# Patent

## Title
Methods and apparatus for the analysis of vitamin D metabolites

## Abstract

The present disclosure relates to CO2-based chromatography for the efficient and precise separation of Vitamin D metabolites.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage Application of International Application No. PCT/US2013/072057, filed Nov. 26, 2013, which claims priority to U.S. Provisional Application No. 61/731,883, filed Nov. 30, 2012, which Each of the foregoing applications is incorporated herein by reference in its entirety.


FIELD OF THE TECHNOLOGY
The present disclosure relates to CO2-based chromatography for use in the analysis and characterization of Vitamin D metabolites.
BACKGROUND
Vitamin D is a fat-soluble secosteroid that is responsible for intestinal absorption of calcium and phosphate. The metabolic pathway for Vitamin D is complex and involves multiple mono-, di- and tri-hydroxy-forms of both Vitamin D3 and Vitamin D2 together with a number of epimer and “pre” isomers. In humans, Vitamin D3 is synthesized in the skin by the action of sun-light while a relatively small amount of vitamin D2 is obtained from dietary sources. In some cases, pharmaceutical Vitamin D2 might be taken as a supplement. In the liver, Vitamin D is converted to 25-hydroxyvitamin D (abbreviated 25-OH-Vit-D) and current clinical practice uses the total serum 25-OH-Vit-D (i.e., 25-OH-Vit-D3 plus 25-OH-Vit-D2) levels to assess a person's Vitamin D status. The 25-OH-Vit-D metabolite is then converted primarily in the kidneys to the hormonally active form 1,25-dihydroxyvitamin D (abbreviated 1,25-(OH)2-Vit-D). 1,25-(OH)2-Vit-D circulates as a hormone in the blood and is responsible for regulating the concentration of calcium and phosphate in the bloodstream to promote healthy growth and remodeling of bone. 25-OH-Vit-D is also converted to 1,25-(OH)2-Vit-D outside of the kidneys for other purposes, such as in the proliferation, differentiation, and apoptosis of cells.
Other metabolites also prove to be informative and relevant to the overall biological effect of Vitamin D. For example, while the metabolite 24,25-(OH)2-Vit-D alone has no known biological activity, it represents the first step in the pathway to degrade 25-OH-Vit-D and is not routinely measured. The enzyme responsible (Vitamin D 24-hydroxylase; CYP24A1) plays an important role in maintaining a normal (“sufficient”) concentration of 25-OH-Vit-D. Too high a concentration of 25-OH-Vit-D can cause hypercalcemia and recent evidence suggests that at least some cases of Idiopathic Infantile Hypercalcemia (IIH) might be the result of natural polymorphisms in the CYP24A1 gene.
It has been recently recognized that a structural isomer of 25-OH-Vit-D3 (the C3-epimer), which was thought only to be present in pediatric samples, is also present in adults. C3-epi-25-OH-Vit-D3 has no known biological relevance and it should therefore not be included in the measurement of Vitamin D status. Recent studies have highlighted that the majority of LC/MS/MS assays used by laboratories to report results to the Vitamin D External Quality Assurance Scheme (DEQAS) are not designed to differentiate between 25-OH-Vit-D3 and the C3-epimer. This is because the two isomers (25-OH-Vit-D3 and the C3-epimer) have identical chemical composition, molecular mass and MS/MS characteristics such that they cannot be separated solely on the basis of mass spectrometry. Instead, both isomers must first be separated from each other before they are introduced into the analyser region of the mass spectrometer. This can be achieved using conventional liquid chromatography. However, this difficult separation can take, if at all, from approximately 6 minutes to approximately 12 minutes per sample, which makes routine adoption of LC/MS based assays in a clinical laboratory impractical. Although some immunoassays are able to differentiate between 25-OH-Vit-D3 and the C3-epimer, there are other interferences and difficulties which result in most immunoassays having relatively poor assay characteristics (accuracy, precision, linearity etc.). Thus, at present there appears to be no single assay for Vitamin D status that provides all the desirable assay characteristics.
LC/MS analyses can quantitatively measure multiple analytes in the same analytical run. For example, in addition to analyzing 25-OH-Vit-D2 and 25-OH-Vit-D3 separately from C3-epi-25-OH-Vit-D3, an LC/MS method can also measure other Vitamin D metabolites (e.g., 24,25-(OH)2-Vit-D, 1,25-(OH)2-Vit-D, etc.) in the same analysis. Such a multi-analyte analysis can provide a much more robust Vitamin D status. However, the analysis times of an LC/MS Vitamin D metabolite panel such as this becomes lengthy and impractical for routine laboratories.
In addition, because some Vitamin D metabolites are present at low concentrations in serum and plasma (e.g., 1,25-(OH)2-Vit-D, 1,24,25-(OH)3-Vit-D, etc), derivatization methods with Cookson-type reagents (e.g., 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and DMEQ-TAD (4-[2-(3,4-Dihydro-6,7-dimethoxy-4-methyl-3-oxo-2-quinoxalinyl)ethyl]-3H-1,2,4-triazole-3,5(4H)-dione)) to form, e.g., PTAD-linked Vitamin D metabolites derivatives, have been used as a way to increase the sensitivity of detecting these metabolites by mass spectrometry. However, this derivatization process results in pairs of isomeric derivatives for each analyte and may create inferences. Therefore, a significant challenge has been to resolve, in a short analysis time compatible with high sample throughput, these low concentration derivatives under current chromatographic techniques.
SUMMARY
Given the importance to accurately determine a person's Vitamin D status, and to further understand the biological implications involved in the metabolic pathway for Vitamin D, e.g., being able to effectively monitor a panel of Vitamin D metabolites to diagnosis conditions at an early stage, there is a need to develop efficient chromatographic methods for the precise analysis of Vitamin D metabolites. For example, an efficient and precise method of analyzing Vitamin D metabolites, such as analyzing 25-OH-Vit-D2 and 25-OH-Vit-D3 separately from C3-epi-25-OH-Vit-D3, would prove useful in providing accurate measurements of a person's Vitamin D status. Also, increasing the separation efficiency of low concentration metabolites (e.g., 1,25-(OH)2-Vit-D or 1,24,25-(OH)3-Vit-D) and/or derivatives thereof provides a superior advancement in the commercial analysis of Vitamin D metabolite panels.
Exemplary embodiments of the present disclosure are directed to rapid and efficient methods for the separation of Vitamin D metabolites. The present disclosure is based, in part, on the discovery that a CO2-based chromatography system (e.g., ACQUITY UPC2®, Waters Corporation, Milford, Mass.) with features, such as, e.g., improved pressure stability, improved sample injection, and superior column chemistry, could reproducibly substantially resolve metabolites of Vitamin D.
Many clinical laboratories have developed routine LC/MS/MS assays to assess Vitamin D status (i.e., the total concentration of 25-OH-Vit-D in serum) to replace automated immunoassay techniques that can suffer from interference. However, commercially efficient and effective separations of certain Vitamin D metabolites, particularly C3-epi-25-OH-Vit-D3, has not yet been achieved. Therefore, one aspect of the present disclosure provides an efficient and precise method for separating C3-epi-25-OH-Vit-D3 using CO2-based chromatography. The methods described herein comprise, at least in part, a CO2-based chromatography method for separating metabolites of Vitamin D, wherein at least a portion of the CO2 used is in a supercritical state (or near supercritical state or STP).
The present disclosure also provides efficient and precise methods for separating low concentration metabolites (e.g., 1,25-(OH)2-Vit-D or 1,24,25-(OH)3-Vit-D) and derivatives thereof, metabolites involved in the determination of a person's Vitamin D status (25-OH-Vit-D2, 25-OH-Vit-D3, and C3-epi-25-OH-Vit-D3), and enzymes and metabolites involved or implicated in Vitamin D metabolic processes.
In addition to other advantages, the CO2-based chromatography methods described herein minimize consumption of mobile phase solvents (e.g., methanol) thereby generating less waste for disposal and reducing the cost of analysis per sample. Also, because relatively short chromatographic run times (less than 5 minutes) are typically achieved with effective separation, the unique speed and resolution provided by the CO2-based chromatography methods described herein serve as a key element in developing high-throughput routine screening assays.



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other features and advantages provided by the present disclosure will be more fully understood from the following description of exemplary embodiments when read together with the accompanying drawings, in which:
FIG. 1 is an exemplary comparison of A) CO2-based chromatography/MS/MS; and B) UPLC/MS/MS separations of 25-OH-Vit-D3 and C3-epi-25-OH-Vit-D3.
FIG. 2 represents a rapid CO2-based chromatography/MS/MS analysis of Vitamin D metabolites using a BEH C18 column (1.7 um, 2.1 mm×50 mm).
FIG. 3 represents a rapid CO2-based chromatography/MS/MS analysis of Vitamin D metabolites including the C3-epi-25-OH-Vit-D3 using a fluorophenyl based column (1.7 um, 2.1 mm×150 mm).



DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present disclosure provides a method of separating one or more metabolites of Vitamin D, comprising: placing a sample in a CO2-based chromatography system comprising a chromatography column; and eluting the sample by a gradient of organic solvent and a mobile phase fluid comprising CO2 to substantially resolve the one or more metabolites. In some embodiments, the retention times for the one or more metabolites are less than 5 minutes, such as, e.g., from about 0.5 to about 3 minutes.
In one embodiment, the chromatography column comprises particles having an average size of about 1.7, about 3.5, or about 5.0 microns. In another embodiment, the chromatography column comprises charged surface hybrid particles having a particle size of about 1.7 microns or about 3.5 microns.
In one embodiment, the chromatography column is a fluorophenyl based chromatography column.
In one embodiment, the one or more metabolites are selected from 25-OH-Vitamin D3; 25-OH-Vitamin D2; C3-epi-25-OH-Vitamin D3; 24, 25-dihydroxy-Vitamin D; 1α,25-dihydroxy-Vitamin D; 23(R), 25-dihydroxy-Vitamin D; 23(S), 25-dihydroxy-Vitamin D; 25,26-dihydroxy-Vitamin D; 4β,25-dihydroxy-Vitamin D; calcitroic acid; and 1,24,25-trihydroxy-Vitamin-D. In another embodiment, the metabolites are 25-OH-Vitamin D3 or C3-epi-25-OH-Vitamin D3.
In one embodiment, the total elution times for the one or more metabolites is about 2 minutes on a chromatography column having a length of about 150 mm.
In one embodiment, the CO2-based chromatography system comprises an operating system pressure of about 1,000 to about 9,000 psi and a backpressure of about 1,000 to about 9,000 psi.
In one embodiment, the CO2-based chromatography system comprises one or more pumps for delivering a flow of the mobile phase fluid comprising CO2; and an injection valve subsystem in fluidic communication with the one or more pumps and the chromatography column.
As described herein, the injection valve subsystem comprises:

    
    
        an auxiliary valve comprising:
        
            an auxiliary valve stator, comprising a first plurality of stator ports, in fluidic communication with the one or more pumps and the chromatography column; and
            an auxiliary valve rotor comprising a first plurality of grooves;
        
        
        an inject valve comprising:
        
            an inject valve stator comprising a second plurality of stator ports; and
            an inject valve rotor comprising a second plurality of grooves;
        
        
        a sample loop fluidically connected to the inject valve stator for receiving a sample slug to be introduced into a mobile phase fluid flow; and
        fluidic tubing fluidically connecting the auxiliary valve stator and the inject valve stator, wherein the auxiliary valve rotor is rotatable, relative to the auxiliary valve stator, between a plurality of discrete positions to form different fluidic passageways within the auxiliary valve; wherein the inject valve rotor is rotatable, relative to the inject valve stator, between a plurality of discrete positions to form different fluidic passageways within the inject valve, and wherein the respective positions of the auxiliary valve rotor and the inject valve rotor can be coordinated in such a manner as to allow the sample loop and the fluidic tubing to be pressurized to a high system pressure with the mobile phase fluid before they are placed in fluidic communication with the chromatography column.
    
    


The methods described herein may, further comprise obtaining a mass spectrometer signal of the one or more metabolites.
In other alternative embodiments, the present disclosure provides a method of separating C3-epi-25-OH-Vitamin D3 from a sample (e.g. Vitamin D or a biological sample from a human specimen), comprising:
placing a sample in a CO2-based chromatography system comprising a fluorophenyl based chromatography column; and
eluting the sample by a gradient of organic solvent and a mobile phase fluid comprising CO2 to substantially resolve C3-epi-25-OH-Vitamin D3, wherein the retention time for C3-epi-25-OH-Vitamin D3 is less than 5 minutes and wherein the CO2-based chromatography system is defined as above and optionally coupled to a mass spectrometer.
In one embodiment, the retention time for the C3-epi-25-OH-Vitamin D3 ranges from about 0 to about 3 minutes, such as, e.g., from about 1 to about 2 minutes using the CO2-based chromatography system described above.
In another embodiment, the present disclosure provides a method of separating low concentration Vitamin D metabolites (e.g., 1,25-(OH)2-Vit-D or 1,24,25-(OH)3-Vit-D), or derivatives thereof (e.g., derivatives generated by derivatisation methods such as with Cookson-type reagents), from a sample (e.g. Vitamin D), comprising:
placing a sample in a CO2-based chromatography system comprising a chromatography column (e.g., a fluorophenyl based column); and
eluting the sample by a gradient of organic solvent and a mobile phase fluid comprising CO2 to substantially resolve the low concentration metabolites, or derivatives thereof, wherein the retention times for the low concentration metabolites, or derivatives thereof, is less than 5 minutes and wherein the CO2-based chromatography system is defined as above and optionally coupled to a mass spectrometer.
Kits for quantifying one or more metabolites of Vitamin D obtained by the methods of any methods described herein are also provided. In one embodiment, a kit may the comprise a first known quantity of a first calibrator, a second known quantity of a second calibrator, and optionally comprising one or more metabolites of Vitamin D, wherein the first known quantity and the second known quantity are different, and wherein the first calibrator, the second calibrator, and the one or more metabolites are each distinguishable in a single sample by mass spectrometry.
The kits as described herein may also comprise instructions for:
(i) obtaining a mass spectrometer signal comprising a first calibrator signal, a second calibrator signal, and one or more metabolites of Vitamin D from the single sample comprising the first known quantity of the first calibrator, the second known quantity of the second calibrator, and optionally comprising one or more metabolites of Vitamin D; and
(ii) quantifying one or more metabolites of Vitamin D in the single sample using the first calibrator signal, the second calibrator signal, and the signal of the one or more metabolites of Vitamin D.
In some embodiments, the first calibrator and the second calibrator are each analogues, derivatives, metabolites, or related compounds of the one or more metabolites of Vitamin D.
Kits may also comprise a third known quantity of a third calibrator and a fourth known quantity of a fourth calibrator, wherein the third known quantity and the fourth known quantity are different, and wherein the first calibrator, the second calibrator, the third calibrator, the fourth calibrator, and the one or more metabolites of Vitamin D are each distinguishable in a single sample by mass spectrometry. These kits may also further comprise instructions for:
(i) obtaining a mass spectrometer signal comprising a third calibrator signal, a fourth calibrator signal, and one or more metabolites of Vitamin D from the single sample comprising the third known quantity of the third calibrator, the fourth known quantity of the fourth calibrator, and optionally comprising one or more metabolites of Vitamin D; and
(ii) quantifying one or more metabolites of Vitamin D in the single sample using the third calibrator signal, the fourth calibrator signal, and the signal of the one or more metabolites of Vitamin D.
The kits described herein may further comprise additional calibrators, such as, e.g., from 5 to 10 calibrators including both nonzero and blank calibrators. Instructions for obtaining a mass spectrometer signal and quantifying one or more metabolites of Vitamin D using these additional calibrators is also contemplated. In one exemplary embodiment, the kit contains 6 nonzero calibrators and a single blank calibrator.
In one embodiment, the kits described herein comprise one or more metabolites selected from 25-OH-Vitamin D3; 25-OH-Vitamin D2; C3-epi-25-OH-Vitamin D3; 24, 25-dihydroxy-Vitamin D; 1α,25-dihydroxy-Vitamin D; 23(R), 25-dihydroxy-Vitamin D; 23(S), 25-dihydroxy-Vitamin D; 25,26-dihydroxy-Vitamin D; calcitroic acid,4β,25-dihydroxy-Vitamin D; C3-epi-1-α,25-dihydroxy-Vitamin D3, and 1,24,25-trihydroxy-Vitamin-D. In other embodiments, the metabolites are 25-OH-Vitamin D3 or C3-epi-25-OH-Vitamin D3.
Computer readable mediums are also provided such that a computer readable medium may comprise computer executable instructions adapted to:
separating one or more metabolites of Vitamin D, or derivatives thereof, as described herein (e.g., 25-OH-Vitamin D3, 25-OH-Vitamin D2, C3-epi-25-OH-Vitamin D3, 1,25-(OH)2-Vit-D, or 1,24,25-(OH)3-Vit-D, etc.);
obtaining a mass spectrometer signal comprising a first known quantity of a first calibrator, a second known quantity of a second calibrator, and optionally comprising one or more metabolites of Vitamin D, wherein the first known quantity and the second known quantity are different, and wherein the first calibrator, the second calibrator, and the one or more metabolites are each distinguishable in a single sample by mass spectrometry.
The computer readable medium may further comprise executable instructions adapted to quantifying one or more metabolites of Vitamin D in the single sample using the first calibrator signal, the second calibrator signal, and the signal of the one or more metabolites of Vitamin D.
EXAMPLES
General Conditions for the Analysis of Vitamin D Metabolites
The autosampler (Acquity UPC2® Autosampler, Waters Corporation, Milford, Mass.) settings set forth in the Table 1 below were used in the Vitamin D metabolite analyses. Injection volumes were controlled by a sample list (MassLynxTM Software, Waters Corporation, Milford, Mass.) and recorded separately for each analysis.










TABLE 1







Parameter
Setting









Load Ahead
Disabled



Injection Mode
Partial Loop With Needle




Overfill



LoopOffline
Disable



Weak Wash Solvent Name



Weak Wash Volume
600 uL



Strong Wash Solvent Name



Strong Wash Volume
200 uL



Target Column Temperature
Off C.



Column Temperature Alarm Band
Disabled



Target Sample Temperature
4.0 C.



Sample Temperature Alarm Band
Disabled



Full Loop Overfill Factor
Automatic



Syringe Draw Rate
Automatic



Needle Placement
Automatic



Pre-Aspirate Air Gap
Automatic



Post-Aspirate Air Gap
Automatic



Column Temperature Data Channel
No



Ambient Temperature Data Channel
No



Sample Temperature Data Channel
No



Sample Organizer Temperature Data
No



Channel



Sample Pressure Data Channel
No



Switch 1
No Change



Switch 2
No Change



Switch 3
No Change



Switch 4
No Change



Chart Out
Sample Pressure



Sample Temp Alarm
Disabled



Column Temp Alarm
Disabled



Run Events
Yes



Needle Overfill Flush
Automatic










Mass spectrometry data was obtained using a tandem quadrupole mass spectrometer (Xevo® TQD tandem quadrupole, Waters Corporation, Milford, Mass.), which was operated under the conditions set forth in Table 2. Eluent from the CO2-based chromatography system (ACQUITY UPC2®, Waters Corporation, Milford, Mass.) was connected to the electrospray source via a splitter device supplied with a methanol make-up flow that was manually controlled. The make-up flow rate was varied to optimise performance. The optimum flow rate was between 0.1 and 0.8 mL/min










TABLE 2







Parameter
Setting









Ionisation
Electrospray +ve



Capillary Voltage (kV)
2.80



Cone Voltage (V)
30.00



Extractor Voltage(V)
3.00



RF Voltage (V)
0.10



Source Temperature (° C.)
150



Desolvation Temperature (° C.)
400



Cone Gas Flow (L/Hr)
50



Desolvation Gas Flow (L/Hr)
750



Collision Gas Flow (mL/Min)
0.15



LM 1 Resolution
6.02



HM 1 Resolution
14.58



Ion Energy 1
0.26



MS Mode Entrance
50.00



MS Mode Collision Energy
20.00



MS Mode Exit
50.00



MSMS Mode Entrance
1.00



MSMS Mode Collision Energy
20.00



MSMS Mode Exit
0.50



LM 2 Resolution
11.57



HM 2 Resolution
14.90



Ion Energy 2
1.73










The multiple reaction monitoring transitions presented in Table 3 were used to monitor the Vitamin D metabolites.












TABLE 3







Dwell
Cone
Collision





Time
Voltage
Energy


Compound
MRM
(S)
(V)
(eV)
Delay







25-OH-D3*
383.35 >
0.017
30.0
15.0
Auto



257.32


25-OH-D2
395.35 >
0.017
30.0
15.0
Auto



269.32


1,25-OH2-
399.40 >
0.017
30.0
20.0
Auto


D3
135.10





*Because 25-OH-Vit-D3 and the C3-epimer have identical chemical composition, this MRM channel detected both analytes.






Example 1: Separation and Analysis of 25-OH-Vitamin D3 and C3-epi-25-OH-Vitamin D3 
Pure standards of C3-epi-25-OH-Vitamin D3 and 25-OH-Vitamin D3 were dissolved in hexane and analysed in separate injections (2 uL per injection, approximately 300 pg of each analyte) using a CO2-based chromatography system (e.g., ACQUITY UPC2®, Waters Corporation, Milford, Mass.). The CO2-based chromatography system was fitted with a fluorophenyl based column (ACQUITY UPC2TM CSH Fluoro-Phenyl Column, 130 Å, 1.7 μm, 2.1 mm×150 mm, Waters Corporation, Milford, Mass.) and coupled to a tandem quadrupole mass spectrometer (Xevo® TQD tandem quadrupole, Waters Corporation, Milford, Mass.). The column was eluted at a flow rate of 1.0 mL/min using CO2 mobile phase with a gradient of methanol from 5% to 20% over 1.5 min.
Retention times of approximately 1.2 and 1.4 minutes for 25-OH-Vitamin D3 and C3-epi-25-OH-Vitamin D3 respectively, with baseline resolution, is shown by FIG. 1a where the two separate analyses are overlayed. The total run time of 2 minutes demonstrated substantial resolution at approximately 3 times faster when compared to conventional UPLC/MS/MS technologies. For comparison, FIG. 1b shows a human plasma sample that was spiked with C3-epi-25-OH-Vitamin D3 and subjected to liquid-liquid extraction before analysis using an existing UPLC/MS/MS method. Only partial resolution of the isomers is obtained in a 6 minute analysis.
Example 2: Separation and Analysis of 25-OH-Vitamin D3, 25-OH-Vitamin D2, and 1,25-(OH)2-Vitamin D3 
Using a C18 UPLC chromatography column (ACQUITY UPLC® BEH C18 Column, 1.7 um, 2.1 mm×50 mm, Waters Corporation, Milford, Mass.), 25-OH-Vit-D2, 25-OH-Vit-D3, and 1,25-(OH)2Vit-D3 were eluted in less than 1.25 minutes using a CO2-based chromatography system (e.g., ACQUITY UPC2®, Waters Corporation, Milford, Mass.). The column was eluted at a flow rate of 2.0 mL/min using CO2 mobile phase with a gradient of 2.5% to 20% methanol over 1.0 min Chromatographic peak widths of approximately 1.5 seconds at the base were observed and shown in FIG. 2. This chromatograph shows that efficient separation of 25-OH-Vit-D2, 25-OH-Vit-D3, and 1,25-(OH)2Vit-D3 can be achieved using a CO2-based chromatography system fitted with a reverse phase C18 column.
Example 3: Separation and Analysis of 25-OH-Vitamin D3, 25-OH-Vitamin D2, 1,25-(OH)2-Vitamin D3, and C3-epi-25-OH-Vitamin D3 
Standards for 25-OH-Vitamin D2, 25-OH-Vitamin D3 and 1,25-(OH)2-Vitamin D3 were combined in hexane and 2 uL (approximately 300 pg of each analyte) was analyzed using a CO2-based chromatography system (e.g., ACQUITY UPC2®, Waters Corporation, Milford, Mass.). The CO2-based chromatography system was fitted with a fluorophenyl based column (ACQUITY UPC2TM CSH Fluoro-Phenyl Column, 130 Å, 1.7 μm, 2.1 mm×150 mm, Waters Corporation, Milford, Mass.). The column was eluted at a flow rate of 1.0 mL/min using CO2 mobile phase with a gradient of methanol from 5% to 20% over 1.5 min. In a separate analysis, 2 uL of a hexane solution of C3-epi-25-OH-Vitamin D3 (approximately 300 pg) was analysed under the same conditions. As shown by FIG. 3, when the chromatograms are overlayed, baseline resolution of 25-OH-Vit-D3 and C3-epi-25OH-Vit-D3, with 25OH-Vit-D2 and 1,25-(OH)2-Vit-D was observed with retention times up to approximately 2 min. It was also noted that additional optimization and the use of other column geometries could further enhance the speed of analysis.
The specification should be understood as disclosing and encompassing all possible permutations and combinations of the described aspects, embodiments, and examples unless the context indicates otherwise. One of ordinary skill in the art will appreciate that the invention can be practiced by other than the summarized and described aspect, embodiments, and examples, which are presented for purposes of illustration, and that the invention is limited only by the following claims.

## Claims

1. A method of separating C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit-D from a sample, comprising:
placing the sample in a CO2-based chromatography system comprising a flurophenyl based chromatography column; and
eluting the sample by a gradient of methanol and a mobile phase fluid comprising CO2 to substantially resolve each of C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit-D, and
wherein the resolved C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, 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-D3, 25-OH-Vit D2, 25-OH-Vit D3, 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-D3, 25-OH-Vit D2, 25-OH-Vit D3, 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-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit D in a sample comprising:
a known quantity of a C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, 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-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit D from the sample using a CO2-based chromatography system, wherein separation includes a gradient of methanol and wherein the resolved C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, 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-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit D signal from the sample comprising the known quantity of C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit D; and
quantifying C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, and 1,25-(OH)2-Vit D in the sample using the C3-epi-25-OH-Vit-D3, 25-OH-Vit D2, 25-OH-Vit D3, 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.