Methods and apparatus for the analysis of vitamin D metabolites

Abstract

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

Claims

1. A method of separating C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit-D from a sample, comprising: placing the sample in a CO.sub.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.sub.2 to substantially resolve each of C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit-D, and wherein the resolved C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.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.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.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.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.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.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit D in a sample comprising: a known quantity of a C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.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.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit D from the sample using a CO.sub.2-based chromatography system, wherein separation includes a gradient of methanol and wherein the resolved C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.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.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit D signal from the sample comprising the known quantity of C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit D; and quantifying C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.2-Vit D in the sample using the C3-epi-25-OH-Vit-D.sub.3, 25-OH-Vit D.sub.2, 25-OH-Vit D.sub.3, and 1,25-(OH).sub.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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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:

(2) FIG. 1 is an exemplary comparison of A) CO.sub.2-based chromatography/MS/MS; and B) UPLC/MS/MS separations of 25-OH-Vit-D.sub.3 and C3-epi-25-OH-Vit-D.sub.3.

(3) FIG. 2 represents a rapid CO.sub.2-based chromatography/MS/MS analysis of Vitamin D metabolites using a BEH C18 column (1.7 um, 2.1 mm×50 mm).

(4) FIG. 3 represents a rapid CO.sub.2-based chromatography/MS/MS analysis of Vitamin D metabolites including the C3-epi-25-OH-Vit-D.sub.3 using a fluorophenyl based column (1.7 um, 2.1 mm×150 mm).

DETAILED DESCRIPTION OF THE INVENTION

(5) In one embodiment, the present disclosure provides a method of separating one or more metabolites of Vitamin D, comprising: placing a sample in a CO.sub.2-based chromatography system comprising a chromatography column; and eluting the sample by a gradient of organic solvent and a mobile phase fluid comprising CO.sub.2 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.

(6) 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.

(7) In one embodiment, the chromatography column is a fluorophenyl based chromatography column.

(8) In one embodiment, the one or more metabolites are selected from 25-OH-Vitamin D.sub.3; 25-OH-Vitamin D.sub.2; C3-epi-25-OH-Vitamin D.sub.3; 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 D.sub.3 or C3-epi-25-OH-Vitamin D.sub.3.

(9) 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.

(10) In one embodiment, the CO.sub.2-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.

(11) In one embodiment, the CO.sub.2-based chromatography system comprises one or more pumps for delivering a flow of the mobile phase fluid comprising CO.sub.2; and an injection valve subsystem in fluidic communication with the one or more pumps and the chromatography column.

(12) 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.

(13) The methods described herein may, further comprise obtaining a mass spectrometer signal of the one or more metabolites.

(14) In other alternative embodiments, the present disclosure provides a method of separating C3-epi-25-OH-Vitamin D.sub.3 from a sample (e.g. Vitamin D or a biological sample from a human specimen), comprising:

(15) placing a sample in a CO.sub.2-based chromatography system comprising a fluorophenyl based chromatography column; and

(16) eluting the sample by a gradient of organic solvent and a mobile phase fluid comprising CO.sub.2 to substantially resolve C3-epi-25-OH-Vitamin D.sub.3, wherein the retention time for C3-epi-25-OH-Vitamin D.sub.3 is less than 5 minutes and wherein the CO.sub.2-based chromatography system is defined as above and optionally coupled to a mass spectrometer.

(17) In one embodiment, the retention time for the C3-epi-25-OH-Vitamin D.sub.3 ranges from about 0 to about 3 minutes, such as, e.g., from about 1 to about 2 minutes using the CO.sub.2-based chromatography system described above.

(18) In another embodiment, the present disclosure provides a method of separating low concentration Vitamin D metabolites (e.g., 1,25-(OH).sub.2-Vit-D or 1,24,25-(OH).sub.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:

(19) placing a sample in a CO.sub.2-based chromatography system comprising a chromatography column (e.g., a fluorophenyl based column); and

(20) eluting the sample by a gradient of organic solvent and a mobile phase fluid comprising CO.sub.2 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 CO.sub.2-based chromatography system is defined as above and optionally coupled to a mass spectrometer.

(21) 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.

(22) The kits as described herein may also comprise instructions for:

(23) (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

(24) (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.

(25) 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.

(26) 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:

(27) (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

(28) (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.

(29) 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.

(30) In one embodiment, the kits described herein comprise one or more metabolites selected from 25-OH-Vitamin D.sub.3; 25-OH-Vitamin D.sub.2; C3-epi-25-OH-Vitamin D.sub.3; 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 D.sub.3 or C3-epi-25-OH-Vitamin D.sub.3.

(31) Computer readable mediums are also provided such that a computer readable medium may comprise computer executable instructions adapted to:

(32) separating one or more metabolites of Vitamin D, or derivatives thereof, as described herein (e.g., 25-OH-Vitamin D.sub.3, 25-OH-Vitamin D.sub.2, C3-epi-25-OH-Vitamin D.sub.3, 1,25-(OH).sub.2-Vit-D, or 1,24,25-(OH).sub.3-Vit-D, etc.);

(33) 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.

(34) 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

(35) General Conditions for the Analysis of Vitamin D Metabolites

(36) 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 (MassLynx™ Software, Waters Corporation, Milford, Mass.) and recorded separately for each analysis.

(37) TABLE-US-00001 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

(38) 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 CO.sub.2-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

(39) TABLE-US-00002 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

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

(41) TABLE-US-00003 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-D.sub.3 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

(42) Pure standards of C3-epi-25-OH-Vitamin D.sub.3 and 25-OH-Vitamin D.sub.3 were dissolved in hexane and analysed in separate injections (2 uL per injection, approximately 300 pg of each analyte) using a CO.sub.2-based chromatography system (e.g., ACQUITY UPC2®, Waters Corporation, Milford, Mass.). The CO.sub.2-based chromatography system was fitted with a fluorophenyl based column (ACQUITY UPC.sup.2™ 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 CO.sub.2 mobile phase with a gradient of methanol from 5% to 20% over 1.5 min.

(43) Retention times of approximately 1.2 and 1.4 minutes for 25-OH-Vitamin D.sub.3 and C3-epi-25-OH-Vitamin D.sub.3 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 D.sub.3 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

(44) 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-D.sub.2, 25-OH-Vit-D.sub.3, and 1,25-(OH).sub.2Vit-D.sub.3 were eluted in less than 1.25 minutes using a CO.sub.2-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 CO.sub.2 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-D.sub.2, 25-OH-Vit-D.sub.3, and 1,25-(OH).sub.2Vit-D.sub.3 can be achieved using a CO.sub.2-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

(45) Standards for 25-OH-Vitamin D.sub.2, 25-OH-Vitamin D.sub.3 and 1,25-(OH).sub.2-Vitamin D.sub.3 were combined in hexane and 2 uL (approximately 300 pg of each analyte) was analyzed using a CO.sub.2-based chromatography system (e.g., ACQUITY UPC2®, Waters Corporation, Milford, Mass.). The CO.sub.2-based chromatography system was fitted with a fluorophenyl based column (ACQUITY UPC.sup.2™ 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 CO.sub.2 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 D.sub.3 (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-D.sub.3 and C3-epi-25OH-Vit-D.sub.3, with 25OH-Vit-D.sub.2 and 1,25-(OH).sub.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.

(46) 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.