Evaluating a direct swabbing method for screening pesticides on fruit and vegetable surfaces using direct analysis in real time (DART) coupled to an Exactive benchtop orbitrap mass spectrometer

Rapid screening of pesticides present on the surfaces of fruits and vegetables has been facilitated by using a Direct Analysis in Real Time (DART(?)) open air surface desorption ionization source coupled to an Exactive(?) high-resolution accurate mass benchtop orbitrap mass spectrometer. The use of cotton and polyester cleaning swabs to collect and retain pesticides for subsequent open air desorption ionization is demonstrated by sampling the surface of various produce to which solutions of pesticides have been applied at levels 10 and 100 times below the tolerance levels established by the United States Environmental Protection Agency (US EPA). Samples analyzed include cherry tomatoes, oranges, peaches and carrots each chosen for their surface characteristics which include: smooth, pitted, fuzzy, and rough respectively. Results from the direct analysis of fungicides on store-bought oranges are also described. In all cases, the swabs were introduced directly into the heated ionizing gas of the DART source resulting in production of protonated pesticide molecules within seconds of sampling. Operation of the orbitrap mass spectrometer at 25,000 full-width half maximum resolution was sufficient to generate high-quality accurate mass data. Stable external mass calibration eliminated the need for addition of standards typically required for mass calibration, thus allowing multiple analyses to be completed without instrument recalibration.     

Suitability of an orbitrap mass spectrometer for the screening of pesticide residues in extracts of fruits and vegetables

For about 500 pesticides, the sensitivity of a benchtop high-resolution mass spectrometer using the Orbitrap for mass separation was compared to that of a widely used (low-resolution) tandem mass spectrometer. Both instruments were coupled to LC and used electrospray ionization. The selectivity of the Orbitrap in the full-scan acquisition mode without fragmentation was evaluated at a resolution of 100 000 full width at half maximum for all pesticides detectable with sufficient sensitivity. For this purpose, quasimolecular ions were extracted within 5 ppm windows from total ion chromatograms of two types of extracts of cucumber, lemon, wheat flour, raisin, and tea. In each of the obtained reconstructed ion chromatograms (individual chromatograms for 500 pesticides, each pesticide in 10 different extracts) the sum of signals not arising from the analyte was used to get a measure on selectivity. In addition, the target analyte list was checked for ions of similar mass. The influence of matrix on the ability to detect low concentrations of fortified pesticides was also studied, with the help of spiked extracts. This part of the survey tested whether analyte peaks were lost because of insufficient mass resolution or an early closing C-Trap (used to control the ion current into the Orbitrap). Finally, the stability of the ion ratio [M+H]+/[M+Na]+ was tested, which may be helpful to confirm the identity of an analyte.     

Full Scan MS in Comprehensive Qualitative and Quantitative Residue Analysis in Food and Feed Matrices: How Much Resolving Power is Required?

In LC full scan based MS screening methods correct mass assignment is essential. Parameters affecting the accuracy of mass assignment, i.e., analyte concentration, complexity of the matrix, and resolving power, were studied using typical examples from the field of residue and contaminant analysis in food and feed. The evaluation was carried out by analyzing samples of honey and animal feed, spiked with 151 pesticides, veterinary drugs, mycotoxins, and plant toxins at levels ranging from 10 to 250 ng/g. Analyses were performed using a single stage Orbitrap with resolving power settings varying from 10,000 to 100,000 (FWHM). For consistent and reliable mass assignment (<2 ppm) of analytes at low levels in complex matrices, a high resolving power (≥50,000) was found to be required. At lower resolving power settings, the error in the assignment of mass increased due to the coelution of analytes with interferences at the same nominal mass. This negatively affected selectivity and quantitative performance due to the inability to use the required narrow mass-extraction windows. In the case of the less complex honey matrix, a resolving power of 25,000 was generally sufficient to obtain a mass assignment error close to the typical instrument mass accuracy (≤2 ppm) down to low concentration levels of 10 ng/g.     

Analysis of β‐blockers in groundwater using large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection and liquid chromatography time‐of‐flight mass spectrometry

Atenolol, nadolol, metoprolol, bisoprolol and betaxolol were simultaneously determined in groundwater samples by large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection (LVI‐LC‐LC‐FD) and liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS). The LVI‐LC‐LC‐FD method combines analyte isolation, preconcentration and determination into a single step. Significant reductions in costs for sample pre‐treatment (solvent and solid phases for clean up) and method development times are also achieved. Using LC‐TOF‐MS, accurate mass measurements within 3 ppm error were obtained for all of the β‐blockers studied. Empirical formula information can be obtained by this method, allowing the unequivocal identification of the target compounds in the samples. To increase the sensitivity, a solid‐phase extraction step with Oasis MCX cartridge was carried out yielding recoveries of 79–114% (n=5) with RSD 2–7% for the LC‐TOF‐MS method. SPE gives a high purification of β‐blockers compared with the existing methods. A 100% methanol wash was allowed for these compounds with no loss of analytes. Limit of quantification was 1–7 ng/L for LVI‐LC‐LC‐FD and 0.25–5 ng/L for LC‐TOF‐MS. As a result of selective extraction and effective removal of coextractives, no matrix effect was observed in LVI‐LC‐LC‐FD and LC‐TOF‐MS analyses. The methods were applied to detect and quantify β‐blockers in groundwater samples of Almería (Spain).     

Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds

Atmospheric pressure photoionization (APPI) is a novel method of ionization in liquid chromatography/mass spectrometry (LC/MS). It was originally developed in order to broaden the range of LC/MS ionizable compounds towards less polar compounds that cannot be analyzed by electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Studies done thus far have shown that non-polar compounds that earlier were not ionizable in LC/MS can indeed be ionized by the use of APPI. However, the best ionization efficiency for low polarity samples has been achieved with low proton affinity (PA) solvents that are not suitable in reversed-phase LC (RP-LC). Here it is demonstrated that the signals for analytes with low proton affinities in acetonitrile can be increased 100-fold by using anisole as the dopant for APPI, which takes the sensitivity to the same level achieved in the analysis of high PA analytes.     

Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma

A sensitive, rapid and specific quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of apomorphine (APO) in canine plasma. The analytes were prepared using one-step liquid-liquid extraction, and analyzed on a Waters Symmetry C(18) column interfaced with triple quadrupole tandem mass spectrometer. A mixture of methanol/0.1% formic acid in water (70: 30, v/v) was employed as the isocratic mobile phase. Positive electrospray ionization was utilized as the ionization source. The analyte and clenbuterol (internal standard) were both detected using multiple reaction monitoring (MRM) mode. The limit of detection (LOD) obtained was 0.03 ng/mL. The assay was linear over the concentration range of 0.1-100 ng/mL, and provided good precision (RSD) and good accuracy (RE). The analyte was stable by using antioxidants throughout the whole study. The experimental results show that LC/MS/MS is a rapid and sensitive method to analyze APO in plasma. Finally, the proposed method was successfully applied to a pharmacokinetic study of APO after intranasal administration of 0.5 mg apomorphine to 10 healthy beagle dogs.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

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.     

Application of hyphenated mass spectrometry techniques for the analysis of urinary free glucocorticoids

Alteration of levels of glucocorticoids in plasma and urine can be related to several diseases. In particular, the determination of endogenous glucocorticoids in urine has been reported to provide information on cortisol and cortisone status, on the activities of steroid hormone enzymes and on glucocorticoid metabolism. In this study, the application of hyphenated mass spectrometry techniques (GC/MS without derivatization and LC/MS) for the simultaneous analysis of free urinary cortisol (F), cortisone (E), tetrahydrocortisol (THF), allo‐tetrahydrocortisol (A‐THF) and tetrahydrocortisone (THE) was evaluated. A sample preparation protocol by solid‐phase extraction, mass spectrometry parameters and chromatographic conditions for both techniques were carefully optimized in terms of extracting phase and solvents, matrix effects, recovery, sensitivity and compound resolution. Baseline separation was achieved for the five underivatized analytes both in GC and LC. The LC/MS/MS technique was more suitable for the analysis of urine samples, being less influenced by matrix effects and showing excellent sensitivity and selectivity. A preliminary application of the reported method for the diagnosis of metabolic diseases was also described. The determination of each analyte in its free form, described for the first time in the paper, offers new perspectives in the application of glucocorticoid analysis for diagnostic purposes. Copyright © 2009 John Wiley & Sons, Ltd.     

Ion suppression and enhancement effects of co-eluting analytes in multi-analyte approaches: systematic investigation using ultra-high-performance liquid chromatography/mass spectrometry with atmospheric-pressure chemical ionization or electrospray ionization

In multi-analyte procedures, sufficient separation is important to avoid interferences, particularly when using liquid chromatography/mass spectrometry (LC/MS) because of possible ion suppression or enhancement. However, even using ultra-high-performance LC, baseline separation is not always possible. For development and validation of an LC/MS/MS approach for quantification of 140 antidepressants, benzodiazepines, neuroleptics, beta-blockers, oral antidiabetics, and analytes measured in the context of brain death diagnosis in plasma, the extent of ion suppression or enhancement of co-eluting analytes within and between the drug classes was investigated using atmospheric-pressure chemical ionization (APCI) or electrospray ionization (ESI). Within the drug classes, five analytes showed ion enhancement of over 25% and six analytes ion suppression of over 25% using APCI and 16 analytes ion suppression of over 25% using ESI. Between the drug classes, two analytes showed ion suppression of over 25% using APCI. Using ESI, one analyte showed ion enhancement of over 25% and five analytes ion suppression of over 25%. These effects may influence the drug quantification using calibrators made in presence of overlapping and thus interfering analytes. Ion suppression/enhancement effects induced by co-eluting drugs of different classes present in the patient sample may also lead to false measurements using class-specific calibrators made in absence of overlapping and thus interfering analytes. In conclusion, ion suppression and enhancement tests are essential during method development and validation in LC/MS/MS multi-analyte procedures, with special regards to co-eluting analytes.     

Some new features of Direct Analysis in Real Time mass spectrometry utilizing the desorption at an angle option

The present study is a first step towards the unexplored capabilities of Direct Analysis in Real Time (DART) mass spectrometry (MS) arising from the possibility of the desorption at an angle: scanning analysis of surfaces, including the coupling of thin‐layer chromatography (TLC) with DART‐MS, and a more sensitive analysis due to the preliminary concentration of analytes dissolved in large volumes of liquids on glass surfaces. In order to select the most favorable conditions for DART‐MS analysis, proper positioning of samples is important. Therefore, a simple and cheap technique for the visualization of the impact region of the DART gas stream onto a substrate was developed. A filter paper or TLC plate, previously loaded with the analyte, was immersed in a derivatization solution. On this substrate, owing to the impact of the hot DART gas, reaction of the analyte to a colored product occurred. An improved capability of detection of DART‐MS for the analysis of liquids was demonstrated by applying large volumes of model solutions of coumaphos into small glass vessels and drying these solutions prior to DART‐MS analysis under ambient conditions. This allowed the introduction of, by up to more than two orders of magnitude, increased quantities of analyte compared with the conventional DART‐MS analysis of liquids. Through this improved detectability, the capabilities of DART‐MS in trace analysis could be strengthened. Copyright © 2011 John Wiley & Sons, Ltd.     

Investigation of uremic analytes in hemodialysate and their structural elucidation from accurate mass maps generated by a multi‐dimensional liquid chromatography/mass spectrometry approach

Historically, structural elucidation of unknown analytes by mass spectrometry alone has involved tandem mass spectrometry experiments using electron ionization. Most target molecules for bioanalysis in the metabolome are unsuitable for detection by this previous methodology. Recent publications have used high‐resolution accurate mass analysis using an LTQ‐Orbitrap with the more modern approach of electrospray ionization to identify new metabolites of known metabolic pathways. We have investigated the use of this methodology to build accurate mass fragmentation maps for the structural elucidation of unknown compounds. This has included the development and validation of a novel multi‐dimensional LC/MS/MS methodology to identify known uremic analytes in a clinical hemodialysate sample. Good inter‐ and intra‐day reproducibility of both chromatographic stages with a high degree of mass accuracy and precision was achieved with the multi‐dimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) system. Fragmentation maps were generated most successfully using collision‐induced dissociation (CID) as, unlike high‐energy CID (HCD), ions formed by this technique could be fragmented further. Structural elucidation is more challenging for large analytes >270 Da and distinguishing between isomers where their initial fragmentation pattern is insufficiently different. For small molecules (<200 Da), where fragmentation data may be obtained without loss of signal intensity, complete structures can be proposed from just the accurate mass fragmentation data. This methodology has led to the discovery of a selection of known uremic analytes and two completely novel moieties with chemical structural assignments made. Copyright © 2009 John Wiley & Sons, Ltd.     

Applicability of gradient liquid chromatography with tandem mass spectrometry to the simultaneous screening for about 100 pesticides in crops

A method was developed for screening crops for a range of pesticide residues by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A complete set of LC, electrospray ionization (ESI), and tandem MS acquisition parameters was established for the determination of 108 analytes; these parameters were used for the simultaneous acquisition of 98 analytes in the positive ESI mode and 10 analytes in an additional MS/MS method in the negative ESI mode. The entire procedure involves extraction of residues with methanol-water and partition into dichloromethane. The utility of the method is demonstrated by the analysis of crops of 5 matrix types (water-containing, acidic, dry, sugar-containing, and fatty). Of 108 pesticides/metabolites tested, 104 showed sufficient stability in most matrixes for determination by LC/MS/MS. These analytes belong to 20 chemical classes, which demonstrate the general applicability of the method for multiclass analysis. By using matrix-matched standards, 67 compounds could be determined in most matrixes with recoveries of 70-120% and a relative standard deviation of < or = 25% at the 0.01 mg/kg level.     

Multiresidue pesticide analysis in ginseng and spinach by nontargeted and targeted screening procedures

Five different mass spectrometers interfaced to GC or LC were evaluated for their application to targeted and nontargeted screening of pesticides in two foods, spinach and ginseng. The five MS systems were capillary GC/MS/MS, GC-high resolution time-of-flight (GC/HR-TOF)-MS, TOF-MS interfaced with a comprehensive multidimensional GC (GCxGC/TOF-MS), an MS/MS ion trap hybrid mass (qTrap) system interfaced with an ultra-performance liquid chromatograph (UPLC-qTrap), and UPLC interfaced to an orbital trap high resolution mass spectrometer (UPLC/Orbitrap HR-MS). Each MS system was tested with spinach and ginseng extracts prepared through a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure. Each matrix was fortified at 10 and 50 ng/g for spinach or 25 and 100 ng/g for ginseng with subsets of 486 pesticides, isomers, and metabolites representing most pesticide classes. HR-TOF-MS was effective in a targeted search for characteristic accurate mass ions and identified 97% of 170 pesticides in ginseng at 25 ng/g. A targeted screen of either ginseng or spinach found 94-95% of pesticides fortified for analysis at 10 ng/g with GC/MS/MS or LC/MS/MS using multiple reaction monitoring (MRM) procedures. Orbitrap-MS successfully found 89% of 177 fortified pesticides in spinach at 25 ng/g using a targeted search of accurate mass pseudomolecular ions in the positive electrospray ionization mode. A comprehensive GCxGC/TOF-MS system provided separation and identification of 342 pesticides and metabolites in a single 32 min acquisition with standards. Only 67 or 81% of the pesticides were identified in ginseng and spinach matrixes at 25 ng/g or 10 ng/g, respectively. MS/MS or qTrap-MS operated in the MRM mode produced the lowest false-negative rates, at 10 ng/g. Improvements to instrumentation, methods, and software are needed for efficient use of nontargeted screens in parallel with triple quadrupole MS.     

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.     

The utility of ultra-performance liquid chromatography/electrospray ionisation time-of-flight mass spectrometry for multi-residue determination of pesticides in strawberry

The utility of ultra-performance liquid chromatography/orthogonal-acceleration time-of flight mass spectrometry (UPLC/TOFMS) for the rapid qualitative and quantitative analysis of 100 pesticides targeted in strawberry was assessed by comparing results with those obtained using a validated in-house UPLC tandem mass spectrometry (MS/MS) multi-residue method. Crude extracts from retail strawberry samples received as part of the 2007 annual UK pesticide residues in food surveillance programme were screened for the presence of pesticide residues using UPLC/TOFMS. Accurate mass measurement of positive and negative ions allowed their extraction following 'full mass range data acquisition' with negligible interference from background or co-eluting species observed during UPLC gradient separation (in a cycle time of just 6.5 min per run). Extracted ion data was used to construct calibration curves and to detect and identify any incurred residues (i.e. pesticides incorporated in or on the test material following application during cultivation, harvest and storage). Calibration using matrix-matched standards was performed over a narrow concentration range of 0.005-0.04 mg kg(-1) with determination coefficients (r2) > or =0.99 for all analytes with the exception of malathion/fenarimol/fludioxanil (r2 = 0.98), quassia/pymetrazine (r2 = 0.97) and fenthion sulfone (r2 = 0.95). Residues found in selected samples ranged from 0.025-0.28 mg kg(-1) and were in excellent agreement with results obtained using UPLC/MS/MS. Mass measurement accuracies of < or =5 ppm were achieved consistently throughout the separation, mass range and concentration range of interest thus providing the opportunity to obtain discrete elemental compositions of target ions.     

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of isoniazid and ethambutol in human plasma

Isoniazid and ethambutol are commonly used in various combination treatments for tuberculosis, and for this reason a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of these two drugs in human plasma. After a simple protein precipitation using methanol, the analytes and the internal standard metformin were chromatographed on a C18 column and detected by MS/MS. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The LC retention times for isoniazid and ethambutol were 2.46 and 2.27 min, respectively. The method was linear in the concentration range of 10.0-5000 ng/mL for each analyte using 100 microL plasma. The intra- and inter-day precisions, expressed as the relative standard deviation (RSD), were less than 5.7 and 6.4%, determined from QC samples for isoniazid and ethambutol, and the accuracies were within +/-2.1% and +/-4.5% in terms of relative error, respectively. The method was successfully employed in a pharmacokinetic study after oral administration of a multicomponent formulation containing 150 mg isoniazid, 500 mg ethambutol, 150 mg rifampicin and 250 mg pyrazinamide.     

An algorithm for thorough background subtraction from high-resolution LC/MS data: application for detection of glutathione-trapped reactive metabolites

A control sample background-subtraction algorithm was developed for thorough subtraction of background and matrix-related signals in high-resolution, accurate mass liquid chromatography/mass spectrometry (LC/MS) data to reveal ions of interest in an analyte sample. This algorithm checked all ions in the control scans within a specified time window around the analyte scan for potential subtraction of ions found in that analyte scan. Applying this method, chromatographic fluctuations between runs were dealt with and background and matrix-related signals in the sample could be thoroughly subtracted. The effectiveness of this algorithm was demonstrated using four test compounds, clozapine, diclofenac, imipramine, and tacrine, to reveal glutathione (GSH)-trapped reactive metabolites after incubation with human liver microsomes supplemented with GSH (30 microM compound, 45-min incubation). Using this algorithm with a +/- 1.0 min control scan time window, a +/- 5 ppm mass error tolerance, and appropriate control samples, the GSH-trapped metabolites were revealed as the major peaks in the processed LC/MS profiles. Such profiles allowed for comprehensive and reliable identification of these metabolites without the need for any presumptions regarding their behavior or properties with respect to mass spectrometric detection. The algorithm was shown to provide superior results when compared to several commercially available background-subtraction algorithms. Many of the metabolites detected were doubly charged species which would be difficult to detect with traditional GSH adduct screening techniques, and thus, some of the adducts have not previously been reported in the literature. Copyright (c) 2008 John Wiley & Sons, Ltd.     

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.