Development of LC/MS/MS methods for cocktail dosed Caco-2 samples using atmospheric pressure photoionization and electrospray ionization

Good reliability of Caco-2 permeability studies requires competent sampling and analytical methods to ensure the comparability of day-to-day experiments. In this work, two n-in-one LC/MS/MS methods based on two different ionization techniques were developed and validated for a group of reference compounds; eight of them are recommended by the Food and Drug Administration (FDA) for the evaluation of oral drug permeability. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), as an interface for quantitative LC/MS analysis was evaluated in comparison to the electrospray ionization (ESI). Generally, the validation parameters, including sensitivity, accuracy, and repeatability, were comparable for the APPI and ESI methods. The main difference was that the linear quantitative range of APPI was 3-4 orders of magnitude (r(2) >/= 0.998) whereas in ESI it was typically 2-3 orders of magnitude (r(2) >/= 0.990). By the APPI and ESI methods, the simultaneous analysis of nine highly heterogeneous compounds was achieved within 5.5-7 min, which leads to significant savings in time and cost of the analyses. The successful validation data indicate the usefulness of both the methods for the rapid and sensitive (LOD values typically 相似文献   

Comparison of atmospheric pressure photoionization and atmospheric pressure chemical ionization for normal-phase LC/MS chiral analysis of pharmaceuticals

In this work, we compared APPI and APCI for normal-phase LC/MS chiral analysis of five pharmaceuticals. Performance was compared both by FIA and by on-column analysis using a ChiralPak AD-H column under optimized conditions. By comparison, APPI generated more reproducible signals and was less susceptible to ion suppression than APCI. APPI generated higher peak area and lower baseline noise, and therefore much higher S/N ratios. APPI sensitivity (i.e., S/N ratio) was approximately 2-130 times higher than APCI by FIA and was approximately 2.6-530 times higher than APCI by on-column analysis depending on specific compounds. The better APPI sensitivity as compared to APCI was more dramatic by on-column analysis than by FIA. APCI sensitivity was degraded by ion suppression caused by LC column bleeding components and by elevated APCI baseline noise relative to APPI. On-column APPI LODs (at S/N = 3) were 83, 16, 17, 95, and 7 pg for enantiomer #1, and 104, 23, 19, 122, and 17 pg for enantiomer #2 for benzoin, naringenin, mianserin, mephenesin, and diperodon, respectively, on a Waters ZQ. APPI offers no concern of explosion hazard relative to APCI corona needle discharge or ESI high voltage discharge when flammable solvents (e.g., hexane) are used as mobile phases. Whether APPI dopants are required depends on the IP(s) of mobile-phase solvent(s) and solvent complexes, and photon energies of VUV lamps. Dopant was not necessary for hexane-based mobile phases due to their self-doping effects. Dopants did enhance Kr lamp APPI sensitivity when MeOH was used as the mobile phase. However, dopants became unnecessary for the MeOH mobile phase when the Ar lamp was used.     

Analysis of phenothiazine and its derivatives using LC/electrochemistry/MS and LC/electrochemistry/fluorescence

The on-line electrochemical conversion of phenothiazine and its derivatives after liquid chromatographic separation has been studied by mass spectrometry and fluorescence spectroscopy. In an electrochemical cell consisting of porous glassy carbon, the phenothiazines are readily converted to oxidized products, which can be detected by on-line fluorescence spectroscopy and mass spectrometry. The method allows rapid investigations on the electrochemical oxidation pathways, as demonstrated for phenothiazine itself. The phenothiazine derivatives are transferred into their strongly fluorescent sulfoxides. Based on this reaction, an LC/electrochemistry/fluorescence method was developed that allows for limits of detection between 5 x 10(-9) and 4 x 10(-8) mol/L and limits of quantification between 2 x 10(-8) and 1 x 10(-7) mol/L for the individual phenothiazines. The linear ranges comprised three decades starting at the limit of quantification.     

Validated comprehensive analytical method for quantification of coenzyme A activated compounds in biological tissues by online solid-phase extraction LC/MS/MS

We report a robust, reliable, and comprehensive analytical method for the identification and quantification of the entire class of coenzyme A (CoA) activated substances, particularly short-, medium-, and long-chain acyl-CoAs derived from various biological tissues. This online SPE-LC/MS/MS-based method is characterized by a simple three-step sample preparation: (1) addition of buffer, organic solvents, and internal standards; (2) homogenization; and (3) centrifugation. The supernatant is injected directly into the SPE-LC/MS/MS system. Identification of CoA activated compounds is performed by accurate mass determination within the HPLC run. Method validation for short-, medium-, and long-chain acyl-CoA fatty acids revealed excellent quality. Accuracy was found to be between 87 and 107% and precision was between 0.1 and 12.8% in mouse skeletal muscle. The lower limit of quantification for all investigated compounds was well below 3.1% of estimated physiological levels in 200 mg of mouse tissue. Comparable results were obtained for mouse liver, mouse brown white adipose tissue and rat liver. For all investigated tissues, no matrix effect was observed.     

Analysis of perchlorate in water and soil by electrospray LC/MS/MS

A method has been developed for the determination of perchlorate in water and soil matrixes using electrospray liquid chromatography/mass spectrometry/mass spectrometry. Perchlorate is quantitated by monitoring the ion signal from mass 83, which is formed by a loss of an oxygen atom from the perchlorate molecular ion. The method was developed to be effective and economical in production laboratory analysis of perchlorate in environmental water and soil samples. Data were gathered to define method sensitivity, performance, selectivity, and robustness. Analyte stability, method susceptibility to interferences, and the reliability of the chlorine isotope ratio as an identification tool were examined. The aqueous method detection limit (MDL) is 0.05 microg/L and was determined using an actual groundwater matrix. The soil MDL is 0.5 microg/kg and was determined using Ottawa sand. The stability study was performed by spiking water samples at 0.25, 10, and 20 microg/L and analyzing them 50 days later. Acceptable recoveries were obtained for all samples. The relative standard deviation (RSD) for the replicate analyses in the stability study indicates that the method is capable of RSD values less than 5% in a relatively clean groundwater matrix. The ionization suppression study was performed by spiking water samples containing 1000 mg/L carbonate, chloride, and sulfate with 0.05 and 0.5 microg/L perchlorate and then measuring the recovery of the spike. The results indicate that the procedure does not have significant suppression effects at the high salt levels tested. Calibration, quality control sample, field sample, and suppression study data were combined to examine isotope ratio reliability. The results of that work show that chlorine isotope ratios can be used to define statistical process control limits for use as an additional analyte identification tool.     

Mass analysis of biological macromolecules at atmospheric pressure using nonresonant femtosecond laser vaporization and electrospray ionization

A nonresonant femtosecond laser pulse, with an intensity of 10(13) Wcm(-2), vaporizes proteins and biomolecules intact, regardless of molecular structure, size or electronic structure for subsequent electrospray ionization and transfer into a mass spectrometer. Rapid, direct analysis from dried sample, aqueous solution and cellular material is demonstrated at atmospheric pressure using laser electrospray mass spectrometry (LEMS). Measurements are presented for lysozyme (14.3 kDa), hemoglobin from human blood, ovalbumin (45 kDa) from hen egg white and phospholipids from hen egg yolk. Mass analysis of biological material is performed without dilution, extraction or sample preparation, other than placing the biological material onto the sample plate.     

Extraction and quantification of phytoestrogens in foods using automated solid-phase extraction and LC/MS/MS

Phytoestrogens are a group of polyphenolic plant metabolites that can induce biological responses. Their bioactivity is based on their similarity to 17beta-estradiol and their ability to bind to the beta-estrogen receptor. Although epidemiological data are inconclusive, phytoestrogens are considered to be beneficial for a variety of conditions, for example, hormone-related cancers like breast and prostate cancer. To investigate the biological effects of these compounds and to assess the exposure of larger cohorts or the general public, reliable data on the phytoestrogen content of food is necessary. Previously, food analysis for phytoestrogens was performed using either HPLC-UV or GC/MS. Here, we describe the development of the first generic method for the analysis of phytoestrogens in food, using automated solid-phase extraction and liquid chromatography-tandem mass spectrometry. The presented method shows a good reproducibility and can be easily adapted to other phytoestrogens if required.     

Analysis of perchlorate in water by reversed-phase LC/ESI-MS/MS using an internal standard technique

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- --> 35ClO3- and 37ClO4- --> 37ClO3- mass transitions. The 35ClO4- --> 35ClO3- transition was quantitated against the internal standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- --> 37ClO3- transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 microg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 microg/L) by 3-18%. The developed reversed-phase LC/ESI-MS/MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 microg/L for deionized reagent water and 0.009 microg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 microg/L.     

Evaluation of triterpene glycoside estrogenic activity using LC/MS and immunoaffinity extraction

We present a study on the mass spectral as well as the binding properties of three triterpene glycosides (cimicifugoside, cimiracemoside F, 27-deoxyactein) contained in black cohosh to the ligand binding domain of estrogen receptor beta (ER-beta). Using affinity ultrafiltration and LC/ MS detection, initial experiments using estradiol and the phytoestrogens daidzein and genistein (compounds known to bind ER-beta) were performed to serve as positive controls. The same affinity techniques and LC/MS procedures were then employed to show that neither the triterpene glycosides nor their enzymatically prepared aglycons bound significantly to ER-beta, except for 27-deoxyactein aglycon, which showed weak binding affinity (4%). Additionally, metabolites of the aglycons were prepared by incubation with female human liver microsomes and subjected to binding experiments with ER-beta. No significant binding of the metabolites to the receptor was observed. Further studies are needed to fully characterize whether these triterpene glycosides as well as other components of black cohosh in this plant extract bind to the estrogen receptor alpha (ER-alpha).     

Atmospheric pressure photoionization for ionization of both polar and nonpolar compounds in reversed-phase LC/MS

Atmospheric pressure photoionization can provide high ionization efficiency simultaneously to both polar and nonpolar compounds delivered in reversed-phase solvent. The method to achieve this utilizes toluene as a dopant and simply requires that the solvent flow be limited so that reactions between toluene photoions and the organic component of the solvent are not driven to completion. Under these conditions, toluene photoions remain in the source for ionizing nonpolar compounds via charge exchange (electron transfer), while protonated solvent ions are available for proton-transfer reactions with polar molecules. The reagent ion mixture is then suitable for ionizing a wide range of both polar and nonpolar compounds. The critical effect of solvent flow rate is demonstrated here with results for a test analyte, 9-methylanthracene, which may be ionized by either charge exchange or proton transfer. For a solvent of 50:50 methanol/water (v/v), lowering the flow from 200 to 50 microL min-1 results in a 10x increase in charge exchange ionization efficiency--further flow reductions provide even greater enhancements. This method is compatible with sample delivery by direct infusion and micro- and narrow-bore LC, as well as conventional LC using a flow splitter.     

Quantification of NTproBNP in rat serum using immunoprecipitation and LC/MS/MS: a biomarker of drug-induced cardiac hypertrophy

Brain natriuretic peptide (BNP) and N-terminal proBNP (NTproBNP) are well established in the clinic as biomarkers of heart failure. BNP hormone and the inactive NTproBNP are predominantly secreted in the ventricles of the heart in response to pressure overload and, consequently, are being investigated as markers of drug-induced cardiac hypertrophy in rat to support drug development. In the work presented here, an immunoaffinity-based LC/MS/MS assay was developed and validated to measure a selective tryptic fragment of NTproBNP in rat serum. The assay covers the range of 13-329 pg/mL of the tryptic fragment LLELIR, corresponding to 0.1-2.5 ng/mL intact NTproBNP. A stable isotope-labeled version of NTproBNP containing the tryptic fragment LLELI[13C615N1]R was prepared by solid-phase peptide synthesis and was used as an internal standard to minimize assay variability. Due to endogenous NTproBNP present in rat serum, human serum was used as the control matrix, and parallelism between rat and human serum was established by standard addition. Assay accuracy (% RE) and precision (% CV) were measured at three concentrations on each of 4 days and did not exceed 4.2 and 14.5%, respectively. Additionally, study data are presented from the application of this assay in which rats demonstrated a significant increase in NTproBNP serum concentration following administration of an agent known to induce cardiac hypertrophy. In this study, the relationship between serum NTproBNP and cardiac hypertrophy was corroborated by increases in heart weight and magnetic resonance imaging of the test subjects' left ventricle. To our knowledge, this represents the first reported assay for NTproBNP in preclinical species for the assessment of cardiac hypertrophy.     

Collection, storage, and filtration of in vivo study samples using 96-well filter plates to facilitate automated sample preparation and LC/MS/MS analysis

The benefits of high-throughput bioanalysis within the pharmaceutical industry are well established. One of the most significant bottlenecks in bioanalysis is transferring in vivo-generated study samples from their collection tubes during sample preparation and extraction. In most cases, the plasma samples must be stored frozen prior to analysis, and the freeze/thaw (F/T) process introduces thrombin clots that are capable of plugging pipets and automated liquid-transfer systems. A new approach to dealing with this problem involves the use of Ansys Captiva 96-well 20-microm polypropylene filter plates to collect, store frozen, and filter plasma samples prior to bioanalysis. The samples are collected from the test subjects, and the corresponding plasma samples are placed directly into the wells of the filter plate. Two Duoseal (patent pending) covers are used to seal the top and bottom of the plate, and the plate is stored at down to -70 degrees C. Prior to sample analysis, the seals are removed and the plate is placed in a 96-well SPE manifold. As the plasma thaws, it passes (by gravity or mild vacuum) through the polypropylene filter into a 96-well collection plate. A multichannel pipet or automated liquid-transfer system is used to transfer sample aliquots without fear of plugging. A significant advantage of this approach is that, unlike other methods, issues related to incomplete pipetting are virtually eliminated. The entire process is rapid since thawing and filtering take place simultaneously, and if a second F/T cycle is required for reanalysis, it is not necessary to refilter the samples (additional clotting was not observed after three F/T cycles). This technique was tested using monkey, rat, and dog plasma and sodium heparin and EDTA anticoagulants. To assess the possibility of nonspecific binding to the polypropylene filter, a variety of drug candidates from diverse drug classes were studied. Validation data generated for two Lilly compounds from distinct classes, before and after filtering, are presented in this paper as practical examples of this technique. While LC/MS/MS is the primary method of bioanalysis in our laboratory, the technique presented in this paper is applicable to other forms of detection as well.     

Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.     

Quantification of o,o'-dityrosine,o-nitrotyrosine,and o-tyrosine in cat urine samples by LC/ electrospray ionization-MS/MS using isotope dilution

Quantification of o-tyrosine, o-nitrotyrosine, and o,o'dityrosine from cat urine samples was achieved by LC/ electrospray ionization-MS/MS (LC/ESI-MS/MS) using an isotope dilution technique in multiple reaction monitoring mode before butylation of o,o'-dityrosine and after butylation of o-tyrosine and o-nitrotyrosine. This novel approach of amino acids butylation enhanced the MS response by a factor of 7-fold for o-tyrosine and 6-fold for o-nitrotyrosine and decreased the overall chemical background noise. Butylation of o,o'-dityrosine resulted in a lower MS response as a result of the formation of both mono- and doubly butylated species. The mean recovery for the oxidized amino acids was estimated at 73 +/- 2%. The limits of quantitation of NO2-Tyr butyl ester, o-Tyr butyl ester, and di-Tyr in cat urine samples were calculated at 14.5, 28.2 and 140.9 nM, respectively. The oxidized amino acids levels in cat urine extracts ranged from 157 to 250 ng/day for o-Tyr and from 3,289 to 11,803 ng/day for di-Tyr. NO2-Tyr was found in only two urine extracts at levels below 58 ng/day. A certain trend of correlation was observed between o,o'-dityrosine and o-tyrosine when comparing these values against their respective creatinine amounts. A comparison of the data gathered from the ThermoFinnigan TSQ 7000 and Micromass Q-TOF instruments revealed several advantages of using the Q-TOF regarding the exact mass measurement, a lower ion suppression effect and the possibility to perform analyses in full scan product ion mode. These results demonstrate that a Q-TOF instrument can be a good alternative to classical triple quadrupole for quantitative purposes on a relatively small linear dynamic range (4 orders of magnitude for the Q-TOF, as compared to 6 for the triple quadrupole).     

LC/MS/MS determination of omapatrilat, a sulfhydryl-containing vasopeptidase inhibitor, and its sulfhydryl- and thioether-containing metabolites in human plasma

Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.     

Pharmacokinetics,tissue distribution,bioavailability, and excretion of nuciferine,an alkaloid from lotus,in rats by LC/MS/MS

Objective: In order to characterize the pharmacokinetics, tissue distribution, bioavailability, and excretion of nuciferine, a reliable gradient LC/MS/MS-based method was developed and validated.

Methods: Sprague-Dawley rats were intravenously injected with a bolus of nuciferine (0.2?mg/kg) and orally given a single dose of nuciferine (10.0?mg/kg). Blood samples were withdrawn via the ocular vein at specific times. Organs, including the liver, kidney, brain, lung, heart, and spleen, were collected at specific times after oral administration of 10.0?mg/kg nuciferine. The plasma and tissue samples were assayed by LC/MS/MS.

Results: The results indicated that nuciferine had rapid distribution and poor absorption into systemic circulation. The value of absolute bioavailability was only 1.9?±?0.8% after administration of 10.0?mg/kg nuciferine by oral and administration of 0.2?mg/kg nuciferine intravenously (IV) to rats. The AUC_0→4?h values in tissues were in the order of kidney?>?lung?>?spleen?>?liver?>?brain?>?heart. The majority of excretion of nuciferine (50.7%) was excreted through kidneys with parent drug after oral administration without liver metabolism.

Conclusion: This study may provide a meaningful basis for clinical application of such a bioactive compound of herbal medicines.     

Comparison of electrospray,atmospheric pressure chemical ionization,and atmospheric pressure photoionization in the identification of apomorphine,dobutamine, and entacapone phase II metabolites in biological samples

The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no site-characteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism.     

Analysis by laser-induced breakdown spectroscopy of complex solids, liquids, and powders with an echelle spectrometer

One of the most promising approaches to laser-induced breakdown spectroscopy (LIBS) experiments involves the use of an echelle spectrometer coupled with an intensified CCD. Even if drawbacks remain with its use, the echelle spectrometer facilitates a multielemental analysis that is more rapid than can be obtained with the more-conventional Czerny-Turner spectrometer and, moreover, does not sacrifice reliability. Quantitative results obtained with such apparatus for solids, liquids, powders, and gases are described and when possible compared with results from Czerny-Turner spectrometers. Liquid analysis by LIBS with echelle spectrometers has allowed a spectral database to be compiled. Once the qualitative spectra of pure elements in aqueous solutions, are obtained, they can be used for qualitative analysis of unknown samples.     

Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides

A method for the simultaneous determination of a series of thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound thiols by tris(2-carboxyethyl)phosphine, the newly formed thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further thiols. The method was successfully applied to the analysis of free and disulfide-bound thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of thiols in urine samples. The thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.     

The importance of chromatographic separation in LC/MS/MS quantitation of drugs in biological fluids: detection,characterization, and synthesis of a previously unknown low-level nitrone metabolite of a substance P antagonist

The observation of an interference peak in plasma samples from dogs dosed with compound I led to the discovery of an unidentified metabolite. The unknown metabolite had the same molecular weight as the parent drug, and their fragmentation profiles were also quite similar. LC/MS/ MS analysis of the plasma extracts of dogs and rats dosed with I and its deuterium-labeled analogue suggested a nitrone structure for the unknown metabolite. Synthesized nitrone matched the unknown metabolite with identical retention time and nearly identical fragmentation profile. The nitrone slowly decomposed in acidic aqueous solution at ambient temperature and also underwent in-source, thermal-induced hydrolysis during electrospray ionization mass spectrometric analysis. The reaction of the nitrone with diethyl acetylenedicarboxylate readily generated a [2 + 3] cycloaddition product. The example shown here clearly demonstrates that precautions must be taken when LC/MS/MS quantitation is conducted in the selected reaction monitoring mode.