chemical metrology for human health...
TRANSCRIPT
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
1960s
1970s
1980s
1990s
2000s
Development of Clinical SRMs
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
Reference Methods & SRMs for Health Status Markers in Blood/UrineReference Methods & SRMs for Health Status Markers in Blood/Urine
Reference Systems Currently in Place for
Many Well-Defined Markers such as:
Characteristics of these markers: • Relatively small well-defined molecular or elemental
species
• Typically, can be determined using methodology well-
studied and characterized by NIST for many years
Marker Disease State Calcium Cancer, Blood Clotting
Chloride Kidney Function
Cholesterol Heart Disease
Creatinine Kidney Function
Glucose Diabetes
Lithium Antipsychotic Treatment
Magnesium Heart Disease
Potassium Electrolyte Balance
Sodium Electrolyte Balance
Triglycerides Heart Disease
Urea Kidney Function
Uric Acid Gout
Glucose
Marker Disease State Troponin-I Myocardial Infarction
Cortisol Endocrine Function
C-Reactive Protein Risk of Heart Attack
Estradiol Hormone Balance
Folates Neural Tube Defects
Glycated Hemoglobin Diabetes Status
Homocysteine Risk of Heart Disease
TSH, T3,T4 Thyroid Function
Progesterone Hormone Balance
Speciated Iron Hemochromatosis
PSA Prostate Cancer
Reference Systems Being Developed for
New Markers such as:
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
Folic Acid
Creatinine
HO
I O
I
O
OH
I
NH2
C-Reactive Protein
Progesterone T3
HO
OH
Estradiol
HIERARCHY OF CLINICAL METHODS
Definitive Methods of Highest Accuracy and Precision; Thoroughly
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.
Refe rence Carefully Tested Method of High Accuracy and Precision;
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
Field Methods Used for Routine Clinical Measure ments; Must be
Sufficiently Accurate and Precise for Accurate Diagnosis;
Must be Simple, Rugged, and Cost Effective
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.”
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 isotope-
dilution gas chromatography/mass
spectrometry (GC/MS)
Methods utilize a stable isotope-
labeled internal standard
Analytes are converted to a stable
derivative for GC/MS
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
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
Tests and Corrections
for Blanks and
Interferences
Calculate Results and
Provide Complete
Uncertainty Statement
CHOLESTEROL DEFINITIVE METHOD
SAMPLE PREPARATION
Weigh serum sample containing 0.5 mg cholesterol, add known mass of
cholesterol-13C3 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 N2, 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 99.8 0.1%) in known mass of ethanol
Add constant mass of cholesterol-13C3 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.
0
50
100
150
200
250
50 100 150 200 250 300 350
Cholesterol, mg/dL
Num
ber
of patients
Bias in Cholesterol Measurement Effects Bias in Cholesterol Measurement Effects
Medical DecisionMedical Decision--MakingMaking
Cholesterol Frequency Cholesterol Frequency
Distribution of >20,000 personsDistribution of >20,000 persons(with +1%, +3% and +10% limits (with +1%, +3% and +10% limits
around 240 mg/dL criteria point)around 240 mg/dL criteria point)
-15-46
-129
+14
+51
+197
If measurement Positives (>240 mg/dL) Predicted Change
bias were: per 1000 in “Positives/1000”
-10% bias 120
-3% bias 203
-1% bias 234
0% bias 249
+1% bias 263
+3% bias 300
+10% bias 446
-15-46
-129
+14
+51
+197
If measurement Positives (>240 mg/dL) Predicted Change
bias were: per 1000 in “Positives/1000”
-10% bias 120
-3% bias 203
-1% bias 234
0% bias 249
+1% bias 263
+3% bias 300
+10% bias 446
ID GC/MS Definitive Methods for
Clinical Analytes
Cholesterol A. Cohen et al., Clin. Chem., 26, 854-860 (1980)
R. Schaffer et al., Clin. Chem., 28 5-8 (1982)
Glucose E. White et al., Biomed. Mass Spectrom., 9, 395-405 (1982)
Urea
M.J. Welch et al., Anal. Chem., 56, 713-719 (1984)
Creatinine M.J. Welch et al., Anal. Chem., 58, 890-894 (1986)
Uric Acid P. Ellerbe et al., Anal. Chem., 62, 2173-2177 (1990)
Total Glycerides and Triglycerides Ellerbe et al, Clin. Chem. 41/3, 397-404 (1995)
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)
Creatinine – Marker for Kidney Disease
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
SRM 967 Creatinine in Human Serum Kidney Failure in the U.S.
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-13C2
LC/MS Reference Measurement
Procedure (RMP) for Creatinine
New LC/MS method developed based on work of Stokes and O’Connor (LGC)
*Internal standard is creatinine-d3
Protein
precipitation,
remove
supernatant
Add IS*,
equilibrate
overnight
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
LC/MS RMP for Creatinine
0
1
2
3
0 5 10 15 20
Time (min)
MS
D T
IC (
x 1
0)
-4
0
1
2
3
0 5 10 15 20
Time (min)
MS
D T
IC (
x 1
0)
-4
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15
Time (min)
MS
D T
IC (
x 1
0)
-4
Creatinine 114m/z
Creatinine-d
1173
m/z
Creatine 132m/z
ID GC/MS and ID LC/MS Method
Comparison
N.G. Dodder, S.S-C. Tai, L.T. Sniegoski, N.F. Zhang, M.J. Welch,
Clin. Chem. 53:1694-1699 (2007)
Pool 1 Pool 2 Pool 1 Pool 2
( m mol/L) ( m mol/L) ( m mol/L) ( m mol/L)
Mean 67.0 346.1 Mean 66.1 346.3
SD 0.6 1.6 SD 0.2 0.8
CV(%) 0.9 0.45 CV(%) 0.2 0.2
LC-MS Method GC-MS Method
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 B6 in Human Serum
SRMs In Progress
SRM 3951 Vitamin B12 in Human Serum
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
Why is Vitamin D Important?
Shedding light on vitamin D deficiency ‘crisis’ Low levels are blamed for many of our ills. But how much is really enough?
Task force recommends against Vitamin D,
calcium supplements
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
Vitamin D
Vitamin D occurs primarily in two forms – vitamin D2 and vitamin D3
Sunlight Food Dietary Supplements
Production of vitamin
D3 in skin
Contains vitamin
D2 or D3
Contains vitamin
D2 or D3
Hydroxylation in liver to 25(OH)D
Hydroxylation in kidney to 1,25(OH)2D
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)
Mass Spectrometry-Based Methods for
Determination of Vitamin D Metabolites
The 3-epimer of 25(OH)D3 co-elutes with 25(OH)D3
on C18 columns
25(OH)D3 and 3-epi-25(OH)D3 have the same
MS/MS fragmentation patterns
Initially the 3-epimer was thought to be only found in
infants (Singh et al.)
25(OH)D2 25(OH)D3 3-epi-25(OH)D3
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 N2,
dilute with
methanol
LC-MS/MS
analysis
* The internal standards were 2H3-25(OH)D2 and 2H3-25(OH)D3
NIST LC-MS/MS Methodology –
25(OH)D3
SRM 972 Level 1
~ 60 nmol/L
APCI MS using cyano
column with methanol:water
mobile phase
3-epi-25(OH)D3 fully
resolved from 25(OH)D3
(separation based on work of
Lensmeyer et al.)
Labeled 3-epi-25(OH)D3
now available for use as
internal standard
Method approved by JCTLM
as Reference Measurement
Procedure Susan Tai et al., Analytical Chemistry, 2010
NIST LC-MS/MS Methodology –
25(OH)D2
SRM 972 Level 3 SRM 972 Level 4
~ 6 nmol/L ~ 65 nmol/L
The “Epi” Question?
1971
25(OH)D3
3-epi-25(OH)D3
The 3-epimer of 25(OH)D3 appears to be
present in nearly all adult sera but its
concentration varies
Design of SRM 972 Vitamin D in Human
Serum
Level 1
65 ± 15 nmol/L 25-hydroxyvitamin D3 (“normal”)
Level 2
Blend of “normal” serum and horse serum to obtain
approximately half the level of 25-hydroxyvitamin D3 in the
“normal” pool (35 ± 5 nmol/L)
Level 3
“Normal” serum spiked with an equivalent amount of 25-
hydroxyvitamin D2
Level 4
“Normal” serum spiked with 3-epi-25-hydroxyvitamin D3
Goal was to have serum pools that presented different analytical challenges
Assigned Values for SRM 972
25(OH)D2 25(OH)D3
3-epi-
25(OH)D3
Level 1 0.59 ± 0.20 23.2 ±
0.8 1.35 ± 0.04
Level 2 1.67 ± 0.08 12.0 ±
0.6 0.74 ± 0.02
Level 3 25.8 ± 1.9 18.1 ±
1.1 1.04 ± 0.03
Level 4 2.35 ± 0.21 32.3 ±
0.8 36.9 ± 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.
Vitamin D Metabolites QA ProgramVitamin D Metabolites QA Program
Results for total 25(OH)D in a non-fortified sample
Data courtesy of M. Bedner and K. Lippa
o Two exercises per year, no
cost for participation
o Not a proficiency program
o NIST value assigned by ID
MS
o Results do not support
common belief that LC-
MS/MS always provides
higher results
Vitamin D Metabolites QA ProgramVitamin D Metabolites QA Program
This sample had been fortified with 25(OH)D2. 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
o Fortification has been used to
prepare multi-level reference
materials for other clinical
analytes (creatinine, etc.)
o Spiking serum with 25(OH)D
may not be an appropriate way
to prepare control materials
o Analyte may bind to other
proteins (HSA) instead of DBP
Human Nutritional Assessment
SRMs – In Progress
SRM 3949 Folate Vitamers in Frozen Human
Serum
SRM 3950 Vitamin B6 metabolites in Frozen
Human Serum
SRM 3951 Vitamin B12 in Frozen Human
Serum
SRM 2378 Fatty Acids in Frozen Human
Serum
NIST
GC-FID
(IS = n-prOH)
NMIA
ID GC/MS
(13C EtOH)
NML-CSIR
Titrimetry
Certified
Value
Mean
stdev
rsd
0.08067
0.00030
0.37%
0.08020
0.00023
0.28%
0.07990
0.00017
0.21%
0.08023 ± 0.00074
0.92%
Mean
stdev
rsd
0.10190
0.00027
0.27%
0.10080
0.00031
0.31%
0.10020
0.00093
0.93%
0.10084 ± 0.00083
0.82%
Mean
stdev
rsd
0.17085
0.00109
0.64%
0.17000
0.00043
0.25%
0.16980
0.00037
0.22%
0.1701 ± 0.0014
0.82%
Is ID GC/MS Required?
Comparison of ID GC/MS vs. GC-FID and Titrimetry
for determination of ethanol in water
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
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
