Quantitative MALDI-MS/MS assay for serum cortisol through charged derivatization

doi:10.1016/j.jpba.2019.112912

Journal of Pharmaceutical and Biomedical Analysis

Volume 178, 30 January 2020, 112912

https://doi.org/10.1016/j.jpba.2019.112912 Get rights and content

Highlights

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A quantitative MALDI-MS/MS method was developed for the serum cortisol.
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Charged derivatization was employed for increasing the sensitivity.
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Reproducible (intra- and inter-assay RSD ≤ 7.4%) quantification using a 20-μL sample.
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The measured values by MALDI-MS/MS agreed well with those by LC/ESI-MS/MS or ECLIA.
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The method was used for the analysis of peripheral and adrenal serum samples.

Abstract

Cortisol (CRT), the main glucocorticoid in humans, plays a crucial role in many physiological processes, therefore, the measurement of its serum level is of great clinical significance. Matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS) might be an effective approach for the quantification of CRT in serum due to its attractive properties, i.e., high specificity, ease of use, ruggedness and rapid analysis. In this study, we developed a method to quantify the serum CRT by MALDI-MS/MS. This method employed the derivatization using a Girard-type reagent, 1-(hydrazinocarbonylmethyl)isoquinolinium chloride, which compensated for the lack of sensitivity. This method enabled the reproducible quantification of the serum CRT using a 20-μL sample (intra- and inter-assay relative standard deviations, 7.4% or lower), and the measurable range was 25–500 ng/mL. The serum CRT concentrations determined by the newly-developed MALDI-MS/MS method agreed well with those by liquid chromatography/electrospray ionization-MS/MS or electrochemiluminescence immunoassay. The MALDI-MS/MS method was used for the analysis of peripheral venous serum samples of healthy subjects and adrenal venous serum samples of patients with primary aldosteronism, and satisfactory results were obtained.

Graphical abstract

Introduction

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) has been extensively used to analyze biopolymers such as proteins, peptides and oligosaccharides. The major attractive properties of MALDI-MS are its high specificity, ease of use, ruggedness and rapid analysis. A variety of small biomolecules, including steroids, can also be measured by MALDI-MS [1,2]. There are some successful applications of MALDI-MS or -tandem MS (MS/MS) for the detection of trace steroids in tissues, including MALDI-MS imaging [[3], [4], [5], [6]]. However, the accurate quantitative analysis of steroids by MALDI-MS is often hampered by matrix-derived interference signals and by the poor reproducibility of the signal intensities. Another problem is the fact that most steroids are not effectively ionized during MALDI due to the absence of acidic or basic functional groups, which compromises their detection sensitivity.

Cortisol (CRT), the main glucocorticoid in humans, plays a crucial role in many physiological processes, including metabolism, immune response and anti-inflammation. The abnormal secretion of CRT leads to life-threatening conditions such as Cushing’s disease [7,8]. Therefore, measurement of the serum/plasma CRT level is of great clinical significance. The reported mean serum/plasma CRT concentrations in the morning are in the range of 100 to 200 ng/mL for healthy subjects [[9], [10], [11]], while several fold higher CRT concentrations are observed in patients with Cushing’s syndrome or Cushing’s disease [10,12]. The serum/plasma CRT has been conventionally measured by immunoassay in clinical practice [7,13]. However, there is a well-known inaccuracy in the immunoassay due to interference from other endogenous and exogenous steroids that are structurally similar to CRT [13]. In some immunoassays, the measured serum/plasma CRT concentrations vary according to the concentrations of the CRT binding proteins, such as corticosteroid-binding globulin [13]. The MS-based method has a high specificity and is a promising alternative to immunoassay for the CRT measurement. Although the initial cost of the MS-based testing, i.e., cost for purchasing an instrument, is very high, the running cost of the MS-based testing is usually lower than the cost of the immunoassay reagents [14]. Many methods using liquid chromatography(LC)/electrospray ionization (ESI)-MS/MS have been developed for the quantification of CRT in serum/plasma [7,8], but to the best of our knowledge, there is no MALDI-MS(/MS) method for this specific purpose. Considering the previously mentioned attractive properties of MALDI-MS(/MS), it might be an effective approach for the accurate quantification of CRT in serum/plasma.

For the two major issues related to the quantification of steroids by MALDI-MS, i.e., low ionization efficiency of the steroids and the matrix-derived interference signals, derivatization can be a solution. It is expected that derivatization enhances the MALDI efficiency of steroids by tagging easily-ionizable moieties and facilitates fragmentation during MS/MS, which lead to an increasing sensitivity and specificity. Several papers have described the successful applications of derivatization for the MALDI-MS(/MS) analysis of steroids [[3], [4], [5], [6],15,16]. For the MALDI-MS/MS of CRT, Girard reagent T and its analogues, which are described as the Girard-type reagents in this study, would be promising derivatization reagents (Fig. 1a). Girard-type reagents rapidly react with a 3-oxo-4-ene-steroid, such as CRT, and a permanently-charged moiety is tagged with the steroid to increase the MALDI efficiency. Furthermore, the resulting derivative is fragmented to produce a characteristic ion during the MS/MS (Fig. 1b) [5]

Based on this background information, the objective of this study was to investigate whether MALDI-MS/MS has the potential for implementation of the quantitative CRT assay. In this study, we developed and validated a method for the quantification of CRT in human serum by MALDI-MS/MS combined with the Girard-type reagent derivatization. The applicability of this method was evaluated by the analysis of peripheral venous serum samples of healthy subjects and adrenal venous serum samples of patients with primary aldosteronism. Based on all the results in this study, we considered the strengths and weaknesses of MALDI-MS/MS for the quantitative analysis of steroids.

Section snippets

Materials and chemicals

CRT and [9,11,12,12-²H₄]-CRT (internal standard, IS) were obtained from Tokyo Chemicals Industry (Tokyo, Japan) and Cambridge Isotope Laboratories (Tewksbury, MA, USA), respectively. Standard solutions of CRT or IS were prepared by dissolving a precisely-weighed quantity of CRT or IS in ethanol and serial dilutions with ethanol. Cortisone, (hydrazinocarbonylmethyl)trimethylammonium chloride (Girard reagent T, GT) and 1-(hydrazinocarbonylmethyl)pyridinium chloride (Girard reagent P, GP) were

MALDI-MS/MS of intact and derivatized CRT

When 5 ng of intact CRT (equivalent to 5 μg/mL of the serum concentration when a 20-μL sample was used) was loaded on the plate and analyzed by MALDI-MS, no characteristic ion was detected; the MALDI efficiency of CRT was very poor. This result prompted us to employ the Girard-type reagent derivatization for increasing the sensitivity for the MALDI-MS(/MS) quantification of the serum CRT. In this study, four Girard-type reagents, two (GT and GP) of which were commercially available and the

Conclusion

In this study, we developed and validated a MALDI-MS/MS method for the quantification of CRT in human serum. The method had a satisfactory precision and provided serum CRT values which well agreed with those by LC/ESI-MS/MS or ECLIA. To the best of our knowledge, this is the first validated quantitative MALDI-MS/MS assay for the serum CRT.

This study demonstrated that MALDI-MS/MS is a working methodology for the quantification of a steroid when a stable isotope dilution method and adequate

Declaration of Competing Interest

The authors have declared no conflict of interest.

Acknowledgments

This study was supported in part by JSPS KAKENHI Grant Number 17K08250.

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View full text

Quantitative MALDI-MS/MS assay for serum cortisol through charged derivatization

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials and chemicals

MALDI-MS/MS of intact and derivatized CRT

Conclusion

Declaration of Competing Interest

Acknowledgments

Biochem. Pharmacol.

Best Pract. Res. Clin. Endocrinol. Metab.

J. Steroid Biochem. Mol. Biol.

Metabolism

Clin. Chim. Acta

Biochem. Biophys. Res. Commun.

J. Steroid Biochem. Mol. Biol.

J. Chromatogr. B

J. Chromatogr. B

MALDI matrices for low molecular weight compounds: an endless story?

Anal. Bioanal. Chem.

Mass spectrometry-based shotgun lipidomics – a critical review from the technical point of view

Anal. Bioanal. Chem.

Microscopic visualization of testosterone in mouse testis by use of imaging mass spectrometry

Anal. Bioanal. Chem.

Spatial localization and quantitation of androgens in mouse testis by mass spectrometry imaging

Anal. Chem.

Aldosterone and 18-oxocortisol coaccumulation in aldosterone-producing lesions

Hypertension

Simultaneous determination of cortisol and cortisone from human serum by liquid chromatography-tandem mass spectrometry

J. Anal. Methods Chem.