934

TH0N4AS ET AL.: JOURNAL OF A O A C INTERNATIONAL VOL. 9 0 , NO. 4 , 2 0 0 7

DIETARY SUPPLEMENTS

Determination of Caffeine and Caffeine-Related Metabolites in

Ephedra-Containing Standard Reference Materials Using Liquid
Chromatography with Absorbance Detection and Tandem Mass
Spectrometry
JEANICE B . THOMAS and KATHERINE E . SHARPLESS

National Institute of Standards and Technology, Analytical Chemistry Division, Gaithersburg, MD 20899-8392
STACI MITVALSKY'

K-Force, 1001 East Palm Ave, Tampa, FL 33605

MARK ROMAN'

Tampa Bay Analytical Research, Inc., 10810 72nd St, Suite 206, Largo, FL 33777
JAMES YEN

National Institute of Standards and Technology, Statistical Engineering Division, Gaithersburg, MD 20899-8980
MARY B . SATTERFIELD

National Institute of Standards and Technology, Biochemical Science Division, Gaithersburg, MD 20899-8392

The concentrations of caffeine and caffeine-related

compounds in 2 ephedra-containing reference
materials have been determined by 3 independent
methods with measurements performed by the
National Institute of Standards and Technology
(NIST) and a collaborating laboratory. Results from
the 3 methods were used for value assignment of
caffeine, theobromine, and theophylline in these
Standard Reference Materials (SRMs). The
methods used at NIST to determine the
concentration levels of caffeine, theobromine, and
theophylline in SRM 3243 Ephedra-Containing
Solid Oral Dosage Form and SRM 3244
Ephedra-Containing Protein Powder used
reversed-phase liquid chromatography with
absorbance detection and tandem mass
spectrometry. These reference materials are part of
the first suite in a series of NIST SRMs that provide
concentration values for multiple components In
dietary supplements. These SRMs are primarily
intended for method validation and for use as
control materials to support the analysis of dietary
supplements and similar materials.

Received December 20, 2006. Accepted by AP March 8, 2007.

Corresponding author's e-mail: jbthomas@nist.gov
' Former address: ChromaDex, Inc., Clearwater, FL 33760.
Certain commercial products are identified to specify adequately the
experimental procedure. Such identification does not imply endorsement or
recommendation by the National Institute of Standards and Technology, nor
does it imply that the materials identified are necessarily the best available
for the purpose.

ultiple-component dietary supplements have been

widely consumed in the United States for a number
of purposes, including weight loss and the
enhancement of athletic performance. Adverse health events
related to ephedra-containing products have been reported to
the U.S. Food and Drug Administration (FDA), resulting in a
federal ban in 2004 on dietary supplements that contain
ephedra (1,2). Many ephedra-containing products also
contained caffeine or another added stimulant. Some
commercially available dietary supplements contain a
botanical source of caffeine known as guaran (Paullinia
cupana). Caffeine and its derivatives (such as theobromine
and theophylline) have received attention because they can
cause various physiological effects. Theophylline and
theobromine are often used as smooth muscle relaxants and
can cause diuresis (3). Caffeine is sometimes used as a cardiac
and respiratory stimulant. When used in combination with
other botanical stimulants such as ephedra, increased health
risks may occur (4, 5).

Dietary supplements are regulated as foods under the

Dietary Supplement Health and Education Act of 1994. The
lack of specific requirements for ingredient quality and
content makes it difficult to accurately evaluate the dietary
intake and the resulting biological effects or potential health
risks posed by these types of stimulants on consumers.
In 2001, the National Institute of Standards and
Technology (NIST) began working with the FDA's Center for
Food Safety and Applied Nutrition (CFSAN) and Center for
Drug Evaluation and Research (CDER), and with the National
Institutes of Health's Office of Dietary Supplements
(NIH/ODS) to produce reference materials for dietary
supplements. NIST, working in collaboration with these
agencies, recently produced a suite of 5 ephedra-containing
Standard Reference Materials (SRMs): SRM 3240 Ephedra

THOMAS ET AL. : JOURNAL OF

sinica Stapf Aerial Parts, SRM 3241 Ephedra sinica Stapf

Native Extract, SRM 3242 Ephedra sinica Stapf Commercial
Extract, SRM 3243 Ephedra-Containing Solid Oral Dosage
Form, and SRM 3244 Ephedra-Containing Protein Powder.
These SRMs, consisting of natural, extracted, and processed
materials, represent a range of analytical challenges.
SRM 3240 was prepared from a cultivated crop of . sinica
Stapf that was harvested in 2002 and processed to yield a
dried, powdered material. SRMs 3241 and 3242 are,
respectively, native and commercial extracts prepared from
portions of further-processed material used to prepare
SRM 3240. SRM 3243 was prepared by grinding and
blending commercially available dietary supplements (tablets
and capsules); SRM 3244 was blended similarly using
commercially available protein drink mixes. Details regarding
the acquisition and preparation of the entire suite of materials
are described elsewhere (6-11). This suite of SRMs is the first
in a series of NIST reference materials that provides
concentration values for multiple components in dietary
supplements (12). These materials are intended for use as
primary controls for assigning values to in-house control
materials and for validation of analytical methods for the
measurement of ephedrine alkaloids, synephrine, toxic trace
elements, caffeine, theobromine, theophylline, and nutrients,
as appropriate, in the materials.
One concern about dietary supplements has been the
disagreement between product content and label claim. Haller
et al. (13) analyzed 35 commercial dietary supplements that

Name

Molecular Weight

Caffeine

194.2

AOAC INTERNATIONAL VOL. 90, No. 4,2007 935

contain ephedrine alkaloids and caffeine using liquid

chromatography/tandem mass spectrometry (LC/MS/MS)
and compared the measured concentrations of these analytes
with the amounts listed on the product labels. Of the products
tested, 31% of the products contained >110% of the total
ephedrine alkaloids listed on the label, and 86% of the product
lots that listed the caffeine concentration contained <90% of
the labeled quantity. This type of product inconsistency, which
is often seen in commercially available dietary supplements,
supports the need for well-characterized matrix-based
materials that can be used to assess product consistency and to
assure the quality of dietary supplements.
This paper describes the analysis of 2 of the
ephedra-containing reference materials, SRM 3243
Ephedra-Containing Solid Oral Dosage Form and SRM 3244
Ephedra-Containing Protein Powder, for the content of
caffeine by 2 independent analytical methods developed at
NIST, LC-UV and LC/MS/MS, and by the method (LC-UV)
of one collaborating laboratory. These methods provide rapid
analysis of the compounds of interest with minimal sample
preparation. The determination of theobromine and
theophylline in these materials using the NIST LC-UV
method is also discussed. Analytical methods used by NIST

SRM 3243 Ephedra-Containing Soiid Oral Dosage

C

SRM 3244 Ephedra-Containing Protein Powder

iS

Theobromine

180.2

Theophyiline

180.2
standards

TP
TB

"Cj-caffeine

197.2

T
0.0

-hydroxyethyltheophylline
(incemal standard for NIST
LC/UV method)

224.2

Figure 1. Structures of caffeine, caffeine-related

metabolites, and the internal standards.

'I'
10.0

Minutes

Figure 2. NiST LC-UV anaiyses of SRIVIs 3243 and

3244 using the method described in the text. Peak
identification: theobromine (TB), theophyiline (TP),
internal standard -hydroxyethyltheophylline (IS), and
caffeine (C).

936

THOMAS ET AL, : JOURNAL OF AOAC INTERNATIONAL VOL, 90, No, 4,2007

Table 1. Summary of results (mass fractions in mg/g) for the analysis of ephedra-containing SRIVIs by each of the
3 methods described in the text^
Sample

NIST LC-UV

ChromaDex LC-UV

NIST LC/MS/MS

Final assigned value*

75,23 (3,95)

76,50 4,12"=

2,89(0,19)

2,99 0,54"

SRM 3243
Caffeine

78,85 (2,60)

75,43(1,30)
SRM 3244

Caffeine

3,24 (0,35)

2,84(0,11)

Theobromine

0,762 (0,024)

0,762 0,026

Theophylline

0,080 (0,003)

0,080 0,003

Results shown represent an average of 6 measurements (n = 6) and are reported on a dry-mass basis. Results have been corrected for
purity and moisture content (refs, 6,10,11 ), Two standard deviations of the mean for the caffeine concentration values are indicated in
parentheses. Caffeine data are also plotted in Figures 4 and 5 for graphical comparison.
The NIST process of assigning values to its SRMs for chemical measurements is discussed in the text (refs, 6,10,11, 23), Concentration
values for caffeine in SRMs 3243 and 3244 are assigned as NIST certified values; concentration values for theobromine and theophylline are
assigned as NIST reference values. The uncertainty in the assigned values is expressed as an expanded uncertainty at the 95% level of
confidence (23, 25),
NIST certified value.

and by collaborating laboratories that participated in the value

assignment for ephedrine alkaloids in this suite of SRMs have
been described elsewhere (14-18), The value assignment
process for these SRMs is also described in detail by Sander et
al, (14) and Sharpless et al, (6),
Experimental
Method 1: LC-UV (NIST)
(a) Calibration standards.Four calibration solutions
containing theobromine (CAS No, 83-67-0, Sigma-Aldrich,
St, Louis, MO); theophylline (CAS No, 58-55-9,
Sigma-Aldrich); and caffeine (CAS No, 58-08-2,
Sigma-Aldrich) were prepared in LC grade methanol (Baker
Omnisolv, J,T, Baker, Phillipsburg, NJ), A stock solution (ca
1 mg/g) of -hydroxyethyltheophylline (CAS No, 519-37-9,
Sigma-Aldrich) in LC grade methanol was used as the internal
standard.
The
internal
standard
(-hydroxyethyltheophylline) selected for the NIST LC-UV method was
a close homolog of the analytes of interest and had similar
retention characteristics and extractability from the matrix.
Working calibration solutions were prepared by weighing ca
1:1 (mass fraction) aliquots of stock calibrants and the internal
standard solution. The purity (mass fraction) of theobromine
(99%), theophylline (>99%), caffeine (>99%), and
-hydroxyethyltheophylline (>99%) was assessed using
LC-UV with detection at 274 nm and was used to correct the
concentration of the calibrants (19), Chemical structures of
these compounds are shown in Figure 1, SRM 2384 Baking
Chocolate was used for quality control and was prepared
according to a previously established protocol reported
elsewhere (19),
(b) Sample preparation of SRMs.Six bottles each of
SRMs 3243 and 3244 were randomly selected and prepared
according to the following protocol: Approximately two
150 mg portions from each of the bottles of SRM 3243 and ca
two 600 to 900 mg portions from each of the 6 bottles of SRM

3244 were weighed in a sample vial. Weighed amounts (ca 7

to 10 g) of the internal standard solution were added to each
vial. The internal standard solution served as the source of
methanol for sample extraction. Each sample was
suhsequently sonicated for 1 h to ensure complete extraction
of the analytes from the matrix. After sonication, the
supernatant from each sample was filtered using a 0,45 ^m
pore nylon syringe filter, A 200 |aL aliquot of the filtered
sample was transferred into a sample vial for LC analysis,
(c) LC-UV conditions.^An isocratic LC method was used
for the LC-UV determination of caffeine in SRMs 3243 and
3244, A 5 |im analytical column (Zorbax R^-Cig, 4,6 x
250 mm; MAC-MOD Analytical, Inc, Chadds Ford, PA)
consisting of an octadecyl stationary phase that has been
chemically bonded to an inactive silica support was used. An
isocratic mobile phase consisting of (volumefractions)11%
acetronitrile and 89% water (pH adjusted to 2,5 using acetic
acid to minimize peak tailing) at 1,5 mL/min with UV
absorbance detection (274 nm) was used for the analysis of
the analytes. All separations were conducted at 28 2C;
15 \iL injections were made.
Method 2: LC-UV (ChromaDex)
(a) Calibration standards.For preparation of a stock
solution, 52,64 mg caffeine reference standard (ChromaDex,
Santa Ana, CA; Lot No, 01-03027-917) was weighed and
transferred into a 50 mL volumetric flask and then dissolved
and diluted to volume with acetonitrile-water (10 + 90, v/v).
Approximately 4 mL of this solution was pipetted into a
50 mL volumetric flask and diluted to volume with
acetonitrile-water (10 + 90, v/v). The final concentration of
the working standard was 0,0826 mg/mL,
(b) Sample preparation of SRMs.Six bottles from each
of SRM 3243 (solid oral dosage form) and SRIVI3244 (protein
powder) were assayed for caffeine using the method described
in the section below. Approximately 1,5 g portions from each

THOMAS ET AL. : JOURNAL OF AOAC INTERNATIONAL VOL. 90, No. 4,2007

Internai standard, " C j - caffeine, in SRM 3244
by naeasurement of the first isotope
199*-> 141*

Table 2. Summary of results (mass fractions in mg/g)

for the analysis of caffeine in SRMs 3243 and 3244 as
measured by monitoring the first isotope of caffeine or
reducing the collision energy^

Caffeine in SRM 3244

by measurement of the first isotope
196*-> 139*

Bottle No.

Theobromine in SRM 3244

181* -> 138*

12

Minutes

1st isotope, mg/g

143-61

74.0

75.6

143-60

73.9

74.9

102-81

74.0

74.0

16-1

75.9

77.6

102-14

72.3

73.8

16-48

78.3

78.5

74.7

75.7

2,1

1.9

2.8

2.6

Average
Standard deviation
Coefficient of variation. %

Caffeine in SRM 3244 with lowered collision energy

195*-> 138*

Theophylline + possible isomer in SRM 3244

181*- 124*

Theobromine in SRM 3244

1 8 1 * ^ 138*

SRM 3244
120-27

3.09

68-90

2.90

2.91

120-64

2.92

2.92

15-81

2.77

2.84

68-36

2.84

2.90

15-80

of 6 bottles of SRM 3243 were transferred into 100 mL

volumetric flasks. The samples were extracted by adding ca
50 mL acetonitrile-water (10 + 90, v/v) to each flask, shaking
for 15 min, and sonicating for an additional 15 min. After
filling each flask to volume with sample diluent, a portion of
each solution was centriaged, and 5 mL of the supernatant
was pipetted into a 50 mL volumetric flask and diluted to
volume with acetonitrile-water (10 + 90, v/v).
Samples of the protein powder SRM were prepared in a
similar manner. About 3 g fi-om each of the 6 bottles of
SRM 3244 were transferred into 50 mL volumetric flasks.
Samples were extracted by adding ca 25 mL acetonitrile to
each flask, shaking for 15 min, and sonicating for an
additional 30 min. Each flask was filled to volume with
acetonitrile, a portion of each solution was centriiged, and
4 mL of the supernatant was pipetted into a 10 mL volumetric
flask and diluted to volume with water.
(c) LC-UV conditions.An isocratic LC-UV method was
used with a 5 (^m C|g column (Nacalai Cosmosil C|g-MS-II,
4.6 X 150 mm; Nacalai Tesque, Inc., Kyoto, Japan) at 25C

3.02

2.75

2.82

Average

2.88

2.90

Standard deviation

0.12

0.070

Coefficient of variation, %

4.3

2.4

Minutes

Figure 3 (A) NIST LC/MS/MS analysis of SRM 3244 in

which the first isotope of caffeine was used to modify
the dynamic range. (B) NIST LC/MS/MS analysis of SRM
3244 in which the collision energy of caffeine was
reduced from the optimized value.

Reduced collision
energy, mg/g

SRM 3243

Theophylline + possible isomer in SRM 3244

1 8 1 * ^ 124*

Internal standard, " C j - caffeine, in SRM 3244

with lowered collision energy
198*-> 140*

937

Results shown represent an average of 6 measurements (n = 6)

and are reported on a dry-mass basis. Results have been
corrected for purity and moisture content (refs. 6,10,11).
Chromatograms of the 2 different methods are shown in Figure 3A
and B.

with a mobile phase consisting of acetonitrile-10 mmol/L

sodium acetate buffer (10 + 90, v/v) at 1.5 mL/min. Detection
was at 273 nm; 20 [xL injections were made.
Mef/70d 3.- LC/MS/MS (NIST)
(a) Calibration standards.Four calibrants were
prepared for the measurement of caffeine in SRMs 3243 and
3244 by LC/MS/MS. '^Cj-Caffeine (CAS No. 78072-66-9,
Sigma-Aldrich) was used as the internal standard. For each
calibrant, ca 11 mg solid caffeine and 0.56 to 5.75 mg
C3-caffeine/g methanol solutions were used.
(b) Sample preparation of SRMs.Sample preparation of
the SRMs for the analysis of caffeine by LC/MS/MS involved
weighing 0.5 g portions from each of 6 bottles of SRM 3243
and 2 g portions from each of 6 bottles of SRM 3244 into
15 mL centrifuge tubes, spiking each sample with the internal
standard (500 |iL), and adding methanol (5 mL to SRM 3243
samples or 15 mL to SRM 3244 samples). The samples were

938

THOMAS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 90, No. 4,2007

Results and Discussion

84

Analysis of SRt^s 3243 and 3244

82
O) 80

S 76
u
g 74

72
70
NISTLC/UV

NIST LC/MS/MS

ChromaDex
LC/UV

Certified Value

Figure 4. Comparison of individual laboratory

measurements of caffeine in SRM 3243. Error bars span
2 repeatability standard deviations about the
participants' means. Dashed lines represent an
approximate 95% confidence level for the analytes.

mixed on a Vortex mixer for 10 s and then sonicated in an

ultrasonic bath for 90 min. After sonication, the samples were
centrifliged for 1 min at 4000 g and filtered with a 0.45 xm
syringe filter into an amber LC vial.
(c) LC/MS/MS conditions.A Waters Corp. (Milford,
MA) 2795 liquid Chromatograph was interfaced with a
Micromass Ultima Quattro triple quadrupole mass
spectrometer in the electrospray ionization positive ion mode.
After optimizing individually on the protonated precursor
ions and the dominant fragments of each of the analytes of
interest, the individual analytes were detected using multiple
reaction monitoring (MRM). Due to the high levels of caffeine
and low levels of theobromine and theophylline, it was not
possible to measure caffeine, theobromine, and theophylline
in the same trial using optimized signals. To lower the caffeine
signal while maintaining optimized theobromine and
theophylline signals, 2 separate approaches were investigated.
The first method involved monitoring the first isotope of
caffeine at '^C instead of '^C. Therefore, the transition 196'*' ->
139"^ was monitored instead ofthe more prevalent 195"^ -^
138"^, reducing the caffeine signal to ca 9% ofthe original and
making possible measurement of all 3 analytes in the same
trial. The second method involved a modification of the
optimal collision energy for caffeine, from 20 to 5 V, which
lowered the fragmentation efficiency of the protontated
precursor ion producing a signal within the dynamic range of
all 3 analytes. Both methods were used independently with the
calibrants and samples.
A YMC phenyl column (5 (jm, 250 x 4.6 mm; Waters
Corp.) at 30 2C was used to separate the analytes. An
isocratic mobile phase consisting of solvent A-acetonitrile
(85 + 15, v/v) was used, with solvent A containing 2%
methanol, 2% glacial acetic acid, and 0.4% ammonium
acetate in water (w/w/w). During the measurement of the
analytes of interest, the flow rate was set at 0.5 mL/min with
the effluent sent to the mass spectrometer from 7 to 16 min.

Measurements
for
caffeine
in
SRM 3243
Ephedra-Containing Solid Oral Dosage Form and for
caffeine, theobromine, and theophylline in SRM 3244
Ephedra-Containing Protein Powder have been made using
the methods described above. These measurements were used
for value assignment of the analyte concentrations in the
SRMs and to determine the homogeneity ofthe matrixes (see
discussion below). Figure 2 shows the simultaneous
separation of the analytes in SRMs 3243 and 3244 using the
NIST LC-UV method. All analytes are well separated from
matrix interferences, with a total elution time of <15 min. The
limit of quantitation for caffeine and theophylline is 50 ng/mL
and for theobromine is 30 ng/mL using the NIST LC-UV
method. SRM 2384 Baking Chocolate was used for method
validation and quality control for the NIST LC-UV method.
This material, one ofthe NIST food-matrix SRMs (19-22), is
characterized for selected nutrients as well as for caffeine,
theobromine, and theophylline. The concentration values
from the quality control measurements were comparable to
the assigned values for SRM 2384.
Table 1 shows the summary of the mean results for the
measurement of each ofthe 6 samples ofthe SRMs using the
3 methods described in this paper. The uncertainty provided
for each mean represents 2 standard deviations (SDs) of
duplicate injections of the 6 samples. An average caffeine
concentration of 75.43 mg/g, with a relative standard
deviation (RSD) of 0.86%, was obtained for SRM 3243 (solid
oral dosage form) when using the LC-UV method developed
by ChromaDex; an average caffeine concentration of
2.84 mg/g, with an RSD of 1.9%, for the 6 samples for
SRM 3244 was also achieved, indicating good content
uniformity in both materials. An analysis of variance using the
NIST LC-UV measurements for caffeine showed a mean
bottle difference, attributed to inhomogeneity, of 1.6% for a
150 mg test portion of SRM 3243; no inhomogeneity was
detected for caffeine (600 mg to 900 mg test portions) in
SRM 3244 (6). The measurement repeatability for caffeine in
the SRMs was <3% for all 3 methods and was about 4% for
theobromine and theophylline in SRM 3244 using the NIST
LC-UV method. The mean results for caffeine in the SRMs
were in agreement for all 3 methods.
Initially, the NIST LC/MS/MS measurements of caffeine,
theobromine, and theophylline in SRM 3244 were made by
using standards for retention time comparisons and the
dominant transitions for monitoring by MRM. The high levels
of caffeine and the low levels of theobromine and
theophylline required modifications in detection of the most
abundant signal in order to reproducibly measure the
low-level analytes. Because the effects of these modifications
on quantitation precision and accuracy were unknown, both
methods were used independently with the calibrants and
samples. Chromatograms of the caffeine extracted from
SRM 3244 using the 2 different methods are shown in
Figure 3A and B. The values determined by each technique

THOMAS ET AL, : JOURNAL OF AOAC INTERNATIONAL VOL, 9 0 , N O . 4 , 2 0 0 7

3.8

3.6

O)

O) 3.4

- 3.2

,2 3.0

2.8
2.6

!S 2.4

^ 2.2
2.0

NIST LC-UV

NIST LC/MS/MS

ChromaDex
LC/UV

Certified Value

Figure 5. Comparison of individual laboratory

measurements of caffeine in SRiVI 3244. Error bars span
2 repeatabiiity standard deviations about the methods
means. Dashed lines represent an approximate 95%
confidence level for the analytes.

(see Table 2) were within 1 SD of each other, and thus

measurements obtained with both methods were used for
assignment of the certified values.
Measurements for theobromine and theophylhne by
LC/MS/MS were not in agreement with those made by
LC-UV. Additional investigations of the separation revealed
the possible presence of a theobromine/theophylline isomer
that could not be unambiguously identified.
Based on the results from the investigations of
theobromine and theophylline in SRM 3244, the initial
LC/MS/MS measurements of these 2 analytes were
withdrawn from consideration for certification. Future studies
may be performed to identify the possible theobromine and
theophylline isomers that were detected in the material using
LC/MS/MS,
Value Assignment of Caffeine, Theobromine, and
Theophylline in SRM 3243 Ephedra-Containing
Solid Oral Dosage Form and SRM 3244
Ephedra-Containing Protein Powder
At NIST, there are 3 categories of assigned
valuescertified, reference, and information valuesand
7 modes for value assigning SRMs for chemical
composition (23). Certified concentration values for caffeine
(Table 1) in these SRMs were assigned based on 2 of the
7 modes of assignment, which involve the use of a
combination of data from NIST and from the collaborating
laboratory (ChromaDex). ANIST-certified value is a value for
which NIST has the highest confidence in its accuracy, in that
all known or suspected sources of bias in the measurements
have been fully accounted for and investigated (23), Each
certified value, expressed as a mass -action on a dry-mass
basis, is the equally weighted mean of results provided by
ChromaDex and the individual NIST methods. Moisture
content of each of the materials was determined in order to

939

report assigned concentration values on a dry-mass basis.

Details regarding the moisture assessment in the SRMs are
reported elsewhere (6, 10, 11), Reference values are provided
for theophylline and theobromine (Table 1) in SRM 3244,
These values are derived from the mean of results obtained by
NIST using the LC-UV method described, A NIST reference
value is a best estimate of the true value provided by NIST
when all known or suspected sources of bias have not been
fully investigated (23). Reference concentration values are not
certified because they are derived from a limited number of
analyses (from a single method, as in this case), the
disagreement among methods is greater than expected, or the
identity of components present in the measured
Chromatographie peak is less certain.
The uncertainty in the certified concentration values is
expressed as an expanded uncertainty, U, and is calculated
according to the method described in the International
Organization for Standardization (ISO) Guide to the
Expression of Uncertainty in Measurement (24, 25). The
expanded uncertainty is calculated as:

where ^ is intended to represent, at the level of 1 SD, the

eombined effect of between-laboratory, within-laboratory,
and drying components of uncertainty, and an inhomogeneity
component, if needed. The coverage factor, k, is determined
from the Student's i-distribution corresponding to the
appropriate associated degrees of freedom and an
approximate 95% confidence for each analyte. Graphical data
of the results in Table 1 for caffeine in the 2 SRMs are plotted
in Figures 4 and 5, Agreement among methods for eaffeine in
both SRMs was good overall, with between-laboratory
uncertainties ranging from 1,5 to 4%. Sources of
disagreement among methods for caflFeine include
intermediate and long-term preeision. Potential sourees of
uncertainty include measurement variability among methods,
heterogeneity (if present), purity correction of ealibrants, and
analyte stability.
Similarly, the uncertainty in the reference value, expressed
as an expanded uneertainty, is ealculated as described above:

The relative expanded uncertainties are 4% for theophylline

and 3% for theobromine in SRM 3244.
Conclusions
The methods deseribed in this paper have been
successfully used to characterize SRMs 3243 and 3244 as part
of a collaborative effort to produce the first suite in a series of
dietary supplement SRMs being offered by NIST, These
SRMs were developed to support analytieal method
validation and quality control for the analysis of similar
materials. The constituents in these SRMs have been
determined by reliable independent methods that support
analytical approaches used for the measurement of seleeted

940

THOMAS ET AL, : JOURNAL OF AOAC INTERNATIONAL VOL. 90, No. 4,2007

compounds. These approaches and reference materials can

also be used to help address labeling accuracy in the dietary
supplement industry as well as the assessment of potential
health risks posed by dietary supplements containing multiple
stimulants. We anticipate that the approaches described for
this work can be applied to the characterization of future
botanical-containing SRMs such as suites consisting of bitter
orange and green tea, which are currently under development.
Acknowledgments

(11)

(12)
(13)
(14)

Support for the development of dietary supplement SRMs

was provided by NIH/ODS, FDA/CDER, and FDA/CFSAN,
References

(15)
( 16)

(1)

(17)

(2)

(3)

(4)
(5)
(6)

(7)

(8)

(9)

( 10)

U,S, Food and Drug Administration (2004) Final Rule

Declaring Dietary Supplements Containing Ephedrine
Alkaloids Adulterated Because They Present an
Unreasonable Risk, 21 CFR Part 119, Beltsville, MD,
Anonymous (2004) Evidence on the Safety and Effectiveness
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