Identity verification of monoclonal antibodies by triple injection capillary zone electrophoresis.

This paper presents an approach based on triple injection capillary zone electrophoresis for identification of monoclonal antibodies. The analyte to be identified is injected between two zones of a known reference. The distances between the reference zones (plug I and III) and the target zone (plug II) are adjusted by partial electrophoresis of the first and second injection plugs. The full migration time of the target analyte is calculated from the observed migration time by considering the migration times of the reference in the first and third injection plugs. The relative migration time, that is, the ratio between the full migration time of the analyte and the migration time of the reference in the third injection plug provides the basis for identification. Here, eight monoclonal antibodies, including a pair of biosimilars, were used interchangeably as both analyte and reference to investigate potential of the method. The relative migration time for a preliminary positive identification were found to vary between 0.994 and 1.006 (1.000 ± 0.006, p = 95%). Beside the relative migration time, isoform distribution, peak profiles, and early migrating peaks, originating from components in the pharmaceutical formulations, were successfully used to verify the identity of all tested monoclonal antibodies.

A 47-Year-Old Woman with a Retained Central Venous Catheter Line Guidewire Presenting with a Right Atrial Thrombus Requiring Removal During Open Heart Surgery: A Case Report.

BACKGROUND A central venous catheter (CVC) is an indwelling catheter that is inserted into a large central vein for different purposes, including hemodynamic monitoring and administration of fluids and medications. This report is of a 47-year-old woman with a retained CVC line guidewire presenting with a large right atrial thrombus requiring removal during open heart surgery. CVC insertion is one of the most frequently attempted procedures in intensive care units, emergency departments, and operation rooms, especially for critically ill patients. Possible complications range from failure to place the catheter to cardiac arrest. One of the rarest complications is missing the guidewire after insertion, which is usually discovered early after inserting it. CASE REPORT We report the case of a 47-year-old woman who had a CVC line inserted following complicated open cholecystectomy. A few years later, she developed shortness of breath, with an incidental finding of a huge right atrial thrombus and a wire shown on transthoracic echocardiography. The right atrial thrombus required open heart surgery to excise the thrombus and the wire, which was done successfully. The thrombus was histopathologically and clinically proven to be an organized right atrial thrombus formed around the CVC guidewire. CONCLUSIONS This case report presents a rare complication of CVC insertion. Because this procedure is increasingly used, clinicians should be aware of the potential complications of retained CVC lines. Moreover, this report outlines different techniques to prevent such fatal complications and emphasizes the significance of radiography after insertion.

Avoid or seek light - a randomized crossover fMRI study investigating opposing treatment strategies for photophobia in migraine.

BACKGROUND: Photophobia, the aberrantly increased sensitivity to light, is a common symptom in migraine patients and light discomfort is frequently found as a trigger for migraine attacks. In behavioral studies, planned exposure to light was found to reduce headache in migraine patients with photophobia, potentially by increasing habituation to this migraine trigger. Here, we aimed to elucidate neurophysiological mechanisms of light exposure versus light deprivation in migraine patients using functional magnetic resonance imaging (fMRI). METHODS: Ten migraine patients (9 female, age = 28.70 ± 8.18 years) and 11 healthy controls (9 female, age = 23.73 ± 2.24 years) spent one hour on 7 consecutive days exposed to flashing light (Flash) or darkness (Dark) using a crossover design with a wash-out period of 3 months. Study participants kept a diary including items on interictal and ictal photophobia, presence and severity of headache 7 days before, during and 7 days after the interventions. One week before and one day after both interventions, fMRI using flickering light in a block design was applied. Functional activation was analyzed at whole-brain level and habituation of the visual cortex (V1) was modeled with the initial amplitude estimate and the corrected habituation slope. RESULTS: Mean interictal photophobia decreased after both interventions, but differences relative to the baseline did not survive correction for multiple comparisons. At baseline, flickering light induced activation in V1 was higher in the patients compared to the controls, but activation normalized after the Flash and the Dark interventions. V1 habituation indices correlated with headache frequency, headache severity and ictal photophobia. In the Flash condition, the individual change of headache frequency relative to the baseline corresponded almost perfectly to the change of the habituation slope compared to the baseline. CONCLUSIONS: On average, light exposure did not lead to symptom relief, potentially due to the short duration of the intervention and the high variability of the patients' responses to the intervention. However, the strong relationship between visual cortex habituation and headache symptoms and its modulation by light exposure might shed light on the neurophysiological basis of exposure treatment effects. TRIAL REGISTRATION: NCT05369910 (05/06/2022, retrospectively registered).

First evidence of long-term effects of transcranial pulse stimulation (TPS) on the human brain.

BACKGROUND: With the high spatial resolution and the potential to reach deep brain structures, ultrasound-based brain stimulation techniques offer new opportunities to non-invasively treat neurological and psychiatric disorders. However, little is known about long-term effects of ultrasound-based brain stimulation. Applying a longitudinal design, we comprehensively investigated neuromodulation induced by ultrasound brain stimulation to provide first sham-controlled evidence of long-term effects on the human brain and behavior. METHODS: Twelve healthy participants received three sham and three verum sessions with transcranial pulse stimulation (TPS) focused on the cortical somatosensory representation of the right hand. One week before and after the sham and verum TPS applications, comprehensive structural and functional resting state MRI investigations and behavioral tests targeting tactile spatial discrimination and sensorimotor dexterity were performed. RESULTS: Compared to sham, global efficiency significantly increased within the cortical sensorimotor network after verum TPS, indicating an upregulation of the stimulated functional brain network. Axial diffusivity in left sensorimotor areas decreased after verum TPS, demonstrating an improved axonal status in the stimulated area. CONCLUSIONS: TPS increased the functional and structural coupling within the stimulated left primary somatosensory cortex and adjacent sensorimotor areas up to one week after the last stimulation. These findings suggest that TPS induces neuroplastic changes that go beyond the spatial and temporal stimulation settings encouraging further clinical applications.

Determination and Pharmacokinetics of Omeprazole Enantiomers in Human Plasma and Oral Fluid Utilizing Microextraction by Packed Sorbent and Liquid Chromatography-Tandem Mass Spectrometry.

In the present work, the determination of omeprazole (OME) enantiomers in oral fluid and plasma samples was carried out utilizing microextraction by packed sorbent (MEPS) and liquid chromatography-tandem mass spectrometry. A chiral column with cellulose-SB phase was used for the first time for enantiomeric separation of OME with an isocratic elution system using 0.2% ammonium hydroxide in hexane-ethanol mixture (70 : 30, v/v) as the mobile phase. OME enantiomers were determined utilizing a triple quadrupole tandem mass spectrometer in positive ion mode (ESI+) monitoring mass transitions: m/z 346.3 ⟶ 198.0 for OME and m/z 369.98 ⟶ 252.0 for internal standard. The limits of detection and quantification of the present method for both enantiomers were 0.1 and 0.4 ng/mL, respectively. The method validation provided good accuracy and precision. The matrix effect factor was less than 5%, and no interfering peaks were observed. The interday precision values ranged from 2.2 to 7.5 (%RSD), and the accuracy of determinations varied from -9.9% to 8.3%. In addition, the pharmacokinetics (PK) of omeprazole enantiomers in healthy subjects after a single oral dose was investigated. (S)-Enantiomers showed higher levels than (R)-enantiomers throughout 24 h. It was found that the mean maximum concentrations of (R)- and (S)-omeprazole in plasma samples were about two times higher than in oral fluid.

A New Rehabilitative Mechanism in Primary Motor Cortex After Peripheral Trauma.

Homuncular organization, i.e., the neuronal representation of the human body within the primary motor cortex, is one of the most fundamental principles of the human brain. Despite this, in rare peripheral nerve surgery patients, the transformation of a monofunctional (diaphragm activation) into a bifunctional motor area (diaphragm and arm activation is controlled by the same cortical area) has previously been demonstrated. The mechanisms behind this transformation are not fully known. To investigate this transformation of a monofunctional area we investigate functional connectivity changes in a unique and highly instructive pathophysiological patient model. These patients suffer from complete brachial plexus avulsion with arm paralysis and had been treated with reconnection of the end of the musculocutaneous nerve to the side of a fully functional phrenic nerve to regain function. Task-based functional connectivity between the arm representations and the diaphragm (phrenic nerve) representations were examined in six patients and 12 aged matched healthy controls at ultra-high field MRI while they either performed or tried isolated elbow flexion or conducted forced abdominal inspiration. Functional connectivity values are considerably increased between the diseased arm and the bilateral diaphragm areas while trying strong muscle tension in the diseased arm as compared to the healthy arm. This effect was not found as compared to the healthy arm in the patient group. This connectivity was stronger between ipsilateral than between corresponding contralateral brain regions. No corresponding differences were found in healthy subjects. Our data suggests that the increased functional connectivity between the deprived arm area and the diaphragm area drives biceps muscle function. From this findings we infer that this new rehabilitative mechanism in the primary motor cortex may establish new intrahemispheric connections within the brain and the motor cortex in particular to reroute the output of a completely denervated motor area. This study extend current knowledge about neuroplasticity within the motor cortex.

Transcranial Pulse Stimulation with Ultrasound in Alzheimer's Disease-A New Navigated Focal Brain Therapy.

Ultrasound-based brain stimulation techniques may become a powerful new technique to modulate the human brain in a focal and targeted manner. However, for clinical brain stimulation no certified systems exist and the current techniques have to be further developed. Here, a clinical sonication technique is introduced, based on single ultrashort ultrasound pulses (transcranial pulse stimulation, TPS) which markedly differs from existing focused ultrasound techniques. In addition, a first clinical study using ultrasound brain stimulation and first observations of long term effects are presented. Comprehensive feasibility, safety, and efficacy data are provided. They consist of simulation data, laboratory measurements with rat and human skulls and brains, in vivo modulations of somatosensory evoked potentials (SEP) in healthy subjects (sham controlled) and clinical pilot data in 35 patients with Alzheimer's disease acquired in a multicenter setting (including neuropsychological scores and functional magnetic resonance imaging (fMRI)). Preclinical results show large safety margins and dose dependent neuromodulation. Patient investigations reveal high treatment tolerability and no major side effects. Neuropsychological scores improve significantly after TPS treatment and improvement lasts up to three months and correlates with an upregulation of the memory network (fMRI data). The results encourage broad neuroscientific application and translation of the method to clinical therapy and randomized sham-controlled clinical studies.

Improving sensitivity, specificity, and reproducibility of individual brainstem activation.

Functional imaging of the brainstem may open new avenues for clinical diagnostics. However, for reliable assessments of brainstem activation, further efforts improving signal quality are needed. Six healthy subjects performed four repeated functional magnetic resonance imaging (fMRI) sessions on different days with jaw clenching as a motor task to elicit activation in the trigeminal motor nucleus. Functional images were acquired with a 7 T MR scanner using an optimized multiband EPI sequence. Activation measures in the trigeminal nucleus and a control region were assessed using different physiological noise correction methods (aCompCor and RETROICOR-based approaches with variable numbers of regressors) combined with cerebrospinal fluid or brainstem masking. Receiver-operating characteristic analyses accounting for sensitivity and specificity, activation overlap analyses to estimate the reproducibility between sessions, and intraclass correlation analyses (ICC) for testing reliability between subjects and sessions were used to systematically compare the physiological noise correction approaches. Masking the brainstem led to increased activation in the target ROI and resulted in higher values for the area under the curve (AUC) as a combined measure for sensitivity and specificity. With the highest values for AUC, activation overlap, and ICC, the most favorable physiological noise correction method was to control for the cerebrospinal fluid time series (aCompCor with one regressor). Brainstem motor nuclei activation can be reliably identified using high-field fMRI with optimized acquisition and processing strategies-even on single-subject level. Applying specific physiological noise correction methods improves reproducibility and reliability of brainstem activation encouraging future clinical applications.

Peripheral Nervous System Reconstruction Reroutes Cortical Motor Output-Brain Reorganization Uncovered by Effective Connectivity.

Cortical reorganization in response to peripheral nervous system damage is only poorly understood. In patients with complete brachial plexus avulsion and subsequent reconnection of the end of the musculocutaneous nerve to the side of a phrenic nerve, reorganization leads to a doubled arm representation in the primary motor cortex. Despite, homuncular organization being one of the most fundamental principles of the human brain, movements of the affected arm now activate 2 loci: the completely denervated arm representation and the diaphragm representation. Here, we investigate the details behind this peripherally triggered reorganization, which happens in healthy brains. fMRI effective connectivity changes within the motor network were compared between a group of patients and age matched healthy controls at 7 Tesla (6 patients and 12 healthy controls). Results show the establishment of a driving input of the denervated arm area to the diaphragm area which is now responsible for arm movements. The findings extend current knowledge about neuroplasticity in primary motor cortex: a denervated motor area may drive an auxilliary area to reroute its motor output.

Analysis of Somatropin by Double-Injection Capillary-Zone Electrophoresis in Polybrene/Chondroitin Sulfate A Double-Coated Capillaries.

Purity determination of somatropin as a recombinant protein is important to ensure its safety and quality. This is carried out by capillary zone electrophoresis in double-injection mode using polybrene/chondroitin sulfate A double-coated capillaries. Modification of the capillary wall eliminates protein-wall interactions which results in improved accuracy and precision of the determinations. In the double-injection mode two somatropin samples are analyzed within a single electrophoretic run. Prior to the second injection, the first injected plug is electrophoresed for a predetermined time period in order to adjust the inter-plug distance. Here, the principle for the separation of somatropin charge variants is described.

Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry.

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150 × 4.6 mm, 5 µm) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8 mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2 mL/min. The total chromatographic run time was 10 min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.08 → 72.09 for metoprolol and m/z 303.3 → 154.3 for IS. The linearity range was 2.5-500 ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5 ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories. Copyright © 2016 John Wiley & Sons, Ltd.

Application of graphitic sorbent for online microextraction of drugs in human plasma samples.

In the present work a new sorbent based on graphitized carbon (CarbonX(®) COA) was evaluated in microextraction by packed sorbent (MEPS) for extraction of lidocaine and ropivacaine from human plasma samples. The new graphitic sorbent showed high recoveries of lidocaine and ropivacaine compared to C18 sorbent. In the present study the G-MEPS (syringe packed with graphitic sorbent) was connect online with liquid chromatography tandem mass spectrometry (LC-MS/MS). In order to obtain a fast and reliable method different factors affecting MEPS performance were investigated. The extraction efficiency of the graphitic sorbent was compared with silica-based sorbents used in MEPS. The G-MEPS was also evaluated for reuse (50-100 times). The recoveries of lidocaine and ropivacaine from plasma samples were 79% and 82%; respectively. The method was validated according to FDA (Food and Drug Administration) guideline for bioanalytical method validation. Linearity was assessed in the range 5-2000nmol/L, with coefficient of determination r(2)>0,995 (n=3) for lidocaine and r(2)>0.997 (n=3) for ropivacaine. The lower limit of quantification (LLOQ) was 5nmol/L and the limit of detection (LOD) was 1nmol/L for studied analytes in plasma samples. For both analytes considered in this study the accuracy values in plasma samples were ranged from 86% to 113%. The Inter-day precisions, expressed as relative standard deviation (%RSD), at three different concentrations (QC-samples) ranged from 8% to 9% for lidocaine, and from 4% to 11% for ropivacaine.

Graphite-based microextraction by packed sorbent for online extraction of ß-blockers from human plasma samples.

In the present work a new graphitic material (Carbon-XCOS) was used as a sorbent for microextraction by packed sorbent (MEPS). The ß-blockers metoprolol and acebutolol in plasma samples were extracted and detected online using Carbon-MEPS syringe and liquid chromatography and tandem mass spectrometry (LC-MS/MS). Factors affecting the MEPS performance such as conditioning, washing and elution solutions were investigated. The validation of the bioanalytical method was performed using human plasma. The standard curve ranged from 10 to 2000nM and the lower limit of quantification (LLOQ) was set to 10nM. The method validation showed good accuracy and precision for the quality control (QC) samples at three concentration levels (30, 800 and 1600nM). The accuracy values of the QC samples were in the range of 86-108% (n=18). The precision values of intra- and inter-day for QC samples ranged from 4.4% to 14.4% (RSD) for the both studied analytes. The coefficient of determination (R(2)) values were ≥0.999 (n=3).

Double-injection capillary electrophoresis for the identification of analytes.

This paper presents a new approach for identifying analytes by CE. The compound to be identified is analyzed together with the corresponding reference standard during a double injection capillary electrophoretic run. The inter-plug distance is regulated by applying an electrical field over the capillary for a predetermined time period (t(PE)). The migration time of an analyte being exposed to the partial electrophoresis was calculated from the partial migration time (t(mig(p))) as described in this paper. The identification is based on the closeness of agreement between the calculated migration time (t(mig(c))) and observed migration time (t(mig)) of the reference standard. The validity of the derived equations was checked by analyzing several substances such as caffeine, melamine, acetyl salicylic acid, paracetamol, ibuprofen, metoprolol, naproxen, somatropin, several insulin analogs, as well as different pharmaceutical and natural products. The migration time ratios for the identified solutes varied between 0.996 and 1.006 (i.e., 1.001 ± 0.005), indicating good agreement between the observed and calculated migration times.

Separation of somatropin charge variants by multiple-injection CZE with Polybrene/chondroitin sulfate A double-coated capillaries.

The performance of dynamic double-coated fused-silica capillaries with Polybrene and chondroitin sulfate A has been compared with uncoated fused-silica capillaries for the determination of recombinant human growth factor (somatropin) charge variants. The separations were carried out under the same electrophoretic conditions as described in the European Pharmacopoeia, i.e. at pH 6.0 and 30°C. The coating significantly reduced the interactions between the proteins and the surface of the fused-silica capillary. The first five separations performed in a new bare fused-silica capillary were discarded because of very poor separation performance as a result of protein-surface interactions. There was an approximate twofold increase in the interday migration time precision (%RSD ≤ 6.5%) in the double-coated capillaries. The method was successfully transferred to a multiple CZE mode where two samples were analyzed in a single electrophoretic run. The average purity of somatropin certified reference standard was 98.0% (%RSD ≤ 0.3%) determined by using uncoated and coated capillaries.

Weight status of the first-degree relatives of patients with type 2 diabetes based on the glucose tolerance test.

BACKGROUND: This study aimed to compare different body mass index (BMI) categories in individuals with diabetes, prediabetes and normal glucose tolerance among the first degree relatives of type 2 diabetic patients. MATERIALS AND METHODS: A cross-sectional study was conducted during 2005-2007 in Isfahan, Iran. It evaluated 3323 first-degree relatives of diabetic patients selected by consecutive convenient sampling method. Participants were classified as diabetic, prediabetic, and normal glucose tolerance test groups according to the results of 75 g oral glucose tolerance test (OGTT). The analysis of variance (ANOVA) was used for comparison of quantitative variables, and chi square test for comparison of categorical parameters. RESULTS: The study population consisted of 3323 individuals including 306 diabetics (98 males and 208 females), 1309 prediabetics (337 males and 972 females), and 1708 normal subjects (430 males and 1278 females). Among diabetic patients, the prevalence of obesity was 48.5% in women and 27.6% in men. Among prediabetics, the corresponding figures were 45.6% and 27.3%, respectively. CONCLUSIONS: Our findings suggest that men are diagnosed with T2DM at lower BMI than women. Moreover, the alarming high prevalence of overweight and obesity among females necessitates preventing and controlling this underlying problem among females.

Association of wrist circumference with cardio metabolic risk factors.

OBJECTIVE: The current study aimed to investigate the association of wrist circumference with major cardio metabolic risk factors. METHODS: This study was conducted in 2005-2007 among 3000 first-degree relatives of diabetic patients in Isfahan, Iran. RESULTS: Overall, 1709 (386 males and 1323 females) participants were enrolled in this study. The association of wrist circumference with cardio- metabolic risk factors was significantly positive with waist circumference (p = 0.001), BMI (p = 0.001), and LDL-C (p = 0.01), but significantly inverse with HDL-C (p = 0.001). The corresponding figure was not significant for triglycerides (p = 0.13), total cholesterol (p = 0.13), systolic BP (p = 0.15), diastolic BP (p = 0.6), and HbA1c (p = 0.4). CONCLUSION: Measurement of wrist circumference can serve as an easy-to-detect clinical marker to identify individuals at risk of cardio metabolic disorders, and can be used in large epidemiological studies.

Principles for different modes of multiple-injection CZE.

This paper introduces four different modes of multiple-injection CZE (MICZE). The validity of these MICZE models was evaluated by the experimental data. Prior to the application of MICZE, the electrophoretic conditions are developed in the single-injection mode by adjusting different experimental parameters such as pH, type and concentration of buffer additives and temperature. Based on the migration time difference (Deltatmig) between the analyte and the internal standard or injection marker, one or more MICZE modes can be employed. The injection marker is added to the sample to compensate for injection-volume fluctuations. The inter-plug distance is regulated by applying an electrical field over the capillary for a short period of time between each injection. After the final injection, the separation is completed by electrophoresis for a time period corresponding to that in the single-injection mode.

Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis.

A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30kV) over the capillary for different time periods, i.e., t(PE1) and t(PE2). The samples in each set were isolated from each other by partial electrophoresis for 2.35min (t(PE1)), while the sample sets were separated for 10.50min (t(PE2)). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8mg salbutamol. The average salbutamol content was determined to 7.8mg (+/-0.3mg) from simultaneous analyses of the 15 different tablets.