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

Abstract

<p>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 <i>m/z</i>. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a two-minute 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.</p>

Portable mass spectrometry system: instrumentation, applications, and path to ‘omics analysis

“…For example, the Microsaic 3500 MiD instrument has been coupled with HPLC, which demonstrated sensitivity that is comparable to that of a traditional bulk MS system in the selected ion monitoring (SIM) mode, enabling the detection of known components at trace levels (ppm) [83,84]. Clinical assays for measuring vitamin D2/D3 have been demonstrated using a portable MS system (Advion Expression CMS) in conjunction with HPLC separation [85].…”

Why tandem mass spectrometry for trace analysis: Concepts of tandem analytical techniques

“…Although one can identify a number of options for additional stages of analysis, perhaps the most popular currently is to add ion mobility to separate mixture components after ionization and before MS/MS, as will be discussed in an example below. 18…”

“…ESI can populate both the closed and open conformations for 25‐OH‐D2 and 25‐OH‐D3, but, for the epimers, the open conformation is not energetically accessible, and the second drift peak is therefore not observed. Thus, ion mobility makes it possible to detect and quantify 25‐OH‐D2 and 25‐OH‐D3 using this second, open conformation peak, without any interference from the epimers 18 …”

“…As mentioned earlier, these can be offline, in front of the LC/MS/MS setup (sample clean‐up with a solid‐phase extraction, for example); but they can be more flexible and more elegant when they are performed online, as part of the instrument. Although one can identify a number of options for additional stages of analysis, perhaps the most popular currently is to add ion mobility to separate mixture components after ionization and before MS/MS, as will be discussed in an example below 18 …”

“…Separation of epimers by LC is challenging at best, and MS and MS/MS generally do not differentiate epimers. In this case, we chose short‐column LC‐drift tube ion mobility‐MS for the rapid quantitation of 25‐hydroxyvitamin D2 and D3 in human serum extracts 18 . Steroids, including secosteroids such as vitamin D, generally are not protonated under electrospray conditions, so the most intense ion is typically the [M + Na] + ion, formed by cationization with trace levels of sodium ions in the sample.…”