The validated micellar electrokinetic chromatography (MEKC) was proposed for the determination of five steroid hormones in human urine samples. That technique allowed for the separation and quantification of cortisol, cortisone, corticosterone, testosterone, and epitestosterone and was sensitive enough to detect low concentrations of these searched steroids in urine samples at the range of 2–300 ng/mL. The proposed MEKC technique with solid-phase extraction (SPE) procedure was simple, rapid, and has been successfully applied as a routine procedure to analyze steroids in human urine samples. The MEKC method offered a potential in clinical routine practice because of the short analysis time (8 min), low costs, and simultaneous analysis of five endogenous hormones. Due to its simplicity, speed, accuracy, and high recovery, the proposed method could offer a tool to determine steroid hormones as potential biomarkers in biomedical investigations, what was additionally revealed with healthy volunteers.
Measuring steroids’ profile can be suitable as a screening test indicating dysfunction of steroid-forming adrenals and gonads, for example, during the Cushing syndrome [
On the other hand, testosterone is an endogenous androgen having both androgenic and anabolic effects in the human body. The normal levels of testosterone and epitestosterone in the urine of healthy male volunteers are 30–60 ng/mL for both [
Currently, capillary electrophoresis (CE) techniques are employed as major very useful separation tools for the analysis of steroids with reliable result [
Based on the our previous experience [
Analyzed substances, cortisol (11
The experiments in steroid separation were performed using a capillary electrophoretic system (P/ACE 2100, Beckman Instruments, Fullerton, CA, USA) equipped with an automatic injector, a filter-carrousel UV detector, a temperature control device, and a data acquisition system supplied by the manufacturer (Beckmann P/ACE System Gold Software). Electrophoretic separation was carried out using a fused silica capillary with the i.d. of 75
Steroids occur in human urine specimens in a conjugated and nonconjugated (free) form. Depending on the relationship between the two forms, a small part of the steroid hormones in urine are found in the free form (below 1%), while the major fraction is contained in glucuronide and sulphate conjugates (99%). To determine the total steroid level, it was necessary to include the hydrolysis step. However, we were aware that if urine samples were taken at random, for example, after or during a stressful situation, the creatinine level would require determination. Therefore, the steroid concentration level was corrected resulting in the steroid-to-creatinine ratio. The creatinine level was evaluated using a diagnostic kit based on the colorimetric method (PZ Cormay, Lublin, Poland). This made the results of the normalized steroid measurements realistic. The urine samples were obtained from adult healthy volunteers between 7.00 and 9.00 AM. All samples were frozen at −20°C until the analysis. Before extraction, the samples were brought to the room temperature and an acid hydrolysis in water bath with 36% hydrochloric acid was performed for 1 hour at 95°C. After the hydrolysis, the urine samples were brought back again to the ambient temperature and subjected to a further extraction process. Before electrophoretic separation the calibration urine specimens (15 mL in volume), with various concentrations of the analytes ranging from 2 to 300 ng/mL and with the internal standard solutions to achieve the final concentrations of 200 ng/mL of dexamethasone, were spiked. SPE was chosen for effective and high recovery of the steroids in the HLB column (6 mL, 200 mg). Urine samples were prepared as detailed above then extracted in several steps. First, the HLB cartridges were conditioned with 5 mL of methanol followed by 10 mL of deionized water. Then, the steroid-spiked urine samples or samples obtained from volunteers were carried to the SPE sorbent. Next, each sample was slowly passed through the SPE column using the Baker system under the vacuum conditions. Once the samples were washed with 4 mL of an acetone-water mixture at the ratio of 25 : 75 (
Acidic hydrolysis was performed before extraction procedure. For that purpose a mineral acid was added to urine to cleave steroids from the conjugation form. We realize that the yield of hydrolysis is strongly influenced by several parameters: the acid concentration, temperature, and time of reaction. In our experience, using high temperature for a relatively short time (1 hour), under strictly controlled conditions, did not cause any significant disruption of the steroids. Moreover, complete hydrolysis was achieved by placement of urine samples using 36% hydrochloric acid under a stream of compressed air in water bath closed with a tight lid at the temperature of 95°C.
In order to develop the best sample preparation procedure, the SPE and liquid-liquid extraction (LLE) were tested. At first, LLE was employed, but many problems were encountered because of the emulsion formation. Moreover, the extraction recovery was not satisfactory, and sample loss during the process was observed. The serious limitations of that particular extraction method come down to the fact that it is a time-consuming process involving high consumption of organic solvents, all of which make the method unfriendly in terms of either the environmental or human health protection. Next, solid-phase extraction (SPE) procedures with silica-based apolar columns including C18, C8, CN, functionalities, and hydrophilic-lipophilic balance (HLB) cartridges were tested. To that aim blank urine samples were spiked with 10, 50, and 100 ng/mL of steroids, and different organic solvents were tested. The extraction efficiency was acceptable when methanol as eluent and an HLB column were used, yielding high recoveries of 95.8% for 10 ng/mL, 94.6% for 50 ng/mL, and 92.1% for 100 ng/mL (Table
Analytical extraction efficiency test of the analyzed steroids after various extraction columns and solvents.
Column extraction | Concentration added (ng/mL) | Dichloromethane | Methanol | ||||
Concentration found (ng/mL) ( | Recovery (%) | RSD (%) | Concentration found (ng/mL) ( | Recovery (%) | RSD (%) | ||
C8 | 10 | 5.5 ± 0.7 | 54.6 | 12.9 | 6.2 ± 0.7 | 62.3 | 11.6 |
50 | 53.1 ± 6.1 | 53.1 | 11.4 | 63.4 ± 6.5 | 63.4 | 10.3 | |
200 | 111.4 ± 10.9 | 55.7 | 9.8 | 123.4 ± 12.2 | 61.7 | 9.9 | |
C18 | 10 | 8.8 ± 0.6 | 88.4 | 6.6 | 9.1 ± 0.6 | 90.9 | 6.4 |
50 | 42.5 ± 3.5 | 85.1 | 8.2 | 44.7 ± 3.2 | 89.4 | 7.1 | |
100 | 81.7 ± 5.9 | 81.7 | 7.3 | 89.8 ± 6.2 | 89.8 | 6.9 | |
CN | 10 | 3.4 ± 0.3 | 33.7 | 8.8 | 3.6 ± 0.3 | 35.6 | 7.6 |
50 | 15.6 ± 1.7 | 31.2 | 10.7 | 17.1 ± 1.4 | 34.2 | 8.2 | |
100 | 30.8 ± 2.1 | 30.8 | 6.9 | 36.3 ± 3.5 | 36.3 | 9.7 | |
HLB | 10 | 8.9 ± 0.2 | 89.6 | 2.2 | 9.6 ± 0.2 | 95.8 | 2.3 |
50 | 45.2 ± 2.4 | 90.4 | 5.4 | 47.3 ± 2.0 | 94.6 | 4.2 | |
100 | 92.1 ± 4.5 | 92.1 | 4.9 | 46.1 ± 1.8 | 92.1 | 3.9 |
A mixture of acetone and water (25 : 75,
Since to steroid hormones are electrically neutral molecules of low hydrophobic (in free form) or hydrophilic (in the form of conjugates) properties, the choice of the running buffer significantly impacts on the resolution of the analytes. The separation principle of the capillary zone electrophoresis (CZE) mode is based on the differences between compounds in terms of their electrophoretic mobility. Unfortunately, the electrophoretic mobility of some steroids is very similar. In effect, the traditional CZE method using borate or phosphate electrolyte only will result in nonseparation. That is why MEKC with SDS, as the most common anionic surfactant at a concentration, greater than its critical micelle concentration is added to the buffer solution and was successfully employed in our study. The SDS micelles enhanced solubility of the analytes and offered excellent resolution. This can be accompanied by differential partitioning of the analytes between the micellar and aqueous phases. The influence of the SDS concentration in the optimized tetraborate buffer was further evaluated for the resolution of all investigated steroids. The concentration of the micelle-forming agent (SDS) was tested from 10 to 50 mM. Raising the concentration of SDS in the running buffer resulted in an increase of the migration times for the compounds of interest. Experimental results indicated that the SDS concentration of 25 mM contributed significantly to the peak quality and migration time and could yield complete baseline separation for all steroids. Thus, the concentration of 25 mM SDS and 20 mM tetraborate in the buffer at pH 9.3 was selected to obtain a good peak shape, low peak width, short migration time, and higher efficiency. Furthermore, the chosen separation conditions eliminated many of the potential interferences, including those ones coming from most endogenous substances of urine biological sample.
It was interesting to study the parameters significantly influencing electrophoretic separation, for example, voltage, injection time, and temperature, in the achievement of good resolution, symmetry, and high peaks of the steroids. To identify the suitable voltage to be applied, its effect was studied within the range of 10–30 kV. Under the described conditions (20 mM tetraborate buffer at pH 9.3 and 25 mM SDS), increased applied voltage shortened the analysis times and sharpened the peaks. Higher separation voltages not only increased the electrophoretic velocity of the analytes, but also increased the current and the Joule heat. This can be mitigated by lowering the ionic strength of the running buffer or by enhancing heat dissipation. Therefore, in order to limit heating inside the capillary, the maximum applied voltage was chosen based on the Ohm’s plot (current versus voltage) and the voltage of 17 kV was finally used.
Moreover, changes of the capillary temperature can cause variation in efficiency, viscosity, electrophoretic mobility, and the migration times of the analytes. The effect of the capillary temperature on selectivity and the migration time was examined over the range of 18–25°C. The best conditions giving sufficient resolution and a good level of baseline noise were achieved at the temperature of 22°C (±0.1). Optimized hydrodynamic injection was employed to introduce the samples into the capillary. The urine extracts were analyzed by MEKC using the injection time of 2, 5, and 7 s. It was observed that the 2 s injection (corresponding approximately to the 8 nL injection volume of the sample) led to achieving the most efficient separation of all steroids. The longer injection times resulted in broadened, overlapping peaks.
The calibration samples were prepared and quality control set by spiking the steroid-free urine samples (charcoal-stripped) with a steroid spiking solution to obtain the final concentrations of 2, 10, 50, 100, 200, 300 ng/mL.
The linearity of the calibration curve was determined for the range of 2 to 300 ng/mL and evaluated by analyzing six different concentrations of steroids. The calibration curve was constructed by plotting the ratios of the peak height against the corresponding concentrations. Each concentration was injected in six replicates. The following regression equations were calculated, including the slopes, the intercepts, and the correlation coefficients, as listed in Table
Results of regression model for the analyzed steroids.
Total cortisol | Total cortisone | Total corticosterone | Total testosterone | Total epitestosterone | |
---|---|---|---|---|---|
Linearity range (ng/mL) | 2–300 | ||||
Slope ± SD | 0.0062 ± 0.00013 | 0.0044 ± 0.00004 | 0.0077 ± 0.00008 | 0.0056 ± 0.00007 | 0.0067 ± 0.00005 |
Intercept ± SD | 0.219 ± 0.0198 | 0.306 ± 0.0060 | 0.192 ± 0.0120 | 0.151 ± 0.0107 | 0.116 ± 0.0083 |
Correlation coefficient ( | 0.9991 | 0.9998 | 0.9997 | 0.9997 | 0.9998 |
6 | |||||
LOD (ng/mL) | 0.5 | ||||
LOQ (ng/mL) | 2.0 | ||||
Total separation time (min) | 8.0 | ||||
Migration time (min) | |||||
Cortisol | 4.95 | ||||
Cortisone | 5.12 | ||||
Corticosterone | 5.97 | ||||
Dexamethasone I.S. | 5.55 | ||||
Testosterone | 6.48 | ||||
Epitestosterone | 6.66 |
The specificity of the method was assessed by conducting a comparative analysis of blank urine samples and urine samples spiked with steroids after the extraction procedure earlier described in this paper. The representative electropherograms are presented in Figures
Typical electropherograms obtained for (a) steroid-free urine sample; (b) male urine sample; (c) female urine sample; (d) urine sample spiked with 50 ng/mL of cortisol (1), 50 ng/mL of cortisone (2), 200 ng/mL of dexamethasone (3) (I.S.), 100 ng/mL of corticosterone (4), 100 ng/mL of testosterone (5), and 100 ng/mL of epitestosterone (6). Conditions of electrophoretic separation: 17 kV, injection 2 s, UV,
Success of any analytical method comprising also CE-based one can be proven when the number of real-world applications increases. The number of CE applications is observed as to be growing, just as its reoccurrence in the reviews published recently and based on clinical, forensic, and biomedical applications.
In the last years many authors demonstrated the application of their methodology developed by the determination of steroid hormones in the analysis of real samples from people [
In the current study, under the optimized experiment conditions, the quantitative evaluation of the endogenous steroid level was carried out using the developed MEKC method. Investigations were performed in compliance with the rules set by the ethics committee, and the study protocol was also approved by the Ethical Committee of the Medical University of Gdańsk, Poland.
All participating volunteers (10 males and 10 females) represented the average age of 23 ± 2.6 years, the body weight of 67 ± 13 kg, and the height of 171 ± 9 cm.
For the evaluation of glucocorticoid concentrations as biomarkers of stress the urine samples should be collected as soon as possible after stress situation. On the other hand, the levels of urinary glucocorticoids could be changed due to the density of this fluid. Because it was confirmed that creatinine, being a product of muscle metabolism, is normally lost in the urine at a relatively steady state, the ratio of urinary corticosteroids to creatinine should be used to gain a correct urinary concentration of compounds of interest. Moreover, abnormal physiological concentrations of creatinine in urine samples may signal renal failure and/or a reduced glomerular filtration. It may cause increasing or decreasing amounts of analyzed hormones in urine. The mean levels of creatinine in urine were between 0.88 and 2.39 mg/dL. These results also confirmed that no participation possessed a dysfunction of kidney. Next, the cortisol, cortisone, and corticosterone levels in all urine samples were determined.
The average total concentrations of cortisol, cortisone, and corticosterone in the urine samples obtained from the searched healthy volunteers were 115.80 ± 49.45 ng/mL, 265.04 ± 141.07, and 64.29 ± 29.65 ng/mL, respectively. The urinary total excretion ranged from 41.67 ng/mL to 193.33 ng/mL for cortisol and from 55.00 ng/mL to 553.50 ng/mL for cortisone, and the urinary total corticosterone was recorded between 24.43 ng/mL and 127.14 ng/mL. The overall average concentration of the urinary steroids is shown in Table
Results of the urinary total steroid levels in healthy volunteers.
Volunteer no. | Sex | Age | Height (cm) | Body mass (kg) | Creatinine level (mg/dL) | Total level (ng/mL) | ||||
1 | 2 | 3 | 4 | 5 | ||||||
1 | F | 23 | 163 | 57 | 1.98 | 46.33 | 121.00 | 73.00 | Nd | Nd |
2 | M | 21 | 192 | 93 | 1.04 | 84.83 | 55.00 | 30.01 | 4.2 | 2.17 |
3 | M | 22 | 182 | 78 | 1.28 | 121.83 | 213.50 | 81.14 | 4.9 | 3.33 |
4 | F | 22 | 166 | 57 | 1.42 | 168.50 | 296.00 | 76.86 | 13.0 | Nd |
5 | F | 23 | 164 | 50 | 0.99 | 121.83 | 321.00 | 36.86 | Nd | Nd |
6 | F | 21 | 158 | 64 | 1.34 | 167.00 | 279.00 | 70.3 | 6.8 | Nd |
7 | F | 22 | 160 | 58 | 2.36 | 121.83 | 321.00 | 36.86 | 2.6 | Nd |
8 | M | 28 | 176 | 74 | 1.45 | 163.50 | 373.50 | 82.57 | 4.5 | 3.83 |
9 | M | 21 | 182 | 86 | 1.49 | 52.83 | 55.75 | 68.57 | 34.4 | 18.67 |
10 | M | 28 | 186 | 95 | 0.88 | 121.83 | 388.50 | 32.00 | 4.7 | 2.30 |
11 | F | 21 | 164 | 59 | 1.09 | 48.50 | 446.00 | 61.14 | Nd | Nd |
12 | F | 30 | 168 | 54 | 2.39 | 158.50 | 398.50 | 69.71 | 10.6 | 7.83 |
13 | F | 24 | 163 | 56 | 1.14 | 193.33 | 553.50 | 51.00 | Nd | Nd |
14 | F | 22 | 166 | 59 | 2.09 | 52.333 | 74.25 | 60.86 | 38.0 | 30.83 |
15 | F | 24 | 168 | 55 | 1.64 | 41.67 | 144.50 | 127.14 | 6.6 | Nd |
16 | M | 23 | 174 | 78 | 2.06 | 94.17 | 235.50 | 35.43 | 9.0 | 8.17 |
17 | M | 22 | 168 | 69 | 1.14 | 86.33 | 82.50 | 24.43 | 6.4 | 2.83 |
18 | M | 23 | 170 | 67 | 1.46 | 138.50 | 363.50 | 40.43 | 4.4 | 2.50 |
19 | M | 21 | 172 | 63 | 0.97 | 172.17 | 246.00 | 112.57 | 11.4 | 3.17 |
20 | M | 21 | 168 | 59 | 1.43 | 160.17 | 332.25 | 115.00 | Nd | Nd |
Average | ||||||||||
SD |
1: cortisol; 2: cortisone; 3: corticosterone; 4: testosterone; 5: epitestosterone; Nd: not determined.
Next, the determination of urinary cortisol/cortisone ratio was used to assess the renal activity of 11
In the case of androgens, the average concentration levels of the endogenous testosterone and epitestosterone were 10.77 ng/mL ± 10.76 and 7.78 ± 9.06 ng/mL, respectively. The urinary total testosterone excretion ranged from 2.6 to 38.00 ng/mL, and the urinary total epitestosterone was recorded between 2.17 ng/mL and 30.83 ng/mL.
An important parameter in terms of doping control is also testosterone/epitestosterone ratio. The norm for urinary testosterone/epitestosterone ratio is below 4. Usually, the amounts of testosterone and epitestosterone are in similar proportions and their normal presence in urine has been found, on an average to be roughly equal to 1 : 1, although it varies on a case-specific basis. Physical effort does not modify this ratio but it can be increased by the use of doping agents. The values of testosterone/epitestosterone ratio obtained in the current experiment from the searched healthy volunteers ranged from 1.1 to 3.6 and no doping with testosterone in the study group could be identified.
The developed and applied novel micellar electrokinetic chromatography method (MEKC) proved to be efficient and allowed for the simultaneous determination of five steroids of interest over a short total time of 8 min.
Full automation, high efficiency, rapidness, low solvent consumption, and low costs made the proposed MEKC method attractive, especially in terms of suitability for routine analysis during clinical investigations. The method presented in this paper appeared to be properly optimized and fully validated, including the proved specificity, linearity, sensitivity, precision, and accuracy. By using the HLB columns for the SPE during the sample treatment procedure, and by an optimization of separation conditions, it was possible to achieve satisfactory detection and quantification limit. The described MEKC method has been successfully applied during the determination of endogenous steroid profiles in human urine obtained from healthy volunteers. The assessment of the proposed methods in terms of detection and evaluation of the steroid level obtained from a real human group of 20 healthy volunteers proved its usefulness in the medical diagnostic practice and made it recommendable for biomedical investigations.
The work was supported by the Polish State Committee for Scientific Research Project no. N405 423839.