chapter 6 spectrophotometric and chromatographic...
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CHAPTER 6
SPECTROPHOTOMETRIC AND CHROMATOGRAPHIC ASSAY OF GLIPIZIDE
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
260
Section 6.0
DRUG PROFILE AND LITERATURE SURVEY
6.0.1 DRUG PROFILE
Glipizide (GPZ), chemically known as N-[2[4[[[(Cyclohexylamino)
carbonyl] amino] sulfonyl] phenyl] ethyl]-5- methylpyrazinecarboxamide is an
oral anti-hyper glycaemic agent [1]. Its molecular weight is 455.5 g mol-1
corresponding to the formula C21H27N5O4S. Its chemical structure is given below:
Figure 6.0.1. Structure of glipizide
GPZ is a white or almost white crystalline powder with a melting point of
200-203ºC, practically insoluble in water, freely soluble in di-methylformamide,
very slightly soluble in methylene chloride and in acetone, and practically
insoluble in ethanol (96%). It dissolves in dilute solutions of alkali hydroxides.
GPZ belongs to sulphonyl urea class of antidiabetics and is indicated for
type II diabetes mellitus [2]. It mainly acts by stimulation of insulin release from
β- cells of the pancreas by blocking the ATP- sensitive K+ channels, resulting in
depolarisation and Ca2+ reduction in hepatic glucose production.
6.0.2 LITERATURE SURVEY ON METHODS FOR GLIPIZIDE IN
PHARMACEUTICALS
Many UV-spectrophotometric, chromatographic and electrochemical
methods were developed for the determination of GPZ in bulk drug and dosage
forms.
UV-spectrophotometric methods
Few UV-spectrophotometric methods were found in the literature. There is
only one direct method described by Mantri and Shanmukhappa [3] when the drug
is present alone in the dosage form. Aruna and Nancey [4] have described the
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
261
simultaneous determination of metformin (MET) and GPZ in solid dosage forms
by two methods: solving simultaneous equations and second derivative mode.
Two more methods were developed for the simultaneous determination of GPZ
and MET in tablet dosage forms by Chungath et al. [5]. Method A involved
solving simultaneous equations were where two wavelengths: 238 nm (for MET)
and 275 nm (for GPZ) were selected for the formation of simultaneous equations.
Method B involved the formation of Q- absorbance equation at isobestic point
(259.5 nm). Linearity was observed in the range 1.2–6.0 µg mL-1 for GPZ in both
the methods. GPZ and MET in combined tablet formulation were assayed by
Sarangi et al. [6] also. The authors used multi component mode at 276 nm (for
GPZ) and 237 nm (for MET) for measurement in methanolic solution. Beer’s law
is obeyed in the concentration range 2-20 µg mL-1 for GPZ. Adhikari et al. [7]
used two methods for the simultaneous determination of pioglitazone (PGT), MET
and GPZ in multi component formulation. The three- wavelength method used
acetonitrile- methanol- water in the ratio (3: 4: 1) with λmax at 236.5, 226.4 and
227.3 nm, for PGT, MET and GPZ, respectively. The isobestic point was found to
be at 254 nm. Method II was based multi wavelength spectroscopy. The Beer’s
law was obeyed over 5-55 µg mL-1 range for GPZ.
HPLC methods
HPLC has been widely used for the determination of GPZ in single and
combined dosage forms. An RP-HPLC method was described by Vijaya et al. [8]
for the determination of GPZ in dosage forms using C18 column (250 × 4.6 mm; 5
µm). The mobile phase consisted of methanol- triethylamine buffer, pH 3 (35: 65)
with a flow rate of 1 mL min-1 and UV- detection at 230 nm. Linearity was found
in the range, 0.1-10 µg mL-1. Mantri and Shanmukhappa [9] developed and
validated an RP-HPLC method with C18 analytical column using methanol-
0.115% w/v ammonium hydrogenphosphate buffer pumped at 1 mL min-1 at
ambient temperature. The calibration graph was linear in the range of 10-70 µg
mL-1. Rapid and sensitive assay of GPZ was achieved by Rahila and Asif [10].
The drug was chromatographed on a RP- C18 column with mobile phase
consisting of 0.05M KH2PO4, pH 7.0- methanol (15: 85 v/v) pumped at a flow
rate of 1 mL min-1. Quantification was achieved by monitoring the UV-
absorbance at 225 nm. The method showed linearity in the range 10-2000
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
262
ng mL-1. An Inertsil ODS- C18 column (250 × 4.6 mm; 5 µm) in isocratic mode
with mobile phase containing methanol– water- 0.01M KH2PO4, (70: 25: 5 v/v/v)
at a flow rate of 1.5 mL min-1 and UV- detection at 270 nm was used by Rayanam
et al. [11] for the assay of GPZ in tablet formulation. The method has been found
to be sensitive with LOD and LOQ values of 15 and 45 µg mL-1, respectively. The
method was also applied for blood serum. Determination of GPZ in sustained-
release tablets by RP-HPLC has been reported by Liping et al. [12]. Separation
and assay were carried out on an Eclipse XDB- C18 column with 0.1M NaH2PO4-
methanol (54: 46) as mobile phase pumped at a flow rate of 1 mL min-1 and UV-
detection at 225 nm. The linear range was from 5 to 250 µg mL-1 GPZ.
Apart from the above methods for GPZ in single component dosage forms,
several workers have applied HPLC for the simultaneous determination of GPZ in
multi-component dosage forms when the drug is present along with glimepiride
[13], metformin [14-16], simvastatin [17], metformin and repaglinide [18],
pioglitazone and rosiglitazone [19], rosiglitazone, pioglitazone, glibenclamide and
glimepiride [20] metformin, pioglitazone, glimeperide, gliclazide and
glibenclamide [21], metformin, pioglitazone, phenformin, gliclazide, glimeperide,
glibenclamide tolbutamide, rosiglitazone and pioglitazone [22].
UPLC
There is only reference in the literature related to determination of GPZ by
UPLC, but it was applied to in vitro study during formulation development and
not to dosage forms [23].
Other methods
Other methods reported for GPZ in dosage forms include TLC [24, 25],
HPTLC [26] and voltammetry [27].
Methods for body fluids
GPZ in body fluids such as blood plasma and serum, and urine have been
determined by HPLC [7, 11, 13, 20 & 28-36], LC-MS/MS [37-41] UPLC-MS/MS
[42,43] and radioimmunoassay [44].
From the literature survey presented in the above paragraphs, it is clear
that except European Pharmacopoeal method [45] no other titrimetric method has
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
263
ever been reported for GPZ in pharmaceuticals. Barring one method [3], all other
UV-spectrophotometric methods are applicable for combined dosage forms.
Though several HPLC methods are available for dosage forms [8-22, 28-36] none
of them is stability-indicating. No UPLC method has ever been developed for
dosage forms.
In the light of the above observations, the author has developed two simple
and direct UV-spectrophotometric, one each of HPLC and UPLC methods for the
drug in dosage form. These details are presented in this Chapter VI .
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
264
Section 6.1
STABILITY-INDICATING UV-SPECTROPHOTOMETRIC
DETERMINATION OF GLIPIZIDE IN PHARMACEUTICALS
6.1.1 INTRODUCTION
The importance of UV-spectrophotometry in pharmaceutical analysis was
presented in Chapter II Section 2.1.1. From the literature survey presented
Section 6.2 it is evident that five UV-spectrophotometric methods [3-7] have been
reported for the quantification of GPZ. There is only one direct method [3]
reported when the drug is present alone in the dosage form. Four methods [4-7]
are based on the measurement of the absorbance of the drug solution in solvent
mixture in combined dosage forms and employ different complicated modes of
UV-spectrophotometry. None of these methods is stability-indicating.
In the present section (Section 6.1), two simple, direct, reproducible, and
stability-indicating UV-spectrophotometric methods for GPZ are described. The
methods are based on the measurement of absorbance of GPZ solution either in
0.1M NaOH at 260 nm in method A, or 0.1M HCl at 255 nm in method B.
Besides, the methods were used to study the degradation of the drug under stress
conditions as per the ICH guidelines.
6.1.2 EXPERIMENTAL
Apparatus
The instrument used for absorbance measurement was the same as
described in Section 2.1.2.
Reagents and materials
All chemicals and reagents used were the same as described in Section
2.1.2.
Preparation of standard GPZ solution
Pure active ingredient sample of GPZ was kindly supplied by Bal Pharma,
Bangalore, India, as gift. Standard stock solutions of 400 µg mL-1 GPZ was
prepared by dissolving 40 mg of pure GPZ in 0.1M NaOH and 0.1M HCl
separately and diluted to 100 mL with the respective solvent, in calibrated flasks.
The solutions were diluted to obtain 80 µg mL-1 each GPZ and used for assay.
GPZ- containing tablets; Dibizide-5 (5 mg) (Micro Labs Limited, Hosur, India),
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
265
Glynase-5 (5 mg) (USV Limited, Aurangabad, India) were procured from the
local market.
6.1.3 Assay procedures
Preparation of standard graphs
Method A (using 0.1M NaOH)
Into a series of 10 mL volumetric flasks, aliquots of GPZ standard solution
equivalent to 4.0-72 µg mL-1 GPZ were accurately transferred and volume was
made up to mark with 0.1M NaOH. The absorbance of each solution was
measured at 260 nm vs 0.1M NaOH.
Method B (using 0.1M HCl)
Varying aliquots (0.5,1.0,….9.0 mL) of working standard solution
corresponding to 4.0-72 µg mL-1 GPZ were taken into a series of 10 mL
volumetric flasks and volume was made up to mark with 0.1M HCl. The
absorbance of each solution was measured at 255 nm vs 0.1M HCl.
In both the cases, calibration curves were plotted and the concentration of
the unknown was computed from the respective regression equation derived using
Beer’s law data.
Procedure for tablets
Weighed amount of tablet powder equivalent to 40 mg of GPZ was
transferred into a 100 mL volumetric flask. The content was shaken well with
about 60 mL of 0.1M NaOH or 0.1M HCl for 20 min. The mixture was diluted to
the mark with the respective solvent. It was filtered using Whatman No 42 filter
paper. First 10 mL portion of the filtrate was discarded and a subsequent portion
was subjected to analysis by following the procedure described earlier after
appropriate dilution.
Procedure for placebo blank and synthetic mixture analyses
A placebo blank of the composition: acacia (15 mg), hydroxyl cellulose
(10 mg), magnesium stearate (15 mg), starch (10 mg), sodium citrate (15 mg), talc
(15 mg) and sodium alginate (10 mg) was made and its solution was prepared by
taking 20 mg as described under ‘procedure for tablets’ and then subjected to
analysis.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
266
A synthetic mixture was prepared by adding pure GPZ (20 mg) to 20 mg
placebo blank and the mixture was homogenized. Its solution was prepared as
described under procedure for tablets. The extract was subjected to assay
following the general procedures and the percentage recovery of GPZ was
calculated.
Procedure for forced degradation studies
For both the methods, a 1 mL aliquot of 400 µg mL-1 GPZ was taken (in
triplicate) in a 10 mL volumetric flask and mixed with 2 mL of 2M HCl (acid
hydrolysis) or 2M NaOH (base hydrolysis) or 5% H2O2 (oxidative degradation)
and boiled for 2 h at 80 °C in a hot water bath. The solution was cooled to room
temperature and diluted to the mark with 0.1M HCl after neutralization with
base/acid. In thermal degradation, solid drug was kept in Petri dish in oven at 100
°C for 24 h. After cooling to room temperature, 100 µg mL-1 GPZ solutions in
0.1M HCl/NaOH were prepared separately and absorbance measured. For UV
degradation study, the stock solutions of the drug (100 µg mL-1) were exposed to
UV radiation of wavelength 254 nm and of 1200K lux intensity for 48 h in a UV
chamber. The solutions after dilution with either 0.1M HCl or 0.1M NaOH were
assayed as described above.
6.1.4 RESULTS AND DISCUSSION
The absorption spectra of 40 µg mL-1 GPZ solution in 0.1M NaOH
(method A) and in 0.1M HCl (method B) were recorded between 200 and 400 nm
and showed absorption maxima at 260 and 255 nm, for method A and method B,
respectively. At these wavelengths, 0.1M NaOH and 0.1M HCl had insignificant
absorbance. Therefore, the analysis of GPZ was carried out at 260 and 255 nm, for
method A and method B, respectively (Figure 6.1.1).
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
267
a) b)
c) d)
Figure 6.1.1. Absorption spectra of: a) 0.1M NaOH blank; b) GPZ in 0.1M NaOH (40 µg mL-1); c) 0.1M HCl blank; d) GPZ in 0.1M HCl (40 µg mL-1)
6.1.5 Method validation
Analytical parameters
The regression parameters calculated from the calibration graphs (Figure
6.1.2), are presented in Table 6.1.1. Beer’s law was obeyed over the concentration
ranges shown in Table 6.1.1, and the linearity of calibration graphs (Figure 6.1.2)
was demonstrated by the high values of the correlation coefficient (r) and the
small values of the y-intercepts of the regression equations. The molar
absorptivity, Sandell sensitivity values of both methods are also shown in Table
6.1.1. The limits of detection and quantification were calculated as per the current
ICH guidelines [46] and are presented in Table 6.1.1.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
268
a) b)
Figure 6.1.2. Calibration curve for GPZ: a) in 0.1M NaOH (method A), b) 0.1M HCl (method B)
Table 6.1.1 Sensitivity and regression parameters
*y=mx+b, where y is the absorbance, x is concentration in µg mL-1, b intercept and m slope.
Precision and accuracy
Accuracy was evaluated as percentage relative error between the measured
and taken concentrations of GPZ (%RE). The results, compiled in Table 6.1.2,
show that the accuracy is good for both methods. Precision of the methods was
calculated in terms of intermediate precision (intra-day and inter-day). Three
different concentration of GPZ (within the working limits) were analyzed in seven
replicates during the same day (intra-day precision) and five consecutive days
(inter-day precision). %RSD values (Table 6.1.2) of the intra-day and inter-day
studies showed that the precision was good for the both methods.
Parameter Method A Method B λmax, nm 260 255 Beer’s law limits (µg mL-1) 4.0–72.0 4.0–72.0 Molar absorptivity (L mol-1 cm-1) 6.06×103 6.13×103 Sandell sensitivity (µg cm-2) 0.0752 0.0743 Limit of detection (µg mL-1) 1.02 0.85 Limit of quantification (µg mL-1) 3.05 2.55 Regression equation, y* Intercept (b) 0.0079 0.0142 Slope (m) 0.0129 0.0127 Correlation coefficient (r) 0.9996 0.9994 Standard deviation of intercept (Sb) 0.0003 0.0012 Standard deviation of slope (Sm) 0.0002 0.0002
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
269
Table 6.1.2 Results of intra-day and inter-day accuracy and precision study
Robustness and ruggedness
Robustness was determined by the analysis of standard solution at three
concentration levels at three wavelengths (λmax ± 1 nm).
Method ruggedness was demonstrated by the analysis done by three
analysts, and also by a single analyst performing analysis with three different
cuvettes in the same laboratory. Intermediate RSD in both instances were in the
range 0.63–1.54% indicating acceptable ruggedness. These results are presented in
Table 6.1.3.
*The wavelengths were 259, 260 and 261 nm (method A) and 254, 255 and 256 nm (method B)
Selectivity
In order to evaluate the selectivity, the effect of the presence of common
excipients described in the previous section was tested for possible interference in
the assay by placebo blank and synthetic mixture analyses. When the synthetic
Method
GPZ taken
(µg mL-1)
Intra-day (n = 5) Inter-day (n = 5) GPZ
founda
(µg mL-1) %RSDb %REc
GPZ founda
(µg mL-1)
%RSDb
%REc
A
20 20.3 1.07 1.50 20.4 0.95 2.00 40 39.5 0.92 1.25 39.3 1.63 1.75 60 58.9 0.63 1.83 61.0 1.12 1.67
B
20 19.8 1.01 1.50 19.7 1.04 1.36 40 39.5 1.09 1.25 40.7 1.25 1.75 60 60.8 1.33 1.17 59.1 0.97 1.50
aMean value of five determinations; bRelative standard deviation (%); cRelative error (%).
Table 6.1.3 Results of ruggedness expressed as intermediate precision
Method
GPZ taken,
µg mL-1
Method
robustness*
Method ruggedness Inter-analysts
%RSD (n = 3)
Inter-cuvettes %RSD (n = 3)
A
20 1.05 1.05 1.03 40 0.92 1.36 1.51 65 1.47 0.91 0.81
B
20 0.69 1.54 1.09 40 1.15 0.75 1.54 60 0.63 0.89 1.21
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
270
mixture solution was subjected to analyses at 40 µg mL-1 concentration levels by
each method, the percent recoveries were 97.42 and 98.25 respectively, with
%RSD being less than 1.9% implying that the assay procedure is free from these
excipients.
Application to tablets
The proposed methods were applied for the quantification of GPZ in
commercial tablets. The results were compared with those of official method [45].
The official method involved the titration of the drug in dimethylformamide with
0.1M lithium methoxide using quinaldine red indicator. The assay was performed
on two different brands of tablets containing 5 mg of active ingredient. Statistical
analysis of the results did not detect any significant difference between the
performance of the proposed methods and reference method with respect to
accuracy and precision as revealed by the Student’s t-value and variance ratio F-
value [47]. The results of this study are presented in Table 6.1.4.
Recovery study by standard-addition procedure
The test was done by spiking the pre-analyzed tablet powder with pure
GPZ at three different levels (50, 100 and 150% of the content present in the tablet
powder (taken) and the total was found by the proposed methods. Each test was
repeated three times. In both the cases, the recovery percentage values ranged
between 97.86 and 102.9% with standard deviation in the range 0.85-1.89%.
Closeness of the results to 100% showed the fairly good accuracy of the methods.
The results are shown in Table 6.1.5.
Table 6.1.4 Results obtained by the analysis of tablets by the proposed methods and statistical comparison of results with the official method
Tablet brand name
Label claim
mg/tablet
Found (Percent of label claim ±SD)a
Official method
Proposed methods Method A Method B
Dibizide-5
5 101.5±1.98
101.7±1.39 t = 0.18 F= 2.03
101.9±1.45 t = 0.36 F = 1.86
Glynase-5
5 102.1±1.29
101.2±1.63 t = 0.97 F= 1.60
102.7±1.21 t = 0.76 F = 1.14
aMean value of five determinations.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
271
Table 6.1.5 Results of recovery study using standard addition method
*Mean value of three determinations
Tablets studied
Method A Method B GPZ
in tablets, µg mL-1
Pure GPZ
added, µg mL-1
Total found,
µg mL-1
Pure GPZ recovered*,
Percent ± SD
GPZ in tablets, µg mL-1
Pure GPZ
added, µg mL-1
Total found,
µg mL-1
Pure GPZ recovered*, Percent±SD
Dibizide-5 20.34 10.0 30.01 98.92±1.05 20.38 10.0 30.87 101.6±1.29 20.34 20.0 40.98 101.6±1.43 20.38 20.0 40.23 99.63±1.63 20.34 30.0 50.89 101.1±1.67 20.38 30.0 50.98 101.2±1.89
Glynase-5 20.24 10.0 30.90 102.2±1.36 20.54 10.0 31.27 102.4±0.54 20.24 20.0 41.01 101.9±0.98 20.54 20.0 41.72 102.9±1.81 20.24 30.0 49.16 97.86±0.85 20.54 30.0 49.97 98.87±1.45
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
272
Results from forced degradation studies
The UV-spectra of 40 µg mL-1 GPZ each in 0.1M NaOH and 0.1M HCl
after forced degradation are shown in Figure 6.1.3 to Figure 6.1.7. The drug was
found to remain intact in method A and method B after acid hydrolysis (Figure
6.1.3). Base hydrolysis resulted in no degradation in both methods (Figure 6.1.4).
The absorption spectra of GPZ solution subjected to H2O2 showed that the drug
experienced slight degradation in method A and significant degradation in method
B (Figure 6.1.5). The drug did not undergo degradation after exposure to heat and
light as revealed by the absorption spectra which are similar to that of unstressed
solution (Figure 6.1.6 and Figure 6.1.7).
Table 6.1.6 Results of stability-indicating study
Stress condition %Degradation
Method A Method B
Acid hydrolysis No degradation No degradation Alkali hydrolysis No degradation No degradation
Oxidation 57.2% 59.8% Thermal (105°C, 3 hours) No degradation No degradation Photolytic (1.2 million lux hours) No degradation No degradation
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
273
a) b) Figure 6.1.3. UV-spectra of 40 µg mL-1 GPZ after subjecting to acid hydrolysis: a) Method A and b) Method B
a) b)
Figure 6.1.4. UV-spectra of 40 µg mL-1 GPZ after subjecting to base hydrolysis: a) Method A and b) Method B
a) b)
Figure 6.1.5. UV-spectra of 40 µg mL-1 GPZ after subjecting to oxidative condition: a) Method A and b) Method B
a) b)
Figure 6.1.6. UV-spectra of 40 µg mL-1 GPZ after subjecting to thermal degradation: a) Method A and b) Method B
a) b)
Figure 6.1.7. UV- spectra of 40 µg mL-1 GPZ solution after subjecting to photolytic condition: a) Method A and b) Method B
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
274
Section 6.2
DETERMINATION OF GLIPIZIDE IN PHARMACEUTICALS BY HP LC
AND FORCED DEGRADATION STUDIES
6.2.1 INTRODUCTION
The importance and advantages of HPLC methods are described in
Chapter IV Section 4.3. None of the previously reported methods [8-12] is
stability-indicating.
By introducing certain changes in respect of column and mobile phase
composition, the author has developed HPLC method which does not require an
internal standard. The stability-indicating power of the method was established by
comparing the chromatograms obtained under optimized conditions before forced
degradation with those after degradation via acidic, basic, oxidative, thermal and
photolytic stress conditions. The optimization parameters and the validation
results in detail are presented in this section (Section 6.2).
6.2.2 EXPERIMENTAL
Apparatus
HPLC analysis was performed on the same instrument used in the Section
4.3.2.
Materials and reagents
Pure active ingredient sample of GPZ and its tablets were obtained as in
Section 6.1.2. HPLC grade methanol was purchased from Merck, potassium
dihydrogenorthophosphate, triethylamine and orthophosphoric acid were from
Qualigens-India. Water purified by the Milli-Q system (Millipore, Milford,
Massachusetts, USA) was used for mobile phase and sample diluent preparation.
An amount equivalent to 10 mM potassium dihydrogenorthophosphate
was dissolved in 1000 mL of water and the pH was adjusted to 3.9 using
triethylamine, or dilute phosphoric acid. A 600 mL portion of this buffer was
mixed with 400 mL of methanol (60:40 v/v), shaken well and filtered using 0.45
µm Nylon membrane filter.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
275
Chromatographic conditions
Chromatographic separation was achieved on an Inertsil ODS 3V (150 mm
× 4.6 mm, 5 µm particle size) column. The flow rate was 0.8 mL min-1, the
detector wavelength was set at 220 nm and the injection volume was 20 µL. The
column temperature was maintained at 35 °C. A solution containing a mixture of
phosphate buffer of pH 3.9 and methanol (60:40) was used as a mobile phase.
Standard GPZ solution
Accurately weighed 100 mg of pure GPZ was dissolved in and diluted to
mark in a 100 mL standard flask with mobile phase to get 1000 µg mL-1 GPZ
stock solution.
6.2.3 General procedures
Procedure for preparation of calibration curve
Working standard solutions containing 1-450 µg mL-1 GPZ were prepared
by serial dilutions of aliquots of the stock solution. Aliquots of 20 µL were
injected (six injections) and eluted with the mobile phase under the reported
chromatographic conditions. The average peak area versus the concentration of
GPZ in µg mL-1 was plotted. Alternatively, the regression equation was derived
using mean peak area-concentration data and the concentration of the unknown
was computed from the regression equation.
Procedure for tablets
Tablet powder equivalent to 100 mg GPZ was transferred into a 100 mL
calibrated flask containing 60 mL of the mobile phase. The mixture was sonicated
for 20 min to achieve complete dissolution of GPZ, and the content was then
diluted to volume with the same solvent to yield a concentration of 1000 µg mL-1
GPZ, and filtered through a 0.45 µm nylon membrane filter. The tablet extract was
injected on to the HPLC column after appropriate dilution.
Procedure for placebo blank and synthetic mixture analyses
A placebo blank of the composition prepared was the same as described in
Section 6.1.3. A 100 mg of the placebo blank was accurately weighed and its
solution was prepared as described under ‘procedure for tablets’, and then
subjected to analysis by following the general procedure.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
276
A synthetic mixture was prepared by adding an accurately weighed 100
mg of pure PZG to 100 mg of placebo mentioned in Section 6.1.3. The solution of
the synthetic mixture equivalent to 1000 µg mL-1 GPZ was prepared as described
under procedure for tablets. The resulting solution was assayed (n= 5) by the
proposed method after dilution to 300 µg mL-1 GPZ with the mobile phase.
Procedure for stress study
A 3 mL aliquot of 1000 µg mL-1 GPZ standard solution was transferred
into four different 10 mL calibrated flasks and the same stress conditions
described in Section 6.1.3 was applied and subjected to HPLC analysis after
suitable dilution.
6.2.4 RESULTS AND DISCUSSION
To obtain good linearity, sensitivity and selectivity, the method was
optimized and validated in accordance with the current ICH guidelines [46]. The
typical chromatograms obtained for blank and pure GPZ in final optimized HPLC
conditions are depicted in Figure 6.2.1.
AU
0.00
0.10
0.20
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00
AU
0.00
0.10
0.20
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00
5.58
9
a) b)
Figure 6.2.1. Chromatograms for; a) Blank (mobile phase)
b) Pure GPZ solution (300 µg mL-1)
6.2.5 Method development
A well defined symmetrical peak and good results were obtained upon
measuring the response of eluent under the optimized conditions after thorough
experimental trials that could be summarized as follows:
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
277
Choice of column
Five different columns were used for performance investigations,
including Chromatopack (250 mm × 4.6 mm, 5 µm particle size) column;
Hypersil BDS C8 (250 mm × 4.0 mm, 5.0 µm particle size) thermo column;
Inertsil ODS 3V (250 mm × 4.0 mm, 5.0 µm particle size); Luna C18 (250 mm ×
4.0 mm, 5.0 µm particle size) and Zorbax XDB (250 mm × 4.0 mm, 5.0 µm
particle size). The experimental studies revealed that the Inertsil ODS 3V column
was more suitable since it gave better sensitivity.
Choice of wavelength
The UV detector response of GPZ was studied and the best wavelength
was found to be 220 nm showing the highest sensitivity.
Mobile phase composition
Several modifications in the mobile phase compositions were tried in order
to study the performance characteristics. These modifications included the change
in the type and ratio of the organic modifier, the pH, the strength of the phosphate
buffer, and the flow rate. The results obtained are shown in Table 6.2.1.
Table 6.2.1 Effect of ratio of organic modifier, pH and ionic strength of buffer on
the number of theoretical plates
Ratio (A/B)a
Number of
theoretical plates (N)
pH of the
medium
Number of
theoretical plates (N)
%H3PO4
Number of
theoretical plates (N)
Flow rate, mL
min-1
Number of
theoretical plates (N)
40/60 50/50 55/45 60/40 70/30
- -
4934 6588 7693 9261 8756
- -
2.0 2.5 3.0 3.9 4.1 4.3 4.5
5789 7866 8679 9956 9867 8954 5724
0.050 0.075 0.100 0.125 0.150 0.200 0.250
8537 9649 9987 9657 8586 8246 8097
0.50 0.60 0.70 0.80 0.90 1.00 1.20
6176 7648 8765 9960 9768 9326 8976
aA- phosphate buffer and B- methanol
Type of organic modifier
Methanol was replaced by other solvents but it did not give good peak.
Methanol was the organic modifier of choice giving nice, elegant and highly
sensitive peak.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
278
Ratio of organic modifier
The effect of ratio of organic modifier on the selectivity and retention time
of the test solute was investigated using mobile phases containing 30-60%
methanol. Table 6.2.1 shows that 40% methanol was the best, giving well defined
peak and the highest number of theoretical plates.
Effect of pH and ionic strength of buffer
The effect of pH of the mobile phase on the selectivity and retention time
of the test solute was investigated using mobile phases of pH ranging from 2.0-
4.5. The results (Table 6.2.1) revealed that pH 3.9 was most appropriate and
giving well defined peak and the highest number of theoretical plates. At lower
and higher pH, non-symmetrical peak and smaller number of theoretical plates
were observed. Therefore pH 3.9 was fixed as optimum. The same trend was
observed after making alteration in the ionic strength of the buffer and 10 mM
phosphate buffer was used as working buffer throughout the investigation. The
results of these observations are presented in Table 6.2.1.
The effect of flow rate
The effect of flow rate on the symmetry, sensitivity and retention time of
the peak was studied and a flow rate of 0.8 mL min-1 was optimal for better
symmetry and reasonable retention time (Table 6.2.1).
6.2.6 Method validation
Linearity
Linearity was studied by preparing standard solutions of different
concentrations from 1 to 450 µg mL-1, plotting a calibration graph of mean peak
area against concentration and determining the linearity by least-square regression
equation. The calibration plot was linear over the concentration range 1-450
µg mL-1 (n= 3) (Figure 6.2.2) and can be described by the equation
y = m x + b
where y is the mean peak area, x is the concentration of GPZ in µg mL-1, m slope
and b intercept. The LOD and LOQ values, slope (m), y-intercept (b) and their
standard deviations were evaluated and presented in Table 6.2.2. These results
confirm the linear relation between concentration of GPZ and the peak areas as
well as the sensitivity of the method.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
279
Table 6.2.2 Linearity and regression parameters
Parameter Value
Linear range, µg mL-1 1 -450
Limits of detection, (LOD), µg mL-1 0.03
Limits of quantification, (LOQ), µg mL-1 0.09
Regression equation, y* Slope (m) 29955 Intercept (b) 86386 Standard deviation of intercept (Sb) 897.9 Standard deviation of slope (Sm) 1681.2 Correlation coefficient (r) 0.9999
*y=mx+b, where y is the mean peak area, x is concentration in µg mL-1, b intercept, m slope.
Figure 6.2.2. Calibration curve
Limits of quantification (LOQ) and detection (LOD)
The limit of quantification (LOQ) was determined by establishing the
lowest concentration that can be measured according to ICH recommendations
[46], below which the calibration graph is non linear and was found to be 0.09
µg mL-1. The limit of detection (LOD) was determined by establishing the
minimum level at which the analyte can be reliably detected and it was found to
be 0.03 µg mL-1.
Precision and accuracy
The percent relative error which is an indicator of accuracy is ≤1.4% and is
indicative of high accuracy. The calculated percent relative standard deviation
(RSD, %) can be considered to be satisfactory. The peak area based and retention
time based RSD values were <1%. The results obtained for the evaluation of
accuracy and precision of the method are compiled in Table 6.2.3.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
280
Table 6.2.3 Results of accuracy and precision study (n=5)
GPZ injected, µg mL-1
Intra-day Inter-day GPZ
found, µg mL-1
% REa
% RSDb
% RSDc
GPZ found,
µg mL-1
% REa
% RSDb
% RSDc
150 151.8 1.20 0.43 0.40
0.63 0.59 152.1 1.40 0.83 0.60
0.49 0.27 300 298.7 0.45 0.36 297.5 0.34 0.31 450 448.2 0.54 0.47 452.7 0.61 0.95
a Relative error b Relative standard deviation based on peak area; c Relative standard deviation based on retention time.
Method robustness
The robustness of the method was evaluated by making small deliberate
changes in the chromatographic conditions. The chromatographic conditions varied
were flow rate (0.8±0.1 mL), wavelength (220±1 nm) and temperature (35±2 °C).
There was no significant change in the retention time (Rt) when the flow rate or
temperature was changed slightly. The values of %RSD (Table 6.2.4) indicate that
the method is robust.
Table 6.2.4 Results of method robustness
Condition altered
Modifi-cation
Mean peak area ± SD*
% RSD
Mean Rt ± SD*
% RSD
Mean theoretical
plates ± SD*
%
RSD
Mean tailing factor ±SD*
% RSD
Actual - 9084249 ± 55609
0.61 5.583± 0.003
0.054 9946± 5.727
0.06 1.258
± 0.004 0.32
Column temperature
35±2 ºC
9126732 ± 91333
1.00 5.612± 0.002
0.036 9889± 6.724
0.07 1.213
± 0.005 0.41
Mobile phase composition
(Buffer: methanol)
9164999 ± 98404
1.07 5.492± 0.003
0.055 9935± 8.114
0.08 1.227
± 0.003 0.25
Flow rate 0.8±0.1 mL min-1
9136736 ± 97336
1.06 5.590± 0.002
0.036 9892± 4.772
0.05 1.221
± 0.004 0.33
Wavelength 220±1 nm
9126632 ± 91313
1.00 5.572± 0.003
0.054 9954± 3.663
0.04 1.211
± 0.005 0.41
*Mean value of three determinations at GPZ concentration of 300 µg mL-1. Method ruggedness
The ruggedness of the method was assessed by comparison of the intra-day
and inter-day results for the assay of GPZ performed by three analysts in the same
laboratory. The RSD for intra-day and inter-day assay of GPZ did not exceed
1.07% indicating the ruggedness of the method (Table 6.2.5).
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
281
Table 6.2.5 Results of method ruggedness (n=3)
Variable
Mean Peak area
± SD*
%
RSD
Mean Rt
± SD*
%
RSD
Mean theoretical
plates ±SD
%
RSD
Mean tailing factor± SD*
%
RSD
Analysts (n=3)
9164999 ± 98404
1.07 5.592± 0.003
0.054 9899± 7.614
0.08 1.221± 0.003
0.25
*Mean value of three determinations for GPZ concentration of 300 µg mL-1. Selectivity
Selectivity of the method was evaluated by injecting the mobile phase,
placebo blank, pure drug solution and tablet extract. No peaks were observed for
mobile phase and placebo blank and no extra peaks were observed for tablet
extracts (Figure 6.2.3). Synthetic mixture when analysed at 300 µg mL-1
concentration level yielded percent recoveries of 97.36 to 102.1% with standard
deviation < 1.2% indicating the absence of interference from the tablet excipients.
AU
0.00
0.10
0.20
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00
AU
0.00
0.10
0.20
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00
5.58
3
a) b)
Figure 6.2.3. Chromatograms obtained for: a) placebo blank and b) tablet extract (300 µg mL-1 GPZ)
Solution stability
The drug solution was injected at different time intervals of 0, 12 and 24 h,
and chromatograms were recorded. At the specified time interval, %RSD for the
peak area obtained from drug solution was within 1.07%. This shows no
significant change in the elution of the peak and its system suitability criteria
(tailing factor, theoretical plates). The results also confirmed that the standard
solution of drug was stable at least for 24 hours during the assay performance.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
282
*Mean value of three determinations for GPZ concentration of 300 µg mL-1 at each time interval.
Application to tablets
The developed method was applied to the determination of GPZ in two
brands of tablets containing GPZ 5 mg per tablet. Quantification was performed
using the regression equation. The same tablet powder used for assay by the
proposed method was used for assay by an official method [45] for comparison.
The results were compared statistically by applying the Student’s test for accuracy
and F-test for precision. As shown by the results compiled in Table 6.2.7, the
calculated t-test and F-values did not exceed the tabulated values of 2.77 and 6.39
for four degrees of freedom at the 95% confidence level, suggesting that the
proposed method and the reference method do not differ significantly with respect
to accuracy and precision.
Table 6.2.7 Results of determination of GPZ in tablet and statistical comparison
with the official method Tablet brand name
Nominal amount,
mg
GPZ found* (%) ± SD t-value
F- value Official
method Proposed method
Dibizide
Glynase
5
5
98.67±0.87
99.45±1.10
99.48±0.56
100.5±0.42
1.75
1.61
2.41
1.34 * Mean value of five determinations. Tabulated t-value at 95% confidence level is 2.77; Tabulated F-value at 95% confidence level is 6.39.
Accuracy by recovery studies
The accuracy of the proposed method was further checked by performing
recovery experiments. Pre-analyzed tablet powder was spiked with pure GPZ at
three different concentration levels and the total was found by the proposed
method. Each determination was repeated three times. The recovery of pure drug
Table 6.2.6 Results of solution stability
Time, hour
Mean peak area ± SD*
%
RSD
Mean Rt ± SD*
%
RSD
Mean theoretical plates ± SD*
%
RSD
Mean tailing
factor ± SD*
%
RSD
0 9084249 ± 55609
0.61 5.592± 0.002
0.035 9885± 8.565
0.09 1.258± 0.004
0.32
12 9126732 ± 91333
1.00 5.581± 0.002
0.036 9989± 9.774
0.10 1.213± 0.005
0.41
24 9164999 ± 98404
1.07 5.603± 0.003
0.054 9835± 9.524
0.10 1.227± 0.003
0.25
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
283
added was quantitative (Table 6.2.8) and revealed that co-formulated substances
did not interfere in the determination.
Table 6.2.8 Results of recovery study by standard addition method
Tablet studied
GPZ in tablet,
µg mL-1
Pure GPZ added,
µg mL-1
Total found,
µg mL-1
Pure GPZ recovered*
(%GPZ ±SD)
Dibizide 99.48 99.48 99.48
50 100 150
150.2 198.7 252.5
100.5±0.97 99.63±0.87 101.2±1.05
Glynase 100.5 100.5 100.5
50 100 150
148.6 199.5 251.5
98.75±0.67 99.49±0.65 100.4±0.90
*Mean value of three determinations
Results from forced degradation studies
All forced degradation studies were analyzed at 300 µg mL-1 concentration
level. The observation was made based on the peak area of the respective sample.
GPZ was found to be more stable under photolytic (1.2 million lux hours), thermal
(80 0C for 2 hours) in solid state, stress conditions. The drug was stable towards
acidic and basic conditions also. The drug degraded up to 60.9% under oxidation
(5% H2O2). The chromatograms obtained for GPZ after subjecting to degradation
are presented in Figure 6.2.4. Assay study was carried out by the comparison with
the peak area of GPZ sample without degradation. The results of this study are
shown in Table 6.2.9.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
284
a) b)
c) d)
e)
Figure 6.2.4. Chromatograms of GPZ (300 µg mL-1) after forced degradation a) acid degradation; b) base degradation; c) peroxide degradation; d) thermal degradation and e) photolytic degradation
Table 6.2.9 Results of degradation study
Stress condition % degradation Acid hydrolysis No degradation
Base hydrolysis No degradation
Oxidation 60.9%
Thermal (105°C, 3 hours) No degradation
Photolytic (1.2 million lux hours) No degradation
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
285
Section 6.3
QUALITY BY DESIGN APPROACH FOR THE DEVELOPMENT
AND VALIDATION OF GLIPIZIDE BY RP-UPLC WITH APPLICA TION
TO FORMULATED FORMS AND URINE
6.3.1 INTRODUCTION
Quality by design (QbD) refers to the achievement of certain
predictable quality with desired and predetermined specifications. This concept
is described in Section 4.4. According to literature survey, there is only reference
in the literature related to determination of GPZ by UPLC, but it was applied to in
vitro study during formulation development and not to dosage forms [23].
Therefore, there is an unmet need to investigate a systematic UPLC
method development approach for pharmaceutical development using QbD
principles to ensure the quality of the method throughout the product lifecycle.
The details of method development, validation and applications are presented in
this section (Section 6.3).
6.3.2 EXPERIMENTAL
Materials and reagents
Pure active ingredient sample of glipizide (GPZ) and its tablets were the
same as described in Section 6.1.2.
HPLC grade acetonitrile was purchased from Merck Ltd., Mumbai, India,
potassium dihydrogenorthophosphate and orthophosphoric acid were from
Qualigens India. Doubly distilled water was used throughout the investigation.
Preparation of buffer: Dissolved 2.2 gram potassium dihydrogenorthophosphate
in 1 litre water containing 1 mL triethylamine, then pH adjusted to 3.5 using dilute
phosphoric acid.
Chromatographic conditions and equipments
Waters Aquity UPLC (Waters Chromatography Division, Milford,
Massachusetts, USA) system with a tunable UV detector was used for the
determination of GPZ. Empower 2 software was used to record and evaluate the
data collected during and following chromatographic analysis. Shimadzu
Pharmaspec 1700 UV/Visible spectrophotometer was used for the initial
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
286
absorbance measurement. Mobile phase was composed of buffer: acetonitrile
(60:40 v/v).
Instrumental parameters
The chromatographic separation was achieved on a Zorbax Extend C18 (50
mm × 4.6 mm × 1.8 µm) column using a mobile phase consisting of acetonitrile:
buffer (40: 60 ratio v/v) at a flow rate of 0.2 mL min-1. The mobile phase was
filtered through 0.22 µm Nylon-66 filter prior to use. The eluent was monitored
using UV detection at a wavelength of 220 nm. The column was maintained at
ambient temperature (25 °C) and an injection volume of 2 µL was used. The run
time was fixed for 5 minutes and the mobile phase used as diluent.
6.3.3 Procedures
Preparation of stock standard solution
A 1000 µg mL-1 stock GPZ solution was prepared by dissolving an
accurately weighed 100 mg of pure drug in diluent and the volume was brought to
100 mL with the same solvent in a volumetric flask and it was stored at 5 °C until
use.
Procedure for preparation of calibration curve
Working solutions containing 0.05-300 µg mL-1 GPZ were prepared by
serial dilutions of aliquots of the stock solution. Aliquots of 2 µL were injected
(six injections) and eluted with the mobile phase under the reported
chromatographic conditions. The mean peak areas were plotted against the
corresponding concentrations of the pure drug to obtain the calibration graphs and
corresponding regression equation was also computed.
Procedure for tablets
Fifty numbers each of Glynase and Dibizide tablets (Each tablet contained
5.0 mg GPZ) were weighed and powdered. Tablet powder equivalent to 20 mg of
GPZ was transferred in to 100 mL volumetric flasks and 60 mL of the mobile
phase were added. The solution was sonicated for 20 min to achieve complete
dissolution of GPZ, made up to the mark with mobile phase and then filtered
through 0.22 µm nylon membrane filter. The resultant solution (200 µg mL-1 in
GPZ) obtained was analysed through the UPLC system.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
287
Procedure for placebo blank and synthetic mixture analyses
A placebo blank of the composition was the same as described in Section
6.1 and its solution was prepared as described under ‘procedure for tablets’ and
then subjected to analysis.
A synthetic mixture was prepared by adding pure GPZ (20 mg) to 15 mg
placebo blank and the mixture was homogenized. Its solution was prepared as
described under procedure for tablets. The extract was subjected to assay
following the general procedure and the percentage recovery of GPZ was
calculated.
Procedure for stress study
A 10 mL aliquot of 1000 µg mL-1 GPZ standard solution was transferred
into three different 50 mL volumetric flasks and added 5 mL of 2M HCl, 2M
NaOH or 5% H2O2 separately, and the flasks were heated for 2 h on a water bath
maintained at 80 °C. Then the solutions were cooled and neutralized by adding
base or acid, the volume in each flask was brought to the mark with mobile phase,
and the appropriate volume (2 µL) was injected for analysis. Solid state thermal
degradation was carried out by exposing pure drug to dry heat at 105 °C for 2 h.
For photolytic degradation studies, pure drug in solid state was exposed to 1.2
million lux hours in a photo stability chamber [48]. The sample after exposure to
heat and light was used to prepare 200 µg mL-1 solutions in mobile phase and the
chromatographic procedure was followed.
Procedure for urine sample
A 1.0 mL of 0.05M hydrochloric acid was added to 0.5 mL of urine,
resulting in a pH of 3.0. The mixture was extracted with 3.0 mL of benzene in a
12 mL glass tube, which was shaken gently for 15 min. After centrifugation for 5
min, the organic phase was transferred to a conical tube for evaporation to dryness
under a stream of a well-ventilated fume chambers. The residue was re dissolved
in mobile phase and an aliquot of 2 µL was injected in to the chromatograph.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
288
6.3.4 RESULTS AND DISCUSSION
Method development
Mobile phases with different organic modifiers were tried, and best results
were obtained with phosphate buffer of pH 3.5 and acetonitrile (60: 40) as shown
in Table 6.3.1 and chromatograms are shown in Figure 6.3.1.
Table 6.3.1 Observations and remarks of method development with Zorbax Extend C18 column
Sl. No.
Trails taken Observations Remarks
1 Buffer (pH 3.5): ACN: (60: 40% v/v)
Peaks found symmetrical Satisfactory
2 Buffer (pH 4.0): ACN: (60: 40% v/v)
Peak eluted before 1 min with less theoretical plates
Not satisfactory
3 Buffer (pH 2.2): ACN: (60: 40% v/v)
Broad peak Not satisfactory
4 Buffer (pH 5.0): ACN: (60: 40% v/v)
Broad peak and late elution Not satisfactory
*ACN- Acetonitrile
a) Buffer (pH 3.5): ACN: (60: 40% v/v) b) Buffer (pH 4.0): ACN: (60: 40% v/v)
c) Buffer (pH 2.2): ACN: (60: 40% v/v) d) Buffer (pH 5.0): ACN: (60: 40% v/v)
Figure 6.3.1. Chromatograms obtained during method development using Zorbax
Extend C18 (50 mm × 4.6mm × 1.8 µm particle size) column
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
289
In order to select the suitable column, trials were made with different
columns and the Zorbax Extend C18 was found to yield symmetrical peak. Hence,
Zorbax Extend C18 was fixed throughout the analysis. The results shown in Table
6.3.2 and chromatograms are shown in Figure 6.3.2.
Table 6.3.2 Observations and remarks of method development with Zorbax Extend C18 column
Sl.No. Column* Observations Remarks
1 Acquity BEH C8 (100 × 2.1 mm, 1.7 µm)
Asymmetrical peak Not satisfactory
2 Acquity BEH C18 (100 × 2.1 mm, 1.7 µm)
Asymmetrical peak with splitting Not satisfactory
3 Acquity HSS Cyano (50 × 2.1 mm, 1.7 µm)
Asymmetrical peak Not satisfactory
4 Acquity HSS BEH Shield RP18 (50 × 2.1 mm, 1.7 µm)
Asymmetrical peak Not satisfactory
5 Zorbax Extend C18 (50 × 4.6 mm, 1.8 µm)
Symmetrical peak Satisfactory
*By keeping one column constant, the mobile phase, temperature, buffer, sample concentration parameters were changed.
a) Acquity BEH C8 column b) Acquity BEH C18 column
c) Acquity HSS Cyano column d) Acquity HSS BEH column
e) Zorbax Extend C18 column
Figure 6.3.2. Chromatograms obtained during method development using different column
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
290
Final selected method conditions: Column : Zorbax Extend C18 (50 × 4.6 mm; 1.8 µm particle size)
Oven temperature : 25 °C
Mobile phase : Buffer (pH 3.5): ACN (60: 40% v/v)
Run time : 5 min Flow rate : 0.2 mL min-1
Diluent : Mobile phase
Injection volume : 2 µL
Blank : Diluent
Wavelength : 220 nm
6.3.5 Method validation
The described UPLC method for the assay of GPZ was validated as per the
current ICH Q2 (R1) Guidelines [46].
Analytical parameters
The response of the drug was found to be linear in the investigation
concentration range from 0.05 to 300 µg mL-1 (Fig. 6.3.3) and the linear
regression equation was y = 11701.20 x + 5660.74 with correlation coefficient of
0.9999, where y is the mean peak area and x concentration in µg mL-1. The LOD
and LOQ values and their standard deviations were evaluated and presented in
Table 6.3.3. These results confirm the linear relation between the mean peak area
and concentration as well as the sensitivity of the method.
Table 6.3.3 Linearity and regression parameters
Parameter Value Linear range, µg mL-1 0.05-300 Limits of detection, (LOD), µg mL-1 0.01 Limits of quantification, (LOQ), µg mL-1 0.03 Regression equation, y* Slope (m) 11701.20 Intercept (b) 5660.74 Standard deviation of m (Sm) 209.19 Standard deviation of b (Sb) 118.29 Correlation coefficient (r) 0.9999
*y=mx+b where y is the mean peak area, x is concentration in µg mL-1, b intercept, m slope
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
291
Figure 6.3.3. Calibration curve
Accuracy and precision
To determine the accuracy and precision, pure GPZ solutions at three
different concentration levels were analyzed in seven replicates during the same
day. These studies were also repeated on different days to determine inter-day
precision. Mobile phase was injected as blank solution before sample injection
and the RSD (%) values of peak area and retention time were calculated. The
percent relative error ≤ 1.55 and is indicative of high accuracy. The calculated
percent relative standard deviation (%RSD) can be considered to be satisfactory.
The peak area based and retention time based RSD values were < 1.0. The results
obtained for the evaluation of precision and accuracy of the method is compiled in
Table 6.3.4 and Table 6.3.5.
Table 6.3.4 Results of accuracy study (n=5) Concentration
of GPZ injected, µg mL-1
Intra-day Inter-day Concentration of GPZ found,
µg mL-1
%REa Concentration of GPZ found,
µg mL-1
%REa
100 99.47 0.53 101.4 1.40 200 202.2 1.11 203.1 1.55 300 302.2 0.73 303.5 1.17
a Relative error
Table 6.3.5 Results of precision study Concentration
injected (µg mL-1)
Intra-day precision (n=7) Inter-day precision (n=5)
Mean area ±SD
% RSDa
Mean Rt±SD
% RSDb
Mean area ±SD
% RSDa
Mean Rt±SD
% RSDb
100 1165237± 11333
0.97 2.13± 0.006
0.28 1161904± 10535
0.91 2.13± 0.007
0.33
200 2347961± 16728
0.71 2.13± 0.002
0.09 2357961± 12728
0.54 2.14± 0.009
0.42
300 3488820± 17850
0.51 2.13± 0.006
0.28 3482365± 23172
0.66 2.13± 0.006
0.28
a Relative standard deviation based on peak area; b Relative standard deviation based on retention time.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
292
Method robustness and ruggedness
To determine the robustness of the method the experimental conditions
were deliberately changed. The flow rate of the mobile phase (0.2±0.02 mL
min-1), column oven temperature (25±1 ºC), mobile phase composition ratio
(65:35, 60:40 & 55:45, buffer: acetonitrile) and detection wavelength (220±1 nm)
were the varied parameters. In each case, the %RSD values were calculated for the
obtained peak area and retention time. The number of theoretical plates and tailing
factors were compared with those obtained under the optimized conditions. Three
different columns of same dimensions were used for the analyses. The studies
were performed on the same day (intra day) and on three different days (inter day)
by three different analysts for three different concentrations of GPZ (triplicate
injections) for ruggedness. The area obtained from each concentration was
compared with that of the optimized one. The relative standard deviation values
were evaluated for each concentration (Table 6.3.6).
Table 6.3.6 Results of method robustness and ruggedness
Condition Modi- fication
Mean peak area
± SD*
% RSD
Mean Rt ± SD*
% RSD
Theoretical plates ± SD*
% RSD
Tailing factor ± SD*
% RSD
Actual - 2351828 ± 11571
0.49 2.130 ± 0.005
0.24 2968 ± 5.16
0.17 1.01 ± 0.005
0.49
Temperature 25±1 ºC 2346161 ± 14060
0.60 2.131 ± 0.004
0.19 2962 ± 20.00
0.68 1.10 ± 0.008
0.73
Mobile phase composition
(Buffer: acetonitrile)
65:35 60:40 55:45
2351828 ±11572
0.49 2.131 ± 0.004
0.19 2970 ± 4.08
0.14 1.10 ± 0.004
0.37
Flow rate 0.20±0.02 mL min-1
2343495 ± 15290
0.65 2.132 ± 0.006
0.28 2968 ± 4.90
0.17 1.11 ± 0.006
0.54
Wavelength 220±1 nm
2348495 ±13198
0.56 2.132 ± 0.005
0.24 2970 ± 4.00
0.14 1.10 ± 0.006
0.54
Analyst - 2351802 ±11535
0.49 2.129 ± 0.005
0.23 2960 ± 24.42
0.82 1.10 ± 0.004
0.37
Column - 2353178 ± 12027
0.51 2.135 ± 0.010
0.47 2968 ± 6.98
0.24 1.11 ± 0.010
0.90
*Mean value of three determinations for GPZ concentration of 200 µg mL-1.
Selectivity
Selectivity of the method was evaluated by injecting the mobile phase,
placebo blank, pure drug solution and tablet extract. No peaks were observed for
mobile phase and placebo blank and no extra peaks were observed for tablet
extracts (Figure 6.3.4). The analysis of the synthetic mixture solution yielded a
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
293
percent recovery of 101.2±0.63 (n=5). It is implied from these studies that there is
no interference from the tablet excipients.
a) b) Figure 6.3.4. Chromatograms obtained for: a) placebo blank and
b) tablet extract
Stability of the solution
Stability of GPZ solution was established by storage of sample solution at
ambient temperature for 24 h. GPZ solution was re-analyzed after 12 and 24 h
time intervals and assay was determined and compared against fresh sample.
Sample solution did not show any appreciable change in assay value when stored
at ambient temperature up to 24 h. At the specified time interval, %RSD for the
peak area obtained from drug solution stability and mobile phase stability were
within 1%. This shows no significant change in the elution of the peak and its
system suitability criteria (%RSD, tailing factor, theoretical plates) (Table 6.3.7).
Table 6.3.7 Results of solution stability
*Mean value of three determinations for GPZ concentration of 200 µg mL-1 at each time interval.
Application to tablet analysis
A 200 µg mL-1 solution of tablets was prepared as per ‘preparation of
tablet extracts and assay procedure’ and was injected in triplicate to the UPLC
system. From the mean peak area, the concentration and hence mg/tablet were
Time, hour
Mean peak
area±SD*
% RSD
Mean Rt ± SD*
% RSD
Mean theoretical plates±SD*
% RSD
Mean tailing factor ±SD*
% RSD
0 2351802 ± 11535
0.49 2.129± 0.005
0.23 2960± 24.42
0.82 1.10± 0.004
0.37
12 2351828 ± 11571
0.49 2.130± 0.005
0.24 2968± 5.16
0.17 1.01± 0.005
0.49
24 2351828 ±11572
0.49 2.131± 0.004
0.19 2970± 4.08
0.14 1.10± 0.004
0.37
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
294
computed; and the results were compared with those of an official method [45].
The accuracy and precision of the proposed method was further evaluated by
applying Student’s t- test (< 2.77) and variance ratio F- test (< 6.39), respectively.
The t- and F- values at 95% confidence level did not exceed the tabulated values
and this further confirms that there is no significant difference between the official
and proposed method with respect to accuracy and precision. Table 6.3.8
illustrates the results obtained from this study.
Table 6.3.8 Results of determination of GPZ in formulations and statistical comparison with the official method
Formulation brand namea
Nominal amount,
mg
% GPZ foundc ± SD t- value
F- value Official
method Proposed method
Dibizidea 5 99.58±0.78 98.88±0.68 1.52 1.32
Glynaseb 5 100.2±0.57 99.94±0.92 0.53 2.61 a Marketed by Micro Labs Limited, Hosur, India; b Marketed by USV Limited, Aurangabad, India; c Mean value of five determinations. Tabulated t-value at 95% confidence level is 2.78; Tabulated F-value at 95% confidence level is 6.39 Recovery study
A standard addition procedure was followed to evaluate the accuracy of
the method. The sample is analyzed for the analyte of interest by adding a
specified amount of this analyte to the sample, thus increasing its concentration.
The analysis is then repeated and the resulting increase in peak area due to
addition of the standard amount is noted. Hence, the concentration of the analyte
in the original sample was calculated. The percentage recovery of GPZ from
pharmaceutical dosage forms ranged from 99.36–102.1%. Detailed results
presented in Table 6.3.9 reveal good accuracy of the proposed method.
*Mean value of three determinations.
Table 6.3.9 Results of recovery study by standard addition method
Tablet studied
GPZ µg mL-1,
tablet
GPZ µg mL-1,
pure
Total GPZ found, µg mL-1
Percent recovery of pure GPZ, (%GPZ±SD*)
Dibizide 98.88 98.88 98.88
50 100 150
147.9 200.1 253.4
99.36±0.36 100.6±0.45 101.8±0.89
Glynase 99.94 99.94 99.94
50 100 150
152.1 200.9 255.2
101.4± 0.75 100.5±1.02 102.1±0.98
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
295
Application to spiked urine
The proposed method was successfully applied to the determination of
GPZ in spiked urine sample with mean percentage recovery in the range of 101.8–
103.1% as shown in Table 6.3.10.
Table 6.3.10 Results of determination of GPZ in spiked urine sample
Spiked concentration µg mL-1
Founda ±SD % Recovery ±
RSD 150.0 154.6±0.63 103.1±1.27 200.0 203.6±0.59 101.8±1.02 250.0 255.4±0.45 102.2±0.98
a Mean value of five determinations; RSD is relative standard deviation
Results of forced degradation studies
GPZ was found to be sensitive towards oxidation and degraded up to 63%
under oxidation condition. The drug was found to be more stable under acidic,
basic, thermal and photolytic stress conditions (Table 6.3.11). No significant
changes (<1%) were observed for the chromatographic responses for the solutions
analysed with other stress conditions except oxidation condition. Figure 6.3.5
shows the degradation chromatograms of GPZ with the corresponding solvent as
blank.
Table 6.3.11 Results of degradation study
Degradation condition % degradation
Acid hydrolysis No degradation Base hydrolysis No degradation Oxidation 63% Thermal (105 °C, 2 hours) No degradation Photolytic (1.2 million lux hours) No degradation
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
296
a) b)
c) d)
e)
Figure 6.3.5. Chromatograms of GPZ (200 µg mL-1) after forced degradation a) acid degradation; b) base degradation; c) peroxide degradation; d) photolytic degradation and e) thermal degradation
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
297
Section 6.4
SUMMARY AND CONCLUSIONS-Assessment of methods
Two UV-spectrophotometric, and one each of high performance liquid
chromatographic and ultra performance liquid chromatographic methods were
developed and validated for the assay of GPZ in pharmaceutical formulations
along with the degradation study. The performance characteristics of the methods
developed and those of the existing methods are compiled in Table 6.4.1 below.
Compared to all reported methods for GPZ, the proposed methods have two
additional advantages of simplicity of operations, extraction-free, no
heating/cooling steps and less analysis run time. These advantageous features
enhance their routine use in quality control laboratories.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
298
Table 6.4.1 Comparison of performance characteristics of proposed methods with the existing methods A. UV-spectrophotometry
Sl. No.
Reagent/s
Methodology
Linear range (µg mL-1)
Molar absorptivity, ∈ (L mol-1 cm-1)
Remarks
Ref.
1 - Absorbance was measured at 274 nm NA NA - 4
2 -
Absorbance of tablet extract was measured at 275 & 259.9 nm
1.2-6.0 1.2-6.0
NA Less sensitive, narrow linear range
5
3 Methanol Absorbance was measured at 276 nm in methanol 2.0-20 NA Narrow linear range 6
4 Acetonitrile- methanol- water
Measurement of absorbance in acetonitrile- methanol- water mixture at 227.3 nm
5-55 NA Mixed solvents system and narrow linear range
7
5
a) 0.1M NaOH
b) 0.1M HCl
Measurement of absorbance at 260 nm in 0.1M NaOH Measurement of absorbance at 255 nm in 0.1M HCl
4.0–72.0
4.0-72.0
6.06×103
6.13×103
Moderately sensitive, wide dynamic linear range, uses a single solvent, stability-indicating
Present work
*NA- Not available
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
299
B. HPLC
Sl. No.
Mobile phase
Column
Detection λmax
(nm) Range (µg mL-1)
LOD & LOQ (µg mL-1)
Remarks Ref.
1 Triethylamine buffer (pH 3.0) : methanol C18 UV 230 0.1-10
NA - 8
2 Ammonium hydrogen phosphate buffer (pH 3.5) : methanol
Hypurity C18 UV 225 10-70
NA Less sensitive 9
3 Phosphate buffer (7.0) : methanol RP C18 UV 225 10-2000
ng/mL - 10
4 Phosphate buffer (pH 2.0) : water: methanol Inertsil ODS-C18 UV 270 1-6
15 & 45 ng/mL Narrow linear range 11
5 Sodium hydrogen phosphate buffer : methanol Eclipse XDB- C18 UV 225 5-250
NA - 12
10 Phosphate buffer (pH 3.9) : methanol Inertsil ODS-3V
UV 220 1-450 0.03 & 0.09
Stability-indicating, wide linear range, less run time, sensitive
Present work
C. UPLC
No UPLC method reported yet for GPZ
1 Phosphate buffer (pH 3.5) : ACN Zorbax Extend C18
UV 220 0.05-300 0.01 & 0.03
Stability-indicating, wide linear range, less run time and sensitive
Present work
ACN-Acetonitrile; NA- Not available
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
300
Table 6.4.1 reveals that the developed UV-spectrophotometric methods
are superior to the reported spectrophotometric methods in terms of linear range,
and stability indicating nature. The method using UPLC with an LOD of 0.01
µg mL-1 is the most sensitive of the four methods developed. All the four methods
are characterized by wide linear dynamic ranges, and the spectrophotometric
methods though moderately sensitive (ε value, 103) are the simplest methods in
terms of experimental variables involved. The developed methods are free from
many experimental variables that would affect their accuracy and precision; and
this is rightly reflected in their high accuracy and precision.
Chapter 6 Spectrophotometric and chromatographic assay of glipizide
301
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