建立毛细管全血中 25-羟维生素 D 和 D 测定的灵敏高效液相色谱-串联质谱法。

Development of a sensitive HPLC-MS/MS method for 25-hydroxyvitamin D and D measurement in capillary blood.

机构信息

Department of Developmental and Behavioral Pediatrics, Pediatric Translation Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Department of Pediatric Cardiology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.

出版信息

J Clin Lab Anal. 2020 Oct;34(10):e23451. doi: 10.1002/jcla.23451. Epub 2020 Jun 26.

BACKGROUND

Measurement of 25-hydroxyvitamin D [25(OH)D)] levels is important. The current method requires a relatively large volume of serum. To minimize the amount of serum needed, we established a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to measure 25(OH)D in capillary serum.

METHODS

Venous blood and fingertip blood were collected from 90 participants. Volumes of 100 µL of venous serum and 20 µL of capillary serum were collected. The serum samples were pretreated by protein removal, extraction and concentration, and an HPLC-MS/MS method based on chromatographic separation and multi reactive ion monitoring was conducted. The intra- and inter-batch variation coefficients were less than 10% for both 25-hydroxyvitamin D [25(OH)D ] and 25-hydroxyvitamin D [25(OH)D )]. For venous specimens, the accuracies were 3.87% and 4.91%, respectively. For capillary specimens, the accuracies were 1.65% and 5.32%, respectively.

RESULTS

The limit of detection (LOD) of 25(OH)D was 0.01 ng/mL, and the LOD of 25(OH)D was 0.05 ng/mL. The results showed that the mean concentration of 25(OH)D in venous blood was 22.56 ± 9.50 ng/mL, while the mean concentration of 25(OH)D in capillary blood was 18.14 ± 7.86 ng/mL. Furthermore, the adjusted capillary blood 25(OH)D level was 22.99 ± 10.24 ng/mL by the correction formula in our study. Similarly, the mean concentration of 25(OH)D in capillary blood was 17.98 ± 7.98 ng/mL. The adjusted capillary blood 25(OH)D level was 22.85 ± 10.42 ng/mL. No difference in the content of 25(OH)D or 25(OH)D was found between venous serum and corrected capillary serum. The correlation coefficients between venous and corrected capillary concentrations of 25(OH)D and 25(OH)D were 0.7941 and 0.8103, respectively, and the areas under the receiver operating characteristic curve were 0.9367 and 0.9565, respectively.

CONCLUSIONS

This capillary blood method requires minimal sample preparation and is suitable for routine use in the 25(OH)D detection.

背景

25-羟维生素 D [25(OH)D] 水平的测量非常重要。目前的方法需要相对较大体积的血清。为了最小化所需的血清量，我们建立了一种高效液相色谱-串联质谱（HPLC-MS/MS）方法，用于测量毛细血管血清中的 25(OH)D。

方法

从 90 名参与者中采集静脉血和指尖血。采集 100μL 静脉血清和 20μL 毛细血管血清。血清样品经蛋白去除、提取和浓缩预处理，采用基于色谱分离和多反应离子监测的 HPLC-MS/MS 方法进行检测。25-羟维生素 D [25(OH)D] 和 25-羟维生素 D [25(OH)D] 的批内和批间变异系数均小于 10%。对于静脉标本，准确度分别为 3.87%和 4.91%。对于毛细血管标本，准确度分别为 1.65%和 5.32%。

结果

25(OH)D 的检测限（LOD）为 0.01ng/mL，25(OH)D 的 LOD 为 0.05ng/mL。结果表明，静脉血中 25(OH)D 的平均浓度为 22.56±9.50ng/mL，而毛细血管血中 25(OH)D 的平均浓度为 18.14±7.86ng/mL。此外，通过本研究中的校正公式，校正后的毛细血管血 25(OH)D 水平为 22.99±10.24ng/mL。同样，毛细血管血中 25(OH)D 的平均浓度为 17.98±7.98ng/mL。校正后的毛细血管血 25(OH)D 水平为 22.85±10.42ng/mL。静脉血清和校正后的毛细血管血清中 25(OH)D 或 25(OH)D 的含量无差异。25(OH)D 和 25(OH)D 的静脉和校正毛细血管浓度的受试者工作特征曲线下面积分别为 0.9367 和 0.9565。