The growing interest in vitamin D has stimulated intensive research activities aiming to address unresolved analytical, clinical and physiological aspects of vitamin D [1][2][3][4]. This work has led to an increasing awareness that our knowledge about vitamin D metabolism and its assessment in clinical practice harbours substantial limitations. For example, Blacks have a markedly lower average 25(OH) D concentration than Whites [2, 5, 6], but exhibit higher bone mineral density (BMD) and a lower risk of fragility fracture [7][8][9]. Also, the relationship between 25(OH)D and parathyroid hormone (PTH) seems to differ between races [2]. These findings have led researchers to look for other markers that are capable of providing more accurate information about the adequacy of patients' vitamin D supply. Several studies suggested that free and bioavailable 25(OH)D reflect vitamin D metabolism better than 25(OH) D [2, 10, 11]. However, both markers require the measurement of vitamin D binding protein (VDBP). Early studies quantified VDBP with either monoclonal or polyclonal immunoassays. However, later studies that employed LC-MS/MS based methods have demonstrated that these immunoassay are strongly biased due to common genetic polymorphisms [4]. The limited number of laboratories that offer VDBP measurement by LC-MS/MS and the lack of a reference measurement procedure hamper a wider use of free and bioavailable 25(OH)D in clinical studies. Another potential surrogate marker of vitamin D metabolism is 24,25(OH) 2 D, the major product of 25(OH)D catabolism. The circulating concentrations of both metabolites are strongly correlated [12] and can reliably be measured by LC-MS/MS [13][14][15][16]. The simultaneous quantitation of 24,25(OH) 2 D and 25(OH)D has been proposed as a dynamic measure of vitamin D metabolism that allows distinguishing CYP24A1 deficiency from vitamin D intoxication and granulomatous disease. However, the interpretation of 25(OH)D and 24,25(OH) 2 D results is still a matter of intensive debate. Previous studies have established reference intervals [17,18] and clinical cut-offs [19][20][21][22]. However, the close relationship between 25(OH)D and 24,25(OH) 2 D implies that a meaningful interpretation is only possible when both metabolites are considered together. This has led to the idea of a ratio between 24,25(OH) 2 D and 25(OH) D, also known as vitamin D metabolite ratio (VMR) [23]. Theoretically, a higher VMR indicates better supply with vitamin D so that excessive 25(OH)D is catabolized to 24,25(OH) 2 D. Several studies have investigated the clinical utility of VMR, but results are inconclusive [3,[24][25][26]. In addition, the VMR cannot be calculated when 24,25(OH) 2 D is below the limit of quantitation. When one measurand has a much lower concentration than the other, calculating the ratio between the two enhances the intrinsic measurement uncertainty. In this issue of Clinical Chemistry and Laboratory Medicine (CCLM) a study by Cavalier et al. has analyzed 24,25(OH) 2 D and 25(OH)D simultaneo...