The present invention relates to an analytical method for the determination of Serratiopeptidase.
More particularly, the present invention relates to estimation of protease enzyme in
Serratiopeptidase tablets as a sum of peptide and amino acids produced upon enzymatic
hydrolysis of Casein by reverse phase high performance liquid chromatographic (HPLC)
method.
BACKGROUND OF THE INVENTION
Serratiopeptidase is a proteolytic enzyme produced by purification from the culture of nonpathogenic enterobacterium Serratia sp. E- 15, having molecular weight of about 52 kDa. It
hydrolyses the Casein (solubilized in buffer), into its constituent peptide and amino acids at a
particular temperature arid p11. It is used to reduce pain and swelling associated with conditions
like back pain, arthritis, tension headaches, migraine headaches etc.
Introducing a pharmaceutical product into the market is a highly intricate and lengthy process
since the drug approval in most countries is regulated by government agencies which require the
applicant to establish safety and efficacy of the pharmaceutical product during the reviewapproval phase and continue to monitor the safety of the drug post-approval. The regulatory
agencies normally require a specification of the pharmaceutical product that specifies the upper
and lower limit ol content ol active ingredient. Alter approval, each batch or lot of the
pharmaceutical product is tested to ensure its compliance with the approved specifications.
Stability tests are also carried out on the pharmaceutical product in order to demonstrate lack of
material change in the formulation over the indicated shelf life. It is also a requisite ofthe good
practices to preserve samples from every commercial batch released into the market so as to
monitor and subsequently correct any defect observed.
It is due to this reason that both the active pharmaceutical ingredient and the pharmaceutical
product are tested during manufacture of the product and subsequently during its shelf life. This
is normally achieved by performing certain analytical tests on the product. Quantitation of active
substance requires assay of both the pharmaceutical product and Active pharmaceutical
substances using validated analytical methods followed by qualitative and quantitative analysis
of the results obtained.
Various analytical techniques are available in the pharmaceutical art for the afore said
quantitation such as nuclear magnetic resonance (NMR), liquid or gaseous chromatography (LC
or GC) techniques coupled with suitable detectors (absorption, fluorescence or mass detectors)
and Infrared (IR), techniques. Amongst these techniques, high performance liquid
chromatography (HPLC) technique coupled with suitable detectors is one of the most frequently
used technique.
Various analytical methods are reported in literature for the analysis of Serratiopeptidase either
alone or iii combination with other active pharmaceutical ingredients in the API or in
pharmaeoutioal composition. In traditional methods, Serratiopeptidase is indirectly estimated by
UVIVisible spectrophotometer as a sum of amino acids generated due to hydrolysis of Casein.
However, the UV method of analysis is reported to be non-specific and yield results having high
variability. This is primarily due to the ulcoLisistent absorption exhibited by the blank employed
during analysis. The present inventors had also tried to optimize a UV based analytical method
for the determination of Serratiopeptidase in tablet formulation. However, readings from the
blank and sample were found to he inconsistent. Similarly, readings from the standard were also
thund to be inconsistent on day to day basis. I his lack of assurance of the results of analytical
tests to be devoid of any error poses significant problem since these analytical tests are extremely
important as critical decisions related to quality of pharmaceutical product are based upon the
results of such tests. From the standpoint of being free from errors, these procedures are not
sufficient.
IIi~h Performance Liquid Clnoumato~maplmy (IIPLC~ is a I~LLLL o[coluLmL LlLL [fLat ~maplmy
whereby a sample mixture is pumped in a stream of solvent (known as the mobile phase) at high
pressure through a column containing chromatographic packing material (known as stationary
phase). HPLC has the ability to separate compounds that are present in sample mixtures, which
can be dissolved in a liquid in trace concentrations as low as parts per billion. Due to this ability,
HPLC is used in a variety of industrial and scientific applications. Depending on the interaction
3
of sample being analyzed with solvent and stationary phase, the retention time (RT) may vary.
When the sample passes through the column, it interacts with the two phases i.e. stationary and
mobile phase, at different rates based on polarity. Molecules that have less interaction with the
stationary phase or more interaction with the mobile phase, elute faster from the column. Main
components of a HPLC system include the solvent reservoir, a high-pressure pump, a column,
injector system and the detector. Reservoir is used to hold the solvent, which is generally
referred to as the mobile phase. Pump is used to generate a specified flow rate of the mobile
phase. A column is the most important part of a chromatographic technique and is filled with the
stationary phase where the separation takes place. I he sample injected in to the column with a
stream of mobile phase through the Injector which can be manual or automated. The detector is
used to visualize the separated compounds as they elute from the column with the stream of
mobile phase. The mobile phase exits the detector and is either sent to a waste or collected, as
(LesLIe(1.
High Performance Liquid Chromatography (HPLC) coupled with a UV-Visible detector has also
been reported in literature for the direct estimation of Serratiopeptidase in combination
formulations. The API and the excipients present, if any, are separated on the HPLC stationary
phase and they can be detected and quantified using the response obtained from the UV-Visible
detector. Most of the reported HPLC methods for the analysis of Serratiopeptidase in published
literature involve solubilisation of the drug in alcohol and separation on hydrophobic C18
stationary phase followed by UV detection. Serratiopeptidase is practically insoluble in alcohol
and due to its high molecular weight, it is not feasible to retain it on hydrophobic stationary
phase which generally have pore size in the range of about 100 A° to 120A°. However, most of
the reported methods involve use of silica based Cl8 columns having pore size in the range of
100 A° to 120 A°. Hydrophobic stationary phases having pore sizes in this range are suitable
OL1IY C([ satnples llaviLl~ L11O16( LLlkIL Wei~lL( ?SO0 Dallotis SLt1(’(’ Wl1~LL SLW1L ‘OlLLLLLLLS ~LO ItS~(l Ioi
analysis of large biomolecules such as Serratiopeptidase (an enzyme), the biomolecule, due to its
high molecular weight, gets excluded partially or entirely from the pores of this sizes and as a
result, interaction with 1% or less of the total column surface is observed leading to poor
separation and peak shape. The reported methods also suffer from the limitation of poor
reproducibility and high variability. Thus, there is an unmet need in the pharmaceutical art of a
4
reproducible and reliable HPLC method for the determination of Serratiopeptidase in
pharmaceutical dosage forms.
The present inventors attempted to develop a HPLC method capable of quantitative estimation of
Serratiopeptidase in pharmaceutical dosage form based on the analysis of hydrolysis of Casein
by Serratiopeptidase. This involved undertaking several trials to separate the peaks of hydrolyzed
Casein. During enzymatic hydrolysis, the protease digests the casein and liberates the amino
acids and peptide fragments. The pattern of generation of peptides is governed by various factors
such as type of casein (homogenous vs non homogenous), purity of casein, time of reaction and
quantity of reactant. In order to resolve the mismatch, the inventors tried different grades of
casein such as alkali soluble casein, fat free casein, Hammarsten casein and bovine milk casein.
The pattern of peptides geneiatioli was 1101 S4fflC and iepiodueible iii all [lie easeili giades
LIlve%EigaEe(I L LLiS [[lay he (ILIe (0 4/dI lal kILL ILL IlOLrLOgCLleiLy 4LL(L [)LLI fEy o[ a%eELL WLLi IL ate E%.4’(I
important factors affecting reproducibility. Eventually, Hammarsten grade casein from Himedia
and VWR were found to be suitable material since these two grades yielded reproducible results.
Development of HPT.X’ method also involved optimization of the reaction time, temperature and
concentration ofreagents as well as of the active pharmaceutical ingredient in order to finalize
the methodology. Further, owing to enzymatic nature of Serratiopeptidase, the method was found
to exhibit a polynomial curve when concentration of Serratiopeptidase was plotted against area
counts. From this curve, hneanty of the analytical procedure was derived and was found to be
linear in a very narrow range of concentration (0.3 j.tg/mL to 0.7 ~.tg/mL) of Serratiopeptidase.
The Applicants have successfully developed a reproducible and reliable HPLC method for the
analysis of Serratiopeptidase in pharmaceutical dosage forms which is not only simple,
reproducible and robust, but is also commercially viable. Further, the analytical method as per
EL[(i eLILLeLLE iLLVtqLiiO[L iS SCIe(EiVC, LILLe4tL aLLd [ILe ise a[LcI ~(e11LaEC
SUMMARY OF THE INVENTION
It is a principal object of the present invention to provide a reproducible and reliable HPLC
method for the analysis of Serratiopeptidase in pharmaceutical dosage forms.
Further object of the present invention is to provide a HPLC method for the analysis of
Serratiopeptidase as a sum of peptide and amino acids produced during hydrolysis of casein in
pharmaceutical dosage forms, wherein the mobile phase comprises two liquids and the relative
concentration of the liquids is varied to a predetermined gradient.
Principally, casein is hydrolyzed at a particular temperature and pH in presence of
Serratiopeptidase. The peptide and amino acids produced upon hydrolysis are quantitated using
reverse phase adsorption chromatography. The amount of peptide and amino acid produced is in
linear correlation with the amount of Serratiopeptidase present in the sample.
The following embodiments further describe the objects of the present invention. However, the
disclosed invention is not restricted to the particular embodiments hereinafter described and
e~ (ettdt.~ (0 ((‘~‘Ct Ike [LLO(LL[L( d(L0LL~ 0Livk’(L~ (0 olLe 01 (~L(LL[1aLy ~.k iLL [LI ILte äL I
In a preferred embodiment, the IIPLU method for the analy~ms of Serratmopeptidase comprises a
mobile phase comprising a first liquid A which is aqueous based.
In an another preferred embodiment, the HPI C’ method for the analysis of Serratiopeptidase
comprises a mobile phase comprising a second liquid B which is an organic solvent.
BluFF lMT~SCRIPTION OF TIUT DRAWINGS
The above and other objects and features of the present invention will become apparent from the
following description of the invention, when taken in conjunction with the accompanying
drawings, in which:
FIG. 1 is a Prototype chromatogram obtained for Blank, Standard and Sample
DETAILED DESCRIPTION OF THE INVENTION
While this specification concludes with claims particularly pointing out and distinctly claiming
that, which is regarded as the invention, it is anticipated that the invention can be more readily
understood through reading the following detailed description of the invention and study of the
included examples.
6
Serratiopeptidase is a protease enzyme which act as a catalyst in the hydrolysis of casein In the
present invention, casein is hydrolyzed in the presence of Serratiopeptidase at a particular
temperature for a particular time. As a result of hydrolysis, the casein breaks down into its
constituent peptide and amino acids. These peptide and amino acids have chromophores along
with other physiochemical properties which helps in analysis of the peptide and amino acids
using HPLC.
As used herein, the term “HPLC”, as in pharmaceutical analysis, is intended to encompass an
analytical method comprising high pressure or high performance liquid chromatography.
The term “composition”, as in pharmaceutical composition, is intended to encompass a drug
product comprising Serratiopeptidase or its pharmaceutically acceptable salts, solvates,
poLyLtLoLplL’., eIIaLLLLOLLL@L s oi LrLIXELLt(is iiLeL(~oI, and oLLL~L L[t~t E LLLgLe(lLeLlL(s) SLiclL iliat LLLaCeLLILCaL
compositions are synonymous with “formulation” and “dosage form”.
The term “about” as used herein, refers to any value which lies within the range defined by a
variation of up to ±10% of the value.
The present invention relates to a reproducible and reliable HPLC analytical method for the
analysis of Serratiopeptidase in pharmaceutical dosage forms. The analytical method to perform
detection and quantification of Sen atiopepticlase complises a colunrn, a suitable detector, and
mohtk~ phasi ALL o(hor acc~~,sson(is which arc.i t~ssin1iaL [or l~r[ornhin~ (he analysis on a I LL’L (
system are well known to a skilled analyst, and are thus assumed to be a part of the general state
of art, and are not being discussed separately.
A first aspect of the present invention provides a HPLC method for the analysis of
Serratiopeptidase in pharmaceutical dosage forms, wherein the mobile phase comprises two
liquids and the relative concentration of the liquids is varied to a predetermined gradient.
It was found by the inventors of the present invention that a gradient I IPLU method comprising
mixture of two liquids as per the present invention exhibited excellent reproducibility, robustness
and reliability.
According to one embodiment of the above aspect, the HPLC method for the analysis of
Serratiopeptidase comprises a mobile phase comprising a first liquid A which is aqueous based.
In an another preferred embodiment, the HPLC method for the analysis of Serratiopeptidase
comprises the first liquid A comprising water or an aqueous solution of a buffer.
Another embodiment of the present invention relates to a HPLC method for the analysis of
Serratiopeptidase comprising the first liquid A comprising a buffer selected from an acid or an
organic salt or an inorganic salt or a mixture thereof.
According to yet another embodiment of the above aspect, the buffer is selected from the group
comprising a phosphate salt, an acetate salt, a formate salt, orthophosphoric acid, trifluoroacetic
acid, acetic acid or a mixture thereof. In a preferred embodiment, the buffer is trifluoroacetic
acid.
According to one embodiment of the above aspect the HPT C method for the analysis of
Serratiopeptidase comprises a mobile phase comprising a second liquid B which is an organic
601 VeLLt.
In accordance with still another embodiment of the above aspect, the HPLC method for the
analysis of Serratiopeptidase comprises a second liquid B selected from the group comprising a
polar protic solvent such as acetic acid, methanol, ethanol, propanol, isopropanol and/or a dipolar
aprotic solvent such as acetone, acetonitrile, dimethoxyethane, Dimethyl Fonnamide (DMF),
Diinethyl suiphoxide (DM50), 1,4-dioxane, pyiidine, ‘Ietiahydio fuian (‘IHF) oi a mixtule
tliereo(. In a prek~rred embodiment, the second liquid B is a dipolar aprotic solvent such as
acetonitrile.
In accordance with another embodiment of the above aspect, the relative concentration of the
liquids A and B is varied to a predetermined gradient as follows:
Time (Minutes) % v/v A % v/v B
0 QO 10
12 90 10
15 75 25
25 75 25
In accordance with still another embodiment of the above aspect, the relative concentration of
the liquids A and B is varied to a predetermined gradient as follows:
Time (Minutes) % v/v A % v/v B
0 90 10
2 90 10
15 75 25
25 75 25
26 90 10
30 90 10
Ac~oidiiig to aiio[liei einbodimeiil of [lie above aspe~[, [lie IIPLC method foi [lie aiialysis of
SeLl at Lopep( L(Ia~e IS (411 Le(1 (‘LIE a I L(~~~’ taLe (‘I (lie LrL(’blLe pILaW bet weeli 4L)OILL 0 0 L LLLI/LLLLLLLL(e
and about 10 ml!minute. More preferably, HPLC method for the analysis of Serratiopeptidase is
carried out a flow rate of the mobile phase between about 0.1 ml!minute and 4 mi/minute. Still
more preferably. HPLC method for the analysis of Sorratiopeptidase is carried out a flow rate of
the mobile phase between 1 mL/minute to 2 mL/minute. Still more preferably, a flow rate of
about 1.5 mi/minute is used.
AecoidiLIg to itll(~Ll[Ot eilrL[~o(LurLeLiL o[ hi al~ov~ ~ELLO ~[uetii iriay be aittls’~ed by a C16te~tOL
such as a UV and! or visible spectrophotometer, a fluorescence spectrophotometer, a differential
refractometer, an electrochemical detector, a mass spectrometer, a light scattering detector or a
radioactivity detector. Preferably, the eluent is analyzed by a UV detector set over a wavelength
range of 200 — 400 mn. Still more preferably, the eluent is analyzed by a UV detector at a
wavelength of 275 nm.
In an embodiment of the above aspect, the ctationary phase used is a gel Pret~rahLy, the
stationary phase used is a silica gel. More preferably, the stationary phase is a reverse phase
stationary phase such as octadecylsilyl silica gel, octylsilyl silica gel, phenylalkyl silica gel,
cyano propyl silica gel, amino propyl silica gel or an aikyl-diol silica gel. Particularly, suitable
stationary phases include octadecylsilyl silica gel or octylsilyl silica gel.
In another embodiment of the above aspect, the chromatography is carried out using a column
having a length between about 10mm and about 5000 mm, or between about 50 mm and about
500 mm, or between about 100 mm and about 250 mm. More preferably, the chromatography is
carried out using a column having a length of about 250 mm.
In yet another embodiment of the above aspect, the chromatography may be carried out using a
column having internal diameter between about 0.01 mm and about 100 mm, or between about
0.1 mm and about 50 mm in internal diameter, or between about 1mm and about 10mm in
internal diameter. More preferably the chromatography is carried out using a column of about 4.6
mm internal diameter.
A particularly preferred stationary phase comprises Zorbax SB Cl 8, 5 ~im column (250 mmx4.6
LrLI CL)
AccoLdLllg to yet aLLotliet einboduiieii[ of [lie above aspeot, [lie oluoiiia[ogiapliy is oaiiied out at a
column temperature between about 15°C and about 40°C. Preferably, the chromatography is
carried out at a column temperature of 30°C.
Another embodiment of the above aspect relates to the use of the HPLC based analytical method
for preparing Serratiopeptidase based composition of acceptable pharmaceutical purity.
In another embodiment of the above aspect, the HPLC method is used for the analysis of a
pharmaceutical composition comprising Serratiopeptidase. The pharmaceutical compositions
which can be analyzed by the current invention include solid and liquid compositions and
optionally comprise one or more pharmaceutically acceptable carriers or excipients. Solid
compositions comprise powders, tablets, pills, capsules, granules, suppositories, dispersible
granules and the like. Liquid compositions comprise solutions, suspensions, emulsions and the
like.
In yet another embodiment of the above aspect, the chrornatographic method is used for the
analysis of a pharmaceutical dosage form comprising Serratiopeptidase as the only active
pharmaceutical ingredient.
According to another embodiment of the above aspect, chromatographic analysis is performed
for a run time ranging between about 30 minutes.
According to another embodiment of the above aspect, all major peaks elutes between about 4
minutes and about 25 minutes ofthe retention time (RT).
According to another embodiment of the above aspect, about 39 peaks elute with area ranging
from about 600 to about 130000 j.tV*seconds.
According to another embodiment of the above aspect, the maj or peak is observed at a retention
time of about 15 minutes and has an area between about 85000 to about 130000 ji.V*seconds.
The current invention is also advantageous as the method is specific, accurate, linear, precise,
arid robust for the analysis of Serratiopeptidase in phairnaceutical dosage foims. Further, the
developed HPLC method is capable of separating about 30 products of enzymatic hydrolysis of
(~L5Oit1 1) ~,/ 5(111 Ut LOIUiLII idti~~ W iLiLLIL ?0 LLLLLLL1Ee~, (‘C Lli~ ((‘till 1 ULL I LLLLC (‘C 10 LrliliLlteS.
I Laviiig clesc’riboct t[ie~ ilivention ~vith referenc~ to c’c’wtain pr~f~rred oinbodiinonts, other
embodiments will become apparent to one skilled in the art from consideration of the
specification. The invention is further defined by reference to the following examples describing
In detail method for the analysis of Serratlopeptidase. It will be apparent to those skilled in the
art that many modifications, both to materials and methods, may be practiced without departing
from the scope of the invention.
EXAMPLES
Following examples are set out to illustrate the invention and do not limit the scope of the present
invention.
Example I
~‘reparation of Liquid A for mobi[~ ph~s~
1 mL oftrifluoroacetic acid was diluted to 1000 mL with distilled water, mixed well and degassed.
Preparation of 1 M Hydrochloric acid:
8.5 mL of concentrated hydrochloric acid was transferred to a 100 mL volumetric flask containing
about 30 mL of water and diluted to volume up to the mark with water.
Preparation of diluent (borate buffer pH 9.0):
19g of disodium tetraborate was accurately weighed and dissolved in 900 mL of distilled water.
The pH of the solution was adjusted to 9.00 ± 0.05 with 1M hydrochloric acid, diluted to 1000 mL
with distilled water and mixed well.
Preparation of trichioroacetic acid solution:
1 Og of trichloroacetic acid, 15 g of sodium acetate trihydrate and 20 mL of glacial acetic acid was
accurately transferred to a 1 OOmL volumetric flask having 50 mL of water. The mixture was
sonicated, allowed to attain room temperature and the volume was made up with distilled water.
Preparation of Casein solution:
L0() ml of diLuc’nt ~va~ added to a )%() rnL gLa~ beaker and ctirred (in a LrLagfletLc’ ctLLTC’L L 1 g of
Hammarsten Casein was accurately transferred to the 250 ml~ glass beaker and stirring was
continued for 30 minutes. The resultant solution was transferred to a 200 mL volumetric flask. The
beaker was rinsed 2-3 times with 20 mL diluent and the rinsing was transferred to the volumetric
flask and diluted to volume with diluent and mixed well.
Preparation of Linearity stock solution-i (Li):
45 mg of Serratiopeptidase working standard was accurately transferred to a 500 mL dried
volumetric flask. ~00 ml oC(liluent was adde(1 and the mixture was sonicated For about 45 minutes
with intermittent vigorous shaking (after every 2 minutes). Froth was allowed to settle down and
the volume was made up to the mark with diluenL3 mL of (his solution was diluLed to 100 mL
with diluent and mixed well.
Preparation of Linearity stock solution-2 (L2):
65 mg of Serratiopeptidase working standard was accurately transferred to a 500 mL dried
volumetric flask. ~0() ml~ of diluent was added and the mLXtULe was sotucated for about 4% mmutes
with intermittent vigorous shaking (after every 2 minutes). Froth was allowed to settle down and
the volume was made up to the mark with diluent. 3 mL of this solution was diluted to 100 mL
with diluent and mixed well.
Preparation of Linearity stock solution-3 (L3):
75 rng of Serratiopeptidase working standard was accurately transferred to a 500 mL dried
12
volumetric flask. 300 mL ofdiluent was added and the mixture was sonicated for about 45 minutes
with intermittent vigorous shaking (after every 2 minutes). Froth was allowed to settle down and
the volume was made up to the mark with diluent. 3 mL of this solution was diluted to 100 mL
with diluent and mixed well.
Preparation of sample stock solution-i (Si):
5 intact tablets were accurately transferred to a 500 mL dried volumetric flask. 300 mL of diluent
was added and the mixture was sonicated for 45 minutes with intermittent vigorous shaking (after
every 2 minutes). Froth was allowed to settle down and the volume was made up to the mark with
diluent and mixed well. The solution was centrifuged at 2500 ipm for 30 minutes. 3 rnL of this
solution was diluted to 100 mL with diluent and mixed well.
Preparation of sample stock solution-2 (S2
5 intact tablets were accurately transferred to a 500 mL dried volumetric flask. 300 mL of diluent
was added and the mixture was sonicated for 45 minutes with intermittent vigorous shaking (after
every 2 minutes). Froth was allowed to settle down and the volume was made up to the mark with
diluent and mixed well. The solution was centriftiged at 2500 rpm for 30 minutes. 3 mL of this
solution was diluted to 100 mL with diluent and mixed well.
1LL~ 14114(L(JLL Pt1ItL(.~.
I) The water bath was maintained at temperature 37.0°C ± 0.1°C.
2) 50 mL glass stoppered Class A glass test tubes were taken.
3) Three test tubes were designated as Lia, Lib for linearity solution-i and Lb for linearity blank
respectively.
4) Two test tubes were designated as L2a, L2b for linearity soiution-2 respectively.
S) Two tost tuhos wore dosi~natod as L ~, I ~ for linearity solution ~ respectively
6) Three test tubes were designated as Tia, Tib for sample solution-I and Tb for sample blank
respectively.
7) Two test tubes were designated as T2a, T2b for sample solution-2 respectively.
8) The solutions were added in the designated test tubes as per scheme mentioned in TABLE 1:
TABLE 1
Test Tube
Lb Lia Lib L2a L2b L3a L3b Tb Tia Tib T2a T2b
Name
Diluent(mL) 1 1 1 1 1 1 1 1 1 1 1
Linearity stock2 2 2 - - - - - - -
1 (ml,)
Linearity stock
- - - 2 2 - - - - - -
2 (niL)
Linearity stock3 (mL)
Sample stock 1
- - - - - - - 2 2 2 -
(mL)
Sample stock-2
2 2
(mL)
Mixed by vortex shaking for 30 seconds. Incubated at 3 7.0°C ± 0.1°C for 10 minutes
Trichioroacetic
acid solution 5 - - - - - - 5 - - -
(mL)
Mixed by vortex shaking for 30 seconds.
Casein Solution
5 5 5 5 5 5 5 5 5 5 5 5
(mL)
N4i’~ed b3~ VOL Le~ siLak LLLg IOL 1() se(o[Lds LLLuL[i~t1ed COL ‘I S LElillutes
Trichioroacetic
acid solution - 5 5 5 5 5 5 - 5 5 5 5
(mL)
Mixed by vortex shaking for 30 seconds. Incubated for 30 minutes
14
The test tubes were removed from the water bath and kept immediately in ice cold water for 10 minutes.
The contents of each tube were filtered through 0.45 ~.tm nylon filter after discarding 3 mL of filtrate.
Chromatographic Conditions:
The chromatographic parameters were as follows -
Mobile Phase : Gradient Program using 0.1% Trifluoro acetic
acid and Acetonitrile as mentioned in table 2
Column
Detection
Lnjectic’n voLuLne
flow mate
Column oven temperature
Sample tray temperature
Run tiiti~
Needle Wash Solution
Zorbax SB C18, 250mm x 4.6 mm, ~
UVat275nm
[00 ~LL
1 ‘ ItLT /L[LiLllLt(1
30°C+5°C
10°C+5°C
10 minutes
Water: Acetonitrile in a ratio of 80:20 (vlv)
Gradient As per Table 2
Table 2
Time (Minutes) Buffer (%v/v) Acetonitrile (%v/v)
0 90 10
2 90 10
15 75 25
?~ 7S
26 90 10
30 90 10
Analysis Procedure:
Evaluation of System Suitability
1) Single injection of linearity blank (Lb) and sample blank (Tb) was injected into the
chromatograph and the chromatogram was recorded.
2) Linearity solutions (Li a, LI b, L2a, L2b L3a and L3b) were injected in to the chromatograph
and the chromatogram was recorded.
3) All the peaks observed after 4 minutes having an area counts of more than 1000 in linearity
blank, sample blank and linearity solutions were integrated.
4) Sum of all peak areas observed in linearity blank, sample blank and linearity solutions was
calculated.
5) The total area counts observed in each injection of linearity solution was subtracted from the
total area counts observed in linearity blank.
6) The HY LH~~ area coLitils of Ewe irietihahons of each lineariEv solttEion after subtraction from
linearity blank was calculated.
1) Intercept, slope and regression coefficient (R2) between area and corrected weight in hneanty
solutions was calculated.
8) System was found to be suitable for analysis if and only the linearity regression coefficient
(R2) was hot be less thaii 0.99000.
Sample Analysis and Evaluation of the Chromatograms
I) Single injection of sample solution (TLd, T[i, T?~ and T?j) was injected into the
chromatograph and the chromatogram was recorded.
2) All the peaks observed after 4 minutes having an area count of more than 1000 were
integrated.
3) The total area counts in each injection of sample solution was measured and the total area
counts observed in sample blank were subtracted from the total area counts of sample
solution
4) Calculate the assay value of each sample solution (Sla, Slb, S2a and S2b) after subtraction
from total area counts observed in sample blank.
5) The average assay value of sample solution-i (average Of Tia&b) and sample solution-2
(average of T2 a &b) was determined.
6) The average assay value of sample solution-i and sample solution-2 was reported.
epresen a ive chromatograms of Blank, Standard and Sample solution analyzed as per the
method of the present invention are presented in Figure 1, Figure 2 and Figure 3 respectively.
Calculations
Area in sample - intercept Serratiopeptidase
(mWtablet) Slope x 5
Area in sample - intercept 100 Serratiopeptidase
= x
(% claiixi) Slope x 5 Claim
Results of assay of three different batches of Serratiopeptidase tablet performed as per the
method of (lie pleseut iiiveiitlon ale pieseuted lii Table 3, Table 4 amid Table 5.
Table 3
% Assay
Sample ID
(with respect to claim)
Ti a 104.0
Tib 1OSQ
T2a 104.3
T2b i03.7
Tablets from Batch-i 104.5
Table 4
% Assay
Sample ID
(with respect to claim)
Ti a 104.8
Tib 107.9
T2a 102.3
T2b 102.9
Tablets from Batch-2 104 S
fable $
% Assay
Sample ID
(with respect to claim)
Ti a 107.6
lib 109.1
T2a 104.8
T2b 112.1
labicts from Batch-J 108.’I
While this invention has been described in detail with reference to certain preferred
embodiments, it should be appreciated that the present invention is not limited to those precise
embodiments. Rather, in view of the present disclosure, which describes the current best mode
for practicing the invention, many modifications and variations would present themselves to
tIlobe bkillcd iii the alt without depaitiiig Iloiii the scope, and spirit of t1ii~ invention.

WE CLAIM:

1. A HPLC method for the assay of Serratiopeptidase, wherein the mobile phase comprises
two liquids and the relative concentration of the liquids is varied to a predetermined
gradient.
2. The HPLC method as claimed in claim 1, wherein the mobile phase comprises a first
liquid A which is aqueous based.
3. The HPLC method as claimed iu claiiii 2, wimemein the first liquid A comprises water or
an aqueous solution of a buffer.
4. Ihe HPLC’ method as claimed in claim 3, wherein the buffer is an acid or an organic salt
or an inorganic salt or a mixture thereof.
5. The HPLC method as claimed in claim 4, wherein the buffer is a phosphate salt, an
acelate salt, a fommale salt, au oUi[opl[osplLoliL aud, ci E1ifluoiod~eEic ~‘tcid, €tceti~.. ?ICL(1 (IL ~t
mixture thereof.
6 Tho HPI C’ mothod as claimed in claim S wherein the buffer is trifluoroacetic acid.
7. The HPLC method as claimed in claim 1, wherein the mobile phase comprises a second
liquid B which is an organic solvent.
8. The HPLC method as claimed in claim 7, wherein the second liquid B is selected from
the group comprising methanol, acetonitrile, propanol or isopropanol or a mixture
thoreof
9. The HPLC method as claimed in claim 8, wherein the second liquid B is acetonitrile.
10. The HPLC method as claimed in claim 1, wherein the gradient is as follows:
Time (Minutes) % v/v A % v/v B
0 90 10
2 90 10
15 75 25
25 75 25
26 90 10
30 90 10
11. The HPLC method as claimed in claim 1, wherein the flow rate of the mobile phase is 1
mL/minute to 2 mL/minute.
12. The’ IIPLC’ iiie[liod u~ ~1uiiiied iii ~luiiii 1. wlieieiii the iuethod fui thei eoiupi i~es ~t ITV
detector set over a wavelength range of 200 — 400 nm.
13. The HPLC method as claimed in claim 1, wherein the method further comprises Zorbax
SB C18, 5 jim (250 mmx4.6 mm) column.
14 The I [P1 C’ method a~ claiLneci iLl claimn I whemeimL the L!lethO(l iS (4L Lied (‘Ut ~tt
te1ripe1atLLL~ beiwecim di)(IUI 15°C’ dLL(l dbout tlO°C’.
15. The HPLC method as claiiiied in claim 1, wherein the method involves analysis of
hydrolyzed Casein.
16. The HPLC method as claimed in claim 1, wherein the method is linear in a range of 0.3
jig/mL to 0.7 j.tg/mL of Serratiopeptidase.