Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/Drift Tube Ion Mobility-Mass Spectrometry

Abstract

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD), only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a 2 min LC/IM-MS method. 25-Hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.

Lipid analysis by ion mobility spectrometry combined with mass spectrometry: A brief update with a perspective on applications in the clinical laboratory

“…Application of TWIMS without LC separation was reported to partially separate several steroid isomer pairs (β-estradiol & α-estradiol, androsterone & trans -androsterone, and testosterone & epitestosterone) only after derivatization with p -toluenesulfonyl isocyanate; the non-derivatized forms not being separable by ion mobility [48] . DTIMS has been demonstrated to be able to separate underivatized 25-hydroxyvitamin D 3 from the interfering epimer 3-epi-25-hydroxyvitamin D 3 in serum without the need for increased LC separation time [49] ; a potentially useful application in the clinical laboratory for removing a possible cause of overestimating total 25-hydroxyvitamin D 3 concentration without increasing sample-to-sample analysis time.…”

Mass Spectrometry-Based Cellular Metabolomics: Current Approaches, Applications, and Future Directions

“…147 The integration of IMS into traditional LC-MS analyses also aids in the separation of isomeric metabolites, which would not be otherwise resolved and be potentially over quantified, as demonstrated by the structural isomers of vitamin D metabolites, which can be separated and accurately quantified by IMS. 148 Without IMS separation, an extensively long chromatographic separation is necessary for the elucidation of isomers, as MS/MS fragmentation patterns of isomers are often similar. 149 Therefore, the integration of IMS in LC-MS workflows can significantly impact the quality of and coverage of metabolites in complex biological samples.…”

“…DTIMS, TWIMS, and TIMS are capable of providing rotationally averaged collision cross section (CCS) values for individual metabolites, although TWIMS and TIMS require extensive calibration with structurally similar calibrant ions to obtain accurate, reproducible CCS measurements. , The benefit of FAIMS, also referred to as differential mobility spectrometry (DMS), for metabolomics studies is its ability to filter out isobaric and often isomeric interferences between the ionization source and MS inlet, further removing chemical noise introduced during sample preparation workflows . The integration of IMS into traditional LC-MS analyses also aids in the separation of isomeric metabolites, which would not be otherwise resolved and be potentially over quantified, as demonstrated by the structural isomers of vitamin D metabolites, which can be separated and accurately quantified by IMS . Without IMS separation, an extensively long chromatographic separation is necessary for the elucidation of isomers, as MS/MS fragmentation patterns of isomers are often similar .…”

Decoding the Metabolome and Lipidome of Child Malnutrition by Mass Spectrometric Techniques: Present Status and Future Perspectives

“…However, elucidating some lipid isomers in complex mixtures by LC-MS is still problematic. Recently, LC coupled with both ion mobility and mass spectrometry (LC-IMS-MS) has emerged as an orthogonal and efficient technique to identify isomeric/isobaric changes of lipids occurring under complex mixtures. , IMS is a gas-phase separation approach that utilized the gas-phase structure to separate ions based on their mobility though an electric field. Therefore, IMS could be a potentially important tool coupled with LC-MS to discern lipid profiles in malnutrition research when LC and MS alone are insufficient.…”