A RP-HPLC Method development and Validation for Simultaneous Estimation of Imipenem, Cilastatin and Relebactum in API and in Pharmaceutical Lyophilized powder for injection

 

Dr. K. Swapna¹, Dr. K. Deepthi², T. Padmini³*

¹Sphoorthy Engineering College, Nadergul, Sagar Road, Saroornagr, R. R. (Dist) Telangana State, 501510.

²Mahaveer Institute of Science and Technology, Vyasapuri, Bandlaguda, Hyderabad.

³Assistant Professor, Megha Institute of Engineering and Technology for Women. Edulabad,

 Ghatkesar, Hyderabad.

 

ABSTRACT:

An accurate, sensitive, precise, rapid and isocratic RP-HPLC method for simultaneous estimation of Cilastatin, Imipenem and Relebactum in the bulk drug and in pharmaceutical metered dose inhalers is developed and validated. The best separation achieved on Hypersil BDS Reverse Phase-C18 column (250mm × 4.6 x 5-μm) with acetonitrile as the organic modifier and dipotassium hydrogen phosphate [0.03M] in water with pH 3.2 adjusted with ortho-phosphoric acid (0.1% v/v) as mobile phase at a flow rate of 1.0mL min⁻¹. UV detection carried out at 265nm. Retention times observed at 3.107 min. for Imipenem, 3.885 min. for Cilastatin and 10.516 min for Relebactum. A linear response observed at concentration range of 20-120 mcg/ml for Cilastatin, 20-120mcg/ml for Imipenem and 10-60 mcg/ml for Relebactum respectively with average correlation coefficient of ≥ 0.999. The percentage assay for Cilastatin, Imipenem and Relebactum were found 98.37, 99.72 and 99.18% respectively. The limit of detection (LOD) was found to be 0.01µg/mL for Cilastatin, 0.02µg/mL for Imipenem and 0.05µg/mL for Relebactum. The limit of Quantification found to be 0.03µg/mL, 0.06µg/mL and 0.15µg/mL for Cilastatin, Imipenem and Relebactum respectively. The excipients present in the formulation did not interfere with the assay procedure. The method is suitable for application in quality-control laboratories, because it is simple and rapid with good accuracy and precision.

 

 

 

INTRODUCTION:

Recarbrio® injection is an antibacterial combination of Imipenem, Cilastatin, and Relebactam used for intravenous administration. Imipenem [1] is a beta lactam antibacterial drug that is crystalline derivative of thienamycin, produced by Streptomyces cattleya. It is nonhygroscopic crystalline compound, sparingly soluble in water. Cilastatin sodium [2] is the sodium salt of a derivatized heptenoic acid that is hygroscopic, amorphous compound, that is highly soluble in water and acts as renal dehydropeptidase inhibitor.

 

Relebactam [3] is a beta lactamase inhibitor that is highly soluble in water and. Cilastatin limits the renal metabolism of imipenem and does not have antibacterial activity. The vials are supplied as a single-dose glass vial, Recarbrio® (imipenem, cilastatin, and relebactam) for injection, 1.25 grams is supplied as a white to light yellow sterile powder for constitution in a single-dose glass vial containing imipenem 500 mg (equivalent to 530 mg imipenem monohydrate), cilastatin 500 mg (equivalent to 531mg cilastatin sodium), and relebactam 250 mg (equivalent to 263 mg relebactam monohydrate). Few UV spectrophotometric methods [4-7], HPLC [8-10] and hydrophilic interaction chromatography/mass spectrometry assay methods [11,12] were reported for determination of drugs individually and simultaneously for any two of the drugs. The current paper is aimed at developing RP-HPLC method for the assay of imipenem/cilastatin and relebactam in fixed dosage form and validated according to ICH guidelines [13,14].

 

EXPERIMENTAL:

Chemicals and Reagents:

Imipenem, 99%, Formula Weight 299.35gms/mol, Relebactum of 99% Formula weight 348.5 are obtained from Hetero Drugs as gift sample and Cilastatin of 99% pure with formula weight 358.5g/mol are acquired from Sum Pharmaceuticals, Mumbai, India. Acetonitrile HPLC Grade from Rankem Fine chemicals of HPLC Grade Potassium Phosphate (Dibasic, KH₂PO₄) [0.03M] from Rankem Fine Chemicals AR grade. Otho-Phosphoric Acid, 85% Quligens Fine chemicals and HPLC Grade water.

 

Chromatography Instrument and conditions:

Quantitative HPLC performed on liquid Chromatography, Waters separation 2996, PDA detector module equipped with automatic injector with the injection volume 20µl, and 2693 pump. A Hypersil BDS C-18 column (250x4.6 mm i.d; particle size 5μm) was used. The HPLC system was equipped with Empower-2 Software. The column was maintained at 40^oC and eluted under gradient conditions over 14.0 min at a flow rate of 1.0mL/min. Mobile phase consisted of acetonitrile as the organic modifier and potassium dihydrogen phosphate [0.03M] in water with pH 3.2 adjusted with ortho-phosphoric acid (0.1% v/v). Before use it was filtered through a 0.45-μm nylon membrane filters and then degassed. UV detection was performed at 265nm.

 

Preparation of the primary standard drug solutions:

50mg of reference standard substance of Cilastatin, 50 mg of Imipenem and 25mg of Relebactum in 50ml volumetric flask containing 25ml of diluent (60:40 acetonitrile: water), sonicated for about 15 min and then made up to 50ml with diluent to get the primary standard stock solution containing Cilastatin [1000µg/ml] Imipenem [1000µg/ml] and Relebactum [500µg/ml]. The prepared stock solutions were storedat 4⁰C and protected from light.

 

Preparation of working standard solution:

5ml of the above stock solution was taken in 50ml volumetric and made made up to mark with diluent to obtain 100mcg/ml of Cilastatin, 100mcg/ml of Imipenem and 50mcg/ml of Relebactum.

 

Preparation of sample solution:

5 vials of Recarbrio® (Merck-India) were emptied, and then powder was mixed gently to get the uniform blend. A sample of the sterile powder for injection, equivalent to 50mg of the Cilastatin, 50mg of Imipenem and 25mg of Relebactum active ingredients, was mixed with 30ml of diluent (60:40 acetonitrile: water) in 50ml of volumetric flask to get the primary standard stock solution from lyophilized sterile powder for injection containing Cilastatin-500mg, Imipenem-500mg and Relebactum 250mg in each vial. The mixture was allowed to stand for 1 hr with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45μm membrane filter, followed by adding diluent up to 50ml obtain a stock solution containing Cilastatin [1000µg/ml], Imipenem [1000µg/ml] and Relebactum [500µg/ml]. 5ml of the above stock solution was taken in 50ml volumetric flask and made up to 50 ml with diluent to get the working standard solution containing 100µg/ml of Cilastatin, 100mcg/ml of Imipenem and 50µg/ml of Relebactum.

 

METHODOLOGY:

Linearity:

Aliquots of working standard solutions of Imipenem, Cilastatin and Relebactum stock solution were taken in 10ml volumetric flasks separately, diluted to mark with the mobile phase to obtain 20-120µg/ml, 20-120µg/ml and 10-60µg/ml concentrations of Imipenem, Cilastatin and Relebactum respectively. 20μL of each drug solution injected thrice into column, for recording retention time and peak area. Calibration graph was obtained by plotting peak areas vs concentration of Imipenem, Cilastatin and Relebactum respectively.

 

Accuracy:

Known amounts of each drug corresponding to 80%, 100%, and 120% of the target test concentrations (10μg/mL of Imipenem, 10μg/mL of Cilastatin and 5μg/mL of Relebactum) were added to a placebo mixture to determine whether the excipients present in the formulation led to positive or negative interferences. Each set of addition repeated thrice at each level.

 

Precision:

The HPLC systems was set up to mention in chromatographic conditions, for system to equilibrate. The working standards containing 100µg/ml of Cilastatin, 100µg/ml of Imipenem and 60µg/ml of Relebactum injected 6 times and recorded the response (peak area). The precision was repeated with the formulated sample Recarbrio® Lyophilized sterile powder for injection of same concentrations was injected the working Sample solution containing 100µg/ml of Cilastatin, 100µg/ml of Imipenem and 60µg/ml of Relebactum. Sample [Recarbrio®, Merck] response in terms of peak areas was recorded 6times. The low value (≤1%) of RSD indicates the repeatability of the method

 

Limits of Detection and Quantification:

Limit of detection (LOD) of the method and limit of quantification (LOQ) was determined by a signal-to-noise (S/N) ratio of about 3 and signal-to-noise (S/N) ratio of about 10 respectively.

Method Applicability:

The method evaluated by applying to pharmaceutical lyophilized powder for injection for the estimation Imipenem, Cilastatin and Relebactum by our research group.

 

Results and Discussion: HPLC Method Development and Optimization:

We examined several HPLC method variables with respect to their corresponding effects on the result of analysis. To optimize the chromatographic conditions, different combinations of methanol- water and acetonitrile- water and acetonitrile- dipotassium phosphate buffer were tested. Acetonitrile with potassium dihydrogen phosphate buffer system [pH 3.2] was chosen as it generated greater resolution of active pharmaceutical ingredients. The other parameters in this factorial design include temperature, flow rate, detection wavelength and volume of injection. Buffer molarity was altered and optimum buffer strength selected as 0.03M on the basis of theoretical plate number. At 265nm, UV response of all three active pharmaceutical analytes was good and free form interferences. Under these conditions, the analyte peaks were well defined and free from tailing, broadening and splitting.

 

Method Validation Tests:

Recommended method validation characteristics including method precision (RSD, %), method accuracy (recovery % and RSD, %), linear range (correlation coefficient), and LOD & LOQ, were investigated.

 

Linearity:

The plot of peak areas of each analyte against respective concentrations was found to be linear in the range of 20-120µg/ml for Imipenem, 20-120µg/ml for Cilastatin and 10-60µg/ml for Relebactum with correlation coefficient of ≥ 0.998Linear regression least square fit data obtained from the measurements are given in Table I. The respective linear regression equation being Y=285992x + 2855.3 for Imipenem, Y= 570782x - 16646 for Cilastatin and Y= 477215x + 31588 for Relebactum. (Figure 1)

 

Accuracy:

Recovery of the individual substances at 80%, 100%, and 120% of specified concentrations found between 97.1% and 104.25%, which proves the accuracy of the method. From these data, RSD < 1% indicates it is obvious that the method is remarkably accurate, produces reliable results (Table 2)

 

Precision:

The intra-day and inter-day variability or precision data are summarized in Table 3. The low value (<1%) of RSD indicates the repeatability of the method. These data indicate a considerable degree of precision and reproducibility for the method both during one analytical run and between different runs (Table 3).

 

Robustness:

Robustness was studied out to evaluate the effect of small but deliberate variations in the chromatographic conditions at three different levels, i.e. –2, 0, +2. To determine the robustness of this method, the experimental conditions were deliberately altered at three different levels and retention time and chromatographic response were evaluated. One factor at a time was changed to study the effect. Variation of the detection wavelength by ±2nm (263nm and 267nm), mobile phase flow rate, mobile phase pH by ±0.2 units (pH 3.0 and 3.4), and mobile phase flow rate by 0.8mL min−1 and 1.2mL min−1) had no significant effect on the retention time and chromatographic response of the method, indicating that the method was robust.

 

Limit of Detection [LOD] and Limit of Quantification:

The limit of detection (LOD) was found to be 0.01µg/mL for Cilastatin, 0.02µg/mL for Imipenem and 0.05µg/mL for Relebactum. The limit of Quantification found to be 0.03µg/mL, 0.06µg/mL and 0.15µg/mL for Cilastatin, Imipenem and Relebactum respectively.

 

Specificity:

From the typical chromatogram, shown in Fig-1, the retention times were found to be 3.117 min for imipenem, 3.8 min. for Cilastatin and 10.5 min for Relebactum. No evidence of signals, in the corresponding times of the chromatogram were monitored as a sign of potential interfering peaks, was found when the powder for injection was tested. Hence, this method can be used reliably for the estimation of respected active pharmaceutical ingredients in a variety of dosage forms.

 

 

 

Table 1: Linearity Studies of Imipenem, Cilastatin and Relebactum

Conc. of the Cilastatin	Peak Areas	Conc. of Imipenem	Peak Areas	Conc. of Relebactum	Peak Areas.
20µg/mL	565566	20 µg/mL	287640	10 µg/mL	501055
40µg/mL	1114978	40 µg/mL	576733	20 µg/mL	989653
60µg/mL	1691999	60 µg/mL	862462	30 µg/mL	1468107
80µg/mL	2260138	80 µg/mL	1141217	40 µg/mL	1940296
100µg/mL	2843669	100 µg/mL	1436750	50 µg/mL	2427538
120µg/mL	3410197	120 µg/mL	1717845	60 µg/mL	2884390

DISCUSSION:

The developed method was applied to the simulatenous determination of (Recarbrio® (imipenem, cilastatin, and relebactam) lyophilized powder for injection. The 1.25 grams dose glass vial containing imipenem 500mg (equivalent to 530mg imipenem monohydrate), cilastatin 500 mg (equivalent to 531mg cilastatin sodium), and relebactam 250mg (equivalent to 263mg relebactam monohydrate). Figure-3 represents the chromatogram of Recarbrio®-Merck powder for injection. The difference between the claimed amount and those assayed was low with minimum R.S.D. The mean percentage recoveries obtained after six repeated experiments were found between 97.53 and 100.98 (Table 8.11), indicating no interference from the common excipients used in the pharmaceutical dosage forms

 

Table 2: Recovery Peak areas of Cilastatin, Imipenem and Relebactum:

Standard drug solution				Spiked drug solution
Concentration of the drug in % of the working standard- in µg/mL	Average Peak Areas			Concentration of the drug in terms of % of working standard and spiked amount in micrograms/mL		Average Peak Areas
	Cilastatin	Imipenem	Relebactum			Cilastatin	Imipenem	Relebactum
				Working Std.	Spiked amount
80% working std	2260112	1146681	1932653	80% working	10% working std	2564419	1296338	2181810
100% working std	2849247	1442483	2434693	100% working std	10% working std	3119427	1570520	2641249
120%	3116313	1574532	2646438	120%	10%	3393518	1712474	2888698

 

Table 3: Performance and Detection characteristics of Proposed HPLC method:

Results of the proposed HPLC method.
Parameter	Cilastatin	Imipenem	Relebactum
Retention time(min)	3.869	3.117	10.539
Theoretical plates(n)	7281	7594	14889
Plates per meter(N)	29122	30387	59675
HETP	3.52x10-5	3.72x10-5	1.12x10-5
Peak Asymmetry(T)	1.41	1.62	1.12
Linearity range(µg/mL]	20-120	201-120	10-60
Limit of Detection[µg/mL]	0.01 µg/mL	0.02µg/mL	0.05µg/mL
Limit of quantification[µg/mL]	0.03 µg/mL	0.06 µg/mL	0.15 µg/mL
% RSD in Precision Studies	0.5	0.7	0.8

 

Table 4: Optical and Regression Characteristics of the proposed HPLC Method:

Results for the proposed HPLC method.
Parameter	Cilastatin	Imipenem	Relebactum
Detection Wavelength (nm)	265	265	265
Linearity range(mcg/mL)	10-120	10-120	5-60
Correlation coefficient	0.9998	0.9999	0.9988
Relative Std. Deviation*	0.13	0.25	0.46

*Average of six determinations ** Average of three determinations.

 

 

Table 5: Results of HPLC assay and Recovery studies of Cilastatin, Imipenem and Relebactum:

Label Claim(mg)

Proposed method

% Found by reference method*

% Recovery by proposed method**

CILASTATIN

IMIPENEM

Relebactum

% found ± S.D

t-Test

F-Test

± S.D.

± S.D.

CILASTATIN

IMIPENEM

Relebactum

CILASTATIN

IMIPENEM

Relebactum

CILASTATIN

IMIPENEM

Relebactum

CILASTATIN

IMIPENEM

Relebactum

CILASTATIN

IMIPENEM

Relebactum

500mg

500 mg

200 mg

99.53 ± 0.214

99.72 ± 0.732

99.18 ± 0.41

1.48

1.29

1.21

1.17

0.45

0.98

99.75± 0.45

99.73 ± 0.68

99.9 ± 0.67

98.18 ± 0.14

98.38 ± 0.92

98.12 ± 0.73

500mg

500 mg

200 mg

99.5 ± 0.235

99.12 ± 0.871

99.3±0.3

1.27

1.68

1.81

0.91

0.72

0.73

99.18 ± 0.85

99.18 ± 0.27

99.8 ± 0.36

98.18 ± 0.30

98.62 ± 0.43

98.29 ± 0.83

500mg

500 mg

200 mg

99.63 ± 0.370

98.83 ± 0.474

99.7±0.2

1.76

0.35

1.09

1.45

0.68

1.05

99.52± 0.57

99.35± 0.91

99.9 ± 0.62

98.75 ± 0.57

98.27 ± 0.86

98.19 ± 1.74

*Average ± standard deviation of six determinations, the t- and F-test values refers to compare the proposed method with the reference method. Theoretical values at 95% confidence limit , t=2.57, F= 5.05

** Average of three determinations.

 

 

 

 

 

Figure 2: Linearity Graphs of (a) Imipenem (b) Cilastatin (c) Relebactum standard solutions

 

 

 

 

CONCLUSION:

A simple and easily available HPLC method was developed and validated for quantification of Imipenem, Cilastatin and Relebactumin pharmaceutical matrices. The method is simple and exhibited relatively wide linear range, acceptable precision and accuracy and practically reliable sensitivity hence can be adopted for routine analysis in pharmaceutical quality control.

 

ACKNOWLEDGEMENTS:

The authors wish to express their gratitude to M/s Sun Pharmaceuticals and M/s Hetero Drugs, Hyderabad for the supply of Imipenem, Cilastatin and Relebactumas gift samples, and to the college management, for providing the necessary facilities to carry out the research work.

 

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Received on 12.02.2020            Modified on 14.04.2020

Accepted on 08.06.2020         © RJPT All right reserved