Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.     

Identification of linoleic acid free radicals and other breakdown products using spin trapping with liquid chromatography-electrospray tandem mass spectrometry

Linoleic acid radical products formed by radical reaction (Fenton conditions) were trapped using 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and analysed by reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS). The linoleic acid radical species detected as DMPO spin adducts comprised oxidized linoleic acid and short-chain radical species that resulted from the breakdown of carbon and oxygen centred radicals. Based on the m/z values, the short-chain products were identified as alkyl and carboxylic acid DMPO radical adducts that exhibited different elution times. The ions identified as DMPO radical adducts were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS spectra of linoleic acid DMPO radical adducts exhibited the fragment ion at m/z 114 and/or the loss of neutral molecule of 113 Da (DMPO) or 131 Da (DMPO + H2O), indicated to be DMPO adducts. The short-chain products identified allowed inference of the radical oxidation along the linoleic acid chain by abstraction of hydrogen atoms in carbon atoms ranging from C-8 to C-14. Other ions containing the fragment ion at m/z 114 in the LC-MS/MS spectra were attributed to DMPO adducts of unsaturated aldehydes, hydroxy-aldehydes and oxocarboxylic acids. The identification of aldehydic products formed by radical oxidation of linoleic acid peroxidation products, as short-chain product DMPO adducts, is a means of identifying lipid peroxidation products.     

Identification of isomeric spin adducts of Leu-Tyr and Tyr-Leu free radicals using liquid chromatography-tandem mass spectrometry

Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.     

Quantification of tizanidine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (MS/MS) method was developed and validated for the assay of tizanidine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the selected reaction monitoring mode. The assay exhibited a linear dynamic range of 50-5000 pg/mL for tizanidine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 13%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry

An analytical method for the determination of total N-acetylcysteine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists of plasma digestion with dithiothreitol in order to reduce all the oxidized forms of N-acetylcysteine, and extraction with ethyl acetate followed by determination of levels by an LC–MS–MS method. The intra- and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 50 ng/ml of plasma. The concentration working range was established between 50 ng/ml and 1000 ng/ml. This method has been used in the analysis of approximately 800 human plasma samples from a clinical study with 24 volunteers; the precision of the quality controls was in the range 8.7 to 13.4% and the accuracy was in the range −5.9 to 8.5%, expressed as the RSD and the relative error, respectively.     

Quantitative and confirmative performance of liquid chromatography coupled to high-resolution mass spectrometry compared to tandem mass spectrometry

The quantitative and confirmative performance of two different mass spectrometry (MS) techniques (high-resolution MS and tandem MS) was critically compared. Evaluated was a new extraction and clean-up protocol which was developed to cover more than 100 different veterinary drugs at trace levels in a number of animal tissues and honey matrices. Both detection techniques, high-resolution mass spectrometry (HRMS) (single-stage Orbitrap instrument operated at 50 000 full width at half maximum) and tandem mass spectrometry (MS/MS) (quadrupole technology) were used to validate the method according to the EU Commission Decision 2002/657/EEC. Equal or even a slightly better quantitative performance was observed for the HRMS-based approach. Sensitivity is higher for unit mass resolution MS/MS if only a subset of the 100 compounds has to be monitored. Confirmation of suspected positive findings can be done by evaluating the intensity ratio between different MS/MS transitions, or by accurate mass based product ion traces (no precursor selection applied). MS/MS relies on compound-specific optimized transitions; hence the second, confirmatory transition generally shows relatively high ion abundance (fragmentation efficacy). This is often not the case in single-stage HRMS, since a generic (not compound-optimized) collision energy is applied. Hence, confirmation of analytes present at low levels is superior when performed by MS/MS. Slightly better precision, but poorer accuracy (fortified matrix extracts versus pure standard solution) of ion ratios were observed when comparing data obtained by HRMS versus MS/MS.     

Implementation of data-dependent acquisitions in the study of melphalan DNA adducts by miniaturized liquid chromatography coupled to electrospray tandem mass spectrometry

The study of defects in DNA caused by xenobiotics, more particularly the study of DNA adducts, is an important field in cancer aetiology. The analysis of low abundance DNA adducts formed in vivo both in animals and humans requires the development and implementation of highly sensitive analytical methods. Since only a minute amount of DNA can be isolated (ca. 100 microg) it is evident that the amount of sample consumed per analysis should be as small as possible in order to gather as much analytical information as possible. In this article an example is given of how this problem can be solved by the implementation of data-dependent acquisitions using capillary liquid chromatography coupled to electrospray tandem mass spectrometry. As a case study the alkylation of DNA by melphalan is presented. Slight modifications of the chromatographic conditions (mobile and stationary phases) can allow the automated analysis of other kinds of DNA adducts.     

Quantification of granisetron in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of granisetron in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/138 for granisetron and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for granisetron in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Re-evaluation of glyphosate determination in water by liquid chromatography coupled to electrospray tandem mass spectrometry

An analytical method based on on-line solid-phase extraction-liquid chromatography coupled to electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) for the determination of glyphosate and aminomethylphosphonic acid (AMPA) residues has been applied to the analysis of water samples within a Round Robin Study. The method had been previously validated in a variety of water samples and it fulfilled all the parameters of precision, accuracy, sensitivity and unequivocal confirmation. The results within the study that we participated were highly satisfactory in all cases with the only exception of glyphosate in groundwater samples, where surprisingly recoveries around 15% were obtained despite the use of isotope-labeled glyphosate as internal standard (I.S.). A slight modification has been introduced in the method, simply consisting of the acidification of sample with hydrochloric acid (HCl) to pH 1. Then, the sample is neutralized and immediately derivatized with 9-fluorenylmethylchloroformate (FMOC) before LC-MS/MS determination. Round Robin Study samples were reanalyzed using this approach, and the recoveries increased up to 98%. A possible explanation might be the slow kinetic interaction between glyphosate and some components of the matrix. These components might act as chelating agents, making glyphosate unavailable for derivatization and therefore for analysis. Several water samples collected at the Mediterranean area of Spain, and previously analyzed and being found to contain glyphosate, were also reanalyzed using this approach, obtaining higher concentrations (between 2 and 14 times) in most of cases.     

Equilenin-2'-deoxynucleoside adducts: analysis with nano-liquid chromatography coupled to nano-electrospray tandem mass spectrometry

The interaction of 4-hydroxy metabolites of estrogens with DNA leads to the formation of DNA adducts. These adducts are believed to play an important role in the incidence of breast and endometrial cancer. In order to be able to analyze these adducts in in vivo samples a method based upon the coupling of miniaturized liquid chromatography (LC) to electrospray tandem mass spectrometry (ES-MS/MS) was developed for the analysis of the adducts formed with 4-hydroxyequilenin. In vitro synthesized adducts obtained by the reaction of 4-hydroxyequilenin with the main 2'-deoxynucleosides were separated on a Hypersyl C(18) BDS nano-HPLC column (15 cm x 75 microm i.d.) at a flow-rate of 300 nl min(-1) using gradient elution with CH(3)OH--0.2% CH(3)COOH in H(2)O. The column was coupled, in combination with a column switching system, to a nano-electrospray interface. Analysis of the low- and high-resolution low-energy collision-activated dissociation product ion spectra of normal and deuterated adducts supported earlier data demonstrating equilenin to form different isomeric adducts, except with thymidine, for which no adducts were found. The nano-HPLC column-switching ES-MS system was tested for its sensitivity on a triple-quadrupole instrument, and detection limits down to 197 fg in the single reaction monitoring mode were obtained for semi-preparatively isolated equilenin--2'-deoxyguanosine adduct.     

Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to Orbitrap and liquid chromatography coupled to tandem mass spectrometry

A simple method for the determination of some anthelmintic drugs and phenylbutazone residues in milk and muscle was developed. Following a fast and easy extraction and evaporation procedure, the extract was injected into an ultra performance liquid chromatography system coupled to a single stage Orbitrap detector. The high mass resolution of 50,000 full width at half maximum and corresponding narrow mass windows permitted a very selective and sensitive detection of analytes without requiring fragmentation of the observed [M+H]⁺ or [M+Na]⁺ ions. This eliminated some difficulties which have plagued the analysis of compounds belonging to the group of avermectins. The analytical method was validated according to the EU commission decision for Orbitrap based, but also for more traditional tandem mass spectrometry based detection and quantification. Equal repeatability but significantly higher sensitivity for critical compounds (avermectins) was obtained for the Orbitrap based detection. A result of this study was the conclusion that analytes with poor fragmentation properties (e.g. sodium-cationized molecules) can be more easily quantified by single stage high resolution mass spectrometry than by tandem mass spectrometry.     

Analysis of tomato glycoalkaloids by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Steroidal glycoalkaloids (SGAs) extracted from tomato leaves and berries (Lycopersicon esculentum Mill.) were separated and identified using optimized reversed-phase liquid chromatography with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The ESI source polarity and chromatographic conditions were evaluated. The ESI spectra contain valuable information, which includes the mass of SGAs, the mass of the aglycones, and several characteristic fragment ions. Cleavage at the interglycosidic bonds proximal to the aglycones is the most prominent process in the ESI process. A protonated molecule, [M+H]+, accompanied by a mixed adduct ion, [M+H+Na]2+, was observed for alpha-tomatine (i.e., m/z 1034.7 and 528.9) and dehydrotomatine (i.e., m/z 1032.6 and 527.9) in positive ion mode spectra. The structures of these tomato glycoalkaloids were confirmed using tandem mass spectrometry. The identification of a new alpha-tomatine isomer glycoalkaloid, named filotomatine (MW 1033), which shares a common tetrasaccharide structure (i.e., lycotretraose) with alpha-tomatine and dehydrotomatine, and soladulcidine as an aglycone, is described for the first time. It occurs in significant amounts in the extracts of wild tomato foliage. Multistage mass spectrometry both of the protonated molecules and of the doubly charged ions was used for detailed structural elucidation of SGAs. Key fragmentations and regularities in fragmentation pathways are described and the fragmentation mechanisms involved are proposed.     

Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry

An analytical method for the determination of total N-acetylcysteine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists of plasma digestion with dithiothreitol in order to reduce all the oxidized forms of N-acetylcysteine, and extraction with ethyl acetate followed by determination of levels by an LC–MS–MS method. The intra- and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 50 ng/ml of plasma. The concentration working range was established between 50 ng/ml and 1000 ng/ml. This method has been used in the analysis of approximately 800 human plasma samples from a clinical study with 24 volunteers; the precision of the quality controls was in the range 8.7 to 13.4% and the accuracy was in the range −5.9 to 8.5%, expressed as the RSD and the relative error, respectively.     

Detection of 22 antiepileptic drugs by ultra-performance liquid chromatography coupled with tandem mass spectrometry applicable to routine therapeutic drug monitoring

The purpose of this study was to develop an ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method of 22 antiepileptics for routine therapeutic monitoring. The antiepileptics used in the analyses were carbamazepine, carbamazepine‐10,11‐epoxide, clobazam, N‐desmethylclobazam, clonazepam, diazepam, N‐desmethyldiazepam, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, N‐desmethylmesuximide, nitrazepam, phenobarbital, phenytoin, primidone, tiagabine, topiramate, valproic acid, vigabatrin and zonisamide. After protein precipitation of 50 μL plasma with methanol, the supernatant was diluted with water or was evaporated to dryness and reconstituted with mobile phase in the case of benzodiazepines. Separation was achieved on an Acquity UPLC BEH C₁₈ column with a gradient mobile phase of 10 mm ammonium acetate containing 0.1% formic acid and methanol at a flow rate of 0.4 mL/min. An Acquity TQD instrument in multiple reaction monitoring mode with ion mode switching was used for detection. All antiepileptics were detected and quantified within 10 min, with no endogenous interference. All the calibration curves showed good linearity in the therapeutic range (r² < 0.99). The precision and accuracy values for intra‐ and inter‐assays were within ±15% except for phenobarbital and tiagabine. A good correlation was observed between the concentration of clinical samples measured by the new method described here and the conventional methods. The values of carbamazepine and phenytoin by UPLC‐MS/MS were lower than those detected by the immunoassays, which might be caused by the cross‐reaction of antibodies with their metabolites. In conclusion, we developed a simple and selective UPLC‐MS/MS method suitable for routine therapeutic monitoring of antiepileptics. Copyright © 2012 John Wiley & Sons, Ltd.     

Pharmacokinetic study of free mangiferin in rats by microdialysis coupled with microbore high-performance liquid chromatography and tandem mass spectrometry

Mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthen-9-one) has been isolated from the herbal root of Anemarrhena asphodeloides Bung showing antioxidative, antiviral, and anticancer effect. An in vivo microdialysis sampling method coupled to microbore high-performance liquid chromatography (HPLC) was employed for continuous monitoring of free mangiferin in rat blood. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and mangiferin at doses of 10, 30 or 100 mg/kg were then administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a microbore HPLC system. Mangiferin was separated by a reversed-phase C18 microbore column (150 x 1 mm) from dialysate within 10 min. The mobile phase consisted of acetonitrile-0.05% phosphoric acid-tetrahydrofuran (10:75:15, v/v/v) with a flow-rate of 0.05 ml/min. The wavelength of the UV detector was set at 257 nm. The limit of quantification for mangiferin was 0.05 microg/ml and in vivo recovery of mangiferin at concentrations of 1, 5 and 10 microg/ml was in range of 37.7-39.8%. The results indicate that the pharmacokinetics of mangiferin at doses of 10-30 mg/kg reveals a linear relation, while doses of 30-100 mg/kg show a nonlinear pharmacokinetic phenomenon. Mangiferin was undetectable in brain dialysate. The proposed method provides a technique for rapid and sensitive analysis of free mangiferin in rat blood and further application in pharmacokinetic study. Furthermore, the metabolites of mangiferin in the rat bile were confirmed by LC electrospray ionization (ESI) tandem mass spectrometry (MS-MS).