Mass spectrometry of steroidal compounds in multiplexed patient samples
10955424 ยท 2021-03-23
Assignee
Inventors
Cpc classification
G01N33/6851
PHYSICS
G01N2560/00
PHYSICS
International classification
Abstract
The invention relates to the quantitative measurement of steroidal compounds by mass spectrometry. In a particular aspect, the invention relates to methods for quantitative measurement of steroidal compounds from multiple samples by mass spectrometry.
Claims
1. A method for determining the amount of one or more vitamin D metabolites in each of a plurality of human samples with a single mass spectrometric assay, the method comprising: i) subjecting each of a plurality of human samples to a different Cookson-type derivatizing agent to generate a differently derivatized one or more vitamin D metabolites in each of the plurality of samples; ii) combining the plurality of samples to form a multiplex sample; and iii) quantifying the amount of the one or more vitamin D metabolites in each sample by mass spectrometry, wherein the method has a limit of quantitation (LOQ) within the range of 1.9 ng/mL to 10 ng/mL, inclusive.
2. The method of claim 1, wherein said different Cookson-type derivatizing agents are isotopic variants of one another.
3. The method of claim 1, wherein said Cookson-type derivatization agents are selected from the group consisting of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD), 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD), 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), and isotopic variants thereof.
4. The method of claim 1, wherein said Cookson-type derivatizing agents are isotopic variants of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD).
5. The method of claim 1, wherein the plurality of samples comprises two samples, wherein a first derivatizing reagent is 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and a second derivatizing reagent is .sup.13C.sub.6-4-phenyl-1,2,4-triazoline-3,5-dione (.sup.13C.sub.6-PTAD).
6. The method of claim 1, wherein said one or more vitamin D metabolites is selected from the group consisting of 25-hydroxyvitamin D.sub.2 (25OHD.sub.2), 25-hydroxyvitamin D.sub.3 (25OHD.sub.3), 1,25-dihydroxyvitamin D.sub.2 (1,25OHD.sub.2), and 1,25-dihydroxyvitamin D.sub.3 (1,25OHD.sub.3).
7. The method of claim 6, wherein said one or more vitamin D metabolites is 25-hydroxyvitamin D.sub.2 (25OHD.sub.2) or 25-hydroxyvitamin D.sub.3 (25OHD.sub.3).
8. The method of claim 1, further comprising subjecting the multiplex sample to an extraction column and an analytical column.
9. The method of claim 8, wherein the extraction column is a solid-phase extraction (SPE) column.
10. The method of claim 8, wherein the extraction column is a turbulent flow liquid chromatography (TFLC) column.
11. The method of claim 8, wherein the analytical column is a high performance liquid chromatography (HPLC) column.
12. The method of claim 1, wherein mass spectrometry is tandem mass spectrometry.
13. The method of claim 12, wherein said tandem mass spectrometry is conducted as multiple reaction monitoring, precursor ion scanning, or product ion scanning.
14. The method of claim 8, wherein the extraction column, analytical column, and the ionization source are connected in an on-line fashion.
15. The method of claim 1, wherein said mass spectrometry comprises laser diode thermal desorption (LDTD).
16. The method of claim 1, wherein said mass spectrometry comprises an electrospray ionization source (ESI) or an atmospheric pressure chemical ionization source (APCI).
17. The method of claim 1, wherein said samples comprise plasma or serum.
18. The method of claim 1, wherein the method has a limit of detection (LOD) of about 4 ng/ml or less.
19. The method of claim 1, wherein the method has a limit of quantitation (LOQ) within the range of 1.9 ng/mL to 5 ng/mL, inclusive.
20. The method of claim 1, wherein the method has a limit of quantitation (LOQ) within the range of 3.3 ng/mL to 5 ng/mL, inclusive.
21. The method of claim 1, wherein the method comprises detecting an ion with a mass/charge ratio of 570.320.50 or 558.320.50.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(17) Methods are described for measuring steroidal compounds, such as vitamin D and vitamin D related compounds, in a sample. More specifically, methods are described for detecting and quantifying steroidal compounds in a plurality of test samples in a single mass spectrometric assay. The methods may utilize Cookson-type reagents, such as PTAD, to generate derivatized steroidal compounds combined with methods of mass spectrometry (MS), thereby providing a high-throughput assay system for detecting and quantifying steroidal compounds in a plurality of test samples. The preferred embodiments are particularly well suited for application in large clinical laboratories for automated steroidal compound quantification.
(18) 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.
(19) The present invention also contemplates kits for quantitation of one or more steroidal compounds. A kit for a steroidal compound quantitation assay may include a kit comprising the compositions provided herein. For example, a kit may include packaging material and measured amounts of 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 steroidal compound quantitation assay.
(20) Calibration and QC pools for use in embodiments of the present invention are preferably prepared using a matrix similar to the intended sample matrix.
(21) Sample Preparation for Mass Spectrometric Analysis
(22) In preparation for mass spectrometric analysis, one or more steroidal compounds 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. These enrichment steps may be applied to individual test samples prior to processing, individual processed samples after derivatization, or to a multiplex sample after processed samples have been combined.
(23) Protein precipitation is one method of preparing a sample, especially a biological 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 one or more steroidal compounds 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, individual test samples, such as plasma or serum, may be purified by a hybrid protein precipitation/liquid-liquid extraction method. In these embodiments, an unprocessed test sample is mixed with methanol, ethyl acetate, and water, and the resulting mixture is vortexed and centrifuged. The resulting supernatant, containing one or more purified steroidal compounds, is removed, dried to completion and reconstituted in acetonitrile. The one or more purified steroidal compounds in the acetonitrile solution may then be derivatized with any Cookson-type reagent, preferably PTAD or an isotopically labeled variant thereof.
(24) 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 steroidal compounds. 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. In preferred embodiments, a multiplex sample may be purified by liquid chromatography prior to mass spectrometry.
(25) In one embodiment, the multiplex 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.
(26) In certain embodiments, analytes may be purified by applying a multiplex sample to a column under conditions where analytes of interest are 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 analytes of interest are 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, analytes may be purified by applying a multiplex sample to a column under mobile phase conditions where the analytes of interest elute at a differential rates in comparison to one or more other materials. Such procedures may enrich the amount of an analyte of interest in the eluent at a particular time (i.e, a characteristic retention time) relative to one or more other components of the sample.
(27) 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.
(28) 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.
(29) In some embodiments, an extraction column may be used for purification of steroidal compounds 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 m) 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.
(30) 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 test samples. The resulting steroidal compounds 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 where the steroidal compounds are selected from the group consisting of vitamin D and vitamin D related compounds, the extraction column is preferably a C-8 extraction column, such as a Cohesive Technologies C8XL online extraction column (50 m particle size, 0.550 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.
(31) Detection and Quantitation by Mass Spectrometry
(32) In various embodiments, derivatized steroidal compounds 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), 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), particle beam ionization, and LDTD. 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.
(33) Derivatized steroidal compounds may be ionized in positive or negative mode. In preferred embodiments, derivatized steroidal compounds are ionized by APCI in positive mode. In related preferred embodiments, derivatized steroidal compounds ions are in a gaseous state and the inert collision gas is argon or nitrogen; preferably argon.
(34) 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.
(35) 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.
(36) 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.
(37) 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).
(38) 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 steroidal compounds. 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 some embodiments, one or more isotopically labeled vitamin D metabolites (e.g., 25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 and 25OHD.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.
(39) 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.
(40) In certain mass spectrometry techniques, such as MS/MS, precursor ions are isolated for further fragmentation though collision activated dissociation (CAD). 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.
(41) Steroidal compounds in a sample may be detected and/or quantified using MS/MS as follows. The samples may first be purified by protein precipitation or a hybrid protein precipitation/liquid-liquid extraction. Then, one or more steroidal compounds in the purified sample are derivatized with a Cookson-type reagent, such as PTAD or an isotopic variant thereof. The purified samples may 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 steroidal compounds such as derivatized vitamin D metabolites), 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. Alternatively, derivatized steroidal compounds in the purified samples may not be subject to liquid chromatography prior to ionization. Rather, the samples may be spotted in a 96-well plate and volatilized and ionized via LDTD.
(42) The ions, e.g. precursor ions, pass through the orifice of a tandem mass spectrometric (MS/MS) instrument and enter the first quadrupole. In a tandem mass spectrometric instrument, 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 molecules with the mass to charge (m/z) ratios of derivatized steroidal compounds 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 the fragment ions of derivatized steroidal compounds of interest are selected while other ions are eliminated.
(43) 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 derivatized steroidal compounds that may be used for selection in quadrupole 3 (Q3).
(44) 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 a particular steroidal compounds. 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.
(45) Processing Patient Samples for Analysis of Multiplex Patient Samples
(46) Following the procedures outlined above, multiple patient samples can be multiplex (i.e., mixed and assayed together) if each patient sample is processed differently. The phrase processed differently means that each patient sample to be included in the multiplex sample is processed in such a way that steroidal compounds in two or more patient samples that are originally indistinguishable by mass spectrometry become distinguishable after processing. This may be accomplished by processing each patient sample with a different agent that derivitizes steroidal compounds. The derivatizing agents selected for use must generate derivatized steroidal compounds that are distinguishable by mass spectrometry. The basis for distinguishing derivatized steroidal compounds by mass spectrometry will be a difference in the mass of ions from the derivatized steroidal compounds. The differences in mass may arise from the use of two or more different derivatizing agents, such as PTAD and DMEQTAD. Differences in mass may also arise from the use of two or more isotopic variants of the same derivatizing agent, such as PTAD and .sup.13C.sub.6-PTAD. These two approaches are not mutually exclusive, and any combination of different derivatizing agents and isotopic variants of the same agent may be used to uniquely label steroidal compounds in each patient sample in the plurality of patient samples to be analyzed. Optionally, one sample from the plurality of patient samples may be processed without a derivatizing agent.
(47) After processing a plurality of patient samples, a particular steroidal compound from one patient sample will have a different mass spectrometric profile than the same steroidal compound in other patient samples. When processed patient samples are mixed to form a multiplex sample which is then analyzed to determine the levels of processed steroidal compounds, the differences in mass spectrometric profiles of the detected processed steroidal compounds allow for each processed steroidal compound to be attributed to an originating patient sample. Thus, the amounts of a steroidal compound in two or more patient samples are determined by a single mass spectrometric analysis of a multiplex sample.
(48) As indicated above, different Cookson-type reagents may be used as derivatizing agents for different patient samples; for example, one patient sample may be derivatized with PTAD, and a second patient sample derivatized with DMEQTAD. Using different Cookson-type reagents generally results in large mass differences between the derivatized analytes. For example, the difference in mass between a steroidal compound derivatized with PTAD and the same compound derivatized with DMEQTAD is about 200 mass units (the mass difference between PTAD and DMEQTAD).
(49) Isotopic variants of the same Cookson-type reagent may also be used to create distinguishable derivatives in multiple patient samples. For example, one patient sample may be derivatized with PTAD, and a second patient sample may be derivatized with .sup.13C.sub.6-PTAD. In this example, the difference in mass between PTAD and .sup.13C.sub.6-PTAD is about 6 mass units.
(50) The following Examples serve to illustrate the invention through processing multiple patient samples with isotopic variants of PTAD. These Examples are in no way intended to limit the scope of the methods. In particular, the following Examples demonstrate quantitation of vitamin D metabolites by mass spectrometry with the use of 25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 or 25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3 as internal standards. Demonstration of the methods of the present invention as applied to vitamin D metabolites does not limit the applicability of the methods to only vitamin D and vitamin D related compounds. Similarly, the use of 25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 or 25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3 as internal standards are not meant to be limiting in any way. Any appropriate chemical species, easily determined by one in the art, may be used as an internal standard for steroidal compound quantitation.
EXAMPLES
Example 1: Hybrid Protein Precipitation/Liquid-Liquid Extraction and Cookson-Type Derivatization
(51) 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.
(52) A Perkin-Elmer Janus robot and a TomTec Quadra Tower robot was used to automate the following procedure. For each sample, 50 L of serum was added to a well of a 96 well plate. Then 25 L of internal standard cocktail (containing isotopically labeled 25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 and 25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3) was added to each well, and the plate vortexed. Then 75 L of methanol was added, followed by additional vortexing. 300 L of ethyl acetate and 75 L of water was then added, followed by additional vortexing, centrifugation, and transfer of the resulting supernatant to a new 96-well plate.
(53) The transferred liquid in the second 96-well plate from Example 1 was dried to completion under a flowing nitrogen gas manifold. Derivatization was accomplished by adding 100 L 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 L of water to the reaction mixture.
Example 2: Extraction of Vitamin D Metabolites with Liquid Chromatography
(54) Sample injection was performed with a Cohesive Technologies Aria TX-4 TFLC system using Aria OS V 1.5.1 or newer software.
(55) The TFLC system automatically injected an aliquot of the above prepared samples into a Cohesive Technologies C8XL online extraction column (50 m particle size, 00550 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 metabolites to the large particles in the column and the passage of excess derivatizing reagent and debris to waste.
(56) Following loading, the sample was eluted off to the analytical column, a Thermo Hypersil Gold Aq analytical column (5 m particle size, 502.1 mm), with a water/ethanol elution gradient. The HPLC gradient was applied to the analytical column, to separate vitamin D metabolites 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 Metabolites by MS/MS
(57) MS/MS was performed 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 ESI.
(58) Ions passed to the first quadrupole (Q1), which selected ions for a derivatized vitamin D metabolite. Ions with a m/z of 570.320.50 were selected for PTAD-25OHD.sub.2; ions with a m/z of 558.320.50 were selected for PTAD-25OHD.sub.3. 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 standards, PTAD-25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 and PTAD-25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3. The following mass transitions were used for detection and quantitation during validation on positive polarity. The indicated mass transitions re not meant to be limiting in any way. As seen in the Examples that follow, other mass transitions could be selected for each analyte to generate quantitative data.
(59) TABLE-US-00001 TABLE 1 Mass Spectrometer Settings for Detection of PTAD-25OHD.sub.2 and PTAD-25OHD.sub.3. Mass Spectrometric Instrument Settings Discharge Current 4.0 A 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
(60) TABLE-US-00002 TABLE 2 Exemplary Mass Transitions for PTAD-25OHD.sub.2, PTAD-25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 (IS), PTAD-25OHD.sub.3, and PTAD-25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3 (IS) (Positive Polarity) Precursor Ion Product Ion Analyte (m/z) (m/z) PTAD-25OHD.sub.2 570.32 298.09 PTAD-25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 (IS) 573.32 301.09 PTAD-25OHD.sub.3 558.32 298.09 PTAD-25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3 (IS) 561.32 301.09
(61) Exemplary chromatograms for PTAD-25OHD.sub.3, PTAD-25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3 (IS), PTAD-25OHD.sub.2, and PTAD-25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 (IS) are found in
(62) Exemplary calibration curves for the determination of 25OHD.sub.2 and 25OHD.sub.3 in serum specimens are shown in
Example 4: Analytical Sensitivity: Lower Limit of Quantitation (LLOQ) and Limit of Detection (LOD)
(63) The 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 five different human serum samples spiked with PTAD-25OHD.sub.2 and PTAD-25OHD.sub.3 at levels near the expected LLOQ and evaluating the reproducibility. Analysis of the collected data indicates that samples with concentrations of about 4 ng/mL yielded CVs of about 20%. Thus, the LLOQ of this assay for both PTAD-25OHD.sub.2 and PTAD-25OHD.sub.3 was determined to be about 4 ng/mL. The graphical representations of CV versus concentration for both analytes are shown in
(64) The LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as three standard deviations from the zero concentration. To determine the LOD, generally, blank samples of the appropriate matrix are obtained and tested for interferences. However, no appropriate biological matrix could be obtained where the endogenous concentration of 25OHD.sub.3 is zero, so a solution of 5% bovine serum albumin in phosphate buffered saline (with an estimated 1.5 ng/mL 25OHD.sub.3) was used for LOD studies. The standard was run in 20 replicates each and the resulting area rations were statistically analyzed to determine that the LOD for 25OHD.sub.2 and 25OHD.sub.3 are about 1.9 and 3.3 ng/mL, respectively. Raw data from these studies is presented in Table 3, below
(65) TABLE-US-00003 TABLE 3 Limit of Detection Raw Data and Analysis Replicate 25OHD.sub.2 (ng/mL) 25OHD.sub.3 (ng/mL) 1 0.0 0.0 2 1.1 2.0 3 0.1 2.4 4 0.3 1.1 5 0.5 1.9 6 0.4 1.8 7 0.2 1.9 8 0.5 2.3 9 1.1 2.3 10 0.5 2.1 11 0.4 1.5 12 1.2 1.9 13 0.4 1.8 14 0.3 1.6 15 0.0 1.3 16 0.9 1.3 17 0.8 1.5 18 0.1 1.9 19 0.5 1.7 20 0.4 1.8 Mean 0.4 1.7 SD 0.5 0.5 LOD (Mean + 3SD) 1.9 3.3
Example 5: Reportable Range and Linearity
(66) Linearity of derivatized vitamin D metabolite detection in the assay was determined by diluting four pools serum with high endogenous concentration of either 25OHD.sub.2 or 25OHD.sub.3 and analyzing undiluted specimens and diluted specimens at 1:2, 1:4, and 1:8, in quadruplicate. Quadratic regression of the data was performed yielding correlation coefficients across the concentration range tested of R.sup.2=0.97. These studies demonstrated that specimens may be diluted at 1:4 with average recovery of 101%, permitting a reportable range of about 4 to about 512 ng/mL. Average measured values for each of the specimen dilution levels and correlation values from linear regression analysis are presented in Table 4A, below. Percent recoveries for each of the specimen dilution levels are presented in Table 4B, below.
(67) TABLE-US-00004 TABLE 4A Linearity Data and Linear Regression Analysis over Reportable Range 25OHD.sub.2 (ng/mL) 25OHD.sub.3 (ng/mL) Dilution Level Pool 1 Pool 2 Pool 1 Pool 2 Undiluted 110.0 75.6 73.3 60.6 1:2 55.5 39.3 35.7 28.7 1:4 26.2 19.4 18.1 16.3 1:8 14.3 10.9 9.7 8.3 R.sup.2 0.9744 0.9721 0.9705 0.9601
(68) TABLE-US-00005 TABLE 4B Percent Recovery at Various Specimen Dilution Levels 25OHD.sub.2 (ng/mL) 25OHD.sub.3 (ng/mL) Dilution Level Pool 1 Pool 2 Pool 1 Pool 2 Undiluted (100%) (100%) (100%) (100%) 1:2 100.9 104 97.4 94.8 1:4 95.5 102.7 98.6 107.3 1:8 104.2 115.0 106.0 109.0
Example 6: Analyte Specificity
(69) The specificity of the assay against similar analytes was determined to have no cross reactivity for any vitamin D metabolite tested with the exception of 3-epi-25OHD.sub.3, which behaves similarly to 25OHD.sub.3 in the assay. The side-chain labeled stable isotopes of 25OHD2 and 25OHD.sub.3 also showed cross-reactivity owing to hydrogen exchange that occurs in the ion source. Thus, side-chain labeled stable isotopes of 25OHD.sub.2 and 25OHD.sub.3 should not be used as internal standards. Table 5, below, shows the compounds tested and the results of the cross-reactivity studies.
(70) TABLE-US-00006 TABLE 5 Cross-Reactivity Studies (Compounds tested and results) Cross- Reac- Analyte 25OHD.sub.2 25OHD.sub.3 tivity 1,25(OH).sub.2D.sub.3 No 1,25(OH).sub.2D.sub.2 No 1,25(OH).sub.2D.sub.3-[6,19,19]-.sup.2H No 1,25(OH).sub.2D.sub.3-[26,26,26,27,27,27]-.sup.2H No 1,25(OH).sub.2D.sub.2-[26,26,26,27,27,27]-.sup.2H No 25OHD.sub.3 (100%) 25OHD.sub.2 (100%) 25OHD.sub.3-IS-[6,19,19]-.sup.2H No 25OHD.sub.2-IS-[6,19,19]-.sup.2H No 25OHD.sub.3-IS-[26,26,26,27,27,27]-.sup.2H 13.8% Yes 25OHD.sub.2-IS-[26,26,26,27,27,27]-.sup.2H 2.7% Yes vitamin D.sub.3 No vitamin D.sub.2 No vitamin D.sub.3-[6,19,19]-.sup.2H No vitamin D.sub.2-[6,19,19]-.sup.2H No vitamin D.sub.3-[26,26,26,27,27,27]-.sup.2H No vitamin D.sub.2-[26,26,26,27,27,27]-.sup.2H No 1-OH-D.sub.3 (Alfacalcidiol) No 1-OH-D.sub.2 (Hectoral) No 24,25(OH).sub.2D.sub.3 No 25,26(OH).sub.2D.sub.3 No 3-epi-25OHD.sub.3 No 3-epi-1,25(OH).sub.2D.sub.3 33.3% Yes Dihydrotachysterol No 1,25(OH).sub.2D.sub.3-26,23-lactone No Paracalcitol (Zemplar) No Calcipotriene (Dovonex) No 7-Dehydrocholesterol No
Example 7: Reproducibility
(71) Six standards at 5, 15, 30, 60, 90, and 120 ng/mL for each analyte were run in every assay as a means as quantitating reproducibility. The day-to-day reproducibility was determined using calibration curves from 19 assays. The data from these 19 assays are presented in Tables 6A (for 25OHD.sub.2) and 6B (for 25OHD.sub.3).
(72) TABLE-US-00007 TABLE 6A Standard curves demonstrate reproducibility of 25OHD.sub.2-PTAD determination. Concentration 5 15 30 60 90 120 Assay ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 1 0.06 0.16 0.36 0.68 0.92 1.23 2 0.08 0.17 0.36 0.61 0.94 1.18 3 0.07 0.17 0.32 0.66 0.92 1.19 4 0.06 0.19 0.29 0.69 0.98 1.16 5 0.07 0.15 0.37 0.60 0.85 1.13 6 0.07 0.16 0.32 0.64 0.95 1.20 7 0.07 0.16 0.35 0.63 0.99 1.18 8 0.06 0.16 0.35 0.60 0.98 1.31 9 0.06 0.18 0.32 0.66 0.96 1.10 10 0.06 0.15 0.35 0.62 0.89 1.22 11 0.05 0.17 0.33 0.65 0.96 1.17 12 0.04 0.17 0.32 0.61 0.97 1.12 13 0.05 0.16 0.34 0.62 0.97 1.30 14 0.06 0.17 0.31 0.61 0.95 1.21 15 0.07 0.16 0.34 0.70 0.94 1.30 16 0.08 0.17 0.39 0.70 1.06 1.27 17 0.06 0.15 0.36 0.65 1.03 1.20 18 0.05 0.18 0.34 0.81 0.91 1.33 19 0.06 0.17 0.30 0.62 1.06 1.21 Avg 0.06 0.16 0.34 0.65 0.96 1.21 SD 0.01 0.01 0.02 0.05 0.05 0.07 CV % 15.4 6.3 7.4 8.0 5.6 5.4
(73) TABLE-US-00008 TABLE 6B Standard curves demonstrate reproducibility of 25OHD.sub.3-PTAD determination. Concentration 5 15 30 60 90 120 Assay ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 1 0.07 0.16 0.36 0.61 0.95 1.19 2 0.07 0.17 0.32 0.66 1.01 1.12 3 0.06 0.16 0.32 0.60 1.00 1.16 4 0.06 0.17 0.31 0.60 0.94 1.09 5 0.05 0.16 0.33 0.65 0.96 1.11 6 0.07 0.17 0.34 0.65 0.87 1.13 7 0.07 0.17 0.31 0.61 0.95 1.21 8 0.06 0.15 0.29 0.58 0.90 1.21 9 0.07 0.17 0.32 0.65 0.88 1.15 10 0.06 0.14 0.30 0.57 1.05 1.16 11 0.06 0.15 0.30 0.56 0.87 1.15 12 0.05 0.15 0.31 0.64 0.85 1.06 13 0.06 0.16 0.33 0.60 0.88 1.08 14 0.06 0.17 0.31 0.61 0.91 1.22 15 0.06 0.18 0.34 0.66 0.96 1.18 16 0.06 0.17 0.35 0.65 0.94 1.21 17 0.06 0.17 0.36 0.64 0.94 1.17 18 0.07 0.17 0.34 0.66 0.98 1.18 19 0.07 0.16 0.34 0.68 0.84 1.27 Avg 0.06 0.16 0.33 0.63 0.93 1.16 SD 0.00 0.01 0.02 0.03 0.06 0.05 CV % 7.9 5.8 5.9 5.5 6.1 4.6
Example 8: Intra-Assay and Inter-Assay Variation Studies
(74) Intra-assay variation is defined as the reproducibility of results for a sample within a single assay. To assess intra-assay variation, twenty replicates from each of four quality control (QC) pools covering the reportable range of the assay were prepared and measured from pooled serum with 25OHD.sub.2 and 25OHD.sub.3 at arbitrary ultralow, low, medium, and high concentrations for each analyte. Acceptable levels for the coefficient of variation (CV) are less then 15% for the three higher concentration, and less than 20% for the lowest concentration (at or near the LOQ for the assay).
(75) The results of the intra-assay variation studies indicate that the CV for the four QC pools are 9.1%, 6.4%, 5.0%, and 5.9% with mean concentrations of 13.7 ng/mL, 30.0 ng/mL, 52.4 ng/mL, and 106.9 ng/mL, respectively, for PTAD-25OHD.sub.2, and 3.5%, 4.9%, 5.1%, and 3.3% with mean concentrations of 32.8 ng/mL, 15.0 ng/mL, 75.4 ng/mL, and 102.3 ng/mL, respectively, for PTAD-25OHD.sub.3. The data from analysis of these replicates is shown in Tables 7A and 7B.
(76) TABLE-US-00009 TABLE 7A PTAD-25OHD.sub.2 Intra-assay variation studies QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # Repli- 090837 090838 090839 090840 cate ng/mL ng/mL ng/mL ng/mL 1 15.2 31.4 49.5 108.9 2 12.3 29.7 53.2 109.3 3 13.8 30.8 50.9 98.9 4 12.4 30.1 50.4 111.5 5 14.6 27.2 49.7 109.0 6 14.6 29.1 47.6 110.3 7 13.6 33.0 53.3 95.6 8 11.4 29.9 53.3 98.5 9 14.0 31.5 55.2 110.7 10 13.7 29.1 49.0 113.5 11 13.7 29.5 56.8 100.4 12 13.0 25.5 54.1 105.4 13 15.6 34.2 53.6 102.0 14 11.7 28.7 52.9 103.2 15 13.5 28.1 49.4 121.0 16 13.6 29.8 52.0 102.9 17 13.1 29.4 56.8 113.4 18 14.4 30.6 54.5 103.3 19 16.2 31.6 53.1 110.8 20 12.7 30.7 110.4 Avg 0.06 0.16 0.33 0.63 SD 0.00 0.01 0.02 0.03 CV % 7.9 5.8 5.9 5.5
(77) TABLE-US-00010 TABLE 7B PTAD-25OHD.sub.3 Intra-assay variation studies QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # Repli- 090837 090838 090839 090840 cate ng/mL ng/mL ng/mL ng/mL 1 34.4 13.7 75.7 101.7 2 35.0 14.2 78.7 101.8 3 33.2 14.7 73.1 103.2 4 34.4 14.9 83.7 104.1 5 32.4 14.5 72.7 107.0 6 33.3 14.3 73.6 107.6 7 33.8 15.0 79.1 97.5 8 32.1 15.8 73.1 98.7 9 32.4 15.5 74.2 106.5 10 31.4 15.4 74.5 106.1 11 31.8 14.7 69.3 105.9 12 31.2 16.8 73.5 97.7 13 34.1 15.4 72.7 104.9 14 33.8 15.3 75.1 99.8 15 32.0 15.7 76.2 102.2 16 33.2 14.7 74.2 102.2 17 32.6 14.7 85.0 100.5 18 31.6 13.9 75.5 101.8 19 31.3 15.6 73.6 99.9 20 32.5 15.3 96.3 Avg 32.8 15.0 75.4 102.3 SD 1.1 0.7 3.8 3.4 CV % 3.5 4.9 5.1 3.3
(78) Five aliquots of each of the same four QC pools were assayed over six days to determine the coefficient of variation (CV) between assays. The results of the intra-assay variation studies indicate that the inter-assay CV for the four QC pools are about 8.3%, 6.2%, 8.1%, and 6.4% with mean concentrations of about 13.1 ng/mL, 29.8 ng/mL, 51.9 ng/mL, and 107.8 ng/mL, respectively, for PTAD-25OHD.sub.2, and about 4.8%, 6.7%, 4.7%, and 6.7% with mean concentrations of about 31.1 ng/mL, 14.5 ng/mL, 75.1 ng/mL, and 108.4 ng/mL, respectively, for PTAD-25OHD.sub.3. The data from analysis of these replicates is shown in Tables 8A and 8B.
(79) TABLE-US-00011 TABLE 8A PTAD-25OHD.sub.2 Inter-assay variation studies QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Assay ng/mL ng/mL ng/mL ng/mL 1 13.6 28.1 51.7 119.6 12.8 30.1 49.4 117.9 14.6 32.0 49.7 105.1 13.0 30.8 52.3 100.2 13.0 29.2 56.6 110.3 2 12.9 31.3 46.3 108.1 13.5 30.3 52.1 117.8 10.9 29.7 46.9 105.8 11.2 30.6 43.6 105.2 12.8 28.7 50.3 104.9 3 12.6 28.8 56.5 115.3 16.4 29.3 63.8 103.0 13.2 26.2 45.5 103.2 11.5 30.8 53.8 113.2 12.4 33.7 51.6 106.9 4 12.1 28.5 58.5 97.0 13.9 26.2 51.8 115.1 14.4 29.6 48.9 112.2 13.1 32.1 52.3 97.9 12.6 30.5 52.2 104.2 5 12.7 29.9 54.5 101.3 14.3 28.3 46.3 102.2 13.9 30.0 56.1 111.4 13.1 32.6 51.2 123.1 12.4 26.2 51.2 98.3 6 12.5 30.6 50.1 104.6 12.9 32.6 51.8 104.8 14.0 28.6 53.7 108.9 14.3 29.1 51.0 113.8 12.9 29.1 56.4 102.2 Avg 13.1 29.8 51.9 107.8 SD 1.1 1.8 4.2 6.8 CV % 8.3 6.2 8.1 6.4
(80) TABLE-US-00012 TABLE 8B PTAD-25OHD.sub.3 Inter-assay variation studies QC (U) QC (L) QC (M) QC (H) Lot # Lot # Lot # Lot # 090837 090838 090839 090840 Assay ng/mL ng/mL ng/mL ng/mL 1 32.6 13.4 76.7 104.9 30.0 12.7 77.6 107.0 34.1 15.4 78.4 107.1 34.0 14.8 76.6 105.1 30.2 15.5 74.8 110.2 2 33.5 13.2 69.8 109.8 32.4 14.3 75.0 106.4 30.2 16.2 73.4 112.1 31.4 16.1 71.9 97.0 31.4 13.7 75.2 117.5 3 31.5 13.3 70.2 112.4 32.1 14.6 82.6 101.5 31.0 15.4 70.8 99.8 28.7 15.6 74.3 103.6 30.7 15.1 79.8 99.1 4 31.9 14.5 76.3 124.2 27.5 14.0 70.5 113.6 27.9 14.8 74.5 112.5 32.1 16.1 74.3 108.8 31.0 14.4 74.5 110.1 5 31.2 13.1 76.7 96.5 31.5 13.5 82.9 106.1 31.5 14.7 70.9 112.9 30.9 14.5 77.6 117.7 31.0 13.9 73.1 101.9 6 29.8 15.6 73.3 110.1 30.5 13.5 71.5 99.3 31.0 13.9 72.6 120.5 30.5 14.6 74.2 109.4 30.7 13.6 81.8 115.9 Avg 31.1 14.5 75.1 108.4 SD 1.5 1.0 3.6 6.9 CV % 4.8 6.7 4.7 6.4
Example 9: Recovery Studies
(81) Two recovery studies were performed. The first was performed using six specimens, spiked with two different concentrations each of 25OHD.sub.2 and 25OHD.sub.3. These spiked specimens were subjected to the hybrid protein precipitation/liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts of the spiked specimens were derivatized with normal PTAD, following the procedure discussed above, and analyzed in quadruplicate. The spiked concentrations were within the workable range of the assay. The six pools yielded an average accuracy of about 89% at spiked levels of greater than about 44 ng/mL and about 92% at spiked levels of greater than about 73 ng/mL. Only two of the 24 experimental recoveries were less than 85%; the remaining 22 assays were within the acceptable accuracy range of 85-115%. The results of the spiked specimen recovery studies are presented in Table 9, below.
(82) TABLE-US-00013 TABLE 9 Spiked Specimen Recovery Studies 25OHD.sub.2 25OHD.sub.3 (% (% Pool Spike Level ng/mL Recovery) ng/mL Recovery) 1 12.0 10.8 44 ng/mL 25OHD.sub.2 48.0 81.2 10.7 73 ng/mL 25OHD.sub.2 79.0 91.6 10.7 44 ng/mL 25OHD.sub.3 12.7 51.9 92.9 73 ng/mL 25OHD.sub.3 11.5 76.5 89.9 2 11.9 10.8 44 ng/mL 25OHD.sub.2 48.0 81.4 10.6 73 ng/mL 25OHD.sub.2 75.6 87.1 11.0 44 ng/mL 25OHD.sub.3 10.0 48.8 85.6 73 ng/mL 25OHD.sub.3 11.6 76.4 89.7 3 13.6 6 44 ng/mL 25OHD.sub.2 52.5 87.8 10.9 73 ng/mL 25OHD.sub.2 76.8 86.4 10.5 44 ng/mL 25OHD.sub.3 13.2 49.6 88.0 73 ng/mL 25OHD.sub.3 12.3 78.0 92.2 4 9.0 12.7 44 ng/mL 25OHD.sub.2 50.3 93.1 13.5 73 ng/mL 25OHD.sub.2 77.6 93.8 13.2 44 ng/mL 25OHD.sub.3 10.0 52.1 89.0 73 ng/mL 25OHD.sub.3 9.5 83.6 97.0 5 21.8 14.0 44 ng/mL 25OHD.sub.2 68.0 104.2 13.3 73 ng/mL 25OHD.sub.2 91.1 94.8 13.6 44 ng/mL 25OHD.sub.3 23.3 53.5 89.1 73 ng/mL 25OHD.sub.3 22.2 86.4 99.1 6 13.8 9.3 44 ng/mL 25OHD.sub.2 50.6 83.0 9.2 73 ng/mL 25OHD.sub.2 83.9 95.9 9.5 44 ng/mL 25OHD.sub.3 13.5 48.6 88.6 73 ng/mL 25OHD.sub.3 13.2 76.5 91.9
(83) The second recovery study was performed again using six specimens. Of these six specimens, three had high endogenous concentration of 25OHD.sub.2 and three had high endogenous concentrations of 25OHD.sub.3. The specimens were paired and mixed at ratios of about 4:1, 1:1, and 1:4. The resulting mixtures were subjected to the hybrid protein precipitation/liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts of the mixed specimens were derivatized with normal PTAD, following the procedure discussed above, and analyzed in quadruplicate. These experiments yielded an average accuracy of about 98% for 25OHD.sub.2 and about 93% for 25OHD.sub.3. All individual results were within the acceptable accuracy range of 85-115%. The results of the mixed specimen recovery studies are presented in Table 10, below.
(84) TABLE-US-00014 TABLE 10 Mixed Specimen Recovery Studies 25OHD.sub.2 25OHD.sub.3 Spec- Meas- Ex- Recov- Meas- Ex- Recov- imen ured pected ery ured pected ery Mixture ng/mL ng/mL (%) ng/mL ng/mL (%) 100% A 45.2 5.5 4:1 A:B 37.1 37.0 100 11.6 13.1 88 1:1 A:B 26.4 24.6 107 24.4 24.4 100 1:4 A:B 12.6 12.3 102 33.9 35.7 95 100% B 4.1 43.3 100% C 46.8 8.3 4:1 C:D 38.1 38.7 98 17.7 18.3 97 1:1 C:D 25.0 26.6 94 32.0 33.4 96 1:4 C:D 14.4 14.4 100 46.5 48.4 96 100% D 6.3 58.5 100% E 38.7 7.4 4:1 E:F 33.4 34.3 97 15.7 17.5 89 1:1 E:F 27.1 27.7 98 27.8 32.6 85 1:4 E:F 18.3 21.0 87 44.0 47.7 92 100% F 16.6 57.8 *Measured values are averages of analysis of four aliquots.
Example 10: Method Correlation Study
(85) The method of detecting vitamin D metabolites following PTAD-derivatization was compared to a mass spectrometric method in which the vitamin D metabolites are not derivatized prior to analysis. Such a method is described in the published U.S. Patent Application 2006/0228808 (Caulfield, et al.). Eight specimens were split and analyzed according to both methods. The correlation between the two methods was assessed with linear regression, deming regression, and Bland-Altman analysis for complete data sets (including calibration samples, QC pools, and unknowns), as well as for unknowns only.
(86) Plots of the linear regression analysis and the Deming regression analysis are shown in
Example 11: Hemolysis, Lipemia, and Icteria Studies
(87) The effect hemolysis, lipemia, and icteria have on the assay was also investigated.
(88) Hemolysis.
(89) The effect of hemolysis was evaluated by pooling patient samples with known endogenous concentrations of both 25OHD.sub.2 and 25OHD.sub.3 to create five different pools with concentrations across the dynamic range of the assay. Then, lysed whole blood was spiked into the pools to generate lightly and moderately hemolyzed samples.
(90) The lightly and moderately hemolyzed samples were analyzed in quadruplicate and the results were compared to levels of samples without whole blood spikes. The resulting comparison indicated a % difference of less than 15% for both 25OHD.sub.2 and 25OHD.sub.3. Therefore, light to moderately hemolyzed specimens are acceptable for analysis.
(91) Lipemia.
(92) The effect of lipemia was evaluated by pooling patient samples with known endogenous concentrations of both 25OHD.sub.2 and 25OHD.sub.3 to create five different pools with concentrations across the dynamic range of the assay. Then, powdered lipid extract was added to the pools to generate lightly and grossly lipemic specimens. Specimens were run in quadruplicate and results were compared to the non-lipemic pool result and the accuracy was calculated. The data shows that determination of 25OHD.sub.2 is unaffected by lipemia (all values were within an acceptable accuracy range of 85-115%), however, 25OHD.sub.3 is affected by lipemia, resulting in determination in lower than expected values. The degree of variance increased with the degree of lipemia. Therefore, light but not grossly lipemic specimens are acceptable.
(93) Icteria.
(94) The effect of icteria was evaluated by pooling patient samples with known endogenous concentrations of both 25OHD.sub.2 and 25OHD.sub.3 to create five different pools with concentrations across the dynamic range of the assay. Then, a concentrated solution of Bilirubin was spiked into the pools to generate lightly and grossly icteric specimens. Specimens were run in quadruplicate and results were compared to the non-icteric pool result and the accuracy was calculated. The data showed that 25OHD.sub.2 and 25OHD.sub.3 are unaffected by icteria (with all values within an acceptable accuracy range of 85-115%). Therefore, icteric specimens are acceptable.
Example 12: Injector Carryover Studies
(95) Blank matrices were run immediately after a specimen with a high concentration of 25OHD.sub.2 and 25OHD.sub.3 in order to evaluate carryover between samples. These studies indicated that the response at the retention time of analyte or internal standard was not large enough to compromise the integrity of the assay. Data from these studies is presented in Table 11, below.
(96) TABLE-US-00015 TABLE 11 Injector Carryover Study Results Specimen 25OHD.sub.2 25OHD.sub.3 Injection Type (ng/mL) (ng/mL) 1 Blank 0.9 1.6 2 High 292.6 356.8 3 Blank 1.0 0.9 4 Blank -0.1 0.5 5 High 290.1 360.1 6 High 299.9 350.5 7 Blank 1.0 1.5 8 Blank 0.6 1.4 9 Blank 1.3 1.4 10 High 285.8 352.1 11 High 303.1 312.1 12 High 293.8 295.1 13 Blank 0.9 0.8 14 Blank 1.0 1.8 15 Blank 1.1 1.4 16 Blank 1.0 1.6 17 High 291.7 371.6 18 High 334.2 360.1 19 High 301.7 328.5 20 High 283.1 382.1 21 Blank 0.6 1.1 22 Blank 0.6 1.3 23 Blank 0.7 1.4 24 Blank 0.4 1.9 25 Blank 0.4 0.9 26 High 300.7 311.7 27 High 279.5 302.0 28 High 317.5 341.0 29 High 261.5 403.4 30 High 288.3 362.6 31 Blank 2.7 1.6 32 Blank 1.7 1.2 33 Blank 0.5 1.3 34 Blank 1.3 1.7 35 Blank 0.3 1.6 36 Blank 0.6 1.4 37 High 311.7 366.2 38 High 314.1 342.0 39 High 325.7 349.1 40 High 289.6 326.6 41 High 291.5 322.3 42 High 278.9 336.5 43 Blank 2.1 2.5 44 Blank 0.6 1.6 45 Blank 0.7 1.4 46 Blank 0.7 1.5 47 Blank 0.1 1.0 48 Blank 0.7 1.1 49 Blank 1.3 1.0 50 High 281.2 345.6 51 High 312.5 348.3 52 High 304.8 329.1 53 High 290.5 353.9 54 High 286.4 344.9 55 High 302.5 330.6 56 High 292.2 388.5 57 Blank 0.8 1.5 58 Blank 1.3 1.4 59 Blank 3.5 2.6 60 Blank 0.4 1.8 61 Blank 1.0 1.4 62 Blank 1.0 1.2 63 Blank 0.7 1.0 64 Blank 1.1 1.4 65 High 285.4 355.4 66 High 318.0 355.0 67 High 285.5 345.7 68 High 303.0 317.1 69 High 276.3 351.4 70 High 321.8 350.4 71 High 279.4 329.6 72 High 299.1 337.9 73 Blank 0.9 1.6 74 Blank 1.7 1.6 75 Blank 1.0 1.1 76 Blank 1.8 2.7 77 Blank 1.0 1.9 78 Blank 0.6 1.1 79 Blank 0.9 0.9 80 Blank 1.2 2.2
Example 13: Suitable Specimen Types
(97) The assay was conducted on various specimen types. Human serum and Gel-Barrier Serum (i.e., serum from Serum Separator Tubes), as well as EDTA Plasma and Heparin were established as acceptable sample types. In these studies, sets of human serum (serum), Gel-Barrier Serum (SST), EDTA Plasma (EDTA), and heparin (Na Hep) drawn at the same time from the same patient were tested for 25OHD.sub.2 (40 specimen sets) and 25OHD.sub.3 (6 specimen sets). Due to the limitations with clot detection/sensing in existing automated pipetting systems, plasma was not tested for automated procedures.
(98) The results of the specimen type studies are presented in Tables 12A and B for 25OHD.sub.2 and 25OHD.sub.3, respectively.
(99) TABLE-US-00016 TABLE 12A Results from Specimen Type Studies for 25OHD.sub.2 Specimen CC Measured Concentration 25OHD.sub.2 (ng/mL) Set ID# Serum SST EDTA Na Hep 1 5804 26.8 25.7 24.3 26.8 2 5207 16.1 17.6 16.1 16.5 3 5235 17.4 17.7 16.8 17.2 4 5333 62.9 62.7 63.7 57.4 5 5336 33.0 32.4 28.8 28.8 6 5339 17.2 17.6 17.8 17.8 7 5340 16.7 17.1 16.8 16.5 8 5342 28.6 27.9 26.9 30.5 9 5344 23.3 23.8 22.3 22.9 10 5351 19.4 20.0 20.4 21.4 11 5355 17.6 16.7 19.4 18.3 12 5362 25.3 25.2 23.5 24.0 13 5365 40.9 44.7 46.8 42.9 14 5406 23.1 20.3 21.5 20.5 15 5408 31.7 33.9 31.6 32.3 16 5414 21.1 21.8 21.2 20.4 17 5422 44.0 47.7 45.5 47.3 18 5432 13.6 14.2 12.3 13.8 19 5463 15.1 15.4 15.6 14.5 20 5493 38.6 42.2 40.1 36.8 21 5366 47.5 48.1 46.7 45.1 22 5368 23.0 23.6 22.3 22.3 23 5392 34.1 33.4 34.4 27.6 24 5451 36.4 42.1 40.0 38.3 25 5455 27.3 29.9 25.1 27.9 26 5476 16.7 17.9 15.8 16.6 27 5483 30.4 28.2 26.5 28.1 28 5484 38.2 37.7 37.2 36.0 29 5537 30.5 30.3 27.2 27.0 30 5547 9.2 9.0 8.7 8.2 31 5560 9.4 10.9 9.8 8.6 32 5571 30.9 31.7 29.6 29.2 33 5572 47.6 50.3 47.7 48.6 34 5577 11.2 11.7 10.4 9.2 35 5606 39.3 38.8 41.0 37.7 36 5611 21.9 25.3 20.7 21.1 37 5650 38.0 34.3 34.6 36.2 38 5651 34.8 32.8 32.4 32.4 39 5653 29.4 32.3 28.1 27.0 40 5668 11.4 12.8 14.2 13.1
(100) TABLE-US-00017 TABLE 12B Results from Specimen Type Studies for 25OHD.sub.3 Specimen CC Measured Concentration 25OHD.sub.3 (ng/mL) Set ID# Serum SST EDTA Na Hep 2 5207 6.6 6.9 7.1 7.2 6 5339 5.8 5.2 4.5 5.6 11 5355 7.8 8.2 8.8 8.2 20 5493 3.9 4.2 4.3 4.2 37 5650 3.7 4.5 4.6 5.2 39 5653 4.7 5.1 4.6 4.7
Example 14: Multiplex Patient Samples with Multiple Derivatizing Agents
(101) Patient sample multiplexing after derivatization with different derivatizing agents was demonstrated in the following crossover experiments.
(102) First, two patients samples (i.e., sample A and sample B) were both subjected to the hybrid protein precipitation/liquid-liquid extraction procedure described in Example 1. Then, aliquots of the extracts from sample A and sample B were derivatized with normal PTAD, following the procedure discussed above. Second aliquots of the extracts from sample A and sample B were also derivatized with .sup.13C.sub.6-PTAD, also according to the procedure discussed above.
(103) After the four derivatization reactions were quenched, a portion of the PTAD-derivatized sample A was mixed with .sup.13C.sub.6-PTAD-derivatized sample B, and a portion of .sup.13C.sub.6-PTAD-derivatized sample A was mixed with PTAD-derivatized sample B.
(104) These mixtures were loaded onto a 96-well plate and analyzed according to the liquid chromatography-mass spectrometry methods described in Examples 2 and 3. Again, 25OHD.sub.2-[6, 19, 19]-.sup.2H.sub.3 and 25OHD.sub.3-[6, 19, 19]-.sup.2H.sub.3 were used as internal standards (shown in Table 13, below, as 25OHD.sub.2-IS and 25OHD.sub.3-IS). The mass spectrometer was programmed to monitor for the PTAD- and .sup.13C.sub.6-PTAD-derivatized vitamin D metabolite conjugates shown in Table 13. The indicated mass transitions re not meant to be limiting in any way. As seen in the Examples that follow, other mass transitions could be selected for each analyte to generate quantitative data.
(105) TABLE-US-00018 TABLE 13 Ions monitored for mass spectrometric determination of multiplex PTAD- and .sup.13C.sub.6-PTAD-derivatized samples (by MRM). Analyte Precursor Fragment PTAD-25OHD.sub.3 558 298 PTAD-25OHD.sub.3-IS 561 301 PTAD-25OHD.sub.2 570 298 PTAD-25OHD.sub.2-IS 573 301 .sup.13C.sub.6-PTAD-25OHD.sub.3 564 304 .sup.13C.sub.6-PTAD-25OHD.sub.3-IS 567 307 .sup.13C.sub.6-PTAD-25OHD.sub.2 576 304 .sup.13C.sub.6-PTAD-25OHD.sub.2-IS 579 307
(106) Derivatized samples A and B and permutations of mixtures of the two described above were analyzed and plotted to evaluate goodness of fit of the data. These results are presented in
(107)
(108)
(109)
(110) Thus, isotopic variation of the PTAD derivatization agent made no meaningful difference even when samples were mixed together and introduced into the mass spectrometer as a single injection. Multiplexing of patient samples was successfully demonstrated.
Example 15: Exemplary Spectra from LDTD-MS/MS Analysis of Native and PTAD Derivatized 25-Hydroxyvitamin D.SUB.2 .and 25-Hydroxyvitamin D.SUB.3
(111) Underivatized and PTAD derivatized 25-hydroxyvitamin D.sub.2 and 25-hydroxyvitamin D.sub.3 were analyzed by LDTD-MS/MS. Results of these analyses are presented below.
(112) Exemplary Q1 scan spectra from analysis of 25-hydroxyvitamin D.sub.2 and 25-hydroxyvitamin D.sub.3 are shown in
(113) Exemplary product ion scans from each of these species are presented in
(114) A preferred MRM transition for the quantitation of 25-hydroxyvitamin D.sub.2 is fragmenting a precursor ion with a m/z of about 395.2 to a product ion with a m/z of about 208.8 or 251.0. A preferred MRM transition for the quantitation of 25-hydroxyvitamin D.sub.3 is fragmenting a precursor ion with a m/z of about 383.2 to a product ion with a m/z of about 186.9 or 257.0. However, as can be seen in the product ion scans in
(115) TABLE-US-00019 TABLE 14 Precursor Ions and Collision Cell Energies for Fragmentation of 25-hydroxyvitamin D.sub.2 and 25-hydroxyvitamin D.sub.3 Collision Precursor Cell Energy Analyte Ion (m/z) (V) 25-hydroxyvitamin D.sub.2 395.2 20 25-hydroxyvitamin D.sub.3 383.2 20
(116) Exemplary Q1 scan spectra from the analysis of samples containing PTAD-25-hydroxyvitamin D.sub.2 and PTAD-25-hydroxyvitamin D.sub.3 are shown in
(117) Exemplary product ion scans from each of these species are presented in
(118) A preferred MRM transition for the quantitation of PTAD-25-hydroxyvitamin D.sub.2 is fragmenting a precursor ion with a m/z of about 570.3 to a product ion with a m/z of about 298.1. A preferred MRM transition for the quantitation of PTAD-25-hydroxyvitamin D.sub.3 is fragmenting a precursor ion with a m/z of about 558.3 to a product ion with a m/z of about 298.1. However, as can be seen in the product ion scans in
(119) TABLE-US-00020 TABLE 15 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-25-hydroxyvitamin D.sub.2 and PTAD-25-hydroxyvitamin D.sub.3 Collision Precursor Cell Energy Analyte Ion (m/z) (V) PTAD-25-hydroxyvitamin D.sub.2 570.3 15 PTAD-25-hydroxyvitamin D.sub.3 558.3 15
Example 16: Exemplary Spectra from LDTD-MS/MS Analysis of PTAD Derivatized 1,25-Dihydroxyvitamin D.SUB.2 .and 1,25-Dihydroxyvitamin D.SUB.3
(120) PTAD derivatives of 1,25-dihydroxyvitamin D.sub.2 and 1,25-dihydroxyvitamin D.sub.3 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 LDTD-MS/MS procedure outlined above.
(121) Exemplary Q1 scan spectra from the analysis of samples containing PTAD-1,25-dihydroxyvitamin D.sub.2 and PTAD-1,25-hydroxyvitamin D.sub.3 are shown in
(122) Exemplary product ion scans generated from three different precursor ions for each of PTAD-1,25-dihydroxyvitamin D.sub.2 and PTAD-1,25-hydroxyvitamin D.sub.3 are presented in
(123) Exemplary MRM transitions for the quantitation of PTAD-1,25-dihydroxyvitamin D.sub.2 include fragmenting a precursor ion with a m/z of about 550.4 to a product ion with a m/z of about 277.9; fragmenting a precursor ion with a m/z of about 568.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 586.4 to a product ion with a m/z of about 314.2. Exemplary MRM transitions for the quantitation of PTAD-1,25-hydroxyvitamin D.sub.3 include fragmenting a precursor ion with a m/z of about 538.4 to a product ion with a m/z of about 278.1; fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 298.0; and fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 313.0. However, as can be seen in the product ion scans in
(124) TABLE-US-00021 TABLE 16 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-1,25-dihydroxyvitamin D.sub.2 and PTAD-1,25- dihydroxyvitamin D.sub.3 Collision Precursor Cell Energy Analyte Ion (m/z) (V) PTAD-1,25-dihydroxyvitamin D.sub.2 550.4, 568.4, 586.4 15 PTAD-1,25-dihydroxyvitamin D.sub.3 538.4, 556.4, 574.4 15
(125) PTAD derivatives of various deuterated forms of dihydroxyvitamin D metabolites were also investigated. PTAD derivatives of 1,25-dihydroxyvitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6, 1,25-dihydroxyvitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3, and 1,25-dihydroxyvitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 were prepared and analyzed as above.
(126) Exemplary MRM transitions for the quantitation of PTAD-1,25-dihydroxyvitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 include fragmenting a precursor ion with a m/z of about 556.4 to a product ion with a m/z of about 278.1; fragmenting a precursor ion with a m/z of about 574.4 to a product ion with a m/z of about 298.1; and fragmenting a precursor ion with a m/z of about 592.4 to a product ion with a m/z of about 313.9.
(127) Exemplary MRM transitions for the quantitation of PTAD-1,25-dihydroxyvitamin D.sub.3-[6, 19, 19]-.sup.2H.sub.3 include fragmenting a precursor ion with a m/z of about 541.4 to a product ion with a m/z of about 280.9; fragmenting a precursor ion with a m/z of about 559.4 to a product ion with a m/z of about 301.1; and fragmenting a precursor ion with a m/z of about 577.4 to a product ion with a m/z of about 317.3. Exemplary MRM transitions for the quantitation of PTAD-1,25-dihydroxyvitamin D.sub.2-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 include fragmenting a precursor ion with a m/z of about 544.4 to a product ion with a m/z of about 278.0; fragmenting a precursor ion with a m/z of about 562.4 to a product ion with a m/z of about 298.2; and fragmenting a precursor ion with a m/z of about 580.4 to a product ion with a m/z of about 314.0.
Example 17: Exemplary Spectra from MS/MS Analysis of PTAD Derivatized Vitamin D.SUB.2 .and Vitamin D.SUB.3
(128) PTAD derivatives of vitamin D.sub.2, and vitamin D.sub.3 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 by MS/MS.
(129) Exemplary Q1 scan spectra from the analysis of samples containing PTAD-vitamin D.sub.2, and PTAD-vitamin D.sub.3 are shown in
(130) Exemplary product ion scans generated from precursor ions for each of PTAD-vitamin D.sub.2 and PTAD-vitamin D.sub.3 are presented in
(131) 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.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. However, as can be seen in the product ion scans in
(132) TABLE-US-00022 TABLE 17 Precursor Ions and Collision Cell Energies for Fragmentation of PTAD-vitamin D.sub.2 and PTAD-vitamin D.sub.3 Collision Precursor Cell Energy Analyte Ion (m/z) (V) PTAD-vitamin D.sub.2 572.2 15 PTAD-vitamin D.sub.3 560.2 15
(133) PTAD derivatives of various deuterated forms of vitamin D were also investigated. PTAD derivatives of 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-[6, 19, 19]-.sup.2H.sub.3, and vitamin D.sub.3-[26, 26, 26, 27, 27, 27]-.sup.2H.sub.6 were prepared and analyzed as above.
(134) 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.
(135) 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.
(136) 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.
(137) 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.
(138) 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.
(139) 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.