Mass Spectrometry Method For Quantitation Research Articles

Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 μL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

CSF neurofilament light chain profiling and quantitation in neurological diseases.

Neurofilament light chain is an established marker of neuroaxonal injury that is elevated in CSF and blood across various neurological diseases. It is increasingly used in clinical practice to aid diagnosis and monitor progression and as an outcome measure to assess safety and efficacy of disease-modifying therapies across the clinical translational neuroscience field. Quantitative methods for neurofilament light chain in human biofluids have relied on immunoassays, which have limited capacity to describe the structure of the protein in CSF and how this might vary in different neurodegenerative diseases. In this study, we characterized and quantified neurofilament light chain species in CSF across neurodegenerative and neuroinflammatory diseases and healthy controls using targeted mass spectrometry. We show that the quantitative immunoprecipitation-tandem mass spectrometry method developed in this study strongly correlates to single-molecule array measurements in CSF across the broad spectrum of neurodegenerative diseases and was replicable across mass spectrometry methods and centres. In summary, we have created an accurate and cost-effective assay for measuring a key biomarker in translational neuroscience research and clinical practice, which can be easily multiplexed and translated into clinical laboratories for the screening and monitoring of neurodegenerative disease or acute brain injury.

A Selective Gas Chromatography–Tandem Mass Spectrometry Method for Quantitation of Ethylene and Diethylene Glycol in Paediatric Syrups

Ethylene Glycol (EG) and diethylene Glycol (DEG) are two contaminants known to cause various human health problems. These glycols might be present in drug syrups that are based on glycerol, sorbitol, or polyethylene glycol. In late 2022, several batches of cough, antipyretics, and antihistamine syrups were reported to contain toxic levels of EG and DEG in multiple countries; this incident concerned the World Health Organization (WHO). From an analytical perspective, several methods of glycols analysis in pharmaceuticals have been reported in the literature, with the majority being dedicated to raw material analysis. This study aims to develop a selective method capable of evaluating a wide range of paediatric syrups in order to assess the safety of commercially available paediatric syrups currently distributed in the local market. This research introduces a method for determining glycols utilizing gas chromatography-tandem mass spectrometry (GC–MS/MS), which offers significantly higher selectivity than conventional single quadrupole gas chromatography-mass spectrometry (GC–MS). The developed method meets the current International Council for Harmonisation (ICH) guidelines for validation. The absence of any interfering peaks in both the unspiked sample of promethazine syrup and the reference standard solutions proved the method's selectivity. Furthermore, 2,2,2-trichloroethanol was used as an internal standard, and a new GC–MS/MS method was developed to analyze it. The calibration curves for EG and DEG were linear within the selected concentration range of 1–10 μg/mL. The detection limit for both EG and DEG was 400 ng/mL, while the quantification limit was 1 μg/mL. Recovery values for both EG and DEG met the accuracy acceptance criterion. Thus, the developed method proved to be efficient and accurate for determining EG and DEG levels in suspected contaminated syrups.

The proteomic landscape of microglia in health and disease.

Microglia are the resident immune cells of the central nervous system (CNS) and as such play crucial roles in regulating brain homeostasis. Their presence in neurodegenerative diseases is known, with neurodegeneration-associated risk genes heavily expressed in microglia, highlighting their importance in contributing to disease pathogenesis. Transcriptomics studies have uncovered the heterogeneous landscape of microglia in health and disease, identifying important disease-associated signatures such as DAM, and insight into both the regional and temporal diversity of microglia phenotypes. Quantitative mass spectrometry methods are ever increasing in the field of neurodegeneration, utilised as ways to identify disease biomarkers and to gain deeper understanding of disease pathology. Proteins are the main mechanistic indicators of cellular function, yet discordance between transcript and proteomic findings has highlighted the need for in-depth proteomic phenotypic and functional analysis to fully understand disease kinetics at the cellular and molecular level. This review details the current progress of using proteomics to define microglia biology, the relationship between gene and protein expression in microglia, and the future of proteomics and emerging methods aiming to resolve heterogeneous cell landscapes.

Quantitative mass spectrometry imaging (qMSI): A tutorial.

Mass spectrometry imaging (MSI) is an analytical technique that enables the simultaneous detection of hundreds to thousands of chemical species while retaining their spatial information; usually, MSI is applied to biological tissues. Combining these elements can create ion images, which allows for the identification and localization of multiple chemical species within the sample. Being able to produce molecular images of biological tissues has already impacted the study of health and disease; however, the next logical step is being able to combine MSI with quantitative mass spectrometry methods to both quantify and determine the localization of disease progression or drug action. In this tutorial, we will detail the main factors to consider when designing a qMSI experiment and highlight the methods that have been developed to overcome these added complexities, specifically for those newer to the field of MSI.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Diverse associations observed between pregnancy complications and RBC or plasma folates determined by an in-house developed LC-MS/MS method

Background As folates are essential for embryonic development and growth, it is necessary to accurately determine the levels of folates in plasma and red blood cells (RBCs) for clinical intervention. The aims of this study were to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of folates in plasma and RBCs and to examine the association between plasma and RBC folate concentrations and gestational diabetes mellitus (GDM), gestational hypertension (GH) and preeclampsia (PE). Methods With the in-house developed LC-MS/MS, a retrospective cross-sectional study was conducted. The healthy pregnant women of first- (n = 147), second- (n = 84) and third-trimester (n = 141) or the women diagnosed with GDM (n = 84), GH (n = 58) or PE (n = 23), that were aged between 22 and 46 years old and registered at our institute, were subjected for measurement of folic acid (FA) and 5-methyltetrahydrofolate (5-MTHF), followed by appropriate statistical association analysis. Results The assay for simultaneous quantitation of FA and 5-MTHF in plasma and RBCs was linear, stable, with imprecision less than 15% and recoveries within ±10%. The lower limits of quantification for FA and 5-MTHF measurement in whole blood were 0.57 and 1.09 nmol/L, and in plasma were 0.5 and 1 nmol/L, respectively. In the association analysis, the patients with lower RBC folate level (<906 nmol/L) presented higher risks of PE development (OR 4.861 [95% CI 1.411–16.505]) by logistic regression and restricted cubic spline (RCS) regression in a nonlinear fashion. In addition, higher level of plasma folates in pregnancy was significantly associated with GH risk but may be protective for the development of GDM. Conclusions The in-house developed LC-MS/MS method for folates and metabolites in plasma or RBC showed satisfactory analytical performance for clinical application. Further, the levels of folates and metabolites were diversely associated with GDM, GH and PE development.

B-201 Development of a Quantitative Gas Chromatography Tandem Mass Spectrometry Method for Metabolic Profiling of Urine Organic Acids

Abstract Background Urine organic acids (UOA) analysis is an essential investigation for inborn errors of metabolism (IEMs). The vast majority of methods use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity, and enables the combination of qualitative screening with quantitative analysis. We describe the development and preliminary validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&amp;W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. The linear concentration ranges of the targeted metabolites within the devised GC-MS/MS protocol were determined. Results The optimal temperature gradient for high-resolution UOA separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N = 38) derivatized UOA. Total analysis time was 33.8 min per injection. The achieved linear concentration ranges for each metabolite was respectively determined. Linear concentration ranges and correlation coefficients of representative UOA: 3-hydroxyisovaleric acid (range = 9 to 708 µmol/L, R2 = 0.9874); lactic acid (range = 15 to 751 µmol/L, R2 = 0.9936); 3-hydroxybutyric acid (range = 15 to 751 µmol/L, R2 = 0.9991); and 2-methyl-3-hydroxybutyric acid (range = 10 to 501 µmol/L, R2 = 0.9996). The correlation coefficients for all remaining UOA ranged from R2 = 0.9458 (4-hydroxyphenylpyruvic acid) to R2 = 0.9998 (3-methylglutanoic acid and ethylmalonic acid). Conclusion The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA used to investigate IEMs. The linear concentration ranges for all analytes were determined and are appropriate for monitoring these markers of inherited enzyme deficiencies. The analytical performance of this UOA GC-MS/MS method must be further validated and its diagnostic performance for selected IEMs characterized.

Determination of plasma concentration of Belimumab by LC-MS/MS: Method development, validation, and clinical application

IntroductionBelimumab is a monoclonal antibody against B cell activating factor (BLyS). This monoclonal antibody (mAb) has been shown to be effective in reducing disease activity in patients with systemic lupus erythematosus (SLE). Belimumab is available in two forms as a lyophilized powder for intravenous (IV) use, or single-dose syringe for subcutaneous (SC) use. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for quantitation of belimumab in human serum. Material and methodsAll analyses relied on nano-surface and molecular-orientation limited (nSMOL) proteolysis coupled with LC-MS/MS. Quantifications was performed in multiple reactions monitoring (MRM) mode, and electrospray ionization was conducted in positive mode. ResultsBelimumab was quantified with signature peptide QAPGQGLEWMGGIPFGTAK and normalized using P14R. The total run time per assay was 10 min. Linearity was measured from 5 to 800 μg/mL (r² > 0.995). Accuracy and precision based on three quality control levels range from 11.2 – 9.51 % and 1.24 – 13.12 % respectively. The carryover was less than 7 %. In all, 87 patient samples were processed (65, IV; 22, SC). Mean concentration of belimumab was significantly higher for SC (93.0 ± 74.0 µg/mL) than for IV (67.4 ± 38.9 µg/mL) administration. ConclusionWe have developed the first method of belimumab quantification combining LC-MS/MS and nSMOL proteolysis. It can be used for future clinical pharmacokinetic studies of belimumab and for investigating the relationship between belimumab concentration, efficacy, and toxicity in SLE patients.

Development, validation, and application of a multimatrix UHPLC-MS/MS method for quantification of Datura-type alkaloids in food

A quantitative ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of tropane alkaloids (TAs), atropine and scopolamine, in a variety of food products. The sample preparation of cereal-based food, oilseeds, honey, and pulses consisted of a solid–liquid extraction with an acidified mixture of methanol and water, while an additional step of solid-phase extraction on a cation-exchange sorbent was introduced in the treatment of teas and herbal infusions, aromatic herbs, spices and food supplements. The limits of quantification of the method varied from 0.5 to 2.5 µg kg−1. Apparent recovery was in the range of 70–120%, and repeatability and intermediate precision were below 20%. The method was successfully applied in a proficiency testing exercise as well as in the analysis of various commercial foods. Only 26% of the analysed food samples contained one or both TAs. The mean concentrations for atropine and scopolamine amounted to 21.9 and 6.5 µg kg−1, respectively, while the maximum concentrations were 523.3 and 131.4 µg kg−1, respectively. Overall, the highest levels of TA sum were found in an herbal infusion of fennel and a spice mix containing fennel and anise seeds.

Collision Induced Unfolding Enables the Quantitation of Isomass Biotherapeutics in Complex Biological Matrices.

Quantitative mass spectrometry has been widely used to evaluate the concentrations of molecules within a variety of biological matrices. Typically, such quantitative mass spectrometry analyses are predicated upon the production of mass-resolved precursor or fragment ions, leading to challenges surrounding the quantification of isomeric or conformationally distinct analytes. As such, new approaches are required for the label-free quantification of isomass proteins. Native ion-mobility MS (nIM-MS) in combination with collision induced unfolding (CIU) is a potentially enabling approach for such quantitative mass spectrometry methods as the technique can rapidly separate and detect many biomacromolecule isoforms. CIU uses collisional activation to capture the unfolding trajectory of ions in the gas phase, producing different intermediate structures that can be leveraged to distinguish protein structures that exhibit identical sizes at lower energies. Here we describe the deployment of quantitative CIU methodology to measure the concentrations of isomass pairs of biotherapeutics and sequence homologues in both standard and biological matrices. Our results cover three antibody pairs and include examples of mixed therapies where multiple biologics are commonly provided to patients. In all cases, CIU enables the production of resolved features for each antibody mixture probed, producing calibration curves with correlation coefficients ranging from 0.92 to 0.99, limits of detection ranging from 300 to 5000 nM and sensitivities ranging from 8.7 × 10-5 nM-1 to 6 × 10-3 μM-1. We conclude our report by projecting the future utility of CIU-enabled quantitative MS methods.

High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS.

Amino acids and acylcarnitines are important biomarkers of the body's energy state and can be used as diagnostic markers of certain inborn errors of metabolism. Few multianalyte methods for high-throughput analysis in serum exist for these compounds, but micromethods suitable for use in young children and infants are lacking. Therefore, we developed a quantitative high-throughput multianalyte hydrophilic interaction liquid chromatography-tandem mass spectrometry method preceded by a derivatization-free sample preparation using minimum amounts of serum (25 μL). Isotopically labeled standards were utilized for quantification. Forty amino acids and amino acid derivatives and 22 acylcarnitines were detected by applying a multiple reaction monitoring mode within a 20 min run. The method was comprehensively validated, comprising linearity, accuracy, (intraday/interday) precision, and quantitation limits, of which the latter ranged from 0.25 to 50 nM for acylcarnitines and from 0.005 to 1 μM for amino acids and their derivatives. Application of the method to 145 serum samples of three- to four-month-old healthy infants showed excellent reproducibility for multiday analyses and enabled simultaneous amino acid and acylcarnitine profiling in this age group.

Validation of circulating histone detection by mass spectrometry for early diagnosis, prognosis, and management of critically ill septic patients

BackgroundAs leading contributors to worldwide morbidity and mortality, sepsis and septic shock are considered a major global health concern. Proactive biomarker identification in patients with sepsis suspicion at any time remains a daunting challenge for hospitals. Despite great progress in the understanding of clinical and molecular aspects of sepsis, its definition, diagnosis, and treatment remain challenging, highlighting a need for new biomarkers with potential to improve critically ill patient management. In this study we validate a quantitative mass spectrometry method to measure circulating histone levels in plasma samples for the diagnosis and prognosis of sepsis and septic shock patients.MethodsWe used the mass spectrometry technique of multiple reaction monitoring to quantify circulating histones H2B and H3 in plasma from a monocenter cohort of critically ill patients admitted to an Intensive Care Unit (ICU) and evaluated its performance for the diagnosis and prognosis of sepsis and septic shock (SS).ResultsOur results highlight the potential of our test for early diagnosis of sepsis and SS. H2B levels above 121.40 ng/mL (IQR 446.70) were indicative of SS. The value of blood circulating histones to identify a subset of SS patients in a more severe stage with associated organ failure was also tested, revealing circulating levels of histones H2B above 435.61 ng/ml (IQR 2407.10) and H3 above 300.61 ng/ml (IQR 912.77) in septic shock patients with organ failure requiring invasive organ support therapies. Importantly, we found levels of H2B and H3 above 400.44 ng/mL (IQR 1335.54) and 258.25 (IQR 470.44), respectively in those patients who debut with disseminated intravascular coagulation (DIC). Finally, a receiver operating characteristic curve (ROC curve) demonstrated the prognostic value of circulating histone H3 to predict fatal outcomes and found for histone H3 an area under the curve (AUC) of 0.720 (CI 0.546–0.895) p < 0.016 on a positive test cut-off point at 486.84 ng/mL, showing a sensitivity of 66.7% and specificity of 73.9%.ConclusionsCirculating histones analyzed by MS can be used to diagnose SS and identify patients at high risk of suffering DIC and fatal outcome.

Comparative proteomics of saliva of healthy and gingivitis individuals from Rio de Janeiro.

In this work, we identified human and bacterial proteomes in the saliva from volunteers with gingivitis or healthy. The reported population consisted of 18 volunteers (six with gingivitis and 12 healthy controls). Proteomics characterization was performed using a quantitative mass spectrometry method. A total of 74 human and 116 bacterial proteins were identified in saliva. The major functional category that was modified in the human proteome was the immune response, followed by transport and protease inhibition. In the bacterial proteome, most of the proteins identified were from the Fusobacteria phylum, followed by Chlamydiae and Spirochaetes. We observed statistically relevant differences in the data between the groups. The 15 most important human proteins affecting the variation between case and control groups included cystatin S, alpha amylase, lactotransferrin, and negative elongation factor E. We found that bacterial proteins from Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum related to the red and orange complexes were closely correlated with the occurrence of periodontal diseases.

Development and application of an enantioselective liquid chromatography – tandem mass spectrometry method for the quantification of enantiomers to evaluate in vivo chiral inversion in beagle dog

To assess whether a suspected metabolic chiral inversion of a new chemical entity (NCE) with a single chiral centre occurred in an in vivo pharmacokinetic (PK) study of UCB-1, a quantitative enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. Chromatographic screening of authentic standards of the two enantiomers was carried out using a range of Phenomenex Lux chiral columns under various isocratic conditions to achieve optimum chromatographic resolution. Protein precipitation was used for analyte extraction from 40 µL of dog plasma. The subjects were given a po dose of either 1, 3 or 10 mg/kg followed by blood sample collection at timepoints over a 72-hour period. For the analysis, a Shimadzu liquid chromatography system was coupled to a Sciex QTRAP 5500 mass spectrometer operated using multiple reaction monitoring (MRM) in positive ion mode. The regression for the two enantiomers was (R2 ≥ 0.999) over the range of 10 – 20,000 ng/mL while employing a quadratic fit, with a lower limit of quantification (LLOQ) of 10 ng/ml. The method was validated for intra- and inter-day precision and accuracy, which were within +/- 20%. The enantiomers were stable in dog plasma at room temperature for 4 h and after three freeze-thaw cycles. The method was successfully applied to a pharmacokinetic study in dog, after po administration of UCB-1 where a significant level of chiral inversion was observed in plasma.

A targeted multiplex mass spectrometry method for quantitation of abundant matrix and cellular proteins in formalin-fixed paraffin embedded arterial tissue

Assessment of proteins in formalin-fixed paraffin-embedded (FFPE) tissue traditionally hinges on immunohistochemistry and immunoblotting. These methods are far from optimal for quantitative studies and not suitable for large-scale testing of multiple protein panels. In this study, we developed and optimised a novel targeted isotope dilution mass spectrometry (MS)-based method for FFPE samples, designed to quantitate 17 matrix and cytosolic proteins abundantly present in arterial tissue. Our new method was developed on FFPE human tissue samples of the internal thoracic artery obtained from coronary artery bypass graft (CABG) operations. The workflow has a limit of 60 samples per day. Assay precision improved by normalisation to both beta-actin and smooth muscle actin with inter-assay coefficients of variation (CV) ranging from 5.3% to 31.9%. To demonstrate clinical utility of the assay we analysed 40 FFPE artery specimens from two groups of patients with or without type 2 diabetes. We observed increased levels of collagen type IV α1 and α2 in patients with diabetes. The assay is scalable for larger cohorts and advantageous for pathophysiological studies in diabetes and the method is easily convertible to analysis of other proteins in FFPE artery samples. SignificanceThis article presents a novel robust and precise targeted mass spectrometry assay for relative quantitation of a panel of abundant matrix and cellular arterial proteins in archived formalin-fixed paraffin-embedded arterial samples. We demonstrate its utility in pathophysiological studies of cardiovascular disease in diabetes.

Evaluation and improvement of a miniature mass spectrometry system for quantitative harm reduction drug checking

The increasing variability of the illicit drug market has imposed serious health risks for people who use drugs (PWUD), particularly due to the increasing co-occurrence of opioids and etizolam (or other benzodiazepines) in some jurisdictions. A miniature mass spectrometer (MS) equipped with paper capillary spray ionization was evaluated for its ability to quantify fentanyl, fluorofentanyl, carfentanil, and etizolam in illicit drug samples. Prior to testing actual drug samples, inter- and intra-day reproducibility, calibration linearity, and analytical sensitivity were assessed. The development of a continuous spray solvent delivery system improved MS/MS quantitation by prolonging ionization stability. A simple, rapid and quantitative tandem mass spectrometry method was developed and employed for the analysis of real-world illicit drug samples provided by PWUD at a harm reduction site in Victoria, British Columbia, Canada. Limits of detection ranged from 0.001 to 0.24% w/w of illicit analyte in the original solid drug samples. Target analytes were detected and quantified via the miniature MS system for samples where conventional on-site drug checking technologies were not always effective. These promising results demonstrate the potential role for miniature mass spectrometer systems for on-site drug checking services that can be both sensitive to trace, toxic levels as well as adaptable/selective for new drug threats as they appear in the illicit drug supply.

Workflow for Quantitative Proteomic Analysis of Intestinal Organoids Using SILAC.

Stable isotope labeling by amino acids in cell culture (SILAC) is a strategic quantitative mass spectrometry method to analyze multiple protein samples in different conditions simultaneously. In recent years, 3D cell growth culture conditions have been developed to establish intestinal organoids from isolated crypts, which mimic the intestine's cell composition and organization. Organoids, isolated from normal or diseased tissues, can be used to compare cell distribution and differentiation, signaling pathways, and cell responses to pharmacological agents, therapeutic drugs, endogenous or exogenous metabolites, and environmental stresses, among others. Here, we describe the process of generating SILAC organoids from the mouse small intestine.

An ultra-high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of 10 alkaloids of Corydalis Decumbentis Rhizoma preparation in dog plasma and its application to a pharmacokinetic study.

A sensitive and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was successfully applied to the determination of 10 alkaloids in beagle dog plasma following a single oral dose of Xiatianwu capsules and enteric-coated capsules, with theophylline serving as the internal standard (IS). Plasma samples were preprocessed using liquid-liquid extraction (LLE) with ethyl acetate ahead to separation using a gradient elution procedure on a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 100 × 2.1 mm). The mobile phase was composed of 0.1% formic acid solution and acetonitrile at the flow rate of 0.3 ml/min. Multiple reaction monitoring (MRM) was used to determine the analytes in the positive ion mode. The calibration curves for 10 analytes demonstrated a high degree of linearity (r ≥ 0.9920). The lower limit of quantification (LLOQ) values for 10 alkaloids were all more than 1.074 ng/ml, and matrix effects varied from 94.25% to 106.15%. The mean extraction recovery of quality control samples at low (LQC), medium (MQC) and high (HQC) concentrations, as well as IS, was all more than 76.60%. The intra-day and inter-day precision (RSD) also satisfied the requirement. Simultaneously, the variation of assay accuracies (RE) was between 13.05% and 9.38%. The test was validated in accordance with regulatory bioanalytical guidelines and was found to be suitable for use in a pharmacokinetic investigation of these compounds in beagle dogs after oral administration of Xiatianwu general capsules and enteric-coated capsules. The Cmax of 10 alkaloids ranged from 52.61 to 192.46 ng/ml after oral administration of Xiatianwu capsules, and from 67.50 to 247.36 ng/ml. The Tmax was between 0.59 and 1.33 h of Xiatianwu capsules, and between 1.08 and 2.00 h of enteric-coated capsules. The t1/2 ranged from 3.18 to 7.47 h of general capsules, and from 6.01 to 11.36 h. AUC0-t ranged from 181.06 to 722.74 ng·h/ml of Xiatianwu capsules, and from 275.03 to 884.17 ng·h/ml of enteric-coated capsules. The Cmax of enteric-coated capsules were significantly increased except for tetrahydropalmatine and berberine. Tmax of general capsules were less than 1 h, and of enteric-coated capsules were less than 2 h. The t1/2 of dehydrocorydaline, palmatine, tetrahydrojatrorrhizine, jatrorrhizine and coptisine in enteric-coated capsules was longer than that in ordinary capsule. The AUC0-t and AUC0-∞ of bicuculline, dehydrocorydaline, protopine, magnoflorine, tetrahydrojatrorrhizine, jatrorrhizine, berberine and coptisine were all significantly higher in enteric-coated capsules.

A quantitative mass spectrometry method to differentiate bovine and ovine heparins from pharmaceutical porcine heparin

Heparin is a polysaccharide extracted from animal tissues and is used widely as an anticoagulant. In most countries, porcine intestine mucosa is the only legal source for producing heparin. It is challenging to differentiate heparins derived from porcine, ovine and bovine, especially when low amounts of ruminant heparin are adulterated into porcine heparin. Herein, we find that two marker saccharides, ΔUA2S-GlcNS6S-HexA2S (ΔISH) and ΔUA2S-GlcNAc6S (ΔIA), show significant differences in the basic building blocks of porcine heparin obtained from ruminant heparin. A quantitative mass spectrometry (MS) method was then established to selectively monitor these two marker saccharides. By using the ΔISH to ΔIA ratio, porcine heparin adulterated with a low amount of ruminant heparin (10 % ovine heparin or 5 % bovine heparin) can be differentiated. This represents a robust and sensitive method for ensuring the authenticity and safety of heparin drugs.