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retention times of the Standard solutions and their

Iron Sucrose Injection molecular weights to generate a third order (cubic)
DEFINITION calibration curve
Iron Sucrose Injection is a sterile, colloidal solution of ferric Analysis
hydroxide in complex with Sucrose in Water for Injection. It Samples: System suitability solution, Standard solutions,
contains NLT 95.0% and NMT 105.0% of the labeled and Sample solution
amount of iron. Sodium Hydroxide may be added to adjust The molecular weight of the complex is calculated from the
the pH. It contains no antimicrobial agent, chelating agent, calibration curve. The molecular weight distribution curve
dextran, gluconate, or other added substances. of the sample is sliced into fractions.
Calculate the weight-average molecular weight (M W) as
IDENTIFICATION follows:
• A. IRON
To 2.5 mL of Injection add 17.5 mL of water and 5 mL of Result = Σ(A T M T)/ΣA T
hydrochloric acid. Mix and heat the solution for 5 min in a
boiling water bath. Cool, add dropwise 13.5 N Calculate the number-average molecular weight (M N) as
ammonium hydroxide until no further precipitation of follows:
ferric hydroxide occurs, and filter. Wash the precipitate
with water to remove excess ammonium hydroxide, Result = Σ(A T)/Σ(A T/M T)
dissolve the precipitate in a minimum volume of 2 N
hydrochloric acid, and add sufficient water to make a AT = area of each fraction of the sample distribution
volume of 20 mL. To 3 mL of the solution add 1 mL of 2 N MT = corresponding mean molecular weight of each
hydrochloric acid and 1 mL of potassium thiocyanate TS: fraction as determined from its retention time on
the calibration curve

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the resulting solution (Solution 1) is red. To 1 mL of
Solution 1 add 5 mL of amyl alcohol or ethyl ether, shake,
and allow to stand: the organic layer is pink. To a separate Acceptance criteria: The molecular weight distribution
1-mL aliquot of Solution 1 add 2 mL of mercuric chloride curve of the Injection conforms to the following
TS: a red color is discharged [iron (III) salts]. parameters.
• B. The retention time of the major peak of the Sample M W: 34,000–60,000 Da
solution corresponds to that of the Standard solution, as
obtained in the Assay for Sucrose.
• C. MOLECULAR WEIGHT DETERMINATION
ci M N: NLT 24,000 Da
M W/M N: NMT 1.7
Mobile phase: Dissolve 7.12 g of dibasic sodium phosphate ASSAY
dihydrate, 5.52 g of monobasic sodium phosphate, and • SUCROSE
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0.40 g of sodium azide in 2 L of water. Mobile phase: Acetonitrile and water (79:21)
System suitability solution: Dissolve 200 mg of high Standard solutions: Individual solutions of 13, 16, 18, 21,
molecular weight dextran and 100 mg of glucose in and 23 mg/mL of sucrose from USP Sucrose RS, in water
20 mL of Mobile phase. Sample solution: Transfer about 1.875 g of Injection to a
Standard solutions: Transfer about 20 mg of each 25-mL flask. Add 1.25 mL of water and mix. Add 1.25 mL
polysaccharide molecular weight standard (5,000– of a monobasic sodium phosphate solution, prepared by
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400,000 Da) to separate 5-mL volumetric flasks. Add dissolving 30 g in 50 mL, and mix. Allow the resulting
4 mL of Mobile phase to each flask, and allow each aliquot solution to stand for 10 min to precipitate the colloidal ferric
to stand at or below 25° for a minimum of 12 h. After the hydroxide. Dilute with water to volume. Centrifuge this
agglomerate particles of each Standard solution have solution at 3000 rpm for 15 min. Pass the resulting solution
swelled to their fullest extent, gently swirl each Standard through a filter, discarding the first 2 mL of the filtrate.
solution until dissolved. Chromatographic system
[NOTE—The chromatograms of freshly prepared (See Chromatography á621ñ, System Suitability.)
Standard solutions regularly show a small, unidentified Mode: LC
secondary peak following the main peak. Discard the Detector: Refractive index
Standard solutions if the secondary peak reaches half Column: 4-mm × 25-cm; packing L8
the height of the main peak.] Temperatures
Sample solution: Transfer 5.0 mL of Injection to a 10-mL Detector: 20–25° (±2°)
volumetric flask, and dilute with Mobile phase to volume. Column: 20–25° (±2°)
Chromatographic system Flow rate: 2 mL/min
(See Chromatography á621ñ, System Suitability.) Injection volume: 20 µL
Mode: LC System suitability
Detector: Refractive index, maintained at a constant Samples: Standard solutions
temperature of 45° [NOTE—The retention time for sucrose is about 8 min.]
Columns: Two 7.8-mm × 30-cm; packing L39 with pore Suitability requirements
sizes of 1000 and 120 Å, respectively Correlation coefficient: NLT 0.998 from the linear
Column temperature: 45 ± 2° regression of the Standard solutions
Flow rate: 0.5 mL/min Analysis
Injection volume: 25 µL Samples: Standard solutions and Sample solution
System suitability Plot the peak area for each Standard solution versus
Samples: System suitability solution and Standard solutions concentration of sucrose in mg/mL, and draw the straight
Suitability requirements line best fitting the five plotted points. From the graph,
Resolution: NLT 4.0 between dextran and glucose, determine the concentration of sucrose, in mg/mL, in the
System suitability solution Sample solution.
Correlation coefficient: NLT 0.98 for the calibration Calculate the quantity of sucrose, in mg, in each mL of
curve generated using a suitable program, plotting the Injection taken:

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Result = (C U × D × G)/W IMPURITIES

• LIMIT OF IRON [FE(II)]
CU = concentration of sucrose in the Sample solution Supplementary electrolyte solution: Dissolve 15.0 g of
(mg/mL) sodium acetate in 100 mL of water and adjust with 0.1 N
D = dilution volume of the Sample solution (mL) acetic acid to a pH of 7.0.
G = density of Injection taken (g/mL) Sample solution: Volume of Injection equivalent to 20–120
W = weight of Injection taken (g) µg/mL of elemental iron
Analysis: Transfer a suitable amount of Supplementary
Acceptance criteria: 260–340 mg/mL electrolyte solution to a polarographic cell equipped with a
• IRON mercury drop electrode. With the electrode submerged in
Solution A: Transfer 2.64 g of calcium chloride to a the liquid, bubble nitrogen through the liquid for 5 min.
1000-mL volumetric flask, add 500 mL of water, and swirl Avoiding any undue exposure to air, immediately transfer
to dissolve. Add 5.0 mL of hydrochloric acid, and dilute with the Sample solution to the polarographic cell. The sample
water to volume. must be analyzed immediately upon opening the
Standard stock solution: 50 µg/mL of iron prepared as container.
follows. Transfer about 350 mg of ferrous ammonium Record the polarogram from 0 mV and −1700 mV. The iron
sulfate to a 1000-mL volumetric flask. Add water to dissolve, [Fe(III)/Fe(II)] peak is detected at −750 ± 50 mV and the
dilute with water to volume, and mix. iron [Fe(II)/Fe(0)] peak is detected at −1400 ± 50 mV.
Standard solutions: Individual solutions containing 2.0, Measure the iron [Fe(II)/Fe(III)] peak responses obtained
4.0, 6.0, 8.0, and 10.0 µg/mL of iron in Solution A from the from the polarogram, and perform a blank determination.
Standard stock solution Calculate the content of iron [Fe(II)], in % w/v, in the
Sample stock solution: Using a “to contain” pipet, transfer volume of Injection taken:
2.0 mL of Injection to a 100-mL volumetric flask. Rinse the

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pipet several times with Solution A. Add 5 mL of Result = [1 − (2/R)] × C T
hydrochloric acid, and swirl until the solution turns yellow.
After the solution has cooled to room temperature, dilute R = peak response ratio of iron [Fe(II)] to iron
with Solution A to volume, and mix. [Fe(III)]
Sample solution: Nominally 8.0 µg/mL of iron prepared as CT = total iron concentration of the Injection (% w/v)
follows. Pipet 2.0 mL of the Sample stock solution to a
100-mL volumetric flask, and dilute with Solution A to
volume.
ci Acceptance criteria: NMT 0.4%
SPECIFIC TESTS
Instrumental conditions
(See Atomic Absorption Spectroscopy á852ñ.) • PH á791ñ: 10.5–11.1 at 20°
• TURBIDITY
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Mode: Atomic absorption spectrophotometry
Analytical wavelength: 248.3 nm iron emission line Sample solution: Transfer 0.5 g of Injection to a 150-mL
Lamp: Iron hollow-cathode beaker. Add 100 mL of water and, with constant stirring,
Flame: Air–acetylene adjust with 0.1 N hydrochloric acid VS to a pH of 6.0.
Blank: Solution A Analysis: Remove the pH electrode from the solution.
Analysis Adjust a light source such that the beam hits the beaker at a
parallel angle 2 cm below the surface of the liquid. The light
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Samples: Standard solutions and Sample solution

Plot the absorbance of each Standard solution versus must shine through to the surface, and the solution must
concentration, in µg/mL, of iron, and draw the straight not have any turbidity. Measurement must be carried out
line best fitting the five plotted points. From the graph, in a room as dark as possible. Slowly add 0.1 N hydrochloric
determine the concentration, in µg/mL, of iron in the acid VS, dropwise, until a slight but lasting turbidity
Sample solution. develops. Record the pH of the solution as the turbidity
Calculate the percentage of the labeled amount of iron in point of the Injection.
each mL of Injection taken: Acceptance criteria: 4.4–5.3
• ABSENCE OF LOW-MOLECULAR WEIGHT IRON [FE(II) AND
Result = (C A/C U) × 100 FE(III)] COMPLEXES: In the polarograms obtained in the
test for Limit of Iron [Fe(II)], no additional peaks are found.
CA = actual concentration of iron in the Sample • ALKALINITY
solution determined from the calibration curve Sample solution: 5 mL of Injection
(µg/mL) Analysis: Titrate the Sample solution with 0.1 N hydrochloric
CU = nominal concentration of iron in the Sample acid VS with constant stirring to a pH of 7.4. Record the
solution (µg/mL) volume of 0.1 N hydrochloric acid VS consumed, and
calculate the alkalinity of the Injection as the volume of acid,
Acceptance criteria: 95.0%–105.0% in mL, consumed per mL of Injection.
Acceptance criteria: 0.5–0.8 mL of 0.1 N hydrochloric acid
OTHER COMPONENTS VS is consumed per mL of Injection.
• CONTENT OF CHLORIDE • OSMOLALITY AND OSMOLARITY á785ñ, Osmolarity
Sample: About 12 g of Injection Sample solution: Dilute Injection in water (1 in 10).
Analysis: Transfer the Sample to a 50-mL beaker. Add 40 mL Acceptance criteria: 1150–1350 mOsmol/L
of water and 0.3 mL of 65% nitric acid, and, while stirring, • SPECIFIC GRAVITY á841ñ: 1.135–1.165 at 20°
titrate with 0.01 N silver nitrate VS, determining the • PARTICULATE MATTER IN INJECTIONS á788ñ, Method 1 Light
endpoint potentiometrically with silver-glass electrodes. Obscuration Particle Count Test
Calculate the content of chloride, in mg, of Injection taken. Sample solution: Prepare a solution of Injection (1 in 40)
Each mL of 0.01 N silver nitrate consumed is equal to using water that has been passed through a filter having a
0.3545 mg of chloride (Cl). 1.2-µm or finer pore size.
Acceptance criteria: 0.012%–0.025% Acceptance criteria: Meets the requirements for
small-volume injections

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• BACTERIAL ENDOTOXINS TEST á85ñ: NMT 3.7 USP Endotoxin • LABELING: Label it to indicate that it is for intravenous use
Units/mg of iron contained in Injection only, and that when administered by intravenous infusion,
• OTHER REQUIREMENTS: Meets the requirements in Injections the Injection must be diluted with 0.9% Sodium Chloride
and Implanted Drug Products á1ñ Injection to a concentration of 1.0–2.0 mg/mL of elemental
iron. Label it also to state the total osmolarity of the solution
ADDITIONAL REQUIREMENTS expressed in mOsmol/L.
• PACKAGING AND STORAGE: Preserve in single-dose • USP REFERENCE STANDARDS á11ñ
containers of Type I glass. Store at controlled room USP Sucrose RS
temperature. Do not freeze.

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