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PART-[A]

ENANTIOSELECTIVE METHOD

DEVELOPMENT AND VALIDATION

OF SOME PHARMACETUICALS

SECTION-4

Enantioselective HPLC Method

Development and Validation of

Ranolazine

Ranolazine Section-4

158

1. Introduction

The increasing availability of single-enantiomer drugs promises to provide

clinicians with safer, better-tolerated, and more efficacious medications for treating

patients. It is incumbent upon the practicing physician to be familiar with the basic

characteristics of chiral pharmaceuticals discussed in this work. In particular, each

enantiomer of a given chiral drug may have its own particular pharmacologic profile, and

a single-enantiomer formulation of a drug may possess different properties than the

racemic formulation of the same drug. When both a single enantiomer and a racemic

formulation of a drug are available, the information from clinical trials and clinical

experience should be used to decide which formulation is most appropriate.

To pharmaceutical companies, the novelty of enantiomers over their racemates is

a favorable rule. As mentioned previously, many chiral drugs have historically been

approved not as single enantiomers, but as racemates. Companies may be able to extend

product life by making a chiral switch, i.e. an industry term for the development of a

single-enantiomer drug when the drug in racemic form is already on the market. It also

means pharmaceutical companies may want to market a drug containing only the more

active enantiomer, even if its mirror image has been disclosed or even previously

patented. For a blockbuster drug based on a single enantiomer, the ability or inability to

acquire a patent can significantly affect profitability by blocking the entrance of generic

competitors [1].

1.1 Description

Ranolazine is an antianginal medication. Ranolazine is designated Chemically as

(RS)-N-(2,6-dimethylphenyl)-2-[4-[2-hydroxy-3-(2-methoxyphenoxy)propyl] piperazin-

1-yl] acetamide. Ranolazine is a white to off white powder with a molecular weight of

427.5 g/mol. Ranolazine is soluble in dichloromethane and methanol, sparingly soluble in

THF, ethanol, acetone and acetonitrile, slightly soluble in ethyl acetate, 2-propanol,

toluene and ethylether, very soluble in water. The empirical formula of Ranolazine is

C24H33N3O4.The CAS number of ranolazine is 142387-99-3.


159

Ranolazine has a chiral center and contains enantiomeric forms i.e. (S)–

ranolazine and (R)- ranolazine (Fig.1). Ranolazine was approved by the U.S. Food and

Drug Administration (FDA) in 2006 as a racemic compound for the treatment of stable

angina pectoris.

(S)-enantiomer

(R)-enantiomer

Figure 1: Ranolazine Enantiomers

1.2 Indication

Ranolazine is helpful for the treatment of chronic angina. It should be used in

combination with amlodipine, beta-blockers or nitrates.

1.3 Mechanism of Action

The mechanism of action of ranolazine is unknown. It does not increase the rate-

pressure product, a measure of myocardial work, at maximal exercise. In vitro studies

suggest that ranolazine is a P-gp inhibitor. Ranolazine is believed to have its effects via

altering the trans-cellular late sodium current. It is by altering the intracellular sodium


160

level that ranolazine affects the sodium-dependent calcium channels during myocardial

ischemia. Thus, ranolazine indirectly prevents the calcium overload that causes cardiac

ischemia.

1.4 Pharmacodynamics

Ranolazine has antianginal and anti-ischemic effects that do not depend upon

reductions in heart rate or blood pressure. It is the first new anti-anginal developed in

over 20 years

1.5 Pharmacology

Ranolazine is a racemic mixture that contains enantiomeric forms (S-ranolazine

and R-ranolazine) that inhibit the INaL. Ranolazine is rapidly metabolized in the liver,

primarily through the cytochrome P-450 3A enzyme (CYP3A) pathway, and in the

intestine. More than 70% of the drug is excreted in the urine. This pharmacokinetic

profile necessitates careful dosage adjustments in patients who are elderly, who weigh

less than 60 kg, and who have mild-to-moderate renal insufficiency or mild hepatic

impairment, and in patients who are in New York Heart Association functional class III–

IV. Ranolazine is contraindicated in patients with severe renal impairment (glomerular

filtration rate, <30 mL/min/1.73 m2) or moderate-to-severe hepatic impairment (Child-

Pugh classes B and C). The use of ranolazine by patients who are undergoing renal

replacement therapy has not been studied.

1.6 Absorption

Ranolazine is well absorbed after oral administration. Absorption is highly

variable. After oral administration of ranolazine as a solution, 73% of the dose is

systemically available as ranolazine or metabolites. The bioavailability of oral ranolazine

relative to that from a solution is 76 %.


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1.7 Metabolism

Ranolazine is metabolized mainly by CYP3A and, to a lesser extent, by CYP2D6.

Following a single oral dose of ranolazine solution, approximately 70% of the dose is

excreted in urine and 25% in feces. Ranolazine is metabolized rapidly and extensively in

the liver and intestine; less than 5% is excreted unchanged in urine and feces. The

pharmacologic activity of the metabolites has not been well characterized. After dosing to

steady state with 500 mg to 1500 mg twice daily, the four most abundant metabolites in

plasma have AUC values ranging from about 5 to 33% that of ranolazine, and display

apparent half-lives ranging from 6 to 22 h.

1.8 Adverse Reaction

The most common side effects of ranolazine are dizziness, nausea, constipation,

and headache. Less than 2% of patients experience these side effects [2, 4]. In most

cases, the symptoms are mild, and they occur within the 1st few weeks of therapy.

Although some patients must discontinue taking the drug, most can tolerate reduced

dosages.

1.9 Macrocyclic Glycopeptide Based Chiral Stationary Phase

Chiral stationary phases (CSPs) prepared by bonding the macrocyclic

glycopeptides vancomycin, teicoplanin, teicoplanin aglycone and ristocetin A, have

demonstrated broad selectivity in LC chiral separations since their introduction by Dr.

D.W. Armstrong in 1994. Their complex structures allow them to interact with chiral

molecules through many different kinds of forces including ionic (electrostatic)

interaction, π‐π interaction, hydrogen bonding, inclusion complexation, hydrophobic

interaction as well as steric (repulsive) hindrance.

One of the important glycopeptide based chiral stationary phase is Teicoplanin

(TE) which is produced by certain strains of Actinoplanes teichomyceticus [5]. It is

applied in the treatment of severe hospital-acquired infections caused by Gram-positive

bacteria.


162

Figure 2: Chemical structure of Teicoplanin

Structurally, TE contains a heptapeptide aglycone that bears three sugar units. The

aglycone consists of four fused medium-size rings, which form a semirigid basket. The

basket contains seven aromatic rings, two of which are chlorosubstituted and four have

ionizable phenolic moieties. In the aglycone moiety, there are also a primary amine (pKa

= 9.2) and a carboxylic acid group (pKa = 2.5).

The three sugar units are d-mannose, d-N-acetylglucosamine, and a residue of d-N-

acylglucosamine. For the presence of the latter hydrophobic residue, TE is considerably

more surface-active than other related glycopeptides. Five main components of the TE

complex have been identified (designated fromA2-1 to A2-5), differing from each other

in the nature and length of the hydrocarbon chain of the N-acylglucosamine moiety.

TEA2–2 (C88H97Cl2N9O33) is the prevalent component (>85%) of the TE complex

(Fig.1), with a molecular mass of 1878 (acyl = 8-methyl-nonanoyl), and contains 23

stereogenic centers.


163

TE was the second macrocyclic antibiotic evaluated as selector for the synthesis

of HPLC CSPs, one year later than vancomycin [6]. Teicoplanin CSP is commercial

available as a Chirobiotic T from astecTM

. Separation normally obtained on chiral crown

ether or ligand exchange type phase are also possible on the Chirobiotic T. This column

is compactible in reverse as well as normal phase mobile phases.

[2] Literature Overview

The literature reviews regarding ranolazine suggest that various analytical methods were

reported for its determination in drug substance, biological sample and pharmaceutical dosage

forms. Analytical methods for the determination of the impurities were also reported.

Tapan Kumar Pal and team have reported a LC-MS/MS method for

determination of ranolazine in human plasma and its application in bioequivalence study.

The method shown linear response over the concentration range of 5-2000 ng/mL for

ranolazine in human plasma. There were satisfactory results for accuracy and precision

studies [7]. Similar work has been reported by Lei Tian and team using LC-MS/MS [8].

Yueqi Liu, and Hanfa Zou have reported enantiomer HPLC separation using

cellulose based CSP. The coated cellulose tris(3,5-dimethylphenyl carbamate) CSP was

used as a chiral stationary phase and they reported base to base enantiomeric separation

using n-hexane and isopropylalcohol as a mobile phase system [9].

Lou X, Zhai Z, Wu X, Shi Y, Chen L, and Li Y have reported analytical and

semi-preparative resolution of ranolazine enantiomers by liquid chromatography using

polysaccharide chiral stationary phase. The authors used cellulose tris(3,5-dimethyl

phenyl carbamate) CSP under both normal-phase and polar organic modes. The

developed method was validated including linearity, LODs, recovery and precision. At

semi-preparative scale, about 14.3 mg/h enantiomers could be isolated [10].


164

[3] Aim of Present Study

Ranolazine is an anti-anginal medication and the clinical anti-anginal

effectiveness of ranolazine is currently being evaluated. However, the mechanism of its

anti-ischaemic action is still unclear. Radioligand binding studies were performed in rat

hearts and guinea-pig lungs for beta1- and beta2-adrenoceptor affinity, respectively.

Ranolazine had micromolar affinity for both beta1- and beta2-adrenoceptors (pKi5.8 and

6.3, respectively) [11]. Collectively, the results from this study demonstrate that

ranolazine behaves as a weak beta1- and beta2-adrenoceptor antagonist in the rat

cardiovascular system.

The recent invention claims that S-ranolazine is a more potent inhibitor of the

beta-adrenoceptor than racemic ranolazine and thus (S)-ranolazine is useful for the

reduction of adverse events as smaller doses of (S)-ranolazine may be therapeutically

equivalent to racemic ranolazine. While this application discusses treatment of all types

of diabetes mellitus including Type I and Type II, it has been unexpectedly discovered

that ranolazine, particularly its R-enantiomer, enhances insulin secretion and is effective

in treating diabetes mellitus in a class of patients that are insulinresponsive and insulin

secretion- deficient. It has also been surprisingly discovered that the (R)-enantiomer of

ranolazine also provides other pharmacokinetic benefits as it provides less inhibition of

the CYP2D6 enzyme [12].

It has become very important to have precise and accurate method for chiral

analysis of ranolazine, which will help to support such biological studies with single

isomer. However, to the best of our knowledge, no report has been published on stability

indicating chiral HPLC method to estimate the enantiomeric purity of ranolazine using

macrocyclic glycopeptide chiral stationary phases in the pharmaceutical formulations.

The major objective of this present work was to develop and validate chiral method using

glycopeptides based CSP. The method should have the application in determining the

enantiomeric estimation of ranolazine in pharmaceutical formulation to support routine

analysis of quality control laboratories. The present work deals with systematic method

development and validation including important specificity, linearity, accuracy, precision,

limit of detection and quantification.


165

[4] Experimental

4.1 Chemicals and Drugs

Standards of Ranolazine, (S)-ranolazine, (R)-ranolazine and capsule formulation

obtained local market. Methanol, ethanol, isopropylalcohol, n-hexane, n-heptane,

trifluoroaceticacid (TFA) and diethyalamine (DEA) were purchased from Merck.

Ranolazine standard and capsule formulation obtained local market.

4.2 High Performance Liquid Chromatography

The method development and validation was performed on an Agilent 1200

HPLC system consist of a quaternary pump, column oven, photo diode array detector and

an auto injector. CHIROBITIC chiral columns were used for method development to

separate ranolazine enantiomers. The HPLC system was controlled and analytical data

were processed using Agilent ChemStation software (Version B.04).

4.3 Chromatographic Conditions

The enantiomeric separation was at 35°C column oven temperature using

Chirobiotic T (250 × 4.6 mm, 5 µm particle size, Astec®

) chiral column. The mobile

phase consisted of n-heptane containing 0.1% TFA, ethanol and methanol (60:35:5,

v/v/v). Flow rate was 1.2 mL/min and injection volume was 5µl and the run time was set

to 50 min. The analyte was detected photometrically at 204 nm.

4.4 Diluent Preparation

Mobile phase was chosen as the diluent to achieve good peak shape and

interference free blank chromatogram.

4.5 Preparation of Stock Solutions

Stock solutions of racemic ranolazine prepared by dissolving appropriate amount

of standard samples in minimum volume of methanol and further dilutions were made in

diluent. A stock solution concentration was fixed at 250 µg/mL.


166

4.6 Preparation of Sample Solutions

For formulation sample, 5 capsules (500 mg of ranolazine label claim) were

opened to a 500 mL volumetric. This was equivalent to 500 mg of ranolazine which was

extracted in to 100 mL of methanol by ultrasonication. The final volume has made up

with methanol and filtered through a 0.45-µm membrane filter. This solution further

dilute in mobile phase to have final analyte concentration of 250 µg/mL. To perform the

recovery study, the placebo corresponding to the capsule formulation was used.

4.7 Method Validation

4.7.1 Selectivity

Analytical techniques that can measure the analyte response in the presence of all

potential sample components should be used for specificity validation. In practice, a test

mixture is prepared that contains the analyte and all potential sample components. The

result is compared with the response of the analyte. In pharmaceutical test mixtures,

components can come from excipients. Selectivity of this method was indicated by the

absence of any endogenous interference at retention times of enantiomeric peaks. The

absence of interfering peak was evaluated by injecting a blank consisting of diluent and

placebo.

4.7.2 Precision

The precision of an analytical procedure as the closeness of agreement between a

series of measurements obtained from multiple sampling of the same homogeneous

sample under the prescribed conditions. The precision of the method was checked by an

analyzing six replicate samples of racemic ranolazine sample. The same exercise repeated

on different day and RSD of area under the peaks was calculated.

4.7.3 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. Detector response linearity was assessed by preparing 9


167

calibration sample solutions covering from 0.10 to 100 µg/mL (0.10, 0.25, 0.50, 1.00,

5.00, 10.00, 25.00, 50.00 and 100.00 µg/mL), Regression curve was obtained by plotting

peak area versus concentration, using the least squares method.

4.7.4 Limit of Detection (LOD)

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

in a sample that can be detected, but not necessarily quantitated as an exact value. The

LOD may be determined by the analysis of samples with known concentrations of

analyte and by establishing the minimum level (lowest calibration standard) at which the

analyte can be reliably detected. LOD was estimated at a signal to noise ratio of 3:1.

4.7.5 Limit of Quantification (LOQ)

The limit of quantification is the lowest amount of the analyte in the sample that

can be quantitatively determined with defined precision under the stated experimental

conditions. The limit of quantification is a parameter of quantitative assays for low levels

of compounds in sample matrices and is used particularly for the determination of

impurities and/or degradation products or low levels of active constituent in a product.

LOQ was estimated at a signal to noise ration of 10:1.

4.7.6 Accuracy

The standard addition and recovery experiments of ranolazine in placebo were

conducted to determine accuracy of the present method. The study was carried out in

triplicate by spiking placebo with three concentrations (400, 500 and 600 ng/mL) of

individual enantiomer’s standards assaying for the chromatographic method.

4.7.7 Ruggedness

In order to evaluate intermediate precision, the precision was repeated using

different instrument and column in other laboratory at different day. To determine the

ruggedness, the racemic ranolazine samples analyzed six times. The % RSD of area and

RT are considered to evaluate ruggedness study.


168

4.7.8 Robustness

The robustness of an analytical method is a measure of its capacity to remain

unaffected by small but deliberate variation in method parameters and provides an

indication of its reliability during normal usage. The following parameters were changed

to establish the robustness of the method. The resolution between ranolazine enantiomers

was determined to evaluate robustness.

(a) Flow Rate Variation:

The flow rate of the mobile phase was changed to 1 mL/min and 1.4 mL/min from

1.2mL/min.

(b) Mobile Phase Composition Variation:

The mobile phase composition of n-heptane containing 0.1% TFA, ethanol and methanol

(60:35:5, v/v/v) was changed to (55: 38:7, v/v/v) and (65:33:2, v/v/v)

(c) Column Oven Temperature Variation:

The temperature of the column oven was changed to 32° C and 38° C from 35° C.

4.7.9 .Solution stability

The sample was analyzed for 24 h at room temperature, i.e., at 25°C. Resolution

and composition of (R)- and (S)- enantiomers were observed during the study period.


169

[5] Result and Discussion

5.1 Method Development

Ranolazine chemically known as (RS)-N-(2,6-dimethylphenyl)-2-[4-[2-hydroxy-

3-(2-methoxyphenoxy)propyl] piperazin-1-yl] acetamide. The racemic sample solution of

100 µg/mL concentration was used for the method development and optimization. To

determine the λmax, the racemic solution was scan between 200 to 350 nm using UV

diode arrays detector and we got two λmax, i.e. at 204 and 274nm (Fig. 3).

Figure 3: UV spectra of Ranolazine

In order to achieve the better sensitivity, the λmax, with higher intensity (i.e.

204nm) has selected for the further study. It has four hydrogen bond acceptor and three

hydrogen bond donor. Ranolazine has logP of 2.83 and has two pKa values, 13.6 and

7.17. It is soluble in dichloromethane and methanol, sparingly soluble in THF, ethanol,

acetone and acetonitrile, and very soluble in water.


170

5.1.1 Development and Optimization of Chromatographic Conditions

Ranolazine exhibits a chiral center and is obtained as a racemic mixture that consists

of a 1:1 ratio of (R) and (S) enantiomers. The objective of this study was to separate two

enantiomers of the ranolazine, using a macrocyclic glycopeptide based chiral column.

Racemic ranolazine of 100 µg/ mL concentration was used for the method development.

In order to achieve the enantiomer separation, different chiral columns namely

Chirobiotic R, Chirobiotic V, Chirobiotic T and Chirobiotic TAG were employed for

primary screening. Initial method development started with polar ionic mode, by keeping

100% methanol as a mobile phase. The trials have been given using various combinations

of various alcohols, like ethanol and isopropylalcohol. The racemic ranolazine analyzed

on all four Chirobiotic columns at different flow rate. There was no sign of separation in

polar ionic mode with given trials on all four Chirobiotic columns (Fig.4).

Figure 4: Chromatograms of primary screening using polar ionic mode


171

We did not evaluate reverse phase mobile phase system as Ranolazine is very

slightly soluble in water. There was a published report which describes the

enantioselective separation of ranolazine in normal phase conditions using cellulose

based CSP, so we preferred to evaluate normal phase solvents as mobile phase.

In typical normal phase conditions, retention is controlled by adjusting the ratio of

nonpolar to polar organic solvents (the greater the polarity, the lower the retention). As a

result of the linear response of solvent composition to resolution, gradients can be run in

the normal phase mode to find the window of separation. For the CHIROBIOTIC phases,

greater peak efficiency and resolution are obtained with ethanol as the polar constituent

instead of the usual isopropanol, although there are a few cases where IPA proved to be a

better modifier. The common starting normal phase conditions are the combination of n-

hexane and ethanol.

The racemic ranolazine was analyzed on all four Chirobiotic columns using the

mixture of n-hexane (0.1% TFA) and ethanol in the proportion of 90:10, v/v. The

primary sign of enantiomeric separation was observed on Chirobiotic T column (Fig.5).

Figure 5: Column: Chirobiotic T,

Mobile phase: n-hexane (0.1% TFA) and ethanol (90:10, v/v)


172

The Chiorobiotic T has unique selectivity for a number of classes of molecules,

specifically underivatized α, β, γ or cyclic amino acids, N-derivatized amino acids, i.e.,

FMOC, CBZ, t-BOC and alpha hydroxy-carboxylic acids, acidic compounds including

carboxylic acids and phenols, small peptides, neutral aromatic analytes and cyclic

aromatic and aliphatic amines. Separations normally obtained on a chiral crown ether or

ligand exchange type phase are also possible on the Chiorobiotic T. In addition, all of the

known beta-blockers (amino alcohols), and dihydrocoumarins have been resolved.

Considering these facts and initial separation of enantiomers, the Chirobiotic T column

prefer for further method development.

During initial trials, the enantiomeric peaks were over retained due to the higher

percentage of n-hexane. In order to elute the peak early, the polarity of mobilephase was

increased by increasing the composition of ethanol. We could achieved sharp peak but

there was loss of resolution due to early elution (Fig. 6).


Mobile phase: n-hexane (0.1% TFA) and ethanol, (50:50, v/v)

min5 10 15

mAU

0

20

40

60

80

100

120

140


173

Various combinations of n-hexane, ethanol and IPA have been tried to optimized

the enantiomeric separation. The optimum resolution could not be achieved more than 1

(Fig 7).


Mobile phase: n-hexane (0.1% TFA), IPA and ethanol, (50:25:25, v/v/v)

In order to retain the peaks for separating the enantiomers, we lost resolution due

to peak broadening. On the other hand, we could sharpen the peaks, but lost the

resolution due to early elution. To achieve better enantiomeric separation, various

combinations of normal phase solvents have been tried along with changing the flow rate

and column thermostat temperature.

The replacement of n-hexane with n-heptane helped to improve the chiral

separation (Fig. 8). The resulted peaks shown the peak fronting, which improved by

adding the polar organic modifier, i.e. methanol into the mobile phase [13]. As shown in

Fig. 9, the optimum enantiomeric separation could achieve with combination of

n-heptane (0.1% TFA), ethanol and methanol (60:35:5, v/v/v).

min0 5 10 15 20

mAU

-50

0

50

100

150

200

250


174

Figure 8: Column: Chirobiotic T

Mobile phase: n-heptane (0.1% TFA), ethanol, (50:50, v/v)

Figure 9: Mobile phase: n-heptane (0.1%TFA), ethanol and methanol (60:35:5, v/v/v)

Column: Chirobiotic T, Flow : 1.2 mL/min Column Temperature: 35 °C

min10 20 30 40

mAU

-20

0

20

40

60

80

100

120

140

160

min10 20 30 40

mAU

0

25

50

75

100

125

150

175

200

33.6

5m

in

37.0

0m

in

(R)-

ranola

zin

e

(S)-

ranola

zin

e


175

Separation achieved at 35°C column oven temperatures using Chirobiotic T (250 ×

4.6 mm, 5 µm particle size). Mobile phase was chosen as the diluent to achieve clean

blank chromatogram without any interference. The flow rate was 1.2 mL min-1

and

injection volume was 5 µl.

To recognize the (R)- and (S)- ranolazine, pure (S)- enantiomer analyzed in final

method. The typical retention time of (R)-ranolazine and (S)-ranolazine were were about

33.6 and 37 min respectively (Fig. 10).

Figure 10: Typical chromatogram of (S)-ranolazine in final method


176

5.2 Results of Method Validation

5.2.1 Results of System suitability

The system suitability results are summarized in Table 1.

Peak T N Rs α

(R)-ranolazine 1.1 5103

1.51 1.1 (S)-ranolazine 1.21 4800

(T: USP tailing factor, N: number of theoretical plates, Rs: USP resolution,

α: enantioselectivity)

Table 1: System suitability results.

5.2.2 Results of Specificity

Peak purity of both the enantiomers was passing using diode array detector. Report of

peak purity is presented in Fig. 11 and 12.The peak purity factor was within the

calculated threshold limit for (R)-ranolazine and (S)-ranolazine enantiomers (Table 2).

Ranolazine Purity Factor Threshold

(R)-enantiomer 999.979 999.840

(S)-enantiomer 999.947 999.791

Table 2: Peak purity results.

Peak purity of both the (R)-ranolazine and (S)-ranolazine passing using diode

array detector. Graphic presentation of peak purity reports are presented in Fig. 11

and 12. To evaluate the selectivity, the chromatogram obtained by analyzing blank

run consisting of diluent and placebo was compared in order to check the absence of

any peaks likely to interfere at RTs of (S)- and (R)- enantiomers.


177

Figure 11:

Peak purity report of

(R)-ranolazine

Figure 12:

Peak purity report of

(S)-ranolazine

Figure 13: Overlay of blank and racemic ranolazine

min32 33 34 35

Calculated

| || |' ' ' ' '

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

min36 38 40

Calculated

| || |' ' ' ' '

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

min0 10 20 30 40

mAU

-20

0

20

40

60

80


178

As can be seen in overlay of racemic and blank chromatogram (Fig. 13),

blank chromatograms is free from any interference at RTs of (R) - and (S)-

enantiomers. The blank sample is consisted of placebo and diluent.

5.2.3 Results of Method Precision

The precision of the method was evaluated by analyzing the six replicate racemic

ranolazine samples. Relative standard deviation (%RSD) of retention time and area

under the peaks were calculated for (S)- and (R)- enantiomers. The results of the study

were also satisfactory, illustrating the excellent precision of the method (Table 3).

Precision Data

Sr. No. (R)-ranolazine (S)-ranolazine

RT Area RT Area

1 33.65 14456 37 14503

2 33.52 14562 36.87 14599

3 33.57 14902 36.92 14892

4 33.98 14864 37.33 14712

5 33.1 14234 36.45 14654

6 33.4 14567 36.75 14231

Average 33.5 14597 36.9 14598

SD 0.29 252.3 0.29 221.9

%RSD 0.86 1.73 0.79 1.52

Table 3: Results of precision study

The intermediate precision was determined in another laboratory by performing

six successive injections. In the intermediate precision study, results showed that %RSD

values were in the same order of magnitude than those obtained for repeatability. The

results for method precision and robustness are summarized in Table 4.


179

Intermediate Precision Data

Sr. No. (R)-ranolazine (S)-ranolazine

RT Area RT Area

1 32.3 15690 35.7 15221

2 32.7 15782 36.1 15444

3 32.5 15132 35.9 15051

4 32.5 15776 35.9 14892

5 32.4 15233 36 15620

6 32.9 15611 36.3 15551

Average 32.5 1537 36 15296

SD 0.24 283.7 0.2 290.1

%RSD 0.74 1.83 0.57 1.9

Table 4: Results of precision study

5.2.4 Results of 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 [14,15]. The results show that good correlation existed between

the peak area and concentration for both enantiomers.

The calibration curve constructed was linear over the concentration range from 0.1

to100 µg/mL. The coefficient values were 0.99950 and 0.9996 for (R)-ranolazine and

(S)-ranolazine respectively (Fig. 14 and 15).


180

Concentration

(ppm) Area

0.1 60

0.25 123

0.50 204

1.00 420

5.00 1600

25.00 7201

50.00 15931

100.00 30124

125.00 1812742

250.00 3293224

Results

Intercept 70.52

Slope 302.9

r2 0.9995

Figure 14 : Linearity results for (R)-ranolazine

0

5000

10000

15000

20000

25000

30000

35000

Pea

k A

rea

Concentration (ppm)

(R)-ranolazine


181

Concentration

(ppm) Area

0.1 72

0.25 139

0.50 209

1.00 502

5.00 1589

25.00 6900

50.00 15234

100.00 31002

Results

Intercept -46.95

Slope 308.1

r2 0.9996

Figure 15 : Linearity results for (S)-ranolazine

0

5000

10000

15000

20000

25000

30000

35000

Pea

k A

rea

Concentration (ppm)

(S)-ranolazine


182

5.2.5 Results of LOD and LOQ

The LOD and LOQ concentration were estimated to be 100 and 200 ng/mL for

both enantiomers respectivey, where singal-to-noise ratio criteria match. The results are

summarized in Table 5.

(R)-ranolazine (S)-ranolazine

LOD (ng/mL) 100 100

S/N 6.7 6.2

LOQ (ng/mL) 200 200

S/N 11.5 12.1

Table 5: Results of Sensibility

5.2.6 Results of Recovery Study in Formulation

The recovery study was carried out in triplicate by spiking placebo with three

concentrations (400, 500 and 600 ng/mL) of individual enantiomer’s standards and

assaying for the chromatographic method. The recovery results are summarized in Table

6.

Added (ng) Recovered (ng) % Recovery % RSD

(R)-ranolazine

400 364.8 91.1 8.2

500 467.5 98.2 6.7

600 564.6 96.1 6.3

(S)-ranolazine

500 362 92.7 7.9

625 467 97.3 8.2

750 573.6 91.5 6.3

Table 6: Recovery result of (R)-ranolazine and (S)-ranolazine


183

5.2.7 Results of Solution Stability

The sample was analyzed for 24 h at room temperature, i.e., at 25°C. Resolution

and composition of (R)- and (S)- enantiomers were observed during the study period.

Time interval (h) % area bias

Resolution (R)-ranolazine (S)-ranolazine

Initial - - 1.51

6 0.04 0.12 1.50

12 0.64 -0.34 1.48

18 -0.24 -0.41 1.50

24 -0.41 0.91 1.50

Table 7: Results of solution stability study

No significant change was observed in resolution and peak area composition of

enantiomers during the solution stability study. The data are presented in Table 7, It can

be seen from the data that % bias of area for enantiomers was less than 1% hence sample

solution and mobile phase are stable for 24h at room temperature, i.e., at 25°C.

5.2.8 Results of Robustness

The chromatographic resolution between both enantiomers was used to

evaluate the method robustness under modified conditions. In robustness study, the

racemic ranolazine sample was analyzed with change of different experimental

conditions as a part of robustness study. The resolution between (R)- and (S)-

enantiomer peaks were remain more than 1.40 for all deliberately changed

chromatographic conditions and this confirmed the robustness of the method. The

results are summarized in Table 8.


184

Parameters Resolution between two enantiomers

Flow rate (mL/min)

1.0 1.52

1.2 1.50

1.4 1.48

Column temperature (°C)

32 1.51

35 1.51

38 1.49

Mobile Phase Content (n-heptane (0.1%TFA), ethanol, methanol)

55: 38:7 (v/v/v) 1.42

60: 35:5 (v/v/v) 1.51

65:33:2 (v/v/v) 1.48

Table 8: Results of robustness study

[6] Conclusion

The develop method is an unique solution for enantioselective analysis of

ranolazine enantiomers in pharmaceuitical formulation. Varioous glycopeptides based

chiral stationary phases were evaluated with combination of mobile phase modes. The

baseline separation was achieved on Chirobiotic T column using mobile phase

consisted of n-heptane (0.1%TFA), ethanol, methanol (60:35:5, v/v/v).

The method was validated showing satisfactory data for all the tested validation

parameters and the method was found to be sensitive, accurate and linear over the tesed

concentration range. The accuracy data proved that the developed method can be used for

the direct quantitative estimation of ranolazine enantiomers. This method can be used for

routine pharmaceutical analysis in quality control laboratories.


185

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