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Multiple analyte adduct formation in liquid chromatography-tandem mass spectrometry - Advantages and limitations in the analysis of biologically-related samples

https://doi.org/10.1016/j.jchromb.2018.03.027Get rights and content

Highlights

  • Multiple analyte adduct formation in LC-MS/MS analysis was investigated.

  • Signal compensation with isotopically labelled analytes was discussed.

  • γ‑Hydroxybutyrate was applied as model drug.

Abstract

Multiple analyte adduct formation was examined and discussed in the context of reproducible signal detection in liquid chromatography-tandem mass spectrometry applied in the analysis of biologically-related samples. Appropriate infusion solutions were prepared in H2O/methanol (3/97, v/v) with 1 mM sodium acetate and 10 mM acetic acid. An API 4000 QTrap tandem mass spectrometer was used for experiments performed in the negative scan mode (−Q1 MS) and the negative enhanced product ion mode (-EPI). γ‑Hydroxybutyrate and its deuterated form were used as model compounds to highlight both the complexity of adduct formation in popular mobile phases used and the effective signal compensation by the application of isotope-labelled analytes as internal standards.

Introduction

Analyte adduct formation became an interesting alternative in the analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The possibility to generate bigger ion fragments and real MS2/MS3 mass transitions for small molecules but also to improve method sensitivity/detection in some applications or even the mass transition specificity of problematic analytes were pointed out in the latest research performed especially in the negative electrospray ionisation (-ESI) [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]]. However, the experiments revealed also that a variety of parameters can influence the analyte adduct formation significantly and as a consequence the appropriate signal detection as well [2,3]. Therefore, it can be stated that without accurate control this process has to be seen as an important analytical pitfall, since it is a possible source of inaccuracy of LC-MS/MS methods [11].

Although, the influence of single adduct ions in quantitative analysis is well known and described, the interest in the process of multiple analyte adduct formation in popular buffer systems can be defined as limited [12]. Recently it was demonstrated that multiple adduct ions have a potential to be applied in a quantification strategy used for problematic small-molecule drugs under controlled analytical conditions [13]. However, the more analyte adducts are formed under given analytical conditions, the more unreliable are the analytical results produced by LC-MS/MS without appropriate compensation [11]. Therefore, the aim of this work was to point out the complexity of analyte adduct formation which has to be considered in popular buffer systems. Since multiple analyte adducts can also be seen as a problem associated with LC-MS/MS application and different methods published are run without appropriate internal standards, the knowledge about this process can be defined as important for a better understanding of possible pitfalls associated with the use of LC-MS/MS in separation science relevant to biology and biomedical research [14]. Therefore, for the purposes of this paper and the examination of multiple adduct formation a small drug analysed in different biological matrices and its deuterated form were applied in infusion solutions. On this basis an appropriate simulation and discussion of the relevance of this process in the analysis of biologically-related samples were possible.

Section snippets

Materials and methods

All experiments were performed with an API 4000 QTrap tandem mass spectrometer (AB Sciex Germany GmbH, Darmstadt, Germany) on the basis of electrospray ionisation performed in the negative mode (-ESI) and chemicals/solvents of analytical/LC-MS grade. The experiments were realised in the negative scan mode (-Q1 MS) and negative enhanced product ion mode (-EPI). Infusion solutions (10 μg/mL) were prepared in H2O/methanol (3/97, v/v) with 1 mM sodium acetate and 10 mM acetic acid from appropriate

Results and discussion

Single ion adducts but especially the process of multiple analyte adduct formation discussed, favoured under different conditions like the presence of sodium ions and acetic acid/acetate salt buffer in the mobile phase, can influence the LC-MS/MS analysis of biologically-related samples negatively. Sodium ions can originate from chemicals/solvents/glassware applied or even be a part of the mobile phase if sodium acetate is used. As a consequence, the formation of relevant amounts of sodium

Conclusions

Liquid chromatography-tandem mass spectrometry became a standard technique in the analysis performed in biologically-related samples. Modern analytical software developed by LC-MS/MS manufacturers and supporting these complex instruments makes the optimisation of detection parameters quite simple and possible without specialised knowledge due to automated compound optimisation algorithms. However, if different sources of inaccuracies together with the problem of multiple adduct formation

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