Methods and apparatus for the analysis of vitamin D metabolites

Patent metadata

Field	Value
Publication number	US-9719970-B2
Application number	US-201314648197-A
Country	US
Kind code	B2
Filing date	Nov 26, 2013
Priority date	Nov 30, 2012
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

The present disclosure relates to CO 2 -based chromatography for the efficient and precise separation of Vitamin D metabolites.

First claim

What is claimed is:

1. A method of separating C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit-D from a sample, comprising:
placing the sample in a CO 2 -based chromatography system comprising a flurophenyl based chromatography column; and
eluting the sample by a gradient of methanol and a mobile phase fluid comprising CO 2 to substantially resolve each of C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit-D, and
wherein the resolved C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit-D elute in under 2 minutes.

2. The method of claim 1 , wherein at least one of C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit-D is a PTAD-linked derivative.

3. The method of claim 1 , further comprising introducing the eluent containing the C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit-D into a mass spectrometer.

4. The method of claim 3 , wherein the mass spectrometer is a tandem quadrupole mass spectrometer.

5. The method of claim 1 , wherein the methanol is present at about 5% at the beginning of the gradient, and the gradient is from about 5% to about 20% over between 1 to 1.5 minutes.

6. A kit for quantifying C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D in a sample comprising:
a known quantity of a C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D calibration standard;
a fluorophenyl based chromatography column having an average particle size of about 1.7 microns, wherein the column dimensions are 2.1 mm by 150 mm; instructions configured for
separating C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D from the sample using a CO 2 -based chromatography system, wherein separation includes a gradient of methanol and wherein the resolved C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D is configured to be eluted in under 2 minutes;
obtaining a mass spectrometer signal comprising a C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D signal from the sample comprising the known quantity of C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D; and
quantifying C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D in the sample using the C3-epi-25-OH-Vit-D 3 , 25-OH-Vit D 2 , 25-OH-Vit D 3 , and 1,25-(OH) 2 -Vit D signal.

7. The method of claim 1 , wherein the fluorophenyl based chromatography column comprises particles having an average size of about 1.7 microns.

8. The method of claim 1 , wherein the fluorophenyl based chromatography column dimensions are 2.1 mm by 150 mm.

9. The method of claim 1 , wherein the mobile phase flow rate is about 1 mL/min.

10. The method of claim 1 , wherein the fluorophenyl based chromatography column comprises particles having an average size of about 1.7 microns, wherein the column dimensions are 2.1 mm by 150 mm, and wherein the mobile phase flow rate is about 1 mL/min.

11. The kit of claim 6 , wherein the methanol is present at about 5% at the beginning of the gradient, and the gradient is from about 5% to about 20% over between 1 to 1.5 minutes.

Assignees

• Waters Technologies Corp

Inventors

• Vukovic John

Classifications

• C12N2500/38

  Vitamins · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N30/7233

  interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04) · CPC title

• G01N30/34 Primary

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title

• B01D11/0203

  with a supercritical fluid · CPC title