High-pressure Liquid Chromatography-tandem Mass Research Articles

Exploiting the beneficial effects of Salvia officinalis L. extracts in human health and assessing their activity as potent functional regulators of food microbiota

Salvia officinalis L. has attracted scientific and industrial interest due to its pharmacological properties. However, its detailed phytochemical profile and its correlation with beneficial effects in the human microbiome and oxidative stress remained elusive. To unveil this, S. officinalis was collected from the region of Epirus and its molecular identity was verified with DNA barcoding. Phytochemical profile for both aqueous and ethanol-based extracts was determined by high-pressure liquid chromatography-tandem mass spectrometry and 103 phytochemicals were determined. The effect of S. officinalis extracts as functional regulators of food microbiota by stimulating the growth of Lacticaseibacillus rhamnosus strains and by suppressing evolution of pathogenic bacteria was verified. Furthermore, we recorded that both extracts exhibited a significant cellular protection against H2O2-induced DNA damage. Finally, both extracts exhibited strong inhibitory effect towards LDL oxidation. This study provides a comprehensive characterization of S. officinalis on its phytochemical components as also its potential impact in human microbiome and oxidative stress.

Pharmacokinetics of Venetoclax Co-Administered with Posaconazole in Patients with Acute Myeloid Leukemia.

The Food and Drug Administration currently approves the combination of hypomethylating agents (HMA), azacytidine or decitabine with venetoclax (VEN) for acute myeloid leukemia (AML) patients aged more than 75 years and for patients unsuitable for intensive chemotherapy. The risk of fungal infection in the early phase of treatment is not negligible; therefore, posaconazole (PCZ) is commonly administered as primary prophylaxis. A drug-drug interaction between VEN and PCZ is well known, but the trend of serum levels of venetoclax when both drugs are overlapped is not clear. In total, 165 plasma samples from 11 elderly AML patients receiving combined treatment with HMA, VEN and PCZ were analyzed by a validated analytical method (high-pressure liquid chromatography-tandem mass spectrometry). Venetoclax trough plasma concentrations were detected during the 3 days of ramp-up as well as on day 7 and day 12 of treatment when the exposure as the area under the plasma concentration-time curve and the accumulation ratio were also calculated. The results were compared with the expected data for 400 mg/dose VEN administered alone-the confirmed high inter-individual variability in pharmacokinetics suggests the need for therapeutic drug monitoring.

25-Hydroxyvitamin D: A potential marker of the incidence of osteoporosis and sarcopenia in diabetic mellitus patients

Purpose: To investigate the feasibility of using the 25-hydroxyvitamin D (25(OH)D) levels in assessing osteoporosis (OP) and sarcopenia in elderly patients with diabetes mellitus (DM).
 Methods: One hundred and forty elderly patients with DM were allocated to DM (n = 40), OP (n = 50), and DM + OP (n = 50) groups. Their clinical data were collected, and muscle function assessed. The levels of fasting insulin, glycated hemoglobin (HbAlc), t-P1NP, and 25(OH)D were determined by chemiluminescent microparticle immunoassay, high-pressure liquid chromatography, electrochemical immunoassay, and liquid chromatography-tandem mass spectrometry, respectively.
 Results: The 25(OH)D levels were lowest in DM + OP group, followed by the OP group, and highest in the DM group (p < 0.05). The DM + OP group showed significantly lower gait speed and grip strength than the OP and DM groups (p < 0.05). The diagnostic AUC of 25(OH)D for OP and sarcopenia were 0.9733 (95 % CI: 0.9274 - 1.000, p < 0.001) and 0.9866 (95 % CI: 0.9632 - 1.000, p < 0.001). Patients with sarcopenia had significantly lower 25(OH)D and t-PINP levels than patients without sarcopenia (p < 0.05). Logistic regression analysis revealed that the 25(OH)D level, duration of DM, and age were independent risk factors for sarcopenia (p < 0.05).
 Conclusion: Patients with DM and OP exhibit increase in the incidence of sarcopenia. Serum 25(OH)D may be used as a potential marker for the incidence of OP and sarcopenia in DM patients.

Multiple Mycotoxin Contamination in Medicinal Plants Frequently Sold in the Free State Province, South Africa Detected Using UPLC-ESI-MS/MS.

Medicinal plants are important in the South African traditional healthcare system, the growth in the consumption has led to increase in trade through muthi shops and street vendors. Medicinal plants are prone to contamination with fungi and their mycotoxins. The study investigated multiple mycotoxin contamination using Ultra High Pressure Liquid Chromatography–Tandem Mass Spectrometry (UPLC-ESI-MS/MS) for the simultaneous detection of Aflatoxin B1 (AFB1), Deoxynivalenol (DON), Fumonisins (FB1, FB2, FB3), Nivalenol (NIV), Ochratoxin A (OTA) and Zearalenone (ZEN) in frequently sold medicinal plants. Medicinal plant samples (n = 34) were purchased and analyzed for the presence of eight mycotoxins. DON and NIV were not detected in all samples analyzed. Ten out of thirty-four samples tested positive for mycotoxins —AFB1 (10.0%); OTA (10.0%); FB1 (30.0%); FB2 (50.0%); FB3 (20.0%); and ZEN (30.0%). Mean concentration levels ranged from AFB1 (15 µg/kg), OTA (4 µg/kg), FB1 (7–12 µg/kg), FB2 (1–18 µg/kg), FB3 (1–15 µg/kg) and ZEN (7–183 µg/kg). Multiple mycotoxin contamination was observed in 30% of the positive samples with fumonisins. The concentration of AFB1 reported in this study is above the permissible limit for AFB1 (5 µg/kg). Fumonisin concentration did not exceed the limits set for raw maize grain (4000 µg/kg of FB1 and FB2). ZEN and OTA are not regulated in South Africa. The findings indicate the prevalence of mycotoxin contamination in frequently traded medicinal plants that poses a health risk to consumers. There is therefore a need for routine monitoring of multiple mycotoxin contamination, human exposure assessments using biomarker analysis and establishment of regulations and standards.

Qualitative and Quantitative Analysis of Phenolic Compounds in Spray-Dried Olive Mill Wastewater.

The processing of olives for oil production generates the most abundant agro-industrial by-products in the Mediterranean area. The three-phase olive oil extraction process requires the addition of a large amount of water to the system, which is difficult to dispose of for its load of toxic pollutants. On the other hand, olive mill wastewater is a rich source of bioactive substances with various biological properties that can be used as ingredients in the food industry for obtaining functional and nutraceutical foods as well as in the pharmaceutical industry. In this study, we present the results relative to the phenolic compounds detected in dried olive mill wastewaters obtained using a spray dryer. Qualitative and quantitative analyses were obtained by high-pressure liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). In particular, the compounds here discussed are: apigenin (9.55 mg/kg dry weight), caffeic acid (2.89 mg/kg dry weight), catecol (6.12 mg/kg dry weight), p-cumaric acid (5.01 mg/kg dry weight), diosmetin (3.58 mg/kg dry weight), hydroxytyrosol (1.481 mg/kg dry weight), hydroxytyrosyl oleate (564 mg/kg dry weight), luteolin (62.38 mg/kg dry weight), luteolin-7-O-glucoside (88.55 mg/kg dry weight), luteolin-4-O-glucoside (11.48 mg/kg dry weight), oleuropein (103 mg/kg dry weight), rutin (48.52 mg/kg dry weight), tyrosol (2043 mg/kg dry weight), vanillin (27.70 mg/kg dry weight), and verbascoside (700 mg/kg dry weight). The results obtained highlighted that the use of dehumidified air as a drying medium, with the addition of maltodextrin, appears to be an effective way to produce a phenol-rich powder to be included in food formulations as well as in pharmaceutical preparations having different biological properties.

Age-dependent reference intervals in children for the disialoganglioside, GD2, a circulating tumor biomarker for the childhood cancer, neuroblastoma

BackgroundThe disialoganglioside GD2 is a circulating tumor biomarker for the childhood cancer, neuroblastoma. This study establishes reference intervals for GD2 concentration in children within the age range where neuroblastoma commonly occurs. MethodsLeftover plasma samples taken for routine clinical laboratory tests from children without cancer were collected and assayed for the 18-carbon fatty acid chain length lipoform of GD2 using a validated high-pressure liquid chromatography tandem mass spectrometry method with a lower limit of quantification of 3 nM. Samples were stratified into 5 age cohorts (0–6 months, 6–12 months, 12–36 months, 3–10 years and > 10 years). Non-parametric statistical methods were used to define the upper bound of the reference interval for each age cohort. ResultsGD2 was measurable in 90% of samples from children < 10 years of age and GD2 concentration was age-dependent, peaking at 9 months followed by a gradual decline. GD2 was below the lower limit of quantification in 55% of samples in the > 10 years cohort. Upper bounds of reference intervals were 15.5 nM in 0–6 month cohort, 35.1 nM in 6–12 month cohort, 24.9 nM in 12–36 month cohort, 18.4 in 3–10 year cohort and 10.4 nM in > 10 year cohort. ConclusionsAge-dependent reference intervals were defined for circulating GD2 in children. GD2 concentration was highest in the 6–12 month age cohort, which is below the age of most children with high-risk neuroblastoma. The peak GD2 concentration at 9 months may reflect neurodevelopmental events in the brain.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

Pharmacokinetics of Synthetic Cathinones Found in Bath Salts in Mouse Brain and Plasma Using High-Pressure Liquid Chromatography-Tandem Mass Spectrometry.

Approximately 10 years ago, "bath salts" became popular as legal alternatives to the psychostimulants cocaine and the amphetamines. These products contained synthetic cathinones, including 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). Most preclinical investigations have only assessed the effects of these synthetic cathinones independently; however, case reports and Drug Enforcement Administration (DEA) studies indicate that bath salts contain mixtures of these substances. In this study, we examine the pharmacokinetic interactions of the drug combination. We hypothesized that combined exposure to MDPV, mephedrone, and methylone would result in increased drug concentrations and enhanced total drug concentrations when compared to individual administration. Adolescent male Swiss-Webster mice were injected intraperitoneally with either 10 mg/kg MDPV, 10 mg/kg mephedrone, 10 mg/kg methylone, or 10 mg/kg combined MDPV, mephedrone, and methylone. Following injection, brains and plasma were collected at 1, 10, 15, 30, 60, and 120 min. Drugs were extracted via solid-phase extraction, and concentrations were determined using a previously published high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. All drugs crossed the blood-brain barrier quickly. For methylone, the maximal concentration (Cmax) and the total drug exposure [as represented by the area under the concentration-time curve (AUC)] were significantly higher when combined with mephedrone and MDPV in both matrices (2.89-fold increase for both Cmax and AUC with combined treatment). For mephedrone, the Cmax was unchanged, but the AUC in brain was increased when in combination by approximately 34%. Interestingly, for MDPV, the Cmax was unchanged, yet the AUC was higher when MDPV was administered individually (there was a 62% decrease in AUC with combined treatment). The pharmacokinetics of methylone, mepedrone, and MDPV are altered when the drugs are used in combination. These data provide insight into the consequences of co-exposure to synthetic cathinones in popular bath salt products.

In situ localization of tris(2,3-dibromopropyl) isocyanurate in mouse organs by MALDI-IMS with auxiliary matrix strategy

Tris(2,3-dibromopropyl) isocyanurate (TBC) is one of the novel brominated flame retardants that has been widely used in consumer goods. Humans may be exposed to TBC daily. Studies showed that TBC can induce significant toxicity. However, there is currently no report on its in situ localization in organs. In this study, we aimed to develop a reliable and reproductive method to determine the in situ localization of TBC in mouse organs by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). As commercially available matrices were not able to detect TBC in tissue section, we then developed a novel MALDI-IMS method based on 1,5-diaminonaphthalene hydrochloride and silver trifluoromethanesulfonate (NDA/AgOTf) as the matrix for the in situ localization of TBC. AgOTf used as the auxiliary matrix in the negative-ion mode showed an excellent MS signal of TBC. The detection limit of [2AgOTf + Br]- was at the μg/mL level. The developed MALDI-IMS method was successfully employed to obtain the TBC spatial distribution in the mouse organs collected from mice exposed to 160 mg/kg/day of TBC for 30 days. High-pressure liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) was also used to evaluate the accumulation of TBC in liver, kidney, heart, and brain. The combination of MALDI-IMS and HPLC-MS/MS showed that TBC can accumulate in mice organs and it is mainly distributed in the renal parenchyma. In summary, an innovative method was developed for the analysis of TBC spatial distribution by MALDI-IMS using a novel NDA/AgOTf matrix, extending the application of MALDI-IMS in environmental pollutants.

Inhibition Kinetics and Mechanism of Glutathione and Quercetin on Acrylamide in the Low-Moisture Maillard Systems

The inhibition kinetics of glutathione (GSH) and quercetin on acrylamide (AA) formation in the low-moisture Maillard systems were investigated at 180°C. The inhibition rates in an equal-molar asparagine-glucose (Asn-Glc) system were higher than those in an asparagine-fructose (Asn-Fru) system, and the maximum inhibition rates for AA were 57.75% with 10-2 mol L-1 GSH and 51.38% with 10-1 mol L-1 quercetin. The Logistic-Index dynamic model and two consecutive simplified first-order kinetic models were well fitted to the changes of AA in the Asn-Glc system. The kinetics results suggested that the predominant inhibition effect of GSH on AA could be attributed to the competitive reaction between GSH and Asn for the consumption of Glc. The kinetic results and high-pressure liquid chromatography-tandem mass spectrometry analysis of the inhibitory effect of quercetin on AA indicated that quercetin might mitigate AA through the binding reaction of quercetin decomposition products and Maillard intermediate products. These experimental results provide theoretical data that may be useful to control the formation of AA during food thermal processing.

Pharmacokinetics of a cytosine arabinoside subcutaneous protocol in dogs with meningoencephalomyelitis of unknown aetiology.

Cytosine arabinoside (CA) is a commonly used treatment for dogs with meningoencephalomyelitis of unknown aetiology (MUE) with various proposed protocols, many requiring 24hours (h) of hospitalization or two visits within 24h. This is a unidirectional study evaluating the pharmacokinetics of a CA subcutaneous (SC) protocol and a standard constant rate infusion (CRI) protocol in 8 dogs with MUE. Dogs received the CRI (200mg/m2 IV over 24h), followed by a SC protocol (50mg/m2 every 2h for 4 treatments) four weeks later. Plasma CA concentrations were measured by high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS). Median peak CA concentration for the SC protocol (3.40µg/ml, range 1.60-9.70µg/ml) was significantly higher than the CRI (1.09µg/ml, range 0.77-1.67µg/ml; p=.02). Median concentration at 1h and 8h following initiation of treatment was significantly higher for the SC protocol (CA1 2.28µg/ml, range 0.97-2.67; CA8 1.83µg/ml, range 0.77-2.84) compared to the CRI (CA1 0.01µg/ml, range 0-0.45; CA8 0.74µg/ml, range 0.67-1.11; p=.01). While the PK properties of CA when administered as a CRI has been previously investigated, this study demonstrated that CA when administered via repeated 50mg/m2 injections every 2h over an 8-h period, provided sustained plasma levels above its therapeutic target and for a significantly longer duration of time than did a standard CRI protocol.

SERUM CONC. OF ANTIHYPERTENSIVE DRUGS MAY REVEAL NON-ADHERENCE IN PATIENTS CONSIDERED TO HAVE TRUE TREATMENT RESISTANT HYPERTENSION ASSESSED BY DIRECTLY OBSERVED THERAPY

Objective: Detecting non-adherence is challenging. Both directly observed intake of antihypertensive medication (DOT) followed by 24 hour-ambulatory blood pressure monitoring and serum conc. measurements of antihypertensive drugs are objective methods considered reliable in revealing drug non-adherence. We aimed to investigate whether serum conc. offers additional information regarding drug adherence compared to DOT in patients with assumed true treatment resistant hypertension. Design and method: Patients referred for renal denervation (RDN) were thoroughly investigated and tested for adherence. 19 patients were defined as true treatment resistant when their daytime systolic BP remained high (&gt; 135 mmHg) after DOT. Blood samples were collected at baseline, 6 months and 3 years after RDN and analyzed using ultra high pressure liquid chromatography-tandem mass spectrometry. Partial non-adherence was defined as at least one undetectable drug (but not all)and non-adherence as no detectable drugs in serum. Results: Ten out of 56 blood samples (17.9%) showed too low levels of one or more drug to be compatible with drug adherence. Six patients were redefined as either non-adherent or partially non-adherent based on their serum conc. results. Their systolic BPs are shown in the Figure. Three of these six patients went through a renal denervation procedure, while the remaining three were randomized to control. One of two patients considered to be RDN responders, became adherent to medication after six months which may have contributed to the fall in BP. At the 3 years visit none of the five antihypertensive agents prescribed were detected, and the patient́s BP was again elevated. Conclusions: Non-adherence was revealed in more patients when serum conc. measurements of antihypertensive drugs were added to directly observed therapy. Serum conc. measurement is objective and quantitative, and since some antihypertensive drugs require regular intake over a certain period of time to have maximal effect, they may offer important additional information.

Integrating liquid chromatography mass spectrometry into an analytical protocol for the identification of organic colorants in Japanese woodblock prints

Three Japanese woodblock prints from the Edo period (1603–1868) underwent a scientific investigation with the aim of understanding the changes in the colorants used in Japanese printing techniques. A multi-analytical approach was adopted, combining non-invasive techniques, such as fiber optic reflectance spectroscopy (FORS), Raman spectroscopy, multispectral imaging (MSI), and macro X-ray fluorescence (MA-XRF) with minimally invasive surface-enhanced Raman spectroscopy (SERS). The results enabled many of the pigments to be identified and their distribution to be studied, apart from two shades of purple of organic composition. Consequently, the potential of high-pressure liquid chromatography tandem mass spectrometry (HPLC–MS/MS) was explored for the first time with application to Japanese woodblock prints. The intrinsic sensitivity of the instrument and an effective extraction protocol allowed us to identify a mixture of dayflower (Commelina communis) blue and safflower (Carthamus tinctorius) red in purple samples constituted of 2–3 single fibers. In addition to the innovative integration of MA-XRF and HPLC–MS/MS to investigate these delicate artworks, the study concluded on the use of traditional sources of colors alongside newly introduced pigments in late Edo-period Japan. This information is extremely important for understanding the printing practices, as well as for making decisions about display, conservation, and preservation of such artworks.

Phosphatidylethanol Reliably and Objectively Quantifies Alcohol Consumption in Adolescents and Young Adults.

Alcohol contributes to numerous annual deaths and various societal problems not just in adult, but also in adolescent, populations. Therefore, it is vital to find methods for reliably detecting alcohol use for early preventative measures. Research has shown phosphatidylethanol (PEth) to be superior to self-report instruments and indirect biomarkers for alcohol consumption in adult populations. However, the transferability onto an adolescent population has not yet been investigated. N=106 adolescents and young adults aged between 13 and 21years were included. PEth analysis using high-pressure liquid chromatography-tandem mass spectrometry was performed on dried blood spot samples. Self-report questionnaires for alcohol consumption (Alcohol Use Disorders Identification Test-Consumption, AUDIT-C, and Timeline Followback, TLFB) and drug and alcohol consumption (Detection of Alcohol and Drug Problems in Adolescents, DEP-ADO) were completed by each participant. AUDIT-C scores showed large correlations with PEth 16:0/18:1 (rs =0.732) and PEth 16:0/18:2 (rs =0.661) concentrations. AUDIT-C with a cutoff value ≥3 was largely correlated with PEth 16:0/18:1 (η=0.411) and showed a medium-sized correlation with PEth 16:0/18:2 (η=0.397) concentrations. Using an AUDIT-C cutoff value ≥5 showed large correlations with both PEth 16:0/18:1 (η=0.510) and PEth 16:0/18:2 (η=0.497) concentrations, respectively. ROC curves indicated higher PEth concentrations are a good model for detecting positive AUDIT-C cutoff values (AUROC range: 0.800 to 0.849). PEth concentrations showed medium to large correlations with DEP-ADO and TLFB subscales (range rs =0.469 to 0.746). The results suggest that PEth is a reliable and objective marker for quantifying alcohol consumption in adolescents and young adults. This could be of importance for early preventative measures against hazardous alcohol consumption, which is increasingly common at younger ages.

Therapeutic Drug Monitoring of Direct Oral Anticoagulants May Increase Their Benefit-Risk Ratio

The attractiveness of direct oral anticoagulants (DOACs) over vitamin K antagonists, in addition to a better benefit-risk ratio, stems from the fact that no therapeutic drug monitoring is deemed necessary. This has been recently mitigated by the fact that increased dabigatran (D) plasma levels have been associated with hemorrhages, and is currently under scrutiny of the European Medicines Agency. We aimed to evaluate, in real conditions of use, whether patients with out-of-range DOAC blood concentrations (too high or too low) were associated with bleeding or thrombosis. Patients treated with D or rivaroxaban (R) were prospectively included in a hospital cohort. D and R plasma levels were measured by high-pressure liquid chromatography-tandem mass spectrometry-at the physician's demand. We defined concentration range as "expected" within the 95% confidence interval of the mean concentration obtained from pivotal trials, and "out of range" when concentrations were outside of that interval. A blind assessment of concentrations versus occurrence of bleeding or thrombosis was performed by means of univariate and multivariate analysis. Three hundred and twenty-two patients (mean age 78.5 years ± 13.1), treated with D or R were included consecutively. They had a mean CHA2DS2-VASc at 4.4 ± 1.7 and a mean HAS-BLED score at 1.7 ± 0.9. Irrespective of the DOAC prescribed, patients presenting with out-of-range concentrations had significantly more bleeding or thrombosis than patients with expected concentrations (P < 0.001). Patients with bleeding were more prone to have concentrations beyond the 95 percentile (N = 62, P < 0.001), whereas patients with thrombosis were more likely to have concentrations below the fifth percentile (N = 26, P < 0.05). The main risks associated with hemorrhages were abnormal concentrations, a high HAS-BLED score, the patient's age, and the creatinine blood level. For thrombosis, a concentration below the fifth percentile was the only risk factor that was significant in our cohort. While D and R under current recommendation have a better benefit-risk ratio than warfarin, their safe usage could be further optimized by some degree of therapeutic monitoring.

Development and Validation of an UHPLC-MS/MS Assay for the Therapeutic Monitoring of Brivaracetam Plasma Concentrations in Patients with Epilepsy.

Brivaracetam is an antiepileptic drug used as an add-on therapy for partial-onset seizures in subjects aged 4 years and older. Owing to potential drug interactions and intersubject variability in plasma concentrations, therapeutic monitoring for brivaracetam may be useful. The aim of this study was to develop a simplified method for measuring brivaracetam plasma concentrations applicable to therapeutic drug monitoring in epilepsy. An ultra high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated according to current guidelines for bioanalytical methods. Sample preparation (100 µL) involved only a simple precipitation step by acetonitrile. Brivaracetam-d7 was used as internal standard. The chromatographic analysis was performed by a Synergi Fusion column using 0.1% formic acid in water/acetonitrile as a binary gradient mobile phase, at a flow rate of 0.3 mL/min. Both brivaracetam and the internal standard eluted at 1.01 minutes. This method was applied to measure trough and 1-hour postmorning dose brivaracetam plasma concentrations of 11 patients with epilepsy. The method was validated over a concentration range of 0.10-10 mcg/mL. The mean recovery was 95%. Both intra- and inter-assay imprecision and inaccuracy were <15% for all quality control samples. The lower limit of quantitation and detection was 0.10 and 0.05 mcg/mL, respectively. No interferences or carry-over was observed. Median (25%-75% quartiles) trough and 1-hour postdosing brivaracetam plasma concentrations were 0.61 mcg/mL (0.47-0.83 mcg/mL) and 1.55 mcg/mL (1.24-2.12 mcg/mL), respectively, at a median dose of 80 mg/d (50-150 mg/d). Large, up to 8-fold, intrasubject fluctuations of brivaracetam concentrations between trough and 1-hour postdosing were observed. The present assay is faster and simpler than previously published analytical reports for brivaracetam in human plasma and is suitable for therapeutic drug monitoring.

Increasing Doses of Blueberry Polyphenols Alters Colonic Metabolism and Calcium Absorption in Ovariectomized Rats.

Blueberries are rich sources of bioactive polyphenols that may provide health benefits when consumed regularly, leading to their increased marketing as dietary supplements. However, the metabolic changes associated with consuming concentrated doses of purified polyphenols, as may be present in dietary supplements, are unknown, especially when considering the colonic metabolites formed. This study aimed to evaluate the pharmacokinetics of high doses of purified blueberry polyphenols. 5-month old, ovariectomized Sprague-Dawley rats are acutely dosed with purified blueberry polyphenols (0, 75, 350, and 1000mg total polyphenols per kg body weight (bw)) and 45 Ca to measure calcium absorption. Blood and urine are collected for 48 h after dosing and phenolic metabolites measured via ultra high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The most prominent metabolites are colonically generated cinnamic and hippuric acids. Smaller amounts of other phenolic acids, flavonols, and anthocyanins are also detected. Most metabolites follow a dose-response relationship, though several show saturated absorption. Maximal metabolite concentrations are reached within 12 h for a majority of compounds measured, while some (e.g., hippuric acid) peaked up to 24 h post-dosing. Calcium absorption is significantly increased in the highest dose group (p = 0.03). These results indicate that increased doses of blueberry polyphenols induce changes in intestinal phenolic metabolism and increase calcium absorption.

Elevated LysoGb3 Concentration in the Neuronopathic Forms of Mucopolysaccharidoses.

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders associated with impaired glycosaminoglycans (GAGs) catabolism. In MPS I, II, III, and VII, heparan sulfate (HS) cannot be degraded because of the lack of sufficient activity of the respective enzymes, and its accumulation in the brain causes neurological symptoms. Globotriaosylsphingosine (LysoGb3), the deacylated form of globotriaosylceramide (Gb3), is described as a highly sensitive biomarker for another lysosomal storage disease—Fabry disease. The connection between MPSs and LysoGb3 has not yet been established. This study included 36—MPS I, 15—MPS II, 25—MPS III, 26—MPS IV, and 14—MPS VI patients who were diagnosed by biochemical and molecular methods and a control group of 250 males and 250 females. The concentration of lysosphingolipids (LysoSLs) was measured in dried blood spots by high pressure liquid chromatography—tandem mass spectrometry. We have demonstrated that LysoGb3 concentration was significantly elevated (p < 0.0001) in untreated MPS I (3.07 + 1.55 ng/mL), MPS II (5.24 + 2.13 ng/mL), and MPS III (6.82 + 3.69 ng/mL) patients, compared to the control group (0.87 + 0.55 ng/mL). LysoGb3 level was normal in MPS VI and MPS IVA (1.26 + 0.39 and 0.99 + 0.38 ng/mL, respectively). Activity of α-galactosidase A (α-Gal A), an enzyme deficient in Fabry disease, was not, however, inhibited by heparan sulfate in vitro, indicating that an increase of LysoGb3 level in MPS I, MPS II, and MPS III is an indirect effect of stored MPSs rather than a direct result of impairment of degradation of this compound by HS. Our findings indicate some association of elevated LysoGb3 concentration with the neuronopathic forms of MPSs. The pathological mechanism of which is still to be studied.

Rapid determination of 12 semi-volatile pesticide residues in vegetables by miniaturized thermal-assisted purge-and-trap coupled with ultra high pressure liquid chromatography-tandem mass spectrometry

A method for the analysis of 12 semi-volatile pesticide residues in vegetables was developed by miniaturized thermal-assisted purge-and-trap coupled with ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Under the optimized conditions, the sample preparation could be completed within 10 min. The linear relationships of the 12 semi-volatile pesticide residues were good within their corresponding concentration ranges. The correlation coefficients were in range of 0.9918-0.9997. The limits of detection and limits of quantification were in range of 0.3-1.3 μg/kg and 1.0-4.3 μg/kg, respectively. The recoveries ranged from 80.0% to 120.0%, with intra-day and inter-day RSDs of less than 10%. This proposed method is quick and simple, and is suitable for the simultaneous and rapid analysis of semi-volatile pesticide residues in vegetables.

Predicting Pharmacokinetics Variation of Faropenem Using a Pharmacometabonomic Approach.

Individual variation in pharmacokinetics of faropenem is significant. We attempted to predict drug response of faropenem using a pharmacometabonomic approach. Metabolic profiling was performed on predose plasma samples from 36 healthy volunteers by gas chromatography-mass spectrometry (GC/MS), with 204 endogenous metabolites detected. Plasma concentration was measured after drug administration, using high pressure liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the pharmacokinetic parameters were calculated. Then a two-stage partial least squares strategy was employed to screen potential biomarkers and predict the pharmacokinetic parameters of faropenem. The results showed a good prediction of AUC and Cmax with the screened biomarkers, and metabolites such as valine, proline, aspartic acid, gluconic acid, glucuronic acid, and 2-ketoisocaproic acid were indicated as candidate biomarkers. Finally, we explored the mechanism of individual variation by pathway enrichment analysis, and it suggested that organic anion transporter 1 (OAT1) and 3 (OAT3) might be responsible for individual variation of faropenem, and this hypothesis was verified by an experiment in rats.