Methods and systems for the quantitative analysis of biomarkers   

The present application is a divisional application of allowed U.S. patent application Ser. No. 11/805,985, filed May 25, 2007, entitled “Methods and Systems for the Quantitative Analysis of Biomarkers which claims priority under 35 U.S.C. §119(e) from U.S. Provisional Patent Application No. 60/808,812, filed May 26, 2006. The disclosure of U.S. patent application Ser. No. 11/805,985 and U.S. Provisional Patent Application No. 60/808,812 are incorporated by reference in their entireties herein.

The presently disclosed subject matter relates to methods and systems for the analysis of biomarkers. In certain embodiments, the biomarkers are endogenous to human subjects such that the measurement may be used for clinical diagnosis.

Biomarkers, such as hormones, vitamins, metabolites, can be used for the clinical diagnosis of multiple disorders and as endogenous biomarkers in endocrinology. For example, the measurement of estrogen compounds, such as estrone and estradiol can be used to evaluate ovarian function and to evaluate excess or diminished estrogen levels in a patient. Also, measurement of thyroxine can be used to quantify thyroid function.

Requirements for the clinical diagnostic testing of endogenous biomarkers in endocrinology may include highly sensitive and specific assays, the ability to analyze small sample volumes (e.g., pediatric sample volumes can be limited to less than about 200 μL), and the ability to screen for multiple analytes to accurately diagnose a disease state, e.g., an endocrine disorder. Historically, radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) methods have been used in such clinical diagnostic testing. Immunoassay methods (IA), such as RIA and EIA, however, may suffer from low throughput, antibody cross-reactivity, which can require extra preparation for specificity, and poor scalability. Also, the analysis of endogenous biomarkers by RIA may require multiple serial dilutions for the analysis of each individual marker, which can lead to the need to make multiple adjustments to normalize sample volumes and/or the need for multiple separate tests. Also, immunoassay testing is not particularly conducive to the analysis of multiple biomarkers in each sample. The analysis for multiple analytes in a single assay can allow for using samples of reduced size which results in assays of increased sensitivity and efficiency per sample.

An important class of hormones are the steroid hormones, such as testosterone and estrogens. Testosterone develops and maintains the male secondary sex characteristics, and promotes growth and development of sperm. Estrogen is the term used for a group of hormones of which there are three principle forms, estrone, estradiol, and estriol.

For example, relatively small variations in estrogen levels may be clinically significant. Generally, the level of estrogen in post-menopausal women, adult males, and prepubescent children is ≦10 pg/mL. Elevated estrogen levels in children may lead to precocious puberty (and short stature). In post-menopausal women, low estrogen levels may require replacement, where as levels greater than 5 pg/mL may be prognostic for certain cancers. In adult males, elevated estrogen levels may be indicative of certain disease states (testicular cancer). In adult females, reduced or elevated levels may also be indicative of certain cancers (e.g., ovarian cancer). A level of serum estrogen of 15 pg/mL is clinically different from 10 pg/mL and thus, measurement of estrogen compounds (e.g., estradiol and estrone) requires an LLOQ of 1-5 pg/mL irrespective of sample type, patient age, gender and diet.

Another important class of hormones are the thyroid hormones. Thyroxine (T4) and triiodothyronine (T3) are examples of thyroid hormones. T4 and T3 enter cells and bind to intracellular receptors. T4 and T3 are important in regulation of a number of factors including growth and development, carbohydrate metabolism, oxygen consumption, and protein synthesis. T4 acts as a prohormone, as the bulk of T3 present in blood is produced by monodeiodination of T4 by intracellular enzymes. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.

Thus, there is a need to develop analytical techniques that can be used for the measurement of endogenous biomarkers, and for methods that provide more sensitivity and higher throughput than RIA. Until recently, however, only GC-MS or LC-MS/MS with derivatization has been successful for small sample volumes. Thus, there is a need in the art for LC-MS/MS techniques for the analysis of endogenous biomarkers for clinical diagnosis in endocrinology capable of providing detection limits at acceptable levels, without the need for the cumbersome derivatization processes.

SUMMARY

In some embodiments, the presently disclosed subject matter provides methods and systems for the quantitative analysis of endocrine biomarkers in a test sample. The quantification of such markers may, in certain embodiments, be used for clinical diagnosis in endocrinology. For example, in some embodiments, the methods and systems of the present invention may be used for the quantitative analysis of total levels of certain hormones, including steroid hormones, such as estrone and estradiol, and their metabolites, such as estrone sulfate. In other embodiments, the methods and systems of the present invention provide for the quantitative analysis of biomarkers that can be difficult to detect in their active state. For example, the systems and methods of the present invention may be used to quantify free (i.e., not bound to protein) serum hormones, such as free thyroxine (T4) in biological samples. Or, in other embodiments, the systems and methods of the present invention may be used to quantify free triiodothyronine (T3) or testosterone. In an embodiment, the methods and systems of the present invention allow for measurement of such hormones without the need for derivation processes.

In some embodiments, the biomarkers of interest are estradiol and/or estrone. Thus, in one embodiment, the present invention comprises a method for determining the presence or amount of estradiol in a sample by tandem mass spectrometry, comprising: (a) generating a dehydrated precursor ion of the estradiol; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the presence or amount of one or more of the ions generated in step (a) or (b) or both, and relating the detected ions to the presence or amount of the estradiol in the sample. In an embodiment, the sample comprises a mixture of estradiol and estrone.

In other embodiments, the biomarker comprises free thyroxine (T4) or triiodothyronine (T3). In certain embodiments, the present invention provides a high-throughput assay for free thyroxine (T4). Thus, in one embodiment, the present invention comprises a method for determining the presence or amount of free thyroxine in a plurality of samples by tandem mass spectrometry, comprising: (a) dialyzing the plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples; (b) generating a precursor ion of the thyroxine; (b) generating one or more fragment ions of the thyroxine; and (c) detecting the presence or amount of one or more of the ions generated in step (b) or (c) or both, and relating the detected ions to the presence or amount of the free thyroxine in the plurality of samples.

In some embodiments, the methods and systems of the present invention comprise liquid chromatography (LC) methods in combination with other analytical techniques as a means to measure such biomarkers with high sensitivity and high throughput. In certain embodiments, the present invention comprises quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of endocrine biomarkers in a test sample. In some embodiments, two-dimensional or tandem LC is used. The method may include, in alternate embodiments, liquid-liquid extractions, dialysis, sample dilution, and/or sample dehydration steps prior to analysis by tandem mass spectrometry.

Accordingly, embodiments of the present invention may provide methods for the quantitative LC-MS/MS and 2D-LC-MS/MS analysis of hormones, including steroid hormones, such as estrone and estradiol. Additionally or alternatively, embodiments of the present invention may provide methods for the quantitative determination of a free (i.e., non-protein bound) hormone or metabolite using dialysis in combination with LC-MS/MS analysis for hormones that in biological samples, may be predominantly protein-bound. Such hormones may include free thyroxine (T4), free triiodothyronine (3), or free testosterone. Certain objects of the present invention, having been stated hereinabove, will become further evident as the description proceeds when taken in connection with the accompanying figures and examples as described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale.

FIG. 1 shows a flow chart of a method for quantitative analysis of a biomarker of interest in accordance with one embodiment of the present invention.

FIG. 2 shows dehydration of estradiol and the effect on mass spectrometry (MS) analysis in accordance with an embodiment of the present invention.

FIG. 3 shows potential isobaric interferences for measurement of estrone and estradiol due to dehydration of dehydroepiandrosterone (DHEA) in accordance with one embodiment of the present invention.

FIG. 4 shows an example of heart-cutting from a primary separation gradient to remove compounds that comprise isobaric interference in the analysis of estrone and estradiol in accordance with one embodiment of the present invention.

FIG. 5 shows a method for the quantification of estrone and estradiol in accordance with an embodiment of the present invention.

FIG. 6 shows a method for the quantification of free thyroxine (T4) in accordance with an embodiment of the present invention.

FIG. 7 shows a system for quantitative analysis of a metabolite in accordance with one embodiment of the present invention (Panel A), and a system for multiplex analysis (Panel B) in accordance with alternate embodiments of the present invention.

FIG. 8 shows a LC-MS/MS chromatogram of estrone sulfate at a limit of quantification of 100 pg/mL in accordance with one embodiment of the present invention.

FIG. 9 shows a LC-MS/MS chromatogram of free thyroxine at a limit of quantification of 2 pg/mL in accordance with one embodiment of the present invention.

FIG. 10 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D2 at a limit of quantification of 1 ng/mL in accordance with one embodiment of the present invention.

FIG. 11 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D3 at a limit of quantification of 1 ng/mL in accordance with one embodiment of the present invention.

FIG. 12 shows a 2D-LC-MS/MS chromatogram of estrone at a limit of quantification of 2.5 pg/mL in accordance with one embodiment of the present invention.

FIG. 13 shows a 2D-LC-MS/MS chromatogram of estradiol at a limit of quantification of 1 pg/mL in accordance with one embodiment of the present invention.

FIG. 14 shows a LC-MS/MS chromatogram of estrone sulfate at an upper limit of quantification of 50 ng/mL in accordance with one embodiment of the present invention.

FIG. 15 shows a LC-MS/MS chromatogram of free thyroxine at an upper limit of quantification of 100 pg/dL in accordance with one embodiment of the present invention.

FIG. 16 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D2 at an upper limit of quantification of 250 ng/mL in accordance with one embodiment of the present invention.

FIG. 17 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D3 at an upper limit of quantification of 250 ng/mL in accordance with one embodiment of the present invention.

FIG. 18 shows a 2D-LC-MS/MS chromatogram of estrone at an upper limit of quantification of 500 pg/mL in accordance with one embodiment of the present invention.

FIG. 19 shows a 2D-LC-MS/MS chromatogram of estradiol at an upper limit of quantification of 500 pg/mL in accordance with one embodiment of the present invention.

FIG. 20 shows a calibration curve obtained by LC-MS/MS for estrone sulfate in accordance with one embodiment of the present invention.

FIG. 21 shows a calibration curve obtained by LC-MS/MS for free thyroxine in accordance with one embodiment of the present invention.

FIG. 22 shows a calibration curve obtained by 2D-LC-MS/MS for 25-hydroxyvitamin D2 in accordance with one embodiment of the present invention.

FIG. 23 shows a calibration curve obtained by 2D-LC-MS/MS for 25-hydroxyvitamin D3 in accordance with one embodiment of the present invention.

FIG. 24 shows a calibration curve obtained by 2D-LC-MS/MS for estrone in accordance with one embodiment of the present invention.

FIG. 25 shows a calibration curve obtained by 2D-LC-MS/MS for estradiol in accordance with one embodiment of the present invention.

FIG. 26 shows cross-validation data for LC-MS/MS as compared to radioimmunoassay (RIA) for estrone sulfate in accordance with one embodiment of the present invention.

FIG. 27 shows cross-validation data for LC-MS/MS as compared to immunoassay (IA) for free thyroxine in accordance with one embodiment of the present invention.

FIG. 28 shows cross-validation data for 2D-LC-MS/MS as compared to a competitive binding protein assay (CBP) (Panel A) or immunoassay (IA) (Panel B) for total 25-hydroxyvitamin D (25-hydroxyvitamin D2+D3) in accordance with alternate embodiments of the present invention.

FIG. 29 shows cross-validation data for 2D-LC-MS/MS as compared to RIA for Estrone in accordance with one embodiment of the present invention.

FIG. 30 shows cross-validation data for 2D-LC-MS/MS as compared to RIA for Estradiol in accordance with one embodiment of the present invention.

FIG. 31 shows a comparison of Estradiol (E2) cross-validation of LC-MS/MS with derivatization to 2D-LC-MS/MS without derivatization in accordance with an embodiment of the present invention.

FIG. 32 shows the measured concentration (pg/mL) of free thyroxine vs. dialysis time (hours). The squares (▪) show dialysis losses and the diamonds (♦) show effective dialysis for free thyroxine using 96-well equilibrium dialysis plates in accordance with one embodiment of the present invention.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying description and drawings, in which some, but not all embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. The disclosure utilizes the abbreviations shown below.

APCI=atmospheric pressure chemical ionization CBP=competitive binding protein

HTLC=high turbulence (throughput) liquid chromatography HPLC=high performance liquid chromatography LLE=liquid-liquid extraction LOQ=limits of quantification LLOQ=lower limit of quantification IA=immunoassay ELISA=enzyme linked immunoassay RIA=radioimmunoassay SST=system suitability test ULOQ=upper limit of quantification 2D-LC-MS/MS=two-dimensional liquid chromatography hyphenated to tandem mass spectrometry (LC)-LC-MS/MS=two-dimensional liquid chromatography tandem hyphenated to mass spectrometry (LC)-MS/MS=liquid chromatography hyphenated to tandem mass spectrometry

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. Other definitions are found throughout the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g. 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference referred to as being “incorporated herein” is to be understood as being incorporated in its entirety.

The terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, unless the context clearly is to the contrary (e.g., a plurality of cells), and so forth.

As used herein, the term “biomarker” is any biomolecule that may provide biological information about the physiological state of an organism. In certain embodiments, the presence or absence of the biomarker may be informative. In other embodiments, the level of the biomarker may be informative. A biomarker may be a hormone, such as an estrogen (e.g., estradiol, estrone), testosterone, thyroxine (T4), triiodothyronine (T3), or a metabolite of a hormone (estrogen sulfate). A biomarker may also be a vitamin or a metabolite of a vitamin. For example, in one embodiment, the measured biomarker may comprise a vitamin D compound such as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

As used herein, the terms “purify” or “separate” or derivations thereof do not necessarily refer to the removal of all materials other than the analyte(s) of interest from a sample matrix. Instead, in some embodiments, the terms “purify” or “separate” refer to a procedure that enriches the amount of one or more analytes of interest relative to one or more other components present in the sample matrix. In some embodiments, a “purification” or “separation” procedure can be used to remove one or more components of a sample that could interfere with the detection of the analyte, for example, one or more components that could interfere with detection of an analyte by mass spectrometry.

As used herein, “derivatizing” means reacting two molecules to form a new molecule. Derivatizing agents may include isothiocyanate groups, dansyl groups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/or phthalaldehyde groups.

As used herein, “chromatography” refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

As used herein, “liquid chromatography” (LC) means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). “Liquid chromatography” includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).

As used herein, the term “HPLC” or “high performance liquid chromatography” refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. 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. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties such as the biomarker analytes quantified in the experiments herein. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18 bonded alkyl groups, preferably C-18 bonded groups. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. In the method, the sample (or pre-purified sample) may be applied to the 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 different analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. In one embodiment, HPLC may performed on a multiplexed analytical HPLC system with a C18 solid phase using isocratic separation with water:methanol as the mobile phase.

As used herein, the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to effect a separation of the components of a test sample matrix. Preferably, the components eluted from the analytical column are separated in such a way to allow the presence or amount of an analyte(s) of interest to be determined. In some embodiments, the analytical column comprises particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.

Analytical columns can be distinguished from “extraction columns,” which typically are used to separate or extract retained materials from non-retained materials to obtained a “purified” sample for further purification or analysis. In some embodiments, the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monolithic silica stationary phase, such as a Poroshell SBC-18 column.

The term “heart-cutting” refers to the selection of a region of interest in a chromatogram and subjecting the analytes eluting within that region of interest to a second separation, e.g., a separation in a second dimension.

The term “electron ionization” as used herein refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

The term “chemical ionization” as used herein refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

The term “field desorption” as used herein refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

The term “matrix-assisted laser desorption ionization,” or “MALDI” as used herein refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

The term “surface enhanced laser desorption ionization,” or “SELDI” as used herein refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

The term “electrospray ionization,” or “ESI,” as used herein refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Upon reaching the end of the tube, the solution may be vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplet can flow through an evaporation chamber which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

The term “Atmospheric Pressure Chemical Ionization,” or “APCI,” as used herein refers to mass spectroscopy methods that are similar to ESI, however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then, ions are typically extracted into a mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N2 gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.

The term “Atmospheric Pressure Photoionization” (“APPI”) as used herein refers to the form of mass spectroscopy where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M+ (see e.g., Robb et al., 2000, Anal. Chem. 72(15): 3653-3659).

The term “inductively coupled plasma” as used herein refers to methods in which a sample is interacted with a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements.

The term “ionization” and “ionizing” as used herein refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those ions having a net negative charge of one or more electron units, while positive ions are those ions having a net positive charge of one or more electron units.

The term “desorption” as used herein refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase.

As used herein, the term “immunoassay” (IA) refers to a method for measuring the amount of an analyte of interest by quantifying the binding, or the inhibition of binding, of a substance to an antibody. Where an enzyme is used to detect the amount of binding of the substance (e.g. antigen) to an antibody, the assay is an enzyme-linked immunoassay (ELISA). As used herein, the term “radioimmunoassay” (RIA) refers to a method for measuring the amount of an analyte of interest by quantifying the binding, or the inhibition, of binding, of a radiolabeled substance to an antibody.

As used herein, the term “hemolysed” refers to the rupturing of the red blood cell membrane, which results in the release of hemoglobin and other cellular contents into the plasma or serum and the term “lipemic” refers to an excess of fats or lipids in blood.

Analysis of Biomarkers by LC-MS/MS

Thus, embodiments of the present invention relate to methods and systems for the quantitative analysis of endogenous biomarkers for clinical diagnosis. The present invention may be embodied in a variety of ways.

In one embodiment, the present invention comprises a method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample believed to contain at least one biomarker of interest; chromatographically separating the at least one biomarker of interest from other components in the sample; and analyzing the chromatographically separated at least one biomarker of interest by mass spectrometry to determine the presence or amount of the at least one biomarker of interest in the sample.

In an embodiment, the at least one biomarker comprises a steroid hormone or a thyroid hormone. For example, in one embodiment, the at least one biomarker comprises estradiol and estrone. Or, the at least one biomarker may comprise free thyroxine (T4) or triiodothyronine (T3).

In certain embodiments, the chromatography may comprise high performance liquid chromatography (HPLC). In an embodiment, the chromatography may comprises extraction and/or analytical liquid chromatography.

In an embodiment, the method may comprise purifying the biomarker of interest prior to chromatography. For example, the sample may be partially purified by at least one of liquid-liquid extraction. Also, the method may comprise the step of diluting the sample into a solvent or solvents used for LS and/or MS.

In some embodiments, the method may comprise the use of two liquid chromatography steps. For example, in certain embodiments, the method for determining the presence or amount of one or more biomarkers in a test sample may comprise the steps of: (a) providing a sample suspected of containing one or more biomarkers of interest; (b) partially purifying the one or more biomarkers of interest from other components in the sample by at least one of liquid-liquid extraction or by diluting the sample; (c) transferring the extracted one or more hormones or metabolites onto an extraction column (i.e., on-line or off-line); (d) transferring the one or more biomarkers of interest from the extraction column onto an analytical column and chromatographically separating the one or more biomarkers of interest from other components in the sample; and (e) analyzing the chromatographically separated biomarkers of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the test sample.

In certain embodiments, the present invention comprises methods for measuring at least one of estradiol and/or estrone in a sample. In certain embodiments, the estradiol is dehydrated to reduce the complexity of the MS/MS spectrum, such that the sensitivity of estradiol detection is increased. For example, in one embodiment, the present invention comprises a method for determining the presence or amount of estradiol in a sample by tandem mass spectrometry, comprising: (a) generating a dehydrated precursor ion of the estradiol; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the presence or amount of one or more of the ions generated in step (a) or (b) or both, and relating the detected ions to the presence or amount of the estradiol in the sample.

In an embodiment, the sample may be subjected to a purification step prior to ionization. For example, in certain embodiments, the purification step may comprises chromatography. As discussed herein, in certain embodiments, the chromatography comprises high performance liquid chromatography (HPLC). The LC step may comprise one LC separation, or multiple LC separations. In one embodiment, the chromatographic separation comprises extraction and analytical liquid chromatography. Additionally or alternatively, high turbulence liquid chromatography (HTLC) (also known as high throughput liquid chromatography) may be used.

The purification may comprise steps in addition to HPLC or other types of chromatographic separation techniques. In alternate embodiments, the method may comprise at least one of liquid-liquid extraction or dilution. In one embodiment, the sample is diluted into a solvent or solvent mixture that may be used for LC and/or MS (e.g., LC-MS/MS or 2D-LC-MS/MS).

In an embodiment, the treatment of estradiol to form a dehydrated form of the compound reduces the molecular weight of the estradiol by about 18 mass units. Thus, in an embodiment, the precursor ion has a mass/charge ratio (m/z) of about 255.2. Also, in an embodiment, treatment of estradiol to form a dehydrated form of the compound reduces the complexity of the mass spectrum. Thus, in a embodiment the fragment ions comprise ions having a mass/charge ratio (m/z) of about 159.0 and 133.0. By reducing the complexity of the spectrum, the sensitivity of the procedure may be increased. The method may comprise detection of estradiol over a range of from a LOQ of about 1 pg/ml to an ULOQ of about 500 pg/mL as a single assay (i.e., as a linear assay without multiple dilution of the samples). Also, the method may comprise detection of estrone over a range of from a LOQ of about 2.5 pg/mL to and ULOQ of about 500 pg/mL as a single assay (i.e., as a linear assay without multiple dilution of the samples).

Also, since the spectrum of the estradiol is simplified, the analysis may further comprise a determination of the amount of other estrogens, such as estrone, in the sample.

The sample may only require heating for a relatively brief period of time to form the dehydrated estradiol. For example, the sample may be heated within the range of 300° C. to 1000° C. In an embodiment, the sample is heated in the interface where the sample is transferred to the mass spectrometer. In alternate embodiments, the heating step is done for less than 1 second, or less than 100 milliseconds (msec), or less than 10 msec, or less than 1 msec, or less than 0.1 msec, or less than 0.01 msec, or less than 0.001 msec.

In other embodiments, the present invention comprises methods for determining the presence or amount of a free thyroxine in a sample or a plurality of samples. In an embodiment, the present invention may comprise a method for determining the presence or amount of free thyroxine in a plurality of samples by tandem mass spectrometry, comprising: (a) dialyzing the plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples; (b) generating a precursor ion of the thyroxine in each sample; (b) generating one or more fragment ions of the thyroxine in each sample; and (c) detecting the presence or amount of one or more of the ions generated in step (b) or (c) or both in each sample, and relating the detected ions to the presence or amount of the free thyroxine in the plurality of samples.

In an embodiment, the method may comprise detection of thyroxine over a range of from a LLOQ of about 2.0 pg/mL to an ULOQ of about 100 pg/mL as a single assay (i.e., without dilution of the samples). In an embodiment, and as described in more detail herein, the dialysing step may comprise the use of a buffer, and wherein the buffer comprises and sufficient salts such that the buffer is isotonic.

In an embodiment, the sample may be subjected to a purification step prior to ionization. For example, in certain embodiments, the purification step may comprises chromatography. As discussed herein, in certain embodiments, the chromatography comprises high performance liquid chromatography (HPLC). The LC step may comprise one LC separation, or multiple LC separations. In one embodiment, the chromatographic separation comprises extraction and analytical liquid chromatography. Additionally or alternatively, high turbulence liquid chromatography (HTLC) may be used.

The purification may comprise steps in addition to HPLC or other types of chromatographic separation techniques. In alternate embodiment, the purification may comprise at least one of liquid-liquid extraction or dilution. In alternate embodiment, the sample may diluted in a solvent or solvents used for LC or MS, rather than undergoing LLE.

In other embodiments, the present invention comprises a system for determining the presence or amount of one or more biomarkers in a sample. In an embodiment, the system for determining the presence or amount of one or more biomarkers in a sample may comprise a station for chromatographically separating the one or more biomarkers from other components in the sample. For example, in some embodiments, the present invention may comprise system for determining the presence or amount of at least one biomarker of interest in a sample, the system comprising: a station for providing a sample believed to contain at least one biomarker of interest; a station for chromatographically separating the at least one biomarker of interest from other components in the sample; and a station for analyzing the chromatographically separated at least one biomarker of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the sample. In an embodiment, the system may comprise a station for partially purifying the at least one biomarker of interest from other components in the sample. In an embodiment, the mass spectrometry is operated in an atmospheric pressure chemical ionization (APCI) mode. In an embodiment, the system may further comprise a station for dialyzing a plurality of samples as a means to separate the at least one biomarker of interest that is bound to proteins in the sample from the portion of the biomarker of interest that is free in solution (i.e., “free”). Also in certain embodiments, at least one of the stations is automated and/or controlled by a computer. For example, as described herein, in certain embodiments, at least some of the steps are automated such that little to no manual intervention is required.

In one embodiment, the station for chromatographic separation comprises at least one apparatus to perform liquid chromatography (LC). In one embodiment, the station for liquid chromatography comprises a column for extraction chromatography. Additionally or alternatively, the station for liquid chromatography comprises a column for analytical chromatography. In certain embodiments, the column for extraction chromatography and analytical chromatography comprise a single station or single column. For example, in one embodiment, liquid chromatography is used to purify the biomarker of interest from other components in the sample that co-purify with the biomarker of interest after extraction or dilution of the sample.

The system may also include a station for analyzing the chromatographically separated one or more biomarkers of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the test sample. In certain embodiments, tandem mass spectrometry is used (MS/MS). For example, in certain embodiments, the station for tandem mass spectrometry comprises an Applied Biosystems API4000 or API5000 or thermo quantum or Agilent 7000 triple quadrupole mass spectrometer.

The system may also comprise a station for extracting the one or more hormones or metabolites from the test sample and/or diluting the sample. In an embodiment, the station for extraction comprises a station for liquid-liquid extraction. The station for liquid-liquid extraction may comprise equipment and reagents for addition of solvents to the sample and removal of waste fractions. In some cases a isotopically-labeled internal standard is used to standardize losses of the biomarker that may occur during the procedures. Thus, the station for liquid-liquid extraction may comprise a hood or other safety features required for working with solvents.

Additionally, the system may comprise a station for dialyzing sample as a means to separate the free hormone or metabolite from a sample that comprises free and protein-bound hormone or metabolite for measurement. The station for dialysis may comprise equipment for aliquoting samples into dialysis chambers. Also, the station for dialysis may comprise a mixing chamber to effect dialysis of the free analyte (e.g., free hormone) from the sample.

In embodiments of the methods and systems of the present invention, the biomarker is a hormone or a metabolite. The methods and systems of the present invention may be used to measure the amount of either total and/or free biomarkers of intersect in serum. In an embodiment, the hormone may comprise a steroid hormone. Or, the hormone may comprise a thyroid hormone. Or, the hormone may comprise a protein or peptide hormone. For example, in alternate embodiments, the steroid hormone may comprise an estrogen, androgen, mineralcorticoid, or glucocorticoid hormone. In certain embodiments, the hormone may comprise at least one of estrone or estradiol. In other embodiments, the hormone may comprise an estrogen metabolite. For example, the hormone may comprise estrone sulfate and/or glucoronidated and sulphated metabolites of estradiol, estrone or estriol. Or, other steroid hormones or steroid hormone metabolites may be measured. For example, the hormone may comprise testosterone. Or, non-steroid hormones may be measured. For example, in certain embodiments, the methods and systems may be used to measure a thyroid hormone, such as free thyroxine (T4) or triiodothyronine (T3). Or, pre-hormones (such as 25 hydroxyvitamin D) may be measured. For examples, the methods and systems of the present invention may be used to measure vitamins or other metabolites. In some embodiments, the metabolite may comprise a vitamin D compound such as 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3, 1,25 dihydroxyvitamin D2 and 1,25 dihydroxyvitamin D3. In yet other embodiments, the methods and systems of the present invention may be used to measure a non-hormone compound.

In certain embodiments, the test samples suitable for analysis by the methods and systems of the present invention can include any liquid sample that can contain one or more target analytes of interest. In an embodiment, the biomarker is endogenous to a subject. For example, in some embodiments, the test sample comprises a biological sample. As used herein, the term “biological sample” refers to a sample obtained from a biological source, including, but not limited to, an animal, a cell culture, an organ culture, and the like. Suitable samples include blood, plasma, serum, urine, saliva, tear, cerebrospinal fluid, organ, hair, muscle, or other tissue sample.

As used herein, a subject may comprise an animal. Thus, in some embodiments, the biological sample is obtained from a mammalian animal, including, but not limited to a dog, a cat, a horse, a rat, a monkey, and the like. In some embodiments, the biological sample is obtained from a human subject. In some embodiments, the subject is a patient, that is, a living person presenting themselves in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. In some embodiments, the test sample is not a biological sample, but comprises a non-biological sample, e.g., obtained during the manufacture or laboratory analysis of a synthetic steroid, which can be analyzed to determine the composition and/or yield of the manufacturing and/or analysis process.

A variety of methods may be used to extract the biomarker of interest from the sample. In certain embodiments, extracting the one or more hormones or metabolites from the test sample comprises a liquid-liquid extraction procedure. For example, for the analysis of estrone and estradiol in serum, a hexane:ethyl acetate is used for extraction. For example, in one embodiment, a 9:1 hexane:ethyl acetate solution may be used.

In certain embodiments, purifying the at least one biomarker of interest from the test sample may also comprise the use of a liquid chromatography extraction column. In one embodiment, the column is on-line. In an embodiment, purification of the biomarker of interest using a extraction column may comprises the steps of: (i) transferring the test sample on an extraction column; and (ii) eluting the biomarker of interest from the extraction column.

In certain embodiments, the methods and systems of the present invention may comprise multiple liquid chromatography steps. Thus, in certain embodiments, a two-dimensional liquid chromatography (LC) procedure is used. For example, in one embodiment, the method and systems of the present invention may comprise transferring the biomarker of interest from the LC extraction column to an analytical column. In one embodiment, the transferring of the at least one biomarker of interest from the extraction column to an analytical column is done by a heart-cutting technique. In another embodiment, the biomarker of interest is transferred from the extraction column to an analytical column by a chromatofocusing technique. Alternatively, the biomarker of interest is transferred from the extraction column to an analytical column by a column switching technique. These transfer steps may be done manually, or may be part of an on-line system.

Various columns comprising stationary phases and mobile phases that may be used for extraction or analytical liquid chromatography are described herein. The column used for extraction liquid chromatography may be varied depending on the biomarker of interest. In some embodiments, the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monolithic silica stationary phase, such as a Poroshell SBC-18 column. The column used for analytical liquid chromatography may be varied depending on the biomarker of interest and/or the column that was used for the extraction liquid chromatography step. For example, in certain embodiments, the analytical column comprises particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.

A variety of methods may be used to quantify the at least one biomarker of interest once the biomarker of interest has been substantially purified (i.e., substantially separated away from other components that may have been present in the sample). In some embodiments, mass spectrometry is used to quantify the at least one biomarker of interest. In certain embodiments, the mass spectrometer may comprise a tandem mass spectrometer (MS/MS). For example, in one embodiment of the methods and systems of the present invention, the tandem MS/MS spectrometry comprises a triple quadrupole tandem mass spectrometer.

The tandem MS/MS may be operated in a variety of modes. In one embodiment, the tandem MS/MS spectrometer is operated in an atmospheric pressure chemical ionization (APCI) mode. In some embodiments, the quantification of the analytes and internal standards is performed in the selected reaction monitoring mode (SRM).

Thus, embodiments of the present invention comprise methods and systems for applying liquid chromatography and mass spectrometry as a means to separate a biomarker analyte of interest from other components that may be present in a biological sample. In certain embodiments, two liquid chromatography (LC) steps are used in tandem. Also, the method may comprise an off-line liquid-liquid extraction and/or sample dilution step as a means to partially purify the sample prior to liquid chromatography. In some embodiments, tandem mass spectrometry is used to quantify the analyte of interest. The methods and systems may be used for clinical diagnosis.

The systems and methods of the present invention may, in certain embodiments, provide for a multiplexed assay. For example, certain embodiments of the present invention may comprise a multiplexed liquid chromatography tandem mass spectrometry (LC-MS/MS) or two-dimensional or tandem liquid chromatography-tandem mass spectrometry (LC)-LC-MS/MS) methods for the quantitative analysis of one or more analytes, including steroid hormones, such as estrone and estradiol and/or thyroid hormones, such as free thyroxine (T4) or triiodothyroine (T3) in biological samples.

An example of a method (2) of the present invention is shown in FIG. 1. Thus, in an embodiment, the method may include a step of providing a biological sample, for example, a serum sample believed to contain one or more analytes of interest (4). In some embodiments, an appropriate internal standard is added to the sample (6). For example, in some embodiments of the presently disclosed method for analyzing estrone and estradiol in serum samples, deuterated D4-estrone and D5-estradiol are added as internal standards. Or, C13-estrone and C13-estradiol stable labeled isotopes may be used. Or, for thyroxine, a deuterated or C13 derivative may be used. For example, in one embodiment, Thyroxine Ring-13C6 may be used. In yet other embodiments, structural analogues of the biomarker of interest may be used. For example, such structural analogues may comprise compounds wherein a first chemical group is replaced with a second chemical group. In general, the groups are of similar chemical reactivity, but different mass, as for example, the replacement of a methyl (—CH3) group with an ethyl (—CH2CH3) group.

In some embodiments, the analytes of interests are partially purified by liquid-liquid extraction of the sample (8). Or, the sample may be diluted (9) in a solvent that can be used for LC or MS in subsequent purification steps.

In an embodiment, the liquid-liquid extraction is used to concentrate and partially purify the analyte. For example, for estradiol/estrone analysis, the liquid extraction may be used to remove conjugated estrogens, such as sulfated and glucoronidated estrogens. Also, the liquid extraction may remove lipids and/or fibrinogen from the samples. In some embodiments, estrone and estradiol can be extracted from a serum sample with an organic solvent that can separate estrone and estradiol from conjugated estrogens. For example, in an embodiment, an alkane mixed with a more polar solvent is used. For example, in certain embodiments, hexane is mixed with a more polar solvent. In an embodiment, the polar solvent comprises ethyl acetate or a similar solvent. In an embodiment, 9:1 hexane:ethyl acetate is used.

Or, other solvents may be used. As is known in the art, the solvents employed may be optimized to separate the analyte of interest from the sample. For example, the solvents used to extract estrone and estradiol from serum may not be the same solvent or solvent mix as used to extract estrone and estradiol from urine. Or, the solvents used to extract estrone and estradiol from serum may not be the same solvent or solvent mix as used to extract thyroxine (T4), triiodothyronine (T3), or vitamin-D compounds from serum. For example, in certain embodiments, acetonitrile is used for liquid extraction of vitamin-D compounds, and ethyl acetate:hexane:methanol is used for extraction of T4.

Certain biomolecules may have a propensity to nonspecifically bind to proteins or other biomolecules. For example, thyroid hormones can non-specifically bind to proteins such as serum albumin, sex hormone binding globulin, and the like. For determination of free thyroxine (T4), the sample may be treated to separate the free thyroxine from thyroxine that is bound to proteins in the biological sample (e.g., serum).

In one embodiment, the sample may initially be dialyzed to separate the free hormone or metabolite from a mixture of free and protein-bound hormone or metabolite (5). In certain embodiments, multiple samples may be processed concurrently. For example, the dialysis may be performed using a multiwell dialysis plate which allows for the dialysis of multiple samples at one time. In certain embodiments 96 well plates are used. In this way, multiple samples are processed to comprise a high throughput assay.

For example, samples of serum that may contain free thyroxine and protein-bound thyroxine may be introduced into the individual sample chambers which are on one side of the membrane and a buffer solution introduced into the diluent chambers on the other side of the membrane from the sample. The 96 well plate is then positioned vertically and rotated to facilitate transfer of the free thyroxine across the membrane.

The dialysis buffer may, in certain embodiments, be isotonic and contain gelatin. The gelatin may be used over a range of concentrations depending upon the nature of the membranes and hardware used for dialysis. In alternate embodiments, the gelatin may be in a range of from about 0.1 to 10 mg/mL. In an embodiment, the gelatin is at about 1 mg/mL. In certain embodiments, the buffer used for dialysis comprises multiple endogenous salts to provide a buffer that is isotonic with the serum sample to thereby negate any potential dilution effects and/or disruptions to the ratio of bound thyroxine to free thyroxine in the sample. Also, gelatin may be include to prevent adsorptive losses of free thyroxine onto the dialysis membrane or the sample chamber. Gelatin may act as a carrier on the dialysate side of the 96-well plate to ensure free thyroxine remains in the dialysate solution. Gelatin does not bind free thyroxine and thus, does not affect the ratio of bound thyroxine to free thyroxine in the sample on the sample side of the membrane.

For the analysis of free thyroxine, a liquid extraction step may be performed after the dialysis. The liquid extraction may be designed to remove residual salts and/or other additives which are used in the dialysis solution and/or remain from the sample, but that may interfere with the MS analysis. Thus, in one embodiment, the dialysate comprising free thyroxine is extracted with 71.25:23.75:5 ethyl acetate:hexane:methanol. In another embodiment, the dialysate may be diluted with a solution of methanol containing a stable labeled internal standard and directly injected onto the LC-MS/MS system for analysis.

Where the sample is extracted, the internal standard addition may include a protein to prevent the free thyroxin from sticking to the walls of the sample container. Addition of protein (e.g., bovine serum albumin) can minimize losses in extraction and recovery for liquid-liquid extraction. Where extraction is not performed, the internal standard may be added in methanol or a similar solvent used for LC.

As is known in the art, in some embodiments, the organic extract may be transferred to a fresh tube and then back-washed. For example, in an embodiment where the analyte of interest is estradiol and/or estrone, the solvent may be back-washed with aqueous sodium hydroxide (pH of about 12) to further purify the sample. Or, for extraction of other biomarkers, back-extraction may employ other solvents. The back-wash may, in certain embodiments, remove additional lipids or interfering analytes from the sample.

The extract supernatant may then be evaporated and the sample reconstituted. For example, for analysis of estradiol and/or estrone, the sample may be reconstituted in 70:30 water:methanol. Or, for analysis of thyroxine, the solvent used for liquid-liquid extraction may be evaporated and the sample reconstituted in 50:50 water:methanol.

Still referring to FIG. 1, the method may further include liquid chromatography as a means to separate the analyte of interest from other components in the sample. In an embodiment, two liquid chromatography steps are used. For example, the method may comprise a first extraction column liquid chromatography (10), transfer of the biomarker of interest to a second analytical column (12), and an analytical column liquid chromatography (16). In other embodiments, only one liquid chromatography step is used.

The first extraction liquid chromatography column may, in certain embodiments, comprise a step whereby the analytes of interest are separated from a majority of contaminants. Thus, in certain embodiments, the first column provides the majority of selectivity for the procedure. The second analytical liquid chromatography column may, in certain embodiments, comprise a step whereby the analytes of interest are concentrated, to thereby increase sensitivity for analysis by mass spectrometry (MS).

For example, the reconstituted extract may be applied onto a high performance liquid chromatography (HPLC) system, wherein the analytes are eluted using an isocratic separation through an extraction column. In certain embodiments, the mobile phase that is used comprises a gradient. For example, in an embodiment for the separation of estradiol and estrone from other components in serum, the stationary phase comprises a Poroshell 300SBC-18 column. Thus, the inventors have found that surprisingly, a stationary phase designed for large molecules such as proteins may be used to separate smaller molecules such as estrone and estradiol. The mobile phase may comprise methanol and water.

Depending upon the biomarker of interest, a variety of analytical columns known in the art may be used as needed to provide good purification. In certain embodiments, the analytical column may comprise particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.

For example, in one embodiment, estrone and estradiol are separated from isobaric substances by separation using a Poroshell 300SBC10 column (7.5 mm by 2.1 mm) with 5 micron particle size using a gradient separation using methanol and water for elution at 1 mL per minute flow rate. Estrone and estradiol are transferred from the extraction column after 2.5 minutes and chromatofocused onto a phenyl-hexyl column (50 mm by 2.1 mm) with 5 micron particles using water for 45 seconds. The transferred and purified analytes are chromatographed using an accelerated gradient employing methanol and water to improve sensitivity prior to introduction into the mass spectrometer interface and subsequent detection.

For liquid chromatography of thyroxin, a single liquid chromatography step may be used. Thus, for liquid chromatography of thyroxin, a phenyl hexyl column (50 mm by 2.1 mm) with 5 micron particle size may be used. Thus, following either: (a) liquid-liquid extraction, evaporation and reconstitution; or (b) post-dialysis sample dilution with internal standard solution; samples are injected onto the liquid chromatography column. The transferred analyte and internal standard are chromatographed using a methanol:water gradient separation at 1 mL per minute. To enable further sensitivity gains, a post-separation additional flow of 90:10 methanol:water containing ammonium carbonate (1 mM) is introduced at 200 microliters per minute prior to introduction into the mass spectrometer (MS) electrospray interface.

If two liquid chromatography steps are employed, the eluted analytes may be transferred to the analytical column in a manner such that the sample is concentrated upon application to the analytical column. In some embodiments, the eluted analytes are transferred to the analytical column via a heart-cutting technique. In some embodiments, a chromato-focusing procedure is used to transfer and focus the analytes on the analytical column. Also in some embodiments, a column-switching procedure is used to transfer the analytes to the analytical column. The analytes may then be separated on the analytical column (16) and the fraction containing the analyte of interest is eluted. In an embodiment, the second column in run in a manner to maximize throughput, and to provide the sample in a reduced volume.

The separated analytes are then introduced into a mass spectrometer (MS) system (20). In some embodiments, a tandem MS/MS system is used. As is known by those of skill in the art, in tandem MS spectrometry, the precursor ion is selected following ionization, and that precursor ion is subjected to fragmentation to generate product (i.e., fragment) ions, whereby one or more product ions are selected for detection. A sample may therefore be analyzed for both estradiol and estrone since the compounds have different precursor and product ions in tandem mass spectrometric methodologies (i.e., different transitions).

The analyte of interest may then be quantified based upon the amount of the characteristic transitions measured by tandem MS. In some embodiments, the tandem mass spectrometer comprises a triple quadrupole mass spectrometer. In some embodiments, the tandem mass spectrometer is operated in a positive ion Atmospheric Pressure Chemical Ionization (APCI) mode. In some embodiments, the quantification of the analytes and internal standards is performed in the selected reaction monitoring mode (SRM). Or, other methods of ionization such as the use of inductively coupled plasma, or MALDI, or SELDI, ESI, or APPI may be used for ionization.

In some embodiments, the back-calculated amount of each analyte in each sample may determined by comparison of unknown sample response or response ratio when employing internal standardization to calibration curves generated by spiking a known amount of purified analyte material into a standard test sample, e.g., charcoal stripped human serum. In one embodiment, calibrators are prepared at known concentrations and analyzed as per the biomarker methodology to generate a response or response ratio when employing internal standardization versus concentration calibration curve.

In one embodiment, the sample may be treated so as to chemically modify the analyte of interest to allow for improved detection in the MS system. For example, in one embodiment, a sample being analyzed for estrone and/or estradiol may be heated to the extent that the estradiol loses a molecule of water thereby converting the estradiol to a dehydrated form of the compound (FIG. 2, Panels A and B, respectively). This conversion can reduce the number of major product ion peaks seen for estradiol from about 60 to 3 (FIG. 2, panels C and D). For MS analysis, the sensitivity of the analysis is generally inversely proportional to the number of product ion peaks. Thus, with fewer peaks, the sensitivity of detection using tandem mass spectrometry is increased. For example, as illustrated in FIG. 2, estradiol may be quantified by measuring the transition from the precursor ion at a mass to charge (m/z) 255.3±0.5 mass units to the two product (fragment) ions at a mass to charge (m/z) of 159.0±0.5 mass units and 133.0±0.5.

In alternate embodiments, the sensitivity obtained for measurement of estradiol is increased more than 10 fold, or more than 20 fold, or more than 50 fold, or more than 100 fold, or more than 150 fold, or more than 200 fold, or more than 500 fold, or more than 1,000 fold. For example, in alternate embodiments, the sensitivity is increased by about 5-1,000 fold, or a by about 20-500 fold, or by about 50-150 fold, or by about 100 fold.

The temperature for heating the sample may, in alternate embodiments range from 300° C. to about 1000° C. and includes all ranges therein. In an embodiment, the dehydration step is performed within the interface of the mass spectrometer employed in APCI or electrospray mode at 500 degrees C.±100 degrees. In an embodiment, the sample is heated for several microseconds at the interface for dehydration to occur. In alternate embodiments, the heating step is done for less than 1 second, or less than 100 milliseconds (msec), or less than 10 msec, or less than 1 msec, or less than 0.1 msec, or less than 0.01 msec, or less than 0.001 msec.

In an embodiment, the tandem liquid chromatography (LC) steps help reduce isobaric interferences. For example, in one embodiment, there are 24 potential isobaric interferences in estradiol (transition m/z 255->159, 133), and 16 potential interferences for estrone (transition m/z 273->159, 133). For example, dehydroepiandrosterone (DHEA) undergoes thermal dehydration forming MH-H20]+ and MH-2H2O]+ (FIG. 3). There may be DHEA concentrations that are about 300-1,500 times the levels of estrone and estradiol in healthy patients. Thus, the M+2 isotopic overlap of dehydrated DHEA may become an isobaric interference. Heart cutting from the primary separation using isocratic or gradient separation resolves most isobaric interferences (FIG. 4). Thus, as shown in FIG. 4, heart cutting combined with chromatofocusing may be used to separate estradiol (E2) and estrone (E1) from all but one potential isobaric contaminant which is separated within the analytical (second) liquid chromatography separation dimension.

An example of a method for measuring estradiol and estrone is provided in FIG. 5. For example, in an embodiment, a method (40) of measuring estrone and estradiol comprise providing a sample believed to contain at least one of estrone and estradiol (44). The method may also comprise adding an internal standard of D4-estrone and D5-estradiol to the sample (46).

Also, the method may optionally comprise partial purification of the estrone and estradiol by liquid-liquid extraction of the estrone and/or estradiol from the serum with 9:1 hexane-ethyl acetate (48). Or, the sample may be diluted (50) as a means to improve sensitivity in subsequent purification and/or analysis steps (e.g. LC and/or MS).

After initial purification by liquid-liquid extraction or dilution, the sample may be further purified by liquid chromatography. Thus, in one embodiment, the solvent is evaporated and the extracted estrone/estradiol is reconstituted in 30:70 methanol water for application to a liquid chromatography extraction column (52). The estradiol/estrone may be eluted from the extraction column. For example, in alternate embodiments, the estradiol/estrone may be eluted by heart cutting, chromatofocusing or column switching. Next, the fraction containing the estrone/estradiol may, in certain embodiments, be applied to an analytical LC column (54). The fraction containing the estrone/estradiol may then be transferred to the LC-MS/MS interface to undergo ionization and dehydration of the estradiol (60) prior to MS/MS detection in SRM mode (62). In an embodiment, heating the estradiol removes a molecule of water, and changes the resultant MS/MS profile such that it comprises only three major product ions.

Thus, the methods provide the ability to quantify estrone and/or estradiol at physiologically relevant levels. As discussed herein, the difference between a serum level of 10 pg/mL and 15 pg/mL may be clinically relevant. In one embodiment, the method is able to measure estrone and/or estradiol at levels of about 2.5 pg/mL and 1 pg/mL respectively.

An example of a method for measuring free thyroxine (T4) (70) is provided in FIG. 6. In an embodiment, the method may comprise providing a sample that includes thyroxine (both free and protein-bound) (74). The method may also comprise dialyzing the sample (76) to separate the free thyroxine from the protein bound thyroxine. Also, the method may comprise adding an internal standard such as 6C13-thyroxine (78) to allow for the measured amount of thyroxine to be correlated to the actual amount present in the sample (i.e., to quantify the amount lost during the extraction and measurement procedures).

The method may also comprise an optional step whereby the free thyroxine present in the dialysate is extracted by liquid-liquid extraction (80). Alternatively, the sample may be diluted into the solvent used for LC-MS/MS as a means to reduce interference from non-T4 or non-T3 analytes (81). At this point, the solvent used for extraction may be evaporated, and the extracted thyroxine reconstituted in 50:50 methanol:water for application to an LC column (82). The free thyroxine may then be eluted from the column (84) and then quantified by MS/MS (86).

Thus, the methods provide the ability to quantify free thyroxine at physiologically relevant levels. The difference between a serum level of 8 pg/mL and 12 pg/mL T4 may be clinically relevant. The method is able to measure free thyroxine (T4) at levels of 2 pg/mL.

Systems for Quantification of Endogenous Biomarkers

FIG. 7A shows an embodiment of a system of the present invention. As shown in FIG. 7, the system may comprise a station for aliquoting a sample (104) that may comprise a biomarker of interest into sampling containers. In one embodiment, the sample is aliquoted into a container or containers to facilitate liquid-liquid extraction or sample dilution. The station for aliquoting may comprise receptacles to discard the portion of the biological sample that is not used in the analysis.

Alternatively or additionally, the sample may be aliquoted into a container for dialysis. As described above, the container for dialysis may comprise a multi-well plate. Thus, in addition to the station for aliquoting, the system may comprise a station for dialysis (106). The station for dialysis may comprise a rotator oven, multi-chamber pipettes for sample transfer, as well as receptacles to discard the portion of the biological sample that is not used in the analysis.

The system may further comprise a station for adding an internal standard to the sample (108). In an embodiment, the internal standard comprises the biomarker of interest labeled with a non-natural isotope. Thus, the station for adding an internal standard may comprise safety features to facilitate adding an isotopically labeled internal standard solutions to the sample. The system may also, in some embodiments, comprise a station (110) for liquid-liquid extraction and/or dilution of the sample.

The system may also comprise a station for liquid chromatography (LC) of the sample. As described herein, in an embodiment, the station for liquid chromatography may comprise an extraction liquid chromatography column (112). The station for liquid chromatography may comprise a column comprising the stationary phase, as well as containers or receptacles comprising solvents that are used as the mobile phase. In an embodiment, the mobile phase comprises a gradient of methanol and water, acetonitrile and water, or other miscible solvents with aqueous volatile buffer solutions. Thus, in one embodiment, the station may comprise the appropriate lines and valves to adjust the amounts of individual solvents being applied to the column or columns. Also, the station may comprise a means to remove and discard those fractions from the LC that do not comprise the biomarker of interest. In an embodiment, the fractions that do not contain the biomarker of interest are continuously removed from the column and sent to a waste receptacle for decontamination and to be discarded.

A variety of extraction LC systems may be used. For example, in the embodiment where the system is being used to measure estrone or estradiol, a Poroshell 300SBC18 extraction column with a phenyl hexyl analytical column, with mobile phases comprising a gradient of methanol and water are used. Or, for measurement of thyroxine, a phenyl hexyl column, with a mobile phase of methanol:water is used with post-column addition of a methanol:water solution containing ammonium carbonate. Or, for vitamin D metabolites, a Fluophase WP extraction column, with a mobile phase of methanol:water is used and an Extent C18 analytical column is used with a mobile phase of methanol:water is used.

The system may also comprise an analytical LC column (114). The analytical column may facilitate further purification and concentration of the biomarker of interest as may be required for further characterization and quantification.

Also, the system may comprise a station for characterization and quantification of the biomarker of interest. In one embodiment, the system may comprise a station for mass spectrometry (MS) of the biomarker. In an embodiment, the station for mass spectrometry comprises a station for tandem mass spectrometry (MS/MS). Also, the station for characterization and quantification may comprise a computer and software for analysis of the MS/MS results. In an embodiment, the analysis comprises both identification and quantification of the biomarker of interest.

In some embodiments, one or more of the purification or separation steps can be preformed “on-line.” As used herein, the term “on-line” refers to purification or separation steps that are performed in such a way that the test sample is disposed, e.g., injected, into a system in which the various components of the system are operationally connected and, in some embodiments, in fluid communication with one another. The on-line system may comprise an autosampler for removing aliquots of the sample from one container and transferring such aliquots into another container. For example, an autosampler may be used to transfer the sample after extraction onto an LC extraction column. Additionally or alternatively, the on-line system may comprise one or more injection ports for injecting the fractions isolated from the LC extraction columns onto the LC analytical column. Additionally or alternatively, the on-line system may comprise one or more injection ports for injecting the LC purified sample into the MS system. Thus, the on-line system may comprise one or more columns, including but not limited to, an extraction column, including an HTLC extraction column, and in some embodiments, an analytical column. Additionally or alternatively, the system may comprise a detection system, e.g., a mass spectrometer system. The on-line system may also comprise one or more pumps; one or more valves; and necessary plumbing. In such “on-line” systems, the test sample and/or analytes of interest can be passed from one component of the system to another without exiting the system, e.g., without having to be collected and then disposed into another component of the system.

In some embodiments, the on-line purification or separation method can be automated. In such embodiments, the steps can be performed without the need for operator intervention once the process is set-up and initiated. For example, in one embodiment, the system, or portions of the system may be controlled by a computer or computers (102). Thus, in certain embodiments, the present invention may comprise software for controlling the various components of the system, including pumps, valves, autosamplers, and the like. Such software can be used to optimize the extraction process through the precise timing of sample and solute additions and flow rate.

Although some or all of the steps in the method and the stations comprising the system may be on-line, in certain embodiments, some or all of the steps may be performed “off-line.” In contrast to the term “on-line”, the term “off-line” refers to a purification, separation, or extraction procedure that is performed separately from previous and/or subsequent purification or separation steps and/or analysis steps. In such off-line procedures, the analytes of interests typically are separated, for example, on an extraction column or by liquid/liquid extraction, from the other components in the sample matrix and then collected for subsequent introduction into another chromatographic or detector system. Off-line procedures typically require manual intervention on the part of the operator.

Liquid chromatography may, in certain embodiments, comprise high turbulence liquid chromatography or high throughput liquid chromatography (HTLC). See, e.g., Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. Traditional HPLC analysis relies on column packings in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. In such columns, separation is a diffusional process. Turbulent flow, such as that provided by HTLC columns and methods, may enhance the rate of mass transfer, improving the separation characteristics provided. In some embodiments, high turbulence liquid chromatography (HTLC), alone or in combination with one or more purification methods, may be used to purify the biomarker of interest prior to mass spectrometry. In such embodiments, samples may be extracted using an HTLC extraction cartridge which captures the analyte, then eluted and chromatographed on a second HTLC column or onto an analytical HPLC column prior to ionization. Because the steps involved in these chromatography procedures can be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. Also, in some embodiments, the use of a high turbulence liquid chromatography sample preparation method can eliminate the need for other sample preparation methods including liquid-liquid extraction. Thus, in some embodiments, the test sample, e.g., a biological fluid, can be disposed, e.g., injected, directly onto a high turbulence liquid chromatography system.

For example, in a typical high turbulence or turbulent liquid chromatography system, the sample may be injected directly onto a narrow (e.g., 0.5 mm to 2 mm internal diameter by 20 to 50 mm long) column packed with large (e.g., >25 micron) particles. When a flow rate (e.g., 3-500 mL per minute) is applied to the column, the relatively narrow width of the column causes an increase in the velocity of the mobile phase. The large particles present in the column can prevent the increased velocity from causing back pressure and promote the formation of vacillating eddies between the particles, thereby creating turbulence within the column.

In high turbulence liquid chromatography, the analyte molecules may bind quickly to the particles and typically do not spread out, or diffuse, along the length of the column. This lessened longitudinal diffusion typically provides better, and more rapid, separation of the analytes of interest from the sample matrix. Further, the turbulence within the column reduces the friction on molecules that typically occurs as they travel past the particles. For example, in traditional HPLC, the molecules traveling closest to the particle move along the column more slowly than those flowing through the center of the path between the particles. This difference in flow rate causes the analyte molecules to spread out along the length of the column. When turbulence is introduced into a column, the friction on the molecules from the particle is negligible, reducing longitudinal diffusion.

The methods and systems of the present invention may use mass spectrometry to detect and quantify the biomarker of interest. The terms “mass spectrometry” or “MS” as used herein generally refer to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m/z.” In MS techniques, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometer where, due to a combination of electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”).

In certain embodiments, the mass spectrometer uses a “quadrupole” system. In a “quadrupole” or “quadrupole ion trap” mass spectrometer, ions in an oscillating radio frequency (RF) field experience a force proportional to the direct current (DC) potential applied between electrodes, the amplitude of the RF signal, and m/z. The voltage and amplitude can be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments can act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.

In certain embodiments, tandem mass spectrometry is used. See, e.g., U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry,” which is hereby incorporated by reference in its entirety. Further, the selectivity of the MS technique can be enhanced by using “tandem mass spectrometry,” or “MS/MS.” Tandem mass spectrometry (MS/MS) is the name given to a group of mass spectrometric methods wherein “parent or precursor” ions generated from a sample are fragmented to yield one or more “fragment or product” ions, which are subsequently mass analyzed by a second MS procedure. MS/MS methods are useful for the analysis of complex mixtures, especially biological samples, in part because the selectivity of MS/MS can minimize the need for extensive sample clean-up prior to analysis. In an example of an MS/MS method, precursor ions are generated from a sample and passed through a first mass filter to select those ions having a particular mass-to-charge ratio. These ions are then fragmented, typically by collisions with neutral gas molecules in a suitable ion containment device, to yield product (fragment) ions, the mass spectrum of which is recorded by a electron multiplier detector. The product ion spectra so produced are indicative of the structure of the precursor ion, and the two stages of mass filtering can eliminate ions from interfering species present in the conventional mass spectrum of a complex mixture.

In an embodiment, the methods and systems of the present invention use a triple quadrupole MS/MS (see e.g., Yost, Enke in Ch. 8 of Tandem Mass Spectrometry, Ed. McLafferty, pub. John Wiley and Sons, 1983). Triple quadrupole MS/MS instruments typically consist of two quadrupole mass filters separated by a fragmentation means. In one embodiment, the instrument may comprise a quadrupole mass filter operated in the RF only mode as an ion containment or transmission device. In an embodiment, the quadrupole may further comprise a collision gas at a pressure of between 1 and 10 millitorr. Many other types of “hybrid” tandem mass spectrometers are also known, and can be used in the methods and systems of the present invention including various combinations of magnetic sector analyzers and quadrupole filters. These hybrid instruments often comprise high resolution magnetic sector analyzers (i.e., analyzers comprising both magnetic and electrostatic sectors arranged in a double-focusing combination) as either or both of the mass filters. Use of high resolution mass filters may be highly effective in reducing chemical noise to very low levels.

For the methods and systems of the present invention, ions can be produced using a variety of methods including, but not limited to, electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization (“MALDI”), surface enhanced laser desorption ionization (“SELDI”), photon ionization, electrospray ionization, and inductively coupled plasma.

In those embodiments, such as MS/MS, where precursor ions are isolated for further fragmentation, collision-induced dissociation (“CID”) may be used to generate the fragment ions for further detection. In CID, 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.

In some embodiments, to attain the required analytical selectivity and sensitivity, the presently disclosed 2D-LC-MS/MS methods include multiplexed sample preparation procedures. For example, in certain embodiments dialysis of the sample is performed using a 96 well plate having a dialysis membrane in each well or multiple sample tubes (FIG. 7B). Additionally or alternatively, the multiplex system may comprise staggered multiplexed LC and MS sample inlet systems. Also, the methods and systems of the present invention may comprise multiple column switching protocols, and/or heart-cutting (LC-LC or 2D-LC) techniques, and/or LC separations prior to MS detection. In some embodiments, the methods and systems of the present invention may include a multiplexed two-dimensional liquid chromatographic system coupled with a tandem mass spectrometer (MS/MS) system, for example a triple quadrupole MS/MS system. Such embodiments provide for staggered, parallel sample input into the MS system.

Thus, as shown in FIG. 7B, four samples (132 A-D) may each be applied to individual extraction columns (134 A-D). Once the samples have each run through the extraction column, they may each be transferred directly (e.g., by column switching) to a second set of analytical columns (136 A-D). As each sample elutes from the analytical column, it may be transferred (138) to the mass spectrometer (140) for identification and quantification.

A plurality of analytes can be analyzed simultaneously or sequentially by the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods. Exemplary analytes amenable to analysis by the presently disclosed methods include, but are not limited to, steroid hormones, such as estradiol, estrone, and metabolites, such as estrone sulfate. In other embodiments, thyroid hormones, such as free thyroxine (T4) and triiodothyronine (T3) can be measured. In the other embodiments, metabolites, such as 25-Hydroxyvitamin D2,25-Hydroxyvitamin D3, may be measured. One of ordinary skill in the art would recognize after a review of the presently disclosed subject matter that other similar analytes could be analyzed by the methods and systems disclosed herein. Thus, in alternate embodiments, the methods and systems may be used to quantify steroid hormones, protein and peptide hormones, peptide and protein biomarkers, drugs of abuse and therapeutic drugs. For example, optimization of key parameters for each analyte can be performed using a modular method development strategy to provide highly tuned bioanalytical assays. Thus, certain steps may be varied depending upon the analyte being measured as disclosed herein.

Also, embodiments of the methods and systems of the present invention may provide greater sensitivity than the sensitivities previously attainable for many of the analytes being measured. For example, through using this optimization procedure, an LOQ of about 1 picogram per milliliter (pg/mL), or less than 5 pg/mL, or less that 10 pg/mL, or less than 25 pg/mL is attained for the analysis of at least one of estradiol, estrone or free thyroxine without the cumbersome derivatization processes historically required for LC-MS/MS analyses of steroids. Importantly, the low levels of detection allow for the analysis of small sample volumes, for example 100 μL, 200 μL, 500 μL, or less than 1 mL, which can be necessary to analyze pediatric sample volumes. Thus, the presently disclosed LC-MS/MS and (LC)-LC-MS/MS methods can be used to measure levels of steroid hormones, such as estrone and estradiol, or other hormones or metabolites (e.g., free thyroxine, vitamin D metabolites and the like) in serum or plasma samples from children, women, and men.

Embodiments of the present invention may provide certain advantages. In certain embodiments, the methods and systems of the present invention may provide greater sensitivity than the sensitivities previously attainable for many of the analytes being measured.

Also, embodiments of the methods and systems of the present invention may provide for rapid throughput that has previously not been attainable for many of the analytes being measured. For example, using the methods and systems of the present invention, multiple samples may be analysed for free thyroxine using 96 well plates and a multiplex system of four LC-MS/MS systems, significantly increasing the throughput.

As another advantage, the specificity and sensitivity provided by the methods and systems of the present invention may allow for the analysis of analytes from a variety of biological materials. For example, the 2D-LC-MS/MS methods of the present invention can be applied to the quantification of analytes of interest in complex sample biological matrices, including, but not limited to, blood, serum, plasma, urine, saliva, and the like. Thus, the methods and systems of the present invention are suitable for clinical applications and/or clinical trials.

As additional potential advantages, in certain embodiments, the systems and methods of the present invention provide approaches for addressing isobaric interferences, varied sample content, including hemolysed and lipemic samples, while attaining low pg/mL limits of quantification (LOQ) of the target analytes. Accordingly, embodiments of the methods and systems of the present invention may provide for the quantitative, sensitive, and specific detection of clinical biomarkers used in the clinical diagnosis of endocrine disorders.

A general strategy for the validation of the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods for endogenous biomarkers is provided in Scheme 1. Thus, Scheme 1 shows the different tests that were used to validate the procedures. Matrix specificity testing was performed by analyzing 6 different lots of charcoal stripped matrix in quadruplicate for the presence of residual analyte, absence of analyte enables the charcoal stripped matrix to be spiked with known concentrations of target analytes to generate calibration curves. Internal standard specificity was performed by spiking the stable labeled internal standard into analyte-free charcoal stripped matrix and measuring for the presence of analyte in quadruplicate. Absence of unlabeled analyte confirms the purity of internal standard materials. Endogenous (hormones) and exogenous (drugs) are spiked into analyte free matrix to confirm the selectivity of the method.

Accuracy and precision was determined using 6 replicates per level in spiked charcoal stripped serum at the LLOQ, 2 levels within the analytical range and the ULOQ in 3 different batches. Precision was determined using 6 replicates in 3 separate runs of pooled matrix samples at concentrations of approximately 3 to 10 times the LLOQ, the mid point of the analytical range and approximately 80% of the ULOQ. Accuracy was determined in pooled matrix samples using spike and recovery (standard addition) at 3 different concentrations throughout the analytical range using 4 replicates per level.

Linearity was confirmed using multi-level calibrators over 5 separate runs. Sample mixing experiments were also undertaken mixing pooled matrix samples with fortified stripped matrix samples to ensure the assays were free of matrix interferences in quadruplicate. Recovery was undertaken using both spiked stripped matrix and pooled matrix samples in quadruplicate as confirmation of linearity and also further proof that the assay was free of matrix effects. The effect of matrix content on measurement was also tested following post-column infusion, addition of lipemia and hemolysis content, alternate sample types (e.g. serum and or plasma) and sample draw-tubes in quadruplicate. Sample stability was undertaken using both spiked stripped matrix samples and pooled matrix samples at storage conditions expected from sample collection to final analysis. Each condition was compared against baseline samples drawn and frozen at −70° C. for comparison and analyzed in quadruplicate at each concentration.

Inter-assay comparison was performed using at least 50 samples representing physiological range during comparison of LC-MS/MS and LC-MS/MS assays with alternate techniques. Reference range generation and/or transference was undertaken using guidance from the National Committee on Clinical Laboratory Standards (NCCLS).

Scheme 1. Bioanalytical Validation Strategy Matrix, Internal Standard, Endogenous/ Specificity Testing: Exogenous Analytes

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