RP-HPLC Method Development and Validation for Assay of Glycopyrrolate in Tablet

MATERIALS AND METHODS

MATERIALS AND METHODS

Chemicals and Reagents

Glycopyrrolate reference standard The HPLC grade solvents used were of E-merck India. All other chemicals and reagents were of analytical reagent grade. HPLC grade water was prepared using Millipore purification system. Maxalt tablets containing 10 mg of Glycopyrrolate was used for analysis purpose.

Equipment

An Agilent liquid chromatography instrument equipped with a pump in isocratic mode. Analytical column used was Bondapak C18 (3.9x300mm), 10m the system was connected with the help of chemistration software in a computer system for data collection and processing

Chromatographic conditions

Chromatographic separation was carried out at room temperature with Bondapak C18 (3.9x300mm), 10m column with 3.5mc size. Mobile phase containing of 1230 volumes of Phosphate buffer, 6 volumes of 1N Sulphuric acid , 470 volumes of Acetonitrile and 300 volumes of Methanol. The ratio pH was found to be 6.5. Then finally filtered using 0.45 nylon membrane filter and degassed in sonicator for 5 minutes. The injection volume for standard and sample were 50 L and eluted at a flow rate of 2.3 mL / min, the eluents were monitored at 222 nm

Standard preparation

Accurately weigh and transfer about 50 mg of Glycopyrrolate into a 50 mL volumetric flask. Add about 30 mL of diluent sonicate for 10 minutes and dilute to the volume with diluent and mix. Pipette 2.0 mL of above solution into 50 mL volumetric flask and dilute to volume with diluent and mix (40 mcg/mL of Glycopyrrolate). Then filter the solution through the 0.45 m membrane filter. This solution was injected into the column and the peak area and retention time was record as shown fig -1

Sample preparation:

Accurately weigh and transfer a quantity of powdered tablets equivalent to 10 mg of Glycopyrrolate (about 1000 mg powder) into a 250 mL volumetric flask, add about 150 mL of diluent, and sonicate for 20 minutes and cool to room temperature then filter the solution through a 0.45 membrane filter. (Duplicate preparation)

Analysis of formulation

Twenty tablets each containing 10mg of Glycopyrrolate were weighed and average weight was calculated. A quantity of fine powder equivalent to 10mg of Glycopyrrolate was weighed accurately, and transferred into 10ml volumetric flask, and made up to volume with mobile phase. Further dilution of this sample stock solution in the linearity range were made using mobile phase and filtered through 0.45 membrane filter. Then 50 L solutions were injected into column and chromatogram was recorded. All the determinations were conducted in triplicate.

METHOD OF VALIDATION

The described method has been validated for the assay of Glycopyrrolate using following parameters

Robustness

To determine the robustness of the developed method, experimental conditions were purposely altered and the retention was examined. In all the deliberate varied chromatographic conditions a flow rate (0.2ml/min+-0.05), mobile phase composition at various ratios (1230:6:470:300 v/v/v/v) showed that the retention time of peak remains unaffected but for small changes (Table-1a, 1b, 1c).

System suitability:

Accurately weighed and transferred about 50 mg of Glycopyrrolate working standard in to the 50 mL volumetric flask. Add bout 30 ml of diluent, sonicated for 10 minutes and diluted to volume with diluent. Transfer 3 ml of the above solution into a 50ml volumetric flask and diluted to volume with diluent. Then transferred the 5ml of the above solution into a volumetric flask and diluted to volume with diluent. Inject 100 L of standard preparation for six times into the chromatograph and recorded the system suitability parameters as per procedure the results are given(table-2)

Linearity:

The linearity of detector response is established by plotting a graph of concentration versus area of Glycopyrrolate standard and determined the correlation coefficient. A series of solutions of Glycopyrrolate standard solutions in the concentration range from about LOQ level (50%) to about 150% of the target concentration were prepared and injected into the HPLC system. The detector response was found to be linear from LOQ level (50%) to150% of target concentration for Glycopyrrolate standard with a correlation coefficient values greater than 0.999698 (Table-3)

Precision

The precision of the analytical method was studied by analysis of multiple (six) sampling of homogeneous sample. The precision expressed as % RSD. The %RSD was found to be 0.50% in the results of precision and the assay range of the six sample preparation is 101.4 to 102.8. (Table-4)

Accuracy:

Method was checked for the accuracy of the analytes covering the range of both Assay and Content Uniformity, % recovery of both analytes has been tabulated in (Table-5)

RESULTS AND DISCUSSION

The Glycopyrrolate peak in the sample was identified by comparing with the Glycopyrrolate standard and the retention time was found to be 4.2 minutes. The estimation of Glycopyrrolate tablets was carried out by RP-HPLC using mobile phase having a composition of 1230 volumes of phosphate buffer, 6 volumes of 1N sulpuricacid 470 volumes of Acetonitrile, 300 volumes methanol. The pH was found to be 6.5.then finally filtered using 0.45 membrane filter and degassed in sonicator for 10 minutes. The column used was Bondapak C18 (3.9x300mm), 10m column with 3.5mc size. Flow rate of mobile phase was 2.3 mL / minute, system suitability parameters such as RSD for six replicate injections were found to be less than 2%, theoretical plates and tailing factor 1.2. The quantitative estimation was carried out on tablete by taking the same concentration as for standard solution and assay results found to be the acceptance criteria of system suitability was RSD should not be more than 2% and the method show system suitability 0.70% which shows that method was repeatable. The acceptance criteria of method repeatability was RSD should not be more than 2.0% and the method show method repeatability 0.50% which shows that the method was precise. The validation of developed method shows that the drug stability is well within the limits. The linearity of the detector response was found to be linear from 50 to 150 g/ml of target concentration for Glycopyrrolate standard with a correlation coefficient value is greater than 0.999698. The correlation coefficient of (r2) = 1, which shows that the method was capable of producing good response in UV-dictator. The accuracy limits is the % recovery should be in the range of 99.0% to 100.2%. The validation of developed method shows that the accuracy is well within the limit, which shows that the method is capable of showing good accuracy.

Influence on Flow Variation Table-1a

S No.

System suitability

Low flow (2.1 mL)

High flow (2.5 mL)

Acceptance criteria

1

%RSD of Peak area

0.1

0.2

NMT 2.0

2

Tailing factor

1.2

1.1

NMT 2.0

Influence on variation of mobile phase Composition

Organic phase Acetonitrile Variation Table-1b

S No.

System suitability

Low organic phase (-5%)

High organic phase (+5%)

Acceptance criteria

1

%RSD of Peak area

0.1

0.1

NMT 2.0

2

Tailing factor

1.1

1.2

NMT 2.0

Organic phase Methanol Variation Table-1c

S No.

System suitability

Low organic phase (-5%)

High organic phase (+5%)

Acceptance criteria

1

%RSD of Peak area

0.1

0.1

NMT 2.0

2

Tailing factor

1.1

1.1

NMT 2.0

System suitability Table-2

System suitability parameters

Results

Limits

%RSD of peak areas

0.7

NMT 2.0

Tailing factor (USP)

1.2

NMT 2.0

Linearity Table-3

% Level

Concentration (mcg/mL)

Peak Area (Average)

Glycopyrrolate

50

20.2

257448

75

30.2

388086

100

40.3

518472

125

50.4

645301

150

60.5

786675

Correlation coefficient (r2)

0.999698

Precision Table -4

Sample No

Day 1 /Analyst 1/ Inst. 1

Day 2 /Analyst 2/

Inst. 2

% of assay

% of assay

01

102.8

99.5

02

101.8

99.7

03

101.7

99.4

04

102.4

99.5

05

102.3

99.6

06

101.4

99.4

Average

102.1

99.5

% RSD

0.5

0.1

Difference (%)

2.6

Accuracy Table -5

Sample No.

Concentration Level

mg added

mg found (recovered)

% recovery

1

50 %

5.0

5.0

100.1

2

5.0

5.0

100.2

3

5.0

5.0

99.9

Average

100.1

4

100%

10.0

10.0

100.0

5

10.1

10.0

99.0

6

9.9

9.9

100.0

Average

99.7

7

150%

14.9

14.9

100.0

8

14.8

14.7

99.3

9

14.9

14.9

100.0

Average

99.8

10

300 %

29.7

29.5

99.3

11

29.6

29.5

99.7

12

29.2

29.1

99.7

Average

99.5

Average

99.8

CONCLUSION:

Based on the above validation data, it is evident that the HPLC method documented in the protocol for the determination of % assay for Glycopyrrolate in Glycopyrrolate Tablets USP 1 mg is treated as a validated method. Hence it is recommended to be use for the determination of % assay of Glycopyrrolate in Glycopyrrolate tablets USP 1 mg

RP-HPLC Method Development and Validation for Assay of Glycopyrrolate in Tablet

By: P PARAMESHWAR

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