HPLC-tandem Mass Spectrometry Research Articles

Limited appearance of apocarotenoids is observed in plasma after consumption of tomato juices: a randomized human clinical trial

Nonvitamin A apocarotenoids occur in foods. Some function as retinoic acid receptor antagonists in vitro, though it is unclear if apocarotenoids are absorbed or accumulate to levels needed to elicit biological function. The aim of this study was to quantify carotenoids and apocarotenoids (β-apo-8'-, -10'-, -12'-, and -14'-carotenal, apo-6'-, -8'-, -10'-, -12'-, and -14'-lycopenal, retinal, acycloretinal, β-apo-13-carotenone, and apo-13-lycopenone) in human plasma after controlled consumption of carotenoid-rich tomato juices. Healthy subjects (n=35) consumed a low-carotenoid diet for 2 wk, then consumed 360 mL of high-β-carotene tomato juice (30.4 mg of β-carotene, 34.5 μg total β-apocarotenoids/d), high-lycopene tomato juice (42.5 mg of lycopene, 119.2 μg total apolycopenoids/d), or a carotenoid-free control (cucumber juice) per day for 4 wk. Plasma was sampled at baseline (after washout) and after 2 and 4 wk, and analyzed for carotenoids and apocarotenoids using high-pressure liquid chromatography (HPLC) and HPLC-tandem mass spectrometry, respectively. The methods used to analyze the apocarotenoids had limits of detection of ∼ 100 pmol/L. Apocarotenoids are present in tomato juices at 0.1-0.5% of the parent carotenoids. Plasma lycopene and β-carotene increased (P<0.001) after consuming high-lycopene and β-carotene tomato juices, respectively, while retinol remained unchanged. β-Apo-13-carotenone was found in the blood of all subjects at every visit, although elevated (P<0.001) after consuming β-carotene tomato juice for 4 wk (1.01±0.27 nmol/L) compared with both baseline (0.37±0.17 nmol/L) and control (0.46±0.11 nmol/L). Apo-6'-lycopenal was detected or quantifiable in 29 subjects, while β-apo-10'- and 12'-carotenal were detected in 6 and 2 subjects, respectively. No other apolycopenoids or apocarotenoids were detected. β-Apo-13-carotenone was the only apocarotenoid that was quantifiable in all subjects, and was elevated in those consuming high-β-carotene tomato juice. Levels were similar to previous reports of all-trans-retinoic acid. Other apocarotenoids are either poorly absorbed or rapidly metabolized or cleared, and so are absent or limited in blood. β-Apo-13-carotenone may form from vitamin A and its presence warrants further investigation. This trial was registered at clinicaltrials.gov as NCT02550483.

Measurement of Succinylacetone Using HPLC-Tandem Mass Spectrometry and Establishment of a Cut-off Value

Measurement of Succinylacetone Using HPLC-Tandem Mass Spectrometry and Establishment of a Cut-off Value

Modular organization analysis of specific naringin/neoeriocitrin related gene expression induced by UVC irradiation in Drynaria roosii

Drynaria roosii is a traditional medicinal fern, whose rhizome is particularly valued for its effective flavonoid components of naringin/neoeriocitrin. UV irradiation can induce flavonoid accumulation in plants, however the molecular mechanism of UVC-mediated naringin/neoeriocitrin synthesis in D. roosii has never been reported. Our HPLC-tandem mass spectrometry (MS/MS) results showed that UVC irradiation increased naringin/neoeriocitrin contents (NNCs) based on irradiation dosage in D. roosii. According to the NGS data, DEG-based heat map analysis revealed up-regulation of naringin/neoeriocitrin synthetic genes was more obvious in 12h UVC irradiation (UV dose = 81.648 kJ m−2) when compared with 24h (UV dose = 163.296 kJ m−2), especially for PAL, C3’H and HCT in old rhizomes. Through systems biology method of modular organization analysis, we clustered 15,678 DEGs into 19 modules, and calculated correlation coefficients between modules and samples, as well as modules and NNCs. Four significant naringin-related modules were achieved and displayed high correlations with specific samples. Moreover, weighted gene co-expression network analysis results discovered that 4CL, CHS and HCT, C3H acted as the hub responsive genes to UVC irradiation involved in naringin and neoeriocitrin synthesis respectively, and presented high co-expression with MYB/bHLH-regulated DEGs. Overall, we demonstrated that UVC irradiation up-regulated the naringin/neoeriocitrin related gene expression in separate new and old rhizomes to enhance NNCs in a dose-dependent manner. Hormetic UVC dose ranges improving NNCs in D. roosii were established for the first time. Our work also provided new insights into the study of secondary metabolites in medicinal plants.

6-Formylindolo[3,2-b]carbazole (FICZ) is a Very Minor Photoproduct of Tryptophan at Biologically Relevant Doses of UVB and Simulated Sunlight.

Exposure to solar UV is at the origin of numerous photodegradation pathways in biomolecules. Tryptophan is readily modified by UVB radiation into ring-opened and oxidized photoproducts. One of them, 6-formylindolo[3,2-b]carbazole (FICZ), has been extensively studied in the recent years because it very efficiently binds to AhR, a major factor in numerous biologic processes, such as metabolism of xenobiotics. Unfortunately, little information is available on the actual yield of FICZ upon exposure to low and biologically relevant doses of UV radiation. In the present work, we used a sensitive and specific HPLC-tandem mass spectrometry assay to quantify a series of photoproducts induced by UVB and simulated sunlight (SSL) in solutions of tryptophan. FICZ represented only a minute amount of the photoproducts (0.02 and 0.03%, respectively). Experiments were repeated in culture medium where the yield of FICZ was also found to be very low, even when Trp was added. Last, no FICZ could be detected in cytosolic fractions of cultured cells exposed to SSL. Altogether, the present results show that FICZ is a very minor photoproduct and that it cannot be considered the only endogenous photoproduct responsible for the induction of AhR-dependent responses in UV-irradiated cells.

Identification of food-derived peptides in human blood after ingestion of corn and wheat gluten hydrolysates

Bioactive peptides in the body after ingestion of plant protein hydrolysates have been speculated but not identified. We aimed to establish an approach to identify small amounts of food-derived peptides in humans after ingestion of non-extracellular matrix protein hydrolysates. Corn and wheat gluten hydrolysates were digested using pancreatin and leucine aminopeptidase; the resultant peptides were identified via size-exclusion chromatography and reverse-phase HPLC-tandem mass spectrometry (MS/MS). Structures of indigestible peptides were confirmed via LC-MS/MS in multi-reaction monitoring mode. All indigestible peptides in the exopeptidase digest were diprolyl and di- and tripyroglutamyl peptides. Blood collected from healthy volunteers (n = 4) before and after ingestion of 9 g of the hydrolysates was assessed for the indigestible peptides via LC-MS/MS. Six peptides (Pro-Ala, Pro-Gly, Pro-Gln, pyroGlu-Pro, pyroGlu-Leu-Pro, and pyroGlu-Gln-Pro) significantly increased in human plasma up to 10–100 nM compared to the baseline. This may hence be a powerful tool for identifying foodderived peptides in blood.

The Free Hormone Hypothesis: Is Free Serum 25-Hydroxyvitamin D a Better Marker for Bone Mineral Density in Older Women?

ABSTRACTIt is presently unclear whether free serum 25‐hydroxyvitamin D (S‐25(OH)D) better reflects bone health than total S‐25(OH)D. We have previously shown that summer total S‐25(OH)D values are more useful to predict bone mineral density (BMD) than winter values. Our objective was therefore to compare the relative importance of free and total S‐25(OH)D for BMD by season. BMD was measured by dual‐energy X‐ray absorptiometry (DXA) in 5002 Swedish women (mean age 68 years) randomly selected from a large population‐based longitudinal cohort study. Free S‐25(OH)D was analyzed by a commercial ELISA and total S‐25(OH)D by HPLC–tandem mass spectrometry (MS/MS). Free and total S‐25(OH)D co‐varied with season, with 26% and 29% higher values in August compared with those in January–March (nadir). There were no differences in mean BMD between categories of free or total S‐25(OH)D in samples collected during winter. Women with higher total S‐25(OH)D measured during summer had higher BMD at the total hip. Compared with women who had total S‐25(OH)D values above 80 nmol/L during summer, adjusted BMD at the total hip was 6% (95% CI, 1% to 11%) lower for S‐25(OH)D concentrations between 30 and 40 mmol/L, and 11% (95% CI, 3% to 19%) lower for those with total S‐25(OH)D <30 nmol/L. In contrast, free S‐25(OH)D measured during summer was not associated with BMD. Compared with women who had highest free S‐25(OH)D measured during summer (>8.8 pmol/L), those with intermediate (2.4–3.5 pmol/L) and lowest (<2.4 pmol/L) free S‐25(OH)D during summer did not have lower total hip BMD values (3% [95% CI, −2% to 7%] and −2% [95% CI, −8% to 4%]). In addition, we found no added value for the prediction of BMD with the combined measurement of total and free S‐25(OH)D during summer or winter. We conclude that vitamin D status assessed by direct measurements of free S‐25(OH)D does not reflect BMD better than total S‐25(OH)D. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Residue concentration of cefquinome after intramammary dry cow therapy and short dry periods

Short dry periods and their effects on milk production, reproductive performance, as well as cow and udder health have been widely studied. A dearth of information is available about the consequences of short dry periods on the residue concentrations of dry cow antibiotics in milk after calving. The objective of our study was to determine the residue concentration of a dry cow antibiotic in milk after short dry periods during the colostrum period and early lactation. Quarters of 19 dry cows were treated with an intramammary (IMM) dry cow antibiotic containing 150 mg of cefquinome on d 21, 14, and 7 before calculated calving date. One quarter of each cow did not receive treatment and served as negative control. After calving, quarter foremilk samples were collected twice daily until 21 d and once daily until 36 d after IMM dry cow treatment (i.e., end of withdrawal period). A total of 588 foremilk samples from odd milking numbers were chosen for the determination of the residue concentration of cefquinome using HPLC-tandem mass spectrometry until the residue concentration fell below the limit of quantification (1 ng/g), which occurred at the latest in milking number 37. The dry period length of the treated quarters was categorized in 3 dry period groups ranging from 1 to 7 d (4.8 ± 2.4), 8 to 14 d (11.5 ± 2.3), and 15 to 26 d (19.5 ± 3.3; ±SEM), in dry period group 1, 2, and 3, respectively. In dry period group 1, the cefquinome concentration increased after calving until the third milking and decreased considerably until the fifth milking. In dry period group 2, the cefquinome concentration peaked at the second milking and decreased considerably until the fifth milking as well. There was no increase in cefquinome after calving in dry period group 3. Up to the 37th milking, the cefquinome concentration was higher in dry period group 1 than in dry period group 2 and 3. On average, 31.3 ± 1.2, 19.0 ± 1.1, and 6.7 ± 0.8 milkings and 19.4 ± 0.4, 20.6 ± 0.5, and 24.1 ± 0.7 d after treatment were necessary for the concentration of cefquinome to fall below the maximum residue limit (MRL) in dry period group 1, 2, and 3, respectively. These results indicate that shorter dry periods lead initially to higher cefquinome residues in milk. The residue concentration after experimental short dry periods still falls below the MRL within the recommended withdrawal period for milk of 36 d after IMM dry cow treatment. For the sake of food safety and economics, these short dry periods should not be used in the dry cow management, as they lead up to a maximum of 31.3 ± 1.2 milkings and 19.4 ± 0.4 d after treatment with cefquinome residues above the MRL. Therefore, a considerable number of milkings have to be discarded due to long withdrawal periods after calving.

Full-Length Transcriptome Sequencing and Modular Organization Analysis of the Naringin/Neoeriocitrin-Related Gene Expression Pattern in Drynaria roosii.

Drynaria roosii (Nakaike) is a traditional Chinese medicinal fern, known as 'GuSuiBu'. The effective components, naringin and neoeriocitrin, share a highly similar chemical structure and medicinal function. Our HPLC-tandem mass spectrometry (MS/MS) results showed that the accumulation of naringin/neoeriocitrin depended on specific tissues or ages. However, little was known about the expression patterns of naringin/neoeriocitrin-related genes involved in their regulatory pathways. Due to a lack of basic genetic information, we applied a combination of single molecule real-time (SMRT) sequencing and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of D. roosii. According to the SGS data, the differentially expressed gene (DEG)-based heat map analysis revealed that naringin/neoeriocitrin-related gene expression exhibited obvious tissue- and time-specific transcriptomic differences. Using the systems biology method of modular organization analysis, we clustered 16,472 DEGs into 17 gene modules and studied the relationships between modules and tissue/time point samples, as well as modules and naringin/neoeriocitrin contents. We found that naringin/neoeriocitrin-related DEGs distributed in nine distinct modules, and DEGs in these modules showed significantly different patterns of transcript abundance to be linked to specific tissues or ages. Moreover, weighted gene co-expression network analysis (WGCNA) results further identified that PAL, 4CL and C4H, and C3H and HCT acted as the major hub genes involved in naringin and neoeriocitrin synthesis, respectively, and exhibited high co-expression with MYB- and basic helix-leucine-helix (bHLH)-regulated genes. In this work, modular organization and co-expression networks elucidated the tissue and time specificity of the gene expression pattern, as well as hub genes associated with naringin/neoeriocitrin synthesis in D. roosii. Simultaneously, the comprehensive transcriptome data set provided important genetic information for further research on D. roosii.

Optimization of HPLC-tandem mass spectrometry for chlortetracycline using response surface analysis

Optimization of HPLC-tandem mass spectrometry for chlortetracycline using response surface analysis

Addition of Exogenous γ-Glutamyl Hydrolase Eliminates the Need for Lengthy Incubation of Whole-Blood Lysate for Quantitation of Folate Vitamers by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC–tandem mass spectrometry (HPLC-MS/MS) in whole-blood lysate (WBL) requires lengthy incubation before analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether the addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated for 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid compared with sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood compared with frozen WBL or with frozen washed RBCs. Samples (n ≤ 15/experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved by using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, the extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to the non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBCs was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsThe use of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBCs may be due to inadequate packing of RBCs, among other unidentified factors. A larger study is required to confirm the lack of differences in folate concentrations determined with and without the use of exoGGH.

An HPLC tandem mass spectrometry for quantification of ET-26-HCl and its major metabolite in plasma and application to a pharmacokinetic study in rats

ET-26-HCl is a new analog of etomidate, a short-acting anesthetic drug, with less adrenal cortex inhibition. The pharmacokinetics of ET-26-HCl in rats needs to be determined for future clinical trials in human subjects. In order to facilitate the pharmacokinetic study, a liquid chromatography based tandem mass spectrometric (HPLC–MS/MS) method was developed and validated for quantification of ET-26-HCl and its major metabolite, ET-26-acid. These two compounds and gabapentin (internal standard) were extracted using a protein precipitation method with methanol and detected by Multiple Reaction Monitoring of m/z transition of 275.6–170.9, 217.7–113.1, and 172.5–154.3 for ET-26-HCl, ET-26-acid, and gabapentin respectively. This method was validated in terms of sensitivity, linearity, reproducibility, and stability. The HPLC–MS/MS method was found linear over the concentration ranges of 21.76–4352ng/mL, and 18.62–3724ng/mL with LLOQ of 21.76 and 18.62ng/mL for ET-26-HCl and ET-26-acid respectively. The mean intra-day and inter-day accuracy was between 94.11–107.78%, while the precision was within the limit of 15.0% for all the quality control samples. A pharmacokinetic study was then conducted in rats following intravenous injection of 2.1, 4.2, and 8.4mg/kg. The linear pharmacokinetics of ET-26-HCl was observed over the dose range of 2.1–8.4mg/kg. The average terminal phase elimination half-lives were 0.87 and 1.03h for ET-26-HCl and ET-26-acid respectively. In summary, an HPLC–MS/MS method for quantification of ET-26-HCl in rat plasma has been developed and successfully applied to a pharmacokinetic study.

Antioxidant, antityrosinase and antibiofilm activities of synthesized peptides derived from Vicia faba protein hydrolysate: A powerful agents in cosmetic application

The aim of this study was to identify peptides with antioxidant, antityrosinase and antibiofilm activities released from Vicia faba seed proteins hydrolysate. The hydrolysis pattern was performed with trypsin. Firstly, the obtained hydrolysate was fractionated by cation exchange chromatography and the fraction exhibiting the highest biological activity was then analyzed by reverse phase HPLC tandem mass spectrometry. After annotation by Peaks software (BSI, Canada) for de novo peptide sequencing, seven peptides were identified, mainly derived from storage proteins: legumin and vicilin. These peptides were further chemically synthesized to assess their antioxidant capacity, antityrosinase activity and antibiofilm ability against Pseudomonas aeruginosa PA14. Results showed that peptides P5, P6 and P7 identified as LSPGDVLVIPAGYPVAIK, VESEAGLTETWNPNHPELR and EEYDEEKEQGEEEIR respectively, displayed the highest DPPH radical scavenging activity (IC50=0.25–1.9mM). P5 was the only peptide able to chelate iron and reduce Fe3+ to Fe2+. Furthermore, peptides P4 (GPLVHPQSQSQSN) and P6 were observed as potent tyrosinase inhibitors with IC50 values of 1 and 0.14mM respectively. P1, P5, P6 and P7 also showed interesting antibiofilm activity against Pseudomonas aeruginosa PA14 with MBIC50 value ranged from 12 to 35μM. Our data suggest that V. faba seed proteins hydrolysate could be potentially used as a source of natural bioactive peptides for cosmetic and pharmaceutical applications.

The effect of allergen-induced bronchoconstriction on concentration of 5-oxo-ETE in exhaled breath condensate of house dust mite-allergic patients.

Arachidonic acid metabolites regulate several aspects of airway function including inflammation, muscle contraction and mucous secretion. The aim of this study was to evaluate concentration of selected 5-lipoxygenase- and cyclooxygenase-derived eicosanoids in exhaled breath condensate (EBC) during allergen-induced bronchoconstriction. The study was performed on 24 allergic rhinitis/asthma patients sensitized to a house dust mite (HDM) Dermatophagoides pteronyssinus (Dp) and 13 healthy controls (HCs). Bronchial challenge with Dp extract was performed only in the allergic patients. EBC samples were collected before (T0 ) and during Dp-induced bronchoconstriction (TEAR ). Eicosanoid concentration was measured using HPLC-tandem mass spectrometry. Significant bronchoconstriction after Dp challenge was demonstrated in 15 patients (Rs), while in 9 patients (NRs) no asthmatic response could be detected. At T0 the most abundant eicosanoids in EBC of HDM-allergic patients were LTB4 and 5-oxo-ETE, while in HCs EBC concentration of LTB4 was significantly greater than that of 5-oxo-ETE. Allergen challenge resulted in significant increase in EBC concentration of 5-oxo-ETE, LTD4 and 8-iso-PGE2 only in Rs. At TEAR , the relative change of 5-oxo-ETE concentration in EBC correlated with decrease of peripheral blood eosinophilia (R=-0.774; P=.0012). Moreover, the relative increase of 5-oxo-ETE in EBC at TEAR significantly correlated with the severity of the subsequent late asthmatic response (R=0.683, P=.007). Our study demonstrates significant up-regulation of 5-oxo-ETE synthesis in HDM-allergic patients and indicates possible involvement of that mediator in the pathogenesis of allergic asthma.

A Daily Dose of 5 mg Folic Acid for 90 Days Is Associated with Increased Serum Unmetabolized Folic Acid and Reduced Natural Killer Cell Cytotoxicity in Healthy Brazilian Adults

The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown. Objective: The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), interferon γ (IFNG), tumor necrosis factor α (TNFA), and interleukin 8 (IL8) genes; and concentrations of serum inflammatory markers. Methods: This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m2) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells. Results: Serum folate concentrations increased by ∼5-fold after the intervention (P < 0.001), and UMFA concentrations increased by 11.9- and 5.9-fold at 45 and 90 d, respectively, when compared with baseline (P < 0.001). UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number (P < 0.001) and cytotoxicity (P = 0.003) of NK cells after 45 and 90 d. Compared with baseline, DHFR mRNA expression was higher at 90 d (P = 0.006) and IL8 and TNFA mRNA expressions were higher at 45 and 90 d (P = 0.001 for both). Conclusion: This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.gov.br as RBR-2pr7zp.

A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.

Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.

Abstract 4205: Intracellular pharmacokinetics of 5FU and palbociclib: Uptake and efflux in disaggregated cells and 3D models

Abstract Introduction: Pre-clinical drug development does not routinely assess drug penetration beyond use of the human intestinal Caco-2 cell line as an in vitro model of the intestinal barrier to evaluate potential drug absorption. Detailed in vivo tumour pharmacokinetic (PK) studies are unusual. However, the ability of a drug to reach its target plays a key role in drug efficacy. Here we present a methodology to study drug PK in disaggregated cells and in a 3D cell culture model (spheroids) using 5FU and palbociclib. The comparison of these 2 drugs is of interest taking into account their different mechanisms and chemical properties, logP 0.58 and 2.12 respectively. Knowledge of cellular uptake and efflux in disaggregated cells and 3D models together with intracellular binding characteristics is vital to optimise a better drug delivery. Experimental procedures: MCF-7 and DLD-1 cells were used for cellular and spheroid experiments. Spheroid growth was optimized to produce a spheroid of 300-400µm diameter at day 4. Drug uptake: 1x106 cells or 30 spheroids/time point were exposed to 10µM 5FU or 0.1µM of palbociclib for different time durations. For 5FU, drug uptake at 100µM was also monitored to detect 5FU metabolites. Drug efflux: 1x106 cells were exposed first to 100µM 5FU or 0.1µM of palbociclib for 60min then, drug media removed, substituted by fresh media and sample over a 4h period. Drug concentration inside cells and in the fresh media was measured. Drug concentrations were measured using HPLC-Tandem Mass Spectrometry. Each experiment was carried out in triplicate. Results: 5FU achieves intracellular steady state within 5-10min in cells and spheroids with equal concentrations inside and outside the cell/spheroids. Intracellular metabolites of 5FU were only seen with 100µM treatment and had a similar PK characteristics to 5FU. 5FU drug efflux experiments showed that efflux occurred rapidly within 5min achieving a new equilibrium with 30% of 5FU remaining inside the cells. 5FUrd was also detected extracellularly after 1h in the fresh media, showing that cells have both 5FU and metabolite efflux. In contrast, palbociclib cellular uptake had an initial intracellular drug concentration peak at 5-10min which then plateaued. Interestingly, total intracellular concentrations of palbociclib were over 30 times higher compared to the external media drug at steady state. Spheroid and efflux experiments of palbociclib are on-going and will be presented. Conclusions: Our results suggest that both drugs penetrate quickly in cancer cells. Palbociclib intracellular concentrations were 30 times higher than the external media suggesting excessive intracellular binding while 5FU achieve equal concentrations between the inside and outside of the cell. These data and methodology may be useful to allow generation of mathematical models to improve drug delivery design. Citation Format: Maria Jove, Paul Loadman, Jade Spencer, Lava Sulayman, Jo Wicks, Amanda Race, Chris Twelves. Intracellular pharmacokinetics of 5FU and palbociclib: Uptake and efflux in disaggregated cells and 3D models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2017-4205

Kinetic analysis of aerobic biotransformation pathways of a perfluorooctane sulfonate (PFOS) precursor in distinctly different soils

With the phaseout of perfluorooctane sulfonate (PFOS) production in most countries and its well known recalcitrance, there is a need to quantify the potential release of PFOS from precursors previously or currently being emitted into the environment. Aerobic biodegradation of N-ethyl perfluorooctane sulfonamidoethanol (EtFOSE) was monitored in two soils from Indiana, USA: an acidic forest silt loam (FRST-48, pH = 5.5) and a high pH agricultural loam (PSF-49, pH = 7.8) with similar organic carbon contents (2.4 and 2.6%) for 210 d and 180 d, respectively. At designated times, triplicate samples were sacrificed for which headspace samples were taken followed by three sequential extractions. Extracts were analyzed using HPLC-tandem mass spectrometry. Measured profiles of EtFOSE degradation and generation/degradation of subsequent metabolites were fitted to the Indiana soils data as well as to a previously published data set for a Canadian soil using an R-based model (KinGUII) to explore pathways and estimate half-lives (t1/2) for EtFOSE and metabolites. EtFOSE degradation ranged from a few days to up to a month. PFOS yields ranged form 1.06–5.49 mol% with the alkaline soils being four to five times higher than the acidic soil. In addition, a direct pathway to PFOS had to be invoked to describe the early generation of PFOS in the Canadian soil. Of all metabolites, the sulfonamidoacetic acids were the most persistent (t1/2 ≥ 3 months) in all soils. We hypothesized that while pH-pKa dependent speciation may have impacted rates, differences in microbial communities between the 3 soils arising from varied soil properties including pH, nutrient levels, soil management, and climatic regions are likely the major factors affecting pathways, rates, and PFOS yields.

Comparison of 2-hydroxyglutarate (2HG) levels in tissue and serum of isocitrate dehydrogenase (IDH)-mutated (MUT) versus wild-type (WT) gliomas.

2037 Background: IDH mutations are common in low-grade gliomas and confer significantly improved prognosis. IDH catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, and subsequently to the oncometabolite 2HG. Mutant IDH leads to preferential accumulation of the R relative to the S enantiomer of 2HG. We analyzed the ratio of R to S enantiomers (rRS) in glioma tissues and matched serum samples, and correlated findings with IDH status, 1p19q codeletion status and survival. Methods: Fresh frozen glioma tissues and matched serum samples were obtained from the University of Toronto Brain Tumor Bank. IDH status was determined by immunohistochemistry and confirmed by 450K methylation profile or direct sequencing. 1p19q codeletion status was determined by loss-of-heterozygosity PCR analysis. R-2HG and S-2HG levels were quantified using HPLC tandem mass spectrometry coupled with a CHIROBIOTIC column. Results: Glioma tissues from 70 patients were analyzed – 52 with IDH MUT and 18 WT. 30 had matched serum samples. Using glioma tissues, rRS clearly distinguished MUT vs WT (median 574 vs 1.3, p &lt; 1x10-9) with only three outliers. In contrast, rRS was not elevated in serum samples (median 1.5 vs 1.2, p = 0.13). Overall survival (OS) was significantly longer for MUT vs WT (median 178 vs 33 months, p &lt; 1×10-7). Stratifying MUT by tissue rRS, median OS was 197, 178, 178 and 122 months for lowest to highest quartiles of rRS respectively. 1p19q codeletion status and tumor latency did not explain this trend, given rRS was similar in codeleted vs non-codeleted MUT, and similar in MUT operated &lt; 3 vs ≥3 months from diagnosis. Progression-free survival results corresponded to OS. Conclusions: rRS from glioma tissues effectively differentiated MUT vs WT, whereas serum samples were unreliable. Unlike current methods, tissue rRS enables real-time determination of IDH status, and thus may guide clinical practice such as extent of surgical resection intraoperatively and upfront selection of adjuvant therapy. rRS potentially stratifies survival within MUT patients, providing detailed correlative information.

Electrochemistry coupled online to liquid chromatography-mass spectrometry for fast simulation of biotransformation reactions of the insecticide chlorpyrifos.

An automated method is presented for fast simulation of (bio)transformation products (TPs) of the organophosphate insecticide chlorpyrifos (CPF) based on electrochemistry coupled online to liquid chromatography-mass spectrometry (EC-LC-MS). Oxidative TPs were produced by a boron doped diamond (BDD) electrode, separated by reversed phase HPLC and online detected by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, EC oxidative TPs were investigated by HPLC-tandem mass spectrometry (LC-MS/MS) and FT-ICR high resolution mass spectrometry (HRMS) and compared to in vitro assay metabolites (rat and human liver microsomes). Main phase I metabolites of CPF: chlorpyrifos oxon (CPF oxon), trichloropyridinol (TCP), diethylthiophosphate (DETP), diethylphosphate (DEP), desethyl chlorpyrifos (De-CPF), and desethyl chlorpyrifos oxon (De-CPF oxon), were successfully identified by the developed EC-LC-MS method. The EC-LC-MS method showed similar metabolites compared to the in vitro assay with possibilities of determining reactive species. Our results reveal that online EC-(LC)-MS brings an advantage on time of analysis by eliminating sample preparation steps and matrix complexity compared to conventional in vivo or in vitro methods.

Quality Evaluation of Lepidium meyenii (Maca) Based on HPLC and LC-MS Analysis of its Glucosinolates from Roots

In order to evaluate the quality of maca samples, 15 batches of maca hypocotyls from different geographical origins were analyzed by using HPLC and liquid chromatography tandem mass spectrometry (LC-MS). Their glucosinolates (GLs) were identified, profiled, and quantified. Three aromatic GLs, glucosinalbin (GSB), glucotropaeolin (GTL), and glucolimnanthin (GLH), were identified from maca roots. It was found that the HPLC profiles of maca samples showed a similar qualitative pattern except for some differences in their contents. The amount ranges (μmol g−1, DW) of GSB, GTL, and GLH were 2.62–5.55, 12.99–57.12, and 3.15–12.12, respectively. Furthermore, a HCA (hierarchical clustering analysis) was used to classify the quality of maca samples according to their cultivated sources and GLs contents. The results showed that the combination of GSB, GTL, and GLH could be a marker for accurate determination and quality control of maca samples. It was concluded that the multi-component analysis in combination with HCA could be a supplement to assess the quality of maca materials.