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    Vitamin C in Plasma Dan Serum

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    dewi

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    © All Rights Reserved
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    of 14
    us) United States 2) Patent Application Publication co) Pub. No.: US 2017/0242044 A1 oy oy ~ ey @) Jiang et al. METHODS FOR DETECTING VITAMIN © BY MASS SPECTROMETRY Applicant: Quest Diagnostics Investments Incorporated, Wilmington, DE (US) Inventors: Qibo Jiang, Los Angeles, CA (US): Richard E Reitz, Las Vegas, NV (US); ‘Sum Chan, San Clemente, CA (US) Appl. Now 15/589,398 File: May & 2017 Related U.S. Application Data Continuation of spplication No, 14/746,037, fled oa Jun, 22, 2015, now Pat, No, 9,685,158, which is a ‘continuation oF application No, 14/305,897, filed on Jun. 16, 2014, now Pat. No. 9,063,119, which is a ‘continuation of application No. 141068,925, led on Oct. 25, 2013, now Pat. No. 8.759.754, which is a continuation of application No, 13/725,049, filed on Dee. 21, 2012, now Pat. No. 8,569,689, which is a continuation of application No, 151078,792, fed oa Apr. 1, 2011, now Pat. No. 8:338,778, which is a ‘ (0) ny 6) on US 2017024204441 43) Pub. Date: Aug. 24, 2017 continuation of application No, 12/269,862, filed on Now. 12, 2008, now Pat, No. 7.982.067 Provisional application No, 61/103,212, filed on Oct. 6, 2008, Publication Classification Int. Cl. GOIN 3382 GOIN 3088 2006.01) (2006.01), OID 1508 (200501), GOIN 3072 (2006.01), US. Cl coc GOIN 33782 (2013.01); GOLN 307233 (2013.01); GOIN 30488 (2013.01); BOID 1508 2013.01}; GOIN 2030/884 (2013.01) GOIN 2560100 (2013.01); GOIN 2800/02 (2013.01), GoiN 2800/32 (2013.01), HOA 49/165 (201301), ABSTRACT Provided are methods for determining the amount of vitamia cin 4 sample using mass spectrometry. The methods genenily involve ionizing vitamin C ina simple and ‘detecting and quantifying the amount of te ion to determine ‘the amount of vitamin © in the sample, srecsez0.0 a = See2-11490) Fev tency at ioeato ot Fev tency Time mig Patent Application Publication Aug. 24, 2017 Sheet 1 of 2 US 2017/0242044 AI Figure 1. Exemplary Chromatograms of Vitamin C and Internal Standard or eo. et eo. Time (mind Tame cin) Patent Application Publication Aug. 24,2017 Sheet 2 of 2. US 2017/0242044 AL Figure 2. Linearity - Typical Calibration Curve Yasoont97ted Rez" 60888 W Ewa prea aa 2 cope nepeneennrn pp rnnninnrnrpenenpeemrreens oy ais eet Seer eRe A royal. US 2017/0242044 AI METHODS FOR DETECTING VITAMIN © BY MASS SPECTROMETRY ‘CROSS-REFERENCE TO RELATED PATENT "APPLICATIONS, 10001) This application is a continuation of U.S. appl 1a See. No, 14746,037, filed Jun. 22, 2015, which is @ ‘continuation of US. application Ser. No. 14/305,897, fled Jun, 16, 2014, which is @ continuation of U.S. application Ser, No, 141063,925, fled Oct. 25, 2013, now US. Pat. No. 8,750,754, which is continuation of US. application Sor No. 13/725,049, fled Dec. 21, 2012, now U.S. Pat. No. 8,560,689, which isa continuation of US. application Sor. No. 131078.702, filed Apr. 1, 2011, now US. Pat. No. 8.338.778, which is continuation of US. application Sor. No. 12/269,862, fled Nov. 12, 2008, now U.S. Pat. No. 7,982,067, which claims benefit of US. Provisional Appli cation No, 61/103,212, fied Oct. 6, 2008, each of which is, Incomporated by reference herein in its entity FIELD OF THE INVENTION [0002] The invention relates to the detection of Vitamin C. Ina particular aspect, the invention relates to methods for detecting Vitamin C by mass spectrometry. BACKGROUND OF THE INVENTION 10003] The following description ofthe background ofthe invention is provided simply as an ad in understanding the invention and isnot admitted to describe or constitute prior fat 10 the invention, 10004} - Vitamin C [2-ox0-L-threo-hexono-1, 4-lactone2, S-enediol] or L-ascorbie acid isa water-soluble vitamin and ‘essential nutrient for humans Its essential i the formation ‘of eollagen, which is require for normal growth and devel ‘opment as well as tisste repair in all parts of the body. Vitamin C also functions as an antioxidant that blocks the damage caused by free radicals and dirclly reduces toxic ‘chemicals and pollutants [0005] "As humans do not produce vitamin Cin the body, itis primarily obtained from dietary sources such a fits ‘and vezetables. Lack of dietary vitamin C may result in vitamin C deficiency. Severe vitamin C deficiency, also know as “scurvy.” leads to the formation of liver spots on skin, spongy gums, and bleeding from mucous membranes, for even death. 10006} Currently, vitamin C js not only use as diotary supplemeat, but also as an adjunet therapy for some viral infections and terminal cancers. The recommended daily Intake of vitamin C for adults to prevent defiiency is 75 mg for females and 90 mg for males, both with tolerable uppet level of 2,000 mg, For therapeutic usaxe in detoxifcati and cancer therapy, vitamin C is given much higher doses. Although vitamin C toxicity is rare clinically, relatively high doses of oral intake may lead t0 somoch upset and diam [0007] - Assays for vitamin C blood levels have been devel- ‘oped and are used by patients and physicians to evaluate firitonal status or to optimize therapeutic dosages SUMMARY OF THE INVENTION 10008] The present invention provides methods for detet- fing the amount of vitamin C ina sample by mass spectom- including tandem mass spectrometry. Aug. 24, 2017 [0009] In one aspect, methods are provided for determine ing the amount of vitamin C ina test sample. Methods ofthis aspect include: (a) ionizing vitamin C from the test sample to pmdce oe or more vitamin C ions detectable by mass spectrometry; and (b) detecting the amount of the vit on(s) by mass spectrometry. Once the amount ofthe one or ‘ore vitamin C fons is measured, the amount of vitamin © jon(s) is related w the amount of vitamin C in the test ‘sumple, In these methods, the one or more vitamin C ions detectable by tandem mass spectrometry include one or ‘more ions selected from the group consisting of ions with 2 sasscharge ratio of 175.0520.5, 114.8520.5, and 86.8520, 5-ln some embodiments the miss spectrometry is tande ‘mass spectrometry. In tome embodiments, the methods further comprise purifying vitamin C in the test samples prior to mass spectrometry. In related embodiments, said purifying comprises purifying by liquid chromatography. In further related embodiments, the liquid chromatography’ is high performance figuid chromatography’ (HPLC). In some embodiments, purifying vitamin C comprises one of more purification steps prior to Tiquid chromatography, In related tembodiniens, the ane or more purification steps preceding Tiquid chromatography may inelude protein precipitation. In some embodiments, the test sample is body uid: preferably plasma oe serum. In some embodiments, the step of ionizing ‘Vitamin C includes generating precursor ion With mass! charge ratio of 175.0520 5, and generating one of more fragment ions selected from the group consisting of ions with a massicharge ratio of 114.8540 5, and 86585205. In some embodiments, a stabilizing agent may be ade t the fest sample prior to mass specttomety. In related embod ‘mens, the stabilizing agent is trichloroacetic acid (TCA). la some embodiments the method has a lower limit of quan- Uitation within the range of 10.0 mi. und 0.1 mpi, inclusive. In some embodiments, the amount of one or more vitamin C ions determined by mass spectrometry is related to the presence or amount of vitamin Cin the test sample by ‘comparison oan internal standard preferably °C,-L-aseor- bie acd, [0010] Ina second aspect, methods are provided for deter- ‘mining the amount of vitamin C ia a body fluid sample by ‘mats spectrometry. Methods of this aspect include: (a) purifying vitamin C in a body uid sample; (b) ionizing Vitamin C from the body fuid sample to produce one or ‘more vitamin C ions detectable By moss spectrometry; and (6) detecting the amount of the vitamin © ion(s) by mass spectrometry: In these methods, the amount of the vitamin © jon(s) determined by mass spectrometry is related to the amount of vitamin C in the tet sample. In some embod ‘ens, the mass spectrometry is andem mass spectrometry. Insome embodiments, the body uid samples are purified by liquid chromatography, In related embodiments, the liquid chromatography may be high performance liguid chroma- tography (HPLC). In some embodiments, the step of puri ‘ving vitamin C in a test sample includes one or more purilication steps prior to iquid chromatography. In elated embodiments, the one or more purification steps preceding Tiquid chromatography may inelude protein precipitation. In some embodiments the test sample is plasma or sorum. In ‘Sone embodiments the vitamin C fons detetable by mass spectrometry include one or more ions selected from the group consisting of ions with a masscharge ratio of 175 (052015, 114.85205, and 86.8520 5, In some embocliments, the step of ionizing vitamin C includes generating @preeur US 2017/0242044 AI sor ion with a masscharge rato of 178.0520 5, and pene ing one of tore fragmcat ions elected fom the Soup ‘consisting of ions with « massicharge rao of 114.8820.3, fund 86.8520.5. In some embodiments, stabilizing agent may be ald fo the test sample prior fo mass spectromety: preferably prior w punijing the test sample. in related ‘embodiments, the stabilizing agent is trichloroacetic acid (ICA) In some embodiments, the method fuser Limit ‘of quantitation within the range of 10.0 mg and 0.1 ‘gl ialinve, ln some embodiments, the amount of one ‘or more vitamin € ions detemined by mass spetromety s relate to the presence or amount of vitamin C inthe test Sample by comparison to an intemal standard: preferably SCoL-asoorbie acid 10011] faa third aspect, methods are provided for deter. mining the amount of vitamin C in a body uid sample by tandem mass speciometry. Methods ofthis aspet inl (@)puniyng vitamin from a body uid sample by liquid chromatography: (b) wenerating a precursor ion of vtaminC having a masscharge ratio of 175.0820; (6) generating one ‘ormore agent ons ofthe precuror ion seleted fom he jnoup of fragment ions having 2 masveharge ratio oF 14 85205, and 86.8520.5: and (d) detecting the amount of one ‘or more ofthe fons generated in step (6) oF () or both and felting the dtemnined ions to the amount of Vitamin C ia the body fd sample. In some embodiments, the method has a lower linit of quantitation within the range of 10.0 mg/dl and 0.1 mpi. incisive. In some embodiments liquid chromatography is hgh perfomance lguid ehroma- tography (HPLC). In some embosimens, the sep of puri {sing vitamin Cina body Mui sample includes one ox nore Puriation steps prior t liguid chromatograph In ated ‘embodiments, the one of more purification steps preceding Ingo chromatography may inelide protein pecpttin. La some embodiments the tes simple is plstna of serum. Ia Some embodiments a stabilizing agen may beaded othe test sample prior to mass spectomstry preferably prior v0 Puslving te test sample. In related embodiments, the Stabilizing goat i wichloretcetic ackd (TCA). In some ‘embodiments, the method has a lower limit of quantitation ‘within the range of 10.0 mgidl-and 0.1 mgt inlsive. In ome embodiments, the amount of one oe more vitamin © ions determined by moss spectrometry is relate to the presence or amount of vitamin C in the test sample by ‘Compason tn intemal standard preferably "CL asco bie acd. 0012] Methods ofthe preset invetion iavole the cme bination of liguid chromatography with mass spectrometry In preferred embodiments, the liguid chromatography #8 HPLC. One prefered embodiment wilzes HPLC slone or in ‘combination with ane or more puriiation methods such as for example HILC or protein precipitation an firation, to pusiy vita C in samples. fn anater prefered embod ment the mass spectrometry is tandem mass spectometry (SMS). 10013} In certain prefered embodiments of the methods dliselosed herein, mass spectrometry is performed in nee five ion mode. Altematively, mass spectrometry is per formed in positive ion made, Various ionization sours, including for example samospheric pressure chemical ion- ization (PCT or electrospray ionization (PSD, may be wed inembodiments ofthe present invention In cera preferred ‘chodimcnts vitamin C is mcosired sing APCI in ee tive mode Aug. 24, 2017 [0014] In prefered embodiments, vitamin € ions detet= able in a mass spectrometer are selected from the group consisting of negative ions with a mass/charge ratio (ni2) of 175.0520-50, 114.8520 50, and 86.8520.50. In particularly preferred embodiments, vitamin C precursor ion has mz (61 175.0820.80, und one or more fragment ions are selected from the group consisting of ions having mi of 114.85s0.50 and 86.8520.50. [0013]. In prefered embodiments, a separately detectable internal vitamin C standard is provided in the sample, the mount of which is also determined in the sample. In these embodiments, all or a portion of both the endogenous ‘vitamin C and the intemal standard present inthe sample is ‘nized to proce a plurality of ions detectable in a mass spectrometer, and one or more ions produced from each are detected by mass spectrometry. [0016] | preferred internal vitamin C standard is °C. ascorbic acid. In prefered embodiments, the intemal vite ‘min C standard ions detectable in a mass spectrometer are slectd from the group consisting of negative ions with m7 (6f 181,120.50, [19.1030.50, and 90,0030.50. In particu lary prefered embodiments, a precursor ion of the intemal vitamin Cstadand has m/z of 181 1020.50, and one or more fragment ions are selected from the soup consisting of ions hhaving mz oF 119.1030.50, and 90.0020 50. [0017] In prefered embodiments, the presence or amount ofthe vitamin C ion is related to the presence or amount of ‘itamnin C in the test sample by comparison to reference such as SC-L-ascorbie acid, [0018] _In certain preferred embodiments, the lower limit ‘of quantitation (LLOQ) of vitamin C is within the range of 10.0 midland 0.1 mg/d, inclusive; preferably within the range of 5.0 mg/dl and 0.1 mds preferably witin the range of 2.5 midland 0.1 mg/dl; preferably within the rnge of 10 mgidl and 0.1 mld preferably within the range of 0.80 md. and 0.1 maid; preferably range of 040 mad and 0.1 mg/dL: preferably range of 0.30 mg/dL. and 0.1 mg/l: preferably range of 0.20 mg/dl and 0.1 mg/dl: preferably about 0.1 mil [0019] As used herein, unless otherwise stated, the singte Jarforms “a."“an," and “the” include plural reference, Thus, for example, reference 10 “a protein” includes a plurality of protein molecules, [0020] As used herein, the term “purification” or “purify- {ng does not eferto removing all materials from the sample cther than the analyt(s) of interest, Instead, purification refers ta procedure that enriches the amount of one or mare analytes of interest relative to other components in the sample that may interfere with detection of the analyte of interest. Purification of the sample by various means may allow relative reduction of one oF more interfering sub- stanees, ea. one oF more substances that may oF may not interfere with the detection of selevted vitamin C parent or daughter ions by mass spectrometry. Relative redetion as ‘is tem is used doesnot require that any substance, present with the analyte of interest i the materia t be purifies entirely moved by purification, [0021] _Asused herein, the tem “test sample” refers to any sample that may contain vitamin C. As used herein the term “body fluid” means any’ fluid that ean be isolated from the body’ of an individual. For example, “body hid” may include blood, plasma, serum, tile, saliva, urine, tear perspiration, and the like US 2017/0242044 AI 10022] As used herein, the term “chromatography” refers to a process in which a chemical mixture carried by liquid ‘or gis is separated info components as result of differential “isiibution of the chemical entities as they flow around oF ‘over a stationary liquid o solid phase. 10023] As used herein, the term “liquid chromatography” ‘or “LC” means a process of selective retardation of one oF ‘more components of a fluid solution asthe uid uniformly pereolates through column ofa finely divided substance, oF through capillary passageways. The retardation resis from the distribution ofthe components ofthe mixture between fone of more stationary phases and the bulk fii, (i ‘mobile psse), as this fluid moves relative tothe stationary Phases). Examples of “Tiguid chromatography” inclade reverse phase liquid chromatography (RPLC), high perfor- ‘mance liquid chromatography (HPLC), and high turbulence Tiguid chromatography (HTLC), 10024] As used herein, the erm “high performance liquid chromatography” oF “HPLC” refers to liquid ebromstograe phy in which the dogree of separation is increased by forcing the mobile phase under pressure through a stationary phase ‘on a support matix, typically a densely packed column. 10025] As used herein, the term “high turbulence liquid chromatography” or “HTLC” refers to a form of chromae togmphy that utilizes turbulent How of the materia being assayed through the column packing as the basis for per Torming the separition, HTLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, ei. Zimmer et al J. Chromatogr. & 854: 28-35 (1999); se also, U.S. Pat. No. 5,968,367, 5,919,368, 8,795,469, and 8,772,874, which fir ther explain HTLC. Persons of ordinary skill in the art understand “turbulent flow". When Muid flows slowly and smoothly, the low is called “Laminar flow”. For example, ‘uid moving through an HPLC columa st low flow rates is laminar. In laminar flow the motion ofthe panicles of fu orderly with particles moving geaerally ia straight lines, At faster velocities, the inertia of the water overcomes Hid Tietional forees and turbulent flow results. Fluid sot ia ‘contact with the iregular boundary “outrans” that which is slowed by liction or deflected by an uneven surfae. When alu i lowing turbulent, it hows in eles snd whirls (or vortices), with more “rag” than when the How is laminae. Many references are available for asisting in detemnining ‘when fluid flow is laminar or turbulent (et, Turbulent Flow Analysis: Measurement and Prediction, BS. Bernard & JM. Wallace, John Wiley & Sons, Inc, 2000}; An Introduction to Turbulent Flow, Jean Mathiew & Julian Seot, Cambridge University Press (2001). 0026] As used herein, the term “gas chromatography” oF "GC" refers to chromatography in whieh the sample mixture js vaporized and injected into a stam of eatrier gas (as nitrogen or helium) moving through a column containing 2 Stationary phase composed ofa liguid or 2 particulate solid ‘and is separated ito its component compounds aecording (0 the affinity ofthe compounds for the stationary phase [0027] _As used herein the term “large particle column” or “extraction column” refers to a chromatography column ‘containing an average particle diameter greater than about 35 jum. As used inthis context, the term “about” micansel0%, 10028] As used herein, the term “analytical column” refers to 8 chromstography column having sufiient chromato- raphic plates to elfet a separation of materials in a sample Aug. 24, 2017 that elute from the column sulicieat to allow a determina- tion ofthe presence or amount of an analyte. Such columns are often distinguished from “extraction columns”, whieh ‘have the general purpose of separating or extracting retained ‘material from non-fetaned materials in order 10 obtain a purified sample for further analysis. As used inthis context, {he term “about” means#10%. In a preferred embodiment the analytical column contains particles of about 3.5 jm in iametor [0029] As wsed herein, the term “on-line” or “inline”, for ‘example as used in “on-line automated fashion” or “on-line fextraction” refers io a procedure performed without the ned Tor operator intervention. [a contrast, the term “of-ine™ as ‘used herein refers to a procedure requiring manual interven- tion of an operator. Thus, if samples are subjected 10 precipitation, and the supernatants are then manually loaded {no an autosampler, the preipitation and loading steps are ‘offline from the subsequent steps. In various embodiments of the methods, one or more steps may be performed in an fon-line automated fashion, [0030] As used herein, the term “mass spectrometry” or "MS" refers to an analytical technique 10 identify com- pounds by their mass, MS refers to methods of filtering. ‘etectng, and meastiing ions based on their mass-o-charge ratio, or “mia”. MS technology generally includes (1) ion- ‘ring the compounds to foam charged compounds; and (2) etecting the molecular weight of the charged compounds land calculating a mass-to-charge ratio, The compounds may be ionized and detected by any suitable means. A “mass spectrometer" generally includes an ionizer and an ion detector. In general, one oF more molecules of interest are jonized, and the ions are subsoquently introduced into a ‘mats spectrogeaphic instrament where, dv to a combination fof magnetic and clectric fields, the ions follow a path in space that is dependent upon mass (*m") and charge ( See, eg. US. Pat. Nos. 6.204.500, enitled “Mass Spee- ‘eometty From Surfaces,” 6,107,623, entitled “Methods and for Tandem Mass Spectrometry:” 6.268.144 » Spectromety: 6.124.137, entitled “Surfuce-Enhanced Photolabile Attach- ‘ment And Release For Desorption And Detection OF Ana- Iytes:" Wripht etal, Prostate Cancer and Prostatic Diseases 2:264-76 (1999): and Merchant and Weinherger, Electro. phoresis 21:1164-67 (2000), [031] As used herein, the term “operating in negative ion mode” refers to those mass spectrometry methods where negative fons are generated and detected. The term “oper- ting in positive ton mode" as used herein, refers to those nass spectrometry methods where positive ons are gener ated and detected. [032] As used herein, the term “ionization” or “ionizing” refers to the process of generating an analyte jon having @ tet electrical charge equal t0 one oF more electron unis [Negative ions are those having a net negative charge of one for more electron units, while positive ions are those having net postive cliarge of one or more electron unis 0033] As used herein, the term “electron ionization” or ET" refers to methods in which an analyte of interest in a sanscous of vapor pase interacts with a How of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique. [0034] As used hercn, the term “chemical ionization” or “CT” refers to methods in Which a reagent yas (eg. ammo- US 2017/0242044 AI nia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules 10035] As sed herein, the term “fst atom bombardment” ‘or “FAB® refers to methods ia which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, \desorbing and ionizing molecules contained in the sample. Test samples ae dissolved ina viscous liquid matrix such as lyeerol, thioglyeerol, mnitrobenzyl aleohol, 18-crow-6 ferown ther, 2-ntrophenylocty! ether, sulfolane, dicthe rnolamine, and wiethanolamine, The choice of an appropriate ‘matrix for @ compound or sample is an enipirical process 10036] As used herein, dhe term “matrix-assisted laser desorption ionization” of "MALDI" refers t methods in Which « non-volatile sample is exposed to laser imadiation, ‘which desorbs and ionizes analytes inthe sample by various ‘onizaton pathways, including photo-enization, protona- tion, deprotonation, and cluster decay. For MALDI, the sample is mixed with an eneray-absorbing matrix, whieh Jaclitates desorption of analyte molecules. [0037] As used herein, the term “surface enhanced laser

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