Michigan State University Veterinary Diagnostic Laboratory, Toxicology Section, Michigan State University, East Lansing, MI, USA.
Department of Pathobiology & Diagnostic Investigation, Michigan State University College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA.
Eur J Mass Spectrom (Chichester). 2021 Feb;27(1):48-62. doi: 10.1177/14690667211000244. Epub 2021 Mar 15.
This report examines the feasibility of determination of Vitamin D3, D2 and their 25-hydroxy metabolites utilizing Gas Chromatography Tandem Mass Spectrometry (GC/MS/MS) as a potential alternative to popular Liquid Chromatography Tandem Mass Spectrometric (LC/MS/MS) methodologies. The GC/MS/MS approach was found to operate reasonably well despite long-standing concerns that gas-liquid chromatography of vitamin D compounds invoke thermal rearrangements owing to the relatively high inlet and capillary column temperatures used. The workup procedure involved incubation of feed samples with concentrated potassium hydroxide for overnight fat saponification, extraction of D Vitamins in n-hexane and reaction with N,O-bis(trimethylsilyl)trifluoroacetamide at 70 °C for 30 mins. In addition to parent compounds, small amounts of pyro-, isopyro-, and iso-vitamin D and isotachysterol3 variants were obtained from each Vitamin D-related compound upon extraction and GC/MS/MS analysis. Mass spectral and chromatographic behavior of these compounds are herein described and interpreted. Multiple Reaction Monitoring settings on GC/MS/MS included 456→351 for Vitamin D3 and 486→363 for Vitamin D2. Trimethylsilylation enabled single predominant peaks for Vitamins D3 and D2, and sample workup in the presence of deuterated Vitamin D analogs enabled accurate and precise sensitivity to 1 ppb (ng/g) in feeds. The method could be extended with reasonable accuracy to 25-hydroxy (25OH) compounds, but accuracies would be significantly improved by inclusion of respective 25OH-specific deuterated internal standards. The method was applied to 27 submissions of suspect dog foods of which 22% were discovered elevated and 44% were discovered to contain toxic levels of Vitamin D3. The described method was thus discovered to provide a suitable mass spectrometric approach for Vitamin D, proving itself here specifically of value in detection of ergocalciferol and cholecalciferol in animal feeds. The specificity and sensitivity of the tandem quadrupole approach can enable suitable applicability to serum determination if desired.
本报告探讨了利用气相色谱串联质谱（GC/MS/MS）测定维生素 D3、D2 及其 25-羟基代谢物的可行性，作为替代流行的液相色谱串联质谱（LC/MS/MS）方法的潜在选择。尽管长期以来一直存在维生素 D 化合物的气-液色谱由于使用相对较高的进样口和毛细管柱温度而引起热重排的担忧，但 GC/MS/MS 方法的操作性能相当不错。该工作流程包括将饲料样品与浓氢氧化钾孵育过夜进行脂肪皂化，用正己烷提取 D 族维生素，然后在 70°C 下与 N,O-双（三甲基硅基）三氟乙酰胺反应 30 分钟。除了母体化合物外，从每种维生素 D 相关化合物中提取和 GC/MS/MS 分析后，还获得了少量的焦维生素 D、异焦维生素 D 和同型维生素 D 以及同型麦角甾醇 3 变体。本文描述并解释了这些化合物的质谱和色谱行为。GC/MS/MS 上的多重反应监测设置包括 456→351 用于维生素 D3 和 486→363 用于维生素 D2。三甲基硅烷基化使维生素 D3 和 D2 都具有单一的主要峰，并且在存在氘代维生素 D 类似物的情况下进行样品处理，可以使饲料中的 1 ppb（ng/g）达到准确和精密的灵敏度。该方法可以以合理的精度扩展到 25-羟基（25OH）化合物，但通过包含各自的 25OH 特异性氘代内标，准确性将显著提高。该方法应用于 27 份可疑狗食的检测，其中 22%的狗食中发现维生素 D3 含量升高，44%的狗食中发现维生素 D3 含量有毒。因此，该方法被发现为维生素 D 提供了一种合适的质谱方法，在动物饲料中特别有助于检测麦角钙化醇和胆钙化醇。串联四极杆方法的特异性和灵敏度如果需要，可适用于血清测定。
