The concentration of steroid hormones in biological fluids of humans is an important indicator of the state of their health. If there is a change in hormone concentrations, one can assume the development of various diseases; therefore, the determination of steroid hormones is of important diagnostic value.

Steroid hormones are traditionally determined in human urine and plasma. In analyzing urine, average concentrations of steroid hormones are obtained over several hours or one day, and the composition of plasma reflects the concentrations of these compounds in real time. A disadvantage of plasma analysis is the invasiveness of sampling and the presence of pain; therefore, saliva is now increasingly considered as an alternative matrix for the determination of steroid hormones. Its main advantages are non-invasiveness [1] and the simplicity of the sampling procedure, the stability of samples at room temperature, and no need in involving qualified personnel for sampling [2].

Studies conducted to date have shown a possibility of using saliva to determine steroid hormones. Thus, a change in the concentration of cortisol in saliva makes it possible to diagnose Cushing’s syndrome [3], the determination of progesterone makes it possible to determine the follicular and luteal phases of the cycle in women [2], and a change in testosterone levels may indicate the development of androgen-dependent diseases in men (androgen deficiency, hypogonadism) [2, 4] and in women (hirsutism, polycystic ovaries) [3, 5].

A possibility of using saliva as an alternative matrix is due to high correlations between the concentrations of a number of steroid hormones in plasma and saliva [3, 6], which is associated with the mechanism of the entry of steroid hormones into human saliva. It was found [7] that unconjugated steroid hormones enter saliva by diffusion through the cells of the salivary glands due to their lipophilic nature, and their concentration does not depend on the rate of saliva secretion, and, therefore, may reflect the concentration of free (not bound to proteins) steroids in plasma [2, 3, 8–10].

The main disadvantage of saliva as a test sample is the need in using highly sensitive methods for the determination of these compounds, because their concentrations in saliva are much lower than those in plasma [11]. The simplest method for analyzing saliva is enzyme immunoassay; however, it is not sufficiently selective, which can lead to overestimated results, especially at low concentration levels. In addition, this method makes it possible to determine only one indicator per analysis, which hinders its application to determining the steroid profile. An alternative way is to use chromatographic methods with mass spectrometric detection, which possess high sensitivity and selectivity, especially with tandem mass spectrometric detection in the multiple reaction monitoring (MRM) mode.

Taking into account the extremely low concentrations of steroid hormones in human saliva, their reliable determination requires preconcentration. This is most often attained using liquid–liquid extraction [3]. At that, most studies are aimed at determining a limited list of compounds [3, 4, 8, 10, 12, 13] and do not provide a complete human steroid profile, which is most effective in diagnosing diseases. The simultaneous determination of steroid hormones of different classes provides more accurate information about the hormonal status compared to a single indicator [14].

The aim of this work was to develop a unified procedure for the determination of steroid hormones of various classes (androgens, progestins, glucocorticoids) in human saliva by ultrahigh performance liquid chromatography with tandem mass spectrometry detection (UHPLC–MS/MS).

EXPERIMENTAL

Materials and methods. Standard samples of testosterone, cortisone, hydrocortisone (cortisol), corticosterone, progesterone, 11α-hydroxyprogesterone (Scheme 1) and methyltestosterone (internal standard) were purchased from Sigma–Aldrich (United States).

Scheme 1 . Structural formulas of the compounds to be determined.

Methyl tert-butyl ether (for HPLC–IR-UV, 99.9%) and methanol (for HPLC) were purchased from PanReac (Spain) and J.T. Baker (United Kingdom), respectively. Dichloromethane, trichloromethane, and carbon tetrachloride, ethyl acetate, hexane (99%) (EKOS-1, Russia), and also potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium tetrahydroborate, and sodium hydroxide (99%) were used to prepare buffer solutions (Vekton, Russia).

Instrumentation. For UHPLC–MS/MS analysis, we used a system consisting of a Dionex Ultimate-3000 ultra-high performance liquid chromatograph and a Thermo TSQ Access Max triple quadrupole mass spectrometer (San-Jose, United States). The analytes were separated on a Phenomenex Kinetex C18 analytical column (100 × 2.1 mm, 1.7 μm) equipped with an appropriate guard column in the gradient elution mode at a thermostat temperature of 40°C (Table 1).

Table 1. Conditions for the gradient elution of analytes (mobile phase flow rate of 0.4 mL/min)
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The studied compounds were ionized in a heated electrospray ionization source in the positive ion detection mode (Table 2). The analytes were detected in the multiple reaction monitoring mode using the collision-induced dissociation of the precursor ion (collision gas argon, pressure 1.5 mTorr) and the determination of product ions (Table 3). The conditions for detection in the MRM mode were optimized by injecting analytes into the chamber of the ionization source using a syringe inlet.

Table 2. Ionization parameters of analytes in a heated electrospray ionization source
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Table 3.   Conditions of mass-spectrometric detection in the MRM mode
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Preparation of standard solutions. Standard solutions of steroid hormones with a concentration of 1 mg/mL were prepared in methanol and stored at 4°C for a month. Calibration solutions with concentrations of 0.5, 1.0, 2.5, 5, 10, 25, 50, 100, 250, and 500 ng/mL were prepared by successive dilutions of standard solutions in methanol and stored at 4°C for a month. Quality control solutions with concentrations of 10, 50, and 250 ng/mL were prepared separately from calibration solutions in methanol. The working solution of the internal standard, methyltestosterone, with a concentration of 200 ng/mL in methanol was stored in a refrigerator at 4°C.

A phosphate buffer solution (pH 8) was prepared using 0.067 M solutions of potassium dihydrogen phosphate and sodium hydrogen phosphate. A borate buffer solution (pH 10) was prepared using a 0.05 M sodium tetrahydroborate solution and a 0.1 M sodium hydroxide solution.

Saliva sampling. Mixed saliva samples were obtained by natural secretion. Before sampling, volunteers (men and women aged 20–45 years) did not eat and refrained from smoking for an hour, and rinsed their mouth with water 10 min prior to sampling. Samples were analyzed immediately after sampling. Saliva samples were stored at −20°C.

RESULTS AND DISCUSSION

Selection of saliva sampling conditions for analysis. As the results of the analysis depend on the conditions of sampling saliva, the influence of various factors on the concentrations of steroid hormones was preliminary considered.

Commercially available containers, such as Salivette® are among the most widely used ones for obtaining saliva samples. However, studies have shown that the swab they contain, soaked with saliva when chewed, can affect the results of hormone determination. Thus, the use of a synthetic swab did not significantly affect the results of the determination of cortisol, while the concentrations of other steroid hormones, in particular testosterone, changed when it was used [1]. The use of a cotton swab led to the sorption of analytes and, accordingly, to the distortion of the results; therefore, direct sampling of saliva was optimal [2].

Direct sampling can be done with or without the stimulation of saliva secretion (e.g., citric acid, sugar-free gum, paraffin). In using chewing gum, an increase in the concentration of testosterone and cortisol was observed, which made it difficult to use it. The stimulation of salivation by various methods led to a change in the concentration of cortisone; therefore, in determining the steroid profile, it was optimal to collect unstimulated saliva in ordinary polypropylene tubes [1].

Another important requirement is the absence of blood in the saliva due to mechanical damage in the oral cavity, e.g., in brushing teeth. As the concentration of steroid hormones in saliva is much lower than their concentration in blood, if it enters the saliva, the results obtained will be significantly overestimated. In the presence of 0.1–0.2% of blood in a saliva sample, it acquires a pink hue, which makes it possible to visually assess the contamination of the analyzed sample [6].

In addition, in determining glucocroticoids, it is necessary to take into account the effect of the enzyme 11β-hydroxysteroid dehydrogenase II, under the action of which cortisol is converted into an inactive keto form (cortisone) in the salivary glands [2]. This leads to discrepancies in the results obtained, so it was necessary to determine not only the active form (cortisol), but also its inactive keto form.

Optimization of liquid–liquid extraction conditions. In optimizing the type of the extractant, the following solvents were considered: di-, tri-, and tetrachloromethane, ethyl acetate, hexane, and methyl tert-butyl ether. One milliliter of the extractant was added to 1 mL of a model sample (distilled water) containing the test compounds in a concentration of 50 ng/mL, stirred for 30 s on a vortex, then the sample was centrifuged for 10 min at 4000 rpm, the extractant phase was taken with a syringe, evaporated to dryness and redissolved in 150 µL of a methanol–water mixture (1 : 1, v/v).

Ethyl acetate, chloroform, and methyl tert-butyl ether provided the quantitative recovery of all analytes (>70%). Methyl tert-butyl ether was chosen for further studies because of its low boiling point (55°C) and lower toxicity compared to chlorinated solvents.

The optimal extractant volume, pH, and vortexing time were determined using multivariate analysis (Box–Behnken design). The results obtained were processed using the STATISTICA 10 software (Statsoft). The levels of factors are presented in Table 4.

Table 4. Levels of factors to be optimized (Box–Behnken plan)
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To find the optimal levels of the studied factors, 0.3 mL of a buffer solution (pH 8 and 10) or 0.3 mL of distilled water (pH 6) were added to a model sample (1 mL, analyte concentration of 50 ng/mL). Then, methyl tert-butyl ether was added to the sample and it was vortexed for 30 s. After that, the aqueous layer was frozen at −35°C, and the ether phase with the analytes was transferred into a test tube for evaporation on a solid-state heater at 60°C, followed by the dissolution of the dry residue in 150 μL of a methanol–water mixture (1 : 1, v/v) for analysis.

All the resulting models turned out to be significant, because the adjusted coefficients of determination (\(R_{{{\text{adj}}}}^{2}\)) exceeded 0.9. As the properties of the compounds were different, the optimal levels of the factors also had different values for the analytes. The volume of methyl tert-butyl ether had the greatest effect on the recovery of analytes: with an increase in the volume of the extractant, the recovery increased; therefore, a volume of 1.5 mL was chosen for the further studies, which, together with other factors, ensured the high recovery of analytes. Stirring time also affects analyte recovery rates, and 30 s was optimal for all analytes. Increasing the pH of the medium did not lead to higher recoveries of analytes; therefore no buffer solution was added in the subsequent experiments.

Thus, sample preparation was carried out under the following conditions: an internal standard (methyltestosterone with a final concentration of 20 ng/mL) was added to 1 mL of a saliva sample. Then, 1.5 mL of methyl tert-butyl ether was added, vortexed for 30 s, centrifuged for 10 min at 4000 rpm, the aqueous phase was frozen out at –35°C, followed by the transfer of the organic phase into another test tube and its evaporation to dryness at 60°C with the dissolution of the dry residue in 150 μL of a methanol–water mixture (1 : 1, v/v). Under these conditions, the recoveries of the analytes were 91–98%. The recoveries were calculated as the ratio of the peak area of a compound in the solution passed through all stages of sample preparation to the peak area of this compound in the model solution.

Validation of the developed method. Steroid hormones are endogenous compounds, there are no matrices for them that do not contain the target analytes; therefore, the procedure was validated on model solutions (distilled water), and also on real samples using the spike-recovery method, taking into account the criteria of the Food and Drug Administration (United States) for the validation of bioanalytical procedures [15].

Calibration curves were constructed in the concentration range of 0.05–50 ng/mL (0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5, 10, 25, 50 ng/mL). The limit of detection corresponded to the concentration detected at a signal-to-noise ratio of 3, while the limit of quantification corresponded to the analyte concentration determined with an error of 15%. The results obtained are presented in Table 5.

Table 5.   Analytical characteristics of the procedure
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The accuracy and reproducibility were controlled by analyzing quality control solutions at three concentration levels: low (1 ng/mL), medium (5 ng/mL), and high (25 ng/mL) for one and several days. Reproducibility was evaluated using relative standard deviation (RSD) and accuracy using equation (1):

$${{e}_{{\text{r}}}} = {\text{ }}(({{c}_{{{\text{det}}}}}-{{c}_{{{\text{theor}}}}}){\text{/}}{{c}_{{{\text{theor}}}}}) \times 100,$$
(1)

where er is the relative error. The results obtained are presented in Table 6.

Table 6. Results of assessing the accuracy and reproducibility of the procedure by the spike-recovery method (n = 15, P = 0.95)
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The stability of real saliva samples containing the analytes was evaluated for a month with two cycles of freezing (−20°C) and thawing to room temperature. The analytes remained stable within this time, as the results obtained differed by less than 15% from the initial ones, which is consistent with the results of [10].

The stability of the solutions in the autosampler was evaluated for 36 h at 5°C. The results showed that the samples remained stable within this time range.

A possibility of cross-contamination was assessed by analyzing a blank solution after analyzing a sample with a concentration of steroid hormones of 50 ng/mL. It was found that the chromatogram of the blank sample did not contain peaks with MRM transitions similar to the target compounds.

Analysis of real samples. The proposed method was used to analyze real samples obtained from volunteers. Before the analysis, the samples were centrifuged for 10 min at 10000 rpm. The analysis was performed by the spike-recovery method by adding quality control solutions of low (1 ng/mL), medium (5 ng/mL), and high (25 ng/mL) concentrations. It was found that the matrix components did not significantly affect the results obtained, as the determination error was <15%.

CONCLUSIONS

Thus, a simple and a sensitive (limits of detection in the range 0.05–0.25 ng/mL) method was developed and validated for the determination of steroid hormones in human saliva by ultrahigh performance liquid chromatography with tandem mass spectrometry detection . A possibility of the simultaneous sensitive determination of steroid hormones of various classes makes saliva a promising matrix for diagnostic purposes, which offers many advantages over blood.