USP Pending Monograph
Draft 1—For Public Comment
BRIEFING
Colesevelam Hydrochloride. Because there is no existing
monograph for this drug substance, a new Pending
Monograph is proposed based on the validated test
methods.
• The liquid chromatographic procedure in the test for
Glycocholate Binding Capacity is based on analyses
performed with the Luna, 5-µm brand of L1 column. The
typical retention time for glycocholic acid to which
Colesevelam Hydrochloride binds is about 7 min.
• The ion-exchange chromatographic procedure in the test
for Limit of Bromide is based on analyses performed with
an IonPac ASII HC, 5-µm brand of L61 column and an
IonPac AG 11 HC brand of guard column. The typical
retention time for the bromide peak is about 17.2 min.
• The ion-exchange chromatographic procedure in the test
for Limit of Allyamine is based on analyses performed with
an IonPac CS14 brand of L53 column and an IonPac CG14 brand of L53 guard column. The typical retention
time for the allylamine peak is about 6.5 min.
• The gas chromatographic procedure in the test for Limit
of Epichlorohydrin is based on analyses performed with a
ZB Wax brand of G16 column. The typical retention time
for the epichlorohydrin peak is about 22.2 min.
• The gas chromatographic procedure in the test for
Organic Impurities is based on analyses performed with a
DB-1 column of packing G1.
Colesevelam Hydrochloride is a white to pale-yellow powder
and is a high-capacity bile acid-binding molecule. It is
hydrophilic and insoluble in water.
(SM2: S. Ramakrishna.) Correspondence Number—C92885
Add the following:
.
Colesevelam Hydrochloride
Draft 1
c
Colesevelam / 1
bromide. Each g binds to NLT 2.0 and NMT 2.5 g of sodium glycocholate, calculated as glycocholate binding capacity on the dried basis.
IDENTIFICATION
• A. NINHYDRIN TEST
Solution A: Isobutanol and 2 M acetic acid (95:5)
Solution B: 2 mg/mL of ninhydrin in Solution A
Analysis: Transfer 50 mg of sample into a 100-mL beaker.
Add 20 mL of water and 5 mL of Solution B. Stir the solution for 5 min, and heat for 10 min on a water bath
maintained at 85°–90°.
Acceptance criteria: Particles in solution appear violet in
color.
PERFORMANCE TESTS
• GLYCOCHOLATE BINDING CAPACITY
Buffer: 10.9 g/L of monobasic potassium phosphate in
water. Adjust with phosphoric acid to a pH of 3.0.
Mobile phase: Acetonitrile and Buffer (35:65)
Diluent: 4.0 g/L of monobasic potassium phosphate and
12.0 g/L of dibasic potassium phosphate in water
Standard solution 1: 30 mg/mL of sodium glycocholate
hydrate in Diluent
Standard solution 2: 1.2 mg/mL of sodium glycocholate
hydrate in water from Standard solution 1
Sample solution: To 100 mg of colesevelam hydrochloride in a 25-mL flask add 15-mL of Standard solution 1,
and stir for 2 h. Pass through a suitable filter of 0.2-µm
pore size. Pipet 5.0 mL of the filtrate into a 50-mL volumetric flask, and dilute with water to volume.
System suitability solution: 0.6 mg/mL of sodium
glycocholate hydrate and 0.3 mg/mL of taurodeoxycholic
acid sodium salt hydrate in water
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 214 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Flow rate: 1.5 mL/min
Injection volume: 50 µL
System suitability
Samples: Standard solution 2 and System suitability
solution
Suitability requirements
Resolution: NLT 1.5 between glycocholic acid and
taurodeoxycholic acid, System suitability solution
Tailing factor: NMT 2.5, Standard solution 2
Relative standard deviation: NMT 1.5%, Standard solution 2
Analysis
Samples: Standard solution 2 and Sample solution
Calculate the amount of unbound sodium glycocholate
(W2), in mg, in the portion of sample taken:
Result = (rU/rS) × (CS2) × D
rU
(C3H7N)m(C3H5ClO)n(C12H27ClN2)o(C13H27N)p · xHCl
2-Propen-1-amine polymer with (chloromethyl)oxirane, N,N,
N-trimethyl-6-(2-propenylamino)-1-hexanaminium chloride,
and N-2-propenyl-1-decanamine, hydrochloride;
Allylamine polymer with 1-chloro-2,3-epoxypropane, [6(allylamino)hexyl]trimethylammonium chloride and N-allyldecylamine, hydrochloride [182815-44-7].
Colesevelam [182815-43-6].
DEFINITION
Colesevelam Hydrochloride is a poly(allylamine hydrochloride)
cross-linked with epichlorohydrin and alkylated with
1-bromodecane and (6-bromohexyl)-trimethylammonium
= peak area of glycocholic acid from the Sample
solution
rS
= peak area of glycocholic acid from Standard
solution 2
CS2
= concentration of sodium glycocholate hydrate
in Standard solution 2 (mg/mL)
D
= dilution, (15/5) × 50 mL
Calculate the glycocholate binding capacity of
Colesevelam Hydrochloride
[(C3H7N)m(C3H5ClO)n(C12H27ClN2)o(C13H27N)p ·
xHCl)] in the portion of sample taken:
Result = [(CS1 × V) − W2]/W1
CS1
V
W2
W1
= concentration of sodium glycocholate hydrate
in Standard solution 1 (mg/mL)
= volume of Standard solution 1 used to prepare
the Sample solution (mL)
= amount of the unbound sodium glycocholate
calculated as above (mg)
= sample weight (mg)
This monograph has been developed under USP‘s Pending Monographs Guideline and is not a USP–NF monograph.
http://www.usp.org
2012 The United States Pharmacopeia. All Rights Reserved.
USP Pending Monograph
Draft 1—For Public Comment
2 / Colesevelam
Acceptance criteria:
2.0–2.5 g/g on the dried basis
OTHER COMPONENTS
• CONTENT OF CHLORIDE
Sample: To about 0.2 g of Colesevelam Hydrochloride in
a 250-mL conical flask add 50 mL of water, 2 mL of nitric
acid, and 25 mL of 0.1 N silver nitrate solution. Boil the
solution for 10 min, cool, and add 3 mL of nitrobenzene.
Shake well.
Blank: 25.0 mL of 0.1 N silver nitrate solution, 2 mL of
nitric acid, and 3 mL of nitrobenzene
Titrimetric system
(See Titrimetry 〈541〉.
Mode: Direct titration.
Titrant: 0.1 N ammonium thiocyanate solution VS
Endpoint detection: Visual, reddish-brown color
Analysis
Calculate the percentage of chloride in the portion of
Colesevelam Hydrochloride
[(C3H7N)m(C3H5ClO)n(C12H27ClN2)o(C13H27N)p · xHCl]
taken:
Result = {[(VS − VB) × N × F]/W} × 100)
VS
= Titrant volume consumed by the Sample (mL)
VB
= Titrant volume consumed by the Blank (mL)
N
= normality of the Titrant (mEq/mL)
F
= equivalency factor, 3.545 mg/mEq
W
= Sample weight (g)
Acceptance criteria: 16.0%–22.0% (w/w), calculated on
the dried basis
IMPURITIES
• RESIDUE ON IGNITION 〈281〉: NMT 0.10%
• HEAVY METALS, Method II 〈231〉: NMT 20 ppm
• LIMIT OF BROMIDE
Mobile phase: 10 mM sodium hydroxide solution
Diluent: 0.2 M sodium hydroxide solution
Standard solution: 0.018 mg/mL of potassium bromide
in Diluent
Sample solution: To 60 mg of Colesevelam
Hydrochloride in a 50-mL volumetric flask add Diluent to
volume, and stir for 2 h. Pass the suspension through a
suitable filter of 0.2-µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: Ion chromatography
Detector: Conductivity with an anion self-regenerating
suppressor
Columns
Guard: 4.0-mm × 5-cm; packing L61
Analytical: 4.0-mm × 25-cm; packing L61
Temperatures
Column oven: 30°
Cell heater: 35°
Flow rate: 1 mL/min
Injection volume: 10 µL
Run time: NLT 2.6 times the retention time of the
bromide peak
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 3000 theoretical plates
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of bromide in the portion of
Colesevelam Hydrochloride taken:
Result = (rU/rS) × (CS/CU) × (Ar/Mr) × 100
rU
rS
CS
= peak response of bromide from the Sample
solution
= peak response of bromide from the Standard
solution
= concentration of potassium bromide in the
Standard solution (mg/mL)
CU
= concentration of Colesevelam Hydrochloride in
the Sample solution (mg/mL)
Ar
= atomic weight of bromine, 79.9
Mr
= molecular weight of potassium bromide,
119.0
Acceptance criteria: NMT 1.0%
• LIMIT OF EPICHLOROHYDRIN
Use freshly prepared samples for solutions containing
colesevelam hydrochloride.
Internal standard solution: 0.05 µg/mL of n-butanol in
methylene chloride
Standard solution: 0.068 µg/mL of epichlorohydrin in
Internal standard solution
Sensitivity solution: 0.02 µg/mL of epichlorohydrin in
Internal standard solution from the Standard solution
Sample solution 1: Add 5 mL of Internal standard solution
to 1000 mg of Colesevelam Hydrochloride in a 25-mL
volumetric flask, stir for 2 h, and pass the suspension
thorough a suitable filter of 0.2-µm pore size.
Sample solution 2: Add 5 mL of Standard solution to
1000 mg of Colesevelam Hydrochloride in a 25-mL
volumetric flask, stir for 2 h, and pass the suspension
through a suitable filter of 0.2-µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: GC
Detector: Flame-ionization
Column: 0.53-mm × 60-m; G16
Temperatures
Injection port: 180°
Detector: 250°
Column: See Table 1.
Table 1
Initial
Temperature
(°)
40
40
Temperature
Ramp
(°/min)
0
6
Final
Temperature
(°)
40
150
150
15
240
Hold Time
at Final
Temperature
(min)
10
0
10, Standard
solution
20, Sample
solution
Carrier gas: Helium
Pressure: Constant pressure at 41.8 kpa
Split mode: Splitless
Injection volume: 2 µL
System suitability
Samples: Sensitivity solution and Sample solution 2
[NOTE—The relative retention times of n-butanol and
epichlorohydrin are 1.00 and 1.13, respectively.]
Suitability requirements
Peak response ratio: The difference in the peak
response ratio of epichlorohydrin to n-butanol between
two injections is ±15%, Sample solution 2.
Signal-to-noise ratio: NLT 3.0, Sensitivity solution
Analysis
Samples: Sample solution 1 and Sample solution 2
Compare the peak response ratios of epichlorohydrin to nbutanol in Sample solution 1 and Sample solution 2.
Acceptance criteria: The difference in the peak response
ratio of epiclorohydrin to n-butanol of Sample solution 2
and Sample solution 1 is less than the peak response ratio
of epiclorohydrin to n-butanol from Sample solution 1
(NMT 0.34 ppm).
• LIMIT OF WATER SOLUBLE AMINES
Ninhydrin solution: 20 mg/mL of ninhydrin in water
Blank: Water
Standard solution: 0.094 mg/mL of USP Polyallylamine
Hydrochloride RS in water
System suitability solution: Add 20 mL of Standard
solution and 20-mL of water to 2.5 g of Colesevelam
Hydrochloride in a 250-mL flask, and stir for 5 h. Pass the
This monograph has been developed under USP‘s Pending Monographs Guideline and is not a USP–NF monograph.
http://www.usp.org
2012 The United States Pharmacopeia. All Rights Reserved.
USP Pending Monograph
Draft 1—For Public Comment
solution first through a suitable filter paper, and then
through a suitable filter of 0.2-µm pore size.
Sample solution 1: Add 40 mL of water to 2.5 g of
Colesevelam Hydrochloride in a 250-mL flask, and stir for
5 h. Pass the solution first through a suitable filter paper,
and then through a suitable filter of 0.2-µm pore size.
Sample solution 2: Add 40 mL of Standard solution to
2.5 g of Colesevelam Hydrochloride in a 250-mL flask,
and stir for 5 h. Pass the solution first through a suitable
filter paper, and then through a suitable filter of 0.2-µm
pore size.
Instrumental conditions
(See Chromatography 〈621〉, System Suitability.)
Mode: UV
Analytical wavelength: 575 nm
Cell: 1 cm, quartz
System suitability
Sample: System suitability solution
To 10 mL of the System suitability solution in a test-tube
add 5-mL of Ninhydrin solution. Shake well, allow to stand
for 15 min, and measure the absorbance.
Suitability requirements: Absorbance is NLT 0.010
Analysis
Samples: Blank, Sample solution 1, and Sample solution 2
To 10 mL each of the Sample solution 1, Sample solution 2,
and Blank in separate test tubes add 5-mL of Ninhydrin
solution. Shake well, allow to stand for 15 min, and
measure the absorbances.
Acceptance criteria: The difference between the
absorbance of Sample solution 2 and Sample solution 1 is
less than the absorbance of Sample solution 1 (NMT
0.15%).
• LIMIT OF ALLYLAMINE
Mobile phase: 0.96 g/L of methane sulfonic acid in
water
Diluent: 0.9 mL/L of hydrochloric acid in water
Standard solution: 5 µg/mL of allylamine in Diluent
Sample solution: To 100 mg of Colesevelam
Hydrochloride in a 10-mL volumetric flask add Diluent to
volume, and stir for 30 min. Centrifuge the suspension,
and pass the supernatant through a suitable filter of 0.2µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: Ion-exchange LC
Detector: Conductivity with a cation self-regenerating
suppressor
Columns
Guard: 4.0-mm × 5.0-cm; L53
Analytical: 4.0 mm × 25-cm; L53
Temperatures
Column oven: 30°
Cell heater: 35°
Flow rate: 1 mL/min
Injection volume: 50 µL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 1000 theoretical plates
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of allylamine in the portion of
Colesevelam Hydrochloride taken:
Result = (rU/rS) × (CS/CU) × 100
rU
rS
CS
CU
= peak response of allylamine from the Sample
solution
= peak response of allylamine from the Standard
solution
= concentration of allylamine in the Standard
solution (mg/mL)
= concentration of Colesevelam Hydrochloride in
the Sample solution (mg/mL)
Colesevelam / 3
Acceptance criteria: NMT 0.05%
• ORGANIC IMPURITIES
Use freshly prepared samples for solutions containing
colesevelam hydrochloride.
Internal standard solution: 0.03 mg/mL of n-heptane in
methylene chloride
Standard stock solution: 0.5 mg/mL each of
1-methoxydecane, 1,6-dibromohexane, and
1-bromodecane in the Internal standard solution
Standard solution: 0.05 mg/mL each of
1-methoxydecane, 1,6-dibromohexane, and
1-bromodecane in Internal standard solution from the
Standard stock solution
Sample solution: Add 10 mL of Internal standard solution
to 1000 mg of Colesevelam Hydrochloride in a 25-mL
flask, and stir for 3 h. Pass the suspension through a
suitable filter of 0.2-µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: GC
Detector: Flame-ionization
Column: 0.53-mm × 60-m; 3-µm coating of G1
Temperatures
Injection port: 220°
Detector: 250°
Column: See Table 2.
Table 2
Initial
Temperature
(°)
50
100
Temperature
Ramp
(°/min)
4
7
Hold Time
at Final
Temperature
(min)
0
10
Final
Temperature
(°)
100
240
Carrier gas: Nitrogen
Flow rates
Column: 3.24 mL/min
Total: 38.6 mL/min
Injection volume: 1 µL
Split ratio: 10:1
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 2.0 between 1,6 dibromohexane and
1-bromodecane
Relative standard deviation: NMT 10.0% for the ratio
of the area of each individual peak to the area of the
internal standard peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each specified impurity in the
portion of Colesevelam Hydrochloride taken:
Result = (RS/RU) × (CU/CS) × 100
RS
= peak response ratio of each impurity to the
internal standard from the Standard solution
RU
= peak response ratio of each corresponding
impurity to the internal standard from the
Sample solution
CS
= concentration of the each corresponding
impurity in the Standard solution (mg/mL)
CU
= concentration of Colesevelam Hydrochloride in
the Sample solution (mg/mL)
Acceptance criteria
Individual impurities: See Table 3.
Table 3
Name
n-Heptane
1-Methoxydecane
Relative
Retention
Time
1.00
2.38
This monograph has been developed under USP‘s Pending Monographs Guideline and is not a USP–NF monograph.
http://www.usp.org
2012 The United States Pharmacopeia. All Rights Reserved.
Acceptance
Criteria,
NMT (%)
—
0.05
USP Pending Monograph
Draft 1—For Public Comment
4 / Colesevelam
Table 3 (Continued)
Name
1,6-Dibromohexane
1-Bromodecane
Relative
Retention
Time
2.55
2.65
Acceptance
Criteria,
NMT (%)
0.05
0.05
SPECIFIC TESTS
• LOSS ON DRYING 〈731〉
Sample: 1 g of Colesevelam Hydrochloride
Analysis: Dry the sample at 105° for 3 h.
Acceptance criteria: NMT 10.0%
• SWELL INDEX
Buffer: 100 mM sodium chloride and 300 mM N,Nbis(hydroxyethyl)-2-aminoethanesulfonic acid (BES) in
water
Sample solution: Add about 0.040 g of Colesevelam
Hydrochloride and 2 mL of Buffer to a filtration unit
containing a stir bar. Cap the unit, invert, and stir the
slurry for 30 min on a magnetic stirrer. Centrifuge at
3000 rpm for an additional 30 min.
Analysis: Note the weight of the filtration unit before
and after sample preparation. Determine the swelling
index of Colesevelam Hydrochloride:
Result= [(W2 − W1) − W3]/W3
W1
W2
= weight of filtration unit (g)
= weight of filtration unit after centrifuging
stage (g)
W3
= sample weight (g)
Acceptance criteria: 2.0–7.5 w/w
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Store at room temperature in
airtight containers.
• USP REFERENCE STANDARDS 〈11〉
USP Colesevelam Hydrochloride RS
USP Polyallylamine Hydrochloride RSb (1-Mar-2013)
This monograph has been developed under USP‘s Pending Monographs Guideline and is not a USP–NF monograph.
http://www.usp.org
2012 The United States Pharmacopeia. All Rights Reserved.