METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYSIS



METHOD DEVELOPMENT AND VALIDATION FOR THE ANALYSIS OF ANTI-DIABETIC DRUGS BY USING HPLC METHOD

a) BRIEF RESUME OF THE INTENDED WORK:

6.1 Need of the Study:

Diabetes is a major disease condition in the world today. Many people are suffering from it all over the world. India is one of the countries which contribute to a wide extent to the diabetic population today.

As it is a major disease many preventive measures are being taken but the prevalence of diabetes is increasing in all the countries and India day by day. For these reasons health care systems are also playing a very important role in this area in many developing countries.

Assay is a test to determine the composition, the content of the ingredients, and the potency unit of a drug by physical, chemical, or biological procedures.

Analytical method development and validation involve a series of activities that are ongoing during the life cycle of a drug product and drug substance.

Method development

Method validation/revalidation

QC laboratory

Analytical method development should be performed to the extent that it is sufficient for its intended purpose.

Upon successful completion of method development, then the method will then be validated to show proof that it is suitable for its intended purpose and to also set up the various in process controls.

Anti-diabetic medications treat diabetes mellitus by lowering glucose levels in the blood. They are also called as oral hypoglycemic agents or oral antihyperglycemic agents. There are different classes of anti-diabetic drugs, and their selection depends on the Type of diabetes (Type I or Type II), age and situation of the person, as well as other factors.

The scope and need of the present project is to develop and validate an analytical method for the analysis of an anti-diabetic drug.

HPLC method is being is used because it is the preferred method for development and validation in the recent trends. Validation of a drug in regulatory requirement and also it ensures that the product is fit for their intended use.1

6.2 Review of Literature

Aryane MS, and co workers carried out the development and validation of RP-HPLC method for the analysis of Metformin. In this a simple RP-HPLC method was developed for the quantification of Metformin hydrochloride in raw materials and in pharmaceutical preparations. Analytical Reverse Phase Column C(18) was used and the mobile phase consisted of methanol-water(30/70v/v) the analytes were then determined by using UV detector. This method was validated according to ICH guidelines. The proposed method is rapid, accurate, economical and selective and it was used for the quantitative analysis of metformin in Neodipar tablets because of its sensitivity and reproducibility.2

Literature shows that a RP-HPLC method was developed and validated for the simultaneous determination of Glipizide, Rosiglitazone, Pioglitazone, Glibenclamide and Glimepiride in Pharmaceutical dosage Forms and human plasma. Elution was performed using a mobile phase mixture of 0.05% triethylamine, acetonitrile and methanol in the ratio of 55:15:30 at a flow rate of 1 ml/min, RP column C (18) was used. The method was successful in detecting the drugs at a concentration of less than 0.1 μg ml-1. This method was validated according to ICH guidelines with suitable accuracy and precision and the results was found to be within acceptable range.3

Onal A, carried out spectrophotometric and HPLC determinations of anti-diabetic drugs, Rosiglitazone maleate and Metformin hydrochloride, in pure form and in pharmaceutical preparations. In this method he developed three Spectrometric method and one HPLC method for analysis of Anti-diabetic drugs. Two Spectrometric methods were based on the reaction of rosiglitazone with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and bromocresol green(BCG), the third Spectrophotometric method consists of a zero-crossing first-derivative Spectrophotometric method for simultaneous analysis of RSG and metformin (MTF) in tablets. The fourth method is a rapid stability indicating HPLC method. The proposed method was successfully applied to the tablet analysis.4

Method development and validation of simultaneous determination of pioglitazone and glimepiride in pharmaceutical dosage form by RP-HPLC was carried out by. Reddy B and team. Here the RP column used was a Phenomenex Luna C18 column. The mobile phase was acetonitrile with a flow rate of 1.5 ml/min. This method was Validated according to ICH guidelines in terms of accuracy, precision, specificity, range, system suitability and robustness.5

The literature survey of the work done by Havaldar F, Vairal D, reveals that a simple, suitable, accurate RP-HPLC method was developed for the simultaneous determination of glimipremide, rosiglitazone and pioglitazone hydrochloride. The column used Nucleodur C-18 and the mobile phase used was orthophosphoric acid and acetonitrile (80:20). Detection was done by using an UV detector. The proposed method was successfully validated and used for the the estimation of glimiprimide, rosiglitazone and pioglitazone hydrochloride.6

A simple, precise and stability indicating RP-HPLC method for the quantitative analysis of glibenclamide present in the tablet formulation and bulk drug was developed. The column used was symmetry C-18 with the mobile phase and detection at 228nms. The proposed model provided linear response with acceptable range. The precision of the method was demonstrated using intra-day assay and RSD% values which were less than 1% in all instances.7

Mallu U, Pyreddy V, Penumajji S, Bobbarala V developed a single and high resolution RP-HPLC method for the determination of six anti diabetic drug products. The objective of this study was to develop a single RP-HPLC method for the simultaneous determination of six active ingredients including Metformin Hydrochloride, Pioglitazone, Glimepiride, Gliclazide, Glibenclamide and Glipizide in pharmaceutical products. The Column used was C-18 containing gradient mobile phase such as Sol A: Phosphate buffer and Sol B: Acetonitrile. This method was successfully validated in terms of specificity, precision, linearity, accuracy, ruggedness and robustness. This RP-HPLC method is simple and reproducible, with high resolution and has been successfully applied for the simultaneous determination of the six components (metformin hydrochloride, pioglitazone, glipizide, gliclazide, glibenclamide and glimepiride) in pharmaceutical drug products.8

A method for the simultaneous estimation of Glibenclamide and Metformin HCl in Bulk and Tablets using UV – visible spectroscopy was developed and validated. This method reveals a spectrophotometric method for the simultaneous estimation of glibenclamide and metformin hydrochloride in combined dosage form using methanol as a solvent, The two wavelengths 229.5 nm and 237 nm were selected for estimation of glibenclamide and metformin HCl respectively. The method was validated successfully as per ICH guidelines, the method can be employed for estimation of pharmaceutical formulations with no interference from any other excipients and diluents.9

Analysis of glimepiride by using derivative UV Spectrophotometric method was done by Altinoz S, Tekeli D, The analysis was performed in the wavelength ranges between 245-290nm. The developed method was applied directly and easily to the pharmaceutical tablet preparations. The devolped method was successfully validated and proven to be rugged.10

Hamdan I and co workers, developed and validated a stability indicating capillary electrophoresis method for the determination of Metformin hydrochloride in tablets. Citrate buffer was used as a background electrolyte with an applied voltage of 15 kv. The capillary used was of 68.5 cm length and detection was carried out at a wave length of 230 nm, the method was validated in accordance with the ICH requirements, which involved accuracy, precision, linearity, selectivity and both limit of detection and limit of quantization. 11

A method was developed and validated for the determination of Repaglinide in human plasma by RP-HPLC and its application in pharmacokinetic studies was also investigated The column used was Purospher® STAR C-18 analytical column and the mobile phase contained acetonitrile-ammonium formate at a flow rate of 1ml/min. this method was successfully validated and applied to determine the repaglinide concentration in human plasma samples for pharmacokinetic study.12

6.3 Objective of the Study:

1) The primary objective of the study is to develope a HPLC method for the analysis of anti-diabetic drugs, to validate it in the manufacturing process and to ensure regulatory compliance as per ICH guidelines.

Method development is performed with an adaptation of ICH Q2 B Guidelines. The method development process of HPLC method involves the following scheme:

2) Method may be developed by changing the temperature of column:

Raising the temperature may lead to increased column efficacy.

Reducing the mobile phase viscosity it may increase the eluent flow rate.

| Important Parameters for HPLC are: |

|a) Solvent(mobile phase) Strength (may be organic mixture) |

|b) Choice of Solvent |

|c) Mobile phase pH |

|d) Silanol activity. |

|In reverse phase, suppression of ionization may be needed |

|Common buffers used: Buffering may be between pH 2-8 |

|Preparation before method validation |

|a) Critical related substances. |

|b) Lower and upper concentration range for method validation. |

|c) Method procedure. |

|d) Critical experimental parameters for robustness. |

|e) System suitability tests. |

2} The secondary objective is to evaluate certain parameters for HPLC method to ensure compliance as per ICH Q2 (R1) Guidelines such as:

6.3.1 Specificity

• Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present.13

• Most related substance methods will be used in a stability study, and therefore they have to be stability indicating.

• Stability indicating means that the method has sufficient specificity to resolve all related substances and the drug substance from each other.

6.3.2 Limit of Quantitation and Limit of Detection.

• The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be determined quantitatively with a suitable precision and accuracy.

• The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not quantitated as an exact value.

• Quantitation limit can be found out using any of the following methods:

1. Signal to noise approach.

2. Visual examination.

3. Standard deviation.

6.3.3 Linearity

• The linearity of an analytical procedure is its ability (within a given range) to obtain test results that are directly proportional to the concentration of analyte in the sample.solution.13

If the linearity does not support such a wide range of concentration, linearity is determined from 50% of the ICH reporting level to 150% of the proposed shelf life specifications of the related substance (for the high–low and external standard methods) as a minimum

6.3.4 Accuracy

• The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or as an accepted reference value and the value found.

• Accuracy for the area percent method should be established from 50% of the ICH reporting limit to the nominal concentration of drug substance in the sample solution.

• For the high–low and external standard methods, determine accuracy from 50% of the ICH reporting level to 150% of the proposed shelf life specification of the related substances. 13

6.3.5 Precision

Precision can be determined by detecting any of the following:

Repeatability:

• Repeatability expresses the precision under the same operating conditions over a short interval of time.

• Repeatability is also termed intra-assay precision.

• Repeatability of a method can be determined by multiple replicate preparations of the same sample.

• This can be done either by multiple sample preparations (n = 6) in the same experiment or by preparing three replicates at three different concentrations.13

Intermediate Precision:

Intermediate precision expresses, within laboratories variations, different days, different analysts, different equipment, and so on.

Reproducibility.

Reproducibility expresses the precision between laboratories (collaborative studies are generally used, for standardization of methodology).

6.3.6 Range:

• The range of an analytical procedure is the interval between the upper and lower concentrations of analytes in the sample.

• Typically, linearity and accuracy determination covers a wide concentration range (e.g., 50% of the ICH reporting limit to 150% of specification).

•

6.3.7. Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal use.13

b) MATERIALS AND METHODS

7.1 Source of Data:

Data will be collected from various related websites, literature surveys, abstracts, journals and related articles from libraries of Krupanidhi College of Pharmacy, ICH Guidelines, FDA Guidelines, and USP etc.

7.2 Method of collection of data:

Data will be collected from the following step wise experimental procedures proposed in the study:

This is to verify that the method performance is not affected by typical changes in normal experiments. Therefore, the variation in method conditions for robustness should be small and reflect typical day-today variation.

1) HPLC conditions:

a) HPLC column (lot, age, brand).

b) Mobile-phase composition (pH ± 0.05 unit, percent organic ± 2%).

c) HPLC instrument (dwell volume, detection wavelength ± 2 nm, column.

d) Temperature ± 5◦C, flow rate).

2) Sample preparation:

a) Sample solvent (pH ± 0.05 unit, percent organic ± 2%).

b) Sample preparation procedure (shaking time, different membrane filters).

c) HPLC solution stability.

Built-in Robustness in Method procedure:

• Weighing error.

• Dilution error.

• Mobile phase as sample solvent.

• Buffer.

7.3 Does the study require any investigations of interventions to be conducted on patients or other human or animals? If so please describe briefly?

• No.

7.4 Has ethical clearance been obtained from your institute in case of as above?

• Not applicable

c) LIST OF REFERENCES

1. Ermer j, Miller J. Method Validation in Pharmaceutical Analysis. Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA: 2005.

2. Arayne MS, Sultana N, Zuberi MH. Development and validation of RP-HPLC method for the analysis of metformin. Pak J Pharm Sci 2006 Jul; 19(3):231-5.

3. Rajesh T, Lakshmi KS. Development and Validation of RP-HPLC Method for Simultaneous Determination of Glipizide, Rosiglitazone, Pioglitazone, Glibenclamide and Glimepiride in Pharmaceutical Dosage Forms and Human Plasma. J Iran Chem 2011 Mar; 8(1):31-37.

4. Onal A. Spectrophotometric and HPLC determinations of anti-diabetic drugs, Rosiglitazone maleate and Metformin hydrochloride, in pure form and in pharmaceutical preparations. Eur J Med Chem 2009 Dec; 44(12):4998-5005.

5. Reddy B, Boopathy D, Mathew B, Prakash M, Perumal P. Method development and validation of simultaneous determination of pioglitazone and glimepiride in pharmaceutical dosage form by RP-HPLC. Pak J Pharm Sci 2008 Oct; 21(4):421-5.

6. Havaldar F, Vairal D. Simultaneous Estimation of Glimepiride, Rosiglitazone and Pioglitazone Hydrochloride in the Pharmaceutical Dosage Form. E-J Chem; 7(4), 1326-1333.

7. Harika SL, Reddy R, Shetty S, Teja S, Teja S, et al. Estimation of glibenclamide present in tablets by RP-HPLC. Int J Pharm Tech 2010 Dec; 2(4).

8. Mallu U, Pyreddy V, Penumajji S, Bobbarala V. Single and high resolution RP-HPLC method for the determination of six anti diabetic drug products. J Pharm Res 2011; 4(4):1209-1212.

9. Patil Sudarshan S, Bonde C. Development and Validation of analytical method for

Simultaneous Estimation of Glibenclamide and Metformin HCl in Bulk and Tablets using UV – visible spectroscopy. Int J Chem Tech Res 2009; 1(4):905-909.

10. Altinoz S, Tekeli D. Analysis of glimepiride by using derivative UV Spectrophotometric method. J Pharm Biomed Anal 2001; 24:507-515.

11. Hamdan I.I, Jaber B, Abushoffa A. Development and validation of a stability indicating capillary electrophoresis method for the determination of Metformin -hydrochloride in tablets. J Pharm Biomed Anal 2010; 53:1254-1257.

12. Ruzilawati AB, Wahab MS, Imran A, Ismail Z, Gan SH. Method development and validation of Repaglinide in human plasma by HPLC and its application in pharmacokinetic studies. J Pharm Biomed Anal 2007 Apr 11; 43(5):1831-5.

13. ICH Harmonized Tripartite Guideline ICH Q2(R1), Impurities in New Drug Products, Nov 2005. Available from (Retrieved on 2011 May 10th: 18:10).

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