Filtration Performance of Biofilm Membrane Bioreactor: Fouling Control by Threshold Flux Operation.

Membrane fouling is the major factor that restricts the furtherly widespread use of membrane bioreactor (MBR). As a new generation of MBR, biofilm membrane bioreactor (BF-MBR) demonstrates high treatment efficiency and low sludge growth rate, however the filtration performance improvement and membrane fouling control are still the challenges for its further development. This work investigated the filtration performance using resistance in series model and membrane fouling control via threshold flux for BF-MBR. At first, the flux behavior and filtration resistance under various operating conditions, including agitation speed, membrane and TMP, were explored by resistance in series model. Because of the desirable anti-fouling capacity, UP100 and UP030 had the high threshold flux (100 and 90 L m-2 h-1) and low irreversible fouling resistance (1 and 1.3×10-10 m). Higher shear stress produced by higher agitation speed could reduce membrane fouling, while greatly promote the threshold flux (138 L m-2 h-1) and membrane cleaning efficiency (96%). Moreover, increasing shear stress or selecting membrane with large pore size could decrease the fouling rate and raise the threshold flux . As for TMP, high TMP reduced the removal rate for organic and nutrient, and enhanced the irreversible fouling. Besides, the aerobic-BF-MBR (101 L m-2 h-1 and 1.3×10-10 m) with lower foulant concentration had a better filtration performance than anoxic-BF-MBR(90 L m-2 h-1 and 1.5×10-10 m). Additionally, the long-term tests with 10 cycles were conducted to evaluate the industrial application value of BF-MBR (45-58 L m-2 h-1). This work provides the technical support for sustainable filtration performance of BF-MBR.

A randomized, crossover, phase I clinical study to evaluate bioequivalence and safety of tofacitinib and Xeljanz® in Chinese healthy subjects.

OBJECTIVE: Tofacitinib is an oral Janus kinase (JAK) inhibitor that has been marketed and approved in the USA for the clinical treatment of rheumatoid arthritis, psoriasis and other inflammatory and autoimmune diseases. A phase I clinical trial was conducted to compare the bioequivalence and safety of tofacitinib (Chia Tai Tianqing Pharmaceutical Group Co., Ltd.) and Xeljanz® (Pfizer Inc.) in healthy Chinese subjects, providing basis for the clinical application of tofacitinib. METHODS: Healthy Chinese subjects (N = 32) were randomly assigned to two groups at a 1:1 ratio. Subjects orally took 5 mg tofacitinib or Xeljanz® per cycle in random sequence. Blood samples were collected at 15 sampling points per cycle, and plasma drug concentrations of tofacitinib or Xeljanz® were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and statistical analysis for the pharmacokinetic (PK) parameters. Subjects' physical indicators were monitored during the whole process to evaluate drug safety. RESULTS: The adjusted geometric mean ratios (GMRs) of the peak concentration (Cmax), area under the curve (AUC) from time zero to the last measurable concentration (AUC0-t) and AUC from time zero to observed infinity (AUC0-∞) were all within the range of 80-125%. The other PK parameter values were similar. The above values were all meeting the bioequivalence criteria with well safety. CONCLUSION: The pharmacokinetic parameters and safety profile of tofacitinib were similar to those of Xeljanz® in healthy Chinese subjects. Therefore, tofacitinib can be considered bioequivalent to Xeljanz®, and the findings of this trial will promote the clinical application of tofacitinib.

Bioanalysis in the Age of New Drug Modalities.

In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.

Detection of sivelestat and its metabolite in small volumes of plasma from Chinese ALI/ARDS patients with SIRS via high-throughput UPLC-MS/MS: A pharmacokinetic study.

In this study, we developed a sensitive and efficient analytical approach combining a 96-well plate-based protein precipitation strategy with ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS) in order to assess the pharmacokinetic (PK) properties of sivelestat and its metabolite XW-IMP-A in samples of plasma from ALI/ARDS patients with SIRS. The samples were separated via gradient elution with a C18 column (Phenomenex Kinetex, C18, 2.6 µm, 100 Å, 50 × 2.1 mm) using 0.1 % formic acid aqueous solution (A) and acetonitrile-methanol (1:1, V:V) (B) as a mobile phase at a 0.6 mL/min flow rate. UPLC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 435.1 → 360.0 for sivelestat, m/z 469.0 → 394.0 for sivelestat-IS, m/z 351.0 → 276.0 for XW-IMP-A, and m/z 384.9 → 310.0 for XW-IMP-A-IS. This assay was run for 2.5 min in total, and achieved lowest limit of quantitation values of 2.0 ng/mL and 0.5 ng/mL for sivelestat and XW-IMP-A, respectively, while remaining highly linear from 2-500 ng/mL for sivelestat (r2 ≥ 0.9900) and from 0.5-125 ng/mL for XW-IMP-A (r2 ≥ 0.9900). These validated data were consistent with US Food and Drug Administration (FDA) and European Medicines Agency (EMA) acceptance criteria. In addition, this method was successfully applied to the steady-state PK evaluation of ALI/ARDS patients with SIRS.

Determination of unbound fraction of dorzagliatin in human plasma by equilibrium dialysis and LC-MS/MS and its application to a clinical pharmacokinetic study.

Dorzagliatin, a novel glucokinase (GK) activator targeting both pancreatic and hepatic GK, is currently in late-stage clinical development for treatment of type 2 diabetes (T2D). For the optimization of dosing regimens to ensure adequate safety and efficacy, it is critical to have a deep understanding of pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the drug in various targeting patient populations, considering the fact that T2D adversely affects a vast patient population who often times also suffer from a wide range of comorbidities including severe liver and/or kidney damage. Since drug efficacy seems to be closely related to unbound drug concentrations at the site of action, therefore, the determination of plasma unbound concentrations/fractions of dorzagliatin is of crucial importance, especially when performing the PK/PD assessment in those special populations. In the current study, a method was developed and validated for determining the unbound fraction (fu) of dorzagliatin in human plasma by using equilibrium dialysis for the separation of the bound and unbound drug, and LC-MS/MS for subsequent quantification. We have successfully addressed two widely recognized challenges for determination of the fu, i.e., the lack of knowledge on the "true fu" and the difficulty in assessing the accuracy and reproducibility of the measurement. Using this method, a 0.2 mL aliquot of human plasma samples were first dialyzed against 0.35 mL of phosphate buffered saline buffer at 37 °C for 5 h in the equilibrium dialysis device to separate the unbound dorzagliatin. Afterwards, post-dialysis samples were extracted by protein precipitation using acetonitrile. Separation of dorzagliatin and potential interferences were achieved using a Gemini C18 column coupled with gradient elution. Subsequent detection was carried out on tandem mass spectrometer operated by multiple reaction monitoring in positive mode using electrospray ionization. The standard curve over the concentration range of 0.125-250 ng/mL exhibits good linearity. The method was fully validated meeting the requirements in current bioanalytical guidance and was successfully applied in a clinical PK study of dorzagliatin in healthy volunteers and patients with renal function impairment. Method reproducibility was demonstrated in incurred sample reanalysis. With demonstrated accuracy, stability and reproducibility, reliable analytical results were obtained from clinical samples for PK/PD interpretation, providing valuable insight for the development of dorzagliatin.

Aerobic granular sludge (AGS) scouring to mitigate membrane fouling: Performance, hydrodynamic mechanism and contribution quantification model.

Aerobic granular sludge (AGS) has been proven to have a low fouling potential in membrane bioreactor (MBR). Nevertheless, AGS scouring effect on mitigating membrane fouling remains poorly investigated. The main objective of this study is to examine AGS-MBR performance, to reveal the AGS scouring mechanism and quantify its contribution rate to membrane fouling mitigation, from the views of theory and experiment. Above all, AGS-MBR exhibited a low fouling rate ((transmembrane pressure (TMP) kept below 20 kPa) without membrane cleaning and a higher removal of organics and nutrients than conventional MBR during 80 days' sludge granulation process. Then, flocculent sludge (FS) with various AGS ratios was applied to simulate the sludge granulation phase. When AGS ratio increased from 0% to 100%, the permeate flux gradually elevated from 40.0 L m-2h-1 to 92.9 L m-2h-1, and fouling resistance decreased from 9.0 × 10-12m-1 to 3.9 × 10-12m-1 benefiting from the loose structure and high porosity of AGS fouling layer. Meanwhile, the scouring effect produced by AGS on the membrane fouling mitigation was investigated. Based on the momentum conservation, a new hydrodynamic model was developed to explain the scouring mechanism of AGS. The scouring stress, proportional to the total amount of AGS depositing on the membrane surface, effectively reinforced the collision between AGS and FS, and reduced their deposition on the membrane surface by friction with the membrane; thus it was further conducive to membrane fouling mitigation. Moreover, a novel contribution quantification model was proposed for analyzing the contribution rate of AGS scouring effect to mitigate membrane fouling. AGS scouring possessed a significant contribution rate (39.9%) for fouling mitigation, compared with AGS structure (50.3%) and hydraulic stress (9.7%). In final, this study provides an in-depth understanding to mitigate the MBR membrane fouling by the unique advantages of sludge granulation.

Biomimetic dynamic membrane (BDM): Fabrication method and roles of carriers and laccase.

Biomimetic dynamic membrane (BDM) has been employed as a promising membrane separation technology regarding water/wastewater treatment (Model pollutant is methylene blue). Given its catalytic function on micro-pollutant removal and fouling control, detailed mechanism for impacts of fabrication method, carriers (CNT and GO) and laccase on the construction of biomimetic layer and enzyme immobilization have not been clear so far. In this work, the BDM performance with various fabrication methods, carriers and laccase were investigated and verified. The BDM fabrication tests demonstrated that BDM with mixed filtration method had better filtration performance (up to 120 L m-2 h-1 flux and 80% removal rate) than BDM with stepwise filtration method. Moreover, the laccases immobilized on GO exhibited a stronger laccase activity than those on CNT. Increasing CNT or GO dosage strengthened removal rate, but lowered flux, meanwhile flux and removal rate exhibited a significant fluctuation with certain laccase dosage. At 25 g m-2 CNT or GO dosage and 50 g m-2 laccase dosage, the optimized flux and removal rate values were obtained. Further study investigated the surface morphology and property of BDM, showing that BDM with mixed filtration method turned out to be the optimized enzyme immobilization mechanism and fabrication method. In addition, during multiple filtration cycles, with the optimized conditions, the removal rate, flux and laccase activity of BDM could maintain at high levels. On account of the finding of the present study, selecting a suitable fabrication method, appropriate CNT or GO dosage and laccase dosage can indeed optimize the structure of biomimetic layer and enzyme immobilization, expanding its possibility on sustainable operation.

High-Throughput Chiral LC-MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study.

A sensitive and high-throughput chiral liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of R-pantoprazole and S-pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid-liquid extraction in 96-well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ-RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 µL/min. The enantiomers were quantified on a triple-quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole-d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r(2)  > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R-pantoprazole and from 92.5% to 96.5% for S-pantoprazole and the IS-normalized matrix factor was 0.98 to 1.07 for R-pantoprazole and S-pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569-575, 2016. © 2016 Wiley Periodicals, Inc.

Identification and quantification of gingerols and related compounds in ginger dietary supplements using high-performance liquid chromatography-tandem mass spectrometry.

Dietary supplements containing preparations of ginger roots/rhizomes (Zingiber officinale Roscoe) are being used by consumers, and clinical trials using ginger dietary supplements have been carried out to evaluate their anti-inflammatory or antiemetic properties with inconsistent results. Chemical standardization of these products is needed for quality control and to facilitate the design of clinical trials and the evaluation of data from these studies. To address this issue, methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) were developed for the detection, characterization, and quantitative analysis of gingerol-related compounds in botanical dietary supplements containing ginger roots/rhizomes. During negative ion electrospray with collision-induced dissociation, the cleavage of the C4-C5 bond with a neutral loss of 194 u and benzylic cleavage leading to the neutral loss of 136 u were found to be class-characteristic fragmentation patterns of the pharmacologically active gingerols or shogaols, respectively. On the basis of these results, an assay using LC-MS/MS with neutral loss scanning (loss of 194 or 136 u) was developed that is suitable for the fingerprinting of ginger dietary supplements based on the selective detection of gingerols, shogaols, paradols, and gingerdiones. In addition, a quantitative assay based on LC-MS/MS with selected reaction monitoring was developed for the quantitative analysis of 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol in ginger dietary supplements. After method validation, the quantities of these compounds in three commercially available ginger dietary supplements were determined. This assay showed excellent sensitivity, accuracy, and precision and may be used to address the need for quality control and standardization of ginger dietary supplements.

Structure-activity relationships of macrolides against Mycobacterium tuberculosis.

Existing 14, 15 and 16-membered macrolide antibiotics, while effective for other bacterial infections, including some mycobacteria, have not demonstrated significant efficacy in tuberculosis. Therefore an attempt was made to optimize this class for activity against Mycobacterium tuberculosis through semisyntheses and bioassay. Approximately 300 macrolides were synthesized and screened for anti-TB activity. Structural modifications on erythromycin were carried out at positions 3, 6, 9, 11, and 12 of the 14-membered lactone ring; as well as at position 4'' of cladinose and position 2' of desosamine. In general, the synthesized macrolides belong to four subclasses: 9-oxime, 11,12-carbamate, 11,12-carbazate, and 6-O-substituted derivatives. Selected compounds were assessed for mammalian cell toxicity and in some cases were further assessed for CYP3A4 inhibition, microsome stability, in vivo tolerance and efficacy. The activity of 11,12-carbamates and carbazates as well as 9-oximes is highly influenced by the nature of the substitution at these positions. For hydrophilic macrolides, lipophilic substitution may result in enhanced potency, presumably by enhanced passive permeation through the cell envelope. This strategy, however, has limitations. Removal of the C-3 cladinose generally reduces the activity. Acetylation at C-2' or 4'' maintains potency of C-9 oximes but dramatically decreases that of 11,12-substituted compounds. Further significant increases in the potency of macrolides for M. tuberculosis may require a strategy for the concurrent reduction of ribosome methylation.

The soy isoflavone daidzein improves the capacity of tamoxifen to prevent mammary tumours.

The aim of this study was to determine how the efficacy of tamoxifen is affected when combined with soy isoflavones. To address this, female Sprague-Dawley rats were placed on diets supplemented with tamoxifen, genistein, daidzein, or a combination of each isoflavone with tamoxifen; a week later mammary tumours were induced by 7,12 dimethylbenzanthracene. The most effective diet was the tamoxifen/daidzein combination. It reduced tumour multiplicity by 76%, tumour incidence by 35%, tumour burden by over 95%, and increased tumour latency by 62% compared with positive controls. The tamoxifen/daidzein combination diet was in all aspects more effective while the tamoxifen/genistein combination was less effective than the tamoxifen diet. The tamoxifen/daidzein diet significantly decreased 8-oxo-deoxyguanosine levels (an indicator of oxidative DNA damage) in the mammary glands. This study conclusively shows for the first time the combination of daidzein with tamoxifen produces increased protection against mammary carcinogenesis, while the combination of genistein with tamoxifen produces an opposing effect when compared with tamoxifen alone.

Ultrafiltration tandem mass spectrometry of estrogens for characterization of structure and affinity for human estrogen receptors.

Although hormone replacement therapy (HRT) is used by post-menopausal women for the relief of menopausal symptoms and the potential reduction of osteoporosis, HRT also increases their risk of Alzheimer's disease, stroke, breast cancer, and endometrial cancer. Since the majority of these effects are associated primarily with estrogen binding to only one of the estrogen receptors (ER), new assays are needed that can more efficiently evaluate ER-binding and identify ligands selective for ER-alpha and ER-beta. High performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) was combined with ultrafiltration as a new method to investigate the relative binding of compounds to the ERs and to evaluate the structures of these estrogens. Mixtures of estradiol and six equine estrogens, including equilin, equilenin, 8,9-dehydroestrone, and their 17beta-hydroxyl derivatives, were assayed simultaneously to determine their relative binding to human ER-alpha and ER-beta. Estrogens containing a 17beta-OH group were found to have higher relative affinities for the estrogen receptors than their ketone analogs. In addition, 17beta-EN showed selectivity for binding to ER-beta over ER-alpha. The results were compared to the IC50 values obtained by using a conventional radiolabled estradiol competitive binding assay. Finally, the utility of negative ion electrospray tandem mass spectrometry for the unambiguous identification of these estrogen isomers was investigated. Several characteristic recyclization pathways during tandem mass spectrometry were identified, which might be useful for distinguishing related estrogens.

Identification of caffeic acid derivatives in Actea racemosa (Cimicifuga racemosa, black cohosh) by liquid chromatography/tandem mass spectrometry.

Caffeic acid derivatives occurring in black cohosh [Cimicifuga racemosa (L.) Nutt., Actaea racemosa (Ranunculaceae)], some of which may have pharmacological activity, were analyzed using high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in a complex plant matrix. Based on these studies, structurally characteristic product ions and neutral molecule losses were identified, which were then used during LC/MS/MS with product ion scanning, precursor scanning and constant neutral loss scanning to detect caffeic acid derivatives in a crude extract of black cohosh. Several caffeic acid derivatives were detected, and the identification of six of them were confirmed by comparison with authentic standards including caffeic acid, ferulic acid, isoferulic acid, fukinolic acid, cimicifugic acid A, and cimicifugic acid B. Four other compounds were detected that appeared to be caffeic acid derivatives based on LC/MS/MS retention times, molecular weights, and fragmentation patterns during MS/MS. Since standards were unavailable for these four compounds, they were tentatively identified using LC/MS/MS as cimicifugic acid E, cimicifugic acid F, dehydrocimicifugic acid A, and dehydrocimicifugic acid B. Dehydrocimicifugic acid A and dehydrocimicifugic acid B have not been reported previously to be constituents of black cohosh.