INTRODUCTION:

Both penem derivatives, carbapenems and thiopenems, have a broad spectrum of activity against bacteria¹. However, their therapeutic use is limited due to the chemical and enzymatic instability². In thiopenems, Faropenem is the only drug approved for therapeutic use and is orally active³. Thiopenems are considerably more stable chemically due to the sulphur atom at C4 into the thiazolidine moiety of the bi-cyclic 4:5 fused ring, in contrast to carbapenems where the sulphur atom was substituted with a carbon atom⁴. The presence of chiral tetrahydrofuran substituent at the C2 position in the faropenem structure is responsible for the reduced neurotoxicity and improved chemical stability^1,5.

Figure 1: Chemical structure of Faropenem

Faropenem is chemically (5R,6S,8R,2'R)-2-(2'-tetrahydrofuryl)-6-(1-hydroxyethyl)-2-penem-3-carboxylic acid1 and the chemical structure is given in figure 1. Faropenem is a novel beta-lactam antibiotic sharing similarities with both penicillin and cephalosporins⁶. Due to the broad spectrum of activity, carbapenems are preferred drugs for pneumonia, chronic bronchitis, acute bacterial sinusitis, urinary tract infections, and skin infections. Similar to other beta-lactam antibiotics, Faropenem's primary mode of action is by binding to penicillin-binding proteins^7-9. Currently available Faropenem oral dry syrup is used with a dose of 15mg/kg/day, divided into three doses to treat a range of pediatric infections, such as urinary tract infections, upper respiratory tract infections, dermatological infections, and bacterial periodontal infections¹⁰.

An extensive survey of literature revealed the few analytical methods, such as spectrophotometric,^11-13HPLC,^14-18 and LC-MS¹⁹for faropenem. The current literature reveals the presence of large number of articles using UPLC as an advanced LC technique.^20-29 As there is no reported UPLC method, the development and validation of an ultra-fast and accurate, LC/MS compatible isocratic RP-UPLC method was developed to analyse Faropenem (FRPM) bulk drug and tablet formulation. From the stress degradation studies, it was proved that the drug undergoes degradation under different stress conditions.

MATERIALS AND METHODS:

Materials:

A gratis sample of Faropenem sodium was obtained from Hetero Labs Limited, Baddi. Ammonium formate AR grade and acetonitrile (HPLC grade) were obtained from Merck (India). Farobact tablets were purchased from a local pharmacy. Triple distilled water was prepared in-house by a distillation system.

UPLC Instrumentation:

Analysis was performed on a UPLC Chromatograph, namely, Waters Acquity UPLC supported by 2996 PDA detector, ACQUITY UPLC Binary Solvent Manager and Sample Manager. A Shimadzu Prominence binary gradient. An End version C18 column (50 X 2.5 mm i.d,1.8μm) was used for the chromatographic separation. Empower 2 software was used for Data acquisition.

Chromatographic Conditions:

Chromatographic analysis was performed on the UPLC system operated isocratically using a mobile phase of acetonitrile and 10mM ammonium formate buffer of pH 3.5 (prepared with 632 mg in 1000mL of triple distilled water ) in the ratio of 35:65v/v at a flow rate of 0.25 mL/min at room temp. The detection wavelength selected was 313nm after scanning the sample with a UV spectrophotometer, and the peak areas were integrated automatically. Peak identity was proved by retention time comparison. Optimised chromatographic conditions are given in Table 1.

Table 1: Chromatographic Conditions, optimised

Sl. No.	Parameter	Optimized condition
1	Chromatograph	A Waters Acquity UPLC having 2996 PDA detector, ACQUITY UPLC Binary Solvent Manager and Sample Manager
2	Column	A End version C18 column (50 X 2.5 mm i.d,1.8μm)
3	Mobile phase	10 mM Ammonium formate buffer (pH=3.5):ACN in the ratio of 65: 35 (v/v) by isocratic mode
4	Flow rate	0.25 mL/min
5	Wavelength	313 nm
6	Injection volume	4 µL

VALIDATION OF THE METHOD:

The method validation parameters were precision, accuracy, linearity, LOD, LOQ and robustness by adopting the guidelines of ICH³⁰. All the solutions required for validation data were prepared with FRPM standard.

Linearity method for FRPM:

The stock solution was prepared by dissolving 10mg of the reference substance in 10mL of methanol (1000 µg/mL). From this, solutions of various concentrations (5–50µg/mL) were prepared by diluting appropriate volumes with the mobile phase. A calibration graph was made by plotting the peak areas versus corresponding concentrations. For linearity assessment, linear regression analysis using the least square method was adopted.

Accuracy method for FRPM:

The accuracy of the developed method was done by the standard addition method. To the analysed sample solution (20µg/mL), a known amount of standard drug was added at 80%, 100% and 120% levels and analysed by the developed method in triplicate. The percentage recovery was calculated from the linear regression equation obtained in the linearity studies.

Precision method for FRPM:

The repeatability (intra-day precision) of the method was determined by intra-day (n=3) analysis of three standard solutions of FRPM at the concentration of 10, 30 and 50µg/mL. Intermediate precision was determined by the inter-day (n=3) analysis of the same concentration as explained above.

Method for LOD and LOQ of FRPM:

LOD and LOQ were determined on samples containing very low concentrations of the analyte. They were estimated at a signal-to-noise ratio of 3:1 and 10:1, respectively, for LOD and LOQ by analysing dilute solutions of known concentration.

Robustness method for FRPM:

The experimental conditions, such as flow rate (0.25mL/min), lambda max (313nm) and percentage of acetonitrile (35), were changed at three levels (-1, 0, +1) for robustness evaluation. Each factor was changed one by one to analyse the impact of the change in the experimental conditions on the assay results. Changes in the assay values and the retention time were noted at each change in the analytical parameters.

ASSAY:

Farobact tablet powder, which is equivalent to 10mg of FRPM, was dissolved in 10mL of methanol with the assistance of an ultrasonic bath and later filtered and diluted using a mobile phase. An aliquot was diluted with the mobile phase to get a final concentration of 30 µg/mL, the peak area of which was used to estimate drug content.

Methodology for stress degradation studies:

Specificity is the ability of a method to measure analytical response in the presence of its potential impurities. We did the stress degradation of the drug by oxidation (3% H₂O₂, 4hrs), photolysis (UV radiations of 254nm exposed to 48 hrs), heat (hot air oven at 100⁰C for 24hrs) and hydrolysis with acid (1N HCl, 48hrs) and alkali (0.1N NaOH, 4hrs), followed by its analysis using the developed method.

RESULTS AND DISCUSSION:

Optimisation of Chromatographic conditions:

A UPLC method was developed with a PDA detector to analyse the drug in the presence of its possible degradation products. Several mobile phase compositions were tried to optimise the UPLC parameters. A symmetric and sharp peak with good separation for the drug from its degradation products was obtained by an isocratic method using a mobile phase of ACN and 10mM ammonium formate buffer in a ratio of 35:65 (v/v), pumped at a flow rate of 0.25 mL/min. The detection was carried out at 313nm, and the retention time was found to be 0.66min. A typical chromatogram of the drug and its tablet formulation is given in Figure 2.

Figure 2: Chromatogram of Faropenem std and sample

Linearity results for FRPM:

The linear regression analysis of the data for the calibration curve (n = 3) showed a good linear relationship over the concentration range of 5–50 µg/mL. The regression equation is y = 14710.38x + 6770.86, and the coefficient of determination, R², is 0.9993 showing a good correlation between the peak area and the concentration. The linearity graph is given in Figure 3.

Figure 3: Linearity graph of Faropenem

Precision results for FRPM:

The precision study was evaluated by calculating %RSD values, and the data is presented in Table 2. The %RSD values are within the acceptance criteria (%RSD <2).

Table 2: Precision data of FRPM

Con. (µg/mL)	Intraday precision		Interday precision
Con. (µg/mL)	Mean con. (µg/mL) AM±SD (n=3)	% RSD	Mean con. (µg/mL) AM±SD (n=9)	% RSD
10.0	9.46±0.07	0.74	9.49±0.05	0.57
30.0	30.40±0.06	0.20	30.42±0.09	0.29
50.0	49.37±0.01	0.02	49.44±0.08	0.15

Accuracy results for FRPM:

The quantitative recovery of FRPM done at three levels was ranged from 99.98 - 100.73% with a low %RSD value. The results of the recovery experiments along with %RSD values are given in Table 3.

Table 3: Accuracy data of FRPM

Spiking level (%)	Actual conc. (µg/mL)	Conc. Recovered (µg/mL) (AM±SD) (n=3)	Recovery (%)	% RSD
80	36	36.18±0.14	100.50	0.38
100	40	39.99±0.37	99.98	0.91
120	44	44.32±0.34	100.73	0.76

LOQ and LOD values of FRPM:

LOQ and LOD values were found to be 638 and 213 ng/mL, respectively, by the proposed method.

Robustness results for FRPM:

The robustness study carried out by the deliberate changes in the chromatographic conditions (flow rate, % acetonitrile in the mobile phase, wavelength) indicated that the results are not much affected by the deliberate variation in the optimised parameters (Table 4).

Table 4: Robustness results

Parameter	R_t in Mts	Peak area (n=3)
A.Flow rate (mL/min)
0.24	0.727	457863
0.25	0.663	455684
0.26	0.637	456791
Mean ± SD	0.676 ± 0.046	456779 ± 1090
B.% of organic phase
34	0.680	465321
35	0.663	455684
36	0.613	466897
Mean ± SD	0.652 ± 0.035	462634 ± 6070
C. Wavelength (nm)
311	0.670	461165
313	0.663	455684
315	0.667	459774
Mean ± SD	0.667+ 0.003	458874 ± 2849

ASSAY OF FAROBACT TABLETS:

The assay of FRPM was done on the marketed formulation (Farobact –200mg). There was no interference due to the excipients in the formulations, which is shown by the absence of extra peaks in the chromatogram. The assay results are tabulated in Table 5.

Table 5: Assay results of Farobact tablets

Brand name

Label claim (mg)

Amount (mg)

(AM±SD) (n=3)

Assay (%)

RSD (%)

Farobact

200

201.65+0.14

100.83

0.07

System suitability tests for FRPM:

For establishing the system suitability, different parameters like theoretical plates, tailing factor, retention time and resolution were evaluated. The results of system suitability tests, shown in Table 6, proved that the method is suitable for the intended purpose.

Table 6: System suitability tests for FRPM

Sr. No.	Parameters	Values
1	Retention time(min)	0.66
2	Theoretical plates	2145
3	Tailing factor	1.69
4	Resolution	>2

RESULTS OF FORCED DEGRADATION STUDIES OF FRPM:

The results of the forced degradation study indicated that the developed method is specific due to the absence of the interfering peaks of degradation products corresponding to the retention time of the drug (0.66 min). The drug undergoes hydrolysis in alkaline, acidic, oxidative and photolytic conditions. The drug was highly susceptible to alkaline degradation, and hence 0.01 N NaOH was used. Chromatograms of forced degradation studies are given in Figures 4 to 7, and the degradation rates are given as a percentage in Table 7.

Figure 4: Chromatogram of alkaline degradation of FRPM

Figure 5: Chromatogram of acidic degradation of FRPM

Figure 6: Chromatogram of oxidative degradation of FRPM

Figure 7: Chromatogram of Photolytic degradation of FRPM

Table 7: Degradation of FRPM (%)

Sl. No.	Stress condition	Degradation of FRPM (%)
1	Acidic (1N HCl, 48 hrs)	52.32
2	Alkaline (0.1N NaOH, 4 hrs)	64.32
3	Oxidative (3%H₂O₂, 4 hrs)	47.13
4	Photolytic (UV radiations of 254 nm exposed to 48 hrs)	61.87

CONCLUSION:

An ultra-fast, accurate, economic and LC-MS compatible isocratic RP-UPLC method is developed to analyse Faropenem bulk drug and its injectable formulation. The stress degradation studies proved that the drug undergoes degradation using alkaline, acidic,oxidative and photolytic stress conditions. The degradation products’ peaks are well resolved from the sample peak. Hence, one can use the method for the routine QC analysis of Faropenem samples and stability samples.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the Principal and the management for the laboratory facilities.

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Received on 06.12.2022 Modified on 16.09.2023

Research J. Pharm. and Tech 2024; 17(4):1498-1502.

DOI: 10.52711/0974-360X.2024.00237