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Caffeine, theobromine, theophylline and paraxanthine quantification by LC-MS/MS in plasma human samples.

Caffeine, theobromine, theophylline and paraxanthine quantification by LC-MS/MS in plasma human samples.

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Caffeine is one of the most widely consumed psycho-stimulants. The study of the beneficial effects of caffeine consumption to decrease the risk of developing several neuropsychiatric pathologies is receiving increasing attention. Thus, accurate and sensitive methods have been developed, mainly by LC-MS/MS, in order to quantify caffeine and its meta...

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... LC-MS/MS quantification methodology described in this paper was tested in human plasma samples for caffeine consumption varying from 0 to 100 mg/day and theobromine consumption varying between 0 to 75 mg/day (Table 4). In samples 1 and 2, where no consumption or a very low amount of caffeine and theobromine was ingested, no caffeine or other metabolite was quantified with the exception of paraxanthine in sample 1. ...
Context 2
... LC-MS/MS quantification methodology described in this paper was tested in human plasma samples for caffeine consumption varying from 0 to 100 mg/day and theobromine consumption varying between 0 to 75 mg/day (Table 4). In samples 1 and 2, where no consumption or a very low amount of caffeine and theobromine was ingested, no caffeine or other metabolite was quantified with the exception of paraxanthine in sample 1. ...

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... Urine caffeine metabolites were analysed by LC-MS/MS, as described [22,23]. A similar protocol was developed for the non-caffeine metabolites. ...
... To date, most studies on the effects of coffee intake on the status of noncommunicable chronic diseases, such as NAFLD and T2D, have relied on self-reported coffee intake; however, within the last few years, there has been an increasing shift to the analysis of coffee metabolites in plasma and urine: this has been facilitated by the development of sensitive and robust LC-and UPLC-MS/MS methods for quantifying coffee metabolites in biofluids such as serum, plasma and cerebrospinal fluid [22,23,29], urine [30,31] and even fingertip sweat [32]. In five out of six measured urinary coffee metabolites (p-coumaric acid being the exception), we found robust and significant Pearson correlations, in the range of 0.29-0.43, ...
... To date, most studies on the effects of coffee intake on the status of non-communicable chronic diseases, such as NAFLD and T2D, have relied on self-reported coffee intake; however, within the last few years, there has been an increasing shift to the analysis of coffee metabolites in plasma, serum and urine: this has been facilitated by the development of sensitive and robust LC-and UPLC-MS/MS methods for quantifying coffee metabolites in biofluids such as serum, plasma and cerebrospinal fluid [22,23,29], urine [30,31] and even fingertip sweat [32]. In five out of six measured urinary coffee metabolites (p-coumaric acid being the exception), we found robust and significant Pearson correlations, in the range of 0.29-0.43, ...
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Coffee may protect against non-alcoholic fatty liver disease (NAFLD), but the roles of the caffeine and non-caffeine components are unclear. Coffee intake by 156 overweight subjects (87% with Type-2-Diabetes, T2D) was assessed via a questionnaire, with 98 subjects (all T2D) also providing a 24 h urine sample for quantification of coffee metabolites by LC–MS/MS. NAFLD was characterized by the fatty liver index (FLI) and by FibroscanÒ assessment of fibrosis. No associations were found between self-reported coffee intake and NAFLD parameters; however, total urine caffeine metabolites, defined as Σcaffeine (caffeine + paraxanthine + theophylline), and adjusted for fat-free body mass, were significantly higher for subjects with no liver fibrosis than for those with fibrosis. Total non-caffeine metabolites, defined as Σncm (trigonelline + caffeic acid + p-coumaric acid), showed a significant negative association with the FLI. Multiple regression analyses for overweight/obese T2D subjects (n = 89) showed that both Σcaffeine and Σncm were negatively associated with the FLI, after adjusting for age, sex, HbA1c, ethanol intake and glomerular filtration rate. The theophylline fraction of Σcaffeine was significantly increased with both fibrosis and the FLI, possibly reflecting elevated CYP2E1 activity—a hallmark of NAFLD worsening. Thus, for overweight/obese T2D patients, higher intake of both caffeine and non-caffeine coffee components is associated with less severe NAFLD. Caffeine metabolites represent novel markers of NAFLD progression.
... Neat and artificial solutions can be used when the study sample matrix is mainly composed of water, such as saliva, urine, tears and cerebrospinal fluid [45]. To assess the application of neat solutions and/or artificial matrices for quantification purposes, a comparison between the slopes of the calibration curves in surrogate matrices and the authentic matrix (standard addition method, SAM) should be performed [100][101][102]. Even if several statistical tests are available for comparing parallelism of curves, the best known is based on analysis of variance. ...
Article
Over the last two decades, liquid chromatography coupled to mass-spectrometry (LC‒MS) has become the gold standard to perform qualitative and quantitative analyses of small molecules. When quantitative analysis is developed, an analyst usually refers to international guidelines for analytical method validation. In this context, the design of calibration curves plays a key role in providing accurate results. During recent years and along with instrumental advances, strategies to build calibration curves have dramatically evolved, introducing innovative approaches to improve quantitative precision and throughput. For example, when a labeled standard is available to be spiked directly into the study sample, the concentration of the unlabeled analog can be easily determined using the isotopic pattern deconvolution or the internal calibration approach, eliminating the need for multipoint calibration curves. This tutorial aims to synthetize the advances in LC‒MS quantitative analysis for small molecules in complex matrices, going from fundamental aspects in calibration to modern methodologies and applications. Different work schemes for calibration depending on the sample characteristics (analyte and matrix nature) are distinguished and discussed. Finally, this tutorial outlines the importance of having international guidelines for analytical method validation that agree with the advances in calibration strategies and analytical instrumentation.
... The analytic recovery of solriamfetol and modafinil was performed by spiking three concentration levels (LQC, MQC and HQC) into blank plasma samples. Analyte recovery was determined by comparing the solriamfetol and modafinil (internal standard) peak area obtained from extracted samples with un-extracted samples 17 . The acceptance criterion was that the relative standard deviation of recovery at each quality control concentration level and for internal standard should be ≤15 %. . ...
... The stability of the solriamfetol in plasma was evaluated under different study conditions; i.e. standing at room temperature over 24 h and storing at −20 °C for one month (long-term stability) 17 . The results of the stability of solriamfetol in plasma at diverse storage conditions were expressed as percentage recoveries and relative standard deviation. ...
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Solriamfetol is a selective dopamine and norepinephrine reuptake inhibitor with wake‐promoting effects. The aim of the present study is to develop a rapid, sensitive and reliable method for the estimation of Solriamfetol in plasma samples using LC-MS. In the present investigation, a rapid, specific, selective and novel method has been optimized for evaluation of solriamfetol in in plasma using modafinil as an internal standard and identification of degradants by LC-MS/MS. The solriamfetol and internal standard were extracted from plasma in a single step using acetonitrile. The principle analytes were eluted with the conditions of mobile phase having the 5mM ammonium format in methanol: 50% Methanol in acetonitrile (90:10%, v/v). The Chromatographic column used is Xterra MS C18, 3.5µ.m, 1mmX150mm analytical column with the 0.5 ml/min flow rate. The detector is CEM array detector. The retention times of solriamfetol and modafinil were 1.50min-1.51min with a total run time of 3 min. The curve indicates correlation coefficient (r2) for modafinil was superior by having the value 1.000 with linear range of 5ng/ml to 500ng/ml. The correlation coefficient (r2) for solriamfetol was found to be 0.999. The LOQ and LOD for the solriamfetol was 33.70pg/ml and 11.12pg/ml respectively The developed method was validated by evaluating system suitability, selectivity, sensitivity, linearity, precision, accuracy, ruggedness and stability in conformity with the guidelines of the United States Food and Drug Administration (US-FDA). The results of validation parameters were found to be within the acceptance limits. Hence, the developed and validated method can be utilized for the routine determination of solriamfetol in plasma samples.
... Therefore, it is critical to select an appropriate surrogate matrix, such as synthetic plasma (Alvi & Hammami, 2011) or fetal bovine serum (Lopez-Sanchez et al., 2018), for quantitative or qualitative analysis of CAF. Recently, Mendes et al. (2019) reported an LC-MS/MS method to quantify CAF and THM using solvent as a surrogate matrix. In that study, however, PAR and THY could not be assayed as they were not well chromatographically separated. ...
... Although CAF is not an endogenous substance, the CAF-free human plasma is not easy to obtain. A variety of surrogate matrices have been tested in LC-MS/MS-based quantification with a lack of blank matrices, including neat solutions (Ko et al., 2021;Mendes et al., 2019), artificial matrices (Virág et al., 2020;Yin et al., 2021), stripped matrices (Ogawa et al., 2016) and animal matrices (Yin et al., 2020). However, most of these reports were not attempts to analyze caffeine in human plasma (Ko et al., 2021;Mendes et al., 2019;Ogawa et al., 2016;Virág et al., 2020;Yin et al., 2020Yin et al., , 2021. ...
... A variety of surrogate matrices have been tested in LC-MS/MS-based quantification with a lack of blank matrices, including neat solutions (Ko et al., 2021;Mendes et al., 2019), artificial matrices (Virág et al., 2020;Yin et al., 2021), stripped matrices (Ogawa et al., 2016) and animal matrices (Yin et al., 2020). However, most of these reports were not attempts to analyze caffeine in human plasma (Ko et al., 2021;Mendes et al., 2019;Ogawa et al., 2016;Virág et al., 2020;Yin et al., 2020Yin et al., , 2021. In this study, Sprague-Dawley rat plasma, UPW, ICR mouse plasma, and beagle dog plasma were successfully evaluated individually as surrogate matrices for the simultaneous determination of CAF, PAR, THM, and THY levels in human plasma. ...
Article
The growing evidence has endorsed the view that therapeutic drug monitoring (TDM) of caffeine for apnea of prematurity is helpful for dose tailoring when the therapeutic response is lacking or toxicity is suspected. However, the plasma without caffeine is difficult to obtain. Therefore, a method was developed and validated to measure caffeine and its three primary metabolites (paraxanthine, theobromine, and theophylline) using LC‐ESI‐MS/MS in human plasma and several surrogate matrices. The chromatographic separation of analytes was finally achieved on a Waters Symmetry C18 (4.6 × 75 mm, 3.5 μm) column. Several strategies were successfully applied to overcome the matrix effects: 1) appropriate dilution for sample cleanup; 2) a starting lower proportion of organic phase; 3) multiple individual stable‐labeled isotopic internal standards. The parallelism between the authentic matrix and surrogate matrices was convincing. The recovery of the analytes in both human plasma and rat plasma was acceptable over the linear range (0.500 to 50.0 μg/mL for caffeine, and 0.0100 to 1.00 μg/mL for three metabolites). The method was successfully applied in 118 samples from 74 preterm infants with apnea of prematurity. The rat plasma or ultrapure water as surrogate matrix is worthy of being recommended for routine TDM of caffeine.
... Compound 1 showed the exact mass of 137.0456 amu and the molecular formula C 5 H 5 N 4 O + , yielding a base peak at m/z 119.0345 as found in hypoxanthine (Cao et al., 2019;Zhang et al., 2019). Compound 9 exhibited an [M + H] + ion at m/z 181.0719 (C 7 H 9 N 4 O 2 + ) and two intense fragment ions at m/z 138.0665 and 110.0701, compatible with theobromine or paraxanthine (Mendes et al., 2019). Thus, compounds 1 and 9 were identified as hypoxanthine and paraxanthine/theobromine, respectively. ...
... Tangutorides were identified in fish and soy sauce, ketchup, and other food products (Hövelmann et al., 2019). Paraxanthine is a metabolite of caffeine and theobromine in animals (Mendes et al., 2019). ...
Article
Ethnopharmacological relevance In Paraguay, healers from the Mbya culture treat cancer with a recipe prepared with the native toad Rhinella schneideri. However, the chemical composition and biological effects of the recipe remain unknown. Aim of the study: The aim is to determine the composition of the traditional preparation made using the toad R. schneideri and to evaluate its effect on human breast cancer (BC) cells. Materials and methods The metabolites contained in the preparation were concentrated using XAD-7 resin, and the concentrate was analyzed by HPLC-MS/MS. The effect of the preparation was assessed in normal (MCF10F) and BC cells (MDA-MB-231 and MCF7). The mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) levels, and cell cycle progression were determined by flow cytometry. The oxygen consumption rate (OCR) was measured by Clark electrode, and fibronectin-dependent migration in normoxia and hypoxia-like conditions were evaluated by transwell assay. Results From the Amberlite-retained extract from the preparation, 24 compounds were identified, including alkaloids, amino acids, bufadienolides, and flavonoids, among others. The crude extract (CE) did not affect cell cycle progression and viability of BC cell lines. Moreover, it did not make cancer cells more sensitive to the cytotoxic effect of the chemotherapeutics doxorubicin and teniposide. On the other hand, the CE reduced the menadione-induced ROS production and increased NADH, Δψm, and the OCR. Respiratory complexes I and III as well as ATP synthase levels were increased in an AMPK-dependent manner. Moreover, the CE inhibited the migration of BC cells in normoxia and a hypoxia-like condition using CoCl2 as a HIF1α-stabilizing agent. This latter effect involved an AMPK-dependent reduction of HIF1α and β1-integrin levels. Conclusions The Paraguayan toad recipe contains metabolites from the toad ingredient, including alkaloids and bufadienolide derivatives. The CE lacks cytotoxic effects alone or in combination with chemotherapeutics. However, it increases mitochondrial bioenergetics and inhibits the cancer cell migration in an AMPK-dependent manner in BC cells. This is the first report of the in vitro anticancer effect of a traditional Rhinella sp. toad preparation based on Mbya tradition.
... This fragmentation pattern is in accordance with that of Carnitine(Sowell et al., 2011). Peak 15 was identified as caffeine from its MS 2 fragments(Mendes et al., 2019). ...
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Green beans (Phaseolus vulgaris L.) are consumed as pods or mature seeds (common beans). The pods were extracted with 95% ethanol and processed to prepare non‐polar and polar fractions. Comparing the antihyperglycemic activity of both fractions, non‐polar fraction (NPF, 200 mg kg⁻¹ day⁻¹) lowered blood glucose in streptozotocin diabetic rats by 65% compared to 57% for the polar fraction at the same dose. When NPF treatment was combined with injection of mesenchymal stem cells (MSC) a 4.4‐fold increase in serum insulin and a 73.6% reduction in blood glucose were observed compared to untreated control. Additionally, a significant decrease in malondialdehyde (76.2%), nitric oxide (68.2%), cholesterol (76.1%), and triglycerides (69.5%) and a 1.75‐fold increase in HDL concentrations were observed in the group treated with this combination compared to diabetic animals. Interestingly, NPF increased homing of MSC in pancreas potentiating their antidiabetic activity. Finally, 26 compounds were identified in NPF using LC/MS analysis and four were isolated in pure form. The isolated compounds namely calotroproceryl acetate, fridelin, calotroproceryl A, and stigmasterol showed good inhibitory activity against pancreatic lipase with IC50 at 1.93, 1.07, 1.34 and 1.44–1 μg/ml, respectively. Additionally, these compounds inhibited α‐amylase, albeit at higher concentration, with IC50 at 248, 212, 254, and 155 μg/ml for calotroproceryl acetate, fridelin, calotroproceryl A, and stigmasterol, respectively. Our results suggest that green beans extract can potentiate effect of MSC in diabetes directly due to its own antidiabetic effect and indirectly by increasing MSC homing in pancreatic tissues. Practical applications It has been suggested in this study that green beans can improve hyperglycemia, oxidative balance in diabetes, so green beans can be promoted as a healthy nutrient for diabetic patients. Green beans also can enhance homing and differentiation of mesnchymal stem cells in the pancreas for future stem cell therapy of type I diabetes.
... Parasitoids are likely at risk of toxicity transfer from their host, as shown for example for the snowdrop lectin entomotoxin transferred from the diet of M. persicae to tissues of Aphidius ervi (Hymenoptera: Braconidae) endoparasitoid pupae (Couty et al., 2001). Next steps of the work will be to perform LC-MS or HPLC analyses (e.g., Kim et al., 2011;Mendes et al., 2019) to try detecting if caffeine-or its main metabolites-has been transferred from aphid to parasitoid tissues. More generally, host quality (size, age, nutritional content, immune defenses) is crucial for proper parasitoid development and survival (Harvey, 2000), but can be altered by the plant quality (Harvey & Gols, 2018). ...
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Secondary metabolites are central to understanding the evolution of plant-animal interactions. Direct effects on phytophagous animals are well-known, but how secondary consumers adjust their behavioral and physiological responses to the herbivore's diet remains more scarcely explored for some metabolites. Caffeine is a neuroactive compound that affects both the behaviour and physiology of several animal species, from humans to insects. It is an alkaloid present in nectar, leaves and even sap of numerous species of plants where it plays a role of chemical defenses against herbivores and pathogens. Caffeine effects have been overlooked in generalist herbivores, that are not specialized on coffee or tea plants. Using a host-parasitoid system, we show that caffeine intake at relatively low dose affects longevity and fecundity of the primary consumer, but also indirectly of the secondary one, suggesting that this alkaloid and/or its effects can be transmitted through trophic levels and persist in the food chain. Parasitism success was lowered by ≈16% on hosts fed with caffeine, and parasitoids of the next generation that have developed in hosts fed on caffeine showed a reduced longevity, but no differences in mass and size were found. This study helps at better understanding how plant secondary metabolites, such as caffeine involved in plant-animal interactions, could affect primary consumers, could have knock-on effects on upper trophic levels over generations, and could modify interspecific interactions in multitrophic systems.
... notably the loss of isocyanic acid and methyl isocyanate, suggests that the demethylation of C 8 -6-I is analogous to N1 and N3 demethylation of caffeine to theobromine and paraxanthine respectively. The neutral loss of isocyanic acid with 43 Da is a neutral loss associated with the fragmentation of theobromine and the neutral loss of methyl isocyanate with 57 Da is associated with fragmentation of paraxanthine (Mendes et al. 2019). 1-Aminoindan is a P450 metabolite of the Parkinson's disease therapeutic rasagiline and, 1-aminoindan can be further metabolized to 3-hydroxy-1-aminoindan (Deftereos et al. 2012;Agundez et al. 2013;de Biase et al. 2014). ...
Preprint
• A challenge in the development of novel ¹⁸F-labelled positron emission tomography (PET) imaging probes is identification of metabolically stable sites to incorporate the ¹⁸F radioisotope. Metabolic loss of ¹⁸F from PET probes in vivo can lead to misleading biodistribution data as displaced ¹⁸F can accumulate in various tissues. • In this study we report on in vitro hepatic microsomal metabolism of novel caffeine containing bifunctional compounds (C8-6-I, C8-6-N, C8-6-C8) that can prevent in vitro aggregation of α-synuclein, which is associated with the pathophysiology of Parkinson’s disease. The metabolic profile obtained guided us to synthesize stable isotope ¹⁹F-labelled analogues in which the fluorine was introduced at the metabolically stable N7 of the caffeine moiety. • An in vitro hepatic microsomal metabolism study of the ¹⁹F-labelled analogues resulted in similar metabolites to the unlabelled compounds and demonstrated that the fluorine was metabolically stable, suggesting that these analogues are appropriate PET imaging probes. This straightforward in vitro strategy is valuable for avoiding costly stability failures when designing radiolabelled compounds for PET imaging.
... Several analytical methods, including GC-MS, high-performance liquid chromatography with ultraviolet, liquid chromatography-mass spectrometry, and LC-MS/MS have been used to measure caffeine concentrations [10][11][12][13]. Among these, LC-MS/MS is a powerful modality for quantifying caffeine concentrations in human serum due to its high selectivity and sensitivity [14]. In sample pretreatment, several sample preparation procedures rely on traditional technologies such as protein precipitation (PPT) and liquid-liquid extraction (LLE). ...
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The number of acute caffeine poisoning cases have increased in Japan. We can use serum caffeine concentrations to evaluate the severity of caffeine poisoning and determine whether or not we should perform hemodialysis. In this study, we sought to develop a rapid method for measuring serum caffeine concentrations. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the new method. We chose caffeine-d9 as the internal standard, and we used the standard addition method to quantify caffeine concentrations. We collected six blood samples from three patients with acute caffeine poisoning to measure serum caffeine concentrations. In our method, retention time for caffeine was 0.4 min, and the time required for the total LC-MS/MS analysis was 1 min per sample. We obtained accurate serum caffeine concentrations 7 min after injection into the LC-MS/MS instrument. Further, time-consuming sample pretreatment was not required because each sample was diluted 10,000-fold. As a result, we could obtain serum caffeine concentrations for each patient in a total of 40 min. Our findings suggest that rapid, accurate measurement of serum caffeine concentrations by LC-MS/MS could contribute to real-time evaluation of poisoning severity and determination of appropriate therapeutic strategies in acute clinical settings.
... Caffeine analysis was performed using Shimadzu 8040 LC-MS/MS (Shimadzu, Kyoto, Japan) equipped with an LC pump (LC-30AD-1 and LC-30AD-2), an autosampler (SIL 30AC), and a column oven (CTO-20AC). The analytical method was modified from Ref [39]. The system was interfaced through electrospray ionization (ESI) in the positive mode. ...
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Dermal absorption of chemicals is a key factor in risk assessment. This study investigated the effects of different amounts of application on dermal absorption and suggested an appropriate application dose for proper dermal absorption. Caffeine and testosterone were chosen as test compounds. An in vitro dermal absorption test was performed using a Franz diffusion cell. Different amounts (5, 10, 25, and 50 mg (or µL)/cm2) of semisolid (cream) and liquid (solution) formulations containing 1% caffeine and 0.1% testosterone were applied to rat and minipig (Micropig®) skins. After 24 h, the concentrations of both compounds remaining on the skin surface and in the stratum corneum, dermis and epidermis, and receptor fluid were determined using LC-MS / MS or HPLC. Dermal absorption of both compounds decreased with increasing amounts of application in both skin types (rat and minipig) and formulations (cream and solution). Especially, dermal absorptions (%) of both compounds at 50 mg (or µL)/cm2 was significantly lower compared to 5 or 10 mg (or µL)/cm2 in both rat and minipig skins. Therefore, a low dose (5 or 10 mg (or µL)/cm2) of the formulation should be applied to obtain conservative dermal absorption.