Report of Ph.D.

THESIS entitled “METHOD DEVELOPMENT & VALIDATION

OF SOME PHARMACEUTICALS BY MODERN ANALYTICAL TECHNIQUES”
By HEMANTKUMAR HIRALAL GADAPE

The thesis is divided in different 10 chapters as follows:

Chapter 1: Introduction
This chapter provides the general information to pharmaceuticals, analytical Chemistry
and broad range of modern analytical techniques with their applications. This information is
useful to choose the most appropriate analytical technique for a particular purpose.

Chapter 2: Modern analytical techniques - NMR and HPLC

More than any other analytical method the NMR spectroscopy provides information
about the chemical structure and the dynamics of organic molecules. The interpretation of the
NMR data depends on a minimum amount of basic information that will be provided in this
chapter. NMR Instrumentation, a practical aspect of the NMR operation, important NMR
parameters and various NMR experiments for structure characterization is discussed.
Chromatographic technique HPLC is also explained briefly at the end of this chapter.

Chapter 3: Quantitative NMR and method validation

NMR spectroscopy is by definition a quantitative spectroscopic tool because the
intensity of a resonance line is directly proportional to the number of resonant nuclei (spins).
This fact enables accurate and precise determinations of the amount of substance. The method
validation of quantitative NMR (qNMR) is discussed on the terms of various parameters like
specificity & selectivity, precision & intermediate precision, linearity, LOD & LOQ, Range,
Accuracy, Stability of analyte in solution and robustness study. With the increase of
sensitivity due to stronger and stronger static magnetic fields, quantitative NMR can be
widely used in various fields of applications.

Chapter 4: Quantification of Pioglitazone Hydrochloride

Rapid, specific and accurate proton nuclear magnetic resonance spectroscopy ( 1H-
NMR) method was developed to determine Pioglitazone antidiabetic drug in pharmaceutical
tablet formulation. The method was based on quantitative NMR spectroscopy (qNMR) using
Maleic acid as an internal standard and deuterated dimethylsulfoxide (DMSO-d6) as an NMR
solvent. For the quantification of the drug, the 1H-NMR signals at 7.95 ppm and 6.26 ppm
corresponding to the analyte proton of Pioglitazone and maleic acid internal reference
standard (IS) respectively were used. The method was validated for the parameters of
specificity, precision, intermediate precision, linearity, range, and accuracy, limit of detection
(LOD), limit of quantification (LOQ), solution stability and robustness. The linearity of the
calibration curve for analyte in the desired concentration range was good (R 2 = 0.9983). The
method was accurate and precise with good recoveries. Range study was also performed up to
saturation level (226.93 mg/0.60 mL) in DMSO-d6. The advantage of the method is that no
reference standard is required for quantification. The method is nondestructive and can be
applied for quantification of Pioglitazone in commercial formulation products.

Chapter 5: Quantification of Metformin Hydrochloride

Rapid, specific and accurate proton nuclear magnetic resonance spectroscopy ( 1H-
NMR) method was developed to determine Metformin hydrochloride antidiabetic drug in
pharmaceutical tablet formulation. The method was based on quantitative NMR
spectroscopy (qNMR) using Maleic acid as an internal standard and deuterium oxide
(D2O) as a diluent. For the quantification of the drug, the 1H-NMR signals at 2.91 ppm
and 6.25 ppm corresponding to the analyte proton of Metformin hydrochloride and
Maleic acid internal reference standard (IS) respectively were used. The method was
validated for the parameters of specificity, precision, intermediate precision, linearity,
range, limit of detection (LOD), limit of quantification (LOQ), accuracy, solution stability
and robustness. The linearity of the calibration curve for analyte in the desired
concentration range was good (R2 = 0.9993). The method was accurate and precise with
good recoveries. Range study was also performed up to saturation level (152.67 mg/0.60
mL) in D2O. The advantage of the method is that no reference standard is required for
quantification. The method is nondestructive and can be applied for quantification of
Metformin hydrochloride in commercial formulation products.

Chapter 6: Quantification of Nateglinide

Rapid, specific and accurate proton nuclear magnetic resonance spectroscopy ( 1H-
NMR) method was developed to determine Nateglinide antidiabetic drug in
pharmaceutical tablet formulation. The method was based on quantitative NMR
spectroscopy (qNMR) using Maleic acid as an internal standard and deuterated dimethyl
sulfoxide (DMSO-d6) as an NMR solvent. For the quantification of the drug, the 1H-
NMR signals at 7.17-7.27 ppm (multiplet) and 6.26 ppm (singlet) corresponding to the
analyte proton of Nateglinide and Maleic acid internal reference standard (IS)
respectively were used. The method was validated for the parameters of specificity,
precision, intermediate precision, linearity, and range, limit of detection (LOD), limit of
quantification (LOQ), accuracy, solution stability and robustness. The linearity of the
calibration curve for analyte in the desired concentration range was good (R 2 = 0.9991).
The method was accurate and precise with good recoveries. Range study was also
performed up to saturation level (224.46 mg/0.60 mL) in DMSO-d6. The advantage of the
method is that no reference standard of analyte drug is required for quantification. The
method is nondestructive and can be applied for quantification of Nateglinide in
commercial formulation products.

Chapter 7: Quantification of Irbesartan

Rapid, specific and accurate proton nuclear magnetic resonance spectroscopy ( 1H-
NMR) method was developed to determine Irbesartan antihypertensive drug in
pharmaceutical tablet formulation. The method was based on quantitative NMR
spectroscopy (qNMR) using Maleic acid as an internal standard and deuterated dimethyl
sulfoxide (DMSO-d6) as an NMR solvent. For the quantification of the drug, the 1H-
NMR signals at 7.08 ppm and 6.25 ppm corresponding to the analyte proton of Irbesartan
drug and Maleic acid internal reference standard (IS) respectively were used. The method
was validated for the parameters of specificity, precision, intermediate precision,
linearity, and range, limit of detection (LOD), limit of quantification (LOQ), accuracy,
solution stability and robustness. The linearity of the calibration curve for analyte in the
desired concentration range was good (R2 = 0.9992). The method was accurate and
precise with good recoveries. Range study was also performed up to saturation level
(90.96 mg/0.60 mL) in DMSO-d6. The advantage of the method is that no reference
standard of analyte drug is required for quantification. The method is fast, sensitive,

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precise, accurate, and nondestructive and can be applied for quantification of Irbesartan in
commercial formulation products.

Chapter 8: Quantification of Carvedilol

New proton nuclear magnetic resonance spectroscopy (1H-NMR) method was
developed to determine Carvedilol antihypertensive drug in pharmaceutical tablet
formulation. The method was based on quantitative NMR spectroscopy (qNMR) using
Maleic acid as an internal standard and deuterated dimethyl sulfoxide (DMSO-d6) as an
NMR solvent. For the quantification of the drug, the 1H-NMR signals at 8.20 ppm and
6.25 ppm corresponding to the analyte proton of Carvedilol and Maleic acid internal
reference standard (IS) respectively were used. The method was validated for the
parameters of specificity, precision, intermediate precision, linearity, and range, limit of
detection (LOD), limit of quantification (LOQ), accuracy, solution stability and
robustness. The linearity of the calibration curve for analyte in the desired concentration
range was good (R2 > 0.9980). The method was accurate and precise with good
recoveries. Range study was also performed up to saturation level (338.24 mg/0.60 mL)
in DMSO-d6. The advantage of the method is that no reference standard of analyte drug
is required for quantification. The method is nondestructive and can be applied for
quantification of Carvedilol in commercial formulation products.

Chapter 9: Quantification of Nifedipine:

Rapid, specific and accurate proton nuclear magnetic resonance spectroscopy ( 1H-
NMR) method was developed to determine Nifedipine antihypertensive drug in
pharmaceutical tablet formulation. The method was based on quantitative NMR
spectroscopy (qNMR) using Maleic acid as an internal standard and deuterated dimethyl
sulfoxide (DMSO-d6) as an NMR solvent. For the quantification of the drug, the 1H-
NMR signals at 7.66-7.69 ppm and 6.25 ppm corresponding to the analyte proton of
Nifedipine and Maleic acid internal reference standard (IS) respectively were used. The
method was validated for the parameters of specificity, precision, intermediate precision,
linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy,
solution stability and robustness. The linearity of the calibration curve for analyte in the
desired concentration range was good (R2 = 0.9995). The method was accurate and
precise with good recoveries. Range study was also performed up to saturation level
(197.91 mg/0.60 mL) in DMSO-d6. The advantage of the method is that no reference
standard of analyte drug is required for quantification. The method is nondestructive and
can be applied for quantification of Nifedipine in commercial formulation products.

Chapter 10: Conclusion

The qNMR method employed herein proved to be rapid as well as easy to
implement. The different aspects of performance of the method, such as linearity,
precision and accuracy satisfied our requirements well. It offers an excellent choice over
previously described procedures and can be used for routine quality control and stability
analysis of various pharmaceutical drugs in solid dosage forms. Assay results obtained by
qNMR were confirmed by comparing with in-house HPLC method. Furthermore, any
modern NMR equipment operating at a field of 300MHz or more may be used, assuming
that suitable processing of data is performed. qNMR has a high potential in analysis of
pharmaceutical products due to the simplicity, reliability, simultaneous identification and
quantification, and the fact that no reference compound of drug is needed.

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 On basis of work done by the candidate, I strongly recommend to award the
degree of Ph.D. of Hemchandracharya North Gujarat University in faculty of
Science (Chemistry subject) to Mr. HEMANTKUMAR HIRALAL GADAPE.