Internal Standard Solution Research Articles

B-171 Iohexol Mass Spectrometry Method Validation and Comparison in Clinical Pediatric Laboratory

Abstract Background Accurate assessment of kidney function is important yet challenging in pediatric patients due to changing muscle mass affecting serum creatinine. There are several key methods to assess kidney function in this population, including estimated glomerular filtration rate (eGFR) from serum creatinine, urine creatinine clearance, and eGFR from serum iohexol clearance, which often provides a more reliable estimate. Iohexol is a non-ionic iodinated contrast agent filtered freely through the glomeruli without tubular secretion, making it an ideal GFR marker.In this study, we validated the LC-MS/MS method to quantitatively measure iohexol in clinical pediatric plasma and serum samples. The clinical performance was compared with the novel Verispray method. Methods A series of iohexol standards (Caymen Chemical) were prepared at 10, 100, 500, 1000 µg/mL in serum (UTAK). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and Verispray method were developed to quantitatively measure iohexol concentrations in spiked pediatric specimens. The internal standard (IS) solution was prepared with Iohexol-D5 (Cayman Chemical) at 9 µg/mL in UPLC grade methanol. Specimen or calibration/QC (50 µL) were mixed with IS solution (450 µL), and then centrifuged at 14,000xg for 3 minutes. The supernatants were analyzed. For LC-MS/MS, 3 µL supernatants were chromatographed on a AccucoreTM Vanquish C18 50 x 2.1mm (Thermo Scientific) using a gradient of water with 0.1% formic acid (FA) against methanol with 0.1% FA. Iohexol and IS were measured by multiple reaction monitoring using a TSQ Quantis Plus Mass Spectrometer (ThermoFisher). For Verispray, 10 µL supernatants were loaded on the Verispray sample plate (ThermoFisher) without LC portion. The samples were extracted from the plate paper using 95% acetonitrile with 5% water and 0.1% FA before directly injected into the mass spectrometer. The LC-MS/MS method was evaluated for linearity, patient comparison, carryover, matrix effect, and stability. 40 patients were analyzed between methods. Results Linearity was assessed over 5 days at 4 calibrator levels, the coefficient of variation (CV) ranged from 3.46 - 5.99%. The patient comparison of 40, 20 serum and plasma respectively, was conducted with Mayo Clinics, and good concordance was observed. Matrix effects were evaluated with spiked samples prepared with 50% mobile phase A and B, the ion enhancement was at 10.7%, within the allowable limits. No significant carryover was seen. The samples were stable at room temperature for 48 hours and 4oC for 7 days. Freezing is not recommended. The comparison between Verispray and LC-MS/MS showed significant bias between and within the performing personnel, the deviation can be up to 40% comparing to LC-MS/MS method. Conclusions The LC-MS/MS method demonstrated stable and consistent analytical performance for iohexol measurement. However, the Verispray method showed significant inter- and intra-user variabilities. As the Verispray sample spot can only be used once, respotting is required for any additional measurement, reducing reproductivity. It is highly recommended to duplicate all samples when using Verispray, to limit preparation to one user per patient run. Overall, our validation indicates the LC-MS/MS method provides reliable performance as a useful alternative to other complex gold standard pediatric tests of kidney function.

B-172 Correlation of Microbiome Sequencing Data and Quantified Short-Chain Fatty Acids by LC-MS/MS in IBD and Non-IBD Stool Samples

Abstract Background SCFAs are the primary metabolites produced by bacterial fermentation of dietary fiber and undigested proteins. They play a crucial role in various metabolic pathways, such as regulating the host’s energy balance and anti-inflammatory processes. The most studied SCFAs are acetic acid, propionic acid and butyric acid, with an approximate molecular ratio of 60:20:20 in healthy adults. A significantly different distribution may indicate a bacterial dysbiosis. While most SCFAs have a positive impact on the host’s health, the impact of branched short-chain fatty acids (bSCFAs), such as isobutyric acid and isovaleric acid, are less well-known. In order to evaluate whether the determination of a broader SCFA panel holds additional diagnostic value, we have developed a sensitive and robust LC-MS/MS method to determine 11 C2 - C7 SCFAs in human fecal samples. Methods The LC-MS/MS method includes acetic acid (AA), propionic acid (PA), butyric acid (BA), valeric acid (VA), caproic acid (CA) and heptanoic acid (HA), as well as the isomers isobutyric acid (iBA), 2-methylbutyric acid (2-MBA), isovaleric acid (iVA), isocaproic acid (iCA) and 2-methylhexanoic acid (2-MHA). After suspending the stool samples in a stabilizing buffer, the tubes were centrifuged and 25 µl of the supernatant was mixed with an internal standard solution (10 isotopically labeled internal standards) and subsequently derivatized. After one hour, the reaction was quenched and samples were injected into an ACQUITY UPLC H-Class (Waters) with an injection cycle of 9.5 min. Analytes were measured in positive mode by ESI-MS/MS on a Xevo TQ-MS (Waters). For quantification, six calibrators and three controls covering the anticipated physiological ranges were used. Results Microbiome sequencing data identified a total of 193 species, most of which belong to the Firmicutes phylum. Next, Actinobacteria and Proteobacteria are in descending order of abundance. In our IBD samples, we see a significant dysbiosis of these ratios and orders in comparison to the screening patients. E.g. in one sample, no Bacteroidetes were found, but a high amount of Actinobacteria (40 %) were sequenced. Corresponding SCFA data detected an AA:PA:BA ratio of 76:11:10, as well as no VA, iCA, CA, 2-MHA and HA, whereas SCFA data from most screening patients showed an AA:PA:BA ratio of 60:20:20 (± 10:10:10). Conclusions After a fast and simple sample preparation and derivatization, the LC MS/MS method allows robust and sensitive measurement of all presented SCFAs from human feces samples. The preliminary analysis of the combined microbiome and SCFA data showed that the detected AA:PA:BA ratio difference could be a valid indicator for a dysbiosis or an altered microbiome, corroborating data from previous studies. While we could detect differences on bSCFAs and longer SCFAs between IBD and non-IBD patient samples in this pilot study, larger patient and control cohorts are needed in order to substantiate our initial promising findings and finally allow a robust evaluation of these parameters as diagnostic tool to monitor and determine the state of the patient microbiome.

Assay for the quantification of abemaciclib, its metabolites, and olaparib in human plasma by liquid chromatography-tandem mass spectrometry

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2% formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 – 1000nM abemaciclib, 0.35 – 1000nM M2 and M18, 0.5 – 1000nM M20, and 0.75 – 1000nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13% and the accuracy was ± 12%, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11% and the accuracy was ± 12% for low, mid, and high and < 19% at LLOQ. The recovery in human plasma was determined to be between 92 and 102% for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers.

Postmortem distribution of ropivacaine and its metabolite in human body fluids and solid tissues by GC-MS/MS using standard addition method.

An analytical method was developed for determining ropivacaine and its main metabolite, 3-hydroxyropivacaine in biomedical samples using gas chromatography-tandem mass spectrometry (GC-MS/MS). Then, this established method was applied to investigate the distribution of ropivacaine and its metabolite in human fluids and solid tissues obtained from an authentic case ropivacaine involved. The fluid sample was added acetonitrile, and solid tissue was homogenized using a freezer mill and then added into acetonitrile. Then, an internal standard solution was added to the mixtures. The mixture was centrifuged at 12,000 × g for 5min, and the upper layer of acetonitrile was transferred to magnesium sulfate and octadecyl silica (C18) gel for cleaning up the sample. After centrifugation, the upper layer was then evaporated to dryness with nitrogen, and dissolved with methanol, then injected into the GC-MS/MS system. The coefficients of determination (r2) of constructed calibration curves were all greater than 0.999. The limits of detection for ropivacaine and 3-hydroxyropivacaine in target samples were 15ng/mL and 10ng/mL, respectively. The recovery rates of ropivacaine and 3-hydroxyropivacaine ranged from 97.6% to 103% and from 96.5% to 104%, respectively. The inter-day precision values of ropivacaine and 3-hydroxyropivacaine were not greater than 6.25% and 7.98%, respectively, and the inter-day trueness values were not greater than 6.90% and 8.33%, respectively; the intra-day precision and trueness values of ropivacaine and 3-hydroxyropivacaine were not greater than 3.20%, 6.78%, 7.84% and 8.99%, respectively. GC-MS/MS method for simultaneous detection and quantification of ropivacaine and 3-hydroxyropivacaine in biological samples was successfully developed. The method could also be applied to samples obtained from an authentic case; their distribution among tested fluids and solid tissues were also measured. This is the first report on the distribution of ropivacaine and its major metabolite 3-hydroxyropivacaine in a human case.

Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Determination of Ethanol in Vinegar and Beverage by Gas Chromatography: A Validated Method for Halal Verification

Validation of the method for determining ethanol in vinegar and beverages has been carried out in the linearity range of 0.05-1% using GC-FID. This research was carried out using an internal standard solution and without an internal standard solution. The research aims to validate the linearity of ethanol determination at low concentration levels. This validation is useful to ensure that the ethanol test method in the sample can be detected at low concentration levels. In the halal authentication process, the maximum limit for ethanol content is 5%. The method validation results confirm that in the concentration range 0.05-1% it has good linearity with a correlation coefficient (R) without the addition of internal standards and with the addition of standard solutions being 0.9972 (R2=0.9943) and 0.9952 (R2=0.9905). The results of the analysis of variance (ANOVA) test with a 95% confidence interval and 9 degrees of freedom F-count (0.0141) &lt; F-critical (0.3146) show that the range 0.05-1% corresponds to the limit of linearity. The results of the ANOVA test with a 95% confidence interval with df=6 show the F-test value (0.0879) &lt; F-critical (0.2334) indicating that the two proposed methods have high precision and no significant differences. Both methods also have high accuracy and no significant differences in accuracy as shown by the results of the ANOVA test with a 95% confidence interval and degrees of freedom = 6 showing the F-test value (1.5204) &lt; F-critical (4.2839). It is recommended that the results of this validation be useful in the halal verification process for vinegar and beverage products.

Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search fordrugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometrymethod, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1ng/mL and 0.02-21ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Comparative Pharmacokinetics Research of 13 Bioactive Components of Jieyu Pills in Control and Attention Deficit Hyperactivity Disorder Model Rats Based on UPLC-Orbitrap Fusion MS.

Jieyu Pills (JYPs), a Chinese medicine consisting of 10 herbal elements, have displayed promising clinical effectiveness and low by-effects in the treatment of depression. Prior investigations mostly focused on elucidating the mechanism and therapeutic efficacy of JYPs. In our earlier study, we provided an analysis of the chemical composition, serum pharmacochemistry, and concentrations of the main bioactive chemicals found in JYPs. However, our precise understanding of the pharmacokinetics and metabolism remained vague. This study involved a comprehensive and meticulous examination of the pharmacokinetics of 13 bioactive compounds in JYPs. Using UPLC-Orbitrap Fusion MS, we analyzed the metabolic characteristics and established the pharmacokinetic parameters in both control rats and model rats with attention deficit hyperactivity disorder (ADHD) following oral administration of the drug. Before analysis, plasma samples that were collected at different time intervals after the administration underwent methanol pre-treatment with Puerarin used as the internal standard (IS) solution. Subsequently, the sample was chromatographed on a C18 column employing gradient elution. The mobile phase consisted of methanol solution containing 0.1% formic acid in water. The electrospray ionization source (ESI) was utilized for ionization, whereas the scanning mode employed was selected ion monitoring (SIM). The UPLC-Orbitrap Fusion MS method was subjected to a comprehensive validation process to assess its performance. The method demonstrated excellent linearity (r ≥ 0.9944), precise measurements (RSD < 8.78%), accurate results (RE: -7.88% to 8.98%), and appropriate extraction recoveries (87.83-102.23%). Additionally, the method exhibited minimal matrix effects (87.58-101.08%) and satisfactory stability (RSD: 1.52-12.42%). These results demonstrated adherence to the criteria for evaluating and determining biological material. The 13 bioactive compounds exhibited unique pharmacokinetic patterns in vivo. In control rats, all bioactive compounds except Ferulic acid exhibited linear pharmacokinetics within the dose ranges. In the ADHD model, the absorption rate and amount of most of the components were both observed to have increased. Essentially, this work is an important reference for examining the metabolism of JYPs and providing guidelines for clinical therapy.

Profiling of Oxylipins as Markers of Oxidative Stress in Biological Samples.

Oxylipins are oxidized metabolites of polyunsaturated fatty acids (PUFAs). They represent a class of risk markers and/or therapeutic targets for diseases associated with inflammation, including cardiovascular disease and brain disorders. Because the biological activities of free PUFAs and oxylipins depend on their chemical structures and concentrations, monitoring PUFAs and oxylipin levels in biological systems is critical for understanding their roles in health and disease. Traditionally, accurate quantification of free PUFAs and oxylipins in biological samples was performed separately, as PUFAs are often 1000-fold more abundant than the derived oxidized fatty acids (oxylipins). This article describes a liquid chromatography multiple reaction monitoring tandem mass spectrometry method for the quantitative analysis of five free PUFAs and 88 oxylipins in various biological fluids, including plasma, platelet supernatants, and tissues. The same approach can also be used in conjunction with an alkaline hydrolysis step to quantify total oxylipins in fish oils. We observed that in some samples, linoleic acid levels in plasma and eicosapentaenoic acid and arachidonic acid levels in brain tissue were above the upper limit of quantification. To address this issue, we developed a data analysis method to obtain PUFA and oxylipin concentrations in these samples without additional sample preparation, thus significantly saving time and labor. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantification of polyunsaturated fatty acids (PUFAs) and oxylipins using liquid chromatography multiple reaction monitoring tandem mass spectrometry Support Protocol 1: Preparation of internal standard mixed working solution Support Protocol 2: Preparation of standard mixed stock solution Support Protocol 3: Preparation of standard mixed working solution Alternate Protocol 1: Extraction and quantitation of free PUFAs and oxylipins from mouse brain tissue Alternate Protocol 2: Extraction and quantitation of total PUFAs and oxylipins from fish oil.

Determination of intracellular tenofovir-diphosphate and emtricitabine-triphosphate concentrations in dried blood spots for pre-exposure prophylaxis adherence

ObjectivesWe measured the intracellular concentrations of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) in dried blood spots (DBS) for pre-exposure prophylaxis (PrEP) adherence using sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS). MethodsA total of 191 DBS were obtained from 85 participants who were receiving tenofovir disoproxil fumarate (TDF; 300 mg) and emtricitabine (FTC; 200 mg) as PrEP at the Sexual Health Clinic, National Center for Global Health and Medicine, Tokyo, Japan. DBS punch (3 mm) added to 25 μL of 50% methanol and 400 μL of internal standard solution was used for solid phase extraction. Chromatographic separation was achieved on an Atlantis Premier BEH C18 AX Column (50 mm × 2.1 mm i.d.; particle size 1.7 μm) using gradient elution (flow rate: 0.6 mL/min); injection volume: 7 μL and run time: 5.5 min. Calibration curves for the two drugs were linear in the range 0.05–12.5 ng/punch. ResultsWe determined the intracellular TFV-DP and FTC-TP concentrations in 191 DBS obtained from 85 patients administered with TDF and FTC as PrEP. The analytical performance data (calibration curve and QC samples) for all the analytical runs met the acceptance criteria. Intracellular concentrations of TFV-DP and FTC-TP in the DBS remained stable for at least 24 h after oral administration. ConclusionsA multiplex LC–MS/MS method was successfully developed for DBS, which can be useful for monitoring the levels of TFV-DP and FTC-TP in individuals receiving PrEP.

Quantitative analysis of fucose in fucoidans from Sargassum spp. in Jeju Island, South Korea using 3-methyl-1-phenyl-5-pyrazolone derivatization and RP-HPLC-UV method

Sargassum species are representative brown algae found worldwide, with 27 species native to the coast of Jeju Island in South Korea. Fucoidans are derived from brown algae and exhibit a variety of biological activities. Analysis of the monosaccharide composition is necessary for an in-depth study of fucoidans. However, quantitative analysis of fucoidans with complex structures according to species and habitat is insufficient. Furthermore, complex pre-processing is often required to analyze sugars, making it less economical. This study aimed to validate high-performance liquid chromatography (HPLC)-UV analysis based on 3-methyl-1-phenyl-5-pyrazolone (PMP) derivatization of monosaccharides. The proposed method had a short running time and could be repeated economically using UV detection. The detected PMP-fucose was confirmed to be uninterfered by comparing the analytical chromatograms of the internal standard solution. This analytical method was characterized for system suitability, precision, accuracy, linearity, limit of detection (LOD), and limit of quantification (LOQ), demonstrating satisfactory results. The quantification was verified by comparison with the quantitative results of commercial fucoidan obtained using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). Furthermore, the antioxidant effects of algal polysaccharides from Sargassum spp. isolated on Jeju Island was evaluated using a zebrafish model. Polysaccharides from Sargassum coreanum (SCpol) and Sargassum autumnale (SApol) showed remarkable free radical scavenging activities. In addition to the results obtained in the zebrafish experiments, all species-derived polysaccharides showed antioxidant potential. In conclusion, this study demonstrates a useful analytical method using HPLC-UV and PMP derivatives for the effective determination of monosaccharide contents, including fucose, in Sargassum spp.

Exposure characteristics and health risk assessment of 97 typical chemical pollutants in human serum

Rapid industrial and agricultural developments in China have led to the wide use and discharge of chemical products and pesticides, resulting in extensive residues in environmental media. These residues can enter the human body through various pathways, leading to high exposure risks and health hazards. Because the human body is exposed to a variety of chemical pollutants, accurately quantifying the exposure levels of these pollutants in the human body and evaluating their health risks are of great importance. In this study, the serum concentrations of 97 typical chemical pollutants of 60 adults in central China were simultaneously determined using solid-phase extraction coupled with gas chromatography-tandem mass spectrometry (SPE-GC-MS/MS). In this method, 200 μL of a serum sample was mixed with 10 μL of an isotope-labeled internal standard solution. The sample was vortexed and refrigerated overnight at 4 ℃. Each sample was then deproteinized by the addition of 200 μL of 15% formic acid aqueous solution and vortexed. The serum sample was loaded into a preconditioned Oasis® PRiME HLB SPE cartridge and rinsed with 3 mL of methanol-water (6∶1, v/v). The SPE cartridge was subsequently vacuumed. The analytes were eluted with 3 mL of dichloromethane followed by 3 mL of n-hexane. The eluent was concentrated to near dryness under a gentle nitrogen stream and reconstituted with 100 μL of acetone. The samples were determined by GC-MS/MS and separated on a DB-5MS capillary column (30 m×0.25 mm×0.25 μm) with temperature programming. The column temperature was maintained at 70 ℃ for 2 min, increased at a rate of 25 ℃/min to 150 ℃, increased at a rate of 3 ℃/min to 200 ℃, and then held for 2 min. Finally, the column temperature was increased at a rate of 8 ℃/min to 300 ℃ and maintained at this temperature for 8 min. The samples were detected in multiple-reaction monitoring (MRM) mode and quantitatively analyzed using the internal standard method. Multiple linear regression models were used to analyze the effects of demographic characteristics, lifestyle habits, and diet on the concentrations of the chemical pollutants in the serum samples, and known biomonitoring equivalents (BEs) and human biomonitoring (HBM) values were combined to compute hazard quotients (HQs) and hazard indices (HIs) and evaluate the health risks of single and cumulative exposures to the chemical pollutants. The results showed that the main pollutants detected in human serum were organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). The detection rates of eight pollutants, including hexachlorobenzene (HCB) (100%), pentachlorophenol (PCP) (100%), p,p'-dichlorodiphenylene (p,p'-DDE) (100%), PCB-138 (100%), PCB-153 (98.3%), β-hexachlorocyclohexane (β-HCH) (91.7%), fluorene (Flu) (85.0%), and anthracene (Ant) (75.0%), were greater than 70%. The serum levels of β-HCH were higher in females than in males, and age was positively correlated with exposure to p,p'-DDE, PCB-138, PCB-153, and β-HCH. Increased exposure levels to p,p'-DDE and β-HCH may be associated with a high frequency of meat intake, whereas increased exposure level to PCP may be associated with a high frequency of vegetable intake. The serum HQ of PCP was greater than 1 in 6.7% of the samples, and no risk was observed for HCB and p,p'-DDE exposure in the study population. Approximately 28.3% of the study subjects had HI values greater than 1. Overall, the general adult population in this region is widely exposed to a wide range of chemical pollutants, and gender, age, and diet are likely to be the main factors influencing the concentration of chemical pollutants. The health risk of single and compound exposures to chemical pollutants should not be ignored.

Development and validation of a simple, fast and sensitive liquid chromatography-tandem mass spectrometry method to establish reference intervals for 24-h urinary free normetanephrine, metanephrine and methoxytyramine

ObjectiveTo develop and validate a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect urinary free metanephrines and methoxytyramine, establishing reference intervals. MethodsUrine samples were diluted with isotope internal standard solution, then analyzed directly using tandem mass spectrometry with multiple reaction monitoring measurement and electrospray ionization source in positive ion mode. Analytical parameters including linearity, lower limit of quantitation, imprecision and accuracy of the method were evaluated. The reference intervals for urinary catecholamine metabolites were established by analyzing 24-h urine samples collected from 81 apparently healthy volunteers. ResultsThe analytical times for MN, NMN, and 3-MT were at 2.79, 2.80, and 2.74 min, respectively. The method displayed excellent linearity (r > 0.99) in the range of 1-1000 ng/mL, with lower limits of quantification (LLOQ) at 0.50 ng/mL for MN and NMN, and 0.25 ng/mL for 3-MT. The method's intra-day and inter-day imprecisions were less than 8 %. The method recovery ranged from 96.8% to 105.8 % for MN, 89.7%–106.4 % for NMN, and 93.5%–106.2 % for 3-MT. No carry-over was observed during the analysis of all analytes. The LC-MS/MS method was used to establish reference intervals in 24-h urine samples from 81 apparently healthy volunteers. There was no association of sex with urinary free metabolites. ConclusionThis study established a novel, fast and sensitive LC-MS/MS method for determining urinary free catecholamine metabolites, which could facilitate screening and diagnosis for catecholamine-related tumors more conveniently and quickly.

Validation of Liquid Chromatography Coupled with Tandem Mass Spectrometry for the Determination of 12 Tyrosine Kinase Inhibitors (TKIs) and Their Application to Therapeutic Drug Monitoring in Adult and Pediatric Populations.

Tyrosine kinase inhibitors (TKIs) are used as targeted cancer therapies in adults and have an off-label pediatric application for the treatment of Langerhans cell histiocytosis. A multitarget LC-MS/MS method was developed and validated for the determination of alectinib, alectinib-M4, binimetinib, cobimetinib, crizotinib, dabrafenib, encorafenib, imatinib, lorlatinib, osimertinib, AZ5104, and trametinib. A total of 150 µL of internal standard methanolic solution was added to 50 µL of plasma sample to precipitate proteins. After centrifugation, 10 µL of the supernatant was injected into the chromatographic system. The chromatographic separation was conducted on a Kinetex C18 Polar column with a gradient of 2 mM ammonium formate in 0.1% formic acid and acetonitrile over 5 min. Limits of detection and quantification, linearity, accuracy, precision, selectivity, carryover, matrix effect, recovery, and stability were evaluated and satisfied EMA guidelines on bioanalytical methods. This method has been successfully applied to the therapeutic drug monitoring (TDM) of adults with melanoma and lung cancer, as well as children with histiocytosis, to improve the pharmacokinetic data for these drugs, with the aim of enhancing the therapeutic management and follow-up of patients. Blood concentrations of trametinib and binimetinib were different in the two groups, highlighting the age-related inter-individual variability of these molecules and the need for TDM.

Precise Determination of Eu Concentration in Coal and Sedimentary Rock Samples Using High-Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS)

Europium (Eu) in coal and sedimentary rocks has important mineral resource potential as well as being a crucial parameter in geochemical studies that can represent changes in the depositional environment during coal deposition and identify the depositional source region. Therefore, it is essential to realize the precise measurement of Eu in coal as this could be a useful parameter for paleoenvironmental reconstruction studies and the exploration of mineral resources. During inductively coupled plasma mass spectrometry (ICP-MS) analysis, polyatomic ions of Ba may interfere with Eu, causing the observed value to be higher than the actual value. This paper develops a new approach for Eu determination by using a high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). The mass spectral interference and correction of Eu in the coal and sedimentary rock samples at low, medium, and high resolutions were investigated. The results showed that in the high-resolution mode (resolution = 10,000 amu), the interference of polyatomic ions of Ba could be distinguished from Eu; hence, Eu was determined under this circumstance. Under the optimal experimental circumstances, the detection limit was 0.006 μg/mL, the relative standard deviation was 0.80%–1.22%, and the linear correlation coefficient of the standard curve was over 0.9999. The recoveries of the 103Rh internal standard solution ranged from 94.41% to 100.10%. This method was verified using standard reference materials and selected samples, which demonstrated its high sensitivity, accuracy, and reliability, and a low detection limit, making it appropriate for detecting Eu in samples of coal and sedimentary rocks.

Quantification of Oxcarbazepine Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Oxcarbazepine (Trileptal®) has been found effective in the treatment of tonic-clonic seizures and partial seizures with or without secondary generalization, with fewer side effects than traditional therapy. Oxcarbazepine is a keto analogue of carbamazepine. It is rapidly reduced to 10-monohydroxy carbamazepine (MHD), its active metabolite. This assay measures concentrations for oxcarbazepine metabolite (MHD), internal standard (MHD 13C6) solution is added to all the patient specimens resulting in precipitation of proteins. The analytes are separated using a Phenomenex Kinetex C18 column and are detected with a mass spectrophotometer utilizing multiple reaction monitoring (MRM). The analytes are qualitatively identified and quantitated from a calibration curve generated from calibrators included in the run.

Quantification of Olanzapine by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Olanzapine (Zyprexa™) is indicated for the treatment of schizophrenia and bipolar disorder. Olanzapine is metabolized to inactive metabolites; therefore, this assay measures the concentrations of olanzapine and internal standard, olanzapine-D3. This procedure provides instructions for quantitating levels of olanzapine in blood serum specimens. An internal standard (IS) solution consisting of the deuterated analogue of olanzapine is added to an aliquot of the patient serum or plasma, standards, and positive controls. The drugs of interest are separated from the biological fluid using solid phase extraction. The dried extracts are reconstituted in mobile phase and separated by liquid chromatography. The solution is introduced into an Agilent 6470A triple quadrupole tandem mass spectrometer via positive mode electrospray ionization (ESI) utilizing multiple reaction monitoring (MRM). Quantitation is performed by comparison to a calibration curve, using Indigo® (Ascent) software.

WITHDRAWN: Mera: A scalable high throughput automated micro-physiological system

Background: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been suggested to play roles in various diseases, yet there is little data on their changes in patients with non-small-cell lung cancer (NSCLC). A simple LC-MS/MS method for EPA and DHA determination is critical to exploring EPA and DHA level changes in NSCLC patients.Method: 25 μL of serum was mixed with 25 μL of internal standard working solution, and then 450 μL of acetonitrile for protein precipitation. After vortex and centrifugation, the supernatant was directly used for LC-MS/MS analysis. The method was well validated with linearity, precision, recovery, and matrix effect. The concentrations of EPA and DHA in serum samples from 211 NSCLC patients and 227 healthy controls were determined by this LC-MS/MS method.Results: Good separation and reliable quantification of EPA and DHA in serum samples were achieved by our method. Compared with healthy controls, serum EPA and DHA were significantly reduced in both adenocarcinoma and squamous cell carcinoma. The concentrations of EPA and DHA showed a progressive decrease in healthy controls, early- and advanced-stage NSCLC patients.Conclusions: This study identified significant reductions in serum EPA and DHA in NSCLC patients through the development of an LC-MS/MS method.

Improved LC-MS/MS method for the simultaneous quantification of tacrolimus and cyclosporine A in human blood and application to therapeutic drug monitoring.

In order to facilitate therapeutic drug monitoring of tacrolimus and cyclosporine A in clinical practice, a simple, rapid, robust, sensitive and specific LC-MS/MS assay was developed and validated for the simultaneous determination of tacrolimus and cyclosporine A in human whole blood. Erythrocytes were destroyed using internal standard solution with 10% (w/v) zinc sulfate in water. The analytes were extracted from 100 μl of whole blood by protein precipitation with acetonitrile. Chromatographic separation was conducted on a Kinetex PFP column (60°C) by a gradient elution with a flow rate of 0.450 ml/min in 2.5 min. Quantitative analysis was performed using electrospray ionization and multiple reaction monitoring in positive ionization mode. The method was fully validated as per current guidelines on bioanalytical methodologies of the US Food and Drug Administration and European Medicines Agency. The method developed was applied successfully in analyzing clinical samples from patients administered tacrolimus or cyclosporine A. The sample treatment procedure was rationalized and improved to fulfill the complete target extraction. The chromatography conditions were optimized to achieve rapid and accurate quantification of both analytes. This method may be beneficial as a constructive input for the therapeutic drug monitoring of tacrolimus and cyclosporine A in obtaining individualized therapy.

B-206 The Determination of Vanillylmandelic Acid (VMA), Homovanillic Acid (HVA) and 5-Hydroxyindolacetic Acid (5-HIAA) by LC-Ms/MS Assay in Urine Samples for Diagnosis of Neuroendocrynes Tumors

Abstract Background The catecholamines dopamine, norepinephrine and epinephrine, as well the neurotransmitter serotonin are biogenic amines that were formed by enzymatic descarboxylation of aminoacids in the body. They realize an important function in central nervous system. Vanillylmandelic acid (VMA) and Homovanillic Acid (HVA) are degradation products of the catecholamines, and 5-Hydroxyindolacetic Acid (5-HIAA) is a serotonine degradation product. They are important to the diagnosis of neuroendocrynes tumors, that produce catecholamines and/or serotonine in excess, resulting in elevated levels of VMA, HVA, and/or 5-HIAA in 24 h collection urine. Objective This study aimed to validate a simple LC-MS/MS method for quantification of VMA, HVA and 5-HIAA in isolated and 24 h urine samples for the diagnosis of neuroendocrines tumors. Methods Using a deep-well microtiter plate, 20 µL of isolated or 24 h urine was diluted in 540 µL of 0.05% of formic acid solution. Then, 20 µL of an internal standard solution containing stable isotopes of the analytes were added (VMA-D3, HVA-13C6,18O and 5-HIAA-13C6). The plate was sealed, homogenized for 30 s at 2500 rpm, and centrifuged for 15 min at 4000 rpm and ambient temperature. Then, 5.0 µL of the supernatant was injected into the LC-MS/MS system. Chromatographic separation was performed on the Waters ACQUITY UPLC system equipped with Kinetex XB-C18 1.7 µm X 50 mm X 2.1 mm column (Phenomenex), and methanol and formic acid 0.2% solution as mobile phase in 3.5 min of gradient mode separation. The detection was performed on a Waters XEVO TQ-S Micro mass spectrometer with electrospray ionization (ESI+). The current method was validated for carryover, linearity, precision, recovery, and limit of quantification, and it was evaluated by comparing the analysis in the developed method and a commercial kit by HCPL-ECD, of 15 real samples from volunteers in the laboratory. Results The method was successfully validated for all the parameters. The linearity was between 0.5–100.0 mg/L. The precision was less than 6.3%, the recovery was between 85.4%–103.4% and the quantification limit was 0.5 mg/L. The comparison with the reference method showed good coefficient correlations (R2 &amp;gt; 0.92) for the three analytes. Conclusion We validate a simple LC-MS/MS method to quantify three metabolites to help the diagnosis of neuroendocrynes tumors. The method represents a simplification of the extraction compared with the commercial kit employing HPLC-ECD, and an important reduction of the per sample production cost, without loss of quality.