Enhanced Separation of Estrogens in Sequential Injection Chromatography Using an Ascentis® Express Column Based on Fused-Core® Technology


The following was generated with the assistance of an outside source using Sigma-Aldrich products. Technical content was generated and provided by:
Petr Chocholouš, Dalibor Šatínský, Petr Solich Charles University, Faculty of Pharmacy, Department of Analytical Chemistry, Heyrovského 1203, Hradec Králové, 500 05, Czech Republic

Introduction

Sequential Injection Chromatography (SIC) introduced in 2003 by Šatinsky et al is a developing technique based on a highly versatile sequential injection analysis (SIC) manifold employing short chromatographic columns1. In SIC, only short monolithic columns for chromatographic separations have been used2. The novel use of an Ascentis Express Fused-Core® particle column (C18, 3 cm x 4.6 mm I.D.) is possible thanks to the compatibility with the medium-pressure SIC manifold. Typical flow rates (0.5 - 1.0 mL/min) and working back-pressures (3-6.9 MPa) enable high performance separations with these columns.

The aim of this work3 was to prove and confirm all the characteristics of the column and test the column with a separation of a mixture of analytes with similar chromatographic properties.

The estrogens are a group of steroid compounds that function as the primary female sex hormones with the primary compound being estradiol. A model mixture of four chosen estrogens for column testing contained ß-estradiol, 17-ß-ethinylestradiol, estrone and 17-ß-estradiol-hemihydrate (Figure 1). The structures are different only in substitutions in position 17 of the steroid structure, thus suited for testing of chromatographic performance of columns. Ethylparaben, a common preservative in foods and drugs was chosen as an internal standard.

Chemical Structures of Estrogens used and Internal Standard Ethylparaben

Figure 1. Chemical Structures of Estrogens used and Internal Standard Ethylparaben

Experimental

SIChrom™ instrument (FIAlab® Instruments Inc., Bellevue, WA, USA) with S17 PDP syringe pump with 4.0 mL reservoir was equipped with a fiber-optic CCD UV–VIS detector USB 4000 (Ocean Optics Inc., Dunedin, FL, USA) (Figure 2).

Scheme of SIC Setup for Determination of 4 Estrogens with Internal Standard

Figure 2. Scheme of SIC Setup for Determination of 4 Estrogens with Internal Standard

  1. Sapphire syringe pump
  2. Eight-port selection valve
  3. Chromatographic column
  4. Z-flow cell
  5. UV lamp
  6. CCD UV-VIS detector
  7. Computer with FIAlab software
  1. Sapphire syringe pump
  2. Eight-port selection valve
  3. Chromatographic column
  4. Z-flow cell
  5. UV lamp
  6. CCD UV-VIS detector
  7. Computer with FIAlab software

 

The mobile phase for the separation of all substances on both particle and monolithic columns was acetonitrile:water (40:60, v/v, no pH adjustment). The sample was a mixture of four estrogens (20.0 µg/mL) and the internal standard ethylparaben (4.0 µg/mL)

Results and Discussion

Mobile phase flow rate was set to 0.48 mL/min with the aim to achieve the highest efficiency of both columns and the fastest analysis time. The system-pressure within the separation was 750 psi. The mobile phase volume of 2.2 mL for the particle column was necessary for the elution of all separated analytes within one analysis cycle. The injected volume was 10.0 µL with regards to the recommended and tested capacity of the columns. From the UV spectra of all analyzed compounds, the optimal detection wavelength of 225 nm was chosen to give the best sensitivity.

Table 1. Characterization of SIC Process Performed on Ascentis Express Column

  EP B-EST A-EST Et-EST ESTN
Retention time (min) 1.55 2.36 2.88 3.24 3.69
Peak symmetry 1.67 1.67 1.5 1.3 1.25
Number of theoretical plates 1288 1875 1930 2482 2377
Height equivalent to a theoretical plate (µm) 23.3 16 15.5 12.1 12.6
Peak resolution 4.06 2.15 1.42 1.58  

 

Table 2. Validation Results and Analytical Parameters of SIC Process on Ascentis Express Column

  EP B-EST A-EST Et-EST ESTN
Calibration range (µg mL-1) 0.6 – 10.0 3.1 – 50.0 3.1 – 50.0 3.1 – 50.0 3.1 – 50.0
Equation of calibration - slope 0.0044 0.0075 0.0055 0.0076 0.0052
Equation of calibration - intercept – 0.0371 0.0275 0.0211 0.0281 0.0180
Correlation coefficient 0.997 0.998 0.998 0.998 0.999
Limit of detection (µg/mL) 0.2 1.0 1.0 1.0 1.0
Limit of quantification (µg/mL) 0.6 3.1 3.1 3.1 3.1
System precision (%)‡ <2.0 <2.0 <2.0 <2.0 <2.0
Repeatability of time tR (%)‡ <2.0 <2.0 <2.0 <2.0 <2.0
‡ Relative standard deviation (R.S.D.) values were calculated for repeated standard injections (n = 6) - concentration 20.0 µg/mL of each estrogen and 4.0 µg/mL of EP.

 

Chromatographic Characteristics and Figures of Merit
Measurements showed good results for all analytical parameters (linearity, sensitivity, repeatability, selectivity and precision). Linearity was established with a series of working solutions prepared by diluting the stock solution with 40% acetonitrile to the final concentrations. It was obvious that the quite low molar absorption coefficients of the estrogens enabled only a narrow linear calibration range and was much lower than the molar absorption coefficient of the ethylparaben. Six working solutions were used for calibration of both methods. Each working solution was injected in triplicate and the mean value of peak height was used for the calibration curve. The characterization of the separation process is given in Table 1. The analytical parameters of measurement are in Table 2. The representative sequential injection chromatograms showing successful separation of all the analytes on the Fused-Core particle column is shown in Figure 3

Separation of Ethylparaben, ß-Estradiol, α-Estradiol, Ethinyl-Estradiol, Estrone using Ascentis Express C18 column

Figure 3. Separation of Ethylparaben, ß-Estradiol, α-Estradiol, Ethinyl-Estradiol, Estrone using Ascentis Express C18 column

column: Ascentis Express, 3 cm x 4.6 mm I.D., 2.7 µm (Product No. 53818-U)
mobile phase: 40:60 (v/v), acetonitrile:water
flow rate: 0.48 mL/min
temp.: ambient
det.: UV at 225 nm
injection: 10 µL
1.
2.
3.
4.
5.
Ethylparaben
ß-Estradiol
17-Estradiolhemihydrate
17-Ethinylestradiol
Estrone
1.55 min
2.36 min
2.88 min
3.24 min
3.69 min

Conclusion

Separation of five analytes was successfully obtained with a short column. The use of short Fused-Core particle columns with low dead volume and with the low mobile phase flow rate results in effective separation of five analytes within 5 minutes. The use of the Fused-Core particle column brings new features to the SIC system – a higher separation efficiency, high mass capacity, wide range of working pH 2 – 9, good choice of column chemistries and column dimensions and the lower price compared to the monolithic columns typically used in SIC.

This work in flow methods can be considered as a broadening of the chromatographic abilities of the SIC and follows the trends in rapid chromatographic separations. Improved performance of chromatography and the typical features of SIC – discontinuous flow, easy sample handling, simple operation and portability of the instrument enables reduction in cost per analysis.

Trademarks

Fused-Core – Advanced Materials Technology, Inc.
SIChrom – FIAlab Instruments, Inc.

Figures 2 and 3 used with permission of Elsevier Limited, Oxford, UK.

Materials

     

 References

  1. D. Šatínský, P. Solich, P. Chocholouš, R. Karlíèek, Anal. Chim. Acta 499 2003 205 - 214.
  2. P. Chocholouš, P. Solich, D. Šatínský, Anal. Chim. Acta 600 2007 129 - 135.
  3. P. Chocholouš, L. Kosarová, D. Šatínský, H. Sklenárová, P. Solich, Talanta 85 (2011) 1129 - 1134.

 

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