Corrigendum: A Hydrogel Drink With High Fructose Content Generates Higher Exogenous Carbohydrate Oxidation and a Reduced Drop in Dental Biofilm pH Compared to Two Other, Commercially Available, Carbohydrate Sports Drinks.

[This corrects the article on p. 88 in vol. 7, PMID: 32596251.].

A Hydrogel Drink With High Fructose Content Generates Higher Exogenous Carbohydrate Oxidation and Lower Dental Biofilm pH Compared to Two Other, Commercially Available, Carbohydrate Sports Drinks.

The purpose of this study was to evaluate the substrate oxidation of three commercially available, 14%-carbohydrate sports drinks with different compositions, osmolality, and pH for their impact on dental exposure to low pH. In a cross-over, randomized double-blinded design, 12 endurance athletes (age 31. 2 ± 7.7 years, V ˙ O2max 65.6 ± 5.0 mL·kg-1) completed 180 min of cycling at 55% Wmax. During the first 100 min of cycling, athletes consumed amylopectin starch (AP), maltodextrin+sucrose (MD+SUC), or maltodextrin+fructose hydrogel (MD+FRU) drinks providing 95 g carbohydrate·h-1, followed by water intake only at 120 and 160 min. Fuel use was determined using indirect calorimetry and stable-isotope techniques. Additionally, dental biofilm pH was measured using the microtouch method in a subsample of participants (n = 6) during resting conditions before, and at different time intervals up to 45 min following a single bolus of drink. Exogenous carbohydrate oxidation (CHOEXO) during the 2nd hour of exercise was significantly (P < 0.05) different between all three drinks: MD+FRU (1.17 ± 0.17 g·min-1), MD+SUC (1.01 ± 0.13 g·min-1), and AP (0.84 ± 0.11 g·min-1). At the end of exercise, CHOEXO and blood glucose concentrations (3.54 ± 0.50, 4.07 ± 0.67, and 4.28 ± 0.47 mmol·L-1, respectively) were significantly lower post MD+FRU consumption than post MD+SUC and AP consumption (P < 0.05). Biofilm acidogenicity at rest demonstrated a less pronounced pH fall for MD+FRU compared to the acidulant-containing MD+SUC and AP (P < 0.05). In conclusion, while total intake of MD+FRU showed signs of completed uptake before end of monitoring, this was less so for MD+SUC, and not at all the case for AP. Thus, this study showed that despite carbohydrates being encapsulated in a hydrogel, a higher CHOEXO was observed following MD+FRU drink ingestion compared to AP and MD+SUC consumption upon exposure to the acidic environment of the stomach. This finding may be related to the higher fructose content of the MD+FRU drink compared with the MD+SUC and AP drinks. Furthermore, a carbohydrate solution without added acidulants, which are commonly included in commercial sport drinks, may have less deleterious effects on oral health.

Alginate and HM-pectin in sports-drink give rise to intra-gastric gelation in vivo.

The addition of gelling polysaccharides to sport-drinks may provide improved tolerability of drinks with high concentration of digestible carbohydrates (CHO), otherwise known to increase the risk of gastro-intestinal complaints among athletes under prolonged exercise. The physico-chemical properties of a drink containing 14 wt% of digestible CHO (0.7 : 1 fructose and maltodextrin-ratio), 0.2 wt% of HM-pectin/alginate and 0.06 wt%. sodium chloride were examined under in vitro gastric conditions using rheology and large deformation testing. The in vivo gelling behaviour of the drink was studied using magnetic resonance imaging of subjects at rest together with blood glucose measurements. The in vivo results confirm gelation of the test drink, with no gel remaining in the stomach at 60 min and blood glucose values were similar to control. The physico-chemical characterisation of the acidified test drink confirms the formation of a weak gel through which low Mw CHO can diffuse.

A sensitive method detecting trace levels of levonorgestrel using LC-HRMS.

OBJECTIVE: To develop a high resolution mass spectrometry (HRMS) method to quantify levonorgestrel (LNG) in serum. STUDY DESIGN: Levonorgestrel was extracted using solid phase extraction and measured using liquid chromatography (LC) HRMS. RESULTS: Low limit of quantification (LLOQ) was 25 pg/mL and low limit of detection (LLOD) was 12.5 pg/mL. Precision and accuracy bias were <10%. LNG in serum samples from Mirena® users ranged between 37 to 219 pg/mL (n=12). In eight out of 22 patients with suspected intrauterine device (IUD) expulsion LNG was detected (26-1272 pg/mL). CONCLUSION: A sensitive, fast and simple LC-HRMS method was developed to detect trace levels of LNG.

Organic bromine compounds produced in sea ice in Antarctic winter.

During polar springtime, active bromine drives ozone, a greenhouse gas, to near-zero levels. Bromine production and emission in the polar regions have so far been assumed to require sunlight. Here, we report measurements of bromocarbons in sea ice, snow, and air during the Antarctic winter that reveal an unexpected new source of organic bromine to the atmosphere during periods of no sunlight. The results show that Antarctic winter sea ice provides 10 times more bromocarbons to the atmosphere than Southern Ocean waters, and substantially more than summer sea ice. The inclusion of these measurements in a global climate model indicates that the emitted bromocarbons will disperse throughout the troposphere in the southern hemisphere and through photochemical degradation to bromine atoms, contribute ~ 10% to the tropospheric reactive bromine budget. Combined together, our results suggest that winter sea ice could potentially be an important source of atmospheric bromine with implications for atmospheric chemistry and climate at a hemispheric scale.

Thermal inactivation of enzymes and pathogens in biosamples for MS analysis.

Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (≈1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

Matrix effect explained by unexpected formation of peptide in acidified plasma.

AIM: Peak distortion and strong signal enhancement was observed when applying a bioanalytical method based on mixed-mode SPE, hydrophilic interaction chromatography and ESI-MS to acidified rabbit plasma samples. RESULTS: High-resolution ESI-MS and N-terminal peptide sequencing revealed a peptide NFQNAL, which was confirmed by H/D exchange ESI-MS. CONCLUSION: The peptide causing the observed matrix effect was formed by enzymatic degradation of serum albumin at pH 3. Degradation required both acidification and presence of other plasma constituents in addition to albumin to take place. The degree of signal enhancement correlated to the level of NFQNAL in the ion source as measured by MS, with a maximal enhancement factor of 3 at intermediate levels of NFQNAL. The interference was eliminated by changing to another type of hydrophilic interaction chromatography column.

Variability of the IS revealed ionization enhancement by bile acid in mouse plasma.

BACKGROUND: Elevated IS response was observed in 22 out of 157 mouse plasma samples in a 3-month toxicity study. This initiated a root cause investigation. RESULTS: Mass spectra revealed that taurocholic acid (TCA) was present in the samples, partially eluted overlapping the analyte peak. An enhanced IS response (> twofold) was reproduced by injecting TCA together with the IS. Tests with five other drug compounds showed compound dependent matrix effects on ESI; enhancement as well as suppression. The matrix effects did not affect the integrity of study results, most likely due to the use of a 13C-labeled IS. CONCLUSION: The variability of TCA levels in plasma as well as the observed instability of the chromatographic retention complicates the evaluation of TCA-induced matrix effects during method development. Thus, monitoring the IS response in incurred samples is a useful tool to evaluate the performance of a validated method.

Capillary microsampling in the regulatory environment: validation and use of bioanalytical capillary microsampling methods.

Capillary microsampling (CMS) has recently been introduced as a response to the demands for more ethical use of laboratory animals according to the 3R principles. In CMS, an exact volume of the blood, plasma or other biofluid is collected in a capillary from which it is washed out, resulting in a diluted sample that can be handled using the existing equipment in the bioanalytical laboratory. CMS differs from traditional large volume sampling as the microsample is diluted before further handling and analysis, and reanalysis is performed using the diluted sample. This has some implications for the validation and this report is an attempt to clarify how to validate and use CMS methods in a regulatory environment. CMS also shows some distinct new opportunities: labile analytes can be immediately stabilized at sample collection and the addition of the internal standard to the whole sample can improve analytical performance. The experiences from 5 years use of CMS of plasma and blood for determination of drug exposure in animal studies are reviewed.

Tracing and separating plasma components causing matrix effects in hydrophilic interaction chromatography-electrospray ionization mass spectrometry.

Matrix effects on electrospray ionization were investigated for plasma samples analysed by hydrophilic interaction chromatography (HILIC) in gradient elution mode, and HILIC columns of different chemistries were tested for separation of plasma components and model analytes. By combining mass spectral data with post-column infusion traces, the following components of protein-precipitated plasma were identified and found to have significant effect on ionization: urea, creatinine, phosphocholine, lysophosphocholine, sphingomyelin, sodium ion, chloride ion, choline and proline betaine. The observed effect on ionization was both matrix-component and analyte dependent. The separation of identified plasma components and model analytes on eight columns was compared, using pair-wise linear correlation analysis and principal component analysis (PCA). Large changes in selectivity could be obtained by change of column, while smaller changes were seen when the mobile phase buffer was changed from ammonium formate pH 3.0 to ammonium acetate pH 4.5. While results from PCA and linear correlation analysis were largely in accord, linear correlation analysis was judged to be more straight-forward in terms of conduction and interpretation.

Heat stabilization of blood spot samples for determination of metabolically unstable drug compounds.

BACKGROUND: Sample stability is critical for accurate analysis of drug compounds in biosamples. The use of additives to eradicate the enzymatic activity causing loss of these analytes has its limitations. RESULTS: A novel technique for sample stabilization by rapid, high-temperature heating was used. The stability of six commercial drugs in blood and blood spots was investigated under various conditions with or without heat stabilization at 95°C. Oseltamivir, cefotaxime and ribavirin were successfully stabilized by heating whereas significant losses were seen in unheated samples. Amodiaquine was stable with and without heating. Artemether and dihydroartemisinin were found to be very heat sensitive and began to decompose even at 60°C. CONCLUSION: Heat stabilization is a viable technique to maintain analytes in blood spot samples, without the use of chemical additives, by stopping the enzymatic activity that causes sample degradation.

Development of a highly sensitive method using liquid chromatography-multiple reaction monitoring to quantify membrane P-glycoprotein in biological matrices and relationship to transport function.

The quantification of P-glycoprotein [P-gp, ABCB1, multidrug resistance 1 (MDR1)] protein in biological matrices is considered a key factor missing for useful translation of in vitro functional data to the in vivo situation and for comparison of transporter data among different in vitro models. In the present study a liquid chromatography (LC)-mass spectrometry method was developed to quantify P-gp membrane protein levels in different biological matrices. The amount of P-gp transporter protein was measured in Caco-2 cell monolayers and in inside-out human embryonic kidney (HEK)-MDR1 vesicles. From both in vitro systems, two preparations with different functionality were used. Transporter function was determined as digoxin efflux in Caco-2 cell monolayers and N-methylquinidine (NMQ) uptake in membrane vesicles, and, in addition, mRNA expression in the Caco-2 monolayers was measured. The results showed an excellent relationship between NMQ uptake functionality in inside-out HEK-MDR1 vesicles and protein contents. Similar concordance between the digoxin efflux and P-gp content in different Caco-2 cell cultures was observed, whereas mRNA levels are indicative of increased P-gp content and activity in older Caco-2 cultures, however, not yielding the same quantitative relationship. The results from both Caco-2 and HEK-MDR1 membrane vesicles confirm that the protein content is directly related to the level of activity in the respective system. The method presented here to quantify P-gp protein by LC-multiple reaction monitoring will facilitate the development of future methodologies to bridge between expression systems and cell/tissue models and to scale from in vitro models to whole organs.

Determination of ximelagatran, melagatran and two intermediary metabolites in plasma by mixed-mode solid phase extraction and LC-MS/MS.

An analytical method was developed for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the two intermediate metabolites, OH-melagatran and ethyl-melagatran in human plasma. Extraction of plasma was carried out on a mixed mode bonded sorbent material (C8/SO(3)(-)). All four analytes, including their isotope-labelled internal standards, were eluted at high ionic strength with a mixture of 50% methanol and 50% buffer (0.25 M ammonium acetate and 0.05 M formic acid, pH 5.3) with an extraction recovery above 80%. The extracts were demonstrated to be clean in terms of a low concentration of albumin and lysoPC. The sample extraction was fully automated and performed in 96-well plates using a Tecan Genesis pipetting robot. Analysis of the extracts were performed with liquid chromatography followed by positive electrospray ionization mass spectrometry. The low organic content and the low pH of the extracts allowed for, after dilution 1:3 with buffer, direct injection onto the LC-column. The four analytes were separated on a C18 analytical LC-column using gradient elution with the acetonitrile concentration varying from 10 to 30% (v/v) and the ammonium acetate and acetic acid concentration kept constant at 10 and 5 mmol/L, respectively, at a flow rate of 0.75 mL/min. Linearity was achieved over the calibrated range 0.010-4.0 micromol/L with accuracy and relative standard deviation in the range 96.9-101.2% and 6.6-17.1%, respectively at LLOQ, and in the range 94.7-102.6% and 2.7-6.8%, respectively at concentrations above 3 x LLOQ. The method replaces a manual method, and displays the advantages of having a fully automated sample clean-up, no evaporation/reconstitution step, high recovery, and complete LC-separation of all four analytes.

Interference from a glucuronide metabolite in the determination of ramipril and ramiprilat in human plasma and urine by gas chromatography-mass spectrometry.

In the course of development and validation of a gas chromatography-mass spectrometry (GC-MS) method for ramipril and its biologically active metabolite ramiprilat, evidence was found for an unknown interfering metabolite. Sample treatment included isolation from plasma or urine by solid-phase extraction, methylation with trimethylsilyldiazomethane and acylation with trifluoroacetic anhydride (TFAA). When liquid chromatography was used to fractionate plasma extracts prior to derivatization, the alkyl, acyl-derivative of ramipril was obtained from two separate LC fractions. Electrospray ionization mass spectral data, together with circumstances for the derivatization, were consistent with the presence of an N-glucuronide of ramipril. Interference from the metabolite was eliminated by including a wash step after extraction/alkylation, prior to acylation. The final assay had a lower limit of quantification at 1.0 nmol/L and a linear range of 1-300 nmol/L. Intra- and inter-batch precision for ramipril and ramiprilat in plasma or urine were better than 10 and 5% at 2 and 80 nmol/L, respectively.

Pharmacokinetics and pharmacodynamics of ximelagatran, a novel oral direct thrombin inhibitor, in young healthy male subjects.

OBJECTIVES: Ximelagatran is a novel, oral direct thrombin inhibitor designed to overcome the low and variable oral absorption of melagatran, its active form. The pharmacokinetics and pharmacodynamics of ximelagatran following single and repeated oral administration were investigated. The primary objectives were to determine the dose linearity and reproducibility of melagatran exposure and the influence of food intake. METHODS: Two open-label studies were performed in healthy male subjects. Study I was a dose-escalation study, in which subjects received single oral doses of ximelagatran (1-98 mg). Study II was a randomised, two-way crossover study consisting of two 5-day treatment periods, in which subjects received a 20-mg oral dose of ximelagatran twice daily, either before breakfast and with dinner, or with breakfast and after dinner. RESULTS: Ximelagatran was rapidly absorbed and converted to melagatran, which was the predominant compound in plasma. The mean (+/- standard deviation) bioavailability of melagatran was 22.2+/-4.3% and 17.4+/-2.8% after single and repeated dosings, respectively. The maximum plasma concentration of melagatran and the area under the melagatran plasma concentration-time curve (AUC) increased linearly with dose. Inter- and intra-subject variability in melagatran AUC was 8% and 12%, respectively, with no relevant food- or time dependence. Anticoagulation, assessed as activated partial thromboplastin time, was correlated with melagatran plasma concentration. There was virtually no increase in capillary bleeding time over the dose range studied, and ximelagatran was well tolerated. CONCLUSION: After oral administration of ximelagatran to healthy male subjects, the pharmacokinetic and pharmacodynamic profile of melagatran is predictable and reproducible.

Absorption, distribution, metabolism, and excretion of ximelagatran, an oral direct thrombin inhibitor, in rats, dogs, and humans.

The absorption, metabolism, and excretion of the oral direct thrombin inhibitor, ximelagatran, and its active form, melagatran, were separately investigated in rats, dogs, and healthy male human subjects after administration of oral and intravenous (i.v.) single doses. Ximelagatran was rapidly absorbed and metabolized following oral administration, with melagatran as the predominant compound in plasma. Two intermediates (ethyl-melagatran and OH-melagatran) that were subsequently metabolized to melagatran were also identified in plasma and were rapidly eliminated. Melagatran given i.v. had relatively low plasma clearance, small volume of distribution, and short elimination half-life. The oral absorption of melagatran was low and highly variable. It was primarily renally cleared, and the renal clearance agreed well with the glomerular filtration rate. Ximelagatran was extensively metabolized, and only trace amounts were renally excreted. Melagatran was the major compound in urine and feces after administration of ximelagatran. Appreciable quantities of ethyl-melagatran were also recovered in rat, dog, and human feces after oral administration, suggesting reduction of the hydroxyamidine group of ximelagatran in the gastrointestinal tract, as demonstrated when ximelagatran was incubated with feces homogenate. Polar metabolites in urine and feces (all species) accounted for a relatively small fraction of the dose. The bioavailability of melagatran following oral administration of ximelagatran was 5 to 10% in rats, 10 to 50% in dogs, and about 20% in humans, with low between-subject variation. The fraction of ximelagatran absorbed was at least 40 to 70% in all species. First-pass metabolism of ximelagatran with subsequent biliary excretion of the formed metabolites account for the lower bioavailability of melagatran.

Determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the intermediate metabolites, in biological samples by liquid chromatography-mass spectrometry.

Analytical methods for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and intermediate metabolites, melagatran hydroxyamidine and melagatran ethyl ester, in biological samples by liquid chromatography (LC) positive electrospray ionization mass spectrometry (MS) using selected reaction monitoring are described. Isolation from human plasma was achieved by solid-phase extraction on octylsilica. Analytes and isotope-labelled internal standards were separated by LC utilising a C(18) analytical column and a mobile phase comprising acetonitrile-4 mmol/l ammonium acetate (35:65, v/v) containing 0.1% formic acid, at a flow-rate of 0.75 ml/min. Absolute recovery was approximately 80% for ximelagatran, approximately 60% for melagatran ethyl ester and >90% for melagatran and melagatran hydroxyamidine. Limit of quantification was 10 nmol/l, with a relative standard deviation <20% for each analyte and <5% above 100 nmol/l. Procedures for determination of these analytes in human urine and breast milk, plus whole blood from rat and mouse are also described.

Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry.

Analytical methods for the determination of melagatran (H 319/68) in biological samples by liquid chromatography (LC)-positive electrospray ionization mass spectrometry using multiple reaction monitoring are described. Melagatran in plasma was isolated by solid-phase extraction on octylsilica, either in separate extraction tubes or in 96-well plates. Absolute recovery of melagatran from plasma was >92%. Melagatran and the internal standard, H 319/68 D2 13C2, were separated from other sample components by LC utilizing a C18 stationary phase and a mobile phase comprising 35% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. After dilution, urine was injected directly onto the LC column and subjected to gradient LC. The relative standard deviation was 1-5% for concentrations above the limit of quantification, which was estimated for plasma at 10 or 25 nmol/l for sample volumes of 500 or 200 microl, respectively, and 100 nmol/l for urine.

Enantioselective determination of felodipine in human plasma by chiral normal-phase liquid chromatography and electrospray ionisation mass spectrometry.

An analytical method was developed for the determination of the enantiomers of felodipine, a dihydropyridine-type calcium antagonist, in human blood plasma. Felodipine was extracted from plasma using toluene as extraction solvent. The enantiomers were separated on a cellulose tris(4-methyl benzoate) stationary phase (Chiralcel OJ-R) using 2-propanol-iso-hexane (11:89) as mobile phase. Post-column addition of ammonium acetate in ethanol-water (95:5) allowed sensitive detection of the ammonium adduct by electrospray ionisation and selected reaction monitoring. Deuterated felodipine racemate was used as internal standard. Within-run repeatability was determined and a coefficient of variation below 2% was achieved at 22 nmol/l and below 10% at 0.27 nmol/l. Between-day precision was evaluated and a coefficient of variation of 3.6% at 4 nmol/l plasma was obtained. Limit of quantification (LOQ) was set at 0.25 nmol/l (0.10 microg/l). The method proved adequate for pharmacokinetic studies of R- and S-felodipine after oral administration of therapeutic doses of felodipine.