Negative Ion Research Articles

Determination of double bond positions in unsaturated fatty acids by pre-column derivatization with dimethyl and dipyridyl disulfide followed by LC-SWATH-MS analysis.

Comprehensive in-depth structural characterization of free mono-unsaturated and polyunsaturated fatty acids often requires the determination of carbon-carbondouble bond positions due to their impact on physiological properties and relevance in biological samples or during impurity profiling of pharmaceuticals. In this research, we report on the evaluation of disulfides as suitable derivatization reagents for the determination of carbon-carbondouble bond positions of unsaturated free fatty acids by UHPLC-ESI-QTOF-MS/MS analysis and SWATH (sequential windowed acquisition of all theoretical mass spectra) acquisition. Iodine-catalyzed derivatization of C = C double bonds with dimethyl disulfide (DMDS) enabled detection of characteristic carboxy-terminal MS2 fragments for various fatty acids in ESI negative mode. The determination of double bond positions of fatty acids with up to three double bonds, the transfer of the method to plasma samples, and its limitations have been shown. To achieve charge-switching for positive ion mode MS-detection, derivatization with 2,2'-dipyridyldisulfide (DPDS) was investigated. It enabled detection of both corresponding characteristic omega-end- and carboxy-end-fragments for fatty acids with up to two double bonds in positive ion mode. It provides a straightforward strategy for designing MRM transitions for targeted LC-MS/MS assays. Both derivatization techniques represent a simple and inexpensive way for the determination of double bond positions in fatty acids with low number of double bonds. No adaptation of MS hardware is required and the specific isotopic pattern of resulting sulfur-containing products provides additional structural confirmation. This reaction scheme opens up the avenue of structural tuning of disulfide reagents beyond DMDS and DPDS using reagents like cystine and analogs to achieve enhanced performance and sensitivity.

Ten questions concerning particle exposure in open electrosurgery and its control strategies

The increasing use of high-frequency electrosurgical pencils in open surgeries has heightened concerns about the adverse implications of electrosurgical smoke (ES) on perioperative personnel. Since 2015, there has been an increasing amount of research on electrosurgical smoke particles annually. Despite data discrepancies, nanoparticles (core particle size <200 nm) generally dominate in number distributions. Particulate matter (PM) concentration is closely related to the type of target tissue. Most studies show that ultrafine particle (UFP) concentrations near the main surgeon’s breathing zone inversely correlate with operating room air changes and positively correlate with the power of the electrosurgical pencil. Local smoke evacuation devices with negative ions, specialized filters and N95 respirators are more effective in protecting perioperative staff health, particularly against UFPs. The objective is to summarize particle exposure knowledge in open electrosurgery and establish a research agenda to protect medical personnel from ES exposure.

Identification of the metabolites of nimbolide in rat by liquid chromatography combined with quadrupole/orbitrap mass spectrometry.

Nimbolide is a major furanoid compound isolated from Azadirachta indica. The aim of this study was to characterize the metabolites of nimbolide in rats and to propose the metabolic pathways. The metabolites were generated by incubating nimbolide (10μM) with rat liver microsomes, nicotinamide adenine dinucleotide phosphate (NADPH), and nucleophiles (glutathione [GSH] or N-acetyl-lysine [NAL]) at 37°C for 60 min. For the in vivo study, nimbolide was intravenously administered to rats at a single dose of 10mg/kg, and the bile and urine were collected. The metabolites were identified by ultra-high-performance liquid chromatography-quadrupole/orbitrap mass spectrometry (UPLC-Q/Orbitrap-MS) using electrospray ionization in positive ion mode. Totally, nine metabolites were detected, and their identities were characterized by accurate MS and MS/MS data. In GSH-supplemented liver microsomes, GSH conjugation was the primary elimination pathway. The furan ring was bioactivated into cis-butene-1,4-dial that can be trapped by GSH. In NAL-supplemented liver microsomes, two NAL conjugates (M4 and M5) derived from cis-butene-1,4-dial were observed. In rat bile and urine, N-acetyl-cysteine, cysteine-glycine, and GSH conjugate were also found. The current study provides an overview of the metabolism and the bioactivation profiles of nimbolide in rats, which aids in understanding its safety and activity.

An Ultra-Fast Green UHPLC-MS/MS Method for Assessing the In Vitro Metabolic Stability of Dovitinib: In Silico Study for Absorption, Distribution, Metabolism, Excretion, Metabolic Lability, and DEREK Alerts.

Background and Objectives: Dovitinib (DVB) is a pan-tyrosine kinase inhibitor (TKI) that can be administered orally. In September 2023, the FDA granted Oncoheroes approval to proceed with an Investigational New Drug (IND) application for dovitinib. This application is intended for the treatment of relapsed or advanced juvenile solid tumors, namely, osteosarcoma. Materials and Methods: The target of the present study was to develop a rapid, green, accurate, and sensitive UHPLC-MS/MS method for measuring DVB levels in human liver microsomes (HLMs). The validations of the HLMs were performed via the established UHPLC-MS/MS approach, as stated in the US FDA reported guidelines for the standards of bioanalytical method validation protocol. The StarDrop in silico software package (version 6.6), which involves the DEREK and WhichP450 in silico modules, was used to check the DVB structure for hazardous alerts and metabolic instability. The DVB and encorafenib (EFB), internal standard, and chromatographic peaks were successfully separated using a reversed phase column (an Eclipse Plus Agilent C8 column) and an isocratic mobile phase. The production of DVB parent ions was accomplished by utilizing the positive ionization mode of an ESI source. The identification and measurement of DVB daughter ions were conducted using the MRM mode. Results: The inter-day accuracy and precision exhibited a spectrum of values in the range of -0.56% to 9.33%, while the intra-day accuracy and precision showcased a range of scores between 0.28% and 7.28%. The DVB calibration curve showed a linear relationship that ranged from 1 to 3000 ng/mL. The usefulness of the currently validated UHPLC-MS/MS method was approved by the lower limit of quantification (LLOQ) of 1 ng/mL. The AGREE findings demonstrate that the UHPLC-MS/MS method had a noteworthy degree of ecological greenness. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of DVB were calculated to be 15.48 min and 52.39 mL/min/kg, respectively, which aligned with the findings from the WhichP450 software (version 6.6). Conclusions: Via the usage of in silico software, it has been observed that making small changes to the structure of the aryl piperazine ring and quinolinone moieties, or replacing these groups in the drug design process, shows potential for enhancing the metabolic safety and stability of newly developed derivatives compared to DVB.

A Comprehensive Spatially Resolved Metabolomics Dataset for Lampreys

As one of the two most ancient groups of extant vertebrates, lamprey has become an important model organism in various fields of biology. In this paper, we present a comprehensive tissue-wide spatial metabolomics dataset for lampreys, where 14 distinct tissues were analyzed using liquid chromatography-mass spectrometry (LC-MS) in both positive and negative ion modes. The dataset has been fully validated using internal standard and pooled quality control samples and is readily accessible at the UCSD Metabolomics Workbench. This dataset serves as a valuable resource for researchers using lampreys as a model organism. Additionally, it acts as a benchmark metabolomics dataset for evaluating new algorithms and software tools and comparing them with previously published results. A lamprey spatial metabolomics database is also provided to support studies utilizing lampreys as an animal model, and to complement and validate other spatial metabolomics studies on lampreys conducted with mass spectrometry imaging or other techniques.

B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase

Abstract Background Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol that are excreted in urine. These two metabolites are often tested for to determine if an individual has recently consumed alcohol. EtG and EtS are very polar analytes, which make them difficult to chromatograph using reversed-phase chromatography. These analytes often elute early, which can lead to irreproducible results. When EtG and EtS are analyzed by LC/MS with a mobile phase that contains formic acid, the signal of these two analytes can be suppressed. This leads to increased LOQ and cutoffs. We examined the perfluorophenyl (PFP) stationary phase for its utility in analyzing EtG and EtS. This stationary phase has been shown to have greater retention of polar compounds than C18 reversed-phase columns. In addition, we compared the effect of formic acid to acetic acid on the separation. Methods Urine samples containing EtG and EtS were diluted 10:1, using 50mM acetic acid or 0.1% formic acid, into a 96-well microtiter filter plate containing a 0.2micron filter. Internal standards were added to the microtiter plate containing the urine samples. The samples were filtered through the filter plate and analyzed by LC/MS using the PFP column (50 x 2.7micron) with a 50mM acetic acid or 0.1% formic acid/acetonitrile mobile phase. The analytes were detected using negative mode ionization. The total analysis time was 4.5minutes. Results EtG and EtS showed improved retention using the PFP column. Retention times for EtG and EtS were 0.75 and 1.20 minutes respectively using the PFP column, compared to 0.4 and 0.3 minutes using the C18 column. The greater retention time improved the LOQ for both analytes.The LOQ for EtS and EtG were determined to be 125 and 250 ng/mL, respectively. Greater retention of EtG was observed by switching the modifier from formic acid to acetic acid. The retention time increased from 0.45 to 0.75 minutes. There was less signal suppression using acetic acid in place of formic acid. Improvement in peak shape was observed when the injection volume was reduced to 5microliters. The method was validated over a range of 250 to 10000 ng/mL for EtG and 125 to 5000 ng/mL for EtS. Acceptable linearity was obtained for both analytes. Accuracy was performed at three concentration levels. All three levels demonstrated acceptable accuracy with the difference between the measured and expected concentration being with in 20% of each other. Conclusions The PFP stationary can be used to test EtG and EtS. This stationary phase gave greater retention of EtS and EtG. The use of acetic acid improved the limit of detection by decreasing the ion suppression, which decreased the limit of detection. The assay was accurate and precise for both analytes, demonstrated in the validation.

B-168 Development and Validation of a Quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) Method for Urine Organic Acid Analysis

Abstract Background Urine organic acid (UOA) analysis is essential for the diagnosis of inborn errors of metabolism (IEMs). Traditionally, UOA analysis is performed with gas chromatography-mass spectrometry (GC-MS) and requires time-consuming sample preparation steps including liquid-liquid extraction and derivatization. The rapid development of Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in the past few years provides the opportunity to perform UOA analysis with a dilute-and-shoot methodology. We describe the development and validation of a quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) method for UOA analysis. Methods Urine specimens were diluted to normalize creatinine concentrations to 1 mmol/L. 20 µL of urine specimen (diluted), calibrator, or quality control (QC) material was mixed with 400 µL of mobile phase A (0.05% formic acid in water) and a mixture of isotope-labeled internal standards. After centrifugation, 10 µL of the supernatant was analyzed using a Xevo G3 QTOF mass spectrometer (Waters) with a ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm column (Waters). Data collection was performed with negative electrospray ionization (ESI) mode using the MSE method to produce fragment ions when applicable. Repeatability, reproducibility, and carryover were assessed using the QC materials. The analytical measuring range (AMR) was assessed using synthetic urine spiked with increasing concentrations of each organic acid. Accuracy was assessed by method comparison with the UOA test performed at Mayo Clinic Laboratory and by spike-recovery study using a pooled urine specimen. Matrix effect was also evaluated with matrix dilution study. Results An optimized LC method was used to enable high-resolution separation of selected UOAs (N = 29) and isomers. Total analytical time was 20 min per injection. Both linear and quadratic regressions were used to build the calibration curves. AMR and correlation coefficients of a few representative UOAs were: orotic acid (3.4 to 214.2 mmol/mol creatinine, R^2 = 0.99, linear regression); 2-methylcitric acid (4 to 189 mmol/mol creatinine, R^2 = 0.99, linear regression); 3-methylcrotonylglycine (0.3 to 18.0 mmol/mol creatinine, R^2 = 0.99, linear regression). Repeatability and reproducibility were mostly &amp;lt;=10% CV and no carryover was observed. Spike-recovery study demonstrated recoveries between 80% and 120%, and method comparison study demonstrated no discrepancies with results from Mayo Clinic Laboratory. Conclusions We have developed and validated a novel UPLC-QTOF method for UOA analysis to support the diagnosis of IEMs with acceptable analytical and clinical performances. Compared with the traditional GC-MS method, the UPLC-QTOF method requires a very small specimen volume and does not require laborious and time-consuming sample preparation steps. Continued optimization of the method will be pursued to measure more UOAs to support the diagnosis of more IEMs.

B-159 Fast Cleanup for 19 Steroid Analytes in Serum Using Supported Liquid Extraction (SLE)

Abstract Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool that holds promise in designing the future of steroid analysis in the clinical laboratory. It is already widely used in steroid analysis in endocrinology and is being utilized increasingly in newer applications for individual steroids and panels relevant in clinical research. LC-MS/MS is gaining user base owing to greater sensitivity and specificity and gradually even replacing some of the legacy test methods. Improved sample preparation, modern chromatography methods, and sensitive, faster scanning mass spectrometers have all played a role in improving LC-MS/MS. Steroid analysis for clinical research can require very low limits of detection which demand high recoveries and very clean extracted samples. Supported Liquid Extraction (SLE) can provide very clean extracted samples with less method development and a streamlined workflow compared to solid phase extraction (SPE). Laboratories desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Chromatographic separation of steroid isomers with the same m/z is necessary for accurate quantitation by LCMS/MS. Meeting these criteria can be challenging in a single LC-MS/MS method. Methods Each 200 µL serum sample was pretreated with 200 µL of pH 6 Ammonium Acetate buffer (aq) before being loaded onto a Novum™ PRO SLE, MAX 96-well plate, following the standard SLE protocol. Samples were eluted using 2 × 900 µL of Dichloromethane or Ethyl Acetate. After evaporation of the elution solvent, samples were reconstituted in 100 µL of 0.5 mM Ammonium Fluoride / Methanol (60:40, v/v). A Kinetex™ 2.6 μm C18, 50 x 3.0 mm column using a gradient of 0.5 mM Ammonium Fluoride (aq) and Methanol on an Agilent® 1290 Infinity UHPLC and SCIEX® 7500 Triple Quad™ were used for quantitation. Results Here, we present an effective sample extraction method and LC-MS/MS analysis method for the quantitation of a 19-analyte steroid panel from serum. The Kinetex 2.6 μm C18 column provided a fast 8-minute chromatographic separation and good selectivity for steroid analytes in both positive ion mode and negative ion mode. Calibration curves with a linear fit and 1/x weighting, showed good linearity with R2 of &amp;gt;0.99 for all analytes except 17-OH-Pregnenolone and DHEAS when using Dichloromethane as the elution solvent, and R2 of &amp;gt;0.980 for all analytes when using Ethyl Acetate as the elution the solvent. Accuracy of samples using Dichloromethane as the elution solvent were 80-112 %, except for 17-OH-Pregnenolone and DHEAS. Accuracy of all samples using the Ethyl Acetate elution solvent were 91-118 %. Conclusions The-96 well plate extraction method was robust, reproducible, and can be automated. SLE using Novum Pro MAX combined with a fast LC method provides a simple, rapid, high-throughput workflow for identification and quantitation of 19 steroids from serum.

B-350 The application of MALDI-TOF imaging to biomarker discovery in a mouse model of papillomavirus associated tumorigenesis

Abstract Background Human papillomavirus (HPV) can cause infection and dysplasia in cutaneous epithelium of immunodeficient patients. An animal model has been developed to study HPV-associated tumorigenesis using Nude mouse (Foxn1nu) and mouse papillomavirus (MmuPV1). This study applies MALDI-TOF imaging to compare the expression pattern of small molecules between viral-infected dysplastic mouse skin and mock-infected control tissue. Three sets of molecules showing distinct patterns are identified in correlation with the upregulation of an enzyme in related biosynthetic pathway. Methods Nude mice were wounded on tail and inoculated with MmPV1 suspension (1×109 viral DNA genomes per infected site) or PBS 24 hours later for treatment and control group, respectively. Tail skins were harvested 4 weeks post-infection and processed for cryosectioning. Tissue sections were analyzed using timsTOF flex (Bruker) under negative ion mode with 9-aminoacridine as the matrix. Viral infection (MmuPV1-L1 antigen) and enzyme expression (3'-phosphoadenosine 5'-phosphosulfate synthetase 1, PAPSS1) were assessed by immunofluorescence on adjacent sections. Results Three peaks demonstrated distinct distribution patterns in mouse skin. One peak at m/Z = 465 was identified as cholesterol sulfate (CS) by tandem MS and comparing with the standard. CS was enriched in keratinocytes, hair follicles, and blood vessels in normal skins, and it increased in dysplastic skins with enhanced angiogenesis. The combined peaks at m/Z = 327 &amp; 329 were tentatively identified as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These long chain polyunsaturated fatty acids (LC-PUFAs) were present in the dermis as foci partially co-localized with CS, and they also increased in virus-infected skins. The peak at m/Z = 426 may represent a combination of ADP and APS. Although these two isobars cannot be separated by tandem MS, APS was revealed by the presence of sulfur isotopes at expected natural abundance (34S and 32S). They were present at a low level in the epidermis of normal skins but significantly increased after virus infection. APS and ADP were closely associated with the proliferating cells, and the signal intensity also increased with the degree of tissue dysplasia. As a single factor, APS &amp; ADP demonstrated a fair differential power for MmuPV1-induced dysplastic skin and normal control (AUC of ROC curve = 0.75). The bifunctional enzyme PAPSS1 catalyzes the conversion of ATP to 3’-phosphonato-5’-adenyl sulfate (PAPS) via APS as an intermediate. Interestingly, immunofluorescence showed the expression of PAPSS1 also increased significantly with the severity of tissue dysplasia and MmuPV1 infection. Conclusions MALDI-TOF imaging reveals three sets of molecules with distinct patterns in mouse skin, and their levels are increased during MmuPV1-induced epithelium dysplasia. As a precursor of the common sulfate donor (PAPS), increased APS and ADP in the epidermis of virus-infected skins indicate an upregulation of sulfation pathway and adenine metabolism. This finding is supported by enhanced expression of enzyme PAPSS1 and an increased sulfation reaction product, CS. In addition, DHA and DPA are also increased in the dermis of virus-infected skins, implying a disturbance in LC-PUFA metabolism. This study demonstrates combined use of MALDI-TOF imaging and immunostaining in the search of novel biomarkers of HPV-associated tumorigenesis.

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100×3mm; 1.7μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8min including column equilibration, which enables the analysis of 160 samples per day.

B-166 Differentiation of Aziridine Functionality from Related Functional Groups in Protonated Analytes by Using Selective Ion-Molecule Reactions

Abstract Background The development of better methods for the rapid and reliable identification of unknown compounds in mixtures is urgently needed in drug development. Aziridines, in particular, are used for drug synthesis but are potentially mutagenic and carcinogenic compounds. However, the detection and identification of these compounds pose challenges for many analytical methods, such as Nuclear Magnetic Resonance (NMR) spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), and X-ray crystallography. These techniques are time-consuming and often require large quantities of highly pure compounds. Methods Therefore, this study demonstrates that selective gas-phase ion-molecule reactions of protonated analytes with neutral tris(dimethylamino)borane (TDMAB), followed by diagnostic collision-activated dissociation (CAD), can be used to differentiate aziridines from structurally related compounds in a modified linear quadrupole ion trap (LQIT) mass spectrometer. The experiments were performed using an atmospheric pressure chemical ionization (APCI) source in positive ion detection mode. TDMAB was purchased from Sigma-Aldrich and used without purification. It was introduced into the ion trap (2 mtorr of helium buffer gas) via an external reagent mixing manifold through a syringe pump at a flow rate of 10 μL h-1 and diluted with helium before entering the ion trap through a variable leak valve. All analytes were prepared at a concentration of 1 mM in methanol. The protonated analytes were transferred into the ion trap, isolated, and allowed to react with TDMAB (MS2 experiments). Results Product ions formed upon reactions of the analyte ions with TDMAB were subjected to a collision-activated dissociation (CAD) experiment (MS3 experiments). CAD on product ions produced diagnostic fragment ions with 25- and 43 m/z-units lower m/z values than the adduct - DMA product ions. Quantum chemical calculations were employed using the Gaussian 16 program calculated at the M06-2X/6-311++G(d,p) level of theory to provide theoretical foundation and to explore the underlying mechanisms of the observed gas-phase reactions. None of the tested structurally related analytes produced a similar fragmentation pattern Conclusions The innovative approach presented in this study offers a promising avenue for enhancing efficiency and accuracy in drug development. The successful differentiation of aziridine is an alternative method in overcoming the limitation of traditional techniques. The findings from the research contributes to the advancement of analytical techniques, addressing critical issues in identifying impurities in drug products. The versatility of the methods extends beyond drug developments, making it applicable in various other fields such as in molecular diagnostics, metabolism and laboratory medicines.

B-021 Impact of Assay Conditions on FT4 Measurement Accuracy of Equilibrium Dialysis LC/MS/MS Clinical Assay

Abstract Background Reliable FT4 measurements are vital for accurate diagnosis and effective management of thyroid disorders. However, currently used FT4 immunoassays (IAs) lack standardization, leading to significant variations between platforms. Equilibrium dialysis (ED) coupled with LC-MS/MS analysis, considered the gold standard for FT4 measurement, has been adopted as a reference measurement procedure (RMP) for IA standardization. This study aimed to establish a high-throughput version of the routine clinical FT4 assay based on ED-LC/MS/MS technology and assess the impact of altered assay conditions on measurement accuracy. Methods A commercially available micro-ED plate was employed for ED-LC/MS/MS analysis. FT4 extraction from the dialysate was performed by utilizing a 96-well C18 SPE plate, followed by FT4 quantitation via LC-MS/MS in positive ion mode. Certified primary reference material was used to calibrate the assay. To evaluate assay robustness, the effects of minor method modifications were studied, including incubation time of ED, serum/buffer ratios in ED inserts, ED incubation temperature, shaker speed during ED, serum sample dilution ratio, and dialysate sample dilution ratio. Additionally, short-term and long-term stability of serum FT4 were assessed. Results The developed ED-LC/MS/MS assay successfully resolved T4, T3, reverse triiodothyronine (rT3), and other interferences within 4 minutes using C18 chromatography. The assay's linear range (0.5-100 pg/mL) encompassed clinically relevant FT4 concentrations across hypo- and hyperthyroid patient samples. Excellent agreement was observed between the routine assay based on ED-LC/MS/MS and CDC RMP with a Deming regression slope near 1 (95%CI: 0.87-1.06). Furthermore, serum/buffer ratios from 1:1 to 1:2.5 in ED inserts, shaker speeds from 75 to 150 rpm, and ED incubation times from 16 to 20 hours did not significantly affect FT4 measurement. However, elevated ED temperature (38°C) led to a more than 10% increase in FT4 values. Serum FT4 remained stable when exposed to room temperature for 12 hours, five freeze-thaw cycles, and -70°C storage for 1.5 years. Additionally, dilution of sera by up to 3-fold before ED did not alter FT4 measurement results. Diluting dialysate samples at least 3 times prior to SPE extraction proved accurate for samples exceeding the calibration curve range. Conclusions In conclusion, we successfully developed a routine clinical FT4 assay based on ED-LC/MS/MS that generates data comparable to the RMP. The assay exhibits robustness, with several altered conditions not significantly impacting measurement accuracy. This high-throughput and accurate assay demonstrates suitability for both clinical laboratories and large-scale epidemiological studies. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 &amp;gt; 97.1 and 287.2 &amp;gt; 109.1 were used while respective quantifier and qualifier m/z values of 331.2 &amp;gt; 97.1 and 331.2 &amp;gt; 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150×2.1mm; 1.7μm) for the separation of lipids from 23 subclasses with a total run time of 25min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

Investigation in improving the Cs-free negative hydrogen ion production with short-pulse low power in the afterglow of pulse-power-modulated plasma sources

Abstract Pulse plasma ion sources have been shown to be capable of achieving high negative hydrogen ion densities ( n H − ) or currents. The fast cooling of the electrons and reduction in the electron density (n e) provide favorable plasma conditions to improve the H− production, with a high density ratio of negative hydrogen ions to electrons during the pulse-off period. The purpose of this research is to further improve the performance of volume-produced H− ion sources based on the pulse power modulation. In this study, a time-modulated negative hydrogen plasma sustained by 10 kHz pulse power at the typical gas pressure of 0.3 Pa was numerically investigated using a global model. The model is compared with measurements obtained in a pulsed radio frequency ion source and shows a reasonable agreement. A steplike low-high power strategy in the active glow period and a short-pulse low power in part of the afterglow period are employed to mitigate overshoot of the electron temperature (T e) in the initial stage of the pulse period and to optimize H− production in the afterglow, respectively. The model predicts that a small-magnitude, short-duration low power operation in the afterglow increases n H − and decreases n e from the onset of the low power until the beginning of the next high power pulse, due to a modest temporary increase in the T e (less than 2 eV). It facilitates dissociative electron attachments and thus H− formation. The reduction in the n e indicates that positive ions lost to the walls cannot be compensated by weak ionization. H− production with a low number of co-extracted electrons can be optimized by adjusting the magnitude and duration of the low power in the afterglow.

Natural volatile organic compounds (NVOCs) and airborne microorganisms in different stands of urban forests

Natural volatile organic compounds (NVOCs) and airborne microorganisms are important elements in urban forest air that affect air quality and human health. In this study, the Zhuyu Bay urban forest in Yangzhou was selected as the research object, and gas chromatography–mass spectrometry (GC–MS) was used to detect the composition of NVOCs in different forest stands. Terpenes, heterocyclic compounds, and esters accounted for the highest proportions. We then explored the effects of NVOCs on the physiological health of each forest stand and used Kyoto Encyclopedia of Genes and Genome enrichment analysis to identify beneficial secondary metabolites. Among the identified compounds, alpha-phellandrene 1, azulene, and other terpenoids were found to possess antibacterial, anti-inflammatory, and antioxidant properties. Heterocyclic compounds, such as 4-Pyridinecarboxylic acid and visnagin, showed significant effects in the treatment of diseases. In addition, we collected and analyzed culturable airborne microorganisms in different forest stands and found that the bamboo forest had the lowest number of culturable airborne microorganisms. To further explore the influence of urban microclimates on air microorganisms and NVOCs, a partial least squares path modelling (PLS-PM) analysis was conducted. Air negative oxygen ion is an important factor affecting NVOCs, and Air moisture has a significant positive effect on bacteria proportion.

Simultaneous metabolomics and lipidomics analysis based on 4in1 online analysis system reveal metabolic signatures in atherosclerotic mice

Developing efficient and comprehensive analysis methods for metabolomics and lipidomics in the biological tissues and body fluids is essential for understanding the disease mechanisms. Although various two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) methods have been proposed to expand metabolite coverage, achieving higher efficiency in integrated metabolomics and lipidomics studies remains a technical challenge. In this work, a novel 4in1 online analysis system with excellent reproducibility and mass accuracy was constructed for metabolomics and lipidomics study in various biological samples from atherosclerotic mice. This system enabled the simultaneous detection in both positive and negative ion modes with extensive polarity separation in a single analytical run. Using the 4in1 online analysis system, we identified distinct but complementary metabolic signatures associated with atherosclerosis in different biological samples. Specifically, a total of 230 and 170 differential metabolites or lipids were detected in mice plasma samples and aortic tissue samples, respectively, including glycerophospholipids, sphingolipids, fatty acyls, glycerolipids, carboxylic acids, and pyrimidine nucleosides. Additionally, atherosclerosis-related metabolic pathways involved in biosynthesis of unsaturated fatty acids, sphingolipid metabolism, cholesterol metabolism, glycerophospholipid metabolism, and choline metabolism further revealed. These findings demonstrate that the novel 4in1 online analysis system is a faithful, stable and powerful tool for comprehensive metabolomics and lipidomics studies in complex biological matrices.

Evaluating the detection of barbiturates in dried blood spots: A comparative analysis using gas chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, and liquid chromatography-tandem mass spectrometry with different extraction methods

Rapid and accurate characterization and quantitation of blood barbiturates and their combination drugs are very important for the clinical treatment of acute barbiturate poisoning. A comparison of dried blood spot (DBS) and traditional liquid-liquid extraction (LLE) in the pre-treatment stage, as well as a comparison of gas chromatography-mass spectrometry (GC–MS), gas chromatography-tandem mass spectrometry (GC–MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) as instrumental analysis methods, revealed differences in the analysis results of barbiturates and their combination drugs under different conditions. Based on these findings, we introduce a DBS-GC–MS/MS method. The developed and validated method showed good selectivity, sensitivity (LOD: 0.1 μg mL−1, LOQ: 0.2 μg mL−1), linearity (R2>0.9992), trueness (<15 %, except for carbamazepine, at 29.4 %), and precision (<15 %). Recovery was also good for most target compounds, but significant matrix effects were evident. Compared with the LLE method, the DBS method has the benefits of easy sample collection, storage, and transport, as well as simple pre-treatment and reduced reagent and energy consumption. Compared to LC-MS/MS, GC–MS/MS requires no switching between positive and negative ion modes and uses the MRM detection mode, meaning that more information about the sample compounds can be obtained in less analysis time. Using actual sample analysis, we have demonstrated the advantages of the DBS-GC–MS/MS method for the qualitative and quantitative analysis of barbiturates and poisoning events due to combinations of these drugs. Comparison of the three instruments and the two treatment methods revealed their analysis characteristics. From the perspective of practical application, the broad practical value and advantages of DBS should be embraced in more applications, and future analytical laboratory development should continue to recognize GC–MS/MS as a useful supplement to LC-MS/MS.

Inferring ion cluster lifetimes from energy-resolved mass spectra of an EMI-BF4 electrospray plume

The fragmentation of ion clusters within the accelerating fields of ionic liquid ion sources (ILISs) is well documented and degrades ILIS performance and lifetime. Some of the most popular ILIS liquids, such as EMI-BF4 (1-Ethyl-3-methylimidazolium tetrafluoroborate) and EMI-Im (1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide), emit clusters with lifetimes as low as ∼1 ns. Studies of fragmentation within the accelerating field typically rely on measuring the plume energy distribution averaged over all plume species and comparing those measurements with numerical simulations to estimate ion cluster lifetimes. Here, for the first time, we estimate EMI-BF4 cluster lifetimes by analyzing the energy distributions of individual plume species. We use this novel analysis method to estimate mean lifetimes of positive EMI-BF4 ion clusters from previously published experimental data. We find that the mean lifetime ranges from τ=3.7ns to τ=124ns for [EMI+][EMI-BF4] dimers and ranges from τ=1.5ns to τ=23ns for [EMI+][EMI-BF4]2 trimers. Fitting those data to an analytical fragmentation model, we estimate the binding energy and temperature as ΔGS0=0.49eV and T=394K for dimers and ΔGS0=0.40eV and T=365K for trimers. Comparing our results with previous studies supports the conclusion that clusters are emitted with a wide distribution of internal energies, contrary to the common assumption of single internal energy for each species.