Introduction

Sex hormones and many other steroids are essential biomolecules in human and animal organisms and have strong biological activities at low concentrations. They regulate maturation and reproduction and are involved in bone metabolism affecting osteogenesis and bone mineral density. The etiologic importance is also understood, as steroids play a fundamental role in the formation of osteoporosis, prostate cancer, and breast cancer, with a positive correlation between the circulating plasma estrogen level and the risk of tumor and metastases [1–4]. The action is mediated not only by intracellular steroid receptors but also by membrane-associated steroid receptors, cross-communicating with growth factors and growth factor receptors [5].

Because of their occurrence at very low, nanomolar or even picomolar concentrations in tissues or body fluids and because of their structural similarity [6], precise and sensitive test methods are required to provide exact information. Human samples analyzed are generally collected from blood, urine, or cerebrospinal fluid [7–11].

A possible and commonly used determination technique is the radioimmunoassay [12, 13], but the method is not reliable in all cases as reported by Dorgan et al. [2], Stanczky et al. [14], and Rauh et al. [15], especially when the samples to be examined are collected from infants. More recently, gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS have been used [16–20] to reveal the steroid composition of complex biological samples to gain further information on steroid content and metabolism [19]. It is important to note, however, that neutral steroids lack thermostability or volatility, so direct GC analysis is not possible. Direct MS analysis, in turn, results in low sensitivity [21, 22], generated by the absence of acidic or basic groups in the structure of steroids [23]. The key to this problem is to convert the neutral steroid compounds into more volatile and stable products, also providing them with chemical groups allowing more effective ionization [24, 25]. There is a wide range of different derivatizing reagents in use, and several protocols have been developed specifically for steroids with distinct types of chemical groups [26–28]. For example, widely used reactants are Girard-T—([carboxymethyl]trimethylammonium chloride hydrazide) [29]—and Girard-P—1-([carboxymethyl]pyridinium chloride hydrazide)—reagents [30]. The reaction results in a specific conversion to Girard-T or Girard-P hydrazone derivatives of oxosteroids containing a positively charged nitrogen atom, ensuring more effective ionization [31]. Trimethylsilylation of alcoholic hydroxy functions or alkylation of phenol functions with pentafluorobenzylbromide also allows improved chromatographic behavior and more sensitive spectrometric detection [32]. For MALDI application, the use of derivatizing reagents has a further advantage, as steroid compounds have their molecular masses in the same ranges as the commonly used matrix substances. Through addition of the mass of the derivatizing agent, the ion in question is moved to a different mass range where common matrix peaks do not interfere. Depending on the objective of the research and the complexity of the matrices analyzed, the use of other catalysts or the combination of derivatizing reagents is conceivable to obtain better results. At the same time, derivatization comes with the drawback that a universal reagent is not yet available and not all steroid compounds form a single derivatized product [33, 34].

The analytical applications of fullerenes cover a wide spectrum owing to their strong optical absorption and their ability to interact with several forms of chemical groups. Therefore, fullerenes can be used as LC stationary phases [35, 36], solid-phase-extraction sorbent materials [37, 38] for analyte preconcentration, and as matrix material for laser desorption/ionization MS. Fullerenes, derivatized fullerenes, and carbon nanotubes are all reported to be efficient matrices. Gholipour et. al. [39] successfully analyzed underivatized plant carbohydrates with carbon nanotubes and found that lower laser intensity was needed to acquire mass spectra compared with conventional 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid matrices. Water-soluble fullerene derivatives can precipitate amino acids, peptides, and proteins from aqueous solutions and also make the addition of any further matrix substances unnecessary [40]. The only disadvantage of the use of fullerenes would be the isobaric interferences between matrix and analyte peaks [41], but steroid compounds are represented in far more different mass ranges than the commonly used fullerene material. In this paper we describe a sensitive MALDI-TOF-MS-based method to analyze natural underivatized steroids. C70 fullerene was picked as the matrix compound to achieve efficient ionization as no derivatization was carried out. The other argument for C70 was its molecular mass of 840.75 Da, which does not interfere with the peaks corresponding to the steroid analyte ions. The method was tested on five general sex steroid hormones (estrone, estradiol, estriol, testosterone, and progesterone), on synthetic steroid compounds [42, 43], and on a urine sample collected from a pregnant individual.

Materials and methods

Chemicals and standard preparation

Estrone [1,3,5(10)-estratrien-3-ol-17-one], β-estradiol [3,17 β,-dihydroxy-1,3,5(10)-estratriene], estriol [1,3,5(10)-estratriene-3,16 α,17β-triol], progesterone (4-pregnene-3,20-dione), and testosterone (17 β-hydroxy-3-oxo-4-androstene) (Sigma-Aldrich, Budapest, Hungary) were used as analytical steroid standards (Fig. 1). The reference solutions were prepared by dissolving 0.1 mg of the steroid hormones in 1.00 mL of methanol (Scharlau Chemie, Barcelona, Spain). Following the complete dissolution, 0.2 mL of Milli-Q water was added to the solution. The C70 fullerene (Gold grade) was purchased from Hoechst (Frankfurt, Germany). The fullerene matrix was prepared as a saturated solution in toluene; the excess was removed by centrifugation. The solutions of the synthetic steroids were prepared by dissolving 0.1 mg of the respective compound in 1.00 mL of dichloromethane (LiChrosolv, Merck, Darmstadt, Germany).

Fig. 1
figure 1

Chemical structures of the five sex steroid hormones: a estrone, b β-estradiol, c estriol, d progesterone, and e testosterone

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Urine sample

The urine sample was collected from a 29-year-old pregnant individual in the third trimester. Hormone extraction was carried out in the following way. First, the sample was centrifuged at 4,000 rpm at 4 C for 20 min. One milliliter of the supernatant was then extracted with 1 mL of dichloromethane three times. The solvent was evaporated under a vacuum and the residue was redissolved in 10 µL of acetonitrile/water (9:1) v/v.

MALDI-TOF mass spectrometry

A thin layer of C70 fullerene crystals was formed on the surface of the target plate (MTP 384 target plate ground steel, Bruker Daltonics, Bremen, Germany) by placing the saturated toluene C70 solution on the corresponding spots. After the evaporation of toluene, 1 µL of each analyte solution was dropped on top of the fullerene crystal layer. The mass spectrometer used was an Autoflex II TOF/TOF (Bruker Daltonics, Bremen, Germany) operated in reflector mode for MALDI-TOF with an automated mode using the FlexControl 2.4 software program. An accelerating voltage of 20.0 kV was used for analysis. The instrument uses a 337 nm pulsed nitrogen laser (model MNL-205MC, LTB Lasertechnik Berlin, Berlin, Germany). External calibration was performed using the monoisotopic quasimolecular and dimer ion peaks of α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid matrices. Hormone masses were acquired with a range of m/z 200 to m/z 500. Each spectrum was produced by accumulating data from 500 consecutive laser shots. The Bruker FlexControl 2.4 software program was used to control the instrument and the Bruker FlexAnalysis 2.4 software program was used for evaluation of spectra.

Results and discussion

MALDI-TOF-MS detection of sex steroid compounds

Nonderivatized, neutral steroids show very low MALDI efficiency when common matrix materials such as α-cyano-4-hydroxycinnamic acid, sinapinic acid, and 2,5-dihydroxybenzoic acid are used. In contrast, the ionization of estrogen and androgen hormones was effective using C60 and especially C70 fullerene as matrices. The mass-spectrometric conditions were optimized using standard solutions of five basic sex steroid hormones, estrone, β-estradiol, estriol, progesterone, and testosterone (Fig. 1). Monoisotopic masses for the respective compounds are summarized in Table 1. All compounds were tested in both positive and negative ionization modes, and different results were obtained. Estrone only provided a significant peak for its molecular ion in the negative ionization mode as in the positive ionization mode the intensity was hardly over the 3 times the noise ratio for the 0.1 mg/mL standard. β-Estradiol and estriol showed more effective ionization in the positive ionization mode than estrone, but it was less than that observed in the negative ionization mode. Progesterone and testosterone, however, behaved the opposite way; ionization was more effective in the positive ionization mode, whereas in the negative ionization mode it was again just above the 3 times noise ratio (data are not shown). Mass spectra of the five sex steroids were finally collected in negative ionization mode for estrone, β-estradiol, and estriol, and in positive ionization mode for progesterone and testosterone (Fig. 2). Estrone gave a prominent peak at m/z 269.16, which corresponds to the monoisotopic peak of the negatively charged molecule ion [M–H]−, and estriol at m/z 287.17 [M–H]− formed the same way as estrone and as described above. β-Estradiol was available as β-estradiol hemihydrate standard. The peak at m/z 271.18 corresponds to the monoisotopic peak of the negatively charged molecular ion after the loss of the hemihydrate unit. Progesterone and testosterone were both detected as positively charged quasimolecular ions [M+H]+ at m/z 315.22 and at m/z 289.21, respectively.

Table 1 The monoisotopic masses and the limits of detection for the steroid hormones investigated
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Fig. 2
figure 2

Mass spectra of the five sex steroid hormones. a estrone, b β-estradiol, c estriol, d progesterone, and e testosterone. Estrone, β-estradiol, and estriol were detected in negative ionization mode, whereas progesterone and testosterone were detected in positive ionization mode

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The limit of detection for each steroid was defined as the amount of the steroid standard giving a peak height 5 times the noise level. The results as amount in nanograms on plate and amount in picomoles on plate are summarized in Table 1.

Detection of synthetic steroid compounds

The method was used for the determination of two synthetic compounds: a steroidal secohydrazone named F-506 (Fig. 3a) and the heterocyclic estrone derivative Ke-47F (Fig. 3b). The latter compound contains two carbonyl functions (at C-17 and C-16a); therefore positive ionization mode was sufficient, but contrary to the previous results, a positively charged molecular ion formed at m/z 562.43 [M]+ (Fig. 4). Two molecular fragments were also identified as a benzyl cation at m/z 91.14 and another fragment at m/z 304.16 (Fig. 3). The secohydrazone, however, is more effectively ionized in the negative ionization mode. The molecule was detected at m/z 508.68 [M–H]− where a proton is lost from the hydrazone nitrogen. Fragments identified include the one at m/z 91.14 discussed above and the most intense one at m/z 137.18, representing the hydrazone group [27].

Fig. 3
figure 3

Chemical structures of a F-506 (monoisotopic mass 509.68 Da) and b KE-47F (monoisotopic mass 562.43 Da) synthetic steroid compounds with structural information on fragmentation

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Fig. 4
figure 4

Mass spectra of a F-506 and b KE-47F synthetic steroid compounds. The spectrum of F-506 was collected in negative ionization mode, whereas the spectrum of KE-47F was collected in positive ionization mode

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Detection of estriol, progesterone, and estradiol during pregnancy

Of the four female sex steroid hormones analyzed, estriol, produced by the fetoplacental unit, progesterone, and estradiol, synthesized by the placenta, are thought to be present in urine and plasma throughout pregnancy [44–46]. After a liquid-liquid extraction step all compounds were detected in the urine sample collected from a 29-year-old pregnant individual (Fig. 5). Detection was performed in both negative and positive ionization modes. In urine, steroids are mostly found in a conjugated form to facilitate urinary excretion [47], but free forms are also present, though in a low concentration. This conjugation reaction is commonly achieved with glucuronide or sulfate units.

Fig. 5
figure 5

Matrix-assisted laser desorption/ionization time-of-flight spectra of the urine sample collected from a pregnant individual. a In negative ionization mode, free estriol at m/z 287.17 and estradiol sulfate at m/z 351.13. b In positive ionization mode, free progesterone at m/z 315.22

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In negative ionization mode (Fig. 5a) no conjugates of estriol were detected; however, free estriol gave a prominent peak at m/z 287.17. Estradiol was found at m/z 351.13 as a sulfate conjugate. In positive ionization mode (Fig. 5b), progesterone was detected in an unconjugated, free form at m/z 315.22.

Conclusion

In this study a rapid, high-throughput, sensitive MALDI-TOF-MS method has been described using C70 fullerene as a matrix compound. High sensitivity (limit of detection proved to be 38–74 pmol) was achieved with fullerene as the matrix compound instead of using common derivatization procedures. The application of the method was extended to the analyses of synthetic steroid compounds and biological samples too. On the basis of our preliminary results, the quantitative analysis of the steroid hormones and their metabolites may also be possible using our technique, but further investigations are needed using stable-isotope-labeled compounds as internal standards.