U.S. patent application number 15/645646 was filed with the patent office on 2017-12-28 for methods for determining total body skeletal muscle mass.
This patent application is currently assigned to GLAXOSMITHKLINE LLC. The applicant listed for this patent is GLAXOSMITHKLINE LLC, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to William J. EVANS, Marc K. HELLERSTEIN.
Application Number | 20170367648 15/645646 |
Document ID | / |
Family ID | 48574852 |
Filed Date | 2017-12-28 |
United States Patent
Application |
20170367648 |
Kind Code |
A1 |
HELLERSTEIN; Marc K. ; et
al. |
December 28, 2017 |
METHODS FOR DETERMINING TOTAL BODY SKELETAL MUSCLE MASS
Abstract
The present invention is based on the finding that enrichment of
D3-creatinine in a urine sample following; oral administration of a
single defined dose of D3-creatine can be used to calculate
total-body creatine pool size and total body skeletal muscle mass
in a subject. The invention further encompasses methods for
detecting creatinine and D3-creatinine in a single sample. The
methods of the invention find use, inter alia, in diagnosing
disorders related to skeletal muscle mass, and in screening
potential therapeutic agents to determine their effects on muscle
mass.
Inventors: |
HELLERSTEIN; Marc K.;
(Kensington, CA) ; EVANS; William J.; (Chapel
Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE LLC
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
WILMINGTON
OAKLAND |
DE
CA |
US
US |
|
|
Assignee: |
GLAXOSMITHKLINE LLC
WILMINGTON
DE
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
OAKLAND
CA
|
Family ID: |
48574852 |
Appl. No.: |
15/645646 |
Filed: |
July 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15098217 |
Apr 13, 2016 |
9737260 |
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15645646 |
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14363779 |
Jun 6, 2014 |
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PCT/US2012/068068 |
Dec 6, 2012 |
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15098217 |
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61708013 |
Sep 30, 2012 |
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61567952 |
Dec 7, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 30/7233 20130101;
A61B 5/15 20130101; A61B 5/4519 20130101; H01J 49/004 20130101;
H01J 49/0036 20130101; A61B 5/4869 20130101; A61B 10/007 20130101;
A61B 5/107 20130101; G01N 33/70 20130101; G01N 33/493 20130101 |
International
Class: |
A61B 5/00 20060101
A61B005/00; G01N 33/70 20060101 G01N033/70; A61B 5/107 20060101
A61B005/107; A61B 10/00 20060101 A61B010/00; A61B 5/15 20060101
A61B005/15; H01J 49/00 20060101 H01J049/00; G01N 30/72 20060101
G01N030/72 |
Claims
1. A method of determining total body skeletal muscle mass in a
subject, comprising: (a) orally administering D.sub.3-creatine to a
subject, wherein the D.sub.3-creatine dilutes in the total body
skeletal muscle pool of creatine and reaches isotopic steady-state
in the total body skeletal muscle pool of creatine; (b) obtaining a
biological sample comprising creatinine and D.sub.3-creatinine from
the subject, wherein the biological sample is selected from the
group consisting of a blood sample, a serum sample, a plasma
sample, and a tissue sample; (c) detecting creatinine and
D.sub.3-creatinine in the biological sample by a method selected
from the group consisting of HPLC/MS, HPLC/MS/MS, LCMS, LC/MS/MS,
and isotope ratio mass spectrometry (IRMS); (d) measuring
enrichment ratio of D.sub.3-creatinine in the biological sample at
a time t based on the creatinine and D.sub.3-creatinine detected in
(c); (e) measuring a total amount of urinary D.sub.3-creatinine
from administration of the D.sub.3-creatine to the time t; (f)
determining total-body creatine pool size in the subject by the
formula [ Amount of D 3 - creatine dosed ( g ) - Total amount of
urinary D 3 - creatine ( o - t ) ( g ) ] enrichment ratio ( t ) =
total - body creatine pool size ; ##EQU00001## and (g) determining
total body skeletal muscle mass in the subject based on the formula
Total body skeletal muscle mass = ( the total - body creatine pool
size creatine concentration in skeletal muscle ) . ##EQU00002##
2. (canceled)
3. The method of claim 1, wherein the biological sample is a blood
sample.
4. The method of claim 1, wherein 5-250 mg D.sub.3-creatine or a
salt or hydrate thereof are administered.
5. The method of claim 1, wherein the D.sub.3-creatine administered
is a hydrate of D.sub.3-creatine.
6. The method of claim 5, wherein the D.sub.3-creatine is
D.sub.3-creatine monohydrate.
7. The method of claim 1, wherein the biological sample is obtained
at least 24 hours after administration of the D.sub.3-creatine.
8. The method of claim 7, wherein the biological sample is obtained
at least 36 hours after administration of the D.sub.3-creatine.
9. The method of claim 7, wherein the biological sample is obtained
at least 48 hours after administration of the D.sub.3-creatine.
10. The method of claim 7, wherein the biological sample is
obtained at least 60 hours after administration of the
D.sub.3-creatine.
11. The method of claim 7, wherein the biological sample is
obtained at least 72 hours after administration of the
D.sub.3-creatine.
12. The method of claim 1, wherein the D.sub.3-creatine is
administered to the subject such that the spillage of
D.sub.3-creatine into the urine is minimized, wherein greater than
99% of the administered D.sub.3-creatine dilutes in the total body
skeletal muscle pool of creatine and reaches isotopic steady-state
in the total body skeletal muscle pool of creatine.
13. The method of claim 1, wherein the creatine concentration in
skeletal muscle is 4.3 g/kg.
14. The method of claim 1, wherein the biological sample is
selected from the group consisting of a blood sample, a serum
sample, and a plasma sample
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/098,217, filed Apr. 13. 2016, which is a
continuation of U.S. patent application Ser. No. 14/363,779,
Internationally filed Dec. 6, 2012, now abandoned, which is a U.S.
National Phase Patent Application under 35 U.S.C. .sctn.371 based
on international Application No. PCT/US2012/068068, filed Dec. 6,
2012, which claims the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Application No. 61/567,952, filed Dec. 7, 2011, and
U.S. Provisional Application No. 61/708,013, filed Sep. 30, 2012,
the disclosures of which are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for determining the total
body pool size of creatine and total body skeletal muscle mass in a
subject by the use of an orally administered tracer dose of
D3-creatine, and encompasses improved methods for determining the
concentration of creatinine in a biological sample.
BACKGROUND OF THE INVENTION
[0003] Skeletal muscle plays a central role in metabolic
adaptations to increasing and decreasing physical activity, in
disease (e.g. cachexia), in obesity, and in aging (e.g.
sarcopenia). Sarcopenia is described as the age-associated loss of
skeletal muscle (Evans (1995) J. Gerontal. 50A:5-8) and has been
associated with mobility disability (Janssen and Ross, (2005) J.
Nutr. Health Aging 9:408-19) and greatly increased healthcare costs
for elderly people (Janssen et al. (2004) J. Am. Geriatr. Soc.
52:80-5). Loss of skeletal muscle with advancing age is associated
with decreased energy requirements and concomitant increase in body
fatness, weakness and disability, insulin resistance and risk of
diabetes. Loss of skeletal muscle associated with an underlying
illness (cachexia) is associated with a greatly increased mortality
(Evans (2008) Clin. Nutr. 27:793-9).
[0004] Because of the important role total body skeletal muscle
mass plays in aging and disease, there is an effort in the
pharmaceutical arts to identify therapeutic agents that will
stimulate muscle protein synthesis and increase muscle mass.
However, current methodologies for quantification of muscle
synthesis and muscle mass often involve invasive procedures (e.g.
muscle biopsies) or rely on expensive equipment (i.e. DEXA, MRI, or
CT) that provides only indirect data on whole body muscle mass.
Because of these limitations, no method is routinely used in the
clinic for estimation of skeletal muscle mass, and no diagnostic
criteria for estimates of muscle mass have been produced. As a
result, there is a no straightforward way to determine the effects
of potential therapeutic agents on muscle protein synthesis
mass.
[0005] Accordingly, there remains a need in the art for reliable,
easily-performed, noninvasive measurements of total body skeletal
muscle mass.
BRIEF SUMMARY OF INVENTION
[0006] The present invention is based on the finding that
steady-state enrichment of D3-creatinine in a urine sample
following oral administration of a single defined tracer dose of
D3-creatine can be used to calculate total-body creatine pool size
and skeletal muscle mass in a subject.
[0007] The invention is further based on the finding that the
concentration of creatinine in a biological sample can he
determined by measuring the concentration of creatinine M+2 isotope
and dividing this concentration by a dilution factor, where the
dilution factor is the ratio of the concentration of creatinine M+2
to the concentration of creatinine M+0 in the biological sample.
Determining the creatinine concentration in a biological sample
according to these improved methods allows for the simultaneous
measurement of the concentration of creatinine and D3-creatinine in
a single sample using widely-available instrumentation.
Accordingly, this improved detection method will facilitate the
wide-spread adaptation of the present methods for use in
determining skeletal muscle mass in patients.
[0008] Accordingly, in one aspect the invention provides a method
for determining the total body skeletal muscle mass in a subject,
where the method comprises the steps of:
[0009] (a) orally administering 10-200 mg D3-creatine or a salt or
hydrate thereof to the subject;
[0010] (b) allowing at least 12 hours to elapse after the
administration of the D3-creatine;
[0011] (c) obtaining a biological sample from the subject,
[0012] (d) determining the concentration of creatinine and
D3-creatinine in said biological sample;
[0013] (e) using the creatinine and D3-creatinine concentrations
determined in step
[0014] (f) to calculate the total body skeletal muscle mass of the
subject.
[0015] In particular embodiments, the biological sample is a urine
sample.
[0016] In certain embodiments, the concentration of creatinine and
D3-creatinine in the urine sample is determined by HPLC/MS/MS.
[0017] In another aspect, the invention provides a method of
determining the concentration of creatinine in a biological sample
from a subject, said method comprising the steps of
[0018] (a) obtaining a biological sample from the subject;
[0019] (b) analyzing the biological sample to determine the peak
area of the creatinine M+2 isotope peak for the biological
sample;
[0020] (c) comparing the peak area determined in step (b) to a
calibration curve generated using D3-creatinine to determine the
concentration of the creatinine M+2 isotope in the biological
sample;
[0021] (d) dividing the concentration obtained in step (c) by a
dilution factor, where the dilution factor is the ratio of the
concentration of creatinine M+2 to the concentration of creatinine
M+0 in the biological sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. Urinary D3-creatinine enrichment and total body
creatine pool size in growing rats. (A) Urinary D3-creatinine
enrichment (determined by isotope ratio mass spectrometry) in 9
week-old (mean body weight 304.+-.11 g, n=10) and 17 week-old (mean
body weight 553.+-.39 g, n=10) rats at the indicated time after a
single oral 0.475mg dose of D3-creatine, showing achievement of
isotopic steady state by 48 h, and clear separation of growing rat
age groups (P<0.001 between groups at all times; within groups,
the difference between 48 and 72 h is not significant; 2-factor
ANOVA and Student's t test). (B) Creatine pool size calculated from
72 h urinary D3-creatinine enrichments for the rat groups in FIG.
1, showing clear separation of age groups (p<0.0001).
[0023] FIG. 2. Correlation between Lean Body Mass by Quantitative
Magnetic Resonance and total body creatine pool size, adjusted for
age effect, for the rat groups in FIG. 1 (r.sub.all rats=0.69;
P<0.001).
[0024] FIG. 3. Even within the rat groups of different age from
FIG. 1, there is a significant correlation of creatine pool size
and lean body mass by either quantitative magnetic resonance (left)
or DEXA (right).
[0025] FIG. 4. Significant correlation between lean body mass
determined by quantitative magnetic resonance and creatine pool
size determined by D3-creatine dilution in 22 week-old rats (n=10
per group) treated the previous two weeks with either vehicle or
dexamethasone (P<0.001 and P=0.01, respectively).
[0026] FIG. 5. Correlation between lean body mass determined by
quantitative magnetic resonance and total-body creatine pool size
determined by D3-creatine dilution for all 40 rats used in the two
cross-sectional studies (y=0.20x+91.6; r=0.9517; P<0.0001).
[0027] FIG. 6. This figure shows a flow chart for one embodiment of
the method of determining total body skeletal mass.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention is based on the finding that
enrichment of D3-creatinine in a urine sample following oral
administration of a single defined dose of D3-creatine can be used
to calculate total-body creatine pool size and skeletal muscle mass
in a subject. Accordingly, the invention provides a non-invasive,
accurate method of determining total body skeletal muscle. The
methods of the invention find use, inter alia, in diagnosing and
monitoring medical conditions associated with changes in total body
skeletal muscle mass, and in screening potential therapeutic agents
to determine their effects on muscle mass.
[0029] According to the method, D3-creatine is orally administered
to a subject. Although the present is not limited by mechanism, it
is believed that the D3-creatine is rapidly absorbed, distributed,
and actively transported into skeletal muscle, where it is diluted
in the skeletal muscle pool of creatine. Skeletal muscle contains
the vast majority (>than 98%) of total-body creatine. In muscle
tissue, creatine is converted to creatinine by an irreversible,
non-enzymatic reaction at a stable rate of about 1.7% per day. This
creatinine is a stable metabolite that rapidly diffuses from
muscle, is not a substrate for the creatine transporter and cannot
be transported back into muscle, and is excreted in urine. As a
result, once an isotopic steady-state is reached, the enrichment of
a D3-creatinine in spot urine sample after a defined oral tracer
dose of a D3 creatine reflects muscle creatine enrichment and can
be used to directly determine creatine pool size. Skeletal muscle
mass can then be calculated based on known muscle creatine
content.
[0030] Accordingly, in one aspect the invention provides a method
of determining the total body skeletal muscle mass in a subject,
where the method comprises the steps of: [0031] (a) orally
administering 10-200 mg D3-creatine or a salt or hydrate thereof to
the subject; [0032] (b) allowing at least 12 hours to elapse after
the administration of the D3-creatine; [0033] (c) obtaining a urine
sample from the subject, [0034] (d) determining the concentration
of creatinine arid D3-creatinine in said urine sample; [0035] (e)
using the creatinine and D3-creatinine concentrations determined in
step [0036] (f) to calculate the total body skeletal muscle mass of
the subject.
[0037] In certain embodiments, a hydrate of D3-creatine is
administered to the subject. In particular embodiments, D3-creatine
monohydrate is administered.
[0038] The dose of D3-creatine to be administered to the subject is
preferably selected such that the labeled creatine is rapidly
absorbed into the bloodstream and spillage of excess label into the
urine is minimized. Accordingly, for a human subject the dose of
D3-creatine is typically 5-250 mgs, such as 20-125 mgs. In
particular embodiments, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or
100 mgs of D3-creatine is administered. In some embodiments, the
dose is adjusted based on the gender of the subject. Thus, in
certain embodiments, the subject is female and 10-50, such as
20-40, or more particularly, 30 mg of D3-creatine is administered
to the subject. In other embodiments, the subject is male and 40-80
mg, such as 50-70, or more particularly, 60 mg or 70 mg of
D3-creatine is administered to the subject.
[0039] Pharmaceutical formulations adapted for oral administration
may be presented as discrete units such as capsules or tablets;
powders or granules; solutions or suspensions, each with aqueous or
non-aqueous liquids; edible foams or whips; or oil-in-water liquid
emulsions or water-in-oil liquid emulsions. For instance, for oral
administration in the form of a tablet or capsule, the active drug
component may be combined with an oral, non-toxic pharmaceutically
acceptable inert carrier such as ethanol, glycerol, water, and the
like. Generally, powders are prepared by comminuting the compound
to a suitable fine size and mixing with an appropriate
pharmaceutical carrier such as an edible carbohydrate, as, for
example, starch or mannitol. Flavorings, preservatives, dispersing
agents, and coloring agents may also be present.
[0040] Capsules can be made by preparing a powder, liquid, or
suspension mixture and encapsulating with gelatin or some other
appropriate shell material. Glidants and lubricants such as
colloidal silica, talc, magnesium stearate, calcium stearate, or
solid polyethylene glycol may be added to the mixture before the
encapsulation. A disintegrating or solubilizing agent such as
agar-agar, calcium carbonate or sodium carbonate may also be added
to improve the availability of the medicament when the capsule is
ingested. Moreover, when desired or necessary, suitable binders,
lubricants, disintegrating agents, and coloring agents may also be
incorporated into the mixture. Examples of suitable binders include
starch, gelatin, natural sugars such as glucose or beta-lactose,
corn sweeteners, natural and synthetic gums such as acacia,
tragacanth, or sodium alginate, carboxymethylcellulose,
polyethylene glycol, waxes, and the like. Lubricants useful in
these dosage forms include, for example, sodium oleate, sodium
stearate, magnesium stearate, sodium benzoate, sodium acetate,
sodium chloride, and the like. Disintegrators include, without
limitation, starch, methyl cellulose, agar, bentonite, xanthan gum,
and the like.
[0041] Tablets can be formulated, for example, by preparing a
powder mixture, granulating or slugging, adding a lubricant and
disintegrant, and pressing into tablets. A powder mixture may be
prepared by mixing the compound, suitably comminuted, with a
diluent or base as described above. Optional ingredients include
binders such as carboxyrnethylcellulose, aliginates, gelatins, or
polyvinyl pyrrolidone, solution retardants such as paraffin,
resorption accelerators such as a quaternary salt, and/or
absorption agents such as bentonite, kaolin, or dicalcium
phosphate. The powder mixture may be wet-granulated with a binder
such as syrup, starch paste, acadia mucilage or solutions of
cellulosic or polymeric materials, and forcing through a screen. As
an alternative to granulating, the powder mixture may be run
through the tablet machine and the result is imperfectly formed
slugs broken into granules. The granules may be lubricated to
prevent sticking to the tablet forming dies by means of the
addition of stearic acid, a stearate salt, talc or mineral oil. The
lubricated mixture is then compressed into tablets. The compounds
of the present invention may also be combined with a free flowing
inert carrier and compressed into tablets directly without going
through the granulating or slugging steps. A clear or opaque
protective coating consisting of a sealing coat of shellac, a
coating of sugar or polymeric material, and a polish coating of wax
may be provided. Dyestuffs may be added to these coatings to
distinguish different unit dosages.
[0042] Oral fluids such as solutions, syrups, and elixirs may be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups may be prepared, for
example, by dissolving the compound in a suitably flavored aqueous
solution, while elixirs are prepared through the use of a non-toxic
alcoholic vehicle. Suspensions may be formulated generally by
dispersing the compound in a non-toxic vehicle. Solubilizers and
emulsifiers such as ethoxylated isostearyl alcohols and polyoxy
ethylene sorbitol ethers may be added. Solubilizers that may be
used according to the present invention include Cremophor EL,
vitamin E, PEG, and Solutol. Preservatives and/or flavor additives
such as peppermint oil, or natural sweeteners, saccharin, or other
artificial sweeteners; and the like may also be added.
[0043] According to the method, the urine sample in preferably
collected after enrichment levels of D3-creatinine in the urine
have reached a steady-state. Thus in one embodiment, at least 6
hours or at least 12 hours is allowed to elapse after the
administration of the D3-creatine but prior to the collection of
the urine sample. In certain embodiments, at least 24 hours is
allowed to elapse. In particular embodiments, at least 36 hours, at
least 48 hours, at least 60 hours, or at least 72 hours are allowed
to elapse after the administration of the D3-creatine and before
the collection of the urine sample.
[0044] The invention also encompasses certain improved analytic
methods for detecting creatinine and D3-creatinine in urine
samples. Specifically, the invention provides for the detection of
creatinine and D3-creatinine in urine samples by HPLC/MS,
particularly HPLC/MS/MS. However, alternate methods know in the art
may also be used to detect creatinine and/or D3 creatinine in urine
samples. Such methods include direct or indirect calorimetric
measurements, the Jaffemethod, enzymatic degradation analysis, or
derivatization of the creatinine followed by GC/MS analysis of HPLC
with fluorescence detection.
[0045] Thus in one aspect, the invention provides a method of
determining the concentration of creatinine in a biological sample
from a subject, said method comprising the steps of:
[0046] (a) obtaining a biological sample from the subject;
[0047] (b) analyzing the biological sample to determine the peak
area of the creatinine M+2 isotope peak for the biological
sample;
[0048] (c) comparing the peak area determined in step (b) to a
calibration curve generated using D3-creatinine to determine the
concentration of the creatinine M+2 isotope in the biological
sample;
[0049] (d) dividing the concentration obtained in step (c) by a
dilution factor, where the dilution factor is the ratio of the
concentration of creatinine M+2 to the concentration of creatinine
M+0 in the biological sample.
[0050] The biological sample may be any appropriate sample
including, but not limited to, urine, blood, serum, plasma, or
tissue. In one particular embodiment, the biological sample is a
urine sample. In another particular embodiment, the biological
sample is a blood sample.
[0051] In a preferred embodiment, the peak area of the creatinine
M+2 isotope peak is determined using liquid chromatography/mass
spectroscopy (LC/MS/MS).
[0052] In one embodiment, the dilution factor is
0.0002142.+-.0.0000214. More particularly, the dilution factor is
0.0002142.+-.0.00001, such as 0.0002142.+-.0.000005.
[0053] The methods of the invention are useful for diagnosing and
monitoring medical conditions associated with changes in total body
skeletal muscle mass. Examples of medical conditions in which loss
of muscle mass plays an important role in function, performance
status, or survival include, but are not limited to frailty and
sarcopenia in the elderly; cachexia (e.g., associated with cancer,
chronic obstructive pulmonary disease (COPD), heart failure,
HIV-infection, tuberculosis, end stage renal disease (ESRD); muscle
wasting associated with HIV therapy, disorders involving mobility
disability (e.g., arthritis, chronic lung disease); neuromuscular
diseases (e.g., stroke, amyotrophic lateral sclerosis);
rehabilitation after trauma, surgery (including hip-replacement
surgery), medical illnesses or other conditions requiring bed-rest;
recovery from catabolic illnesses such as infectious or neoplastic
conditions; metabolic or hormonal disorders (e.g., diabetes
mellitus, hypogonadal states, thyroid disease); response to
medications (e.g., glucocorticoids, thyroid hormone); malnutrition
or voluntary weight loss. The claimed methods are also useful in
sports-related assessments of total body skeletal muscle mass.
[0054] The methods of the invention are also useful for screening
test compounds to identify therapeutic compounds that increase
total body skeletal muscle mass. According to this embodiment, the
total body skeletal mass of a subject is measured according to the
method before and after a test compound is administered to the
subject. The assessment of total body skeletal muscle mass can be
repeated at appropriate intervals to monitor the effect of the test
compound on total body skeletal muscle mass.
EXPERIMENTAL
Use of the D3-Creatine Tracer Dilution Method to Determine Total
Body Skeletal Muscle Mass in a Pre-Clinical Model
[0055] A dose of 0.475 mg D3-creatine per rat was determined to be
rapidly and completely absorbed and reach the systemic circulation
with minimal urinary spillage, such that >99% of the D3-creatine
tracer dose should be available to equilibrate with the body
creatine pool.
[0056] The creatine dilution method was then used to determine
urinary D3-creatine enrichment and the time to isotopic steady
state in growing rats. In a cross-sectional study, a single oral
dose of 0.475 mg D3-creatine per rat was given to two groups of
rats, 9 and 17 weeks of age, and urine was collected at 24, 48, and
72 hour time points after dosing. As expected, the larger, older
rats had lower urinary D3-creatinine enrichment (expressed as mole
percent excess, MPE) at all time points than the younger, smaller
rats, reflecting greater dilution of the D3-creatine tracer in the
total body creatine pool. For both age groups, urinary enrichment
was highest at 24 h and stable between 48 and 72 h, indicating
isotopic steady state was achieved between 24 and 48 h after the
tracer D3-creatine dose. (FIG. 1A).
[0057] Total body creatine pool size was then calculated using a
formula for determination of pool size based on enrichment of a
tracer, assuming a single creatine pool (Wolfe and Chinkes (2005)
Calculation of substrate kinetics: Single-pool model. 2nd ed.
Isotope tracers in metabolic research. Hoboken, N.J.: John Wiley
& Sons, Inc. 21-9): the D3-creatine dose (0.475 mg) was divided
by the D3-creatinine enrichment (MPE/100). FIG. 1B shows the total
body creatine pool sizes calculated from urinary enrichment 72 h
after the tracer dose for the 9 and 17 week-old rat groups and
indicates the creatine pool size for the larger, older rats is
significantly larger than for the smaller, younger rats.
[0058] The day before giving the tracer dose of D3-creatine, lean
body mass (LBM) in all rats was assessed by either quantitative
magnetic resonance (QMR) or DEXA. FIG. 2 shows that after
accounting for age effect, LBM by QMR and creatine pool size are
significantly correlated. LBM by QMR and creatine pool size are
also significantly correlated within each age group, and LBM by
DEXA and creatine pool size are significantly correlated within the
17 week-old age group (FIG. 3).
[0059] In a second cross-sectional study, an older rat age group
(still within the rat growth phase of 22 weeks of age) was treated
once daily subcutaneously with either saline vehicle, or
dexamethasone to induce skeletal muscle atrophy for 2 weeks prior
the administration of D3-creatine. As with the first
cross-sectional study with 9 and 17 week-old rats, isotopic steady
state was reached between 48 and 72 h.
[0060] Compared to vehicle-treated controls, dexamethasone induced
a significant reduction in LBM (353.+-.32 vs. 459.+-.45 g,
P<0.001) and a significant reduction in total body creatine pool
size (1216.+-.227 vs. 1853.+-.228 mg, P<0.001). As in the first
study, LBM and creatine pool size were significantly correlated
within the two individual treatment groups (FIG. 4).
[0061] FIG. 5 show the correlation between LBM and creatine pool
size for all 40 rats used in the two cross-sectional studies
(r=0.95; P<0.001).
Use of the D3-Creatine Tracer Dilution Method to Determine Total
Body Skeletal Muscle Mass in Human Subjects
[0062] Human subjects are orally administered a single dose of 30,
60, or 100 rugs of D3 creatine-monohydrate. Urine samples are then
collected 1, 2, 3, 4, 5,or 6 days after administration of the
D3-creatine monohydrate dose.
[0063] Urine pharmacokinetic analyses for each collection interval
may include quantitation of MPE ratio by IRMS, ratio of
deuterium-labeled creatine+deuterium-labeled creatinine to total
creatine+total creatinine by LCMS, total creatinine, creatine pool
size, and % of deuterium-labeled creatine dose excreted in
urine.
[0064] Steady-state enrichment (MPE) can be assessed both visually
and from the estimate of the slope from the linear regression of
enrichment (MPE) vs time (midpoint of each urine collection
interval). A mixed effect ANOVA model can be fit with time
(continuous variable) as a fixed effect and subject as a random
effect. The coefficient for the slope of the time effect can be
used to evaluate steady-state. The 90% confidence intervals for the
slope can be calculated.
[0065] Creatine pool size can be estimated once steady-state
enrichment has been achieved a for each collection interval during
steady-state according to the formula:
[Amount of D3 Cr dosed(g)-total Amount of urinary D3
Cr(0-t)(g)]/enrichment ratio(t) where t is the urine collection
interval during steady-state.
[0066] Muscle mass can be estimated from the creatine pool size by
assuming that the creatine concentration is 4.3 g/kg of whole wet
muscle mass (WWM) (Kreisberg (1970) J Appl Physiol 28:264-7).
Muscle mass=creatine pool size/Cr concentration in muscle
[0067] Creatine pool size can also be estimated by total urine
creatinine (moles/day) divided by K (1/day).
[0068] The excretion rate constant (K) can be estimated using a
rate excretion method by estimating the declining slope of the line
for the log of the amount of D3-creatine in urine collection
interval vs. time (midpoint of that urine collection interval) for
each collection interval over time. This estimate of K can be used
in calculating creatine pool size from 24 hr urinary creatinine
excretion rather than using an estimate of turnover form the
literature.
Analytic Methods for Quantitating D3-Creatine and D3-Creatinine in
Urine Samples From Clinical Subjects
[0069] Reference Standards of D3-Creatine monohydrate and
D3-creatinine were purchased from C/D/N Isotopes, Montreal
Canada.
HPLC-MS/MS Analysis
[0070] The separation of D3-creatine was carried out using an
Acquity UPLC (Waters Corp., Milford, Mass.) equipped with a Zorbax
Hilic Plus silica analytical column (50.times.2.1 mm, Rapid
Resolution HD 1.8 .mu., Agilent Corp., Santa Clara Calif.).
Injection volume is typically 8 .mu.L.
[0071] Mobile phase A (MP A) consisted of 10 mM ammonium formate in
water and mobile phase B is acetonitrile. Gradient chromatography
was employed with initial mobile phase composition of 2% 10 mM
ammonium formate with a flow rate of 0.7 mL/min. This was held for
0.5 minute and then a linear gradient to 50% MPA was achieved at
2.3 minutes. This was immediately increased to 80% and held for 0.4
minutes and then returned to starting conditions at 2.9 minutes.
The total run time was 3.5 minutes. This gradient allowed baseline
separation of the D3-creatine from interfering compounds.
[0072] The detection of D3-creatine was carried out using a Sciex
API5000 (Applied Biosystems, Foster City, Calif.). The HPLC system
was connected to the API5000 through a turbo ion spray source
operating in positive ionization mode using the following
parameters: ionization temperature of 650.degree. C., ionspray
voltage of 2500 V, curtain gas setting of 45 (N.sub.2), nebulizer
gas setting was 65 (N.sub.2), drying gas setting was 70 (N.sub.2),
collision gas setting of 3 (N.sub.2). All other mass spectrometer
parameters were optimized for the individual transitions. The
following ion transitions (MRM) were acquired: D3-creatine is
m/z=135 to m/z=47 with a typical retention time of 1.99 min. The
creatine standard is monitored with an ion transition of m/z=139 to
m/z=50 with a typical retention time of 1.99 min.
[0073] The separation of the creatinine and D3-creatinine analytes
were carried out using an Acquity FPLC (Waters Corp., Milford,
Mass.) equipped with a Zorbax Hilic Plus silica analytical column ,
dimensions of 50.times.2.1 mm (Rapid Resolution HD 1.8 .mu.,
Agilent Corp., Santa Clara Calif.). Injection volume was typically
5 .mu.L.
[0074] Mobile phase A consisted of 5 mM ammonium formate and mobile
phase B was acetonitrile. Gradient chromatography was employed with
initial mobile phase composition of 2% 5 mM ammonium formate with a
flow rate of 0.7 mL/min. This was held for 0.4 minute and then a
linear gradient to 40% MPA was achieved at 2.1 minutes. This was
immediately increased to 50% at 2.2 minutes and held for 0.4
minutes and then returned to starting conditions at 2.7 minutes.
The total run time was 3.5 minutes. This gradient allowed baseline
separation of the d3-creatinine and creatinine from interfering
compounds.
[0075] The detection of the creatinine and D3-creatinine analytes
was carried out using a Sciex API5000 (Applied Biosystems, Foster
City, Calif.). The HPLC system was connected to the API5000 through
a turbo ion spray source operating in positive ionization mode
using the following parameters: ionization temperature of
350.degree. C., ionspray voltage of 5500 V, curtain gas setting of
45 (N.sub.2), nebulizer gas setting was 60 (N.sub.2), drying gas
setting was 65 (N.sub.2), collision gas setting of 3 (N.sub.2). All
other mass spectrometer parameters were optimized for the
individual transitions. The following ion transitions (MRM) were
acquired: D3-creatinine is m/z=117 to m/z=47 and for creatinine
(M+2 isotope) was m/z=116 to m/z=44 with a typical retention time
of 1.5 min. The creatine standard is monitored with an ion
transition of m/z=121 to m/z=51 with a typical retention time of
1.5 min. For creatinine, the M+2 isotope version was acquired to
avoid diluting the sample with buffer.
[0076] Endogenous creatinine concentration values are determined in
human urine clinical samples using a D3-creatinine calibration
standard curve. The D3-creatinine isotope behaves similarly to
creatinine throughout the extraction and HPLC-MS/MS procedures,
thus allowing clean urine matrix to prepare standards and QC
samples.
[0077] The amount of endogenous creatinine (m/z=114) in the human
clinical samples is much greater (.about.1000 fold) than the levels
of D3-creatinine. Therefore, instead of diluting the sample, the
M+2 isotope of creatinine (m/z=116) will be monitored, thus
allowing the simultaneous measurement of creatinine and
D3-creatinine from one sample analysis. The MRM of (M+2) endogenous
creatinine (116/44) is monitored. A correction factor that
represents the ratio of the MRM of 116/44 to 114/44, is used to
correct the calculated concentrations determined from the
d3-creatinine calibration curve. The isotope ratio (M+2) MRM/(M+0)
MRM or correction factor is 0.00286. Therefore, the amount of
D3-creatinine, which would come from the D3 creatine dose and the
endogenous creatinine, can be quantitated from the single
D3-creatinine calibration curve.
EXAMPLE
[0078] Chemical and Reagents: Acetonitrile and Water (all HPLC
grade or better) purchased from Sigma Aldrich (St. Louis, Mo.).
Ammonium Formate purchased from Sigma Aldrich (St. Louis, Mo.).
Reference Standards of d3-Creatine (monohydrate) and d3-creatinine
were purchased from CDN Isotopes, Montreal Canada.
[0079] Stock solutions of d3-creatine and d3-creatinine are
prepared at 1.0 mg/mL in water and confirmation of equivalence is
performed. Dilute solutions ranging from 0.1 .mu.g/mL to 100
.mu.g/mL and 0.2 .mu.g/mL to 200 .mu.g/mL are prepared in water and
used to prepare calibration standards and quality control (QC)
samples in human urine for d3-creatine and d3-creatinine,
respectively. Isotopically labelled internal standards for creatine
(SIL) .sup.13C.sub.3.sup.2H.sub.3 .sup.15N.sub.1creatine) and
creatinine (SIL) (.sup.13C.sub.3.sup.2H.sub.4 .sup.15
N.sub.1-creatinine) are prepared at 1.0 mg/mL in water. Dilute
solutions of these are prepared at 500 ng/mL in acetonitrile and
used as an extraction solvent for the urine standards, quality
controls and study samples.
[0080] Sample Preparation: (d3-creatine, creatinine and
d3-creatinine in urine) A 200 .mu.L aliquot of the internal
standard working solution (500 ng/mL) in acetonitrile is added to
each well, except double blank samples, acetonitrile is added. A 40
.mu.L aliquot of sample, standard or QC is transferred to the
appropriate wells in the plate containing the SIL. The plate is
sealed and vortex mixed for approximately 3 minutes. The plate is
centrifuged at approximately 3000 g for 5 minutes. Supernatant is
transferred to a clean 96 well plate and then injected onto the
HPLC-MS/MS system for analysis. D3-creatine and d3-creatinine are
analyzed from separate human urine samples.
HPLC-MS/MS Analysis
[0081] The separation of d3-creatine, d3-creatinine and creatinine
is carried out using an Acquity UPLC (Waters Corp., Milford, Mass.)
equipped with a Agilent Zorbax Hilic Plus silica analytical column
, dimensions of 50.times.2.1 mm (Rapid Resolution HD 1.8.mu.,
Agilent Corp., Santa Clara Calif.). Injection volume is typically 2
.mu.L.
[0082] D3-creatine: mobile phase A consists of 10 mM ammonium
formate and mobile phase B is acetonitrile. Gradient chromatography
is employed with initial mobile phase composition at 2% 10 mM
ammonium formate with a flow rate of 0.7 mL/min. This is held for
0.5 minute and then a linear gradient to 50% MPA is achieved at 2.3
minutes. This is increased to 80% over 0.2 minutes and held for 0.4
minutes and then returned to starting conditions at 3.0 minutes.
The total run time is 3.5 minutes.
[0083] The detection of d3-creatine is carried out using a Sciex
API5000 (Applied Biosystems, Foster City, Calif.). The HPLC system
is connected to the API5000 through a turbo ion spray source
operating in positive ionization mode using the following
parameters: ionization temperature of 650.degree. C., ionspray
voltage of 2500 V, curtain gas setting of 45 (N.sub.2), nebulizer
gas setting is 65 (N.sub.2), drying gas setting is 70 (N.sub.2),
collision gas setting of 3 (N.sub.2). All other mass spectrometer
parameters are optimized for the individual transitions. The
following ion transitions (MRM) are acquired: d3-creatine is
m/z=135 to m/z=47 with a typical retention time of 2 min. The SIL
is monitored with an ion transition of m/z=139 to m/z=50 with a
typical retention time of 2 min.
[0084] D3-creatinine: mobile phase A consisted of 5 mM ammonium
formate, and mobile phase B is acetonitrile. Gradient
chromatography is employed with initial mobile phase composition at
2% 5 mM ammonium formate with a flow rate of 0.7 mL/min. This is
held for 0.4 minute and then a linear gradient to 60% acetonitrile
is achieved at 2.1 minutes. This is immediately increased to 50%
acetonitrile and held for 0.4 minutes and then returned to starting
conditions at 2.7 minutes. The total run time is 3.5 minutes.
[0085] The detection of the creatinine and d3-creatinine analytes
is carried out using a Sciex API5000 (Applied Biosystems, Foster
City, Calif.). The HPLC system was connected to the API5000 through
a turbo ion spray source operating in positive ionization mode
using the following parameters: ionization temperature of
350.degree. C., ionspray voltage of 5500 V, curtain gas setting of
45 (N.sub.2), nebulizer gas setting was 60 (N.sub.2), drying gas
setting was 65 (N.sub.2), collision gas setting of 3 (N.sub.2). All
other mass spectrometer parameters are optimized for the individual
transitions. The following ion transitions (MRM) are acquired:
d3-creatinine is m/z=117 to m/z=47 and for creatinine (M+2 isotope)
is m/z=116 to m/z=44 with a typical retention time of 1.5 min. The
SIL is monitored with an ion transition of m/z=121 to m/z=51 with a
typical retention time of 1.5 min. For creatinine, the M+2 isotope
MRM is acquired to avoid diluting the sample with a surrogate
matrix (a creatinine free control urine is not available). These
isotopes will behave similarly throughout the extraction and
HPLC-MS/MS procedures, thus allowing clean urine matrix to prepare
standards and QC samples as well as allowing for the quantification
of endogenous creatinine using a calibration curve that was
generated from the deuterated form of creatinine. Therefore, the
amount of d3-creatinine and the endogenous creatinine, can be
quantitated from the single d3-creatinine calibration curve.
[0086] HPLC-MS/MS data were acquired and processed (integrated)
using Analyst.TM. software (Version 1.4.2, MDS Sciex, Canada). A
calibration plot of area ratio versus d3-creatinine concentration
was constructed and a weighted 1/x.sup.2 linear regression applied
to the data.
RESULTS
[0087] To perform bioanalytical quantification of biomarkers using
LC/MS/MS, a surrogate matrix or a surrogate analyte must be used.
In this assay, human urine can be used since d3-creatinine is not
found endogenously and the quantification of creatinine can be
determined from the d3-creatinine calibration curve. The
equivalency of d3-creatinine and creatinine is shown.
D3-Creatinine and Creatinine Equivalence Determination
[0088] A number of experiments were performed in order to verify
that d3 creatinine can be used as a surrogate analyte to quantitate
creatinine and that the MRM transition of 116/44 (M+2) can be used
with the isotope ratio correction factor.
[0089] To confirm that d3 creatinine can be used as a surrogate
analyte for creatinine; two concentration levels of creatinine and
d3 creatinine neat standard solutions were prepared to show
equivalent LC-MS/MS response. The peak areas of 200 ng/mL and 40
ng/mL of both creatinine and d3 creatinine standard solutions were
compared using the MRM transitions of 114/44 and 117/47,
respectively. The results showed that the two solutions gave
equivalent responses with mean percent difference and percent CV of
less than 7.5%. See Table 1.
TABLE-US-00001 TABLE 1 D3 creatinine and creatinine equivalence
using LC/MS/MS CRN vs Percent Std Creatinine d3 CRN of D3 (ng/mL)
(MRM of 114/44) % difference Response d3-Creatinine (MRM of 117/44)
40 791648 717010 10.4 90.6 40 804513 780182 3.1 97.0 40 774228
717528 7.9 92.7 40 776144 823064 -5.7 106.0 40 766927 828642 -7.4
108.0 40 741290 758937 -2.3 102.4 Mean 1.0 99.4 % CV 7.2
d3-Creatinine (MRM of 117/47) 200 3296195 3336107 -1.2 101.2 200
3469440 3325274 4.3 95.8 200 3416181 3428709 -0.4 100.4 200 3363696
3185389 5.6 94.7 200 3335259 3390463 -1.6 101.7 200 3255799 3321365
-2.0 102.0 Mean 0.8 99.3 % CV 3.2
[0090] These results show that d3 creatinine and creatinine give
equivalent LC/MS/MS responses and d3-creatinine can be used as a
surrogate analyte for creatinine. This is not surprising since
deuterated compounds are used routinely as stable label internal
standards, in regulated environments to validate assays. These
deuterated standards have been shown to correct LC/MS/MS response
of analyte from matrix effects as well as other extraction and
chromatographic related effects. Since the only difference is an
extra proton at three hydrogen atoms on the methyl group, we would
expect the two compounds to behave almost identically throughout
the extraction, chromatographic separation and mass spectral
detection.
Determination of Isotope Ratio
[0091] This method is used to determine the amount of d3 creatinine
in human urine that has been converted from a dose of d3 creatine.
Additionally, the amount of endogenous creatinine will be
determined using the d3 creatinine standard curve. The amount of
endogenous creatinine is much greater (.about.1000 fold) than the
levels of d3-creatinine in the human clinical urine samples,
therefore instead of diluting the sample, the M+2 isotope of
creatinine will be monitored. This will allow the simultaneous
measurement of creatinine and d3-creatinine from one sample using a
urine matrix calibration curve. The peak area of the MRM of (M+2)
endogenous creatinine (116/44) is monitored along with the d3
creatinine MRM of 117/47. A correction factor that represents the
ratio of the MRM of 116/44 to 114/44, is used to correct the
calculated concentrations determined from the d3-creatinine
calibration curve.
[0092] The isotope ratio (response ratio) or difference in peak
area response from the naturally abundant form of creatinine (M+0)
or m/z=114 to the much less abundant form of creatinine (M+2) or
m/z=116 is calculated experimentally. The isotope ratio is
determined using two different experimental procedures. The
original experimental design uses one standard concentration, a 200
ng/mL creatinine solution (Table 2a).
[0093] The peak area of the creatinine is monitored at both the M+0
and M+2 MRM transitions (114/44 and 116/44), respectively. One
solution was used to reduce variation which may occur from separate
injections and preparation of separate solutions. This
concentration is chosen because it allows the peak area of both
MRMs to be in the detector range, and with adequate signal to noise
for the smaller peak. However, some variability in the day to day
measurements is observed (.+-.10%) as shown in Table 3.
[0094] Therefore, an additional experiment to generate this
response ratio was performed. In the second approach, the response
ratio is experimentally determined using two separate solutions. A
separate solution for each MRM transition is prepared which gives
peak areas that are closer in magnitude to each other. A 10 ng/mL
solution of creatinine is used to acquire the MRM transition of
114/144 and a 500 ng/mL solution is used to acquire the MRM
transition of 116/44. These solutions are injected on the LC/MS/MS
system in replicates of 10 and the mean peak area ratio (PAR) for
each solution is determined. The response ratio is then calculated
by dividing the mean PAR of 116/44 by the corrected PAR of 114/44.
In order to compare the PARs from the two MRMs, the PAR from the 10
ng/mL solutions is multiplied by 50 (since 500 ng/mL is 50 times
larger than the 10 ng/mL), an example is shown in Table 2b. This
allows the peak area of both solutions to be closer in value and
potentially eliminating errors associated with integrating peaks
with vastly different signal to noise values.
TABLE-US-00002 TABLE 2 Creatinine Response Ratio (M + 2/M + 0)
Determination using LC/MS/MS 2a. Determined using a single
creatinine standard solution Peak Area Ratio STD Creatinine (M + 2)
Creatinine (M + 0) Response (ng/mL) MRM of 116/44 MRM of 114/44
Ratio 200 0.0209 9.19 0.00227 200 0.0216 9.42 0.00229 200 0.0195
9.53 0.00205 200 0.0208 9.42 0.00221 200 0.0202 9.37 0.00216 200
0.0199 9.46 0.00210 200 0.0188 9.22 0.00204 200 0.0201 9.64 0.00209
200 0.0202 9.33 0.00217 200 0.0188 9.49 0.00198 200 0.0200 9.45
0.00212 200 0.0198 9.32 0.00212 Mean 0.0201 9.4 0.00213 % CV 4.05
1.35 3.10 2b. Determined using separate creatinine concentrations
Peak Area Ratio Creatinine (M + 2) Creatinine Creatinine MRM of (M
+ 0) (M + 0)* Response 116/44 MRM of 114/44 MRM of 114/44 Ratio
0.0460 0.4240 21.2 0.00217 0.0467 0.4240 21.2 0.00220 0.0466 0.3990
20.0 0.00234 0.0471 0.4110 20.6 0.00229 0.0477 0.4000 20.0 0.00239
0.0459 0.3850 19.3 0.00238 0.0453 0.3990 20.0 0.00227 0.0452 0.4000
20.0 0.00226 0.0443 0.3920 19.6 0.00226 0.0442 0.3780 18.9 0.00234
Mean 0.0459 0.4 20.1 0.00229 % CV 2.53 3.75 3.75 3.15 * = corrected
for concentration difference
[0095] The corrected peak area ratio would be equivalent to a 500
ng/mL creatinine standard monitoring the peak area of the MRM
transition of 114/44.
[0096] The isotope ratio (response ratio) was determined on
multiple occasions over a four month time span and on two different
triple quadrapole instruments. The mean of these nine values was
determined and the inverse of this response ratio is the dilution
factor used to correct the creatinine values in the LIMS system.
See Table 3.
TABLE-US-00003 TABLE 3 Summary of Response Ratio (M + 2/M + 0)
Determined using LC/MS/MS Date Response Ratio Instrument Name
22-Nov-11 0.00206 RTP12 29-Nov-11 0.00213 RTP12 AM 7-Dec-11 0.00193
RTP12 PM 7-Dec-11 0.00193 RTP12 * 14-Jan-12 0.00221 RTP12 *
16-Jan-12 0.00229 RTP12 * 17-Jan-12 0.00195 RTP12 * 7-Feb-12
0.00229 RTP12 * 10-Feb-12 0.00249 RTP52 Mean 0.002142 % CV 9.09 *=
performed using two concentrations of creatinine
[0097] This experimentally determined response ratio is used to
correct peak areas of creatinine M+2 (MRM of 116/44) and these
corrected peak areas of creatinine were compared to peak areas run
for the same concentration of d3 creatinine standard (MRM of
117/47). The comparison of the corrected creatinine peak area to
the peak area obtained from the d3 creatinine standards gave
equivalent responses with percent difference and percent CV of less
than 10%. See Table 4.
TABLE-US-00004 TABLE 4 Creatinine (M + 2) Response Corrected using
Response Ratio CRN CRN M + 2* d3 CRN STD (MRM 116/44) Corrected as
to (MRM 117/47) Percent (ng/mL) M + 2 M + 0 Peak Area Difference
200 7856.2 3667693.7 3599411.5 98.1 200 7422.7 3465312.8 3682013.9
106.3 200 6101.3 2848412.7 3516609.5 123.5 200 7490.2 3496825.4
3330922.4 95.3 200 7288.0 3402427.6 3359823.2 98.7 200 6625.6
3093183.9 3264518.6 105.5 Mean 7130.7 3328976.0 3458883.2 104.6 %
CV 9.0 9.0 4.8 9.8 *= corrected peak area (divided by the mean
response ratio of 0.002142)
* * * * *