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Fondaparinux Sodium Injection

DEFINITION

Fondaparinux Sodium Injection is a sterile solution of Fondaparinux Sodium in Water for Injection with sodium chloride added for isotonicity. It is a clear, colorless to slightly yellow solution.

IDENTIFICATION

• A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

• B. Identification Tests—General, Chloride

191

: Proceed as directed in the chapter. Meets the requirements of the Chloride and Sulfate

221

test.

ASSAY

• Procedure

5 mM phosphate solution: 0.210 g of monobasic sodium phosphate dihydrate and 0.650 g of dibasic sodium phosphate dihydrate. Dissolve in and dilute with water to 1000 mL. pH is approximately 7.3.

Solution A: 15 ± 10 ppm of dimethylsulfoxide (DMSO) in 5 mM phosphate solution (1 in 67000, v/v)

Solution B: 2.0 M sodium chloride solution in 5 mM phosphate solution

Mobile phase: See Table 1. [Note—Make adjustments to Solution A as necessary, and degas the Mobile phase before use. Dissolved gas in the injected solution may lead to baseline interference. Degassing of the Mobile phase is critical to obtain a suitable signal-to-noise ratio and higher sensitivity. An eluant generator1 installed between the injector and the column may reduce the baseline interference.

]

Table 1

Time (min)	Solution A (%)	Solution B (%)
0	50	50
5	50	50
25	5	95
30	5	95
35	50	50
50	50	50

System suitability solution: 5.0 mg/mL of USP Fondaparinux Sodium System Suitability Mixture B RS

Standard solution: 5.0 mg/mL of USP Fondaparinux Sodium for Assay RS in water. Prepare in duplicate.

Sensitivity check solution: 0.01 mg/mL of USP Fondaparinux Sodium for Assay RS in water from the Standard solution

Sample solution: Transfer the contents of prefilled syringes to a suitable container, and mix well. Dilute with water, if needed, to obtain a 5.0-mg/mL solution of fondaparinux sodium.

Blank: Water

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4-mm × 25-cm; packing L46

Column temperature: 25

Flow rate: 1.0 mL/min

Injection volume: 100 µL

System suitability

Samples: System suitability solution, Standard solution, Sensitivity check solution, and Blank

Inject the Blank in duplicate, the Sensitivity check solution, and the System suitability solution. Inject the Standard solution at least six times consecutively.

Suitability requirements

Specificity and baseline drift: The chromatogram of a second Blank injection shows a baseline drift between 0.00 and 0.02 AU over 30 min. If necessary, adjust the DMSO content of the Mobile phase until an acceptable baseline is achieved. The chromatogram of a second Blank injection does not contain peaks between 3.00 and 30.00 min.

Chromatogram similarity: The chromatogram of the System suitability solution corresponds to that of the reference chromatogram provided with USP Fondaparinux Sodium System Suitability Mixture B RS.

Signal-to-noise ratio: NLT 10 for the fondaparinux peak in the chromatogram of the Sensitivity check solution

Resolution: NLT 1.2 between fondaparinux related compound C and fondaparinux related compound D, System suitability solution; NLT 1.1 between fondaparinux related compound F and fondaparinux related compound G (see Table 2), System suitability solution

Standard agreement: The difference in the mean response factors for each Standard solution is NMT 2.0%.

Relative standard deviation: For six consecutive injections of the Standard solution the calculated % RSD of the area of the fondaparinux peak is NMT 2.0%. The retention time of the fondaparinux peak should be ±5% of the mean value. The calculated % RSD of the response factors for six consecutive injections of the Standard solution is NMT 2.0%. The calculated % RSD of the pooled response factors for all injections of the Standard solution is NMT 2.0%. The % RSD of the mean response factors for the duplicate preparations of the duplicate Standard solutions is NMT 2.0%.

Analysis

Samples: Standard solution and Sample solution

Inject the Standard solution at least six times consecutively. Inject duplicate preparations of the Sample solution. Record the chromatograms, and measure the retention times and areas for the major peaks (excluding peaks before 3.00 and after 30.00 min).

Calculations: For each injection of the Standard solution calculate a response factor (FR):

FR = (CS/rS)

CS	=	= concentration of fondaparinux sodium in the Standard solution (mg/mL)
rS	=	= peak response of fondaparinux sodium from the Standard solution

Relative retention times (RRT) are calculated by dividing the retention time of the peak by the retention time of fondaparinux established by the Standard solution. Using the mean response factor (FM), calculate the concentration (mg/mL) of fondaparinux sodium in each injection of the Sample solution:

Result = FM × rU × DU

FM	=	= mean response factor from the Standard solution
rU	=	= peak response of fondaparinux sodium in the Sample solution
DU	=	= dilution factor for the Sample solution, if needed

Acceptance criteria: 90%–105% (for the 2.5-mg/0.5-mL injection) or 95%–105% (for the 5.0-mg/0.4-mL, 7.5-mg/0.6-mL, and 10-mg/0.8-mL injections)

IMPURITIES

• Free Sulfate Determination

[Note—Regenerate the anion-exchange column for 15 min with 0.1 M sodium hydroxide after each injection of fondaparinux sample, followed by equilibration with Mobile phase for 21 min.

]

Mobile phase: 3 mM carbonate solution using 0.106 g of sodium carbonate and 0.168 g of sodium hydrogen carbonate in 1000 mL of water

Standard solution 1: Prepare a 1000-ppm sulfate solution, using anhydrous sodium sulfate in water.

Standard solution 2: Prepare a 10-ppm sulfate solution by diluting Standard solution 1 in water.

Sensitivity check solution: Dilute 1.0 mL of Standard solution 2 with water to 5.0 mL.

Resolution solution: 0.100 g of anhydrous sodium sulfate and 0.100 g of sodium chloride. Dissolve in and dilute with water to 100.0 mL. Dilute 1.0 mL with water to 100.0 mL.

Sample solution: In triplicate, combine and mix the contents of a suitable number of syringes. Dilute 0.8 mL (strengths of 5.0 mg/0.4 mL, 7.5 mg/0.6 mL, and 10.0 mg/0.8 mL) or 2.0 mL (strengths of 1.5 mg/0.3 mL and 2.5 mg/0.5 mL) with water to 5.0 mL.

Blank: A sample of the water used to prepare other solutions

Chromatographic system

(See Chromatography

621

, System Suitability).

Mode: LC

Detector: Conductivity; range 200 µS, suppressor current 300 mA

Column: 4.6-mm × 5-cm; packing L23, coupled with a neutralization micromembrane suppressor2

Column temperature: Ambient

Regenerating solvent for the suppressor: Ultrapurified water in a counter current direction

Flow rate: 1.0 mL/min

Injection volume: 50 µL

Run time: 24 min

System suitability

Samples: Standard solution 2, Sensitivity check solution, Resolution solution, and Blank

Suitability requirements

Specificity: The chromatogram of a second Blank injection does not contain a peak corresponding to the sulfate ion.

Signal-to-noise-ratio: NLT 10, Sensitivity check solution

Resolution: NLT 10 between the sulfate and chloride peaks, Resolution solution

Relative standard deviation: NMT 5% of the response factors for six consecutive injections of Standard solution 2

Standard agreement: NMT 5% difference in the mean response factors for each Standard solution 2 injection

Analysis: Inject the Blank in duplicate, the Sensitivity check solution, and the Resolution solution. Inject Standard solution 2 at least six times consecutively. Inject triplicate preparations of the Sample solution. Record the chromatograms, and measure the retention times and areas for the sulfate peaks found.

Calculations: For each injection of Standard solution 2, calculate a response factor (F):

F = (CS/rS)

CS	=	= concentration of sodium sulfate in Standard solution 2 (mg/mL)
rS	=	= peak response of the sulfate peak from Standard solution 2

Using the mean response factor (FM), calculate the concentration (% w/w) of free sulfate in each injection of the Sample solution:

Result = FM × rU × DU × (Mr1/Mr2) × (100/C)

FM	=	= mean response factor from Standard solution 2
rU	=	= peak response of the sulfate ion in the Sample solution
DU	=	= dilution factor for the Sample solution
Mr1	=	= molecular weight of the sulfate ion, 96.1
Mr2	=	= molecular weight of sodium sulfate, 142.0
C	=	= nominal concentration of fondaparinux sodium in the content of the syringe

Acceptance criteria: NMT 0.50% (w/w)

• Organic Impurities

System suitability solution, Standard solution, Sensitivity check solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Samples: System suitability solution, Standard solution, Sensitivity check solution, Sample solution, and Blank

Calculate the percentage (area/area) of each individual unspecified impurity for each injection of the Sample solution:

Result = [rU/(rT + rS)] × 100

rU	=	= peak response of each impurity from the Sample solution
rT	=	= sum of all the peak responses for degradation impurities from the Sample solution
rS	=	= peak response of fondaparinux sodium from the Sample solution

Taking into account the response factors for specified impurities (see Table 2), calculate the individual content (% w/w) of specified fondaparinux related compounds B, C, and G:

Result = (rU × Fi × 100)/{[S(rU × Fi)] + rS }

rU	=	= peak response of each impurity from the Sample solution
Fi	=	= relative response factor for the individual impurity peak (response factor of fondaparinux sodium/response factor of individual impurity [see Table 2])
rs	=	= peak response of fondaparinux sodium from the Sample solution

Calculate the total degradation product content by summing the mean unrounded content values for the following peaks: fondaparinux related compounds A, B, C, D, F, and G and any unspecified impurities that are not synthetic impurities. Exclude peaks below the LOQ (0.003% w/w for fondaparinux related compound B, 0.002% w/w for fondaparinux related compound G, and 0.200% for all other degradation products and fondaparinux related compound E).

Individual impurities: See Table 2.

Table 2

Name	Relative Retention Time	Relative Response Factor	Acceptance Criteria, NMT (%)
Fondaparinux related compound A	0.35	1.0	1.0 (a/a)
Fondaparinux related compound Ba	0.48	70	0.150 (w/w)
Fondaparinux related compound Cb	0.76	1.0	0.8 (w/w)/0.4 (w/w)c
Fondaparinux related compound D	0.80	1.0	0.8 (a/a)
Fondaparinux related compound Ed	0.93	—	—
Fondaparinux related compound Fe	1.29	1.0	2.0 (a/a)
Fondaparinux related compound Gf	1.34	100	0.10 (w/w)
Fondaparinux sodium	—	1.0	—
Total impurities	—	—	5.0
a Methyl-O-(4-deoxy-2-O-sulfo--l-threo-hex-4-enopyranosyluronate)-(1®4)-O-(2-deoxy-6-O-sulfo-2-sulfamino--d-glucopyranoside), tetrasodium salt. b Methyl O-(2-deoxy-6-O-sulfo-2-(sulfoamino)--d-glucopyranosyl)-(1®4)-O-(-d-glucopyranosyluronate)-(1®4)-O-(2-deoxy-3,6-di-O-sulfo-2-amino--d-glucopyranosyl-(1®4)-O-2-O-sulfo--l-idopyranosyluronate)-(1®4)-(2-deoxy-6-O-sulfo-2-(sulfoamino)--d-glucopyranoside), nonasodium salt. c 0.8 (w/w) for Injection at 12.5 mg/mL and 0.4% (w/w) for Injection at 5 mg/mL. d Synthetic impurity included for identification purposes only and excluded from impurities calculations. e The fondaparinux related compound F peak can appear as a complex set of peaks in the region RRT 1.2 to RRT 1.24. These peaks, which may not be fully resolved from each other, appear before the fondaparinux related compound G peak. In such a case, the integration should be performed so that all such peaks are combined. Specified degradation products can be assigned by reference to the specimen chromatogram of the System suitability solution associated with USP Fondaparinux Sodium System Suitability Mixture B RS. f 2-Deoxy-6-O-sulfo-2-(sulfoamino)--d-glucopyranosyl-(1®4)-O-(-d-glucopyranosyluronate)-(1®4)-O-(2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)--d-glucopyranosyl)-(1®4)-O-(2-O-sulfo--l-idopyranosyluronate)-(1®4)-(1,2-dideoxy-6-O-sulfo-2-(sulfoamino)-d-enoglucopyranoside), decasodium salt.

SPECIFIC TESTS

• Bacterial Endotoxins Test

: NMT 3.3 USP Endotoxin Units/mg of fondaparinux sodium

• Particulate Matter in Injections

788

: Meets the requirements for small-volume injections

• Sterility Tests

: Where it is labeled as sterile, it meets the requirements.

• pH

791

: 5.0–8.0, in a solution, at 20

–25

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in single-dose or in multiple-dose containers in Type I glass or other validated container-closure system. Store at or below 25

• Labeling: Label it to indicate the amount, in mg, of fondaparinux sodium in the total volume of contents.

• USP Reference Standards

USP Endotoxin RS

USP Fondaparinux Sodium for Assay RS

USP Fondaparinux Sodium System Suitability Mixture B RS

USP38

1 One suitable eluant generator is Dionex DEGAS EG40/50 (12 × 17 cm, thickness 2.2 cm).

2 One suitable suppressor is Dionex ASRS 300 4 mm.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Anita Y. Szajek, Ph.D. Principal Scientific Liaison (301) 816-8325	(BIO12010) Monographs - Biologics and Biotechnology 1
85	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
71	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP38–NF33 Page 3607

Pharmacopeial Forum: Volume No. 39(5)