Steroid hormones analysis with surface-assisted laser desorption/ionization mass spectrometry using catechin-modified titanium dioxide nanoparticles

Abstract

This paper describes the application of catechin-modified titanium dioxide nanoparticles (TiO₂ NPs) as matrices to analyze four steroid hormones by surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The catechin-modified TiO₂ NPs have high absorbance at 337 nm and are effective SALDI matrices when using a nitrogen laser. Four test steroid hormones—cortisone, hydrocortisone, progesterone, and testosterone—were directly analyzed by SALDI-MS. The limits of detection at a signal-to-noise ratio of 3 for cortisone, hydrocortisone, progesterone, and testosterone were 1.62, 0.70, 0.66, and 0.23 μM, respectively. This approach provides good quantitative linearity for the four analytes (R² > 0.986) with good reproducibility (the shot-to-shot and batch-to-batch variations for the four analytes were less than 10% and 15%, respectively). We validated the practicality of this approach—considering its advantages in sensitivity, repeatability, rapidity, and simplicity—through the analysis of testosterone in a urine sample.

Introduction

Steroids are compounds that contain cyclopental[a]phenalthrene ring systems. Substitutions in this ring system form diverse classes of steroids that have distinct physiological actions that serve important biological functions such as producing hormones during chemical signaling (e.g., testosterone) and forming structural compounds in cell membranes (e.g., cholesterol) [1]. Steroid derivatives are also used widely as drugs for the treatment of a variety of disorders, as well as substances of abuse (e.g., anabolic steroids) in sports medicine [1]. These compounds promote the development of secondary male sexual characteristics (androgenic effects) and accelerate muscle growth (anabolic effects). The determination of testosterone levels in body fluids such as urine and plasma are of great importance for the medical diagnosis of hirsutism, polycystic ovary disease, and virilization [1], [2].

Since it was first introduced by Karas et al. in 1988, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a powerful analytical tool for bioanalysis [3], [4]. As a result of rapid energy transfer from UV-absorbing matrices, analytes undergo soft and efficient desorption/ionization with a minimum degree of fragmentation. Although MALDI-MS is successfully used for the analyses of a variety of molecules, especially peptides and proteins, it has not been employed extensively for the characterization of low-molecular weight compounds (<500 Da) [5]. This is partly because of the presence of a variety of abundant matrix-related ions in the low-mass region of the MS spectra, and partly because the inhomogeneous co-crystallization of analytes with traditional organic matrices such as 2,5-dihydroxybenzoic acid and sinapinic acid usually leads to the existence of “sweet spots” on the sample probe, resulting in quantitative errors and irreproducibility. These disadvantages have hindered efforts to utilize the full power of MALDI-MS for the high-throughput analyses of drugs and their metabolites in complex mixtures containing high concentrations of salts. To overcome these problems, surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has been implemented for eliminating matrix-ion interferences and for improving sample homogeneity. Useful SALDI matrices include gold nanoparticles (NPs) [6], [7], [8], silver NPs [9], [10], [11], silicon oxide NPs [12], titanium dioxide (TiO₂) NPs [13], [14], [15], [16], zinc oxide NPs [17] and nanowire [18], zinc sulfide NPs [19], zinc selenium quantum dots [20], iron (III) oxide NPs [21], [22], platinum NPs [23] and nanoflowers [24], [25], and HgTe nanostructures [26].

Although SALDI-MS was developed nearly two decades ago, it was not as widely used as MALDI-MS for the analysis of biomolecules, mainly because its accessible mass range is limited to ca. 20 kDa and its mass resolution is limited to around a few hundred or less [24]. However, owing to the recent increased interest in nanoscience, SALDI-MS has become a popular technique. Monodisperse nanomaterials have been prepared as efficient SALDI-MS matrices through several synthetic routes. Compared with conventional micro-nanosized particles and/or organic matrices, monodisperse nanomaterials offer several advantages including simple and facile surface modification, large surface-area-to-volume ratios, flexibility of sample deposition under specific conditions, and high extinction coefficients in the UV range. Several bioconjugated nanomaterials such as gold [8] and titanium dioxide [13] serve not only as efficient matrices but also as effective media for the required concentration of analytes and for minimizing sample-matrix interferences.

In the UV region, TiO₂ NPs exhibit strong absorption characteristics (band gap of bulk anatase TiO₂: 3.2 eV) that mainly depend on their size, shape, and composition. TiO₂ NPs are usually prepared through sol–gel reactions and by carefully controlling parameters such as the reaction solution pH, water/alkoxide molar ratios, reaction temperature, and the nature of the solvents and additives—the size and shape of TiO₂ NPs can be fine tuned. In this study, catechin-modified TiO₂ NPs are used as matrices for the analysis of steroid hormones and are analyzed by SALDI-MS, which provides advantages such as easy and quick sample preparation and high repeatability.