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CHAPTER 4

DETERMINATION OF DRUGS IN SINGLE AND MULTI-COMPONEN T DOSAGE FORMS

4.1. Method development and optimization [36-38]

4.1.1. Selection of chromatographic method

Proper selection of the method depends upon the nature of the sample (ionic

or non-ionic or neutral molecule) its molecular weight and solubility. The drugs

selected for the present study are polar in nature and hence either reverse phase or

ion pair or ion exchange chromatography can be used. Reverse Phase (RP) HPLC

has been selected for the initial separation because of simplicity and suitability.

4.1.2. Selection of initial separation conditions

A gradient run was performed for the initial separation. From this the

approximate ratio of the organic phase in the buffer solution required to elute the

drugs from the column would be determined. An aliquot of the mixed standard

solutions containing 50 µg/ml of each drug (drug combinations) are prepared and

chromatographed using the following chromatographic conditions:

Stationary phase: C8/ C18, (5µ, 15cm x 4.6mm i.d)

Mobile phase: Aqueous phase: water/ acetate/ phosphate buffer/organic modifiers

Organic phase: Methanol/ Acetonitrile

Solution ratio: Gradient run, 10 to 100% solution of B for 30 min

Flow rate: 0.25 to 1.0 ml/min

Sample injector: Rheodyne syringe

61

Sample size: 20 µl

Temperature: Ambient to 40 oC

From the above gradient run the approximate percentage of acetonitrile/

methanol in the aqueous phase required to elute the drugs form the column is

determined. The ratio would be used for subsequent isocratic separation and

chromatograms are recorded.

To optimize the chromatographic conditions, the effect of chromatographic

variables such as mobile phase pH, solvent strength, addition of peak modifiers,

flow rate, solvent ratio and nature of stationary phase on the peak separation will

be studied. The resulting chromatograms are recorded and the chromatographic

parameters such as capacity factor, asymmetric factor, resolution and column

efficiency are calculated. The conditions that give the best resolution, symmetry

and capacity factor will be selected for the estimation.

4.1.3. Effect of pH

The mixed standard solutions are chromatographed using acetonitrile/

methanol in buffer solutions of different pH ranging from 2-7 (phosphate buffers

pH 2, 3, 6, 7, acetate buffers pH 3, 5) as the mobile phase at a flow rate of 0.25 to

1.0 ml/min.

4.1.4. Effect of peak modifier

With above chromatographic conditions, a peak modifier such as 0.1-0.5%

Triethylamine or 0.5% Acetic acid or 0.05-0.1% heptane sulfonic acid is added to

the mobile phase in order to improve the peak shapes and the chromatograms were

recorded.

62

4.1.5. Effect of nature of stationary phase

Different reverse phase stationary columns (C4, C8 and C18) are used and the

chromatograms are recorded. When C4 and C8 columns used, the retention time

and separation time between the peaks will be reduced. To increase the separation

between the peaks, the ratio of the organic solvents in the mobile phase is reduced

by 5% from that of the initial separation conditions and the standard drug solutions

were chromatographed.

4.1.6. Effect of solvent strength

Different mobile phases, namely, acetonitrile (ACN), methanol (METH),

Tetra Hydro Furan (THF) and a mixture of THF/METH (1:1) in aqueous phase are

used at a flow rate of 0.25-1.0 ml/min, the strength of water, ACN, METH, THF

and the mixture of THF and METH in the reverse phase chromatography may be

0.0, 3.2, 2.6, 4.5 and 3.55 respectively, for the initial separation ACN/ METH is

used. The ratio of ACN/ MET/ THF/ THF and MET is calculated using solvent

strength and used as mobile phase. When ACN/ MET is substituted by the other

solvents, their solvent to buffer ratios were calculated. The analyte was injected

into the HPLC system using the resulting ratios of the mobile phase and the

chromatograms were obtained.

4.1.7. Effect of mobile phase ratio

The standard solutions were chromatographed with the mobile phases of

different ratios containing organic and aqueous phases at a flow rate of 0.25-1.0

ml/min.

4.1.8. Effect of flow rate

The standard solutions were chromatographed at different flow rates

namely 0.25, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 ml/min etc.,

63

4.2. Determination of Voglibose

4.2.1. Experimental Part

4.2.1.1. Materials

Voglibose (Pure and formulations), Acetonitrile and methanol (HPLC

grade), Aqueous solutions were prepared with Milli Q (Millipore, USA) grade

water and all the other reagents used were of analytical reagent grade.

4.2.1.2. Instrumentation

The Shimadzu HPLC system consisted of CBM-20A Prominence

communication bus module with DGU-20A5 prominence degasser and SIL-

10Advp auto injector connected to a RID-10A refractive index detector. The data

were acquired and processed with LC solution version 1.22 SP1 software. The

analytical column was Waters C18-, 4.6 x 250 mm, 5 µm particle size. The cell

temperature was maintained at 40 oC.

4.2.1.3. Mobile phase

The isocratic mobile phase consisted of acetonitrile as solvent A and water

as solvent B in the ratio of 50:50, v/v. Flow rate of 0.5 ml/min was set for the

analysis.

The mobile phase was filtered through 0.45 µm millipore membrane filters

and degassed by sonication in an ultrasonic bath before use.

4.2.1.4. Standard and sample solutions

Stock standard solution of VGB (1 mg/ml) was prepared in water, and

working standard solutions were prepared by diluting the stock standard solution

with the mobile phase.

64

4.2.1.5. Preparation of sample solutions

Twenty tablets were finely powdered and the equivalent of one tablet (0.2

and 0.3 mg as VGB) was accurately weighed and extracted with 10 ml of the

mobile phase. It was sonicated for 30 min with vortex mixing at 10 min intervals

to avoid aggregation of the powdered samples. After centrifugation (2000×g for 10

min), supernatant was collected and filtered through a 0.22 µm filter and injected

into HPLC system.

4.2.1.6. Validation [36, 99]

Once the chromatographic method had been developed and optimized, it

has to be validated. The validation of an analytical method verifies that the

characteristics of the method satisfy the requirements of the application domain.

The proposed method was validated in the light of ICH Guidelines for linearity,

intra- and inter-day precision, LOD, LOQ, selectivity and specificity, stability and

recovery.

4.2.1.7. Recovery of VGB from formulations

The recovery of an analyte is the extraction efficiency of an analytical

process, reported as a percentage of the known amount of an analyte obtained

during the sample extraction. The mobile phase selected was most efficient in

extracting VGB from formulation. Samples were prepared in triplicate at three

concentrations 10, 50, and 100 µg/ml of VGB and assayed as described above. The

extraction efficiency of VGB was determined by comparing the peak areas

measured after analysis of samples from formulation with those found after direct

injection of unextracted (pure) samples into the chromatographic system at the

same concentration levels.

65

4.2.2. Results and discussion

4.2.2.1. Optimization and method development

VGB was determined using HPLC with Refractive Index (RI) detector and

found to be well suited technique for the analysis of non-UV absorbing compound

without derivatization. RI detector increased the sensitivity of detection. Fig. 4.1

shows an LC elution of VGB. During development phase, the mobile phase

containing methanol-water resulted in broad and asymmetric peak with a greater

tailing factor (>2). The successful use of acetonitrile and water resulted in drastic

reduction of peak tailing, which was found to be within the acceptable limit (1.5)

resulting good peak symmetry and resolution. The mobile phase optimized

contained water and acetonitrile (50: 50 v/v) at 0.5 ml/min flow rate. The retention

time was found to be 3.26 min for VGB. There were no interferences at retention

time of the analyte.

Fig-4.1 Chromatogram showing (a) VGB (80µg/ml) in pure form (b) VGB

(30µg/ml) in formulations) and (c) blank run devoid of sample

66

4.2.2.2. Validation of the proposed method

4.2.2.2.1. Linearity and Calibration curve

Calibration curve (peak area) (Fig 4.2) was constructed by spiking six

different concentrations of VGB. The chromatographic responses were found to be

linear over an analytical range of 10-100 µg/ml and found to be quite satisfactory

and reproducible with time. The linear regression equation was calculated by the

least squares method using Microsoft Excel® program. The correlation coefficient

equals 0.9994, indicating a strong linear relationship between the variables. The

variance of response variable S2Yx calculated was 1.863, indicates low variability

between the estimated and calculated values. This further confirms negligible

scattering of the experimental data points around the line of regression and good

sensitivity of the proposed method.

Fig- 4.2 Calibration curve of VGB

4.2.2.2.2. Precision and accuracy

Inter-day as well as intra-day replicates of VGB gave an R.S.D. below 2.12

which revealed that the proposed method is highly precise. Accuracy data (%bias)

in the present study ranged from -1.86 to +0.11 and indicated no interference from

67

formulation excipients. Accuracy and precision calculated during the intra- and

inter-day run are given in Table 4.1.

Table 4.1 Precision and accuracy data for VGB (from formulation) obtained for

the developed method

Nominal Concentration (µg/ml)

10 50 100

Day 1

Mean 9.92 50.02 99.25

S.D. 0.14 1.01 2.11

%R.S.D 1.41 2.02 2.12

%Bias -0.8 +0.04 -0.75

Day 2

Mean 9.89 49.41 100.11

S.D. 0.13 1.04 1.97

%R.S.D 1.31 2.10 1.96

%Bias -1.1 -1.18 +0.11

Day 3

Mean 9.86 49.07 99.11

S.D. 0.14 1.04 2.07

%R.S.D 1.42 2.12 2.09

%Bias -1.4 -1.86 -0.89

Each mean value is the result of triplicate analysis

%R.S.D= (S.D/mean) x100, %Bias= [(measured value-true value)/true value]x100

4.2.2.2.3. LOD and LOQ

The LOD and LOQ were found to be 2.91 and 9.70 µg/ml, respectively.

When this method is applied to formulation samples, its sensitivity was found to be

adequate for assay of the formulations.

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4.2.2.2.4. Specificity and selectivity

Any potential interference (overlapping peaks) due to formulation

excipients were within 2 min, later on there was no significant interference from

blank that affected the response of VGB (Fig 4.1a-4.1c).

4.2.2.2.5. Stability

The study indicated that samples were stable for 24 h (short-term) at room

temperature and samples stored at -4 oC, were injected over a period of 1 month it

did not suffer any appreciable changes in assay value and met the criterion

mentioned above. The solutions were found to be stable even after three freeze–

thaw cycles and the results were found to be with the range of 90-110% (Table

4.2).

Table 4.2 Stability studies of Voglibose (n=3)

S. No Concentration (µg/ml)

Short-term Long-term Freeze-thaw

Mean ± S.D. Mean ± S.D. Mean ± S.D.

1 10 9.91 ± 0.16 9.89 ± 0.19 9.92 ± 0.21

2 50 50.12 ± 0.97 48.24 ± 1.30 49.01 ± 1.41

3 100 98.61 ± 1.88 98.12 ± 1.96 100.39 ± 2.3

4.2.2.2.6. System suitability

A system suitability test according to USP was performed on the

chromatograms obtained from standard and test solutions to check the above

mentioned parameters and the results obtained from six replicate injections of the

standard solution are summarized in the Table 4.3.

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Table 4.3 System suitability parameters

Parameter Obtained valuea

Retention time, Rt (min) 3.264 ± 0.07

Capacity factor (k’) 4.834 ± 0.1

Theoretical plates (USP) 7928 ± 149.2

Tailing factor (Tf) 1.15 ± 0.02

aMean ± S.D.

4.2.2.3. Recovery of VGB from formulations

Extraction efficiency was performed to verify the effectiveness of the

extraction step and the accuracy of the proposed method. The extraction efficiency

of VGB from formulation samples was satisfactorily ranged from 98.35 to

100.62% (R.S.D. was less than 1.55) at all three concentration levels, which

confirm no interference due to the excipients in the formulation (Table 4.4).

Table 4.4 Results of VGB recovery from formulations

Formulation Label claim (mg)

Amount recovered (mg)

% recovery %RSD

VGBF 1 0.3 0.297 99.0 1.37

VGBF 2 0.2 0.199 99.5 1.71

VGBF 3 0.3 0.295 98.3 2.06

VGBF 4 0.2 0.201 100.5 2.11

VGBF1- Volix (RANBAXY LABS), VGBF 2- Volvo (ZYDUS PHARMACEUTICALS),

VGBF 3- Vocarb (GLENMARK PHARMACEUTICALS), VGBF 4- Volix (RANBAXY LABS).

70

4.3. Determination of drugs in multi-component dosage forms

Drugs are typically developed and manufactured into dosage forms prior to

their use by patients. Dosage forms require a variety of tests and standards to

assure therapeutic benefit. These intricacies of drug delivery system complicate

efforts to develop control assays and tests. Very often administration of two or

more drugs at a time becomes imperative for several therapeutic reasons and there

exist a number of drug combinations or multi-component dosage forms which

have proved to have better therapeutic effect due to their additive or synergistic

effect, ease of administration as a single dose, economic in production, distribution

and treatment costs.

Analysis of dosage forms containing a single ingredient is easier when

compared to the analysis of multi-component dosage forms containing more than

two drugs. Interference from the excipients can no doubt occur. These excipients

can be avoided by adopting suitable sample preparation. Analysis of multi

component dosage forms by extraction of individual drugs, however, is

cumbersome and very often results in errors due to incomplete extraction. Multi

component dosage forms are complex in nature because of the presence of not

only the different additives, but also two or more drug components present in

them. In the process of estimation it is important to confirm that the estimation of

one component does not interfere with the estimation of the other. The complexity

of multi component dosage forms thus presents challenges during the development

of assay methods [100-103]. The general steps in optimization of chromatographic

conditions for the separation of all the selected combinations are as follows:

Combination 1: Metformin and Pioglitazone

Combination 2: Metformin and Nateglinide

Combination 3: Rosiglitazone and Gliclazide

71

Combination 4: Pioglitazone and Glimepiride

Combination 5: Metformin, Pioglitazone and Glimepiride

4.3.1. Experimental

4.3.1.1. Materials

Pure drug samples and formulations (combinations) - Metformin,

Pioglitazone, Gliclazide, Rosiglitazone, Nateglinide, Glimepiride. HPLC grade

methanol and acetonitrile. Analytical reagent grade Ortho-phosphoric acid,

Ammonium acetate, Glacial acetic acid, Potassium dihydrogen phosphate,

Dipotassium hydrogen phosphate. MilliQ water was used for preparation of buffer

solutions.

4.3.1.2. Instrumentation

The Shimadzu HPLC system consists of Shimadzu Class LC-10AT vp and

LC-20AD pumps connected with SPD-10A vp UV-Visible detector. The data

acquisition was performed by Spincotech 1.7 version software. The elution was

performed on Gemini C18 (150 x 4.6 mm, 5µ) with a guard column.

4.3.1.3. Mobile Phase

Solvent A: Acetonitrile or methanol or both

Solvent B: Water/ buffer (pH 3 to 4 adjusted with glacial aceticacid or ortho

phosphoric acid).

The mobile phase was filtered through 0.45 µm millipore membrane filters

and degassed by sonication in an ultrasonic bath before use.

72

4.3.1.4. Standard and sample solutions

The standard stock solutions were prepared with methanol to get the final

concentration 1000 µg/ml. The working standard solutions of drug combinations

were prepared by taking suitable aliquots of drug solution from the standard

solutions and the volume was made up to 10 ml with mobile phase. A mixed

standard solution was prepared by transferring 0.2 ml of each from the stock (1000

µg/ml) into 10 ml volumetric flask and made up the volume with mobile phase to

get 20 µg/ml each solution.

For the analysis of pharmaceutical dosage forms, ten tablets were weighed

and powdered. A quantity equivalent to one tablet was transferred into extraction

flask, to this suitable amount of methanol was added and the mixture was

subjected to vigorous shaking for 30 min for complete extraction of drugs, and

then centrifuged at 5000 rpm for 20 min (Remi R8C laboratory centrifuge).

Supernatant was collected from each set and diluted with the mobile phase and

injected into HPLC system for the analysis.

4.3.1.5. Validation

The validation of an analytical method verifies that the characteristics of the

method satisfy the requirements of the application domain. The proposed method

was validated in the light of ICH Guidelines [99] for linearity, intra- and inter-day

precision, LOD, LOQ, selectivity and specificity, stability and recovery.

4.3.1.6. Recovery of selected drugs from the formulations

Samples were prepared in triplicate and assayed as described. The

extraction efficiency was determined by comparing the peak areas measured after

analysis of samples from formulation with those found after direct injection of

unextracted (pure) samples into the chromatographic system at the same

concentration levels.

73

4.3.2. Results and discussion

4.3.2.1. Optimization of chromatographic conditions

Optimization of the chromatographic conditions were intended to take into

account the various goals of method development and to weigh each goal

(resolution, run time, sensitivity, peak symmetry etc.,) accurately according to the

requirements of the high performance liquid chromatographic method being used

for the simultaneous estimation of multi component dosage forms. Reverse phase

HPLC method was chosen because the drugs selected in the present study are

weakly acidic/ basic in nature.

4.3.2.2. Selection of wavelength

The sensitivity of the HPLC method that uses UV detection depends upon

the proper selection of the wavelength. The standard solutions were scanned from

200-400nm and the overlaid UV spectra obtained were recorded. From the

overlaid UV spectra, the detection wavelengths were selected for the methods to

estimate simultaneously two or more drugs, the drugs in multi component dosage

forms used in the present study gave good peak response at the wavelength

selected.

4.3.2.3. Initial LC conditions

Acetonitrile/ methanol was selected as organic phase in the mobile phase to

elute the drugs from the stationary phase because of its favorable UV transmittance

(UV cut off wavelength is less than 210nm), low viscosity and better solubility for

the selected drugs. The pH of the initial mobile phase selected was 2.0 because a

low pH protonates column silanols (free hydroxyl group in reverse phase column)

and reduces their chromatographic activity i.e., it forms H-bonds with the polar

groups leading to peak tailing. Further, a low pH (less than 3) is quite different

from the pKa values of the selected (weakly acidic) drugs under study. At low pH,

74

therefore, the retention of the drugs will not be affected by slow changes in pH and

the reverse phase HPLC methods will not be more rugged.

The mixed standard solutions were chromatographed using the initial

chromatographic conditions. To improve the resolution or symmetry of the peaks

or to study the effect of the other chromatographic conditions, the chromatographic

variables like pH, stationary phase, mobile phase, flow rate, were optimized. The

typical chromatograms of the mixed standard and sample solutions were recorded

using optimized conditions are presented in Fig 4.4-4.8.

Several trials with ammonium formate, ammonium acetate, potassium and

sodium phosphates were made before the selection of suitable buffer.

To perform the initial separation, methanol was used because of its

favorable UV transmittance, low viscosity and better solubility. When methanol

was substituted by other solvents, the solvents to buffer ratios were calculated

using solvent strength. The resulting ratios of the mobile phase were prepared and

the drugs chromatographed. These mobile phases gave well retained and

symmetrical peaks. Tetrahydrofuran was not selected due to its UV cut off

wavelength of (215 nm). Methanol or acetonitrile was used as the mobile phase for

further studies in combination with aqueous phase (water/ buffer). The optimized

composition was used for isocratic separation with the above conditions and the

chromatograms were recorded. To improve separation between analyte peaks, the

percentage of acetonitrile or methanol in the mobile phase was modified and the

chromatograms were recorded.

A well resolved, retained and symmetrical peak was obtained at pH 3 and 4.

A lower pH was avoided as it might hydrolyse the alkyl chain from the reverse

phase column (column bleeding). A pH of 3 to 4 was selected for further studies.

75

Flow rates of 0.3-1.0 ml/min were used and the chromatograms were

recorded. These flow rates gave symmetrical and well retained peaks.

Different reverse phase columns (C8 and C18) were used and the

chromatograms were recorded. When C8 column was used, poor separation

between analytes was observed. The resolution improved when acetonitrile in

mobile phase was decreased, well resolved peaks were eluted after 4.0-8.0 min.

When C18 column was used well resolved and symmetric peaks were eluted in less

than 10 min. Hence C18 was used for the study.

Based on the studies, the following chromatographic conditions were

optimized for the simultaneous estimation of Anti-diabetic drugs in multi

component dosage forms.

4.3.2.3.1. Combination 1 (Metformin and Pioglitazone)

Stationary phase: Reverse Phase C18 Column

Mobile phase: Acetonitrile and ammonium acetate buffer (pH 3)

Ratio: 42: 58

Wavelength: 255nm

Flow rate: 0.3 ml/min

Pressure: 97 Kgf

Temperature: Ambient (25±2 oC)

Retention time: 5.17 (MET) and 8.1 (PIO) min

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4.3.2.3.2. Combination 2 (Metformin and Nateglinide)

Stationary phase: Reverse Phase C18 Column

Mobile phase: Methanol and Phosphate buffer (pH 3)

Ratio: 60: 40

Wavelength: 235 nm

Flow rate: 0.8 ml/min

Pressure: 112 Kgf

Temperature: Ambient (25±2 oC)

Retention time: 2.63 (MET) and 4.51 (NAT) min

4.3.2.3.3. Combination 3 (Rosiglitazone and Gliclazide)

Stationary phase: Reverse Phase C18 Column

Mobile phase: Acetonitrile and water (pH 3 with ortho phosphoric acid)

Ratio: 70: 30

Wavelength: 250 nm

Flow rate: 0.6 ml/min

Pressure: 59 Kgf

Temperature: Ambient (25±2 oC)

Retention time: 2.41 (ROS) and 5.22 (GLC) min

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4.3.2.3.4. Combination 4 (Pioglitazone and Glimepiride)

Stationary phase: Reverse Phase C18 Column

Mobile phase: Methanol and ammonium acetate buffer (pH 3.5)

Ratio: 55: 45

Wavelength: 252 nm

Flow rate: 0.5 ml/min

Pressure: 131 Kgf

Temperature: Ambient (25±2 oC)

Retention time: 5.63 (PIO) and 7.18 (GLM) min

4.3.2.3.5. Combination 5 (Metformin, Pioglitazone and Glimepiride)

Stationary phase: Reverse Phase C18 Column

Mobile phase: Acetonitrile and phosphate buffer (pH 3)

Ratio: 65: 35

Wavelength: 245 nm

Flow rate: 0.5 ml/min

Pressure: 89 Kgf

Temperature: Ambient (25±2 oC)

Retention time: 2.75 (MET), 4.35 (PIO) and 8.75 (GLM) min

78

4.3.2.4. Validation of the method

Specificity was evaluated by preparing a solution of an analytical placebo

(containing all the ingredients of the formulation except the analyte) and injected.

To identify the interference by these excipients, a mixture of the inactive

ingredients (placebo), before and after being spiked with standards, standard

solutions and the commercial pharmaceutical preparations including analytes were

analyzed by the proposed method. The representative chromatograms show no

other peaks which confirm the specificity of the method. Peaks obtained in QC

samples may be due to excipients present in the formulations. These peaks

however did not interfere with the standard peaks. These observations show that

the developed assay methods are specific.

Calibration curves (Fig 4.3) were acquired by plotting the peak area

analytes against the nominal concentration of calibration standards. Linearity

solutions were injected in triplicate and the calibration graphs were plotted as peak

area of the analyte against the concentration of the drug in µg/ml. The calibration

curve show a linear response over the range of concentrations used in the assay

procedure and also pass close to the origin, which justifies the use of single point

calibration. The numerical values of the slope, intercept, correlation co-efficient,

and proximity of all the points of the calibration line demonstrate that the methods

developed have adequate linearity of response to the concentration of the analytes.

The linearity ranges were given in Table 4.5.

The accuracy of the proposed method was also tested by recovery

experiments. Recovery experiments were performed by adding known amounts of

analytes to the analytical placebo solution. They were spiked at three different

concentrations according to label claim in the pharmaceutical preparations. Six

samples were prepared for each recovery level. Samples were treated as described

in the procedure for sample preparation. An analysis of the results shows that the

79

% recovery and % R.S.D. values were with in the acceptable limits thus

establishing the developed methods are accurate and reliable.

In order to measure repeatability of the system (injection repeatability), ten

consecutive injections were made with a standard solutions of analytes. The results

were evaluated by considering retention time, peak area, capacity factor, peak

asymmetry and resolution values of analytes. Three different concentrations (in the

linear range) were analyzed in three independent series in the same day (intra-day

precision) and three consecutive days (inter-day precision) within each series every

sample was injected three times. The mean and S.D and % CV were calculated and

presented. The results reveal a low standard deviation and % CV was with in the

limit showing that the developed methods are precise (Table 4.6).

The LOD/ LOQ values obtained show that the developed methods have

adequate sensitivity. These values are affected by the separation conditions,

instrumentation, data systems, detectors and use of the contaminated reagents and

low grade (other than HPLC grade for LC) can result in large changed in S/n ratio

due to baseline noise and drift. (Table 4.7)

Robustness was carried out by deliberate variations into the method

parameters, like mobile phase ratio (±2%), buffer pH (±0.2), and flow rate (±0.1

ml/min), were varied around the value set in the method to reflect changes likely to

arise in different test environments. Analyses were carried out in triplicate and

only one parameter was changed in the experiments at a time. Each mean value

was compared with the mean value obtained by optimum conditions. It was

observed that there were no marked changes in the chromatograms and in the

parameters demonstrating that the HPLC methods developed are rugged and

robust.

80

Stability of the standard solutions of analytes was evaluated under different

storage conditions. For short-term stability, working standard solutions were kept

at room temperature for 24 h. The long-term stability was assessed after storage of

stock solutions at 4 oC for 2 months. The stability results were evaluated by

comparing peak areas of analytes with those of freshly prepared standard solutions.

The solvents, buffers, mobile phase additives and drug solutions were subjected to

stability studies by performing experiments and looking for changes in the peak

pattern. The studies were carried out as short-term (bench-top), long-term

(refrigerator). The peaks of the drugs were compared with the freshly prepared

solutions, the drugs samples were found stable at all the chosen conditions and the

results are found to be in acceptable limit (Table 4.8). The stocks, solutions,

buffers and additives were stable up to 3 days when these were stored around 5 oC.

At room temperature, phosphate and acetate buffers provide good media for

microbial growth, hence these buffers were filtered using 0.45 µ membrane filters

before use. 10-12% acetonitrile in buffers increased the stability of the solutions.

0.1% sodium azide may be added in the buffers to inhibit the growth of

microorganisms at room temperature.

The requirements for system suitability are usually developed after the

completion of method development and validation (Table 4.5). The data obtained,

demonstrate the suitability of the system for the analysis of the drugs in

combinations (two or more) under study. The system suitability parameters might

fall within ±3% S.D. range during routine performance of the methods.

4.3.2.5. Recovery from formulations

Estimation of the selected formulations by chromatographic methods was

carried out using the optimized chromatographic conditions. The standard and

sample solutions were injected and the chromatograms recorded. The peak area or

the response factor of the standard and the sample solutions were calculated. The

assay procedure was repeated six times and mean peak area, response factor,

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percentage recovery of the drugs, mean, standard deviation were calculated. The

results of the analysis show that the amount of each drug was in good agreement

with the label claim of the formulations. The results were summarized in Table

4.9.

Table 4.5 Data showing linearity range and system suitability parameters

Combination Drugs Linearity

range (µg/ml)

Retention time

Tailing factor

LOQ (µg/ml)

N

1 MET 0.5-50 5.17 1.31 0.01 8432

PIO 0.3-30 8.1 1.21 0.02 8257

2 MET 0.5-50 2.63 1.26 0.48 9120

NAT 0.06-6 4.51 1.38 0.056 7812

3 ROS 0.025-2.5 2.41 1.21 0.02 7999

GLC 0.08-8 5.22 1.39 0.025 9094

4 PIO 0.025-25 5.63 1.19 0.023 9199

GLM 0.01-10 7.18 1.41 0.01 8457

5

MET 0.25-25 2.75 1.20 0.19 10112

PIO 0.25-25 4.35 1.31 0.21 9123

GLM 0.25-25 8.75 1.12 0.20 7983

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Table 4.6 Data showing precision (Intra- and Inter-day) [n=6]

Combination Drugs Amount Added (µg/ml)

Intra-day Inter-day

Amount Found

%RSD Amount Found

%RSD

1

MET

25 24.72 1.8 24.63 1.6

50 48.77 2.2 48.74 1.7

100 98.39 1.8 98.31 2.04

PIO

1.5 1.46 1.4 1.46 1.21

3 2.91 1.7 2.87 1.6

6 5.83 2.15 5.89 1.9

2

MET

10 9.92 1.64 9.87 3.64

20 19.85 0.54 19.82 2.17

50 50.37 1.28 49.89 1.31

NAT

1.2 1.19 1.09 1.18 2.76

3 2.89 2.13 3.01 0.89

6 6.03 0.69 5.93 1.47

3

ROS

1 0.96 1.19 0.96 2.11

1.5 1.47 2.11 1.51 1.89

2 1.89 2.31 1.83 1.76

GLC

40 39.71 1.31 39.63 1.75

60 58.84 2.11 58.68 1.34

80 78.51 1.78 78.19 1.67

4

PIO

15 14.81 1.86 14.53 1.95

30 28.70 2.20 28.82 2.31

45 44.52 2.05 44.23 2.05

GLM

2 1.99 1.14 1.93 1.00

4 3.92 1.21 3.94 1.09

6 5.81 1.96 5.91 1.90

83

(Table 4.6 continued…………….)

5

MET 500 100.11 0.26 100.02 0.25

PIO 15 99.89 0.25 99.61 0.23

GLM 1 99.87 0.25 99.51 0.21

Table 4.7 Data showing sensitivity of selected combination of drugs using proposed HPLC methods

Combination Drugs LOD (µg/ml) LOQ (µg/ml)

1 MET 0.003 0.01

PIO 0.061 0.02

2 MET 0.14 0.48

NAT 0.016 0.056

3 ROS 0.006 0.02

GLC 0.008 0.025

4 PIO 0.01 0.023

GLM 0.004 0.01

5

MET 0.052 0.19

PIO 0.061 0.21

GLM 0.058 0.20

84

Table 4.8 Data showing short-term and long-term stability of the selected drug combinations (µg/ml) n=3

Combination Drugs Short-term Long-term

Mean %RSD Mean %RSD

1 MET 25.21 1.24 24.98 1.37

PIO 14.56 1.87 15.02 1.75

2 MET 25.23 2.08 25.02 1.89

NAT 2.92 1.91 3.01 0.86

3 ROS 0.97 1.11 0.97 0.78

GLC 4.07 0.97 4.12 1.09

4 PIO 9.87 1.35 9.85 1.21

GLM 5.12 2.11 4.97 2.01

5

MET 9.89 2.13 9.91 1.11

PIO 10.03 1.08 9.95 1.89

GLM 9.91 0.89 10.11 2.31

85

Table 4.9 Data showing recovery from formulations

Combination Formulation Drugs Labeled claim (mg)

Amount Found (mg)

% Recovery %RSD

1

MPF-1 MET 500 491.76 98.4 1.37

PIO 30 29.79 99.3 1.51

MPF-2 MET 500 487.32 97.4 1.12

PIO 15 28.67 95.6 1.08

2

MNF-1 MET 500 496.27 99.25 2.78

NAT 60 59.08 98.47 3.01

MNF-2 MET 500 495.01 99.0 1.86

NAT 120 120.45 100.37 2.34

3

RGF-1 ROS 2 2.03 101.5 1.97

GLC 80 78.76 98.45 2.22

RGF-2 ROS 2 1.97 98.5 1.51

GLC 80 79.32 99.15 1.84

4

PGF-1 PIO 15 14.79 98.6 1.87

GLM 2 1.92 96.0 1.91

PGF-2 PIO 15 14.81 98.7 2.03

GLM 2 1.94 97.0 2.11

5

MPGF-1

MET 500 491.23 98.25 2.02

PIO 15 14.87 99.13 1.86

GLM 1 0.99 99.0 1.24

MPGF-2

MET 500 490.11 98.02 1.37

PIO 15 15.02 100.12 2.12

GLM 2 1.98 99.0 1.09

MPF1- Pio-M (Systopic labs), MPF2- GTase (Unichem labs), MNF1- Glinate MF (Glenmark Pharmaceuticals), MNF2- Glinate MF (Glenmark Pharmaceuticals), RGF1- Rosinorm-G (Micro labs), RGF2- Glyroz-2 (Aristo Pharma), PGF1- Euglim (Zydus Pharmaceuticals), PGF2- Glimy (DRL), MPGF1- PIOZ-MPG- (USV), MPGF2- Matce-PG 2 (Xeena Pharmaceuticals)

86

Fig 4.3 Calibration curves of five selected combination

87

Fig 4.4 Chromatograms of Combination 1 (Metformin and Pioglitazone)

88

Fig 4.5 Chromatograms of Combination 2 (Metformin and Nateglinide)

89

Fig 4.6 Chromatograms of Combination 3 (Rosiglitazone and Gliclazide)

90

Fig 4.7 Chromatograms of Combination 4 (Pioglitazone and Glimepiride)

91

Fig 4.8 Chromatograms of Combination 5 (Metformin, Pioglitazone and

Glimepiride)