separation and estimation of five imidazoles by packed column supercritical fluid chromatography

Separation and estimation of ®ve imidazoles by packedcolumn supercritical ¯uid chromatography

Yagnesh Patela, U.J. Dhordaa, M. Sundaresanb,*, A.M. Bhagwatb

a I.Y.College of Arts, Science and Commerce, Jogeshwari (East), Mumbai-400 060, Indiab C.B.Patel Research Centre for Chemistry and Biological Sciences, Mithbai College Building, Vile Parle (West), Mumbai-400 056, India

Received 6 June 1997; received in revised form 14 November 1997; accepted 5 January 1998

Abstract

An isocratic, isothermal and isobaric supercritical ¯uid chromatographic method using packed columns has been developed

for the separation and estimation of 5 imidazole, viz., metronidazole, tinidazole, secnidazole, albendazole and mebendazole.

Supercritical CO2 doped with methanol has been used as the mobile phase and separations were carried out on a reverse phase

octadecyl column packed with 5 mm particles. Detection was with a UV multiwavelength spectrophotometer equipped with a

16 ml cell with a pathlength of 5 mm. The wavelength used was 254 nm. The internal standard method was used, with

albendazole serving as internal standard for the other four imidazoles and secnidazole for albendazole (2 sets). A full scale

validation of the method of estimation of drugs has been carried out and the viability of the method has been established. The

method was successfully extended to pharmaceutical dosage forms available locally. # 1998 Elsevier Science B.V.

Keywords: Supercritical ¯uid chromatography; Imidazole derivatives

1. Introduction

The present interest in packed column supercritical

¯uid chromatography (PC-SFC) for the separation and

estimation of drugs and pharmaceuticals stems from a

series of publications [1±8] in which the authors have

claimed superior, or at least, equal chromatographic

®gures of merit for this technique as compared to

liquid chromatographs (LC). The low viscocities and

high diffusivities of supercritical ¯uids enhance the

chromatographic ef®ciencies. Further, SFC generates

less volumes of disposable solvent waste and employs

a cheap non-toxic gas, CO2. The pioneering work in

this ®eld has been due to Berger and Wilson who used

this method to separate 10 phenothiazine-based anti-

psychotics [1]. This work was followed by the separa-

tion of ten antidepressant drugs [2] and stimulants [3].

The separation of benzodiazepines was demonstrated

by Takaichi et al. [4]. The utility of SFC for the

estimation of drugs in biological ¯uids has also been

demonstrated [5±9]. The work was further extended to

commercial tablets [5]. The claims of SFC for equality

or superiority to LC is still a matter of dispute and

there arises a need for more work to either establish the

claim or to deny it. In this context the present work

aims at an isobaric, isothermal and isocratic separation

of a series of imidazole derivatives, using modi®ed

supercritical carbon dioxide and packed columns.

Analytica Chimica Acta 362 (1998) 271±277

*Corresponding auther. Fax: +91 022 613 3400.

0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.

P I I S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 0 0 6 - 3

Imidazole or 1,3-diazole is a weak base from

which several derivatives have been synthesised

and used as a drugs. Metronidazole and tinidazole

are 5-nitroimidazole derivatives with activity against

anaeriobic protozoa and bacteria. Secnidazole is

also a 5-nitroimidazole derivative which is used

in the treatment of amaoebiasis and Trichomona

infections. Albendazole and membendazole are

benzimidazole derivatives which are antitheliminitic

against most nematodes and some cesatode worms.

From a total of nine imidazole derivatives which

were investigated by this technique it was possible

to separate and estimate only the 5 derivatives identi-

®ed above, by the use of methanol doped carbon

dioxide. The structures of the ®ve drugs are given

in Fig. 1.

Fig. 1. Structures of imidazole derivatives. (1) Albendazole, (2) Secnidazole, (3) Tinidazole, (4) Metronidazole, (5) Mebendazole, (6)

Clotrimazole, (7) Econazole, (8) Miconazole, (9) Ketoconazole.

272 Y. Patel et al. / Analytica Chimica Acta 362 (1998) 271±277

2. Experimental

2.1. Apparatus

A JASCO-900 series SF chromatograph was

employed for the study. The apparatus was equipped

with 2 pumps (PU-980) for pumping supercritical CO2

and the modi®er. The pumping rates could be adjusted

from 0.001±10 ml minÿ1 for CO2 and the modi®er. A

Rheodyne model 7125 injection valve was used with

an external 20 ml loop. Detection was at 254 nm with a

UV multiwavelength spectrophotometer equipped

with a 16 ml high pressure cell with a path length of

5 mm. The analytes were eluted using a JASCO-RP-

18 Column (250�4 mm) packed with 5 mm particles.

2.2. Reagents and chemicals

Carbon dioxide used was 99.9% pure obtained from

Bombay Carbon Dioxide, Mumbai. Methanol was

HPLC grade from S.D.Fine Chemicals. The samples

of the drugs were obtained from reputed ®rms with

certi®cate of analysis. The samples were assayed in

this laboratory also. Individual drug solutions were

prepared by weighing appropriate quantities and dis-

solving them in methyanol to give stock solutions of

500 mg mlÿ1. Mixtures were obtained by mixing the

individual solutions. Where necessary, when solution

volumes exceeds the desired volume, the mixtures

were evaporated to dryness under nitrogen at 558C and

the residue reconstituted in the desired volume of

methanol. The stock solutions were diluted in ten-fold

stages to the appropriate required concentrations.

3. Results and discussion

Supercritical ¯uid chromatography (SFC) offers

three degrees of freedom, viz, pressure, temperature

and modi®er concentration, for the optimisation of

chromatographic parameters. While the ®rst two para-

meters regulate the density of the gaseous ¯uid, the

third parameter can be regulated by both the rate of

¯ow of CO2 and that of the modi®er. The process of

optimisation thus involves both the density and modi-

®er concentration programming with four variables. A

set of preliminary experiments with individual drugs

and then with mixtures could establish a separation

strategy involving the four variables. In these experi-

ments, analytes were detected at 254 nm. Retention

times were measured as a function of outlet pressure

(9.117±12.660 MPa), temperature (40±558C) and

modi®er concentration (7.5±15%). The ¯ow rate of

CO2 was varied from 1.5 to 4 ml minÿ1 and ®nally

®xed at 2.0 ml minÿ1. Table 1 lists the retention times

of the ®ve drugs as a function of temperature, pressure

and modi®er concentration.

A perusal of Table 1 reveals that the effect of

pressure and temperature on the retention times and

hence on separation is minimal. Pressures�8.13 MPa

did not provide proper separation. At 8.13 MPa the

retention times of albendazole and secnidazole were

too close to provide separation. For tinidazole and

metronidazole, at <8.13 MPa no discernible pattern

was obtained. This behaviour was repeated above

12.66 MPa, where, too, the retention times were too

near to offer satisfactory capacity factors.

In contrast to reports [1] that temperatures have the

largest effects on selectivity, no dramatic effect was

noticed in this case (Table 1). For a 158C change, a

shift of ca. 1 min was observed in retention time.

Retention times with respect to modi®er concentra-

tions showed a monotonous decrease with increase in

the concentration range 7±15%. At 15%, the retention

times were so near for all the drugs that all the curves

almost merged.

Thus, from Table 1 the parameters of pressure,

temperature and modi®er concentration for optimum

separation and quantitation of the 5 drugs could be

ascertained as 9.80 MPa, 458C and 11.1%, respec-

tively. The mobile phase which was run at

2.0 ml minÿ1 consisted of 11% methanol in carbon

dioxide at 9.80 MPa outlet pressure and 458C. A

representative chromatogram, obtained under these

conditions, of a mixture of the solutions of the 5

drugs, each at 100 mg mlÿ1 is shown in Fig. 2. The

injection volume was 20 ml and the other conditions

were given as in the Figure captions. With these

parameters, steady state conditions were found that

produced baseline resolution of all the drugs in the

mixture. The chromatographic ®gures of merit for

these drugs on a RP-18 column are given in Table 2.

For linearity studies, nine different concentrations

of each drug were assayed. The concentration ranges

for each drug are given in Table 2. The detector

responses are expressed in peak heights. Both peak

Y. Patel et al. / Analytica Chimica Acta 362 (1998) 271±277 273

areas and heights were found to show a linear relation-

ship with concentration of the 5 drugs. As the internal

standard method was employed, calibration graph

were obtained by plotting the added drug concentra-

tion (mg mlÿ1) vs. peak height ratios (drug/internal

standard). Two sets of experiments were carried out,

one with albendazole (2 mg) as the internal standard

for the other four derivatives and secnidazole (3 mg) as

the internal standard for albendazole. The linear

regression (least-squares ®t) calibration data are pre-

sented in Table 3 together with the correlation coef®-

cients. For convenience, only peak height ratios are

mentioned. The Syx term in Table 3 refers to the

standard deviation of the residuals from the linear

Table 1

Effect of temperature, pressure and modifier concentrations on capacity factors of the drugs

Modifier

conc. (%)

Temp

(8C)

Pressure

(MPa)

Capacity factor (k0)

Alben Secni Tini Metro Meben

1. 11.11 45 7.84 ± 5.6 ± 7.8 10.8

11.11 45 8.82 4.2 5.1 6.3 7.1 8.5

11.11 45 9.80 3.7 4.1 5.6 5.9 7.3

11.11 45 10.78 3.2 4.1 4.8 5.8 6.2

11.11 45 12.25 2.9 3.8 4.4 5.2 5.5

2. 11.11 40 9.80 4.3 5.5 6.4 7.5 8.1

11.11 45 9.80 3.7 4.1 5.6 5.9 7.3

11.11 50 9.80 3.7 4.8 5.6 7.1 7.8

11.11 55 9.80 4.3 5.9 6.5 8.0 8.8

3. 6.67 45 9.80 6.5 9.5 10.8 13.5 16.3

8.89 45 9.80 4.6 5.9 7.1 7.9 10.2

11.11 45 9.80 3.7 4.1 5.6 5.9 7.3

13.33 45 9.80 3.7 4.6 5.4 6.2 7.4

Fig. 2. Typical SFC separation of drugs eluted out from a JASCO RP-18(250�4.0 mm) 5 mm column under optimised conditions. Sequence of

the peaks as in Fig. 1. Conditions: CO2 flow rate: 2.0 ml minÿ1; Modifier (methanol) flow rate: 0.25 ml minÿ1; Temperature: 4508C; Pressure:

9.80 MPa; Retention time (min): (1) 6.71 (2) 7.89 (3) 9.13 (4) 10.32 (5) 11.98.


least-squares regression. The statistical evaluation has

been done according to Gordus [10].

The absorption-elution spectra of all ®ve drugs as a

function of wavelength from 210±320 nm are shown

in Fig. 3. As can be seen from Fig. 3 the limits of

quantitation of the individual drugs can be much

improved (2±7 times) by choice of an appropriate

wavelength. The conditions for this experiment used

the optimised parameters of pressure, temperature and

modi®er concentration, with 100 mg mlÿ1 for each

drug in the mixture.

The accuracy and precision of the method was

assessed from analytical recoveries of each drug from

spiked concentrations. Table 4 lists the data obtained

over three (low, medium and high) ranges of concen-

tration of the drugs. As can be seen from the Table 4

the errors are about 2% in the higher ranges and ca. 8%

in the lowest range. The reproducibility of the method,

as assessed by inter- and intra-day determinations,

showed a coef®cient of variation well below 5%, as

shown in Table 5. The separation of clotrimazole,

ketoconazole, econazole nitrate and meconazole

nitrate also from the 5 drugs under study was inves-

tigated under the same conditions. Of these ketoco-

nazole was not eluted at all, while the peaks of

clotrimazole and mebendazole merged with each

other. Both the nitrates were not eluted using the

present mobile phase and the reversed phase column.

Elution from cyano, phenyl and silica columns did not

offer any advantage with respect to capacity, selectiv-

ity factor and faster elution. The use of a binary

modi®er, i.e., methanol and butanol in different pro-

portions, also failed to offer any better separation or

elution of these three imidazole derivatives.

Table 2

Chromatographic figures of merit. Concentration of each component in the mixture was 100 mg mlÿ1

Drug RRT a

(min)

Symmetry

factor (T)

Capacity

factor (�K 0)No. of theoretical

Plates (N)

HETP

�h� � LN

Albendazole 1.00 1.17 3.65 706 0.035

Secnidazole 1.19 1.17 4.60 1024 0.024

Tinidazole 1.41 1.20 5.50 1878 0.013

Metronidazole 1.58 1.08 6.30 2018 0.012

Mebendazole 1.78 1.25 7.40 1129 0.022

a RRT�relative retention time.

Table 3

Linear (least-squares fit) regression data for calibration

Drug Conc.range (mg mlÿ1) Slope m�tcl,vSm a Intercept b�tcl,v Sb a R2 Syx

Albendazole 1.0±20.0 0.034�0.004 0.004�0.019 0.999 0.007

20.0±150.0 0.047�0.002 ÿ0.039�0.025 0.999 0.088

Secnidazole 2.0±30.0 0.021�0.009 0.03�0.095 0.992 0.032

30.0±270.0 0.032�0.005 ÿ0.039�0.839 0.999 0.313

Tinidazole 2.0±40.0 0.019�0.002 0.003�0.027 0.999 0.01

40.0±370.0 0.024�0.001 ÿ0.11�0.338 0.999 0.117

Metronidazole 1.0±20.0 0.022�0.005 0.011�0.042 0.998 0.015

20.0±210.0 0.029�0.002 0.031�1.070 0.999 0.123

Mebendazole 0.50±8.0 0.087�0.021 0.032�0.046 0.998 0.018

8.0±64.0 0.116�0.001 0.032�0.072 0.999 0.026

a Two-tailed confidence coefficients for Student's t distribution with V degrees of freedom.


The method was successfully extended to pharma-

ceutical dosage forms of individual drugs available

locally. For this purpose, 20 tablets were crushed to a

®ne powder and homogenised. An appropriate quan-

tity was dissolved in 20 ml of methanol and the

solution ®ltered. After adequate dilutions, 20 ml of

the solution was injected into the column. The relevant

information of the total drug in the tablet, amount of

Fig. 3. Conditions same as in Fig. 2. Peak responses vs. wavelength. 1±5 as in Fig. 1.

Table 4

Accuracy and precision of the method (n�5)

Drug Conc.

(mg mlÿ1)

Mean found

conc. (mg mlÿ1)

Error

(%)

CV

(%)

Albendazole 1.20 1.10 8.3 1.79

38.5 41.6 8.1 0.69

153.8 150.6 2.1 0.44

Secnidazole 2.10 1.97 6.2 2.39

67.3 70.3 4.5 2.42

269.2 262.7 2.4 0.71

Tinidazole 2.40 2.50 4.2 4.21

38.5 40.9 6.3 0.83

307.7 301.2 2.1 0.64

Metronidazole 1.62 1.72 6.2 2.92

25.6 26.8 4.6 1.73

205.1 200.6 2.2 1.02

Mebendazole 0.50 0.48 4.0 6.30

16.0 17.1 6.7 1.03

64.1 62.8 2.1 1.59

CV�coefficient of variation.

Table 5

Performance data

Drug Conc.

(mg mlÿ1)

Mean CV

within day (%)

CV between

day (%)

Albendazole 1.20 1.14 1.00

38.5 0.09 0.10

153.5 0.09 0.02

Secnidazole 2.10 3.50 1.40

67.3 0.41 0.39

269.2 0.07 0.002

Tinidazole 2.40 0.99 0.015

38.5 0.35 0.054

307.7 0.05 0.002

Metronidazole 1.62 0.04 1.49

25.6 0.60 0.18

205.1 0.08 0.07

Mebendazole 0.50 4.52 1.55

16.0 0.30 0.16

64.1 0.11 0.02


drug injected and amount recovered is given in

Table 6. None of the excipients was found to interfere

as is evidenced by the data in Table 6. A typical

chromatogram of the tablet extract for albendazole

is given in Fig. 4. Table 6 thus amply con®rms the

viability of empolying packed column SFC for the

assay of these ®ve imidazoles in dosage forms.

4. Conclusions

Packed column SFC is shown to be a viable tech-

nique for the analysis of mixtures of some imidazole

derivatives. Even though it was not possible to sepa-

rate a further four imidazole derivatives from the ®ve

under investigation, all the compounds under study

produced symmetrical, ef®cient peaks. The modi®er

concentration has been shown to have the largest

effect on both retention and selectivity for the separa-

tion of these imidazole derivatives. Quantitation limits

are lower than those in LC and can be improved by a

factor of 2±7 times by the choice of an appropriate

wavelength of detection. The advantages over LC are

the speed and different selectivity of SFC.

Acknowledgements

The authors thank Prof. C.P. Kelkar, Director

(Admn) and Dr. R. Kalyanaraman, Prof. Emeritus,

C.B. Patel Research Centre, Mumbai-56, Dr. Mrs.

Devyani Dave, Principal, Ismail Yusuf College,

Mumbai-60, and coworkers I.C. Bhoir and V.R. Bari

for encouragement in carrying out this work.

References

[1] T.A. Berger, W.H. Wilson, J. Pharm. Sci. 83 (1994) 281.



[4] K. Takaichi, T. Shinohara, T. Nagano, Hochudoku 13 (1995)

132.

[5] B.R. Simmons, N.K. Jagota, J.T. Stewart, J. Pharm. Biomed.

Anal. 13 (1995) 59.

[6] M.S. Smith, J. Oxford, M.B. Evans, J. Chromatogr. 683

(1994) 402.

[7] C.J. Bailey, R.J. Ruane, I.D. Wilson, J. Chromatogr. Sci. 32

(1994) 426.

[8] J.T.B. Strode, L.T. Taylor, A.L. Howard, M.A. Brooks, J.

Pharm. Biomed. Anal. 12 (1994) 1003.

[9] D.L. Mount, G.D. Todd, V. Navaratnam, J. Chromatogr. 666

(1995) 183.

[10] A.A. Gordus, Analytical Chemistry, Schaum's Outline Series

in Science, NY, USA, 1987, p. 30.

Table 6

Analysis of imidazole dosage forms using packed column SFC (n�5)

Drug Labelled amount (mg) Amount of drug injected (mg) Amount of drug found (mg) Recovery (%)

Albendazole 200 2.0 199.1 99.55

Secnidazole 500 2.0 498.8 99.76

Tinidazole 300 2.0 298.1 99.37

Metronidazole 200 2.0 198.6 99.30

Mebendazole 100 1.0 99.2 99.20

Fig. 4. A typical chromatogram of the tablet extract for

albendazole.


separation and estimation of five imidazoles by packed column supercritical fluid chromatography

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