Identification of 1‐palmitoyl‐2‐linoleoyl‐phosphatidylethanolamine modifications under oxidative stress conditions by LC‐MS/MS

Phosphatidylethanolamines are a major class of phospholipids found in cellular membranes. Identification of the alterations in these phospholipids, induced by free radicals, could provide new tools for in vivo diagnosis of oxidative stress. In this study, 1‐palmitoyl‐2‐linoleoyl‐phosphatidylethanolamine oxidation products, induced by the hydroxyl radical, were studied using LC‐MS and LC‐MS/MS. Data obtained allowed the identification and separation of isomeric oxidative products with modifications in the sn‐2 acyl chain, attributed to long‐ and short‐chain products. Among long‐chain products keto, keto‐hydroxy, hydroxy, poly‐hydroxy, peroxy and hydroxy–peroxy derivatives were identified. Product ions formed by loss of two H₂O molecules vs loss of HOOH, allowed the identification of, respectively, di‐ (or poli‐) hydroxy vs peroxy derivatives. Location of functional groups was determined by the product ions formed by cleavage of C–C bonds, in the vicinity of the oxidation positions, allowing the identification of C9, C12 and C13 as the predominant substituted positions. Short‐chain products identified comprised aldehydes, hydroxy‐aldehydes and carboxylic derivatives, with modified sn‐2 acyl lengths of C7–C9 and C11, C12. Among the short‐chain products identified, C9 products showed higher relative abundance. Copyright © 2009 John Wiley & Sons, Ltd.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites in urine by LC‐MS after derivatization and its application for differentiation between positional isomers of prilocaine

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites by GC‐EI‐MS and LC‐ESI‐MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine‐containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential “in‐vial” instantaneous derivatization processes at ambient temperature followed by LC‐ESI‐MS/MS analysis was developed. 2,2,2‐Trichloro‐1,1‐dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI‐MS and ESI‐MS/MS spectra, enabling their structural elucidation in a single LC‐ESI‐MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of LC‐MS and LC‐MS‐MS to the analysis of photo‐decomposed crystal violet in the investigation of cultural heritage materials aging

In this work, the accurate liquid chromatography‐ultraviolet‐visible (LC‐UV‐Vis), LC‐mass spectrometry (MS) and LC‐MS‐MS analysis of the photo‐degradation products of crystal violet (CV) is reported. CV is a light fugitive early synthetic dye which had a widespread diffusion into the market starting from the end of the XIX century and was used among others by V. Van Gogh and P. Gauguin in their writings, drawings or paintings. On‐line photodiode array detector enabled simultaneous UV‐Vis spectra acquisition. Many degradation compounds were identified through their exact mass (2 ppm accuracy) and MS‐MS technique. In particular, all CV demethylated products, demethylated Michler's ketone and particularly some compounds that most likely contain oxygen, such as N‐oxides, were found. Fragmentation products are all justified by the proposed fragmentation scheme, in term of precursor exact mass and isotopic profile, characteristic losses in fragmentation and rebuilt structure formula. In particular, we hypothesized the presence of N‐imido oxides and hydroxylamine derivates, never reported before, together with the demethylated derivatives of the studied dyes. All these compounds, although at trace level in our samples, contribute to the discoloration and fading of works of arts made with CV. In particular, demethylation of CV by UV light leads to formation of compounds absorbing at shorter wavelengths than CV (blue shift) or no‐absorbing in visible range (yellow‐colourless) with an overall effect that may appear reddish‐brown. This phenomenon justifies drawings appearing grey or brown on aged yellowed paper, when CV‐based inks or paints were used. The final aim was to better characterize the photo‐degradation of early synthetic dyes (in particular of CV) and to gain a better insight into the discoloration and fading of purple ink strokes made of CV. Copyright © 2012 John Wiley & Sons, Ltd.     

Solid‐Phase Synthesis of Polyamine Spider Toxins and Correlation with the Natural Products by HPLC‐MS/MS

A recently developed new and divergent approach for the solid‐phase synthesis of polyamines and polyamine derivatives was extended to the preparation of linear pentamines, and it was applied to the synthesis of three quartets of isomeric polyamine spider toxins. The twelve synthetic acylpolyamines were investigated by HPLC‐UV(DAD)‐MS and HPLC‐UV(DAD)‐MS/MS and compared with the natural products in the complex mixture of the venom of Agelenopsis aperta. The comparative investigation supported the structures and assignments of seven previously found toxins and allowed the identification of an additional five polyamine derivatives in the natural sample. The MS/MS study of the isomerically pure polyamine derivatives revealed furthermore a characteristic pattern for the fragmentation of these compounds, which can possibly be used as evidence in the trace analysis of other polyamine derivatives.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Tandem mass spectrometry of intact oxidation products of diacylphosphatidylcholines: evidence for the occurrence of the oxidation of the phosphocholine head and differentiation of isomers

Three glycerophosphatidylcholine (GPC) phospholipids (oleoyl-, linoleoyl- and arachidonoylpalmitoylphosphatidylcholine) were oxidized under Fenton reaction conditions (H(2)O(2) and Fe(2+)), and the long-chain oxidation products were detected by electrospray mass spectrometry (ES-MS) and characterized by ES-MS/MS. The intact oxidation products resulted from the insertion of oxygen atoms into the phospholipid structure. The tandem mass spectra of the [MNa](+) molecular ion showed, apart from the characteristic fragments of GPC, fragment ions resulting from neutral losses from [MNa](+), and combined with loss of 59 and 183 Da from [MNa](+). These ions resulted from cleavage of the bond near the hydroxy group by a charge-remote fragmentation mechanism, allowing its location to be pinpointed. The fragments thus formed reflected the positions of the double bonds and of the derivatives along the unsaturated fatty acid chain, giving very useful information, as they allowed the presence of structural isomers and positional isomers to be established. The identification of the fragment ion at m/z 163, which is 16 Da higher than the five-membered cyclophosphane ion (m/z 147), in some tandem mass spectra, is consistent with the oxidation of the phosphocholine head. Some ions were found to occur with the same m/z value; in two of the phospholipids and based on the MS/MS data, structural and positional isomers were differentiated. Our findings indicate that MS/MS is a valuable tool for the identification of the wide complexity of structural features occurring in oxidized phosphatidylcholines during lipid peroxidation in cellular membranes.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation pathways and differentiation of positional isomers of sorafenib and structural analogues by ESI‐IT‐MSn and ESI‐Q‐TOF‐MS/MS coupled with DFT calculations

Sorafenib is an orally active multikinase inhibitor for the treatment of renal cell carcinoma. A series of sorafenib structural analogues were investigated in this work for their gas‐phase fragmentation behaviors using electrospray ionization ion trap mass spectrometry and quadrupole time‐of‐flight mass spectrometry in the positive mode. The possible fragmentation pathways were proposed based on ESI‐MS/MS data and theoretical calculation. Different from the typical α‐cleavage of amide, consecutive reactions that involved elimination of H₂O and CH₃NC were observed for 2‐pyridinecarboxamide derivatives, which were followed by the formation of a stabilized 7‐membered ring carbocation by loss of CO. Two possible protonation sites occurred at carbonyl oxygen atoms for aryl‐urea derivatives and the α‐cleavage of urea was the main fragmentation pathways, which was followed by the formation of stable benzo [d] oxazole ring characteristic to aryl‐urea derivatives. The relative abundance of characteristic fragment ions and the energy‐resolved breakdown curves were used to distinguish the 4 sets of positional isomers of sorafenib and analogues. The methodology and results of the present work would contribute to the chemical structure identification of other structural analogues and the potential impurities presented in active pharmaceutical ingredients and drug formulations.     

Neutral and acidic products derived from hydroxyl radical‐induced oxidation of arabinotriose assessed by electrospray ionisation mass spectrometry

The oxidation of α‐(1 → 5)‐l ‐arabinotriose (Ara₃), an oligosaccharide structurally related to side chains of coffee arabinogalactans, was studied in reaction with hydroxyl radicals generated under conditions of Fenton reaction (Fe²⁺/H₂O₂). The acidic and neutral oxidation products were separated by ligand exchange/size‐exclusion chromatography, subsequently identified by electrospray ionisation mass spectrometry (ESI–MS) and structurally characterised by tandem MS (ESI–MS/MS). In acidic fraction were identified several oxidation products containing an acidic residue at the corresponding reducing end of Ara₃, namely arabinonic acid, and erythronic, glyceric and glycolic acids formed by oxidative scission of the furanose ring. In neutral fractions were identified derivatives containing keto, hydroxy and hydroperoxy moieties, as well as derivatives resulting from the ring scission at the reducing end of Ara₃. In both acidic and neutral fractions, beyond the trisaccharide derivatives, the corresponding di‐ and monosaccharide derivatives were identified indicating the occurrence of oxidative depolymerisation. The structural characterisation of these oxidation products by ESI–MS/MS allowed the differentiation of isobaric and isomeric species of acidic and neutral character. The species identified in this study may help in detection of roasting products associated with the free radical‐mediated oxidation of coffee arabinogalactans. Copyright © 2014 John Wiley & Sons, Ltd.     

An isomer-specific high-energy collision-induced dissociation MS/MS database for forensic applications: a proof-of-concept on chemical warfare agent markers

Spectra database search has become the most popular technique for the identification of unknown chemicals, minimizing the need for authentic reference chemicals. In the present study, an isomer‐specific high‐energy collision‐induced dissociation (CID) MS/MS spectra database of 12 isomeric O‐hexyl methylphosphonic acids (degradation markers of nerve agents) was created. Phosphonate anions were produced by the electrospray ionization of phosphonic acids or negative‐ion chemical ionization of their fluorinated derivatives and were analysed in a hybrid magnetic‐sector–time‐of‐flight tandem mass spectrometer. A centre‐of‐mass energy (E_com) of 65 eV led to an optimal sequential carbon–carbon bond breakage, which was interpreted in terms of charge remote fragmentation. The proposed mechanism is discussed in comparison with the routinely used low‐energy CID MS/MS. Even‐mass (odd‐electron) charge remote fragmentation ion series were diagnostic of the O‐alkyl chain structure and can be used to interpret unknown spectra. Together with the odd‐mass ion series, they formed highly reproducible, isomer‐specific spectra that gave significantly higher database matches and probability factors (by 1.5 times) than did the EI MS spectra of the trimethylsilyl derivatives of the same isomers. In addition, ionization by negative‐ion chemical ionization and electrospray ionization resulted in similar spectra, which further highlights the general potential of the high‐energy CID MS/MS technique. Copyright © 2011 John Wiley & Sons, Ltd.     

GC‐MS of amaryllidaceous galanthamine‐type alkaloids

Galanthamine‐type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC‐EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high‐resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC‐MS/MS) and accurate mass measurements (GC‐HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC‐MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine‐N‐oxide and N‐formylnorgalanthamine, the galanthamine‐type compounds showed abundant [M]^+. and [M‐H]⁺ ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine‐type alkaloids, including 3‐O‐(2′‐butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC‐MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

Analysis of dammar resin with MALDI‐FT‐ICR‐MS and APCI‐FT‐ICR‐MS

Comprehensive analysis of high‐resolution mass spectra of aged natural dammar resin obtained with Fourier transform ion cyclotron resonance mass spectrometer (FT‐ICR‐MS) using matrix‐assisted laser desorption/ionization (MALDI) and atmospheric pressure chemical ionization (APCI) is presented. Dammar resin is one of the most important components of painting varnishes. Dammar resin is a terpenoid resin (dominated by triterpenoids) with intrinsically very complex composition. This complexity further increases with aging. Ten different solvents and two‐component solvent mixtures were tested for sample preparation. The most suitable solvent mixtures for the MALDI‐FT‐ICR‐MS analysis were dichloromethane‐acetone and dichloromethane‐ethanol. The obtained MALDI‐FTMS mass spectrum contains nine clusters of peaks in the m/z range of 420–2200, and the obtained APCI‐FTMS mass spectrum contains three clusters of peaks in the m/z range of 380–910. The peaks in the clusters correspond to the oxygenated derivatives of terpenoids differing by the number of C₁₅H₂₄ units. The clusters, in turn, are composed of subclusters differing by the number of oxygen atoms in the molecules. Thorough analysis and identification of the components (or groups of components) by their accurate m/z ratios was carried out, and molecular formulas (elemental compositions) of all major peaks in the MALDI‐FTMS and APCI‐FTMS spectra were identified (and groups of possible isomeric compounds were proposed). In the MALDI‐FTMS and APCI‐FTMS mass spectrum, besides the oxidized C₃₀, triterpenoids also peaks corresponding to C₂₉ and C₃₁ derivatives of triterpenoids (demethylated and methylated, correspondingly) were detected. MALDI and APCI are complementary ionization sources for the analysis of natural dammar resin. In the MALDI source, preferably polar (extensively oxidized) components of the resin are ionized (mostly as Na⁺ adducts), whereas in the APCI source, preferably nonpolar (hydrocarbon and slightly oxidized) compounds are ionized (by protonation). Either of the two ionization methods, when used alone, gives an incomplete picture of the dammar resin composition. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a mass spectrometric hydroxyl‐position determination method for the hydroxyindole metabolites of JWH‐018 by GC‐MS/MS

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH‐018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography‐electron ionization‐tandem mass spectrometry (GC‐EI‐MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole‐type SC JWH‐081, and speculated that the same approach could be used for the metabolite isomers. Using JWH‐018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC‐MS/MS. Standard compounds of JWH‐018 and its hydroxyindole metabolite positional isomers were first analyzed by GC‐EI‐MS in full scan mode, which was only able to differentiate the 4‐hydroxyindole isomer. Further GC‐MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH‐018 administered mice and determined the hydroxyl positions to be at the 6‐position on the indole ring. GC‐EI‐MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH‐018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.     

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC‐CI‐MS/MS: a methandienone case study

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC‐CI‐MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long‐term metabolite (18‐nor‐17β‐hydroxymethyl,17α‐methyl‐androst‐1,4,13‐trien‐3‐one) could be detected up to 26 days by using GC‐CI‐MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long‐term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.