HPLC Determination of Three Cephalosporins (Cefepime, Cefotriaxone and Cefotaxime) in Their Bulk and Dosage Forms

 

Sobhy M. El-Adl1, Abdallah A. El-Shanawany1, Lobna M. Abdel-Aziz1, Ali F. Hassan2*

1Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazige University, Zagazig, Egypt.

2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assuit, Egypt.

*Corresponding Author E-mail: dr_a_n@yahoo.com

 

ABSTRACT:

An isocratic HPLC method had been developed for rapid simultaneous separation and determination of three ceohalosporins including Cefepime, Cefotriaxone and Cefotaxime in pure form or in presence of some impurities within 8 minutes. Separation was carried out on a Hypersil gold C18 (10um, 100x4.6mm) column. Effect of pH and composition of mobile phase was studied. Beer’s law was obeyed in the range of 1-70 µg/ml for all drugs. The method was applied for the determination of drugs in both bulk and pharmaceutical forms and was validated when obtained results were compared with reference methods. 

 

KEYWORDS: Cefepime, Cefotriaxone, Cefotaxime, Hypersil gold, mobile phase.

 

1. INTRODUCTION:

Cephalosporins, like all β-lactam antibiotics, inhibit bacterial growth by interfering with a specific step in bacterial cell wall synthesis [1]. Cephalosporins consist of a fused β-lactam-A -dihydrothiazine two-ring system, known as 7-ACA, and vary in their side chain substituents at C3 (R2), and C7 (acylamido, Ri) [2]. The later generation agents, with their better spectrum of activity against gram-negative bacteria make them useful for hospital-acquired infections or complicated community-acquired infections.

 Several methods have been developed for their determination, including  UV specrootomtric methds[3-7], Visible specrootomtric methds[8-14], spectroflurometry [15], high-performance thin layer chromatography (HPTLC) [16,17], high-performance liquid chromatography (HPLC) [3,18- 28], electro chemical methods(voltammetry) [29-31], capillary electrophoretic methods [32].

 

Hypersil Gold columns are Endcapped, ultrapure, silica-based columns with exceptional peak shape and resolution for HPLC and LC/MS Significant reduction in peak tailing while retaining C18 selectivity, Excellent resolution, efficiency and sensitivity Confidence in the accuracy and quality of analytical data well suited to extended pH applications. Hypersil GOLD columns have been shown to produce robust assays at high pH. At low pH, excellent column stability and reproducibility are illustrated.

 

Some few HPLC methods using Hypersil gold columns has been reported for Recent developments in liquid chromatography—Impact on qualitative and quantitative performance [33], Evaluation of ultra performance liquid chromatography [34], Use of ultra-performance liquid chromatography in pharmaceutical development [35] and determination of some pharmaceutical drugs such as Miconazole, primaquine phosphate and its impurities[36], and Determination of Cotinine in Urine[37]. However, No HPLC method for determination of Cephalosporins using Hypersil gold columns has been reported till date.

 

In this part, an isocratic HPLC method had been developed for rapid simultaneous separation and determination of three cephalosporins including cefepime, cefotaxime and cefotriaxone in pure form within 8 minutes. Separation was carried out on a Chromolith® Performance RP-18e (100 x 4.6 mm) using a mobile phase of MeOH : 0.025M KH2PO4 adjusted to pH 7.5 using trimethyl amine (16:84, v/v) at ambient temperature. The flow rate was 1 ml/min and maximum absorption was measured at 270nm.

 

2. EXPERIMENTAL:

2.1. Apparatus:

HPLC apparatus equipped with a Surveyor quaternary pump with Intel vacuum degasser (Thermo Scientific Co. USA) , Surveyor autosampler plus (Thermo Scientific Co., USA), Surveyor photodiode array detector (PAD) (Thermo Scientific Co. USA). Computer with a software chromo quest 5 (Surveyor Thermo Scientific Co. USA), for data collection and analysis, Hypersil gold C18 (10um, 100x4.6mm) column (Thermo Scientific Co. USA). Autosampler vials 1.8 ml screw cap, Thermo Scientific, USA.

 

Consort P400® digital pH-meter for pH adjustment.

 

2.2. Materials and reagents:

All solvents and reagents were of an HPLC analytical grade (methanol, potassium dihydrogen phosphate and triethylamine were supported from Romil, England).

 

Cefepime (Adwia, Egypt) purity 97.19%, Cefotaxime (EPICO, Egypt) purity 95.06% and Cefotriaxone (EIPICO, Egypt) purity 98.11%.  Standard solutions 100 µg.ml-1 was prepared individually by dissolving 10 mg of each pure drug in 100 ml of the mobile phase.

 

Mobile phase was a freshly prepared binary mixture of methanol: 0.025M potassium dihydrogen phosphate adjusted to pH 7.5 using triethylamine (16:84, v/v), filtered and degassed using 0.45µm membrane filter.

 

2.3. Pharmaceutical preparations:

The following available vial preparations were analyzed.

Wincef® vials labeled to contain 1000 mg cefepime per vial.  Batch No. 090235\9869 (Adwia, Egypt).

 

Cefotax® vials labeled to contain 1000 mg cefotaxime per vial.  Batch No.1202601 (Eipico, Egypt).

 

Ceftriaxone® vials labeled to contain 200 mg cefotriaxone per vial.  Batch No.1280325 (Kahira, Egypt).

 

2.4. Procedures:

2.4.1. Procedure for authentic powder:

An accurately amounts of the powder equivalent to 10 mg of each drug were dissolved in 25 ml of the mobile phase, filtered into 100 - ml measuring flask and completed to volume with the mobile phase, Appropriate mixed dilution of the standard stock solutions was done in 10 - ml volumetric flask to get final concentration of 50 µg.ml-1 for all drugs.. A 10 μl of the mixture was injected into the column and the chromatogram was obtained at 254 nm.

 

2.4.2. Procedure for marketed vials:

A vial of each formulation were powdered and weighed. An accurately amounts of the powder equivalent to 10 mg of each drug were dissolved in 25 ml of the mobile phase, filtered into 100 - ml measuring flask and completed to volume with the mobile phase. The procedure was then completed as previously mentioned under the Procedure for authentic powder 2.4.1.

 

3. RESULTS AND DISCUSSION:

3.1. Optimization of Chromatographic Conditions:

All chromatographic conditions are illustrated in table 1. the chromatographic detection was performed at 254nm using Surveyor photodiode array detector (PAD) (Thermo Scientific Co. USA). The method was performed on a Hypersil gold® C18 (10um, 100x4.6mm) column (Thermo Scientific Co. USA). It was observed that when a combination of all the three drugs was injected, Cefepime and Cefotriaxone together gave a mixed peak. Chromatographic conditions were optimized by changing the mobile phase composition and buffers used in the mobile phase. Different experiments were performed to optimize the mobile phase but adequate separation of drugs could not be achieved. By altering the pH, mobile phase and flow rate, a good separation was achieved (figs. 1, 2, and 3). The optimized mobile phase was determined as a mixture of methanol: 0.025M potassium dihydrogen phosphate adjusted to pH 7.5 using triethyl amine (16:84, v/v) at a flow rate of 1 ml/min. Under these conditions, Cefepime, Cefotriaxone and Cefotaxime were eluted at1.9, 3.04, and 7.6 minutes respectively with a run time of 10 minutes.

 

A typical chromatogram for simultaneous estimation of the three drugs obtained by using the aforementioned mobile phase is illustrated in figures (4, 5) for authentic mixture and vial formulations respectively.

 

3.2. Method Validation:

The developed methods were validated according to international conference on harmonization guidelines [38].

 

3.2.1. Linearity:

Nine different concentrations of a mixture of all three drugs were prepared for linearity studies. The response was measured as peak area. The calibration curves obtained by plotting peak area against concentration showed linearity in the concentration range of 0.8 -70 µg.ml-1in case of ceftriaxone and 1 - 70 µg.ml-1 for other drugs. Linear regression equations of Cefepime, Cefotriaxone and Cefotaxime were found to be y = 27918x + 21730,  y = 26863x + 29305 and y = 24883x + 26634, respectively and the regression coefficient values (r) were found to be 0.999, 0.999 and 0.999, respectively indicating a high degree of linearity for all drugs.

 

3.2.2. Accuracy:

The accuracy of the method was determined by investigating the recovery of drugs at concentration levels covering the specified range (three replicates of each concentration). The results showed excellent recoveries (table 2).

 

3.2.3. Precision:

Intra - day precision was evaluated by calculating standard deviation (SD) of five replicate determinations using the same solution containing pure drug. The SD values revealed the high precision of the methods. For inter - day reproducibility on a day - to - day basis, a series was run, in which the standard drug solutions were analyzed each for five days. Results showing in (table 3).

 

 

 

3.2.4. Specificity:

The specificity studies revealed the absence of any excipent or impurity interference, since none of the peaks appeared at the same retention time of Cefepime, Cefotriaxone and Cefotaxime as shown in figure 6.

 

3.2.5. L.D. and L.Q.:

For determining the limit of detection (L.D.) and limit of quantitation (L.Q.), the method based on signal – to - noise ratio (3:1 for L.D. and 10:1 for L.Q.) was adopted. The limit of detection for the three drugs was 0.250µg.ml-1 while the limit of quantitation was 0.8 µg.ml-1  (table 2).

 

3.2.6. Robustness:

The robustness of the method was evaluated by making small changes in one parameter keeping the other chromatographic conditions constant such as the flow rate (0.9, 1, and 1.1), pH of mobile phase within a range of ± 0.1 unit of the optimized pH and mobile phase ratio where the effect of the changes was studied on the percent recovery of drugs. The changes had negligible influence on the results as revealed by small SD as shown in (table 4).

 

3.3. Applications:

Some Pharmaceutical formulations containing stated drugs have been successfully analyzed by the proposed method. Results obtained were compared to those obtained by applying reported reference methods [94] where Student’s t-test and F-test were performed for comparison. The calculated t and F values were less than tabulated values for the four drugs which in turn indicate that there is no significant difference between proposed method and reference ones relative to precision and accuracy.

 

4. CONCLUSION:

An RP-HPLC method for rapid simultaneous estimation of Cefepime, Cefotriaxone and Cefotaxime within 8 minutes was developed and validated. The amounts obtained by the proposed method are between 98.3% and 101.6%, within the acceptance level of 95% to 105%. The results obtained indicate that the proposed method is rapid, accurate, selective, and reproducible. Linearity was observed over a concentration range of 1 to70 ug.ml-1 for all three drugs. The method has been successfully applied for the analysis of marketed vials.  It can be used for the routine analysis of formulations containing any one of the above drugs or their combinations without any alteration in the assay. The main advantage of the method is the common chromatographic conditions adopted for all formulations in addition to reduced analysis time due to column.

Fig.(2)  HPLC Chromatogram of authentic mixture of 50 µg.ml-1 Cefepime(A), Cefotriaxone(B) and Cefotaxime(C) at different pH.

Column:  Hypersil gold C18 (10um, 100x4.6mm) column.

Mobile phase:   MeOH : 0.025M  KH2PO4  adjusted to  pH  using  Triethylamine  (16:84, v/v).

Flow rate  :   1 ml/min.

pH :    6(a), 7(b) and 7.5(c).

 

Fig.(3)  HPLC Chromatogram of authentic mixture of 50 µg.ml-1 Cefepime(A), Cefotriaxone(B) and Cefotaxime(C) at different percentages of mobile phase.

Column          :  Hypersil gold C18 (10um, 100x4.6mm) column.

Mobile phase:   MeOH : 0.025M  KH2PO4  adjusted to  pH  7.5  using  Triethylamine  20:80, v/v (a), 15:75, v/v (b) and 16:84, v/v (c).

Flow rate       :   1 ml/min.

pH                 :    7.5

 

Fig.(4)  HPLC Chromatogram of authentic mixture of 50 µg.ml-1 Cefepime(A), Cefotriaxone(B) and Cefotaxime(C) at different flow rates.

Column          :  Hypersil gold C18 (10um, 100x4.6mm) column.

Mobile phase:   MeOH : 0.025M  KH2PO4  adjusted to  pH  7.5  using   Triethylamine  (16:84, v/v).

Flow rate       :   1.5 ml/min (a), 0.8 ml/min (b) and 1 ml/min(c).

pH                 :    7.5

 

 

Fig.(5)  HPLC Chromatogram of authentic mixture of 50 µg.ml-1 Cefepime(A), Cefotriaxone(B) and Cefotaxime(C).

Column          :  Hypersil gold C18 (10um, 100x4.6mm) column.

Mobile phase  :   MeOH : 0.025M  KH2PO4  adjusted to  pH  7.5using Triethylamine  (16:84, v/v).

Flow rate       :   1 ml/min.

pH                 :    7.5

 

Fig.(6)  HPLC Chromatogram of mixture of 50 µg.ml-1 marketed Cefepime(A), Cefotriaxone(B) and Cefotaxime(C).

Column          :  Hypersil gold C18 (10um, 100x4.6mm) column.

Mobile phase   :   MeOH : 0.025M  KH2PO4  adjusted to  pH  7.5using  Triethylamine  (16:84, v/v).

Flow rate        :   1 ml/min.

pH                  :    7.5

Table(1).Chromatographic Conditions for the proposed method.

Parameters

Conditions

Column

Hypersil gold® C18 (10um, 100x4.6mm) column

Mobile phase

Isocratic binary mobile phase of MeOH : 0.025M KH2PO4 adjusted to pH 7.5 using triethylamine (16:84, v/v), filtered and degassed using 0.45µm membrane filter

UV detection, nm

254

Flow rate, ml/min

1

Injected volume, µl

10

Pressure, MPa

11

Temperature

Ambient (25±5  ͦC)

 

 

Table(2).Results of the analysis for the proposed method.

parameters

Cefepime*

Cefotriaxone*

Cefotaxime*

Taken µg/ml

Found µg/ml

Recovery %

Taken µg/ml

Found

µg/ml

Recovery

%

Taken µg/ml

Found

µg/ml

Recovery %

 

1

0.98

98.113

0.8

0.787

98.44

1

0.9967

99.67

 

5

4.994

99.88

5

4.917

98.34

5

5.081

101.624

 

10

10.086

100.86

10

9.999

99.99

10

10.182

101.82

 

20

20.366

101.83

20

20.13

100.68

20

19.954

99.77

 

30

29.708

99.028

30

30.08

100.28

30

29.82

99.40

 

40

39.509

98.77

40

39.89

99.74

40

40.188

100.47

 

50

50.124

100.25

50

50.07

100.14

50

49.49

98.98

 

60

60.214

100.36

60

59.88

99.8

60

59.81

99.68

 

70

70.017

100.03

70

70.03

100.06

70

70.48

100.69

Mean

 

 

99.90

 

 

99.72

 

 

100.235

±SD

 

 

1.13

 

 

0.80

 

 

0.988

±RSD

 

 

1.13

 

 

0.805

 

 

0.986

±SE

 

 

0.340

 

 

0.242

 

 

0.285

Variance

 

 

1.278

 

 

0.6456

 

 

0.976

Slope

 

 

27918.2

 

 

26709.51

 

 

24883.5

L.D.

 

 

0.239

 

 

0.250

 

 

0.269

L.Q.

 

 

0.798

 

 

0.834

 

 

0.895

* Average of three independent procedures.

 

 

 

Table (3). Intra – day and interday precision of three drugs.

Drug

Conc. µg/ ml

Intraday

Interday

Mean± SD

RSD

Mean± SD

RSD

Cefepime

50

101.2 ± 0.179

0.177

100.8± 0.57

0.56

Cefotriaxone

50

99.9 ± 0.122

0.122

100.034 ± 0.23

0.23

Cefotaxime

50

100.34 ± 0.26

0.26

100.06± 0.48

0.48

 

Table(4). Robustness

 

Parameters

% Of recovery ± SD

Cefepime

Cefotriaxone

Cefotaxime

Flow rate 0.9

101.8±1.31

101.9±0.98

100.55±0.87

Flow rate 1.1

98.15±1.26

99.2±0.73

98.06±1.16

Methnol:Buffer17:83

101.5±1.26

101.7±0.93

101.4±0.93

Methnol:Buffer 15:85

98.45±1.22

98.5±0.79

98.3±1.11

pH 7.4

101.85±1.26

101.4±0.88

101.8±0.99

pH 7.6

98.7±1.18

98±1.86

98.7±1.05

 

 

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Received on 07.07.2014       Accepted on 20.08.2014     

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