MASS SPECTROMETRIC DETERMINATION OF NON-DERIVATIZED, NON-METABOLIZED VITAMIN D
20230146212 · 2023-05-11
Inventors
Cpc classification
H01J49/004
ELECTRICITY
G01N30/7233
PHYSICS
International classification
Abstract
The invention relates to the detection of non-metabolized vitamin D. In a particular aspect, the invention relates to methods for detecting underivatized non-metabolized vitamin D by mass spectrometry.
Claims
1. A method for determining the amount of non-metabolized vitamin D.sub.3 in a sample by tandem mass spectrometry, the method comprising the steps of: (i) subjecting non-metabolized vitamin D.sub.3 from a sample to an ionization source under conditions suitable to generate one or more precursor ions comprising a mass to charge ratio (m/z) of 367.2±0.5; (ii) fragmenting at least one of said precursor ions to generate one or more fragment ions detectable by mass spectrometry, wherein the fragment ions comprise one or more ions selected from the group consisting of ions with m/z of 172.2±0.5, 145.0±0.5, and 119.1±0.5; (iii) determining the amount of one or more of the fragment ions generated in step (ii) by mass spectrometry; and (iv) relating the presence of non-metabolized vitamin D.sub.3 ions determined in step (iii) to the presence of non-metabolized vitamin D.sub.3 in the sample.
2. The method of claim 1, wherein the sample is subjected to an extraction column prior to ionization.
3. The method of claim 2, wherein the extraction column is a solid phase extraction (SPE) column.
4. The method of claim 2, wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column.
5. The method of claim 1, wherein the sample is further subjected to an analytical column prior to ionization.
6. The method of claim 5, wherein the analytical column is a high performance liquid chromatography (HPLC) column.
7. The method of claim 5, wherein the extraction and analytical columns and the ionization source of step (i) are connected in an on-line fashion.
8. The method of claim 1, wherein said ionization source is an atmospheric pressure chemical ionization (APCI) source.
9. The method of claim 1, wherein said mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning.
10. The method of claim 1, further comprising detecting non-metabolized vitamin D.sub.2 in the sample, wherein the non-metabolized vitamin D.sub.2 and non-metabolized vitamin D.sub.3 are ionized simultaneously.
11. The method of claim 1, wherein the sample comprises a biological sample, and wherein said biological sample is from a human, and the amount of non-metabolized vitamin D.sub.2 determined in the sample is the amount present in the sample when taken from the human.
12. The method of claim 1, wherein the sample comprises serum or plasma.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
DETAILED DESCRIPTION OF THE INVENTION
[0080] Methods are described for measuring vitamin D in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying vitamin D in a sample. The methods may utilize Cookson-type reagents, such as PTAD, to generate derivatized vitamin D. However, in some methods, no derivatizing agent is used, and underivatized vitamin D.sub.2 and/or vitamin D.sub.3 are detected by mass spectrometry.
[0081] The methods may use an extraction chromatography technique, such as turbulent flow liquid chromatography (TFLC), to perform a purification of underivatized or derivatized vitamin D.sub.2 and/or vitamin D.sub.3, combined with methods of mass spectrometry (MS), thereby providing a high-throughput assay system for detecting and quantifying vitamin D.sub.2 and/or vitamin D.sub.3 in a sample. Alternatively, in some methods, no chromatography, including extraction chromatography, is necessary for sample analysis. In these methods, the underivatized or derivatized vitamin D.sub.2 and/or vitamin D.sub.3 is ionized with LDTD. Preferred embodiments are particularly well suited for application in large clinical laboratories for automated vitamin D quantification.
[0082] Suitable test samples for use in methods of the present invention include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc. In certain preferred embodiments, samples are obtained from a mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably male or female humans. Preferred samples comprise bodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid, or tissue samples; preferably plasma (including EDTA and heparin plasma) and serum; most preferably serum. Such samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition.
[0083] The present invention also contemplates kits for a vitamin D quantitation assay. A kit for a vitamin D quantitation assay may include a kit comprising the compositions provided herein. For example, a kit may include packaging material and measured amounts of a Cookson-type reagent and an isotopically labeled internal standard, in amounts sufficient for at least one assay. Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged reagents for use in a vitamin D quantitation assay.
[0084] Calibration and QC pools for use in embodiments of the present invention are preferably prepared using a matrix similar to the intended sample matrix.
Sample Preparation for Mass Spectrometric Analysis
[0085] In preparation for mass spectrometric analysis, vitamin D may be enriched relative to one or more other components in the sample (e.g. protein) by various methods known in the art, including for example, liquid chromatography, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including ethyl acetate or methanol extraction, and the use of chaotropic agents or any combination of the above or the like.
[0086] Protein precipitation is one method of preparing a test sample, especially a biological test sample, such as serum or plasma. Protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatography B 2003, 785:263-275, describes protein precipitation techniques suitable for use in methods of the present invention. Protein precipitation may be used to remove most of the protein from the sample leaving vitamin D in the supernatant. The samples may be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively the samples may be filtered to remove precipitated proteins. The resultant supernatant or filtrate may then be applied directly to mass spectrometry analysis; or alternatively to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, samples, such as plasma or serum, may be purified by a hybrid protein precipitation/liquid-liquid extraction method. In these embodiments, a sample is mixed with methanol, ethyl acetate, and water, and the resulting mixture is vortexed and centrifuged. The resulting supernatant is removed, dried to completion and reconstituted in acetonitrile. The purified vitamin D may then be derivatized with any Cookson-type reagent, preferably PTAD or an isotopically labeled variant thereof.
[0087] Another method of sample purification that may be used prior to mass spectrometry is liquid chromatography (LC). Certain methods of liquid chromatography, including HPLC, rely on relatively slow, laminar flow technology. Traditional HPLC analysis relies on column packing in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. The skilled artisan will understand that separation in such columns is a diffusional process and may select LC, including HPLC, instruments and columns that are suitable for use with derivatized vitamin D. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles, or may include a monolithic material with porous channels. A surface of the medium typically includes a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded, cyano bonded surface, or highly pure silica surface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferred embodiments, the column is a highly pure silica column (such as a Thermo Hypersil Gold Aq column). The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. The sample may be supplied to the inlet port directly, or from an extraction column, such as an on-line SPE cartridge or a TFLC extraction column.
[0088] In one embodiment, the sample may be applied to the LC column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the analyte(s) of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a “mobile phase”), elution mode, gradient conditions, temperature, etc.
[0089] In certain embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. Such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample.
[0090] In one preferred embodiment, HPLC is conducted with an alkyl bonded analytical column chromatographic system. In certain preferred embodiments, a highly pure silica column (such as a Thermo Hypersil Gold Aq column) is used. In certain preferred embodiments, HPLC and/or TFLC are performed using HPLC Grade water as mobile phase A and HPLC Grade ethanol as mobile phase B.
[0091] By careful selection of valves and connector plumbing, two or more chromatography columns may be connected as needed such that material is passed from one to the next without the need for any manual steps. In preferred embodiments, the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system. Thus, an operator may place a tray of samples in an autosampler, and the remaining operations are performed under computer control, resulting in purification and analysis of all samples selected.
[0092] In some embodiments, an extraction column may be used for purification of vitamin D metabolites prior to mass spectrometry. In such embodiments, samples may be extracted using a extraction column which captures the analyte, then eluted and chromatographed on a second extraction column or on an analytical HPLC column prior to ionization. For example, sample extraction with a TFLC extraction column may be accomplished with a large particle size (50 μm) packed column. Sample eluted off of this column may then be transferred to an HPLC analytical column for further purification prior to mass spectrometry. Because the steps involved in these chromatography procedures may be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature may result in savings of time and costs, and eliminate the opportunity for operator error.
[0093] In some embodiments, protein precipitation is accomplished with a hybrid protein precipitation/liquid-liquid extraction method which includes methanol protein precipitation and ethyl acetate/water extraction from serum. The resulting vitamin D metabolites may be derivatized prior to being subjected to an extraction column. Preferably, the hybrid protein precipitation/liquid-liquid extraction method and the extraction column are connected in an on-line fashion. In preferred embodiments, the extraction column is a C-8 extraction column, such as a Cohesive Technologies C8XL online extraction column (50 μm particle size, 0.5×50 mm) or equivalent. The eluent from the extraction column may then be applied to an analytical LC column, such as a HPLC column in an on-line fashion, prior to mass spectrometric analysis. Because the steps involved in these chromatography procedures may be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature may result in savings of time and costs, and eliminate the opportunity for operator error.
Detection and Quantitation by Mass Spectrometry
[0094] In various embodiments, derivatized vitamin D may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), Laser diode thermal desorption (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand that the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.
[0095] Derivatized vitamin D may be ionized in positive or negative mode. In preferred embodiments, derivatized vitamin D is ionized by APCI or LDTD in positive ion mode.
[0096] In mass spectrometry techniques generally, after the sample has been ionized, the positively or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio. Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion traps analyzers, and time-of-flight analyzers. Exemplary ion trap methods are described in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.
[0097] The ions may be detected using several detection modes. For example, selected ions may be detected, i.e. using a selective ion monitoring mode (SIM), or alternatively, mass transitions resulting from collision induced dissociation or neutral loss may be monitored, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a “quadrupole” or “quadrupole ion trap” instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge ratio. The voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.
[0098] One may enhance the resolution of the MS technique by employing “tandem mass spectrometry,” or “MS/MS”. In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of ionization/fragmentation conditions, the MS/MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.
[0099] Alternate modes of operating a tandem mass spectrometric instrument include product ion scanning and precursor ion scanning. For a description of these modes of operation, see, e.g., E. Michael Thurman, et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez-Alba, ed., Elsevier 2005) (387).
[0100] The results of an analyte assay may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, external standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of vitamin D. Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. For example, in preferred embodiments one or more isotopically labeled vitamin D (e.g., vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3 and vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3) may be used as internal standards. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.
[0101] One or more steps of the methods may be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed in an on-line fashion.
[0102] In certain embodiments, such as MS/MS, where precursor ions are isolated for further fragmentation, collision activated dissociation (CAD) is often used to generate fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as “unimolecular decomposition.” Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.
[0103] In some preferred embodiments, vitamin D in a sample is detected and/or quantified using MS/MS as follows. The samples are first purified by protein precipitation or a hybrid protein precipitation/liquid-liquid extraction. Then, vitamin D in the purified sample is optionally derivatized with a Cookson-type reagent, such as PTAD. The purified samples are then subjected to liquid chromatography, preferably on an extraction column (such as a TFLC column) followed by an analytical column (such as a HPLC column); the flow of liquid solvent from a chromatographic column enters the nebulizer interface of an MS/MS analyzer; and the solvent/analyte mixture is converted to vapor in the heated charged tubing of the interface. The analyte(s) (e.g., derivatized or underivatized vitamin D) contained in the solvent, are ionized by applying a large voltage to the solvent/analyte mixture. As the analytes exit the charged tubing of the interface, the solvent/analyte mixture nebulizes and the solvent evaporates, leaving analyte ions. The analyte ions, e.g. precursor ions, pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3, respectively) based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. The first quadrupole of the mass spectrometer (Q1) selects for ions with the mass to charge ratios of interest. Precursor ions with the correct mass/charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. The fragment ions generated are passed into quadrupole 3 (Q3), where derivatized or underivatized vitamin D fragment ions are selected while other ions are eliminated.
[0104] The methods may involve MS/MS performed in either positive or negative ion mode; preferably positive ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of a derivatized vitamin D that may be used for selection in quadrupole 3 (Q3).
[0105] As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC-MS methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, may be measured and correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of vitamin D. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard.
EXAMPLES
Example 1: Hybrid Protein Precipitation/Liquid-Liquid Extraction and Cookson-Type Derivatization
[0106] The following automated hybrid protein precipitation/liquid-liquid extraction technique was conducted on patient serum samples. Gel Barrier Serum (i.e., serum collected in Serum Separator Tubes) as well as EDTA plasma and Heparin Plasma have also been established as acceptable for this assay.
[0107] A Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used to automate the following procedure. For each sample, 50 μL of serum was added to a well of a 96 well plate. Then 25 μL of internal standard cocktail (containing isotopically labeled vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3) was added to each well, and the plate vortexed. Then 75 μL of methanol was added, followed by additional vortexing. 300 μL of ethyl acetate and 75 μL of water was then added, followed by additional vortexing, centrifugation, and transfer of the resulting supernatant to a new 96-well plate.
[0108] The transferred liquid in the second 96-well plate was dried to completion under a flowing nitrogen gas manifold. Derivatization was accomplished by adding 100 μL of a 0.1 mg/mL solution of the Cookson-type derivatization agent PTAD in acetonitrile to each well. The derivatization reaction was allowed to proceed for approximately one hour, and was quenched by adding 100 μL of water to the reaction mixture.
Example 2: Extraction of Vitamin D with Liquid Chromatography
[0109] Sample injection was performed with a Cohesive Technologies Aria TX-4 TFLC system using Aria OS V 1.5.1 or newer software.
[0110] The TFLC system automatically injected an aliquot of the above prepared samples into a Cohesive Technologies C8XL online extraction column (50 μm particle size, 005×50 mm, from Cohesive Technologies, Inc.) packed with large particles. The samples were loaded at a high flow rate to create turbulence inside the extraction column. This turbulence ensured optimized binding of derivatized vitamin D to the large particles in the column and the passage of excess derivatizing reagent and debris to waste.
[0111] Following loading, the sample was eluted off to the analytical column, a Thermo Hypersil Gold Aq analytical column (5 μm particle size, 50×2.1 mm), with a water/ethanol elution gradient. The HPLC gradient was applied to the analytical column, to separate vitamin D from other analytes contained in the sample. Mobile phase A was water and mobile phase B was ethanol. The HPLC gradient started with a 35% organic gradient which was ramped to 99% in approximately 65 seconds.
Example 3: Detection and Quantitation of Derivatized Vitamin D by MS/MS
[0112] MS/MS was performed on the above generated samples using a Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation). The following software programs, all from Thermo Electron, were used in the Examples described herein: Quantum Tune Master V 1.5 or newer, Xcalibur V 2.07 or newer, LCQuan V 2.56 (Thermo Finnigan) or newer, and ARIA OS v1.5.1 (Cohesive Technologies) or newer. Liquid solvent/analyte exiting the analytical column flowed to the nebulizer interface of the MS/MS analyzer. The solvent/analyte mixture was converted to vapor in the tubing of the interface. Analytes in the nebulized solvent were ionized by APCI.
[0113] Ions passed to the first quadrupole (Q1), which selected vitamin D.sub.2 and vitamin D.sub.3 precursor ions with a mass-to-charge ratio of 572.3±0.5 m/z and 560.3±0.5 m/z, respectively. Ions entering quadrupole 2 (Q2) collided with argon gas to generate ion fragments, which were passed to quadrupole 3 (Q3) for further selection. Mass spectrometer settings are shown in Table 1. Simultaneously, the same process using isotope dilution mass spectrometry was carried out with internal standard, vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3. The mass transitions used for detection and quantitation during validation on positive polarity and at the indicated collision energies are shown in Table 2.
TABLE-US-00001 TABLE 1 Mass Spectrometer Settings for Detection of PTAD-vitamin D.sub.2, PTAD-vitamin D.sub.3, and vitamin D.sub.3-[6,19,19]-.sup.2H.sub.3 (internal standard) (Positive Polarity) Mass Spectrometric Instrument Settings Discharge Current 4.0 μA Vaporizer Temperature 300° C. Sheath Gas Pressure 15 Ion Sweep Gas Pressure 0.0 Aux Gas Pressure 5 Capillary Temperature 300° C. Skimmer Offset −10 V Collision Pressure 1.5 mTorr Collision Cell Energy 15 V
TABLE-US-00002 TABLE 2 Mass Transitions for PTAD derivatized vitamin D.sub.2, vitamin D.sub.3, and vitamin D.sub.3-[6,19,19]-.sup.2H.sub.3 (internal standard) (Positive Polarity) Precursor Product Analyte Ion (m/z) Ions (m/z) PTAD-vitamin D.sub.2 572.3 ± 0.5 298.1 ± 0.5 PTAD-vitamin D.sub.3 560.3 ± 0.5 298.1 ± 0.5 PTAD-vitamin D.sub.3- 563.3 ± 0.5 301.1 ± 0.5 [6,19,19]-.sup.2H.sub.3
[0114] Exemplary chromatograms for PTAD-vitamin D.sub.2, PTAD-vitamin D.sub.3, PTAD-vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3 (internal standard), are shown in
[0115] Exemplary calibration curves for the determination of vitamin D.sub.2 and vitamin D.sub.3 in serum specimens are shown in
Example 4: Linearity of Response for Detection of Derivatized Vitamin D MS/MS
[0116] Linearity was determined by diluting four pools of serum with high endogenous concentration of either vitamin D.sub.2 or vitamin D.sub.3 and analyzing dilutions of 75%, 50%, and 25% in duplicate according to the methods of Examples 1-3. Specimens may be diluted 1:4 with average recovery of 102%, permitting a Clinically Reportable Range (CRR) of 2-240 ng/mL within precision limits of 85%-115% CV. Measured values and percent recoveries from these studies are shown in Table 3.
TABLE-US-00003 TABLE 3 Data Demonstrating Linearity of Response over Dilution Range Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 Nutritional Vitamin D.sub.2 (ng/mL (percent recovery %)) Concen- tration 100% 25.0 24.5 28.1 24.4 28.1 26.7 (100) (100) (100) (100) (100) (100) 75% 17.8 17.5 21.9 17.3 21.2 20.1 (95.3) (95.4) (103.7) (94.5) (100.4) (100.5) 50% 12.8 13.0 14.0 13.8 14.8 11.8 (102.3) (106.2) (99.4) (112.8) (105.7) (88.9) 25% 6.7 6.2 7.7 6.7 7.3 7.4 (107.9) (101.4) (109.6) (109.6) (104.1) (110.7) Nutritional Vitamin D.sub.3 (ng/mL (percent recovery %)) Sample concen- tration 100% 31.2 33.5 33.4 29.1 29.6 30.6 (100) (100) (100) (100) (100) (100) 75% 22.1 23.6 25.8 22.0 24.8 24.1 (94.2) (93.8) (102.8) (100.8) (111.9) (104.8) 50% 15.9 16.1 16.5 15.7 15.9 15.0 (101.9) (96.1) (98.5) (107.8) (107.8) (98.0) 25% 7.6 7.9 8.5 7.3 8.0 8.0 (97.6) (94.3) (101.8) (100.4) (108.4) (105.0)
Example 5: Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) and Limit of Detection (LOD)
[0117] The lower limit of quantitation (LLOQ) is the point where measurements become quantitatively meaningful. The analyte response at this LLOQ is identifiable, discrete and reproducible with a precision (i.e., coefficient of variation (CV)) of greater than 20% and an accuracy of 80% to 120%. The LLOQ was determined by assaying samples with known analyte concentrations (2 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 40 ng/mL, and 60 ng/mL) in quadruplicate five times according to the methods of Examples 1-3, then evaluating the reproducibility. Analysis of the collected data indicates that samples with concentrations of less than 2 ng/mL yielded CVs of less than 20% for both analytes. Thus, the LLOQ of each analyte was determined to be <2 ng/mL. Data generated for the determination of LLOQ of PTAD-vitamin D.sub.2 and PTAD-vitamin D.sub.3 are shown in Tables 4 and 5, respectively. The graphical representations of CV versus concentration for both analytes are shown in
TABLE-US-00004 TABLE 4 Determination of Lower Limit of Quantitation of Vitamin D.sub.2 Vitamin D.sub.2 concentration Assay Assay Assay Assay Assay (ng/mL) 1 2 3 4 5 2 2.1 1.9 2.1 1.9 2.0 Average 2.04 1.7 2.0 2.1 1.9 2.0 Std Dev 0.2 1.9 2.0 2.2 2.2 2.3 CV (%) 8.5 1.9 1.9 2.1 2.5 2.2 Accuracy (%) 101.8 5 4.0 6.2 4.3 4.7 4.0 Average 4.7 NC 4.6 4.6 4.6 4.6 Std Dev 0.6 4.6 5.6 5.1 4.7 4.3 CV (%) 12.8 5.7 4.1 4.6 4.6 5.3 Accuracy (%) 94.8 10 9.8 11.2 10.8 11.3 10.3 Average 10.3 11.3 9.6 8.6 10.0 10.9 Std Dev 0.7 10.9 10.6 9.9 9.6 10.1 CV (%) 6.9 10.0 9.7 9.6 10.2 10.9 Accuracy (%) 102.6 15 13.6 14.7 15.6 15.0 14.5 Average 14.6 13.0 16.2 15.6 12.7 17.4 Std Dev 1.2 13.6 13.4 13.6 14.1 13.4 CV (%) 8.2 14.8 14.6 16.0 14.8 14.6 Accuracy (%) 97.1 20 19.4 19.1 20.3 19.9 19.1 Average 20.2 22.0 18.6 20.7 21.6 19.4 Std Dev 1.0 19.0 21.1 21.9 20.8 20.1 CV (%) 4.9 20.0 19.6 19.3 20.7 20.4 Accuracy (%) 100.8 40 42.2 41.5 39.1 42.5 41.8 Average 40.3 39.2 40.6 39.8 39.0 37.8 Std Dev 1.6 40.2 40.0 41.3 40.4 37.6 CV (%) 3.9 41.1 39.6 38.9 39.6 43.6 Accuracy (%) 100.7 60 64.4 57.8 59.1 59.3 57.0 Average 59.8 57.4 61.4 62.3 60.8 65.2 Std Dev 2.5 58.9 59.2 56.8 59.0 62.0 CV (%) 4.2 58.1 61.1 61.8 59.7 55.6 Accuracy (%) 99.7
TABLE-US-00005 TABLE 5 Determination of Lower Limit of Quantitation of Vitamin D.sub.3 Vitamin D.sub.3 concentration Assay Assay Assay Assay Assay (ng/mL) 1 2 3 4 5 2 2.1 1.6 2.2 2.0 1.8 Average 2.03 2.1 1.8 2.2 1.8 1.8 Std Dev 0.3 1.6 2.3 2.0 2.3 1.7 CV (%) 14.3 2.0 2.5 2.0 2.0 2.7 Accuracy (%) 101.4 5 4.7 5.6 4.6 5.0 4.9 Average 5.0 7.1 4.9 4.7 5.3 4.4 Std Dev 0.6 4.6 5.0 5.4 5.5 5.0 CV (%) 12.2 4.6 4.7 4.3 4.7 5.1 Accuracy (%) 99.7 10 9.4 10.2 10.1 9.9 11.0 Average 10.0 11.6 9.6 9.3 10.3 10.0 Std Dev 0.7 10.1 10.2 9.2 9.0 11.3 CV (%) 7.5 9.5 8.6 10.0 9.5 10.2 Accuracy (%) 99.5 15 14.5 14.9 14.4 15.7 15.8 Average 15.0 14.2 16.6 15.7 12.8 15.5 Std Dev 0.9 15.3 13.8 14.2 15.1 14.4 CV (%) 6.3 15.7 15.1 16.1 16.0 14.2 Accuracy (%) 100.0 20 19.8 18.4 18.2 22.2 17.5 Average 19.5 19.0 19.1 25.0 19.9 20.9 Std Dev 1.7 17.8 20.1 20.0 20.1 18.3 CV (%) 8.9 17.8 19.7 19.7 18.6 18.6 Accuracy (%) 97.7 40 44.3 41.7 40.6 39.1 42.0 Average 41.0 39.3 40.3 38.5 40.2 38.6 Std Dev 1.8 42.6 42.6 42.0 41.7 40.1 CV (%) 4.4 42.2 42.1 37.8 40.3 44.0 Accuracy (%) 102.5 60 65.0 57.0 60.3 60.6 55.7 Average 59.5 57.6 61.5 61.1 59.3 62.6 Std Dev 2.7 55.4 55.6 56.9 60.0 61.6 CV (%) 4.5 58.2 62.4 61.7 59.3 58.1 Accuracy (%) 99.2
[0118] The limit of detection (LOD) is the point where a measured value is larger than the uncertainty associated with it and is defined arbitrarily as four standard deviations (SD) from the zero concentration. Selectivity is the ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample. A blank was analyzed in 20 replicates according to the methods of Examples 1-3 and the resulting area ratios were statistically analyzed to determine that the LOD for both vitamin D.sub.2 and vitamin D.sub.3 are 0.4 ng/mL. Data collected to determine LOD for each analyte is shown in Table 6.
TABLE-US-00006 TABLE 6 Determination of Limit of Detection of Vitamin D.sub.2 and Vitamin D.sub.3 Replicate # NVD.sub.2 (Response Ratio) NVD.sub.3 (Response Ratio) 1 0.012 0.014 2 0.004 0.005 3 0.008 0.011 4 0.005 0.004 5 0.018 0.03 6 0.004 0.001 7 0.007 0.006 8 0.006 0.002 9 0.014 0.006 10 0.015 0.006 11 0.003 0.008 12 0.005 0.004 13 0.009 0.002 14 0.017 0.016 15 0.007 0.005 16 0.006 0.012 17 0.003 0.001 18 0.002 0.001 19 0.011 0.001 20 0.023 0.009 Mean 0.009 0.007 SD 0.0058 0.0070 CV 65.1 97.0 Mean + 4SD 0.032 0.035 LOD 0.4 ng/mL 0.4 ng/mL
Example 7: Specificity of Detection
[0119] Several samples were prepared with vitamin D.sub.2, vitamin D.sub.3, and spiked amounts of potentially interfering species (including vitamin D metabolites and related compounds) and analyzed according to the methods of Examples 1-3. The compounds tested for potential interference are listed in Table 7. None of the tested compounds demonstrated cross-reactivity with detection of vitamin D.sub.2 or vitamin D.sub.3 according to the methods of Examples 1-3.
TABLE-US-00007 TABLE 7 Compounds Tested for Possible Interference with Detection of Vitamin D.sub.2 or Vitamin D.sub.3 Compounds Tested 1,25(OH).sub.2D.sub.3 1,25(OH).sub.2D.sub.2 1,25(OH).sub.2D.sub.3-[6,19,19′]-2H 1,25(OH).sub.2D.sub.3-[26,26,26,27,27,27]-2H 1,25(OH).sub.2D.sub.2-[26,26,26,27,27,27]-2H 25OHD3 25OHD2 25OHD.sub.3-IS-[6,19,19′]-.sup.2H 25OHD.sub.2-IS-[6,19,19′]-.sup.2H 25OHD.sub.3-IS-[26,26,26,27,27,27]-.sup.2H 25OHD.sub.2-IS-[26,26,26,27,27,27]-.sup.2H vitamin D.sub.3-[6,19,19′]-.sup.2H vitamin D2-[6,19,19′]-.sup.2H vitamin D3-[26,26,26,27,27,27]-.sup.2H vitamin D.sub.2-[26,26,26,27,27,27]-.sup.2H 1-OH-D.sub.3 (Alfacalcidiol) 1-OH-D.sub.2 (Hectoral) 24,25(OH).sub.2D.sub.3 25,26(OH).sub.2D.sub.3 3-epi-25OHD.sub.3 3-epi-1,25(OH).sub.2D.sub.3 Dihydrotachysterol 1,25(OH).sub.2D.sub.3-26,23-lactone Paracalcitol (Zemplar) Calcipotriene (Dovonex) 7-Dehydrocholesterol
Example 8: Reproducibility of Quantitation of Vitamin D.SUB.2 .and Vitamin D.SUB.3
[0120] The intra-assay variation is defined as the reproducibility of a sample within an assay and was determined by assaying 20 replicates of a sample from each of three QC pools according to the methods of Examples 1-3. Data collected from these analyses are shown in Tables 8 and 9 for vitamin D.sub.2 and vitamin D.sub.3, respectively. The concentrations of the analytes in the QC pools were determined to be 6.6 ng/mL, 20.6 ng/mL, and 52.6 ng/mL for vitamin D.sub.2, and 4.9 ng/mL, 20.5 ng/mL, and 48.6 ng/mL for Vitamin D.sub.3. Statistics performed on the results yielded reproducibility for the three QC pools at 5.1%, 4.6%, and 3.9% for vitamin D.sub.2, and 6.4%, 4.0%, and 4.5% for vitamin D.sub.3.
TABLE-US-00008 TABLE 8 Intra-Assay Variation Determination for Vitamin D.sub.2 Low QC Medium QC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Lot 120709-L Lot 120709-M Lot 120709-H 1 6.7 20.3 52.5 2 6.6 21.0 54.7 3 7.0 21.1 51.5 4 6.2 19.5 52.1 5 6.4 20.0 52.0 6 6.2 22.4 53.3 7 7.0 20.4 48.9 8 6.7 21.5 54.6 9 6.7 21.5 52.4 10 6.9 20.9 52.5 11 7.0 19.2 50.2 12 6.2 20.8 52.3 13 7.0 19.6 57.5 14 6.6 20.6 54.7 15 6.2 19.8 53.0 16 6.2 20.6 49.2 17 6.2 22.9 52.5 18 6.2 20.9 51.4 19 7.0 19.6 52.2 20 6.5 20.3 55.2 Mean 6.6 20.6 52.6 SD 0.3 1.0 2.0 CV % 5.1 4.6 3.9
TABLE-US-00009 TABLE 9 Intra-Assay Variation Determination for Vitamin D.sub.3 Low QC Medium QC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Lot 120709-L Lot 120709-M Lot 120709-H 1 4.3 20.0 48.3 2 5.1 21.8 47.2 3 4.8 21.7 45.4 4 4.5 21.3 49.9 5 5.0 20.5 49.8 6 4.7 21.0 48.2 7 5.8 20.4 46.3 8 4.9 21.2 50.8 9 4.7 22.0 49.1 10 4.7 20.1 48.9 11 4.8 19.1 47.3 12 4.7 19.9 49.1 13 4.9 19.5 52.9 14 5.4 20.3 52.7 15 4.7 19.6 51.3 16 4.8 19.9 46.7 17 4.8 21.0 47.5 18 4.7 21.1 46.9 19 5.0 19.7 45.3 20 5.1 20.4 48.8 Mean 4.9 20.5 48.6 SD 0.3 0.8 2.2 CV % 6.4 4.0 4.5
[0121] The inter-assay variation is defined as the reproducibility (CV) of a sample between assays. Using the three QC pools covering the reportable range of the assay, evaluated over 5 assays according to the methods of Examples 1-3, the inter-assay variation (CV) for the pools was determined for vitamin D.sub.2 and vitamin D.sub.3. For Vitamin D.sub.2, the CVs were determined to be 6.7%, 5.6%, and 4.0% with mean concentrations of 6.5 ng/mL, 21.1 ng/mL, and 50.5 ng/mL, respectively. For Vitamin D.sub.3, the CVs were determined to be 6.5%, 5.9%, and 4.2% with mean concentrations of 4.7 ng/mL, 20.8 ng/mL, and 46.8 ng/mL, respectively. Data collected from these analyses are shown in Tables 10 and 11 for vitamin D.sub.2 and vitamin D.sub.3, respectively. All pools met with acceptable reproducibility requirements of ≤15% CV.
TABLE-US-00010 TABLE 10 Inter-Assay Variation Determination for Vitamin D.sub.2 Low QC Medium QC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Assay Lot 120709-L Lot 120709-M Lot 120709-H 1 6.7 20.3 52.5 6.6 21.0 54.7 7.0 21.1 51.5 6.2 19.5 52.1 6.4 20.0 52.0 2 7.3 23.3 49.4 6.8 22.1 51.6 6.7 20.6 48.6 6.7 20.6 47.2 7.0 21.1 50.4 3 6.3 22.3 49.7 6.1 23.0 52.2 6.6 24.0 49.5 5.6 22.1 49.6 6.1 19.1 51.7 4 6.1 20.5 47.5 7.0 21.1 50.1 6.9 22.2 50.2 7.3 22.1 48.3 6.6 20.2 46.8 5 5.8 21.1 52.4 6.9 19.6 49.6 6.0 20.8 49.1 6.4 20.6 56.1 6.3 20.1 50.8 6 6.3 21.8 50.0 7.0 22.5 49.8 6.2 21.6 51.2 5.9 20.1 50.3 6.9 20.1 50.9 Mean 6.5 21.1 50.5 SD 0.4 1.2 2.0 CV % 6.7 5.6 4.0
TABLE-US-00011 TABLE 11 Inter-Assay Variation Determination for Vitamin D.sub.3 Low QC Medium QC High QC Pool (ng/mL) Pool (ng/mL) Pool (ng/mL) Assay Lot 120709-L Lot 120709-M Lot 120709-H 1 4.3 20.0 48.3 5.1 21.8 47.2 4.8 21.7 45.4 4.5 21.3 49.9 5.0 20.5 49.8 2 4.5 23.0 46.3 4.2 22.8 47.8 4.5 19.8 45.1 4.4 20.1 44.4 4.7 21.1 48.7 3 4.9 21.8 44.9 5.1 20.9 46.8 5.2 23.9 45.5 4.4 22.1 45.7 4.6 19.3 47.1 4 4.5 20.1 43.7 4.8 21.2 44.6 5.2 21.9 46.1 4.7 21.3 44.1 4.7 20.5 42.8 5 4.5 20.5 49.1 5.1 20.8 46.9 4.5 21.3 47.3 4.8 20.6 45.8 4.2 19.7 46.1 6 4.7 19.9 49.6 4.7 18.9 47.6 4.4 20.2 48.9 4.4 18.6 48.5 5.2 19.4 49.4 Mean 4.7 20.8 46.8 SD 0.3 1.2 2.0 CV % 6.5 5.9 4.2
Example 9: Method Correlation Studies for Quantitation of Vitamin D.SUB.2
[0122] A method correlation study was performed for quantitation of vitamin D.sub.2 according to the methods of Examples 1-3 by comparing 20 split samples analyzed according to the tandem mass spectrometric methods described herein with extensive off-line extraction followed by HPLC with UV detection. Specimens were analyzed in singles for each method. Data was analyzed by Linear and Deming regressions. Correlation analyses are summarized in Table 12.
TABLE-US-00012 TABLE 12 Correlation Analyses for Method Comparison Vitamin D.sub.2 (n = 20) Linear Regression y = 1.26x − 0.55 R.sub.2 = 0.96 Deming Regression y = 1.29x − 1.31
Example 10: Interference Studies
[0123] Hemolysis Interference: The effects of hemolysis in the assay described in Examples 1-3 were evaluated by spiking hemoglobin into serum pools containing elevated vitamin D.sub.2 and vitamin D.sub.3. A fresh blood sample was centrifuged to yield packed red blood cells. The cells were reconstituted in deionized water and frozen to achieve cell lysis. This crude hemoglobin solution was then was spiked into the pools to generate lightly (100 mg/dL) and moderately (500 mg/dL) hemolyzed samples. Specimens were analyzed in duplicate according the method in Examples 1-3 and results were compared to the control pool result and a percent difference was calculated. The data shows that none of the hemoglobin spikes was >15% different than control, for either analyte). Therefore, light to moderate hemolyzed specimens are acceptable. For raw data see Table 13 (% Difference=(Spiked−UnSpiked)/UnSpiked×100%).
TABLE-US-00013 TABLE 13 Hemolysis Interference Studies Vitamin D.sub.2 Vitamin D.sub.3 ng/mL % Diff. ng/mL % Diff. Pool 1 Control 22.1 9.7 31.2 8.5 Light Hemolysis 24.3 7.6 33.8 10.0 Moderate Hemolysis 23.8 34.3 Pool 2 Control 22.3 −4.8 34.8 −6.4 Light Hemolysis 21.3 −7.2 32.6 −4.5 Moderate Hemolysis 20.7 33.3 Pool 3 Control 27.3 −2.2 34.6 0.2 Light Hemolysis 26.7 −9.8 34.7 −3.5 Moderate Hemolysis 24.6 33.4 Pool 4 Control 23.7 −3.8 30.4 3.6 Light Hemolysis 22.8 −10.6 31.5 −2.0 Moderate Hemolysis 21.2 29.8 Pool 5 Control 27.7 −3.4 33.9 −0.4 Light Hemolysis 26.7 −7.5 33.7 −1.9 Moderate Hemolysis 25.6 33.2 Pool 6 Control 24.2 0.3 33.6 −0.4 Light Hemolysis 24.3 0.5 33.5 1.3 Moderate Hemolysis 24.1 34.1
[0124] Icteria Interference: The effects of icteria in the assay described in Examples 1-3 were evaluated by spiking bilirubin into serum pools containing elevated vitamin D.sub.2 and vitamin D.sub.3. A concentrated solution of bilirubin was spiked into the pools to generate lightly (10 mg/dL) and moderately (50 mg/dL) icteric specimens. Specimens were analyzed in duplicate according to the method in Examples 1-3 and results were compared to the non-icteric pool result and the accuracy was calculated. The data shows that both analytes are unaffected by icteria (all values within acceptable accuracy range of 85-115%). Therefore, icteric specimens are acceptable. For raw data see Table 14 (% Difference=(Spiked−UnSpiked)/UnSpiked×100%).
TABLE-US-00014 TABLE 14 Icteria Interference Studies Vitamin D.sub.2 Vitamin D.sub.3 ng/mL % Diff. ng/mL % Diff. Pool 1 Control 22.1 0.3 31.2 4.8 Light Icteria 22.2 −2.6 32.6 −1.6 Moderate Icteria 21.5 30.7 Pool 2 Control 22.3 −1.3 34.8 −4.0 Light Icteria 22.0 −8.2 33.4 −6.9 Moderate Icteria 20.5 32.4 Pool 3 Control 27.3 3.8 34.6 1.8 Light Icteria 28.3 −0.7 35.2 1.0 Moderate Icteria 27.1 35.0 Pool 4 Control 23.7 −6.3 30.4 4.0 Light Icteria 22.2 −8.7 31.7 1.0 Moderate Icteria 21.7 30.7 Pool 5 Control 27.7 −3.7 33.9 −2.8 Light Icteria 26.7 −2.0 32.9 −4.4 Moderate Icteria 27.1 32.4 Pool 6 Control 24.2 −5.8 33.6 −5.5 Light Icteria 22.8 −6.4 31.8 −9.4 Moderate Icteria 22.6 30.5
[0125] Lipemia Interference: The effect of lipemia in the assay described in Examples 1-3 was evaluated by spiking porcine brain extract into serum pools containing elevated vitamin D.sub.2 and vitamin D.sub.3. Powdered lipid sample (Avanti Polar Lipids) was dissolved into each pool to generate lightly (400 mg/dL) and moderately (2000 mg/dL) lipemic specimens. Specimens were analyzed in duplicate according to the method in Examples 1-3 and results were compared to the control pool result and the accuracy was calculated. The data shows that both analytes are unaffected by lipemia (all values within acceptable accuracy range of 85-115%). For raw data see Table 15 ((% Difference=(Spiked−UnSpiked)/UnSpiked×100%).
TABLE-US-00015 TABLE 15 Lipemia Interference (Porcine Brain Extract) Studies Vitamin D.sub.2 Vitamin D.sub.3 ng/mL % Diff. ng/mL % Diff. Pool 1 Control 22.1 −0.6 31.2 4.8 Light Lipemia 22.0 3.8 32.7 5.0 Moderate Lipemia 23.0 32.7 Pool 2 Control 22.3 3.2 34.8 1.6 Light Lipemia 23.0 8.3 35.4 −0.2 Moderate Lipemia 24.2 34.7 Pool 3 Control 27.3 5.4 34.6 2.9 Light Lipemia 28.7 3.3 35.7 5.3 Moderate Lipemia 28.1 36.5 Pool 4 Control 23.7 7.4 30.4 9.0 Light Lipemia 25.5 −2.5 33.2 7.3 Moderate Lipemia 23.2 32.7 Pool 5 Control 27.7 −2.2 33.9 1.5 Light Lipemia 27.1 −3.4 34.4 0.1 Moderate Lipemia 26.7 33.9 Pool 6 Control 24.2 8.0 33.6 2.5 Light Lipemia 26.1 −0.7 34.5 −0.2 Moderate Lipemia 24.0 33.6
[0126] The effects of lipemia in the assay described in Examples 1-3 were also evaluated by spiking Intralipid emulsion into serum pools containing elevated vitamin D.sub.2 and vitamin D.sub.3. To the serum pools, Intralipid (20% emulsion) was added to generate lightly (400 mg/dL) and moderately (2000 mg/dL) lipemic specimens. Specimens were analyzed in duplicate according to the method in Examples 1-3 and results were compared to the control pool result and the accuracy was calculated. The data shows that both analytes are unaffected by lipemia (all values within acceptable accuracy range of 85-115%). For raw data see Table 16.
TABLE-US-00016 TABLE 16 Lipemia Interference (Intralipid) Studies Vitamin D.sub.2 Vitamin D.sub.3 ng/mL % Diff. ng/mL % Diff. Pool 1 Control 22.1 1.6 31.2 6.8 Light Lipemia 22.5 −10.4 33.3 −7.6 Moderate Lipemia 19.8 28.8 Pool 2 Control 22.3 0.4 34.8 −3.6 Light Lipemia 22.4 −13.8 33.5 −12.3 Moderate Lipemia 19.2 30.5 Pool 3 Control 27.3 0.6 34.6 −0.3 Light Lipemia 27.4 −8.6 34.5 −6.1 Moderate Lipemia 24.9 32.5 Pool 4 Control 23.7 −0.6 30.4 3.0 Light Lipemia 23.6 −16.0 31.3 −2.0 Moderate Lipemia 19.9 29.8 Pool 5 Control 27.7 −1.1 33.9 −5.6 Light Lipemia 27.4 −6.9 32.0 −5.9 Moderate Lipemia 25.8 31.9 Pool 6 Control 24.2 1.2 33.6 0.6 Light Lipemia 24.5 −5.9 33.8 −10.7 Moderate Lipemia 22.8 30.0
[0127] Based upon the two lipemia experiments using porcine brain extract and Intralipid, lipemic specimens are acceptable.
Example 11: Specimen Type Studies
[0128] Specimens were collected from 10 sources into four different Vacutainer® containers. The Vacutainers used were Red-Top (silicon-coated serum tubes), SST (Serum Separator Tubes, which result in gel-barrier serum), EDTA tubes, and Sodium Heparin tubes.
[0129] These 40 samples were analyzed for nutritional vitamin D.sub.3 according to the method in Examples 1-3. Comparative results are presented in
Example 12: Demonstration of Routine Range for Vitamin D.SUB.3
[0130] Serum specimens from 140 patients were analyzed according to the method described in Examples 1-3 to quantitate vitamin D.sub.3. The results ranged from <2 ng/mL to about 63 ng/mL vitamin D.sub.3, with 95% of the results falling within the range of <2 ng/mL to about 20 ng/mL. The results of these analyses are presented in Table 17.
TABLE-US-00017 TABLE 17 Routine Range Studies for Vitamin D.sub.3 (in descending order) Vit D.sub.3 Patient (ng/mL) 1 62.8 2 35.8 3 24.3 4 20.2 5 15.6 6 15.6 7 15.5 8 14.6 9 14.3 10 12.7 11 12.7 12 12.5 13 10.5 14 10.3 15 9.8 16 9.6 17 9.6 18 7.9 19 7.4 20 6.6 21 6.5 22 6.4 23 5.9 24 5.8 25 5.5 26 5.4 27 5.3 28 5.1 29 5.1 30 5.0 31 4.9 32 4.8 33 4.6 34 4.6 35 4.4 36 4.3 37 4.2 38 4.2 39 4.0 40 3.9 41 3.9 42 3.9 43 3.7 44 3.6 45 3.4 46 3.3 47 3.2 48 3.2 49 3.2 50 3.2 51 3.2 52 3.1 53 2.8 54 2.8 55 2.7 56 2.6 57 2.6 58 2.5 59 2.3 60 2.2 61 2.2 62 2.2 63 2.2 64 2.1 65 2.0 66 2.0 67 2.0 68 2.0 69 <2 70 <2 71 <2 72 <2 73 <2 74 <2 75 <2 76 <2 77 <2 78 <2 79 <2 80 <2 81 <2 82 <2 83 <2 84 <2 85 <2 86 <2 87 <2 88 <2 89 <2 90 <2 91 <2 92 <2 93 <2 94 <2 95 <2 96 <2 97 <2 98 <2 99 <2 100 <2 101 <2 102 <2 103 <2 104 <2 105 <2 106 <2 107 <2 108 <2 109 <2 110 <2 111 <2 112 <2 113 <2 114 <2 115 <2 116 <2 117 <2 118 <2 119 <2 120 <2 121 <2 122 <2 123 <2 124 <2 125 <2 126 <2 127 <2 128 <2 129 <2 130 <2 131 <2 132 <2 133 <2 134 <2 135 <2 136 <2 137 <2 138 <2 139 <2 140 <2
Example 13: Recovery Studies
[0131] Mix recovery studies were performed by analysis of specimens with naturally elevated levels of 25-hydroxyvitamin D.sub.2 or 25-hydroxyvitamin D.sub.3, and therefore also had some endogenous circulating vitamin D.sub.2 or vitamin D.sub.3. Six pairs of specimens were selected for the studies. From each pair of specimens (generically referred to as specimen A and specimen B), five samples were prepared and analyzed in quadruplicate according to the method in Examples 1-3. The samples corresponded to 100% A, 80% A—20% B, 50% A—50% B, 20% A—80% B, and 100% B. The results of the recovery studies are presented in Tables 18 and 19.
TABLE-US-00018 TABLE 18 Mixed Specimen Recovery Studies Vitamin D.sub.2 Vitamin D.sub.3 Spike Measured Expected Recovery Measured Expected Recovery Pool Level (ng/mL) (ng/mL) (%) (ng/mL) (ng/mL) (%) 1 100% A 49.0 2.1 80/20 A/B 37.8 39.3 103.8 3.3 3.5 106.1 50/50 A/B 26.0 24.7 94.8 5.6 5.5 96.9 20/80 A/B 10.5 10.1 96.5 7.6 7.5 98.5 100% B 0.4 8.8 2 100% A 5.0 2.6 80/20 A/B 4.9 4.2 85.5 6.6 6.6 99.4 50/50 A/B 2.9 2.9 101.7 11.8 12.5 106.2 20/80 A/B 1.6 1.7 106.2 17.7 18.5 104.3 100% B 0.9 22.4 3 100% A 8.8 3.0 80/20 A/B 7.6 7.4 96.7 6.6 6.6 101.2 50/50 A/B 5.4 5.2 95.8 11.3 12.1 106.7 20/80 A/B 3.1 3.0 99.1 16.3 17.5 107.6 100% B 1.6 21.2 *Measured values are averages of analysis of four aliquots.
TABLE-US-00019 TABLE 19 Summary of Results of Mixed Specimen Recovery Studies Pool Vitamin D.sub.2 Vitamin D.sub.3 1 103.8% 106.1% 94.8% 96.9% 96.5% 98.5% 2 85.5% 99.4% 101.7% 106.2% 106.2% 104.3% 3 96.7% 101.2% 95.8% 106.7% 99.1% 107.6% Avg 97.8% 103.0% Avg 100.4%
Example 14: Exemplary Spectra from MS/MS Analysis of Vitamin D.SUB.2 .and Vitamin D.SUB.3
[0132] Exemplary Q1 scan spectra from the tandem mass spectrometric analysis of vitamin D.sub.2, vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3, and vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are shown in
[0133] Exemplary product ion scans generated from two different precursor ions for each of vitamin D.sub.2, vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3, and vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are presented in
[0134] Exemplary MRM transitions for the quantitation of vitamin D.sub.2 include fragmenting a precursor ion with a m/z of about 397.2 to a product ion with a m/z of about 159.0; and fragmenting a precursor ion with a m/z of about 379.2 to a product ion with a m/z of about 158.9. Exemplary MRM transitions for the quantitation of vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3 include fragmenting a precursor ion with a m/z of about 400.2 to a product ion with a m/z of about 147.0; and fragmenting a precursor ion with a m/z of about 382.2 to a product ion with a m/z of about 312.2. Exemplary MRM transitions for the quantitation of vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 include fragmenting a precursor ion with a m/z of about 403.2 to a product ion with a m/z of about 159.1; and fragmenting a precursor ion with a m/z of about 385.2 to a product ion with a m/z of about 159.0. However, as can be seen in the product ion scans in
TABLE-US-00020 TABLE 20 Precursor Ions and Collision Cell Energies for Fragmentation of vitamin D.sub.2, vitamin D.sub.2-[6, 19,19]-.sup.2H.sub.3, and vitamin D2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 Precursor Collision Cell Analyte Ion (m/z) Energy (V) vitamin D.sub.2 397.2, 379.2 25 vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3 400.2, 382.2 25 vitamin D.sub.2-[26, 26, 26, 403.2, 385.2 25 27, 27, 27]-.sup.2H.sub.6
[0135] Exemplary Q1 scan spectra from the tandem mass spectrometric analysis of vitamin D.sub.3, vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3, and vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are shown in
[0136] Exemplary product ion scans generated from two different precursor ions for each of vitamin D.sub.3, vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3, and vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are presented in
[0137] Exemplary MRM transitions for the quantitation of vitamin D.sub.3 include fragmenting a precursor ion with a m/z of about 385.2 to a product ion with a m/z of about 147.0; and fragmenting a precursor ion with a m/z of about 367.2 to a product ion with a m/z of about 159.0. Exemplary MRM transitions for the quantitation of vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3 include fragmenting a precursor ion with a m/z of about 388.2 to a product ion with a m/z of about 147.0; and fragmenting a precursor ion with a m/z of about 370.2 to a product ion with a m/z of about 162.0. Exemplary MRM transitions for the quantitation of vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 include fragmenting a precursor ion with a m/z of about 391.2 to a product ion with a m/z of about 159.1; and fragmenting a precursor ion with a m/z of about 373.2 to a product ion with a m/z of about 159.0. However, as can be seen in the product ion scans in
TABLE-US-00021 TABLE 21 Precursor Ions and Collision Cell Energies for Fragmentation of vitamin D.sub.3, vitamin D.sub.3-[6, 19,19]-.sup.2H.sub.3, and vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 Precursor Collision Cell Analyte Ion (m/z) Energy (V) vitamin D.sub.3 385.2, 367.2 25 vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3 388.2, 370.2 25 vitamin D.sub.3-[26, 26, 26, 27, 27, 391.2, 373.2 25 27]-.sup.2H.sub.6
Example 15: Exemplary Spectra from MS/MS Analysis of PTAD Derivatized Vitamin D.SUB.2 .and Vitamin D.SUB.3
[0138] PTAD derivatives of vitamin D.sub.2, vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3, vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6, vitamin D.sub.3, vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3, and vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 were prepared by treating aliquots of stock solutions of each analyte with PTAD in acetonitrile. The derivatization reactions was allowed to proceed for approximately one hour, and were quenched by adding water to the reaction mixture. The derivatized analytes were then analyzed according to the procedure outlined above in Examples 2-3.
[0139] Exemplary Q1 scan spectra from the analysis of samples containing PTAD-vitamin D.sub.2, PTAD-vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3, and PTAD-vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are shown in
[0140] Exemplary product ion scans generated from precursor ions for each of PTAD-vitamin D.sub.2, PTAD-vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3, and PTAD-vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are presented in
[0141] An exemplary MRM transition for the quantitation of PTAD-vitamin D.sub.2 includes fragmenting a precursor ion with a m/z of about 572.2 to a product ion with a m/z of about 297.9. An exemplary MRM transition for the quantitation of PTAD-vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3 includes fragmenting a precursor ion with a m/z of about 575.2 to a product ion with a m/z of about 301.0. An exemplary MRM transition for the quantitation of PTAD-vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 includes fragmenting a precursor ion with a m/z of about 578.2 to a product ion with a m/z of about 297.9. However, as can be seen in the product ion scans in
TABLE-US-00022 TABLE 22 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-vitamin D.sub.2, PTAD-vitamin D.sub.2-[6, 19,19]-.sup.2H.sub.3, and PTAD-vitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 Precursor Collision Cell Analyte Ion (m/z) Energy (V) PTAD-vitamin D.sub.2 572.2 15 PTAD-vitamin D.sub.2-[6, 19, 19]-.sup.2H.sub.3 575.2 15 PTAD-vitamin D.sub.2-[26, 26, 26, 578.2 15 27, 27, 27]-.sup.2H.sub.6
[0142] Exemplary Q1 scan spectra from the analysis of samples containing PTAD-vitamin D.sub.3, PTAD-vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3, and PTAD-vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are shown in
[0143] Exemplary product ion scans generated from precursor ions for each of PTAD-vitamin D.sub.3, PTAD-vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3, and PTAD-vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 are presented in
[0144] An exemplary MRM transition for the quantitation of PTAD-vitamin D.sub.3 includes fragmenting a precursor ion with a m/z of about 560.2 to a product ion with a m/z of about 298.0. An exemplary MRM transition for the quantitation of PTAD-vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3 includes fragmenting a precursor ion with a m/z of about 563.2 to a product ion with a m/z of about 301.0. An exemplary MRM transition for the quantitation of PTAD-vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 includes fragmenting a precursor ion with a m/z of about 566.2 to a product ion with a m/z of about 298.0. However, as can be seen in the product ion scans in
TABLE-US-00023 TABLE 23 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-vitamin D.sub.3, PTAD-vitamin D.sub.3-[6, 19,19]-.sup.2H.sub.3, and PTAD-vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 Precursor Collision Cell Analyte Ion (m/z) Energy (V) PTAD-vitamin D.sub.3 560.2 15 PTAD-vitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3 563.2 15 PTAD-vitamin D.sub.3-[26, 26, 26, 566.2 15 27, 27, 27]-.sup.2H.sub.6
[0145] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
[0146] The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
[0147] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0148] Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.