DETERMINATION OF ANALYTES IN LIQUID SAMPLES BY MASS SPECTROMETRY

Abstract

The present invention relates to compositions and methods for analyzing analytes of interest in liquid samples by mass spectrometry, and preferably in patient samples. Preferred analytes of interest include sirolimus (rapamycin), corticosteroids, bile acids and lamotrigine (lamictal). In one embodiment, by careful selection of target ions, a number of corticosteroids can be analyzed simultaneously and without interference from closely related molecules. In another embodiment, the present methods combine high turbulence liquid chromatography with mass spectrometry performed in positive and negative mode in a single assay to enable the detection and quantification of the compositon of bile acid pools. By combining mass spectrometry and high-throughput chromatography, the methods and compositions described herein can provide a rapid, sensitive, and accurate assay for use in large clinical laboratories.

Claims

1. A method for determining the amount of sirolimus in a test sample by tandem mass spectrometry, comprising: (a) purifying sirolimus from said test sample; (b) ionizing sirolimus purified from said sample to produce lamotrigine precursor ions detectable by mass spectrometry; (c) fragmenting said precursor ion into one or more fragment ions detectable by mass spectrometry; and (d) detecting the amount of one or more of the ions of steps (b) and (c) ions by mass spectrometry, wherein the amount of ions detected is related to the amount of lamotrigine in the test sample.

2. The method of claim 1, wherein the purifying in step (a) comprises subjecting said test sample to high performance liquid chromatography (HPLC).

3. The method of claim 2, wherein said HPLC is conducted with a phenyl analytical column.

4. The method of claim 1, wherein the purifying in step (a) comprises extracting lamotrigine from said test sample with a high turbulence liquid chromatography (HTLC) column.

5. The method of claim 4, wherein said HTLC extraction column is a C-18 extraction column.

6. The method of claim 4, wherein said HTLC extraction column comprises a styrene-divinylbenzene cross-linked copolymer packing material.

7. The method of claim 1, wherein said ionizing of step (b) is conducted in positive ion mode.

8. The method of claim 1, wherein the purified sirolimus is ionized by electrospray ionization.

9. The method of claim 1, wherein said ionizing of step (b) is conducted in positive ion mode.

10. The method of claim 1, wherein the test sample comprises blood, plasma, or serum.

11. The method of claim 1, wherein the test sample is obtained from a patient receiving sirolimus as an immunosuppressive agent.

12. The method of claim 1, wherein said precursor ions comprise a mass to charge ratio (m/z) of about 931.70.

13. The method of claim 1, wherein said fragment ions comprise a mass to charge ratio (m/z) of about 864.76.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0052] FIG. 1 depicts the general skeleton of sirolimus.

[0053] FIG. 2 depicts a product ion scan obtained from a 4.4 ng/mL blood sample containing sirolimus.

[0054] FIG. 3 depicts a diagram of the general pump set-up for HTLC purification of an analyte of interest.

[0055] FIG. 4 depicts the pump set up for HTLC purification of sample containing an analyte of interest prior to elution.

[0056] FIG. 5 depicts the pump set up for HTLC reverse elution of an analyte of interest and delivery to mass spectrometer.

[0057] FIG. 6 depicts the pump set up for HTLC column purification.

[0058] FIG. 7 depicts the general cyclopentanophenanthrene skeleton of corticosteroids, together with several exemplary corticosteroids.

[0059] FIG. 8 depicts a product ion scan obtained from a 50 ng/mL urinary cortisol sample run in positive mode, comparing the cortisol signal to that obtained from dexamethasone, and prednisolone.

[0060] FIG. 9 depicts a product ion scan obtained from a 50 ng/mL urinary cortisol sample run in negative mode.

[0061] FIG. 10 depicts the general skeleton of various bile acids.

[0062] FIG. 11 depicts a product ion scan obtained from a blood sample containing various bile acids in positive mode.

[0063] FIG. 12 depicts a product ion scan obtained from a blood sample containing various bile acids in negative mode.

[0064] FIG. 13 depicts a product ion scan obtained from a 10 mcg/mL sample containing lamotrigine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The present invention describes methods and compositions that can provide the ability to unambiguously detect an analyte of interest, and/or its metabolite(s) or precursor(s), and/or combinations thereof (e.g., multiple bile acids) present in a test sample. By combining mass spectrometry with a multiplexed chromatography system to perform the initial purification of the selected analytes, the present invention can provide a high-throughput assay system particularly well suited to the large clinical laboratory.

[0066] Mass Spectrometry

[0067] The terms mass spectrometry or MS as used herein refer to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or m/z. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (m) and charge (z). See, e.g., U.S. Pat. No. 6,204,500, entitled Mass Spectrometry From Surfaces; U.S. Pat. No. 6,107,623, entitled Methods and Apparatus for Tandem Mass Spectrometry; U.S. Pat. No. 6,268,144, entitled DNA Diagnostics Based On Mass Spectrometry; U.S. Pat. No. 6,124,137, entitled Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes; Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21:1164-67 (2000), each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims.

[0068] For example, in a quadrupole or quadrupole ion trap instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and 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.

[0069] Moreover, one can often enhance the resolution of the MS technique by employing tandem mass spectrometry, or MS/MS. In this technique, a first, or parent, ion generated from a molecule of interest can be filtered in an MS instrument, and these parent ions subsequently fragmented to yield one or more second, or daughter, ions that are then analyzed in a second MS procedure. By careful selection of parent ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collision with atoms of an inert gas to produce these daughter ions. Because both the parent and daughter ions are produced in a reproducible fashion under a given set of ionization/fragmentation conditions, the MS/MS technique can provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation can be used to eliminate interfering substances, and can be particularly useful in complex samples, such as biological samples.

[0070] Ions can be produced using a variety of methods including, but not limited to, electron ionization, chemical ionization, fast atom bombardment, field desorption, photon ionization, electrospray ionization, and inductively coupled plasma.

[0071] The term electron ionization as used herein refers to methods in which an analyte of interest in a gaseous or vapor phase is interacted with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectroscopy technique.

[0072] 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.

[0073] The term fast atom bombardment as used herein refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile test sample, desorbing and ionizing molecules contained in the sample. Samples are dissolved in a viscous liquid matrix, such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The choice of an appropriate matrix for a compound or sample is an empirical process.

[0074] 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.

[0075] 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. Solution reaching the end of the tube, is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows 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.

[0076] The term Atmospheric Pressure Chemical Ionization, or APCI, as used herein refers to 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 the 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.

[0077] 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.

[0078] The term ionization 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.

[0079] The term operating in negative ion mode refers to those mass spectrometry methods where negative ions are detected. Similarly, operating in positive ion mode refers to those mass spectrometry methods where positive ions are detected.

[0080] 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.

[0081] In those embodiments, such as MS/MS, where parent ions are isolated for further fragmentation, collision-induced dissociation, or CID, is often used to generate the ion fragments for further detection. In CID, parent 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 parent ion so that certain bonds within the ion can be broken due to increased vibrational energy.

[0082] In exemplary embodiments described herein, corticosteroids are analyzed by mass spectrometry. Corticosteroids are a class of steroid hormones and their synthetic relatives constructed on a hydrogenated cyclopentanophenanthrene skeleton. Exemplary corticosteroids are depicted in FIG. 7. While cortisol and cortisone are the primary corticosteroids produced by the adrenal cortex, other metabolites of these parent molecules are often seen in patient samples, including 20--dihydrocortisone, 20--dihydrocortisone, 20--dihydrocortisol, and 20--dihydrocortisol. Additionally, closely related steroid hormones, such as prednisolone, estrogen, testosterone, prednisone, etc., may also be present in such samples in significant amounts.

[0083] Methods that can distinguish one or more of these corticosteroids in such a background of very closely related molecules generally require extensive purification steps and/or multiple assay runs in order to isolate individual steroid molecules. Because of its high resolution and sensitivity, mass spectrometry can offer the ability to qualitatively or quantitatively measure both parent compounds and metabolites. The methods developed to date, however, can still suffer from interference by closely related steroid molecules.

[0084] The present invention describes methods and compositions that can provide the ability to unambiguously detect one or more corticosteroids in a test sample. By careful selection of negative mode ions for detection and/or isolation and fragmentation, assays have been developed that can identify a plurality of corticosteroids simultaneously. Moreover, by combining mass spectrometry with a multiplexed chromatography system to perform the initial purification of the selected analytes, the present invention can provide a high-throughput corticosteroid assay system particularly well suited to the large clinical laboratory.

[0085] In other exemplary embodiments, the present invention also describes methods and compositions that can provide the ability to unambiguously detect bile acids, and preferably combinations of bile acids, and/or their metabolite(s), present in a test sample. By combining unique methods of mass spectrometry with a multiplexed chromatography system to perform the initial purification of the selected analytes, the present invention can provide a high-throughput assay system for detecting the presence and amount of bile acids, and is particularly well suited to the large clinical laboratory. The present invention also allows for the separation and detection of combinations of bile acids that have not heretofore been detectable without multiple assay procedures. For example, using the methods of the present invention, one may successfully separate, detect, and quantify many bile acids in a single assay run. The present methods also enable the separation of the dihydroxy bile acids, which are known for being difficult to separate.

[0086] In the present invention, it was also discovered unexpectedly that valuable information could be obtained by switching the mass spectrometer from negative to positive ion mode (or vice versa) during analysis of fragment ions. Preferably, the switch between modes takes place just prior to injection into the mass spectrometer for detection. Thus, in MS/MS detection, each injection isolates a selected precursor ion, fragments that precursor ion, and detects a single bile acid. This process is repeated, switching the ion mode as necessary for each detection. It had previously been believed that switching from one mode to the other during the mass spectrometry analysis results in unreliable, inaccurate data. But in the present case of the analysis of bile acids, switching ion modes was found to yield useful and reliable data and result in a substantial savings in time and resources.

[0087] Sample Preparation for Mass Spectrometry

[0088] Numerous methods have been described to purify analytes of interest from samples prior to assay. For example, high performance liquid chromatography (HPLC) has been used for sample clean-up. See, e.g., Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation of blood samples, followed by manual C18 solid phase extraction, injection into an HPLC for chromatography on a C18 analytical column, and MS/MS analysis); Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000) (manual precipitation of blood samples, followed by manual C18 solid phase extraction, injection into an HPLC for chromatography on a C18 analytical column, and MS/MS analysis); and Hallensleben et al., J. Am. Soc. Mass Spectrom. 11:516-25 (2000) (incubation of Sirolimus with microsomes to generate metabolites, followed by off-line HPLC chromatography and manual collection of metabolites for analysis by MS).

[0089] Taking corticosteroids as an example, numerous methods have been described to purify corticosteroids from samples prior to assaying for one or more corticosteroids. For example, chromatography, particularly high performance liquid chromatography (HPLC), thin layer chromatography (TLC), with and without an extraction step, have been used for sample clean-up. See, e.g., Tang et al., J. Chromatogr. B. 742:303-13 (2000) (multiple extractions followed by reverse phase HPLC); Antignac et al, Rapid Comm. Mass Spectrom. 14:33-9 (2000) (hydrolysis of corticosteroids, followed by C-18 reverse phase HPLC and liquid/liquid extraction); Dodds et al., Anal. Biochem. 247:342-7 (1997) (C-18 reverse phase HPLC followed by centrifugation); Dodds et al., J. Steroid Biochem. Mol. Biol. 62:337-43 (1997) (C-18 reverse phase HPLC); Gaillard et al., Forensic Sci. Intl. 107:361-79 (2000) (C-18 reverse phase HPLC, hydrolysis, and liquid-liquid extraction); Nassar et al., J. Chromatogr. Sci. 39:59-64 (2001) (C-18 analytical reverse phase HPLC).

[0090] Numerous methods have been described to purify bile acids from samples prior to assay. For example, high performance liquid chromatography (HPLC) has been used for sample purification and analysis. See, e.g., Yoshida et al., J. Chromatogr. 431:27-36 (1988).

[0091] Purification in this context does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to one or more other components of the sample. In preferred embodiments, purification can be used to remove one or more interfering substances, e.g., one or more substances that would interfere with detection of an analyte ion by mass spectrometry.

[0092] Recently, high turbulence liquid chromatography (HTLC) has been applied for sample preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. 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, each of which is hereby incorporated by reference in its entirety. 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 test sample. The skilled artisan will understand that separation in such columns is a diffusional process. In contrast, it is believed that turbulent flow, such as that provided by HTLC columns and methods, may enhance the rate of mass transfer, improving the separation characteristics provided.

[0093] Additionally, the commercial availability of HTLC apparatuses that permit multiplexing of columns and direct integration with MS instruments makes such instruments particularly well suited to high-throughput applications.

[0094] Numerous column packings are available for chromatographic separation of samples, and selection of an appropriate separation protocol is an empirical process that depends on the sample characteristics, the analyte of interest, the interfering substances present and their characteristics, etc. For HTLC, polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, and C-18 columns are commercially available. Similar columns are also available for traditional HPLC and low pressure separations. During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a mobile phase), choice of gradient elution and the gradient conditions, temperature, etc.

[0095] The particles of a typical column packing material are typically greater than 3 m in average diameter, more preferably greater than 5 m in average diameter, and preferably may be about 3-10 m in diameter. Columns can also contain packing material comprising spherical alumina, titania, carbon, and other materials. The person of ordinary skill will realize that other columns may be utilized successfully by following the selection guidelines provided herein. Such packing material may exhibit an extremely narrow particle size and pore size distribution.

[0096] For purification of lamotrigine, it has been discovered that a column packed with a styrene-divinylbenzene cross-linked copolymer produces an advantageous separation. Most preferably, the HTLC may be followed by HPLC on a C18 column with a porous spherical silica. During chromatography, the separation of materials is effected by variables such as choice of eluant (also known as a mobile phase), choice of gradient elution and the gradient conditions, temperature, etc.

[0097] In certain embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. The second mobile phase may be phased in gradually, usually under computer control directing the composition of mobile phase over time, or by an immediate change in the mobile phase. The retained materials may also be removed from the column by backflushing the column, or reversing the direction of flow of the mobile phase. This may be particularly convenient for material that is retained at the top of the column. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. As discussed above, such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0098] The terms phenyl, C-2, C-8, and C-18 as used herein refer to functional groups present on a column packing material. For example, a phenyl column exposes the material flowing through the column to unsubstituted phenyl groups, while a C-18 column exposes the material flowing through the column to unsubstituted straight or branched chain 18-carbon alkyl groups.

[0099] The term analytical column as used herein refers to a chromatography column having sufficient chromatographic plates to effect a separation of materials in a sample that elute from a column sufficient to allow a determination of the presence or amount of an analyte. Such columns are often distinguished from extraction columns, which have the general purpose of separating, or extracting, retained from non-retained materials. Examples of analytical columns are C-18 columns.

[0100] Preferred analytical columns in the present invention may be monofunctional with polar and bulky end-capping. The columns may be highly retentive and particularly useful for the separation of non-polar to medium polarity compounds using organic/aqueous mobile phases. Columns sold under the name Advantage Lancer C18 (Cohesive Technologies, Franklin, Mass.), or Hypersil (company, city, state) columns or similar columns utilizing porous spherical silica are examples of suitable analytical columns for use in the present invention.

[0101] Additionally, workers have described the use of affinity binding for sample purification in mass spectrometry. For example, U.S. Pat. Nos. 6,020,208 and 6,153,389, each of which is hereby incorporated by reference in its entirety, disclose the use of antibodies directed against a particular analyte as a ligand receptor to extract and concentrate the analyte.

[0102] In preferred embodiments, one or more of the purification and/or analysis steps can be performed in an on-line fashion. The term on-line as used herein refers to steps performed without the need for operator intervention. For example, by careful selection of valves and connector plumbing, two or more chromatography columns can be connected as needed such that material is passed from one to the next without the need for any manual steps. In preferred embodiments, the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system. Thus, an operator may place a tray of samples in an autosampler, and the remaining operations are performed under computer control, resulting in purification and analysis of all samples selected. The commercial availability of HTLC apparatuses that permit multiplexing of columns and direct integration with HPLC and MS instruments makes such instruments particularly well suited to on-line and high-throughput applications.

[0103] In contrast, the term off-line as used herein refers to a procedure requiring manual intervention of an operator. Thus, if samples are subjected to precipitation, and the supernatants are then manually loaded into an autosampler, the precipitation and loading steps are off-line from the subsequent steps.

EXAMPLES

[0104] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

Example 1. Determination of Sirolimus by Mass Spectrometry

[0105] Sample Collection.

[0106] Human whole blood was collected in a sterile container and stored refrigerated or at room temperature until analysis. Samples were stable in non-coagulated samples for up to 7 days refrigerated, or 5 days at room temperature. Steady-state (0.5-1 hour pre-oral sirolimus dosage, 2 weeks following beginning of administration of drug) are preferred samples. A minimum volume of 1 mL was collected for an assay.

[0107] Sirolimus Assay Procedure.

[0108] Samples (0.2 mL) were precipitated by mixing with 0.4 mL ZnSO.sub.4, 0.4 mL acetone, and 0.05 mL internal standard extraction solution (32-desmethoxyrapamycin in 50% aqueous methanol). Following mixing and collection of the supernatant by centrifugation through a PVDF filter, each sample was placed into a well of a standard 96-well plate (MicroLiter Analytical Supplies, Cat. #07-3000). Samples at this stage should be maintained at 4-8.

[0109] 96-well plates were loaded into a HTS PAL autosampler (LEAP Technologies) for injection into the HTLC apparatus. At this point, no further operator handling of samples is required, as the HTLC may be computer-controlled to perform the subsequent purification and analysis steps in a fully on-line configuration.

[0110] 155 uL of each sample was injected onto a PolarPlus (C-18 variety) extraction column (Cohesive Technologies No. 952311) in a Cohesive Technologies model 2300 HTLC system synchronized to an API 3000 LC/MS/MS system. Sirolimus is retained by this column, while various other sample substances are eluted.

[0111] Flow through the extraction column is reversed, and the column is flushed with a solvent that elutes Sirolimus from the column. This flush solution is injected into a Metachem C-18-A analytical column (Ansys Technologies Cat. #2000-050x020) fitted with a Polaris C18-A guard column (Cat, #2000-MG2) and eluted with a solvent gradient. A portion of the eluent from this column, representing the Sirolimus peak, is injected into the MS/MS instrument for analysis. Total run time is approximately 3 minutes.

[0112] Chromatography was performed under the following conditions:

For low pressure mixing pump,

Solvent A: 5 mM Ammonium Acetate

Solvent B: Methanol

[0113]

TABLE-US-00001 Time (min) Solvent B Solvent A Flow (mL/min) 0.00 10% 90% 5.0 0.49 10% 90% 5.0 0.50 65% 35% 0.2 1.49 65% 35% 0.2 1.50 100% 0% 5.0 2.25 100% 0% 5.0 2.26 10% 90% 5.0
For high pressure mixing pump,

Solvent A: 5 mM Ammonium Acetate

Solvent B: Methanol

[0114]

TABLE-US-00002 Time (min) Solvent B Solvent A Flow (mL/min) 0.00 30% 70% 0.40 0.49 30% 70% 0.40 0.50 30% 70% 0.20 1.00 30% 70% 0.20 1.01 95% 5% 0.20 1.50 95% 5% 0.40 2.00 95% 5% 0.40 2.01 30% 70% 0.40
MS/MS parameters (can be modified for optimal result)

TABLE-US-00003 Mode: Positive Mode Collision Gas N.sub.2 Cycle time 0.420 sec Res Q1 unit Res Q2 unit Curtain Gas 8.0 CAD Gas 6.0 IS 5500 Temp 375 NEB 9.00 TURBO GAS 7000 DP 41 (Sirolimus)/46(Int Std) FP 250 (Sirolimus)/240(Int Std) EP 10 (Sirolimus)/10 (Int Std) CE 23 (Sirolimus)/21 (Int Std) Note: Int Std = Internal Standard

[0115] The following mass transitions were used:

[0116] Sirolimus: precursor ion ([NH.sub.3].sup.+) 931.70 m/z; fragment ion 864.76 m/z; 32-Desmethoxyrapamycin: precursor ion: 901.68 m/z; fragment ion 834.67 m/z.

Example 2. Determination of Corticosteroids by Mass Spectrometry

[0117] Sample Collection.

[0118] Human urine was collected in a sterile container and stored refrigerated or at room temperature until analysis. Samples were stable for up to 7 days refrigerated, or 2 days at room temperature. In addition, samples may be frozen for up to 5 months if necessary. A minimum volume of 0.5 mL was used for an assay.

[0119] Cortisol Assay Procedure Using Positive-Mode MS.

[0120] Samples were loaded into a Perkin Elmer series 200 autosampler, together with low, medium, and high concentration controls (urine samples spiked with 10-20 ng/mL, 60-100 ng/mL, and 140-180 ng/mL cortisol). 450 uL of each sample was mixed with 450 uL 1% formic acid and vortexed. Samples (50 L) were injected onto a TurboFlow PolarPlus extraction column (Cohesive Technologies No. 952242) in a Cohesive Technologies model 2300 HTLC system synchronized to a Perkin Elmer Sciex API 2000 LC/MS/MS system which used a set of four quadrupoles to select and measure ions. Ions were generated by atmospheric pressure chemical ionization (APCI).

[0121] Chromatography was performed under the following conditions:

[0122] eluant: 0.1% formic acid in methanol;

[0123] 5.3 min, flow rate HTLC 5 mL/min, analytical column 0.6 mL/min direct to MSMS.

[0124] The following mass transitions are used to measure cortisol in the positive mode:

[0125] Precursor ion: 363.1 m/z; fragment ion 121.247 m/z.

[0126] The total time necessary for each assay is 5.25 minutes. Limitations to the positive mode assay include the fact that certain cortisol metabolites (e.g., 18-hydroxycortisol) cannot be measured, and that high levels of prednisolone and estrogen interfere in specifically detecting the cortisol fragment ion.

[0127] Cortisol Assay Procedure Using Negative-Mode MS.

[0128] Samples were loaded into a Perkin Elmer series 200 autosampler, together with low, medium, and high concentration controls (urine samples spiked with 10-20 ng/mL, 60-100 ng/mL, and 140-180 ng/mL cortisol). 100 uL of each sample was mixed with 400 uL 5 mM ammonium acetate and vortexed. Samples were injected onto a TurboFlow PolarPlus extraction column (Cohesive Technologies No. 952242) in a Cohesive Technologies model 2300 HTLC system synchronized to a Perkin Elmer API 2000 LC/MS/MS system.

[0129] Chromatography was performed under the following conditions:

eluant 0.1% ammonium acetate in methanol;
5.25 min, flow rate HTLC 5 mL/min, analytical column 0.6 mL/min direct to MSMS.

[0130] The following mass transitions are used to measure cortisol in the negative mode:

[0131] Cortisol: 361 m/z; fragment ion 331 m/z.; cortisone, 359.0/329.1; 18-hydroxycortisol, 377.2/317.2; 6--hydroxycortisol, 377.1/347.2.

[0132] Selected MS/MS Parameters.

TABLE-US-00004 Positive Mode Negative Mode Dwell time 200 msec 200 msec Res Q1 unit unit Res Q2 low low Curtain Gas 35 35 CAD Gas 3 3 NC 2 2 Temp 485 485 GS1 60 60 GS2 15 15 DP 40 21 FP 350 350 EP 10 11.5 CE 33 12

[0133] By detecting cortisol in negative mode, the assay is free of interference from all substances commonly present in human urine, including prednisolone and estrogen. Additionally, cortisone, 18-hydroxycortisol, and 6--hydroxycortisol can be measured simultaneously with cortisol, thus reducing the number of assays that must be performed on each sample in order to determine these molecules. The total time necessary for each assay is 5.25 minutes.

[0134] Additionally, the HTLC system can be operated with from 1 to up to 4 columns in parallel. Given that a single assay requires 5.25 minutes to traverse the column, by staggering the start time on each column, a 4-fold multiplexed system can inject four times as many test samples into the MS/MS instrument. Thus, a set of 200 samples may be assayed for four different corticosteroids in 263 minutes in both positive and negative modes, as opposed to 1500 minutes by HPLC. Furthermore, following transfer of samples to the autosampler, no further operator handling of samples is required, as the HTLC may be computer-controlled to perform the subsequent purification and analysis steps in a fully on-line configuration.

Example 3. Determination of Bile Acids by Mass Spectrometry

[0135] Sample Collection.

[0136] Human whole blood was collected in containers that do not contain anticoagulant, and permitted to clot. A minimum of 0.5 mL serum was used for each assay. Samples were stable for 7 days at room temperature, 14 days at 2-8 C., and 1 month at 20 C.

[0137] The pH was adjusted to pH 4.0 with a 5 mM ammonium acetate solution adjusted to pH 4.00.1 with formic acid. The samples were then loaded onto a Perkin Elmer Series 200 autosampler for analysis using an on-line purification/analysis system.

[0138] Bile Acid Assay Procedure.

[0139] The bile acid samples were analyzed using a HTLC/MS/MS procedure. The samples were first purified by loading them onto a HTLC extraction column (Cohesive Technologies Polar Plus, Cat #952242; a C-18 reverse phase packing). Following a wash step, the column was backflushed to elute bound bile acids, which were directly loaded on an analytical column (Metachem Technologies Cat #2000-050x020; a C-18 reverse phase packing). The purified bile acids eluted from the analytical column were detected by MS/MS.

[0140] Chromatography was performed under the following conditions:

[0141] Extraction column wash solution: 5 mM ammonium acetate, 100%; 1.4 minutes, 5.0 mL/min flow rate.

[0142] Extraction column eluant: 60:40% Methanol: 5 mM ammonium acetate; 1.0 minutes, 600 L/min flow rate.

[0143] Analytical column eluant: gradient 60% to 100% methanol; 3.0 min, 600 L/min flow rate.

[0144] Selected MS/MS Parameters.

TABLE-US-00005 Curtain gas 30.0 (negative) 35.0 (Positive) Collision gas 4 (negative) 4 (Positive) Nebulizer current 2.0 (negative) 5.0 (Positive) Temperature 485 (negative) 485 (Positive) GS1 80.0 (negative) 80.0 (Positive) GS2 15.0 (negative) 15.0 (Positive) Dwell time 200 msec (negative) 200 msec (Positive)

TABLE-US-00006 Analyte DP FP EP CEP CE CXP MODE cholic acid 66 290 10 21.07 44 12.34 negative taurocholic acid 101 350 8.5 25.32 122 7.9 negative glycocholic acid 61 350 10.5 23.34 64 7.77 negative chenodeoxycholic acid 11 350 11 30.21 17 27.81 positive taurochenodeoxycholic acid 116 340 9 24.69 125 7.9 negative glycochenodeoxycholic acid 61 310 9 22.70 66 7.774 negative deoxycholic acid 76 350 10.5 20.43 40 13.72 negative taurodeoxycholic acid 116 340 9 24.69 125 7.9 negative glycodeoxycholic acid 61 310 9 22.70 66 7.774 negative

[0145] The following mass transitions were used:

TABLE-US-00007 Analyte precursor ion fragment ion cholic acid 407.2 289.3 taurocholic acid 514.2 80.1 glycocholic acid 464.4 74.1 chenodeoxycholic acid +393.2 +359.2 taurochenodeoxycholic acid 498.3 80.1 glycochenodeoxycholic acid 448.3 74.1 deoxycholic acid 391.1 354.3 taurodeoxycholic acid 498.3 80.1 glycodeoxycholic acid 448.3 74.1

[0146] During the analysis, the mass spectrometer was switched from negative ion mode to positive ion mode. This was found unexpectedly to result in the detection of both negative and positive ions in a single assay. This was surprising because it is known in the art that switching from one mode to another during an assay leads to poor, unreliable, and difficult to decipher results. Surprisingly, in the case of the bile acids of the present invention, they were found to be readily detectable with accuracy and reliability, and therefore to save the cost and effort of performing a second assay. Instead, only detection of ions in a second detection mode need be performed. The quantitation of the level of bile acids was based on the abundance of the final fragment ions.

Example 4. Determination of Lamotrigine by Mass Spectrometry

[0147] Sample Collection.

[0148] Human whole blood was collected in a sterile container and permitted to clot at room temperature for 20-30 minutes. Serum was collected by centrifugation and collection of supernatant fluid. Samples were stored frozen, refrigerated, or at room temperature until analysis. Samples were stable for up to 2 weeks frozen, 5 days refrigerated, or 2 days at room temperature. A minimum volume of 0.5 mL was collected for this assay.

[0149] Lamotrigine Assay Procedure.

[0150] Human serum samples suspected of containing lamotrigine, and internal standard solution were aliquoted into a Captiva 96 well filter plate, which was placed over a 96 well collection plate. Filtration was performed by applying positive pressure (5-10 psi) for 1-2 minutes. Filtered samples were loaded into an autosampler for injection onto a Cyclone HTLC extraction column (Cohesive Technologies, Inc., Franklin, Mass.). At this point, no further operator handling or attention was required as the process was in-line and automated. Injected sample volume was 15 L+/3 L. An Advantage Lancer phenyl analytical column (Analytical Sales and Services, Mahwah, N.J., Cat # ADV5976) was used for analysis. The Lancer HPLC column further purified the sample and provided greater separation of the lamotrigine and internal standard peaks. The sample was eluted using a fast linear gradient to separate the lamotrigine from the internal standard. The sample with the internal standard was then injected onto a tandem mass spectrometer. The HTLC, HPLC, and MS/MS systems were inline and the analysis was performed in an automated fashion.

[0151] Chromatography was performed under the following conditions:

[0152] Extraction column: Cyclone, Cat. #952434, Cohesive Technologies, Franklin, Mass.

[0153] Extraction and Chromatography Parameters.

[0154] (settings can be modified for optimal results)

TABLE-US-00008 Start Sec Flow Grad % B % A Tee Loop Flow Grad % B % A Comments 0/1 0.00 30 5.00 Step 0 100 out 1.00 Step 0 100 Load Sample into First (HTLC) Column 2 0.50 45 0.20 Step 0 100 T in 1.00 Step 0 100 Transfer Sample to Second Column 3 1.25 20 5.00 Step 100.0 0 out 1.00 Ramp 30.0 70 Elute Sample to Second Column 4 1.58 20 5.00 Step 100.0 0 in 1.00 Ramp 60.0 20 Elute Sample to Second Column 5 1.92 30 5.00 Step 100.0 0 out 1.00 Ramp 90.0 10 Elute Sample to Second Column 6 2.42 20 5.00 Step 100.0 0 in 1.00 Step 90.0 10 Elute Sample to Second Column 7 2.75 30 5.00 Step 0 100 out 1.00 Step 0 100 Re-equilibrate System

[0155] Selected MS/MS Parameters.

TABLE-US-00009 Mode: Positive Mode Collision Gas N.sub.2 Dwell time 100 msec Res Q1 unit Res Q2 unit Curtain Gas 40.0 CAD Gas 4.0 Temp 350 GS1 50 GS2 20 DP 60 FP 360 EP 10 CE 35 CEP 14.92 CXP 0

[0156] The following mass transitions were used:

[0157] lamotrigine: precursor ion 255.9 m/z; fragment ion 210.8 m/z;

[0158] hydroxyzine: precursor ion: 375.2 m/z; fragment ion 201.0 m/z

[0159] The reportable range of the assay was about 0.5 to about 25 g/mL. The following drugs were tested as possible interfering substances, all with negative results: acetaminophen, amikacin, aminoguanidine, amiodarone, amitryptyline, amoxicillin, asprin, benztropine, carbamazepine, chlorpromazine, clomipramine, norclomipramine, clonazepam, clozapine, N-desmethylclozapine, cyclosporine A, cyclosporine C, desipramine, N-desethyl amiodarone, diazepam, 5,5-diphenylhydrantoin, disopyramide, doxepin, nordoxepin, ethambutanol, felbamate, flecainide, flunitrazepam, fluoxetine, norfluoxetine, gabapentin, gemfibrozil, haloperidol, ibuprofen, imipramine, lidocaine, maprotiline, mesoridazine, methylphenidate, mexiletine, nordiazepam, norfluoxetine, nortriptyline, prazepam, propafenone, propranolol, protryptyline, pyrazinamide, rifampicin, risperidone, 9-hydroxyrisperidone, sertraline, N-desmethylsertraline, sulfamethoxazide, thiothixene, thioridazine, trazodone, trimethoprim, and trimipramine.

[0160] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention.

[0161] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[0162] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0163] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0164] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of and consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0165] Other embodiments are set forth within the following claims.