Tandem Mass Spectrometry Method Research Articles

Comprehensive profiling of ceramides in human serum by liquid chromatography coupled to tandem mass spectrometry combining data independent/dependent acquisition modes

Ceramides are sphingolipids with a structural function in the cell membrane and are involved in cell differentiation, proliferation and apoptosis. Recently, these chemical species have been pointed out as potential biomarkers in different diseases, due to their abnormal levels in blood. In this research, we present an overall strategy combining data-independent and dependent acquisitions (DIA and DDA, respectively) for identification, confirmation, and quantitative determination of ceramides in human serum. By application of liquid chromatography–tandem mass spectrometry (LC–MS/MS) method in DIA mode we identified 49 ceramides including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. Complementary, quantitative determination of ceramides was based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC–MS/MS in DDA to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL−1 level, the intra-day and between-days variability were below 20 and 25 %, respectively; and the accuracy, expressed as bias, was always within ±25 %. The proposed method was tested with the CORDIOPREV cohort in order to obtain a qualitative and quantitative profiling of ceramides in human serum. This characterization allowed identifying d18:1 ceramides as the most concentrated with 70.8% of total concentration followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. Less concentrated ceramides, d16:1, d17:1 and t18:0, reported a 7.1 % of the total content. Combination of DIA and DDA LC–MS/MS analysis enabled to profile qualitative and quantitatively ceramides in human serum.

The effect of icotinib or apatinib on the pharmacokinetic profile of oxycodone in rats and the underlying mechanism

This study aimed to investigate the interactions between icotinib/apatinib and oxycodone in rats and to unveil the underlying mechanism. An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine oxycodone and its demethylated metabolite simultaneously. In vivo, Sprague–Dawley (SD) male rats were administered oxycodone with or without icotinib or apatinib. Blood samples were collected and subjected to UPLC-MS/MS analysis. An enzyme incubation assay was performed to investigate the mechanism of drug–drug interaction using both rat and human liver microsomes (RLM and HLM). The results showed that icotinib markedly increased the AUC(0–t) and AUC(0–∞) of oxycodone but decreased the CLz/F. The Cmax of oxycodone increased significantly upon co-administration of apatinib. In vitro, the Km value of oxycodone metabolism was 101.7 ± 5.40 μM and 529.6 ± 19.60 μM in RLMs and HLMs, respectively. Icotinib and apatinib inhibited the disposition of oxycodone, with a mixed mechanism in RLM (IC50 = 3.29 ± 0.090 μM and 0.95 ± 0.88 μM, respectively) and a competitive and mixed mechanism in HLM (IC50 = 22.34 ± 0.81 μM and 0.48 ± 0.05 μM, respectively). In conclusion, both icotinib and apatinib inhibit the metabolism of oxycodone in vitro and in vivo. Therefore, the dose of oxycodone should be reconsidered when co-administered with icotinib or apatinib.

SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.

Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS)methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometrymethod was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.

A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis.

Comprehensive Stereoselectivity Assessment of Toxicokinetics, Tissue Distribution, Cytotoxicity, and Environmental Fate of Chiral Pesticide Propiconazole.

Propiconazole (PRO) has been widely used in the treatment of fungal infection in fruits, vegetables, cereals, and seeds. In this study, a newly established chiral liquid chromatography tandem mass spectrometry method was applied to the systemic stereoselectivity evaluation of PRO enantiomers, including toxicokinetics, tissue distributions, cytotoxicity, accumulation, and degradation. Our results showed that both trans (+)-2S,4S-PRO and cis (-)-2S,4R-PRO had lower Cmax and AUC0-∞ and higher CLz/F values in plasma and lower accumulation concentrations in the liver, heart, and brain. In cytotoxic assays, cis (-)-2S,4R-PRO exhibited the lowest cytotoxicity in PC12 neuronal, N9 microglia, SH-SY5Y neuroblastoma, and MRC5 lung fibroblast cell lines. Moreover, the Eisenia fetida incubation experiment revealed that the accumulations of both trans (+)-2S,4S-PRO and cis (-)-2S,4R-PRO were higher than those of their antipodes in E. fetida. In summary, our findings first suggested that the application of cis (-)-2S,4R-PRO for agriculture would hugely reduce the environmental risk.

Mechanochemical synthesis ionic covalent organic frameworks/cotton composites for pipette tip solid-phase extraction of domoic acid in seafood

Pipette tip solid-phase extraction (PT-SPE) as a miniaturized solid-phase extraction technique have a wide range of applications in the field of sample pretreatment. In this study, ionic covalent organic frameworks@cotton (iCOF@cotton) were facilely synthesized by mechanochemical grinding method only in half an hour, and used as the adsorbents of PT-SPE. The synthesized iCOF@cotton not only had high specific surface area, suitable pore structure and cationic charge groups of iCOF that can extract polar targets quickly, but also reduced the problem of high back pressure of PT-SPE by the addition of cotton, thus accelerating extraction time. Combined with high performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), an efficient and sensitive method was established for detection of domoic acid (DA, a toxin produced by algae). Under the optimal conditions, the proposed analysis method displayed excellent analytical performance, including broad range of linearity (10–1000 pg mL−1), low limit of detection (LOD, 5 pg mL−1), high correlation coefficient (0.9993), satisfactory precision (RSDs ≤6.4 %). In addition, the developed method was applied to the detection of DA in marine samples, and detected trace DA (18.6 pg mL−1) with satisfactory recovery (85.7%–107.2 %). The above results indicated that the prepared iCOF@cotton have great potential as the adsorbents for PT-SPE.

A comprehensive LC-MS/MS method for the simultaneous measurement of 24 adrenal steroids: From research to clinical practice

Steroids are essential in the differential diagnosis of congenital adrenal hyperplasia (CAH) subtypes; however, they may confuse physicians with multifarious results. In this study, we established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of 24 steroids and developed a steroid metabolite pathway-based report to aid physicians in understanding these results. Solid-phase extraction was used to concentrate and purify target plasma steroids. The linearity, precision, recovery, and matrix effects were thoroughly evaluated. PowerBuilder was used to transfer the results from LC-MS/MS to the graphic report in a laboratory information management system (LIS) and was applied to different subtypes of CAH. Twenty-four steroids were separated and analyzed in one sample preparation and two injections using LC-MS/MS. The linearity of the steroids was excellent, with coefficients of linear regression greater than 0.99. The relative recovery ranged from 90.0 to 107.1 %, whereas the intra- and total coefficient variations were 1.6 ∼ 8.7 % and 2.0 ∼ 9.9 %, respectively. Matrix effects were compensated after internal standard correction. A graphic combination report mode was established and used to effectively identify CAH subtypes. In conclusion, a useful LC-MS/MS method and graphic combination report of 24 steroids based on their metabolite pathways were established.

Inhibitory effect of napabucasin on arbidol metabolism and its mechanism research

As a broad-spectrum antiviral, and especially as a popular drug for treating coronavirus disease 2019 (COVID‐19) today, arbidol often involves drug–drug interactions (DDI) when treating critical patients. This study established a rapid and effective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method to detect arbidol and its metabolite arbidol sulfoxide (M6-1) levels in vivo and in vitro. In this study, a 200 μL incubation system was used to study the inhibitory effect of the antitumor drug napabucasin on arbidol in vitro, with IC50 values of 2.25, 3.91, and 67.79 μM in rat liver microsomes (RLMs), human liver microsomes (HLMs), and CYP3A4.1, respectively. In addition, we found that the mechanism of inhibition was non-competitive inhibition in RLM and mixed inhibition in HLM. In pharmacokinetic experiments, it was observed that after gavage administration of 48 mg/kg napabucasin and 20 mg/kg arbidol, napabucasin inhibited the metabolism of arbidol in vivo and significantly changed the pharmacokinetic parameters of arbidol, such as AUC(0-t) and AUC(0-∞), in rats. We also found that napabucasin increased the AUC(0-t) and AUC(0-∞) of M6-1, the main metabolite of arbidol. This study provides a reference for the combined use of napabucasin and arbidol in clinical practice.

Separation of bile acid isomer plays a pivotal role in bioequivalence evaluation of ursodeoxycholic acid

Based on our experiences in bile acid profiling, this work developed and validated a liquid chromatography electrospray ionization tandem mass spectrometry method to separate endogenous bile acid isomers and quantitatively determine ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. The separation was performed on a CORTECS C18 column with the mobile phase consisting of 1.0 mM ammonium acetate and acetonitrile-methanol (80:20, v/v). UDCA, GUDCA and TUDCA were detected in the negative mode on a triple-quadrupole mass spectrometer at the ion transitions of m/z 391 > 391, m/z 448 > 74, m/z 498 > 80, respectively. Phosphate buffer was employed as the surrogate matrix to establish the isotope internal standard corrected calibration curves of analytes. The background-method with a linearity range of 10–200 ng/mL was partially validated to determine the endogenous levels of analytes in blank human plasma, which was incorporated into the validation of bioequivalence-method with a linearity range of 50–10000 ng/mL. The bioequivalence (BE)-method was fully validated with special focus on matrix effects, which have been critically evaluated using the precision and accuracy of quality control samples prepared from the blank human plasma of 12 individuals. It is disclosed for the first time that the BE results of UDCA formulation may yield false results when the method is insufficient to separate UDCA from isoursodeoxycholic acid, a microbial metabolite of both endogenous and exogenous UDCA. The present method has established a milestone for the evaluation of UDCA formulations and is expected to provide a valuable reference for the bioanalytical development of endogenous medicinal products.

483. Pharmacokinetics of Nirmatrelvir and Ritonavir in COVID-19 Patients with Hemodialysis

Abstract Background Nirmatrelvir/ritonavir (Paxlovid) is one of the few therapeutic options for COVID-19. It is not recommended to use in patients with end-stage renal disease (ESDR), and large-scale clinical trials are needed to support its application in this population. We investigated the pharmacokinetics of nirmatrelvir/ritonavir (150 mg/100 mg twice a day) in COVID-19 patients with hemodialysis (HD). Methods SARS-Cov-2 tested positive patients with HD were included and given treatment with nirmatrelvir/ritonavir (150 mg/100 mg twice a day) for 5 days. For most patients, a 4 h-HD started 2 h after the morning dose on day 2. Blood samples were collected 4 h and 12 h after the first dose on day 1 to obtain peak and trough concentrations, respectively. And the blood samples at the start and finish time points of HD were drawn to observe the effect of dialysis on drug clearance. A tandem mass spectrometry (LC-MS/MS) method was conducted to quantify the nirmatrelvir and ritonavir concentrations. Results A total of 14 COVID-19 patients undergoing dialysis were enrolled, with 4 females and 10 males. The age was 66.6 ± 8.0 years and the body mass index (BMI) was 22.3 ± 3.5. The peak concentration of nirmatrelvir after the first dose was 4960 ± 1889 ng/mL. And the trough concentration was 2940 ± 1305 ng/mL, which was approximately 10 times above the EC90 (292 ng/mL). Nirmatrelvir concentration fell by 42% from 10426 ± 3191 ng/mL to 6035 ± 2402 ng/mL during 4 h-HD. The peak and trough concentrations of ritonavir were 816 ± 445 ng/mL and 326±201 ng/mL after the first dose, respectively. And the concentration fell by 17% during 4 h-HD. Based on the assumption of a first-order elimination kinetic, nirmatrelvir/ritonavir 150 mg/100 mg, once a day dose regimen was simulated which showed that the plasma concentration of nirmatrelvir could also achieve the target EC90 value.Figure 1.Plasma concentration of nirmatrelvir (blue circles) and ritonavir (orange circles) in COVID-19 patients with hemodialysis during the course of 150 mg/100 mg twice a day treatment. 4 h- hemodialysis was performed at 26 - 30 h after the first dose. Data are presented as the mean ± standard deviation. The open circles indicate the individual concentration data.Figure 2.Simulated plasma concentration data in dialysis patients following a single dose of 150 mg/100 mg nirmatrelvir/ritonavir. Long dashed line represents patients not on dialysis. Short dashed line represents patients receiving a 4 h - dialysis. Conclusion 150 mg/100 mg twice a day dose regimen of nirmatrelvir/ritonavir produced sufficient concentrations to reach the effective target in COVID-19 patients with HD. Simulation data suggested that a modified dose with 150 mg/100 mg once a day could also reach effective concentration. Our data provide evidence for the application of nirmatrelvir/ritonavir in patients with ESRD and further validation in large-scale investigation was needed. Disclosures All Authors: No reported disclosures

Mobility-Modulated Sequential Dissociation Analysis Enables Structural Lipidomics in Mass Spectrometry Imaging.

Spatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure-resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility-modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility-enabled data-independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18 : 1_18 : 1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.

Mobility‐Modulated Sequential Dissociation Analysis Enables Structural Lipidomics in Mass Spectrometry Imaging

AbstractSpatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure‐resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility‐modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility‐enabled data‐independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18 : 1_18 : 1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.

Fast and Sensitive Analysis of Fosfomycin in Human Plasma Microsamples Using Liquid Chromatography-Tandem Mass Spectrometry for Therapeutic Drug Monitoring.

Fosfomycin is an antibiotic recently repurposed as a potential combination treatment for difficult-to-treat Gram-negative bacterial infections. The pharmacokinetic features of fosfomycin have demonstrated that different pathophysiologic alterations may affect its exposure. Therapeutic drug monitoring may improve real-time management of fosfomycin therapy in different clinical scenarios. To develop and validate a fast and sensitive liquid chromatography - tandem mass spectrometry method for measuring fosfomycin in human plasma microsamples (3 µL). Analysis was preceded by a user-friendly pre-analytical single-step process performed via a rapid chromatographic run of 2.5 minutes, followed by negative electrospray ionization and detection on a high-sensitivity triple quadrupole tandem mass spectrometer operated in the multiple reaction monitoring mode. European Medicines Agency guidelines were used to validate the specificity, sensitivity, linearity, precision, accuracy, matrix effects, extraction recovery, limits of quantification, and stability of the analytical method. The new assay produced accurate (BIAS%: 0.9-9.1) and precise (coefficient of variation [CV]%: 8.1-9.5) measurements of fosfomycin over a concentration range of 1-1000 mg/L. Overall, analyte recovery was consistent (mean values: 91.2%-97.2%) at all tested concentration levels. The analyte was also stable in human plasma and the final extract under various storage conditions. The clinical applicability of the assay was confirmed through quantitation of plasma samples obtained from patients. A sensitive liquid chromatography - tandem mass spectrometry method for measuring fosfomycin in plasma was developed and validated according to the European Medicines Agency criteria. Quantitation of fosfomycin in clinical plasma samples confirmed that the assay is suitable for therapeutic drug monitoring in clinical scenarios.

Determination of Multi-Class Antimicrobial Residues and Antimicrobial Resistance in Cow Milk and Feces Samples during Withdrawal Period

The use of antimicrobials in livestock production and their effect on the development of antimicrobial resistance (AMR) is a global health problem for humans, animals and the environment. The aim of this study was to determine antimicrobial residue levels in milk and feces samples during the withdrawal period in dairy cattle administrated with a single dose of the drug, as well as to characterize the antimicrobial resistance patterns of Escherichia coli cultured from feces samples. In the study, dairy cows from three different farms in North Macedonia were included. Raw milk and feces samples were collected before drug administration (0 day) and on the 1st, 2nd, 3rd, 7th and 21st day after drug administration. The antimicrobial residues of oxytetracycline, enrofloxacin, amoxicillin, trimethoprim and procaine-benzylpenicillin were determined using a validated liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method involving stable isotopes. According to results obtained, the highest levels of analyzed antimicrobial residues were determined on the first day after drug administration, which then gradually decreased until their elimination (7th day). The highest AMR of E. coli (100%) was found in β-lactam antimicrobials. Less exposure to broad-spectrum antimicrobials could be an important factor for reduction of AMR on dairy farms.

Identification of degradation products of brivaracetam using liquid chromatography quadrupole time-of-flight tandem mass spectrometry: Degradation pathway elucidation.

Pyrrolidone-based drugs find widespread use in treating conditions such as epilepsy and Alzheimer's disease, and in various other medical applications. Brivaracetam, the latest generation of pyrrolidone drugs, has exhibited significant promise owing to chemical structure modifications. Its affinity to the SV2A receptor is double that of the previous-generation drug, levetiracetam. Consequently, brivaracetam holds substantial potential for diverse applications. As a novel drug not yet included in the pharmacopeias of developed nations, comprehensive analysis and research are necessary to guarantee its safe utilization in clinical settings. A liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC/QTOFMS) method has been developed to effectively separate, identify and characterize both the degradation products and process-related substances of brivaracetam. Stress testing of the sample was carried out following the guidelines outlined in ICH Q1A(R2). The structures of these impurities were identified through positive electrospray ionization QTOF high-resolution MS and NMR spectroscopy. Additionally, the formation mechanism of each degradation product is thoroughly discussed. Under the analytical conditions outlined in this paper, brivaracetam and its degradation products were effectively separated. Thirteen degradation products were detected and characterized, shedding light on their origins and degradation pathways. Among these, three degradation products align with previously reported impurities, and two unreported degradation products were synthesized and confirmed through NMR spectroscopy. The stress testing results revealed the instability of brivaracetam under acidic, alkaline, oxidative and thermal stress conditions, while it exhibited relative stability under photolytic stress conditions. The study developed an analytical method for brivaracetam that enabled the effective detection and separation of brivaracetam and its 13 degradation products. This method addresses a gap in both current domestic and foreign drug standards. The structures of all the major degradation products were characterized by high-resolution LC/QTOFMS, which is essential for quality control during the drug production process, stability evaluation and the establishment of proper storage conditions.

Dolutegravir quantification in wistar rat tissues following chronic administration

Dolutegravir (DTG) has been introduced into first line combination antiretroviral therapy (ART) for HIV/AIDS. Penetration of ART into HIV reservoirs is essential to prevent continuous replication of HIV. However, accumulation of DTG in HIV reservoirs could be contributing to the adverse effects reported. We have developed and applied liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to quantify DTG in wistar rat biological matrices following chronic DTG administration. DTG was detected using a Shimadzu 8040 triple quadrupole-mass spectrometer. The methods developed were in the concentration ranges of 17.5–8000 ng/mL for plasma and 15.5–16 680 ng/g for tissue matrices. Mean plasma DTG concentrations in the current study closely corresponded to plasma DTG levels reported in humans after chronic treatment. Plasma and tissue DTG concentrations were generally higher in females compared to male wistar rats, but these differences were nullified after correcting for body and organ size. Plasma DTG levels correlated with tissue DTG concentrations in the liver and gastrocnemius muscle tissue. Data suggest that body size – rather than sex – may be a major risk factor determining adverse outcomes of patients on the current DTG dosing strategy which does not account for differences in body mass. Furthermore, plasma DTG was not correlated with adipose tissue DTG concentration. This suggests that adipose may be a primary site for longer term inflammatory dysregulation and adverse outcome following DTG treatment.

Vitamin D Status in Bipolar Disorder.

Vitamin D status may impact acute affective symptomatology and the severity of symptoms in patients with bipolar disorder (BD). Therefore, this cross-sectional study analyzed 25(OH)D, 24,25(OH)2D, and the vitamin D metabolite ratio (VMR) in BD and correlated the results with clinical affective symptomatology and functionality. The inactive precursor 25(OH)D, and its principal catabolite 24,25(OH)2D, were measured simultaneously with a validated liquid chromatography-tandem mass spectrometry method in 170 BD outpatients and 138 healthy controls. VMR was calculated as follows: VMR = 100×(24,25(OH)2D/25(OH)D). The psychometric assessment comprised: Beck Depression Inventory-II, Hamilton Depression Rating Scale, Young Mania Rating Scale, Global Assessment of Functioning, and number of suicide attempts. We did not find a significant difference between patients and controls in the concentrations of 25(OH)D and 24,25(OH)2D. Additionally, the VMR was comparable in both groups. The calculations for the clinical parameters showed a negative correlation between the Young Mania Rating Scale and 24,25(OH)2D (r = -0.154, p = 0.040), as well as the Young Mania Rating Scale and the VMR (r = -0.238, p = 0.015). Based on the small effect size and the predominantly euthymic sample, further exploration in individuals with manic symptoms would be needed to confirm this association. In addition, long-term clinical markers and an assessment in different phases of the disease may provide additional insights.

A liquid chromatography electrospray ionization tandem mass spectrometry method for quantification of up to eighteen cannabinoids in hemp-derived products

A LC-ESI/MS/MS method was developed for quantification of up to eighteen cannabinoids, the maximum number published so far. A thorough study of published LC-ESI/MS/MS methods using triple quadrupole mass spectrometers revealed a possible misconception that multiple reaction monitoring (MRM) was able to definitively differentiate structural isomers of cannabinoids, especially Δ8-/Δ9-tetrahydrocannabinol (THC), which explained why many of those methods were developed for a limited number of cannabinoids, as small as two, and did not include Δ8-THC. In this study, the use of a quadrupole time-of-flight (QTOF) mass spectrometer for targeted analysis indicated that MRM could not definitively distinguish structural isomers of Δ9-THC, with a possible exception of cannabicyclol (CBL) for less accurate quantification, so their baseline separation was essential for their accurate quantification. After the developed method was successfully validated according to the ISO 17025 guidelines, it was further applied for the analysis of eighteen hemp-derived products, including drinks, water-soluble oils, topical serum, body lotion, face cream, lip balm, gummies, hard candy, coffee, snacks, and pet treats. The LOQ was 0.00008% (w/w) for drinks with the analysis of 12.5 mg/mL extracts, while the LOQ was 0.008% (w/w) for other samples because 125 μg/mL extracts were analyzed due to higher content of cannabinoids in non-drink samples. For the first-time, extraction recovery and matrix effect were tracked in real-time for each sample being analyzed, obtaining 92.9–106.3% and 91.3–120.2% in triplicate measurements, respectively, by spiking abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp, into each sample before extraction and ACBD-d3 into each sample after extraction.

Development of a novel UHPLC-MS/MS method for the quantification of corynoxeine: Application to pharmacokinetics and tissue distribution studies in normal and chronic unpredictable mild stress-induced depression rats

Corynoxeine, a natural active alkaloid found in Genus Uncaria, has been reported to have anti-depressant effects. In this study, a sensitive and efficient ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying corynoxeine in rat plasma and tissues was established, validated and applied to investigate the pharmacokinetics and tissue distribution differences between normal rats and chronic unpredictable mild stress (CUMS)-induced depression model rats following oral administration. All bio-samples were prepared by methanol protein precipitation method with theophylline as internal standard (IS). Chromatographic separation was conducted on an Agilent ZORBAX Eclipse Plus C18 column using mobile phase A (acetonitrile) and B (0.1% formic acid in water) in gradient elution mode with a flow rate of 0.3 mL/min. Mass spectrometric detection was performed in multiple-reaction monitoring mode with positive electrospray ionization source. The transitions of m/z 383.0→160.2 for corynoxeine and m/z 181.1→124.0 for IS were chosen for quantification. The method showed good linearity, stability, accuracy, precision, recovery, and non-significant matrix effect, which were within the acceptable ranges. The pharmacokinetic results revealed that the absorption and bioavailability of corynoxeine in depression rats decreased compared to normal rats. The tissue distribution of corynoxeine trended to be mostly in the intestine and stomach and the distribution of this compound in intestine tissue of depression rats was significantly increased compared to the normal rats. The pharmacokinetics and tissue distribution profiles of corynoxeine were altered in CUMS-induced depression rats compared to normal rats and these experimental findings could provide beneficial information to the mechanism research and clinical applications of corynoxeine.

338 A Survey of Mycotoxin Occurrence in Us Corn and Corn By-Product Feed Ingredients from 2018 to 2022 Harvest Years

Abstract The aim of this survey was to compare the occurrence and contamination levels of six major mycotoxin categories found in corn and corn by-product feed ingredients harvested in the United States from 2018 through 2022. Mycotoxins are secondary fungal metabolites that can negatively impact animal health and performance and have been routinely found in a variety of feedstuffs including, but not limited to, cereal grains, by-products, silages, dry hays and pasture grasses. Mycotoxin categories evaluated in this survey included: aflatoxins (AFLA), type A trichothecenes (A-Trich), type B trichothecenes (B-Trich), fumonisins (FUM), zearalenone (ZEN), and ochratoxin A (OTA). This study includes data from corn samples labeled as 2022 harvest and corn b-product feed ingredients submitted from October 2022 through March 2023 and were analyzed for 17 different metabolites via the Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method. Samples that tested above the limit of detection for each mycotoxin group were analyzed using the generalized linear mixed model procedure of SAS (SAS 9.4, Cary, NC) using harvest year as a fixed effect. The effect of harvest year on average contamination level for each mycotoxin is presented in Table 1. For 2022 harvest year, 260 corn samples and 66 corn by-product samples have been evaluated to date. Mean B-Trich, ZEN, FUM and AFLA levels (parts per billion, ppb) for corn grain samples were similar in 2022 as previous years (P > 0.05). However, for corn grain samples, FUM prevalence and concentration were numerically greater in 2022 than in 2021 and 2020. In the 66 samples of corn by-products, FUM was found in all the samples (100%) and B-Trich were found in the majority of the samples (97%). Mean concentration of FUM in 2022 corn by-product samples was greater (P < 0.001) compared with 2021. Preliminary results of this survey suggest mycotoxin occurrence and contamination remains similar to previous harvest years except for greater mean FUM concentration levels in corn and corn by-products feed ingredients.