Development and Validation of a Quantification Method of Cotinine in Urine Using Two Innovative Technologies: Supported Liquid Extraction and QDa Detection

Objective: Cotinine is the best biomarker of tobacco smoke exposure because of its long half-life. Several methods are developed to quantify cotinine in biological fluids, including high performance liquid chromatography. This method requires an extraction step in order to clean-up the sample and concentrate the analyte of interest. The aim of this study was to optimise the extraction step and to develop an easy, reproducible and specific method to measure cotinine in urine. Methods: An extraction of neutrals was chosen and performed by Supported Liquid Extraction (SLE). SLE consists in liquidliquid extraction in the presence of a sorbent enabling efficient extraction with less organic solvent and without any emulsion formation. Urine was basified by treatment with NH4OH in order to neutralise cotinine before loading on SLE plate. After drying, neutrals were eluted with a mix containing dichloromethane and isopropyl alcohol. Solvent was then evaporated and samples were reconstituted with water. Detection of cotinine was performed by mass detection using a QDa detector after UHPLC separation with a C18 column at a flow rate of 0.4 ml/min. A gradient elution of H2O +0.1% NH4OH and CH3CN was used. The method was validated based on linearity, precision, recovery and limits of detection and quantification. Results: The range of linearity was 0.001 μg/ml -5 μg/ml with a determination coefficient of 0.997. The precision was evaluated by the Relative Standard Deviation (RSD) for intra- and interassay and was below 5% and 10% respectively. 3 levels of concentration were tested to assess the recovery rate which was consistent and higher than 96%. Conclusion: Cotinine concentration can be measured in urine by SLE extraction and UHPLC-QDa detection. This method is easy, reproducible and allows quantification of low concentrations. It is a good solution to assess patients’ tobacco smoke exposure in medical laboratory

Development of an Ultra-High Performance Liquid Chromatography method for the simultaneous mass detection of tobacco biomarkers in urine.

The quantification of tobacco exposure biomarkers is relevant to follow the patients' tobacco use. They allow to discriminate between tobacco users, non-users, passive smokers, and nicotine products users, such as in nicotine replacement therapy. The aim of this study was to develop and validate a quantification method of tobacco biomarkers of choice - nicotine, cotinine, trans-3'-hydroxycotinine, anatabine and anabasine - in urine. The challenge was to develop an easy and rapid liquid chromatography method requiring only one extraction step and allowing simultaneous detections. Some methods are described in the literature but need specific investment in terms of instrumentation and users training. Here, the developed method had to be carried out with instrumentation easily accessible for medical laboratories. The extraction of the analytes was performed by Supported Liquid Extraction (SLE), which consists in liquid-liquid extraction but supported by a sorbent. It allows to insure efficient neutrals extraction with less organic solvent and without any emulsion formation. 200 µl of basified urine - analytes of interest are neutral in this condition - were loaded on Novum SLE 96-Well Plates (Phenomenex) and analytes were eluted with 1 % formic acid in dichloromethane/propan-2-ol (95/5). After solvent evaporation, samples were reconstituted with 100 µl of water for injection. A mass detector (QDa, Waters) was used to detect analytes, this pre-optimised quadrupole mass analyser being less expensive and requiring less adjustments than traditional mass spectrometers while benefiting of the reliability of mass spectral data. This detector was integrated after an Ultra-high performance liquid chromatography (UHPLC) separation on a BEH C18 column (Waters) at a flow rate of 0.5 ml/min. A gradient elution of HO (pH 10 with NHOH) and CHCN was used. Finally, the developed method was validated. This new method is conclusive to assess the patients' tobacco exposure and is easy to implement in medical laboratories

Alteration of the Condylar Oral Bone in Obese and Gastric Bypass Mice

Obesity is the main cause of type 2 diabetes mellitus (T2DM). Roux-en-Y gastric bypass (RYGB) surgery is an effective treatment for this obesity-related health problem. However, the adverse effects of T2DM on bone tissue persist or even aggravate after this surgical procedure. As studies on the mandibular condyle bone are scarce, the aim of the present study was to assess its compositional characteristics in T2DM and RYGB conditions. Thirty-two male C57BL/6 mice at 8 weeks of age were randomly assigned to receive either a high-fat or low-fat diet. After 14 weeks of high-fat diet intake, seven obese mice were subjected to RYGB surgery. All animals were euthanized at the age of 30 weeks. Mandibular bones were removed and the trabecular condyle region was assessed using Raman spectroscopy. A decreased mineralization was observed for both T2DM and RYGB condyle bones when compared to controls, with elevated carbonate substitutions for the RYGB group. No compositional differences in crystallinity and presence of advanced glycation end products were found between the groups, with the exception of an increased presence of N-carboxymethyl-lysine in RYGB bone compared to their T2DM counterpart. Site-specific measurements revealed a non-uniform bone composition, with increasing mineralization and carbonate substitutions towards the centre of the mandibular condyle. T2DM and RYGB surgery affect the mandibular condyle bone quality, as investigated at compositional level. Assessment of bone structural properties and remodelling should be carried out to further explore the effects of T2DM and RYGB surgery on this skeleton area

Visibility, location, and morphology of the primary maxillary sinus ostium and presence of accessory ostia: a retrospective analysis using cone beam computed tomography (CBCT)

OBJECTIVES: This retrospective study evaluated the visibility, location, and morphology of the primary maxillary ostium (PMO), as well as the presence and number of accessory maxillary ostia (AMO) in the maxillary sinus using cone beam computed tomography (CBCT). MATERIALS AND METHODS: CBCT scans with a large field of view with both maxillary sinuses entirely visible, acquired from February 2016 to February 2018, were initially screened. Patients were included if there was no history of surgical intervention/trauma in the sinus region. Two observers evaluated the CBCTs for PMO and AMOs independently. PMO and AMOs were evaluated in axial, coronal, and sagittal CBCT views. In case of disagreement, a third observer served as a referee. The findings were correlated with age, gender, condition of the sinus mucosa, and status of the dentition to assess for potential influencing factors. RESULTS: A total of 184 patients (368 maxillary sinuses) were included. PMO was present and patent in 346 (94.0%) of the 368 analyzed sinuses. Most of the PMOs were located above the attachment of and in the middle third of the inferior turbinate (76.1%) and exhibited a slit shape (71.1%). An AMO was present in 167 (45.5%) of the 368 analyzed sinuses, and 66 (17.9%) sinuses had multiple AMOs. Gender and sinus mucosa morphology were found to be influencing factors for the patency of the PMO. Furthermore, gender seems to be influencing the presence of an AMO. CONCLUSIONS: Most of the analyzed maxillary sinus cavities in the present population had a patent PMO. Being male and having morphological changes of the sinus mucosa were factors associated with a reduced prevalence of a patent PMO. CLINICAL RELEVANCE: A maxillary sinus with pathological findings of the mucosa seems to have a reduced prevalence of patent PMOs. Therefore, clinicians should take care to assess any clinical and radiographical sign indicating a potential maxillary sinusitis prior to surgical interventions in this region, especially in cases with planned sinus floor elevation.status: publishe

Alteration of the Condylar Oral Bone in Obese and Gastric Bypass Mice

Obesity is the main cause of type 2 diabetes mellitus (T2DM). Roux-en-Y gastric bypass (RYGB) surgery is an effective treatment for this obesity-related health problem. However, the adverse effects of T2DM on bone tissue persist or even aggravate after this surgical procedure. As studies on the mandibular condyle bone are scarce, the aim of the present study was to assess its compositional characteristics in T2DM and RYGB conditions. Thirty-two male C57BL/6 mice at 8 weeks of age were randomly assigned to receive either a high-fat or low-fat diet. After 14 weeks of high-fat diet intake, seven obese mice were subjected to RYGB surgery. All animals were euthanized at the age of 30 weeks. Mandibular bones were removed and the trabecular condyle region was assessed using Raman spectroscopy. A decreased mineralization was observed for both T2DM and RYGB condyle bones when compared to controls, with elevated carbonate substitutions for the RYGB group. No compositional differences in crystallinity and presence of advanced glycation end products were found between the groups, with the exception of an increased presence of N-carboxymethyl-lysine in RYGB bone compared to their T2DM counterpart. Site-specific measurements revealed a non-uniform bone composition, with increasing mineralization and carbonate substitutions towards the centre of the mandibular condyle. T2DM and RYGB surgery affect the mandibular condyle bone quality, as investigated at compositional level. Assessment of bone structural properties and remodelling should be carried out to further explore the effects of T2DM and RYGB surgery on this skeleton area.status: publishe

Facts and Fallacies of Radiation Risk in Dental Radiology

nrpages: 26status: publishe

Novel proposed cutoff values for anatabine and anabasine in differentiating smokers from non-smokers.

Anatabine and anabasine are two tobacco alkaloids used to differentiate between tobacco users and abstainers, including users of nicotine replacement therapy. Cutoff values (>2 ng/mL for both alkaloids) have not been revised since their implementation in 2002. These values may be too high, leading to increased likelihood of misclassification between smokers and abstainers. This results in major consequences, especially adverse outcomes of transplantation when smokers were incorrectly identified as being abstinent. This study proposes that a lower threshold for anatabine and anabasine will better distinguish tobacco users from non-users and thereby improve patients' care. A new and more sensitive analytical method by liquid chromatography-mass detection was developed to allow the quantification of low concentrations. Anatabine and anabasine were measured in urine samples of 116 self-reported daily smokers and 47 long-term non-smokers (confirmed by the analysis of nicotine and its metabolites). The best compromise between sensitivity and specificity allowed us to determine new cutoff values. The thresholds >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine were associated with a sensitivity of 97% (anatabine) and 89% (anabasine) and a specificity of 98% for both alkaloids. These cutoff values greatly increased the sensitivity given that it dropped to 75% (anatabine) and 47% (anabasine) when using the reference value (>2 ng/mL). The cutoff values >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine appear to better differentiate tobacco users from abstainers than the current reference threshold (>2 ng/mL for both alkaloids). It may considerably impact patients' care, especially in transplantation settings in which smoking abstinence is essential to avoid adverse outcomes of transplantation

Effect of the nicotine replacement therapy on biomarkers of inflammation, endothelial dysfunction, oxidative stress and lipids in smokers who quit smoking.

Our previous study showed major changes in biomarkers on quitting compared to the smoking state. They reflected a decrease of inflammation, endothelial activation, and oxidative stress, as well as an improved lipid profile. Nicotine replacement therapy (NRT) is effective to increase the rate of successful quitting, but health care professionals may have concerns to prescribe this first line smoking cessation treatment because its effect on inflammation and related processes is controversial. Therefore, the present study assessed the influence of NRT on biomarkers of inflammation, endothelial function, oxidative stress, and lipids, in people who quit smoking. Sixty-five subjects who daily smoke cigarettes were recruited and followed on quitting. Thirty-five quit using NRT and thirty quit without NRT. Biomarkers of inflammation, endothelial function, oxidative stress, and lipids were quantified at baseline when actively smoking and after cessation in the presence of NRT or not. Changes in biomarkers on quitting did not differ according to the treatment used. No difference was found when comparing participants who were exposed to NRT and those who were not. These results may indicate that NRT has no effect on inflammation, endothelial function, oxidative stress, and lipids, when used as a medication aid for quitting smoking. This study provides new evidence to support the safety profile of nicotine replacement therapy products regarding the biomarkers of endothelial function, oxidative stress, inflammation, and lipids

Changes in biomarkers of endothelial function, oxidative stress, inflammation and lipids after smoking cessation: A cohort study.

Tobacco use is known to be involved in the development of cardiovascular diseases, which leads to premature mortality. Endothelial dysfunction, the first step in this process, was shown induced by smoking. It is reported that quitting smoking could reduce the risk of diseases, but the implied mechanisms are still unclear. This study aimed to evaluate the biological markers of endothelial function in smokers when actively smoking and after cessation. Quantification of several biomarkers reflecting inflammation, endothelium activation, oxidative stress, and lipids was performed in 65 smokers when actively smoking and after cessation (median abstinence duration of 70 days). A possible decrease of inflammation was observed through the concentration reduction of a proinflammatory cytokine (interleukine-6) on quitting. A decrease of endothelium activation was visible by the reduced level of the soluble intercellular adhesion molecule. Two antioxidants, uric acid and vitamin C, were found at higher concentration than before the cessation, potentially reflecting the decrease of oxidative stress on quitting. Lipid profile was improved post-quit since HDL level was increased and LDL level was decreased. All these effects were visible at short term with abstinence duration less than 70 days. No sex-specific difference was observed and no additional changes were observed for longer abstinence duration. These observations suggest that some adverse effects of smoking on endothelial function could be reversible on quitting smoking. It could encourage smokers to enter a cessation program to reduce the risk for cardiovascular diseases development

Long-term stability of an infusion containing paracetamol, alizapride, ketorolac and tramadol in glass bottles at 5±3°C

Background and objective Infusion containing paracetamol, alizapride, ketorolac and tramadol is used after a general anaesthesia in order to limit pain, fever and nausea. Currently, these infusions are prepared according to demand in the anaesthesia unit, but the preparation in advance could improve quality of preparation and time management. The aim of this study was to investigate the long-term stability of this infusion in glass bottles at 5°C ± 3 °C. Method Five bottles of infusion were stored at 5°C ± 3 °C for 60 days. A visual and microscope inspection were performed periodically to observe any particle appearance or colour change. pH and absorbance at three wavelengths were measured. The concentrationswere measured by ultra-high performance liquid chromatography – diode array detection. Results Multiple verifications were performed during the first 35 days and no crystal, impurity or colour change were observed. At the next time point (42nd day), crystals were visible to the naked eye. pH and absorbance at 350 nm and 550 nm were stable. A slight increase in the absorbance at 410 nm was observed during the study, suggesting that a degradation product could be formed and absorb at this wavelength. The infusion was considered chemically stable while the lower one-sided prediction limit at 95% remains superior to 90% of the initial concentration. Concentration measurements demonstrated that ketorolac and alizapride remained stable in the infusion for 35 days. The stability of tramadol was 28 days. However, degradation of paracetamol was much faster given that concentration has fallen below 90% of the initial concentration after 7 days. Conclusion Infusion of paracetamol, alizapride,ketorolac and tramadol remains stable for 7 days in glass bottles at 5°C ± 3 °C and could be prepared in advance with these storage conditions