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TNE.NO.09W.(1)16.800.JUN.TR

TM

OHpak SB-800 HQ series

Analysis of the polymer regarding pharmaceuticals using

No.9TECHNICAL NOTEBOOK

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TM

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Contents

1. Introduction

2. Basic characteristic of ShodexTM OHpak SB-800 HQ

2-1. Specification of SB-800 HQ series

2-2. Calibration curves

2-3. Target molecular weight range and exclusion limit of SB-800 HQ series

3. Separation mechanism of size exclusion chromatography (SEC)

3-1. Separation mechanism

3-2. Precautions for polar polymer analysis

4. Analysis of pharmaceutical excipients

4-1. Type of pharmaceutical excipients

4-2. Cellulose-type excipient

4-3. Other polysaccharides

4-4. Macrogol

4-5. Gelatin

4-6. Polyvinylpyrrolidone

4-7. Cellulose acetate

5. Mucopolysaccharide (Glycosaminoglycan)

5-1. Mucopolysaccharide

5-2. Heparin

5-3. Chondroitin sulfate

5-4. Hyaluronic acid

6. Information for the analysis of vaccines

1

1

1

2

2

3

3

4

5

5

6

10

13

13

14

16

17

17

17

18

18

19

2-1. Specification of SB-800 HQ series

Column Size (mm)I.D. x Length

Particle Size(µm)

MaximumPore Size

(Å)

MaximumPore Size

(Å)

Product Code Product Name

F6429100

F6429101

F6429102

F6429103

F6429104

F6429105

F6429106

F6709430

Shipping solvent : 0.02% NaN3 aq.

≥ 12,000

≥ 16,000

≥ 16,000

≥ 16,000

≥ 12,000

≥ 12,000

≥ 12,000

(guard column)

OHpak SB-802 HQ

OHpak SB-802.5 HQ

OHpak SB-803 HQ

OHpak SB-804 HQ

OHpak SB-805 HQ

OHpak SB-806 HQ

OHpak SB-806M HQ

OHpak SB-G 6B

8.0 x 300

8.0 x 300

8.0 x 300

8.0 x 300

8.0 x 300

8.0 x 300

8.0 x 300

6.0 x 50

8

6

6

10

13

13

13

10

100

200

800

2,000

7,000

15,000

15,000

—

Plate Number(TP/column)

Column Size (mm)I.D. x Length

Particle Size(µm)

Product Code Product Name

F6429108

F6709431

Shipping solvent : H2O(Common)

Base material : Polyhydroxymethacrylate

Usable pH range : pH 3 - 10

≥ 1,500

(guard column)

OHpak SB-807 HQ

OHpak SB-807G

8.0 x 300

8.0 x 50

35

35

30,000

—

Plate Number(TP/column)

Table 1. Specification of SB-800 HQ series

Product NameMethanol

The maximum usable concentration (%)

SB-802 HQ

SB-802.5 HQ, SB-803 HQ

SB-804 HQ ~ SB-806M HQ

SB-G 6B

SB-807 HQ, SB-807G

0

100

75

75

30

Acetonitrile

0

75

75

75

30

DMF

0

100

100

100

0

Table 2. Usable concentration of organic solvents

- 1 -

1. Introduction

2. Basic characteristic of ShodexTM OHpak SB-800 HQ

Pharmaceutical-relevant polymers are active ingredients comparable to chondroitin sulfate or heparin, as well as pharmaceutical excipients. The pharmaceutical excipients are classified into several classes: binder, excipient, disintegrant, thickening agent and coating agent. Many different polymers are used as pharmaceutical excipients.Size exclusion chromatography (SEC) is useful for the analysis of polymers. This technical notebook introduces pharmaceutical applications with polymer-based aqueous SEC columns: ShodexTM OHpak SB-800 HQ series.


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54 6 7 8 9 10 11 12

103

102

104

105

106

107

Elution volume (mL)4 5 6 7 8 9 10 11 12

Elution volume (mL)

Mol

ecul

ar w

eigh

t (P

EG

/PE

O)

Mol

ecul

ar w

eigh

t (P

ullu

lan)

103

102

104

105

106

107

- 3 -- 2 -

SB-806 HQ

SB-805 HQ

SB-804 HQ

SB

-806M H

Q

SB-803 HQ

SB-802.5 HQSB-802 HQ

SB-806 H

Q

SB-805 HQ

SB-804 HQ

SB

-806M H

Q

SB-803 HQ

SB-802.5 HQ

Figure 1. Calibration curves by pullulan Figure 2. Calibration curves by PEG/PEO

Figure 3. The principle of SEC

Column : Shodex OHpak SB-800 HQ seriesEluent : H2OFlow rate : 1.0mL/minDetector : RIColumn temp. : 30˚C

Column : Shodex OHpak SB-800 HQ series Eluent : DMFFlow rate : 1.0mL/minDetector : RIColumn temp. : 40˚C

2-2. Calibration curves

3-1. Separation mechanism

2-3. Target molecular weight range and exclusion limit of SB-800 HQ series

Product Name Exclusion LimitTarget Molecular Weight Range

SB-802 HQ

SB-802.5 HQ

SB-803 HQ

SB-804 HQ

SB-805 HQ

SB-806 HQ

SB-806M HQ

SB-807 HQ

*( ) Estimated value

*( ) Estimated valueDMF can not be used for SB-802 HQ

200 - 1,000

500 - 10,000

1,000 - 100,000

5,000 - 400,000

100,000 - 1,000,000

100,000 - *(20,000,000)

500 - *(20,000,000)

500,000 - *(500,000,000)

1000

10000

100000

1000000

*(4,000,000)

*(20,000,000)

*(20,000,000)

*(500,000,000)

Table 3. Molecular range of Pullulan (Eluent : H2O)

Product Name Target Molecular Weight Range

SB-802.5 HQ

SB-803 HQ

SB-804 HQ

SB-805 HQ

SB-806 HQ

SB-806M HQ

100 - 2,000

200 - 40,000

500 - 300,000

50,000 - 700,000

70,000 - *(20,000,000)

200 - *(20,000,000)

Table 4. Molecular range PEG/PEO (Eluent : DMF)

3. Separation mechanism of size exclusion chromatography (SEC)

The packing material has pores or small cavities on the surface allowing the analytes elute by entering through the pores. The separation mechanism is shown in the figure 3. The analytes are diffused through the pores while larger substances cannot enter into the pores. Samples above the exclusion limit pass outside the packing material and elute from the column over the exclusion limit. Samples smaller than the pores can diffuse into the pores and move to the outside of the packing material. Within the cone like opening, the pore sizes decrease deeper into the opening. The SEC mechanism described above allows the analytes elute in order of size.Assuming that there is no interaction between the packing material and the analyte, the separation mode of SEC depends on the molecular size of the substances making it possible to measure the molecular size and the molecular distribution of polymers.

• Substances are eluted in decreasing order of molecular size.• There is no interaction with the packing material.

Elution volume

The largest substance

The smallest substance

Packing material

SEC : Size Exclusion Chromatography

Exclusion limitvolume


log

MW

(A)

(C) (D)

(B)

- 5 -- 4 -

Figure 4. Interfering interactions likely to be observed

4-1. Type of pharmaceutical excipients

• Excipient

4. Analysis of pharmaceutical excipients

In USP 39 (General Information Chapter, <1078> Good Manufacturing Practices for Bulk Pharmaceutical Excipients), “Pharmaceutical excipients are substances other than the active pharmaceutical ingredient (API) that have been appropriately evaluated for safety and are intentionally included in a drug delivery system. For example, excipients can do the following :

- aid in the processing of the drug delivery system during its manufacture,- protect, support, or enhance stability, bioavailability, or patient acceptability,- assist in product identification, and- enhance any other attribute of the overall safety, effectiveness, or delivery of the drug during storage or use.”

“Type” and “purpose of use” of typical pharmaceutical excipients are shown below.

When the dose of an active ingredient is small, it is added for giving a certain size and a weight for the preparation. Most of cases, the excipient is used not only for increasing the size and the weight but also for the purpose of a binder and a disintegrant.

(Main polymer)Starch, Dextrin, Microcrystalline cellulose, Hydroxypropyl cellulose, Carmellose, Calcium carmellose, Macrogol 20000, etc.

3-2. Precautions for polar polymer analysis

Interactions between the analyte and the packing materials

Hydrophobic interaction The analyte is adsorbed into the packing material. This delays the analyte elution, and thus results in under estimation of its molecular weight (Figure B, D).

Ionic interaction(1) Ion Exclusion The analyte is repelled from the packing material. This accelerates the analyte elution, and thus results in over estimation of its molecular weight (Figure A, C).(2) Ion Exchange The analyte is adsorbed onto the packing material. This delays the analyte elution, and thus results in under estimation of its molecular weight (Figure B, D).

Size exclusion chromatography analysis of polar polymers can be influenced by unexpected interactions in the column.These interactions may change elution patterns and results in an invalid molecular weight calculation.It is important to reduce them in order to obtain the accurate molecular weight distribution.

Interaction within and between the analyte

Ionic repulsion effects observed within the multivalent macromolecules causes structure expansion This accelerates the analyte elution, and thus results in over estimation of its molecular weight (Figure A).

Association between the molecules Associated molecule detected as a larger molecule (Figure A).

Interactions between the analyte and the solvent

The multivalent ion of the solvent works as a bridge to bind ionic molecules (analyte).

Elution will be retarded

Elution will beaccelerated

Exclusion limitmolecular weight

Exclusionlimit volume

Permeationlimit volume

• Binder It is added for giving the binding power with an active ingredient and an excipient for the preparation.

(Main polymer)Hydroxypropyl cellulose, Microcrystalline cellulose, Hydroxypropyl methylcellulose, Methylcellulose, Sodium carboxymethyl cellulose, Poly(vinyl alcohol), Macrogol, Sodium alginate, Dextrin, Gelatin, Arabic gum, etc.

• Disintegrant It is added for promoting the disintegration and the dispersion of a solid prepared granular agent and tablet.

(Main polymer)Starch, Carmellose, Calcium carmellose, Microcrystalline cellulose, Hydroxypropyl starch, Hydroxypropyl cellulose, etc.

• Coating agent Coating agent has a film coating, a sugar coating, and a dry coating. It has the function of masking, stabilization, sustained release, and etc.

(Main polymer)Hypromellose, Hydroxypropyl cellulose, etc.


5 10 15

min

20 25 30

5 10 15

min

20 25 30

5 10 15

min

20 25 30

5 10 15

min

20 25 30

- 7 -- 6 -

4-2. Cellulose-type excipient

Carmellose Other name : Carboxymethyl cellulose (CMC)

Carmellose (Carboxymethyl cellulose) can be used as a binder for tablets, disintegrant or a stabilizer. With these uses, it is widely used in cosmetics and foods. Carmellose is an anionic polysaccharide with carboxyl groups. Anionic samples may elute earlier due to ion exclusion interaction between the sample and packing materials hence leading to a larger than expected calculated molecular weight. The addition of salt to the eluent will decrease the ion exclusion interactions. The effect of increased salt concentration in the eluent is shown for the analysis of carmellose. It was confirmed that peak shape stabilized at a concentration ≥ 50mM NaCl.

Chromatograms of carboxymethyl cellulose of three different viscosities is shown in Figure 6.

Sample : 50µLCarboxymethyl cellulose (400 – 800 cP) 0.1%

CH2OR

Column : Shodex OHpak SB-806M HQ x 2Eluent : H2O or NaCl aq. Flow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

100mM NaCl aq.

50mM NaCl aq.

20mM NaCl aq.

10mM NaCl aq.

5mM NaCl aq.

1,500 - 3,000 cP

Mn : 201,300Mw : 6,940,500Mw/Mn : 34.47

H2O

O O

O OR

OR

R = H or CH2COONa

n

Sample : 50µLCarboxymethyl cellulose 0.1% each

*Molecular weight was determined from the calibration curve of pullulan. *Molecular weight was determined from the calibration curve of pullulan.

1,500 – 3,000 cP

400 – 800 cP

50 – 200 cP

Column : Shodex OHpak SB-806M HQ x 2Eluent : 0.1M NaCl aq. Flow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

Figure 5. Effect of salt concentration for the analysis of carboxymethyl cellulose

Hydroxyethyl cellulose

Hydroxyethyl cellulose, a nonionic polysaccharide, can be used as a binder, thickening agent, as well as other applications. When analyzing a highly polar polymer, the chromatogram may be abnormal due to intermolecular hydrogen bonding. The addition of salt to the eluent will decrease the hydrogen bonding interactions, improving the chromatogram. When water is used as the eluent, hydroxyethyl cellulose is quickly eluted and the chromatogram shows three peaks. The peak shape of hydroxyethyl cellulose stabilizes best at low salt concentration.

Figure 7. Effect of salt concentration for the analysis of hydroxyethyl cellulose

Figure 6. Analysis of carboxymethyl celluloses

Chromatograms of hydroxyethyl cellulose of three different viscosities are shown in Figure 8.

Figure 8. Analysis of hydroxyethyl celluloses

4,500 - 6,500 cP

Mn : 149,400Mw : 3,829,200Mw/Mn : 25.64

800 - 1,500 cP

Mn : 130,900Mw : 2,003,800Mw/Mn : 15.30

200 - 300 cP

Mn : 100,600Mw : 1,018,600Mw/Mn : 10.12

400 - 800 cP

Mn : 105,400Mw : 2,126,000Mw/Mn : 20.18

50 - 200 cP

Mn : 64,600Mw : 402,000Mw/Mn : 6.22

Sample : 50µLHydroxyethyl cellulose (800 – 1,500 cP) 0.1%

CH2OR

Column : Shodex OHpak SB-806M HQ x 2Eluent : H2O or NaCl aq. Flow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

100mM NaCl aq.

50mM NaCl aq.

20mM NaCl aq.

10mM NaCl aq.

5mM NaCl aq.

H2O

O

O OR

OR

R = H or (CH2CH2O)mH

n

O

Sample : 50µLHydroxyethyl cellulose 0.1% each

4,500 – 6,500 cP

800 – 1,500 cP

200 – 300 cP



5 10 15

min

20 25 30

CH2OCH3

HH

O O

n

O OCH3

OCH3H

HH

5 10 15

min

20 25 30

5 10 15

min

20 25 30

- 9 -- 8 -

Methylcellulose

Methylcellulose is a long chain cellulose with ~30 % substitution of the hydroxyl groups as a methyl ether. High viscosity methylcellulose is used as a thickener and emulsifier, while medium and low viscosity methylcellulose are used as a tablet binder.

*Molecular weight was determined from the calibration curve of pullulan.



Figure 9. Analysis of methylcelluloses

Hypromellose Other name : Hydroxypropyl methylcellulose

Hypromellose (hydroxypropyl methylcellulose) is an ether of cellulose which is partially hydroxymethylated and hydroxypropylated. Classes of hypromellose differ in their viscosity and substitution degree, allowing its widely usage in oral and topical formulations and is used as coating agents, film-forming agents, binders, thickening agents, etc.

Figure 11. Analysis of hydroxypropyl methylcelluloses

400 cP

Mn : 57,800Mw : 457,300Mw/Mn : 7.90

80 - 120 cP

Mn : 45,200Mw : 390,800Mw/Mn : 8.65

2,600 - 5,600 cP

Mn : 140,500Mw : 1,351,800Mw/Mn : 9.62

4,000 cP

Mn : 69,100Mw : 978,300Mw/Mn : 14.15

Mw > 100,000

Mn : 8,800Mw : 123,600Mw/Mn : 14.06

Mw > 1,000,000

Mn : 123,500Mw : 2,395,600Mw/Mn : 19.40

Sample : 50µLMethylcellulose 0.1% each


400 cP

4,000 cP

Hydroxypropyl cellulose

Hydroxypropyl cellulose is an ether of cellulose in which hydroxyl groups are hydroxypropylated. There are many classes of hydroxypropyl cellulose which differ in viscosity. Hydroxypropyl cellulose is used as coating agents, stabilizers, binders, etc.

Figure 10. Analysis of hydroxypropyl celluloses

R = H or (CH2CH(CH3)O)mH

Sample : 50µLHydroxypropyl cellulose 0.1% each


Mw > 100,000

Mw > 1,000,000

CH2OR

CH2OR

O

OO OR

OR

OR

OR

n

O

Sample : 50µLHydroxypropyl methylcellulose 0.1% each


80 – 120 cP

2,600 – 5,600 cP

R = H, CH3 or CH3CH(OH)CH2

CH2OR

CH2OR

O

OO OR

OR

OR

OR

n

O


50 10 15

min

20 25 30

50 10 15

min

20 25 30

50 10 15

min

20 25 30

50 10 15

min

20 25 30

- 11 -- 10 -

Dextran

Dextran is a polysaccharide which is composed with only glucose, and contains a large amount of α-1,6 glycosidic linkages. Dextran is used as a stabilizer or a thickening agent.


Figure 12. Analysis of dextrans

Carrageenan

Carrageenans are a family of sulfated polysaccharides extracted from red algae. It can be found in three different forms: Lambda (λ)-, Kappa (κ)-, and Iota (ι)-. These three types are different in the feature of gelation.

Figure 14. Analysis of carrageenans

Mn : 78,500Mw : 210,800Mw/Mn : 2.68

Arabic gum

Arabic gum, isolated from acacia sap, is an acidic heteropolysaccharide which is constituted arabinose, galactose, rhamnose and glucuronic acid. The viscosity of arabic gum is much lower than that of other water-soluble polysaccharides allowing it to be used as a binder or coating agent.

Figure 13. Analysis of arabic gum

Sodium alginate

Sodium alginate is a polyuronic acid which consists of D-mannuronic acid and L-guluronic acid. Sodium alginate is used as a stabilizer, a binder, as well as many other applications.

Figure 15. Analysis of sodium alginates

4-3. Other polysaccharides

Sample : 0.1% each, 200µL

Column : Shodex OHpak SB-806M HQ x 2Eluent : 0.1M NaNO3 aq. Flow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

Dextran 10000

Dextran 40000

Dextran 500000

CH2

CH2

OHO

HOHO

HO

OHOH

n

O O

O

Sample : 50µLArabic gum 0.1%




κ- Carrageenan

CH2OH

CH2OH CH2OSO3-

OSO3- OSO3

-

CH2-O3SO

n

O O

O

HO

O

OO

OH OH

λ- Carrageenan

OH

n

OO

O

O

O

100 – 150 cP

500 – 600 cP

Sample : 100µLSodium alginate 0.1% each



50 10 15min

20 25 3050

1

2

3

4

5

10 15min

20 25

5 10 15min

20 25 30

5 10 15min

20 25 30

- 13 -- 12 -

Pectin

Pectin is a heteropolysaccharide which has a backbone composed of α-1,4- glycosidic bound galacturonic acids; a majority of the carboxyl groups of galacturonic acids are esterified with methoxy group. Pectin is extracted from the endothelial of citrus or the pomace of apples with dilute acid. Pectin is used as a binder, suspending agent and adhesive.

*Molecular weight was determined from the calibration curve of pullulan. *Molecular weight was determined from the calibration curve of pullulan.

*After dissolving sample with 50mM acetic acid aq., add sodium nitrate to a final concentration of 0.3M.

Figure 16. Analysis of pectins

Macrogols with low molecular weights (PEG 200 - 600) are liquid and high molecular weights (larger than PEG 1000) are solid. The characteristic and the use of macrogol are different according to the molecular weight. Macrogols with different molecular weights were separated simultaneously. (Figure 18)

Figure 18. Analysis of macrogols

Chitosan

Chitosan is a polysaccharide containing D-glucosamine and N-acetyl-D-glucosamine. Chitosan is used as a coating agent, a disintegrant, a binder, etc. Although chitosan has limited solubility in water it is easily dissolved in many organic acids, such as acetic acid. Chitosan may carry a positive charge in acetic acid solution and it may be adsorbed to the packing materials. Therefore, for reducing ion suppression between chitosan and the packing materials, sodium nitrate was added to the eluent.

Figure 17. Analysis of chitosans

4-4. Macrogol Other name : Polyethyleneglycol (PEG)

Sample : 10µL1. PEG 200002. PEG 40003. PEG 10004. PEG 4005. Ethylene glycol

Column : Shodex OHpak SB-803 HQEluent : H2OFlow rate : 0.5mL/min Detector : RI Column temp. : 30˚C

HO OHC ( )O mCH2 CH2

H

H

C

H

H

Gelatin is a purified protein which is made by partial acid hydrolysis or partial alkaline hydrolysis of animal collagen, and is used for stabilizer, lubricant, binder, brightening agent, or coating agent. Figure 19 shows the analysis of gelatins from animals.

Figure 19. Analysis of gelatins

4-5. Gelatin

Column : Shodex OHpak SB-806M HQ x 2Eluent : 0.1M KH2PO4 aq./0.1M Na2HPO4 aq. =50/50 Flow rate : 1.0mL/min Detector : RIColumn temp. : 40˚C



Pectin from Citrus

Pectin from Apple


Column : Shodex OHpak SB-806M HQ x 2Eluent : 50mM CH3COOH + 0.3M NaNO3 aq. Flow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

CH2OHO

O

NHR n R=H or CH2COONa

5 - 20 Pa •S

Mn : 19,400Mw : 187,900Mw/Mn : 9.69

Chitosan ( 5 - 20 Pa·S*)

Chitosan ( 50 - 100 Pa·S*)

Chitosan (200 - 600 Pa·S*)

From bovine skin

Mn : 21,400Mw : 108,100Mw/Mn : 5.05

From porcine skin

Mn : 11,500Mw : 83,700Mw/Mn : 7.28

50 - 100 Pa •S

Mn : 165,200Mw : 896,100Mw/Mn : 5.42

200 - 600 Pa •S

Mn : 314,000Mw : 3,309,600Mw/Mn : 10.54


Gelatin from bovine skin (Acid treatment, Gel strength : 225g)

Gelatin from porcine skin(Alkali treatment, Gel strength : 90-100g)


5 10 15min

20 25 30

5 10 15min

20 25 30

- 15 -

Povidone

Povidone is a linear aqueous polymer composed of N-vinyl-2-pyrolidone, and is divided from K-10 to K-120 by viscosity. When povidone is analyzed using an aqueous eluent, the retention time is delayed or the peak area is lessened due to the hydrophobic interaction between povidone and the packing material. To compensate for the hydrophobic interaction, the addition of acetonitrile into the eluent has been found effective. Figure 20 shows the comparison of the peak shapes by changing the acetonitrile concentration of the eluent from 0% (only 0.1M NaCl solution) to 50%. In case of K-30, best results were obtained from 30% to 45% concentration, but the retention time was delayed with 50% acetonitrile because of the hydrophilic interaction between K-30 and the packing materials.

Figure 20. Analysis of povidone with the dependence of acetonitrile concentration

Figure 21 shows the chromatograms of several kinds of povidone.

Figure 21. Analysis of povidones

Figure 22. Analysis of test drug sample Figure 23. Analysis of HPC sample

Polyvinylpyrrolidone is divided into three classes by the structure: Povidone, Crospovidone and Copovidone. In this part, povidone and copovidone have been studied.

4-6. Polyvinylpyrrolidone (PVP)

Sample : 100µLPolyvinylpyrrolidone 0.1% each

Column : Shodex OHpak SB-806M HQ x 2Eluent : 0.1M NaCl aq./CH3CN=55/45Flow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

Column : Shodex OHpak SB-806M HQEluent : 1.5% DMF/H2OFlow rate : 1.0mL/min Detector : DiscovIR-LC™Column temp. : 40˚C

Column : Shodex OHpak SB-806M HQEluent : 1.5% DMF/H2OFlow rate : 1.0mL/min Detector : DiscovIR-LC™Column temp. : 40˚C

Excipients are pharmacologically inactive substances used as carriers for the active pharmaceutical ingredient (API). A significant amount (30-80% solid) of polymeric excipients such as hydroxypropyl methylcellulose (HPMC), hydroxylpropyl cellulose (HPC), and povidone are generally used. Those excipients have very large molecular weights (MW) which is not suitable for the commonly used drug QC method by LC-MS. Whereas LC-IR is feasible of characterizing any polymeric excipients in all drug forms. OHpak SB-806M HQ was used to separate excipients and API based on their sizes. Then size-separated components were detected by IR which provides compositional fingerprinting information. The method is useful in the areas such as drug production quality control, drug deformulation, and/or counterfeit drug testing.Figure 22 shows there were cellulose (Peak A; MW=~485K Da) and two different MW povidones (Peak A; MW=~485K Da and Peak B; MW=~49K Da) used as excipients in the tested drug sample.Figure 23 shows there were HPC excipient having two different MW distributions (MW information not available).

Sample : 100µLPolyvinylpyrrolidone (K-30) 0.1%

Column : Shodex OHpak SB-806M HQ x 2Eluent : 0.1M NaCl aq./CH3CNFlow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

50 / 50

55 / 45

60 / 40

70 / 30

75 / 25

80 / 20

85 / 15

90 / 10

95 / 5

100 / 0

0.1M NaCl aq./CH3CN

N

nCH CH2

Figure 22 and 23 Data was provided by Dr. Ming Zhou, Spectra Analysis Instruments, Inc. www.spectra-analysis.com

K-15

K-30

K-60

K-90

*Molecular weight was determined from the calibration curve of PEG/PEO.

K-15

Mn : 1,300Mw : 2,800Mw/Mn : 2.18

K-30

Mn : 5,500Mw : 15,800Mw/Mn : 2.88

K-60

Mn : 21,500Mw : 154,000Mw/Mn : 7.17

K-90

Mn : 63,900Mw : 530,400Mw/Mn : 8.29

- 14 -


5 10 15min

20 25

5 10 15min

20 25

5 10 15

min

20 25 30

- 17 -

Cellulose acetate can be used for coating agent or sustained release agent. Cellulose acetate has very low water solubility, but it is easily dissolved into DMF or dioxiane. DMF was used as the eluent and LiBr was added for analyzing cellulose acetate in Figure 25.

4-7. Cellulose acetate

*Molecular weight was determined from the calibration curve of PEG/PEO.

Copolymer 7:3

Mn~30,000

Mn~50,000

Mn : 2,000Mw : 14,400Mw/Mn : 7.40

Mn : 20,200Mw : 31,200Mw/Mn : 1.54

Copolymer 3:7

Mn : 6,400Mw : 28,900Mw/Mn : 4.53

- 16 -

Copovidone

Copovidone is co-polymer made by N-vinyl-2-pyrolidone and vinyl acetate, commonly used for coating agent or binder. Copovidone is insoluble in water, so dimethyl formamide (DMF) was used as the eluent with the addition of salt (lithium bromide) for optimal results. (Figure 24)

Figure 24. Analysis of copovidones

Sample : 100µLPoly(1-vinylpyrrolidone-co-vinyl acetate) 0.1% each

Column : Shodex OHpak SB-806M HQ x 2Eluent : 20mM LiBr in DMFFlow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

Copolymer 7:3

Copolymer 3:7


Figure 25. Analysis of cellulose acetates

Figure 26. Analysis of heparin

Sample : 100µLCellulose acetate 0.1% each

Column : Shodex OHpak SB-806M HQ x 2Eluent : 20mM LiBr in DMFFlow rate : 1.0mL/min Detector : RI Column temp. : 40˚C

Mn ～30,000

Mn ～50,000

5. Mucopolysaccharide (Glycosaminoglycan)

5-1. Mucopolysaccharide

Mucopolysaccharide is a long unbranched polysaccharide composed of a repeating disaccharide unit with sulfate groups and it is found in connective tissue in animals. The repeating disaccharide consists of amino sugar, uronic acid, or galactose. Mucopolysaccharide is usually present as a proteoglycan.Mucopolysaccharide has been suggested to have function-retaining effect of organizational structure, lubricating function, etc. Common mucopolysaccharides are heparin, hyaluronic acid, chondroitin sulfate, etc. Constituent sugars of mucopolysaccharides are shown in table 3.

5-2. Heparin

Heparin is an acidic mucopolysaccharide which consists of D-glucosamine and D-glucuronic acid (or L-iduronic acid) prepared from bovine lungs or pig intestinal mucosa and it is used for treatment and recurrence prevention of disseminated intravascular coagulation (DIC), treatment and prevention of phlebothrombosis and inhibition of blood coagulation when using extracorporeal circulation apparatus or catheter.

Mucopolysaccharides Amino sugars Uronic acids

Heparin

Chondroitin-4-sulfate (Chondroitin sulfate A)

Chondroitin-6-sulfate (Chondroitin sulfate C)

Dermatan sulfate (Chondroitin sulfate B)

Hyaluronic acid

D- Glucosamine

N-Acetyl-D-galactosamine




D-Glucuronic acid or L-Iduronic acid

D-Glucuronic acid

D-Glucuronic acid

L-Iduronic acid

D-Glucuronic acid

Sample : 50µLHeparin sodium 0.1%


Table 5. List of constituent sugars of mucopolysaccharides


50 10 15

min

min

20 25 30

50 10 15 20 25 30

- 18 -

Figure 27. Analysis of chondroitin sulfates


6. Information for the analysis of vaccines

OHpak SB-800 HQ series has been used for the analysis of various conjugate vaccines for preventing bacterial infection.

5-3. Chondroitin sulfate

Chondroitin sulfate is an acidic mucopolysaccharide which has a chain alternating disaccharide units composed of N-acetyl-D-galactosamine and D-glucuronic acid; found in animal tissues. There are seven kinds of chondroitin sulfate (A, B, C, D, E, H and K) which differ in the positions and the number of sulfate groups. Chondroitin sulfate B, dermatan sulfate, contains the L-iduronic acids in place of D-glucuronic acids as uronic acids.

Figure 28. Analysis of hyaluronic acids

5-4. Hyaluronic acid

Hyaluronic acid is an anionic mucopolysaccharide which has a chain alternating disaccharide units composed of N-acetyl-D-glucosamine and D-glucuronic acid; it is found in synovial fluid, and umbilical cord, skin and eye tissue. Unlike other mucopolysaccharides, hyaluronic acid does not attach to proteins to form proteoglycans. Hyaluronic acid is used to improve joint function, an ophthalmological operation adjuvant, an endoscopic submucosal injectant and ophthalmic solution.



Chondroitin sulfate A(Chondroitin-4-sulfate)

COOHHO3SO

CH2OH

CH2OSO3H

HO3SO

CH2OH

n

OO

OH

OO

OH NHAc

Chondroitin sulfate B(Dermatan sulfate)

Chondroitin sulfate C(Chondroitin-6-sulfate)

n

n

O

O

O

O

O

OCOOH

COOH

OH

OH

HO

OH

OH

NHAc

NHAc

CH2OH

n

OO O

OOCOONa

HOOH

OH

NHCOCH3

OO

from Pig Skin

Mn : 153,600Mw : 507,800Mw/Mn : 3.31

Sample : 50µLHyaluronic acid sodium salt 0.1% each

from Pig Skin

from Rooster Comb

fron Human Ymbilical Cord


from Rooster Comb

Mn : 648,700Mw : 4,685,300Mw/Mn : 7.22

from Human Umbilical Cord

Mn : 884,600Mw : 5,817,000Mw/Mn : 6.58

O

[ Example ]

• Column : OHpak SB-804 HQ, SB-805 HQ, SB-G

• Reference : Biologicals. 2014, vol.42, p.160-168.

• Title : Process development and immunogenicity studies on a serogroup ‘X’ Meningococcal polysaccharide conjugate vaccine

• Author : Srinvas Reddy Chilukuri etc. (Serum Institute of India Ltd., India)

I. Neisseria meningitides

[ Example ]

• Column : OHpak SB-804 HQ, SB-805 HQ, SB-G

• Reference : Vaccine. 2012, vol.30, p.4897-4906.

• Title : Development and technology transfer of Haemophilus influenzae type b conjugate vaccines for developing countries

• Author : Michel Beurret etc. (National institute for public Health and the Environment, Netherlands)

II. Haemophilus influenzae type b

[ Example ]

• Column : OHpak SB-806M HQ

• Reference : Vaccine. 2014, vol.32, p.5755-5760.

• Title : Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumonia as the carrier protein

• Author : Neha Kothari(International Vaccine Institute, Korea), Kristopher R. Genschmer (University of Alabama, USA) etc.

III. Streptococcus pneumonia

- 19 -


- 20 -

Sadao Mori. Saizuhaijyokuromatogurafi – Kobunshi no kosokuekitai kuromatogurafi.

Kyoritsu Shuppan Co., Ltd., 1992, 192p.

“USP39-NF34”, The United States Pharmacopeial Convention, Rockville,MD, 2016, p. 680-683.

International Pharmaceutical Excipients Council Japan. Handbook of PHARMACEUTICAL

EXCIPENTS Fifth Edition. YAKUJI NIPPO LIMITED, 2007, 1150p.

International Pharmaceutical Excipients Council Japan. Iyakuhintenkabutsujiten 2007.

YAKUJI NIPPO LIMITED, 2007, 462p.

Takashi Sugawara. Iyakuhin·keshohinbunya ni okeru tenkazai no sentaku·shohorei to

saishinkiseishinseitaio. Technical information institute Co,. Ltd, 2008, 228p.

Yutaka Yahaba. Tenkazai no tokusei·erabikata·tsukaikata nouhaushu.

Technical information institute Co,. Ltd, 2012, 357p.

References

“The Japanese Pharmacopoeia Sixteenth Edition”. Pharmaceuticals and Medical Devices

Agency(PMDA)．2011-3-24

http://www.pmda.go.jp/kyokuhou/YAKKYOKUHOU16.pdf (Reference 2014-08-25)

“Mukotato ni tsuite [About mucopolysaccharide]”． Japan Food Research Laboratories．2009-04

http://www.jfrl.or.jp/jfrlnews/files/news_vol3_no4.pdf (Reference 2014-08-25)

Reference websites

[Caution]

1. Please read the operating manual included on the product carefully before use.

2. For improvement purposes, some specifications are subject to change without notice.

3. Provided to help you select the appropriate column, the figures and descriptions in this technical notebook are not guaranteed and do not warrant suitability for your applications.

4. It is essential to take normal precautions when handling reagents and other chemical products even if the safety information is not included on the operating manual.

5. Products described in this brochure are not intended for medical use or medical applications including medical diagnosis.

The following names are trademarks or registered trademarks of SHOWA DENKO K.K.

Shodex, AFpak, Asahipak, AXpak, CLNpak, CXpak, HILICpak, MSpak, ODP, ODSpak,

OHpak, ORpak, RSpak, SUGAR, USPpak


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