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Synthetic opioids are a class of compounds that are of particular concern due to their high potency and potential health impacts. With the relentless emergence of new synthetic opioid derivatives, non-targeted screening strategies are required that do not rely on the use of library spectra or reference materials. In this study, product ion searching, and Kendrick mass defect analysis were investigated for non-targeted screening of synthetic opioids. The estimated screening cut-offs for these techniques ranged between 0.05 and 0.1 ng/mL. These techniques were designed to not be reliant on a particular vendor's software, meaning that they can be applied to existing drug screening protocols, without requiring the development and validation of new analytical procedures. The efficacy of the developed techniques was tested through blind trials, with spiked samples inserted amongst authentic plasma samples, which demonstrated the usefulness of these methods for high-throughput screening. The use of a non-targeted screening workflow that contains complementary techniques can increase the likelihood of detecting compounds of interest within a sample, as well as the confidence in detections that are made., (© 2020 John Wiley & Sons, Ltd.)

Rationale: A calibration solution for mass spectrometry needs to cover the range of interest with intense and sufficiently narrowly spaced peaks. Limited options fulfilling this may lead to compromises between performance and ease of use. SpheriCal ® -ESI was designed to combine high calibration performance for electrospray ionization (ESI) mass spectrometric analysis of peptides in positive mode with quick and easy use., Methods: The developed calibration solution was tested using three mass spectrometers: two ion traps and one tandem quadrupole. The m/z errors of SpheriCal ® -ESI itself and of a tryptic digest of cytochrome C were measured after calibration. The results were compared with those achieved with ESI Tuning Mix. The memory effects of the dendrimers, and contamination from Na + in the calibration solution, were evaluated., Results: SpheriCal ® -ESI showed good shelf life as powder and was quickly reconstituted for use. Achieving intense and stable signals was straightforward. The accuracies and precisions were as expected for the instruments. SpheriCal ® -ESI was more precise and at least as accurate as ESI Tuning Mix. The memory effects and Na + contamination were found to be negligible in typical peptide solvents. In addition, the dendrimers showed predictable dissociations with product ions common to collision-induced dissociation in both ion trap and tandem quadrupole mass spectrometers., Conclusions: SpheriCal ® -ESI provided easily accessible calibration by showing intense signals at low infusion rates and at source settings equal or similar to those used in peptide analysis. Nine calibration points in the range of interest gave precise and accurate results. Memory effects and contamination were negligible even without rinsing., (© 2020 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

This study demonstrates the utility of electrospray ionization inlet mass spectrometry (ESII-MS/MS) for the quantitative determination of analytes in complex animal matrices without chromatographic separation. Veterinary drugs including flunixin, its metabolite 5-hydroxyflunixin, and zilpaterol and persistent organic perfluoroalkyl compounds were determined in incurred plasma, urine, and/or tissue samples. Limits of detection (LOD) of zilpaterol in kidney, liver, lung, and muscle ranged from 0.02 to 0.06 ng/g, whereas the limit of quantitation (LOQ) for zilpaterol in all tissues was 0.1 ng/g. For urinary or plasma flunixin, 5-hydroxyflunixin, and PFOS/PFHxS, LODs ranged from 0.1 to 0.7 ng/mL while the LOQs ranged from 0.4 to 50 ng/mL. Regression coefficients for matrix-matched standard curves were 0.993-0.997, 0.977-0.999, and 0.999 for plasma, tissues, and urine, respectively. Correlations between quantitative results obtained by ESII-MS/MS and LC-MS for flunixin, 5-hydroxyflunixin, and zilpaterol ranged from 0.930 to 0.985. ESII-MS/MS provided rapid, sensitive, and accurate analyses of veterinary drugs and environmental contaminants from complex matrices without chromatographic separation.

LC/ESI/MS is the technique of choice for qualitative and quantitative food monitoring; however, analysis of a large number of compounds is challenged by the availability of standard substances. The impediment of detection of food contaminants has been overcome by the suspect and non-targeted screening. Still, the results from one laboratory cannot be compared with the results of another laboratory as quantitative results are required for this purpose. Here we show that the results of the suspect and non-targeted screening for pesticides can be made quantitative with the aid of in silico predicted electrospray ionization efficiencies and this allows direct comparison of the results obtained in two different laboratories. For this purpose, six cereal matrices were spiked with 134 pesticides and analysed in two independent labs; a high correlation for the results with the R 2 of 0.85., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Paralytic shellfish toxins (PSTs) are a complex class of analogs of the potent neurotoxin saxitoxin (STX). Since calibration standards are not available for many PSTs, including C -11 hydroxyl analogs called M-toxins, accurate quantitation by liquid chromatography-mass spectrometry (LC-MS) can be challenging. In the absence of standards, PSTs are often semiquantitated using standards of a different analog (e.g., STX), an approach with a high degree of uncertainty due to the highly variable sensitivity between analytes in electrospray ionization. Here, relative molar response factors (RMRs) were investigated for a broad range of PSTs using common LC-MS approaches in order to improve the quantitation of PSTs for which standards are unavailable. First, several M-toxins (M1-M6, M9 and dcM6) were semipurified from shellfish using preparative gel filtration chromatography and quantitated using LC-charged aerosol detection (LC-CAD). The RMRs of PST certified reference materials (CRMs) and M-toxins were then determined using selective reaction monitoring LC-MS/MS and full scan LC-high-resolution MS (LC-HRMS) methods in positive and negative electrospray ionization. In general, RMRs for PSTs with similar chemical structures were comparable, but varied significantly between subclasses, with M-toxins showing the lowest sensitivity. For example, STX showed a greater than 50-fold higher RMR than M4 and M6 by LC-HRMS. The MS instrument, scan mode and polarity also had significant impacts on RMRs and should be carefully considered when semiquantitating PSTs by LC-MS. As a demonstration of their utility, the RMRs determined were applied to the semiquantitation of PSTs in contaminated mussels, showing good agreement with results from calibration with CRMs.

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recently, ion chromatography coupled with mass spectrometry has been used for the determination of anionic metabolites. However, connection with a mass spectrometer in this method is not straightforward because backpressure produced by the addition of a make-up solution often affects the peak resolutions of the target metabolites. To overcome this problem, we developed a capillary ion chromatography-mass spectrometry method utilizing a double coaxial electrospray ionization sprayer. This method was not affected by backpressure and the number of theoretical plates was about three times that of a conventional sprayer. Under optimized conditions, 44 anionic metabolites, including organic acids, sugar phosphates, nucleotides, and cofactors, were successfully separated and selectively detected with a Q Exactive mass spectrometer. The calibration curves of the tested metabolites showed excellent linearity within the range of 1-100,000 nmol/L and the correlation coefficient was greater than 0.991. The detection limits for these metabolites were between 1 and 500 nmol/L (0.4 and 200 fmol). The developed method was applied to the quantitation of anionic metabolites in cultured cancer cell samples with tumor necrosis factor (TNF)-α stimulation. This allowed for the successful determination of 105 metabolites. The levels of tricarboxylic acid cycle intermediates changed significantly after TNF-α stimulation. These results demonstrate that the developed method is a promising new tool for comprehensive analysis of anionic metabolites., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Huangqin Qinfei Decoction (HQD) is a traditional Chinese medicine that is administered for acute pneumonia, bronchial inflammation, acute bronchitis and acute lung infection. In this study, we used liquid chromatography linked with tandem mass spectrometry (LC-MS/MS) for the concurrent identification of 11 bioactive compounds; namely, baicalin, baicalein, wogonoside, scutellarin, wogonin, oroxylin A, geniposide, genipin, geniposidic acid, chlorogenic acid, and crocin-I, for the quality control of HQD. The evaluation was conducted on an Agilent Poroshell 120 EC-C18 (2.1mm×100mm, 2.7μm) with gradient elution in the mobile phase with 0.1% formic acid and 1mM/L ammonium acetate in water as solvent A and methanol as solvent B at a flow rate of 0.3mL/min in under 12 min. Mass spectrometric detection was conducted in the selected reaction monitoring mode utilizing electro spray ionization in the positive and negative modes. Every one of the calibration curves had good linearity with R2 >0.9992. Intra-day and inter-day accuracies for every one of the evaluated components were expressed as the relative standard deviation (RSD) from 1.72%-5.02% and 0.63%-5.99%, respectively. The recuperation of the 11 compounds that were measured at the three concentrations was within 94.05%-105.18%, with the RSD ≤ 6.26%. The use of this method was determined through the effective evaluation of 11 compounds in 5 batches of HQD. The confirmed method is precise, sensitive, and effective for identifying the contents of the chosen compounds in HQD for quality control.

Human exposures to fentanyl analogs, which significantly contribute to the ongoing U.S. opioid overdose epidemic, can be confirmed through the analysis of clinical samples. Our laboratory has developed and evaluated a qualitative approach coupling liquid chromatography and quadrupole time-of-flight mass spectrometry (LC-QTOF) to address novel fentanyl analogs and related compounds using untargeted, data-dependent acquisition. Compound identification was accomplished by searching against a locally-established mass spectral library of 174 fentanyl analogs and metabolites. Currently, our library can identify 150 fentanyl-related compounds from the Fentanyl Analog Screening (FAS) Kit), plus an additional 25 fentanyl-related compounds from individual purchases. Plasma and urine samples fortified with fentanyl-related compounds were assessed to confirm the capabilities and intended use of this LC-QTOF method. For fentanyl, 8 fentanyl-related compounds and naloxone, lower reportable limits (LRL 100 ), defined as the lowest concentration with 100 % true positive rate (n = 12) within clinical samples, were evaluated and range from 0.5 ng/mL to 5.0 ng/mL for urine and 0.25 ng/mL to 2.5 ng/mL in plasma. The application of this high resolution mass spectrometry (HRMS) method enables the real-time detection of known and emerging synthetic opioids present in clinical samples., (Published by Elsevier B.V.)

Traditionally, for a liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC-MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification. This methodology has great potential in many applications, for example biomarker measurement, quantitative proteomics and clinical diagnosis. Here, we assessed the feasibility of applying this ISCC-LC-MS/MS methodology in regulated bioanalysis using BMS-984478, a drug candidate, as the model compound. We also proposed method validation procedures/processes for this new approach for industry peers' consideration and feedback. A LC-MS/MS method using the ISCC strategy was successfully developed and validated for the quantitative analysis of BMS-984478 in human plasma over the range of 1.33-993.42 ng/mL. The validated ISCC-LC-MS/MS method was compared with a previously validated method using the conventional external calibration curve approach, and the two methods showed equivalent performance. Critical considerations and practical approaches in method development, validation and sample analysis were also discussed. Our work demonstrated that the ISCC-LC-MS/MS methodology is a promising approach for regulated LC-MS/MS bioanalysis. ISCC-LC-MS/MS methodology has its unique advantages and has great potential to be widely applied for various quantitative applications, and may even change the landscape of quantitative analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cyclic-1,N 2 -propano-2'-deoxyguanosine-d 7 (CPr-dG-d 7 ) was prepared as an isotopic internal standard (IS) for electrospray ionization tandem mass spectrometry (ESI-MS/MS) quantification of CPr-dG in DNA as a candidate cancer risk marker of acetaldehyde intake, mainly from drinking. The deuterated compound was reasonably synthesized from acetaldehyde-d 4 and 2'-deoxyguanosine in deuterium oxide (D 2 O), preventing the deuterium atoms of acetaldehyde-d 4 from being substituted by hydrogen atoms, which occurred seriously in aqueous synthesis media via keto-enol tautomerism. Furthermore, another deuterium atom was added from D 2 O to form CPr-dG-d 7 . After four weeks of storage in H 2 O at 10°C, CPr-dG-d 7 was found to be sufficiently stable for practical use. The calibration curve of CPr-dG by using a hydrophilic interaction chromatography-ESI-MS/MS system with CPr-dG-d 7 as the IS showed sufficient linearity from 1.0 × 10 -10 to 4.0 × 10 -9 M with r 2 = 0.998.

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated., Competing Interests: Ludger Ltd is a commercial bioscience company specialising in development and validation of glycoprofiling technology. The following authors declare competing financial interest(s): MK, DIRS, RPK are employed by Ludger Ltd. In addition, products from Ludger were used in this research. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: Non-targeted metabolic profiling using high-resolution mass spectrometry (HRMS) is a standard approach for pathway identification despite technical limitations., Objectives: To assess the performance of combining targeted quadrupole (QQQ) analysis with HRMS for in-depth pathway profiling., Methods: Serum of exercising patients with type 1 diabetes (T1D) was profiled using targeted and non-targeted assays., Results: Non-targeted analysis yielded a broad unbiased metabolic profile, targeted analysis increased coverage of purine metabolism (twofold) and TCA cycle (three metabolites)., Conclusion: Our screening strategy combined the benefits of the unbiased full-scan HRMS acquisition with the deeper insight into specific pathways by large-scale QQQ analysis.

Introduction: Polyherbal formulations are an integral part of various indigenous medicinal systems such as Traditional Chinese Medicine (TCM) and Ayurveda. The presence of a very large number of compounds makes the quality control of polyherbal formulations very difficult., Objectives: To overcome this problem, we have developed a comprehensive strategy for the dereplication of natural products in polyherbal formulations by using Adhatoda vasica as a case study., Methods: The strategy is based on five major steps: the collection of plant samples from different locations to observe the effects of environmental variables; LC-ESI-MS/MS-based untargeted metabolite profiling of the plant samples to identify marker compounds using extensive chemometric analysis of the obtained data; the identification of marker compounds in polyherbal products; the isolation, purification and characterization of the marker compounds; and MRM-based quantitative analysis of the isolated marker compounds using LC-ESI-MS/MS., Results: Using this strategy, we identified a total of 51 compounds in the methanolic extract of A. vasica plants from 14 accessions. Chemical fingerprinting of the plant led to the identification of characteristic peaks that were used to confirm the presence of A. vasica in complex polyherbal formulations. Four quinazoline alkaloids (marker compounds) were isolated, purified and quantified in various herbal formulations containing A. vasica., Conclusion: This method demonstrates a comprehensive strategy based on untargeted and targeted metabolite analysis that can be used for the standardization of complex polyherbal formulations.

Dried blood spots (DBSs) have gained increasing attention recently with their growing importance in precision medicine. DBS-based metabolomics analysis provides a powerful tool for investigating new biomarkers. Until now, very few studies have discussed measures for improving analytical accuracy with the consideration of the special characteristics of DBSs. The present study proposed a postcolumn infused-internal standard (PCI-IS) assisted strategy to improve data quality for DBS-based metabolomics studies. An efficient sample preparation protocol with 80% acetonitrile as the extraction solvent was first established to improve the metabolite recovery. The PCI-IS assisted liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was used to simultaneously estimate the blood volume and correct the signal change caused by ion source contamination and the matrix effect to evaluate the spot volume effect and hematocrit (Hct) variation effect on target metabolites. Phenylalanine- d 8 was selected as the single PCI-IS to correct the matrix effect. For calibration of errors caused by the blood volume difference, 75% of the test metabolites showed good correlation ( R 2 ≥ 0.9) between the spot volume and the signal intensity after PCI-IS correction compared to less than 50% metabolites with good correlation before calibration. The spot volume was further calibrated by the same PCI-IS. Investigation of the Hct variation effect on target metabolites revealed that it affected the concentrations of metabolites in the DBS samples depending on their abundance in the red blood cell (RBC) or plasma; it is essential to preinvestigate the distribution of metabolites in blood to minimize the comparison bias in metabolomics studies. Finally, the PCI-IS assisted method was applied to study acetaminophen-induced liver toxicity. The results indicated that the proposed PCI-IS strategy could effectively remove analytical errors and improve the data quality, which would make the DBS-based metabolomics more feasible in real-world applications.

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC-MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC-MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The accurate and timely identification of the causative organism of infection is important in ensuring the optimum treatment regimen is prescribed for a patient. Rapid evaporative ionisation mass spectrometry (REIMS), using electrical diathermy for the thermal disruption of a sample, has been shown to provide fast and accurate identification of microorganisms directly from culture. However, this method requires contact to be made between the REIMS probe and microbial biomass; resulting in the necessity to clean or replace the probes between analyses. Here, optimisation and utilisation of ambient laser desorption ionisation (ALDI) for improved speciation accuracy and analytical throughput is shown. Optimisation was completed on 15 isolates of Escherichia coli, showing 5 W in pulsatile mode produced the highest signal-to-noise ratio. These parameters were used in the analysis of 150 clinical isolates from ten microbial species, resulting in a speciation accuracy of 99.4% - higher than all previously reported REIMS modalities. Comparison of spectral data showed high levels of similarity between previously published electrical diathermy REIMS data. ALDI does not require contact to be made with the sample during analysis, meaning analytical throughput can be substantially improved, and further, increases the range of sample types which can be analysed in potential direct-from-sample pathogen detection.

Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A 2 (PDx) and its subunits were characterized as calibrants for TWIM-MS. Conditions for acquisition of native-like TWIM (Synapt G1 HDMS) and DTIM (Agilent 6560 IM-Q-TOF) mass spectra were optimized to ensure the spectra exhibited similar charge state distributions. CCS measurements (DTIM-MS) for ubiquitin, cytochrome c, holo-myoglobin, serum albumin and glutamate dehydrogenase were in good agreement with other recent results determined using this and other DTIM-MS instruments. PDx and its β and γ subunits were stable across a wide range of cone and trap voltages in TWIM-MS and were stable in the presence of organic solvents. The CCS of PDx and its subunits were determined by DTIM-MS and were used as calibrants in determination of CCS of native-like cytochrome c, holo-myoglobin, carbonic anhydrase, serum albumin and haemoglobin in TWIM-MS. The CCS values were in good agreement with those measured by DTIM-MS where available. These experiments demonstrate conditions for analysis of native-like proteins using a commercially available DTIM-MS instrument, characterize robust calibrants for TWIM-MS, and present CCS values determined by DTIM-MS and TWIM-MS for native proteins to add to the current literature database. Graphical Abstract ᅟ.

Background: Comprehensive serum sex hormone profiling is essential for monitoring the occurrence and development of many related diseases. However, the current methods for multi-class sex hormone detection were always lack of Standard Reference Material (SRM) certification and suffered from large sample consumption. For improvement, we developed a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method focused on SRM certification and minimization of serum consumption for simultaneous quantification of seven mainstream serum sex hormones including estrogens (estrone E1, estradiol E2 and estriol E3), androgens (testosterone T, androstenedione AD, dehydroepiandrosterone DHEA) and progestogens (progesterone P)., Methods: To achieve one-batch analysis, a straightforward strategy was designed and carefully optimized. Schematically, serum was firstly spiked with isotope-labeled internal standards. Then, liquid-liquid extraction was performed with methyl tert-butyl ether. After drying under nitrogen, dansyl chloride was introduced for derivatization. Finally, the mixture was submitted to LC-MS/MS for quantification., Results: The limit of quantification was 0.005 ng/mL for E1, E2 and E3, 0.01 ng/mL for T, P and AD, 0.25 ng/mL for DHEA. Inter- and intra-assay CVs were less than 11.8%. The selectivity was proved satisfactory by interference spiking tests. With systematical SRM validation, the mean bias of -5.4 to 4.7% was observed, which indicated excellent method reliability. We found significant positive bias in chemiluminescence immunoassay (CLIA) detection comparing with current method, which promoted us to reconsider our previous results on sex hormone regulation in male patients with coronary atherosclerotic disease. After redetecting the related samples, modified and improved conclusions were proposed., Conclusions: A LC-MS/MS method for multi-class serum sex hormone profiling was developed with SRM certification and minimized serum consumption. Taking advantages of such reliable method, the previous CLIA-based research findings on sex hormone regulation in male patients with coronary atherosclerosis were modified and improved after redetecting the same sample-pool., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run. We show that this LC/ESI/DMS/MS/MS method exhibits robust quantitative performance within an analyte concentration range of 2.8-355 nM. We further report the simultaneous quantification of nine GlcCers (16:0, 18:0, 20:0, 22:0, 23:0, 24:1, 24:0, 25:0, and 26:0) and five GalCers (16:0, 22:0, 23:0, 24:1, and 24:0) molecular species in human plasma, as well as six GalCers (18:0, 22:0, 23:0, 24:1, 24:0 and 25:0) and two GlcCers (24:1 and 24:0) in human cerebrospinal fluid. Our method expands the potential of DMS technology in the field of glycosphingolipid analysis for both biomarker discovery and drug screening by enabling the unambiguous assignment and quantification of cerebroside lipid species in biological samples., (Copyright © 2019 Xu et al.)

Growth hormone releasing peptides (GHRPs) are synthetic peptides with the ability to stimulate human growth hormone (hGH) secretion. Several GHRPs have been developed as drug candidates; however, only one of them, GHRP-2 (Pralmorelin), has received a clinical approval. Nevertheless, they are distributed on the black market and misused by cheating athletes, due to their performance-enhancing effects. Hence, GHRPs have been included in the World-Anti-Doping-Agency's Prohibited List as forbidden substances in sport. Predominantly, analytical methods for detection and unequivocal identification of doping substances are based on mass spectrometry. Therefore, in the present work, a qualitative analysis by liquid chromatography coupled to high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyzer was performed to identify a new heptapeptide (MW = 874.02 Da) - a glycine analogue of GHRP-2. Structure determination using de novo sequencing is described here in detail. The results of this study may indicate a new approach to circumvent a detection of doping practices., (© 2018 John Wiley & Sons, Ltd.)

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

This study analyzed the performance of different molecular technologies along with blood culture (BC) in the diagnosis of bloodstream infections (BSI) in patients from internal medicine wards - including intensive care units (ICUs) - and the emergency room. Patients with systemic inflammatory response syndrome were prospectively included. BCs and EDTA whole blood were obtained simultaneously. The latter was analyzed by PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS; IRIDICA BAC BSI assay, Abbott) and by SeptiFast (Roche). Cases were classified as BSI according to adapted European Centre for Disease Prevention and Control criteria. Out of 462 analyzed episodes, 193 with valid test results fulfilled the inclusion criteria and were further evaluated. Sixty-nine (35.8%) were classified as BSI. PCR/ESI-MS showed a significantly better overall performance than BC (p = 0.004) or SeptiFast (p = 0.034). Only in patients from the ICU the performance of SeptiFast was comparable to that of PCR/ESI-MS. Mainly due to the negative effect of antimicrobial pre-treatment on BC results, the cumulative performance of each of the molecular tests with BC was significantly higher than that of BC alone (p < 0.001). SeptiFast and in particular the broad-range pathogen detection system PCR/ESI-MS proved to be an essential addition to BC-based diagnostics in BSI.

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE-enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2-2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale., (© 2018 John Wiley & Sons, Ltd.)

Introduction: Paper spray mass spectrometry has provided a rapid, quantitative ambient ionization method for xenobiotic and biomolecule analysis. As an alternative to traditional sample preparation and chromatography, paper spray demonstrates the sampling ionization of a wide range of molecules and significant sensitivity from complex biofluids. The amenability of paper spray with dried blood spots and other sampling types shows strong potential for rapid, point-of-care (POC) analysis without time-consuming separation procedures. Areas covered: This special report summarizes the current state and advances in paper spray mass spectrometry that relate to its applicability for clinical analysis. It also provides our perspectives on the future development of paper spray mass spectrometry and its potential roles in clinical settings. Expert commentary: Paper spray has provided the fundamental aspects of ambient ionization needed for implementation at the POC. With further clinical management and standardization, paper spray has the potential to replace traditional complex analysis procedure for rapid quantitative detection of illicit drugs, therapeutic drugs and metabolites. Surface and substrate modifications also offer significant improvement in desorption and ionization efficiencies, resulting in enhanced sensitivity. Comprehensive analysis of metabolites and lipids will further extend the implementation of paper spray ionization mass spectrometry into clinical applications.

Objectives: This study aimed to explore the residue levels of multiclass mycotoxins in medicinal and edible lotus seeds., Methods: A rapid and reliable isotope-labelled internal standard-based UPLC-MS/MS method was developed and validated for sensitive and accurate analysis of multiclass mycotoxins including aflatoxins (AFB 1 , AFB 2 , AFG 1 and AFG 2 ), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FB 1 and FB 2 ), T-2 and HT-2 toxins in lotus seeds. Some critical conditions such as extract solution with the addition of isotope-labelled internal standard, type of mobile phase and the elution condition were scientifically optimized. The 11 mycotoxins obtained satisfactory resolution and sensitive detection in multiple reactions monitoring scanning mode combined with the ion switching technology in positive and negative ion switching mode., Key Findings: The developed isotope-labelled internal standard-based UPLC-MS/MS method exhibited an approving linearity (r ≥ 0.9984), high sensitivity (limit of detection in the range of 0.015-30.05 μg/kg), acceptable precision (RSDs ≤6.3%) and good recovery (76.0-116.0%) for 11 analytes, respectively. Ten batches of real lotus seed samples were tested, and three batches out of which were contaminated with AFB 1 , FB 2 , T-2 and ZEN. AFB 1 showed the highest occurrence rate (30%) with contents of 10.50 and 8.32 μg/kg in two samples over the official limit (5.0 μg/kg)., Conclusions: The monitoring of multiclass mycotoxins in Chinese herbal medicines is in great urgency to ensure the security of consumers. The proposed method could be further utilized for simple, sensitive and rapid detection of more mycotoxins in other complex matrices to compensate for matrix effects., (© 2018 Royal Pharmaceutical Society.)

When developing a quantitative assay for exogenous or endogenous compounds, guidelines for method validation normally recommend that the biological specimens should be prepared in corresponding authentic matrices, yet "analyte-free authentic matrices" is in general not available. It is generally known that GSH and CYS are endogenous compounds and present in both prokaryotes and eukaryotes. Herein, an efficient approach for the quantitative analysis of endogenous substances in biological specimens was developed, and glutathione (GSH) & cysteine (CYS) were chosen as model endogenous substances. Activated carbon (AC), a common adsorbent for the adsorption of environmental pollutants, was used to remove the endogenous GSH and CYS and prepare "GSH&CYS-free biological matrix". The endogenous GSH and CYS in mouse plasma, blood and liver homogenate were found can be almost removed via incubating with 100 mg of AC for 2 h. After optimizing the derivatization reagents, internal standard and analytical parameters, a reliable quantitative assay of GSH and CYS in mouse plasma, blood and liver homogenate was developed and validated on LC-ESI-MS/MS using corresponding AC-adsorbed mouse biological matrices. The validation results indicated that the developed method provided suitable accuracy, sensitivity, specificity and high throughput for the analysis of GSH and CYS. Finally, the developed LC-ESI-MS/MS assay was successfully applied to measure the concentrations of GSH and CYS in liver injury mice. The presently developed methodology could be widely applied in the quantitative analysis of endogenous compounds in various complex mixtures such as biological, herbal and environmental samples., (Copyright © 2018. Published by Elsevier B.V.)

Pharmaceutical drugs are potential molecules with specific biological activity. However, long-term use of these chemical molecules can affect the human physiological system because of their increased levels in the human body. Therefore, identification and structure elucidation of impurities or degradation products should be taken into consideration in order to assure drug safety. The present study assessed the degradation behaviour of dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin under different stress conditions as per ICH guidelines Q1A (R2) followed by elucidation of the structure of degradation products. All the stress samples were analysed by using UPLC/PDA. The superior separation of drug from its degradation products was attained with time programmed gradient elution on BEH C18 (100 mm × 2.1 mm, 1.7 μm) column using 10 mM ammonium formate (aqueous) and acetonitrile (organic) as the mobile phase components. All the degradation products of anagliptin were characterized using LC/QTOF/MS/MS. In addition, the activity and toxicity of degradation products were determined through molecular docking and in silico toxicity prediction studies, respectively. The developed UPLC/PDA method was validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity and robustness., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A new and sensitive ultra fast liquid chromatography coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method was developed to evaluate the quality of Red ginseng (RG) and to find out its chemical markers by comparing with multi-batches of RG and white ginseng (WG). This innovative method could quantify sixty-six saponins and their six aglycones including 10 pairs of 20(S) and 20(R) epimers within 35 min simultaneously. All compounds could be determined in individual multiple-reaction monitoring channel without interference, and the optimized method was rapid, accurate, precise, reproducible and efficient. Using the orthogonal partial least squared discriminant analysis, ginsenosides Rg 5 , Rh 4 , Rk 1 , Rs 4 , F 4 , and 20(S)-Rg 3 were found to be the characteristic components of RG, the six compounds should be suggested as quality control markers to distinguish RG from WG. These findings will be significant for standardizing the processing procedures of RG and ensuring the consistent quality, as well as consequently the efficacy of RG in clinical applications. Results will be helpful in providing crucial chemical profiles of RG., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rationale: The importance of saccharides, the most abundant biomolecules on Earth, extends beyond their biological roles and to consumer products and industrial processes. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is an attractive tool for the analysis of underivatized saccharides (US), but they tend to have relatively low sensitivities due to their low surface activities and lack of easily protonable or deprotonable chemical groups. An understanding of the influences that solvent parameters have on their signal intensities would enhance the usefulness of ESI-MS/MS for their analysis., Methods: Solutions of glucose, a model analyte for US, in various combinations of solvent, additive, additive concentration, and pH were analyzed by flow injection analysis ESI-MS/MS in both the positive and negative ionization mode. The blank-corrected signal intensities of the solvent parameter combinations were then compared., Results: The addition of acetonitrile led to severe ionization suppression in the positive ionization mode through its competition with glucose for cation adduction. High signal intensity was achieved under wide pH and concentration ranges for methanol: water solutions containing ammonium trifluoroacetate in the positive ionization mode. The highest signal intensities for acetonitrile: water solutions were those containing ammonium formate or lithium fluoride in the negative ionization mode., Conclusions: An understanding of the influence of solvent parameters on the signal intensity of a given analyte is useful for guiding the selection process of mobile phases/flow solvents that lead to low limits of detection or the minimization of matrix effects by allowing its detection at high dilution factors., (Copyright © 2018 John Wiley & Sons, Ltd.)

We report the development and availability of a mass spectral reference library for oligosaccharides in human milk. This represents a new variety of spectral library that includes consensus spectra of compounds annotated through various data analysis methods, a concept that can be extended to other varieties of biological fluids. Oligosaccharides from the NIST Standard Reference Material (SRM) 1953, composed of human milk pooled from 100 breastfeeding mothers, were identified and characterized using hydrophilic interaction liquid chromatography electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS) and the NIST 17 Tandem MS Library. Consensus reference spectra were generated, incorporated into a searchable library, and matched using the newly developed hybrid search algorithm to elucidate unknown oligosaccharides. The NIST hybrid search program facilitates the structural assignment of complex oligosaccharides especially when reference standards are not commercially available. High accuracy mass measurement for precursor and product ions, as well as the relatively high MS/MS signal intensities of various oligosaccharide precursors with Fourier transform ion trap (FT-IT) and higher energy dissociation (HCD) fragmentation techniques, enabled the assignment of multiple free and underivatized fucosyllacto- and sialyllacto-oligosaccharide spectra. Neutral and sialylated isomeric oligosaccharides have distinct retention times, allowing the identification of 74 oligosaccharides in the reference material. This collection of newly characterized spectra based on a searchable, reference MS library of annotated oligosaccharides can be applied to analyze similar compounds in other types of milk or any biological fluid containing milk oligosaccharides.

Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY* 411 TKKVPQVSTPTL (pepsin) and as the tripeptide Y* 411 TK (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (μLC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS). Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at +37 °C and -30 °C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 °C for at least 24 h. Limits of identification of adducts varied between 0.25 μM and 4.0 μM with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recent advances in analytical capabilities allowing for the identification and quantification of drugs and metabolites in small volumes at low concentrations have made oral fluid a viable matrix for drug testing. Oral fluid is an attractive matrix option due to its relative ease of collection, reduced privacy concerns for observed collections and difficulty to adulterate. The work presented here details the development and validation of a liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone in neat oral fluid. The calibration range is 0.4-150 ng/mL for 6-acetylmorphine and 1.5-350 ng/mL for all other analytes. Within-run and between-run precision were <5% for all analytes except for hydrocodone, which had 6.2 %CV between runs. Matrix effects, while evident, could be controlled using matrix-matched controls and calibrators with deuterated internal standards. The assay was developed in accordance with the proposed mandatory guidelines for opioid confirmation in federally regulated workplace drug testing. The use of neat oral fluid, as opposed to a collection device, enables collection of a single sample that can be split into separate specimens.

A liquid chromatography-tandem mass spectrometry method for the determination of four Alternaria toxins, i.e. alternariol, alternariol monomethyl ether, tentoxin, and tenuazonic acid in tomato-based and fruit-based products was developed using a QuEChERs approach for the extraction of the mycotoxins. To optimise the QuEChERs extraction, several parameters were tested: types of QuEChERs pouches, sample weights, quantities of added water, use of dispersive SPE as a purification step, types of solvent and conditions of shaking. The method showed good linearity (R 2  > 0.997) and precision (RSD% < 10) for all analytes. Tenuazonic acid showed very good recovery (98.8%-108.9%) for tomato-based products, as well as for fruit-based products. The method was successfully applied to 57 samples collected from the Italian market. Tenuazonic acid was found in appreciable concentrations in some products. The highest value was found in a tomato sauce sample (814 μg/kg)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Postmortem blood samples may not accurately reflect antemortem drug concentrations, as the levels of some drugs increase due to postmortem redistribution (PMR). The brain has been suggested as an alternative sampling site. The anatomically secluded site of the brain limits redistribution and prolongs the detection window, thereby enabling sampling from deceased individuals where blood is no longer suitable for analysis. We report concentrations in brain tissue and blood from 91 cases for the four antidepressants citalopram, duloxetine, mirtazapine and sertraline. The cases were classified according to their role in the cause of death, as follows: (A) concentrations where the drug was the sole cause of fatal intoxication; (B) concentrations where the drug contributed to a fatal outcome; and (C) concentrations where the drug was not related to the cause of death. The analytical method was successfully validated in brain tissue in terms of linearity, process efficiency, precision and accuracy. Quantification of analytes was performed by ultra-performance liquid chromatography with tandem mass spectrometry. Correlations between blood and brain concentrations were achieved with R2-values between 0.67 and 0.91. The following median brain-blood ratios were obtained: 3.71 for citalopram (range: 1.4-5.9), 11.0 for duloxetine (range: 5.0-21.6), 1.53 for mirtazapine (range: 1.02-4.71) and 7.38 for sertraline (range: 3.2-14.2). The S/R ratio of racemic citalopram was the same in brain (0.80) and blood (0.85), whereas the median citalopram/N-desmethylcitalopram ratio was higher in brain (9.1) than blood (4.1). The results of this study may serve as reference concentrations in brain for forensic cases.

The aim of this paper is to present the development and validation of a high-resolution full scan (HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine according to World Antidoping Agency (WADA) requirements. The method was also validated for quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample preparation comprised a combination of a hydrolyzed urine liquid-liquid extraction and the dilute & shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity Switching was applied in combination of the Quadrupole-Orbitrap mass filter. The HR-FS acquisition of analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable with LC-MS for antidoping investigations to identify the use of known or novel prohibited substances and metabolites after electronic data files' reprocessing. The method has been validated to be fit-for-purpose for the antidoping analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Top-down mass spectrometry (MS) strategies allow in-depth characterization of proteins by fragmentation of the entire molecule(s) inside a mass spectrometer without requiring prior proteolytic digestion. Importantly, the fragmentation techniques on commercially available mass spectrometers have become more versatile over the past decade, with different characteristics in regards to the type and wealth of fragment ions that can be obtained while preserving labile protein post-translational modifications. Due to these and other improvements, top-down MS has become of broader interest and has started to be applied in more disciplines, such as the quality control of recombinant proteins, analysis and characterization of biopharmaceuticals, and clinical biochemistry to probe protein forms as potential disease biomarkers. This article provides a technical overview and guidance for data acquisition strategies on the Orbitrap platform for single proteins and low complexity protein mixtures. A protein standard mixture composed of six recombinant proteins is also introduced and analysis strategies are discussed in detail., Biological Significance: The article provides a detailed overview and guidance on how to choose from the variety of available methods for protein characterization by top-down analysis on the Orbitrap platform. Technical details are provided explaining important observations and phenomena when working with intact proteins and data from a number of different samples should serve to provide a solid understanding on how experiments were and should be setup and to set the right expectations on the outcome of these types of experiments. Additionally, a new intact protein standard sample is introduced that will help as a QC sample to check the instrument's hardware and method setup conditions as a requirement for obtaining high quality data from biologically relevant samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Annotation of signals of interest represents a key point in mass spectrometry-based metabolomics studies. The first level of investigation is the elemental composition, which can be deduced from accurately measured masses and isotope patterns. However, accuracy of these two parameters remains to be evaluated on last generation mass spectrometers to determine the level of confidence that can be used during the annotation process. In this context, we evaluated the performance of the Orbitrap Fusion mass spectrometer for the first time and demonstrated huge potential for metabolite annotation via elemental composition determination. This work was performed using a set of 50 standard compounds analyzed under LC/MS conditions in solvent and biological media. Accurate control of the number of trapped ions proved mandatory to avoid space charge effects, ensure sub-ppm mass accuracy (using external calibration), and reliable measurement of isotopic patterns at 500,000 resolution. On the basis of the results, we propose standard optimized experimental conditions for performing robust and accurate untargeted metabolomics on the Orbitrap Fusion at high mass measurement and mass spectral accuracy.

Carotenoids and tocopherols were characterised in the meso- and exocarp of wild-growing Costa Rican Acrocomia aculeata fruits. Comprehensive profiling of these lipophilic micronutrients in fruits of three varying maturity stages was conducted for the first time. A method for the simultaneous extraction and quantitation of carotenoids and α-tocopherol was developed and validated. Detailed HPLC-DAD-APCI/ESI-MS n analyses enabled the identification of α-tocopherol and 25 carotenoids. The latter comprised antheraxanthin, β-carotene, lutein, luteoxanthin, neoxanthin, phytoene, phytofluene, violaxanthin, zeaxanthin, and several (Z)-isomers of the aforementioned compounds. Quantitation by HPLC-DAD/FLD revealed total carotenoid concentrations of 872±178 and 3075±407μg/100g fresh weight in the meso- and exocarp of fully ripe fruits, respectively. In both fruit fractions, progressing maturation resulted in the accumulation of phytoene, phytofluene, (all-E)-zeaxanthin, (all-E)-antheraxanthin, and (all-E)-violaxanthin. Carotenoid profiling was supported by multivariate data analysis. Carotenoid precursors and xanthophyll cycle pigments characterised Macauba fruits of full maturity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Quantitative mass spectrometry imaging (MSI) is a robust approach that provides both quantitative and spatial information for drug candidates' research. However, because of complicated signal suppression and interference, acquiring accurate quantitative information from MSI data remains a challenge, especially for whole-body tissue sample. Ambient MSI techniques using spray-based ionization appear to be ideal for pharmaceutical quantitative MSI analysis. However, it is more challenging, as it involves almost no sample preparation and is more susceptible to ion suppression/enhancement. Herein, based on our developed air flow-assisted desorption electrospray ionization (AFADESI)-MSI technology, an ambient quantitative MSI method was introduced by integrating inkjet-printing technology with normalization of the signal extinction coefficient (SEC) using the target compound itself. The method utilized a single calibration curve to quantify multiple tissue types. Basic blue 7 and an antitumor drug candidate (S-(+)-deoxytylophorinidine, CAT) were chosen to initially validate the feasibility and reliability of the quantitative MSI method. Rat tissue sections (heart, kidney, and brain) administered with CAT was then analyzed. The quantitative MSI analysis results were cross-validated by LC-MS/MS analysis data of the same tissues. The consistency suggests that the approach is able to fast obtain the quantitative MSI data without introducing interference into the in-situ environment of the tissue sample, and is potential to provide a high-throughput, economical and reliable approach for drug discovery and development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC-MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement., (Copyright © 2017 Elsevier B.V. All rights reserved.)

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ± 15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An analytical method that utilizes isotope-dilution ultrahigh-performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS or called UHPLC-HRMS) was developed, and validated to be highly precise and accurate for the detection of nine parabens (methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, hexyl-, and benzyl-parabens) in human urine samples. After sample preparation by ultrasound-assisted emulsification microextraction (USAEME), the extract was directly injected into UHPLC-HRMS. By using negative electrospray ionization in the multiple reaction monitoring (MRM) mode and measuring the peak area ratios of both the natural and the labeled-analogues in the samples and calibration standards, the target analytes could be accurately identified and quantified. Another use for the labeled-analogues was to correct for systematic errors associated with the analysis, such as the matrix effect and other variations. The limits of quantitation (LOQs) were ranging from 0.3 to 0.6 ng/mL. High precisions for both repeatability and reproducibility were obtained ranging from 1 to 8%. High trueness (mean extraction recovery, or called accuracy) ranged from 93 to 107% on two concentration levels. According to preliminary results, the total concentrations of four most detected parabens (methyl-, ethyl-, propyl- and butyl-) ranged from 0.5 to 79.1 ng/mL in male urine samples, and from 17 to 237 ng/mL in female urine samples. Interestingly, two infrequently detected pentyl- and hexyl-parabens were found in one of the male samples in this study., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The objective of this study was to identify the metabolites of rotundic acid after oral administration to rats and compare the similarities with its biotransformation by Syncephalastrum racemosum AS 3.264 using ultra-high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. A total of fourteen metabolites were determined based on the mass spectrometry and chromatographic behaviors, among which eleven (M1-M3, M7-M14) and six (M2, M4-M8) metabolites were identified in rats and S. racemosum, respectively. Three identical metabolites (M2, M7 and M8) were found in rats and S. racemosum, indicating that there were metabolic similarities. Moreover, to confirm the results of mass spectrometry, three (M2, M4 and M7) metabolites were obtained by the means of amplifying incubation and their structures were determined by various spectroscopic analyses, and M4 was proved to be a previously undescribed compound. This results showed that in vitro assisted preparation by microbial transformation is a feasible and effective method of obtaining metabolites which are in low amounts and difficult to be prepared in vivo., (Copyright © 2017 Elsevier B.V. All rights reserved.)

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50μL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A rapid and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of a novel anti-HBV compound Y101 and its metabolites M8 and M9 in human plasma. The plasma samples were deproteinated with acetonitrile after addition of Peramivir (internal standard, IS) and separated on a 40 °C ACQUITY UPLC BEH C 18 column (2.1 mm × 50 mm, 1.7 μm). The mobile phase consisted of water (containing 5 mM ammonium acetate and 0.1% formic acid) and acetonitrile (74:26, v/v) at a flow rate of 0.3 mL/min. The detection was performed on a Triple Quad 5500 tandem mass spectrometer coupled with electrospray ionization (ESI) source in positive mode. Quantification was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 490.1 → 339.0 for Y101, m/z 357.2 → 105.2 for metabolite M8, m/z 373.1 → 105.1 for metabolite M9 and m/z 329.1 → 270.2 for IS, respectively. The method was validated over the calibration curve range of 1.000-1000 ng/mL for Y101, 2.000-2000 ng/mL for metabolite M8 and 0.3000-300.0 ng/mL for metabolite M9, using linear regression and 1/x 2 weighting. No matrix effect and carryover effect was observed. The intra- and inter-batch precision and accuracy of Y101, metabolite M8 and M9 were all within the acceptable criteria. This method allows a rapid and simple determination of Y101 and its metabolites M8 and M9 in human plasma. It was successfully applied in a pharmacokinetic study in human for the first time., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ambrisentan is a highly selective endothelin-1 type A receptor antagonist indicated for use in the treatment of pulmonary hypertension. In this study an assay was developed and validated for the quantification of total and unbound (free) concentrations of ambrisentan in human plasma. Plasma samples were dialysed against phosphate buffered saline in a rapid equilibrium dialysis device to obtain dialysate and plasma for unbound and total ambrisentan, respectively. Subsequently, ambrisentan and deuterated ambrisentan (internal standard) were extracted from plasma or plasma dialysate by solid-phase extraction and separated by ultra performance liquid chromatography using on a reversed-phase C 18 column. Detection was conducted with a tandem mass spectrometer with an electrospray ionization source and analysed in positive ion mode with multiple reaction monitoring. Calibration curves were generated over a linear concentration range of 0.1-200 ng/mL in plasma and 0.1-10 ng/mL in plasma ultrafiltrate; with a recovery for ambrisentan of 69.4% and 77.5%, respectively. This assay has been shown to be reproducible and sensitive. The lower limit of quantification in both cases was 0.1 ng/mL; reaching a sensitivity not previously described in the literature. The inter- and intra-batch precision and accuracy were in both cases ≤±15%. The procedure was applied to assess total and free plasma concentrations of ambrisentan in healthy volunteers. Plasma protein binding of ambrisentan was approximately 99%., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Seven unknown impurities in Repaglinide bulk drug batches at below 0.1% (ranging from 0.05 to 0.10%) were detected by an ultra-performance liquid chromatographic (UPLC) method. These impurities were isolated from the crude sample of Repaglinide using preparative high performance liquid chromatography (prep-HPLC). Based on liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI/MS) study, the chemical structures of seven new impurities (8, 9, 10, 11, 13, 14, and 16) were presumed and characterized as 4-(cyanomethyl)-2-ethoxybenzoic acid (8), 4-(cyanomethyl)-2-ethoxy-N-(3-methyl-1-(2-(piperidin-1-yl)phenyl)butyl)benzamide (9), 4-(2-amino-2-oxoethyl)-2-ethoxy-N-(3-methyl-1-(2-(piperidin-1-yl)phenyl)butyl) benzamide (10) and 2-(3-ethoxy-4-((3-methyl-1-(2-(piperidin-1-yl)phenyl)butyl) carbamoyl) phenyl) acetic acid (11) and 4-(cyanomethyl)-N-cyclohexyl-2-ethoxybenzamide (13), 2-(4-(cyclohexylcarbamoyl)-3-ethoxyphenyl) acetic acid (14) and N-cyclohexyl-4-(2-(cyclohexylamino)-2-oxoethyl)-2-ethoxybenzamide (16). The complete spectral analysis, proton nuclear magnetic resonance ( 1 H NMR), 13 C NMR, MS, and infrared (IR) confirmed the proposed chemical structures of impurities. Identification, structural characterization, formation, and their synthesis was first reported in this study. The impurity 11 was crystallized and structure was solved by single crystal X-ray diffraction. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)