High-throughput LC Research Articles

Analysis of Gut Microbiome Dysbiosis in Acute Myocardial Infarction Patients

Background and Aim: This study aims to detect the structure of gut microbiota in acute myocardial infarction (AMI) patients complicated with newly diagnosed atrial fibrillation (AF) through high-throughput sequencing and LC–MS Based Metabolomics so as to explore the characteristic changes of gut microbiota in these patients. Methods and Results: This prospective research recruited AMI patients, in the Fourth Affiliated Hospital of Soochow University. Patients who underwent physical examination were also included in this study. The characteristic of gut microbiota was conducted by high-throughput sequencing and LC–MS Based Metabolomics. At genus classification level, the abundance of Prevotella (P=0.009), Parabacteroides (P=0.036) in AMI patients with newly diagnosed AF were lower than that in AMI patients, while the abundance of Bifidobacterium (P=0.018), Dorea (P=0.036) Eggerthella (P=0.013), Peptoniphilus (P=0.039), Acinetobacter (P=0.016) tended to be higher. The species diversity of AMI patients with newly diagnosed AF was lower than that of AMI patients (P=0.037). Beta diversity analysis revealed a highly significant separation. The results of correlation analysis between differential metabolites and differential microbiota showed that the decrease of Prevotella and Parabacteroides in AMI patients with newly diagnosed AF may lead to an increase in some metabolites such as N-Acetyltyrosine, L-Isoleucine, 7-Methylguanine, L-Tyrosine, Sphingosine, 6-methoxyquinoline and 4-Hydroxybenzaldehyde. These changes will influence multiple metabolic pathways, thus affecting the prognosis of patients. Conclusions: There was an imbalance of gut microbiota in AMI patients, including AMI patients with newly diagnosed AF.

Deciphering putative protein profile of a photomorphogenic high pigment mutant of Solanum lycopersicum (hp-1) by high-throughput LC–MS/MS analysis

Deciphering putative protein profile of a photomorphogenic high pigment mutant of Solanum lycopersicum (hp-1) by high-throughput LC–MS/MS analysis

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC–MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic’s (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin

Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC–MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones.

Complementary effects of biochar, secondary metabolites, and bacteria biocontrol agents rejuvenate ratoon sugarcane traits and stimulate soil fertility

Bacteria biological control agents (BCAs) against diseases and pests are widely deemed a sustainable approach in agriculture. However, the extent to which the multifunctionality of plant secondary metabolites and BC interactions shape soil chemistry (e.g., C and N cycling enzymes) and BCAs communities in complex environments, especially under a cropping system (e.g., ratooning) prone to nutrient depletion, disease, etc., is not well established. Besides, the role metabolites-BCAs-BC interactions play in rejuvenating growth conditions necessary for ratoon sugarcane and suppressing plant pathogens/soil-borne diseases remains unclear. Here, we leveraged high-throughput sequencing and LC–MS/MS analyses to illuminate the regulatory effects of BC and plant secondary metabolites interactions on soil chemistry and BCAs communities. Additionally, we explored the roles metabolites-BCAs-BC interactions play in rejuvenating growth conditions necessary for ratoon sugarcane and suppressing plant pathogens/soil-borne diseases. Our finding revealed a pronounced rejuvenation of sugarcane ratoon traits and edaphic factors under the BC amendment soil, primarily due to the role metabolites-BCAs-BC interactions play in remediating soil fertility and health. We also proved that the BC-amended soils were worthy of enriching essential BCAs communities, including Pseudomonas, Bacillus, Streptomyces, Pantoea, Serratia, Weissella, and Leuconostoc. Abscisic aldehyde had a positive correlation with BCAs genera, including Bacillus, Pseudomonas, Serratia, Pantoea, and Ligilactobacillus. Whereas Pseudomonas, Pseudonocardia, Weissella, Bacillus, and Pantoea responded strongly and positively to jasmonic acid. Additionally, benzoate exhibited a strong positive association with BCAs genera, namely, Pseudomonas, Pseudonocardia, Weissella, Bacillus, Pantoea, and Ligilactobacillus in the different plant tissues. In the rhizosphere zone, BCAs genera such as Pseudomonas and Ligilactobacillus responded strongly to phenyllactic acid. Plant secondary metabolites, including apigenin and naringenin, demonstrated a strong positive relationship with N-cycling enzymes, including urease and phosphatase, respectively. Whereas palmitic acid, jasmonic acid, apiin, and abscisic aldehyde exhibited a significant positive correlation with soil TN, urease, pH, and AP, respectively. BugBase functional analysis confirmed that potentially pathogenic bacterial predicted phenotypes were low in the BC amendment. This work demonstrates how the multifunctionality of plant secondary metabolites-BCAs-BC interactions remediate soil fertility and suppress the presence of pathogens/parasites (e.g., Exserohilum, Aphis gossypii Glover, and Colletotrichum), thereby rejuvenating ratoon sugarcane growth and productivity.

Omic-Scale High-Throughput Quantitative LC-MS/MS Approach for Circulatory Lipid Phenotyping in Clinical Research.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

Rapid LC–MS assay for targeted metabolite quantification by serial injection into isocratic gradients

Liquid chromatography mass spectrometry (LC–MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC–MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC–MS is throughput. Clinical LC–MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which—when analyzing complex metabolomics samples—are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC–MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC–MS tool for quantifying small sets of target biomarkers across large cohorts.Graphical

Comparison of intact protein and digested peptide techniques for high throughput proteotyping of ApoE

IntroductionApolipoprotein E (ApoE) genotyping has been shown to have diagnostic value in the evaluation of cardiovascular diseases and neurodegenerative disorders such as Alzheimer’s disease. Although genetic testing is well established for this application, liquid chromatography-mass spectrometry (LC–MS) has the potential to provide a high throughput, low-cost alternative for ApoE evaluation.MethodsSerum samples were analyzed by peptide, intact protein, and genomic techniques. For peptide analysis, samples were digested with trypsin followed by liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) using a high-throughput multichannel LC system coupled to a Sciex 7500 mass spectrometer. For intact protein analysis, ApoE was immuno-purified using a monoclonal antibody immobilized on magnetic beads followed by high-resolution LC–MS analysis using an Exploris 480. DNA was extracted and evaluated using Sanger sequencing as a reference method.Results and discussionThe peptide measurement method produced one discrepant result when compared to genomic sequencing (out of 38 sequenced samples), whereas the intact protein analysis followed by deconvolution resulted in two discrepant results and when the intact protein data was processed with chromatographic integration there were three discrepant results. Therefore, the intact protein method proved slightly less accurate, required longer analysis time, and is substantially more costly, while providing only a 30 min improvement in sample preparation time.ConclusionsWith current MS technology clinical laboratories appear to be better served to utilize trypsin digest sample preparation and LC–MS/MS as opposed to high-resolution LC–MS intact protein analysis techniques for evaluation of ApoE proteotype. Peptide analysis methods are capable of producing accurate results with high throughput and minimal cost.

A simple, high-throughput and validated LC-MS/MS method for the determination of azithromycin in human plasma and its application to a clinical pharmacokinetic study.

A sensitive, specific and rapid liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated to quantify azithromycin concentrations in human plasma. Azithromycin (AZI) is the most common outpatient prescribed antibiotic in the US and clinical studies have demonstrated the efficacy and safety of AZI in many bacterial infections. To support a clinical study, we developed a high‐throughput LC–MS/MS method to process up to 250 samples per day to quantify AZI in human plasma. Samples were prepared by solid‐phase extraction. Separation was achieved with an ACE C18 column (2.1 × 100 mm, 1.7 μm) equipped with a C18 guard column. The mobile phase consisted of 0.1% formic acid and methanol–acetonitrile (1:1, v/v) at a flow rate of 0.25 ml/min. The ionization was optimized with positive electrospray source using multiple reaction monitoring transition, m/z 749.50 > 591.45 for AZI and m/z 754.50 > 596.45 for AZI‐d5. Extraction recoveries were approximately 90% for AZI. The assay was linear from 0.5 to 2,000 ng/ml and required only 100 μl of plasma with a total analysis time of 4.5 min. The method was successfully applied to pharmacokinetic studies of a weight‐based dosing protocol for AZI.

Plasma metabolite profiles related to plant-based diets and the risk of type 2 diabetes.

Plant-based diets, especially when rich in healthy plant foods, have been associated with a lower risk of type 2 diabetes. However, whether plasma metabolite profiles related to plant-based diets reflect this association was unknown. The aim of this study was to identify the plasma metabolite profiles related to plant-based diets, and to evaluate the associations between the identified metabolite profiles and the risk of type 2 diabetes. Within three prospective cohorts (Nurses' Health Study, Nurses' Health Study II and Health Professionals Follow-up Study), we measured plasma metabolites from 10,684 participants using high-throughput LC MS. Adherence to plant-based diets was assessed by three indices derived from the food frequency questionnaire: an overall Plant-based Diet Index (PDI), a Healthy Plant-based Diet Index (hPDI), and an Unhealthy Plant-based Diet Index (uPDI). Multi-metabolite profiles related to plant-based diet were identified using elastic net regression with a training/testing approach. The prospective associations between metabolite profiles and incident type 2 diabetes were evaluated using multivariable Cox proportional hazards regression. Metabolites potentially mediating the association between plant-based diets and type 2 diabetes risk were further identified. We identified multi-metabolite profiles comprising 55 metabolites for PDI, 93 metabolites for hPDI and 75 metabolites for uPDI. Metabolite profile scores based on the identified metabolite profiles were correlated with the corresponding diet index (Pearson r = 0.33-0.35 for PDI, 0.41-0.45 for hPDI, and 0.37-0.38 for uPDI, all p<0.001). Metabolite profile scores of PDI (HR per 1 SD higher = 0.81 [95% CI 0.75, 0.88]) and hPDI (HR per 1 SD higher = 0.77 [95% CI 0.71, 0.84]) showed an inverse association with incident type 2 diabetes, whereas the metabolite profile score for uPDI was not associated with the risk. Mutual adjustment for metabolites selected in the metabolite profiles, including trigonelline, hippurate, isoleucine and a subset of triacylglycerols, attenuated the associations of diet indices PDI and hPDI with lower type 2 diabetes risk. The explainable proportion of PDI/hPDI-related diabetes risk by these metabolites ranged between 8.5% and 37.2% (all p<0.05). Plasma metabolite profiles related to plant-based diets, especially a healthy plant-based diet, were associated with a lower risk of type 2 diabetes among a generally healthy population. Our findings support the beneficial role of healthy plant-based diets in diabetes prevention and provide new insights for future investigation.

Metabolite Identification Using Infrared Ion Spectroscopy─Novel Biomarkers for Pyridoxine-Dependent Epilepsy.

Untargeted liquid chromatography–mass spectrometry (LC–MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification—confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC–MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics–IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

ITRAQ-based quantitative glutelin proteomic analysis reveals differentially expressed proteins in the physiological metabolism process during endosperm development and their impacts on yield and quality in autotetraploid rice

Autotetraploid rice, which is developed through chromosome set doubling using diploid rice, produces high-quality kernels that are rich in storage proteins. However, little information is available about the content of different proteins in autotetraploid rice and their proteomic analysis. The dynamic changes in four storage proteins, namely, albumin, globulin, prolamin, and glutelin, were analyzed in the endosperm of autotetraploid rice (AJNT-4x) and in that of its diploid counterpart (AJNT-2x) for comparison. The contents of the four proteins were all higher during endosperm development in AJNT-4x than in AJNT-2x, but their change and composition were almost the same in the two materials. Then, iTRAQ was employed to analyze the glutelin profiles of AJNT-4x and AJNT-2x at 10 DAF, 15 DAF, and 20 DAF. A total of 1326 proteins were identified in AJNT-4x and AJNT-2x using high-throughput LC–MS/MS. Among the 1326 identified proteins, there were 362 DEPs in AJNT-4x compared with AJNT-2x and 372 DEPs between different developmental stages in AJNT-4x. Eight important upregulated proteins were identified by qRT-PCR, including B8AM24, B8ARJ0, B8AQM6, A2ZCE6, and P37833. Among them, B8AM24 and B8ARJ0 were related to the lysine biosynthesis process. GO enrichment analysis revealed that the critical functions of DEPs exhibited little overlap between the 10, 15, and 20 DAF groups. Endosperm glutelin accumulation was regulated mainly by different DEPs during the early stage, and 15 DAF was a critical regulating point for glutelin accumulation. KEGG pathway analysis showed that ribosomal proteins were significantly higher in AJNT-4x than in AJNT-2x at 10 DAF, and protein processing, biosynthesis, and metabolism of amino acids were higher and more active in AJNT-4x at 15 DAF, while the peroxisome was richer in AJNT-4x at 20 DAF. The PPI network showed that ribosomal proteins gradually decreased with increasing endosperm development. These results provide new insights into dynamic glutelin expression differences during endosperm development in autotetraploid rice, which will aid in the development of rice cultivars with increased yield and improved grain nutritional quality.

From Proteomics to Personalized Medicine: The Importance of Isoflavone Dose and Estrogen Receptor Status in Breast Cancer Cells.

Continuing efforts are directed towards finding alternative breast cancer chemotherapeutics, with improved safety and efficacy profiles. Soy isoflavones represent promising agents but, despite extensive research, limited information exists regarding their impact on the breast cancer cell proteome. The purpose of this study was to compare the proteomic profiles of MCF-7 (estrogen responsive) and MDA-MB-231 (estrogen non-responsive) breast cancer cells exposed to different concentrations of genistein, daidzein, and a soy seed extract, using a high throughput LC–UDMSE protein profiling approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed the dual activity of soy isoflavones on MCF-7 cells and the inhibitory effect on MDA-MB-231 cells. Proteome profiling of paramagnetic beads prepared peptides by nano-LC UDMSE and pathway enrichment analysis revealed that isoflavones affected distinct molecular pathways in MCF-7 and MDA-MB-231 cells, such as tyrosine kinases signaling pathway, cytoskeleton organization, lipid and phospholipid catabolism, extracellular matrix degradation and mRNA splicing. Also, in MCF-7 cells, low and high isoflavone doses induced different changes of the proteome, including cell cycle alterations. Therefore, the expression of estrogen receptors and the isoflavone dose are determinant factors for the molecular impact of isoflavones and must be taken into account when considering adjuvant breast cancer therapy towards personalized medicine.

Development and Clinical Evaluation of a High-Throughput LC-MS/MS Assay for Vitamin B6 in Human Plasma and Serum.

Pyridoxal 5'-phosphate (PLP) is the primary circulatory form of vitamin B6, an essential cofactor for numerous biochemical enzymatic reactions. Conventional PLP analysis using high-performance liquid chromatography (HPLC) with fluorescence requires derivatization and long injection-to-injection time. Development of high-throughput LC-MS/MS assays is desirable. Stable isotope labeled internal standard was added to aliquots of samples, proteins were precipitated using trichloroacetic acid, and supernatants were analyzed by multiple reaction monitoring using LC-MS/MS in positive ion mode. Analysis time for PLP was 3.0 min using single column HPLC separation and 2.4 min using alternating column regeneration (ACR). Clinical evaluation of the method included review of results (n = 102 386) from routine performance of the assay. The assay was linear to 500 nmol/L; limit of quantification was 5 nmol/L. Imprecision (CV) of the assay was <5%. Equivalent performance was observed for single HPLC column and ACR. In 62% of routinely analyzed patient samples, PLP concentrations were within the reference interval; higher PLP concentrations were observed in samples from males than from females. Vitamin B6 deficiency was lowest in children and highest in elderly adults. Lower PLP concentrations were observed in samples collected during winter/spring than during summer/fall. We observed lower concentrations in plasma collected in lithium heparin tubes, suggesting PLP degradation caused by the anticoagulant. This LC-MS/MS method allows PLP determination using simple sample preparation and short analysis time. We observed association of PLP concentrations with age, sex, and season of sample collection. Our data indicate that lithium heparin anticoagulant tubes reduce measured PLP concentration.

High-throughput protein precipitation method with 96-well plate for determination of doxepin and nordoxepin in human plasma using LC-MS/MS.

The aim of this study was to establish a high-throughput and sensitive LC-MS/MS method for the determination of doxepin and its major active metabolite nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of doxepin. Doxepin and nordoxepin were extracted from human plasma samples by protein precipitation with acetonitrile by using protein precipitation 96-well plates. The analyte was separated using a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm) using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 v:v (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 μL. The detection was performed using a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 280.4 → 107.0 and 283.4 → 235.0 for doxepin and doxepin-D3, respectively, and 266.3 → 106.9 and 269.3 → 235.0 for nordoxepin and nordoxepin-D3, respectively, in positive electrospray ionization mode. The total run time was 3.5 min. The method was validated over a concentration range of 50-10,000 pg/mL using a Triple Quad 4500 MS System (Sciex) for both analytes. The developed and validated method can be successfully used to study the bioequivalence/pharmacokinetics of doxepin and nordoxepin.

Quantitative analysis of underivatized 17 β-estradiol using a high-throughput LC–MS/MS assay – Application to support a pharmacokinetic study in ovariectomized guinea pigs

Difference in female sex hormone, β-estradiol (E2), levels can contribute to sex differences in biological processes that underlie target tissue functions (QT interval), vulnerability to diseases (hepatitis or HIV), and response toward therapies. Accurate quantification of plasma E2 level is thus an important aspect in both basic science research examining hormone-regulated physiological mechanisms and in clinical settings to support patient care associated with altered E2 levels. Due to lack of a high-throughput high-sensitivity analytical method, we developed and validated a LC–MS/MS assay for accurate low-level quantification of E2 and demonstrated its application to a guinea pig pharmacokinetic study in which guinea pigs were treated with 10 or 40 μg/kg E2 subcutaneously and blood samples collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post-dosing. E2 was extracted using 90 μL ovariectomized guinea pig plasma by liquid-liquid extraction. The method was robust, sensitive with linear range from 3.9 to 1000 pg/mL, and the assay met acceptance criteria for validation parameters listed in the current FDA Guidance on Bioanalytical Method Validation. Compared to the 10 μg/kg dose, more than dose proportional increase in maximum E2 plasma concentration (Cmax) and AUC0-∞ and correspondingly longer half-life were observed after 40 μg/kg dose. This assay is a significant improvement over existing E2 quantification methods in bioanalytical field, with high precision and accuracy, low sample and injection volumes, no derivatization, and short assay run time of 3 min. This assay is amenable in high-throughput settings requiring low-level E2 quantitation in basic science research and clinical settings.

Advancement in steroid hormone analysis by LC–MS/MS in clinical routine diagnostics – A three year recap from serum cortisol to dried blood 17α-hydroxyprogesterone

Steroid analysis by LC–MS/MS in daily clinical routine diagnostics requires high-throughput conditions including fast chromatographic separation. Hereby, signal interferences may occur due to limited specificity in complex biologic matrices. During the last three years of routine steroid analysis in our laboratory and roughly 50,000 measurements, about 1% was affected by interferences, mainly serum cortisol (>90%) and dried blood 17α-hydroxyprogesterone (17-OHP). To overcome specificity problems, enhanced chromatography, ionization polarity switching, and detection via two-stage fragmentation (MS3) using a quadrupole linear ion trap were investigated in our study. Signal interferences of serum cortisol were eliminated by applying a protocol for automated method switching without changing the basic high-throughput LC–MS/MS setup. This approach includes negative ionization and extended chromatography from 4 to 6.6 min using the fourfold column length. From 9 samples affected by cortisol interference using the high-throughput method, 8 could be reliably analyzed applying the method switching protocol. Moreover, the applicability of the high-throughput method as second tier analysis in congenital adrenal hyperplasia (CAH) diagnostics from dried blood was verified with 100% diagnostic specificity. In addition, the combination of fast LC and MS3 detection enables specific quantitation of 17-OHP from dried blood spots on a screening time scale. This approach may be an alternative to the newborn screening for CAH by immunoassay due to its higher specificity, reducing the number of false positive results by 90%. In this work we recap experiences from three years of clinical routine steroid analysis via LC–MS/MS and present a unique analytical setup that enables both high-throughput and enhanced resolution analysis of steroid hormones in serum and dried blood.

Rational selection of reverse phase columns for high throughput LC-MS lipidomics.

Natural lipidomes are characterized by extremely high complexity and dynamic range of lipid concentrations. Furthermore, high diversity of lipid physicochemical properties requires high resolving powers for both chromatographic and mass spectrometric analytical platforms. Reverse-phase chromatography coupled with data-dependent MS/MS acquisition is one of the most popular techniques in untargeted lipidomics. Optimal method should provide good chromatographic separation and resolution, reproducibility, selectivity and sensitivity. Here, we developed and set-up a RPLC-MS/MS workflow capable of resolving complex mixtures of lipids in 32 min of analysis. Human blood plasma was chosen as a representative complex natural lipidome with large variance of lipid classes, species and lipid concentrations. Lipids were separated by RPLC on five different reverse phase columns with different types of stationary phase particles, size and chemistry. High mass accuracy MS analysis and data-dependent MS/MS analysis were performed using a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer to identify individual lipid molecular species. This workflow was applied to evaluate the separation capability of each column and to identify the lipidomics profile in highly complex biological samples. As a result, we report more than 600 lipid species covering 18 lipid classes in human blood plasma and provide suggestions to the selection of the appropriate reverse phase column for the analysis of specific lipidomes.

Somatic proteome of Haemonchus contortus

Currently, there is a dearth of proteomic data to underpin fundamental investigations of parasites and parasitism at the molecular level. Here, using a high throughput LC–MS/MS-based approach, we undertook the first reported comprehensive, large-scale proteomic investigation of the barber's pole worm (Haemonchus contortus) – one of the most important parasitic nematodes of livestock animals worldwide. In total, 2487 unique H. contortus proteins representing different developmental stages/sexes (i.e. eggs, L3s and L4s, female (Af) and male (Am) adults) were identified and quantified with high confidence. Bioinformatic analyses of this proteome revealed substantial alterations in protein profiles during the life cycle, particularly in the transition from the free-living to the parasitic phase, and key groups of proteins involved specifically in feeding, digestion, metabolism, development, parasite-host interactions (including immunomodulation), structural remodelling of the body wall and adaptive processes during parasitism. This proteomic data set will facilitate future molecular, biochemical and physiological investigations of H. contortus and related nematodes, and the discovery of novel intervention targets against haemonchosis.

Delapril and Indapamide: Development and Validation of a Stability-Indicating Core-Shell LC Method and Its Application for Simultaneous Tablets Assay.

Background: The combination of delapril (DEL) and indapamide (IND) may be regarded as an optimal drug treatment for hypertensive patients. However, there is no published study concerning the suitable stability conditions and evaluation of drugs in the raw material and commercial product. Objective: The aim of the present study was to develop an innovative, high-throughput, and stability-indicating LC method for the simultaneous analysis of DEL and IND in combined dosage form. Methods: Analyses were performed using a core-shell C18 column (100 mm × 4.6 mm id, 2.6 μm) at 45°C using isocratic elution for the mobile phase composed of triethylamine solution (0.3%, pH 5.0)-acetonitrile-methanol (58 + 35 + 7, v/v/v). The separation was obtained within 3.5 min at a flow rate of 1.0 mL/min and UV detection set at 213 nm. Results: The specificity and stability-indicating capability of the method were proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that the peak is free from any co-eluting peak. Conclusions: The method showed adequate precision, with relative standard deviation values lower than 1.85%. Excellent values of accuracy were obtained, with a mean value of 98.64% for IND and 98.65% for DEL. Experimental design was used during validation to calculate and prove the method robustness. Highlights: The proposed LC method was successfully validated according to International Conference on Harmonisation requirements and applied for the simultaneous determination of DEL and IND in tablets, presenting suitability for stability studies and contributing to improve the QC of pharmaceuticals.