Spectrophotometric Determination Of Penems In Bulk And Injection Formulations By MBTH reagent

Spectrophotometric Determination Of Penems In Bulk And Injection Formulations By MBTH reagent

Dr. K. Raghu Babu 1, N. Aruna Kumari 2, Dr. A.Vasundhara 3, R. Vijaya Lakshmi 4

1Department of Engg.Chem, Andhra University , Visakhapatnam, India.

2Department of HBS, GIET, Rajahmundry, India.

3Department of Chemistry, SKR College for Women, Rajahmundry, India.

4Department of Analysis, GIET School of Pharmacy, Rajahmundry, India.

Abstract

Three simple and cost effective spectrophotometric methods were described for the determination of Imipenem, Meropenem and Biapenem in pure form and in pharmaceutical formulations. The method is based on the formation of green colored chromogen when the drug reacts with 3-methyl-2- benzothiazolinone hydrazone (MBTH) in presence of an oxidizing agent FeCl3. The colored species has an absorption maximum at 608 nm for Imipenem (Method A), 397 nm for both Meropenem (Method B), Biapenem (Method C) and obeys beers law in the concentration range 0.02 0.012 mg/mL of Imipenem, 0.04 0.012 mg/mL Meropenem and 0.04

0.16 mg/mL of Biapenem. The apparent molar absorptivities were 0.034, 0.0106 and 0.0144 and sandells sensitivity were 2.5×10-5, 2.96×10-3 and 1×10-3 respectively for Imipenem, Meropenem and Biapenem. The slopes were 0.4379 ± 0.01829, 0.2048

± 0.003599 and 0.2008 ± 0.001171 and intercept of the equation of the regression line are 0.06607 ± 0.03298, -0.0009786 ± 0.006970, 0.003963 ±

0.002950 for Imipenem, Meropenem and Biapenem respectively. The optimum experimental parameters for the reaction have been studied and the validity of the described procedure was assessed. Statistical analysis of the results has been carried out revealing high accuracy and good precision. The proposed method was successfully applied for the determination of Imipenem, Meropenem and Biapenem in pharmaceutical formulations.

Key words: Imipenem, Meropenem, Biapenem, MBTH, Spectrophotometry.

1. Introduction

   Imipenem[1] is a broad spectrum beta-lactam antibiotic belonging to the carbapenem class. Chemically it is (5R,6S)-6-[(1R)-1-hydroxyethyl]-3- ({2-[(iminomethyl)amino]ethyl}thio)-7-oxo-1- azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.

   Imipenem acts by interfering with their ability to form cell walls, and therefore the bacteria break up and die. It is a broad spectrum antibiotic with activity against many aerobic and anaerobic gram-positive and gram-negative organisms.

   Meropenem[2] is an ultra-broad spectrum injectable antibiotic used to treat a wide variety of infections. It

   is a beta-lactam and belongs to the subgroup of carbapenem, similar to imipenem and ertapenem. It penetrates well into many tissues and body fluids including the cerebrospinal fluid, bile, heart valves, lung, and peritoneal fluid. Chemically it is 3-[5- (dimethylcarbamoyl) pyrrolidin-2-yl] sulfanyl-6- (1- hydroxyethyl)-4-methyl-7-oxo- 1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid.

   In contrast to other beta-lactams, it is highly resistant to degradation by beta-lactamases or cephalosporinases.

   Biapenem[3] is a carbapenem antibiotic. It has in vitro activity against anaerobes.

   Biapenem is a new parenteral carbapenem antibacterial agent with a broad spectrum of in vitro antibacterial activity encompassing many Gram- negative and Gram-positive aerobic and anaerobic bacteria, including species producing beta- lactamases.

   Literature survey reveals that the drugs were determined by using HPLC and some

   [4-

   solution was measured at 608 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus the concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation.

   2.2.2 Method B

   Into 10 mL volumetric flask, different aliquots of working standard solution (1.0 3.0 mL) of Meropenem were transferred to provide final concentration range of 0.04 0.12 mg/mL. To each flask, 2.5 mL of freshly prepared FeCl3 and 2.5 mL

   of MBTH reagent were successively added and kept

   spectrophotometric methods for Imipenem 9] and

   Meropenem[10-23]. According to the literature survey there is no method reported for Biapenem with MBTH reagent by visible spectrophotometry. Hence an attempt made to develop simple and sensitive spectrophotometric methods for the estimation of the above named penems in pure drug and in pharmaceutical formulations. The method uses the well known oxidation reaction involving MBTH reagent and penems resulting in the formation of a blue /green chromogen that could be measured at 300

   800 nm.

2. Experimental

   1. Apparatus

      All spectral characteristics and absorbance measurements were made on Perkin Elmer, LAMBDA 25 double beam UV-Visible spectrophotometer with 10 mm matched quartz cells. All chemicals used were of analytical reagent grade and double distilled water was used throughout. MBTH supplied by SD Fine chemicals ltd., India, was used and 0.2% MBTH solution was prepared by dissolving 200 mg MBTH in 100 mL distilled water. FeCl3 solution (0.5%) was prepared by dissolving 500 mg of FeCl3 in 100 mL double distilled water. 10 mg/mL stock reference solution was freshly prepared from pure sample of penems by dissolving 100 mg in 100 mL of double distilled water.

   2. General procedure

      2.2.1 Method A

      Into 10 mL volumetric flask, different aliquots of working standard solution (0.5 3.0 mL) of Imipenem were transferred to provide final concentration range of 0.02 0.12 mg/mL. To each flask, 2 mL of freshly prepared FeCl3 and 2 mL of MBTH reagent were successively added and kept aside for 5 minutes. The solutions were made up to volume with distilled water. The absorbance of each

      aside for 5 minutes. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 397 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus the concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation.

      2.2.3 Method C

      Into 10 mL volumetric flask, different aliquots of working standard solution (1.0 4.0 mL) of Biapenem were transferred to provide final concentration range of 0.04 0.16 mg/mL. To each flask, 1.5 mL of freshly prepared FeCl3 and 2.0 mL of MBTH were successively added and kept aside for 5 minutes. The solutions were made up to volume with distilled water. The absorbance of each solution was measured at 397 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus the concentration of the drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation.

3. Procedure for Injections

   An amount of powder equivalent to 100 mg of penems were weighed into a 100mL volumetric flask,

   50 mL of distilled water was added and shaken thoroughly for about 10 minutes, then the volume was made up to the mark with the distilled water, mixed well and filtered. Further dilutions were made and the assay of injections was completed according to general procedure.

4. Results and Discussion

   3-methylbenzthiazolinone-2(3H)-hydrazone (MBTH) originally introduced as a reagent for aldehydes and ketones. Later its use was extended to a variety of organic compound (example: Phenols,

   aryl amines and different N- and S- heterocyclic compound).

   investigated. All conditions tudied were optimized at room temperature (32±20C).

   Aldehydes and ketones are partially oxygenated organic compounds containing carbonyl group. An aldehyde functional group consists of a carbon atom bonded to a hydrogen atom and double-bonded to an oxygen atom (O=CH-). Whereas a ketone functional group contains a carbonyl group (C=O) bonded to two other carbon atoms.

   MBTH reacts with aldehyde/ketones first to form an azine. Only if there is remaining MBTH, it is oxidized to another species which combines with the azine to form formazan. However, if there is enough aldehyde/ketone, all the MBTH is converted to azine and there is no formation of blue color. Thus, by using the limiting agent MBTH to test the amount of aldehyde/ketone around the point of interest, then less aldehyde/ketone would produce more blue /green color and more aldehyde/ketone would produce less blue/green color. The end color may be different depending upon the order of addition of the reactants. For example, if an oxidizing agent and MBTH are mixed before adding the aldehyde/ketone, a light green to green/blue color results. This method could be used could be used for measurements with a device or instrument such as a color reader and used in combination with a second aldehyde/ketone tester and a pH tester. With Phenols under reaction

5. Selection of reaction medium

   To find a suitable medium for the reaction, different aqueous bases were used, such as Sodium meta periodate, Ammonium Ceric Sulphate, Ferric chloride. The best results were obtained when Ferric chloride was used. In order to determine the optimum concentration of Ferric chloride, different volumes of 0.5% Ferric chloride solution (1 2.5 mL) were used to a constant concentration of Imipenem (25 mg/mL), (1 3.0 mL) were used to a constant concentration of both Meropenem and Biapenem (25 mg/mL) and the results of the observation were plotted. From the figure it is evident that 2 mL of 0.5% Ferric chloride solution for Imipenem, 2.5 mL of 0.5% Ferric chloride solution for Meropenem and 1.5 mL of 0.5% Ferric chloride solution for Biapenem were found optimum. Larger volumes had no effect on the absorbance of the colored species.

6. Effect of order of addition of reactants

Few trials were performed to ascertain the influence of order of addition of reactants on the color development and the results are presented in Table 1. The order of addition of serial number (ii) is recommended for all the three samples.

Table 1. Effect of order of addition of drug reactants on color development.

Reco-

condition MBTH loses two electrons and one proton to form the electrophilic intermediate, which has been identified as the active coupling species that undergoes electrophilic substitution with phenol and

S.

No.

Drug Order

of Addition

Absor bance

mended order of Addition

other groups to form the colored product.

i D+MBTH+ FeCl3 0.50

a 3

a 3

1. Imi ii D + FeCl + MBTH 0.70 ii

   penem

   a

   a

2. Meropen em

   iii FeCl3+ MBTH + D 0.10

   1. D + MBTH + FeCl3 0.41

   2. D + FeCl3+ MBTH 0.57 ii

   3. FeCl3+ MBTH + D 0.32

   i D + MBTH + FeCl3 0.25

   Optimization of conditions on absorption spectrum of the reaction product

   The Conditions under which reaction of penems with MBTH fulfills the essential requirements was

3. Bia ii D + FeCl + MBTH 0.38 ii

a 3

a 3

penem

iii FeCl3+ MBTH + D 0.22

aFor 40 µg/mL of Drug samples

1. Effect of MBTH concentration

   Several experiments were carried out to study the influence of MBTH concentration on the color development by keeping the concentration of drug and Ferric chloride to constant and changing reagent concentration. It was apparent that 2.0 mL of reagent gave maximum color for Imipenem, 2.5 mL for Meropenem and 2.0 mL for Biapenem.

2. Reaction time and stability of the colored species

The color reaction was not instantaneous. Maximum color was developed within 5 minutes of mixing the reactants and was stable for 60 minutes thereafter.

Fig 2 Calibration graph of Meropenem

Meropenem with MBTH

0.8

Absorbance

Absorbance

0.6

0.4

0.2

9. Absorption spectrum and calibration graph

Absorption spectrum of the colored complex was scanned at 400-900 nm against a reagent blank. The reaction product showed absorption maximum at 608 nm for Imipenem, 397 nm for both Meropenem and Biapenem. Calibration graph was obtained according to the above general procedure. The linearity

0.0

0 1 2 3 4

Concentration

replicates for six different concentrations of Imipenem and Meropenem, seven different concentrations of Biapenem with MBTH were checked by a linear least – squares treatment. All the spectral characteristics and the measured or calculated factors and parameters were summarized in Table 2.

Fig 1. Calibration graph of Imepenem

Fig 3 Calibration graph of Biapenem

Biapenem with MBTH

1.0

Absorbance

Absorbance

0.8

0.6

0.4

0.2

0.0

0 1 2 3 4 5

Concentration

10. Sensitivity, accuracy and precision

Sandells sensitivity, molar absorptivity, precision and accuracy were found by performing eight replicate determinations containing 3/4th of the amount of upper Beers law limits. The measured standard deviation (S.D), relative standard deviation (RSD), and confidence limits (Table 2) were considered satisfactory.

Table 2. Optical and regression characteristics, precision and accuracy of the proposed method for penems.

Hence, the method is devoid of error due to above substances.

Parameters

Values

Imipenem Meropenem Biapenem

12. Application to formulation

The proposed procedures were applied for the determination of penems in commercially available injections. Table 3 summarized the results.

max nm 608 nm 397 nm 397 nm

Beers law

Table 3. Results of analysis of injection formulations containing penems

limits, µg/mL 0.02 0.03 0.04 0.12 0.04

0.16

Injection Imi penem

Mero penem

Bia penem

Molar

absorptivity, L/mol.cm

Sandells

0.034 0.0106 0.0144

2.5×10-5 2.96×10-3 1×10-3

Company Name

Troika Pharma

Neon Pharma

Novachem

sensitivity (µg/cm2/0.001

Formulation Inj Inj Inj

absorbance unit)

Labeled

amount, mg

1000 1000 1000

Regression equation

% Recovery 99.8 99.56 98.92

Slope(b)

0.066 ±

-0.0009 ±

0.003963

13. Conclusion

The proposed methods were found to be simple, rapid and inexpensive, hence can be used for routine analysis of penems in bulk and in injection formulations.

Limit of

0.1385

Dept., of Analysis, GIET School of Pharmacy,

11. Interference

Aldehydes and ketones are partially oxygenated organic compounds containing carbonyl group. An aldehyde functional group consists of a carbon atom bonded to a hydrogen atom and double-bonded to an oxygen atom (O=CH-). Whereas a ketone functional group contains a carbonyl group (C=O) bonded to two other carbon atoms. MBTH reacts with aldehyde/ketones first to form an azine. Only if there is remaining MBTH, it is oxidized to another species which combines with the azine to form formazan. However these substances are seldom present in the reagents and used in the pharmaceutical formulations.

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