Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant.

IF 2.3 Q3 BIOCHEMICAL RESEARCH METHODS
Christian Ezequiel Olivetti, María Florencia Fernández, Jana Stojanova, Silvina Ruvinsky, Andrea Mangano, Paula Schaiquevich
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Abstract

3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is a paucity of data regarding microbiome health and outcomes in the pediatric HSCT setting. We developed and thoroughly validated an affordable high-performance liquid chromatography/fluorescence detector (HPLC-FLD) method to quantify 3-IS in urine for use in the pediatric setting. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm × 5 μm) with a mobile phase consisting of pH 4.0 acetic acid-triethylamine buffer and acetonitrile (88:12, v/v), eluted isocratically at 1 mL/min. 3-IS fluorescence detection was set at excitation/emission of 280 and 375, respectively. The method was fully validated according to FDA-specified limits including selectivity, linearity (0.10 to 10.00 mg/L, r2 > 0.997), intra- and inter-day accuracy, and precision. 3-IS stability was confirmed after three freeze-thaw cycles, for short- and medium-term on a benchtop and at 4 °C and for long-term up to 60 days at -20 °C. The validated method was used to quantify 3-IS in urine samples from HSCT pediatric patients.

用于评估小儿造血干细胞移植患者尿液中 3-吲哚硫酸酯含量的高效液相色谱法的全面验证及在临床研究中的应用。
3-吲哚硫酸盐(3-IS)是由肠道共生细菌产生的色氨酸降解产物吲哚经肝脏转化而成。这种代谢物有望成为成人造血干细胞移植(HSCT)后菌群失调和临床结果的生物标志物。然而,有关儿科造血干细胞移植环境中微生物组健康和预后的数据却很少。我们开发并全面验证了一种经济实惠的高效液相色谱/荧光检测器(HPLC-FLD)方法,用于定量检测儿科尿液中的3-IS。色谱分离采用 C18 色谱柱(250 × 4.6 mm × 5 μm),流动相为 pH 值为 4.0 的醋酸-三乙胺缓冲液和乙腈(88:12, v/v),以 1 mL/min 的速度等度洗脱。3-IS 荧光检测器的激发/发射光谱分别为 280 和 375。该方法的选择性、线性(0.10 至 10.00 mg/L,r2 > 0.997)、日内和日间准确度和精密度均符合 FDA 规定的限值。3-IS 的稳定性在经过三次冻融循环后得到确认,短期和中期在台式机上和 4 °C,长期在 -20 °C,长达 60 天。验证后的方法用于定量检测造血干细胞移植儿科患者尿样中的3-IS。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods and Protocols
Methods and Protocols Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (miscellaneous)
CiteScore
3.60
自引率
0.00%
发文量
85
审稿时长
8 weeks
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