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Detection of hypoglycin A and MCPA-carnitine in equine serum and muscle tissue: Optimisation and validation of a LC-MS-based method without derivatisation.

检测马血清和肌肉组织中的低甘氨酸A和MCPA-肉碱: 优化和验证基于lc-ms的无衍生化方法。

  • 影响因子:1.86
  • DOI:10.1111/evj.13303
  • 作者列表:"González-Medina S","Hyde C","Lovera I","Piercy RJ
  • 发表时间:2021-05-01
Abstract

BACKGROUND:Measurement of hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA), in equine serum confirms a diagnosis of atypical myopathy (AM), a pasture-associated toxic rhabdomyolysis with high mortality linked to the ingestion of Acer trees plant material. Supportive diagnostic tests include plasma acyl-carnitine profiling and urine organic acid testing, but these are not specific for AM. Previously reported HGA and MCPA analytical techniques used liquid chromatography-mass spectrometry (LC-MS) with a derivatising step, but the latter prolongs testing and increases costs. OBJECTIVES:To develop a rapid LCMS method for detection of serum and tissue HGA and MCPA that enables expedited diagnosis for horses with AM. STUDY DESIGN:Analytical test validation. METHODS:Validation parameters to industry standards using as criteria precision, accuracy, linearity, reproducibility and stability in analyte-spiked samples were calculated on 9-calibration points and 3 different validation concentrations in both serum and muscle tissue. RESULTS:The test was successfully validated for the detection of HGA and MCPA-carnitine in equine serum and muscle. Test linearity was excellent (r2  = .999), accuracy was very good for both analytes (93%-108%), precision did not exceed 10% coefficient of variation and reproducibility met the requirements of the Horwitz equation. Stability was unaffected by storage at a range of temperatures. MAIN LIMITATIONS:The spectrum of the tested analytes was limited to only two relevant analytes in favour of a quick and easy analysis. Linearity of the muscle method was not evaluated as calibration curves were not produced in this matrix. CONCLUSION:We report an optimised, simplified and validated method for detection of HGA and MCPA-carnitine in equine serum and muscle suitable for rapid diagnosis of suspected AM cases. The serum-based test should also enable risk assessment of toxin exposure in cograzing horses and assessment of horses with undiagnosed myopathies, while the tissue detection test should help to confirm cases post-mortem and to determine toxin distribution, metabolism and clearance across different tissues.

摘要

背景: 在马血清中测定低甘氨酸A (HGA) 及其毒性代谢产物亚甲基环丙基乙酸 (MCPA),证实了非典型肌病 (AM) 的诊断,AM是一种牧场相关的毒性横纹肌溶解症,高死亡率与摄入枫树植物材料有关。支持性诊断测试包括血浆酰基肉碱分析和尿液有机酸测试,但这些不是AM特异性的。先前报道的HGA和MCPA分析技术使用具有衍生化步骤的液相色谱-质谱 (lc-ms),但后者延长了测试并增加了成本。 目的: 开发一种快速LCMS方法来检测血清和组织HGA和MCPA,从而能够快速诊断患有AM的马。 研究设计: 分析测试验证。 方法: 使用作为标准的工业标准的验证参数在9个校准点和血清和肌肉组织中的3种不同的验证浓度上计算掺入分析物的样品中的精密度、准确度、线性、再现性和稳定性。 结果: 本试验成功用于马血清和肌肉中HGA和MCPA-肉碱的检测。测试线性良好 (r2 = .999),两种分析物的准确度都非常好 (93%-108%),精密度不超过10%,变异系数和再现性符合Horwitz方程的要求。稳定性不受在一定温度范围内储存的影响。 主要限制: 测试的分析物的光谱仅限于两种相关分析物,有利于快速和容易的分析。没有评价肌肉方法的线性,因为在该矩阵中没有产生校准曲线。 结论: 我们报告了一种优化、简化和验证的方法,用于检测马血清和肌肉中HGA和MCPA-肉碱,适用于疑似AM病例的快速诊断。以血清为基础的试验还应能够对共放牧马的毒素暴露进行风险评估,并对患有未确诊肌病的马进行评估,而组织检测试验应有助于在死后确认病例,并确定毒素在不同组织中的分布、代谢和清除情况。

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影响因子:2.68
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DOI:10.1080/14656566.2020.1814255
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DOI:10.1007/s11033-021-06299-9
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