Patent Application
20070259445
The invention description below refers to the accompanying drawings, of which:
Features in the drawings are not, in general, drawn to scale.
The prepared solid substrate of the invention comprises an internal standard joined to a supporting material.
The supporting slab 12 of substrate 10 includes paper, glass, textiles, ceramics, metals, or plastics such as polystyrene, polyethylene glycol, divinylbenzene; methacrylate, polymethacrylate, polyacryloylmorpholide, polyamide, poly(tetrafluoroethylene), polyethylene, polypropylene, poly(4-methylbutene), poly(ethylene terephthalate), nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde. Silicate agarose, cellulose acetate, nitrocellulose, cotton, rayon, and natural plastics are also candidate materials for the supporting material.
The invention does not limit the manner in which the internal standard is joined to the supporting material in the substrate 10. The internal standard can be dried on all or part of a face of the supporting material, infused or diffused into a portion of or throughout the supporting material, chemically linked to the supporting material, or otherwise bound covalently, noncovalently, via hydrogen bonding, capillary forces or surface tension to the supporting material. Joining to the supporting material can be effected by methods such as spraying the internal standard onto a face of the supporting material; soaking a supporting material in a solution containing the internal standard; or by forming the substrate from a slurry containing the internal standard along with the precursor from which the supporting material is formed. Methods for impregnating paper with chemical materials, for example, are well known to those skilled in the art, as described, in U.S. Pat. No. 6,890,481.
The device 40 includes a spray capillary 43 through which a liquid solvent 44 is fed. A nebulizer capillary 45 surrounds the spray capillary 43 to form an annular space through which a nebulizing gas 46 is fed at high velocity. Nitrogen is a typical candidate for the nebulizing gas 46. Aqueous methanol has been used for the liquid solvent 44.
A power supply 47 applies a high voltage to the liquid solvent 44. The interaction between the fast-flowing nebulizing gas 46 and the liquid 44 leaving the capillary 43 forms the desorptive, ionizing spray 42 comprising liquid droplets. The spray 42 also may include neutral atmospheric molecules, nebulizing gas, and gaseous ions.
The spray 42 is directed onto the sample material S which is supported on a prepared substrate 10 incorporating an internal standard. The substrate 10 may be on a platform moveable by well known drive means to desorb and ionize different areas of sample S over time, for example to effect a raster of the entire substrate surface. Electric potential and temperature of such a platform may also be controlled by known means.
An ion transfer line 52 collects the desorbed ions 54 leaving the substrate 10 and introduces them into the atmospheric inlet or interface 56 of a mass spectrometer for analysis. Any atmospheric interface that is normally found in mass spectrometers is suitable for use in a DESI-type system. Interfaces that have been found to work well include a typical heated capillary atmospheric interface and an atmospheric interface that samples via an extended flexible ion transfer line made either of metal or an insulator.
Considerations informing the selection of an internal standard incorporated in substrate 10 for assessment of a particular analyte by a particular experimental configuration are well known to those skilled in the art. In general a suitable internal standard is chemically similar to the analyte, which is what is meant by an internal standard “corresponding” to the analyte. Further, the internal standard must be resolvable from the analyte using mass spectrometry. Finally, the internal standard does not react chemically with the analyte and contains substantially no trace amount of the analyte.
A stable isotopically labeled form of the analyte is commonly found to fulfill these requirements. Extensive published references provide guidance for selecting an internal standard to those skilled in the art. (See, for example, Liu et al., “Selecting an appropriate isotopic internal standard for gas chromatography/mass spectrometry analysis of drugs of abuse—pentobarbital example,” J. Forensic Sci.; November 1995; 40(6): 938-9.) The absolute amount of internal standard detected during a sample analysis can be predetermined by empirical testing of the particular internal standard incorporated into a particular substrate under specified ionization conditions. Typically, the amount of the internal standard is well above the limit of quantitation but not so high as to suppress the ionization of the analyte.
A variety of types of samples can be analyzed using the methods described herein, including biological, medical, industrial, agricultural, laboratory and food samples. For biological and medical applications, samples can include any biological fluid, cell, tissue, or fraction thereof, that includes molecules corresponding to the selected internal standards. A sample can be, for example, a specimen obtained from a subject (e.g., a mammal such as a human) or can be derived from such a subject. For example, a sample can be a tissue section obtained by biopsy, or cells that are placed in or adapted to tissue culture. Exemplary samples therefore include cultured fibroblasts, cultured amniotic fluid cells, and chorionic villus sample. A sample can also be a biological fluid specimen such as urine, blood, plasma, serum, saliva, semen, sputum, cerebral spinal fluid, tears, mucus, and the like. A sample can be further fractionated, if desired, to a fraction containing particular cell types. For example, a blood sample can be fractionated into serum or into fractions containing particular types of blood cells such as red blood cells or white blood cells (leukocytes). If desired, a sample can be a combination of samples from a subject such as a combination of a tissue and fluid sample, and the like. Methods for obtaining samples that preserve the activity or integrity of molecules in the sample are well known to those skilled in the art. Such methods include the use of appropriate buffers and/or inhibitors, including nuclease, protease and phosphatase inhibitors, which preserve or minimize changes in the molecules in the sample. Such inhibitors include, for example, chelators such as ethylenediamne tetraacetic acid (EDTA), ethylene glycol bis(Paminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain and the like, and phosphatase inhibitors such as phosphate, sodium fluoride, vanadate and the like. Appropriate buffers and conditions for isolating molecules are well known to those skilled in the art and can be varied depending, for example, on the type of molecule in the sample to be characterized (see, for example, Ausubel et al. Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds. W. B. Saunders, Philadelphia, (1999)).
The invention is well suited to newborn blood screening, which generally involves assaying more than twenty analytes in a sample. Tables 1 and 2 list analytes typically tested in a newborn blood assay. For many of the disorders diagnosable using newborn blood levels of these analytes, several criteria for diagnosis have been reported in the literature. For example, phenylketonuria may be indicated by the level of phenylalanine alone (as reported by CDC, U.S. Department of Health and Human Services, “Using Tandem Mass Spectrometry for Metabolic Disease Screening Among Newborns,” MMWR_Apr. 13, 2001; Vol. 50, No. RR-3; Rashed et al., Clinical Chemistry; 1997; 43(7):1129-41; and The Wisconsin NBS Laboratory—Wisconsin State Laboratory of Hygiene, “Health Professionals Guide to Newborn Screening,” retrieved Oct. 28, 2003, from the website of The Board of Reagents of the University of Wisconsin System). Alternatively, the level of tyrosine may be additionally considered (ACMG/ASHG Test and Technology Transfer Committee Working Group, Tandem Mass Spectrometry in Newborn Screening, Genetics in Medicine; July/August 2000; 2(4); and Schulze et al., Pediatrics; 2003; 111(6):1399-1406). A third approach considers the level of phenylanaline and the Phe/Tyr ratio (Zytkovicz et al., Clinical Chemistry; 2001; 47(11):1945-55).
Six criteria have been reported for diagnosing the fatty acid oxidation disorder known as medium-chain acyl-CoA dehydrogenase deficiency (MCAD), none of which relies on a single indicator. One paradigm uses levels of C8 and C10:1. (ACMG/ASHG Test and Technology Transfer Committee Working Group). A second additionally uses levels of C10 and C6 (CDC). A third considers the ratio C8/C10 in addition to the four individual levels (Chace et al., Clinical Chemistry; 2001; 47:1166-82). A fourth approach considers only levels of C6, C8, C10:1 (Rashed et al. and Zytkovicz et al.). A fifth approach considers individual levels of C6, C8, C10, and the ratios C8/C2, C8/C10 and C8/C12 (Schulze et al.) A sixth selects individual levels of C6, C8, and C10:1 and the ratio C8/C10 (Wisconsin NBS Laboratory). The substrate of the invention is able to incorporate internal standards for all of these several analytes.
It will therefore be seen that the foregoing represents a highly advantageous approach to quantitative surface-interrogation mass spectrometry, especially for quantitation of blood components. The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/797,993, which was filed on May 5, 2006, by Blas Cerda for Quantitative Analysis of Surface-Derived Samples using Mass Spectrometry and is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60797993 | May 2006 | US |
