Precursor Ion Scan Method Research Articles

Unveiling distinct influencing mechanisms of Br- on NDMA formation during ozonation of actual water Matrixes: Identification of precursors and brominated substance

Bromide ion (Br-) in water sources has been found to affect the formation of carcinogenic non-brominated N-nitrosodimethylamine (NDMA) during ozonation of typical amines. The increasing bromide levels bring more concern due to climate change and industrial discharge, but its effects on actual water matrixes were still unclear. In this study, NDMA formation from three actual water matrixes during ozonation as the Br- concentration increased from 0 to 100 µM were investigated (promotion (100.4 ∼ 231.6 ng/L), promotion then inhibition (399.5 ∼ 461.2 ∼ 428.2 ng/L), and subtle influence (51.9 ± 6.9 ng/L)); from which the NDMA precursors were screened and 8 precursors were selected for exploring the influencing mechanisms. The results indicated that the roles of Br- on NDMA formation significantly depended on the water characteristics of actual matrixes. A total of 16 typical NDMA precursors, which were mainly derived from industrial, agricultural and pharmaceutical sources, were identified. Moreover, 6 brominated substance (Br-DMA, Br-UDMH, Br-DMSC, Br-DMS, Br-TMDS, Br2-TMDS), which were thought to play a crucial role in promoting NDMA formation, were also identified in actual water by precursor ion scan (PIS) method. Hypobromous acid (HOBr), which has been proved to be the culprit to the generation of brominated substances, tend to attack the electron-donating groups of amines, such as acylamino (–NHCO-R) or amidogen (–NH2) groups. However, the reactive bromine species (∙Br and ∙Br2-) was inclined to attack the aromatic and anti-oxidant moieties then result in Br- release, as no bromine substitution but free radical transfer reaction occurs. Therefore, decreasing HOBr formation is the crucial step to reduce NDMA generation. This work would provide technical guide for NDMA risks control during ozonation of Br--amines co-containing water matrixes.

Effects of ozone dose on brominated DBPs in subsequent chlor(am)ination: A comprehensive study of aliphatic, alicyclic and aromatic DBPs

Ozone‒chlor(am)ine is a commonly used combination of disinfectants in drinking water treatment. Although there are quite a few studies on the formation of some individual DBPs in the ozone‒chlor(am)ine disinfection, an overall picture of the DBP formation in the combined disinfection is largely unavailable. In this study, the effects of ozone dose on the formation and speciation of organic brominated disinfection byproducts (DBPs) in subsequent chlorination, chloramination, or chlorination‒chloramination of simulated drinking water were investigated. High-molecular-weight, aliphatic, alicyclic and aromatic brominated DBPs were selectively detected and studied using a powerful precursor ion scan method with ultra performance liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (UPLC/ESI-tqMS). Two groups of unregulated yet relatively toxic DBPs, dihalonitromethanes and dihaloacetaldehydes, were detected by the UPLC/ESI-tqMS for the first time. With increasing ozone dose, the levels of high-molecular-weight (m/z 300–500) and alicyclic and aromatic brominated DBPs generally decreased, the levels of brominated aliphatic acids were slightly affected, and the levels of dihalonitromethanes and dihaloacetaldehydes generally increased in the subsequent disinfection processes. Despite different molecular compositions of the detected DBPs, increasing ozone dose generally shifted the formation of DBPs from chlorinated ones to brominated analogues in the subsequent disinfection processes. This study provided a comprehensive analysis of the impact of ozone dose on the DBP formation and speciation in subsequent chlor(am)ine disinfection.

Toward Machine Learning-Driven Mass Spectrometric Identification of Trichothecenes in the Absence of Standard Reference Materials.

While a significant body of work exists on the detection of commonly known trichothecene toxins, biological, environmental, and other transformational processes can generate many under-characterized and unknown modified trichothecenes. Lacking both analytical reference standards and associated mass spectral databases, identification of these modified compounds reflects both a challenge and a critical gap from forensic and public health perspectives. We report here the application of machine learning (ML) techniques toward identification of discriminative fragment ions from mass spectrometric data that can be exploited to detect evidence of type A and B trichothecenes. The goal of this work is to establish a new method for the identification of unknown, though structurally similar trichothecenes, by leveraging objective ML techniques. Discriminative fragments derived from a series of gradient-boosted machine learners are then used to develop ML-driven precursor ion scan (PIS) methods on a triple quadrupole mass spectrometer (QQQ) for screening of "unknown unknown" trichothecenes. Specifically, we apply the PIS method to a laboratory-synthesized trichothecene, a first step in demonstrating the power of alternative, machine learning-driven mass spectrometric methods.

Untargeted detection of the carbonyl metabolome by chemical derivatization and liquid chromatography-tandem mass spectrometry in precursor ion scan mode: Elucidation of COVID-19 severity biomarkers

Metabolomics (both targeted and untargeted) has become the gold standard in biomarker discovery. Whereas targeted approaches only provide information for the selected markers, thus hampering the determination of out-of-the-box markers, the common bottleneck of untargeted metabolomics is the identification of detected biomarkers. In this study, we developed a strategy based on derivatization and LC-MS/MS detection in a precursor ion scan for the untargeted determination of a specific part of the metabolome (carbonyl-containing metabolites). The usefulness of this guided metabolomics approach has been demonstrated by elucidating carbonyl-containing biomarkers of COVID-19 severity. First, the LC-MS/MS behavior of 63 model compounds after O-benzylhydroxylamine derivatization was studied. A precursor ion scan of m/z 91 was selected as a suitable approach for the untargeted detection of carbonyl-containing metabolites. The method was able to detect ≈300 potential carbonyl-containing molecules in plasma, including mono-/di-/tricarbonylic compounds with satisfactory intra-day and inter-day repeatability and RSDs commonly <15%. Additionally, the semiquantitative nature of the precursor ion scan method was confirmed by comparison with a fully validated targeted method. The application of the guided metabolomics method to COVID-19 plasma samples revealed the presence of four potential COVID-19 severity biomarkers. Based on their LC-MS/MS behavior, these biomarkers were elucidated as 2-hydroxybutyrate, 2,3-dihydroxybutyrate, 2-oxobutyrate and 2-hydroxy-3-methylbutyrate. Their structures were confirmed by comparison with reference materials. The alterations of these biomarkers with COVID-19 severity were confirmed by a target analysis of a larger set of samples. Our results confirm that guided metabolomics is an alternative approach for the untargeted detection of selected families of metabolites; this approach can accelerate their elucidation and provide new perspectives for the establishment of health/disease biomarkers.

Detecting Fentanyl Analogs in Urine Using Precursor Ion Scan Mode.

The opioid crisis has led many providers to inquire about the capabilities of urine drug testing to detect contemporary compounds such as fentanyl and fentanyl analogs. However, current methods for clinical urine drug testing, including immunoassays and targeted liquid chromatography tandem mass spectrometry, are not designed to broadly screen for the variety of fentanyl analogs that may be encountered. In this proof-of-principle study we developed a precursor ion scan method to enable semi-targeted data acquisition for structurally related fentanyl analogs. Based on the knowledge that many analogs fragment to m/z=188 and m/z=105, data was acquired on all precursor ions 250-400 Da that fragmented to these product ions. Using a tandem mass spectrometer we analyzed 102 residual urine specimens, in which we identified fentanyl, acetylfentanyl and acrylfentanyl. In 30 contrived urine samples, the precursor ion scan was also able to identify furanylfentanyl, butryrlfentanyl, 4-fluroisobutrylfentanyl, and despropionylfentanyl with accuracy ranging from 83-100%.

Rapid and on-site detection of multiple fentanyl compounds by dual-ion trap miniature mass spectrometry system

With the increasing abuse of fentanyl and its derivatives, it is urgent to develop techniques that can rapidly detect these compounds in different types of matrices. In this work, we developed a miniature mass spectrometer-based method for the fast and on-site analysis of fentanyl compounds. Optimization of several direct sampling procedures such as paper capillary spray cartridge with a miniature mass spectrometry system enables sensitive analysis of multiple fentanyl compounds. This system was evaluated by analysis of fentanyl and its derivatives in several types of beverage, showing limits of detection (LODs) as low as 10 ppb. It has also been applied into analysis of fentanyl compounds on the surface of a dusty plastic bag, showing LODs of 1 ng/cm2. A precursor ion scan method was also developed for fast screening of multiple fentanyl compounds. This system has also been applied in the analysis of fentanyl in urine samples.

Detection of new emerging type-A trichothecenes by untargeted mass spectrometry

Mycotoxins occur naturally as agricultural contaminants all over the world. The toxic effects of some of their metabolites are known and their presence regulated in food and feed. This paper describes two methods for the detection of toxins of type-A trichothecenes group, and their modified forms, using mass spectrometry. Ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight (UPLC-MS-IT-TOF) was employed to characterize the fragmentation pathways of 10 type-A trichothecenes, and characteristic ions were tentatively identified in scan mode through their accurate masses. Unknown signals were detected in a F. sporotrichioides extract, which afterwards were identified as seven modified forms of neosolaniol (NEO) and T-2 toxin. Then, UPLC coupled to tandem mass spectrometry (MS/MS) was employed to develop a precursor ion scanning method that can be used as a screening tool to detect any modified type-A trichothecenes.

Use of liquid chromatography hybrid triple-quadrupole mass spectrometry for the detection of emodin metabolites in rat bile and urine.

Emodin is the representative form of rhubarb, which is widely used in traditional Chinese medicine for the treatment of purgative, anti-inflammatory, antioxidative and antiviral, etc. Previous reports demonstrated that emodin glucuronide was the major metabolite in plasma. Owing to the extensive conjugation reactions of polyphenols, the aim of this study was to identify the metabolites of emodin in rat bile and urine. Neutral loss and precursor ion scan methods of triple-quadrupole mass spectrometer revealed 13 conjugated metabolites in rat bile and 22 metabolites in rat urine, which included four phase I and 18 phase II metabolites. The major metabolites in rat biosamples were emodin glucuronoconjugates. Moreover, rhein monoglucuronide, chrysophanol monoglucuronide and rhein sulfate were proposed for the first time after oral administration of emodin. Overall, liquid chromatography hybrid triple-quadrupole mass spectrometry analysis leads to the discovery of several novel emodin metabolites in rat bile and urine and underscores that conjugated with glucuronic acid is the main metabolic pathway.

Constant Ion Loss Method for the Untargeted Detection of Bis-sulfate Metabolites.

The untargeted detection of phase II metabolites is a key issue for the study of drug metabolism in biological systems. Sensitive and selective mass spectrometric (MS) techniques coupled to ultrahigh performance liquid chromatographic (UHPLC) systems are the most effective for this purpose. In this study, we evaluate different MS approaches with a triple quadrupole instrument for the untargeted detection of bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were synthesized and their MS behavior was comprehensively studied. Bis-sulfates ionized preferentially as the dianion ([M - 2H]2-) with a small contribution of the monoanion ([M - H]-). Product ion spectra generated from the [M - 2H]2- precursor ions were dominated by the loss of HSO4- to generate two product ions, that is, the ion at m/z 97 (HSO4-) and the ion corresponding to the remaining monosulfate fragment. Other product ions were found to be specific for some structures. As an example, the loss of [CH3 + SO3]- was found to be important for several compounds with unsaturation adjacent to the sulfate. On the basis of the common behavior of the bis-sulfate metabolites two alternatives were evaluated for the untargeted detection of bis-sulfate metabolites (i) a precursor ion scan method using the ion at m/z 97 and (ii) a constant ion loss (CIL) method using the loss of HSO4-. Both methods allowed for the untargeted detection of the model compounds. Eight steroid bis-sulfates were synthesized in high purity in order to quantitatively evaluate the developed strategies. Lower limits of detection (2-20 ng/mL) were obtained using the CIL method. Additionally, the CIL method was found to be more specific in the detection of urinary bis-sulfates. The applicability of the CIL approach was demonstrated by determining progestogens altered during pregnancy and by detecting the bis-sulfate metabolites of tibolone.

前駆イオンスキャンと精密質量分析を組み合わせたLC/MS/MS によるヒト尿中のパラベン抱合体の分離と同定

A precursor ion scanning method in liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed to identify metabolites of 4-hydroxybenzoic acid esters (parabens) in urine as a first step of paraben-metabolites determination in urine. The identification was confirmed by analyzing elemental compositions of conjugation group ions and those fragment ions with LC/Q-ToF MS/MS. Two milliliters of urine diluted with the same volume of acetonitrile was passed through an ODS cartridge column. After the urine sample passed through the ODS cartridge, the cartridge was added with 6mL of methanol elute, which was concentrated to 4mL. The concentrate was filtrated with a polytetrafluoroethylene (PTFE) filter of 0.45 mm pore size. Then the filtrates with PTFE was prepared to a sample for LC/MS/MS and LC/Q-ToF/MS after being concentrated to 1mL. Precursor ion scan by monitoring product ions of [M-H]－ of paraben molecules was applied for finding paraben conjugates. Precursor ions of m/z245 and 341 were found as candidate ions that produce [M-H]－ ion of ethyl-4-hydroxybenzoate (ethyl paraben). In order to confirm elemental compositions of the candidates, accurate masses of the m/z245 and 341 were respectively determined to be m/z245.0138 and 341.0875 in the mass error within 1.5ppm by LC/MS/MS. The elemental compositions of C9H9SO6 and C15H17O9 were respectively confirmed from the accurate masses of m/z245.0138 and 341.0875 within mass error of 1.5ppm. If the ions of C9H9SO6－ and C15H17O9－ are conjugates of ethylparaben, those would be respectively produced by esterization with chemicals of H2SO4 and C6H10O7 in elemental composition. From the compositions of H2SO4 and C6H10O7, sulfuric acid and glucuronic acid were retrieved as a top scored chemicals in a database(ChemSpider). The retrieved chemicals are well known as conjugating chemicals to parabens, and the retention times of the C9H9SO6 and C15H17O9 peaks were respectively agreed with those estimated from the logKow values of ethylparaben conjugates of sulfuric acid and glucuronic acid. Then we concluded that the ions of C9H9SO6－ and C15H17O9－ were [M-H]－ of ethylparaben conjugates of sulfuric acid and glucuronic acid, respectively. With the similar manner, some of other conjugating chemicals were estimated.

Formation and toxicity of halogenated disinfection byproducts resulting from linear alkylbenzene sulfonates

Linear alkylbenzene sulfonates (LAS) are an important group of organic pollutants in urban wastewater effluents. The practice of using seawater for toilet flushing results in saline wastewater effluents, which contain high levels of bromide ions. Chlorine is most commonly used in wastewater disinfection. During chlorination of freshwater or saline wastewater effluents, some halogenated disinfection byproducts (DBPs) resulting from LAS could be formed. In this study, the overall formation of halogenated DBPs resulting from LAS was quantified by total organic halogen (TOX) measurement. Polar halogenated DBPs resulting from LAS were detected with a novel precursor ion scan method. The structures and formation pathways of the major ones were tentatively proposed. The overall toxicity of different scenarios of LAS samples was evaluated with embryos of a marine polychaete Platynereis dumerilii. The results demonstrate that chlorinated DBPs were generated during chlorination of LAS without bromide, while brominated DBPs were generated during chlorination of LAS with bromide. The TOX concentrations were relatively low, indicating that LAS were not quite reactive with halogen. The major polar chlorinated and brominated DBPs resulting from LAS were proposed to be 2,6-dichloro-3,5-dihydroxy-4-dodecylbenzenesulfonic acid and 2,6-dibromo-3,5-dihydroxy-4-dodecylbenzenesulfonic acid, which belong to a group of DBPs with similar structures but different halogen atoms, and their formation pathways were tentatively proposed. The results also reveal that the undisinfected LAS sample was the least toxic, followed by the chlorinated LAS sample without bromide, and the chlorinated LAS sample with bromide was the most toxic.

Detection and characterization of prednisolone metabolites in human urine by LC-MS/MS.

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β-dihydro-PRED and 20α-dihydro-PRED, 20β-dihydro-prednisone and 20α-dihydro-prednisone, 6β-hydroxy-PRED and 6α-hydroxy-PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21-pentahydroxypregnan-1,4-diene-3-one, 6α,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one and Δ(6) -PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6-hydroxy-metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6).

Detection and characterization of betamethasone metabolites in human urine by LC-MS/MS.

Glucocorticosteroids are prohibited in sports when administered by systemic routes and allowed using other administrations for therapeutic reasons. Therefore, markers to distinguish between routes of administration through the analysis of urine samples are needed in anti-doping control. As a first step to achieve that goal, the metabolism of betamethasone (BET) was investigated in the present work. Urine samples obtained after BET intramuscular injection were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Common open screening methods for fluorine containing corticosteroids (precursor ion scan method of m/z 121, 147, 171, and neutral loss (NL) scan methods of 20 and 38 Da in positive ionization, and 46 and 76 Da in negative ionization) were applied to detect BET metabolites. Moreover, an NL method was applied to detect A-ring reduced metabolites of BET, which are ionized as [M+NH4 ](+) (NL of 55, 73, and 91 Da, corresponding to the consecutive losses of NH3 , HF and one, two and three water molecules, respectively). BET and 24 metabolites were detected. Six metabolites were identified by comparison with standards, and for ten, feasible structures were proposed based on mass spectrometric data. Eleven of the characterized metabolites had not been previously reported. Metabolites resulting from 11-oxidation, 6-hydroxylation, C20 or 4-ene-3-one reduction and combination of some of them were detected. Moreover one metabolite resulting from cleavage of the side chain with subsequent oxidation of carbon at C17 was also detected.

Unambiguous characterization of analytical markers in complex, seized opiate samples using an enhanced ion mobility trace detector-mass spectrometer.

Ion mobility spectroscopy (IMS)-based trace-compound detectors (TCDs) are powerful and widely implemented tools for the detection of illicit substances. They combine high sensitivity, reproducibility, rapid analysis time, and resistance to dirt with an acceptable false alarm rate. The analytical specificity of TCD-IMS instruments for a given analyte depends strongly on a detailed knowledge of the ion chemistry involved, as well as the ability to translate this knowledge into field-robust analytical methods. In this work, we introduce an enhanced hybrid TCD-IMS/mass spectrometer (TCD-IMS/MS) that combines the strengths of ion-mobility-based target compound detection with unambiguous identification by tandem MS. Building on earlier efforts along these lines (Kozole et al., Anal. Chem. 2011, 83, 8596-8603), the current instrument is capable of positive and negative-mode analyses with tightly controlled gating between the IMS and MS modules and direct measurement of ion mobility profiles. We demonstrate the unique capabilities of this instrument using four samples of opium seized by the Canada Border Services Agency (CBSA), consisting of a mixture of opioid alkaloids and other naturally occurring compounds typically found in these samples. Although many analytical methods have been developed for analyzing naturally occurring opiates, this is the first detailed ion mobility study on seized opium samples. This work demonstrates all available analytical modes for the new IMS-MS system including "single-gate", "dual-gate", MS/MS, and precursor ion scan methods. Using a combination of these modes, we unambiguously identify all signals in the IMS spectra, including previously uncharacterized minor peaks arising from compounds that are common in raw opium.

Detection, identification and formation of new iodinated disinfection byproducts in chlorinated saline wastewater effluents

The use of seawater for toilet flushing introduces high levels of inorganic ions, including iodide ions, into a city's wastewater treatment systems, resulting in saline wastewater effluents. Chlorination is widely used in disinfecting wastewater effluents owing to its low cost and high efficiency. During chlorination of saline wastewater effluents, iodide may be oxidized to hypoiodous acid, which may further react with effluent organic matter to form iodinated disinfection byproducts (DBPs). Iodinated DBPs show significantly higher toxicity than their brominated and chlorinated analogues and thus have been drawing increasing concerns. In this study, polar iodinated DBPs were detected in chlorinated saline wastewater effluents using a novel precursor ion scan method. The major polar iodinated DBPs were identified and quantified, and their organic precursors and formation pathways were investigated. The formation of iodinated DBPs under different chlorine doses and contact times was also studied. The results indicated that a few polar iodinated DBPs were generated in the chlorinated saline primary effluent, but few were generated in the chlorinated saline secondary effluent. Several major polar iodinated DBPs in the chlorinated saline primary effluent were proposed with structures, among which a new group of polar iodinated DBPs, iodo-trihydroxybenzenesulfonic acids, were identified and quantified. The organic precursors of this new group of DBPs were found to be 4-hydroxybenzenesulfonic acid and 1,2,3-trihydroxybenzene, and the formation pathways of these new DBPs were tentatively proposed. Both chlorine dose and contact time affected the formation of iodinated DBPs in the chlorinated saline wastewater effluents.

Halopyrroles: a new group of highly toxic disinfection byproducts formed in chlorinated saline wastewater.

Utilizing seawater for toilet flushing is an effective way to conserve freshwater in coastal cities. During chlorination for disinfecting saline wastewater effluents, the high levels of bromide from seawater are oxidized to hypobromous acid which may then react with effluent organics to form brominated disinfection byproducts (DBPs). In this research, by applying a new precursor ion scan method, we detected and identified a group of halopyrroles in a chlorinated saline wastewater effluent, including tetrabromopyrrole, tribromochloropyrrole, tribromoiodopyrrole, and tribromopyrrole, with tetrabromopyrrole as the predominant species. It is the first time that this group of halopyrroles were identified as wastewater DBPs (though 2,3,5-tribromopyrrole has been found to be a DBP in drinking water before). Detection of halopyrroles was problematic as these compounds in the pretreated samples were found to convert to halonitropyrroles; the problem was successfully solved by diluting the pretreated samples. The formation, occurrence, precursor, and toxicity of tetrabromopyrrole were investigated. This DBP showed significantly higher developmental toxicity than any of the haloaliphatic and haloaromatic DBPs previously tested.

Detection and characterization of triamcinolone acetonide metabolites in human urine by liquid chromatography/tandem mass spectrometry after intramuscular administration.

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. intramuscular, IM), whereas they are allowed using other ways of administration. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of triamcinolone acetonide (TA), one of the most used glucocorticosteroids, was studied using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Urine samples obtained after IM administration of TA were analyzed using two sample treatments: (a) hydrolysis with β-glucuronidase enzymes and liquid-liquid extraction under alkaline conditions, and (b) liquid-liquid extraction under acidic conditions. The extracts were analyzed by LC/MS/MS. TA, commercially available metabolites (6β-hydroxytriamcinolone acetonide, 6β-OH-TA, and triamcinolone), and their C20-reduced derivatives showed characteristic fragmentation behavior. Besides common product ions and neutral losses for corticosteroids containing fluorine, additional characteristic neutral losses (58 Da, loss of acetone; 44 Da, loss of acetaldehyde) were observed in positive electrospray ionization. Based on that behavior, two complementary approaches were applied to detect TA metabolites: (a) open detection by precursor ion and neutral loss scan methods and (b) targeted detection by selected reaction monitoring methods (SRM) containing theoretical ion transitions of the potential metabolites. Two main compounds, TA and 6β-OH-TA, and nine minor potential metabolites, were detected by open screening methods. Using SRM, two additional metabolites were detected. Some of the metabolites were characterized using reference standards and, for the rest of metabolites, feasible structures were proposed based on mass spectrometric data. Metabolites resulting from hydroxylation in C-6, oxidation of the 11-hydroxyl group, reduction of the Δ(4) double bond and oxidation of the side chain were detected. Some of them have not been previously described. Excretion profiles of the detected metabolites after IM administration are presented.

Application of precursor ion scanning method in rapid screening of illegally added phosphodiesterase-5 inhibitors and their unknown derivatives in Chinese traditional patent medicines and health foods

The compounds with similar structure often have similar pharmacological activities. So it is a trend for illegal addition that new derivatives of effective drugs are synthesized to avoid the statutory test. This bring challenges to crack down on illegal addition behavior, however, modified derivatives usually have similar product ions, which allow for precursor ion scanning. In this work, precursor ion scanning mode of a triple quadrupole mass spectrometer was first applied to screen illegally added drugs in complex matrix such as Chinese traditional patent medicines and healthy foods. Phosphodiesterase-5 inhibitors were used as experimental examples. Through the analysis of the structure and mass spectrum characteristics of the compounds, phosphodiesterase-5 inhibitors were classified, and their common product ions were screened by full scan of product ions of typical compounds. Then high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method with precursor ion scanning mode was established based on the optimization of MS parameters. The effect of mass parameters and the choice of fragment ions were also studied. The method was applied to determine actual samples and further refined. The results demonstrated that this method can meet the need of rapid screening of unknown derivatives of phosphodiesterase-5 inhibitors in complex matrix, and prevent unknown derivatives undetected. This method shows advantages in sensitivity, specificity and efficiency, and is worth to be further investigated.

A new sulphate metabolite as a long-term marker of metandienone misuse

Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26days after the administration of a single dose of metandienone (5mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).

Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed.