Rapid analysis of steroids and steroid derivatives

Patent metadata

Field	Value
Publication number	US-9733222-B2
Application number	US-201314432313-A
Country	US
Kind code	B2
Filing date	Oct 2, 2013
Priority date	Oct 3, 2012
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

The subject technology is directed to a CO 2 -based chromatography system and method for rapid determination of the levels and/or the presence or absence of steroids or steroid derivatives in a sample.

First claim

What is claimed is:

1. A chromatography method for detecting one or more steroids or steroid derivatives in a sample comprising the steps of:
providing a sample comprising one or more steroids or steroid derivatives for analysis; wherein the sample is prepared with, extracted or dissolved in a diluent comprising at least about 60% organic solvent, with the proviso that the sample is analyzed without a solvent exchange step;
applying the sample to a chromatography column with a solid stationary phase comprising inorganic or hybrid particles having a mean particle size of about 0.5 to about 3.5 microns; wherein said hybrid particles comprise an inorganic portion and an organic portion wherein the organic portion of the hybrid particles comprises substituted or unsubstituted C1-C18 alkane, alkylene, alkenylene, alkynylene or arylene moiety bonded to one or more atoms of the inorganic portion; and wherein said inorganic or hybrid particles have a polar or polar/non-polar surface functionality and retain said one or more steroids or steroid derivatives;
eluting the one or more steroids or steroid derivatives from the chromatography column by a mobile phase comprising a mixture of CO 2 , and a modifier to form one or more eluted steroids or steroid derivatives; and
detecting said one or more eluted steroids or steroid derivatives.

2. The method of claim 1 , wherein the sample comprises a biological sample or a non-biological sample or a mixture thereof.

3. The method of claim 1 , wherein the particles have a mean particle size of about 0.5 to about 2 microns.

4. The method of claim 1 , wherein the particles have a mean pore volume in the range of about 0.1 to about 2.5 cm 3 /g.

5. The method of claim 1 , wherein the particles have a mean pore diameter in the range of about 100 to about 1000 Angstroms.

6. The method of claim 1 , wherein the inorganic particles comprise silicone, aluminum, titanium, cerium, zirconium, barium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silver, titanium, diamond, zinc, boron or oxides thereof, silicon carbide, carbon black, carbon nanotubes, ceramic, glass, metallic materials or mixtures thereof.

7. The method of claim 1 , wherein the inorganic portion of the hybrid particles comprises comprise silicone, aluminum, titanium, cerium, zirconium, barium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silver, titanium, diamond, zinc, boron or oxides thereof, silicon carbide, carbon black, carbon nanotubes, ceramic, glass, metallic materials or mixtures thereof.

8. The method of claim 1 , wherein the organic portion of the hybrid particles comprises substituted or unsubstituted C1-C18 alkylene, alkenylene, alkynylene or arylene moiety bridging two or more atoms of the inorganic portion.

9. The method of claim 1 , wherein the particles comprise free surface hydroxyl groups, free surface silanol groups or surface modifications with embedded polar functional groups.

10. The method of claim 1 , wherein the particles comprise free surface hydroxyl groups or free surface silanol groups and surface modifications with non-polar functional groups.

11. The method of claim 1 , wherein the chromatography column is kept in temperature range of about 5° C. to about 85° C.

12. The method of claim 1 , wherein the modifier is mixed with the CO 2 under a constant or gradient condition or both over an elution period or a fraction thereof.

13. The method of claim 1 , wherein the modifier is a polar water-miscible organic solvent comprising at least one of methanol, ethanol, isopropanol, acetonitrile, acetone, tetrahydrofuran or water.

14. The method of claim 12 , wherein the gradient condition comprises increasing or decreasing flow volume of the modifier over the elution period or a fraction thereof.

15. The method of claim 14 , wherein the elution period is about 2 min.

16. The method of claim 14 , wherein the gradient condition comprises increasing the flow volume of the modifier from about 0% to about 40% (v/v CO 2 ) or any intervals therebetween.

17. The method of claim 14 , wherein the gradient condition comprises increasing the flow volume of the modifier from about 1% to about 17% (v/v CO 2 ).

18. The method of claim 1 , wherein the CO 2 is liquid CO 2 in subcritical or supercritical state or both.

19. The method of claim 1 , wherein the detection comprises determining the level or the presence or absence of the one or more steroids or steroid derivatives.

20. The method of claim 1 , wherein the detection is by way of a UV detector; a mass spectrometer; Evaporative Light Scattering (ELS) detector or a photodiode array detector (PDA).

21. The method of claim 1 , wherein the sample is not subject to a derivatization step.

22. The method of claim 1 , wherein the chromatography column has a length of about 50 to 150 mm and an internal diameter about 2 to 4 mm.

23. The method of claim 1 , wherein the chromatography column is part of a chromatography system comprising a pre-column mobile phase dwell volume of about 75 to about 500 μL; wherein said pre-column mobile phase dwell volume is the volume of the mobile phase present in a fluidic connection between a junction at which the CO 2 and the modifier are mixed and the head of the chromatography column.

24. The method of claim 1 , wherein the one or more steroids or steroid derivatives are eluted from the chromatography column by the mobile phase with a flow rate of about 1 to 4 mL/min.

25. A chromatography method for detecting one or more steroids or steroid derivatives in a sample comprising the steps of:
providing a sample comprising one or more steroids or steroid derivatives for analysis; wherein the sample is prepared with, extracted or dissolved in a diluent comprising at least about 60% organic solvent, with the proviso that the sample is analyzed without a solvent exchange step;
applying the sample to a chromatography column with a solid stationary phase comprising inorganic or hybrid particles having a mean particle size of about 0.5 to about 3.5 microns; wherein said particles retain said one or more steroids or steroid derivatives and wherein said particles comprise 1) free surface hydroxyl groups, free surface silanol groups or surface modifications with embedded polar functional groups; or 2) free surface hydroxyl groups or free surface silanol groups and surface modifications with non-polar functional groups;
eluting the one or more steroids or steroid derivatives from the chromatography column by a mobile phase comprising a mixture of CO 2 , and a modifier to form one or more eluted steroids or steroid derivatives; and
detecting said one or more eluted steroids or steroid derivatives.

26. A chromatography method for detecting one or more steroids or steroid derivatives in a sample comprising the steps of:
providing a sample comprising one or more steroids or steroid derivatives for analysis; wherein the sample is prepared with, extracted or dissolved in a diluent comprising at least about 60% organic solvent, with the proviso that the sample is analyzed without a solvent exchange step;
applying the sample to a chromatography column with a solid stationary phase comprising inorganic or hybrid particles having a mean particle size of about 0.5 to about 3.5 microns; wherein said particles have a polar or polar/non-polar surface functionality and retain said one or more steroids or steroid derivatives;

Assignees

• Waters Technologies Corp

Inventors

• Hudalla Christopher J
• Fairchild Jacob N
• Hill Jason F

Classifications

• B01D15/322 Primary

  Normal bonded phase · CPC title

• B01J20/3251

  comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur · CPC title

• G01N2030/484

  Physics · mapped topic

• B01J20/3293

  Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers · CPC title

• B01J20/20

  comprising free carbon; comprising carbon obtained by carbonising processes · CPC title