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