You are on page 1of 5

Accessed from 128.83.63.

20 by nEwp0rt1 on Thu Dec 01 22:26:35 EST 2011

136 207 1,6-Anhydro Derivative / Chemical Tests USP 35

207 TEST FOR 1,6-ANHYDRO


DERIVATIVE FOR ENOXAPARIN
SODIUM Figure 2. Reduction of oligosaccharides by sodium
borohydride
The following procedure is used to determine the levels of
1, 6-anhydro forms in enoxaparin sodium. [NOTEThe test The sodium borohydride reduction eliminates the
for the 1,6-anhydro derivative is conducted only where anomeric effect by opening the terminal oligosaccha-
specified in the individual monograph.] ride ring. The four 1,6-anhydro derivatives (see Appendix 2.)
are not reduced by sodium borohydride because the ring
opening is blocked by the 1,6-anhydro bridge. The reduc-
INTRODUCTION tion of the oligosaccharides decreases their retention time,
whereas the retention time of the 1,6-anhydro derivatives
The disaccharides specified in this general chapter are remains unchanged. Thus, it is possible to separate the two
listed by name and structure in Appendix 1; the oligosaccha- compounds1,6-anhydro IIS and 1,6-anhydro IISepi
rides are listed in Appendix 2. from the reduced IIA disaccharide peak. [NOTE1,6-
Depolymerization of heparin into enoxaparin sodium pro- Anhydro IIS and 1,6-anhydro IISepi are eluted as two non-
duces a partial but characteristic conversion of glucosamines resolved peaks and are quantitated together as a single
at the reducing termini of oligosaccharide chains with termi- compound, 1,6-anhydro IIS. Therefore, for the purpose of
nal glucosamine 6-O sulfate, yielding 1,6-anhydro deriva- simplification, the epimeric form is not referred to in the
tives (see Figure 1). remaining text.]

PROCEDURE
USP Reference Standards 11USP Enoxaparin Sodium
RS.
Solutions
Solution ADissolve 0.280 g of monobasic sodium phos-
phate in 950 mL of water, adjust with phosphoric acid to a
pH of 3.0, and dilute with water to 1000 mL.
Solution BDissolve 140 g of sodium perchlorate in 950
mL of Solution A, adjust with phosphoric acid to a pH of
3.0, and dilute with Solution A to 1000 mL.
Mobile PhaseUse variable mixtures of filtered and
degassed Solution A and Solution B as directed in Chromato-
Figure 1. Structure of enoxaparin sodium containing a 1,6- graphic System.
anhydro derivative on the reducing end of the chain. Sodium/Calcium Acetate pH 7.0 SolutionDissolve 10 mg
of bovine serum albumin and 32 mg of calcium acetate in
The percentage of oligosaccharide chains that are cyclized 60 mL of water. Add 580 L of glacial acetic acid, and ad-
in a 1,6-anhydro ring is a characteristic of enoxaparin just with 2 M sodium hydroxide to a pH of 7.0. Transfer to a
sodium. 100-mL volumetric flask, and dilute with water to volume.
Pass the solution through a filter having a porosity of 0.45
or 0.22 m.
DEPOLYMERIZATION OF ENOXAPARIN Potassium Phosphate pH 7.0 BufferDissolve 68 mg of
SODIUM BY HEPARINASES AND RESULTING monobasic potassium phosphate and 10 mg of bovine se-
OLIGOSACCHARIDES rum albumin in 30 mL of water in a 50-mL volumetric flask.
Adjust with potassium hydroxide, if necessary, to a pH of
The assay involves HPLC analysis of a depolymerized 7.0, and dilute with water to volume. Pass the solution
enoxaparin sodium solution by a mixture of heparinases. Af- through a filter having a porosity of 0.45 or 0.22 m.
ter enzymatic depolymerization, the main 1,6-anhydro resi- Sodium Borohydride SolutionDissolve 12 mg of sodium
dues of enoxaparin sodium observed are 1,6-anhydro IIS borohydride in 400 L of water, and mix on a vortex mixer.
and 1,6-anhydro IISepi, and 1,6-anhydro IS and 1,6- [NOTEPrepare fresh immediately before use.]
anhydro ISISepi (see Appendix 2.). Heparinase 1 SolutionDissolve heparinase 1 (see Reagent
The 1,6-anhydro ISISepi tetrasaccharide (2-O-sulfated Specifications under Reagents, Indicators, and Solutions) [ref-
mannosamine form) is not completely cleaved by the erence: heparin lyase I, EC 4.2.2.7] in Potassium Phosphate
heparinases. The two disaccharides (1,6-anhydro IIS and pH 7.0 Buffer to obtain a solution having an activity of 0.4
1,6-anhydro IISepi), which generally co-elute, are poorly re- IU per mL. Store the solution at 20 until ready to use.
solved with respect to IIA (see Appendix 1), especially be- [NOTEHeparinase solutions can be stored for 3 months at
cause the latter occurs as two anomers: and . To allow 20.]
quantitation of 1,6-anhydro IIS and 1,6-anhydro IISepi, the Heparinase 2 SolutionDissolve heparinase 2 (see Reagent
enoxaparin sodium sample already depolymerized by Specifications under Reagents, Indicators, and Solutions [no
heparinases is then reduced by sodium borohydride (see Fig- EC number] in Potassium Phosphate pH 7.0 Buffer to obtain a
ure 2). solution having an activity of 0.4 IU per mL. Store the solu-
tion at 20 until ready to use.
Heparinase 3 SolutionDissolve heparinase 3 (see Reagent
Specifications under Reagents, Indicators, and Solutions [refer-
ence: heparitinase I, EC 4.2.2.8] in Potassium Phosphate pH

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 22:26:35 EST 2011

USP 35 Chemical Tests / 207 1,6-Anhydro Derivative 137

7.0 Buffer to obtain a solution having an activity of 0.4 IU Time Solution A Solution B
per mL. Store the solution at 20 until ready to use. (minutes) (%) (%) Elution
Heparinases 1, 2, 3, SolutionPrepare a 1:1:1 (v:v:v) mix- 020 9765 335 Linear gradient
ture of Heparinase 1 Solution, Heparinase 2 Solution, and 2050 650 35100 Linear gradient
Heparinase 3 Solution. 5060 0 100 Isocratic
Peak Identification Solutions[NOTEThe depolymer- 6061 097 1003 Linear gradient for
ized test solutions and Standard solutions must be prepared re-equilibration
at the same time. Depolymerized test solutions are stable for 6179 97 3 Isocratic for re-equili-
1 month at 20. Also, the reduced test solutions and Stan- bration
dard solutions must be prepared at the same time. Reduced
solutions are also stable for 1 month at 20.] Chromatograph the reduced Test Solution 3 and the reduced
Disaccharide SolutionsSeparately prepare a 0.25 mg per Standard Solution 3, and record the peak responses as di-
mL solution of each disaccharide1 IA, IIA, IIIA, IVA, IS, rected for Procedure.
IIS, IIIS, IVS (see Appendix 1). Chromatograph each di- Depolymerization Suitability TestThe ratio of the peak
saccharide solution, and record the peak responses. area of 1,6-anhydro-IS-IS to that of 1,6-anhydro IS is not
Reduced Disaccharide SolutionsTo 60 L of each Disac- more than 1.15 for the depolymerized Standard Solution 2.
charide Solution, add 10 L of freshly prepared Sodium Boro- Column Performance Suitability TestIdentify the peaks
hydride Solution. Mix on a vortex mixer, and allow to stand corresponding to reduced IA and 1,6-anhydro-IS for the
at room temperature for at least 4 hours. Chromatograph Standard Solution 3: the retention time of reduced IS is
each solution, and record the peak response. between 27 and 33 minutes for the depolymerized and re-
Blank SolutionPrepare a mixture of 20 L of water, 70 duced Standard Solution 3; and the resolution, R, between
L of Sodium/Calcium Acetate pH 7.0 Solution, and 100 L of reduced IA and 1,6-anhydro-IS is not less than 1.5.
the Heparinases 1, 2, 3 Solution. Mix gently by inversion, Reduction Suitability TestThe ratio of the peak area of
and allow to stand for at least 48 hours in a 25 water bath. IS disaccharide to that of reduced IS in the depolymer-
Prepare a mixture of 60 L of this depolymerized solution ized and reduced Standard Solution 3 and Test Solution 3 is
with 10 L of freshly prepared Sodium Borohydride Solution. not more than 0.02%.
Homogenize, and allow to stand at room temperature for at Procedure/CalculationSeparately inject equal volumes of
least 4 hours. Chromatograph the resulting solution, and the reduced Test Solutions 3 and the reduced Standard Solu-
record the peak responses. tion 3 into the chromatograph. Use the normalized area
Test Solution 1Prepare two solutions, each containing percentage method for calculation. Each peak is integrated
20 mg of enoxaparin sodium in 1 mL of water. from the dwell volume peak to the last detected peak.
Standard Solution 1Prepare one solution containing 20 Measure the area of each analyte peak after excluding sol-
mg of USP Enoxaparin Sodium RS in 1 mL of water. vent peaks at the beginning of the chromatogram and in
Test Solution 2For each solution, prepare a mixture of the Blank Solution. Using the previously obtained chromato-
20 L of Test Solution 1, 70 L of Sodium/Calcium Acetate pH grams of the Reduced Disaccharide Solutions, identify peaks
7 Solution, and 100 L of Heparinases 1, 2, 3 Solution. Mix belonging to the eight reduced disaccharides in the chro-
gently by inversion, and allow to stand for at least 48 hours matograms for Test Solution 3 and Standard Solution 3. The
in a 25 water bath. After 48 hours of depolymerization, peaks belonging to 1,6-Anhydro IS, 1,6-Anhydro IIS, and
chromatograph the solution, and record the peak responses. 1,6-Anhydro IS-ISepi are identified from the relative reten-
Standard Solution 2 Prepare a mixture of 20 L of Stan- tion times provided in Table 1 and the Reference chromato-
dard Solution 1, 70 L of Sodium/Calcium Acetate pH 7 Solu- gram provided with USP Enoxaparin Sodium RS. Once the
tion, and 100 L of Heparinases 1, 2, 3 Solution. Mix gently, peaks have been identified, use the values in Table 1 to cal-
and allow to stand for at least 48 hours in a 25 water bath. culate the (w/w) percentage of the three main 1,6-anhydro
After 48 hours of depolymerization, chromatograph the so- derivatives obtained after depolymerization of enoxaparin
lution, and record the peak responses. sodium using the following formula:
Test Solution 3For each depolymerized test solution, % 1,6-anhydro i (w/w) = (100 MWi Ai)/ (MWx Ax)
prepare a mixture of 60 L of Test Solution 2 and 10 L of
freshly prepared Sodium Borohydride Solution. Homogenize, in which MWi and Ai are the molecular weight and the area
and allow to stand loosely capped at room temperature for of the 1,6-anhydro peak i, respectively; and MWx and Ax are
at least 4 hours before injecting into the chromatograph. the molecular weight and the area, respectively, of either
Test Solution 3 is stable for 48 hours at room temperature. the peak X or the zone X specified by its retention time.
Standard Solution 3Prepare a mixture of 60 L of Stan- [NOTEOnce the method is established, the peaks belong-
dard Solution 2 and 10 L of freshly prepared Sodium Boro- ing to the different di- and tetrasaccharides can be easily
hydride Solution. Homogenize and mix on a vortex mixer, identified using the USP Enoxaparin Sodium RS chromato-
and allow to stand loosely capped at room temperature for gram. Thus, the use of the disaccharide Standards is only
at least 4 hours before injecting into the chromatograph. needed during the method-implementation stage.]
Standard Solution 3 is stable for 48 hours at room Calculate the molar percentage of components containing
temperature. a 1,6-anhydro structure at the reducing end of their chain
Chromatographic System (see Chromatography 621) in the enoxaparin sodium test sample according to the fol-
The liquid chromatograph is equipped with a 234-nm de- lowing formula:
tector and a 3-mm 25-cm column that contains 5-m
packing L14. A guard column packed with the same mate-
rial should also be used. The flow rate is 0.45 mL per min-
ute, the column temperature is maintained at 50, and the
injection volume is 10 L. The chromatograph is pro-
grammed as follows.
1Suitable disaccharides are available from Grampian Enzymes (GE-H1001, GE-
in which MW is the mass-average molecular mass (see Iden-
G1002, GE-H1003, GE-H1004, GE-H1005, GE-H1006, GE-H1007, GE-H1008), tification test D under Enoxaparin Sodium); MWx and Areax
Nisthouse, Harray, Orkney, KW17 2LQ, United Kingdom, Tel: 01856 771771, are the molecular weight and the area, respectively, of ei-
Scottish Local Authority: Orkney Islands. ther the peak X or the range X specified by its retention
time. The molar percentage of components having a 1,6-
anhydro structure at the reducing end of their chain is be-

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 22:26:35 EST 2011

138 207 1,6-Anhydro Derivative / Chemical Tests USP 35

tween 15% and 25%. Typical retention times and molecular Table 1. Typical Relative Retention Times (tRR) and Molecular
masses attributed to different oligosaccharide structures are Masses Attributed to Different Compounds* (Continued)
provided in Table 1. Molecular
Mass
Table 1. Typical Relative Retention Times (tRR) and Molecular Compound tRR (Daltons)
Masses Attributed to Different Compounds* Reduced IA 0.88 605
Molecular 0.88 < tRR < 0.90 635
Mass 1,6-Anhydro IS 0.90 545
Compound tRR (Daltons) 0.90 < tRR <0.98 635
< 0.25 741 Reduced IIA- 0.98 1066
Reduced IVA 0.25 401 IVSglu
0.25 <tRR < 0.51 741 0.98< tRR <1.00 635
Reduced IVS 0.51 461 Reduced IS 1.00 665
0.51 < tRR < 0.55 483 1.00< tRR <1.04 665
Reduced IIA 0.55 503 IS 1.04 665
0.55 < tRR < 0.59 503 1.04< tRR <1.10 1228
1,6-Anhydro IIS 0.59 443 Reduced IIA-IIS- 1.10 1168
0.59 < tRR < 0.64 503 glu
Reduced IIIA 0.64 503 1.10< tRR <1.27 1228
0.64 < tRR < 0.72 533 1,6-Anhydro IS-IS 1.27 1210
Reduced IIS 0.72 563 tRR >1.27 1228
0.72 < tRR < 0.80 563 * Relative retention times were obtained with a depolymerized and
Reduced IIIS 0.80 563 reduced batch of enoxaparin sodium. They are expressed relative to
0.80 < tRR < 0.88 583 the retention time of the main peak corresponding to reduced IS.
Note that according to the quality of the column, relative retention
* Relative retention times were obtained with a depolymerized and times can change slightly.
reduced batch of enoxaparin sodium. They are expressed relative to
the retention time of the main peak corresponding to reduced IS.
Note that according to the quality of the column, relative retention
times can change slightly.

APPENDIX 1: STANDARD DISACCHARIDE STRUCTURES


IVA UA-(14)-GlcNAc

IVS UA-(14)-GlcN(NS)

IIA UA-(14)-GlcNAc(6S)

IIIA UA-2S-(14)-GlcNAc

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 22:26:35 EST 2011

USP 35 Chemical Tests / 207 1,6-Anhydro Derivative 139

APPENDIX 1: STANDARD DISACCHARIDE STRUCTURES (Continued)


IIS UA-(14)-GlcN (NS,6S)

IIIS UA-2S-(14)-GlcN (NS)

IA UA-2S-(14)-GlcNAc(6S)

IS UA-2S-(14)-GlcN (NS,6S)

APPENDIX 2: OLIGOSACCHARIDE STRUCTURES


1,6-Anhydro IS or
1,6-Anhydro IS glucose

1,6-Anhydro IIS or
1,6-Anhydro IIS glucose

1,6-Anhydro IIS epi or


1,6-Anhydro IIS mannose

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 22:26:35 EST 2011

140 207 1,6-Anhydro Derivative / Chemical Tests USP 35

APPENDIX 2: OLIGOSACCHARIDE STRUCTURES (Continued)


IIA-IVSglu

IIA-IISglu

1,6-Anhydro IS-IS epi or


1,6-Anhydro IS-IS mannose

211 ARSENIC
This procedure is designed to determine the presence of
trace amounts of arsenic (As) by converting the arsenic in a
substance under test to arsine, which is then passed
through a solution of silver diethyldithiocarbamate to form a
red complex. The red color so produced is compared, either
visually or spectrophotometrically, to the color produced
similarly in a control containing an amount of arsenic equiv-
alent to the limit given in the individual monograph. Limits
are stated in terms of arsenic (As). The content of arsenic
does not exceed the limit given in the individual
monograph.
Two methods are provided, the methods differing only in
the preliminary treatment of the test substance and the
standard. Generally, Method I is used for inorganic materials,
while Method II is used for organic materials.
Apparatus
The apparatus (see illustration) consists of an arsine gener-
ator (a) fitted with a scrubber unit (c) and an absorber tube
(e) with standard-taper or ground glass ball-and-socket Arsenic Test Apparatus
joints (b and d) between the units. However, any other suit-
able apparatus, embodying the principle of the assembly
described and illustrated, may be used. Arsenic Trioxide Stock SolutionDissolve 132.0 mg of
arsenic trioxide, previously dried at 105 for 1 hour and
accurately weighed, in 5 mL of sodium hydroxide solution
(1 in 5) in a 1000-mL volumetric flask. Neutralize the solu-
tion with 2 N sulfuric acid, add 10 mL more of 2 N sulfuric
acid, then add recently boiled and cooled water to volume,
and mix.
Standard Arsenic SolutionTransfer 10.0 mL of Arsenic
Trioxide Stock Solution to a 1000-mL volumetric flask, add 10
mL of 2 N sulfuric acid, then add recently boiled and cooled
water to volume, and mix. Each mL of Standard Arsenic So-
lution contains the equivalent of 1 g of arsenic (As). Keep
this solution in an all-glass container, and use within 3 days.

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.