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Automation of analytical sample preparation and testing is always attractive due to its high efficiency and continuous operation, but it can also be very challenging due to the operational complexity and method development. Studies focused on method development in automated systems are extremely rare and little has been published on the subject. In this paper, we investigate several unique and advanced aspects of this subject. The example product is selected containing two APIs to be tested by a single method to be productive. The product strengths of the two APIs are significantly different, making development of a single method much more challenging. In addition, we use a "green chemistry" approach, avoiding any organic solvent in the dissolving media in order to achieve an environmentally friendly solution. The selection and optimization of several key operational parameters are then discussed. The successful method is achieved using a new strategy called "Automated Online Standard (AO-STD)" methodology. This novel approach is compared to the manual operation procedure. The proposed approach can substantially reduce the method transfer time, generally considered the biggest obstacle to practical deployment of automated systems. The automated system applied in this paper is most suitable for content uniformity testing, but is also applicable for potency assays, blend uniformity testing, and degradation product tests., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The development of photodynamic therapy (PDT) is severely limited by short half-life of singlet oxygen ( 1 O 2 ) and the hypoxic microenvironment. In this work, a plasma membrane targeted photodynamic O 2 economizer (designated as P-POE) is developed to improve the subcellular delivery of photosensitizers and alleviate the tumor hypoxia for enhanced PDT effect. After self-assembly into nanomicelles, P-POE has a relatively high stability and a favorable photochemical performance, which are conducive to boosting the 1 O 2 production. Besides, the plasma membrane anchoring of P-POE contributes to enhancing the preferential retention and cellular accumulation of photosensitizers on tumor tissues and cells. More importantly, P-POE-induced mitochondrial respiratory depression is demonstrated to reduce the O 2 consumption of tumor cells to relieve the hypoxia. Consequently, P-POE still exhibits a robust PDT effect against hypoxic tumors, which greatly inhibits the proliferation of breast cancer with low adverse reactions. This innovative combination of subcellular targeting and hypoxic alleviation would advance the development of individualized drug delivery systems for photodynamic therapy against hypoxic tumors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cardiovascular diseases are the leading cause of death and morbidity worldwide. Atherosclerotic cardiovascular disease (ASCVD) is affected by both environmental and genetic factors. Microenvironmental disorders of the human gut flora are associated with a variety of health problems, not only gastrointestinal diseases, such as inflammatory bowel disease, but also extralintestinal organs. Hydrogen sulfide (H 2 S) is the third gas signaling molecule other than nitric oxide and carbon monoxide. In the cardiovascular system, H 2 S plays important roles in the regulation of blood pressure, angiogenesis, smooth muscle cell proliferation and apoptosis, anti-oxidative stress, cardiac functions. This review is aiming to explore the potential role of gut microbiota in the development of atherosclerosis through hydrogen sulfide production as a novel therapeutic direction for atherosclerosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Relative Response Factors (RRFs) can be used for quantitation of one compound against another and it is widely used for Impurity analysis of pharmaceutical products; however, the application in potency assay is limited. Through an extensive study shown in this paper, it can be concluded that using the "RRF methodology" for potency assay is much more challenging compared to impurity analysis, due to the much tighter criteria required for potency analysis. The effects of instrument settings, which are rarely discussed or recognized in current HPLC analytical method development and quality release testing, are discussed. These factors impact the RRF just as much as other commonly recognized HPLC parameters. The effects of UV detector settings, i.e. Slit Width, Step Width, Band Width, and Data Collection Module, have been explored. This phenomenon has been demonstrated using three compounds to observe the impact of their quantitation due to the significant RRF variations. Finally, principles to reduce RRF variations have been discussed, and practical considerations of RRF application to method development and method transfer are provided., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

A sensitive and rapid liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for simultaneous quantification of guanfu base A (GFA) and its metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) in human plasma with phenoprolamine hydrochloride (DDPH) as the internal standard. The analytes were extracted from human plasma by using liquid-liquid extraction with ethyl acetate and the LC separation was performed on a Diamonsil C(18) analytical column (150 mm x 2.1 mm i.d., 5 microm). The MS acquisition was performed in selected ion monitoring (SIM) mode of positive ions. Analysis was carried out in SIM mode at m/z 430.25 for GFA [M+H](+), m/z 388.25 for GFI [M+H](+), m/z 346.25 for AA [M+H](+) and m/z 344.20 for the IS DDPH [M+H](+). The calibration curves were linear over the range of 50-5000 ng/mL for GFA and 5-1000 ng/mL for GFI and AA, with coefficients of correlation above 0.999. The lower limit of quantification for GFA was 1 ng/mL, while for GFI and AA were both 5 ng/mL. The intra- and inter-day precisions (CV) of analysis were within 9%, and the accuracy ranged from 91% to 108%. The overall recoveries for GFA, GFI and AA were about 94.2%, 87.8% and 80.6%, respectively. The total LC-MS run-time was only 5.5 min. This quantitation method was successfully applied to the simultaneous determination of GFA and its metabolites in human plasma for the metabolic study and pharmacokinetic evaluation.

A new type of collagen-hyaluronate (COL/HA) matrix was synthesized by cross-linking collagen fibers with modified hyaluronate polymers bearing active formyl groups. The resulting matrix is a three-dimensional scaffold consisting of interconnected pores with an average size of 40 microm and a high pore volume/surface area ratio. The covalent nature of the bond between the collagen fibers and the modified hyaluronate was demonstrated by extended elution with phosphate buffered saline and by extraction in increasing ionic gradients. The fraction of covalently bound hyaluronate in the matrix ranged from 5 to 25 w%. The total hyaluronate content of the COL/HA matrix affected both the in vitro non-enzymatic and enzymatic degradation as well as the in vivo turnover. When implanted in cranial defects in rats, the COL/HA matrix demonstrated good biocompatibility and exhibited greater osteoconductive potential than matrices composed of either cross-linked collagen or cross-linked hyaluronate alone.

Synthetic fluorescent substrates for thrombin were immobilized on a polyurethane membrane. The interaction of thrombin with the immobilized substrates was investigated. Upon hydrolysis of immobilized substrates by thrombin, fluorescence was emitted, intensity increasing linearly with time. From a Lineweaver-Burk plot, kinetic parameters of the enzyme reaction on the membrane surface were determined. The dissociation constant (K) was increased and the catalytic constant (k) decreased markedly by immobilization. More reactive thrombin substrates in solution were also more reactive in the immobilized state. More thrombin was adsorbed and adsorbed thrombins were inactivated more strongly on the surface with more reactive substrates when immobilized. The immobilization of thrombin substrate markedly suppressed fibrin formation on the membrane surface and made the membrane non-thrombogenic.

The fibrinolytic enzyme urokinase was immobilized by encapsulation or cross-linking to poly(2-hydroxyethyl methacrylate) networks. The immobilized urokinase was more thermally stable, more stable against pH change and more resistant to inactivation by plasma protease inhibitors. These stabilities were improved with increasing degree of cross-linking. On the other hand, the enzymatic activity of immobilized urokinase decreased with increasing degree of cross-linking. A suitable degree of cross-linking is needed for the maintenance of high biological activity of immobilized urokinase over a long period.

Heparin was immobilized to polyetherurethaneurea membrane by covalent or ionic bondings with intervening spacer chains having different lengths and different terminal functional groups. The amount of immobilization of heparin and the release rate of immobilized heparin were controlled by the nature and the mode of bonding of spacer chains. The heparinized polyetherurethaneurea membranes became more in vitro antithrombogenic and suppressed more strongly the adhesion and activation of platelets, as the amount of immobilization increased. It was also shown that the membrane to which the low-molecular-weight fraction of heparin was immobilized was less stimulating to platelets.