A New Simple RP – HPLC Method for Simultaneous Estimation of Meropenem and VaborbactaminTablet Dosage Form

 

B. Balaswami^1*,Dr.P.Venkata Ramana¹, B.Subba Rao², P.Sanjeeva³

Department of Chemistry, Sri Krishnadevaraya University, Ananthapuramu-515003 A.P, India.

*Corresponding Author E-mail: balaswamypgt@gmail.com

A simple, accurate, economical, rapid, selective, reverse phase high performance liquid chromatography (RP-HPLC) was developed for simultaneous estimation of Meropenem and Vaborbactam in its tablet dosage form. The separation was carried out using a mobile phase of buffer and acetonitrile in the ratio of 50:50 pumped at a flow rate of 1 ml/min along with 250nm as a UV detection wavelength. The stationary phase used was column Kromasil 250 x 4.6 mm, 5m. Meropenem and Vaborbactam were eluted at a retention time of Meropenem 2.47min and Vaborbactam 3.31 min. The method was developed and validated as per ICH guidelines by considering the parameters such as precision, accuracy, linearity, specificity, robustness and degradation studies. The developed RP-HPLC method can be used for routine analysis of Meropenem and Vaborbactam in combinational dosage form.

 

 

 

INTRODUCTION:

VABOMERE  drug (Meropenem and Vaborbactam) for injection is a combination product that contains Meropenem, a synthetic penmen antibacterial drug[1] and the chemical name meropenem trihydrate is (4R,5S,6S)-3-[[(3S,5S)-5-(dimethylcarbamoyl)-3pyrrolidinyl]thio]-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, trihydrate[2]. The structure is shown below. Vaborbactam is a cyclic boronic acid beta-lactamase inhibitor, for endovenous administration. The chemical name is (3R, 6S)-2-hydroxy-3-[[2-(2-thienyl) acetyl] amino]-1,2-oxaborinane-6-acetic acid[3-5]. The chemical structure is shown below. Meropenem and Vaborbactam are official in US pharmacopeia. There is no official method for this combination.  As per literature survey several methods have been reported for the estimation of Meropenem and Vaborbactam individually or with the combination of some other drugs. The present proposed method estimates both Meropenem and Vaborbactam in a simple and economical process.

 

Fig. 1 Meropenem

 

Fig. 2 Vaborbactam

 

MATERIAL AND METHODS:

CHROMATOGRAPHIC CONDITIONS:

TheanalysiswasperformedonaHPLC instrument Waters HPLC 2965 System with Auto Injector and PDA Detector. Software used is Empower 2.0. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance for Meropenem  and Vaborbactam solutions. HPLC grade acetonitrile, water purchased from Ranbaxy, India, and ortho phosphoric acid AR grade purchased from SD Fine Chem. Mumbai, India were used in the study and supported by literature [6]

 

DRUG SAMPLES:

The reference samples and formulation samples were secured from Spectrum pharmaceuticals Pvt. Ltd, Hyderabad, India.

 

REAGENTS AND SOLUTIONS:

A mixture of ortho phosphoric acid buffer and acetonitrile in the ratio of 50:50%V/V was used as a mobile phase at a pH 2.5 adjusted with ortho phosphoric acid and it is also used as a diluents for preparing the working solution of drug. The mobile phase was deaerated in ultrasonic water bath for 5 minutes and filtered through 0.45μm filter under vacuum filtration

 

PREPARATION OF THE MEROPENEM AND VABORBACTAM STANDARD AND SAMPLE SOLUTION:

BUFFER: (0.1%OPA) (2.5pH):

1ml of ortho phosphoric acid solution in a 1000 ml of volumetric flask added about 100ml of milli-Qwater and final volume make up to 1000 ml with milli-Q water.

 

MOBILE PHASE:

Buffer and acetonitrile in the ratio 50:50 was used as the mobile phase

 

STANDARD SOLUTION PREPARATION:

Accurately weighed the samples using Electronicbalance [BL-220H, Shimadzu Corporation, Japan] and transferred 10 mg of Meropenem and 10 mg of Vaborbactam working standards into a 10 ml and 10 ml clean dry volumetric flasks, add 7 ml of diluents, sonicated for 5 minutes and made up to the final volume with diluents. These concentrations were selected based on recommended guidelines by the International Conference on Harmonization (ICH) and Food and Drug Administration (FDA) for analytical methods validation [7-9].

 

SAMPLE SOLUTION PREPARATION:

5 tablets were weighed by using Electronicbalance [BL-220H, Shimadzu Corporation, Japan] and calculated the average weight of each tablet, and then the weight equivalent to 1 tablet was transferred into a 500 ml volumetric flask, 250 ml of diluents added and sonicated for 25 min, further the volume made up with diluents and filtered. From the filtered solution 1 ml was pipetted out into a 10 ml volumetric flask and made up to 10 ml with diluents.

 

Label Claim:1000 mg of Meropenem + 1000 mg of Vaborbactam

 

METHOD DEVLOPMENT:

Three trials were executed for the method development and the best peaks with least fronting factor was elevated for Meropenem and Vaborbactam with RT= 2.473min, RT= 3.316 min. accordingly. The resultant chromatogram is shown in the Figure-1 and the values are presented in Table-1.

 

Figure-3: Chromatogram of Meropenem and Vaborbactam

 

Table-1

	Peak Name	RT	Area	% Area	USP Plate Count	USP Tailing	USP Resolution
1	Meropenem	2.473	572999	45.40	7639	1.34
2	Vaborbactam	3.316	689000	54.60	8635	1.22	6.6

 

 

METHOD VALIDATION:

The method was validated according to ICH guidelines [10-14]. Thedifferentvalidation characteristics whichwereperformed arefollowing: precision, accuracy, linearity, specificity,limitofdetection,limitof quantification, robustness, degradation studies andthestabilityindicating capability.

 

SYSTEM SUITABILITY TEST:

Six repeat injections of standard solution were injected and the chromatograms were recorded. The system was suitable for analysis if the % relative standard deviation (%RSD) of area counts in six replicate injections should be not more than 2.0%. USP tailing factor for Meropenem and Vaborbactam peak should be not more than 2.0. USP resolution factor between the peaks corresponding to Meropenem and Vaborbactam should be more than 2.0. The results are shown in  Table-2

 

Table-2: System suitability parameters

 

PRECISION:

The standard Meropenem and Vaborbactam solutions were injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six repeat injections was established to be within the specific limits. The data is shown in the Table-3

 

Acceptance Criteria:

The % RSD should not be more than 2%

 

 

Table-3: Precision study

 

ACCURACY:

Injected the standard solutions of accuracy -50%, 100% and 150% and calculated the amount found, Amount added for Meropenem and Vaborbactam and the individual recovery and mean recovery values are shown in the Table-4.

Acceptance Criteria:

The % Recovery for meropenem and vaborbactam at each level should be between 99 to 101%.

 

 

 

Table-4: Accuracy data (Triplicate values at 50, 100 and150 percent levels)

 

RECOVERY STUDIES:

To determine the accuracy and precision of the proposed method recovery studies were carried out. A fixed amount of sample was taken and reference drugs were added at 50%, 100% and 150% levels. The results were analyzed and found within the limits.

 

LINEARITY AND CALIBRATION CURVE:

Working dilutions of Meropenem and Vaborbactam in the range of 25-150 ppm were prepared by considering suitable aliquots of working standard solutions of drugs in different 10 ml volumetric flask and diluting up to the mark with mobile phase and these are shown in the Table-5. 20μl quantity of every dilution was injected in to the column at a flow rate of 1ml/min. The drug in the elute was monitored at 250 nm and the resultant chromatograms were recorded. From these, the mean peak areas were computed and shown in the Table-6. A plot of concentration vs. peak areas was constructed and shown in the Figure-2and3 for Meropenem and Vaborbactam respectively. The regression of the plot was calculated by least square regression method. The slope and intercept value for calibration curve for Meropenem and Vaborbactam was y=12657.x+692.2 (R²=0.999) and y=15296x+918.5 (R²=0.999) respectively.

 

Table-5: Calibration data of the proposed method

 

Table-6: Linearity mean peak area values

*Each value is a mean of three readings

 

 

Figure-2: Linearity graph for Meropenem

 

X-Axis = Concentration

Y-Axis = Peak area

Figure-3: Linearity graph for Vaborbactam

 

SPECIFICITY:

The specificity of the HPLC method is provided, where complete separations of Meropenem and Vaborbactam were detected in presence of other inactive excipients used in tablets. In addition, there was no any deterrence at the retention time in the chromatogram of placebo solution. In peak purity analysis with PDA, purity angle was always less than purity threshold for the analyte. This shows that the peaks of analyte were pure and excipients in the formulation does not interfere the analyte. The data are presented in the Table-7.

 

Table-7: Specificity study

S. No.	Name	No.of Injections	Meropenem	Vaborbactam
			Area	Area
1	Blank	1	-	-
2	Placebo	1	-	-
3	Standard	1	1152278	1571924
4	Sample	1	572999	689000

 

LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION:

Limit of Detection (LOD) is the lowest concentration of an analyte in a sample that can be detected but not quantified. LOD is expressed as a concentration at a specified signal to noise ratio. The LOD will not only depend on the procedure of analysis but also on the type of instrument. In chromatography, detection limit is the injected amount that results in a peak with a height at least twice or thrice as high as baseline noise level.

 

The LOD for Meropenem was found to be 0.06

The LOD for Vaborbactam was found to be 0.01

 

Limit of Quantification (LOQ) is defined as lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and reliability by a given method under stated experimental conditions. LOQ is expressed as a concentration at a specified signal to noise ratio. In chromatography, limit of quantification is the injected amount that results in a peak with a height, ten times as high as base line noise level.

 

The LOQ for Meropenem was found to be 0.17

The LOQ for Vaborbactam was found to be 0.02

 

ROBUSTNESS:

Robustness is determined by making deliberate changes in the chromatographic conditions like change in temperature, flow rate, and mobile phase composition and evaluated for the impact on the method. It was observed from the chromatograms that the results were within the limits. This indicates that the method developed is robust and shown in the Table-8.

 

Table-8: Robustness study

Parameter		Meropenem	Vaborbactam
Temperature±50C	250C	1353934	1622582
	350 C	1369867	1635985
Flow rate±0.1ml	0.9 ml	1476959	1773758
	1.1 ml	1192213	1433388
Mobile Phase change ±5 pH	45:55	1306936	1565083
	55:45	1308333	1569628

DEGRADATION STUDIES:

The forced degradation studies were conceded out to ensure the effective separation of Meropenem and Vaborbactam in the current study from degradation products. The degradation was observed by reducing the peak areas of the drug substances with same drug molecules of degraded peak areas. The percentage assay of degradation was calculated from the peak area obtained in degradation conditions and it was compared with assay of nondegraded conditions. The percentage assay degradation in both acidic and alkali conditions was found to be within the limits. Oxidative degradation studies were performed by using peroxide solution and the results showed that there was no degradation products formed. The drug solutions were placed in oven at 105°C for 6 h for thermal stress studies and then injected into HPLC system and photo stress testing was carried out by keeping the drug solutions in UV chamber for 7 days. The purity of angle is found to be less than that of purity of threshold in all the conditions which indicates that the developed method was stability indicating. The forced degradation studies were performed without planning to recognize the degradation products but only to show that they are not interfering with active molecules if any present. The data of stress studies are shown in Table-9.

 

 

Table-9: Degradation studies of Meropenem and Vaborbactam

Sample Name	Total purity	Meropenem			Vaborbactam
		% of Purity	% of Degradation	Purity of peak area	% of Purity	% of Degradation	Purity of peak area
Acid	100	92.99	7.01	1072054	94.86	5.14	1487557
base	100	95.01	4.99	1095361	96.18	3.82	1508377
peroxide	100	94.02	5.98	1083949	96.74	3.26	1517149
thermal	100	95.87	4.13	1105245	95.79	4.21	1502279
uv	100	97.02	2.98	1118510	96.36	3.64	1511176
water	100	99.58	0.42	1148042	99.45	0.55	1559554

 

Table-10: Results of all parameters

 

RESULTS AND DISCUSSION:

A simple, rapid and precise method has been developed and validated for the drug Meropenem and Vaborbactam. The estimation was carried out with a mixture of buffer and acetonitrile at pH 2.5 adjusted with ortho phosphoric acid in the ratio of 50:50%V/V. Precision of the methods were studied by making repeated injections of the samples and system precision values were determined. The retention time was 2.473 min and 3.316 min. The calibration curve was linear over the concentration range of 25-150ppm. The LOD values were 0.06, 0.01 and LOQ values were found to be 0.17, 0.2  The high percentage of recovery and low percentage coefficient of variance confirm the suitability of the method and the forced degradation studies shows that the  developed method was stability indicating. Hence it was concluded that the RP-HPLC method developed was highly suitable for routine analysis and all the parameters result data are shown in the below Table-10

 

CONCLUSION:

The current study describes new and simple RP-HPLC method for the simultaneous estimation of Meropenemand Vaborbactam. The method validated was found to be simple, accurate and precise. Therefore the proposed method can be used for quantification of Meropenemand Vaborbactam in bulk and pharmaceutical dosage form.

 

REFERENCES:

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Received on 06.11.2017         Modified on 28.11.2017

Accepted on 08.12.2017         © AJRC All right reserved