Detection of anabolic steroids via cyclodextrin-promoted fluorescence modulation

Reported herein is the detection of anabolic steroids through the use of cyclodextrin-promoted interactions between the analyte of interest and a high quantum yield fluorophore, which lead to measurable, analyte-specific changes in the fluorophore emission signal. By using a variety of β-cyclodextrin derivatives (unmodified β-cyclodextrin, methyl-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin) in combination with high quantum yield fluorophore rhodamine 6G, we detected five anabolic steroid analytes with 100% differentiation between structurally similar analytes and micromolar level limits of detection. Overall, these results show significant potential in the development of practical, fluorescence-based steroid detection devices.


MATERIALS AND METHODS
The anabolic steroid analytes, chemicals required to make buffer solutions, fluorophore Rhodamine 6G, and solvent tetrahydrofuran were obtained from Sigma-Aldrich Chemical company and the cyclodextrins were obtained from Tokyo Chemical Industry (TCI). All chemicals were used as received without further purification. All fluorescence measurements were performed using a Shimadzu RF 6000 spectrophotometer. The excitation and emission slit widths were set to 3.0 nm. All fluorescence spectra were integrated vs. wavenumber on the X-axis using OriginPro 2019 Version 9.60. All arrays were generated using SYSTAT Version 13.1. Figure S1: Structure of anabolic analytes (compound 1: Mesterolone; compound 2: Oxandrolone; compound 3: Oxymetholone; compound 4: Stanozolol; compound 5: Trenbolone) and fluorophore Rhodamine 6G (compound 6) All analyte samples were prepared at a concentration of 1.0 mg/mL in THF. The fluorophore solution was prepared at a concentration of 0.1 mg/mL in THF. An 0.1 M citrate buffer was prepared by combining 2.409 grams of sodium citrate and 0.347 grams of citric acid in a 1.0 L volumetric flask and diluting to the mark with distilled water. The pH of the buffer was measured at 6.1, and remained consistent throughout the experimental procedures. Cyclodextrin solutions were prepared at a concentration of 10 mmol in the citrate buffer. The final concentrations of the analytes and fluorophore are shown in Table S1, below:

Experimental Procedure for Fluorescence Modulation Experiments
Fluorescence modulation experiments were done with 5 µL, 10 µL, and 20 µL sequential additions of analyte.
The fluorescence modulation values for each analyte-cyclodextrin combination were determined according to the following procedure: 1. 100 µL of fluorophore 6 solution (0.1 mg/mL) in THF was measured into six 15 mL glass vials (vial 1 for THF, vial 2 for analyte 1, vial 3 for analyte 2, etc.). 2.00 mL of a 10 mM cyclodextrin in citrate buffer and 0.40 mL 0.1 M citrate buffer were added to each (citrate buffer was at pH 6.1). The vials were capped and left to stabilize for 48 hours in a dark drawer.
2. After the 48 hours, the contents of one vial and 5.0 µL of analyte were added to the cuvette and stirred thoroughly to ensure homogeneity. The solution was excited at 490 nm and recorded from 500-800 nm. Four repeat measurements were taken. This step was repeated for each analyte and an additional time, adding 5.0 µL of THF instead of an analyte solution to use as a control. 3.
Step 1 and 2 were repeated for each analyte-cyclodextrin combination (18 trials in total). In all cases, the solution was excited at the same wavelength (490 nm) and the emission spectra from 500 to 800 nm was recorded four times.
4. To conduct an experiment with no cyclodextrin present, step 1 was repeated but citrate buffer solution was substituted in place of the cyclodextrin solution. The final contents of the vials were then 100 µL of fluorophore 6 solution and 2.4 mL of 0.1 M citrate buffer.
5. After 48 hours, step 2 was repeated using this set of solutions containing no cyclodextrin for each analyte. The solutions were excited at the same wavelength (490 nm) and the emission spectra from 500 to 800 nm was recorded four times.
6. Emission spectra were integrated versus wavenumber on the X-axis using OriginPro software and fluorescence modulation ratios were determined according to Equation 1, below: Fluorophore Modulation Ratios = Fl analyte / Fl blank (Eq. S1) where Fl analyte represents the integrated fluorescence emission of the fluorophore in the presence of the analyte and Fl blank represents the integrated fluorescence emission of the fluorophore in the absence of the analyte.
Step 2 and 5 were repeated with sequential additions for total addition amounts of 10 µL and 20 µL of analyte. The final concentrations of the analytes and the fluorophore in solution using each of the three addition protocols are summarized in Table S2, below:

Experimental Procedure for Limit of Detection Experiments
The limit of detection (LOD), defined as the lowest concentration of the analyte that can be detected, was obtained using the calibration curve method, following procedures reported by Loock et. al. 1 The limit of quantification (LOQ) is the lowest concentration of analyte that can be reliably and accurately quantified. The limit of detection and quantification experiments were conducted following literature-reported procedures. 1,2 LOD experiments were done with sequential 5 µL additions of analyte, according to the procedures listed below: 1. 100 µL of fluorophore 6 solution (0.1 mg/mL) in THF was measured into a 15 mL glass vial. 2.00 mL of a 10 mM cyclodextrin in citrate buffer and 0.40 mL 0.1 M citrate buffer were added (citrate buffer was at pH 6). The vial was capped and left to stabilize for 48 hours.
2. The solution was transferred to a quartz cuvette and then excited at 490 nm and the fluorescence emission spectra was recorded from 500 to 800 nm. Each fluorescence measurement was repeated six times 3. 5.0 µL of the analyte solution in THF was added to the cuvette and stirred thoroughly to ensure homogeneity. The solution was excited at the same wavelength (490 nm) and the emission was measured between 500 nm and 800 nm. Six repeat measurements were taken.

4.
Step 2 was repeated four times for total addition volumes of 10 µL, 15 µL, 20 µL, and 25 µL of the analyte solution. In all cases, the solution was excited at the same wavelength (490 nm) and the emission spectra from 500 to 800 nm was recorded six times.
5. Emission spectra were integrated versus wavenumber on the X-axis using OriginPro software, and were used to generate calibration curves with analyte concentration on the X-axis and integrated fluorescence emission on the Y-axis. The curve was fitted with a linear trendline and the equation of the line was determined.
6. The measurements from Step 1, the emission spectra of the combination of the Rhodamine solution and β-cyclodextrin solution with no addition of analyte, are referred to as the blank in the following calculations.
7. The limit of the blank (LOD blank ) is defined according to the following equation: where m blank is the mean of the blank integrations and SD blank is the standard deviation of those measurements.

The LOD blank was then entered into the equation determined in
Step 4 as the y-value. The corresponding x-value was calculated. This value is the LOD of the analyte in µM in the system. 9. The LOQ (LOQ blank ) was calculated in a similar manner to the LOD. The limit of quantification of the blank is defined according to the following equation: This value is then entered as the y-value from step 4 and the corresponding x-value was calculated. This is the value of the LOQ of the analyte for the system in µM.
The summary tables of these results for each analyte-cyclodextrin combination are shown in Tables S4-S6 (vide infra).

Experimental Procedure for Array Generation Experiments
Linear discriminant analysis was performed using SYSTAT 13 statistical computing software with the following software settings 3 : These experiments were then repeated using only two predictors (i.e. cyclodextrins) instead of all three, and the results of array-based analysis for each pair of predictors is reported herein as well.

Experimental Procedure for Computational Experiments
Spartan software version '18 was used to calculate the equilibrium molecular conformations of each analyte in their ground states in the gas phase using a semi-empirical PM3 model for each analyte. This allowed an electrostatic potential map surface to be overlaid over the molecules, using the mesh overlay function.

SUMMARY TABLES
Summary Tables for Fluorescence Modulation Experiments   Table S3. Fluorescence modulation results obtained for analytes 1-5 with various cyclodextrins in the presence of fluorophore 6 a Addition amount Analyte , and results reported represent an average of at least four trials.

Summary Tables for Array Generation Experiments
With 5 µL analyte additions Table S7. Linear discriminant analysis results with β-CD, Me-β-CD, and 2-HPCD as predictors Table S8. Linear discriminant analysis results using β-CD and Me-β-CD as predictors Table S9. Linear discriminant analysis results using β-CD and 2-HPCD as predictors S11 Table S10. Linear discriminant analysis results using Me-β-CD and 2-HPCD as predictors With 10 µL analyte additions Table S11. Linear discriminant analysis results with β-CD, Me-β-CD, and 2-HPCD as predictors Table S12. Linear discriminant analysis results using β-CD and Me-β-CD as predictors S12 Table S13. Linear discriminant analysis results using β-CD and 2-HPCD as predictors Table S14. Linear discriminant analysis results using Me-β-CD and 2-HPCD as predictors With 20 µL analyte additions Table S15. Linear discriminant analysis results using β-CD, Me-β-CD, and 2-HPCD as predictors S13 Table S16. Linear discriminant analysis results using β-CD and Me-β-CD as predictors Table S17. Linear discriminant analysis results using β-CD and 2-HPCD as predictors Table S18. Linear discriminant analysis results using Me-β-CD and 2-HPCD as predictors S14 All Additions with THF Table S19. Linear discriminant analysis results with β-CD, Me-β-CD, and 2-HPCD as predictors All Additions excluding THF Table S20. Linear discriminant analysis results with β-CD, Me-β-CD, and 2-HPCD as predictors S15

Summary Figures for Individual Fluorescence Modulation Experiments Using Fluorophore 6
With 5 µL analyte addition β-CD

Summary Figures for Computational Experiments
Spartan '18 Electrostatic Potential Map Diagrams Figure S97. Electrostatic potential map of analyte 1 in the gas phase at its most stable (i.e. "ground state" configuration) Figure S98. Electrostatic potential map of analyte 2 in the gas phase at its most stable (i.e. "ground state" configuration) S48 Figure S99. Electrostatic potential map of analyte 3 in the gas phase at its most stable (i.e. "ground state" configuration) Figure S100. Electrostatic potential map of analyte 4 in the gas phase at its most stable (i.e. "ground state" configuration)