bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase
Authors: Simms P 
Published: October 2024

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-169 Combining the EXENT System and LC-MS for the Detection of Monoclonal Immunoglobulins at Low Levels
Authors: Barnidge D R , Troske D , North S , Wallis G , Perkins M , Harding S 
Published: October 2024

Abstract

Background
The EXENT® system is an automated platform designed to quantify monoclonal immunoglobulins in serum. EXENT® combines immunoprecipitation and MALDI-TOF mass spectrometry for the detection of monoclonal immunoglobulins at levels lower than traditional gel based methods. This study was designed to demonstrate if EXENT® prepared samples that were negative could be reflexed to a more sensitive method based on capillary flow Liquid Chromatography- Mass Spectrometry (LC-MS) without any modifications to the EXENT® prepared sample.

Methods
Patient samples containing a monoclonal immunoglobulin (ranging from 20 to 40 g/L) were diluted into pooled polyclonal serum and the samples were prepared and analyzed by EXENT®. Samples that were negative by EXENT® were reflexed by directly loading EXENT® eluates onto the LC-MS instrument. The abundance of the monoclonal immunoglobulin was defined by the signal-to-noise (S/N) of the light chain peak observed after deconvolution.

Results
Baseline samples were diluted down to two levels, 0.100 g/L and 0.001 g/L. LC-MS detected the same light chain from the monoclonal immunoglobulin as the EXENT® system in all reflexed samples that were negative by EXENT®.

Conclusions
LC-MS can detect monoclonal immunoglobulins that can no longer be detected using the EXENT® system by tracking the unique molecular mass of the monoclonal light chain. Reflexing samples to LC-MS did not require additional sample handling resulting in a faster time-to-result than current NGS, NGF, and clonotypic peptide approaches. Furthermore, monitoring the intact monoclonal light chain circumvents enzymatic cleavage to create a unique clonotypic peptide, alleviating a situation where no unique peptide can be generated as has been shown previously by Bergen et al. As a consequence, this reflex-methodology results in a consistent way of measuring the presence of malignant B-cells with high sensitivity.
B-175 Development of a Rapid and Sensitive LC-MS/MS Assay for Dolutegravir Quantitation in Breastmilk
Authors: Rackow A R , Pandey A , Marzinke M A 
Published: October 2024

Abstract

Background
Approximately 1.5 million HIV positive individuals experience pregnancy annually. The goal of HIV treatment in pregnancy is to achieve viral suppression prior to delivery, minimizing the risk of vertical transmission. Dolutegravir (DTG) is an integrase strand inhibitor that prevents insert of HIV DNA into host cells, thus decreasing viral load. In a clinical trial conducted in conjunction with 60 pregnant women initiating anti-retroviral therapy (ART) in the third trimester, dolutegravir (DTG) achieved viral suppression approximately two months faster than the standard of care at the time (28d vs. 84d). Secondary endpoints from this study demonstrate transplacental and breastmilk transfer of dolutegravir to the infant and delayed clearance. The pharmacokinetics of dolutegravir in infancy remain uncharacterized, with important implications for the development of HIV and resistance if there is intermittent medication compliance and/or barriers to care. The development of a robust DTG breastmilk assay will allow future pharmacokinetic studies to quantify pharmacologic exposure and relate this to infant health, namely HIV transmission and development of resistance. The objective of this study was to develop and validate a rapid LC-MS/MS assay in breastmilk with a dynamic measuring range, suitable for application in pharmacology studies.

Methods
Breastmilk was purchased from commercial vendors and spiked with dolutegravir and an isotopically labeled internal standard. Samples were prepared by protein precipitation and injected into an LC-MS/MS system consisting of a Shimadzu LC-40, a C8 column, and a Sciex QTRAP 5500. Peaks were identified by monitoring two transitions for DTG and the presence of the internal standard. All data analysis was performed in Analyst and Excel.

Results
The DTG breastmilk assay has an analytical measuring range of 0.5-1,000 ng/mL with a final method time of 2.2 minutes. Breastmilk was prepared by protein precipitation with a required sample volume of 30 µL and an injection volume of 3 µL. There was no evidence of ion enhancement or suppression, nor was there any evidence of carryover or crosstalk. DTG was reliably detected across the analytical measuring range and among different lots of breastmilk.

Conclusions
This assay meets performance criteria put forth by the European Medical Association and the Food and Drug Administration. Upon completion of all validation experiments, this assay may be applied to assess the pharmacokinetics of DTG in mother-infant pairs.
B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis
Authors: Nakagawa T , Homma K , Shimojo S , Yokota H , Wakui M , Matsushita H 
Published: October 2024

Abstract

Background
Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities.

Methods
The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (&gt;0.06ng/g Creatinine).

Results
Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney.

Conclusions
The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.
B-180 A Component Equation Approach to Resolving Calibration Curve Nonlinearity When Using Stable Isotope-Labeled Internal Standards for Quantifying Amino Acids With Mass Spectrometry
Authors: Kim Y , Miller A , Vicente F , Haymond S 
Published: October 2024

Abstract

Background
Stable isotope-labeled internal standards (SIL-IS) have been widely used with mass spectrometry to accurately quantify analytes. However, over an extended dynamic range, calibration curves calculated with the relative ratio of analytes to their corresponding SIL-IS are typically nonlinear. This makes accurate estimation of sample concentration difficult, especially at lower concentrations. Highly multiplexed clinical mass spectrometry assays routinely face this challenge. The component equation assumes interaction between an analyte and its SIL-IS causes the nonlinearity. Modeling this resulted in a linear calibration curve and high accuracy across the dynamic range. We compared the component equation with other calibration methods to quantify amino acids.

Methods
A total of 47 compounds were serially diluted and added with extraction solution containing a SIL-IS mixture to prepare 7 levels of calibrators. Calibrators were chromatographically separated using a Sciex ExionLC system with an Imtakt Intrada Amino Acid column and injected into a Sciex Citrine triple-quadrupole mass spectrometer. Seven different fitting methods were compared for calibration of each amino acid: non-weighted linear, quadratic and cubic models, linear models with 1/X and 1/X2 weighting, Padé[1,1] approximation, and component equation.

Results
Results for an example amino acid, tyrosine, are shown in the table below. Most models yield high accuracy at high concentrations, however, all models recorded significantly higher bias for the lowest calibrator than the component equation (≥55.7% vs 11.8%). The component equation outperformed all other models for mean bias (≥16.6% vs 5.9%). Similar results were obtained for the complete set of analytes.

Conclusions
When compared to other popular calibration methods to quantify a panel of amino acids, the component equation generated a linear curve and the greatest accuracy across all concentrations. The application of this simple equation could overcome the limitations of existing weighting and non-linear fitting approaches when using SIL-IS for quantifying analytes with mass spectrometry.
B-185 A Comparison Between Two Oral Sample Collection Devices on Preserving High-Quality Genomic DNA
Authors: Wood A , Lam V , Wang Y 
Published: October 2024

Abstract

Background
High-quality genomic testing significantly enhances the knowledge on gene-disease association, and greatly benefits future biomedical research and precision medicine. However, widely used saliva sample collection presents challenges with high-quality and quantity of human DNA and high bacterial DNA ratio. In this study, we validated the performance of two commercially available oral collection devices for their ability to preserve high-quality genomic DNA long-term at room temperature.

Methods
Fifty-one buccal samples in total from twelve donors were collected using two kinds of commercially available oral sample collection devices (iSWAB®-DNA (Device 1) and OCD-100 (Device 2)). All of the collected samples were stored at ambient temperature for up to 1 year. Collected samples were extracted with QIAamp Blood Mini Kit (Qiagen) at baseline, 30 days, 60 days, and 1 year. All DNA yields were measured using a Nanodrop One Microvolume UV-Vis Spectrophotometer; TapeStation System was used for determining the integrity of DNA. The bacterial DNA ratio was estimated by quantitative PCR with primers targeting the 16S rRNA gene known to be present in prokaryotes but not eukaryotes.

Results
Device 1 had higher yield (Device 1: 18.59 ± 8.98; Device 2: 6.44 ± 4.62), lower bacterial DNA ratio (Device 1: 2.06 ± 1.93; Device 2: 3.79 ± 4.58), and higher overall DNA integrity (Device 1: 6.1 ± 0.31; Device 2: 5.42 ± 0.96) compared to Device 2. Device 1 demonstrated longer post-collection sample stability at ambient temperature compared with Device 2 regarding DNA yield, bacterial DNA ratio and DNA integrity. No significant changes in DNA yield, integrity, and bacterial DNA ratio were observed after one year of storage in Device 1. No significant changes in DNA yield, integrity, and bacterial DNA ratio were observed after 60 days of storage in Device 2. However, Device 2 showed a significant increase in DNA yield, bacterial DNA ratio, and DNA integrity comparing baseline to 1 year.

Conclusions
Device 1 preserves higher quantity and quality of DNA from oral samples at ambient temperature for up to 1 year. These advantages significantly enhance efficiency and cost-effectiveness in downstream applications such as genotyping and sequencing, making it a valuable tool in genomics research with a commendable success rate.
B-191 Genomic Profile of Circulating Tumor DNA in Advanced Lung Cancer: Comparison with Tissue DNA
Authors: Park J , Choi Y , Jeon S , Kee S , Na E , Shin M 
Published: October 2024

Abstract

Background
The most common cancer in Korea is thyroid cancer, followed by lung cancer, and the most common cancer in men is lung cancer. The cancer with the highest mortality rate is lung cancer. Recently, the precision medical approach is becoming a major treatment strategy for lung cancer. For precision medical approach, the usefulness of next-generation sequencing (NGS) analysis targeting circulating tumor DNA (ctDNA) is being emphasized. Therefore, in this study, NGS analysis was performed on blood and tissues of advanced lung cancer patients to compare and analyze genetic mutation patterns to confirm the usefulness of ctDNA NGS assay.

Methods
A total of 101 patients with advanced lung cancer (stage 3 or 4) were recruited, and ctDNA NGS assays were performed on their plasma using a Pan100 panel (Dxome). Tissues from primary lung cancer, metastatic lymph nodes, or metastatic organs of 22 patients were analyzed using ONCOaccuPanel (NGeneBio). All variants were classified into four tiers based on the level of clinical significance, according to the standards and guidelines established by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists.

Results
In the ctDNA NGS assay of 101 patients, 70 (69.3%) harbored 154 tier 1 or 2 variants. Mutations in TP53 (45, 29.2%) and EGFR (23, 14.9%) were the most frequently detected, followed by mutations in RB1, CHEK2, ATM, JAK2, ATR, DNMT3A, and KRAS. Among the 22 patients whose tissue was examined, tier 1 or 2 variants were detected in both the plasma and tissue of 14 patients and only in the tissue of six patients. All genes for these variants were targeted using both methods. Identical variants were detected in the plasma and tissue of six of the 14 patients, and discordant results were observed in the remaining eight patients. Twenty copy number variations were detected in 13 of the 101 patients, with copy number gain of EGFR being the most common (6, 30%). Fusion genes were detected in five of the 101 patients, two of which were not detected in the tissue. Overall, 74 (73.3%) of the 101 patients had positive ctDNA NGS assay results.

Conclusions
Although the NGS assay with ctDNA is less sensitive than that with tissue DNA, some genetic alterations are detected only in ctDNA NGS assays; therefore, it is useful to apply the ctDNA NGS assay as a complement.
B-195 Accelerating Molecular Diagnostics: Development of a novel Rapid, Integrated PCR System for Acute Respiratory Infections Detection
Authors: Ma T , Dong L , Chen H , Lv Z , Chen J , Yang Y 
Published: October 2024

Abstract

Background
The COVID-19 pandemic and acute respiratory infections have underscored the necessity for high-throughput and timely molecular diagnostics. Existing molecular diagnostic methods are labor-intensive, a challenge particularly evident in large-scale testing. This study aims to streamline the nucleic acid testing process by integrating extraction and detection in PCR testing, thereby optimizing efficiency.

Methods
We developed 'EXP-160R', an integrated PCR system capable of rapid thermal cycling. This innovation employs advanced temperature control technology to markedly reduce heating, cooling, and annealing-extension times. The PCR reagent-kit (Zybio Inc.) was optimized to complement the instrument's capabilities, aiming for pathogen nucleic acid detection in acute respiratory infections within 25 minutes. The precision of in-tube temperature during rapid amplification was validated by using temperature-sensitive molecular probes in system development stage. Performance evaluation used the WHO International Standard for SARS-CoV-2 RNA to assess sensitivity and detection limits. We also analyzed 500 clinical samples from multiple Chinese hospitals, including 295 COVID-19 positives and 205 negatives, comparing the EXP-160R system's performance with an NMPA-approved PCR kit.

Results
The innovative system significantly improved PCR cycle times, enabling detection of 16 samples in 22 minutes and a throughput of 2400 tests per day, without compromising sensitivity or specificity. Validation confirmed a detection limit of 200 copies/mL, and the system achieved a 100% detection rate for medium and high viral load samples, with a coefficient of variation (CV) for Ct values ≤ 5%. Clinical trial comparisons with clinical standard diagnostics revealed a sensitivity of 98.05%, specificity of 100%, accuracy of 99.2%, and a Kappa coefficient of 0.984. When compared to the established PCR kit, our system showed comparable efficacy, with a sensitivity of 98.50%, specificity of 98.67%, accuracy of 98.6%, and a Kappa of 0.971.

Conclusions
The rapid PCR system represents a significant advancement in molecular diagnostics, offering rapid, high-throughput, and accurate detection of SARS-CoV-2. This system is particularly beneficial in urgent testing scenarios, such as airports and fever clinics. It addresses current pandemic needs and sets a new benchmark for future respiratory and infectious disease diagnostics, enhancing both efficiency and user experience.
B-199 Rapid Molecular Detection of Meningitis Agents in Diverse Brazilian Regions (2019-2023): A Multicenter Study
Authors: de Araujo C M , Kappel H B , Barra G B , Jacomo R , Abdalla L F 
Published: October 2024

Abstract

Background
Meningitis, caused by viral and bacterial agents, is a significant public health issue globally, with a substantial economic impact. In Brazil, the incidence of meningitis varies by region, making the identification of the causative agent crucial for effective treatment. This study aims to evaluate the epidemiology of meningitis agents diagnosed using a rapid molecular detection panel in samples from various hospital units of a nationwide diagnostic and healthcare company.

Methods
Samples from several hospitals across different regions of Brazil were analyzed using the FilmArray® rapid molecular panel for meningitis and encephalitis, capable of identifying up to 14 agents, including bacteria, viruses, and fungi/yeasts. Gender, age group, regional distribution, and agent prevalence from January 2019 to April 2023, were retrieved from the database ensuring compliance with the Brazilian Data Privacy and Protection (LGPD) and local Research Ethics Committee guidelines.

Results
The study included 485 patients from 41 cities across 11 Brazilian states. The age range was 0 to 96 years, with an average age of 45 years, and a majority of female participants (59%). The highest number of test requests occurred in 2022 (17.5%), followed by 2021 (10%), with 2019, 2020, and 2023 accounting for less than 5% of requests each. The overall positivity rate was 10.5% (51/485), with 92.8% (13/14) of the possible agents identified. Viral agents were detected in 60.7% of positive cases, bacterial agents in 39.2%, and one mixed infection (human herpesvirus 6 and Escherichia coli K1) in a 2-year-old child. No fungal/yeast infections were detected. The most common agents were varicella-zoster virus (18.2%), Haemophilus influenzae (12.7%), herpes simplex viruses type 1 and 2 (12.7% each), and enterovirus (12.7%).

Conclusions
The study highlights the importance of rapid molecular diagnostic methods in Brazilian clinical-laboratory practice, offering fast, safe, and sensitive detection of meningitis-causing agents, which can contribute to improved patient outcomes.