SIMULTANEOUS DETERMINATION OF IMIDAZOLE IN 2 METHYL IMIDAZOLE BY REVERSE PHASE LIQUID CHROMATOGRAPHY

About Authors:
Chandorkar J.G.
Head, Analytical development laboratory
Indofil Industries Limited
Thane.
jchandorkar-icc@modi.com

Abstract-
2 methyl Imidazole is the active raw material in the manufacturing of  Micanazole Nitrate, Ketokenazole type Active pharma ceuticals. Imidazole is the Impurity which get form during manufacturing of 2Methyl Imidazole.
The Proposed Method is to determine the Impurity as well as Content in 2 Methyl Imidazole.

Reference Id: PHARMATUTOR-ART-1457

Introduction.
Methyl Imidazole is basically Imidazole ring.Imidazole is a heterocyclic compound of five-membered diunsaturated ring structure composed of three carbon atoms and two nitrogen atoms at nonadjacent positions.  They have antibacterial, antifungal, antiprotozoal, and anthelmintic activity. Most commomnly used derivative of Imidazole is 2 Methyl Imidazole.2-Methylimidazole is used as an intermediate for pharmaceuticals, agrochemicals, dyes, textile auxiliaries, pigments and other organic chemicals.

2-Methylimidazole is chiefly used to produce the intermediate of metronidazole, used as the curing agent for epotherm and the auxiliary  agent for colourants of fibre textile as well.

In addition, it can work as the additive agent in the production of poly. Several Methods are cited in Literature to determine  2 Methyl Imidazoe by U.V,Gas Chromatography like Instruments. Proposed method is simple fast accurate using Reversible Phase H.P.L.C. Disclosed is a novel method for  simultaneous determination of 2 Methyl Imidazole and Imidazole  by High Performance Liquid chromatography

Experimental
Reagent and Chemicals -
2 Methyl Imidazole and Imidazole   were procured from Aarti Drugs Limited, Mumbai along with certificate of analysis.

Di Ammonium Hydrogen Orthophosphate Water,  were used of HPLC grade of S.D.Fine chem, make. Sodium Hydroxide was used of Analytical Grade.

Apparatus

A Perkin Elmer make  HPLC- 785  Series Chromatograph equipped with  200Series  Pump, UV Detector.

The column used was Stainless less steel C- 18, 250 x 4.6mm, 5 Micron, Inertsil ODS – 3, operating at room Temperature. The elution was carried out isocratically at flow rate of 0.7 Ml/min. using   0.1 M Diammonium Hydrogen orthophosphate buffer and pH adjusted with 0.1 N Sodium Hydroxide  as Mobile phase. The detector was set at 204 Nm. The response of peak area were recorded and integrated using Delta Chromatographic Software version 5.0 .

Stationary Phase-
Inter Sil ODS -3 ,of C18,250mm Length,Internal Diameter 4.6 mm, and particle size 5.0Micron was used.

HPLC conditions - A mixture of 1.3208 volumes of 0.01 M Di Ammonium Hydrogen Orthophosphate and 1000 ml Water adjusted to pH 3.0 with 01 N Sodium Hydroxide and sonicate and filter it through 0.45 Micron filter membrane and degas it by sonication.

Preparation of Test Solution for the analysis of 2 Methyl Imidazole

Amber Coloured glassware must be used when preparing these solution Reference

Test Solution –
Dissolve 0.10 g of the substance to be examined in the mobile phase & dilute to 50 ml with the mobile phase.

Reference Solution A –
Dissolve a quantity of Imidazole 5.0 mg in the mobile phase & dilute to 50 ml with mobile phase.

Reference Solution B –
Dilute 2.0 ml of Reference Solution [A] to 50 ml with the mobile phase.

Reference Solution C –
To  5.0 ml of Reference Solution A, add 0.25 ml of the test solution & dilute to 50.0 ml with the mobile phase [100 ppm]

System suitability
As Per USP 34 System suitability tests were carried out on freshly prepared. Reference solution B to check the various parameters. Such as efficiency, retention time, and peak tailing which was found to comply with USP requirements.

The instrumental precision as determined by six successive injections of the reference solution B give RSD below 2% of Retention Time, And area and resolution between the these two peaks more than 1.0,Column  efficiency for many impurity was 10352.20, and for  2 Methyl Imidazole 3838 theoretical plates.

CALCULATION  OF  RESULTS  :

1. IMIDAZOLE CONTENT   :
                                      A_Samp.  X   W_Std.
IMIDAZOLE Content    =   ----------------------     X   P
                                     A_Std.    X   W_Samp.

A_Samp. = Area of IMIDAZOLE Peak in an injection of sample.
A_Std.      =  Mean Area of IMIDAZOLE peak in injections of analytical standard solution.
W_Samp. =  Weight of the sample taken to prepare relevant sample solution. ( in mg )
W_Std.     = Weight of IMIDAZOLE Reference Standard taken to prepare analytical standard solution. (in mg)
P        = Potency of IMIDAZOLE Reference Standard.

METHYL IMIDAZOLE CONTENT  :
                                                    A_Samp. X  W_Std.
2-METHYL IMIDAZOLE Content   = ----------------------   X  P
                                                   A_Std.    X  W_Samp.

A_Samp.   = Area of 2-METHYL IMIDAZOLE peak in an injection of sample.
A_Std.        =  Mean Area of 2-METHYL IMIDAZOLE peak in injection of analytical standard solution.
W_Samp.   =  Weight of the sample taken to prepare relevant sample solution. (in mg)
W_Std.       = Weight of 2-METHYL IMIDAZOLE Reference Standard taken to prepare analytical standard sol.
P          = Potency of 2-METHYL IMIDAZOLE Reference Standard.

Reference solution C is tested for Recovery and Reproducibility study and results are as follows-
RECOVERY
The Table 1 Show recovery is more than 98% as well impurity.

Reproducibility-
The data in table II shows reproducibility is more than 99% for the product as  impurity on analysis of 3 Consecutive days in Triplicate. Assay value of each run (For the product and impurity) is taken in to consideration and SD &RSD is calculated.

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Table- 1 : Recovery

Where A = Actual Concentration taken for analysis: B- Recovered Concentration, X = B/A; (%Recovery = X*100); Mean – Average of 3 (x) Injection; SD Standard Deviation, CV Coefficient Of Variation; 95%C.L.= Confidence Limit at 95%.

Table II: Reproducibility

Where- SD- Standard Deviation, RSD- Relative standard deviation ,2 V- 2 x Coefficient of variation.

Table III System Suitability Parameter

 SD- Standard Deviation, RSD- Relative Standard Deviation, T.P.- Theoritical Plates

R.F. Resolution Factor , T.F. – Tailing Factor.

RESULTS AND DISCUSSSIONSS-
As per USP XXXIV system suitability was carried out freshly prepared reference solution B  to check various parameters such as efficiency, resolution and peak tailing which found to comply with USP  requirements.

Limit of Detection- and Limit of Quantitation
The limit of detection (LOD) and Limit of Quantitation(LOQ)  For the impurity were found  o.121ppm.

The optimum mobile phase of 0.01 M Di Ammonium Hydrogen Orthophosphate and 1000 ml Water adjusted to pH 3.0 with 01 N Sodium Hydroxideas mobile phase was selected because it was found to be ideally resolve peak of impurity from2 Methyl Imidazole . Wavelength was selected by scanning standard drug over the wide range of wavelength 200 nm to 400 nm. The analysis was performed at 204nm detector setting.

The content of an impurity in 2 Methyl Imidazole by proposed method. The lower values of reproducibility  indicate that the method is precise and accurate. The mean recoveries of Impurity was in the range of 98% to 100% which shows that there is no interference from the mobile phase, which also confirm the reproducibility and reliability of the method.

CONCLUSION-
The proposed method is simple rapid and selective. Percent Relative standard Deviation was very slow, below 2.0% which indicate that method id highly precise.

Short Analysis time (≤15 min.) coupled with simplicity and ease of operation warrants use of the method for analysis  of 2 Methyl Imidazole along with its impurity stated above in Bulk. Therefore method can be useful in routine quality Control analysis in bulk.

ACKNOWLEDGEMENT-
Authors are thankful to the Management of  Aarti Drugs Ltd. for providing the standard of 2 Methyl Imidazole and its impurity (Imidazole along with certificate of analysis.

References-
i) Chan P, Mahler J, Travlos G et al. (2006). Induction of thyroid lesions in 14-week toxicity studies of 2 and 4-ethylimidazole in Fischer 344/N rats and B6C3F1mice. Arch Toxicol, 80: 169–180. doi:10.1007/s00204-005-0018-4 PMID:16180012
ii) Chan PC, Sills RC, Kissling GE et al. (2008). Induction  of thyroid and liver tumors by chronic exposure to 2-methylimidazole in F344/N rats and B6C3F1 mice.Arch Toxicol, 82: 399–412. doi:10.1007/s00204-007-0249-7 PMID:17924096
iii) GESTIS (2010). 2-Methylimidazole. GESTIS-database on hazardous substances. Available at: dguv.de/ifa/en/gestis/stoffdb/index.jspiv)Hargreaves MB, Jones BC, Smith DA, Gescher A (1994).
iv) Inhibition of p-nitrophenol hydroxylase in rat livermicrosomes by small aromatic and heterocyclic molecules.Drug Metab Dispos, 22: 806–810. PMID:7835233

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