Atmospheric pressure ionization–tandem mass spectrometry of the phenicol drug family

In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H]⁺ dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M–H]^?, while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high‐resolution mass spectrometry in a quadrupole‐Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N‐(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS³ and MS⁴ spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high‐performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg. Copyright © 2013 John Wiley & Sons, Ltd.     

Mass spectrometric behavior of anabolic androgenic steroids using gas chromatography coupled to atmospheric pressure chemical ionization source. Part I: Ionization

The detection of anabolic androgenic steroids (AAS) is one of the most important topics in doping control analysis. Gas chromatography coupled to (tandem) mass spectrometry (GC–MS(/MS)) with electron ionization and liquid chromatography coupled to tandem mass spectrometry have been traditionally applied for this purpose. However, both approaches still have important limitations, and, therefore, detection of all AAS is currently afforded by the combination of these strategies. Alternative ionization techniques can minimize these drawbacks and help in the implementation of a single method for the detection of AAS. In the present work, a new atmospheric pressure chemical ionization (APCI) source commercialized for gas chromatography coupled to a quadrupole time‐of‐flight analyzer has been tested to evaluate the ionization of 60 model AAS. Underivatized and trimethylsylil (TMS)‐derivatized compounds have been investigated. The use of GC–APCI–MS allowed for the ionization of all AAS assayed irrespective of their structure. The presence of water in the source as modifier promoted the formation of protonated molecules ([M+H]⁺), becoming the base peak of the spectrum for the majority of studied compounds. Under these conditions, [M+H]⁺, [M+H‐H₂O]⁺ and [M+H‐2·H₂O]⁺ for underivatized AAS and [M+H]⁺, [M+H‐TMSOH]⁺ and [M+H‐2·TMSOH]⁺ for TMS‐derivatized AAS were observed as main ions in the spectra. The formed ions preserve the intact steroid skeleton, and, therefore, they might be used as specific precursors in MS/MS‐based methods. Additionally, a relationship between the relative abundance of these ions and the AAS structure has been established. This relationship might be useful in the structural elucidation of unknown metabolites. Copyright © 2014 John Wiley & Sons, Ltd.     

Fast liquid chromatography/multiple‐stage mass spectrometry of coccidiostats

Drugs that are used as medicines and also as growth promoters in veterinary care are considered as emerging environmental contaminants and in recent years concern about their potential risk to ecosystems and human health has risen. In this paper we used a method based on liquid chromatography/electrospray tandem mass spectrometry to analyze eight coccidiostatic compounds: diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, lasalocid, monensin, salinomycin, maduramicin and nasarin. Multiple‐stage mass spectrometry (MSⁿ) based on the precursor ions [M+Na]⁺ (polyether ionophores), [M+H]⁺ (robenidine) and [M–H]^? (diclazuril and dinitrocarbanilide) was used to study the fragmentation of these compounds. MSⁿ data and genealogical relationships were used to propose a tentative assignment of the different fragment ions. Loss of water, decarboxylations, ketone β‐cleavages and rearrangement of cyclic ethers and amide groups were some of the fragmentations observed for these compounds. Liquid chromatography with a sub‐2 µm particle size column was coupled to tandem mass spectrometry (LC/MS/MS) allowing the separation of these compounds in less than 7 min. Method detection limits ranging from 11 to 71 ng L^?1 and run‐to‐run values in terms of relative standard deviation (RSD) (up to 12%) were obtained. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid separation and characterization of diterpenoid alkaloids in processed roots of Aconitum carmichaeli using ultra high performance liquid chromatography coupled with hybrid linear ion trap‐Orbitrap tandem mass spectrometry

The lateral root of Aconitum carmichaeli, a popular traditional Chinese medicine, has been widely used to treat rheumatic diseases. For decades, diterpenoid alkaloids have dominated the phytochemical and biomedical research on this plant. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap tandem mass spectrometry was developed to characterize the diterpenoid alkaloids in Aconitum carmichaeli. Based on an optimized chromatographic condition, more than 120 diterpenoid alkaloids were separated with good resolution. Using a systematic strategy that combines high resolution separation, highly accurate mass measurements and a good understanding of the diagnostic fragment‐based fragmentation patterns, these diterpenoid alkaloids were identified or tentatively identified. The identification of these chemicals provided essential data for further phytochemical studies and toxicity research of Aconitum carmichaeli. Moreover, the ultra high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry platform was an effective and accurate tool for rapid qualitative analysis of secondary metabolite productions from natural resources.     

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization‐multiple stage tandem mass spectrometry (ESI‐MSⁿ) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na]⁺ and [M + NH₄]⁺ ions, a characteristic [M‐glycosyl + H]⁺ ion was observed in full‐scan mode with in‐source fragmentation. Sequential in‐trap collision‐induced dissociation of [M + Na]⁺ ions from 11,12‐diesters revealed consistent preferred losses of substituents first from C‐12, then from C‐11, followed by losses of monosaccharide fragments from the C‐3 tri‐ and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high‐performance liquid chromatography coupled to ESI‐MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C₄H₇O C‐11/C‐12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in‐source fragmentation and in‐trap collision‐induced dissociation and supports the usefulness of LC‐ESI‐MSⁿ not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Semi-targeted residue screening in complex matrices with liquid chromatography coupled to high resolution mass spectrometry: current possibilities and limitations

Some twenty cultured fish samples were analyzed for possible residues of veterinary drugs with high resolution mass spectrometry (single stage Orbitrap) coupled to ultra performance liquid chromatography. Quantitative analysis based on external standards covered 110 analytes. Some 116 additional compounds were monitored without having access to reference materials. Detection was based on calculated exact masses and narrow mass windows. Furthermore, a number of semi-targeted techniques were evaluated and compared to corresponding triple quadrupole precursor scan experiments. Single stage high resolution mass spectrometry was used to monitor compound specific product ions (without relying on a previous precursor selection). The capabilities of neutral loss searches based on exact masses were shown by detecting small concentrations of incurred oxytetracycline residues. High resolution mass spectrometry provided more sensitivity and selectivity than corresponding tandem quadrupole precursor and neutral loss scans. The currently limiting factor is the not adequate performance of the available software used for data mining. The high number of false positives that were produced, when searching for chlorine isotopic patterns, was clearly linked to the fact that the utilized software does not perform a peak deconvolution, but simply investigates one individual spectrum after another.     

Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography–tandem mass spectrometry

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography–tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1′‐carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]⁺ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M‐CO₂+H]⁺ and [M‐ΙmCO₂+H]⁺ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti‐Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.     

Characterization of the potential new phthalides in Ligusticum chuanxiong Hort. using ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra‐performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFI^TM platform based on the in‐house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty‐five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry‐oriented method for the discovery of the potential new compounds.     

Rapid characterization of triterpene saponins from Conyza blinii by liquid chromatography coupled with mass spectrometry

Conyza blinii Le'vl is a medicinal herb used for the treatment of inflammation in Chinese folk medicine. Its major bioactive constituents are triterpene saponins, most of which contain 6–8 sugar residues. In this report, electrospray ionization tandem mass spectrometry fragmentation behaviors of bisdesmosidic triterpene saponins (conyzasaponin A, B, and C) were studied in both positive and negative ion modes with an ion‐trap mass spectrometer. In full scan mass spectrometry, these saponins gave predominant [M–H]^? and [M+Na]⁺ ions, which determined the molecular weights. In tandem mass spectrometry (MSⁿ, n = 2–4), the [M–H]^? and [M+Na]⁺ ions yielded fragments [Y_0α–H]^? and [B_α+Na]⁺, which were diagnostic for the structures of the triterpene skeleton and sugar chains. The structural elucidation was approved by accurate mass data using IT‐TOF‐MS. An interpretation guideline based on MSⁿ (n = 2–4) diagnostic ions was proposed in order to elucidate the chemical structures of unknown triterpene saponins in C. blinii extract. The saponins in C. blinii were separated by liquid chromatography with a methanol/acetonitrile/water solvent system, and then analyzed by ion‐trap and IT‐TOF mass spectrometers. Based on the interpretation guideline, a total of 35 triterpenoid saponins were tentatively identified. Among them, 15 saponins had been previously reported, and the other 20 saponins were reported from Conyza species for the first time. This study indicates that LC/MS is a powerful technology for the rapid characterization of complicated saponins in herbal extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

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.     

Determination of Rosiglitazone by Direct Analysis in Real-Time Mass Spectrometry

The determination of rosiglitazone in dietary supplements by direct analysis in real-time mass spectrometry normally provides low repeatability. The [M+H]⁺ signal sharply decreased in the presence of strong-base and weak-acid ionic compounds because rosiglitazone decomposition occurred due to the hydrolysis of strong-base and weak-acid anions. The repeatability was improved and the influence of ionic compounds was minimized by the use of pioglitazone as an internal standard. Orbitrap mass spectrometry was used to provide high resolution in which isotopic interferences from M?+?1 of pioglitazone upon M of rosiglitazone were eliminated. This approach was used to determine rosiglitazone in tablet and dietary supplements in 1?min per sample.     

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.     

Analytical performance of the various acquisition modes in Orbitrap MS and MS/MS

Quadrupole Orbitrap instruments (Q Orbitrap) permit high‐resolution mass spectrometry‐based full scan acquisitions and have a number of acquisition modes where the quadrupole isolates a particular mass range prior to a possible fragmentation and high‐resolution mass spectrometry‐based acquisition. Selecting the proper acquisition mode(s) is essential if trace analytes are to be quantified in complex matrix extracts. Depending on the particular requirements, such as sensitivity, selectivity of detection, linear dynamic range, and speed of analysis, different acquisition modes may have to be chosen. This is particularly important in the field of multi‐residue analysis (eg, pesticides or veterinary drugs in food samples) where a large number of analytes within a complex matrix have to be detected and reliably quantified. Meeting the specific detection and quantification performance criteria for every targeted compound may be challenging. It is the aim of this paper to describe the strengths and the limitations of the currently available Q Orbitrap acquisition modes. In addition, the incorporation of targeted acquisitions between full scan experiments is discussed. This approach is intended to integrate compounds that require an additional degree of sensitivity or selectivity into multi‐residue methods.     

Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry

The accurate and precise measurement of endogenous corticosteroids in urine is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate, and sensitive method based on liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the quantification of 67 endogenous gluco- and mineralo-corticosteroids and progestins has been developed and validated. Sample preparation, chromatographic separation, and mass spectrometric detection were optimized. Urine samples (0.5 mL) were hydrolyzed with β-glucuronidase and the released analytes were extracted by liquid–liquid extraction. The chromatographic separation was performed in 20 min after redisolution of the extract. MS behavior of endogenous corticosteroids was evaluated in order to select the most specific precursor ion ([M+H]⁺, [M+NH₄]⁺, or [M+H-nH₂O]⁺) for the detection. MS/MS determination was performed under selected reaction monitoring mode using electrospray ionization in positive mode. The method was shown to be linear (r > 0.99) in the range of endogenous concentrations for all studied metabolites. Limits of detection (LOD) below 1 ng mL⁻¹ were typically obtained for analytes with a 3-oxo-4-ene structure whereas LODs below 15 ng mL⁻¹ were common for the rest of analytes. Recoveries were higher than 80% and intra-assay precisions below 20%, evaluated at three concentration levels, were found for most steroids. No significant or moderate matrix effect, ranging from 54 to 155%, was observed for most of the analytes. The applicability of the method was confirmed by analyzing 24 h urine samples collected from twenty healthy volunteers and comparing the results with previously established normal ranges. The wide coverage of corticosteroid metabolism, together with short analysis time, low sample volume, simple sample preparation, and satisfactory quantitative results make this method useful for clinical purposes.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of C-21 steroidal glycosides from the roots of Cynanchum chekiangense by high-performance liquid chromatography/tandem mass spectrometry

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) was used to identify C-21 steroidal glycosides with immunological activities in roots of Cynanchum chekiangense. In the MS/MS spectra, fragmentation reactions of the [M + Na]⁺ were recorded to provide structural information about the glycosyl and aglycone moieties. To further confirm the fragments structures, off-line Fourier transform ion cyclotron resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. In the study, four known steroidal glycosides cynascyroside C, chekiangensosides A and B, glaucoside H, and four novel steroidal glycosides chekiangensosides C, D, E and chekiangensoside A isomer were identified based on mass spectral data, NMR spectral data and standards. This is the first report on identifying steroidal glycosides in roots of C. chekiangense by HPLC/ESI-MS/MS directly, which could save time and material consuming efforts in traditional phytochemistry analysis.     

Development and validation of a qualitative screening method for the detection of exogenous anabolic steroids in urine by liquid chromatography-tandem mass spectrometry

A screening method for the urinary detection of 34 exogenous anabolic steroids has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography-tandem mass spectrometry. The use of some adducts such as [M+NH₄]⁺, [M+CH₃COO]⁻ and [M+H+MeOH]⁺ was necessary in order to detect some analytes at the required level (lower than 10 ng/ml). Two transitions were selected for each analyte. Different concentration factors have been studied in order to increase the sensitivity. A concentration factor of 50 was selected for the screening method although the high ion suppression observed under these conditions can hamper its application as a quantitative method. The method was validated and limits of detection were obtained by spiking ten different blank urine samples at five different concentration levels. Up to 29 analytes were detected in all spiked urines at the required level. Limits of detection between 1 and 10 ng/ml were obtained for most analytes which fulfil current requirements. The applicability of the method was shown by analysing positive samples.     

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.