On the isobaric space of 25‐hydroxyvitamin D in human serum: potential for interferences in liquid chromatography/tandem mass spectrometry, systematic errors and accuracy issues

Abstract

It was shown in this proof-of-concept study that DMS-MS has the potential to significantly decrease systematic errors, and thus improve accuracy of vitamin D measurements using LC/MS/MS.

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

“…TOF-MS experiments (Figure 1a and b) showed high abundances of isobaric compounds, some of which were very close to the m/z value of the precursor ion for 25(OH)D 3 (m/z 401.3414), similar to previous FTICR-MS results [6]. Several of these isobars were of an exogenous nature (Figure 1a), others originated from the serum matrix (Figure 1b).…”

“…The intensities of some of these isobars were much higher than the signals obtained for 25(OH)D 3 at relevant concentration levels. Although the instrumental resolving power of the QqTOF initially appeared to be sufficiently high, there was a small overlap of the main interference at m/z 401.2666 (C 21 H 33 N 6 O 2 + ; mass measurement accuracy, 1.5 ppm; the same interference was previously observed in the isobaric space of m/z 401 by FTICR analysis of serum samples [6]) from the tail-end of the peak into the 25(OH)D 3 signal. This effect caused isobaric cross-talk from the slightly skewed peaks, which then irreproducibly co-contributed to signal intensities of 25 + ion at m/z 401.3414 and no interference occurred [18].…”

“…We considered this comparison very important, as we previously encountered a significant co-eluting isobaric interference in the low resolution mass spectrometric (LRMS) selected reaction monitoring (SRM) analysis of 25(OH)D 3 that was introduced by the HPLC system [6]. This particular isobaric interference was impossible to resolve in the triple quadrupole CID spectra and required either differential ion mobility spectrometry or increased collision energy in the CID stage for removal to avoid systematic errors [6]. We were able to reduce and eventually remove the interference signal by systematic exchange of those pump parts that were exposed to the mobile phase flow.…”

“…Furthermore, endogenous isobars of 25(OH)D 3 must be separated using proper chromatographic separation, combined with the application of appropriate product ions in the selected reaction monitoring (SRM) mode of the QqQ instrument. As recently demonstrated [6], there is a large number of isobaric endogenous and exogenous compounds present during the LC-MS/MS analysis of 25(OH)D 3 . Furthermore, additional complications arise from isomeric contributions, where the C-3 epimer of 25(OH)D 3 presents a particular challenge [2].…”

“…Several experimental approaches have been developed to overcome some of these problems, including derivatization techniques to improve ionization efficiency [10][11][12], ion mobility spectrometry to remove isobars [6], isotope standards, and certified reference materials to improve intra-and interlaboratory precision and accuracy [2], and high resolution mass spectrometry (HRMS) [2]. Mass analyzers of medium/high mass resolving power such as time-of-flight (TOF), Orbitrap, and Fourier transform ion cyclotron resonance (FTICR) have not yet routinely entered clinical practice, even though their potential has been clearly recognized [13,14].…”