There is need for a single assay able to quantify the most biologically active metabolite, 1α,25-dihydroxy-vitamin-D3, and the recently discovered biologically distinct C3-epimers of 25OHD, in addition to traditional vitamin D metabolites. We developed a method of chromatographic separation and absolute quantification of the following ten forms of vitamin D: 3-epi-25OHD3, 25OHD3, 3-epi-25OHD2, 25OHD2, 1α,25(OH)2D3, 24R,25(OH)2D3, 23R,25(OH)2D3, 1a,25(OH)2D2, D3, and D2 by single extraction and injection. Chemical derivatization followed by liquid chromatography using a charged surface hybrid C18 column and subsequent tandem mass spectrometry was utilized to detect and quantify each metabolite. This method is remarkable as a cooled column was required to achieve chromatographic resolution of epimers. Validation of each metabolite was performed at four concentrations and revealed inter- and intra-day precision and accuracy below 15% across three consecutive days of analysis. After validation, this method was applied to analyze the blood plasma from 739 samples from 352 subjects (8mo to 20 yr), 79 pooled plasma samples, and 10 NIST SRM972a samples. Healthy control samples (n = 357) were used to investigate developmentally associated changes in vitamin D metabolite concentrations during early life. This method yields excellent linearity (R2 ≥ 0.99) across concentrations encompassing the biological range of many metabolites including 1α,25(OH)2D3. Concentrations of 25OHD2 and 24R,25(OH)2D3 were significantly (q ≤0.05) lower in infants compared to both children and adolescents. The percentage of 3-epi-25OHD3 in total 25OHD3 was significantly lower (q ≤ 0.009) in post-puberty subjects. Here we present a single assay capable of separating and quantifying ten vitamin D metabolites including C3-epimers of 25OHD, and quantifying 1α,25-dihydroxy-vitamin-D3 at and below concentrations observed in human plasma (LLOQ < 10 pM).

中文翻译：

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利用冷却的液相色谱和化学衍生化来分离和量化25-羟基维生素D和低丰度1α，25（OH）2D3的C3表位：在儿科人群中的应用。

除了传统的维生素D代谢物外，还需要一种能够定量分析最具生物活性的代谢物1α，25-二羟基维生素D3和最近发现的生物学上不同的25OHD C3表位的方法。我们开发了以下十种形式的维生素D的色谱分离和绝对定量方法：3-epi-25OHD3、25OHD3、3-epi-25OHD2、25OHD2、1α，25（OH）2D3、24R，25（OH）2D3 ，23R，25（OH）2D3、1a，25（OH）2D2，D3和D2通过单次萃取和注射获得。化学衍生化，然后使用带电荷的表面杂化C18色谱柱进行液相色谱分析，随后进行串联质谱分析，以检测和定量每种代谢物。该方法非常出色，因为需要冷却的色谱柱才能实现差向异构体的色谱分离。在四个浓度下对每种代谢物进行验证，并在连续三天的分析中显示日间和日内精度以及低于15％的精度。验证后，该方法用于分析来自352位受试者（8个月至20年）的739个样品，79个合并血浆样品和10个NIST SRM972a样品的血浆。健康对照样品（n = 357）用于研究生命早期维生素D代谢物浓度的发育相关变化。此方法在包括1α，25（OH）2D3在内的许多代谢物的生物学范围内的浓度范围内均具有出色的线性（R2≥0.99）。与儿童和青少年相比，婴儿中25OHD2和24R，25（OH）2D3的浓度显着降低（q≤0.05）。在青春期后受试者中，3-epi-25OHD3在总25OHD3中的百分比显着降低（q≤0.009）。在这里，我们提出了一种能够分离和定量分析包括25OHD的C3受体在内的十种维生素D代谢物，并定量检测和降低人体血浆中所观察到的浓度（LLOQ <10 pM）中的1α，25-二羟基维生素D3的一种测定方法。