CHAPTER 6 SPECTROPHOTOMETRIC AND CHROMATOGRAPHIC...

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


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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


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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


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 .


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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),


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.


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).


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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.


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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


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


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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.


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


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


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


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.


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.


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.


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.


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.


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:


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


%H3PO4

Number of


Flow rate, mL

min-1

Number of


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.


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).


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.


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.


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


(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).


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.


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


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.


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


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


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.


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.


287


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.


288


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


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)


4 Acquity HSS BEH Shield RP18 (50 × 2.1 mm, 1.7 µm)


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


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


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


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.


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


(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


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


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


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


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


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.


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


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


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.


301

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