SUMMARY AND CONCLUSION

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SUMMARY

The work presented in the thesis was aimed to develop and validate analytical and

bioanalytical methods for the determination of drugs from the dosage forms and

biological matrices respectively using High Performance Liquid chromatography

(HPLC) and Liquid Chromatography coupled to mass spectrometry (LC-MS/MS).

The thesis consists of preface and 11 chapters.

In Chapter I, a general introduction about the drugs, types of analytical techniques,

HPLC technique & LC-MS/MS techniques, method development approach and

method validation for analytical and bioanalytical methods are discussed.

In Chapter II and III development and validation of analytical methods for the

determination of Varenicline and Dipyridamole & Aspirin in pharmaceutical dosage

forms using HPLC were discussed.

In Chapter IV and V development and validation of bioanalytical methods for the

determination of Theophylline and Ranitidine in human plasma using HPLC were

discussed.

In Chapter VI to XI development and validation of bioanalytical methods for the

determination of Imatinib, Azithromycin, Donepezil, Gliclazide, Zileuton and

Deferasirox in human plasma using LC-MS/MS were discussed.

CONCLUSION

The present thesis compiled with the development and validation of analytical and

bioanalytical methods for the selected drugs demonstrate the applicability for routine

sample analysis during quality control and bioequivalence/pharmacokinetic studies

respectively. The developed and validated methods are novel for some of the drugs

like Varenicline, Dipyridamole & Aspirin combination, Zileuton and Deferasirox. For

other drugs, the methods have improvements when compared to reported methods.

For bioanalytical methods, both HPLC and LC-MS/MS techniques are used and even

though both the techniques are suitable, LC-MS/MS technique have the advantage

due to more selective and sensitive determination. In LC-MS/MS technique, power of

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separation of liquid chromatography and supremacy of selective detection of mass

spectrometry are combined for the selective determination of intended drug from the

biological matrices. LC-MS/MS technique also have the advantage of shorter runtime.

From the developed bioanalytical methods, sensitivity of around 10 ng/ml was

achieved with HPLC and with LC-MS/MS sensitivity of around 50 pg/ml was

achieved with good reproducibility. Even though sensitivity of around 10ng/ml was

achieved with HPLC, it was required more sample cleanup and more effective

processing using selective solid phase extraction cartridges leading to increase in the

cost of the processing activity. For the LC-MS/MS methods, simpler and cost

effective extraction procedures like protein precipitation and liquid-liquid extraction

are used and application of these simpler extraction procedures are cost effective for

the routine application.

For bioanalytical methods, internal standard usage is recommended to control the

processing variations. Usage of labeled or structural analogue internal standards is

preferable to match the properties of the analyte and with the usage of such internal

standards matrix effects will be controlled. For the developed bioanalytical methods

labeled internal standards are used for Imatinib, Donepezil, Zileuton, Deferasirox

methods and structural analogue internal standards are used for Theophylline and

Azithromycin methods. Similar therapeutic category drugs are used as internal

standards for Ranitidine and Gliclazide methods. Usage of internal standards other

than labeled and structural analogues led to the usage of more effective and costlier

processing method of solid phase extraction.

Bioanalytical methods are developed with the limit of quantification and linearity

range suitable for bioavailability/bioequivalence and pharmacokinetic studies.

Generally bioequivalence studies are performed using higher available dose of the

drugs. Both analytical and bioanalytical methods are validated in compliance to

international regulatory guidelines and have shown high degree of selectivity,

sensitivity, reproducibility and stability.

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Characteristics of analytical and bioanalytical methods:

Drug Analytical

technique Matrix Extraction method Internal Standard LOQ

Linearity / Calibration

curve range Application

Analytical methods

Varenicline

HPLC Dosage

form Aqueous extraction Not Applicable

Not Applicable 2.5 to 7.5 µg/ml

For Quality Control Dipyridamole

and Aspirin Not Applicable

Dipyridamole:

4 to 80 µg/ml

Aspirin:

0.5 to 10 µg/ml

Bioanalytical methods

Theophylline HPLC

Human

Plasma

Protein precipitation β-hydroxyethyl

theophylline 0.251µg/ml 0.251 to 16.071 µg/ml

For Bioequivalence/

Pharmacokinetic

studies

Ranitidine Solid phase extraction Nizatidine 10.0 ng/ml 10.0 to 1000.5 ng/ml

Imatinib

LC-MS/MS

Protein precipitation Imatinib D8 10.0 ng/ml 10.0 to 4000.1 ng/ml

Azithromycin Liquid-liquid extraction Azithromycin

N-ethyl 5.01 ng/ml 5.01 to 602.25 ng/ml

Donepezil Liquid-liquid extraction Donepezil D7 50.1 pg/ml 50.1 to 25052.5 pg/ml

Gliclazide Solid phase extraction Tolazamide 10.3 ng/ml 10.3 to 2055.2 ng/ml

Zileuton Liquid-liquid extraction Zileuton D4 50.5 ng/ml 50.5 to 10012.7 ng/ml

Deferasirox Liquid-liquid extraction Deferasirox D4 0.201 µg/ml 0.201 to 25.229 µg/ml