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Quantification and clinical performance of serum parathyroid hormone 1–84 via immunocapture coupled to LC–MS/MS in chronic renal failure
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-14 DOI: 10.1016/j.jpba.2025.116678
Haiwei Cao , Yuting Jin , Yingwu Wang , Hao Wang , Yijia Qin , Xinhua Guo , Suyan Tian , Jing Huang , Yanyan Li
Accurate measurement of serum parathyroid hormone (PTH) is crucial for diagnosing and managing endocrine and osteological diseases. Conventional immunoassay methods struggle with cross reactivity issues between full-length PTH and truncated fragments or post-translationally modified forms. Both the standardization of PTH assays and the peptide's stability are concerning. This study addresses these issues by establishing an immunocapture coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to precisely quantify PTH1–84. PTH1–84 was isolated from one mL serum samples by immunocapture on a polystyrene bead and eluted from matrix, then quantitated by LC–MS/MS. The results from 268 serum samples were compared to an intact PTH immunoassay. The assay's linear range was 5.0–1000.0 pg/mL. The intra-assay coefficients of variation (CVs) ranged from 3.2 % to 6.8 %, and the inter-assay CVs ranged from 4.6 % to 9.5 %. The extraction efficiencies were 98.0 %–100.5 %, with no significant matrix effects observed after internal standard correction. The correlation coefficient between LC–MS/MS and immunoassay was 0.989, but the bias between the methods was substantial. Nevertheless, the immunocapture purification coupled LC–MS/MS method offers a promising approach for accurate PTH measurement.
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引用次数: 0
Rapid determination of etomidate and its structural analogues in e-liquid by probe electrospray ionization quadrupole time-of-flight mass spectrometry 探针电喷雾电离四极杆飞行时间质谱法快速测定电子液体中依托咪酯及其结构类似物。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-13 DOI: 10.1016/j.jpba.2025.116677
Meiting Lin , Zhen Zhang , Qun He , Hongyuan Hao , Ping Xiang , Junbo Zhao
Etomidate and its structural analogues, which have anesthetic effects, are classified as controlled psychotropic drugs. Electronic cigarettes (e-cigarettes) have become more and more popular. With the increase of adding etomidate and its analogues to electronic liquids (e-liquids), there is a trend of abuse, which is a tough problem urgently need to be solved. This seriously affects the health and security of the public and the development of society. A simple, rapid and effective screening method is very crucial for their identification. In this study, we applied a newly developed method, probe electrospray ionization quadrupole time-of-flight mass spectrometry (PESI-QTOF-MS) with DPiMS QT ion source to analyze etomidate and its analogues in e-liquids. It allowed identification in 0.3 min with lower sample usage. Isomers can be distinguished by ion abundance ratios at collision energy (CE) 15 eV, which provided possibility for distinguishing more isomers by in-situ mass spectrometry. Limit of detection (LOD) and limit of quantitation (LOQ) of four substances were 20 ng/mL and 50 ng/mL, respectively. Good linear relationships were obtained in the concentration range of 50–5000 ng/mL with little matrix effect. The accuracy, precision, dilution effect and carryover of the method were also validated. Positive specimens (n = 38) were analyzed by both PESI-QTOF-MS and gas chromatography-mass spectrometry (GC-MS). There were five impurities including nicotine, cooling agent and flavorings were investigated by PESI-QTOF-MS, which provided the possibility for tracing the origin of illegal e-liquids. This study will help solve the backlog of cases and improve work efficiency effectively by reducing analysis time. Furthermore, it meets the need of addressing current situation of drug control and can assist forensic laboratories in investigating cases. It also demonstrates the application prospects of rapid screening in new drugs.
依托咪酯及其结构类似物具有麻醉作用,被列为管制精神药物。电子烟(e-cigarette)已经变得越来越流行。随着依托咪酯及其类似物在电子液体中添加量的增加,有滥用的趋势,这是一个亟待解决的棘手问题。这严重影响了公众的健康安全和社会的发展。一种简单、快速、有效的筛选方法对其鉴别至关重要。在这项研究中,我们采用了一种新的方法,探针电喷雾电离四极杆飞行时间质谱(PESI-QTOF-MS)与DPiMS QT离子源分析电子液体中的依托咪酯及其类似物。它可以在0.3 min内以较低的样本使用量进行鉴定。用碰撞能(CE) 15 eV的离子丰度比可以区分异构体,这为原位质谱法区分更多异构体提供了可能性。4种物质的检出限和定量限分别为20 ng/mL和50 ng/mL。在50 ~ 5000 ng/mL浓度范围内呈良好的线性关系,基质效应较小。验证了该方法的准确度、精密度、稀释效果和延展性。阳性标本(n = 38)采用PESI-QTOF-MS和气相色谱-质谱(GC-MS)分析。利用PESI-QTOF-MS分析了电子烟中尼古丁、冷却剂和调味剂等5种杂质,为非法电子烟的来源溯源提供了可能。通过减少分析时间,有助于解决积压的案例,有效提高工作效率。此外,它满足了解决药物管制现状的需要,并可协助法医实验室调查案件。同时也展示了快速筛选技术在新药中的应用前景。
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引用次数: 0
Chromatographic analysis and pKa evaluation of active pharmaceutical ingredients in anti-metastatic breast cancer: Green vs. conventional RPLC 抗转移性乳腺癌有效药物成分的色谱分析和pKa评价:绿色与传统RPLC。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-11 DOI: 10.1016/j.jpba.2025.116671
Fatmanur Kalkir , Ebru Çubuk Demi̇ralay , Yaşar Doğan Daldal , Hülya Yilmaz
This study aimed to determine the chromatographic retention and dissociation/protonation constant (pKa) values of lapatinib and tamoxifen, key drugs used in metastatic breast cancer treatment, at 37°C using both conventional and green high-performance liquid chromatography (HPLC) methods. Qualitative analysis was conducted on an XTerra C18 column (250 ×4.6 mm I.D., 5 μm particle size) at a flow rate of 1 mL/min. Hydroorganic mixtures with 45 %, 50 %, 55 %, and 60 % (v/v) organic modifiers were used to evaluate the retention times of the compounds. The compatibility of pKa values pKass of the compounds in water-organic solvent mixtures obtained from these studies, which were carried out without any significant change in liquid chromatography performance, with the values obtained by the conventional method is remarkable. The pKass values determined in this study were correlated with the macroscopic parameters of acetonitrile, methanol, ethanol and the pKa (pKaww)values of lapatinib and tamoxifen in water were calculated. The pKaww values calculated from these studies are compatible with each other and with the literature values. The environmental impact of the study, which was carried out using the green method and the conventional RPLC method, was evaluated using the Green Solvent Selection Tool (GSST), Green Analytical Procedures Index (GAPI), and Analytical Greenness Metric Approach (AGREE).
本研究旨在采用传统高效液相色谱法和绿色高效液相色谱法测定转移性乳腺癌治疗的关键药物拉帕替尼和他莫昔芬在37℃下的色谱保留和解离/质子化常数(pKa)值。定性分析采用XTerra C18色谱柱(250 ×4.6 mm内径,5 μm粒度),流速为1 mL/min。采用含45 %、50 %、55 %和60 % (v/v)有机改性剂的水有机混合物评价化合物的保留时间。从这些研究中得到的化合物在水-有机溶剂混合物中的pKa值pKass与传统方法得到的值的相容性是显著的,这些研究在液相色谱性能没有任何明显变化的情况下进行。本研究测定的pKass值与乙腈、甲醇、乙醇的宏观参数相关,并计算拉帕替尼和他莫昔芬在水中的pKa (pKaww)值。这些研究计算的pKaww值相互吻合,也与文献值吻合。本研究采用绿色方法和常规RPLC方法进行,并使用绿色溶剂选择工具(GSST)、绿色分析程序指数(GAPI)和分析绿色度度量方法(AGREE)对研究的环境影响进行评估。
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引用次数: 0
Hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of oligosaccharides and iridoid glycosides in rat plasma: Pharmacokinetic characterization of previously overlooked oligosaccharides from Radix Rehmanniae 大鼠血浆中低聚糖和环烯醚萜苷的亲水相互作用-液相色谱-串联质谱分析:以前被忽视的地黄低聚糖的药代动力学表征。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-09 DOI: 10.1016/j.jpba.2025.116670
Hanyang Wang , Yue Du , Chunwei Zhou , Yan Wang , Xin Wang
Radix Rehmanniae (RR) is a widely used herb in traditional Chinese Medicine with properties of tonifying the kidneys and nourishing the blood. Both raw and processed RR are effective for the treatment of diabetes in clinical practice. Oligosaccharides and iridoid glycosides are the primary active components responsible for the anti-diabetic effects of RR. In this study, a rapid and sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC−MS/MS) method was developed for simultaneous determination of oligosaccharides (raffinose, manninotriose and stachyose) and iridoid glycosides (catalpol and ajugol) in rat plasma. Significant analytical challenges were encountered during method development, including distinct retention behaviors of oligosaccharides and iridoid glycosides, low ionization and extraction efficiency of oligosaccharides, thermal instability of catalpol and reduced column performance. The strategies to overcome these challenges were presented by optimizing chromatographic separation, mass spectrometric detection and sample preparation. The best separation was achieved using an Accucore-150-Amide-HILIC column (100 mm × 2.1 mm, 2.6 μm) at 50 °C with mobile phase consisted of acetonitrile and ammonium acetate (2.5 mM) under gradient elution. Ammonium adduct ions produced by positive electrospray ionization were chosen as precursor ions for multiple reaction monitoring transitions. The established HILIC−MS/MS method exhibited good linearity (r > 0.9937) with the lower limits of quantification of 0.01–0.2 μg/mL using only 50 µL of plasma sample. The method was successfully applied to pharmacokinetic characterization of oligosaccharides and iridoid glycosides in normal and type 2 diabetic rats following intragastric administration of raw and processed RR extracts.
地黄(RR)是一种广泛使用的中药,具有补肾养血的特性。在临床实践中,生的和加工的红霉素对糖尿病的治疗都是有效的。低聚糖和环烯醚萜苷是RR抗糖尿病作用的主要活性成分。本研究建立了快速、灵敏的亲水相互作用液相色谱-串联质谱(HILIC-MS/MS)同时测定大鼠血浆中低聚糖(棉子糖、甘露糖和水苏糖)和环烯醚萜苷(catalpol和ajugol)的方法。在方法开发过程中遇到了重大的分析挑战,包括低聚糖和环烯醚萜苷的独特保留行为,低聚糖的低电离和提取效率,catalpol的热不稳定性以及色谱柱性能降低。通过优化色谱分离、质谱检测和样品制备,提出了克服这些挑战的策略。Accucore-150-Amide-HILIC色谱柱(100 mm × 2.1 mm, 2.6 μm),温度为50 °C,流动相为乙腈和乙酸铵(2.5 mm),梯度洗脱,分离效果最佳。采用正电喷雾电离产生的铵加合物离子作为前体离子,用于多反应监测跃迁。所建立的HILIC-MS/MS方法具有良好的线性关系(r > 0.9937),定量下限为0.01 ~ 0.2 μg/mL,仅需50 µL血浆样品。该方法成功地应用于正常和2型糖尿病大鼠灌胃RR提取物后低聚糖和环烯醚萜苷的药动学表征。
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引用次数: 0
Cellulose filter paper immobilized acetylcholinesterase for rapid screening of enzyme inhibitors in Phyllanthus emblica L. 纤维素滤纸固定化余甘子乙酰胆碱酯酶抑制剂的快速筛选。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-08 DOI: 10.1016/j.jpba.2025.116669
Yangzom Dawa , Yong-Chen Hua , Fang-Di Hu , Juan Chen
Acetylcholinesterase (AChE) is widely recognized as a promising therapeutic target enzyme for Alzheimer's disease (AD). The screening of AChE inhibitors (AChEIs) holds great significance for the treatment of AD. In this study, cellulose filter paper (CFP) -immobilized AChE was prepared and firstly applied to screening AChEIs from 30 % ethanol extract of Phyllanthus emblica L. fruits combined with ultra-high performance liquid chromatography quadrupole time-of-fight mass spectrometry (UHPLC-Q-TOF-MS/MS). Using CFP-immobilized AChE as the bait, AChEIs were harvested and the instantaneous separation characteristics of CFP were utilized to further facilitate the separation of the complex from the inactive components. Ultimately, 27 compounds specifically bound with AChE were screened and identified using UHPLC-Q-TOF-MS/MS. Additionally, molecular docking was employed to explore the binding mechanisms between screened potential inhibitors and AChE. The results show that, most of the screened compounds were found to exhibit higher affinity that of the positive control (huperzine A), and all the compounds expect mucic acid to be well embedded into the active pocket of AChE. To verify the reliability of the screening method and molecular docking, two commercial standards geraniin and ellagic acid were experimented with an AChE inhibition assay in vitro. The results showed that both compounds were found to effectively inhibit AChE with IC50 values of 42.42 ± 7.10 μM, 172.43 ± 9.22 μM. The developed method exhibits the advantages of rapidness and effectiveness in screening of AChEIs from complex herbal extracts.
乙酰胆碱酯酶(AChE)被广泛认为是治疗阿尔茨海默病(AD)的一种有前景的靶酶。AChE抑制剂(AChEIs)的筛选对AD的治疗具有重要意义。本研究制备了纤维素滤纸(CFP)固定化乙酰氨基甲酸乙酯(AChE),并首次应用超高效液相色谱-四极杆对抗时间质谱(UHPLC-Q-TOF-MS/MS)技术从30 %乙醇的余甘子果实提取物中筛选乙酰氨基甲酸乙酯。以CFP固定的AChE为诱饵,收获AChEIs,利用CFP的瞬时分离特性进一步促进配合物与非活性组分的分离。最终,通过UHPLC-Q-TOF-MS/MS筛选和鉴定了27个与AChE特异性结合的化合物。此外,采用分子对接的方法探索筛选出的潜在抑制剂与AChE的结合机制。结果表明,筛选的大部分化合物都比阳性对照石杉碱A具有更高的亲和力,并且所有化合物都期望乙酸能很好地嵌入AChE的活性口袋中。为了验证筛选方法和分子对接的可靠性,我们对两种商业标准天竺葵苷和鞣花酸进行了体外AChE抑制实验。结果表明,两种化合物均能有效抑制AChE, IC50值分别为42.42 ± 7.10 μM和172.43 ± 9.22 μM。该方法对复方草药提取物中乙酰胆碱酯类化合物的筛选具有快速、有效的优点。
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引用次数: 0
Investigation and elimination of noncovalent artificial aggregates during non-reduced capillary electrophoresis-sodium dodecyl sulfate analysis of a multi-specific antibody 非还原毛细管电泳过程中非共价人工聚集体的研究与消除——一种多特异性抗体的十二烷基硫酸钠分析。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-08 DOI: 10.1016/j.jpba.2025.116673
Jianhui Cheng , Qianchuan Lv , Yuanzhao Ji , Chunling Zhou , Jifen Guo , Xinxin Li , Jianzhong Hu
Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is widely used in the biopharmaceutical industry for monitoring purity and analyzing impurities. The accuracy of the method may be compromised by artificial species resulting from sample preparation or electrophoresis separation due to suboptimal conditions. During non-reduced CE-SDS analysis of a multispecific antibody (msAb), named as multispecific antibody C (msAb-C), a cluster of unexpected peaks was observed after the main peak. The corrected peak area ratio of these peaks showed a strong dependence on loaded protein concentration, which affected the accurate assessment of the purity of msAb-C. After investigation, the unexpected peaks were identified as artifacts produced during electrophoresis separation. These artifacts can be mitigated by three different strategies: 1) adding a more hydrophobic surfactant, sodium hexadecyl sulfate (SHS), to the sample and/or sieving gel buffer; 2) reducing the sample loading amount; and 3) increasing the capillary separation temperature to above 40 ℃. We adopted strategy 1) and strategy 3), and successfully developed an optimal non-reduced CE-SDS method for the accurate and reliable purity assessment of msAb-C samples. These strategies of optimizing non-reduced CE-SDS can be used in developing quality control methods for other therapeutic bispecific/multispecific antibodies.
毛细管电泳-十二烷基硫酸钠(CE-SDS)广泛应用于生物制药行业的纯度监测和杂质分析。由于次优条件,该方法的准确性可能因样品制备或电泳分离产生的人工物种而受到损害。在对多特异性抗体C (msAb-C)进行非还原CE-SDS分析时,在主峰后观察到一簇意想不到的峰。校正后的峰面积比与载蛋白浓度有很大关系,影响了对msAb-C纯度的准确评价。经过调查,意外的峰被确定为在电泳分离过程中产生的伪影。这些伪影可以通过三种不同的策略来减轻:1)在样品和/或筛分凝胶缓冲液中添加更疏水的表面活性剂,十六烷基硫酸钠(SHS);2)减少样品装填量;3)提高毛细管分离温度至40℃以上。我们采用策略1)和策略3),成功开发了一种最佳的非还原CE-SDS方法,可以准确可靠地评估mabs - c样品的纯度。这些优化非还原CE-SDS的策略可用于开发其他治疗性双特异性/多特异性抗体的质量控制方法。
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引用次数: 0
A reliable LC-MS/MS method for the quantification of natural amino acids in human plasma and its application in clinic 可靠的LC-MS/MS定量人血浆中天然氨基酸的方法及其临床应用。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-08 DOI: 10.1016/j.jpba.2025.116672
Junhuan Lin , Yibo Song , Yangrui Zhang , Tao Ke , Fengting Ou , Kui Zeng , Debo He , Li Li , Lushan Yu
A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 L-amino acids (AAs) in human plasma. Chromatographic separation was achieved on an Agilent AdvanceBio Hilic column within 15 min via gradient elution with an aqueous solution containing 5 mM ammonium formate, 5 mM ammonium acetate and 0.1 % formic acid and an organic mobile phase containing 0.1 % formic acid, 5 mM ammonium formate and 5 mM ammonium acetate acetonitrile-water (90:10, v/v) at the flow rate of 0.25 mL/min. Individual AAs and internal standard were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. The method was thus utilized to compare the dynamics of individual plasma AAs between healthy females and patients with ovarian tumors. Our results revealed that, in cancer group, plasma 3-Methyl-L-Histidine, L-Proline, L-Phenylalanine and L-Lysine concentrations were significantly increased in patients with malignant ovarian tumors while L-Leucine and L-Isoleucine levels were sharply decreased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for ovarian cancer.
建立了一种简便、快速的LC-MS/MS同时定量人血浆中20种l -氨基酸(AAs)的方法。在Agilent AdvanceBio Hilic色谱柱上,以含有5 mM甲酸铵、5 mM乙酸铵和0.1 %甲酸的水溶液和含有0.1 %甲酸、5 mM甲酸铵和5 mM乙酸铵的有机流动相(90:10,v/v)梯度洗脱,在15 min内实现色谱分离,流速为0.25 mL/min。在优化条件下,采用正离子模式多反应监测(MRM)对单个原子吸收剂和内标进行分析。方法验证包括线性度、灵敏度、准确度和精密度、回收率、基质效应和稳定性,结果表明该LC-MS/MS方法具有特异性、准确性和可靠性。因此,该方法被用于比较健康女性和卵巢肿瘤患者个体血浆AAs的动态。结果显示,恶性卵巢肿瘤患者血浆3-甲基- l-组氨酸、l-脯氨酸、l-苯丙氨酸和l-赖氨酸浓度显著升高,l-亮氨酸和l-异亮氨酸水平显著降低。这些发现支持了LC-MS/MS方法的实用性,以及特异性AAs作为卵巢癌生物标志物的前景。
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引用次数: 0
Research on the metabolites and key metabolic enzymes of allocryptopine in chicken liver microsomes via stable isotope tracing technology 利用稳定同位素示踪技术研究异隐碱在鸡肝微粒体中的代谢产物及关键代谢酶。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-06 DOI: 10.1016/j.jpba.2025.116667
Zhiyong Wu , Xinhao Wang , Lin Wang , Na Sun , Zihui Yang , Jianguo Zeng
Allocryptopine (ALL), a principal active component of the novel veterinary medicine Bopu Powder®, has gained widespread application in the poultry farming sector for the effective management of Escherichia coli (E. coli) diarrhea. In order to explore the metabolites and the pivotal enzymes associated with ALL, this study was conducted employing an in vitro chicken liver microsomal incubation. The metabolites of ALL were analyzed and identified by combining isotope tracing technology with the application of mass spectrometry fragmentation patterns. The key metabolic enzymes involved in the biotransformation of ALL were explored using the CYP450 recombinant enzyme method, which facilitated the identification of the enzymes contributing to ALL's metabolic pathway. The liver microsomal metabolism investigation revealed a total of five metabolites, with the predominant being M2 (harmol or 3-hydroxy-4-methoxy-6-methyl-5,7,8,15-tetrahydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-g]benzo[c]azecin-14(6 H)-one). The recombinant enzyme analysis conclusively identified CYP2D6 as the pivotal CYP450 isoenzyme that plays a central role in the metabolic pathway of the principal ALL metabolite, M2. This research not only expands our comprehension of the biotransformation process of ALL but also provides significant scientific evidence for the clinical safety of ALL, which was of great importance for guiding the application of ALL in the field of veterinary medicine.
异隐碱(ALL)是新型兽药博普粉®的主要活性成分,已广泛应用于家禽养殖部门,用于有效管理大肠杆菌(E. coli)腹泻。为了探索与ALL相关的代谢产物和关键酶,本研究采用鸡肝微粒体体外孵育。采用同位素示踪技术和质谱破碎图谱相结合的方法对ALL的代谢物进行了分析鉴定。利用CYP450重组酶法对ALL生物转化过程中涉及的关键代谢酶进行了探索,从而促进了ALL代谢途径中相关酶的鉴定。肝微粒体代谢调查共发现5种代谢物,主要代谢物为M2 (harmol或3-羟基-4-甲氧基-6-甲基-5,7,8,15-四氢-[1,3]二唑罗[4',5':4,5]苯并[1,2-g]苯并[c]azecin-14(6 H)-one)。重组酶分析最终确定CYP2D6是关键的CYP450同工酶,在ALL主要代谢物M2的代谢途径中起核心作用。本研究不仅扩大了我们对ALL生物转化过程的认识,而且为ALL的临床安全性提供了重要的科学依据,对指导ALL在兽药领域的应用具有重要意义。
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引用次数: 0
Characterization of main degradation products from dendrobine under stress conditions by multistage cleavage of UPLC-ESI-IT-TOF UPLC-ESI-IT-TOF多级解理表征应力条件下石斛主要降解产物。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-05 DOI: 10.1016/j.jpba.2025.116663
Hengju Zhou , Meiling Zeng , Keyong Geng , Zaipeng Chen , Zhijia Tang , Jianwei Xu , Xiaoyan Zhang , Wei Zhou
Dendrobine is a sesquiterpene alkaloid primarily used in the treatment of inflammatory diseases, immune system disorders, and conditions related to oxidative stress. To understand the possible degradation pathways of dendrobine for its quality control, we conducted an in-depth investigation of its degradation products using forced degradation methods. The separation of dendrobine and its degradation products was achieved on a Shim-pack XR-ODS III (75 mm × 2 mm, 1.6 µm) column with a methanol-water mixture as the mobile phase under isocratic conditions, the isolated compounds were examined in positive ion mode with an ion trap-time of flight mass spectrometer (IT-TOF). In order to obtain in-depth structural information about the degradation products, mass spectrometry was performed using a five-stage fragmentation approach. This method allowed for thorough structural clarification via several rounds of selective fragmentation and high-resolution detection. System control and data acquisition were managed using LCMSsolution 3.81 software. The results showed that dendrobine undergoes significant degradation under oxidative, acidic, hydrolytic and thermal conditions, resulting in the formation of several degradation products with notable structural changes. Under oxidative conditions, dendrobine primarily generates two degradation products with mass increases of 16 Da and 32 Da, indicating mono-oxidation and di-oxidation reactions. Acidic degradation led to the identification of three degradation products, including a novel compound with an 18 Da mass increase, suggesting potential hydrolysis or dehydration reactions. Hydrolytic and thermal conditions resulted in the formation of two and three degradation products, respectively, with structural changes indicating possible molecular cleavage and reorganization mechanisms. In contrast, dendrobine exhibited strong stability under alkaline and photolytic conditions, with no significant degradation products detected. Detailed characterization of the degradation products via multi-stage mass spectrometry revealed key reaction pathways and mechanisms involved in dendrobine's degradation, providing critical insights for assessing its chemical stability and optimizing storage conditions.
石斛碱是一种倍半萜生物碱,主要用于治疗炎症性疾病、免疫系统紊乱和与氧化应激相关的疾病。为了了解石斛石可能的降解途径并进行质量控制,我们采用强制降解方法对石斛石的降解产物进行了深入研究。采用Shim-pack XR-ODS III(75 mm × 2 mm, 1.6 µm)色谱柱,甲醇-水混合物为流动相,等压条件下对石斛碱及其降解产物进行分离,用离子捕获飞行时间质谱仪(IT-TOF)在正离子模式下对分离产物进行检测。为了获得降解产物的深入结构信息,质谱分析采用五段破碎法进行。这种方法允许通过几轮选择性破碎和高分辨率检测来彻底澄清结构。系统控制和数据采集采用LCMSsolution 3.81软件进行管理。结果表明,石斛石在氧化、酸性、水解和热条件下都发生了明显的降解,形成了几种结构变化明显的降解产物。在氧化条件下,石斛石主要生成两种降解产物,质量分别增加16 Da和32 Da,分别为单氧化和双氧化反应。酸性降解鉴定出三种降解产物,包括一种Da质量增加18 的新化合物,表明可能发生水解或脱水反应。水解和热条件下分别形成2种和3种降解产物,其结构变化表明可能的分子裂解和重组机制。相比之下,石斛石在碱性和光解条件下表现出很强的稳定性,没有检测到明显的降解产物。通过多级质谱分析对降解产物进行了详细表征,揭示了石斛降解的关键反应途径和机制,为评估石斛的化学稳定性和优化储存条件提供了重要见解。
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引用次数: 0
Implementation of a novel method for the determination of plasma protein binding of highly bound compounds using the equilibrium dialysis method coupled with extraction to the organic phase and its comparison to known methods 一种利用平衡透析法耦合萃取到有机相来测定血浆蛋白与高结合化合物结合的新方法的实施及其与已知方法的比较。
IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL Pub Date : 2025-01-05 DOI: 10.1016/j.jpba.2025.116665
Dawid Gogola , Sanja Novak Ratajczak , Ewelina Gabor-Worwa , Anna Kowal-Chwast , Nilesh Gaud , Gniewomir Latacz , Krzysztof Brzózka , Kamil Kuś
Accurate determination of plasma protein binding (PPB) is crucial in understanding the pharmacokinetics and pharmacodynamics of drugs, particularly for highly bound compounds where traditional methods may fall short. In this study, we present a pioneering approach for the precise determination of PPB that takes advantage of the lipophilicity of highly bound compounds. Twenty four highly bound compounds (with a fraction unbound (fu) from 10−1 to 10−6) were tested with the most commonly used method, i.e., the rapid equilibrium dialysis (RED) method, along with its modifications adapted especially for strongly bound compounds, including dilution, presaturation, competition, and flux-dialysis methods. The results of these methods were compared to data obtained with the modification of RED coupled with extraction to organic phase, and the correlations between them were presented. Comparison studies demonstrate the accuracy of our approach across a set of highly bound compounds within a twofold range. Moreover, PPB values were determined for venetoclax, amiodarone, montelukast, and fulvestrant, for which, to the best of our knowledge and after extensive review of the literature, it has not been possible with the standard RED method until now. In conclusion, our new method can be a big step forward in finding the PPB of strongly plasma protein-bound compounds. It gives us a better understanding of how drugs and proteins interact, which helps us make safer and more effective medicines.
血浆蛋白结合(PPB)的准确测定对于理解药物的药代动力学和药效学至关重要,特别是对于传统方法可能达不到的高结合化合物。在这项研究中,我们提出了一种开创性的方法来精确测定PPB,利用高结合化合物的亲脂性。用最常用的方法,即快速平衡透析(RED)方法,对24种高结合化合物(未结合分数(fu)从10-1到10-6)进行了测试,并对其进行了专门针对强结合化合物的修改,包括稀释、预饱和、竞争和通量透析方法。将这些方法得到的结果与RED改性和萃取有机相得到的结果进行了比较,并给出了它们之间的相关性。比较研究证明了我们的方法在两倍范围内跨越一组高度结合的化合物的准确性。此外,还测定了venetoclax、胺碘酮、孟鲁司特和氟维司汀的PPB值,据我们所知,经过广泛的文献回顾,迄今为止,标准的RED方法还无法测定这些药物的PPB值。总之,我们的新方法在寻找强血浆蛋白结合化合物的PPB方面迈出了一大步。它让我们更好地了解药物和蛋白质是如何相互作用的,这有助于我们制造更安全、更有效的药物。
{"title":"Implementation of a novel method for the determination of plasma protein binding of highly bound compounds using the equilibrium dialysis method coupled with extraction to the organic phase and its comparison to known methods","authors":"Dawid Gogola ,&nbsp;Sanja Novak Ratajczak ,&nbsp;Ewelina Gabor-Worwa ,&nbsp;Anna Kowal-Chwast ,&nbsp;Nilesh Gaud ,&nbsp;Gniewomir Latacz ,&nbsp;Krzysztof Brzózka ,&nbsp;Kamil Kuś","doi":"10.1016/j.jpba.2025.116665","DOIUrl":"10.1016/j.jpba.2025.116665","url":null,"abstract":"<div><div>Accurate determination of plasma protein binding (PPB) is crucial in understanding the pharmacokinetics and pharmacodynamics of drugs, particularly for highly bound compounds where traditional methods may fall short. In this study, we present a pioneering approach for the precise determination of PPB that takes advantage of the lipophilicity of highly bound compounds. Twenty four highly bound compounds (with a fraction unbound (f<sub>u</sub>) from 10<sup>−1</sup> to 10<sup>−6</sup>) were tested with the most commonly used method, i.e., the rapid equilibrium dialysis (RED) method, along with its modifications adapted especially for strongly bound compounds, including dilution, presaturation, competition, and flux-dialysis methods. The results of these methods were compared to data obtained with the modification of RED coupled with extraction to organic phase, and the correlations between them were presented. Comparison studies demonstrate the accuracy of our approach across a set of highly bound compounds within a twofold range. Moreover, PPB values were determined for venetoclax, amiodarone, montelukast, and fulvestrant, for which, to the best of our knowledge and after extensive review of the literature, it has not been possible with the standard RED method until now. In conclusion, our new method can be a big step forward in finding the PPB of strongly plasma protein-bound compounds. It gives us a better understanding of how drugs and proteins interact, which helps us make safer and more effective medicines.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"255 ","pages":"Article 116665"},"PeriodicalIF":3.1,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142965574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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Journal of pharmaceutical and biomedical analysis
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