Int. Res J Pharm. App Sci., 2012; 2(6): 196-206 ISSN: 2277-4149

Naga Raveendra et.al, 2012 196

International Research Journal of Pharmaceutical and Applied Sciences (IRJPAS)

Available online at www.irjpas.com

Int. Res J Pharm. App Sci., 2012; 2(6): 196-206

METHOD DEVELOPMENT AND VALIDATON FOR DETERMINATION OF ANTIFACTOR IIA

POTENCY OF HEPARIN SODIUM BY CHROMOGENIC METHOD 1S.L.NagaRaveendra.M,

2Nandan K Peddi,

3Krishnakar.M

1School of Pharmaceutical sciences and Technology, JNTU, Kakinada, A.P -533003 2University College of Pharmaceutical sciences, Warangal, A.P- 506009 3College of Technology, Osmania University, Hyderabad, A.P-500007

(Received: 11 November2012; Accepted: 24 November, 2012; Published: 29 December, 2012)

Corresponding Author’s email: raveendra.mudrageda@gmail.com

Abstract: A novel, precise, rapid, economical, and Accurate Chromogenic method for determination of

Antifactor IIA potency of Heparin sodium.Tris-buffer preparation(PH 8.4) and 6%w/v solution of citric acid are

the reagents used. Anti IIA reagents (AntithrombinIIIreagent (R1), Bovine thrombin IIa reagent (R2)

,Spectrozyme TH substrate(R3)) are reconstituted. Switch on the versamax instrument and wait for 15min for

the temperature to attain 37ºc±0.2ºc. Use new microplates for every test and perform pre-read of empty

plate.Heparin reference standard solution and test solution (API) are prepared. sample and standard preparations

are incubated with different reagents at 37ºc. Incubation time depends on the potency of different lots of

reagents(anti IIa) used.On the receipt of the reagents the preliminary assays are carried out over different

incubation time to determine the suitable time combination to get a linear standard curve for USP heparin

standard and use the corrected incubation time in sub seeding steps.

Key words: heparin sodium, antifactor IIA, versamax, instrument, chromogenic method.

INTRODUCTION

K T Goodall, C CChooi, and A S Gallus1have

studied the effect of diluent, heparin activity after

dilution, container, and pH on the stability of heparin solutions. Heparin activity was measured

by activated partial thromboplastin time and

thrombin clotting time. Heparin activity was stable

for 6 hours regardless of storage conditions. After

24 hours heparin activity was stable when the drug

was diluted in 0.9% saline and stored in plastic, but

a small loss of activity was observed in several

studies after dilution in 5% dextrose or storage in

glass. A more extensive comparison confirmed a 3-

5% loss in heparin activity over 24 hours after

dilution in 5% dextrose. Changing the pH to 3.5 or 10.0 had little effect on storage stability. They

concluded that heparin activity in vitro remains

stable during short infusions but recommend

dilution in 0.9% saline and a plastic container when

a heparin solution is infused over 24 hours.Eddy H.

H. Li, John W. Fenton, II and Richard D. Feinman2

reported the effect ofheparin on the Kinetics of

inactivation of thrombinbyantithrombin III (AT) to

distinguish between two possible

mechanisms.Either heparin activates AT to make it

a more effective inhibitor, or heparin makes thrombin more susceptible to inhibition by AT. The

resultswere consistent only with mechanism 1. The

experimental approach was to premix heparin with

either thrombin or AT and then to measure the rate

of association of the two proteinsin the rapid-

mixing Stop-flow spectrophotometer. Reactions

were followed spectrophotometrically by observing displacement of the dye proflavine from the active

site of thrombin as AT binds. Only premixing AT

with heparin accelerated the reaction compared to

control (no heparin); the observed second-order

rate constant was enhanced by a factor of 200–400.

JF Pierson-Perry, DM Obzansky and JP

Mizzer3developedan automated assay for

determining effective heparinactivity in plasma,

based on heparin-catalyzed inhibition of Factor Xa

byantithrombin III (AT III). Residual Factor Xa is

determinedkinetically by the Du Pont discrete clinical analyzer with achromogenic substrate and

is inversely related to heparinactivity. Becausethe

test plasma is the sole source of AT III, the assay

result is dependenton AT III activity and reflects

effective rather than total heparinactivity. The

assay range is 20-1200 USP units/L, and the assay

showsequivalent sensitivity to standard and low-

molecular-mass heparins. Within-run

reproducibility is 1.6% at 390 units/L. There was

nointerference from common blood components or

drugs. Results agreed wellwith those by the Coatestheparin kit (r = 0.85, n = 122). GF Kapke,

RD Feld, DL Witte and WG Owen4proposed an

esterolytic method for determination of heparin in

Research Article

Int. Res J Pharm. App Sci., 2012; 2(6): 196-206 ISSN: 2277-4149

Naga Raveendra et.al, 2012 197

plasma. This assay for heparin is based on the

heparin-accelerated rate of alpha-thrombin III. The

rate or product formation from the residual active

thrombin is inversely proportional to plasma

heparin content. The assay is insensitive to

concentrations of anti thrombin III in plasma. Acorrelation coefficient of 0.90 was obtained (n =

62). GF Kapke, RD Feld, DL Witte and GF

Johnson 5 have developed a single-stage assay for

heparin, using reagents . The single-stage assay

involves simultaneous mixing of a plasma sample,

an anti thrombin III source, alpha-thrombin, and

the alpha- thrombin fluorogenic substrate. The

synthetic substrate, anti thrombin III, and heparin-

anti thrombin III complex compete for the alpha-

thrombin active site. The alpha-thrombin is

inactivated by the heparin- anti thrombin complex

while substrate is being hydrolyzed, so that total product formation decreases with heparin

concentration. Day-to-day CV was 9.3% at a

heparin concentration of 246 USP units/L.

Comparison of results of the single-stage heparin

assay with those of a two-stage esterolytic assay

yielded the linear regression equation: esterolytic =

0.834(single-stage)--7 USP units/L (r = 0.94, n =

47). H ten Cate, RJ Lamping, CP Henny, A Prins

and JW ten Cate6developed an automated an

amidolytic assay for heparin Using the

chromogenic substrate. The assay is based on the detection of anti-Xa activity generated by heparin

in plasma. The method is reproducible(intra- and

inter assay CVs of 2.4 and 3.3%, respectively) and

reliable in anti thrombin III-deficient plasma.

Results of this assay, obtained forplasma samples

from patients and volunteers treated with heparin,

correlate well (r = 0.899) with those of the test for

activated partial thromboplastin time. This

automate damidolytic assay for heparin is suitable

not only for monitor in gstandard therapy with

heparin but also for measuring the activity

ofrecently developed heparin fractions. Drozd NN, Tolstenkov AS, Bannikova GE, Miftakhova NT,

Lapikova ES, Makarov VA, Varlamov VP7 The

anticoagulant activity of low-molecular weight

heparins (LMWH-PC) with average distribution of

molecular weights within 3.4-5.8 kD was

investigated. The samples of LMWH-PC were

obtained from unfractionated heparin using

immobilized enzyme complex of protease C. The

LMWH-PC derivatives inhibited the activity of

blood coagulation factors IIa (thrombin) and Xa.

The LMWH-PC derivatives had an anti-factor-Xa activity up to 131-208 IU/mg and anti-factor-IIa

activity up to 81-175 IU/mg. Philip Band and

Aaron Lukton8 , developed a sensitive and specific

chemical assay for heparin is herein presented. It is

based on heparin's ability to catalyze the acidic

hydrolysis of the cationic dye Auramine 0. The

assay can detect as little as 1 μg of heparin, and is

specific in that it distinguishes heparin from all

other glycosaminoglycans tested. The applicability

of the assay and interference due to the presence of

proteins, organic cations, and inorganic salts is

discussed.

In all the above studies ,they had some problems

like no rapidity, economical, lack of accuracy etc. So to rectify those problems , we discussed a novel

chromogenic method for the determination of anti

factor IIA potency factor for heparin sodium.

EXPERIMENTAL WORK

Heparin Sodium:

Principle:

HEPARIN+AT AT HEP

AT HEP+FIIa F IIa-AT-HEP+ RESIDUAL F

IIa

RESIDUAL F IIa + SUBSTRATE

PEPTIDE+ PARA NITRO ANILINE

Equipment and materials:

Microplate reader (model-versamax 190, mfr-

molecular devices), Microplate-96wells, Test tubes

(5ml RIA vial), Micropipette (1000µl, 100 µl, 50 µl

and tips), Multi channel pipette (5-50 µl),

Volumetric flasks, Timer, Analytical balance,

Vortex shaker, Heating block.

Reagents:

Anti-thrombinIII, Bovine thrombin

IIa,,Spectrozyme TH substrate, TRIS buffer,USP heparin sodium RS,6%w/v solution of citric acid.

Preparation of reagents:

Tris buffer preparation(PH 8.4): Dissolve 6.056g

of Trisbuffer{(hydroxyl methy) amino methane}

(0.05), 10.227g of Nacl(0.175M), 2.7918g

EDTA(0.0075M), and 1g of PEG-6000(0.1%) in

water and dilute it with950ml distilled water.

Adjust PH to 8.4 with dilute Hcl and make up to

1litre. Filter through 0.45 microns filter disc before

use.

6%w/v solution of citric acid: weighed accurately

6g of citric acid and dissolved in 100ml with water and filter through 0.45 microns filter disc.

Reconstitution of anti IIa reagents:

AntithrombinIIIreagent (R1): Dissolve the

reagent with 1ml of filterd distilled water and 4ml

buffer using calibrated pipette, dissolve reagent

gently.DO NOT SHAKE VIGOUROUSLY. Use

reagent after 5min of reconstitution.

Bovine thrombin IIa reagent (R2): Dissolve the

reagent with 1ml of filtered distilled water and 4ml

buffer using calibrated pipette, dissolve reagent gently.DO NOT SHAKE VIGOUROUSLY. Use

reagent after 5min of reconstitution.

Spectrozyme TH substrate(R3): Dissolve the

reagent with 5ml of filtered distilled water using

calibrated pipette, dissolve reagent gently.DO NOT

SHAKE VIGOUROUSLY. Use reagent after 5min

of reconstitution.

Int. Res J Pharm. App Sci., 2012; 2(6): 196-206 ISSN: 2277-4149

Naga Raveendra et.al, 2012 198

Preparation of heparin reference standard:

Standard stock solution: Break USP heparin

sodium reference standard vial with cutter and

transfer contents of vial into 10ml volumetric flask.

Make up the volume to 10ml with water. Dilute

2ml of this solution to 50ml with distilled water. Mix properly before use. The stock standard shall

have a potency of 8.576 USP heparin units/ml.

Further dilute 1.17 to 20ml to yield final

concentration of 1.0034 USP units/ml.

Heparin standard solution:

Dilute stock standard solution with buffer to get the

below standard concentrations.

Standard stock-1.0034 USP heparin units/ml-SD

↓

0.2ml SD +1.8ml buffer (0.1003 USP heparin

units/ml) - SD1

↓ 0.55ml SD1+ 1.45ml buffer (0.0276 USP heparin

units/ml) - S1

↓ 0.44ml SD1+1.56ml buffer (0.025 USP heparin

units/ml) - S2

↓

0.32ml SD1+ 1.68ml of buffer (0.0175 USP

heparin units/ml) - S3

↓

0.2ml SD1+ 1.8ml of buffer (0.0105 USP heparin

units/ml) - S4

Preparation of test solution (API):

NOTE: For a satisfactory assay the assumed

potency of test sample must be close to true

potency (225.68 USP heparin units/ml). on the

basis of this assumed potency and the assigned

potency of standard , equipotent dilutions are

prepared i.e corresponding doses of standard and

unknown are expected to give same response.

Accurately weighed 95mg of API on weighing

balance and transfer contents into 100ml volumetric flask. Dissolve contents in distilled

water and volume is made up to the mark. Shake

thoroughly. This solution has a concentration of

1000µg/ml. dilute 2ml of this to 50ml with water.

Further dilute 1.17ml of this to10ml water,

Sample Stock solution-4.446 µg/ml –TD

↓

0.2ml SD +1.8ml buffer (0.4446 µg/ml) - TD1

↓

0.55ml SD1+ 1.45ml buffer (0.1222 µg/ml) - T1

↓ 0.44ml SD1+1.56ml buffer (0.0978 µg/ml) - T 2

↓

0.32ml SD1+ 1.68ml of buffer (0.0711 µg/ml) - T 3

↓

0.2ml SD1+ 1.8ml of buffer (0.0446 µg/ml) - T 4

PROCEDURE:

Switch on the versamax instrument and wait for

15min for the temperature to attain 37ºc±0.2ºc. Use

new plates for every test and perform pre-read of

empty plate. Arrange standard and test dilutions S1, S2, S3, S4,

T1, T2, T3, T4 in duplicate.

Drop the dilutions of standard solutions and sample

solutions in microplate as

(Table 1)

Activate the microplate reader:

Wipe the microplate with tissue paper. Check the

microplate for any scratches of dirt against light

with naked eye. Ensure that there are no dust

particles of any cracks in the microplate. If

observed the dust and cracks in the use new plate. Measure the absorbance of empty microplate at

405nm

Standard preparation: Add 25µl of each of the

standard aliquot in four replicates as indicated in

chart to the respective wells designated as S1, S2,

S3, and S4 in 96 wells microplate. Use different

tips for each of the standard concentration.

Blank preparations: Add 25µl of TRIS buffer

PH8.4 into well designated as blank.

Test preparations: Add 25µl of each of test

aliquot four replicates as indicated in dilution charts to the respective wells T1, T2, T3 and

T4.Use different tips for each of sample

concentration.

RB: Add 25µl of TRIS buffer PH8.4 in duplicate to

the well designated as RB.Allow to equilibrate at

37ºc for about 10min, add 80µl of citric acid to

wells designated as RB.

ADDITION OF REAGENTS IN THE

MICROPLATE:

Add 40µl anti thrombin-III reagent to all the wells

at interval of 15sec and simultaneously mix the

content of the microplate gently and allow to

incubate for exactly 2:45 min at 37ºc.

Add 40µl of bovine thrombin IIa reagent to all the wells at end of exactly 2:45min at interval of 15sec

and simultaneously mix content of the microplate

gently and incubate for exactly 3:15min at 37ºc.

Add 40µl spectrozyme TH substrate reagent to all

wells at end of 6min at interval pf 15sec and

simultaneously mix content of microplate gently

and incubate for exactly 2min at 37ºc.

Stop reaction after exactly 8min by adding 80ml of

citric acid to all the wells except RB at interval of

15sec and mix gently.

The potency of sample is calculated using parallel-line assay: for each series, calculate the regression

of the absorbance against log concentration of

sample solution and standard solution and calculate

potency of heparin sodium in USP units/mg using

statistical methods for parallel-line assays. Express

potency of heparin sodium/mg, calculated on dried

basis.

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Naga Raveendra et.al, 2012 199

SUPPLEMENTARY INFORMATION:

Table 1:

B1 S1 S2 S3 S4

B2 T1 T2 T3 T4

B3 T1 T2 T3 T4

B4 S1 S2 S3 S4

B5 RB1 RB2

Table 2:%RSD for five blank OD’S of Repeatability

S.NO

EXPERIMENT

.NO BLANK OD'S AVG STDEV %RSD

B1 B2 B3 B4 B5

1 HS-RP-001 0.780 0.769 0.782 0.762 0.770 0.773 0.0083 1.07

2 HS-RP-002 0.776 0.765 0.778 0.766 0.768 0.771 0.0060 0.78

3 HS-RP-003 0.774 0.764 0.776 0.764 0.763 0.768 0.0063 0.82

4 HS-RP-004 0.767 0.757 0.770 0.757 0.755 0.761 0.0068 0.89

5 HS-RP-005 0.763 0.753 0.767 0.753 0.751 0.757 0.0071 0.94

6 HS-RP-006 0.816 0.824 0.809 0.808 0.811 0.814 0.0066 0.81

Table 3:%RSD for six Anti-factor IIavalues of Repeatability

S.NO.

EXPERIMENT

NO.

SAMPLE

NAME

ANTI-FACTOR IIa(USP HEPARIN

UNITS/MG)

1 HS-RP-001 Sample at 100% 215

2 HS-RP-002 Sample at 100% 214

3 HS-RP-003 Sample at 100% 214

4 HS-RP-004 Sample at 100% 213

5 HS-RP-005 Sample at 100% 214

6 HS-RP-006 Sample at 100% 217

Average 215

Standard deviation 1.378

%RSD 0.643

Fig: 1 :Microplate of sample preparation 1 at 405nm

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Naga Raveendra et.al, 2012 200

Fig: 2: Linearity graph 0f sample preparation 1

Fig 3: Potency result 0f sample preparation 1

Fig 4: Microplate of sample preparation 2 at 405nm

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Naga Raveendra et.al, 2012 201

Fig 5: Linearity graph of sample preparation 2

Fig 6: Potency result of sample preparation 2

Fig 7: Microplate of sample preparation 3 at 405nm

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Naga Raveendra et.al, 2012 202

Fig 8:Linearity graph of sample preparation 3

Fig 9: Potency result of sample preparation 3

Fig 10: Microplate of sample preparation 4 at 405nm

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Naga Raveendra et.al, 2012 203

Fig 11: Linearity graph of sample preparation 4

Fig 12: Potency result of sample preparation 4

Fig 13: Microplate of sample preparation 5 at 405nm

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Naga Raveendra et.al, 2012 204

Fig 14: Linearity graph of sample preparation 5

Fig 15: Potency result of sample preparation 5

Fig 16: Microplate of sample preparation 6 at 405nm

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Naga Raveendra et.al, 2012 205

Fig 17: Linearity graph of sample preparation 6

Fig 18: Potency result of sample preparation 6

NOTE: The time allowed to incubate the sample

and standard preparations with different reagents at

37ºc is an important criteria and incubation time

described here may be changed depending on potency of different lots of reagents(anti IIa) used.

On the receipt of the reagents the preliminary

assays are carried out over different incubation

time to determine the suitable time combination to

get a linear standard curve for USP heparin

standard and use the corrected incubation time in

sub seeding steps.

System suitability:

The % RSD over blank readings should be < 10%

The zero concentration aliquot (B1 TO B5) OD

value should be higher than the lower concentration

of samples or standard (S1 TO S4) OD values Correlation coefficient R2 must be higher than 0.99

for standard and sample preparations.

VALIDATION :

validation of analytical methods mentioned for the

determination of Anti IIa potency of heparin

sodium by chromogenic method using microplate

reader .

It will be determined by analyzing six individual

preparations at100% test concentration level from same drug substance lot.Standard and samples

solutions are prepared as per the test method.

Calculate anti factor IIa potency of six preparations

using prefixed excel calculation sheet.

Calculate % relative standard deviation for anti

factorIIa potency. Tables indicating the values for

repeatability for calculating the potency of Anti IIa

are mentioned in supplementary information(

Tables 2,3 and Figures in supplementary

information).

Acceptance criteria:

Individual anti-factor IIa potency value should be greater than 180USP heparin units/mg. The %RSD

for five blank ODS should not be more than 10.The

%RSD for six anti-factor IIa potency values should

not be more than 5.

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Naga Raveendra et.al, 2012 206

RESULTS AND DISCUSSION

The proposed method for the estimation of Anti IIa

potency in Heparin sodium was a simple method

and been validated.

Different trials have been done for developing the

method for estimation of Anti IIa potency by chromogenic method. It deals with the procedures

to carry out the estimation of Anti IIa potency.

Also deals with the validation of the

developed method and been found that the method

is valid.

ACKNOWLEDGEMENT

„Words‟ are very poor comforters to express the

deep debt of gratitude which one feels in one‟s

inner corner of the heart when one is helped to

achieve the goal in this boundless and endless field

of research work, but since there is no way which

can better express ones feeling of love and gratitude than this.

First and foremost I express my deepest sense of

gratitude and faithfulness to God‟s grace which has

enabled me to finish my project work successfully.

On the occasion of presenting this dissertation, I

would like to thank the Almighty and My Parents,

brothers for their co-operation offered in reaching

every milestone of my life.

Last but not least I express my deep sense at

magnitude to all of our Classmates for their

heartful cooperation and timely help in submission of my thesis. Above all I submit and surrender my

selfless and humble prostration to the almighty

whose spiritual blessings inspired me to deal with

every odds and complete my dissertation in time.

REFERENCES

1. K T Goodal,C C Chooi,A S Gallus, “Heparin

stability: effects of diluent, heparin activity,

container, and pH” J ClinPathol 1980,33:1206-

121

2. Eddy H. H. Li, John W. Fenton, II and Richard

D. Feinman,“The role of heparin in the

thrombin-antithrombin III reaction”, Biochimica et Biophysica Acta , 1979; 585(3-4): 405-415.24

3. JFPierson-Perry, characterization of an

automated assay of effective heparinactivity in

plasma”, Archives of Biochemistry and

Biophysics,1976; 175(1):153-159 .

4. GF Kapke, RD Feld, DL Witte and WG Owen, “

Esterolytic method for determination of heparin

in plasma”, Clinical chemistry 1981: 27(4) 526-

9.

5. GF Kapke, RD Feld, DL Witte and GF Johnson ,

“Single-stage automatedassay for heparin”,

Clinical chemistry-1982; 28(7) :1521-4.

6. H ten Cate, RJ Lamping, CP Henny, A Prins and

JW ten Cate“Automatedamidolytic method for

determining heparin, a heparinoid, and a low-Mr

heparin fragment, based on their anti-Xa activity

”, Clinical chemistry 1984: 30(6) 860-4.

7. Drozd NN, Tolstenkov AS, Bannikova GE, Miftakhova NT, Lapikova ES, “Anticoagulant

activity of low-molecular-weight heparins

obtained using a hydrolase complex” – Eksp

Klin Farmakol. 2007:70(6):19-24.

8. Philip Bandand Aaron Lukton, developed a

sensitive and specific chemical assay for

Heparin, Analytical biochemistry, 1982;

120(1):19-24.