Certificate of Analysis – Certified Reference Material

FD&C YELLOW NO. 6

Product no.: PHR2263-100MG HO
+ +
Na Na
Lot no.: LRAC3604
O N
Description of CRM: Red to Orange Solid -
O S N
Expiry date: 31 May 2024
O
Storage: Refrigerator
O
Certificate version: LRAC3604.2 (Note: Certificates may be updated due to S
O -
Pharmacopeial Lot Changes or the availability of new data. O
Check our website at: www.sigma-aldrich.com for the most
current version.)

Chemical formula: C16H10N2Na2O7S2

Molecular mass: 452.36
CAS No.: 2783-94-0

Certified Purity ± associated uncertainty U, U=k . u (k=) (Mass Balance/

Analyte
basis)

FD&C YELLOW NO. 6 92.7 % Ucrm = ± 0.3 %, k = 2.0 (as is basis)

Metrological traceability: Traceable to the SI and higher order standards from NIST through an unbroken
chain of comparisons. Additional traceability to Primary Standards is established
through comparative assay determinations. See “Details on metrological
traceability” on page 2.
Measurement method: Where applicable, the certified value is based on a purity determination by mass
balance. See “Certification process details” on page 2.
Intended use: Intended for R&D and Analytical Use only. Not for drug, household or other uses.
Minimum sample size: 10 mg
Instructions for handling Do not dry, use on the as is basis. The internal pressure of the container may be
and correct use: slightly different from the atmospheric pressure at the user’s location. Open
slowly and carefully to avoid dispersion of the material. Attachment of a 20 mm
aluminum crimp seal recommended for unused portions.
Health and safety All chemical reference materials should be considered potentially hazardous and
information: should be used only by qualified laboratory personnel. Please refer to the Safety
Data Sheet for detailed information about the nature of any hazard and appropriate
precautions to be taken.
Accreditation: Sigma-Aldrich RTC is accredited by the US accreditation authority ANAB as a
registered reference material producer AR-1470 in accordance with ISO 17034.
Certificate issue date: 13 April 2022

ISO 17034
AR-1470 [Andy Ommen; Quality Control] [Mark Pooler; Quality Assurance]

Sigma-Aldrich RTC, 2931 Soldier Springs Rd. Laramie, WY 82070, USA;

Tel. 1 307-742-5452; Fax 1 307-855-831-9211; www.sigmaaldrich.com
Sigma-Aldrich RTC is a subsidiary of Merck KGaA, Darmstadt, Germany.

Certificate Page 1 of 5 Certificate version 02

 Packaging: 100MG in amber vial
Details on metrological This standard has been gravimetrically prepared using balances that have been
traceability: fully qualified and calibrated to ISO 17025 requirements. All calibrations utilize
NIST traceable weights which are calibrated externally by a qualified ISO 17025
accredited calibration laboratory to NIST standards. Qualification of each
balance includes the assignment of a minimum weighing by a qualified and ISO
17025 accredited calibration vendor taking into consideration the balance and
installed environmental conditions to ensure compliance with USP tolerances of
NMT 0.10% relative error. Fill volume to predetermined specifications is
gravimetrically verified throughout the dispensing process using qualified and
calibrated balances. Further traceability to a corresponding Primary Standard
may be achieved through a direct comparison assay. Where a Primary Standard
is available, the assay value will be included in the specified section of the COA.
Associated uncertainty: Uncertainty values in this document are expressed as Expanded Uncertainty
(UCRM) corresponding to the 95% confidence interval. UCRM is derived from the
combined standard uncertainty multiplied by the coverage factor k, which is
obtained from a t-distribution and degrees of freedom. The components of
combined standard uncertainty include the uncertainties due to characterization,
homogeneity, long term stability, and short term stability (transport). The
components due to stability are generally considered to be negligible unless
otherwise indicated by stability studies.

METHOD: UV (ref.: Appendix III, Total Color Method I)

Solvent: 0.04 N Ammonium Acetate
Cell Pathlength: 1 cm
Wavelength: 484 nm

Total color: 85.3 %

Certification process details:

The certified purity is determined by mass balance and calculated as

(100 − 𝑇𝑇𝑇𝑇𝑇𝑇) (100 − 𝐿𝐿𝐿𝐿𝐿𝐿) (100 − 𝐻𝐻2𝑂𝑂) (100 − 𝑅𝑅𝑅𝑅𝑅𝑅) (100 − 𝑅𝑅𝑅𝑅)
% 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 = � ∗ ∗ ∗ ∗ � ∗ 100%
100 100 100 100 100

• TCI = Total Chromatographic Impurities

• LOD = Loss on Drying
• H2O = Water content determined by Karl Fischer analysis
• ROI = Residue on Ignition
• RS = Residual Solvents

Methods for impurity determination may be added or deleted as required. The following techniques are applied:

CHROMATOGRAPHIC IMPURITY ANALYSIS

METHOD: HPLC (ref.: Adapted from Food Dyes, Current Compendial Monographs)
Column: Ascentis Express C18, 4.6 x 150mm, 2.7 µm particle size
Mobile Phase A: 10mM Ammonium Acetate in water
Mobile Phase B: ACN
Mobile Phase C: MeOH
Gradient:
Time (min) %A %B %C
0 97 1 2
4.53 97 1 2
5.18 90 1 9
23.97 50 25 25
24.29 2 49 49
27.53 2 49 49
27.85 97 1 2
32.39 97 1 2

Certificate Page 2 of 5 Certificate version 02

 Flow Rate: 1.0 mL/min
Column Temperature: 30 °C
Cooled Autosampler: 16 °C
Injection Volume: 2 µL
Detector: UV/DAD/VWD, Wavelength: 484 nm

Impurities Detected:

Impurity 1: 0.107 % Impurity 2: 0.257 %

Impurity 3: 0.595 %

Total Impurities: 0.96 %

Representative Chromatogram from Lot: LRAC3604 Impurity Analysis

RESIDUAL SOLVENTS
Method: GC-MS Headspace (ref.: Adapted from Residual Solvents USP <467>)
Column: SPB-624
Carrier gas: He
Flow: 1.2 mL/min
Split Ratio: 1:5
Injection/Temperature: 1 mL/220 °C
Temperature Program: 40 °C for 5 min, 8 °C/min to 200 °C, hold 5 min

Solvents Detected: None

WATER DETERMINATION
Method: Karl Fischer (ref.: Current Compendial Monographs)
Mean of three measurements, Water Content = 6.36 %

CERTIFIED PURITY BY MASS BALANCE

92.7 % Ucrm = ± 0.3 %, k = 2.0

(as is basis)

Homogeneity assessment: Homogeneity was assessed in accordance with ISO Guide 35. Completed units
were sampled using a random stratified sampling protocol. The results of
chemical analysis were then compared by Single Factor Analysis of Variance
(ANOVA). The uncertainty due to homogeneity was derived from the ANOVA.
Heterogeneity was not detected under the conditions of the ANOVA.
Analytical method: HPLC
Sample size: 10 mg
Stability assessment: Significance of the stability assessment will be demonstrated if the analytical
result of the study and the range of values represented by the Expanded
Uncertainty do not overlap the result of the original assay and the range of its
values represented by the Expanded Uncertainty. The method employed will
usually be the same method used to characterize the assay value in the initial
evaluation.

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 Long Term Stability Evaluation - An assessment, or re-test, versus a
Compendial Reference Standard may be scheduled, within the 3 year
anniversary date of a release of a Secondary Standard. The re-test interval will
be determined on a case-by-case basis. Short Term Stability Study - It is useful
to assess stability under reasonably anticipated, short term transport conditions
by simulating exposure of the product to humidity and temperature stress. This
type of study is conducted under controlled conditions of elevated temperature
and humidity.

Identification Test:
INFRARED SPECTROPHOTOMETRY (For Information Purpose Only)

MilliporeSigma Lot: LRAC3604

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 1H NMR (Data provided by an external laboratory; not in scope of accreditation)

Consistent with structure

Certificate of analysis revision history:

Certificate version Date Reason for version
LRAC3604.1 26 June 2020 Original Release
LRAC3604.2 13 April 2022 Added NMR

Disclaimer:
The purchaser is required to determine the suitability of this product for any particular
application. Sigma-Aldrich RTC makes no warranty of any kind, express or implied, other than
its products meet all quality control standards set by Sigma-Aldrich RTC. We do not guarantee
that the product can be used for any particular application.

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Detailed information on trademarks is available via publicly accessible resources.
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Certificate Page 5 of 5 Certificate version 02