Biomedical Chromatography

Zhang, Yun; Wang, Jun; Wang, Bo; Gao, Yanting; Lin, Shengyun; Zhou, Yuhong; Wu, Liqiang

doi: 10.1002/bmc.5963pmid: 39030833

The aim of this work was to investigate the therapeutic effect of modified Shisiwei Jianzhong Decoction (SJD) on aplastic anemia (AA) and its potential pharmacological mechanism from the perspective of mitophagy. A comprehensive approach combining network pharmacology, mendelian randomization, molecular docking and animal experiments was applied to evaluate the properties of SJD against AA. By integrating multiple databases, it was determined that SJD exerted its therapeutic effect on AA by targeting three key targets [mammalian target of rapamycin (MTOR), poly(ADP‐ribose) polymerase 1 (PARP1) and Sirtuin 1 (SIRT1)] through four core compounds (quercetin, resveratrol, genistein and curcumin). Mendelian randomization analysis identified MTOR as a risk factor for AA occurrence while PARP1 was a protective factor. Results of animal experiments showed that SJD improved peripheral blood counts and promoted the proliferation of hematopoietic stem cells. Mechanistically, SJD, especially at high dose, played a therapeutic role in AA by activating mitophagy‐related proteins PTEN induced kinase 1 (PINK1)/Parkin and inhibiting the phosphatidylinositol 3‐kinase (PI3K)/protein kinase (AKT)/MTOR pathway. This study revealed for the first time the core chemical composition of SJD and its pharmacological effects against AA, which can restore hematopoietic function by activating mitophagy. The results provide inspiration for the clinical application of traditional Chinese medicine in AA treatment.

Badgujar, Devendra; Bawake, Sanket; Chawathe, Ashwini; Sharma, Nitish

doi: 10.1002/bmc.5983pmid: 39113387

Peptide aggregation is one of the key challenges associated with the development of therapeutic peptides. Peptide and protein aggregates are considered as one of the most important critical quality attributes (CQA). Therapeutic liraglutide (LGT) is proteinaceous in nature, and aggregation can be triggered by various environmental stress condition. Therefore, it is essential to separate and identify aggregation states of such drugs. In this study, we have established size exclusion chromatography‐liquid chromatography‐ultraviolet/high resolution mass spectrometry (SEC‐LC‐UV/HRMS) method to separate and identify the stress induced LGT aggregates. LGT samples were subjected to photolytic, thermal, freeze thaw and shaking stress conditions. Additionally, LGT solution was incubated with surfactant and excipient that are commonly used in peptide formulation, to evaluate their impact on aggregation level and physicochemical stability over time. The developed SEC method was also validated for specificity, accuracy, precision and linearity. The results of this study will be useful for investigators to monitor LGT aggregates during product development.

Hollander, Esther M.; Zimmerman, Nachel M. C.; Piet, Berber; Heuvel, Michel M.; Burger, David M.; Brake, Lindsey H. M.; Heine, Rob

doi: 10.1002/bmc.5986pmid: 39136165

Small molecule inhibitors (SMIs) are increasingly being used in the treatment of non‐small cell lung cancer. To support pharmacokinetic research and clinical treatment monitoring, our aim was to develop and validate an ultra‐performance liquid chromatography–mass spectrometry (UPLC‐MS/MS) assay for quantification of eight SMIs: adagrasib, alectinib, brigatinib, capmatinib, crizotinib, lorlatinib, selpercatinib, and sotorasib. Development of the UPLC‐MS/MS assay was done by trying different columns and eluents to optimize peak shape. The assay was validated based on guidelines of the European Medicines Agency. Chromatographic separation was performed with a gradient elution using ammonium formate in water and methanol. Detection was performed using a triple quadrupole tandem mass spectrometer with electrospray ionization. Validation was performed in a range of 10–2500 μg/L for lorlatinib, 25–6250 μg/L for alectinib and crizotinib, 25–10,000 μg/L for capmatinib and selpercatinib, 50–12,500 μg/L for brigatinib, and 100–25,000 μg/L for adagrasib and sotorasib. Imprecision was <8.88% and inaccuracy was <12.5% for all compounds. Seven out of eight compounds were stable for 96 h at room temperature. Sotorasib was stable for 8 h at room temperature. A sensitive and reliable method has been developed to quantify eight SMIs with a single assay, enhancing efficacy and safety of targeted therapies.

Sun, Shu‐ding; Zhao, Di; Liu, Xue‐fang; Zhang, Wei‐wei; Dong, Hao‐ran; Tian, Yan‐ge; Feng, Su‐Xiang

doi: 10.1002/bmc.5969pmid: 39126348

This study aimed to explore the pharmacodynamics and mechanisms of different processing methods of Ligustrum lucidum Ait. (LLA) in addressing kidney‐yin deficiency (KYD). Forty‐eight Sprague–Dawley rats were divided into eight groups based on their weight. The KYD model was established by intragastric administration of levothyroxine sodium. Each group was administered the corresponding treatment for 15 consecutive days. The general condition of the rats during the treatment period was observed. In addition, the levels of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and the ratio of cAMP to cGMP in the serum of rats from different groups were measured. Serum samples were analyzed using the ultra‐performance liquid chromatography (UPLC)‐Orbitrap Fusion MS technique for metabolomics analysis. Compared with the model group, the general condition of the rats in the wine‐steamed L. lucidum group (WL) and salt‐steamed L. lucidum group (SSL) groups showed significant improvement. The serum levels of cAMP, cGMP, and the cAMP‐to‐cGMP ratio tended to return to normal. Metabolic analysis identified 38 relevant biomarkers and revealed 3 major metabolic pathways: phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; and sphingolipid metabolism. The different processing methods of LLA demonstrated therapeutic effects on KYD in rats, likely related to the restoration of disturbed metabolism by adjusting the levels of endogenous metabolites in the kidney. The SSL demonstrated significantly superior effects compared with the other four types of LLA processed products.

Temiz, Ebru; Akmese, Sukru; Koyuncu, Ismail; Barut, Dursun

doi: 10.1002/bmc.5970pmid: 39090031

Hashimoto's thyroiditis (HT) is an autoimmune disease caused by the immune system attacking healthy tissues. However, the exact pathogenesis of HT remains unclear. Metabolomic analysis was performed to obtain information about the possible pathogenic mechanisms and diagnostic biomarkers of HT. The amino acid profile was analyzed using an LC–MS/MS method using serum samples obtained from 30 patients diagnosed with ultrasonographic imaging and laboratory markers (thyroid stimulating hormone) free thyroxine and thyroid peroxidase) and 30 healthy individuals. There were statistically significant changes in 27 amino acids out of 32 amino acids analyzed (p < 0.05). Based on the receiver operating characteristic curve analysis, the six amino acid (1‐methylhistidine, cystine, norvaline, histidine, glutamic acid and leucine) biomarkers showed high sensitivity, specificity (area under the curve > 0.98), positive likelihood ratio and low negative likelihood ratio. Also, according to pathway analysis, degradation of phenylalanine, tyrosine and tryptophan biosynthesis was the highest metabolic pathway according to the impact value (p < 0.001 and impact value = 1.0). We provide serum amino acid profiles of patients with Hashimoto's thyroiditis and identify five potential biomarkers for early diagnosis by clinicians.

Leoni, Laura; Rosmini, Federica; Ledda, Francesca; Parasiliti‐Caprino, Mirko; Settanni, Fabio; Nonnato, Antonello; Ghigo, Ezio; Moghetti, Paolo; Mengozzi, Giulio; Ponzetto, Federico

doi: 10.1002/bmc.5982pmid: 39149929

Biochemical confirmation of ovulation typically involves measuring serum progesterone levels during the mid‐luteal phase. Alternatively, this information could be obtained by monitoring urinary excretion of conjugated metabolites of ovarian steroids such as pregnanediol 3‐glucuronide (PDG) using immunoassay techniques that have methodological limitations. The aim of the present study was to develop a mass spectrometry (MS)‐based method for the rapid and accurate measurement of urinary PDG levels in spot urine samples. A “dilute and shoot” ultra‐high‐performance liquid cromatography tandem mass spectrometry (UHPLC‐MS/MS) method was developed for measuring PDG urinary concentration with a 6‐min analysis time. The method underwent validation in accordance with ISO 17025 documentation for quantitative methods, proving an efficient separation of PDG from other structurally similar glucuro‐conjugated steroid metabolites and ensuring a sufficient sensitivity for detecting the target analyte at concentrations as low as 0.01 μg/mL. The validation protocol yielded satisfactory results in terms of accuracy, repeatability, intermediate precision, and combined uncertainty. Additionally, the stability of both the samples and calibration curves was also conducted. The application to real urine samples confirmed the method's capability to measure PDG levels throughout an entire menstrual cycle and detecting ovulation. The rapidity of the analytical platform would therefore enable high throughput analysis, which is advantageous for large cohort clinical studies.

Sher, Ayaz Ali; Iqbal, Arshad; Adil, Muhammad; Shah, Zamarud; Butt, Zahid Ali; Ullah, Sami; Nafees, Muhammad; Sohni, Saima

doi: 10.1002/bmc.5968pmid: 39039695

Withania somnifera belongs to the family Solanaceae, commonly called ashwagandha, and is traditionally used as an astringent, hepatoprotective and antioxidant, and as a treatment for rheumatism. Therefore the current study aimed to explore the dichloromethane fraction of W. somnifera whole plant (DCFWS) and ethyl acetate fraction of W. somnifera (EAFWS) using gas chromatoghraphy–mass spectrometry (GC–MS) analysis and to find the acetylcholinesterase inhibition potential along with spasmolytic activity. The GC–MS‐detected phytochemicals were 2,4‐bis(1,1‐dimethylethyl), hexadecanoic acid, 1‐nonadecene and 11‐octadecenoic acid. The DCFWS and EAFWS exhibited acetylcholinesterase inhibitory potential with significant inhibitory concentration values. The acute toxicity results of both fractions showed high toxicity, causing emesis at 0.5 g and both emesis and diarrhea at 1 g/kg. Both fractions exhibited significant (p ≤ 0.01) laxative activity against metronidazole (7 mg/kg) and loperamide hydrochloride (4 mg/kg) induced constipation. Both DCFWS (66.8 ± 3.85%) and EAFWS (58.58 ± 3.28%) significantly (p ≤ 0.05) increased charcoal movement compared with distal water (43.93 ± 4.34%). Similarly the effect of DCFWS on KCl‐induced (80 mm) contraction was more significant as compared with EAFWS. It was concluded that the plant can be used in the treatment of gastrointestinal tract diseases such as constipation. Furthermore, additional work is required in the future to determine the bioactive compounds that act as therapeutic agents in W. somnifera.

Al Azzam, Khaldun M.; Al‐Areer, Nadeen Waleed; Al Omari, Rima H.; Al‐Deeb, Ibrahim; Bounoua, Nadia; Negim, El‐Sayed; Al‐Samydai, Ali; Aboalroub, Adam A.; Said, Rana

doi: 10.1002/bmc.5993pmid: 39152776

Sharma, Sakshi; Katna, Sapna; Sharma, Ajay; Istatu, Pankaj Sharma; Devi, Nisha; Kumar, Arvind; Singh, Shubhra

doi: 10.1002/bmc.5962pmid: 39014970

Residue behaviour and dietary risk assessment of cyantraniliprole, flubendiamide and acetamiprid in broccoli were carried out using the QuEChERS (quick, easy, cheap, effective, rugged and safe) technique coupled with LC–MS/MS. The QuEChERS technique was validated on parameters such as linearity, accuracy, precision, robustness, matrix effects, limit of quantification (LOQ), specificity, retention time and ion ratio as per SANTE (Directorate General for Health and Food Safety) guidelines to attest to the specificity, accuracy and precision of the analytical method in estimating insecticide residues in and on broccoli heads and cropped soil. The LOQ of the method for all three insecticides was 0.01 mg/kg. The initial deposits of cyantraniliprole, flubendiamide and acetamiprid reduced to half of its concentration in 1.873–2.354, 1.975–2.484 and 1.371–1.620 days, respectively. No residues were detected in broccoli‐cropped soil at harvest time (30 days after last spray). The proposed maximum residue limits (MRLs) of 1.5, 0.5–0.9 and 2.0–3 mg/kg for cyantraniliprole, flubendiamide and acetamiprid were calculated using the Organisation for Economic Co‐operation and Development MRL calculator. The acute and chronic dietary risk assessment of the tested insecticides identified no appreciable dietary risk to the Indian population from the consumption of broccoli heads. The findings of no dietary risk highlight the importance of informed pesticide usage in broccoli and the proposed MRL derived from this study offers crucial guidelines for the regulatory authorities, ensuring the safety of broccoli consumption.

Kim, Taeyoung; Han, Dong‐Gyun; Yoon, In‐Soo

doi: 10.1002/bmc.5959pmid: 39039810

Scopoletin, a coumarin class natural phytoalexin, is present in medicinal plants such as noni (Morinda citrifolia). It exhibits diverse pharmacological properties, including antioxidant, anti‐hyperuricemic, and anti‐inflammatory effects. The objective of this study was to develop a novel HPLC‐fluorescence (HPLC‐FL) method for the quantitative analysis of scopoletin in the plasma and to investigate its pharmacokinetics in rats. Sample preparation involved a methanol‐based protein precipitation method, and chromatographic separation was conducted using a C18 column with an isocratic mobile phase composed of water and acetonitrile containing 0.1% trifluoroacetic acid. The eluent was detected using an FL detector set to optimized excitation/emission wavelengths of 337/453 nm. Method validation encompassed assessments of selectivity, linearity (1–500 ng/mL), precision, accuracy, recovery, matrix effect, and stability in accordance with the prevailing Food and Drug Administration (FDA) guidelines. The developed method was successfully applied for pharmacokinetic study in rats. To the best of our knowledge, this study is the first application of a simple and sensitive HPLC‐FL method for the quantification of scopoletin in a pharmacokinetic study. This method offers a promising alternative for preclinical pharmacokinetic investigations with appropriate modifications and validations and holds potential for clinical applications.