Next generation β-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices. Each enzyme shows distinct substrate preferences, pH optima, and variability across clinical specimens. These results demonstrate how reliance on a single glucuronidated substrate as the internal hydrolysis control cannot ensure performance across a broader panel of analytes. Moreover, sample specific urine properties compromise β-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. A minimum of 3-fold dilution of urine with buffer yields measurable improvements in achieving target pH and reducing the impact of endogenous compounds on enzyme performance. After subjecting the enzymes to pH extremes and compromising chemicals, one particular β-glucuronidase was identified that addressed many of these challenges and greatly lower the risk of failed hydrolyses. In summary, we present strategies to evaluate glucuronidases that aid in higher accuracy urine drug tests with lower potential for false negatives.

中文翻译：

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尿液药物检测中影响葡萄糖醛酸酶性能的因素可能导致假阴性

下一代 β-葡萄糖苷酸酶可在室温下有效切割尿液中的葡萄糖苷酸。然而，在发现研究期间，确定了跨生物学相关的极端 pH 值和患者尿液样本的尿液药物测试的其他挑战。在临床尿液样本和市售的尿液对照基质中评估了不同的酶。每种酶在临床标本中表现出不同的底物偏好、最佳 pH 值和可变性。这些结果表明，依赖单一的葡萄糖醛酸化底物作为内部水解控制无法确保在更广泛的分析物组中的性能。此外，样本特定的尿液特性会影响 β-葡萄糖醛酸酶到不同的水平，对于某些酶来说更为明显，从而以酶特异性方式降低某些药物分析物的回收率。用缓冲液将尿液稀释至少 3 倍可显着改善达到目标 pH 值并减少内源性化合物对酶性能的影响。在使酶经受极端 pH 值和损害化学品后，确定了一种特殊的 β-葡萄糖醛酸酶，它解决了许多这些挑战并大大降低了水解失败的风险。总之，我们提出了评估葡萄糖醛酸酶的策略，这些策略有助于提高尿液药物测试的准确性，降低假阴性的可能性。在使酶经受极端 pH 值和损害化学品后，确定了一种特殊的 β-葡萄糖醛酸酶，它解决了许多这些挑战并大大降低了水解失败的风险。总之，我们提出了评估葡萄糖醛酸酶的策略，这些策略有助于提高尿液药物测试的准确性，降低假阴性的可能性。在使酶经受极端 pH 值和损害化学品后，确定了一种特殊的 β-葡萄糖醛酸酶，它解决了许多这些挑战并大大降低了水解失败的风险。总之，我们提出了评估葡萄糖醛酸酶的策略，这些策略有助于提高尿液药物测试的准确性，降低假阴性的可能性。