Negative Ion Research Articles

Nutrient content and ultra-performance liquid chromatography tandem mass spectrometry metabolomic comparative analysis of Lithocarpus corneus and Lithocarpus pachylepis.

China possesses abundant species resources in genus Lithocarpus Blume, commonly known as "acorns." These nuts are traditional foods in China, holding significant potential for local specialty food and rural economic development. To explore their edible value, we compared the nutritional components and metabolites between the fruits of Lithocarpus corneus (YD) and Lithocarpus pachylepis (HL), which are cultivated widely in south of China. Non-targeted metabolomics analysis of YD and HL was performed using ultra-performance liquid chromatography tandem mass spectrometry technology. A total of eight classes of differential metabolites (DAMs) were detected: alkaloids, amino acids and peptides, carbohydrates, fatty acids, polyketides, shikimates and phenylpropanoids, terpenoids, and other metabolites. In the positive ion mode, 225 DAMs were identified, whereas in the negative ion mode, 116 DAMs were identified. KEGG pathway enrichment analysis showed that the differentially abundant metabolites were generally enriched in three pathways: aminoacyl-tRNA biosynthesis, alanine, aspartate, and glutamate metabolism, and ABC transporters. The nutritional content of YD has higher moisture, fructose, and total flavonoid levels, but lower starch, protein, fat, glucose, sucrose, and total sugar content than HL. Additionally, YD contains more Ca, K, P, and Fe, but less Mg and Zn compared to HL. Analyses indicate that YD is nutrient-rich and beneficial, making it suitable for further development. Two species of Lithocarpus contain nandrolone, suggesting a new direction for specialty food development. The unique chemical composition and rich nutritional content of Lithocarpus fruits provide a theoretical basis for their development and application as food resources. PRACTICAL APPLICATION: L. corneus and L. pachylepis are traditional Chinese nuts, rich in starch, minerals, and flavonoids, making them suitable for consumption or use as food constituents. Their fruits contain various unique natural chemical compounds, indicating high application value and development potential.

Nonlinear Ion Acoustic Waves in Multicomponent Plasmas with Nonthermal Electrons-positron and Bipolar Ions

Abstract This paper studied the propagating characteristics of (2+1) dimensional nonlinear ion acoustic waves in a multicomponent plasma with nonthermal electrons, positrons and bipolar ions. The dispersion relations are initially explored by using the small amplitude wave's dispersion relation. Then, the Sagdeev potential method is employed to study large amplitude ion acoustic waves. The analysis involves examining the system's phase diagram, Sagdeev potential function, and solitary wave solutions through numerical solution of an analytical process in order to investigate the propagation properties of nonlinear ion acoustic waves under various parameters. It is found that the propagation of nonlinear ion acoustic waves is subject to the influence of various physical parameters, including the ratio of number densities between the unperturbed positrons, electrons to positive ions, nonthermal parameters, the masses ratio of positive ions to negative ions, and the charge numbers ratio of negative ions to positive ions, the ratio of the electrons’ temperature to positrons’ temperature. In addition, the multicomponent plasma system has a compressive solitary waves with amplitude greater than zero or a rarefactive solitary waves with amplitude less than zero, in the meantime, compressive and rarefactive ion acoustic wave characteristics depend on the charge numbers ratio of negative ions to positive ions.

Pulsational mode dynamics in the presence of negions

The stability dynamics, of the gravito-electrostatically driven pulsational mode of gravitational collapse (PMGC) excitable in spherically symmetric dust molecular clouds (DMCs), is systematically theorized. It actively incorporates the interplay of dust viscosity, dust charge fluctuation dynamics, and negionic (negative ionic) effects. We take the DMC constitutive electrons, positive ions, and negions as the Boltzmann species (lighter). The negatively charged dust grains behave as viscous fluids (heavier). A generalized linear cubic dispersion equation, involving a new and unique set of multi-factorial dispersion coefficients, results from the applied spherical normal mode treatment in the DMC model. The diverse growth characteristics of the PMGC dynamics are investigated illustratively. It is found mainly that the cloud size, dust charge, equilibrium electronic number density, and equilibrium dust number density play stabilizing roles in the PMGC. However, the dust mass, equilibrium positive ionic number density, equilibrium negionic number density, and dust viscosity play destabilizing roles against self-gravity. The reliability of our study is ensured by the fact that the diversified stabilizing features explored here are consistent with the previous results. The applicability of our study in astrophysical gravity-driven structure formation processes moderated actively with the negionic effects is finally outlined.

Metabolomic profiling of human semen in patients with oligospermia using high performance liquid chromatography-tandem mass spectrometry

Male infertility is one of the most common reproductive dysfunctions. Despite oligospermia being a cause of infertility, few studies have been conducted on it. This study aimed to investigate differences in semen metabolic patterns in patients with oligospermia and to identify potential biomarkers associated with oligospermia. Semen samples from oligospermia patients (20 cases) and healthy controls (20 cases) were detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and 72 and 89 metabolites were identified as potential markers in positive and negative ion modes, respectively. In addition, the results identified multiple metabolic pathways in patients with oligospermia, such as glycine serine and threonine metabolism, Synthesis and degradation of ketone bodies, Valine, leucine, and isoleucine degradation. These results described unique metabolic characteristics of semen in patients with oligospermia and provided novel insights into the mechanism of the semen disorder.

Development of a rapid and robust hydrop interaction liquid chromatography tandem mass spectrometry method for the detection of 13 endogenous amino acids as well as trimethylamine oxide in serum and tissues of the mice.

This work aimed to establish an HILIC-MS/MS method to simultaneously determine the levels of 13 endogenous amino acids and trimethylamine oxide in the biological samples from the mice. Electrospray ion source was used for the analysis of mass spectrometry. The 20 min separation was applied in a Dikma Inspire Hilic column (2.1 × 100.0mm, 3μM). Positive ion mode under an MRM model gave a satisfying response value. The limits of quantitation were evaluated by accuracy from -12.59% to 7.89% and precision from 1.77% to 14.00% as well as acceptable interday and intraday precision, matrix effect, recovery, and stability. Later, the assay was successfully used to measure the concentrations of the determinands in the biological samples. Individual and tissue distribution differences for these metabolites were observable. The amino acids had a consistent highest content in the spleens, while the lowest levels were found in the livers. Alanine was the most abundant amino acid in the serum, and taurine kept the highest content in all of the tissues. Trimethylamine oxide remained low level, especially in the liver samples.

Collision cross sections of large positive fullerene molecular ions and their use as ion mobility calibrants in trapped ion mobility mass spectrometry.

Positive-ion laser desorption/ionization (LDI) of fullerenes contained in soot as produced by the Krätschmer-Huffman process delivers a wide range of fullerene molecular ions from C56+• to above C300+•. Here, the collision cross section (CCS) values of those fullerene molecular ions are determined using a trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) instrument. While CCS values in the range from C60+• to C96+• are already known with high accuracy, those of ions from C98+• onward had yet to be determined. The fullerene molecular ions covered in this work have CCS values from about 200 to 440Å2. The fullerene molecular ion series is evenly spaced at C2 differences in composition, and thus, small CCS differences of just 2.2-3.5Å2 were determined across the entire range. Fullerene M+• ions may be employed as mobility calibrants, in particular, when very narrow 1/K0 ranges are being analyzed to achieve high TIMS resolving power. In addition, due to the simple elemental composition, M+• ions of fullerenes could also serve for mass calibration. This study describes the determination of CCS values of fullerene molecular ions from C56+• to C240+• and the application of ions from C56+• to C220+• to calibrate the ion mobility scale of a Bruker timsTOFflex instrument in any combination of LDI, matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) modes in the CCS range from about 200 to 420Å2. This use was exemplified along with ions from Agilent Tune Mix, leucine-enkephalin, angiotensin I, angiotensin II, and substance P.

Determination of the extremolyte ectoine in plasma and a pharmacokinetic study in rats by a validated and BAGI-evaluated UPLC-MS/MS method

Ectoine (ECT) has recently gained considerable interest in the healthcare sector due to its promising therapeutic benefits in a variety of human disorders. This research aimed to quantify the ECT plasma level in rats by creating and optimizing a sensitive and validated UPLC-MS/MS method. Prior to analysis, ECT extraction from the plasma samples was conducted via a protein precipitation procedure, using hydroxyectoine as an internal standard (IS). A 1.7 μm UPLC C8 column (100 mm × 2.1 mm) was selected for the chromatographic separation, using a gradient mobile phase consisting of acetonitrile and 0.05% formic acid. The electrospray ionization mass spectrometry (ESI-MS) was used to detect ECT in the positive ion mode. To determine the specific precursor and the product ions of ECT, multiple reaction monitoring (MRM) methods were carried out. The selected ion pair of ECT was 143.1 > 97 and 159.1 > 113.13 for the IS. The ECT’s linearity range in rat plasma was found to be 1-1000 ng/mL, with a recovery rate of 96.48–97.37%. Consistent with FDA guidelines for bio-analytical method validation, the suggested method was validated. The method was efficiently employed to quantify the studied drug in spiked rat plasma with good accuracy and precision with no significant matrix effects. Furthermore, it was effectively used to investigate the pharmacokinetic behavior of ECT in rats after a single oral dose of 30 mg/kg.

Collating the dependence of temperature on the bioactivity of various silicate compounds in orthopedic applications

Various ceramics have been used over the years for the treatment of bone-related issues and finding a suitable ceramic for bone repair is still a question since it is an arduous task to find a synthetic alternative for natural bone that can meet all the required criteria. Silica and its various composites have been used over the years for bone repair and regeneration due to its excellent biocompatibility and bioactivity. This paper briefly analyses the temperature dependence on the bioactivity and other characteristics of various silicate compounds such as calcium silicate and zinc silicate considering various properties. XRD and FTIR analysis of the samples proved the incorporation of Ca2+ and Zn2+ into the silica structure. The composites prepared with 20 % Ca and Zn provided better bioactivity than pure silica even on the third day and the ICP-OES analysis proved that dissociation of ions played an important role in the biological properties of the compounds. Among all the samples, CS600 and CS900 had a thicker layer of apatite formation within three days of SBF treatment. Antibacterial analysis of the samples showed better activity against E. coli than S. aureus due to the ability of cell wall rupture of E. coli by the positive ions present in the samples. Temperature dependence on the cell viability of the samples were observed and samples sintered at 900 °C had an elevated rate of cell viability than those sintered at 600 °C. Increasing the sintering temperature of the sample provided stabilization of the product and desired biological properties.

6651 A Traceable Clinical Free Thyroxine Assay Based on Equilibrium Dialysis ID-LC/MS/MS

Abstract Disclosure: D. Spector: None. L. Zhang: None. A. Ribera: None. A. Doty: None. C. Tse: None. O. Sugahara: None. N. Vazquez: None. U. Danilenko: None. H.W. Vesper: None. Accurate diagnosis and effective treatment of thyroid diseases rely on reliable thyroid function tests. Serum free thyroxine (FT4) immunoassays (IAs) are most commonly used for FT4 measurements in clinical practice. However, FT4 IAs have not been standardized with large variations between different assay platforms. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for CDC standardization program to support the standardization of clinical assays for patient care. In the current study, we aim to develop a routine clinical FT4 assay based on ED-LC/MS/MS that is traceable to the RMP. ED-LC/MS/MS analysis was performed using a commercially available micro-ED plate. FT4 in dialysate was extracted with a 96-well C18 SPE plate. FT4 in the samples was quantitated by using LC/MS/MS in positive ion mode. The assay was calibrated with the certified primary reference material. By using RMP as a reference, we compared the analysis of 40 single donor sera between the ED-LC/MS/MS assay and FT4 IA on Roche e401platform. ED-LC/MS/MS assay could resolve T4, T3, reverse triiodothyronine (rT3), and other interferences in the samples with C18 chromatography in 4 min. The linear range of the routine FT4 assay was from 0.5-100 pg/mL covering clinically relevant FT4 concentrations including hypo-, hyperthyroid patient samples. The intra and inter-run CV% of the assay in 20 days was 3.72 and 5.25%, respectively. The effect of minor changes to the method conditions was studied. It was determined that serum/buffer ratios from 1:1 to 1:2 in the ED inserts, speeds of shaker for ED, and ED incubation time, did not influence the FT4 measurement. However, elevated ED temperature at 38°C could result in a more than 10% increase in FT4 values. A microplate format for the procedures of ED and SPE is amiable to the application of an automation system which significantly improves the throughput of the procedure. Method comparison showed good agreement between ED-LC/MS/MS assay and RMP in Deming regression analysis with a slope close to 1, while there was significant bias of IA from RMP. ED-LC/MS/MS assay was also applied for pregnancy serum samples with minimal difference on the FT4 measurement from RMP. We have developed a routine clinical FT4 assay that is traceable to RMP. The accuracy, precision, and sensitivity of the assay are suitable for high-throughput FT4 measurements in clinical laboratories and large epidemiologic studies. We demonstrate that the developed method can provide accurate FT4 measurements in patients including pregnant women. Presentation: 6/3/2024

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.

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 <=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 >0.99 for all analytes except 17-OH-Pregnenolone and DHEAS when using Dichloromethane as the elution solvent, and R2 of >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 & 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 & 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.