4.A.1 Discussion. - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/76847/11/11_chapter-4.pdfby...

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4.A.1 Discussion.

Assay method development and Validation of Ziprasidone Hydrochloride

4.A.1.1. HPLC method development and optimization

In the present study the main target for the development of chromatographic method was to get

the reliable method for the quantification of Ziprasidone hydrochloride from bulk drug and can

be applied for the degradable products ,as very few researcher as carried and reported

systematic study of HPLC and UV methods for estimation of Ziprasidone Hydrochloride

Monohydrate which were sophisticated, but time consuming. Thus ,the present study was aimed

for development of speedy and cost effective HPLC technique for determination of Ziprasidone

Hydrochloride Monohydrate as bulk and in dosage forms. The chromatographic method was

optimized by changing various parameters, such as the mobile phase composition , pH of the

buffer used in the mobile phase. Retention time and separation of peak of Ziprasidone

Hydrochloride Monohydrate were dependent on pH of the buffer and the percentage of

methanol. Various blends of solvent systems in varying proportions were tried as mobile phase

by G.Srinubabu,1-3

Different mobile phases were tried, but satisfactory separation and good

symmetrical peak were obtained with mobile phases having triethylamine buffer PH = 3.0 with

orthophosphuric acid .530 ml of buffer mixed with 150 ml acetonitrile and 320 ml of

methanol,the volume was 53:15:32,v/v/v ,it was filtered and degased . and Column used YMC

pack C18, 250 x 4.6 mm, 5µm found to comparatively better and gave the graph with better

gaussian shape at retention time 9.80 min .

4.A.1.2. Method validation

Validation of an analytical method is the process by which it is established by laboratory studies,

that the performance characteristics of the method meet the requirements for the intended

analytical application.4

Validation is required for any new or amended method to ensure that it is

capable of giving reproducible and reliable results, when used by different operators employing

the same equipment in the same or different laboratories. The type of validation programme

required depends entirely on the particular method and its proposed applications. The proposed

analytical method was validated for the parameters such as linearity, precision, accuracy,

specificity, limit of quantification [LOQ], limit of detection [LOD] ,Forced degradation and

robustness.5-7

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4.A.1.3. System precision:

Precision is the measure of the degree of repeatability of an analytical method under normal

operation and is normally expressed as the percent relative standard deviation for a statistically

significant number of samples. The two most common precision measures are 'repeatability' and

'reproducibility'.These are expression of two extreme measure of precision which can be

obtained.

In the present finding Standard solution of Ziprasidone Hydrochloride Monohydrate were

prepared as per testing procedure and injected into the HPLC system in six replicates. The values

of % relative standard deviation (NMT 2.0%) for peak area obtained in six replicate injections

were reported in Table-3.A.1.1. thus showing that the equipment used for the study worked

correctly for the developed analytical method, and being highly repetitive 8.

Figure -4.A.1. A chromatogram of the Ziprasidone Hydrochloride standard

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4.A.1.4. Linearity

A linear study identifies a specified concentration range where analytes response is linearly

proportional to the concentration. The present study reveals that standard curve found to be

linear over the concentration range of 1000-3000 ppm. The results are depicted in Table 3.A.1.2

of chapter -3. The equation of the standard curve relating the peak area to the Ziprasidone

concentration in this range was y = 480x + 560. The drug showed good linearity in the range of

1000-3000 ppm with coefficient of correlation value 0.999 for peak area (Table-3.A.1.2)

Figure-4. A.2. Linearity graph of Ziprasidone HCl

y = 480x + 560

R² = 0.993

0

500

1000

1500

2000

2500

3000

3500

0 1 2 3 4 5 6

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4.A.1.5.Accuracy

Accuracy is popularly used to describe the measure of exactness of an analytical method, or the

close of an agreement between the value, which is accepted as a conventional, true value or as an

accepted reference value, and the value found.It is properly a qualitative concept and the correct

term is 'bias'.The bias of a method is an expression of how close the mean of asset of results

(produced by the method) is to the true value. Bias is usually determined by study of relevant

reference materials or by spiking studies 9

Accuracy , sometimes also referred to as recovery is an indicator of the trueness of the test

measurements. To determine the accuracy of the method three quality control samples were used.

The samples chosen were such to represent the entire range of the standard curve i e lower,

middle and higher concentration of the range.10

In the present finding recovery studies were

conducted after addition of standard drug solution at three different levels i.e. 50 %, 100 %, and

150 % to pre-analyzed sample solution and to check the accuracy of the method, Similar work

as been also reported by Chudasama, 11-12

. The results of the finding are reported in chapter 3

and in Table-3.A.1.4.

4.A.1.6. Robustness

The robustness of an analytical procedure refers to its capability to remain unaffected by small

and deliberate variations in method parameters. 13-14

The percentage recovery of Ziprasidone Hydrochloride Monohydrate was good under most

conditions and didn’t show any significant change when the critical parameters were modified.

The tailing factor for Ziprasidone Hydrochloride Monohydrate was always less than 2.0 and it

was well separated under all the changes carried out. Considering the modifications in the system

suitability parameters and the specificity of the method it can conclude that the method is robust.

Similar result for other drugs were reported by Ahmed et.al 15

4.A.1.7. Forced degradation :

Forced degradation studies are used to facilitate the development of analytical methodology, to

gain a better understanding of active pharmaceutical ingredient (API) and drug product (DP)

stability, and to provide information about degradation pathways and degradation products16

.

In present finding Forced degradation studies of Ziprasidone Hydrochloride Monohydrate were

carried under different stress condition such as acid, alkali , peroxide ,thermal and water

according to ICH guideline shown in table- 3.A.1.10 .22.97% degradation observed in 1.0 N

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HCl. The peaks of the degradation products were well resolved.17

The degradation study showed

that Ziprasidone Hydrochloride was stable to chemical oxidation study, dry heat and alkali

hydrolysis where as it was highly susceptible to acid hydrolysis .

Figure -4. A.3.A chromatogram of the Ziprasidone Hydrochloride alkali

degradation

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4.A.2. Assay method development and Validation of Duloxetine Hydrochloride.

4.A.2.1 HPLC method development and optimization

In present investigation the main target was for the development of chromatographic method to

get the reliable method for the quantification of Duloxetine hydrochloride from bulk drug and

which is applicable for the degradable products, for that number of systematic trials were

performed to optimize the chromatographic conditions for developing a sensitive, precise and

accurate RP-HPLC method for the analysis of Duloxetine HCl in pharmaceutical dosage forms.

Various blends of solvent systems in varying proportions were tried as mobile phase by O’Neil

and others 18-19

The present method contains mobile phase 0.1% Orthophosphuric acid buffer as

A and acetonitrile as mobile phase B , gradient program mention in expermential details and

column used Kromasil C18, 250 x 4.6 mm, 5µm found to comparatively better and gave the

graph with better gaussian shape at retention time 17.72 min.

4.A.2.2 Method validation

The validation of analytical procedures is based on the four most common types of analytical

procedures:- Identification tests, Quantitative tests for impurities' content,Limit tests for the

control of impurities, Quantitative tests of the active moiety in samples of drug substance or drug

product or other selected component(s) in the drug product 20

In the present study the developed method was validated, as described below, for various

parameters like linearity and range, accuracy, precision, ruggedness, system suitability,

specificity, LOQ,and LOD.

4.A.2.3 Precision

The precision of an analytical procedure expresses the closeness of agreement (degree

of scatter) between a series of measurements which is obtained from multiple sampling of the

same homogeneous sample under the prescribed conditions. Precision may be considered at three

levels: repeatability,intermediate precision and reproducibility.

Precision should be investigated using homogeneous, authentic samples. However, if it

is not possible to obtain a homogeneous sample, it may be investigated using artificially prepared

samples or a sample solution. The precision of an analytical procedure is usually expressed as the

variance, standard deviation or coefficient of variation of a series of measurements 21

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In our study instrument precision was determined by performing repeatability test and the %RSD

(NMT 2.0%) values for Doluxtine hydrochloride and are reported in Chapter 3, Table-3.A.2.2

Figure -3.A.4. A chromatogram of the Doluxetine Hydrochloride standard

4.A.2.4. LOD and LOQ

The Quantitation limit of an individual analytical procedure is the lowest amount of analyte in a

sample which can be quantitatively determined with suitable precision and accuracy. The

Quantitation limit is a parameter of quantitative assays for low levels of compounds in sample

matrices and is used particularly for the determination of impurities or degradation products. 21

The detection limit of an individual analytical procedure is the lowest amount of analyte in a

sample which can be detected but not necessarily quantitated as an exact value.21

LOD and LOQ

of the drug were calculated using the following equations according to International Conference

on Harmonization (ICH) guidelines. 22

LOD = 3.3 × /S LOQ = 10 × /S where = the

standard deviation of the response and S = the slope of the regression equation and were found to

be 0.026 and 0.078 µg/ml.(Table-3.A.2.1). The present study reveals that the method is sensitive

for the determination of Doluxtine hydrochloride.

4.A.2.5. Linearity and Range

Linearity is the method's ability to obtain results which are either directly, or after mathematical

transformation proportional to the concentration of the analyte within a given range. The range of

the method is the interval between the upper and lower levels of an analyte that have been

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determined with acceptable precision, accuracy and linearity. It is determined by calculating the

regression line using a mathematical treatment of the results (ie least mean squares) vs analyte

concentration. . 23

In present study linearity of the method was evaluated at five concentration levels by diluting the

standard stock solution. The calibration curve for Doluxetine hydrochloride was prepared by

plotting area v/s concentration. Calibration data for Doluxtine hydrochloride are given in Table

Table-3.A.2.3. The linearity plot of Doluxetine hydrochloride was found to be linear and

correlation coefficient 1.000 for Doluxetine hydrochloride i.e y = 747.3 x + 1412 .The present

study reveals that linearity observed were in the expected concentration range, demonstrating

suitability of the method for analysis, which may be attributed to the method linear in the

specified range for the analysis of Doluxtine hydrochloride .Our findings are in good aggrement

with the earlier reported work on other drugs24

Figure-4. A.5. Linearity graph of Duloxetine Hydrochloride

y = 747.3x + 1412.

R² = 1

0

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1500000

2000000

2500000

0 500 1000 1500 2000 2500 3000 3500

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4.A.2.6. Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the value,

which is accepted either as a conventional true value or an accepted reference value and the

value found. This is sometimes termed trueness.



The samples chosen were such to represent the entire range of the standard curve i.e lower,

middle and higher concentration of the range 10

The recovery experiments were carried out by the standard addition method. The recoveries

obtained by the RPHPLC method for Doluxetine hydrochloride are depicted in Table 3.A.2.4.

The method was found to be accurate with % recovery 99% - 100.50% and found to be in

acceptable %RSD of not more than 2% at each level.

4.A.2.7. 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 usage.21.

The present study reveals that the method found to be robust, which may

be attributed to the small but deliberate changes in the method and low value of relative standard

deviation. These parameters have no detrimental effect on the method performance. Similar

results are also reported by different research during their study. 21

4.A.2.8. Forced degradation

Stress testing of the active substance can help to identify the likely degradation products, which

can in turn help to establish the degradation pathways and the intrinsic stability of the molecule

and validate the stability indicating power of the analytical procedures used. The nature of the

stress testing depends on the individual active substance and the type of Pharmaceutical product

involved.25

In our present investigation forced degradation study were carried out by subjecting

the drug to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photolytic

(sun light) conditions was carried. . The peaks of the degradation products were well resolved.

The study reveals that chromatograms of alkali degraded sample showed 19.55 % degradation in

0.2 N NaOH. The Doluxetine hydrochloride was found to be stable to rest of the conditions like

oxidative stress degradation, dry heat degradation and acid hydrolysis. Our findings showed that

304

Doluxtine hydrochloride was stable to chemical oxidation study, dry heat and alkali hydrolysis

while it was highly susceptible to alkali hydrolysis which is in good aggrement with the work

published earlier.26

Figure-4. A.6. A chromatogram of the Doluxetine Hydrochloride alkali degradation

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4.A.3. Assay method development and Validation of carmustine


In our present work the main target was for the development of chromatographic method was to

get the reliable method for the quantification of Carmustine from bulk drug and which will be

also applicable for the degradable products. Few HPLC and UV methods have been reported for

estimation of Carmustine which are sophisticated, but time consuming. The present study was

aimed at development of speedy and cost effective HPLC technique for determination of

Carmustine as bulk and in dosage forms. The chromatographic method was optimized by

changing various parameters, such as the mobile phase composition , pH of the buffer used in the

mobile phase. Retention time and separation of peak of Carmustine were dependent on pH of the

buffer and the percentage of methanol. Various blends of solvent systems in varying proportions

were tried as mobile phase by Nataranjan et al 27

. The present study reveals that during use of

different mobile phases 0.01 M Potassium dihydrogen phosphate at pH 3.2 with Orthophosphuric

acid and acetonitrile in the ratio of (70:30,v/v) and column temperature 30 °C showed better peak

shap, finally mobile phase filtered and degased. The Column used YMC ODS-A C18, 250 x 4.6

mm, 5µm found to comparatively better and gave the graph with better gaussian shape at

retention time 14.70 min min .


The Method was validated based on the International Conference on Harmonization (ICH)

Guidelines 28-30

. The method validation parameters checked were specificity, linearity,

accuracy, precision, limit of detection, limits of quantitation and robustness. The analytical

method was validated as per ICH guidelines with respect to parameters such as linearity,

precision, accuracy, specificity ,forced degradation and robustness.


The precision of an analytical method is the closeness of a series of individual measurements of

an analyte when the analytical preocedure is applied repeatedly to multiple aliquots of a single

homogeneous volume of biological matrix. The precision is calculated as coefficient of variation

(C. V.), i.e., relative standard deviation (RSD). The measure RSD can be subdivided into three

categories: repeatability (intra-day precision) and reproducibility (between laboratories

precision).31

306

In the present finding Standard solution of Carmustine were prepared as per testing procedure

and injected into the HPLC system in six replicates.The study reveals that the values of %

relative standard deviation 0.20 (NMT 2.0%) for peak area obtained in six replicate injections

indicates that the equipment used for the study worked correctly for the developed analytical

method, and being highly repetitive. It is in good agrrement with shinde et.al and others32-33.

The

results are reported in chapter 3, Table-3.A.3.1.

Figure -4.A.7. A chromatogram of the carmustine standard

4.A.3.4. Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain test results,

which are directly proportional to the concentration (amount) of analyte in the sample. 34-35

The

equation of the standard curve relating the peak area to the carmustine concentration were in the

range of y = 751x + 1252 .In our findings the drug showed good linearity in the range of 1000-

3000 ppm with coefficient of correlation value 1.0000 for peak area (Table-3.A.3.2). . The

standard curve found to be linear over the concentration range of 1000-3000 ppm.

307

Figure-4. A.8. Linearity graph of Carmustine

4.A.3.5. Accuracy


measurements. In our present study to determine the accuracy of the method by taking three

samples were used. The samples chosen were such that they represent the entire range of the

standard curve i.e lower, middle and higher concentration of the range.

Recovery studies are carried after addition of standard drug solution at three different levels i.e.

50 %, 100 %, and 150 % to pre-analyzed sample solution to varify the accuracy of method

similar study for different drugs was also carried by Susan et al and others 36-37

. The results are

given in table-3.A.3.3, 3.A.3.4 and 3.A.3.5.

4.A.3.6. Robustness

In the present findings the percentage recovery of Carmustine was good under most conditions

and didn’t show any significant change when the critical parameters were modified. The tailing

factor for Carmustine was always less than 2.0 and it was well separated under all the changes

carried out. The modifications made in the system suitability parameters and the specificity of

the method shows that the method is robust. Robustness parameter flow plus,flow minus,pH plus

and pH minus results are given in table-3.A.3.6, 3.A.3.7 and 3.A.3.8.

y = 751x + 1252.

R² = 1

0

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0 500 1000 1500 2000 2500 3000 3500

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In present investigation the forced degradation of Carmustine under different stress condition

such as acid, alkali , peroxide ,thermal and water according to ICH guidline shown in table-

3.A.3.10 .

21.55% degradation observed in 0.1 N NaOH. The peaks of the degradation products were well

resolved shown in fig-3A.17. The degradation study reveals that Carmustine was stable to

chemical oxidation study, dry heat and acid hydrolysis while it was highly susceptible to alkali

hydrolysis .

Figure-4. A.9. A chromatogram of the carmustine alkali degradation.

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4.A.4. Assay method development and Validation of Irinotecan Hydrochloride


In our present work the main target for the development of chromatographic method was to get

the reliable method for the quantification of Irinotecan hydrochloride from bulk drug and which

are also applicable for the degradable products. It was also observed that few HPLC and UV

methods have been reported for estimation of Irinotecan hydrochloride which are sophisticated,

but time consuming. The present study was aimed at development of speedy and cost effective

HPLC technique for determination of Irinotecan hydrochloride as bulk and in dosage forms. The

chromatographic method was optimized by changing various parameters, such as the mobile

phase composition, pH of the buffer used in the mobile phase. Retention time and separation of

peak of Irinotecan hydrochloride were dependent on pH of the buffer and the percentage of

methanol. HPLC study carried out by using various blends of solvent systems in varying

proportions were tried as mobile phase as reported earlier 41-42

Different mobile phases were

tried, but satisfactory separation and good symmetrical peak were obtained with the mobile

2.0gm Sodium dihydrogen phosphate and 1.0gm 1-Octane Sulphonic acid salt in 1000 ml 550 ml

buffer ,170 ml acetonitrile and 280 ml methanol mixed filtered and degased . The column

temperature was maintained at 45 °C have better peak shape. and Column used were Inertsil

ODS 3V C18(250X4.6)mm,5µ found to comparatively better and gave the graph with better

gaussian shape at retention time 13.33 min.


Method validation of an analytical procedure is to demonstrate suitability for intended purpose.

To meet current pharmaceutical regulatory guidelines i.e. USP , ICH and EP , a number of

criteria such as specificity, linearity, precision, accuracy, sensitivity and robustness are

determined investigated in order to validate analytical methods.5


Precision which was an important aspect of method validation were carried and used to ensure

adequate performance of the chromatographic system. Retention time (RT), number of

theoretical plates (N) and tailing factor (T) Standard solution of Irinotecan hydrochloride was

prepared as per testing procedure and injected into the HPLC system in six replicates.171

The

values of % relative standard deviation 0.14 (NMT 2.0%) for peak area obtained in six replicate

injections are reported in Table-3.A.4.1. In our finding the the low value of relative standard

310

deviation attributes that the method is robust.Similar results are also reported by different

research during their study.Thus showing that the equipment used for the study worked correctly

for the developed analytical method, and being highly repetitive .43

Figure -3.A.10. A chromatogram of the Irinotecan Hydrochloride standard

prepration.

4.A.4.4. Linearity

linearity is a highly successful parameter for method accuracy and defined as the method's

ability to obtain results which are either directly, or after mathematical

transformation proportional to the concentration of the analyte within a given range. It is

determined by calculating the regression line using a mathematical treatment of the results (i.e

least mean squares) vs analyte concentration.A linear study identifies a specified concentration

range where analytes response is linearly proportional to the concentration. The standard curve

found to be linear over the concentration range of 1000-3000 ppm. The equation of the standard

curve relating the peak area to the Irinotecan Hydrochloride concentration in this range was y =

813x + 1523 . The present study reveals that ,the drug showed good linearity in the range of

1000-3000 ppm with coefficient of correlation value 0.9999 for peak area (Table-3.A.4.2).

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Figure- 4. A.11. Linearity graph of Irnotecan Hydrochloride

4.A.4.5. Accuracy

Accuracy methods have been described as a measure of the closeness of test results obtained by

a method to the true value. Accuracy indicates the deviation between the mean value found and

the true value. lt is determined by applying the method to samples to which known amounts of

analyte have been added. It is analysed against standard and blank solutions to ensure that no

interference exists. The accuracy is then calculated from the test results as a percentage of the

analyte recovered by the assay. Accuracy ,it is sometimes also referred to as recovery is an

indicator of the trueness of the test measurements. In our present investigation to determine the

accuracy of the method three quality control samples were used. The samples chosen were such

to represent the entire range of the standard curve i.e. lower, middle and higher concentration of

the range To check the accuracy of the method, recovery studies were conducted after addition

of standard drug solution at three different levels i.e. 50 %, 100 %, and 150 % to pre-analyzed

sample solution. Our findigs good aggrement with the earlier reported work on other drugs by

Herben et al 44-46

. The results of the findings are given in table-3.A.4.3 3.A.4.4 and 3.A.4.5.

y = 813x + 1523.

R² = 1

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0 500 1000 1500 2000 2500 3000 3500

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4.A.4.6. Robustness

Robustness is the ability of a method to tolerate variations of parameters without significant

change in the result47

.In the present findings recovery of Irinotecan hydrochloride was good

under most conditions and didn’t show any significant change when the critical parameters were

modified. The tailing factor for Irinotecan hydrochloride was always less than 2.0 and it was well

separated under all the changes carried out.48-51

Considering the modifications in the system


Robustness parameter flow plus,flow minus, Temperature plus and Temperature minus results

are depicted in table-3.A.4.6, 3.A.4.7 and 3.A.4.8.


In present investigation the forced degradation of Irinotecan hydrochloride under different stress

condition such as acid, alkali , peroxide ,thermal and water according to ICH guidline reported

in table- 3.A.4.10 . The present study reveals that 15.88% degradation observed in 0.2 N NaOH.

Which is an indicative of development of new chromatographic analytical method to resolve the

problem. The chromatogram are shown in figure-4.A.8 .The peaks of the degradation products

were well resolved, shown in fig-3A.21 The degradation study thereby indicated that Irinotecan

hydrochloride were stable to chemical oxidation study, dry heat and alkali hydrolysis while it

was highly susceptible to alkali hydrolysis .Acid and thermal degradation study tried by Kono

et al and others 52-54

Figure -4. A.12. A chromatogram of the Irinotecan Hydrochloride alkali

degradation.

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4.A.5. Assay method development and Validation of Cyclophosphamide


In present investigation the main target for the development of chromatographic method was to

get the reliable method for the quantification of Cyclophosphamide from bulk drug and which

will be also applicable for the degradable products.55

It was also observed that few HPLC and

UV methods have been reported for estimation of Cyclophosphamide from plasma which are

sophisticated, but time consuming. The present study was aimed at development of speedy and

cost effective HPLC technique for determination of Cyclophosphamide as bulk and in dosage

forms. The chromatographic method was optimized by changing various parameters, such as the

mobile phase composition, pH of the buffer used in the mobile phase. Retention time and

separation of peak of Cyclophosphamide were dependent on pH of the buffer and the percentage

of methanol. Various blends of solvent systems in varying proportions were tried as mobile phase

by Alsarra et al and others 56-57

. Different mobile phases were tried, but satisfactory separation

and good symmetrical peak were obtained with the mobile phases containg 800 ml of water and

200 ml methanol mixed filtered and degased and column temperature was maintained at 25 °C

have better peak shape. The Column used Hypersil BDS C8, 250 x 4.6 mm, 5µm found to

comparatively better and gave the graph with a much better peak shape at retention time 6.21

min.


Validation is required for any new or amended method to ensure that it is capable of giving

reproducible and reliable results, when used by different operators employing the same

equipment in the same or different laboratories.The analytical method was validated as per ICH

guidelines with respect to parameters such as linearity, precision, accuracy, specificity, Forced

degradation and robustness.


"The precision of an analytical method is the degree of agreement among individual test results

obtained when the method is applied to multiple sampling of a homogenous sample. It is a

measure of the reproducibility of the whole analytical method (including sampling, sample

preparation and analysis) under normal operating circumstances. It can be determined by using

the method to assay a sample for a sufficient number of times to obtain statistically valid results

314

and is then expressed as the relative standard deviation. In the present study Standard solution of

Cyclophosphamide was prepared as per testing procedure and injected into the HPLC system in

six replicates. The values of % relative standard deviation 0.11 (NMT 2.0%) for peak area

obtained in six replicate injections are reported in Table-3.A.5.1. In our finding the the low value

of relative standard deviation attributes that the method is robust.Similar results are also reported

by different researchers Casale et al during their studyWhich is an indicative of the equipment

used for the study worked correctly for the developed analytical method, and being highly

repetitive. 58-59

Figure -3.A.13. A chromatogram of the Cyclophosphamide Standard

4.A.5.4. Linearity

Linearity is determined by calculating the regression line using a mathematical treatment of the

results (ie least mean squares) vs analyte concentration.

In our present investigation a linear study identifies a specified concentration range where

analytes response is linearly proportional to the concentration. The standard curve found to be

linear over the concentration range of 1000-3000 ppm. The equation of the standard curve

relating the peak area to the Cyclophosphamide concentration in this range was y = 699x + 1359

The drug showed good linearity in the range of 1000-3000 ppm with coefficient of correlation

value 0.9993 for peak area (Table-3.A.5.2). Our findings are in good aggrement with the work

published earlier by Compagnon et al 60

315

Figure : 4. A.14. Linearity graph of cyclophosphamide

4.A.5.5. Accuracy

Accuracy indicates the deviation between the mean value found and the true value. lt is

determined by applying the method to samples to which known amounts of analyte are added,

And are analysed against standard and blank solutions to ensure that no interference exists. The

accuracy is then calculated from the test results as a percentage of the analyte recovered by the

assay.. In the present study to determine the accuracy of the method three samples were used.

The samples chosen to represented the entire range of the standard curve i.e lower, middle and

higher concentration of the range. Recovery studies were also carried after addition of standard

drug solution at three different levels i.e. 50 %, 100 %, and 150 % to pre-analyzed sample

solution for conformation of methods accuracy61

. The results are given in table-3.A.5.3, 3.A.5.4

and 3.A.5.5

4.A.5.6. Robustness

"The robustness of an analytical procedure is a measure of its capacity to remain unaffected by


during normal usage".62

In present investigation the percentage recovery of Cyclophosphamide was good under most

conditions and didn’t showed no significant change when the critical parameters were modified.

y = 699x + 1359.

R² = 1

0

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0 500 1000 1500 2000 2500 3000 3500

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The tailing factor for Cyclophosphamide was always less than 2.0 and well separated under all

the changes carried out. Considering the modifications in the system suitability parameters and

the specificity of the method it can conclude that the method is robust. Robustness parameter

flow plus,flow minus, Temperature plus and Temperature minus results are given in table-

3.A.5.6, 3.A.5.7 , 3.A.5.8 and 3.A.5.9.


Analytical specificity of a method may be defined as the ability to measure accurately and

specifically the analyte in the presence of components that may be expected to be present in the

sample matrix.Specificity for an assay ensures that the signal measured comes from the

substance of interest, and that there is no interference from excipient degradation products and/or

impurities. 63

In our present study the forced degradation of Cyclophosphamide under different stress condition

such as acid, alkali , peroxide ,thermal and water according to ICH guideline are reported in

table- 3.A.5.10 . However this is the first time reported that 12.44% degradation observed in 0.1

N NaOH. Typical chromatogram show in figure-4.A.10 The peaks of the degradation products

were well resolved. The degradation study thereby indicated that Cyclophosphamide was stable

to chemical Our findings are in good aggrement with the work published earlier for different

drug by Arayne et al and others 64-68

oxidation study, dry heat and alkali hydrolysis while it was

highly susceptible to alkali hydrolysis .

Figure -4. A.15. A chromatogram of the Cyclophosphamide alkali degradation.

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4.A.6. Assay method development and Validation of Thiamine Hydrochloride



the reliable method for the quantification of Thiamine hydrochloride from bulk drug and which

will be also applicable for the degradable products. HPLC method by Thomas 69

and UV

methods have been reported for estimation of Thiamine hydrochloride which are sophisticated,

but time consuming. UV assay method reported by Yantih et al and others. 70-72

The present

study was aimed at development of speedy and cost effective HPLC technique for determination

of Thiamine hydrochloride as bulk and in dosage forms. The chromatographic method was


buffer used in the mobile phase. Retention time and separation of peak of Thiamine

hydrochloride were dependent on pH of the buffer and the percentage of methanol. Various

blends of solvent systems in varying proportions were tried as mobile phase 73

. Different mobile

phases were tried, but satisfactory separation and good symmetrical peak were obtained with the

mobile phases 0.01 M Ammonium acetate buffer adjust PH = 4.5 with acetic acid . 100 ml of

buffer ,600 ml acetonitrile and 300 ml of methanol mixed filtered and degased .The Column used

Water Symmetry C18(250X4.6),5 m found to comparatively better and gave the graph with

better gaussian shape at retention time 12.17 min.


Method validation of analytical procedures is based on the four most common types of analytical

procedures:- Identification tests,Quantitative tests for impurities' content,Limit tests for the


product or other selected component(s) in the drug product. 20

The analytical method was validated as per ICH guidelines with respect to parameters such as

linearity, precision, accuracy, specificity, forced degradation and robustness.


In the present findings Standard solution of Thiamine hydrochloride was prepared as per testing

procedure and injected into the HPLC system in six replicates. The values of % relative standard

deviation 0.08 (NMT 2.0%) for peak area obtained in six replicate injections are reported in

Table-3.A.6.1. thus showing that the equipment used for the study worked correctly for the

318

developed analytical method, and being highly repetitive. In our finding the low value of relative

standard deviation attributes that the method is robust.Similar results are also reported by

different researchers during their study Amidzic et al 74

.

Figure -3.A.16. A chromatogram of the Thiamine Hydrochloride standard

Prepration.

4.A.6.4. Linearity

Analytical linearity study identifies a specified concentration range where analytes response is

linearly proportional to the concentration. In our finding the standard curve found to be linear

over the concentration range of 1000-3000 ppm. The equation of the standard curve relating the

peak area to the Thiamine Hydrochloride concentration in this range was y = 733x+1389


value 0.9999 for peak area (Table-3.A.6.2). Although the initial results obtained in this study are

very promising.

Figure- 4. A.17.Linearity graph of Thiamine Hydrochloride

y = 733x + 1389.

R² = 1

0

500000

1000000

1500000

2000000

2500000

0 500 1000 1500 2000 2500 3000 3500

319

4.A.6.5. Accuracy




term is 'bias'. In present investigation to check the accuracy of the method, recovery studies were

carried after addition of standard drug solution at three different levels i.e. 50 %, 100 %, and 150

% to pre-analyzed sample solution for conformation of methods accuracy75

. The results are given

in table-3.A.6.3, 3.A.6.4 and 3.A.6.5. Hence this present study investigates the various abilities.

4.A.6.6. Robustness

In our finding the percentage recovery of Thiamine hydrochloride was good under most


The tailing factor for Thiamine hydrochloride was always less than 2.0 and it was well separated

under all the changes carried out. Considering the modifications in the system suitability

parameters and the specificity of the method it can conclude that the method is robust.


are given in table-3.A.6.6, 3.A.6.7 , 3.A.6.8 and 3.A.6.9. Our findings are in good aggrement

with the work published earlier by Ditjen et al 76-77


Selectivity is the ability to measure accurately and specifically the analyte in the presence of

components that may be expected to be present in the sample matrix.

In present investigation the forced degradation of Thiamine hydrochloride under different stress

condition such as acid, alkali , peroxide ,thermal and water according to ICH guideline shown in

table- 3.A.6.10 .However this present study investigates the various abilities ,In present

investigation 11.89% degradation observed in 0.2 N NaOH. Typical chromatogram of degredable

compound are shown in figure-4.A.12 The peaks of the degradation products were well resolved.

The degradation study thereby indicated that Thiamine hydrochloride was stable to chemical

oxidation study, dry heat and acid hydrolysis while it was highly susceptible to alkali hydrolysis

shown in figure. Our findings are in good aggrement with the work published earlier by Ibrahim

et al 78

320

Figure- 4. A.18.A chromatogram of the Thiamine Hydrochloride alkali degradation

321

4.A.7. Assay method development and Validation of Topotecan Hydrochloride


In present investigation the Topotecan is freely soluble in isopropyl alcohol. The drug can be

separated on a Inertsil ODS 3V C18(250X4.6)mm,5µ column in reverse phase mode The main

target for the development of chromatographic method was to get the reliable method for the

quantification of Topotecan hydrochloride from bulk drug and which will be also applicable for

the degradable products. We also observed a trend where few HPLC and UV methods have been

reported for estimation of Topotecan hydrochloride which are sophisticated, but time consuming.

The present study was aimed at development of speedy and cost effective HPLC technique for

determination of Topotecan hydrochloride as bulk and in dosage forms. The chromatographic

method was optimized by changing various parameters, such as the mobile phase composition ,

pH of the buffer used in the mobile phase. Retention time and separation of peak of Topotecan

hydrochloride were dependent on pH of the buffer and the percentage of methanol. Various

blends of solvent systems in varying proportions were tried as mobile phase by Hsiang et al and

others 79-80

. Different mobile phases were tried, but satisfactory separation and good symmetrical

peak were obtained with the mobile phases 0.02M Potassium dihydrogen phosphate buffer pH =

3.0 with orthophosphuric acid , 300 ml of buffer and 700 ml acetonitrile are mixed,filtered and

degassed.The column temperature were maintained at 35 °C and gave the graph with better

gaussian shape at retention time 13.26 min .


The objective of validation of an analytical procedure is to demonstrate that it is suitable for its

intended purpose. To meet current pharmaceutical regulatory guidelines i.e. USP , ICH and EP ,

a number of criteria such as specificity, linearity, precision, accuracy, sensitivity and robustness

must be investigated in order to validate analytical methods.


Precision was carefully tested ,System suitability test as an integral part of method development

was used to ensure adequate performance of the chromatographic system. Retention time (RT),

number of theoretical plates (N) and tailing factor (T) .Standard solution of Topotecan

hydrochloride was prepared as per testing procedure and injected into the HPLC system in six

replicates. The values of % relative standard deviation 0.10 (NMT 2.0%) for peak area obtained

322

in six replicate injections were reported in Table-3.A.7.1. In our finding the the low value of

relative standard deviation attributes that the method is robust.Similar results are also reported by

different research during their study Underberg et al 81

.The present study reveals that the

equipment used for the study worked correctly for the developed analytical method, and being

highly repetitive.

Figure -3.A.19. A chromatogram of the Topotecan Hydrochloride standard

prepration

4.A.7.4. Linearity

. Linearity is determined by calculating the regression line using a mathematical treatment of the

results (i.e least mean squares) vs analyte concentration. In the present findings a linear study

identifies a specified concentration range where analytes response is linearly proportional to the

concentration. The standard curve found to be linear over the concentration range of 1000-3000

ppm. The equation of the standard curve relating the peak area to the Topotecan Hydrochloride

concentration in this range was y = 812x+1544. This is an important observation. Therefore,in

this thesis, developments of new chromatographic analytical methods were established to solve

this problem. The drug showed good linearity in the range of 1000-3000 ppm with coefficient of

correlation value 0.9995 for peak area (Table-3.A.7.2).

323

Figure- 4. A.20.Linearity graph of Topotecan Hydrochloride

4.A.7.5. Accuracy

Analytical accuracy , sometimes also referred to as recovery is an indicator of the trueness of the

test measurements. To determine the accuracy of the method three quality control samples were

used. The samples chosen were such to represent the entire range of the standard curve i.e. lower,

middle and higher concentration of the range

In present investigation to check the accuracy of the method, recovery studies were conducted

after addition of standard drug solution at three different levels i.e. 50 %, 100 %, and 150 % to

pre-analyzed sample solution Hence these evidence support the hypothesis that result are good

aggriments with Pearson et al 82-83

. The results are given in table-3.A.7.3, 3.A.7.4 and 3.A.7.5

4.A.7.6. Robustness

The ICH guidelines also recommend that "one consequence of the evaluation of robustness

should be that a series of system suitability parameters (e.g. resolution tests) is established to

ensure that the validity of the analytical procedure is maintained whenever used".84

In the present findings the percentage recovery of Topotecan hydrochloride was good under most


The tailing factor for Topotecan hydrochloride was always less than 2.0 and it was well separated

y = 812x + 1544.

R² = 1

0

500000

1000000

1500000

2000000

2500000

0 500 1000 1500 2000 2500 3000 3500

324

under all the changes carried out.85-88

Considering the modifications in the system suitability



are given in table-3.A.7.6, 3.A.7.7 , 3.A.7.8 and 3.A.7.9.



components that may be expected to be present in the sample matrix.Our present work the forced

degradation of Topotecan hydrochloride under different stress condition such as acid, alkali ,

peroxide ,thermal and water according to ICH guidline shown in table- 3.A.7.10 .In present

findings 18.78% degradation observed in 0.5 N NaOH. Typical chromatogram of degredable

compound show in figure-4.A.14. The peaks of the degradation products were well resolved.89-92

The degradation study thereby indicated that Topotecan hydrochloride was stable to chemical

oxidation study, dry heat and alkali hydrolysis while it was highly susceptible to alkali hydrolysis

. These results facilitate the detection of that impurity in presence of the main drug by using the

developed method.

Figure- 4. A.21. A chromatogram of the Topotecan Hydrochloride alkali

degradation

325

4.A.8. Assay method development and Validation of Tenofovir Disoproxil fumarate



get the reliable method for the quantification of Tenofovir Disoproxil fumarate from bulk drug

and which will be also applicable for the degradable products. This is an important observation

few HPLC and UV methods have been reported for estimation of Tenofovir Disoproxil fumarate

which are sophisticated, but time consuming. The present study was aimed at development of

speedy and cost effective HPLC technique for determination of Tenofovir Disoproxil fumarate as

bulk and in dosage forms. The chromatographic method was optimized by changing various

parameters, such as the mobile phase composition , pH of the buffer used in the mobile phase.

Retention time and separation of peak of Tenofovir Disoproxil fumarate were dependent on pH

of the buffer and the percentage of methanol. Various blends of solvent systems in varying

proportions were tried as mobile phase by Sweetman et al 93-94

. Different mobile phases were

tried, but satisfactory separation and good symmetrical peak were obtained with the mobile

phases 0.01M Potassium dihydrogen phosphate buffer pH = 4.5 with orthophosphuric acid 900

ml buffer and 100 ml acetonitrile mixed, filtered anddegassed.The column temperature was

maintained at 25°C . Column used are Inertsil ODS 3V C18(150X4.6)mm,5µ found to

comparatively better and gave the graph with better gaussian shape at retention time 25.54 min .



Guidelines 28-30

. The method validation parameters checked were specificity, linearity, accuracy,

precision, limit of detection, limits of quantitation and robustness. The analytical method was

validated as per ICH guidelines with respect to parameters such as linearity, precision, accuracy,

specificity ,forced degradation and robustness


In present investigation Tenofovir Disoproxil fumarate was prepared as per testing procedure and

injected into the HPLC system in six replicates. The values of % relative standard deviation 0.14

(NMT 2.0%) for peak area obtained in six replicate injections are reported in Table-3.A.8.1. In

our finding the the low value of relative standard deviation attributes that the method is

robust.Similar results are also reported by different researchers during their study. 95

326

thus showing that the equipment used for the study worked correctly for the developed analytical

method, and being highly repetitive 96

.

Figure -4.A.22. A chromatogram of the tenofovir Desoproxill fumarate standard

4.A.8.4. Linearity

Linearity corresponds to the capacity of the method to supply results directly proportional to the

concentration of the substance being determined within a certain interval of concentration A

linear study identifies a specified concentration range where analytes response is linearly

proportional to the concentration. In present findings the standard curve found to be linear over

the concentration range of 1000-3000 ppm. The equation of the standard curve relating the peak

area to the tenofovir Desoproxill fumarate concentration in this range was y = 712x+1388.


value 1.0000 The linearity was evaluated by linear regression analysis calculated by the least

square method (Table-3.A.8.2). Our findings are in good aggrement with the earlier reported

work on other drugs by Gish et al. 97

327

Figure- 4. A.23. Linearity graph of Tenofovir Disoproxil Fumarate

4.A.8.5. Accuracy

Analytical accuracy of a method may be defined as , an indicator of the trueness of the test


The samples chosen were such to represent the entire range of the standard curve i.e. lower,

middle and higher concentration of the range 98

In the present findings to check the accuracy of the method, recovery studies were conducted


pre-analyzed sample solution. The results are given in table-3.A.8.3, 3.A.8.4 and 3.A.8.5

4.A.8.6. Robustness

In the present findings the percentage recovery of Tenofovir Disoproxil fumarate was good under

most conditions and didn’t show any significant change when the critical parameters were

modified. The tailing factor for Tenofovir Disoproxil fumarate was always less than 2.0 and it




are given in table-3.A.8.6, 3.A.8.7 , 3.A.8.8 and 3.A.8.9. 99

y = 712.8x + 1388.

R² = 1

0

500000

1000000

1500000

2000000

2500000

0 500 1000 1500 2000 2500 3000 3500

328


In our finding the forced degradation of Tenofovir Disoproxil fumarate under different stress

condition such as acid, alkali , peroxide ,thermal and water according to ICH guidline shown in

table- 3.A.1.10 . Our results have shown that 12.77% degradation observed in 0.2N M NaOH

and 0.1N M HCl shown 10.44% The peaks of the degradation products were well resolved.

Typical chromatogram of degredable compound show in figure-4.A.16. The degradation study

thereby indicated that Tenofovir Disoproxil fumarate was stable to chemical oxidation study, dry

heat and alkali hydrolysis while it was highly susceptible to acid and alkali hydrolysis. Our

findings are in good aggrement with the work published earlier by Mangoankar et al .100-101

Figure- 4. A.24. A chromatogram of the tenofovir Desoproxill fumarate acid

degradation

329

Figure- 4. A.25. A chromatogram of the tenofovir Desoproxill fumarate alkali

degradation

330

4.A.9. Assay method development and Validation of Atazanavir



the reliable method for the quantification of Atazanavir from bulk drug and which will be also

applicable for the degradable products. It was also observed that few HPLC and UV methods

have been reported for estimation of Atzanavir which are sophisticated, but time consuming. The

present study was aimed at development of speedy and cost effective HPLC technique for

determination of Atazanavir as bulk and in dosage forms. The chromatographic method was


buffer used in the mobile phase. Retention time and separation of peak of Atazanavir were

dependent on pH of the buffer and the percentage of methanol. Various blends of solvent systems

in varying proportions were tried as mobile phase by Cateau et al 102-103

. Different mobile phases

were tried, but satisfactory separation and good symmetrical peak were obtained with the mobile

phases 0.01M Potassium dihydrogen phosphate buffer pH = 3.2 with orthophosphuric acid .800

ml of buffer and 200 ml acetonitrile mixed filtered and degased. The column temperature was

maintained at 25°C . The Column used YMC ODS-A(150X4.6) mm, 3.5 found to

comparatively better and gave the graph with better gaussian shape at retention time 29.08 min .







Precision was carefully tested the precision of an analytical method is the degree of agreement

among individual test results obtained when the method is applied to multiple sampling of a

homogenous sample.Precision is a measure of the reproducibility of the whole analytical method

(including sampling, sample preparation and analysis) under normal operating circumstances.

Precision is determined by using the method to assay a sample for a sufficient number of times to

obtain statistically valid results . The precision is then expressed as the relative standard

deviation.

331

Standard solution of Atazanavir was prepared as per testing procedure and injected into the

HPLC system in six replicates. The values of % relative standard deviation 0.07 (NMT 2.0%) for

peak area obtained in six replicate injections were reported in Table-3.A.9.1. In our finding the

low value of relative standard deviation attributes that the method is robust.Similar results are

also reported by different research during their study thus showing that the equipment used for

the study worked correctly for the developed analytical method, and being highly repetitive

Loregian et al 104

.

Figure -4.A.26. A chromatogram of the atazanavir standard prepration

4.A.9.4. Linearity

Linearity is determined by calculating the regression line using a mathematical treatment of

the results (ie least mean squares) vs analyte concentration.In our present work a linear study

identifies a specified concentration range where analytes response is linearly proportional to the

concentration. In present findings standard curve found to be linear over the concentration range

of 1000-3000 ppm. The equation of the standard curve relating the peak area to the Atazanavir

concentration in this range was y = 1933x + 588.7 . The drug shown good linearity in the range

of 1000-3000 ppm with coefficient of correlation value 0.9996 for peak area (Table-3.A.9.2).

332

Figure- 4. A.27. Linearity graph of Atazanavir

4.A.9.5. Accuracy

Accuracy was measured as the percent of deviation from the nominal concentration Accuracy ,

sometimes also referred to as recovery is an indicator of the trueness of the test measurements.

To determine the accuracy of the method three quality control samples were used. The samples

chosen were such to represent the entire range of the standard curve i.e. lower, middle and higher

concentration of the range

It was sometimes also referred to as recovery is an indicator of the trueness of the test


The samples chosen were such to represent the entire range of the standard curve i e lower,

middle and higher concentration of the range. In the present finding recovery studies were

conducted after addition of standard drug solution at three different levels .i.e. 50 %, 100 %, and

150 % to pre-analyzed sample solution105

. The results are given in table-3.A.9,3, 3.A.9,4 and

3.A.9,5

4.A.9.6. Robustness


change in the result In the present findings the percentage recovery of Atzanavir was good under


y = 1933.x + 588.7

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

333

modified. The tailing factor for Atzanavir was always less than 2.0 and it was well separated




are given in table-3.A.9.6, 3.A.9.7 , 3.A.9.8 and 3.A.9.9.


In the present findings Different force degradation samples were analyzed and it was found that

the drug peaks in acid, alkali, peroxide, UV and photo degraded drug sample solutions have

passed the purity test. Purity angle for the selected drug components in all stress conditions were

found to be less than the threshold angle. This study confirms the specificity of the developed

method.The overall degradation in basic condition was found to be around 16.22% The peaks of

the degradation products were well resolved. Our findings are in good aggrement with the work

published earlier by Raymond et al 106-107

Typical chromatogram of degredable compound show

in figure-4.A.19.The degradation study thereby indicated that Atzanavir was stable to chemical

oxidation study, dry heat and alkali hydrolysis while it was highly susceptible to alkali

hydrolysis.

Figure- 4. A.28. A chromatogram of the Atazanavir alkali degradation.

334

4.B Discussion of Related substance method development and Validation

4.B.1 Related Substance method development and Validation of Carmustine

4.B.1.1. HPLC method development and optimization


get the reliable method for the quantification of Carmustine from bulk drug and which will be

also applicable for the degradable products. The elution gradient and influence of mobile phase

pH were studied in order to optimize the analytical performance. A short analytical column and

elution gradient ,buffer and organic modifier were chose as the best compromise between

retention time of analyte. This suggests that the few HPLC and UV methods have been reported

for estimation of Carmustine which are sophisticated, but time consuming. The present study

was aimed at development of speedy and cost effective HPLC technique for determination of

Carmustine as bulk and in dosage forms. The chromatographic method was optimized by


mobile phase. Retention time and separation of peak of Carmustine were dependent on pH of the


were tried as mobile phase by Kirtikar et al and others 108-109

. Different mobile phases were tried,

but satisfactory separation and good symmetrical peak were obtained with the mobile phases

0.01 M Potassium dihydrogen phosphate buffer PH = 3.2 with orthophosphuric acid as A and

Methanol as mobile phase B, gradient program mention in expermential details and column

temperature maintained at 30 °C. The Column used YMC ODS-A C18, 250 x 4.6 mm, 5µm

found to comparatively better and gave the graph with better gaussian shape at retention time

36.11min .

4.B.1.2. Method validation


Guidelines 28-30

. The method validation parameters checked were specificity, linearity, accuracy,

precision, limit of detection, limits of quantitation and robustness. The analytical method was

validated as per ICH guidelines with respect to parameters such as linearity, precision, accuracy,

specificity ,Forced degradation and robustness

335

4.B.1.3. LOD and LOQ

Analytical LOD of a method may be defined as the lowest concentration in a sample that can be

detected, but not necessarily quantitated, under the stated experimental conditions. The limit of

detection is important for impurity tests..LOQ is the lowest concentration of analyte in a sample

that can be determined with acceptable precision and accuracy.lt is quoted as the concentration

yielding a signal-to-noise ratio of 1 0: 1 and is confirmed by analyzing a number of samples near

this value. In the present findings these data show that the method is sensitive for the

determination of carmustine . The LOD and LOQ were measured by using an equation and were

found to be 0.1ppm. (Table-3.B.1.1)

4.B.1.4. System precision:





obtained.

In present investigation Standard solution of Carmustine was prepared as per testing procedure

and injected into the HPLC system in six replicates. The values of % relative standard deviation

1.94 (NMT 5.0%) for peak area obtained in six replicate injections were reported in Table-

3.B.1.2. In our finding the the low value of relative standard deviation attributes that the method

is robust.Similar results are also reported by different research during their study. Zuolian et al 110

thus showing that the equipment used for the study worked correctly for the developed

analytical method, and being highly repetitive .

336

Figure -4.B.1. A chromatogram of carmustine standard prepration

4.B.1.5. Linearity

Linearity is the ability of a method to elicit test results that are directly proportional to analyte

concentration within a given range. Range is the interval between the upper and lower levels of

analyte that have been demonstrated to be determined with precision, accuracy, and linearity

using the method as written. The accepted criteria for linearity is that the correlation coefficient

(R2) is not less than 0.999 for the least squares method of analysis of the line 111-112

In present investigation the linear study identifies a specified concentration range where analytes

response is linearly proportional to the concentration. The standard curve found to be linear over

the concentration range of 5.0-15.0 ppm. The equation of the standard curve relating the peak

area to the Carmustine concentration in this range was y = 4008x – 899 . The drug showed

good linearity in the range of 5.0-15.0 ppm with coefficient of correlation value 0.9996 for peak

area (Table-3.B.1.3). Therefore,in this thesis, developments of new chromatographic analytical

methods were established to solve this problem.

337

Figure -4.B.2. Linearity graph of Carmustine

Figure -4.B.3.Linearity graph of Impurity-A

y = 4008x 899

R² = 0.998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 2948x +2269

R² = 0.998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

338

4.B.1.6. Accuracy

Accuracy of a method is expressed as percentage of analyte recovered by spiking samples in

placebo of the drug formulation . To document accuracy, a minimum of nine determinations over

a minimum of three concentration levels covering the specified range (for example, three

concentrations, three replicates for each) were collected. It was performed at 50, 100, and 150%

levels of label claim. The RSD of the replicates provides the analysis variation and gives an

indication of the precision of the test method.

In our present investigation to check the accuracy of the method, recovery studies were

conducted after addition of standard drug solution at three different levels i.e. 50 %, 100 %, and

150 % to pre-analyzed sample solution214

. The results are given in table-3.B.1.4.

4.B.1.7. Robustness

The assessment of the robustness of a method is not required yet by the ICH guidelines, but it

can be expected that in the near future it will become obligatory. The definition for

robustness/ruggedness applied is "The robustness/ruggedness 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 usage" .

In our present finding the percentage recovery of Carmustine was good under most conditions


factor for Carmustine was always less than 2.0 and it was well separated under all the changes

carried out. Considering the modifications in the system suitability parameters and the specificity

of the method it can conclude that the method is robust. Our findigs good aggrement with the

earlier reported work on other drugs by Yang et al 113

4.B.1.8. Forced degradation :

Specificity is the ability to assess unequivocally the analyte in the presence of components that

may be expected to be present, such as impurities, degradation products, and matrix components

114-116. It is a measure of the degree of interferences from such components, ensuring that a peak

response is due to a single component only. Specificity is measured and documented in a

separation by the resolution, plate count (efficiency), and tailing factor. Specificity of the current

method. In present investigation the forced degradation of Carmustine under different stress


339

table- 3.B.1.9. In present findings have shown that 23.19% degradation observed in 0.2N NaOH,

and 12.78 % degradation observed in 0.5 N HCl , UV degradation 10.78% and thermal

degradation 11.56 % were observed,the peaks of the degradation products were well resolved.

Typical chromatogram of degredable compound shown in figure-4.B.3, 4.B.4 4.B.5 .The

degradation study thereby indicated that Carmustine was stable to chemical oxidation study and

water while it was highly susceptible to alkali hydrolysis . Our findings are in good aggrement

with the work published earlier by Mahmood et al and others. 117-118

Figure -4.B.4. A chromatogram of the Carmustine alkali degradation

Figure -4.B.5. A chromatogram of the Carmustine acid degradation

340

Figure -4.B.6. A chromatogram of the Carmustine Thermal degradation

341

4.B.2. Related Substance method development and Validation of Irinotecan

Hydrochloride


In our present work the elution gradient and influence of mobile phase pH were studied in order

to optimize the analytical performance. A short analytical column and elution gradient ,buffer

and organic modifier were chose as the best compromise between retention time of analyteThe

main target for the development of chromatographic method was to get the reliable method for

the quantification of Irinotecan hydrochloride from bulk drug and which will be also applicable

for the degradable products. We also observed a trend where Few HPLC and UV methods have

been reported for estimation of Irinotecan hydrochloride which are sophisticated, but time

consuming. The present study was aimed at development of speedy and cost effective HPLC

technique for determination of Irinotecan hydrochloride as bulk and in dosage forms. The


phase composition , pH of the buffer used in the mobile phase. Retention time and separation of

peak of Irinotecan hydrochloride were dependent on pH of the buffer and the percentage of


by Ganjali et al and others 119-123

. Different mobile phases were tried, but satisfactory separation

and good symmetrical peak were obtained with the mobile phases 1.8 gm Potassium dihydrogen

phosphate in 1000 ml water and and adjust PH 3.3 with orthophosphuric acid as A and mixture of

600 ml of acetonitrile and 400ml of methanol as mobile phase B . The column temperature was

maintained at 30 °C, and Column used Water symmetry RP18 (250 x 4.6)mm , 5 found to

comparatively better and gave the graph with better Gaussian shape at retention time 13.20 min

.124



procedures:- Identification tests, Quantitative tests for impurities' content,Limit tests for the






342


Limit of detection (LOD) is the lowest concentration of analyte in a sample that can be detected,

but not necessarily quantitated, under the stated experimental conditions .

In the present findings these data show that the method is sensitive for the determination of

Irinotecan hydrochloride . The LOD and LOQ were measured by using an equation and were

found to be 0.25ppm and 0.5ppm .(Table-3.B.2.1). Our findigs good agrrement with the earlier

reported work on other drugs by Dwyer et al. 125



operation and is normally expressed as the RSD for a statistically significant number of samples.

RSD for the peak areas of the samples should not be greater than 1.5% .System suitability test as

an integral part of method development was used to ensure adequate performance of the

chromatographic system. Retention time (RT), number of theoretical plates (N) and tailing factor

(T). Standard solution of Irinotecan hydrochloride was prepared as per testing procedure and

injected into the HPLC system in six replicates. The values of % relative standard deviation of

Related Compound-A, Related Compound-B, 7-Desethyl Irinotecan Camptothecine, -Ethyl

Camptothecine and 7,11-Diethyl-10-hydroxycamptothecine are 1.61, 1.79, 0.50, 1.41 and 1.32

respectively. (NMT 5.0%) for peak area obtained in six replicate injections were reported in

Table-3.A.1.1. the present study reveals that that the equipment used for the study worked

correctly for the developed analytical method, and being highly repetitive 126

.

Figure -4.B.7. A chromatogram of the Irinotecan and impurity prepration.

343

4.B.2.5. Linearity





(R2) is not less than 0.990 for the least squares method of analysis of the line.

127

In the present findings the standard curve found to be linear over the concentration range of 5.0-

15.0 ppm. The equation of the standard curve relating the peak area to the Irinotecan

Hydrochloride concentration in this range was y = 1190x – 885. The drug showed good linearity

in the range of 5-15 ppm with coefficient of correlation value of Related Compound-A, Related

Compound-B, 7-Desethyl Irinotecan Camptothecine, -Ethyl Camptothecine and 7,11-Diethyl-10-

hydroxycamptothecine are 0.9997, 0.9998, 0.9997, 0.9998, 0.9997 and 0.9998 resp.for peak area

(Table-3.B.2.3). Our findings good agrrement with the earlier reported work on other drugs. 128

Figure -4.B.8.Linearity graph of Irinotecan Hydrochloride

y = 1190.x 885

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

344

Figure -4.B.9.Linearity graph of Related Comp-A

Figure -4.B.10.Linearity graph of Related Comp-B

y = 982x 224

R² = 1.0

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1447.x 1075

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

345

Figure -4.B.11.Linearity graph 7-Desethyl Irinotecan

Camptothecine

Figure -4.B.12.Linearity graph of 7-Ethyl Camptothecine

y = 1277.x 949

R² = 0.998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1196x 273

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

346

Figure -4.B.13.Linearity graph of 7,11-Diethyl-10-hydroxycamptothecine

4.B.2.6. Accuracy


measurements. In our present study to determine the accuracy of the method by taking three

samples were used. The samples chosen were such that they represent the entire range of the

standard curve i.e lower, middle and higher concentration of the range.

In present investigation three solutions were prepared for this study having a concentration of

0.1 mg/ml of Irinotecan hydrochloride and three different concentrations of Irinotecan

hydrochloride related compounds: 50%, 100%, and 150%. Low concentration of Irinotecan

hydrochloride related compounds relative to Irinotecan hydrochloride was employed to check if

this low concentration of Irinotecan hydrochloride related compounds can be recovered in the

presence of high concentration of Irinotecan hydrochloride Percentage recovery of Irinotecan

hydrochloride related compounds at these levels was found consistent as can be seen in table

3.B.2.4, 3.B.2.5, 3.B.2.6, 3.B.2.7 and 3.B.2.8.Our findings similar with other researchers. 129

y = 2913.x 94.77

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

347

4.B.2.7. Robustness

The robustness of the HPLC method was evaluated according to the variety of conditions such as

small changes in the percentage of mobile phase and buffer concentration, pH and flow rate of

the mobile phase .

In present investigation the percentage recovery of Irinotecan hydrochloride was good under

most conditions and should no significant change when the critical parameters were modified.

The tailing factor for Irinotecan hydrochloride was always less than 2.0 and it was well separated


parameters and the specificity of the method it can conclude that the method is robust.130-131


In present investigation the forced degradation of Irinotecan hydrochloride under different stress


table- 3.A.1.10 . Our findings shown that 23.56% degradation observed in 0.01 N NaOH. The

peaks of the degradation products were well resolved. Typical chromatogram of degredable

compound show in figure-4.B.12. The degradation study thereby indicated that Irinotecan

hydrochloride was stable to chemical oxidation study, dry heat and alkali hydrolysis while it was

highly susceptible to acid hydrolysis . Our findings are in good aggrement with the work

published earlier by Amidzic et al and others.132-135

Figure -4.B.14. A chromatogram of the Irinotecan Hydrochloride alkali degradation

348

4.B.3. Related Substance method development and Validation of Cyclophosphamide



the reliable method for the quantification of Cyclophosphamide from bulk drug and which will

be also applicable for the degradable products. We also observed a trend where few HPLC and

UV methods have been reported for estimation of Cyclophosphamide which are sophisticated,

but time consuming. The present study was aimed at development of speedy and cost effective

HPLC technique for determination of Cyclophosphamide as bulk and in dosage forms. The



peak of Cyclophosphamide were dependent on pH of the buffer and the percentage of methanol.

Various blends of solvent systems in varying proportions were tried as mobile phase by Danesi et

al 136-137

. Different mobile phases were tried, but satisfactory separation and good symmetrical

peak were obtained with the mobile phases 900 ml of water 100 ml acetonitrile as A and 300 ml

of water 700 ml acetonitrile as mobile phase B, gradient program mentioned in experimental

details and column temperature was maintained at 30 °C. The Column used YMC Pack ODS

C18, 250 x 4.6 mm, 5µm found to comparatively better and gave the graph with better gaussian

shape at retention time 24.96 min .138



linearity, precision, accuracy, specificity, limit of quantification [LOQ], limit of detection

[LOD],Forced degradation and robustness.


The Quantitation limit of an individual analytical procedure is the lowest amount of analyte in a

sample which can be quantitatively determined with suitable precision and accuracy. The

Quantitation limit is a parameter of quantitative assays for low levels of compounds in sample

matrices and is used particularly for the determination of impurities or degradation products. The

detection limit of an individual analytical procedure is the lowest amount of analyte in a sample

which can be detected but not necessarily quantitated as an exact value.21

LOD and LOQ of the

drug were calculated using the following equations according to International Conference on

349

Harmonization (ICH) guidelines. 22

LOD = 3.3 × /S LOQ = 10 × /S where = the standard

deviation of the response and S = the slope of the regression equation .

In our present work these data show that the method is sensitive for the determination of

Cyclophosphamide . The LOD and LOQ were measured by using an equation and were found to

be 0.1ppm. (Table-3.B.3.1). Our findigs good aggrement with the earlier reported work on other

drugs by Vainchtein et al139



obtained when the method is applied to multiple sampling of a homogenous sample. Precision is

a measure of the reproducibility of the whole analytical method (including sampling, sample

preparation and analysis) under normal operating circumstances. Precision is determined by

using the method to assay a sample for a sufficient number of times to obtain statistically valid

results . The precision is then expressed as the relative standard deviation. In the present

investigation Standard solution of Cyclophosphamide was prepared as per testing procedure and

injected into the HPLC system in six replicates. The values of % relative standard deviation of

Monochlorocyclophosphamide is 1.81 and Impurity-A is 2.27 (NMT 5.0%) for peak area

obtained in six replicate injections were reported in Table-3.B.3.2. the present study reveals that

the equipment used for the study worked correctly for the developed analytical method, and

being highly repetitive. 140-143

.

Figure -4.B.15. A chromatogram of the Cyclophosphamide and impurity

prepration.

350

4.B.3.5. Linearity


proportional to the concentration.In present investigation the standard curve found to be linear

over the concentration range of 5.0-15.0 ppm. The equation of the standard curve relating the

peak area to the Cyclophosphamide concentration in this range was y = 1378x +323 . The drug

showed good linearity in the range of 5.0-15.0 ppm with coefficient of correlation value for

Monochlorocyclophosphamide is 0.9998 and Impurity-A is 0.9998 (Table-3.B.3.3). Our

findings are good aggrement with the earlier reported work on other drugs by Ibrahim et al and

others. 144-147

Figure -4.B.16.Linearity graph of Cyclophosphamide

y = 1378x +323

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

351

Figure -4.B.17.Linearity graph of Monochlorocyclophosphamide


y = 1606x +497

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1743x + 639

R² = 0.999

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5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

352

4.B.3.6. Accuracy




term is 'bias'.The bias of a method is an expression of how close the mean of asset of results

(produced by the method) is to the true value. Bias is usually determined by study of relevant

reference materials or by spiking studies

To determine the accuracy of the method three quality control samples were used. The samples


concentration of the range Our findings shown that recovery studies were conducted after

addition of standard drug solution at three different levels i.e. 50 %, 100 %, and 150 % to pre-

analyzed sample solution148-150

. The results are given in table-3.B.3.4 for

Monochlorocyclophosphamide and for Impurity-A in table-3.B.3.5.

4.B.3.7. Robustness

"The robustness of an analytical procedure is a measure of its capacity to remain unaffected by


during normal usage".

In present investigation the percentage recovery of Cyclophosphamide was good under most

conditions and didn’t showed no significant change when the critical parameters were modified.

The tailing factor for Cyclophosphamide was always less than 2.0 and well separated under all

the changes carried out. Considering the modifications in the system suitability parameters and

the specificity of the method it can conclude that the method is robust. Considering the

modifications in the system suitability parameters and the specificity of the method it can

conclude that the method is robust.151


In present investigation the forced degradation of Cyclophosphamide under different stress

condition such as acid, alkali , peroxide ,thermal and water according to ICH guideline are

reported in table- 3.B.3.10. Our results have shown that 21.44% degradation observed in 0.5 N

353

NaOH. Typical chromatogram of degredable compound show in figure-4.B.16. The peaks of the

degradation products were well resolved. The degradation study thereby indicated that

Cyclophosphamide was stable to chemical oxidation study, dry heat and acid hydrolysis while it

was highly susceptible to alkali hydrolysis .152-154

Figure -4.B.19. A chromatogram of the Cyclophosphamide acid degradation

Figure -4.B.20. A chromatogram of the Cyclophosphamide alkali degradation.

354

4.B.4. Related Substance method development and Validation of Thiamine

Hydrochloride


Our present investigation the main target for the development of chromatographic method was

to get the reliable method for the quantification of Thiamine hydrochloride from bulk drug and

which will be also applicable for the degradable products. Few HPLC and UV methods have

been reported for estimation of Thiamine hydrochloride which are sophisticated, but time


technique for determination of Thiamine hydrochloride as bulk and in dosage forms.155-157

The



peak of Thiamine hydrochloride were dependent on pH of the buffer and the percentage of


by Rivory et al 158-160

. Different mobile phases were tried, but satisfactory separation and good

symmetrical peak were obtained with the mobile phases 0.1 M Ammonium acetate buffer PH =

4.5 with acetic acid , 800 ml buffer and 200 ml water as A and mobile phase-B as 200 ml of

water and 800 ml acetonitrile mixed filtered and degased and final mobile phase is a homogenus

mixture of 700 ml of A and 300 ml of B and column temperature was maintained at35 °C . The

Column used Xterra C18, 250 x 4.6 mm, 5µm found to comparatively better and gave the graph

with better Gaussian shape at retention time 16.27 min . Hence this present study investigates the

various abilities these shown in ICH Harmonised Tripartite Guideline. 161








but not necessarily quantitated, under the stated experimental conditions . LOD can be

determined by preparing a solution that is expected to produce a response that is approximately 3

to 10 times of the base line noise. The solution is injected three times, and the signal and the

355

noise for each injection are recorded. Each signal to noise ratio (S/N) is then calculated, and

averaged.

In the present study the LOD and LOQ were measured by using an equation were found to be

0.1ppm .(Table-3.B.4.1). Our findigs good aggrement with the earlier reported work on other

drugs by Sawada et al 162-164


Precision was carefully tested ,System suitability test as an integral part of method development

was used to ensure adequate performance of the chromatographic system. Retention time (RT),

number of theoretical plates (N) and tailing factor (T) Standard solution of Thiamine

hydrochloride Hydrochloride was prepared as per testing procedure and injected into the HPLC

system in six replicates. The values of % relative standard deviation for Related Compound-B is

1.75 and Related Compound-C is 2.53 (NMT 5.0%) for peak area obtained in six replicate

injections were reported in Table-3.B.4.2. thus showing that the equipment used for the study

worked correctly for the developed analytical method, and being highly repetitive 165-166

.

Figure -4.B.21. A chromatogram of the Thiamine Hydrochloride and impurity

preparation.

356

4.B.4.5. Linearity

In present investigation linearity of the method was determined by constructing calibration

curves. Standard solutions of Thiamine at different concentrations level (50%, , 80%, 100%,

120% and 150%) were used for this purpose. Before injection of the solutions, the column was

equilibrated for at least 30 min with the mobile phase. Each measurement was carried out in six

replicates to verify the reproducibility of the detector response at each concentration level.167-168


proportional to the concentration. The standard curve found to be linear over the concentration

range of 5.0-15.0 ppm. The equation of the standard curve relating the peak area to the Thiamine

Hydrochloride concentration in this range was y = 3990x – 569 . In our investigation first time

reported that the drug showed good linearity in the range of 5.0-15.0 ppm with coefficient of

correlation value for Thiamine HCl is 0.9995, Related Compound-B is 0.9993 and Related

Compound-C is 0.9992 for peak area (Table-3.B.4.3).

Figure -4.B.22.Linearity graph of Thiamine Hydrochloride

y = 3990x 569

R² = 1.0

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

357

Figure -4.B.23.Linearity graph of Related compound-B

Figure -4.B.24.Linearity graph of Related compound-C

y = 2873x 101

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

y = 2088x + 367

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

358

4.B.4.6. Accuracy

Accuracy is generally assessed by analyzing a sample with known concentration and comparing

the measured value with the true value. The measured value was obtained by recovery

test.Accuracy , sometimes also referred to as recovery is an indicator of the trueness of the test

measurements. To determine the accuracy of the method three samples were used. The samples


concentration of the range. In present investigation recovery studies were conducted after

addition of standard drug solution at three different levels i.e. 50 %, 100 %, and 150 % to pre-

analyzed sample solution. The results are given in table-3.B.4.4for Related Comp-B and table-

3.B.4.5 for Related Comp-C.169-172

.4.B.4.7. Robustness


change in the result. The robustness of the HPLC method was evaluated according to the variety

of conditions such as small changes in the percentage of mobile phase and buffer concentration,

pH and flow rate of the mobile phase . In present findings the percentage recovery of Thiamine

hydrochloride was good under most conditions and didn’t show any significant change when the

critical parameters were modified. The tailing factor for Thiamine hydrochloride was always less

than 2.0 and it was well separated under all the changes carried out. Considering the

modifications in the system suitability parameters and the specificity of the method it can

conclude that the method is robust.


Analytical specificity of a method may be defined as a measure of the degree of interferences

from such components, ensuring that a peak response is due to a single component only. . In

present investigation the forced degradation of Thiamine hydrochloride under different stress

condition such as acid, alkali , peroxide ,thermal and water according to ICH guidline shown in

table- 3.B.4.10. Our results have shown that 16.86% degradation observed in 0.5 N NaOH.

Typical chromatogram of degredable compound show in figure-4.B.21. The peaks of the

degradation products were well resolved. The degradation study thereby indicated that Thiamine

hydrochloride was stable to chemical oxidation study, dry heat and acid hydrolysis while it was

highly susceptible to alkali hydrolysis . findings are in good aggrement with the work published

earlier by Takasago et al 173-176

359

Figure -4.B.25. A chromatogram of the Thiamine Hydrochloride acid degradation

Figure -4.B.26. A chromatogram of the Thiamine Hydrochloride alkali degradation

360

4.B.5. Related Substance method development and Validation of Topotecan

Hydrochloride



get the reliable method for the quantification of Topotecan hydrochloride from bulk drug and

which will be also applicable for the degradable products. However few HPLC and UV methods

have been reported for estimation of Topotecan hydrochloride which are sophisticated, but time


technique for determination of Topotecan hydrochloride as bulk and in dosage forms. The



peak of Topotecan hydrochloride were dependent on pH of the buffer and the percentage of


by Faouzi et al 177-178

. Different mobile phases were tried, but satisfactory separation and good

symmetrical peak were obtained with the mobile phases 0.02M Potassium dihydrogen phosphate

buffer pH = 3.0 with orthophosphuric acid as A and 300 ml of water and 700 ml acetonitrile as

mobile phase B mixed filtred and degassed. The column temperature was maintained at 35°C ,

and Column used Inertsil ODS 3V C18(250X4.6)mm,5µ found to comparatively better and gave

the graph with better gaussian shape at retention time 20.37 min .









361





to 10 times of the base line noise.

In the present findings these data show that the method is sensitive for the determination of

Topotecan hydrochloride . The LOD and LOQ were measured by using an equation and were

found to be 0.1ppm .(Table-3.B.5.1). Our findigs good aggrement with the earlier reported work

on other drugs 179






obtained.

Our findings are Standard solution of Topotecan hydrochloride was prepared as per testing

procedure and injected into the HPLC system in six replicates. The values of % relative standard

deviation for Impurity-A is 1.74 and Impurity-B is 1.62 (NMT 5.0%) for peak area obtained in

six replicate injections were reported in Table-3.B.5.2. thus showing that the equipment used for

the study worked correctly for the developed analytical method, and being highly repetitive. In

our finding the low value of relative standard deviation attributes that the method is

robust.Similar results are also reported by different research during their study Draviam et al 180-

181.

362

Figure -4.B.27. A chromatogram of the Topotecan Hydrochloride Standard

prepration.

4.B.5.5. Linearity

Linearity is a highly successful parameter for method accuracy and is defined as the ability of a

method to elicit test results that are directly proportional to analyte concentration within a given

range. Range is the interval between the upper and lower levels of analyte that have been

demonstrated to be determined with precision, accuracy, and linearity using the method as

written. The accepted criteria for linearity is that the correlation coefficient (R2) is not less than

0.999 for the least squares method of analysis .In our finding the linear study identifies a

specified concentration range where analytes response is linearly proportional to the

concentration. The standard curve found to be linear over the concentration range of 5.0-15.0


concentration in this range was y = 2300x – 448

In present findings the drug showed good linearity in the range of 5.0-15.0 ppm with

coefficient of correlation value for Topotecan is 0.9999, Impurity-A is 0.9992 and Impurity-B is

0.9997 for peak area (Table-3.B.5.3)

363

Figure -4.B.28.Linearity graph of Topotecan Hydrochloride


y = 2300x + 448

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

y = 1710x 291

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

364

Figure -4.B.30.Linearity graph of Impurity-B

4.B.5.6. Accuracy

Accuracy of a method is expressed as percentage of analyte recovered by spiking samples in

drug formulation . The RSD of the replicates provides the analysis variation and gives an

indication of the precision of the test method. It was sometimes also referred to as recovery is an

indicator of the trueness of the test measurements. To determine the accuracy of the method three

quality control samples were used. The samples chosen were such to represent the entire range of

the standard curve i.e. lower, middle and higher concentration of the range



pre-analyzed sample solution. The results are given in table-3.B.5.4for Impurity-A and table-

3.B.5.5 for Impurity-B.182-183

y = 1602x 652

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

365

4.B.5.7. Robustness :

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by


during normal usage..The present study reveals that the method found to be robust, which may

be attributed to the small but deliberate changes in the method and low value of relative standard

deviation. These parameters have no detrimental effect on the method performance. Similar

results are also reported by different research during their study.

In the present findings the percentage recovery of Topotecan hydrochloride was good under most


The tailing factor for Topotecan hydrochloride was always less than 2.0 and it was well separated


parameters and the specificity of the method it can conclude that the method is robust.184



components that may be expected to be present in the sample matrix. In our present investigation

Forced degradation study was carried out by subjecting the drug to stress condition such as acid,

alkali , peroxide ,thermal and water according to ICH guidline shown in table- 3.B.5.10. Our

findings shown that 18.69% degradation observed in 0.2N NaOH. Typical chromatogram of

degredable compound show in figure-4.B.26 .The peaks of the degradation products were well

resolved. The degradation study thereby indicated that Topotecan hydrochloride was stable to

chemical oxidation study, dry heat and acid hydrolysis while it was highly susceptible to alkali

hydrolysis .185-186

366

Figure -4.B.31. A chromatogram of the Topotecan HydrochlorideAcid degradation.

Figure -4.B.32. A chromatogram of the Topotecan Hydrochloride Alkali

degradation.

367

4.B.6. Related Substance method development and Validation of

TenofovirDisoproxil fumarate



get the reliable method for the quantification of TenofovirDisoproxil fumarate from bulk drug

and which will be also applicable for the degradable products. We also observed a trend where

few HPLC and UV methods have been reported for estimation of TenofovirDisoproxil fumarate

which are sophisticated, but time consuming. The present study was aimed at development of

speedy and cost effective HPLC technique for determination of TenofovirDisoproxil fumarate as

bulk and in dosage forms.265

The chromatographic method was optimized by changing various

parameters, such as the mobile phase composition , pH of the buffer used in the mobile phase.

Retention time and separation of peak of TenofovirDisoproxil fumarate were dependent on pH of

the buffer and the percentage of methanol. Various blends of solvent systems in varying

proportions were tried as mobile phase by Alderden-Los et al 187-189

. Different mobile phases

were tried, but satisfactory separation and good symmetrical peak were obtained with the mobile

phases 0.02M Potassium dihydrogen phosphate buffer pH = 3.0 with ortho-phosphuric acid as A

and 300 ml of water and 700 ml acetonitrile as mobile phase B mixed filtered and degassed.The

column temperature was maintained at 35 °C , and Column used YMC ODS -A

C18(150X4.6)mm,5µ found to comparatively better and gave the graph with better gaussian

shape at retention time 47.00 min .



linearity, precision, accuracy, specificity, limit of quantification [LOQ], limit of detection [LOD]

,Forced degradation and robustness.However,"The precision of an analytical method is the

degree of agreement among individual test results obtained when the method is applied to

multiple sampling of a homogenous sample. Precision is a measure of the reproducibility of the

whole analytical method (including sampling, sample preparation and analysis) under normal

operating circumstances. Precision is determined by using the method to assay a sample for a

sufficient number of times to obtain statistically valid results . The precision is then expressed as

the relative standard deviation.

368


Analytical LOD and LOQ of a method may be defined as the lowest concentration in a sample

that can be detected, but not necessarily quantitated, under the stated experimental conditions.

The limit of detection is important for impurity tests. In the present findings,these data show that

the method is sensitive for the determination of TenofovirDisoproxil fumarate . The LOD and

LOQ were measured by using an equation and were found to be 0.1ppm .(Table-3.B.6.1). Our

findings are in good aggrement with the work published earlier of Weller et al.190-191


In our present investigation Standard solution of TenofovirDisoproxil fumarate was prepared as

per testing procedure and injected into the HPLC system in six replicates. The values of %

relative standard deviation for Mono poc PMPA is 1.58, Moc poc PMPA is 2.00 , Ipr poc pmpa is

1.16 , Dec poc PMPA is 0.44 , n-poc poc PMPA is 0.80 , Mixed Dimer is 0.50 and Dimer is 0.70

(NMT 5.0%) for peak area obtained in six replicate injections were reported in Table-3.B.6.2.In

our finding the low value of relative standard deviation attributes that the method is

robust.Similar results are also reported by different research during their study. thus showing that

the equipment used for the study worked correctly for the developed analytical method, and

being highly repetitive 192-193

.

Figure -4.B.33. A chromatogram of Tenofovir Disoproxil Fumarate Sample

4.B.6.5. Linearity





369

(R2) is not less than 0.990 for the least squares method of analysis. In present investigation a

linear study identifies a specified concentration range where analytes response is linearly

proportional to the concentration. The standard curve found to be linear over the concentration

range of 5.0-15.0 ppm. The equation of the standard curve relating the peak area to the Tenofovir

Disoproxil Fumarate concentration in this range was y = 3931x – 591 . Our findings are in good

aggrement with the work published earlier by Rossella et al 194

In present investigation the drug shown good linearity in the range of 5.0-15 ppm with

coefficient of correlation value for Tenofovir is 0.9998 Mono poc PMPA is 0.9993, Moc poc

PMPA is 0.9992, Ipr poc pmpa is 0.9993, Dec poc PMPA is 0.9993, n-poc poc PMPA is 0.9992,

Mixed Dimer is 0.9999 and Dimer is 0.9999 for peak area (Table-3.B.6.3, 3.B.6.4)

Figure -4.B.34.Linearity graph of Tenofovir Disoproxil Fumarate

y = 3931x 591

R² = 0.998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

370

Figure -4.B.35.Linearity graph of Mono

poc PMPA

Figure -4.B.36.Linearity graph of Mono poc PMPA

y = 1798x +519

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1961x + 414

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

371

Figure -4.B.37.Linearity graph of Ipr poc pmpa

Figure -4.B.38.Linearity graph of Dec poc PMPA

y = 2330x +573

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1645x + 411

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

372

Figure -4.B.39.Linearity graph of n-poc poc PMPA

Figure -4.B.40.Linearity graph of Mixed Dimer

y = 1402x +214

R² = 0.998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1678x +168

R² = 0.998

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

373

Figure -4.B.41.Linearity graph of Dimer

4.B.6.6. Accuracy

Accuracy indicates the deviation between the mean value found and the true value. lt is

determined by applying the method to samples to which known amounts of analyte have been

added. These should be analysed against standard and blank solutions to ensure that no

interference exists. To determine the accuracy of the method three samples were used. The

samples chosen were such to represent the entire range of the standard curve i.e. lower, middle

and higher concentration of the range in present study investigates the various abilities to check

the accuracy of the method, recovery studies were conducted after addition of standard drug

solution at three different levels i.e. 50 %, 100 %, and 150 % to pre-analyzed sample solution[5-

8]. The results are given in table-3.B.6.5for Mono poc PMPA , table-3.B.6.6for Ipr poc pmpa,

table-3.B.6.7for Dec poc PMPA, table-3.B.6.8for n-poc poc PMPA, table-3.B.6.9 for Mixed

Dimer and table-3.B.6.10for Dimer. 195

y = 1585x 238

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

374

4.B.6.7. Robustness

In the present findings the percentage recovery of Tenofovir Disoproxil fumarate was good under


modified. The tailing factor for TenofovirDisoproxil fumarate was always less than 2.0 and it


suitability parameters and the specificity of the method it can conclude that the method is

robust.196


Purity angle for the selected drug components in all stress conditions were found to be less than

the threshold angle. In our present investigation the forced degradation of TenofovirDisoproxil

fumarate under different stress condition such as acid, alkali , peroxide ,thermal and water

according to ICH guidline shown in table- 3.B.6.14. Our results have shown that15.74%

degradation observed in 0.5N NaOH and 10.89% degradation observed in UV Light degradation.

Typical chromatogram of degredable compound show in figure-4.B.36. The peaks of the

degradation products were well resolved. The degradation study thereby indicated that

TenofovirDisoproxil fumarate was stable to chemical oxidation study, dry heat and acid

hydrolysis while it was highly susceptible to alkali hydrolysis. Our findings are in good

aggrement with the work published earlier by Vainchtein et al and others.197-200

Figure -4.B.42. A chromatogram of the Tenofovir Disoproxil Fumarate acid

degradation

375

Figure -4.B.43. A chromatogram of the Tenofovir Disoproxil Fumarate Peroxide

degradation

Figure -4.B.44. A chromatogram of the Tenofovir Disoproxil Fumarate alkali

degradation.

376

Figure -4.B.45. A chromatogram of the Tenofovir Disoproxil Fumarate Thermal

degradation.

Figure -4.B.46. A chromatogram of the Tenofovir Disoproxil Fumarate Photolytic

degradation.

377

4.B.7. Related Substance method development and Validation of Atazanavir



get the reliable method for the quantification of Atazanavir from bulk drug and which will be

also applicable for the degradable products. Few HPLC and UV methods have been reported for

estimation of Atazanavir which are sophisticated, but time consuming.201

The present study was

aimed at development of speedy and cost effective HPLC technique for determination of

Atazanavir as bulk and in dosage forms. The chromatographic method was optimized by


mobile phase. Retention time and separation of peak of Atzanavir were dependent on pH of the


were tried as mobile phase by D'Avolio et al 202-204

. Different mobile phases were tried, but

satisfactory separation and good symmetrical peak were obtained with the mobile phases 0.01M

Potassium dihydrogen phosphate buffer pH = 3.2 with orthophosphuric acid as A and acetonitrile

as mobile phase B mixed filtered and degased. The Column used YMC ODS-A(150X4.6) mm,

3.5 µ , column temperature 25 °C found to comparatively better and gave the graph with better

Gaussian shape at retention time 19.79 min .


Method validation of analytical procedures is based on the four most common types of analytical



product or other selected component(s) in the drug product.


linearity, precision, accuracy, specificity, forced degradation and robustness.






378


averaged. The concentration of the solution is used for determination of the detection limit if the

average S/N ratio is between 3 and 10. If it is not between 3 and 10, the solution concentration is

modified as necessary and the experiment is repeated. LOD may be expressed as: LOD = 3.3 /S

Where is the standard deviation of the response, and S is the slope of the calibration curve.

Limit of quantitation can be determined in the same manner but using the formula 10 /S.

In present investigation these data show that the method is sensitive for the determination of

Atzanavir . The LOD and LOQ were measured by using an equation and were found to be

0.1ppm .(Table-3.B.7.1).Our findigs good aggrement with the earlier reported work on other

drugs by Zhou et al 205-206


Precision is a measure of the reproducibility of the whole analytical method (including sampling,

sample preparation and analysis) under normal operating circumstances. Precision is determined

by using the method to assay a sample for a sufficient number of times to obtain statistically

valid results (ie between 6 - 1 0). Precision is determined by using the method to assay a sample

for a sufficient number of times to obtain statistically valid results . The precision is then

expressed as the relative standard deviation as

std dev x 100%

%RSD = --------------------

mean

Standard solution of Atazanavir was prepared as per testing procedure and injected into the

HPLC system in six replicates. The values of % relative standard deviation is 1.24 (NMT 2.0%)

for peak area obtained in six replicate injections were reported in Table-3.B.7.2. In our finding

the the low value of relative standard deviation attributes that the method is robust.Similar results

are also reported by different research during their study by Carpenter et al and others 208-210

379

Figure -4.B.47. A chromatogram of the Atazanavir Sample degradation sample

4.B.7.5. Linearity





(R2) is not less than 0.990 for the least squares method of analysis of the line

211

In present investigation a linear study identifies a specified concentration range where analytes


the concentration range of 5.0-15.0 ppm. The equation of the standard curve relating the peak

area to the Atazanavir concentration in this range was y = 2856x – 265 . Our findigs good

aggrement with the earlier reported work on other drugs by McCormick et al and others.212-213

The drug showed good linearity in the range of 5.0-15.0 ppm with coefficient of correlation

value 0.9999 for peak area of Atzanavir and 0.9997 for peak area of Impurity-A (Table-3.B.7.3)

380

Figure -4.B.48.Linearity graph of Atzanavir


y = 2856x +265

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16

y = 1934x + 592

R² = 0.999

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8 10 12 14 16

381

4.B.7.6. Accuracy




middle and higher concentration of the range. In our findings the accuracy of the method,

recovery studies were conducted after addition of standard drug solution at three different levels

i.e. 50 %, 100 %, and 150 % to pre-analyzed sample solution280-284

. The results are given in

table-3.B.7.4.Hence this present study investigates the various abilities.214-216

4.B.7.7. Robustness

In the present findings the percentage recovery of Atazanavir was good under most conditions


factor for Atzanavir was always less than 2.0 and it was well separated under all the changes

carried out. Considering the modifications in the system suitability parameters and the specificity

of the method it can conclude that the method is robust.217


In our present investigation Forced degradation study was carried out by subjecting the drug to

different stress condition such as acid, alkali , peroxide ,thermal and water according to ICH

guidline shown in table- 3.B.7.9.Our results have shown that 12.86% degradation observed in 0.5

N NaOH. Typical chromatogram of degredable compound show in figure-4.B.43 The peaks of

the degradation products were well resolved. The degradation study thereby indicated that

Atzanavir was stable to chemical oxidation study, dry heat and acid hydrolysis while it was

highly susceptible to alkali hydrolysis .218-219

382

Figure -4.B.50. A chromatogram of the Atazanavir Alkali degradation sample

383

4.B.8. Enantiomer content method Development and validation of Irinotecan

Hydrochloride



get the reliable method for the quantification of Irinotecan HCl from bulk drug. The elution

gradient and influence of mobile phase were studied in order to optimize the analytical

performance. A short analytical column and isocratic and organic modifier were chose as the best

compromise between retention time of analyte.Method development of Irinotecan HCl

enantiomer content also done by Sweetman et al.220

For separating conditions HPLC as an

effective tool. In HPLC three important variables influencing separation are the organic modifier

, basic modifier and Temperature.As a means of quantifying resolution, the peak capacity of the

separation was determined. 221-222

Enantiomer content method development of Irinotecan HCl

from bulk drug and which is applicable for accurate quantification. Initially, the effort for the

development of Irinotecan HCl from bulk drug HPLC method of R and S isomer seperation. For

this purpose, we have used Chiralpak ADH, 250 x 4.6 mm, 5µm and Chiralcel, 250 x 4.6 mm,

5µm. Out of these used HPLC column, Chiralcel, 250 x 4.6 mm, 5µm found to comparatively

better and gave the graph with better gaussian shape at retention time for R and S isomer is 10.64

and 15.22 min. To improve the shape and width of the graph, for the above columns different

solvent used such as n-hexane and IPA(70:30),n-hexane and IPA(68:32),.Finally tried for459 ml

n-hexane and 505 ml ethanol mixed and degased and column temperature was maintained at30

°C gave better peak shape. Method developed for Irinotecan HCl accurate short. and

enantioselective method.






384


This is the lowest concentration in a sample that can be detected, but not necessarily

quantitated, under the stated experimental conditions. The limit of detection is important for

impurity tests. The limit of detection is generally quoted as the concentration yielding a signal-

to-noise ratio of 2:1 and is confirmed by analyzing a number of samples near this value using

the following equation. The signal-to-noise ratio is determined by:s = H/h Where H = height of

the peak corresponding to the component. h = absolute value of the largest noise fluctuation from

the baseline of the chromatogram of a blank solution.In our present finding, LOQ determination

at 0.1 ppm level (Table-3.B.8.1)and it should good agreement with Perry et al 223-224

4.B.8.4. Method Precision

The system suitability was determined by injecting racemic mixture containing equal quantity

of (R) and (S)-enantiomers. Since the enantiomers form a critical pair of peaks in the

chromatogram, the qualification criteria was resolution between two enantiomers, shown to be

not less than 2 and tailing factor should not exceed 1.5. "The precision of an analytical method

is the degree of agreement among individual test results obtained when the method is applied to

multiple sampling of a homogenous sample. Precision is a measure of the reproducibility of the

whole analytical method (including sampling, sample preparation and analysis) under normal

operating circumstances. Precision is determined by using the method to assay a sample for a

sufficient number of times to obtain statistically valid results . The precision is then expressed as

the relative standard deviation.

In present investigation Table-3.B.8.2 showed Method precision data in which % RSD of six

replicate were mentioned.In our finding the low value of relative standard deviation attributes

that the method is robust.Similar results are also reported by different research during their study

225-227

385

Figure -4.B.51. A chromatogram of the Irinotecan Hydrochloride System suitability

prepration

Figure -4.B.52. A chromatogram of the Irinotecan Hydrochloride standard

prepration

4.B.8.5. Linearity

In present investigation a linear study identifies a specified concentration range where analytes



area to the Irinotecan Hydrochloride concentration in this range was y = 2491x – 130 and y =

2773x + 360

Calibration curves showed high linearity over the concentration range with correlation

coefficient 0.999 (Table-3.B.8.3). In our finding the low value of relative standard deviation

386

attributes that the method is robust.Similar results are also reported by different research during

their study Shirkhedkar et al228-229

Figure -4.B.53. Linearity graph of Irinotecan Hydrochloride ( R- Isomer)

Figure -4.B.54. Linearity graph of Irinotecan Hydrochloride ( R- Isomer)

y = 2491.x 130

R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16

y = 2773x + 360

R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16

387

4.B.8.6. Accuracy

Analytical accuracy , sometimes also referred to as recovery is an indicator of the trueness of the

test measurements. To determine the accuracy of the method three quality control samples were

used. The samples chosen were such to represent the entire range of the standard curve i.e. lower,




pre-analyzed sample solution. The recovery was determined using impurity spiked with sample

at three different concentration level show in table-3.B.8.4 (82-101%).

4.B.8.7. Robustness

"The robustness/ruggedness 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 usage"

In the present findings the robustness study flow rate change,pH change result showen in

table. Finally both the temperature and the egree of organic modifier were explored as a possible

means of improving resolution. It was found that increasing the temperature did not improve

peak capacity.230

Figure -4.B.55. A chromatogram of the Irinotecan Hydrochloride sample

prepration.

388

4.B.9. Enantiomer content method Development and validation of Topotecan

Hydrochloride


get the reliable method for the quantification of Topotecan HCl from bulk drug. The elution

isocratic and influence of mobile phase were studied in order to optimize the analytical

performance 231

. Analytical column and elution depand on organic modifier, acidic or basic

modifier ,were chose as the best compromise between retention time of analyte. For separating

conditions HPLC as an effective tool. In HPLC three important variables influencing separation

are the organic modifier and Temperature.As a means of quantifying resolution, the peak

capacity of the separation was determined. Method development of Topotecan HCl enantiomer

content done by Venkataramanan et al 232-233

Enantiomer content method development of

Topotecan HCl from bulk drug and which is applicable for accurate quantification. Initially, we

took the effort for the development of Topotecan HCl from bulk drug HPLC method of R and S

isomer seperation. For this purpose, we have used Chiralpak ADH, 250 x 4.6 mm, 5µm and

Chiralcel, 250 x 4.6 mm, 5µm. Out of these used HPLC column, Chiralpak ADH, 250 x 4.6 mm,

5µm found to comparatively better and gave the graph with better gaussian shape at retention

time for R and S isomer is 9.50 and 14.15 min. To improve the shape and width of the graph, for

the above columns different solvent used such as acetonitrile and methanol (62:38), acetonitrile

and methanol (65:35),.Finally tried for 700 mlMethanol, 300 ml acetonitrile and 2.0 ml diethyl

amine mixed and degassed. The column temperature was maintained at 30 °C gave better peak

shape. Method developed for Topotecan HCl accurate short. and enantioselective method.



intended purpose. To meet current pharmaceutical regulatory guidelines i.e. USP , ICH and EP , a

number of criteria such as specificity, linearity, precision, accuracy, sensitivity and robustness




but not necessarily quantitated, under the stated experimental conditions. LOD can be

389


to 10 times of the base line noise.

In the present findings,the limits of detection (LOD) and LOQ were determined by analyzing

Working Standard .LOD and LOQ were found to be 0.1ppm .(Table-3.B.9.1).

4.B.9.3. Method Precision

"The precision of an analytical method is the degree of agreement among individual test

results obtained when the method is applied to multiple sampling of a homogenous sample.

Precision is a measure of the reproducibility of the whole analytical method (including sampling,

sample preparation and analysis) under normal operating circumstances. Precision is determined

by using the method to assay a sample for a sufficient number of times to obtain statistically

valid results . The precision is then expressed as the relative standard deviation.

In present investigation Standard solution of Topotecan was prepared as per testing procedure

and injected into the HPLC system in six replicates. The values of % relative standard deviation

of R-Isomer was 1.81 and S-Isomer was 2.27 (NMT 5.0%) for peak area obtained in six

replicate injections are reported in Table-3.B.9.2 showed Method precision data in which % RSD

of six replicate were mentioned.234

Figure -4.B.56. A chromatogram of the Topotecan Hydrochloride system suitability

solution.

390

4.B.9.4. Linearity


concentration within a given range. In present study investigates a linear study identifies a

specified concentration range where analytes response is linearly proportional to the

concentration. The standard curve found to be linear over the concentration range of 1000-3000


concentration in this range was y = 1256x +220 and

y = 1715x +449. Calibration curves showed high linearity over the concentration range with

correlation coefficient 0.999 (Table-3.B.9.3). Our findings are in good aggrement with the work

published earlier by Delahunty et al 235

Figure -4.B.57. Linearity graph of Topotecan Hydrochloride ( R-Isomer)

y = 1256x + 220

R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16

391

Figure -4.B.58. Linearity graph of Topotecan Hydrochloride( S-Isomer)

4.B.9.5. Accuracy



The samples chosen were such to represent the entire range of the standard curve i.e lower,


In present investigation the recovery was determined using impurity spiked with sample at

three different concentration level show in table-3.B.9.4 (83-101%).Our findigs good aggrement

with the earlier reported work on other drugs by Massaki et al and others 236-237

4.B.9.6. Robustness

The ICH guidelines also recommend that "one consequence of the evaluation of robustness

should be that a series of system suitability parameters (e.g. resolution tests) is established to

ensure that the validity of the analytical procedure is maintained whenever used".

In the present findings, robustness study flow rate change result showen in table-3.B.9.5.

Finally both the temperature and the organic modifier were explored as a possible means of

improving resolution. It was found that increasing the temperature did not improve peak

capacity.238

y = 1715x + 449

R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16

392

Figure -4.B.59. A chromatogram of the Topotecan Hydrochloride Sample

prepration.

393

4.B.10. Enantiomer content method Development and validation of

TenofovirDisoproxil fumarate


get the reliable method for the quantification of Tenofovir Disoproxil fumarate from bulk drug.

The elution gradient and influence of mobile phase were studied in order to optimize the

analytical performance. A short analytical column and organic modifier were chose as the best

compromise between retention time of analyte. For separating conditions HPLC as an effective

tool. In HPLC three important variables influencing separation are the organic modifier and

Temperature.As a means of quantifying resolution, the peak capacity of the separation was

determined. Method development of TenofovirDisoproxil fumarate enantiomer content done by

Unnam et al.239-240

Enantiomer content method development of Tenofovir Disoproxil fumarate

from bulk drug and which is applicable for accurate quantification.308-312

Initially, the effort for

the development of TenofovirDisoproxil fumarate from bulk drug HPLC method of R and S

isomer seperation. For this purpose, we have used Chiralpak ADH, 250 x 4.6 mm, 5µm and

Chiralcel, 250 x 4.6 mm, 5µm. Out of these used HPLC column, Chiralpak AD-RH (150 X 4.6)

mm, 5!. found to comparatively better and gave the graph with better gaussian shape at

retention time for R and S isomer is 2.31 and 3.39 min. To improve the shape and width of the

graph, for the above columns different solvent used such as acetonitrile and methanol (45:55),

acetonitrile and methanol (50:50),.Finally tried for Methanol,acetonitrile and diethyl amine

(850:150:0.8,v/v/v) and column temperature was maintained at 35 °C have better peak shape.

Method developed for TenofovirDisoproxil fumarate accurate short. and enantioselective

method.Therefore,in this thesis, developments of new chromatographic analytical methods were

established to solve this problem.









394




averaged. The concentration of the solution is used for determination of the detection limit if the

average S/N ratio is between 3 and 10. If it is not between 3 and 10, the solution concentration is

modified as necessary and the experiment is repeated. LOD may be expressed as: LOD = 3.3 /S

Where is the standard deviation of the response, and S is the slope of the calibration curve.

Limit of quantitation can be determined in the same manner but using the formula 10 /S.

In present investigation the limits of detection (LOD) and LOQ were determined by analyzing

Working Standard .

LOD and LOQ were found to be 0.1 ppm .(Table-3.B.10.1).Similar findings were reported by

researchers in different drugs. 242-243

4.B.10.3.Method Precision


obtained when the method is applied to multiple sampling of a homogenous sample. Precision is

a measure of the reproducibility of the whole analytical method (including sampling, sample

preparation and analysis) under normal operating circumstances. Precision is determined by

using the method to assay a sample for a sufficient number of times to obtain statistically valid

results . The precision is then expressed as the relative standard deviation.

Table-3.B.10.2 showed Method precision data in which % RSD of Assay of six replicate were

mentioned.In our finding the low value of relative standard deviation attributes that the method is

robust.Similar results are also reported by different research during their study by Weller et al

and others.244-248

395

Figure -4.B.60. A chromatogram of the Tenofovir Disoproxil Fumarate System

suitability prepration.

4.B.10.4.Linearity

In the present findings a linear study identifies a specified concentration range where analytes



area to the Tenofovir Disoproxill Fumarate concentration in this range was y = 1430x +313 and

y = 1244x +302. 249-253

Calibration curves showed high linearity over the concentration range

with correlation coefficient 0.999 (Table-3.B.10.3).

Figure -4.B.61. Linearity graph of Tenofovir Disoproxill Fumarate( R-Isomer)

y = 1296x + 326

R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16

396

Figure -4.B.62. Linearity graph of Tenofovir Disoproxill Fumarate( S-Isomer)

4.B.10.5.Recovery





In present investigation the recovery was determined using impurity spiked with sample at

three different concentration level show in table-3.B.10.4 (82-101%).Our findigs good

aggrement with the earlier reported work on other drugs by Manogaokar et al. 254-255

4.B.10.6.Robustness

In our present work the robustness study flow rate change,pH change result showen in table.

Finally both the temperature and the egree of organic modifier were explored as a possible means

of improving resolution. It was found that increasing the temperature did not improve peak

capacity.256

y = 1387x + 352

R² = 0.999

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2 4 6 8 10 12 14 16

397

Figure -4.B.63. A chromatogram of the Tenofovir Disoproxil Fumarate sample

prepration.

398

References:

1]. G.Srinubabu, B. Sudha Rani, J.V.L.N. Seshagiri Rao. Spectrophotometric Determination of

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January 2006.

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