quantitative estimation of dextran conjugated ppi

www.wjpps.com Vol 6, Issue 4, 2017.

1260

Manisha et al. World Journal of Pharmacy and Pharmaceutical Sciences

QUANTITATIVE ESTIMATION OF DEXTRAN CONJUGATED PPI

DENDRIMER FOR DELIVERY OF DOXORUBICIN

HYDROCHLORIDE AS AN ANTICANCER DRUG

Kumari Manisha*1, Dr. Agrawal Amit

1, Mishra T. S.

1, Kumari Mamta

2 and

Bhawarker Swati3

*1Patel College of Pharmacy, Ratibad, Bhopal, Madhya Pradesh, India-462044.

2Lakshmi Narain College of Pharmacy, Raisen, Bhopal, Madhya Pradesh, India-462044.

3Ravishankar College of Pharmacy, Bhanpura, Bhopal, Madhya Pradesh, India-462044.

ABSTRACT

The major drawbacks associated with anticancer drugs are their toxic

effects and non-availability of appropriate dose at the desired site.

Conventional mode of administration of such bioactives leads to their

interaction with cancerous as well as normal cells. This not only

precipitates toxicity but also adds to considerable loss of dose before

reaching the desired site. Any attempt to slightly modify the chemical

structure of the drug to achieve the results may reduce its efficacy. To

avoid the possible hazards in cancer chemotherapy we selected novel

dextran conjugated PPI dendritic systems as drug carriers. The drug

was identified for any impurities by chemical tests, UV scanning and

IR spectroscopy. The absorption maxima of the drug Doxorubicin Hydrochloride in methanol

and was found to be 480.2 nm. The various peaks obtained in the FTIR spectrum matched

with the IR spectrum. The standard curves of Doxorubicin HCl were prepared in different

media and the absorbance data obtained was subjected to linear regression. The equation of

line obtained were Y = 0.0161X + 0.0001, Y = 0.0161X - 0.0044, Y = 0.0179X + 0.0017 and

the correlation coefficients were found to be 0.9977, 0.9942, 0.9987, respectively for standard

curve of drug in water, PBS (pH 7.4) and phosphate buffer (pH 6.4) which are close to 1.0

indicating good linearity. The partition coefficient values obtained were 0.520 in n-

octanol:distilled water and 0.441 in n-octanol:PBS (pH 7.4). This concludes that the drug in

its salt form is aqueous soluble in nature and can be assayed for various parameters in water

as base using the standard curve made in UV range. The outcome of the present study

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 4, 1260-1273 Research Article ISSN 2278 – 4357

*Corresponding Author

Kumari Manisha

Patel College of

Pharmacy, Ratibad,

Bhopal, Madhya Pradesh,

India-462044.

Article Received on

30 Jan. 2017,

Revised on 19 Feb. 2017, Accepted on 12 March 2017

DOI: 10.20959/wjpps20174-8893


1261


suggests that the macromolecule conjugated dendrimers extended the circulation time and the

tumor-specific delivery of the bioactive.

KEYWORD: DENDRIMERS, PPI, ANTICANCER, UV SPECTROSCOPY, IR

SPECTRUM PARTITION COEFFICIENT.

INTRODUCTION

Cancer is the state that is characterized by spontaneous outgrowth of abnormal mass of cells.

The unpredictable microenvironment of the cancerous cells in all of its existing forms i.e.

leukemic cells, solid tumors and sarcomas is well documented. This phenomenon expressed

by cancerous sites in the body poses various obstacles towards drug’s efficacy. Under normal

conditions, the cells reproduce, grow, divide, multiply and eventually undergo apoptosis. This

maintains proper balance and functioning of the organs. Cancer is caused in all instances

either by mutation or by some other abnormal activation of cellular genes (oncogenes) that

control cell growth and cell mitosis.[1]

Macromolecules accumulate in tumor tissues because

these tissues have vascular network characterized by both enhanced vascular permeability of

the neovasculature and a lack of lymphatic recovery system[2]

Albumin, globulin and dextran

are examples of natural macromolecules, while synthetic macromolecules include styrene-

maleic anhydride and poly-ethylene glycol.[3]

Dextran conjugates are polysaccharides

(Mol.Wt. > 40 KDa), have low clearance and relatively long plasma half life, display passive

targeting to the tumor tissues due to Enhanced Permeation and Retention of macromolecules

by tumors.[4]

Doxorubicin conjugated to dextran have been evaluated as potential carrier for

delivery of these drugs to tumor sites.[5]

MATERIALS AND METHODS

1. DRUG PROFILE

Doxorubicin hydrochloride[6-12]

Doxorubicin hydrochloride is an antibiotic that is a part of highly colored Streptomyces

products known as rhodomycins. In general, these compounds have planar anthraquinone

nucleus attached to an amino sugar. The drug has quinone-hydroquinone, phenolic

functionalities and planar ring structure. The drug has amino-sugar that confers added

stability to binding with DNA through its interaction with sugar-phosphate backbone of

DNA.


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1.1 Properties of drug

Indication According to B.P. it contains not less than 98% and not more than

the equivalent of 102% of Doxorubicin.

Mechanism of Action

Doxorubicin can intercalate with DNA and hence the functions of

DNA are affected including DNA and RNA synthesis and

transcription. The anthracyclins intercalate between nucleotide

pairs, and the amino sugar binds tightly to DNA. Scission of DNA

is believed to be mediated by drug binding to DNA and

topoisomerase II, an action that prevents releasing of DNA breaks

created by the enzyme. Another theory suggests that the

anthracyclins are activated to free radicals that promote generation

of highly super-active superoxides, and these mediate DNA

damage. The doxorubicin radicals thus participate in oxygen-

mediated single strand breakage.

pKa 3.18

Solubility in water Sparingly Soluble.

1.2. Pharmacokinetic data

Bioavailability 5%

Half life After I.V. injection 1.520.3 hrs

Protein Binding 76%

Absorption Gastrointestinal absorption is unpredictable

Metabolism glucuronide conjugates of parent aglycone

Excretion Urinary excretion < 7%

Chemical Data

Chemical Formula C27H29NO11.HCl

IUPAC name

(8S,10S)-10-(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)

oxy-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-hydroxyacetyl-1-

methoxy-5,12-napthacenedione

Chemical Structure

HCL

Molecular mass 580.0g/mol

1.3. Therapeutic considerations

Legal Status Prescription only

Route available Doxorubicin Injection. USP

Method of Analysis FT-IR Analysis( 220 to 550 nm)

Conventional Dose 1.2 – 2.4 mg/kg body weight or 60-75 mg/sq. meter

of body

OMe O

O

OOH

OH

OH

C

O

CH2

OH

O

CH3

H

OHNH

2


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METHODOLOGY

Preformulation study is performed in order to establish optimum conditions for developing

suitable drug delivery system. The drug Doxorubicin Hydrochloride was obtained as a gift

sample from M/s Sun Pharmaceutical Advanced Research Centre (Baroda, INDIA) for the

present study.

2. PHYSICAL APPEARANCE AND MELTING POINT

2.1 Physical Appearance

The drug (Doxorubicin Hydrochloride) was found as red orange, odorless, crystalline powder.

2.2 Melting Point

The melting point was determined by melting point apparatus (Superfit, India) and was found

to be 205-2080C.

2.3 SOLUBILITY STUDIES[13]

Solubility is defined in quantitative terms as the concentration of solute in a saturated solution

at a certain temperature and in qualitative terms it may be defined as the spontaneous

interaction of two or more substances to form a v/v homogeneous molecular dispersion.

Solubility of Doxorubicin Hydrochloride was determined in water, phosphate buffer saline

(PBS), methanol, ethanol, ether, chloroform and benzene. Accurately weighed amount of

Doxorubicin Hydrochloride (10 mg) was suspended in 10 ml of these solvents in screw cap

test tubes. These tubes capped after being tightly closed and packed were shaken for about 72

h using a wrist shaker equilibrated for 6h, the supernatant was removed, filtered and

estimated spectrophotometrically. (Table1).

2.4. IDENTIFICATION OF DRUG

Chemical test for Identification

Accurately weighed amount of drug was dissolved (10 mg) in 0.5 ml of nitric acid in a test

tube and then add 0.5 ml of water was added to it. The solution was heated on the flame for 2

min and allowed to cool. Finally 0.5 ml of silver nitrate solution was added dropwise with

shaking until white precipitate was produced.

Ultraviolet Spectroscopy[14]

The UV scanning was performed in the photometric mode in methanol, PBS (pH 7.4) and

phosphate buffer (pH 6.4). The max at 480.2 nm, 480.2 nm and 480.2 nm respectively was


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obtained (Fig. 1,2 and 3). This matches the standard max as reported in official literature for

the drug, Doxorubicin Hydrochloride.

FTIR Spectroscopy

FTIR spectrum of Doxorubicin was obtained using IR spectrophotometer and the obtained

peaks were interpreted and compared with standard spectra of drug. FT-IR of the sample was

matched with the reference. The results of interpretation are shown in (Table 2).

PARTITION COEFFICIENT

The partition coefficient is defined as the ratio of unionized drug distributed between the

organic and aqueous phase at equilibrium.

Po/w = [Corg/Caq] equilibrium

Ten mg of Doxorubicin Hydrochloride was accurately weighed and transferred in

volumetric flask of 25 ml containing 10 ml each of two immiscible phases, n-octanol and

aqueous phase (PBS, pH 7.4 or distilled water). The vials were placed on a wrist action

shaker for 24 h. Phases were separated in a separation funnel and aqueous phase was

analyzed spectrophotometrically for the amount of drug after suitable dilution. The

partition coefficient was calculated. The concentration in the n-octanol phase was

determined by difference. Similarly, partition coefficient of Doxorubicin Hydrochloride was

estimated in PBS (pH 7.4) and n-octanol (Table 3).

2.5 METHOD FOR QUANTITATIVE ESTIMATION

A spectrophotometric method based on UV-visible absorption provided convenient, precise

and accurate mode to estimate the drug concentration in the range of 2.0 to 20.0 µg/ml in

water, PBS (0.1 M, pH 7.4) and phosphate buffer (0.1 M, pH 6.4).

Standard Curve of Doxorubicin Hydrochloride in Water

Accurately weighed 10 mg of Doxorubicin HCl was transferred into a clean and dried 10 ml

stoppered volumetric flask, a minimum required volume of methanol was added, the

volumetric flask was shaken gently to dissolve whole amount of the drug. The volume was

made to 100 mL with water to obtain 100 g/ml stock solution. The aliquots of 0.2 ml, 0.4

ml, 0.6 ml....2.0 ml were taken in 10 ml volumetric flasks and volumes were made up to the

mark with distilled water. The resulting concentrations ranged from 2-20 g/ml. The

absorbance of each concentration was determined at 480nm against methanol as blank. The


1265


standard curve was prepared between absorbance and concentration, which was linearly

regressed. The standard curve procedure was repeated three times and statistical parameters

related to it were derived (Table 4, Fig. 4).

Standard Curve of Doxorubicin Hydrochloride in Phosphate Buffer Saline (pH 7.4)[15]

All dilutions and measurements were made as above in phosphate buffer saline (pH 7.4)

which was prepared according to the following official method.

Composition of PBS (0.1 M, pH 7.4)

Disodium hydrogen phosphate 2.38g

Potassium dihydrogen phosphate 0.19 g

Sodium chloride 8.0 g

Distilled water 1000 ml

The pH of the solution was adjusted to 7.4 before use. The standard curve was linearly

regressed and statistical parameters related to it were desired.

Preparation of standard curve

Accurately weighed 10 mg of Doxorubicin HCl was transferred into a clean and dried 10 ml

stoppered volumetric flask, a minimum required volume of PBS was added, the volumetric

flask was shaken gently to dissolve whole amount of the drug. The volume was made to 100

ml with PBS to obtain 100 g/ml stock solution. The aliquots of 0.2 ml, 0.4 ml, 0.6 ml....2.0

ml were taken in 10 ml volumetric flasks and volumes were made up to the mark with PBS.

The resulting concentration ranged from 2-20 g/ml. The absorbance of each concentration

was determined at 480nm using Cintra 10 GBC UV visible spectrophotometer against PBS as

blank. The standard curve was prepared between absorbance and concentration, which was

linearly regressed. The standard curve procedure was repeated three times and statistical

parameters related to it were derived Observations are recorded in Table 5. The standard

curve is presented in Fig. 5.

Standard Curve of Doxorubicin Hydrochloride in Phosphate Buffer (pH 6.4)[16]

All dilutions and measurements were made as above in phosphate buffer saline (pH 7.4)

which was prepared according to the following official method.


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Composition of PBS (0.1 M, pH 6.4)

Disodium hydrogen phosphate 1.79 g

Potassium dihydrogen phosphate 1.36 g

Sodium chloride 7.02 g

Distilled water 1000 ml

The pH of the solution was adjusted to 7.4 before use. The standard curve was linearly

regressed and statistical parameters related to it were desired.

Preparation of standard curve

Accurately weighed 10 mg of Doxorubicin HCl was transferred into a clean and dried

10 ml stoppered volumetric flask, a minimum required volume of phosphate buffer was

added, the volumetric flask was shaken gently to dissolve whole amount of the drug.

The volume was made to 100 ml with phosphate buffer to obtain 100 g/ml stock

solution. The aliquots of 0.2 ml, 0.4 ml, 0.6 ml....2.0 ml were taken in 10 ml volumetric

flasks and volumes were made up to the mark with phosphate buffer. The resulting

concentration ranged from 2-20 g/ml. The absorbance of each concentration was

determined at 480nm against phosphate buffer as blank. The standard curve was

prepared between absorbance and concentration, which was linearly regressed. The

standard curve procedure was repeated three times and statistical parameters related to

it were derived Observations are recorded in Table 6. The standard curve is presented in

Fig. 6.

RESULTS AND DISCUSSION

From identification studies it was inferred that the drug procured as gift sample matched

with the standard as prescribed in I.P., 1996 and B.P., 2004 for identity and purity. The

drug was found to be red orange, odorless, crystalline powder that was similar in

physical appearance as mentioned in I.P., 1996 Melting point of Doxorubicin

Hydrochloride was near to that reported value.Solubility profile of the drug in different

solvents at room temperature (25°C) depicted its solubility in methanol, ethanol,

sparingly soluble in water and practically insoluble in non-polar solvents such as ether

chloroform and benzene (Table 1). The absorption maxima at 480.2 nm for solubilized

drug in methanol. The maxima matched with standards reported standards. Similarly, an

absorption maximum of drug in PBS (pH 7.4) and in phosphate buffer (pH 6.4) was


1267


found to be at 480.2 nm. FTIR spectrum of Doxorubicin HCl confirmed the presence of

different groups. The various peaks obtained in the IR spectrum matched with the IR

spectrum given in the official books of reference Florey (2005). The standard curves of

Doxorubicin HCl were prepared in different media and the absorbance data obtained

was subjected to linear regression. The equation of line obtained were Y = 0.0161X +

0.0001, Y = 0.0161X - 0.0044, Y = 0.0179X + 0.0017 and the correlation coefficients

were found to be 0.9977, 0.9942, 0.9987, respectively for standard curve of drug in

water, PBS (pH 7.4) and phosphate buffer (pH 6.4) which are close to 1.0 indicating

good linearity. All the curves were found to be linear and a straight line was obtained in

range of 2-20 μg/mL in all cases. The partition coefficient value obtained was 0.520 in

n-octanol:distilled water and 0.441 in n-octanol:PBS (pH 7.4). This concludes that the

drug in its salt form is aqueous soluble in nature and can be assayed for various

parameters in water as base using the standard curve made in UV range.

Table 1. Solubility of Doxorubicin HCl in different Solvent Systems

Solvents Solubility Found

Water ++

PBS (pH 7.4) +++

Methanol +++

Ethanol +++

Ether -

Chloroform -

Benzene -

+++ ; Soluble, ++ ; Sparingly soluble, - ; Practically insoluble.

Table 2: IR Spectral Analysis of Doxorubicin HCl

Named Group Reported Band

Frequency cm-1

Band Frequency

Obtained cm-1

O-H stretch 3560-3160 3301.2

NH3+

stretch 3160-2300 3150.1-2192.5

C=O stretch (ketone) 1724 1702.3

C=O stretch (intra hydrogen bonded quinone) 1613-1580 1600.4

C-O-C stretch (ether) 1282 1261.8

C-O (tertiary alcohol) 1115 1125.4

C-O (secondary alcohol) 1071 1065.1

C-O (primary alcohol) 1008 1002.9


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Table 3: Partition Coefficient of the Drug Doxorubicin HCl

Medium

Amount of the drug Partition coefficient (K)

(n-octanol/aq.phase) Aqueous

Phase n-Octanol

Distilled Water 66..5588 mmgg 33..4422 mmgg 00..552200

Phosphate Buffer Saline (pH 7.4) 66..9944 mmgg 33..0066 mmgg 00..444411

Table 4: Standard Curve of Doxorubicin HCl in Water at max 480.2 nm

Concentration

(g/ml) Absorbance

Regressed

Absorbance Statistical Parameters

2 0.0291 0.0332

Correlation coefficient

r2 = 0.9983

Equation of Line

Y = 0.0178X + 0.0022

4 0.0601 0.0644

6 0.0937 0.0966

8 0.1335 0.1288

10 0.1684 0.1610

12 0.2003 0.1932

14 0.2224 0.2254

16 0.2584 0.2576

18 0.2877 0.2898

20 0.3172 0.3220

Table 5: Standard Curve of Doxorubicin HCl in PBS (pH 7.4) at max 480.2 nm

Concentration

(g/ml) Absorbance

Regressed


2 0.0341 0.0279


r2 = 0.9942

Equation of Line

Y = 0.0161X - 0.0044

4 0.0652 0.0598

6 0.0929 0.0918

8 0.1245 0.1246

10 0.1510 0.1563

12 0.1779 0.1889

14 0.2101 0.2201

16 0.2534 0.2543

18 0.2905 0.2865

20 0.3292 0.3253

Table 6: Standard Curve of Doxorubicin HCl in Phosphate Buffer (pH 6.4) at max 480.2

nm

Concentration

(g/ml) Absorbance

Regressed


2 0.0334 0.0650


r2 = 0.9987

Equation of Line

Y = 0.0179X + 0.0017

4 0.0732 0.0962

6 0.1075 0.1274

8 0.1466 0.1586

10 0.1856 0.1898

12 0.2216 0.2210

14 0.2543 0.2522


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16 0.2857 0.2834

18 0.3169 0.3146

20 0.3612 0.3458

Fig. 1: UV Visible scan of Doxorubicin HCl in Water at max 480.2 nm

Fig. 2: UV Visible scan of Doxorubicin Hydrochloride in PBS (pH 7.4) at max 480.2 nm


1270


Fig. 3: UV Visible scan of Doxorubicin HCl in Phosphate Buffer (pH 6.4) at max 480.2

nm

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20

Concentration (µg/ml)

Reg

ressed

Ab

so

rban

ce

Fig. 4: Standard curve of Doxorubicin HCl in Water at max 480.2 nm


1271


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 15 20 25


Reg

ressed

ab

so

rban

ce

Fig. 5: Standard curve of Doxorubicin HCl in PBS (pH 7.4) at max 480.2 nm

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 15 20 25


Reg

ressed

ab

so

rban

ce

Fig. 6: Standard curve of Doxorubicin HCl in Phosphate Buffer (pH 6.4) at max 480.2

nm

CONCLUSION

The major drawback associated with anticancer drugs are their toxic effects and non-

availability of appropriate dose at the desired site. Conventional mode of administration of

such bioactives leads to their interaction with cancerous as well as normal cells. This not only

precipitates toxicity but also adds to considerable loss of dose before reaching the desired


1272


site. Any attempt to slightly modify the chemical structure of the drug to achieve the results

may reduce its efficacy. To avoid the possible hazards in cancer chemotherapy we selected

novel dextran conjugated PPI dendritic systems as drug carriers. The system is hypothesized

to have the drug within its internal cavities and the system is expected to selectively enter

cells where it is intended to release the drug. The drug was found to be red orange, odorless,

crystalline powder & Melting point of Doxorubicin Hydrochloride was near to that reported

value. Solubility profile of the drug in different solvents at room temperature (25°C) depicted

its solubility in methanol, ethanol, sparingly soluble in water and practically insoluble in non-

polar solvents such as ether chloroform and benzene. The drug was identified for any

impurities by chemical tests, UV scanning and IR spectroscopy. The absorption maxima of

the drug Doxorubicin Hydrochloride in methanol was measured and was found to be 480.2

nm. The above maxima matched with standards. Infra red spectrum of Doxorubicin

Hydrochloride confirmed the presence of different groups. The various peaks obtained in the

FTIR spectrum matched with the IR spectrum given in the official books of reference Florey

(2005). The standard curves of Doxorubicin HCl were prepared in different media and the

absorbance data obtained was subjected to linear regression. The equation of line obtained

were Y = 0.0161X + 0.0001, Y = 0.0161X - 0.0044, Y = 0.0179X + 0.0017 and the

correlation coefficients were found to be 0.9977, 0.9942, 0.9987, respectively for standard

curve of drug in water, PBS (pH 7.4) and phosphate buffer (pH 6.4) which are close to 1.0

indicating good linearity. The partition coefficient values obtained were 0.520 in n-

octanol:distilled water and 0.441 in n-octanol:PBS (pH 7.4). This concludes that the drug in its

salt form is aqueous soluble in nature and can be assayed for various parameters in water as base

using the standard curve made in UV range.

REFERENCES

1. Armspach, D., Cattalini, M., Coontable, E.C., Housecroft, C.E., Phillips, D., (1996) J.

Chem. Soc. Commum., 12: 1823-1824.

2. Barar, F.S.K., (2000) Essentials of Pharmacotherapeutics, S. Chand and Company Ltd.,

New Delhi, 482.

3. Barth, R.F., Adams, D.M., Soloway, A.H., Alam, F. Darby, M.V., (1994) Biconj. Chem.,

5(1): 58.

4. Breskin, A., Chechik, R., Abraham, S., Warshawsky, N. M., (1998) X-Ray imaging of

tumors with Dextran carrier of platinum compounds, “US patent application”.


1273


5. Florey, K., (2005), "Analytical Profiles of Drug Substances", Reed Elsevier India Pvt.

Ltd., New Delhi, 2: 221-244.

6. Fainaro, R.S., Puder, M., Davies, J.W., Tran, H.T., Sampson, D.A., Greene, A.K., Corfas,

G., Folkman, J., (2004) Nature (medicine), 10(3): 255-261.

7. Jain, N.K., (2001) Advances in controlled and novel drug delivery, CBS Publishers and

Distributors, New Delhi, 1st ed., 40-69.

8. Martin, A., Bustamante, P., Chun, A.H.C., (1995), "Physical Pharmacy", Waverly Pvt.

Ltd., New Delhi, 4: 212, 404.

9. Martindale Extra Pharmacopoeia, (2005) London, Royal Pharmaceutical Society, 31:

572-575.

10. Freshney R.I., (2000), Culture of animal cells, a manual of basic techniques, 4th

Ed., A

John Willey and Sons Inc. Publications, NY, USA.

11. Kohle, P., Misra, E., Kannan, R., Kannan, S., Lieh- Lai, M., (2003) Int. J., Pharm., 259:

143-160.

12. Kramer, E.D., Haasen, P., Cohn, R.W., (2004) Polymers Sci. Tech., 686-688.

13. Reddy, L.H., Meda, N., Murthy, R.S.R., (2005), Biomed. Papers, 55: 81-91.

14. Reddy, L.H., Murthy, R.S.R., (2004), Biomed. Papers, 148(2): 161-166.

15. Indian Pharmacopoeia, (1996) Govt. of India, Ministry of Health and Family Welfare,

New Delhi, I: 323.

16. British Pharmacopoeia, (2004) Her Majesty’s Stationary Office, Dept. of Health, Scottish

Office, London, I: 849-850.

quantitative estimation of dextran conjugated ppi

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