Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital The Netherlands Cancer Institute, Amsterdam, the Netherlands; Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands.
Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital The Netherlands Cancer Institute, Amsterdam, the Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 15;1211:123494. doi: 10.1016/j.jchromb.2022.123494. Epub 2022 Oct 7.
Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.
采用超高效液相色谱-串联质谱法，建立并验证了定量检测人皮肤组织中抗寄生虫药物巴龙霉素的生物分析测定方法。巴龙霉素是一种氨基糖苷类药物，肌肉注射给药，用于治疗多种临床表现的被忽视热带病利什曼病。目前，该药正在治疗卡拉-阿扎尔后皮肤利什曼病，这种疾病中利什曼原虫在皮肤中分裂和生存。我们提出了一种针对靶部位的生物分析方法，能够准确地定量检测人皮肤组织中的巴龙霉素，其临床目的是定量检测来自苏丹的卡拉-阿扎尔后皮肤利什曼病患者的皮肤活检中的巴龙霉素。采用胶原酶 A 在 37°C 孵育过夜的酶消化方法作为均质化方法，制备皮肤组织匀浆。进一步通过三氯乙酸进行蛋白沉淀和稀释步骤进行样品制备。最终提取液注入 C18 分析柱，采用庚氟丁酸离子对分离和洗脱进行等度洗脱。该色谱系统与三重四极杆质谱仪联用进行检测。该方法在消化液中的线性范围为 5 至 1000ng/mL（r≥0.9967），其准确度和精密度的测定性能符合 EMA 指南规定的可接受标准值。此外，在人皮肤组织中观察到基质效应，并通过多重氘代巴龙霉素内标进行校正。同时，未检测到明显的 IS 归一化基质效应，且具有较高的样品制备回收率。因此，消化液基质可以作为校准标准品的制备替代人皮肤组织基质，这在控制基质有限的情况下非常方便。最后，在来自苏丹临床试验的卡拉-阿扎尔后皮肤利什曼病患者的皮肤中准确地定量了巴龙霉素。
