Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Impacts of preoperative anxiety and depression on pain and range of motion after arthroscopic frozen shoulder release: a cohort study.

PURPOSE: We aimed to evaluate the impact of preoperative anxiety and depression levels on baseline and postoperative pain in patients who underwent arthroscopic frozen shoulder release. METHODS: The study included 59 patients with more than three months of idiopathic frozen shoulder. All patients had arthroscopic frozen shoulder release. Two patients were excluded from statistical analysis. Therefore, the statistical analysis was performed on the remaining 57 patients. The patients were divided into two groups according to HADS scores: group 1 which included 28 patients with a healthy psychological status (anxiety ≤ 7 and depression ≤ 7), and Group 2, which included 29 patients with psychological distress ( anxiety ≥ 8 or depression ≥ 8). RESULTS: The hallmark finding of this study is that patients complaining of frozen shoulder symptoms and having psychological distress (HADS ≥ 8) experienced higher pain scores preoperatively and at one-year follow-up after arthroscopic release. All patients showed significant improvement between the preoperative period and the one year follow-up regarding the abduction, forward flexion, external rotation at the side and the VAS pain score with a P value of 0.001. CONCLUSIONS: Arthroscopic frozen shoulder release significantly lowers the VAS pain score over the 12-month.

Recent advances in miniaturization of portable liquid chromatography with emphasis on detection.

Liquid chromatography is a prominent analytical technique in separation science and chemical analysis, applied across numerous fields of research and within industrial applications. Over the past few decades, there has been a growing interest in the miniaturization of this technique, which has been particularly enabled through new miniature and portable detection technologies for in-field, at-site, and point-of-need (collectively 'out-of-lab') analyses. Accordingly, significant advances have been made in recent years in the development of miniaturized liquid chromatography with photometric, electrochemical, and mass spectrometric detection, enabling the development of field-deployable and portable instruments for various applications. Herein, recent developments in the miniaturization of detection systems for inclusion within, and/or coupling with, portable liquid chromatographic systems, are reviewed in detail together with critical comments and expected future trends in this area.

Small-Footprint, Field-Deployable LC/MS System for On-Site Analysis of Per- and Polyfluoroalkyl Substances in Soil.

Per- and polyfluoroalkyl substances (PFASs) are emerging environmental pollutants of global concern. For rapid field site evaluation, there are very few sensitive, field-deployable analytical techniques. In this work, a portable lightweight capillary liquid chromatography (capLC) system was coupled with a small footprint portable mass spectrometer and configured for field-based applications. Further, an at-site ultrasound-assisted extraction (pUAE) methodology was developed and applied with a portable capLC/mass spectrometry (MS) system for on-site analysis of PFASs in real soil samples. The influential variables on the integration of capLC with MS and on the resolution and signal intensity of the capLC/MS setup were investigated. The important parameters affecting the efficiency of the pUAE method were also studied and optimized using the response surface methodology based on a central composite design. The mean recovery for 11 PFASs ranged between 70 and 110%, with relative standard deviations ranging from 3 to 12%. In-field method sensitivity for 12 PFASs ranged from 0.6 to 0.1 ng/g, with wide dynamic ranges (1-600 ng/g) and excellent linearities (R2 > 0.991). The in-field portable system was benchmarked against a commercial lab-based LC-tandem MS (MS/MS) system for the analysis of PFASs in real soil samples, with the results showing good agreement. When deployed to a field site, 12 PFASs were detected and identified in real soil samples at concentrations ranging from 8.1 ng/g (for perfluorooctanesulfonic acid) to 2935.0 ng/g (perfluorohexanesulfonic acid).

Miniature Multiwavelength Deep UV-LED-Based Absorption Detection System for Capillary LC.

A new miniature deep UV absorbance detector has been developed using low-cost and high-performance LEDs, which can be operated in both scanning (230 to 300 nm) and individual wavelength (240, 255, and 275 nm) detection modes. The detector is mostly composed of off-the-shelf components, such as LEDs, trifurcated fiber optic assembly, a capillary Z-type flow cell, and photodiodes. It has been characterized for use with a standard capillary LC system and was benchmarked against a standard variable wavelength capillary LC detector. The detector shows very low levels of stray light (<0.4%), utilization of up to 99.0% of the effective path length of the flow cell, a wide dynamic range (0.5 to 200 µg/mL for sulfamethazine, carbamazepine, and flavone), and low noise levels (at 300 µAU level). The detector was applied within a miniaturized LC system.

Computer-assisted multifactorial method development for the streamlined separation and analysis of multicomponent mixtures in (Bio)pharmaceutical settings.

The (bio)pharmaceutical industry is rapidly moving towards complex drug modalities that require a commensurate level of analytical enabling technologies that can be deployed at a fast pace. Unsystematic method development and unnecessary manual intervention remain a major barrier towards a more efficient deployment of meaningful analytical assay across emerging modalities. Digitalization and automation are key to streamline method development and enable rapid assay deployment. This review discusses the use of computer-assisted multifactorial chromatographic method development strategies for fast-paced downstream characterization and purification of biopharmaceuticals. Various chromatographic techniques such as reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and supercritical fluid chromatography (SFC) are addressed and critically reviewed. The most significant parameters for retention mechanism modelling, as well as mapping the separation landscape for optimal chromatographic selectivity and resolution are also discussed. Furthermore, several computer-assisted approaches for optimization and development of chromatographic methods of therapeutics, including linear, nonlinear, and multifactorial modelling are outlined. Finally, the potential of the chromatographic modelling and computer-assisted optimization strategies are also illustrated, highlighting substantial productivity improvements, and cost savings while accelerating method development, deployment and transfer processes for therapeutic analysis in industrial settings.

Valorization of Eichhornia crassipes for the production of cellulose nanocrystals further investigation of plethoric biobased resource.

Chemical processing is among the significant keys to tackle agro-residues utilization field, aiming to obtain value-added materials. Extraction of cellulose nanocrystals (CNCs) is an emerging route to valorize lignocellulosic wastes into high value particles. In this investigation, effect of acidic hydrolysis duration was monitored on size and morphology of obtained crystals; namely: CNCs from Nile roses fibers (NRFs) (Eichhornia crassipes). Different acidic hydrolysis duration range or different characterization techniques set this article apart from relevant literature, including our group research articles. The grinded NRFs were firstly subjected to alkaline and bleaching pretreatments, then acid hydrolysis process was carried out with varied durations ranging from 5 to 30 min. Microcrystalline cellulose (MCC) was used as reference for comparison with NRFs based samples. The extracted CNCs samples were investigated using various techniques such as scanning electron microscopy (SEM), Atomic force microscopy (AFM), Raman spectroscopy, and thermogravimetric (TGA) analysis. The figures gotten from SEM and AFM depicted that NRFs based CNCs appeared as fibril-like shapes, with reduced average size when the NRFs underwent pulping and bleaching processes. This was indicated that the elimination of hemicellulose and lignin components got achieved successfully. This outcome was proven by chemical composition measurements and TGA/DTG curves. On the other hand, AFM-3D images indicated that CNCs topology and surface roughness were mostly affected by increasing hydrolysis durations, besides smooth and homogeneous surfaces were noticed. Moreover, Raman spectra demonstrated that the particle size and crystallinity degree of NRFs based CNCs can be affected by acidic hydrolysis durations and optimum extraction time was found to be 10 min. Thermal stability of extracted CNCs-NRFs and CNCs-MCC was measured by TGA/DTG and the kinetic models were suggested to identify the kinetic parameters of the thermal decomposition of CNCs for each acid hydrolysis duration. Increasing hydrolysis duration promoted thermal stability, particularly for NRFs based CNCs. Results showcased in this article add new perspective to Nile rose nanocellulose and pave down the way to fabricate NRFs based humidity nano-sensors.

Cellulose nanocrystals from agricultural residues (Eichhornia crassipes): Extraction and characterization.

Extraction of cellulose nanocrystals (CNCs) from agro-residues has received much attention, not only for their unique properties supporting a wide range of potential applications, but also their limited risk to global climate change. This research was conducted to assess Nile roses (Eichhornia crassipes) fibers as a natural biomass to extract CNCs through an acid hydrolysis approach. Nile roses fibers (NRFs) were initially subjected to alkaline (pulping) and bleaching pretreatments. Microcrystalline cellulose (MCC) was used as control in comparison to Nile rose based samples. All samples underwent acid hydrolysis process at a mild temperature (45 °C). The impact of extraction durations ranging from 5 to 30 min on the morphology structure and crystallinity index of the prepared CNCs was investigated. The prepared CNCs were subjected to various characterization techniques, namely: X-ray diffraction (XRD), FT-IR analysis, Transmission electron microscopy (TEM), and X-ray Photoelectron spectroscopy (XPS). The outcomes obtained by XRD showed that the crystallinity index increased as the duration of acid hydrolysis was prolonged up to 10 min, and then decreased, indicating optimal conditions for the dissolution of amorphous zones of cellulose before eroding the crystallized domains. These data were confirmed by FT-IR spectroscopy. However, a minor effect of hydrolysis duration on the degree of crystallinity was noticed for MCC based samples. TEM images illustrated that a spherical morphology of CNCs was formed as a result of 30 min acid hydrolysis, highlighting the optimal 20 min acid hydrolysis to obtain a fibrillar structure. The XPS study demonstrated that the main constituents of extracted CNCs were carbon and oxygen.

Multi-wavelength deep-ultraviolet absorbance detector based upon program-controlled pulsing light-emitting diodes.

A novel approach for multi-wavelength ultraviolet (UV) absorbance detection has been introduced employing a single board computer (SBC) with a field programmable gate array (FPGA), Red Pitaya SBC, to generate separated micro pulses for three deep-ultraviolet light-emitting diodes (DUV-LEDs), λmax = 235, 250, and 280 nm, along with data acquisition and processing via a custom-made program. The pulse set generation and data acquisition were synchronized using the SBC. The outputs of the three pulsing DUV-LEDs were combined and transmitted to the flow cell via a solarisation resistant trifurcated optical fiber (OF). An ultra-fast responding photodiode was connected to the optical-fiber-compatible flow cell to record the intensity of the DUV pulses. Upper limit of detector linearity (A95 %) was found to be 1917 mAU, 2189 mAU, and 1768 mAU at 235 nm, 250 nm, and 280 nm, respectively, with stray light ≤0.9 %. In addition, the effective path length (Leff) was estimated to be ≥98.0 % of the length of the used flow cell (50 mm). The new pulsed multi-LEDs absorbance detector (PMLAD) has been successfully coupled with a standard liquid chromatograph and utilized for the analysis of pharmaceuticals. Paracetamol, caffeine, and aspirin were simultaneously determined at 250, 280, and 235 nm, respectively, using the PMLAD. The absorbance ratios between the different wavelengths were applied to further confirm the identity of the studied compounds. Excellent linearity was achieved over a range of 0.1-3.2 µg/mL for paracetamol, 0.4-6.4 µg/mL for caffeine, and 0.8-12.8 µg/mL for aspirin with a regression correlation coefficient (r2) ≥ 0.99996. The quantitation limits (LOQs) were 0.10 µg/mL, 0.38 µg/mL, and 0.66 µg/mL for paracetamol, caffeine, and aspirin, respectively.

Incidence and Outcomes of Acute Kidney Injury in Critically Ill Patients with Coronavirus Disease 2019.

Patients with more severe cases of coronavirus disease-19 (COVID-19) may be at greater risk for developing acute kidney injury (AKI). The aim of our study was to analyze incidence and outcomes of AKI in critically ill patients with COVID-19. Our study prospectively followed about 198 patients with COVID-19 admitted to intensive care unit (ICU), Al Adan Hospital, Kuwait, for developing AKI and outcomes. Age, gender, nationality, history of hypertension, diabetes mellitus, ischemic heart disease, congestive heart failure, bronchial asthma, and chronic obstructive pulmonary disease were analyzed. The need for mechanical ventilation (MV), extracorporeal membrane oxygenation, inotropes, and medications was recorded. Causes of AKI, indication of dialysis, dialysis modality, dialysis outcomes, and mortality were analyzed. Our study reported that61 out of 198 (30.8%) ICU patients positive for COVID-19, developed AKI according to the Kidney Disease Improving Global Outcomes definition of AKI. Forty-eight out of 61 (79%) patients need continuous renal replacement therapy using continuous venovenous hemodiafiltration. Thirty-seven (61%) out of 61 patients were with severe sepsis syndrome. The most common cause of AKI was sepsis, cytokine storm, hypovolemia, heart failure, MV, and nephrotoxic drugs. Twenty-four patients (39%) out of 61 patients died, and the most common cause of death was sepsis, cytokine storm with respiratory failure, heart failure, and AKI. The outcome of AKI was as follows: six patients (10%) had complete recovery, five patients had partial recovery (8%), and 26 (43%) patients became dialysis dependent. Incidence of AKI is high in ICU COVID-19 patients and is associated with poor outcomes and high mortality. Early detection and specific therapy of kidney changes, including adequate hemodynamic support and avoidance of nephrotoxic drugs, may help to improve critically ill patients with COVID-19.

Small footprint liquid chromatography-mass spectrometry for pharmaceutical reaction monitoring and automated process analysis.

Liquid chromatography (LC) has broad applicability in the pharmaceutical industry, from the early stages of drug discovery to reaction monitoring and process control. However, small footprint, truly portable LC systems have not yet been demonstrated and fully evaluated practically for on-line, in-line or at-line pharmaceutical analysis. Herein, a portable, briefcase-sized capillary LC fitted with a miniature multi-deep UV-LED detector has been developed and interfaced with a portable mass spectrometer for on-site pharmaceutical analysis. With this configuration, the combined small footprint portable LC-UV/MS system was utilized for the determination of small molecule pharmaceuticals and reaction monitoring. The LC-UV/MS system was interfaced directly with a process sample cart and applied to automated pharmaceutical analysis, as well as also being benchmarked against a commercial process UPLC system (Waters PATROL system). The portable system gave low detection limits (∼3 ppb), a wide dynamic range (up to 200 ppm) and was used to confirm the identity of reaction impurities and for studying the kinetics of synthesis. The developed platform showed robust performance for automated process analysis, with less than 5.0% relative standard deviation (RSD) on sample-to-sample reproducibility, and less than 2% carryover between samples. The system has been shown to significantly increase throughput by providing near real-time analysis and to improve understanding of synthetic processes.

Miniature and fully portable gradient capillary liquid chromatograph.

A robust, portable and miniature battery powered gradient capillary liquid chromatograph (total weight ∼2.7 kg, without battery ∼2.0 kg), with integrated microfluidic injection, column heating and high sensitivity low-UV absorbance detection is presented. The portable capillary chromatograph, was applied with a packed reversed-phase capillary column (100 mm × 300 µm I.D., 5 µm ODS), housed within an integrated capillary column heater controlled by a proportional-integral-derivative (PID) chip module. The system delivered retention time and peak area relative standard deviation in isocratic mode of <0.7% (n = 10) and <3.3% (n = 10), respectively, and <0.1% (n = 10) and <2.3% (n = 10) respectively, for gradient elution mode. Detection was based upon a 255 nm light-emitting diode (LED) using one of two commercial capillary flow-cell options, namely a high sensitivity 12 nL Agilent capillary z-cell (HSDC) and a 45 nL Thermo Fisher Scientific UZ-View™ flow cell (UZFC). The HSDC, housed within a 3D printed detector arrangement, gave an effective pathlength of 1.01 mm (84% of nominal pathlength) and stray light of only 0.2%. Limits of detection for four test small molecule pharmaceuticals ranged from 65 to 101 µg L-1 based upon a 316 nL injection volume, with separation efficiencies of between 18,000 and 29,700 N m-1, with sub-4 min run times. The portable capillary LC system was successfully coupled to a small footprint portable mass spectrometer (Microsaic 4500 MiD) to demonstrate compatibility and 'point-of-need' miniaturised LC-MS capability.

Tibio-calcaneal fusion by retrograde intramedullary nailing in charcot neuroarthropathy.

INTRODUCTION: Charcot's neuroarthropathy of ankle leads to instability, destruction of the joint with significant morbidity that may require an amputation. Aim of surgical treatment is to achieve painless stable plantigrade foot through arthrodesis. Achieving surgical arthrodesis in Charcot's neuroarthropathy has a high failure rate. We assess the outcomes of retrograde intramedullary interlocked nailing in tibio-talar arthrodesis for Charcot's neuroarthropathy. MATERIALS AND METHODS: 42 diabetic patients with a mean age of 49 year underwent ankle tibio-talar arthrodesis using retrograde nailing for Charcot's neuroarthropathy. The postoperative complications have been discussed and their management outlined. The outcomes were measured radiologically and clinically. Follow up was done after 6 weeks, 3 months, 6 months and 1 year. RESULTS: 14 patients (33.3%) achieved uneventful uncomplicated fusion. Thirty three patients had varus deformity. Nine patients had valgus deformity. Using Paired t test, it showed statistically significant improvement in the functional outcomes (AOFAS& EQ-5D-5L) over the follow up time, despite of the mild deterioration of radiological angles in the final follow up visit compared to the postoperative radiological findings. CONCLUSION: Retrograde nailing is one of the best options for tibio-talo-calcaneal arthrodesis in the high-risk Charcot's neuroarthropathy population. It could be done through small incisions with lower soft tissue complications, its load-sharing properties allows a considerable compression across the ankle and talocalcaneal joints with early weight bearing and with satisfactory functional outcomes.