Ultrasensitive Quantification of Serum Vitamin D Metabolites Using Selective Solid-Phase Extraction Coupled to Microflow Liquid Chromatography and Isotope-Dilution Mass Spectrometry

Abstract

The capacity for quantification of active metabolites of vitamin D (VitD) is highly valuable to evaluate the risks and therapies for numerous diseases such as multiple sclerosis. However, the extremely low circulating levels and poor detectability of some dihydroxyl metabolites such as the 1alpha,25-dihydroxy-VitD(3) constitute a daunting challenge. Based on the combination of a selective solid-phase extraction (SPE) and a microflow liquid chromatography tandem mass spectrometry (microLC-MS/MS), we developed an ultrasensitive method for the robust, selective, and accurate quantification of four key VitD metabolites, including 25-hydroxy-VitD(2), 25-hydroxy-VitD(3), 24(R),25-dihydroxy-VitD(3), and 1alpha,25-dihydroxy-VitD(3), in serum samples. A one-step derivatization was employed to improve the ionization efficiency of the metabolites. The SPE procedure was optimized so that the analytes were selectively extracted from serum, while the sample matrix was substantially simplified. By eliminating majority of undesirable compounds from the matrix, the selective SPE enabled a high sample loading volume on the microLC column without causing overcapacity of the microLC column and thus helped to achieve ultralow detect limits in serum. An on-column sample focusing approach was employed to prevent band-broadening, and a sufficient microLC separation was achieved to eliminate endogenous interferences and to minimize ion suppression effect. Detect limits of the four metabolites ranged from 0.5-1 pg/mL, and the linearity was excellent for all compounds. The method showed high quantitative accuracy (error < 13.8%) and precision (CV < 14.1%). For 1alpha,25-dihydroxy-VitD(3), a lower limit of quantification (LLOQ) of 5 pg/mL was validated. This high level of sensitivity, for the first time, enabled the robust and consistent LC/MS/MS-based analysis of the four metabolites in a large-scale clinical investigation. Serum samples from 281 multiple sclerosis patients and 22 healthy subjects were analyzed, and it was discovered that the levels of both 24(R),25-dihydroxy-VitD(3) and 1alpha,25-dihydroxy-VitD(3) were significantly lower in patients than healthy subjects (P < 0.05). This novel observation may imply that the incidence of multiple sclerosis is inversely associated with the levels of the two metabolites. Moreover, the method was highly robust and reproducible as evaluated extensively in the clinical analysis; therefore, it could serve as a more selective and accurate alternative to immunoassay for large-scale clinical studies.

Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25-Hydroxyvitamin D

“…The authors used adduct formation with ammonium to achieve a lower limit of quantification (LLOQ) of 48 pmol/L from 1 mL of serum (25 ) (27 ). The method exhibited accurate (error Ͻ13.8%) and precise (CV Ͻ14.1%) concentration data, with an LLOQ of 5 pg/mL using an ESI triple-quadrupole mass spectrometer.…”

Assessment of vitamin D status – a changing landscape

“…However, this method lacked sensitivity and thus was not feasible for use in clinical practice. The first LC-MS/MS method with clinically useful limits of quantification (LOQ) was described almost 20 years later by Duan et al [170], with validated LOQs for 1,25-(OH) 2 D 3 and 24,25-(OH) 2 D 3 of 5 pg/mL and 50 pg/mL, respectively.…”

“…Another potential interference is the occurrence of the epimeric forms 1α,25-(OH) 2 -3-epi-D 3 and 24,25-(OH) 2 -3-epi-D 3 [174], which need to be separated by chromatography. An extensive list of potentially interfering compounds can be consulted in the Supplementary Information of the work of Duan et al [170]. Recently, it has been observed that some lubricants used in LC-MS instruments can also hamper an accurate quantification of some monohydroxylated compounds including 25-OHD as they can share the same transitions.…”

“…Two-dimensional UHPLC (2D-LC) with column switching systems [182,183] has also been tested, but the sophisticated configuration of a 2D-LC is rather difficult to set up. Another possibility is to use micro-flow UHPLC [170] with flow rates of 1-10 μL/min. However, such slow flow rates tend to be unstable.…”

“…The vast majority of sample pre-treatments described in the literature are based on protein precipitation, followed by solid phase extraction (SPE) with OASIS HLB cartridges and derivatization with a Cookson-type reagent [170,175,185,188]. Liquid-liquid extraction (LLE) is another option that is significantly cheaper due to the low cost of solvents compared to SPE cartridges.…”