Product Ion Scan Research Articles

Triterpenoid saponins profiling by adducts-targeted neutral loss triggered enhanced resolution and product ion scanning using triple quadrupole linear ion trap mass spectrometry

Triterpenoid saponins (TSs) are a unique class of high molecular weight glycosides and have been frequently used in cosmetic and phytotherapy industry. There is a great need to comprehensively profile these plant metabolites for studying their functions. In the present study, a novel adducts targeted neutral loss (NL), triggered enhanced resolution (ER) and enhanced product ion (EPI) scanning approach were described for TSs profiling using a triple quadrupole linear ion trap mass spectrometry. This approach circumvented the disadvantages of poor glycosidic bond cleavage of TSs by monitoring the NH3 (NL17) and HCOOH (NL46) loss of their abundant ammonium and formate adducts, respectively. The sugar-loss independent NL scanning served as a sensitive survey scan and triggered information-dependent ER and EPI scans to increase peak assignment confidence. NL17 was superior to NL46 for TSs characterization due to the better fragmentation of ammonium adducts than formate adducts. For those TSs undetectable by NL17, precursor ion (PI) scan for sapogenin fragments could be used to screen out non-adducted TSs. The NL/PI-ER-EPI approach was applied for TSs profiling in Astragali Radix, a famous medicinal and nutritional plant widely used in Asian countries and United States. In total, 136 TSs were detected while previous research using high resolution mass spectrometry based full scan only detected 22 TSs in this herb.

Metabolomic approaches for orange origin discrimination by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

In this work, hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) coupled to ultra high performance liquid chromatography (UHPLC) has been used for biomarkers identification for correct authentication of Valencia (Spain) oranges. Differentiation from foreign Argentinean, Brazilian and South African oranges has been carried out using XCMS application and multivariate analysis to UHPLC-(Q)TOF MS data acquired in both, positive and negative ionisation modes. Several markers have been found and corroborated by analysing two seasons samples. A seasonal independent marker was found and its structure elucidated using accurate mass data and MSE fragmentation spectrum information. Empirical formula was searched in Reaxys database applying sub-structure filtering from the fragments obtained. Three possible structures were found and citrusin D, a compound present in sweet oranges, has been identified as the most plausible as it fits better with the product ion scan performed for this compound. As a result of data obtained in this work, citrusin D is suggested as a potential marker to distinguish the geographic origin of oranges.

Metabolism of synthetic cannabinoids PB-22 and its 5-fluoro analog, 5F-PB-22, by human hepatocyte incubation and high-resolution mass spectrometry

PB-22 (1-pentyl-8-quinolinyl ester-1H-indole-3-carboxylic acid) and 5F-PB-22 (1-(5-fluoropentyl)-8-quinolinyl ester-1H-indole-3-carboxylic acid) are new synthetic cannabinoids with a quinoline substructure and the first marketed substances with an ester bond linkage. No human metabolism data are currently available, making it difficult to document PB-22 and 5F-PB-22 intake from urine analysis, and complicating assessment of the drugs' pharmacodynamic and toxicological properties. We incubated 10μmol/lPB-22 and 5F-PB-22 with pooled cryopreserved human hepatocytes up to 3h and analyzed samples on a TripleTOF 5600+ high-resolution mass spectrometer. Data were acquired via TOF scan, followed by information-dependent acquisition triggered product ion scans with mass defect filtering (MDF). The accurate mass full scan MS and MS/MS metabolite datasets were analyzed with multiple data processing techniques, including MDF, neutral loss and product ion filtering. The predominant metabolic pathway for PB-22 and 5F-PB-22 was ester hydrolysis yielding a wide variety of (5-fluoro)pentylindole-3-carboxylic acid metabolites. Twenty metabolites for PB-22 and 22 metabolites for 5F-PB-22 were identified, with the majority generated by oxidation with or without glucuronidation. For 5F-PB-22, oxidative defluorination occurred forming PB-22 metabolites. Both compounds underwent epoxide formation followed by internal hydrolysis and also produced a cysteine conjugate. Human hepatic metabolic profiles were generated for PB-22 and 5F-PB-22. Pentylindole-3-carboxylic acid, hydroxypentyl-PB-22 and PB-22 pentanoic acid for PB-22, and 5'-fluoropentylindole-3-carboxylic acid, PB-22 pentanoic acid and the hydroxy-5F-PB-22 metabolite with oxidation at the quinoline system for 5F-PB-22 are likely the best targets to incorporate into analytical methods for urine to document PB-22 and 5F-PB-22 intake.

Development of a New Method for the Identification of Degradation Products of V-Type Nerve Agents by Liquid Chromatography–Tandem Mass Spectrometry

Degradation products of V-type nerve agents are important markers of these toxic chemical warfare agents; hence their detection and identification are of high importance from verification point of view of Chemical Weapons Convention. The new analytical technique using quantitation-enhanced data-dependent (QED) method has been developed for the analysis of the degradation products, 2-(N,N-dialkylamino)ethanesulfonic acids of V-type nerve agents, by using liquid chromatography-tandem mass spectrometry (Thermo-Scientific Vantage triple stage quadrupole mass spectrometer, Thermo Finnigan Surveyor, San Jose, CA, USA) via an atmospheric pressure ionization source/interface operated in eletrospray ionization mode. With a single analytical run, we could perform the quantitative analysis of the 2-(N,N-dialkylamino)ethanesulfonic acids by the selected reaction monitoring scan mode with limit of detection at 0.1 ng/mL and identify their isomeric compounds by product ion scan mode, simultaneously. The QED method will be applicable to the trace analysis of degradation products of V-type nerve agents in the environmental matrices in the Organisation for the Prohibition of Chemical Weapons proficiency test.

A lipidomic platform establishment for structural identification of skin ceramides with non-hydroxyacyl chains

The stratum corneum (SC) is the outermost layer of skin that functions as a barrier and protects against environmental influences and transepidermal water loss. Its unique morphology consists of keratin-enriched corneocytes embedded in a distinctive mixture of lipids containing mainly ceramides, free fatty acids, and cholesterol. Ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine (dS), sphingosine (S), phytosphingosine (P), and 6-hydroxysphingosine (H), and the fatty acid acyl chains are composed of non-hydroxy fatty acid (N), α-hydroxy fatty acid (A), ω-hydroxy fatty acid (O), and esterified ω-hydroxy fatty acid (E). The 16 ceramide classes include several combinations of sphingoid bases and fatty acid acyl chains. Among them, N-type ceramides are the most abundant in the SC. Mass spectrometry (MS)/MS analysis of N-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, which could be applied to structural identification of ceramides. Based on the MS/MS fragmentation patterns of N-type ceramides, comprehensive fragmentation schemes were proposed. In addition, mass fragmentation patterns, which are specific to the sphingoid backbone of N-type ceramides, were found in higher m/z regions of tandem mass spectra. These characteristic and general fragmentation patterns were used to identify N-type ceramides in human SC. Based on established MS/MS fragmentation patterns of N-type ceramides, 52 ceramides (including different classes of NS, NdS, NP, and NH) were identified in human SC. The MS/MS fragmentation patterns of N-type ceramides were characterized by interpreting their product ion scan mass spectra. This information may be used to identify N-type ceramides in the SC of human, rat, and mouse skin.

Pseudo-MS3Approach Using Electrospray Mass Spectrometry (ESI-MS/MS) to Characterize Certain (2E)-2-[3-(1H-Imidazol-1-yl)-1-phenylpropylidene]hydrazinecarboxamide Derivatives

An approach for the use of in-source fragmentation with electrospray ionization followed by product ion scan in a triple quadrupole mass spectrometer system is described. This approach is based on the elucidation of the various fragmentation pathways by further dissociation of each fragment ion in the ion spectrum. This can be achieved predominately, by combining fragmentor voltage induced dissociation (in-source fragmentation) with subsequent collision-induced dissociation; this process can be referred to as pseudo-MS3scan mode. This technique permitted unambiguous assignment and provided sufficient sensitivity and specificity. It is advantageous for structure elucidation of unknown compounds. We investigate the possibility of using in-source fragmentation with the diverse novel chemical entities encompassing different substituents. This process was intended to improve the qualitative capability of tandem mass spectrometry simulating the MS3of ion trap for studying fragmentation mechanisms. The approach is to implement the investigated technique as a well established tool for the characterization of new pharmacologically important chemical entities. The data presented in this paper provided useful information on the effect of different substituents on the ionization/fragmentation processes and can be used in the characterization of (2E)-2-[3-(1H-imidazol-1-yl)-1-phenylpropylidene]-hydrazinecarboxamide derivatives3a–h.

Augmentation of blood lipid glycation and lipid oxidation in diabetic patients

Lipid oxidation plays a role in the pathophysiology of several diseases, including diabetes. Patients with type 2 diabetes show abnormally high plasma levels of phosphatidylcholine hydroperoxide (PCOOH). However, little is known about the biochemical processes that increase plasma PCOOH in diabetes. We hypothesized that "glycated lipid moieties" may form in diabetic plasma and cause oxidative stress resulting in PCOOH formation. To evaluate this hypothesis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed to analyze Amadori-glycated phosphatidylethanolamine (Amadori-PE, an early stage Maillard product), as well as the advanced glycation end products (AGE) carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE). The product ion scan, neutral loss scanning, and multiple reaction monitoring provide useful structural and quantitative information about Amadori-PE, CM-PE, and CE-PE in diabetic plasma and erythrocytes. We found that plasma and erythrocyte Amadori-PE concentrations were significantly higher in diabetic patients (757±377 nM plasma, 2793±989 nM packed cells) than in normal subjects (165±66 nM plasma, 712±52 nM packed cells), and that Amadori-PE concentrations were positively correlated with PCOOH. By contrast, no significant differences were observed in blood AGE-PE concentrations between diabetic patients (CM-PE: 7.7±3.5 nM plasma, 528±83 nM packed cells; CE-PE: 2.5±1.1 nM plasma, 82±24 nM packed cells) and normal subjects (CM-PE: 6.6±3.1 nM plasma, 705±533 nM packed cells; CE-PE: 4.2±1.5 nM plasma, 68±16 nM packed cells). These results suggest that Amadori-PE is more prone to accumulation in the blood with diabetes than CM-PE or CE-PE. This review describes the involvement of blood lipid glycation and lipid oxidation in the development of diabetes.

An effective integrated method for comprehensive identification of eighty-five compounds in Zhi-Zi-Da-Huang decoction by HPLC-DAD-ESI-MS (TOF) and HPLC-DAD-ESI-MS/MS (QqQ) without the help of reference standards

Zhi-Zi-Da-Huang decoction (ZZDHD) is an effective formula to treat alcoholic liver disease in China for nearly 2000 years. However, compounds in the formula are still not clear due to the complexity of the coexisting constituents. In this study, a sensitive integrated method based on HPLC-DAD-ESI-MS (TOF) and HPLC-DAD-ESI-MS/MS (QqQ) was developed and 85 chemical compounds in ZZDHD were identified. By TOF/MS, the quasi-molecular ions of the constituents were deduced according to the accurate mass of corresponding positive and negative ions, and product ion scan was then carried out using QqQ/MS. The fragments of each compound were observed by TOF/MS and/or QqQ/MS. Compounds with different chemical structures and concentration levels were tentatively identified according to their chromatographic retention time, UV absorption and MS data. Fragmentation patterns of six types were proposed to verify the identification results. It was found that at TOF/MS mode, a retro diels-alder (RDA) reaction occurred at bonds 1 and 3 in C-ring of flavanone 7-O-diglycosides when 4′-OH exists at B-ring. While the reaction was not observed if a methoxyl group appeared at the same site of flavanone skeleton. Another important phenomenon was noticed that 3-sinapoyl-5-caffeoylquinic acid, 4-sinapoyl-3-caffeoylquinic acid, and sinapylglucoside were detected only in ZZDHD while they were not found in four component herb extracts. The phenomenon revealed that changes of compounds happened when the component herbs were decocted together. The results in the paper are favorable of the clinic dosage and have laid a foundation for the subsequent research of ZZDHD in vivo.

Simultaneous determination of nine beta-blockers in porcine tissues by ultra-fast liquid chromatography coupled with quadrupole/linear ion trap mass spectrometry

A highly sensitive method using ultra-fast liquid chromatography coupled with quadrupole/linear ion trap mass spectrometry (UFLC-Q/Trap MS) was developed to simultaneously screen and confirm nine beta-blockers (BBs) in porcine tissues (porcine muscle, liver and kidney). The method was used for trace determination of atenolol, pindolol, acebutolol, metoprolol, carazolol, labetalol, bisoprolol, propranolol and penbutolol. The homogenized tissues were hydrolyzed by beta-glucuronidase/aryl sulfatase and extracted with acetonitrile, followed by continuous purification procedures of disperse solid phase extraction (d-SPE) with diatomaceous earth and BondElut cartridge. The ultra-fast chromatographic separation was conducted on a Kinetex C18-XB column (150 mm x 2.1 mm, 2.6 microm) using 0.1% (v/v) formic acid aqueous solution and methanol as mobile phases in gradient elution. The optimized ion transitions were mployed in the mixed-mode of scheduled multiple reaction monitoring (sMRM) -information dependent acquisition (IDA)-enhanced product ion (EPI) scan. Qualification analysis was performed through spectra-matching with on-line lab-built MS/MS library. For quantification stable isotope-labelled analogues of the analytes were used as internal standards. As a result, in porcine liver, kidney and muscle, the nine BBs showed good linearity with all the correlation coefficients (r) more than 0.995 in the range of 0.1-20 microg/L. The limits of quantification (LOQ, S/N > or = 10) were 0.5 kg/kg for all the analytes. The developed method gave average recoveries of 87.5%-111.8% spiked at 0.5, 1.0 and 5.0 microg/kg with the relative standard deviations of 4.0%-12. 5%. The proposed method can be used to screen and confirm the nine BBs in a single run, which makes it effective in surveillance and detection of the BBs residues in porcine tissues.

Detection of efaproxiral (RSR13) and its metabolites in equine by liquid chromatography tandem mass spectrometry

Efaproxiral (RSR 13) is an experimental synthetic allosteric modifier of haemoglobin (Hb) that acts by increasing the release of oxygen from Hb to the surrounding tissues. It has been shown to increase maximum oxygen uptake (VO(2max)) in a canine skeletal muscle model. The ability to increase maximal muscle oxygen uptake makes efaproxiral a potential performance-enhancing agent and is therefore prohibited by the World Anti-Doping Agency. In this study, a method for the detection and elimination of efaproxiral in equine plasma and urine after a 2.5 g intravenous administration of efaproxiral is described. Post administration plasma and urine samples were collected up to 120 h. Efaproxiral was detected up to 120 h in urine and up to 78 h in plasma. In plasma, the peak concentration was 42 µg/ml and detected at 5 min post administration. In urine, the peak concentration was 2.8 mg/ml and detected at 0-1 h post administration. A validated liquid chromatography tandem mass spectrometry method was used for the quantitation of efaproxiral in equine plasma and urine. The limit of detection of the method is 0.05 ng/ml in plasma and 0.1 ng/ml in urine. The method is highly sensitive and specific with good precision, accuracy and recovery. The manuscript also describes the systematic identification of efaproxiral metabolites detected in post administration equine urine samples. The metabolites were identified by use of enhanced mass spectra and enhanced product ion scans. Both positive and negative mode ionizations were utilized for metabolite identification and plausible fragmentation pathways were proposed for the phase 1 metabolite identified. In addition to free efaproxiral, one phase 1 metabolite and two phase 2 metabolites were identified in post administration urine.

High throughput analysis of cerebroside molecular species from sea cucumber Parastichopus californicus by liquid chromatography-quadrupole-time-of-flight mass spectrometry

Cerebrosides from sea cucumber Parastichopus californicus were identified by using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF MS/ MS). The samples were extracted with chloroform-methanol (2: 1, v/v) solution and purified by a SPE cartridge. In positive ion mode electrospray ionization (ESI), the precursor ion scan mass spectra and product ion scan mass spectra were obtained through the automatic MS/MS mode. Cerebroside molecules were selected according to the neutral loss fragments of 180 Da, and then were identified according to long-chain base (LCB) fragments and fatty acid (FA) fragments. One hundred and twenty-three cerebroside molecular species were identified. There are 18 species of LCB, and the relative content ratio of phytosphingosines and sphingosines is 1: 2. The carbon numbers of fatty acids are mainly 18 - 25, of which 24 carbon fatty acids are predominant. The relative content ratio of saturated fatty acids and monounsaturated fatty acid is about 1: 3, and the presence of 2-hydroxy fatty acids is about 58.62%. LC-Q-TOF MS/MS method is sensitive, accurate and simple. At the same time, this study provided a theoretical basis for structure-activity relationship studies and functional food development of Parastichopus californicus as well.

Multistage Fragmentation of Ion Trap Mass Spectrometry System and Pseudo-MS3of Triple Quadrupole Mass Spectrometry Characterize Certain (E)-3-(Dimethylamino)-1-arylprop-2-en-1-ones: A Comparative Study

A new approach was recently introduced to improve the structure elucidation power of tandem mass spectrometry simulating the MS3 of ion trap mass spectrometry system overcoming the different drawbacks of the latter. The fact that collision induced dissociation in the triple quadrupole mass spectrometer system provides richer fragment ions compared to those achieved in the ion trap mass spectrometer system utilizing resonance excitation. Moreover, extracting comprehensive spectra in the ion trap needs multistage fragmentation, whereas similar fragment ions may be acquired from one stage product ion scan using the triple quadrupole mass spectrometer. The new strategy was proven to enhance the qualitative performance of tandem mass spectrometry for structural elucidation of different chemical entities. In the current study we are endeavoring to prove our hypothesis of the efficiency of the new pseudo-MS3 technique via its comparison with the MS3 mode of ion trap mass spectrometry system. Ten pharmacologically and synthetically important (E)-3-(dimethylamino)-1-arylprop-2-en-1-ones (enaminones 4a–j) were chosen as model compounds for this study. This strategy permitted rigorous identification of all fragment ions using triple quadrupole mass spectrometer with sufficient specificity. It can be used to elucidate structures of different unknown components. The data presented in this paper provide clear evidence that our new pseudo-MS3 may simulate the MS3 of ion trap spectrometry system.

Predicted multiple selected reaction monitoring to screen activated drug-mediated modifications on human serum albumin

Metabolic activation of drugs frequently generates electrophilic products that may undergo covalent binding to biological macromolecules, such as proteins and DNA. The resulting covalent adducts are of considerable concern in drug discovery and development. Several strategies for assessing the potential risks of candidate drugs have been reported. Of these, glutathione trapping is the most commonly used method together with mass spectrometry. Furthermore, drug-mediated protein modifications have been studied using serum albumin and CYP enzymes to clarify target amino acids and mechanism-based inhibition, respectively. In this article, we introduce a practical way to screen drug-mediated protein modifications. The method, referred to as “predicted multiple selected reaction monitoring,” is based on the selected reaction monitoring (SRM) strategy, but targets all possible chemically modified tryptic peptides. The creation of SRM lists may require patience; however, this strategy could facilitate more sensitive screening compared with the common strategy of data-dependent product ion scanning. Ketoprofen-N-hydroxysuccinimidyl ester (equivalent to glucuronide) and N-acetyl-p-benzoquinone imine (NAPQI) were allowed to react with human serum albumin as a model experiment. Using this strategy, 11 ketoprofen-adduction sites (at Lys137, 195, 199, 212, 351, 402, 432, 436, 525, 536, and 541) and 1 NAPQI-adduction site (at Cys34) were easily identified.

Gas-Phase Separation of Drugs and Metabolites Using Modifier-Assisted Differential Ion Mobility Spectrometry Hyphenated to Liquid Extraction Surface Analysis and Mass Spectrometry

The present work describes an alternative generic approach to LC-MS for the analysis of drugs of abuse as well as their metabolites in post-mortem tissue samples. The platform integrates liquid extraction surface analysis (LESA) for analytes tissue extraction followed by differential ion mobility spectrometry (DMS) mass spectrometry for analytes gas phase separation. Detection is performed on a triple quadrupole linear ion trap using the selected reaction monitoring mode for quantification as well as product ion scan mode for structural confirmatory analyses. The major advantages of the platform are that neither chromatographic separation nor extensive sample preparation are required. In DMS the combination of a high separation voltage (i.e., up to 4 kV) together with organic modifiers (e.g., alcohols, acetonitrile, acetone) added in the drift gas is required to achieve the separation of isomeric metabolites, such as the ones of cocaine and tramadol. DMS also separates morphine from its glucuronide metabolites, which allows for preventing the overestimation of morphine in case of fragmentation of the glucuronides in the atmospheric-to-vacuum interface of the mass spectrometer. Cocaine, opiates, opioids, amphetamines, benzodiazepines and several of their metabolites could be identified in post-mortem human kidney and muscle tissue based on simultaneous screening and confirmatory analysis in data-dependent acquisition mode using an analyte-dependent compensation voltage to selectively transmit ions through the DMS cell to the mass analyzer. Quantitative performance of the LESA-DMS-MS platform was evaluated for cocaine and two of its metabolites spotted onto a tissue section using deuterated internal standard. Analyte's responses were linear from 2 to 1000 pg on tissue corresponding to a limit of detection in the order of nanograms of analyte per gram of tissue. Accuracy and precision based on QC sample was found to be less than 10%. Replicate analyses of cocaine and its metabolites in forensic samples showed an intra- and inter-sections variability of less than 25%.

Specificity enhancement by electrospray ionization multistage mass spectrometry – a valuable tool for differentiation and identification of ‘V’‐type chemical warfare agents

The use of chemical warfare agents has become an issue of emerging concern. One of the challenges in analytical monitoring of the extremely toxic 'V'-type chemical weapons [O-alkyl S-(2-dialkylamino)ethyl alkylphosphonothiolates] is to distinguish and identify compounds of similar structure. MS analysis of these compounds reveals mostly fragment/product ions representing the amine-containing residue. Hence, isomers or derivatives with the same amine residue exhibit similar mass spectral patterns in both classical EI/MS and electrospray ionization-MS, leading to unavoidable ambiguity in the identification of the phosphonate moiety. A set of five 'V'-type agents, including O-ethyl S-(2-diisopropylamino)ethyl methylphosphonothiolate (VX), O-isobutyl S-(2-diethylamino)ethyl methylphosphonothiolate (RVX) and O-ethyl S-(2-diethylamino)ethyl methylphosphonothiolate (VM) were studied by liquid chromatography/electrospray ionization/MS, utilizing a QTRAP mass detector. MS/MS enhanced product ion scans and multistage MS(3) experiments were carried out. Based on the results, possible fragmentation pathways were proposed, and a method for the differentiation and identification of structural isomers and derivatives of 'V'-type chemical warfare agents was obtained. MS/MS enhanced product ion scans at various collision energies provided information-rich spectra, although many of the product ions obtained were at low abundance. Employing MS(3) experiments enhanced the selectivity for those low abundance product ions and provided spectra indicative of the different phosphonate groups. Study of the fragmentation pathways, revealing some less expected structures, was carried out and allowed the formulation of mechanistic rules and the determination of sets of ions typical of specific groups, for example, methylphosphonothiolates versus ethylphosphonothiolates. The new group-specific ions elucidated in this work are also useful for screening unknown 'V'-type agents and related compounds, utilizing precursor ion scan experiments.

A qualitative/quantitative approach for the detection of 37 tryptamine-derived designer drugs, 5 β-carbolines, ibogaine, and yohimbine in human urine and plasma using standard urine screening and multi-analyte approaches

The first synthetic tryptamines have entered the designer drug market in the late 1990s and were distributed as psychedelic recreational drugs. In the meantime, several analogs have been brought onto the market indicating a growing interest in this drug class. So far, only scarce analytical data were available on the detectability of tryptamines in human biosamples. Therefore, the aim of the presented study was the development and full validation of a method for their detection in human urine and plasma and their quantification in human plasma. The liquid chromatography-linear ion trap mass spectrometry method presented covered 37 tryptamines as well as five β-carbolines, ibogaine, and yohimbine. Compounds were analyzed after protein precipitation of urine or fast liquid-liquid extraction of plasma using an LXQ linear ion trap coupled to an Accela ultra ultra high-performance liquid chromatography system. Data mining was performed via information-dependent acquisition or targeted product ion scan mode with positive electrospray ionization. The assay was selective for all tested substances with limits of detection in urine between 10 and 100ng/mL and in plasma between 1 and 100ng/mL. A validated quantification in plasma according to international recommendation could be demonstrated for 33 out of 44 analytes.

First Metabolic Profile of XLR-11, a Novel Synthetic Cannabinoid, Obtained by Using Human Hepatocytes and High-Resolution Mass Spectrometry

Since the mid-2000s synthetic cannabinoids have been abused as recreational drugs, prompting scheduling of these substances in many countries. To circumvent legislation, manufacturers constantly market new compounds; [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), the fluorinated UR-144 analog, is one of the most recent and widely abused drugs, and its use is now linked with acute kidney injury. Our goal was to investigate XLR-11 metabolism for identification of major urinary targets in analytical methods and to clarify the origin of metabolites when one or more parent synthetic cannabinoids can be the source. We incubated 10 μmol/L XLR-11 with pooled human hepatocytes and sampled after 1 and 3 h. Samples were analyzed by high-resolution mass spectrometry with a TOF scan followed by information-dependent acquisition triggered product ion scans with dynamic background subtraction and mass defect filters. Scans were thoroughly data mined with different data processing algorithms (Metabolite Pilot 1.5). XLR-11 underwent phase I and II metabolism, producing more than 25 metabolites resulting from hydroxylation, carboxylation, hemiketal and hemiacetal formation, internal dehydration, and further glucuronidation of some oxidative metabolites. No sulfate or glutathione conjugation was observed. XLR-11 also was defluorinated, forming UR-144 metabolites. On the basis of mass spectrometry peak areas, we determined that the major metabolites were 2'-carboxy-XLR-11, UR-144 pentanoic acid, 5-hydroxy-UR-144, hydroxy-XLR-11 glucuronides, and 2'-carboxy-UR-144 pentanoic acid. Minor metabolites were combinations of the biotransformations mentioned above, often glucuronidated. These are the first data defining major urinary targets of XLR-11 metabolism that could document XLR-11 intake in forensic and clinical investigations.

Separation and identification of underivatized plasma acylcarnitine isomers using liquid chromatography–tandem mass spectrometry for the differential diagnosis of organic acidemias and fatty acid oxidation defects

A simple HPLC–MS/MS method has been established to separate and identify underivatized acylcarnitine isomers. Human plasma samples were deproteinized and concentrated. Acylcarnitines were separated on a reverse phase column and detected with triple quadrupole linear ion trap mass spectrometry. Deuterium-labeled internal standards were used for quantitation. To identify acylcarnitines without pure standards, information-dependent acquisition linking to enhanced product ion scan mode was used. 112 acylcarnitines, including stereoisomers, were found in samples of patients. Dicarboxylic acylcarnitines, such as methylmalonylcarnitine and glutarylcarnitine, were detected with high sensitivity. Three stereoisomers of (R,S)2-methyl-3-hydroxy butyrylcarnitine were detected in samples of patients with β-ketothiolase deficiency. Validation results revealed excellent precision and accuracy of the method. In general the within- and between-run coefficients of variation (CV%) were less than 15%, and recoveries were in the range of 92.7–117.5%. In addition, the reference intervals of acylcarnitines for children aged 3-day to13-year old were established. Using the new method and reference intervals, we have correctly diagnosed 49 patients with fatty acid oxidation defects or organic acidemias in 176 high-risk patients.

Plasma lipid profiling in a large population-based cohort

We have performed plasma lipid profiling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 μl of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identified statistically significant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was significantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk.

Simultaneous identification and detection of 16 anabolic steroid hormones in muscle using liquid chromatography coupled to quadrupole/linear ion trap mass spectrometry

A comprehensive method for simultaneous identification and detection of 16 anabolic steroid hormones (ASs, including andorgens, gestagens and their esters) in muscle samples was developed with liquid chromatography coupled to quadrupole/linear ion trap mass spectrometry (LC-Q/Trap-MS). The ASs in muscle samples were extracted with acetonitrile under ultrasonic assistance. The extract was defatted by n-hexane with liquid-liquid partitioning and followed by clean-up with NH2 solid phase extraction (SPE) cartridge. The separation of analytes was carried out on a CAPCELL PAK C18 MG II column (150 mm x 2.0 mm, 5.0 microm) using mobile phases of 0.1% (v/v) formic acid in acetonitrile and 0.1% (v/v) formic acid-5 mmol/L ammonium formate aqueous solution with gradient elution. A scheduled multiple reaction monitoring (sMRM) in positive mode as survey scan and an enhanced product ion (EPI) scan as dependent scan in an information-dependent acquisition (IDA) experiment was adopted in mass spectrometry acquisition. On-line lab-built MS/MS library and internal standards were employed for the identification and quantification. As a result, the 16 ASs showed good linearity with all correlation coefficients (r) no less than 0. 999 0 within the linear ranges. The limits of quantification (LOQs, S/N > or = 10) for the 16 ASs were in the range of 0.029-0.36 microg/kg. At the three spiked levels (0.5, 2.0 and 20 microg/kg), the overall recoveries ranged from 89.9% to 118% with the relative standard deviations (RSDs) no more than 16.2% under within--laboratory reproducibility conditions. The proposed method can be used to identify and detect the 16 ASs in a single run, which makes it effective in residue surveillance of anabolic hormones in muscle samples.