Harahap Yahdiana, Steven Steven, Suryadi Herman
Bioavailability/Bioequivalence Laboratory, Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia.
Faculty of Military Pharmacy, Republic of Indonesia Defense University, Sentul, Indonesia.
Front Pharmacol. 2022 Aug 11;13:928721. doi: 10.3389/fphar.2022.928721. eCollection 2022.
Cyclophosphamide (CP) is an anti-cancer alkylating prodrug, metabolized by CYP450 into its active metabolite 4-hydroxycyclophosphamide (4-OHCP). Its therapeutic effectiveness is determined by the 4-OHCP concentration. Several analytical methods in plasma and dried blood spots have been developed to analyze cyclophosphamide and 4-OHCP; however, there are many disadvantages. The objective of this study was to develop and validate the ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method by volumetric absorptive microsampling (VAMS) and 4-hydroxycyclophosphamide-d4 (4-OHCP-d4) as an internal standard. VAMS requires small sample volumes, and it is not affected by the hematocrit values; therefore, it is an efficient sampling method. The samples were derivatized with 5 μL semicarbazide hydrochloride (SCZ) and 25 μL of the resulting 4-OHCP-SCZ; 4-OHCP-d4-SCZ derivatives were absorbed by VAMS and extracted by protein precipitation. The optimum conditions were obtained using the Waters Acquity UPLC BEH C18 (2.1 × 100 mm; 1.7 μm) column; flow rate 0.15 ml/min; mobile phase 0.01% formic acid and methanol; gradient elution mode for 6 min by positive electrospray ionization; and multiple reaction monitoring of m/z 260.7 > 140.0 for CP, 333.7 > 221.0 for 4-OHCP-SCZ, and 337.7 > 225.1 for 4-OHCP-d4-SCZ. The method met the validation requirements set by the FDA. The cyclophosphamide LLOQ value was 5 ng/mL, and the calibration curve range was 5-60,000 ng/ml. Furthermore, the 4-OHCP LLOQ value was 2.5 ng/ml, and the calibration curve range was 2.5-1,000 ng/ml.
环磷酰胺（CP）是一种抗癌烷化前体药物，经细胞色素P450代谢为其活性代谢物4-羟基环磷酰胺（4-OHCP）。其治疗效果由4-OHCP浓度决定。已开发出几种用于分析血浆和干血斑中环磷酰胺和4-OHCP的分析方法；然而，这些方法存在许多缺点。本研究的目的是开发并验证一种采用体积吸收微量采样（VAMS）和以4-羟基环磷酰胺-d4（4-OHCP-d4）作为内标的超高效液相色谱-串联质谱（UPLC-MS/MS）方法。VAMS所需样本体积小，且不受血细胞比容值影响；因此，它是一种高效的采样方法。样本用5μL盐酸氨基脲（SCZ）和25μL所得的4-OHCP-SCZ进行衍生化；4-OHCP-d4-SCZ衍生物通过VAMS吸收并通过蛋白沉淀法提取。使用沃特世Acquity UPLC BEH C18（2.1×100mm；1.7μm）色谱柱获得最佳条件；流速0.15ml/min；流动相为0.01%甲酸和甲醇；通过正电喷雾电离进行6分钟的梯度洗脱模式；对CP进行m/z 260.7>140.0的多反应监测，对4-OHCP-SCZ进行m/z 333.7>221.0的多反应监测，对4-OHCP-d4-SCZ进行m/z 337.7>225.1的多反应监测。该方法符合美国食品药品监督管理局（FDA）设定的验证要求。环磷酰胺的定量下限（LLOQ）值为5ng/mL，校准曲线范围为5-60,000ng/ml。此外，4-OHCP的LLOQ值为2.5ng/ml，校准曲线范围为2.5-1,000ng/ml。
