatography-mass spectrometry and NMR.

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) exhibits the highest aryl hydrocarbon receptor (AhR) binding affinity reported so far. In different cells, in vitro, both extracts of UV-irradiated tryptophan and the synthesized pure compound FICZ induce a rapid and transient expression of AhR-regulated genes. The transient induction suggests that the biotransformation gene battery induced by AhR activation takes part in a metabolic degradation of the ligand, whereby a low steady-state level is regained. The down-regulation of AhR-regulated gene expression was previously shown to be dependent on cytochrome P450 1A1 (CYP1A1). Metabolism of FICZ generates five major metabolites, which appeared as three peaks (M1-M3) in the high performance liquid chromatography. The aim of the present study was to use rat liver S9 from Aroclor-pretreated rats to produce large enough quantities of FICZ metabolites for structure characterization and to determine their product precursor relationship. NMR analysis of large combined fractions of the metabolites indicated that M3 and M2 contained 2 isomers, respectively. By means of liquid chromatography-mass spectrometry (negative ion electrospray mode) and NMR spectroscopy (by (1)H-NMR, correlation spectroscopy, and nuclear Overhauser effect spectroscopy techniques) five metabolites of FICZ were identified, and their structures were elucidated. The molecular weights of the two M3 isomers were 300 and both M2 and M1 compounds demonstrated molecular weights of 316, corresponding to addition of one (M3) and of two oxygen (M2 and M1), respectively. The structures were assigned as 2- and 8-hydroxy (M3), 2,10- and 4,8-dihydroxy (M2) and 2,8-dihydroxy derivatives of indolo[3,2-b]carbazole-6-carboxaldehyde (6-formylindolo[3,2-b]carbazole).     

Toxin profile of Alexandrium ostenfeldii (Dinophyceae) from the Northern Adriatic Sea revealed by liquid chromatography-mass spectrometry.

This paper reports on the first occurrence of fairly high numbers of Alexandrium ostenfeldii along the Emilia Romagna coasts (Italy). Detailed liquid chromatography-mass spectrometry (LC-MS) analyses of the toxin profile were performed on a strain of the organism collected in November 2003, isolated during the event and grown in culture. Selected ion monitoring (SIM) and multiple reaction monitoring (MRM) experiments were carried out for detection of spirolides and paralytic shellfish poisoning (PSP) toxins. They revealed that the Adriatic A. ostenfeldii produces mainly spirolide 13-desmethyl C at levels of 3.7 pg/cell but not PSP toxins. Interestingly, low levels of some spirolide isomers that have not been reported so far in other strains of the dinoflagellate were also detected. This represents the first report of spirolide-type toxins in the Adriatic Sea.     

基于液相色谱-质谱技术的乳腺癌转移相关 代谢标志物的筛选

摘要：目的 应用代谢组学技术筛选与乳腺癌转移相关的代谢标志物。方法 收集100例乳腺癌患者和50例 健康志愿者的血清标本,采用高效液相色谱-轨道离子阱质谱联用（HPLC-LTQ Orbitrap XL MS）代谢组学研究平台分 析乳腺癌未转移患者、乳腺癌转移患者和健康人群血清标本的代谢轮廓,并通过模式识别方法结合非参数检验对数 据进行分析。结果 由乳腺癌未转移组、乳腺癌转移组和健康对照组的代谢轮廓构建的正交偏最小二乘判别分析 （OPLS-DA）模型具有很好的判别能力（R2=95.2%,Q2=86.7%）,可以鉴别出用于区分乳腺癌转移与否的8个代谢标志 物,包括溶血磷脂酸［18∶1（9Z）/0∶0］、溶血磷脂酰胆碱（18∶0）、溶血磷脂酰胆碱［20∶3（5Z,8Z,11Z）］、胆碱、磷酸二羟 丙酮（18∶0e）、2R,3S-番石榴酸、芥酸、L-氢化乳清酸。结论 利用代谢组学方法获得的血清代谢轮廓可以用来构建 区分模型和寻找乳腺癌转移相关的代谢标志物,为乳腺癌的早期诊治、预后评估和药物治疗靶点的选择提供支持和 依据。     

An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.     

Applications of liquid chromatography-mass spectrometry in doping control

This paper reviews liquid chromatographic-mass spectrometric (LC-MS) procedures for the screening, identification and quantification of doping agents in urine and other biological samples and devoted to drug testing in sports. Reviewed methods published approximately within the last five years and cited in the PubMed database have been divided into groups using the same classification of the 2004 World Anti-Doping Agency (WADA) Prohibited List. Together with procedures specifically developed for anti-doping analysis, LC-MS applications used in other fields (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been included when considered as potentially extensible to doping control. Information on the reasons for potential abuse by athletes, on the requirements established by WADA for analysis, and on the WADA rules for the interpretation of analytical findings are provided for the different classes of drugs.     

Chromatographic screening for zopiclone and metabolites in urine using liquid chromatography and liquid chromatography-mass spectrometry techniques

Zopiclone is a benzodiazepine-like hypnotic that was believed not to have any abuse potential. Nevertheless, during the past few years there have been an increasing number of reports on the abuse and misuse of zopiclone. Despite this, methods for screening analysis in urine are lacking. To investigate whether UV detection would be possible to use for this purpose, a liquid chromatography method with ultraviolet detection for analyzing zopiclone and its urinary metabolites was developed, with liquid chromatography-mass spectrometry for confirmation. The method was used for analyzing samples from subjects receiving methadone. The limits of detection were approximately 100 ng/mL in control urine samples and 500 ng/mL in urine samples from subjects receiving methadone. However, due to the high background in these patients' urine, a single therapeutic dose was impossible to detect.