Biomedical Chromatography

Javeed, Rabia; Hussain, Dilshad; Jabeen, Fahmida; Saeed, Adeela; Fatima, Batool; Ashiq, Muhammad Naeem; Najam‐ul‐Haq, Muhammad

doi: 10.1002/bmc.4693pmid: 31465544

High‐density lipoproteins (HDLs) have anti‐inflammatory and antioxidant properties and are potentially cardio‐protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE‐based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS–PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI–MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum‐based proteomics approach gives insight into the functional role of HDL. HDL‐associated phospholipids have also been analyzed by LDI–MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.

Nuland, Merel; Rosing, Hilde; Schellens, Jan H.M.; Beijnen, Jos H.

doi: 10.1002/bmc.4623pmid: 31215049

Therapeutic drug monitoring (TDM) has shown to benefit patients treated with drugs of many drug classes, among which is oncology. With an increasing demand for drug monitoring, new assays have to be developed and validated. Guidelines for bioanalytical validation issued by the European Medicines Agency and US Food and Drug Administration are applicable for clinical trials and toxicokinetic studies and demand fully validated bioanalytical methods to yield reliable results. However, for TDM assays a limited validation approach is suggested based on the intended use of these methods. This review presents an overview of publications that describe method validation of assays specifically designed for TDM. In addition to evaluating current practice, we provide recommendations that could serve as a guide for future validations of TDM assays.

Anđelković, Darko; Branković, Milica; Kocić, Gordana; Mitić, Sanja; Pavlović, Radmila

doi: 10.1002/bmc.4720pmid: 31634409

Apple peel is frequently exposed to pesticides, especially in the last stages of the pesticide treatment practice. For a certain period, peel retains most of the applied pesticides, which precisely indicates the presence of pesticide residues. This study was conducted to establish and evaluate the method for the residue analysis of pyrimethanil, cyprodinil, trifloxystrobin, bifenthrin and boscalid by GC–MS in peel of Granny Smith, Golden Delicious and Idared varieties. The method consisted of few steps, without the routine usage of sorbents, and resulted in efficient removal of waxes, targeted as main interferences in GC–MS analysis. The matrix effect, boscalid selectivity issue, trend of lower trueness for bifenthrin in all matrices and for all analytes in Granny Smith matrix were in some way associated with the waxes. Finally, the method was applied to the analysis of three varieties of apple orchard samples, after 9 and 5 months of treatment with commercial formulations that contain pyrimethanil, cyprodinil and boscalid as active ingredients. Since the developed method consisted of few steps and used nontoxic and economic reagents, it could be implemented as a fast, economic and reliable screening method.

Ahmad, Rizwan; Ahmad, Niyaz; AlHudaithi, Nourah; AlHebshi, Asmaa; Bukhari, Areej

doi: 10.1002/bmc.4698pmid: 31762077

Han, Xiao; Wang, Shuai; Yang, Xin‐xin; Li, Tian‐jiao; Zhao, Huan‐jun; Zhou, Li‐ping; Zhao, Lin; Bao, Yong‐rui; Meng, Xian‐sheng

doi: 10.1002/bmc.4701pmid: 31596954

Patrinia villosa (Thunb.) Juss. (PVJ) is described as pungent, bitter and slightly cold in Chinese medicine, and is associated with the large intestine, stomach and liver meridians. The preliminary experiments of our research team proved that PVJ total flavonoids have excellent inhibitory effects on liver cancer cells. The present experiment uses the UPLC–Q‐TOF–MS technology and serum pharmacochemistry methods to analyze the chemical components in vitro and in vivo of PVJ antiliver tumors. A total of 14 chemical components were identified in the total flavonoids extract of PVJ, and it is mainly composed of flavonoids, flavonones, flavonols and phenolic acids. At the same time, seven prototypical components and seven metabolic components were detected in the drug‐containing plasma. Hydrocaffeate and scutellarein are the phase I metabolites of caffeic acid and scutellarin, respectively. Sulfated apigenin, sulfated luteolin, sulfated kaempferol and methylated kaempferol are the II phase metabolites of apigenin, luteolin, kaempferol, respectively. The experiment provides a reference for the research and development of antitumor drug candidates, and provides a basis for revealing the bioactive components of PVJ and the antitumor mechanism.

Furtado, Danielle Zildeana Sousa; Leite, Fernando Brunale Vilela de Moura; Jedlicka, Leticia Dias Lima; Souza, Danilo Santos; Barreto, Cleber Nunes; Silva, Heron Dominguez Torres; Assunção, Nilson Antonio

doi: 10.1002/bmc.4673pmid: 31385327

Cri du Chat or 5p minus (5p−) syndrome is characterized by a deletion located on the chromosome 5 short (−p) arm and has an incidence rate of 1 in 50,000 individuals worldwide. This disease manifests in disturbances across a range of systems biochemicals. Therefore, a targeted metabolomics analysis was evaluated in patients with 5p− syndrome to help unravel the biochemical changes that occur in this disease. Urine samples were collected from people of both sexes aged 1–38 years old and analyzed by ultra‐performance liquid chromatography coupled to mass spectrometry. Student' statistical test, metabolomic pathway analysis and metabolite set enrichment analysis were applied to the data. Alterations of some amino acids and amine biogenics levels were found in Cri du Chat Syndrome individuals. The alteration of most of these metabolites is associated with energy recuperation and glycolysis. In general, we found the catabolism of some metabolic pathways to be affected in 5p− patients.

Shakouri, Amir; Parvan, Reza; Adljouy, Nasim; Abdolalizadeh, Jalal

doi: 10.1002/bmc.4709pmid: 31630417

Hyaluronidase (Hyal) can be employed to accomplish a diversity of complications related to hyaluronic acid (HA). Hyal contains some classes of catalysts that cleave HA. This enzyme is detected in several human tissues as well as in animal venoms, pathogenic organisms and cancers. Destructive cancer cells regularly increase the CD44 receptor existing in a cell membrane. This receptor acts as an exact receptor for HA, and HA is recognized to motivate the migration, spread, attack and metastasis of cancer cells. Nearly all of the methods used to purify Hyal are highly costly and not proper for industrial applications. This survey aims to review different methods of Hyal purification, which acts as an anticancer agent by degrading HA in tissues and thus inhibiting the CD44–HA interaction. Hyal can be successfully employed in the management of cancer, which is associated with HA–CD44. This review has described different methods for Hyal purification to prepare an origin to develop a novel purification technique for this highly appreciated protein. Using multiple columns is not applicable for the purification of Hyal and thus cannot be used at the industrial level. It is better to use affinity chromatography of anti‐Hyal for Hyal with one‐step purification.

Meng, Ying; Ding, Lin; Wang, Yuan; Nie, Qi‐ting; Xing, Yang‐yang; Ren, Qiang

doi: 10.1002/bmc.4705pmid: 31629370

Lithocarpus polystachyus leaves exhibit antidiabetic activity and is consumed as a herbal tea in China. In this study, phytochemical profiles of L. polystachyus leaves were identified and characterized by ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight–MS in both positive and negative ion modes. A total of 17 compounds were tentatively characterized and identified by accurate mass and characteristic fragment ions. The total phenolic contents in the leaf extracts ranged from 9.0 to 13.4 g gallic acid equivalents/100 g of dry weight (DW). In addition, the effect of these extracts on inhibiting the activities of α‐glucosidase and protein tyrosine phosphatase 1B (PTP1B) were evaluated. L. polystachyus extracts demonstrated significant inhibition of α‐glucosidase (more than 88.1% at a concentration of 1.25 mg/mL) and acarbose (93.6% at a concentration of 5 mg/mL) while the PTP1B inhibition rate was over 84.3%. The antioxidant capacities of the leaf extracts were determined using 2,2‐diphenyl‐1‐picrylhydrazyl, ABTS, and ferric reducing ability of plasma methods and ranged from 50.5 to 72.5 g trolox, from 43.2 to 77.7 g trolox, and from 5.0 to 10.6 g butylated hydroxytoluene (BHT; equaling trolox or BHT per 100 g of DW), respectively. Based on these results, L. polystachyus can be considered as a functional food owing to its antidiabetic and antioxidative activities, which are attributed to its rich phenolic and dihydrochalcone contents.

Bartlett, Michael G.

doi: 10.1002/bmc.4764pmid: 31884691

Patel, Shefali R.

doi: 10.1002/bmc.4640pmid: 31256423

Bioanalysis of unstable compounds such as acyl glucuronide metabolites represents a great analytical challenge owing to poor analyte stability in biological matrices. The primary goal for bioanalytical assay development is to minimize the breakdown of acyl glucuronide metabolite into its parent aglycone during sample collection, transportation, storage and analysis. Samples need to be stabilized ex vivo immediately after sample collection to minimize potential breakdown and thus to ensure accurate concentration measurement of both acyl glucuronide metabolite and its parent aglycone. In this review paper, formation of acyl glucuronide metabolites, the importance of establishing acyl glucuronide exposure measurement and safety coverage, optimization of sample pretreatment to stabilize the acyl glucuronide metabolites, current analytical strategy of assaying them as well as considerations for regulatory filings are discussed. It is important to identify acyl glucuronide metabolites that are capable of undergoing hydrolysis and pH‐dependent intra‐molecular migration as well as covalently binding to plasma and tissue proteins which can cause toxicity in vivo in the early stages of drug development. Carefully planning analytical experiments, identifying structures of acyl glucuronides and monitoring their concentrations in early drug development can help assess the risks associated with their exposures and potentially predict their concentrations in human circulation.