chapter-8 method development and validation of...

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Chapter-8

Method development and validation of

pitavastatin and its impurities by RP-

HPLC method

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8.1 Introduction

Pitavastatin chemically know as

quinolin-3-yl]-3, 5-dihydroxyhept

medication class of statins marketed in the Unit

chemical structure of pitavastatin shown in Figure

and its molecular weight is 421.46

enzyme that catalyses the first step of

is used for controlling hypercholesterolaemia (elevated cholesterol) and for the prevention of

cardiovascular disease [2].

Figure-8.1: Chemical structure of pitavastatin

Pitavastatin is odourless and occurs as white to pale

in pyridine, chloroform, dilute hydrochloric acid, and tetrahydrofuran, soluble in ethylene

glycol, sparingly soluble in octanol, slightly soluble in metha

water or ethanol, and practically insoluble in acetonitrile or diethyl ether. Pitavastatin is

hygroscopic and slightly unstable in light.

Pitavastatin chemically know as (3R, 5S, 6E)-7-[2-cyclopropyl-4-(4-

dihydroxyhept-6-enoic acid, is a member of the blood cholesterol lowering

marketed in the United States under the trade name Livalo

chemical structure of pitavastatin shown in Figure-8.1. Its empirical formula is

421.46g/mol. It is an inhibitor of HMG-CoA reductase

that catalyses the first step of cholesterol synthesis. Like the other statins, pitavastatin

hypercholesterolaemia (elevated cholesterol) and for the prevention of

8.1: Chemical structure of pitavastatin

Pitavastatin is odourless and occurs as white to pale-yellow powder. It is freely soluble


glycol, sparingly soluble in octanol, slightly soluble in methanol, very slightly soluble in


hygroscopic and slightly unstable in light.

-fluorophenyl)

is a member of the blood cholesterol lowering

ed States under the trade name Livalo[1]. The

is C25H24FNO4

CoA reductase, the

synthesis. Like the other statins, pitavastatin

hypercholesterolaemia (elevated cholesterol) and for the prevention of

yellow powder. It is freely soluble


nol, very slightly soluble in


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Each film-coated tablet of LIVALO contains 1.045 mg, 2.09 mg, or 4.18 mg of

pitavastatin calcium, which is equivalent to 1 mg, 2 mg, or 4 mg, respectively of free base and

the following inactive ingredients such as lactose monohydrate, low substituted

hydroxypropylcellulose, hypromellose, magnesium aluminometasilicate, magnesium stearate,

and film coating containing the inactive ingredients like hypromellose, titanium dioxide,

triethyl citrate, and colloidal anhydrous silica. Each tablet has “KC” debossed on one side and

a code number specific to the tablet strength on the other. The different dosage is shown in

Figure-8.2.

Figure-8.2: Different dosage of pitavastatin

Pitavastatin calcium was discovered by Nissan chemical industries limited Japan [3] and

developed further by kowa pharmaceuticals Tokyo, Japan. This is a novel member of the

medication class of statins. Several methods for the preparation of pitavastatin calcium are

reported in literatures [4-16]. The main highlight of any process in this case would be to

maintain the desired stereochemistry in the final product and control the formation of side

products, in this case the lactone. Further, the electrochemical behavior associated with the

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compound i.e reduction or oxidation of functional group in aqueous media will be studied

[17-18].

It is available in Japan since 2003, and is being marketed under licence in South Korea

and in India [19]. It is likely that pitavastatin will be approved for use in hypercholesterolemia

(elevated levels of cholesterol in the blood) and for the prevention of cardiovascular disease

outside South and Southeast Asia as well [20]. In the US, it has received FDA approval in

2009 [21].

Most statins are metabolised in part by one or more hepatic cytochrome P450 enzymes,

leading to an increased potential for drug interactions and problems with certain foods (such

as grapefruit juice). Pitavastatin appears to be a substrate of CYP2C9, and not CYP3A4

(which is a common source of interactions in other statins). As a result, pitavastatin is less

likely to interact with drugs that are metabolized via CYP3A4, which might be important for

elderly patients who need to take multiple medicines. Like the other statins, pitavastatin is

indicated for hypercholesterolemia (elevated cholesterol) and for the prevention of

cardiovascular disease. A 2009 study showed that pitavastatin increased HDL cholesterol

(24.6%), especially in patients with HDL lower than 40 mg/dl, in addition to greatly reducing

LDL cholesterol (–31.3%). As a consequence, pitavastatin is most likely to be appropriate for

patients with metabolic syndrome with high LDL, low HDL and diabetes mellitus. The

metabolic activity of different statins are shown in Figure-8.3

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Figure-8.3: Metabolic path of different stains in liver and intestineetabolic path of different stains in liver and intestine

etabolic path of different stains in liver and intestine

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Literature survey revealed that several analytical methods such as spectrophotometry

[22-24] high performance thin layer chromatography (HPTLC) [25-26], column switching

high performance liquid chromatography (HPLC) [27-35], ultra performance liquid

chromatography (UPLC) [36] and LC-MS/MS methods [37-42] have been reported for the

determination of pitavastatin in pharmaceutical dosage forms and in biological samples.

Virupaxappa et.al developed a spectrophotometric method for the determination of

pitavastatin calcium by using acidic potassium permanganate. The method was based on the

reduction of permanganate by pitavastatin in acidic medium, and the unreacted oxidant was

measured at 550 nm. [24]. Satheesh et.al proposed a high performance thin layer

chromatography method was developed for the determination of pitavastatin calcium in tablet

dosage form by aluminum backed silica gel 60F254 plate, washed with methanol and ethyl

acetate-methanol‐ammonia‐one drop, formic acid (7:2:0.8) as mobile phase with UV

detection at 245 nm[25]. Hiral J. Panchal et.al developed and validated a quantitative high-

performance thin-layer chromatographic method for the determination of pitavastatin calcium

in pharmaceutical preparations. The drug from the formulations was separated and identified

on silica gel 60F254 HPTLC plates with toluene–methanol–glacial acetic acid 7.6:2.36:0.04

(v/v) mixture as mobile phase with UV detection at 238nm[26]. Neelima et.al proposed

stability indicating RP-HPLC method for quantitative determination of pitavastatin in bulk

and pharmaceutical dosage form [30]. The chromatographic separation was achieved with

Agilent Eclipse XDB, C18, (150 x 4.6 mm, 5µ) column. The optimized mobile phase used

consists of consisting phosphate buffer: acetonitrile (65:35% v/v) whose pH was adjusted to

3.5 with ortho phosphoric acid. The flow rate was maintained at the 0.9 mL/min and the

eluent was detected at 244nm using PDA detector.

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Antony Raj Gomas et.al developed a novel ultra performance liquid chromatography

method for the determination of pitavastatin using degradation path way [36]. The efficient

chromatographic separation was achieved on a BEH C18 stationary phase with simple mobile

phase combination delivered in gradient mode and quantification is carried at 245 nm with a

flow rate of 0.3 mL/min.

Though large numbers of assay methods are available in literature for pitavastatin, only

very few of them are standard, sensitive and selective. In view of the importance of

pitavastatin in drug formulation in the treatment of blood cholesterol lowering, a more simple,

sensitive, selective and robust method is needed for its validation in formulations. All the

reported methods were used for the determination of pitavastatin in bulk and formulations but

no method was reported for the determination of pitavastatin and its related impurities. We

are now reporting a simple sensitive and selective RP-HPLC method for the validation of

pitavastatin and its related impurities which is a robust and rugged method.

8.2 Experimental:

8.2.1 Chemicals, Reagents and samples:

The standard and samples of pitavastatin and known related impurities of drug were

received from local analytical Labs. HPLC grade sodium acetate, acetic acid and acetonitrile

were purchased from Merck, Mumbai, India. High purity water was prepared by using

Millipore Milli-Q plus water purification system. The purity of all samples and drug related

impurities used in this study was greater than 99%.

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8.2.2 Instrumentation

For initial method development studies Waters prominence HPLC system was

employed. This was equipped with a quaternary UFLC LC-20AD pump, DGU-20A5

degasser, SPD-M20A diode array detector, SIL-20AC auto sampler, CTO-20AC column oven

and CBM-20A communications bus module. Agilent 1200 series with high pressure liquid

chromatographic instrument provided with Auto sampler and VWD UV detector with

thermostatted column compartment connected with EZ Chrom software was employed for the

validation of the drug and its related impurities. The analysis was carried out on phenomenex,

kinetex C18 75mm x 4.6 mm column with 2.6µm particle size.

8.2.3 Standard and sample solutions:

8.2.3.1. Preparation of standard solution:

Accurately weighed and transferred 10.0 mg of pitavastatin standard into 100 ml amber

colored Volumetric flask dissolved and diluted to the Volume with acetonitrile: water in the

ratio of (50:50v/v) used as diluent. 1 ml of this solution was further diluted to 100 ml with the

diluent.

8.2.3.2 Sample preparation:

10 pitavastatin tablets were weighed accurately into 50 ml amber colored Volumetric flask

and added 30 ml of diluent. The resultant mixture was sonicated for 20 minutes to dissolve

and diluted to the Volume with diluent. The solution was filtered through 0.22 µm Nylon

filter and the first 5ml were discarded.

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8.3 Evaluation of system suitability:

The system suitability was evaluated by injecting a known Volume of sample containing

a known amount of pitavastatin into the chromatograph and calculated the resolution between

pitavastatin and its anti isomer, the number of theoretical plates, relative standard deviation

(RSD) for six injections and asymmetry of pitavastatin peak. The resolution was found to be

2.87; the number of theoretical plates was calculated 30794. The relative standard deviation

was calculated as 0.32 % and the asymmetry of pitavastatin peak was found to be 0.96 which

showed that the selected column is suitable for the analysis.

8.4 Results and discussion:

8.4.1 Method development and optimization:

The method development was initiated with the solubility study of pitavastatin.

Based on the solubility studies, acetonitrile: water mixture in the ratio 1:1 v/v was chosen as

solvent and diluent for the preparation of sample solutions. Pitavastatin is polar in nature due

to the presence of -OH groups. Hence, non polar silica based C18 column was selected for

developing reverse phase high performance liquid chromatogram. From the molecular

structure, it was observed that there are chromophore groups i.e. double bonds and –C=O

group present in pitavastatin. Hence there is possibility for its UV–Visible detection. The UV

experiment was performed on pitavastatin, which showed maximum absorbance at 250nm.

To arrive at the optimal chromatographic conditions suitable for the validation of

pitavastatin and its related impurities, various trail chromatograms were recorded with

different conditions.

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Trail-1

The first trail method was performed by using isocratic mode and mobile phase as

buffer and acetonitrile in the ratio of 80: 20 v/v. The injection Volume was 20µL.

Column : Phenomenex,Kinetex C18 75mm x 4.6 mm with 2.6 µm size

Pump mode : Isocratic

Flow rate : 1.0 ml/min

Detection wavelength : UV , 250 nm

Injection Volume : 20µL

Column temperature : 25.00C

Sampler temperature : 5.00C

Run time : 30 min

Buffer : Dissolved 0.82 g of sodium acetate in 1000 mL of water and

adjusted the pH to 3.8 with acetic acid.

Mobile phase : Buffer : Acetonitrile (80:20 v/v)

In this trail, there was no elution of impurity peaks up to run time of 30 minutes and tailing of

pitavastatin peak was more than 2.0

Trail-2

To over the limitations of the above trail method, the experiment was repeated by

changing the mode of mobile phase keeping the remaining conditions same as above.


Pump mode : Gradient






Run time : 30 min


adjust pH to 3.8 with acetic acid.

Mobile phase-A : Buffer : Acetonitrile (90:10 v/v)

Mobile phase-B : Acetonitrile : water (80:20 v/v)

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In this trail there was elution of pitavastatin and its known impurities but peak shapes of

pitavastatin and impurities were not symmetrical.

Trail -3

To achieve symmetrical peaks of pitavastatin and its impurities, the injection

Volume was changed from 20 µL to 10 µL, the composition of mobile phase-B was changed

from 80: 20v/v to 90:10 v/v and the experiment was repeated under the optimal conditions.


Pump mode : Gradient






Run time : 30 min


adjust pH to 3.8 with acetic acid.

Mobile phase-A : Buffer : Acetonitrile (90:10 v/v)

Mobile phase-B : Acetonitrile : water (90:10 v/v)

In this trail pitavastatin peak was eluted at 9.13 minutes and the impurities were well

separated with main pitavastatin peak .All peaks were obtained with excellent symmetry.

Finally, satisfactory separation with better peak shape was achieved within a reasonable

retention time with gradient mode with flow rate of 1.0mL/min at temperature 250C.

8.5 Method validation:

In order to determine the related substances of pitavastatin, the method was validated as

per the ICH guidelines [43-50] individually in terms of system suitability, specificity,

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precision, accuracy, linearity, robustness, limit of detection and limit of quantification (LOD

and LOQ) and solution stability.

8.5.1 System suitability test:

The system suitability was studied by injecting diluted blank, reference solution and

standard six replicate injections. The resolution between pitavastatin and anti isomer, the

number of theoretical plates, relative standard deviation (RSD) for six injections and

asymmetry of pitavastatin peak were evaluated. The system suitability results are given in the

Table-8.1

Table-8.1: Results of system suitability

System suitability parameters

Result

Acceptance criteria

Theoretical plates 30794 >3000

% RSD for six injections 0.32 < 5.0%

Resolution 2.87 >1.5

Asymmetry 0.96 <2.0

.

Since all the system suitability results were within the acceptance criteria, the system

developed is suitable for separation and validation of pitavastatin and its impurities.

8.5.2 Specificity of method with related substances:

For specificity determination, solution containing diluent and all related substances of

pitavastatin was prepared by mixing in suitable proportions. Then diluent, standard

preparation, sample preparation, sample spiked with impurities were injected into the

chromatograph and the peak homogeneity was verified for haloperidol and its related

substances using EZ Chrom software. The summary of specificity experiment results are

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shown in Table-8.2.The standard and specificity chromatograms were given in figures-8.3 and

8.4

Table-8.2: Results of specificity experiment

S. No. Compound Name Peak Purity

1 Pitavastatin 1.00000

2 Desfluoro impurity 1.00000

3 Anti isomer 1.00000

4 Z-isomer impurity 1.00000

5 Methyl ester impurity 1.00000

6 Lactone impurity 1.00000

7 Tertiary butyl ester impurity 1.00000

The results in Table 8.2 and Figure 8.4 indicate that under the optimal conditions developed

in the present method, pitavastatin and all its related impurities in the tablet formulations are

well separated with high peak purity. This shows that the proposed method is highly selective

for the quantitative determination of pitavastatin and its impurities without prior separation.

8.5.3 Precision:

Six replicate aliquots of sample solution of pitavastatin spiked with impurities were

injected into RP-HPLC system and the chromatograms were recorded for checking the

performance of the system under the chromatographic conditions on the day tested and

impurity areas of these samples were determined. The precision of the method was evaluated

by computing the relative standard deviation of impurity results. The system precision results

are shown in Tables 8.3-8.5

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Table-8.3: System precision of pitavastatin for 1 mg

S. No Desfluoro Impurity

Anti isomer Z-isomer Impurity

Methyl

ester

Impurity

Lactone

Impurity

Tertiary butyl

ester Impurity

1 0.98 1.00 1.00 1.01 1.02 1.00

2 0.98 0.99 1.00 1.01 1.02 1.00

3 0.99 1.00 1.01 1.02 1.02 1.01

4 0.99 0.98 1.01 1.02 1.02 1.01

5 0.98 0.99 1.00 1.01 1.01 0.99

6 0.99 0.98 1.01 1.02 1.02 1.01

Avg 0.99 0.99 1.01 1.02 1.02 1.00

SD 0.01 0.01 0.01 0.01 0.01 0.01

% RSD 0.54 1.03 0.52 0.71 0.51 0.84




Methyl

ester

Impurity

Lactone

Impurity

Tertiary butyl

ester Impurity

1 0.99 1.00 1.01 1.02 1.01 1.01

2 0.99 1.00 1.01 1.02 1.02 1.01

3 0.99 1.00 1.01 1.01 1.01 1.00

4 0.99 1.00 1.00 1.00 0.99 0.98

5 0.99 1.00 1.01 1.01 1.01 1.00

6 1.00 1.01 1.02 1.02 1.01 1.01

Avg 0.99 1.00 1.01 1.01 1.01 1.00

SD 0.00 0.00 0.00 0.01 0.01 0.01

% RSD 0.42 0.50 0.49 0.82 0.76 1.09

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Methyl ester

Impurity

Lactone

Impurity

Tertiary butyl

ester Impurity

1 0.99 0.99 1.01 1.01 1.00 1.00

2 0.99 0.99 1.01 1.00 0.99 0.99

3 0.99 0.99 1.00 1.00 1.01 0.99

4 0.98 0.98 1.00 0.99 0.99 0.98

5 0.99 0.99 1.01 0.99 0.99 0.99

6 0.98 0.98 1.00 0.99 1.00 0.98

Avg 0.99 0.99 1.00 1.00 1.00 0.99

SD 0.01 0.01 0.01 0.01 0.01 0.01

% RSD 0.53 0.73 0.63 0.67 0.61 0.71

8.5.4 Accuracy:

The accuracy of the proposed method was tested by preparing sample solutions with

known quantities of impurities of pitavastatin at the level of LOQ, 50%, 100%, 150% and

200% of target concentration. The chromatograms were recorded and the recovery

percentages were evaluated from the peak areas. The results are shown in Table 8.6

Table 8.6: Accuracy results of pitavastatin impurities

Levels

% Mean recovery

Desfluoro Impurity

Anti

isomer

Z-isomer Impurity

Methyl

ester

Impurity

Lactone

Impurity

Tertiary butyl

ester Impurity

LOQ 99.19 98.32 95.53 99.55 103.27 101.47

50% 106.67 101.28 104.97 95.94 101.26 104.92

100% 99.58 99.35 98.91 92.19 94.82 100.40

150% 98.66 97.49 97.90 97.33 100.53 101.17

200% 97.76 98.44 98.90 97.03 100.05 100.59

% RSD 2.67 1.20 2.36 1.84 2.15 1.55

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The RSD values for all the pitavastatin impurities are in the range 1.20-2.67% indicating that

the accuracy of the method is quite good.

8.5.5 Linearity:

The concentrations ranges in which pitavastatin and its impurities can be determined

with good accuracy were evaluated by preparing calibration plots between the concentration

of the analyte and peak areas. Six different aliquots of standard solutions were injected into

the RP-HPLC chromatograph and the chromatograms were recorded. Peak areas of the

resultant chromatograms were plotted against the concentration of the analyte. The

experimental data obtained are shown in Table -8.7 and the resultant linear plots obtained for

these data are given in figure-8.4.

Table 8.7 Linearity results for pitavastatin and its impurities

a) Pitavastatin b) Desfluoro impurity

Linearity

level

Amount of

pitavastatin

(ppm)

Average areas of

pitavastatin

LOQ 0.018 132019

25.0% 0.57 3781518

50.0% 1.14 7017721

100.0% 2.28 14638741

150.0% 3.43 22603326

200.0% 4.57 29593556

Linearity

level

Amount of desfluoro

impurity(ppm)

Average areas of

desfluoro

impurity

LOQ 0.021 126480

25.0% 0.47 2838171

50.0% 0.94 5494869

100.0% 1.88 11689496

150.0% 2.82 16909797

200.0% 3.76 22262397

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c) Anti isomer impurity d) Z-isomer impurity

e) Methyl ester impurity f) Lactone impurity

Linearity

Level

Amount of anti

isomer

impurity(ppm)

Average areas of

anti isomer

impurity

LOQ 0.023 152184

25.0% 0.51 3460179

50.0% 1.03 6490783

100.0% 2.05 14357773

150.0% 3.08 19554820

200.0% 4.10 26251866

Linearity

Level

Amount of

Z-isomer

impurity(ppm)

Average areas of

Z-isomer

impurity

LOQ 0.019 98125

25.0% 0.49 2437988

50.0% 0.98 4721605

100.0% 1.95 9953256

150.0% 2.93 14385597

200.0% 3.90 19298391

Linearity

Level

Amount of lactone

impurity(ppm)

Average areas of

lactone

impurity

LOQ 0.012 98376

25.0% 0.48 3437621

50.0% 0.96 6955773

100.0% 1.92 14294017

150.0% 2.88 21049798

200.0% 3.84 27799258

Linearity

Level

Amount of methyl ester

impurity(ppm)

Average areas of

methyl ester

impurity

LOQ 0.019 101848

25.0% 0.53 2850607

50.0% 1.05 5712943

100.0% 2.11 11831534

150.0% 3.16 17183171

200.0% 4.21 23040719

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g) Tertiary butyl ester impurity

Table-8.8: Summarized statistical results of pitavastatin and its impurities

Compound Name

Correlation

coefficient

Slope

Y-Intercept

Residual sum

square

Residual

standard

deviation

Pitavastatin 0.9998 6519577 -86701 2.8084 x 1011

264970

Desfluoro impurity 0.9997 5955400 69392 2.5691 x 1011

253434

Anti isomer 0.9987 6396207 205557 1.3685 x 1012

584922

Z-isomer impurity 0.9998 4948762 12688 1.2063 x 1011

173657

Methyl ester impurity 0.9999 5474794 10929 9.5204 x 1010

154272

Lactone impurity 0.9999 7271965 41028 1.3058 x 1011

180681

Tertiary butyl ester

impurity

0.9995 5933905 -23418 3.6593 x 1011

302462

The data was subjected to statistical analysis and the results of these analysis are presented in

table 8.8.The values of different statistical analyses like correlation coefficient intercept,

residual sum square and related standard deviation confirm high precision and accuracy of the

proposed method.

Linearity

Level

Amount of tertiary

butyl ester

impurity(ppm)

Average areas of

tertiary butyl

ester impurity

LOQ 0.016 103947

25.0% 0.46 2646500

50.0% 0.91 5357731

100.0% 1.82 11078742

150.0% 2.74 15763287

200.0% 3.65 21851031

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8.5.6 Robustness:

The robustness study was carried out with respect to flow rate, column and buffer pH.

The chromatographic conditions were maintained same as per test method in each case. From

the obtained results, it was observe that there was no much variation in retention time,

theoretical plates and asymmetry of pitavastatin peak, obtained at different deliberately

varied conditions from the test method. Hence the method is robust for all the varied

conditions. The complete robustness results are shown in Table 8.9.

Table 8.9: Robustness results of pitavastatin

8.5.7 Limit of Detection and Limit of Quantification:

The detection limit and limit of quantification of pitavastatin and related substances

were determined by diluting known concentrations. The simplest method to calculate limit of

detection and limit of quantification is signal to noise ratio method. The quantification limits

and detection limits of each known impurity and pitavastatin are given in the Tables 8.10 and

8.11.

Robustness Condition

Theoretical plates

Asymmetry

Normal Condition 3.29 42857

Flow changed to 1.1ml/min 3.29 37174

Flow changed to 0.9ml/min 3.25 49037

Column Temperature changed to 30°C 3.03 39859

Column Temperature changed to 20°C 3.33 40600

Buffer pH changed to 4.0 1.96 9411

Buffer pH changed to 3.6 1.97 6594

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Table-8.10: LOQ values of pitavastatin and impurities

S. No Name of the Component S/N ratio Quantification limit(mg/ml)

1 Pitavastatin 10.25 0.0091

2 Desfluoro impurity 9.93 0.0104

3 Anti isomer impurity 10.20 0.0113

4 Z-isomer impurity 10.10 0.0098

5 Methyl ester impurity 10.06 0.0095

6 Lactone impurity 9.72 0.0058

7 Tertiary butyl ester impurity 9.64 0.0082

Table-8.11: LOD values of pitavastatin and impurities

S. No Name of the Component S/N ratio Detection limit(mg/ml)

1 Pitavastatin 2.63 0.0027

2 Desfluoro impurity 2.87 0.0031

3 Anti isomer impurity 2.64 0.0034

4 Z-isomer impurity 2.51 0.0029

5 Methyl ester impurity 2.43 0.0028

6 Lactone impurity 2.80 0.0017

7 Tertiary butyl ester impurity 2.78 0.0025

8.6 Conclusion

The liquid chromatographic method with gradient elution developed for the

determination of pitavastatin and its impurities in the injections was fully validated and

proved to be reliable, sensitive, accurate and precise .The method has higher sensitivity

towards the determination of impurities and it is the first time that such a method is being

reported and can be useful for routine analysis and quality control of pitavastatin in the

relevant forms. Thus, the method can be used for quality assurance of pitavastatin in tablets.

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