Tissue distribution of flufenacet in rats determined by liquid chromatography-tandem mass spectrometry

Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography–mass spectrometry (LC–MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended.

The objectives of this study were to define the phenolic and fatty acid profiles, anticholinesterase, antioxidant, antimicrobial activities, and total phenolic-flavonoid contents of Lycopsis orientalis and Tragopogon latifolius var. angustifolius which have been used as food source and food supplement in Anatolia and have never been examined before. Rosmarinic and quinic acids (21.11 and 11.46 mg g–1 extract, respectively) were found to be the most abundant constituents in L. orientalis and T. latifolius var. angustifolius among the studied 27 compounds by liquid chromatography tandem mass spectrometry. In the fatty acid compositions of L. orientalis and T. latifolius var. angustifolius that were determined by gas chromatography mass spectrometry, oleic (29.1%) and palmitic (28.7%) acids were identified as the major components, respectively. The high antioxidant activity of the methanol extract of L. orientalis shows parallelism to its rosmarinic acid content. Besides, this extract showed medium anticholinesterase activity. The results of the present study proves that the L. orientalis might also be used as a food source due to its high phenolic acid content and strong antioxidant property.

Without constant development and progress, such words as progress, accomplishment, and achievement have no importance. Journal of Bioanalysis and Biomedicine (ISSN: 1948-593X) is developing persistently. It is our pleasure to declare that during year 2020, all issues of volume 12 were distributed online on schedule and the print issues were additionally brought out and dispatched inside 30 days of distributing the issue on the online. LBA and LC-MS are two stages broadly utilized in pharmacokinetic bioanalysis. In this audit, we think about LBA and LC-MS and sum up their qualities and constraints. Methodologies for stage determination are given by the investigation reason, study stage, analyte types, assay requirements and different components. Driven by an expansion in the geriatric populace and the predominance of constant ailments, for example, malignant growth, diabetes, fiery and immune system maladies, huge particle based therapeutics are the quickest developing class of medications being worked on in both scholastic and modern divisions in the previous ten years. During drug advancement, the exact measurement of the helpful medication fixation is vital to depicting the connection between tranquilize presentation and security or/and adequacy of the particle. As a result of this need, critical endeavors have been spent in creating and improving bioanalytical strategies to help test examination. Among regularly utilized test stages, Ligand Binding Assays (LBA) and Liquid Chromatography and Mass Spectrometry (LC-MS) remain the two most well known choices for Pharmacokinetic (PK) measure improvement. In this study, the key boundaries of both bioanalytical stages are checked on and toward the end, the suggestions are given for choosing a strategy as to various kinds of therapeutics, including oligo onucleotides, proteins, counter acting agent medicate conjugates and bispecific antibodies. LC-MS is a stage which joins two investigative advancements. LC gives a straightforward strategy to the physical detachment of an objective substance from the biofluid which contains an unpredictable blend of parts. Solubilized analytes in the portable stage are gone through a section stuffed with the fixed stage which isolates the mixes dependent on size, fondness, charge or hydrophobicity. In the interface among LC and MS the isolated analytes are divided and ionized, after which they can be recognized by MS with high particularity. A significant impediment of LC/MS-based measurement in contrast with LBAs is the requirement for test refinement/extraction and compound absorption preceding the investigation. Evaluation by MS likewise requires the measurement of chose signature or substitute peptides got from the unadulterated objective analyte to be utilized as reference standard. Drug development is an exceptionally serious business. Normally there is high strain to abbreviate the courses of events for tranquilize applicants both in the real turn of events and in the assessment stage. Contrasted with LBA, LC-MS is less restricted by reagent accessibility and quality (e.g., the requirement for high partiality and explicitness catch and discovery reagents). Accordingly, LC-MS is supported particularly in early disclosure stages at whatever point the investigation purposes can be served. A few profoundly touchy LC-MS approaches have been set up, yet much of the time the affectability for bio macromolecules is at ng/mL level. Notwithstanding the flawless medication, LC-MS likewise fit for estimating its metabolites or truncations. As a result of these favorable circumstances, LC-MS has been broadly used to evaluate a wide assortment of therapeutics, including peptides, proteins, monoclonal antibodies, oligonucleotides, and bispecific antibodies. One primary impediment of conventional LC-MS techniques is powerlessness to separate the deliberate medication at various status (e.g., free or bound). Immunocapture approaches have been created for expanding LC-MS application for these reasons. The unpredictability and decent variety of huge particle remedial operators a work in progress require an enhanced utilization of bioanalytical measure stages to help the pharmacokinetic concentrates from early revelation to preclinical and clinical stages. In this paper, both LBA and LC-MS, the two significant bioanalytical examine stages, have been looked into and their qualities and constraints relatively summed up in the table above. The key measure boundaries have been talked about and proposals have been given on the two stages with regards to various sorts of remedial particles. I take this opportunity to acknowledge the contribution by author Dr: Xiaolong Zhang, Linglong Zou and during the final editing of articles published and the support rendered by the editorial assistant in bringing out issues of JBABM in time. I would also like to express my gratitude to all the authors, reviewers, the publisher, the advisory and the editorial board of JBABM, the office bearers for their support in bringing out yet another volume of JBABM 13 and look forward to their unrelenting support to bring out the Volume 12 of JBABM in scheduled time.

Structural annotation of small molecules in biological samples remains a key bottleneck in untargeted metabolomics, despite rapid progress in predictive methods and tools during the past decade. Liquid chromatography–tandem mass spectrometry, one of the most widely used analysis platforms, can detect thousands of molecules in a sample, the vast majority of which remain unidentified even with best-of-class methods. Here we present LC-MS2Struct, a machine learning framework for structural annotation of small-molecule data arising from liquid chromatography–tandem mass spectrometry (LC-MS2) measurements. LC-MS2Struct jointly predicts the annotations for a set of mass spectrometry features in a sample, using a novel structured prediction model trained to optimally combine the output of state-of-the-art MS2 scorers and observed retention orders. We evaluate our method on a dataset covering all publicly available reversed-phase LC-MS2 data in the MassBank reference database, including 4,327 molecules measured using 18 different LC conditions from 16 contributors, greatly expanding the chemical analytical space covered in previous multi-MS2 scorer evaluations. LC-MS2Struct obtains significantly higher annotation accuracy than earlier methods and improves the annotation accuracy of state-of-the-art MS2 scorers by up to 106%. The use of stereochemistry-aware molecular fingerprints improves prediction performance, which highlights limitations in existing approaches and has strong implications for future computational LC-MS2 developments.

Tumors of the oral cavity account for 8% of all canine neoplasia. Malignant oral mass such as melanoma, oral squamous cell carcinoma (OSCC) and fibrosarcoma covers approximately 59% of canine oral neoplasia. Among these, canine oral melanoma (COM) is the most common oral tumors in dogs, with approximately 30–40% of all oral tumors. With high recurrence and/or distant metastasis rate, biomarkers are required for early detection, diagnosis, prognosis, and monitoring treatment response. Since saliva directly contacts tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are suitable to support the evaluation of oral diseases. This study aimed to investigate novel salivary peptide mass fingerprints (PMFs), clusters, and feasible biomarkers in dogs with oral tumors using proteomic techniques. A total of 62 canine salivary samples from 5 early-stage oral melanoma (EOM), 24 late-stage oral melanoma (LOM), 10 OSCC, 11 oral benign tumors (BN), 7 normal and 5 periodontitis (age range 7–14 years) were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) and in-gel digestion coupled with liquid chromatography mass spectrometry/mass spectrometry (GeLC-MS/MS). The principle component analysis (PCA) plot of MALDI-TOF MS showed a combined cluster of normal and periodontitis groups as a control group (CP), whereas other clusters of tumors were separated as EOM, LOM, OSCC and BN. Moreover, the PMFs of MALDI-TOF MS revealed specific peptide peaks which their peptide sequences were identified by LC-MS/MS and were compared with mammalian protein databases using a Basic Local Alignment Search Tool (BLAST) program. Western blot analysis was used to verify the protein expressions of sentrin-specific protease 7 (SENP7) in OSCC, toll-like receptor 4 (TLR4) and its related protein, nuclear factor kappa B (NF-kB), in LOM and OSCC. The protein bands of SENP7, TLR4 and NF-kB from western blot analysis were verified by LC-MS/MS. Western blot analysis was then carried on in individual samples, showing enhanced expression of SENP7, TLR4 and NF-kB in LOM and OSCC. For the GeLC-MS results, the levels of tyrosine phosphatase non-receptor type 5 (PTPN5) were significantly increased in all tumor groups with prominent expression in LOM and OSCC. Western blot analysis was used to confirm the expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) and tumor protein p53 (p53), associated with PTPN5 in a PTPN5-chemotherapy drug interaction, in all tumor groups with marked expression in LOM. Peptide sequences of PTPN5 and p53 western blot analysis were verified by LC-MS/MS. Furthermore, in-solution digestion and LC-MS/MS were used to investigate biomarkers for monitoring treatment response in 12 COM with the pre-surgery stage (PreS), with post-surgery stage (PostS), after treatment with carboplatin for 1–6 times [After treatment (AT)1–AT6] and during follow-up care for 1–4 times (C1–4). Patients were then divided into those with short-term survival (STS) (less than 12 months after surgery) and long-term survival (LTS) (more than 12 months after surgery). With LC-MS/MS, the expression of a potential salivary biomarker, ubiquitin D (UBD), was observed in PreS, PostS, AT1–6, and C1–4. Western blot analysis verified the increased ratio of free UBD (fUBD) to conjugated UBD (cUBD) expression in PreS in the STS group. Moreover, the levels of fUBD was significantly enhanced in PreS compared with AT2 in the STS group. In conclusion, different clusters and peptide fragment masses in PMF were observed among EOM, LOM, OSCC, BN and CP, showing the potential for rapid screening of the diseases. Using MALDI-TOF MS coupled with LC-MS/MS approaches, SENP7, TLR4, and NF-kB were shown to be conceivable salivary biomarkers. Using GeLC-MS/MS technique, PTPN5 and its partner, p53 were suggested to be candidate salivary biomarkers. In addition, with in solution digestion and LC-MS/MS method, a ratio of fUBD to cUBD in PreS was discovered to be a plausible prognostic biomarker for survival in canine oral melanoma. The expression of innovative potential salivary biomarkers was confirmed by western blot analysis and peptide sequences from the western blot analysis was verified by LC-MS/MS.

Internal standards for quantitative LC-MS bioanalysis.- Method development, validation and sample analysis for regulated quantitative bioanalysis using LC-MS/MS.- Impact of sampling paper/cards on bioanalytical quantitation via dried blood spots by liquid chromatography - mass spectrometry.- Highly sensitive pharmaceutical and clinical analysis using selective solid-phase extraction coupled to microflow liquid chromatography and isotope-dilution mass spectrometry.- Pitfalls of LC-MS/MS in the clinical laboratory.- Therapeutic drug monitoring to support clinical pharmacogenomics.- Liquid chromatography - mass spectrometry for the determination of antidepressants and some of their major metabolites in human biological matrices.- The analysis of antipsychotic drugs in human biosamples by LC-MS.- Therapeutic drug monitoring of targeted anticancer therapy - tyrosine kinase inhibitors and selective oestrogen receptor modulators: a clinical pharmacology laboratory perspective.- Applications of mass spectrometry in analyses of steroid hormones.- Liquid chromatography - mass spectrometric analysis of tropane alkaloids in mammalian samples: techniques and applications.- Analysis of illicit drugs in human biological samples by LC-MSn.- Use of matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) in the development of novel small molecule drugs.- A planar integrated micro mass spectrometer.

A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5-50.0 ng/ml (r(2) > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30-40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration.

The combination of high-performance liquid chromatography and mass spectrometry (LC/MS) has had a significant impact on drug development over the past decade. Continual improvements in LC/MS interface technologies combined with powerful features for structure analysis, qualitative and quantitative, have resulted in a widened scope of application. These improvements coincided with breakthroughs in combinatorial chemistry, molecular biology, and an overall industry trend of accelerated development. New technologies have created a situation where the rate of sample generation far exceeds the rate of sample analysis. As a result, new paradigms for the analysis of drugs and related substances have been developed. The growth in LC/MS applications has been extensive, with retention time and molecular weight emerging as essential analytical features from drug target to product. LC/MS-based methodologies that involve automation, predictive or surrogate models, and open access systems have become a permanent fixture in the drug development landscape. An iterative cycle of "what is it?" and "how much is there?" continues to fuel the tremendous growth of LC/MS in the pharmaceutical industry. During this time, LC/MS has become widely accepted as an integral part of the drug development process. This review describes the utility of LC/MS techniques for accelerated drug development and provides a perspective on the significant changes in strategies for pharmaceutical analysis. Future applications of LC/MS technologies for accelerated drug development and emerging industry trends are also discussed.

Monitoring pain management drugs and frequently abused drugs is important for physicians to assess patient compliance. Liquid chromatography-tandem mass spectrometry offers high specificity needed for this purpose. In this report, a novel liquid chromatography-tandem mass spectrometry method for simultaneously monitoring 19 drugs/metabolites in urine was developed and validated. Sample preparation included hydrolysis, dilution and turbulent flow online extraction. Analysis was achieved by reverse phase liquid chromatography and triple-quadruple tandem mass spectrometry. Two fragment ions, one for quantification and the other for assuring identification, were monitored for each analyte. No matrix effect or interference was observed. Lower limits of quantification ranged from 5 to 25 ng/mL. Within the linear range, analytical recovery was between 85.8% and 119.4%. Intra-assay and total coefficient of variations were between 0.2% and 12.7%. This method was compared with mass spectrometry methods offered by two other laboratories using 82 patient samples and 60 spiked urine samples showing 60%-100% agreement. The current method identified more positive samples for all analytes except THCA. The discrepancy in detection rates was primarily due to the different cut-offs used by the other laboratories. A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed for measuring 19 drugs/metabolites in urine important for pain management clinics.

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.

There is no standardized assay of testosterone in women. Liquid chromatography mass spectrometry (LC/MS) has been proposed as the preferable assay by an Endocrine Society Position Statement. The aim was to compare assay results from a direct RIA with two LC/MS. We conducted a blinded laboratory study including masked duplicate samples at three laboratories--two academic (University of Virginia, RIA; and Mayo Clinic, LC/MS) and one commercial (Quest, LC/MS). Baseline testosterone levels from 596 women with PCOS who participated in a large, multicenter, randomized controlled infertility trial performed at academic health centers in the United States were run by varying assays, and results were compared. We measured assay precision and correlation and baseline Ferriman-Gallwey hirsutism scores. Median testosterone levels were highest with RIA. The correlations between the blinded samples that were run in duplicate were comparable. The correlation coefficient (CC) between LC/MS at Quest and Mayo was 0.83 [95% confidence interval (CI), 0.80-0.85], between RIA and LC/MS at Mayo was 0.79 (95% CI, 0.76-0.82), and between RIA and LC/MS at Quest was 0.67 (95% CI, 0.63-0.72). Interassay variation was highest at the lower levels of total testosterone (≤50 ng/dl). The CC for Quest LC/MS was significantly different from those derived from the other assays. We found similar correlations between total testosterone levels and hirsutism score with the RIA (CC=0.24), LC/MS at Mayo (CC=0.15), or Quest (CC=0.17). A testosterone RIA is comparable to LC/MS assays. There is significant variability between LC/MS assays and poor precision with all assays at low testosterone levels.

Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry

Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review

Zearalenone-14-glucoside (ZEN-14G), a key modified mycotoxin, has attracted a great deal of attention due to the possible conversion to its free form of zearalenone (ZEN) exerting toxicity. In this study, the toxicokinetics of ZEN-14G were investigated in rats after oral and intravenous administration. The plasma concentrations of ZEN-14G and its major five metabolites were quantified using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The data were analyzed via non-compartmental analysis using software WinNonlin 6.3. The results indicated that ZEN-14G was rapidly hydrolyzed into ZEN in vivo. In addition, the major parameters of ZEN-14G following intravenous administration were: area under the plasma concentration–time curve (AUC), 1.80 h·ng/mL; the apparent volume of distribution (VZ), 7.25 L/kg; and total body clearance (CL), 5.02 mL/h/kg, respectively. After oral administration, the typical parameters were: AUC, 0.16 h·ng/mL; VZ, 6.24 mL/kg; and CL, 4.50 mL/h/kg, respectively. The absolute oral bioavailability of ZEN-14G in rats was about 9%, since low levels of ZEN-14G were detected in plasma, which might be attributed to its extensive metabolism. Therefore, liquid chromatography high-resolution mass spectrometry (LC-HRMS) was adopted to clarify the metabolic profile of ZEN-14G in rats’ plasma. As a result, eight metabolites were identified in which ZEN-14-glucuronic acid (ZEN-14GlcA) had a large yield from the first time-point and continued accumulating after oral administration, indicating that ZEN-14-glucuronic acid could serve a potential biomarker of ZEN-14G. The obtained outcomes would prompt the accurate safety evaluation of ZEN-14G.

Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. Serum samples (100 μL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 μm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 μmol/L, and lower limit of quantification was 0.07 and 0.26 μmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with ultraviolet detection methodology, and is suitable for routine clinical monitoring of patients predisposed to fat-soluble vitamin malabsorption.