{'fraction_non_alphanumeric': 0.035238095238095235, 'fraction_numerical': 0.025238095238095237, 'mean_word_length': 5.524844720496894, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Candidate reference method for the quantification of circulating 25-hydroxyvitamin D3 by liquid chromatography-tandem mass spectrometry.

Quantification of 25-hydroxyvitamin D3 (25-hydroxycholecalciferol) in serum is the best-established indicator of vitamin D status (1). Vitamin D3 (cholecalciferol) is absorbed from the diet, and, given sufficient ultraviolet irradiation, nutritionally adequate amounts of vitamin D3 are formed in the skin from its precursor, 7-dehydrocholesterol. In the liver, vitamin D3 undergoes hydroxylation to 25-hydroxyvitamin D3, which is further metabolized in the kidney to form the active metabolite 1,25-dihydroxyvitamin D3. Vitamin D2 (ergocalciferol) is derived solely from plant sources; relevant serum concentrations of 25-hydroxyvitamin D2 are observed only after ingestion of vitamin D2 drug preparations, and biological equivalence to vitamin D3 has never been demonstrated conclusively.

The prevalence of hypovitaminosis D has been recognized as substantial (2)(3), even in regions with high sun exposure (4), contributing not only to osteoporosis (5)(6) but possibly to a loss of muscle strength in aging as well (7).

Various assays are used for the quantification of circulating 25-hydroxyvitamin D3 that incorporate either vitamin D-binding globulin or anti-vitamin D antibodies for analyte recognition (8)(9)(10). Fully automated tests have become available during recent years (11). Several HPLC methods with ultraviolet detection have been described as well (12)(13), but their routine use is limited by complex sample preparation requirements.

A key problem for the quantification of circulating 25-hydroxyvitamin D3 is the strong binding of the molecule to vitamin D-binding globulin. Precipitation of serum constituents by use of organic solvents or acids can lead to variable coprecipitation of the analyte. Release of 25-hydroxyvitamin D3 from its bonds to the binding protein is technically challenging in automated assays in particular. In light of these analytical problems, a reference method for the quantification of 25-hydroxyvitamin D …