Liquid Chromatography-tandem Mass Spectrometry Assay Research Articles

Pharmacokinetics, pharmacodynamics, and safety of single- and multiple-dose intravenous ceftobiprole in healthy Chinese participants

BackgroundCeftobiprole is a novel β-lactam cephalosporin with activity against Gram-positive and -negative bacteria. The aim of the present study was to investigate the pharmacokinetics (PK), pharmacokinetics/pharmacodynamics (PK/PD), safety and tolerance of ceftobiprole in Chinese participants, to evaluate this dosage regimen for the treatment of community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP) in China.MethodsThe use of ceftobiprole was investigated in a single-center, open-label, single- and multiple-dose study using 12 healthy Chinese participants (6 males and 6 females). Ceftobiprole plasma and urine concentrations were analyzed using a validated liquid chromatography-tandem mass spectrometry assay. The PK/PD characteristics of 500 mg ceftobiprole every 8 h at 1.5-, 2-, 3-, or 4-h infusion time were analyzed by Monte Carlo simulations (MCS).ResultsThe maximum plasma concentration of ceftobiprole was observed 2 h after dosage; its terminal half-life was about 3 h. Ceftobiprole was predominantly eliminated in urine, and the cumulative excretion in 24 h was >90%. There was no accumulation after multiple dosing. Both single and multiple doses were well tolerated, with no severe or serious adverse events (AEs). PK/PD analysis indicated that Staphylococcus pneumoniae (S. pneumoniae) and Staphylococcus aureus (S. aureus) were sensitive to ceftobiprole. About half of extended-spectrum β-lactamase (ESBL) non-producing Enterobacteriaceae are sensitive to ceftobiprole, according to PK/PD results of ceftobiprole. For Pseudomonas aeruginosa (P. aeruginosa), no regimen was found to be effective against strains.ConclusionsThe PK/PD results indicated that 500 mg ceftobiprole every 8 h at 2-h infusion time is expected to achieve good microbiological efficacy in the treatment of CAP and HAP in China.

Evaluation of potential metabolomic-based biomarkers of protein, carbohydrate and fat intakes using a controlled feeding study.

Objective biomarkers of dietary exposure are needed to establish reliable diet-disease associations. Unfortunately, robust biomarkers of macronutrient intakes are scarce. We aimed to assess the utility of serum, 24-h urine and spot urine high-dimensional metabolites for the development of biomarkers of daily intake of total energy, protein, carbohydrate and fat, and the percent of energy from these macronutrients (%E). A 2-week controlled feeding study mimicking the participants' habitual diets was conducted among 153 postmenopausal women from the Women's Health Initiative (WHI). Fasting serum metabolomic profiles were analyzed using a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for aqueous metabolites and a direct-injection-based quantitative lipidomics platform. Urinary metabolites were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy at 800MHz and by untargeted gas chromatography-mass spectrometry (GC-MS). Variable selection was performed to build prediction models for each dietary variable. The highest cross-validated multiple correlation coefficients (CV-R2) for protein intake (%E) and carbohydrate intake (%E) using metabolites only were 36.3 and 37.1%, respectively. With the addition of established dietary biomarkers (doubly labeled water for energy and urinary nitrogen for protein), the CV-R2 reached 55.5% for energy (kcal/d), 52.0 and 45.0% for protein (g/d, %E), 55.9 and 37.0% for carbohydrate (g/d, %E). Selected panels of serum and urine metabolites, without the inclusion of doubly labeled water and urinary nitrogen biomarkers, give a reliable and robust prediction of daily intake of energy from protein and carbohydrate.

Effects of Maternal Sucrose Consumption on Inflammation and Steroids in the Placenta and Fetal Brain

Consumption of sucrose (table sugar) is high in much of the world. The effects of a maternal diet high in sucrose on the placenta and fetal brain remain unknown. In rats, maternal consumption of sucrose at a human-relevant level has effects on the mother’s physiology and steroids, as well as long-lasting and sex-specific effects on the adult offspring’s brain and behavior. In the mothers, there are metabolic effects of sucrose intake, such as impaired glucose tolerance, increased liver lipids, and increased adipose inflammation. In rat dams, sucrose intake also decreases corticosterone levels in the blood but not in the brain. In the adult male offspring, preference for a high-sucrose diet and a high-fat diet increases due to maternal sucrose intake. In addition, maternal sucrose intake increases motivation for sugar rewards in a progressive ratio schedule of reinforcement in adult male offspring. In adult female offspring, corticosterone levels increase in the blood and brain as a result of maternal sucrose intake. In this study, we investigated the underlying mechanisms of the observed behavioral and endocrine effects in the adult offspring. Here, we examined cytokines and anti-inflammatory steroids in the placenta, amniotic fluid, and fetal blood and brain. In our model, we feed rat dams either a high-sucrose diet (26% of kCal) or an isocaloric, matched, control diet (1% sucrose) 10 weeks prior to and during gestation. At embryonic day 19 (E19), we collected maternal blood, placenta, amniotic fluid, fetal blood, and fetal brain. We use Palkovits punch to microdissect the placenta and fetal brain. Next, we use a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is highly precise and specific, to measure multiple steroids (e.g. corticosterone, progesterone, estradiol, allopregnanolone). The method is highly sensitive, and we can measure neurosteroids in multiple regions of the fetal brain (e.g. prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus). Moreover, we will examine steroidogenic enzymes and cytokines in the fetal brain and placenta. Preliminary data show distinct steroid patterns in amniotic fluid and fetal blood, as well as in different parts of the placenta.

A Sensitive LC-MS/MS Method for the Determination of Afatinib in Human Plasma and Its Application to a Bioequivalence Study.

A high performance liquid chromatography-tandem mass spectrometry assay for the determination of afatinib (AFT) in human plasma was established. A simple sample preparation of protein precipitation was used and separation was achieved on a C18 column by the gradient mixture of mobile Phase A of water (containing 0.1% ammonia) and the mobile Phase B of acetonitrile and water (V:V=95:5, containing 0.2% ammonia). The multiple reaction monitoring mode was used to monitor the precursor-to-production transitions of m/z 486.2→m/z 371.4 for AFT and m/z 492.2→m/z 371.3 for AFT-d6 (internal standard) at positive ionization mode. The calibration curve ranged from 0.100 to 25.0ng·mL-1 and the correlation coefficient was greater than 0.99. The intra- and inter-batch precision was less than or equal to 10.0%. Accuracy determined at four concentrations was in the range of 92.3-103.3%. In summary, our method was sensitive, simple and reliable for the quantification of AFT and was successfully applied to a bioequivalence study.

Establishment, Validation, and Initial Application of a Sensitive LC-MS/MS Assay for Quantification of the Naturally Occurring Isomers Itaconate, Mesaconate, and Citraconate.

Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that have been linked to metabolic disorders, but their functional relationships with itaconate are unknown. We aimed to establish a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of itaconate, mesaconate, citraconate, the pro-drug 4-octyl-itaconate, and selected TCA intermediates. The assay was validated for itaconate, mesaconate, and citraconate for intra- and interday precision and accuracy, extended stability, recovery, freeze/thaw cycles, and carry-over. The lower limit of quantification was 0.098 µM for itaconate and mesaconate and 0.049 µM for citraconate in 50 µL samples. In spike-in experiments, itaconate remained stable in human plasma and whole blood for 24 and 8 h, respectively, whereas spiked-in citraconate and mesaconate concentrations changed during incubation. The type of anticoagulant in blood collection tubes affected measured levels of selected TCA intermediates. Human plasma may contain citraconate (0.4–0.6 µM, depending on the donor), but not itaconate or mesaconate, and lipopolysaccharide stimulation of whole blood induced only itaconate. Concentrations of the three isomers differed greatly among mouse organs: Itaconate and citraconate were most abundant in lymph nodes, mesaconate in kidneys, and only citraconate occurred in brain. This assay should prove useful to quantify itaconate isomers in biomarker and pharmacokinetic studies, while providing internal controls for their effects on metabolism by allowing quantification of TCA intermediates.

Association of adalimumab trough concentrations and treatment response in patients with juvenile idiopathic arthritis

To assess the relationship of adalimumab trough concentrations and treatment response in paediatric patients with juvenile idiopathic arthritis (JIA). Monocentric cohort study of JIA patients treated with adalimumab. Clinical data and samples were collected during routine follow-up. Adalimumab trough concentrations were quantified by a novel liquid chromatography-tandem mass spectrometry assay. Anti-adalimumab antibodies were measured in samples with trough concentrations ≤5mg/l. Disease activity was evaluated using the clinical Juvenile Arthritis Disease Activity Score with 71 joint-count (cJADAS71). Response to adalimumab was defined according to recent international treat-to-target guidelines. 35 adalimumab trough samples were available from 34 paediatric patients with JIA. Although there was no significant difference in adalimumab dose, trough concentrations were significantly lower in patients with secondary failure (median 1.0 mg/l; IQR 1.0-5.3) compared with patients with primary failure (median 13.97 mg/l; IQR 11.81-16.67) or an adequate response (median 14.94 mg/l; IQR 10.31-16.19) to adalimumab. Adalimumab trough concentrations were significantly lower in JIA patients with secondary failure compared with patients with primary failure or an adequate response to adalimumab. Results suggest that trough concentration measurements could identify JIA patients who require increased adalimumab doses to achieve or maintain therapeutic drug concentrations.

A data-driven approach for the detection of internal standard outliers in targeted LC-MS/MS assays

Heavy-labelled internal standard (IS) compounds are commonly used in liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to control for stochastic and systematic variation. Identifying samples that suffer from unwanted variation is critically important in order to avoid factitiously inaccurate results. Current approaches for outlier detection typically employ arbitrary thresholds and ignore systematic drift. To improve this, we applied robust linear mixed-effects models (LMMs) to capture the within- and between-run variability in IS signal and generate data-driven acceptance ranges for routine use.Data from in-house LC-MS/MS assays for 25-hydroxyvitamin D3and D2 and prednisolone were retrospectively collected. The variation in the percentage deviation of the internal standard area from the mean of the calibrators was modelled through the use of robust LMMs. The fitted LMMs revealed significant positive drift in IS signal over the analytical runs for vitamin D, with slope coefficients of 0.118 (95% CI: 0.098, 0.138) and 0.192 (0.168, 0.215) for D3 and D2, respectively. In contrast, the models for prednisolone demonstrated a significant negative drift in IS signal, with a slope coefficient of −0.164 (−0.297, −0.036). Non-parametric, cluster bootstrap resampling enabled us to define acceptance ranges for use in future assays.Here, we have described a computational approach to extensively characterise the variation in IS signal in routinely-performed LC-MS/MS assays. This approach facilitates a robust quality assessment of IS outliers in routine practice and thus has the potential to improve patient safety. Importantly, this approach is applicable to other MS assays where linear variation in IS signal is observed.

Co-culture fermentation of Pediococcus acidilactici XZ31 and yeast for enhanced degradation of wheat allergens

Previous researchers have shown the potential of sourdough and isolated lactic acid bacteria in reducing wheat allergens. As the interactions of lactic acid bacteria with yeast is a key event in sourdough fermentation, we wished to investigate how yeast affects metabolism of lactic acid bacteria, thereby affecting protein degradation and antigenic response. In this study, three strains isolated from sourdough were selected for dough fermentation, namely Pediococcus acidilactici XZ31, Saccharomyces cerevisiae JM1 and Torulaspora delbrueckii JM4. The changes in dough protein during the fermentation process were studied. Protein degradation and antigenic response in dough inoculated with Pediococcus acidilactici XZ31 monoculture and co-culture with yeast were mainly evaluated by SDS-PAGE, immunoblotting, ELISA and Liquid chromatography-tandem mass spectrometry assay. The whole-genome transcriptomic changes in Pediococcus acidilactici XZ31 were also investigated by RNA sequencing. The results showed that water/salt soluble protein and Tri a 28/19 allergens content significantly decreased after 24 h fermentation. Co-culture fermentation accelerated the degradation of protein, and reduced the allergen content to a greater extent. RNA-sequencing analysis further demonstrated that the presence of yeast could promote protein metabolism in Pediococcus acidilactici XZ31 for a certain period of time. These results revealed a synergistic effect between Pediococcus acidilactici XZ31 and yeast degrading wheat allergens, and suggested the potential use of the multi-strain leavening agent for producing hypoallergenic wheat products.

Simultaneous liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application

A rapid and easy liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed, validated, and used to quantify amlodipine besylate and metoprolol succinate in human plasma. Hydrochlorothiazide was used as the internal standard. LC–MS/MS with electrospray ionization in the positive ionization mode was performed under multiple reaction monitoring to examine analytes, which were drawn from human plasma via liquid–liquid extraction using a mixture of diethyl ether and dichloromethane (6:4). The extracts were dried under a nitrogen stream at 40°C, after which the samples were dissolved in a mixture of methanol and water (80:20). The re-dissolved samples were injected onto a C18 column and eluted using a mixture of methanol and 0.4% formic acid (80:20) as the mobile phase. Linear standard calibration was performed, and lower limits of quantification were determined for each analyte. The developedmethod was fully validated with respect to selectivity and carryover, as well as intra-and inter-day accuracy and precision in accordance with guidelines of the United StateFood and Drug Administration and the European Medicines Agency. Mean analyte recovery was between 71.72% and 97.55%, and matrix effects exerted only a minor influence on precision.The validated method was applied in aclinicalbioequivalence study to evaluate the in vivo bioequivalence of twocommer-cialproducts containing 5 mg amlodipine and 50 mg metoprolol. This randomized, single-dose, two-treatment, three-period, two-sequence, open-label, crossover study involved 14 healthy subjects, and a two-week washout period between the two phases of the research was implemented. Standard pharmacokinetic parameters were calculated to compare a test product with Selomax®as the reference. How to Cite this Article Pubmed Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP. 2019; 10(2): 1-7. doi:10.5530/srp.2019.2.01 Web Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. http://www.sysrevpharm.org/?mno=302644896 [Access: March 31, 2021]. doi:10.5530/srp.2019.2.01 AMA (American Medical Association) Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP. 2019; 10(2): 1-7. doi:10.5530/srp.2019.2.01 Vancouver/ICMJE Style Tuyen NTL, Nghiem LQ, Tuan ND. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP. (2019), [cited March 31, 2021]; 10(2): 1-7. doi:10.5530/srp.2019.2.01 Harvard Style Tuyen, N. T. L., Nghiem, . L. Q. & Tuan, . N. D. (2019) liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. SRP, 10 (2), 1-7. doi:10.5530/srp.2019.2.01 Turabian Style Tuyen, Nguyen Thi Linh, Le Quan Nghiem, and Nguyen Duc Tuan. 2019. liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and Reviews in Pharmacy, 10 (2), 1-7. doi:10.5530/srp.2019.2.01 Chicago Style Tuyen, Nguyen Thi Linh, Le Quan Nghiem, and Nguyen Duc Tuan. Simultaneous liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. Systematic Reviews in Pharmacy 10 (2019), 1-7. doi:10.5530/srp.2019.2.01 MLA (The Modern Language Association) Style Tuyen, Nguyen Thi Linh, Le Quan Nghiem, and Nguyen Duc Tuan. Simultaneous liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and application. Systematic Reviews in Pharmacy 10.2 (2019), 1-7. Print. doi:10.5530/srp.2019.2.01 APA (American Psychological Association) Style Tuyen, N. T. L., Nghiem, . L. Q. & Tuan, . N. D. (2019) liquid chromatography-tandem mass spectrometry assay of amlodipine besylate and metoprolol succinate in human plasma: Development, validation, and Reviews in Pharmacy, 10 (2), 1-7. doi:10.5530/srp.2019.2.01

A validated surrogate analyte LC-MS/MS assay for quantification of endogenous cortisol in human whole blood

Cortisol is a steroid hormone that is frequently measured as a marker of stress, inflammation, and immune function. While commonly analyzed in saliva, hair, blood plasma and urine, a recent trend towards whole blood-based at-home collection devices has emerged, which necessitates development of more sensitive assays for cortisol in whole blood. To support the implementation of a patient-centric sampling approach in a drug development program, a fit-for-purpose surrogate analyte-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for cortisol in whole blood was developed using 13C3-cortisol as a surrogate analyte and cortisol-d6 as the internal standard. The surrogate analyte approach was chosen due to a lack of available cortisol-free whole blood and the absence of appropriately representative surrogate matrices. Samples were prepared using supported liquid extraction, and the LC-MS/MS analysis consisted of a 4.00 min analytical run. The method demonstrated linearity between 0.500 and 500 ng/mL of 13C3-cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and 13C3-cortisol-based quality control (QC) samples when quantified against a 13C3-cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a 13C3-cortisol-based calibration curve also suggests parallelism between the analytes. These results indicate a viable surrogate analyte method, that is fit-for-purpose to analyze whole blood cortisol levels using a surrogate analyte LC-MS/MS approach. Evaluation of patient samples showed very promising comparability between whole blood and plasma cortisol concentrations, suggesting that whole blood could be used in place of or in addition to a plasma-based sampling protocol in clinical trials analyzing cortisol. Overall, this method presents a novel tool that is a first step in supporting the trend towards sample miniaturization and at-home sample collection, and may be readily used in clinical and diagnostic settings.

An Ultraperformance Liquid Chromatography Tandem-Mass Spectrometry Method for Determination of Multiclass Pharmaceuticals in Water Sample by Dispersive Liquid-Liquid Microextraction Combined with Ultrasound Assisted Reverse Extraction from Solidified Floating Organic Droplets.

A novel, simple, and reliable ultraperformance liquid chromatography tandem-mass spectrometry (UPLC–MS/MS ) assay based on dispersive liquid–liquid microextraction followed by ultrasound-assisted reverse extraction from solidified floating organic droplets was established for determination of multiclass pharmaceuticals in the water sample. Six commonly used drugs of various therapeutic classes: ibuprofen, ketorolac, lamotrigine, propranolol, pantoprazole, and losartan were extracted from water samples by using 50 μL 1-undecanol as extracting solvent and 400 μL acetonitrile as dispersive solvent. After collecting the floating organic droplets by cold centrifugation, an ultrasound-assisted back extraction procedure was performed to make the sample compatible for UPLC–MS/MS analysis. Acquity BEH C18 column (2.1 × 100; 1.7 μm) was used for separation of target analytes that were eluted by a gradient mobile phase composition of 15 mM ammonium acetate and acetonitrile at a flow rate of 0.25 mL/min. The sample ionization was performed by using electrospray ionization in positive mode, and multiple reaction monitoring was used for quantification of target analytes. After optimizing the assay conditions, all calibration curves were found to be linear with limit of detection and limit of quantification were ranged in between 0.06–0.15 and 0.16–0.41 ng/mL, respectively. The enrichment factor was found to be 172–192-fold and the relative recovery was ranged between 93.1 and 109.4% between target analytes. These satisfactory results confirmed that the proposed method is specific and reliable for application of trace analysis of target analytes in waste water samples.

Development and validation of a combined liquid chromatography tandem-mass spectrometry assay for the quantification of aprepitant and dexamethasone in human plasma to support pharmacokinetic studies in pediatric patients

A pharmacokinetic study was set up to investigate the pharmacokinetics of the anti-emetic agents aprepitant and dexamethasone and the drug-drug interaction between these drugs in children. In order to quantify aprepitant and dexamethasone, a liquid chromatography-tandem mass spectrometry assay was developed and validated for the simultaneous analysis of aprepitant and dexamethasone. Protein precipitation with acetonitrile-methanol (1:1, v/v) was used to extract the analytes from plasma. The assay was based on reversed-phase chromatography coupled with tandem mass spectrometry detection operating in the positive ion mode. The assay was validated based on the guidelines on bioanalytical methods by the US Food and Drug Administration and European Medicines Agency. The calibration model was linear and a weighting factor of 1/concentration2 was used over the range of 0.1–50 ng/mL for aprepitant and 1–500 ng/mL for dexamethasone. Intra-assay and inter-assay bias were within ±20% for all analytes at the lower limit of quantification and within ±15% at remaining concentrations. Dilution integrity tests showed that samples exceeding the upper limit of quantification can be diluted 100 times in control matrix. Stability experiments showed that the compounds are stable in the biomatrix for 25 h at room temperatures and 89 days at −20 °C. This assay is considered suitable for pharmacokinetic studies and will be used to study the drug-drug interaction between aprepitant and dexamethasone in pediatric patients.

Ultra performance liquid chromatography–tandem mass spectrometry assay for the quantification of RNA and DNA methylation

Previous studies have reported that nucleic acid methylation is a critical element in cardiovascular disease, and most studies mainly focused on sequencing and biochemical research. Here we developed an Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method for the quantification analysis of the dissociative epigenetic modified nucleosides (5mdC, 5mrC, m6A) in Myocardial Infarction (MI) SD rats from different periods (1 week, 4 weeks, 8 weeks) after the surgery. The samples for analysis were obtained from heart tissue and blood of the rats. All the quantification results are compared with the sham-operated group. Total RNA and DNA were isolated by enzymatic hydrolytic methods before the UPLC-MS/MS analysis. The statistical analysis demonstrates the dynamic changes of modified nucleosides in MI rats, and it showed good specificity, accuracy, stability and less samples were needed in the method. In this paper, we discovered that the concentration of 5mdC, 5mrC, m6A from heart tissue significantly increased at 8 weeks after the surgery. Furthermore, UPLC-MS/MS helps us observe the similar change of the concentration of those 3 methylated biomarkers in peripheral blood after 8 weeks. The result shows that the dynamic process of those 3 methylated biomarkers in peripheral blood is related to the content of methylated biomarkers from the heart tissue. Based on the scientific evidence available, we proved that the methylation of genetic materials in peripheral blood is similar to myocardial infarction tissue. The relation between them indicates that peripheral blood could be a promising alternative to the heart tissue which monitor the level of methylation and MI diagnosis-aided.

Simultaneous analysis of E1 and E2 by LC-MS/MS in healthy volunteers: estimation of reference intervals and comparison with a conventional E2 immunoassay

Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC–MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4–1500 pmol/L for E1 and 4–2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC–MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC–MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.

Abstract PO029: Oral contraceptive use and postmenopausal sex steroid hormone metabolism

Abstract OBJECTIVE: Oral contraceptive (OC) use is common in the United States and high-profile articles have renewed research interest in OC use and cancer risk. Several mechanisms have been proposed to explain how OC use influences postmenopausal cancer risk; one of the leading hypotheses is that OC use fundamentally alters feedback in the hypothalamic-pituitary-gonad axis, leading to long term changes in sex steroid hormone metabolism. However, women who use OCs are also unique with respect to factors that may affect both hormone metabolism and cancer risk. No studies have comprehensively commented on hormone levels in postmenopausal women who did and did not use OCs. DESIGN: We examined differences in circulating markers of sex steroid hormone metabolism associated with prior use of OCs among a subcohort of postmenopausal women from the Women’s Health Initiative Observational Study (983 women with at least one intact ovary and not currently using menopausal hormone therapy). MATERIALS AND METHODS: Using highly sensitive liquid chromatography–tandem mass spectrometry assays, we measured over 30 markers of estrogen and androgen metabolism in study serum samples. We used linear regression (adjusted and weighted) to estimate geometric mean hormone levels, which were then converted into relative percent differences. We controlled for potential confounders and stratified by: lifetime ovulatory cycles, body mass index, and parity (the focus of this abstract). RESULTS: Women with a history of OC use (n=346) had lower levels of estrogen metabolites than women who never used OCs (n=637), particularly when limiting this comparison among parous women (n=158 nulliparous women, n=825 parous women). In parous women, OC use was associated with reductions in total estrone (-18.5%, 95% confidence interval [CI] -31.1, -3.7%) and in estrogen metabolism overall: 2-hydroxylation metabolites were reduced by -18.5% (CI -28.2, -7.7%), 4-hydroxylation metabolites by -19.9% (CI -29.4, -9.1%), and 16-alpha-hydroxylation metabolites by -24.0% (CI -34.2, -12.2%). Hormone differences associated with OC use among nulliparous women were imprecise. When we made comparisons to nulliparous women who did not use OCs, women who both used OCs and were parous had lower testosterone (-21.2%, CI -33.1, -7.2%) and higher dihydrotestosterone relative to testosterone (its metabolic precursor; 16.5%, CI% -0.6, 36.6). However, aside from lower total estrone (-25.5%, CI -44.6, 0.2%), estrogen metabolism was not different between these groups. CONCLUSIONS: We saw evidence suggesting estrogen metabolism, as reflected in circulating markers, may be lower in postmenopausal women who previously used OCs. These effects were strongest among parous women, likely due to sufficient sample size, rather than any synergistic effect attributable to both using OCs and giving birth. These results support the idea that OC users have lower estrogen levels, but this should be evaluated in other age groups, as differences in hormone metabolism early in the lifecourse may lead women to use OCs. Citation Format: Kara A. Michels, Sally B. Coburn, Garnet Anderson, Louise A. Brinton, Chu Chen, Roni T. Falk, Margery L. Gass, Ashley M. Geczik, JoAnn E. Manson, Ruth M. Pfeiffer, Kerryn Reding, Gloria E. Sarto, Nicolas Wentzensen, Robert A. Wild, Xia Xu, Britton Trabert. Oral contraceptive use and postmenopausal sex steroid hormone metabolism [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO029.

Diagnostic accuracy of a liquid chromatography-tandem mass spectrometry assay in small hair samples for rifampin-resistant tuberculosis drug concentrations in a routine care setting

BackgroundTreatment monitoring of drug-resistant tuberculosis (DR-TB) in resource-limited settings is challenging. We developed a multi-analyte assay for eleven anti-TB drugs in small hair samples as an objective metric of drug exposure.MethodsSmall hair samples were collected from participants at various timepoints during directly observed RR-TB treatment at an inpatient tertiary referral facility in South Africa (DR-TB cohort). We assessed qualitative determination (i.e., detection above limit of detection) of bedaquiline, linezolid, clofazimine, pretomanid, levofloxacin, moxifloxacin, pyrazinamide, isoniazid, ethambutol, ethionamide, and prothionamide in an LC-MS/MS index panel assay against a reference standard of inpatient treatment records. Because treatment regimens prior to hospitalization were not available, we also analyzed specificity (for all drugs except isoniazid) using an external cohort of HIV-positive patients treated for latent TB infection with daily isoniazid (HIV/LTBI cohort) in Uganda.ResultsAmong the 57 DR-TB patients (58% with pre-XDR/XDR-TB; 70% HIV-positive) contributing analyzable hair samples, the sensitivity of the investigational assay was 94% or higher for all drugs except ethionamide (58.5, 95% confidence interval [CI], 40.7–99.9). Assay specificity was low across all tested analytes within the DR-TB cohort; conversely, assay specificity was 100% for all drugs in the HIV/LTBI cohort.ConclusionsHair drug concentrations reflect long-term exposure, and multiple successive regimens commonly employed in DR-TB treatment may result in apparent false-positive qualitative and falsely elevated quantitative hair drug levels when prior treatment histories within the hair growth window are not known.

Absolute quantitative analysis of endogenous neurotransmitters and amino acids by liquid chromatography-tandem mass spectrometry combined with multidimensional adsorption and collision energy defect

Considering that neurotransmitters (NTs) and amino acids (AAs) exert pivotal roles in various neurological diseases, global detection of these endogenous metabolites is of great significance for the treatment of nervous system diseases. Herein, a workflow that could cope with various challenges was proposed to establish an extendable all-in-one injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for analyzing these small molecular metabolites with high coverage. To obtain a qualified blank biological matrix for the preparation of standard curves and quality control samples, different absorption solvents, including activated carbon (AC), calcite (Cal) and montmorillonite (Mnt) were systematically evaluated for efficient absorption of endogenous substances with minimum residue. We also firstly proposed a “Collision Energy Defect (CED)” strategy to solve the huge difference of mass signal strength caused by different properties and concentrations of 11 NTs and 17 AAs. The quantitative results were validated by LC-MS/MS. Sensitivity, accuracy, and recovery meeting generally accepted bioanalytic guidelines were observed in a concentration span of at least 100 to 500 times for each analyte. Then the temporal changes of intracerebral and peripheral NTs and AAs in ischemic stroke model and sham operated rats were successfully produced and compared using the described method. All these results suggested that the currently developed assay was powerful enough to simultaneously monitor a large panel of endogenous small molecule metabolites, which was expected to be widely used in the research of various diseases mediated by NTs and AAs.

A Sensitive and Specific Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Quantification of Salivary Melatonin and Cortisol: Development and Comparison With Immunoassays.

Melatonin and cortisol are clinically important for diagnosing sleep and mood disorders. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of salivary melatonin and cortisol concentrations according to the Clinical and Laboratory Standards Institute guidelines. Additionally, we compared the LC-MS/MS assay with immunoassays, ELISA (Direct Salivary Melatonin Elisa EK-DSM, Bühlmann Laboratories AG, Schönenbuch, Switzerland) and electrochemiluminescence immunoassay (Cortisol II, Roche, Mannheim, Germany), using 121 saliva samples. The LC-MS/MS assay exhibited good performance in terms of linearity, precision, accuracy, limit of detection, lower limit of quantification, extraction recovery, carry-over, and matrix effect. The LC-MS/MS assay and immunoassays showed strong correlation (Pearson’s r=0.910 for melatonin, r=0.955 for cortisol), but demonstrated a significant mean bias of 23.2% (range 54.0–143.7%) for melatonin and 48.9% (range 59.7–184.7%) for cortisol. Our LC-MS/MS assay provided more sensitive and reliable salivary melatonin and cortisol quantification results compared with immunoassays.

Hexosamine biosynthetic pathway promotes the antiviral activity of SAMHD1 by enhancing O-GlcNAc transferase-mediated protein O-GlcNAcylation.

Rationale: Viruses hijack the host cell machinery to promote viral replication; however, the mechanism by which metabolic reprogramming regulates innate antiviral immunity in the host remains elusive. Herein, we explore how the hexosamine biosynthesis pathway (HBP) and O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulate host antiviral response against hepatitis B virus (HBV) in vitro and in vivo.Methods: We conducted a metabolomics assay to evaluate metabolic responses of host cells to HBV infection. We systematically explored the role of HBP and protein O-GlcNAcylation in regulating HBV infection in cell and mouse models. O-linked N-acetylglucosamine (O-GlcNAc) target proteins were identified via liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation assays. Additionally, we also examined uridine diphosphate (UDP)-GlcNAc biosynthesis and O-GlcNAcylation levels in patients with chronic hepatitis B (CHB).Results: HBV infection upregulated GLUT1 expression on the hepatocyte surface and facilitated glucose uptake, which provides substrates to HBP to synthesize UDP-GlcNAc, leading to an increase in protein O-GlcNAcylation. Pharmacological or transcriptional inhibition of HBP and O-GlcNAcylation promoted HBV replication. Mechanistically, O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation of sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) on Ser93 stabilizes SAMHD1 and enhances its antiviral activity. Analysis of clinical samples revealed that UDP-GlcNAc level was increased, and SAMHD1 was O-GlcNAcylated in patients with CHB.Conclusions: HBP-mediated O-GlcNAcylation positively regulates host antiviral response against HBV in vitro and in vivo. The findings reveal a link between HBP, O-GlcNAc modification, and innate antiviral immunity by targeting SAMHD1.

An occurrence of neurotoxic shellfish poisoning by consumption of gastropods contaminated with brevetoxins

Brevetoxins were confirmed in urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) after consumption of gastropods that were recreationally harvested from an area previously affected by a Karenia brevis bloom. Several species of gastropods (Triplofusus giganteus, Sinistrofulgur sinistrum, Cinctura hunteria, Strombus alatus, Fulguropsis spirata) and one clam (Macrocallista nimbosa) from the NSP implicated gastropod collection area (Jewfish Key, Sarasota Bay, Florida) were examined for brevetoxins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). All gastropods and the clam were contaminated with brevetoxins. Composite B-type toxin concentrations in gastropods ranged from 1.1 to 198 μg BTX-3 equiv./g by ELISA, levels likely capable of causing NSP in consumers. Several brevetoxin metabolites previously characterized in molluscan shellfish were identified in these gastropods. Brevetoxin analog profiles by ELISA were similar in the gastropod species examined. This work documents the occurrence of NSP through consumption of a type of seafood not typically monitored in Florida to protect human health, demonstrating the need to better assess and communicate the risk of NSP to gastropod harvesters in Karenia brevis endemic areas.