simultaneous determination of sitagliptin phosphate...
TRANSCRIPT
126
CHAPTER-3
Simultaneous determination of Sitagliptinphosphate monohydrate and Metforminhydrochloride in tablets by validated ultraperformance liquid ChromatographyMethod
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3.1 Introduction
Sitagliptin phosphate monohydrate (SP) chemically, (7-[(3R)-3-amino-
1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoro meth-
yl)-1,2,4-triazolo[4,3-a] pyrazine phosphate monohydrate) [1] is an oral
anti-diabetic, which is available in 25 mg, 50 mg and 100 mg tablets for
oral administration. SP is indicated for the improvement of glycemic
control in patients with type II diabetes mellitus as monotherapy or
combination therapy with metformin or a peroxisome proliferator-
activated receptor gamma (PPAR) agonist (e.g., thiazolidinediones) when
the single agent does not provide adequate glycemic control. Sitagliptin
phosphate monohydrate is a white to off white crystalline powder and
soluble in water, slightly soluble in methanol, very slightly soluble in
ethanol, acetone, acetonitrile, insoluble in isopropanol and isoproprl
acetate. The empirical formula of sitagliptin phosphate monohydrate is
C16H15F6N5O.H3PO4.H2O. The molecular weight of sitagliptin phosphate
monohydrate is 523.33.
Metformin hydrochloride (MH) chemically, N,N-dimethylimidodi-
carbonimidic diamide hydrochloride [2] is an antidiabetic agent [3]. It is
first line drug for choice for the treatment of type II diabetes, particularly
in over weight and obese people and those with normal kidney function.
It works by lowering the blood sugar and helping the body to use insulin
more efficiently. It is available in 500 mg, 850 mg and 1000 mg tablets
(immediate release) and in 500 mg, 750 mg (slow release) for oral
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administration. It is a white to off-white crystalline powder and freely
soluble in water and is practically insoluble in acetone, ether, and
chloroform. The empirical formula of metformin hydrochloride is
C4H11N5.HCl. The molecular weight of metformin hydrochloride is 165.63.
Merck and Co. market sitagliptin in combination with metformin in a
single dosage form as JANUMET TM [4]. JANUMET is available for oral
administration as tablets containing 64.25 mg sitagliptin phosphate
monohydrate and metformin hydrochloride equivalent to 50 mg
sitagliptin as free base and 500 mg metformin hydrochloride (JANUMET
50 mg/500 mg) or 1000 mg metformin hydrochloride (JANUMET 50
mg/1000 mg). Each film-coated tablet of JANUMET contains the
following inactive ingredients: microcrystalline cellulose,
polyvinylpyrrolidone, sodium lauryl sulfate, and sodium stearyl
fumarate. In addition, the film coating contains the following inactive
ingredients: polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide,
red iron oxide, and black iron oxide.
Metformin works by decreasing glucose (sugar) production in the liver
and decreasing absorption of glucose by the intestines. Sitagliptin works
by regulating the levels of insulin your body produces after eating.
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Fig. 3.1.F1: Chemical structures of Sitagliptin phosphate andMetformin hydrochloride
(a) Sitagliptin phosphateF
F
F
N
NN
N
CF3
. H3PO4
H NH2O
Molecular formula: C16H15F6N5O.H3PO4
Molecular weight: 505.3
(b) Metformin hydrochloride
Molecular formula: C4H11N5.HCl
Molecular weight: 165.63
The literature reveals that there are some of the methods have been
reported for metformin. Few UV spectrophotometric methods [5-6], HPLC
[7-11] and ion-pair HPLC [12] method have been reported for the
estimation of metformin. SP is not yet official in any of the
pharmacopoeia but MH is official in IP [13], BP [14] and USP NF [15].
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Literature survey reveals that only LC‐MS [16-19] methods were reported
for the determination of sitagliptin phosphate in plasma and urine of
humans, rats and dogs. Literature indicated spectroflourometric and
spectrophotometric methods for the determination of sitagliptin in
pharmaceutical dosage forms [20]. Described the mass spectrometry
method for simultaneous quantitation of metformin and sitagliptin from
mouse and human dried blood spots using laser diode thermal
desorption tandem mass spectrometry [21].
The review of the literature revealed that ultra performance liquid
chromatographic (UPLC) method is not reported for the simultaneous
estimation of the SP and MH in combined pharmaceutical dosage form.
Therefore, it was thought worthwhile to develop a simple, precise,
accurate reverse phase Ultra performance liquid chromatographic
method for the simultaneous estimation of SP and MH in combined
tablet dosage form.
Forced degradation or stress studies of drug substance and products
play an integral role in the development of pharmaceuticals. The results
of degradation studies facilitate the stability indicating method
development. The current ICH guidelines requires that the analysis of
stability samples should be done by using stability-indicating assay
methods (SIAM’s), developed and validated after stress testing on drug
under variety of conditions, including hydrolysis, oxidation, photolysis
and thermal degradation.
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The target is to develop a suitable stability-indicating LC assay
method for simultaneous determination of sitagliptin phosphate and
metformin hydrochloride in combined dosage forms. In this chapter
described a stability indicating UPLC method which can separate
metformin and sitagliptin impurities along with separation of sitagliptin
and metformin.
3.2 Experimental
3.2.1 Materials:
Bulk sample of sitagliptin phosphate monohydrate and metformin
hydrochloride were received from research development department of
Dr. Reddy’s laboratories limited, Hyderabad, India. Commercially
available Janumet [4] tablets manufactured by Merck & Co., Inc., NJ,
USA. Hexane-1- sulfonic acid sodium salt and acetonitrile (HPLC grade)
were purchased from Merck specialties private limited, India. Water was
de-ionized and purified on a Milli-Qwater purification system (Millipore,
Bedford, MA, USA) and used to prepare all solutions.
3.2.2 Instrumentation and Chromatographic Conditions:
The UPLC system was Waters alliance 2695 including pump, auto
sampler and PDA detector 2996. Aquity UPLC BEH C8 (100 x 2.1 mm,
1.7 µm) was used as stationary phase. The mobile phase composition
used was the buffer 10mM potassium dihydrogen phosphate buffer and
2mM hexane-1- sulfonic acid sodium salt (pH adjusted to 5.5 with
diluted phosphoric acid) and acetonitrile with gradient program
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(Time(min)/% acetonitrile): 0/8, 2/8, 4/45, 6/45, 8/8, 10/8). Prior to
use, the mobile phase was filtered by using 0.2 µm filter and degassed.
The mobile phase pumped at 0.2 mL min-1 and water was used as
sample diluent. The column temperature was 27°C (ambient) and eluants
were monitored at 210 nm. The injection volume for samples and
standards was 0.5 µL. The total analysis run time was 10 min.
3.2.3 Standard preparation:
A standard solution containing 50 µg mL-1 of sitagliptin phosphate
and 500 µg mL-1 of metformin hydrochloride were prepared by dissolving
in diluent.
3.2.4 Sample Preparation:
One tablet containing 50 mg of SP and 500 mg of MH was transferred
to a 1000 mL standard volumetric flask and dissolved in1000 mL of
diluent. The solution was filtered through 0.2 µm Millipore PVDF filter.
Then 0.5 µL of these solutions were injected in the column. Prepared
impurity blend solution of metformin impurity1 & 2 and sitagliptin
impurity with 0.10 mg mL-1 concentration in diluent.
3.2.5 Generation of stress samples:
One lot of Junumet tablets were selected for stress testing. From the
ICH Stability guideline: “stress testing is likely to be carried out on a
single batch”. Different kinds of stress degradation conditions (like heat,
humidity, acid, base, oxidative and light) were performed on one lot of
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sitagliptin phosphate and metformin hydrochloride tablets based on the
guidance available from ICH Stability Guideline (Q1AR2). The details of
the stress conditions applied are as follows:
a) Acid hydrolysis: Drug solution in 0.1N HCl at 70° C for 48 h.
b) Base hydrolysis: Drug solution in 0.1N NaOH at 70°C for 48 h.
c) Study in Neutral solution: Drug product dissolved in water was heated
at 70°C for 48 h.
d) Oxidative stress: Drug solution in 3% hydrogen peroxide at room
temperature for 4 h.
e) Thermal stress: Tablets were subjected to dry heat at 105°C for 10 d.
f) Photolytic degradation (254 nm): for 10 days.
3.3 Method development and optimization of chromatographic
conditions:
Forced degradation studies were performed to develop a stability
indicating UPLC method for the simultaneous quantitative determination
of sitagliptin phosphate and metformin hydrochloride in combined
pharmaceutical dosage forms.
Sitagliptin phosphate (pKa = 7.7) and Metformin hydrochloride (pKa =
12.4) are basic in nature. The main target of the chromatographic
method is to get the separation of metformin impurities (met impurity-1
and met impurity-2) from metformin and sitaglipin impurity from
sitagliptin. The impurities blend solution was analyzed using different
stationary phases like C18, C8, Cyano and mobile phases, containing
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buffers like phosphate, sulphate and acetate with different pH (2-7) and
using organic modifiers like acetonitrile and methanol in the mobile
phase.
3.3.1 Selection of wavelength:
The two drugs sitagliptin phosphate and metformin hydrochloride
spectrums were collected using Water PDA system. The two drugs were
having UV maxima at around 210 nm (Fig 3.3.F1). Hence detection at
210 nm was selected for method development purpose.
Fig. 3.3.F1: Typical UV spectrums of Sitagliptin phosphate andMetformin hydrochloride.
(a) UV Spectrum of Sitagliptin phosphate
7.075 Sitagliptin190.3
267.2
AU
0.00
0.05
0.10
0.15
nm200.00 250.00 300.00 350.00
(b) UV Spectrum of Metformin hydrochloride2.200 Metformin
195.8 232.9
AU
0.00
1.00
2.00
nm200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
Wavelength (nm)
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3.3.2 Column Selection:
Two different C18 (Aquity BEH C18, 100 x 2.1 mm, 1.7 microns and
Zorbax C-18, 50 x 4.6 mm, 1.8 microns) Columns, one C8 Column
(Aquity BEH C8, 100 x 2.1 mm; 1.7 microns) and Zorbax SB-CN, 50 x
4.6 mm, 1.8 microns columns were used for method development. The
stationary phase had a great influence on the retention time, resolution
and peak shape.
The main difficulty of the chromatographic method was to get the
separation of metformin impurities from metformin. When Aquity BEH
C18, 100 x 2.1 mm; 1.7 micron and Zorbax C18, 50 x 4.6 mm, 1.8
microns columns were used in initial experiment with a mobile phase
composition of potassium dihydrogen phosphate buffer adjusted to pH
5.5 and acetonitrile in the various ratios in gradient mode. The flow rate
was 0.3 ml/min. The separation was achieved between metformin and
sitagliptin. But metformin imputiy-1 and metformin impurity-2 were not
separated from metformin. The typical chromatograms on BEH C18 and
Zorbax C18 are shown in Fig. 3.3.F2 & F3.
Fig. 3.3.F2: Trial chromatogram on BEH C18 column
Metforminimp1-0.599
Metformin+Metforminimp2-0.780
Sitagliptin-13.377
Sitagliptinimp-14.810
AU
0.00
1.00
2.00
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
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Fig. 3.3.F3: Trial chromatogram on Zorbax C18 column
Metformin+Metimps-3.140
Sitagliptin-5.630
SitagliptinImpurity-5.909
AU
0.00
0.02
0.04
0.06
Minutes0.00 2. 00 4.00 6.00 8.00 10 .00
X-axis: Retention time in min and Y-axis: Peak response in AUWhen Zorbax SB-CN, 100 x 4.6 mm, 1.8 microns column was used
with the mobile phase consists buffer (10mM potassium dihydrogen
phosphate whose pH was adjusted to 5.50 with diluted phosphoric acid)
and acetonitrile in a gradient program metformin impurities were not
separated properly from metformin. But sitagliptin impurity was
separated from sitagliptin (Fig. 3.3.F4).
Fig. 3.3.F4: Trial chromatogram on Zorbax SB-CN column
Metformin-1.359
MetImpuirty1-1.505
MetImpurity2-1.608
Sitagliptin-5.582
SitagliptinImpurity-6.061
AU
0.00
0.20
0.40
0.60
Minutes0.00 2.00 4.00 6.00 8.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AU
When Aquity UPLC BEH C8, 100 x 2.1 mm, 1.7 microns column
used as stationary phase with buffer (10mM potassium dihydrogen
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phosphate whose pH was adjusted to 5.50 with diluted phosphoric acid)
and acetonitrile in a gradient program, the resolution was not achieved
between metformin impurities from metformin.
The further trail was carried out by using the same column with
mobile phase consists buffer (10mM potassium dihydrogen phosphate
and 2mM hexane-1- sulfonic acid sodium salt, pH was adjusted to 5.50
with diluted phosphoric acid) and acetonitrile in a gradient program,
metformin impurities are well separated from metformin along with the
separation of sitagliptin impurity from sitagliptin. After adding hexane-1-
sulfonic acid sodium salt as ion pair reagent to buffer solution, the
complete separation was achieved between metformin impurities and
metformin. The blend chromatogram of sitagliptin and metformin with
impurities (metformin impurity1, metformin impurity2 and sitagliptin
inpurity) is shown in Fig.3.3.F5.
Fig. 3.3.F5: Trial chromatogram on UPLC BEH C-8 column
MetforminImp1-1.705
MetforminImp2-1.988
Metformin-2.185
Sitagliptin-6.969
SitagliptinImp-7.332
AU
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AU3.3.3 Effect of Organic solvent:
When methanol was used as a solvent in the mobile phase, methanol:
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buffer in the ratio 85: 15 (v/v) the retention time of metformin was
around 5 min with broad peak and resolution was not achieved between
metformin and metformin impurities. The sitagliptin was not retained
properly. The acetonitrile was instead of methanol, improved the peak
shape for metformin and sitagliptin.
3.3.4 Effect of pH:
The method was optimized to get the gussion peaks for metformin
and sitagliptin under varies pH of buffer. The pH 5.5 was found more
appropriate for peak shape for metformin, sitagliptin and related
impurities.
Based on method development trails it indicated that the gradient
elution with ion pair mobile phase was successful in separating drugs,
related impurities and all chromatographic degradation products.
3.3.5 Optimized chromatographic conditions:
Column: Aquity UPLC BEH C8 (100 x 2.1 mm, 1.7 µm)
Mobile phase: The mobile phase composition used was the buffer 10mM
potassium dihydrogen phosphate buffer and 2mM hexane-1- sulfonic
acid sodium salt (pH adjusted to 5.5 with diluted phosphoric acid) and
acetonitrile with gradient program.
Flow rate : 0.2 ml min-1
Column temperature : 27 °C
Wavelength : 210 nm
Injection volume : 0.5 µL
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Run time : 10 min
Diluent : Water
Gradient program (Time(min)/%acetonitrile): 0/8, 2/8, 4/45, 6/45, 8/8
and 10/8.
Retention time: sitagliptin is about 7 min and metformin is about 2 min .
The interference of inactive ingredients microcrystalline cellulose,
polyvinylpyrrolidone, sodium lauryl sulfate, and sodium stearyl fumarate
was also checked by injecting sample solutions of excipients. There was
no interference of excipients with sitagliptin and metformin peaks.
Fig. 3.3.F6: Typical tablet chromatogram
Metformin-2.199
Sitagliptin-7.033
AU
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AU3.4 Degradation behavior:
Degradation studies on sitagliptin phosphate and metformin
hydrochloride tablets under different stress conditions suggested the
following degradation behavior.
3.4.1 Degradation in acidic solution:
Sitagliptin phosphate and metformin hydrochloride drugs were
exposed to 0.1 N HCl for 48 hours reflux at 70°C. Slight degradation was
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observed (Fig. 3.4.F1 (a) and (b)).
Fig. 3.4.F1(a): Typical Blank chromatogram of acid hydrolysis
1.822
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AU
Fig. 3.4.F1(b): Typical chromatogram of acid hydrolysis
Metformin-2.212
Peak2-2.668
Peak3-6.392
Sitagliptin-7.135
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Metformin 2.21 0.46 1.00 No pass
Sitagliptin 7.13 0.55 1.02 No pass3.4.2 Degradation in Basic solution:
Sitagliptin phosphate and metformin hydrochloride drugs were
exposed to 0.1 N NaOH for 48 hours reflux at 70°C. Major degradation
products were observed (Fig 5.4.F2(a) and (b)).
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Fig. 3.4.F2(a): Typical Blank chromatogram of base hydrolysis
1.480
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Fig. 3.4.F2(b): Typical chromatogram of base hydrolysis
Metformin-2.212
Peak2-2.668
Peak3-6.392
Sitagliptin-7.135
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Metformin 2.21 0.77 1.00 No pass
Sitagliptin 7.15 0.80 1.07 No pass
3.4.3 Degradation in peroxide (Oxidative degradation):
Sitagliptin phosphate and metformin hydrochloride drugs were
exposed to 3 % peroxide, for 4 hours reflux at room temperature. Major
degradation products observed (Fig. 3.4.F3(a) and (b)).
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Fig. 3.4.F3(a): Typical Blank chromatogram of oxidative degradation
1.566
1.732
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Fig. 3.4.F3(b): Typical chromatogram of oxidative degradation
Peak1-1.979
Metformin-2.194
Peak3-2.617
Peak4-3.369
Peak5-4.839
Sitagliptin-7.111
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Metformin 2.19 0.72 1.00 No passSitagliptin 7.11 0.53 1.02 No pass
3.4.4 Degradation in Neutral (Water) solution:
No major degradation products were observed when stressed
conditions were applied (in water 48 hours reflux at 70 ºC). Minor
degradation products observed (Fig. 3.4.F4(a) and (b)).
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Fig. 3.4.F4(a): Blank chromatogram of Water degradation
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Fig. 3.4.F4(b): Typical chromatogram of Water degradation
Peak1-1.753
Metformin-2.212
Peak3-2.627
Peak4-6.425
Sitagliptin-7.169
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Metformin 2.21 0.65 1.00 No pass
Sitagliptin 7.17 0.83 1.02 No pass
3.4.5 Photolytic conditions:
When the sitagliptin phosphate and metformin hydrochloride drugs
powder was exposed to UV and visible light for 10 days, no degradation
was observed (Fig. 3.4.F5). The drugs were stable to photolysis.
144
Fig. 3.4.F5 : Typical chromatogram of photolytic degradation
Metformin-2.194
Sitagliptin-7.056
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Metformin 2.19 0.64 1.00 No pass
Sitagliptin 7.05 0.80 1.03 No pass
3.4.6 Thermal Degradation:
No significant degradation was observed when the sitagliptin
phosphate and metformin hydrochloride drugs powder was exposed to
heat at 105°C for 10 days (Fig. 3.4.F6 ).
Fig. 3.4.F6: Typical chromatogram of thermal degradation
145
Metformin-2.216
Sitagliptin-7.057
AU
-0.10
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 2.00 4.00 6.00 8.00 10.00
X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Metformin 2.22 0.78 1.00 No pass
Sitagliptin 7.06 0.41 1.04 No pass
Peak purity test performed for the sitagliptin and metformin peaks
using photodiode array (PDA) detector data confirmed the purity of the
peak for all the stressed samples. In all the samples the peaks of
sitagliptin phosphate and metformin hydrochloride were pure and
homogenous. Assay of all stressed samples were calculated using
qualified standards of sitagliptin phosphate and metformin
hydrochloride. Considering the purities from the respective
chromatograms of stressed samples, mass balance (%assay + %
degradation products + % impurities) was calculated for each stress
sample. The mass balance of stressed samples was close to 99.0%. This
clearly demonstrates that the developed HPLC method was found to be
146
specific for sitagliptin phosphate and metformin in presence of its
degradation products.
3.5 Method Validation
The developed and optimized HPLC method was taken up for
validation. The analytical method validation was carried out in
accordance with ICH guidelines [22-24].
3.5.1 System Suitability Test:
Standard containing sitagliptin phosphate and metformin
hydrochloride was injected into UPLC system for 5 times and the system
suitability results were tabulated (Table 3.5.T1)
Table 3.5.T1: System Suitability results
Compound(n=5)
USPTailing factor (T)
% RSD for 5replicate injections
Metforminhydrochloride 1.2 0.5%
Sitagliptinphosphate
1.1 0.2%
n = Number of determinations
3.5.2 Precision:
The precision of an analytical procedure expresses the closeness of
agreement between a series of measurements obtained from multiple
sampling of the same homogenous sample under the prescribed
conditions. Assay method precision study was evaluated by carrying out
six independent assays of sitagliptin phosphate and metformin
147
hydrochloride test sample against qualified reference standards and RSD
of six consecutive assays was 0.60% and 0.60% for sitagliptin phosphate
and metformin hydrochloride respectively (intra-day) (Table 3.5.T2) and
0.50% and 0.72% for sitagliptin phosphate and metformin hydrochloride
respectively (inter-day) (Table 3.5.T3 and Table 3.5.T4). Results showed
no significant variation in measured response, which demonstrated that
the method was repeatable with RSDs below 1.0 %.
Table 3.5.T2: Precision results of the assay method (intra-day)
Preparation
% Assay
Sitagliptin phosphate Metformin hydrochloride
1 98.56 98.38
2 99.12 99.17
3 99.44 98.65
4 100.10 97.59
5 99.80 98.14
Average 99.40 98.39
SD 0.60 0.59
% RSD 0.60 0.60
148
Table 3.5.T3: Precision results of the assay method (inter-day)
Preparation% Assay
Sitagliptin phosphate Metformin hydrochloride
1 99.45 99.56
2 98.92 98.49
3 99.16 99.18
4 99.53 97.93
5 98.29 98.05
Average 99.07 98.64
SD 0.50 0.71
% RSD 0.50 0.72
Table 3.5.T4: Results of repeatability and intermediate precision
S.No Parameter Variation
% RSDfor Assay
Sitagliptinphosphate
Metforminhydrochloride
1
Intermediate
precision
(intra-day)
(a) Analyst-2
(b) Waters AcquityUPLC system withUV detector
(c) Day-2
0.6 0.6
2
Repeatability
(inter-day)
(a) Analyst-1
(b) Waters AcquityUPLC system withPDA detector
(c) Day-1
0.5 0.7
149
3.5.3 Linearity of the assay method:
The linearity of an analytical procedure is its ability (within a given
range) to obtain test results which are directly proportional to the
concentration (amount) of analyte in the sample. The linearity of the
assay method was established by injecting test sample from 50% to
150% of two drugs sitagliptin phosphate and metformin hydrochloride
assay concentrations (i.e. 50 µg/ml of sitagliptin phosphate and 500
µg/ml of metformin hydrochloride). Each solution injected into UPLC and
from that calculated the correlation coefficient (Table 3.5.T5 and Table
3.5.T6). The slope and Y-Intercept of the metformin peak in the linearity
study was found to be 8545 and 41987 respectively and the slope and Y-
Intercept of the sitagliptin peak in the linearity study was found to be
3975 and -17863 respectively.
Calibration curve obtained by least square regression analysis
between average peak area and the concentration showed (Fig. 3.5.F1 to
Fig. 3.5.F2) linear relationship with a regression coefficient of 0.999. The
best fit linear equation obtained was Y =3975 Con - 17863 for sitagliptin
phosphate and Y =18545 Con + 41987 for metformin hydrochloride.
150
Table 3.5.T5: Linearity results of the assay method for Sitagliptinphosphate
Concentration (µg mL-1) Peak area
25 81392
37.5 131354
50 180767
62.5 230935
75 280056
Correlation Coefficient(r) 0.999
Slope 3975
Intercept -17863
Fig. 3.5.F1: Linearity plot for Assay Method for Sitagliptinphosphate
X-axis: Concentration in µg mL-1 and Y-axis: Peak area mAU
151
Table 3.5.T6: Linearity results of the assay method for Metforminhydrochloride
Concentration (µg mL-1) Peak area
250 2129272
375 3265303
500 4338078
625 5476560
750 6364618
Correlation Coefficient(r) 0.998
Slope 8545
Intercept 41987
Fig. 3.5.F2: Linearity plot for Assay Method for Metforminhydrochloride
X-axis: Concentration in µg mL-1 and Y-axis: Peak area mAU
152
3.5.4 Accuracy:
The accuracy of an analytical procedure expresses the closeness of
agreement between the value, which is accepted either as a conventional
true value or an accepted reference value and the value found.
Accuracy of the assay method was established by injecting three
preparations of test sample using tablets at 50%, 100% and 150% of
analyte concentration (i.e. 50 µg/ml of sitagliptin phosphate and 500
µg/ml of metformin hydrochloride). Each solution was injected thrice
(n=3) into UPLC and the mean peak area was calculated. % Assay of test
solution was determined against three injections (n=3) of qualified
sitagliptin phosphate and metformin hydrochloride standards (Table
3.5.T7). The method showed consistent and high absolute recoveries at
all three concentration (50, 100 and 150%) levels with mean absolute
recovery ranging from 99.1 to 100.5% for sitagliptin phosphate and from
99.0 to 100.8% for metformin hydrochloride.
Table 3.5.T7: Recovery of the assay method
S.No Concentration(%)Mean recovery (%) % RSD
SP MH SP MH
1 50 99.75 100.6 0.45 0.50
2 100 101.25 100.0 0.26 0.47
3 150 100.18 98.32 0.29 0.61
SP = Sitagliptin phosphate
MH= Metformin hydrochloride
153
3.5.5 Solution stability:
The solution state stability of sitagliptin phosphate and metformin
hydrochloride in diluent in the assay method was carried out by leaving
the test solutions of sample in tightly capped volumetric flasks at room
temperature for two days. The same sample solution were assayed for
every six hours interval up to the study period, each time freshly
prepared reference standard was used to estimate the assay of test
sample. The % RSD of assay of sitagliptin phosphate and metformin
hydrochloride during solution stability experiments was within 0.5% for
two drugs. Hence sample solutions are stable for at least 48 hours in the
developed method. Standard and test solution injected at each 0 h, 6 h,
12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h.
Table 3.5.T8: Solution stability results of the assay method
S.No. Interval% Assay
Sitagliptinphosphate
Metforminhydrochloride
1 0 h 99.62 99.312 6 h 99.63 98.893 12 h 99.11 99.213 18 h 99.55 98.764 24 h 99.44 99.135 30 h 98.99 98.496 36 h 99.14 99.127 42 h 99.01 98.658 48 h 99.26 99.17
Average 99.31 98.97SD 0.259 0.284
% RSD 0.261 0.287
154
3.5.6 Robustness:
To determine the robustness of the developed method experimental
conditions were purposely altered and the tailing factor for metformin
peak and sitagliptin peak and also % RSD for 5 replicate injections of
metformin and sitagliptin were evaluated. In each of the deliberately
altered chromatographic condition (flow rate 0.18 mL min-1 and 0.22 mL
min-1, column temperature 20 ºC and 30 ºC and pH of the buffer 5.30
and 5.50) the tailing factor, and % RSD for 5 replicate injections of
metformin and sitagliptin peaks were within the limits illustrating the
robustness of the method (Table 3.5.T9).
Table 3.5.T9: Results of robustness study
Parameter Temperature
(± 5 0C of settemperature)
Flow rate(± 0.02 mL min-1
of the set flow) pH of the buffer(± 0.2 of pH)
Variation 200C 300C 0.18mL min-1
0.22mL min-1 5.30 5.50
USP Tailingfactor (T) SP 1.1 1.0 1.0 1.1 1.1 1.0
MH 1.0 1.1 1.1 1.0 1.1 1.1% RSD for
5 replicateinjections
SP 0.2 0.3 0.3 0.2 0.4 0.3
MH 0.3 0.4 0.4 0.3 0.5 0.6
SP= Sitagliptin phosphate ,
MH= Metformin hydrochloride
155
3.5.7 Tablet application
Analysis was performed for commercially available innovator tablets.
The Mean assay (n = 6) for SP and MH was 100.2% and 99.6%,
respectively. The percentage RSD value for the six assay values was
0.5%, 0.6% for SP and MH, respectively.
3.6 Summary and Conclusion
Validated stability-indicating UPLC method was developed for
simultaneous determination of sitagliptin phosphate and metformin
hydrochloride in combined tablets formulation. The RP-UPLC method
developed is rapid, precise, accurate and selective. The method was
completely validated showing satisfactory results for all the method
validation tested parameters. The developed method was found “specific”
to the drugs and for dosage form in combined tablets, as the peaks of the
degradation products did not interfere with the two drugs. Thus the
proposed method can be employed for assessing the stability of
sitagliptin phosphate and metformin hydrochloride for its individual
dosage form and for its combined dosage forms.
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