Differentiation of Alkyl- and Plasmenyl-phosphatidylcholine by Endogenous Sphingomyelin RT-XLOGP3 Regression for Coronary Artery Disease Plasma Lipidomics Analysis.

Accurate identification between alkyl- and plasmenyl-phosphatidylcholine (PC(O-) and PC(P-)) isomers is a major analytical challenge in lipidomics studies due to a lack of structure-specific ions in conventional tandem mass spectrometry (MS/MS) methods and the absence of universal retention time (RT) references. Given the importance of PC(O-) and PC(P-), an easy-to-apply method for current research is urgently needed. In this study, we present a quadratic RT-XLOGP3SM regression model that uses endogenous sphingomyelin (SM) species in blood samples as retention time (RT) indicators to predict the RTs of PC(O-) and PC(P-) species by coupling their calculated partition coefficients based on XLOGP3. The prediction results were obtained with a root-mean-square error (RMSE) of 0.12 min (1.3%) for the RRHD (rapid resolution high definition) nonlinear LC condition. A lipidomic analysis with RT-XLOGP3SM regression was used to study lipid regulation in coronary artery disease (CAD) outpatient plasma samples, and we found that the types of exhibited regulation were highly dependent on the lipid subclasses in comparison to the healthy control group. In conclusion, given that the quadratic RT-XLOGP3SM regression model predicts the RTs of PC species based on the relative value of XLOGP3 and the RTs of endogenous SM species, it can be expected that most of the C18-based lipidomics analyses could apply this method to increase the identification ability of the PC(O-) and PC(P-) subclasses and to improve the understanding of their physiological functions.

Development of an Efficient and Sensitive Chemical Derivatization-Based LC-MS/MS Method for Quantifying Gut Microbiota-Derived Metabolites in Human Plasma and Its Application in Studying Cardiovascular Disease.

Recently, the gut microbiota has been found to be associated with many diseases, such as inflammatory bowel disease, depression, Parkinson's disease, cancer, metabolic syndrome, and cardiovascular disease (CVD). Among various gut microbiota-derived metabolites (GMs), short-chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) metabolites are the most frequently discussed metabolites. LC-MS/MS shows advantages in quantifying the levels of metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time-consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with CVD. The results showed a significant reduction in plasma butyrate levels in CVD patients compared with healthy controls, suggesting its potentially protective role in CVD. In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.

Using post-column infused internal standard assisted quantitative metabolomics for establishing prediction models for breast cancer detection.

RATIONALE: Breast cancer is one of the most common cancers among women and its associated mortality is on the rise. Metabolomics is a potential strategy for breast cancer detection. The post-column infused internal standard (PCI-IS)-assisted liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been demonstrated as an effective strategy for quantitative metabolomics. In this study, we evaluated the performance of targeted metabolomics with the PCI-IS quantification method to identify women with breast cancer. METHODS: We used metabolite profiling to identify 17 dysregulated metabolites in breast cancer patients. Two LC/MS/MS methods in combination with the PCI-IS strategy were developed to quantify these metabolites in plasma samples. Detection models were built through the analysis of plasma samples from 176 subjects consisting of healthy volunteers and breast cancer patients. RESULTS: Three isotope standards were selected as the PCI-ISs for the metabolites. The accuracy was within 82.8-114.16%, except for citric acid and lactic acid at high concentration levels. The repeatability and intermediate precision were all lower than 15% relative standard deviation. We have identified several metabolites that indicate the presence of breast cancer. The area under the receiver operating characteristics (AUROC) curve, sensitivity and specificity of the linear combinations of metabolite concentrations and age with the highest AUROC were 0.940 (0.889-0.992), 88.4% and 94.2% for pre-menopausal woman, respectively, and 0.828 (0.734-0.922), 73.5% and 85.1% for post-menopausal women, respectively. CONCLUSIONS: The targeted metabolomics with PCI-IS quantification method successfully established prediction models for breast cancer detection. Further study is essential to validate these proposed markers.

Vortex-assisted liquid-liquid microextraction of strontium from water samples using 4',4″(5″)-di-(tert-butylcyclohexano)-18-crown-6 and tetraphenylborate.

A vortex-assisted liquid-liquid microextraction method was developed for the chromatographic determination of strontium in aqueous samples. In the method, strontium was complexed with 4',4″(5″)-di-(tert-butylcyclohexano)-18-crown-6 in the presence of tetraphenylborate as the counter anion, which increased the hydrophobicity of the ion-association complex, resulting in its improved extraction into 1-octanol. Strontium from the organic phase was stripped with nitric acid back to aqueous solution and determined by ion chromatography. The optimum microextraction conditions were as follows: 2.0 mL aqueous samples with 3 mM tetraphenylborate; 150 µL of 1-octanol as the extractant phase with 10 mM DtBuCH18C6; vortex extraction time for 10 s; centrifugation at 6000 rpm for 4 min; stripping by 0.1 M nitric acid. Under the optimum conditions, the detection limit for strontium was 0.005 mg/L. The calibration curves showed good linearity over the range between 0.01 and 2.5 mg/L. Intra- and interday precisions of the present method were satisfactory with relative standard deviations of 1.7 and 2.1%, respectively.

Molecular Detection of Candida auris Using DiaSorin Molecular Simplexa® Detection Kit: A Diagnostic Performance Evaluation.

Candida auris is a globally emerging fungal pathogen that is associated with healthcare-related infections. The accurate and rapid detection of C. auris is crucial for effective infection prevention, control, and patient management. This study aimed to validate the analytical and diagnostic performance of the DiaSorin Molecular C. auris Detection Kit. The analytical specificity, sensitivity, and reproducibility of the assay were evaluated. The limit of detection (LOD) was determined to be 266 CFU/µL using the ZeptoMetrix Candida auris Z485 strain and standard calibration curves. The assay demonstrated high analytical specificity and showed no amplification against a diverse panel of bacteria and fungi. Clinical validation was conducted using deidentified residual axillary/groin surveillance culture specimens from C. auris culture-positive and culture-negative patients. The DiaSorin Molecular Detection Kit exhibited 100% agreement in sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) when compared to cultures coupled with MALDI-TOF identification. Intra- and inter-reproducibility testing demonstrated consistent and reliable diagnostic performance. This validated assay offers rapid and accurate detection of C. auris, facilitating timely implementation of infection control measures and appropriate patient care. The DiaSorin Molecular C. auris Detection Kit has the potential to aid in controlling the outbreaks caused by this emerging fungal pathogen. Providing a reliable diagnostic tool can contribute to the effective management and containment of C. auris infections in healthcare settings and ultimately improve patient outcomes.

Sensitivity enhancement in the fluorometric determination of aliphatic amines using naphthalene-2,3-dicarboxaldehyde derivatization followed by vortex-assisted liquid-liquid microextraction.

A highly sensitive liquid chromatographic method was developed for the fluorometric determination of trace amounts of linear aliphatic primary amines. Prior to extraction, amines were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion (CN) and extracted by vortex-assisted liquid-liquid microextraction (VALLME). The optimum conditions were as follows: derivatization reaction time for 5 min in 2.0 mL aqueous donor samples with 50 µM NDA/CN, and 10mM borate buffer at pH 9; vortex extraction time for 20s in the VALLME step with 50 µL of isooctane as the extractant phase; centrifugation for 1 min at 6000 rpm. Under the optimum conditions, the limits of detection (LOD) were between 0.01 and 0.04 nmol L(-1). The calibration curves showed good linearity in the range of 0.1-20 nmol L(-1). In comparison with previous work using o-phthalaldehyde/2-mercaptoethanol derivatization, the method has much more stable fluorescent derivatives, higher fluorescence intensities, and greater extraction efficiencies. The sensitivity enhancement factors (SEF) were between 2 and 70, which is in good agreement with the theoretical values calculated from partition coefficients in VALLME system.

Vortex-assisted liquid-liquid microextraction coupled with derivatization for the fluorometric determination of aliphatic amines.

A new one-step derivatization and microextraction technique was developed for the fluorometric determination of C(1)-C(8) linear aliphatic primary amines in complex sample solutions containing high levels of amino acids. In this method, amines were derivatized with o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) in aqueous solution and extracted simultaneously by vortex-assisted liquid-liquid microextraction (VALLME). Parameters affecting the extraction efficiency were investigated in detail. The optimum conditions were as follows: 50 µL of isooctane as the extractant phase; 2.0 mL aqueous donor samples with 12 mM OPA, 24 mM 2-ME, and 0.1 M borate buffer at pH 10; 1 min vortex extraction time; centrifugation for 4 min at 6000 rpm. After centrifugation, the enriched analytes in the floated extractant phase were determined by HPLC-FL in less than 14 min. Under the optimum conditions, the limits of detection were of the order of 0.09-0.31 nM. The calibration curves showed good linearity over the investigated concentration range between 0.4 and 40 nM. The proposed method has been applied to the determination of aliphatic amines in acidophilus milk, beer, and Cu(II)/amino acid solution.