Vitamin D Determination Tested Using Liquid Chromatography–Tandem Mass Spectrometry


Scientists from Hangzhou Medical College in Hangzhou, Zheijiang, China recently recorded the effectiveness of novel quality control strategies for the determination of vitamin D using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Their findings were published in the Journal of Pharmaceutical and Biomedical Analysis (1).

Close up capsules of fish fat oil in the sun shape, omega 3, vitamin e on the yellow background. Healthy food diet. Nutritional supplement | Image Credit: © MagicalKrew -

Close up capsules of fish fat oil in the sun shape, omega 3, vitamin e on the yellow background. Healthy food diet. Nutritional supplement | Image Credit: © MagicalKrew -

Vitamin D is a prohormone that is essential for immune systems, brain health, and regulating inflammation, among other benefits (2). The body produces vitamin D as a response to sun exposure, though certain food and supplements can boost vitamin D intake. Symptoms of vitamin D deficiency include bone weakness, bone softness, and fractures (3). Different diseases have some association with vitamin D deficiency, such as rickets or osteoporosis. Additionally, vitamin D abnormalities can indicate bone disorders, organ damage, or other medical conditions, making it important to check for these phenomena.

Unfortunately, vitamin D deficiency and insufficiency are very common among children and adolescents in China. According to a 2021 study on vitamin D status in China, the reported prevalence of low vitamin D status varied from 22.3 to 81.9% in adults and from 19.6 to 78.1% in children and adolescents (4). The major circulating form of vitamin D is 25-hydroxyvitamin D (25(OH)D), with various technologies being used to perform detection, from competitive-protein binding assays to high-pressure liquid chromatography-based assays. The latest technology to be used in this regard is liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Mass spectrometry technology has become a very important detection technology in clinical laboratories, with various LC–MS/MS instruments being developed to make the technique’s clinical application more extensive. However, clinical MS technology is still a highly complex technology, with there being large differences between laboratories, affecting the clinical application of this method. As such, before using MS technology to detect clinical analytes, laboratories must perform methodological confirmation or performance verification of the established methods or commercial kits that are used.

Read More: UHPLC–MS/MS Determines Forms of Vitamin K and Their Distribution in Human and Rat Serum

In this study, LC–MS/MS 25-hydroxyvitamin D (25(OH)D) was used to establish internal quality control strategies to ensure the accuracy of clinical vitamin D results. 141 batches of samples were analyzed, with sample internal standard peak area variability, ion pair ratio, and physical examination population data being monitored as quality control strategies for 25(OH)D results. Analytical performance was evaluated by calculated Sigma metrics. Using their quality control strategies, the scientists noticed and monitored several abnormal data points in their routine analysis. The daily peak area cyclic voltammetry(CV) of 25(OH)D fluctuated within a certain range; the scientists selected P99 CV as a control target, finding two abnormal batches in the process. The ratio of 25(OH)VD3 ion pairs was mostly stable, with batch20230120 having a high CV value, which may be due to the bias caused by the limited number. However, batch20220913 and batch20220919 were found to exceed the alarm limit. The sigma level of 25(OH)VD3 in the laboratory was 6.52, indicating “excellent” performance. Altogether, the scientists were able to establish comprehensive quality control strategies for the determination of 25(OH)D by LC-MS/MS. Now, these techniques exhibit high analytical performance and can provide more accurate reports for clinics.

“The proposed method has demonstrated great potential for the early differential diagnosis of pancytopenia related diseases,” the authors wrote in the study. “It has significant clinical importance for the timely and rational guidance on subsequent treatment to improve patient survival” (1).


(1) Zhou, Y.; Du, J.; Liu, Y.; Xia, J. Novel Quality Control Strategies for the Determination of 25-Hydroxyvitamin D by LC-MS/MS. J. Pharm. Biomed. Anal. 2024, 239, 115908. DOI:

(2) Bernstein, A. What is Vitamin D and Why Does the Body Need It? Healthline Media 2024. (accessed 2024-5-2)

(3) Why Is Vitamin D Testing Important? Campbell County Health 2022. (accessed 2024-5-2)

(4) Liu, W.; Hu, J.; Fang, Y.; Wang, P.; et al. Vitamin D Status in Mainland of China: A Systematic Review and Meta-Analysis. EClinicalMedicine 2021, 38, 101017. DOI: 10.1016/j.eclinm.2021.101017

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