Development and validation of an LC‒MS/MS method for the determination of cyclocreatine phosphate and its related endogenous biomolecules in rat heart tissues

IF 4.3 2区 化学 Q2 CHEMISTRY, MULTIDISCIPLINARY BMC Chemistry Pub Date : 2024-11-04 DOI:10.1186/s13065-024-01304-1
Ibrahim F. Abo-Elmagd, Amr M. Mahmoud, Medhat A. Al-Ghobashy, Marianne Nebsen, Mostafa A. Rabie, Ahmed F. Mohamed, Lamiaa A. Ahmed, Nesrine S. El Sayed, Reem K. Arafa, Robert Todd, Salwa A. Elgebaly
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Abstract

The cardioprotective drug cyclocreatine phosphate has been awarded Food and Drug Administration-orphan drug designation for the prevention of ischemic injury to enhance cardiac graft recovery and survival in heart transplantation. Cyclocreatine phosphate is the water-soluble derivative of cyclocreatine. Estimating the levels of Cyclocreatine phosphate, Adenosine triphosphate, Creatine Phosphate, Creatine and Cyclocreatine helps us in understanding the energy state as well as evaluating the heart cells’ function. The quantification of endogenous compounds imposes a challenging task for analysts because of the absence of a true blank matrix, whose use is required according to international guidelines. Recently, the International Council for Harmonization issued a new guideline that contains guidance on the validation of methods used to quantify endogenous components, such as the background subtraction approach that was employed in our current study. Specifically, we developed and validated a sensitive, reliable and accurate liquid chromatography-tandem mass spectrometry assay to determine simultaneously the levels of mentioned endogenous compounds in rat heart tissue. Tissue samples were prepared by protein precipitation extraction using water: methanol (1:1). Using Ultra Performance Liquid Chromatography, Chromatographic separation was achieved with ZORBAX Eclipse Plus C18 4.6 × 100 mm,3.5 μm column and conditions as following: ammonium acetate (pH 8.5): acetonitrile, 70:30 mobile phase, 0.7 mL/min flow rate and 25 °C temperature. Electrospray ionization mass detector with Multiple reaction monitoring mode was then employed, using both positive and negative modes, Analysis was carried out using 5.00–2000.00 ng/mL linear concentration range within 2 min for each analyte. According to Food and Drug Administration guidelines for bioanalytical methods, validation was carried out. We investigated the matrix effect, recovery efficiency and process efficiency for the analyte in neat solvent, postextraction matrix and tissue. The results stated mean percentage recoveries higher than 99%, accuracy 93.32–111.99%, and Relative Standard Deviation (RSD) below 15% within the concentration range of our study which indicated that target analytes’ stability in their real matrix is sufficient under the employed experimental conditions.

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开发并验证测定大鼠心脏组织中环磷酸肌酸及其相关内源性生物大分子的 LC-MS/MS 方法。
心脏保护药物磷酸环肌肽已被美国食品药品管理局认定为非专利药物,用于预防缺血性损伤,以提高心脏移植手术中的心脏移植物恢复和存活率。磷酸环肌酸是环肌酸的水溶性衍生物。估算磷酸环肌酸、三磷酸腺苷、磷酸肌酸、肌酸和环肌酸的水平有助于我们了解能量状态和评估心脏细胞的功能。由于缺乏真正的空白基质,内源性化合物的定量对分析人员来说是一项极具挑战性的任务。最近,国际协调理事会发布了一份新指南,其中包含对用于量化内源性成分的方法进行验证的指导,例如我们目前研究中采用的背景减去法。具体来说,我们开发并验证了一种灵敏、可靠、准确的液相色谱-串联质谱测定法,用于同时测定大鼠心脏组织中提及的内源性化合物的水平。组织样本采用水-甲醇(1:1)沉淀提取法制备。采用 ZORBAX Eclipse Plus C18 4.6 × 100 mm、3.5 μm 色谱柱,以乙酸铵(pH 8.5):乙腈(70:30)为流动相,流速 0.7 mL/min,柱温 25 ℃,进行超高效液相色谱分离。采用电喷雾离子化质量检测器和多反应监测模式,同时使用正离子和负离子模式,在 2 分钟内对每种分析物在 5.00-2000.00 纳克/毫升的线性浓度范围内进行分析。根据食品和药物管理局生物分析方法指南进行了验证。我们研究了分析物在纯溶剂、萃取后基质和组织中的基质效应、回收率和过程效率。结果表明,在我们研究的浓度范围内,平均回收率高于 99%,准确度为 93.32-111.99%,相对标准偏差(RSD)低于 15%。
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来源期刊
BMC Chemistry
BMC Chemistry Chemistry-General Chemistry
CiteScore
5.30
自引率
2.20%
发文量
92
审稿时长
27 weeks
期刊介绍: BMC Chemistry, formerly known as Chemistry Central Journal, is now part of the BMC series journals family. Chemistry Central Journal has served the chemistry community as a trusted open access resource for more than 10 years – and we are delighted to announce the next step on its journey. In January 2019 the journal has been renamed BMC Chemistry and now strengthens the BMC series footprint in the physical sciences by publishing quality articles and by pushing the boundaries of open chemistry.
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