Validated Method for the Simultaneous Determination of Lisinopril, Pravastatin, Atorvastatin and Rosuvastatin in API, Formulations and Human Serum by RP-HPLC

Validated Method for the Simultaneous Determination of Gemifloxacin and

H2-receptor Antagonists in Bulk, Pharmaceutical Formulations and Human

Serum by RP-HPLC; In-vitro Applications

Najma Sultana,a M. Saeed Arayne,b Sana Shamima* and Asia Naza

aResearch Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, University of Karachi, Karachi-75270, PakistanbDepartment of Chemistry, University of Karachi, Karachi-75270, Pakistan

Received January 14, 2011; Accepted May 13, 2011; Published Online May 25, 2011

Simple, isocratic and rapid RP-HPLC method has been developed for the simultaneous analysis of

gemifloxacin and H2-receptor antagonists i.e. Cimetidine, Famotidine and Ranitidine, in bulk, pharma-

ceutical formulation and human serum. Separation was achieved on the RP-Mediterranea column [C18

(250 � 4.6 mm, 5 µm)] at ambient temperature using mobile phase consisting of acetonitrile: methanol:

water (20:28:52 v/v/v pH 2.8 adjusted by phosphoric acid). Flow rate was 1.0 mL/min with an average op-

erating pressure of 180 kg/cm2. Gatifloxacin (GATI) was used as an internal standard (IS). Quantitation

was achieved with UV detection at 221, 256 and 267 nm, respectively. Linear calibration curves, at con-

centration ranges of 0.05-37.5 �gmL-1 with a correlation coefficient of ±0.9994. The detection and quanti-

fication limits were in the ranges of 0.023-0.250 µgmL-1 and 0.071-0.756 µgmL-1, respectively. Fried-

man’s and Student’s t-test were applied to correlate these results. Method was validated in terms of selec-

tivity, linearity, precision, robustness, recovery, limits of detection and quantitation and is applicable to

the routine analysis of GFX and H2-receptor antagonists, alone or in combination.

Keywords: Gemifloxacin; H2-receptor antagonists; RP-HPLC; Human serum; Student’s t-test.

INTRODUCTION

Gemifloxacin (GFX) Fig. 1 is a fourth generation

fluoroquinolone antibacterial compound with enhanced af-

finity for bacterial topoisomerase IV and is being used for

the treatment of respiratory and urinary tract infections.1-3

It is particularly active against Gram-positive organisms

including penicillin, macrolide, and quinolone-resistant

Streptococcus pneumoniae,4 4-folds more potent than

moxifloxacin against S. pneumoniae.5-7 Furthermore, the

compound has shown potent activity against many organ-

isms that cause urinary tract infections and bronchitis.8

Few analytical methods have been reported for the es-

timation of GFX; including tandem mass-HPLC,9,10 micro-

chip electrophoresis,11 chiral HPLC12 and chiral counter-

current chromatography.13,14 Ion-pair spectrophotometric

method was described for the assay of gemifloxacin mesylate

by Marothu et. al.15 Barbosa et al studied dissociation con-

stants of series of compounds including diuretics and quin-

olones in several acetonitrile: water mixtures.16 Our re-

search group has also developed RP-HPLC methods of

gemifloxacin, alone17 and with diuretics.18

H2-receptor antagonists may alter the absorption, me-

tabolism, or renal excretion of concurrently administered

drugs.19 Literature survey also revealed that in-vivo or

in-vitro availability of a quinolone is affected by concomi-

tant administration (oral or parentral route) of H2-receptor

antagonists.20-23 Indicating that the increased bioavailabil-

ity of quionolones is due to the presence of H2-receptor an-

tagonists.24,25

To conduct our study we have selected three H2-re-

ceptor antagonists (Fig. 1) i.e. Cimetidine (CIME) which

produces its action by inhibiting the hepatic cytochrome

P450 and its isoforms,26 Famotidine (FAMO) is indicated

for the treatment of duodenal ulcer, gastric ulcer, gastro

esophageal reflux disease, Zollinger–Ellison syndrome,27

as well as secretion stimulated by food and pentagastrin.28

Ranitidine hydrochloride (RANI) has a furan ring structure

and inhibits gastric acid secretion induced by various stim-

Journal of the Chinese Chemical Society, 2011, 58, 629-636 629

* Corresponding author. E-mail: [email protected]

uli.29

Therefore, here we report a simple, easy, quick and

inexpensive isocratic RP-HPLC method for the simulta-

neous determination of gemifloxacin and H2-receptor an-

tagonists. The method is equally valid for the determina-

tion in bulk materials, pharmaceutical dosage formulations

and human serum and is no where else reported before.

EXPERIMENTAL

Chemicals and reagents

All chemicals and reagents were of analytical grade.

Gemifloxacin mesylate was a kind gift from PharmEvo

(99.63%) (Pvt) Ltd, Pakistan. HPLC grade methanol was

obtained from Merck Schuchardt OHG, Darmstadt, Ger-

many. Gemixa™ (Gemifloxacin 320 mg tablets by Bosch

Pharmaceuticals (Pvt) Ltd) While, the H2 receptor antago-

nists used were cimetidine HCl (99.78%) (cimet 400 mg

tablet), ranitidine (99.82%) (acidonil 150 mg tablet) and

famotidine (99.79%) (Hiler 40 mg tablet) of Ferozsons lab-

oratories Ltd., Global Pharmaceuticals Pakistan, and Mul-

ler & Phipps Pakistan (PVT) LTD, respectively. Each prod-

uct was labeled and expiry dates were not earlier than two

years, at the time of study. HPLC grade methanol were

supplied by Tedia company, Inc. (USA).

Statistical study

Standard regression curve analysis was performed by

use of STATISTICA version 7.0 (USA), without forcing

through zero. Linearity graphs were obtained by use of Mi-

cro-soft Excel 2007 software. SPSS software version 10.0

(Carry, NC, USA) was used for the calculation of means,

standard deviations, homoscedasticity of the calibration

plots, and Student’s t-test.

Instrumentation

HPLC system consisted of an LC-20 AT VP Shimadzu

pump, SPD-20AV VP Shimadzu UV visible detector, a

Separation was achieved on a Mediterranea column [C18

(250 � 4.6 mm, 5 µm)]. The chromatographic and inte-

grated data were recorded using a CBM-102 communica-

tion Bus Module Shimadzu. Sonicated by DGU-14 AM,

and filtered through 0.45-micron membrane filter. Cali-

brated Pyrex glassware was used for the solution and mo-

bile phase preparation.

Calibration standards and quality control samples

Standard preparation

Calibration standard solutions of GFX and H2-recep-

tor antagonists were prepared by dissolving 1 mgmL-1 of

drug using mobile phase as solvent, in 100 mL volumetric

flasks. Working solutions were prepared separately by

making serial dilutions from the standard solution to achieve

630 J. Chin. Chem. Soc., Vol. 58, No. 5, 2011 Sultana et al.

Fig. 1. Gemifloxacin mesylate, Cimetidine, Famoti-

dine, Ranitidine and Gatifloxacin.

the desired concentrations between 0.05-37.5 (0.05, 0.1,

1.0, 3.125, 6.25, 12.5, 18.75, 20, 25, 30, 37.5), 1.0-37.5

(1.0, 3.125, 6.25, 12.5, 18.75, 20, 25, 30, 37.5), 0.05-37.5

(0.05, 0.1, 1.0, 3.125, 6.25, 12.5, 18.75, 20, 25, 30, 37.5)

and 1.0-37.5 (1.0, 3.125, 6.25, 12.5, 18.75, 20, 25, 30,

37.5) �gmL-1 for GFX, CIME, FAMO and RANI, respec-

tively.

Procedure for tablets

For quality control samples (QC), twenty tablets of

each formulation were powdered finely and an amount

equivalent to 10 mg of GFX and H2-receptor antagonists

were weighed and then dissolved in the mobile phase. So-

lutions with high, medium and low concentrations i.e.; 80,

100 and 120% were prepared, and then filtered through a

0.45 �m millipore filter, in order to separate out the insolu-

ble excepients. Serial dilutions were prepared by the same

procedure as calibration standards.

All these solutions, calibration and QC were stored at

20 ºC. Once prepared, analyzed daily for inter and intra-

day variations of the method. 20 µL of these solutions were

injected into LC system and chromatographed.

Procedure for human serum

Plasma sample, obtained from healthy volunteers,

was collected and stored at -20 °C. To an aliquot of 1.0 mL

plasma, 10 mL of acetonitrile was added and the mixture

was vortexed for one minute followed by centrifugation at

10,000 rpm for 10 minutes. It was then alienated super-

natant by filtration (0.45 µ pore size membrane filter). An

aliquot human serum sample was fortified with gemi-

floxacin and H2-receptor antagonists to get the final con-

centrations of 0.05-37.5 �gmL-1.

Preparation of 0.1 M hydrochloric acid (simulated gas-

tric juice)

9 mL HCl of analytical grade (36%, 11 N) was taken

in a liter volumetric flask and the volume was made up to

the mark with de-ionized water.

Preparation of Buffer of pH 4 (chloride buffer)

It was prepared by dissolving 3.725 g of potassium

chloride in deionized water in one liter; the pH was ad-

justed by 0.1 N HCl.

Preparation of Buffer of pH 7.4 (phosphate buffer)

0.6 g of potassium dihydrogen O-phosphate, 6.4 g of

disodium hydrogen O-phosphate and 5.85 g of sodium

chloride was dissolved in sufficient deionized water to pro-

duce 1000 mL and pH adjusted if necessary.

Preparation of Buffer of pH 9 (ammonia buffer)

Dissolve 4.98 g of ammonium chloride in 1000 mL of

deionized water and then pH was adjusted to 9 with 10%

ammonia.

Procedure for in-vitro interaction studies

For interaction study stock solutions of GFX and

H2-receptor antagonists were prepared by dissolving 10 mg

of the drug in 100 mL of simulated gastric juice, using buff-

ers of pH 1, 4, 7.4 and 9 as solvents, individually, then

sonicated. Gemifloxacin solution was mixed with solution

of Cimetidine in a flask, to give the final concentration of

50 �g mL-1 which was then kept in water bath at 37 ± 5 �C

for 3 hours. Aliquots of 5 mL were withdrawn at an interval

30 min for 180 minutes, following filtration through nor-

mal filter paper then 0.45 �m filter paper to avoid any hin-

drance and then subjected to assay by RP-HPLC. Same

procedure was repeated with every H2-receptor antagonists

for interaction study.

RESULTS AND DISCUSSION

Method development

The aim of the present study was to develop a simple,

isocratic, accurate and sensitive RP-HPLC method for the

simultaneous determination of GFX and H2-receptor an-

tagonists. Mediterranea column [C18 (250 � 4.6 mm, 5

µm)] was selected among all the studied columns as this

column provides the best peak shapes, efficiencies and re-

producible separations of non-polar compounds while min-

imizing solvent usage. A UV scan showed a maximal

absorbance at 221, 256 and 267 nm, for Cime, Famo and

Rani, respectively. The chromatographic conditions, espe-

cially the composition of the mobile phase, were optimized

through several trials to achieve symmetric peak shapes for

GFX and H2-receptor antagonists as well as shorter run

time. It was found that a mobile phase containing a certain

proportion of methanol and water gave symmetric peak

shapes for all drugs. Inclusion of acetonitrile in the mobile

phase was crucial in obtaining high signal intensity. There-

fore the final mobile phase composed of acetonitrile, meth-

anol and water in ratio of (20:28:52, v/v/v) provides good

resolution.

In order to keep constant pH of the mobile phase,

85% Phosphoric acid was added into mobile phase to

achieve the desire pH. The mobile phase pH had a little im-

pact on resolution and the best separations were observed

at pH 2.8. The chromatographic conditions were optimized

to achieve best separation and to get best resolution be-

tween analytes and to optimize chromatographic parame-

Simultaneous Determination of GMFX and H2 Receptor Antagonists J. Chin. Chem. Soc., Vol. 58, No. 5, 2011 631

ters like resolution, tailing factor and retention time.

Peaks were identified using retention times compared

with those of standards. Retention times were 2.66, 2.85,

2.75, 3.81 and 4.67 minutes for CIME, FAMO, RANI,

GFX and GATI (IS), respectively. For validation of analyti-

cal methods, the guidelines of the International Conference

on the Harmonization of Technical Requirements for the

Registration of Pharmaceuticals for Human Use30 and USP

200231 were followed for the accuracy tests, precision, spe-

cificity, linearity, work strip and robustness of the method.

Method validation

System suitability

It is an imperative module of method validation to

make certain that the operational system is running appro-

priately throughout the analysis. The system was equili-

brated with the initial mobile phase composition, followed

by 10 injections of the same standard. These 10 consecu-

tive injections were used to evaluate the system suitability

on each day of method validation (Table 1).

Calibration curves

Calibration curves were characterized by different

linear segments for all the drugs. These curves were ob-

tained using the linear least squares regression procedure

as shown in Table 2. These analysis results reveals good

linear correlations between all the drugs having correlation

coefficient (r) value >0.999. The homoscedasticity of the

calibration plots, tested by Friedman’s tests were found to

be significantly linear over the tested ranges.

Recovery studies

The accuracy of the method was evaluated from the

recovery results of spiked placebo samples. Appropriate

portions of stock solution of drugs were spiked into blank

placebo matrix to produce concentrations of 4.0, 5.0 and

6.0 µgmL-1 of the theoretical concentration. Mean recovery

of spiked samples were in the ranges of 98.34-100.59%.

Recovery tests were performed by adding known amounts

of standard solutions to sample followed by analysis using

proposed method. Three runs were performed for every

concentration and then peak area was calculated (Table 3).

The average recovery for each level was calculated as indi-

cated by Association of Official Analytical Chemists Inter-

national.32-35

Precision

Instrumental precision was determined by six repli-

cate determinations of standard solution i.e. repeatability.

Method precision or intra-assay precision was performed

by preparing six different samples involving different

weightings. Each solution was injected in triplicate under

the same conditions and the mean values of peak area re-

sponses for each solution were taken. The precision of the

method was analyzed as % RSD throughout the linear

range of concentrations30-33 (Table 4). All the results were

correlated and found insignificant by student’s t-tests indi-

cating no remarkable difference in intra and inter day

632 J. Chin. Chem. Soc., Vol. 58, No. 5, 2011 Sultana et al.

Table 1. System suitability parameters

DrugsRetention time

(tR)

Capacity factor

(K')

Tailing factor

(T)Resolution (RS)

Theoretical

plates (N)

Separation

factor (�)

GFX 3.75 2.57 1.27 1.91 2018 2.16

CIME 2.66 2.61 1.32 2.26 2167 5.17

FAMO 2.80 4.19 1.29 1.94 2545 2.28

RANI 2.75 3.16 1.77 1.89 1933 2.20

Table 2. Regression characteristics

Drugs Conc. (µgmL-1) r2 Slope Intercept S.E.E S.E LOD (µgmL-1) LOQ (µgmL-1)

GFX 0.05-37.5 0.9991 53311 1.27 0.44 0.21 0.244 0.741

CIME 1.0-25 0.9992 57953 2.03 0.61 0.45 0.250 0.756

FAMO 0.05-37.5 0.9992 25759 3.35 7.57 3.24 0.142 0.431

RANI 1.0-37.5 0.9997 9217 1.982 0.23 0.15 0.023 0.071

Conc., Concentration; S.E.E, Standard error of estimate; S.E, Standard error; LOD, Limit of Detection and LOQ,

Limit of Quantitation.

precision.

LOD and LOQ

LOD and LOQ values ranging from 0.023-0.250

µgmL-1 and 0.071-0.741 µgmL-1 respectively. LOD and

LOQ were calculated as three and ten times of the noise

level, respectively and can be expressed as:

LOD = 3.3 d/S and LOQ= 10 d/S

where:

d = the standard deviation of y-intercept of the regres-

Simultaneous Determination of GMFX and H2 Receptor Antagonists J. Chin. Chem. Soc., Vol. 58, No. 5, 2011 633

Table 3. Recovery studies of gemifloxacin and H2 receptor antagonists

Assay Assay in human serum (spiking method)Parameters

Conc. (µgmL-1)

Spiked GFX CIME FAMO RANI GFX CIME FAMO RANI

4.0 3.998 3.90 3.994 3.934 3.998 3.90 3.994 3.934

5.0 4.985 4.99 4.979 4.970 4.985 4.99 4.979 4.970Conc. found

6.0 6.035 6.01 6.033 6.011 6.035 6.01 6.033 6.011

4.0 99.96 98.4 99.85 98.34 99.96 98.4 99.85 98.34

5.0 99.70 99.9 99.58 99.40 99.70 99.9 99.58 99.40Recovery %

6.0 100.59 100.1 100.56 100.19 100.59 100.1 100.56 100.19

Table 4. Precision of gemifloxacin and H2 receptor antagonists

Pharmaceutical

Formulation (%RSD)

Human Serum

(%RSD)

Pharmaceutical

Formulation (%RSD)

Human Serum

(%RSD)Drug Conc. (µgmL-1)

D1 D2 D1

Drug Conc. (µgmL-1)

D1 D2 D1

0.05 0.47 0.42 1.42 0.05 0.65 0.64 1.67

0.1 0.29 0.25 0.25 0.1 0.10 0.12 0.13

1 0.18 0.15 0.85 1 0.28 0.30 0.25

3.125 0.38 0.35 0.35 3.125 0.54 0.56 0.55

6.25 0.31 0.41 0.41 6.25 0.12 0.14 0.11

12.5 0.38 0.36 1.36 12.5 0.48 0.50 0.45

18.75 0.16 0.54 1.54 18.75 0.47 0.51 1.49

20 0.12 0.11 1.11 20 0.54 0.57 0.55

25 0.16 0.15 0.85 25 0.56 0.57 0.59

30 0.16 0.14 0.14 30 0.33 0.36 0.35

GFX

37.5 0.17 0.18 0.68

FAMO

37.5 0.20 0.23 1.25

1 0.22 0.23 0.23 1 0.65 0.68 0.68

3.125 0.65 0.67 0.67 3.125 1.19 1.20 1.20

6.25 0.18 0.15 0.15 6.25 1.01 1.10 1.10

12.5 1.15 1.14 1.14 12.5 1.17 1.19 1.19

18.75 0.50 0.55 0.55 18.75 0.91 0.93 0.93

20 0.85 0.88 0.88 20 0.35 0.39 0.39

25 1.08 1.05 1.05 25 0.75 0.76 0.76

30 0.84 0.87 0.87 30 1.25 1.24 1.24

CIME

37.5 0.52 0.56 0.56

RANI

37.5 0.42 0.45 0.45

t-Test: paired two sample for precision

df t P(T > t) two tail

GFX 10 -6.8 0.512

FAMO 10 -5.333 0

CIME 8 -1.244 0.249

RANI 8 -2.874 0.021

D1 = Intra-day and D2 = Inter-day variations, df = Degree of freedom

sion line.

S = the average slope of regression lines.

Specificity and selectivity

Fig. 2 showed the typical chromatograms of drugs

alone and with spiked human serum. No significant inter-

ference was observed from endogenous substances in drug

free human plasma at the retention time of all drugs under

study.

Ruggedness

The ruggedness of the method was established by de-

termining GFX and H2 receptor antagonists, in bulk, dos-

age formulation and in human serum in two different labs.

Lab 1 was Research Institute of Pharmaceutical Sciences,

Department of Pharmaceutical Chemistry, Faculty of Phar-

macy, University of Karachi while other lab was lab 9, De-

partment of Chemistry, Faculty of science, University of

Karachi. Using, two different instruments of same configu-

ration; LC 20, on different days by different analytes (Table

4). All the results were in good limits.

Robustness

Robustness of the method was accomplished by de-

signed modifications made to the method parameters such

as composition, flow rate and pH of the mobile phase (Ta-

ble 5) and it was found that the % R.S.D values did not ex-

ceed more than 1.5%.

In-vitro Application of the proposed method

The developed method was successfully employed

for the quantization of gemifloxacin and H2 antagonists.

The drugs were analyzed by measuring the area under

curve (AUC) and % recovery. Interactions of gemifloxacin

with H2-receptor antagonists were studied in simulated

body fluids at 37°C employing high performance liquid

chromatographic techniques (Table 6).

Availability of all the gemifloxacin was found to be

69.36-99.55% at pH 1, 4, 7.4 and 9. Drifts in the availabil-

ity values of gemifloxacin in presence of cimetidine and

ranitidine was observed at every pH. In presence of cimeti-

dine, gemifloxacin was 73.22-99.55% available. In case of

famotidine the % availability of gemifloxacin was in the

range 71.47-96.4; whereas with ranitidine the % availabil-

ity was found to be 69.36-98.28.

On the basis of present results and according to litera-

ture it can be inferred that the elevation in availability val-

ues may be due to an addition of functional group being at-

tached to the pharmacophore of gemifloxacin and these in-

teractions were pH dependent.

CONCLUSION

The proposed HPLC method is simple, isocratic,

rapid, specific, accurate and precise for simultaneous de-

634 J. Chin. Chem. Soc., Vol. 58, No. 5, 2011 Sultana et al.

Fig. 2. Representative chromatograms of CIME (A), FAMO (B), RANI (C) with GFX and GATI as an internal standard in

mobile phase of acetonitrile: methanol: water (20:28:52 v/v/v) and human serum.

termination of GFX and H2 receptor antagonists, in bulk,

pharmaceutical dosage formulation and human serum has

been developed for the first time. Hence, it can be recom-

mended for the routine quality control and evaluation of

clinical data as well, of these drugs.

ACKNOWLEDGEMENT

The authors wish to thanks Higher Education Com-

mission (H.E.C) Pakistan for their financial support.

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Table 5. Robustness of the proposed method (n=6)

DrugsRetention time

(tR)

Capacity factor

(K')

Tailing factor

(T)Resolution (Rs)

Theoretical

plates (N)

Separation factor

(�)

A: pH of mobile phase 2.8 ± 0.2

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CIME 2.66 ± 0.41 2.61 ± 0.90 1.32 ± 0.40 2.26 ± 0.74 2167 ± 32 5.17 ± 0.11

FAMO 2.80 ± 0.50 4.19 ± 0.21 1.29 ± 0.14 1.94 ± 0.29 2545 ± 25 2.28 ± 0.09

RANI 2.75 ± 0.82 3.16 ± 0.20 1.77 ± 0.15 1.89 ± 0.53 1933 ± 43 2.20 ± 0.23

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at pH 01 at pH 4

Time

(min)Gemi + Cime Gemi + Famo Gemi + Rani

Time

(min)Gemi + Cime Gemi + Famo Gemi + Rani

0 100 100 100 100 100 100 0 100 100 100 100 100 100

30 99.55 94.1 93.1 98 85.5 92 30 90.3 97.3 84.6 99.8 90.1 91.3

60 96.73 87 92.7 91.7 78 87 60 93.6 82.2 86.8 93.1 84.4 94.9

90 94.02 80.4 83.7 94.7 79.1 86 90 85.5 84.3 80 91 85 83.8

120 99.16 80.6 86.5 98.2 75.2 79 120 73.8 87.1 76.8 82.9 82 89.2

150 83.74 81.9 77.4 84.1 69.4 77 150 76.6 88.3 74.8 79.5 83 88

180 81.32 74.9 79.4 87.4 69.5 76 180 77.8 86.7 71.5 79 85.5 83.3

at pH 7.4 at pH 9

Time

(min)Gemi + Cime Gemi + Famo Gemi + Rani

Time

(min)Gemi + Cime Gemi + Famo Gemi + Rani

0 100 100 100 100 100 100 0 100 100 100 100 100 100

30 94.32 96.1 100 100 98.3 98 30 93.7 96.8 98.9 97.4 95.3 94.9

60 97.17 94.3 96.8 101 92.8 98 60 93.8 93.7 97.4 93.3 94.5 94.4

90 93.38 91.3 96.4 93.3 91.8 96 90 89.9 87 94.1 87.9 94.4 87.5

120 92.38 89.7 97.7 94.3 84.1 90 120 89.2 89.9 94.8 85.5 89.1 83.4

150 92.75 85.7 94.8 99.2 82.8 90 150 79.8 84.8 87.5 84.1 88.4 79.1

180 94.02 83.8 94.2 98.9 80.8 82 180 73.2 86.3 84.2 84.1 77.6 79.1

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