Abo-Elmagd Ibrahim F, Mahmoud Amr M, Al-Ghobashy Medhat A, Nebsen Marianne, Rabie Mostafa A, Mohamed Ahmed F, Ahmed Lamiaa A, El Sayed Nesrine S, Arafa Reem K, Todd Robert, Elgebaly Salwa A
Bioanalysis Research Group, School of Pharmacy, New Giza University, Giza, 12256, Egypt.
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt.
BMC Chem. 2024 Nov 4;18(1):214. doi: 10.1186/s13065-024-01304-1.
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.
心脏保护药物环磷酸肌酸已被美国食品药品监督管理局授予孤儿药称号，用于预防缺血性损伤，以提高心脏移植中心脏移植物的恢复和存活率。环磷酸肌酸是环肌酸的水溶性衍生物。估算环磷酸肌酸、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以及评估心脏细胞的功能。由于缺乏真正的空白基质，而根据国际指南又需要使用空白基质，因此对内源性化合物进行定量分析对分析人员来说是一项具有挑战性的任务。最近，国际协调理事会发布了一项新指南，其中包含有关用于定量内源性成分的方法验证的指南，例如我们当前研究中采用的背景扣除法。具体而言，我们开发并验证了一种灵敏、可靠且准确的液相色谱 - 串联质谱分析法，用于同时测定大鼠心脏组织中上述内源性化合物的水平。组织样品通过使用水：甲醇（1:1）的蛋白质沉淀萃取法制备。使用超高效液相色谱，采用ZORBAX Eclipse Plus C18 4.6×100 mm，3.5μm色谱柱进行色谱分离，条件如下：醋酸铵（pH 8.5）：乙腈，70:30流动相，0.7 mL/min流速，25°C温度。然后采用电喷雾电离质谱检测器，采用多反应监测模式，同时使用正模式和负模式，对每种分析物在2分钟内使用5.00 - 2000.00 ng/mL线性浓度范围进行分析。根据美国食品药品监督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%，这表明在所采用的实验条件下，目标分析物在其实际基质中的稳定性足够。
