Use of high‐resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose‐dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high‐resolution mass spectrometry led to unequivocal identification of the interfering peak as an N‐desmethyl metabolite of the parent analyte. High‐resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the ¹³C‐isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Isobaric metabolite interferences and the requirement for close examination of raw data in addition to stringent chromatographic separations in liquid chromatography/tandem mass spectrometric analysis of drugs in biological matrix

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS. In the first case, two sequential metabolic reactions, demethylation followed by oxidation of a primary alcohol moiety to a carboxylic acid, produced an isobaric metabolite that exhibits a MRM transition identical to the parent drug. Because the drug compound was rapidly metabolized in rats and completely disappeared in plasma samples, the isobaric metabolite appeared as a single peak in the total ion current (TIC) trace and could easily be quantified as the drug since it was eluted at a retention time very close to that of the drug in a 12-min LC run. In the second example, metabolism via the ring-opening of a substituted isoxazole moiety led to the formation of an isomeric product that showed an almost identical collision-induced dissociation (CID) MS spectrum as the original drug. Because two components were co-eluted, the isomeric product could be mistakenly quantified and reported by data processing software as the parent drug if the TIC trace was not carefully inspected. Nowadays, all LC/MS data are processed by computer software in a highly automated fashion, and some analysts may spend much less time to visually examine raw TIC traces than they used to do. Two examples described in this article remind us that quality data require both adequate chromatographic separations and close examination of raw data in LC/MS/MS analyses of drugs in biological matrix.     

Strategies to avoid false negative findings in residue analysis using liquid chromatography coupled to time-of-flight mass spectrometry

Liquid chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (LC/TOF) provides an attractive alternative to liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS/MS) in the field of multiresidue analysis. The sensitivity and selectivity of LC/TOF approach those of LC/MS/MS. TOF provides accurate mass information and a significantly higher mass resolution than quadrupole analyzers. The available mass resolution of commercial TOF instruments ranging from 10 000 to 18 000 full width at half maximum (FWHM) is not, however, sufficient to completely exclude the problem of isobaric interferences (co-elution of analyte ions with matrix compounds of very similar mass). Due to the required data storage capacity, TOF raw data is commonly centroided before being electronically stored. However, centroiding can lead to a loss of data quality. The co-elution of a low intensity analyte peak with an isobaric, high intensity matrix compound can cause problems. Some centroiding algorithms might not be capable of deconvoluting such partially merged signals, leading to incorrect centroids.Co-elution of isobaric compounds has been deliberately simulated by injecting diluted binary mixtures of isobaric model substances at various relative intensities. Depending on the mass differences between the two isobaric compounds and the resolution provided by the TOF instrument, significant deviations in exact mass measurements and signal intensities were observed. The extraction of a reconstructed ion chromatogram based on very narrow mass windows can even result in the complete loss of the analyte signal. Guidelines have been proposed to avoid such problems. The use of sub-2 microm HPLC packing materials is recommended to improve chromatographic resolution and to reduce the risk of co-elution. The width of the extraction mass windows for reconstructed ion chromatograms should be defined according to the resolution of the TOF instrument. Alternative approaches include the spiking of the sample with appropriate analyte concentrations. Furthermore, enhanced software, capable of deconvoluting partially merged mass peaks, may become available.     

Negative ion electrospray and tandem mass spectrometric analysis of platelet activating factor (PAF) (1-hexadecyl-2-acetyl-glycerophosphocholine)

The analysis of 1-hexadecyl-2-acetyl-glycerophosphocholine (platelet activating factor, PAF) by negative ion and normal-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) was investigated as an alternative technique to the currently used gas chromatography/MS and positive ion LC/MS/MS procedures. The positive ion [M + H]+ derived from PAF and generated by electrospray ionization is abundant, but the potential presence of isobaric 1-octadecanoyl-2-lyso-glycerophosphocholine (stearoyl-lyso-GPC) and 1-hexadecanoyl-2-formyl-glycerophosphocholine (PFPC) in biological samples limits the use of the most abundant collision-induced decomposition (CID) transition (formation of the phosphocholine ion, m/z 524-->184) if chromatographic separation is not achieved. Less abundant CID product ions, such as loss of the neutral ketene molecule derived from the respective fatty acyl groups, provide the requisite specificity, but the intensity of these transitions yields a signal-to-noise ratio that greatly diminishes the analytical sensitivity. With negative ion LC/MS/MS, however, the molecular anions [M - 15]- derived from PAF, stearoyl-lyso-GPC and PFPC decompose to the carboxylate anions at m/z 59, 283 and 255, respectively, permitting discrimination of these isobaric molecules even without chromatographic separation. In addition, the CID of [M - 15]- was favorable, yielding ion currents of sufficient intensity to permit the measurement of PAF when isolated from small quantities of biological material. With the use of a stable isotopically labeled variant of PAF and isotope dilution, negative ion LC/MS/MS was found to measure PAF reliably even in the presence of the isobaric stearoyl-lyso-GPC and permitted the use of non-chlorinated mobile phases for normal-phase high-performance LC.     

Importance of MS selectivity and chromatographic separation in LC‐MS/MS‐based methods when investigating pharmaceutical metabolites in water. Dipyrone as a case of study

Pharmaceuticals are emerging contaminants of increasing concern because of their presence in the aquatic environment and potential to reach drinking‐water sources. After human and/or veterinary consumption, pharmaceuticals can be excreted in unchanged form, as the parent compound, and/or as free or conjugated metabolites. Determination of most pharmaceuticals and metabolites in the environment is commonly made by liquid chromatography (LC) coupled to mass spectrometry (MS). LC coupled to tandem MS is the technique of choice nowadays in this field. The acquisition of two selected reaction monitoring (SRM) transitions together with the retention time is the most widely accepted criterion for a safe quantification and confirmation assay. However, scarce attention is normally paid to the selectivity of the selected transitions as well as to the chromatographic separation. In this work, the importance of full spectrum acquisition high‐resolution MS data using a hybrid quadrupole time‐of‐flight analyser and/or a suitable chromatographic separation (to reduce the possibility of co‐eluting interferences) is highlighted when investigating pharmaceutical metabolites that share common fragment ions. For this purpose, the analytical challenge associated to the determination of metabolites of the widely used analgesic dipyrone (also known as metamizol) in urban wastewater is discussed. Examples are given on the possibilities of reporting false positives of dypirone metabolites by LC‐MS/MS under SRM mode due to a wrong assignment of identity of the compounds detected. Copyright © 2012 John Wiley & Sons, Ltd.     

Distinguishing a phosphate ester prodrug from its isobaric sulfate metabolite by mass spectrometry without the metabolite standard

A phosphate prodrug of a phenolic or alcoholic drug is isobaric with the putative sulfate metabolite of the drug. During liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of biological samples obtained after the administration of a phosphate prodrug, a product ion arising from the parent drug portion of the prodrug molecule is commonly used in selected reaction monitoring (SRM) utilized for the simultaneous quantitation of the prodrug and the in vivo generated parent drug. While the advantage of using a drug moiety‐specific LC‐SRM method is obvious, one drawback is that the sulfate metabolite will also respond to such an SRM transition since the metabolite will invariably yield the same product ion as the prodrug. Thus, the sulfate metabolite could be mistaken for the prodrug unless chromatographic separation between the two is achieved. In the absence of a reference standard for the sulfate metabolite to demonstrate chromatographic separation, it is important to establish a procedure that can ascertain the absence of the sulfate metabolite in the study samples to ensure the specificity of the method for the prodrug. To this end, we studied the MS/MS behavior of model phosphate and sulfate ester compounds and developed a procedure based on phosphate‐specific and sulfate‐specific product ions for distinguishing the phosphate prodrug from the sulfate metabolite. Copyright © 2009 John Wiley & Sons, Ltd.     

ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode

The diagnosis of inherited disorders of amino acids (AA) metabolism is usually performed on automated analysers by ion-exchange chromatography and quantification after ninhydrin derivatisation of about 50 different AA. A single run liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for these molecules can be an alternative to this time-consuming technique. The first step of this development is the infusion study of the fragmentation of 79 molecules of biological interest in electrospray ionisation tandem mass spectrometry (ESI-MS/MS), in positive and in negative ionisation mode. Among them, three molecules can be detected only in negative ionisation mode, 38 only in positive mode and 38 in the two modes. All the most abundant fragmentations are presented, with optimisation of the MS/MS parameters. The positive ionisation mode was retained for the simultaneous analysis of 76 molecules. One sensitive and/or specific transition is proposed for the monitoring of each molecule. Improvement in sensitivity of detection was obtained with the use of an acidic mobile phase. Flow injection analysis studies led us to highlight a number of interferences-due to isobaric molecules, to in-source collision-induced dissociation, or to natural isotopic distribution of the elements-which are listed. For a reliable quantification method, these molecules have to be separated by LC before analysis in the tandem mass spectrometer. Ion-pairing reversed-phase liquid chromatography (RPLC) using perfluorinated carboxylic acids as ion-pairing agents has already been found suitable for analysis of AA in MS/MS positive ionisation mode and is under development.     

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.     

Application of 1-alkylamines to a liquid chromatographic/turbo ionspray tandem mass spectrometric method for quantifying metabolites of a new bone anabolic agent,TAK-778, in human serum

We investigated the application of alkylamines, as additives to the mobile phase, to a quantification method for the metabolites, M-III and M-IV, of TAK-778, which is a new bone anabolic agent, in human serum using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Prior to setting up the analytical method, we found that 1-alkylamines co-existing with M-III and M-IV in the turbo ionsprayed solution formed 1-alkylammonium adduct molecules of these metabolites during the ionization process, and the abundance of the adduct ions was considerably higher than that of protonated molecules ([M + H](+)s) of these metabolites. Based on these findings, we investigated a variety of 1-alkylamines and their spiked concentrations in the mobile phase for LC/MS/MS analysis to obtain higher sensitivities for the quantification of these metabolites. After these examinations, we found that 1-hexylamine at a final concentration of 0.05 mmol l(-1) was the most suitable additive for the mobile phase, and set the selected reaction monitoring (SRM) ions for the 1-hexylammonium adduct molecule and [M + H](+), allowing about a fivefold gain in the SRM chromatographic peak compared with that without 1-hexylamine. The adduct ion was considered to be formed by interaction between the amino group of 1-hexylamine and the phosphoryl group of M-III and M-IV. The internal standard (I.S.) used was deuterated M-III for each metabolite. The analytes and I.S. were extracted with diethyl ether from serum samples at neutral pH and injected into the LC/MS/MS system with a turbo ionspray interface. The limit of quantification for both analytes was 0.5 ng ml(-1) when 0.1 ml of serum was used, and the calibration curves were linear in the range 0.5-100 ng ml(-1). The method was precise; the intra- and inter-day precisions of the method were not more than 5.6%. The accuracy of the method was good, with deviations between added and calculated concentrations of M-III and M-IV being typically within 16.6%. This method provided reliable pharmacokinetic data for M-III and M-IV after the intramuscular administration of TAK-778 sustained-release formulation in humans.     

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 μM up to 2.5 μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.     

Targeted quantitative bioanalysis in plasma using liquid chromatography/high-resolution accurate mass spectrometry: an evaluation of global selectivity as a function of mass resolving power and extraction window, with comparison of centroid and profile modes

There is a growing interest in exploring the use of liquid chromatography coupled with full-scan high resolution accurate mass spectrometry (LC/HRMS) in bioanalytical laboratories as an alternative to the current practice of using LC coupled with tandem mass spectrometry (LC/MS/MS). Therefore, we have investigated the theoretical and practical aspects of LC/HRMS as it relates to the quantitation of drugs in plasma, which is the most commonly used matrix in pharmacokinetics studies. In order to assess the overall selectivity of HRMS, we evaluated the potential interferences from endogenous plasma components by analyzing acetonitrile-precipitated blank human plasma extract using an LC/HRMS system under chromatographic conditions typically used for LC/MS/MS bioanalysis with the acquisition of total ion chromatograms (TICs) using 10 k and 20 k resolving power in both profile and centroid modes. From each TIC, we generated extracted ion chromatograms (EICs) of the exact masses of the [M + H](+) ions of 153 model drugs using different mass extraction windows (MEWs) and determined the number of plasma endogenous peaks detected in each EIC. Fewer endogenous peaks are detected using higher resolving power, narrower MEW, and centroid mode. A 20 k resolving power can be considered adequate for the selective determination of drugs in plasma. To achieve desired analyte EIC selectivity and simultaneously avoid missing data points in the analyte EIC peak, the MEW used should not be too wide or too narrow and should be a small fraction of the full width at half maximum (FWHM) of the profile mass peak. It is recommended that the optimum MEW be established during method development under the specified chromatographic and sample preparation conditions. In general, the optimum MEW, typically ≤ ±20 ppm for 20 k resolving power, is smaller for the profile mode when compared with the centroid mode.     

Analysis of a commercial preparation of erythromycin estolates by tandem mass spectrometry and high performance liquid chromatography/electrospray ionization tandem mass spectrometry using an ion trap mass spectrometer

Because of the lack of a UV chromophore and their much smaller abundances in comparison with the major component, the minor components in erythromycin estolate preparations are difficult to analyze by high performance liquid chromatography ultraviolet (HPLC-UV). Tentative assignment of the major and minor components can be achieved with the combination of full scan and ZoomScan using an ion trap mass spectrometer. Tandem mass spectrometry (MS/MS) provided an effective method to quickly identify most components without chromatographic separation, and all the related compounds, except the isobaric pair ECE and PdMeEA, could be identified in this way. The best result was obtained by using liquid chromatography/tandem mass spectrometry (LC/MS/MS) operated in selected reaction monitoring mode. The major compound, the estolate of erythromycin A (EAE), and seven other minor components, could be separated and identified, with semiquantitative estimates of relative concentrations.     

Sensitive and rapid method to determine trimetazidine in human plasma by liquid chromatography/tandem mass spectrometry

A simple, sensitive, and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method, using electrospray ionization, was developed and validated to quantify trimetazidine in human plasma using propranolol hydrochloride as an internal standard (IS). Samples were prepared by solid-phase extraction and analyzed without drying and reconstitution. The analyte and IS were chromatographed on a C18 reversed-phase column under isocratic conditions using 2 mM ammonium acetate (pH 3.5)-acetonitrile (40 + 60, v/v) as the mobile phase with a run time of 2.0 min. Quantitation was done on a triple-quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring mode to detect parent --> product ion (m/z 267.2 --> 181.4) transition. The method was validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, and stability. Linearity in plasma was observed over the concentration range of 1.5-300 ng/mL. Lower limit of quantification achieved was 1.5 ng/mL with precision < 10% using 10 microL injection volume. The mean relative recovery of analyte (97.36%) and IS (99.93%) was consistent and reproducible. Interbatch and intrabatch precision was < 8.0% and the accuracy determined was within +/- 8% in terms of relative error.     

Strategies for ascertaining the interference of phase II metabolites co‐eluting with parent compounds using LC–MS/MS

LC‐MS/MS is currently the most selective and efficient tool for the quantitative analysis of drugs and metabolites in the pharmaceutical industry and in clinical assays. However, phase II metabolites sometimes negatively affect the selectivity and efficiency of the LC‐MS/MS method, especially for the metabolites that possess similar physicochemical characteristics and generate the same precursor ions as their parent compounds due to the in‐source collision‐induced dissociation during the ionization process. This paper proposes some strategies for examining co‐eluting metabolites existing in real samples, and further assuring whether these metabolites could affect the selectivity and accuracy of the analytical methods. Strategies using precursor‐ion scans and product‐ion scans were applied in this study. An example drug, namely, caffeic acid phenethyl ester, which can generate many endogenous phase II metabolites, was selected to conduct this work. These metabolites, generated during the in vivo metabolic processes, can be in‐source‐dissociated to the precursor ions of their parent compounds. If these metabolites are not separated from their parent compounds, the quantification of the target analytes (parent compounds) would be influenced. Some metabolites were eluted closely to caffeic acid phenethyl ester on LC columns, although long columns and relatively long elution programs were used. The strategies can be utilized in quantitative methodologies that apply LC‐MS/MS to assure the performance of selectivity, thus enhancing the reliability of the experimental data.     

Application of a linear ion trap/orbitrap mass spectrometer in metabolite characterization studies: Examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.     

Rapid and sensitive method for simultaneous determination of six carcinogenic aromatic amines in mainstream cigarette smoke by liquid chromatography/electrospray ionization tandem mass spectrometry

Aromatic amines are one of the sources of carcinogenicity in cigarette and tobacco smoke. Accurate quantification of these chemicals is needed to assess public health risk. A new validated rapid, sensitive and analyte specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/MS/MS) method has been developed for the simultaneous determination of six aromatic amines in mainstream cigarette smoke using research reference cigarette 2R4F. Three popular Indian brand cigarettes were also analyzed using the same procedure. The limit of detection of this method ranged from 0.04 to 0.59 ng/cig using an injection volume of 7 μl. The identification of each amine was established by chromatographic retention times, analyte specific fragmentation pattern and relative peak area ratios of two product/precursor ion pairs. The method showed excellent reproducibility and was also rapid, selective and robust for aromatic amine determination from cigarette smoke.     

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: Applications and constraints

LC coupled to single (LC–MS) and tandem (LC–MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC–MS/MS. Analyses were performed using a previously published LC–MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT–tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their “enhanced” product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.     

Simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene using high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

A simple and rapid method using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene in human urine specimens and standard solutions is described. A hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer was compared for the determination of metabolite of aromatic solvents in urine samples. The metabolites selected were: trans,trans-muconic acid, hippuric acid, o-, m- and p-methylhippuric acid and phenylglyoxylic acid. The compounds were well separated from each other on narrow-bore 1-mm i.d. reversed-phase LC C-18 columns. Average recoveries for loading 100 microL of urine samples varied from 88-110% and the quantification limits were less than 30 ng/mL for each analyte (3 ng/mL for trans,trans-muconic acid). The qualitative information obtained (mass accuracy, resolution and full-scan spectra) with the QqTOF mass spectrometer allows a secure identification of analytes in biological matrices.     

In vivo metabolite detection and identification in drug discovery via LC-MS/MS with data-dependent scanning and postacquisition data mining

An important aspect in drug discovery is the early structural identification of the metabolites of potential new drugs. This gives information on the metabolically labile points in the molecules under investigation, suggesting structural modifications to improve their metabolic stability, and allowing an early safety assessment via the identification of metabolic activation products. From an analytical point of view, metabolite identification still remains a challenging task, especially for in vivo samples, in which they occur at trace levels together with high amounts of endogenous compounds. Here we describe a method, based on LC-ion trap tandem MS, for the rapid in vivo metabolite identification. It is based on the automatic, data-dependent acquisition of multiple product ion MS/MS scans, followed by a postacquisition search, within the entire MS/MS data set obtained, for specific neutral losses or marker ions in the tandem mass spectra of parent molecule and putative metabolites. One advantage of the method is speed, since it requires minimum sample preparation and all the necessary data can be obtained in one chromatographic run. In addition, it is highly sensitive and selective, allowing detection of trace metabolites even in the presence of a complex matrix. As an example of application, we present the studies of the in vivo metabolism of the compound MEN 15916 (1). The method allowed identification of monohydroxy ([M + H](+) = m/z 655), dihydroxy ([M + H](+) = m/z 671), and trihydroxy ([M + H](+) = m/z 687) metabolites, as well as some unexpected biotransformation products such as a carboxylic acid ([M + H](+) = m/z 669), a N-dealkylated metabolite ([M + H](+) = m/z 541), and its hydroxy-analog ([M + H](+) = m/z 557).     

A sensitive liquid chromatography–tandem mass spectrometry method for quantitative bioanalysis of fingolimod in human blood: Application to pharmacokinetic study

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of fingolimod in human blood. The analyte and internal standard fingolimod-d4 were extracted from 300 μl of human blood using protein precipitation coupled with solid-phase extraction method. The chromatographic separation was achieved with a Kinetex biphenyl column (100 × 4.6 mm, 2.6 μm) under isocratic conditions at the flow rate of 0.8 ml/min and column temperature was maintained at 45°C. The detection of analyte and internal standard was carried out by tandem mass spectrometry, operated in positive ion and multiple reaction monitoring acquisition mode. The method was fully validated for its selectivity, precision, accuracy, linearity, stability, detection and quantification limit. The extraction recovery of fingolimod in human blood ranged from 98.39 to 99.54%. The developed method was linear over the concentration range of 5–2500 pg/ml with a detection limit of 1 pg/ml. The developed method was validated and successfully applied for pharmacokinetic study after oral administration of fingolimod capsules.