Preparation and Biophysical Characterization of Quercetin Inclusion Complexes with ß-Cyclodextrin Derivatives to be Formulated as Possible Nose-to-Brain Quercetin Delivery Systems.

Quercetin (Que) is a flavonoid associated with high oxygen radical scavenging activity and potential neuroprotective activity against Alzheimer's disease. Que's oral bioavailability is limited by its low water solubility and extended peripheral metabolism; thus, nasal administration may be a promising alternative to achieve effective Que concentrations in the brain. The formation of Que-2-hydroxypropylated-ß-cyclodextrin (Que/HP-ß-CD) complexes was previously found to increase the molecule's solubility and stability in aqueous media. Que-methyl-ß-cyclodextrin (Que/Me-ß-CD) inclusion complexes were prepared, characterized, and compared with the Que/HP-ß-CD complex using biophysical and computational methods (phase solubility, fluorescence and NMR spectroscopy, differential scanning calorimetry (DSC), and molecular dynamics simulations (MDS)) as candidates for the preparation of nose-to-brain Que's delivery systems. DSC thermograms, NMR, fluorescence spectroscopy, and MDS confirmed the inclusion complex formation of Que with both CDs. Differences between the two preparations were observed regarding their thermodynamic stability and inclusion mode governing the details of molecular interactions. Que's solubility in aqueous media at pH 1.2 and 4.5 was similar and linearly increased with both CD concentrations. At pH 6.8, Que's solubility was higher and positively deviated from linearity in the presence of HP-ß-CD more than with Me-ß-CD, possibly revealing the presence of more than one HP-ß-CD molecule involved in the complex. Overall, water solubility of lyophilized Que/Me-ß-CD and Que/HP-ß-CD products was approximately 7-40 times and 14-50 times as high as for pure Que at pH 1.2-6.8. In addition, the proof of concept experiment on ex vivo permeation across rabbit nasal mucosa revealed measurable and similar Que permeability profiles with both CDs and negligible permeation of pure Que. These results are quite encouraging for further ex vivo and in vivo evaluation toward nasal administration and nose-to-brain delivery of Que.

Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography-tandem mass spectrometry.

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography-tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1'-carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]+ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M-CO2 +H]+ and [M-ΙmCO2 +H]+ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti-Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.

Hyperhydration using different hydration agents does not affect the haematological markers of the athlete biological passport in euhydrated volunteers.

Athlete Biological Passport (ABP) is an indirect approach, implemented by WADA, aimed at detecting blood manipulation based on abnormal changes in haematological markers. Cases report the use of hyperhydration as masking method during anti-doping urine sample collection which could potentially mask suspicious fluctuations on ABP profiles. This study investigated the hyperhydration effect on haemoglobin concentration, reticulocyte percentage and OFF-hr score (an algorithm based on haemoglobin concentration and reticulocyte percentage), with and without recombinant human erythropoietin (rHuEPO) administration. A five-week clinical study performed; Baseline and rHuEPO Phase. Water and a sports drink were used as hyperhydration agents. To examine the hyperhydration effect on the normal ABP profile per volunteer, hyperhydration was implemented at 0, 24 and 48 hours during the baseline. During the rHuEPO phase, volunteers received Epoetin beta (3000 IU) with hyperhydration to be implemented at 0, 24 and 48 hours after drug administration. Blood and urine samples were collected and analysed according to WADA guidelines. No significant effect on ABP markers was observed due to hyperhydration at any time during the study. Pre- and post-hyperhydration data were not statistically different compared to individual baseline data. In conclusion, hyperhydration does not affect the ABP haematological markers under the examined conditions.

Effect of hyperhydration on the pharmacokinetics and detection of orally administered budesonide in doping control analysis.

The aim of the present study was to investigate if hyperhydration could influence the excretion and subsequent detection of budesonide (BDS) and its main metabolites (6ß-hydroxy-budesonide and 16α-hydroxy-prednisolone) during doping control analysis by leading to concentrations below the WADA reporting level (30 ng/mL). The influence of hyperhydration on the plasma and urinary pharmacokinetic (PK) profiles of BDS and metabolites was also examined. Seven healthy physically active non-smoking Caucasian males participated in a 15-day clinical study. BDS was administered orally at a single dose of 9 mg on Days 1, 7, and 13. Hyperhydration was applied in the morning on two consecutive days, that is, 0 and 24 hours after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Results showed no significant difference (P > 0.05, 95% CI) on plasma or urinary PK parameters under hyperhydration conditions for all the analytes. However, significant differences (P < 0.05, 95% CI) due to hyperhydration were observed on the urinary concentrations of BDS and metabolites. To compensate the dilution effect due to hyperhydration, different adjustment methods were applied based on specific gravity, urinary flow rate, and creatinine. All the applied methods were able to adjust the concentration values close to the baseline ones for each analyte; however, specific gravity was the optimum method in terms of effectiveness and practicability. Furthermore, no masking of the detection sensitivity of BDS or its metabolites was observed due to hyperhydration either in plasma or urine samples.

Hyperhydration-Induced Decrease in Urinary Luteinizing Hormone Concentrations of Male Athletes in Doping Control Analysis.

Low urinary luteinizing hormone (LH) values have been discussed as a marker to detect steroid abuse. However, suppressed LH concentrations related to highly diluted urine samples could be a misleading indication of anabolic steroid abuse. One aim of the present study was to examine the effect of hyperhydration on the interpretation of LH findings during doping control analysis and to investigate different possibilities to correct volume-related changes in urinary LH concentrations. Seven healthy, physically active, nonsmoking White males were examined for a 72-hr period, using water and a commercial sports drink as hyperhydration agents (20 ml/kg body weight). Urine samples were collected and analyzed according to the World Anti-Doping Agency's technical documents. Baseline urinary LH concentrations, expressed as the mean ± SD for each individual, were within the acceptable physiological range (7.11 ± 5.42 IU/L). A comparison of the measured LH values for both hyperhydration phases (Phase A: 4.24 ± 5.60 IU/L and Phase B: 4.74 ± 4.72 IU/L) with the baseline ("normal") values showed significant differences (Phase A: p < .001 and Phase B: p < .001), suggesting the clear effect of urine dilution due to hyperhydration. However, an adjustment of urinary LH concentrations by specific gravity based on a reference value of 1.020 seems to adequately correct the hyperhydration-induced decrease on the LH levels.

Horseradish-peroxidase-conjugated anti-erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept.

Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non-specific cross-reactivity with secondary antibodies. An in-house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in-house anti-EPO-HRP conjugate was performed as well as the current two-step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in-house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control.

Hyperhydration Effect on Pharmacokinetic Parameters and Detection Sensitivity of Recombinant Human Erythropoietin in Urine and Serum Doping Control Analysis of Males.

Excessive fluid intake, that is, hyperhydration, may be adopted by athletes as a masking method during antidoping sample collection to influence the excretion patterns of doping agents and, therefore, manipulate their detection. The aim of this exploratory study was to assess the hyperhydration effect on the detection sensitivity of recombinant human erythropoietin (rHuEPO) by sodium N-lauroyl sarcosinate ("sarkosyl") polyacrylamide gel electrophoresis analysis. The influence of hyperhydration on the serum and urinary pharmacokinetic (PK) profiles of rHuEPO was also investigated. Seven healthy physically active nonsmoking Caucasian males participated in a 31-day clinical study comprising a baseline (days 0, 1-3, and 8-10) and a drug phase (days 15-17, 22-24, and 29-31). Epoetin beta was administered subcutaneously at a single dose of 3000 IU on days 15, 22, and 29. Hyperhydration was applied in the morning on 3 consecutive days (days 1-3, 8-10, 22-24, and 29-31), that is, 0, 24, and 48 h after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Serum and urinary concentration-time profiles were best described by a one-compartment PK model with zero-order absorption. Delayed absorption was observed after hyperhydration and, therefore, lag time was introduced in the PK model. Results showed no significant difference (p > 0.05) on serum or urinary erythropoietin concentrations under hyperhydration conditions. A trend for decreasing volume of distribution and increasing clearance after hyperhydration was observed, mainly after sports drink consumption. However, no significant differences (p > 0.05) due to hyperhydration for any of the serum PK parameters calculated by noncompartmental PK analysis were observed. Renal excretion of endogenous erythropoietin and rHuEPO, as reflected on the urinary cumulative amount, was increased approximately twice after hyperhydration and this supports the nonsignificant difference on the urinary concentrations. Analysis of serum and urine samples was able to detect rHuEPO up to 72 h after drug administration. The detection window of rHuEPO remained unaffected after water or sports drink ingestion. Hyperhydration had no effect on the detection sensitivity of EPO either in serum or urine samples.

Population reference ranges of urinary endogenous sulfate steroids concentrations and ratios as complement to the steroid profile in sports antidoping.

The population based Steroid Profile (SP) ratio of testosterone (T) and epitestosterone (E) has been considered as a biomarker approach to detect testosterone abuse in '80s. The contemporary Antidoping Laboratories apply the World Antidoping Agency (WADA) Technical Document (TD) for Endogenous Androgenic Anabolic Steroids (EAAS) in the analysis of SP during their screening. The SP Athlete Biological Passport (ABP) adaptive model uses the concentrations of the total of free and glucuronide conjugated forms of six EAASs concentrations and ratios measured by GC/MS. In the Antidoping Lab Qatar (ADLQ), the routine LC/MS screening method was used to quantitatively estimate the sulfate conjugated EAAS in the same analytical run as for the rest qualitative analytes. Seven sulfate EAAS were quantified for a number of routine antidoping male and female urine samples during screening. Concentrations, statistical parameters and selected ratios for the 6 EAAS, the 6 sulfate EAAS and 29 proposed ratios of concentrations from both EAAS and sulfate EAAS, which potentially used as SP ABP biomarkers, population reference limits and distributions have been estimated after the GC/MSMS analysis for EAAS and LC/Orbitrap/MS analysis for sulfate EAAS.

Predominantly post-transcriptional regulation of activation molecules in chronic lymphocytic leukemia: the case of transferrin receptors.

Transcriptional and post-transcriptional control mechanisms have a differential impact on cellular physiology depending on activation status. Several lines of evidence suggest that chronic lymphocytic leukemia (CLL) malignant B cells resemble antigen-experienced and activated B cells. In the present study, we investigated the expression of transferrin receptor 1 (TfR1, CD71), one of the "classical" markers up-regulated upon B-cell activation, and TfR2, a novel receptor for transferrin, in peripheral blood CD19+ B cells from ten healthy individuals and 76 patients with CLL so as to gain insight into potential disease-related differences in underlying regulatory mechanisms. Marked differences in the production and expression of these receptors were detected in malignant but not in normal B cells. Specifically, TfR1 mRNA and protein levels were significantly higher in comparison to TfR2, both in normal and malignant B cells. Furthermore, discrepancies between TfR mRNA and protein expression were observed in CLL; in contrast, mRNA and protein expression levels were generally concordant in normal B cells. Exposure to actinomycin D decreased TfR1 and TfR2 mRNA levels in normal CD19+ B cells but had no effect on CLL malignant cells. The protein synthesis inhibitor cycloheximide had opposing effects in normal vs. CLL malignant B cells: thus, TfR1 and TfR2 mRNA levels were increased in normal B cells, whereas they were unaffected or even suppressed in CLL malignant B cells. These results allude to differential regulation of TfR1 and TfR2 expression in normal B cells vs. CLL. In normal B cells, transcriptional mechanisms exert a critical control over TfR1 and TfR2 expression, whereas in CLL post-transcriptional mechanisms seem to play a complementary and perhaps more important role. This type of control appears to be especially suited for modulation of genes implicated in proliferation of activated cells, like CLL malignant B cells.

Transferrin receptor-1 and 2 expression in chronic lymphocytic leukemia.

Transferrin receptor (TfR)-1 and 2 mRNA and CD71 (TfR1) expression was analyzed in 118 CLL patients. Ninety-five out of 109 analyzed cases expressed CD71, mostly at a high level; 60% of CD71 (+) cases were IGH-mutated. All samples were TfR1 mRNA (+); TfR2-alpha/beta mRNA was detected in, respectively, 52/102 and 100/109 cases. Competitive RT-PCR showed widely divergent levels of TfR1 mRNA in cases with high CD71 expression, alluding to post-transcriptional control of TfR1 expression in CLL. The almost uniformly high CD71 expression in CLL is in keeping with the activated status of neoplastic cells, regardless of IGH mutational load.

Analytical progresses of the World Anti-Doping Agency Olympic laboratories: a 2016 update from London to Rio.

The 2016 Olympic and Paralympic Games, the biggest event in human sports, was held in Rio de Janeiro with more than 10,500 athletes from 206 countries over the world competing for the highest of sports honors, an Olympic medal. With the hope that the Olympic ideal accompanies all aspects of the XXXI Olympiad, WADA accredited antidoping laboratories use the spearhead of analytical technology as a powerful tool in the fight against doping. This review summarizes the main analytical developments applied in antidoping testing methodology combined with the main amendments on the WADA regulations regarding analytical testing starting from the 2012 London Olympics until the 2016 Olympic Games in Rio de Janeiro.

Current status and recent advantages in derivatization procedures in human doping control.

Derivatization is one of the most important steps during sample preparation in doping control analysis. Its main purpose is the enhancement of chromatographic separation and mass spectrometric detection of analytes in the full range of laboratory doping control activities. Its application is shown to broaden the detectable range of compounds, even in LC-MS analysis, where derivatization is not a prerequisite. The impact of derivatization initiates from the stage of the metabolic studies of doping agents up to the discovery of doping markers, by inclusion of the screening and confirmation procedures of prohibited substances in athlete's urine samples. Derivatization renders an unlimited number of opportunities to advanced analyte detection.

Gas chromatographic-mass spectrometric quantitation of busulfan in human plasma for therapeutic drug monitoring: a new on-line derivatization procedure for the conversion of busulfan to 1,4-diiodobutane.

A simplified gas chromatographic-mass spectrometric (GC-MS) analytical method, involving a novel derivatization procedure was developed for monitoring busulfan (Bu) plasma concentrations in populations undergoing bone marrow transplantation. Plasma samples (500 µL) containing Bu-d8 as internal standard were extracted with ethyl acetate (2 mL) followed by centrifugation (1800 rpm, 5 min) and evaporation of the organic layer under nitrogen flow (50 °C). The dry residue was reconstituted with 100 µL iodine solution in acetonitrile (0.25%, w/v) and 3 µL were injected into the GC-MS system at 250 °C. Conversion of Bu to 1,4-diiodobutane was accomplished on-line without the need of an extra derivatization step. MS was operated at selected ion monitoring mode at m/z 183 and 191 corresponding to Bu and Bu-d8 derivatives. Total analysis time was 11.5 min. Calibration curves were linear (mean r=0.9996) over a concentration range of 25-3651 ng/mL using a (1/x)-weighted scheme. Limit of detection and lower limit of quantitation were 10.6 and 25 ng/mL, respectively. Overall accuracy Er (%) was ranging from -5.10% to 10.5%. Within- and between-run RSD (%) were lower 4.51% and 2.15%, respectively. Overall recovery of Bu was equal to 69.3±4.56% (RSD (%)). The present method is sensitive and specific, requiring a simple sample preparation procedure and short analysis time, advantages crucial for therapeutic drug monitoring of Bu in clinical practice and application in pharmacokinetic studies.