Chemical Metrology for Human Health Assessment

Transcription

1 Chemical Metrology for Human Health Assessment Metrology and Physical Constants International School of Physics Enrico Fermi Stephen A. Wise Analytical Chemistry Division Material Measurement Laboratory National Institute of Standards and Technology (NIST) Gaithersburg, Maryland USA

2 Development of Clinical SRMs 1960s 1970s 1980s 1990s 2000s Development of pure, crystalline standards for calibration Development of highly accurate and precise, isotope dilution GC/MS Definitive Methods for clinical analytes in serum Human serum-based SRMs certified for metabolites and electrolytes, e.g., SRM 909 Human Serum (lyophilized) New serum-based SRMs are frozen to reduce matrix effects. New efforts focus on reference methods for toxic metals and protein-based health markers Expanded efforts to develop reference methods and SRMs to address EU IVD Directive. New methods focus on isotope dilution with LC/MS and LC/MS/MS

3 Reference Methods & SRMs for Health Status Markers in Blood/Urine Reference Systems Currently in Place for Many Well-Defined Markers such as: Marker Calcium Chloride Cholesterol Creatinine Glucose Lithium Magnesium Potassium Sodium Triglycerides Urea Uric Acid Disease State Cancer, Blood Clotting Kidney Function Heart Disease Kidney Function Diabetes Antipsychotic Treatment Heart Disease Electrolyte Balance Electrolyte Balance Heart Disease Kidney Function Gout Characteristics of these markers: Relatively small well-defined molecular or elemental species Typically, can be determined using methodology wellstudied and characterized by NIST for many years Folic Acid Creatinine HO Estradiol OH C-Reactive Protein Reference Systems Being Developed for New Markers such as: Marker Troponin-I Cortisol C-Reactive Protein Estradiol Folates Glycated Hemoglobin Homocysteine TSH, T3,T4 Progesterone Speciated Iron PSA Disease State Myocardial Infarction Endocrine Function Risk of Heart Attack Hormone Balance Neural Tube Defects Diabetes Status Risk of Heart Disease Thyroid Function Hormone Balance Hemochromatosis Prostate Cancer Characteristics of some new markers: Proteins, peptides or DNA-based Heterogeneity and instability of analyte form Low concentration in blood or urine Cannot all be standardized using conventional analytical chemistry approaches HO I I O I T3 O NH 2 OH Glucose Progesterone

4 HIERARCHY OF CLINICAL METHODS Definitive Refe rence F ield Methods of Highest Accuracy and P recision; Tho roughly Tested for Bias; Generally not within Capabilities of Clinical Laboratories; Used to Assign Analyte Concentrations to Primary Refe rence Mate rials and to Validate Accuracy of Refe rence Methods. Carefully Tested Method of High Accuracy and P recision; Within Capabilities of Most Clinical Laboratories, but too Time Consuming for Routine Use; Used to Assign Analyte Concentrations of Secondary Reference Materials and Validate the Accuracy of Field Methods Methods Used for Routine Clinical Measure ments; Must be Sufficiently Accurate and P recise for Accurate Diagnosis; Must be Simple, Rugged, and Cost Effective

5 Definitive Methods and Traceability Definitive Method is defined as: A method of exceptional scientific status, which is sufficiently accurate to stand alone in the determination of a given property for the Certification of a Reference Material. Such a method must have a firm theoretical foundation so that systematic error is negligible relative to the intended use. Analyte masses (amounts) or concentrations must be measured directly in terms of the base units of measurements, or indirectly related through sound theoretical equations. Definitive methods, together with Certified Reference Materials, are primary means for transferring accuracy -- i.e., establishing traceability. Traceability is defined as: The property of a result or measurement whereby it can be related to appropriate standards, generally international or national standards, through an unbroken chain of comparisons.

6 Definitive Methods for Clinical Analytes NIST developed a series of definitive methods for clinical analytes (e.g., cholesterol, glucose, uric acid, etc.) during the 1980s and 1990s Methods are based upon isotopedilution gas chromatography/mass spectrometry (GC/MS) Methods utilize a stable isotopelabeled internal standard Analytes are converted to a stable derivative for GC/MS

7 Isotope Dilution/Mass Spectrometry-based Definitive Methods Addition of Known Mass of Isotope labeled Material to Known Mass of Serum (or other matrix) Isolation of the Analyte from the Matrix Further Separation From Potential Interferences Tests and Corrections for Blanks and Interferences Precise Isotope Ratio Measurements of the Labeled and Unlabeled Forms Calibration of the Mass Spectrometer With Known Mixtures of Primary Reference Material and Labeled Material Calculate Results and Provide Complete Uncertainty Statement

8 CHOLESTEROL DEFINITIVE METHOD SAMPLE PREPARATION Weigh serum sample containing 0.5 mg cholesterol, add known mass of cholesterol- 13 C 3 in ethanol, and equilibrate All alcoholic KOH and heat at 37 deg for 3 h to saponify esters Extract with 10 ml hexane, evaporate under N 2, and dissolve in 1 ml methanol Take 0.1 ml, evaporate methanol, redissolve in BSA, and heat at 60 deg for 0.5h CALIBRATION STANDARDS Prepare primary standard solution by dissolving known mass of SRM 911b Cholesterol (purity %) in known mass of ethanol Add constant mass of cholesterol- 13 C 3 in ethanol to series of tubes and add masses of primary standard solution to the tubes such that the unlabeled/labeled cholesterol ratio ranges from 0.8 to 1.2.

9 Number of patients Bias in Cholesterol Measurement Effects Medical Decision-Making Cholesterol, mg/dl Cholesterol Frequency Distribution of >20,000 persons (with +1%, +3% and +10% limits around 240 mg/dl criteria point) If measurement Positives (>240 mg/dl) Predicted Change bias were: per 1000 in Positives/ % bias % bias % bias % bias % bias % bias % bias

10 ID GC/MS Definitive Methods for Clinical Analytes Cholesterol A. Cohen et al., Clin. Chem., 26, (1980) R. Schaffer et al., Clin. Chem., (1982) Glucose E. White et al., Biomed. Mass Spectrom., 9, (1982) Urea M.J. Welch et al., Anal. Chem., 56, (1984) Creatinine M.J. Welch et al., Anal. Chem., 58, (1986) Uric Acid P. Ellerbe et al., Anal. Chem., 62, (1990) Total Glycerides and Triglycerides Ellerbe et al, Clin. Chem. 41/3, (1995)

11 New Methods for Clinical Analytes Recent trend toward development of liquid chromatography/mass spectrometry (LC/MS and LC/MS/MS) methods for clinical analytes Sample preparation often simplified Better suited for thermally labile analytes Derivatization usually not required Mass spectrometry methods increasingly adopted by clinical laboratories Reference Measurement Procedures (RMPs)

12 Creatinine Marker for Kidney Disease SRM 967 Creatinine in Human Serum Kidney Failure in the U.S. Creatinine in serum is a diagnostic marker for chronic kidney disease (CKD) Physicians use serum creatinine levels to calculate egfr GFR (glomerular filtration rate) is indicative of ability of kidneys to filter wastes from blood Errors in creatinine measurement will affect egfr and accuracy of positive or negative diagnosis of CKD

13 GC/MS Definitive Method for Creatinine Definitive method based on isotope-dilution GC/MS Add IS*, equilibrate overnight Ion-exchange resin to remove creatine Elute creatinine, remove water Derivatize GC/MS analysis *Internal standard is creatinine- 13 C 2

14 LC/MS Reference Measurement Procedure (RMP) for Creatinine New LC/MS method developed based on work of Stokes and O Connor (LGC) Add IS*, equilibrate overnight Protein precipitation, remove supernatant Solvent exchange, filter, LC/MS analysis C18 Chromatographic column Mobile phase is 10 mm ammonium acetate/acetonitrile Positive mode electrospray ionization Selective ion monitoring of (M+H) + ions at m/z 114 and 117 *Internal standard is creatinine-d 3

15 -4 MSD TIC (x 10 ) -4 MSD TIC (x 10 ) -4 MSD TIC (x 10 ) LC/MS RMP for Creatinine 3 2 Creatinine m/z Time (min) 3 2 Creatinine-d m/z Time (min) Time (min) Creatine m/z 132

16 ID GC/MS and ID LC/MS Method Comparison GC-MS Method LC-MS Method Pool 1 Pool 2 Pool 1 Pool 2 ( m mol/l) ( m mol/l) ( m mol/l) ( m mol/l) Mean Mean SD SD CV(%) CV(%) N.G. Dodder, S.S-C. Tai, L.T. Sniegoski, N.F. Zhang, M.J. Welch, Clin. Chem. 53: (2007)

17 Recent SRMs for Health Status Markers SRMs Recently Issued SRM 1951b Cholesterol in Human Serum SRM 965b Glucose in Human Serum SRM 956b Electrolytes in Human Serum SRM 2721 Cardiac Troponin I SRM 1955 Homocysteine and Folate in Human Serum SRM 967a Creatinine in Frozen Human Serum SRM 927d Bovine Serum Albumin SRM 971 Hormones in Human Serum SRM 972 Vitamin D Metabolites in Serum SRM 3950 Vitamin B 6 in Human Serum SRMs In Progress SRM 3951 Vitamin B 12 in Human Serum

18 Why are Nutritional Biomarkers Important? Identifying individuals with vitamin deficiencies Not everyone responds the same way to nutrient exposure Difficult to quantify intake based on diet and self-reporting Understanding biochemical pathways Population surveys and public health policies Data is meaningful only if the measurement methods used are accurate

19 Why is Vitamin D Important? Task force recommends against Vitamin D, calcium supplements Shedding light on vitamin D deficiency crisis Low levels are blamed for many of our ills. But how much is really enough? Vitamin D is essential for maintaining calcium homeostasis Both Calcium and vitamin D are needed for bone health Vitamin D deficiency associated with rickets and osteomalacia Potential link between vitamin D deficiency and increased disease risk

20 Vitamin D Vitamin D occurs primarily in two forms vitamin D 2 and vitamin D 3 Sunlight Food Dietary Supplements Production of vitamin D 3 in skin Contains vitamin D 2 or D 3 Contains vitamin D 2 or D 3 Hydroxylation in liver to 25(OH)D Hydroxylation in kidney to 1,25(OH) 2 D

21 Measurement Techniques for Vitamin D Immunoassay Antibody specificity is high, cross-reactivity may occur No independent confirmation of analyte identity Gas Chromatography (GC-MS) Liquid Chromatography LC-UV Mass Spectrometry (LC-MS) Tandem Mass Spectrometry (LC-MS/MS)

22 Mass Spectrometry-Based Methods for Determination of Vitamin D Metabolites The 3-epimer of 25(OH)D 3 co-elutes with 25(OH)D 3 on C18 columns 25(OH)D 3 and 3-epi-25(OH)D 3 have the same MS/MS fragmentation patterns Initially the 3-epimer was thought to be only found in infants (Singh et al.) 25(OH)D 2 25(OH)D 3 3-epi-25(OH)D 3

23 LC-MS/MS Methodology for 25(OH)D Add water and ISTDs* Equilibrate 1 hr, adjust ph to 9.8 Extract with hexane:ethyl acetate Dry with N 2, dilute with methanol LC-MS/MS analysis * The internal standards were 2 H 3-25(OH)D 2 and 2 H 3-25(OH)D 3

24 NIST LC-MS/MS Methodology 25(OH)D 3 SRM 972 Level 1 ~ 60 nmol/l APCI MS using cyano column with methanol:water mobile phase 3-epi-25(OH)D 3 fully resolved from 25(OH)D 3 (separation based on work of Lensmeyer et al.) Labeled 3-epi-25(OH)D 3 now available for use as internal standard Susan Tai et al., Analytical Chemistry, 2010 Method approved by JCTLM as Reference Measurement Procedure

25 NIST LC-MS/MS Methodology 25(OH)D 2 SRM 972 Level 3 SRM 972 Level 4 ~ 65 nmol/l ~ 6 nmol/l

26 The Epi Question? (OH)D 3 3-epi-25(OH)D 3 The 3-epimer of 25(OH)D 3 appears to be present in nearly all adult sera but its concentration varies

27 Design of SRM 972 Vitamin D in Human Serum Level 1 65 ± 15 nmol/l 25-hydroxyvitamin D 3 ( normal ) Level 2 Blend of normal serum and horse serum to obtain approximately half the level of 25-hydroxyvitamin D 3 in the normal pool (35 ± 5 nmol/l) Level 3 Normal serum spiked with an equivalent amount of 25- hydroxyvitamin D 2 Level 4 Normal serum spiked with 3-epi-25-hydroxyvitamin D 3 Goal was to have serum pools that presented different analytical challenges

28 Assigned Values for SRM (OH)D 2 25(OH)D 3 3-epi- 25(OH)D 3 Level ± 0.20 Level ± 0.08 Level ± 1.9 Level ± ± ± ± ± ± ± ± ± 1.1 Certified and reference values obtained from combination of results from multiple methods: LC-MS (NIST), LC- MS/MS (NIST) and LC-MS/MS (CDC). Certified values are shown in bold. All data in ng/g.

29 Vitamin D Metabolites QA Program o o o o Two exercises per year, no cost for participation Not a proficiency program NIST value assigned by ID MS Results do not support common belief that LC- MS/MS always provides higher results Results for total 25(OH)D in a non-fortified sample Data courtesy of M. Bedner and K. Lippa

30 Vitamin D Metabolites QA Program o o o Fortification has been used to prepare multi-level reference materials for other clinical analytes (creatinine, etc.) Spiking serum with 25(OH)D may not be an appropriate way to prepare control materials Analyte may bind to other proteins (HSA) instead of DBP This sample had been fortified with 25(OH)D 2. Results are grouped into two distinct populations, suggesting not all techniques see the sample the same way. Data courtesy of M. Bedner and K. Lippa

31 Human Nutritional Assessment SRMs In Progress SRM 3949 Folate Vitamers in Frozen Human Serum SRM 3950 Vitamin B 6 metabolites in Frozen Human Serum SRM 3951 Vitamin B 12 in Frozen Human Serum SRM 2378 Fatty Acids in Frozen Human Serum

32 Is ID GC/MS Required? Comparison of ID GC/MS vs. GC-FID and Titrimetry for determination of ethanol in water NIST GC-FID (IS = n-proh) NMIA ID GC/MS ( 13 C EtOH) NML-CSIR Titrimetry Certified Value Mean stdev rsd % % % ± % Mean stdev rsd % % % ± % Mean stdev rsd % % % ± %

33 ID GC/MS vs. ID LC/MS (or MS/MS) Requirements for quantification using internal standards for GC/MS vs. LC/MS are different For GC/MS internal standard does not need to elute with the analyte for good precision For LC/MS (or MS/MS) internal standard should elute with or near analyte to achieve best precision Precision for both can be similar under optimal conditions

34 Isotope Dilution MS - Conclusions Isotope dilution MS-based GC or LC analyses are the recommended approach for organic analysis in complex matrices if labeled standards are available In simpler matrices ID MS methods may not be required Use of ID MS methods does not guarantee that the results are accurate

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