Development and Validation of RP-HPLC Method for Estimation of Zinc-Sparfloxacin 1,10-Phenanthroline Metal Complex.

 

Prafulla M. Sabale1*, Jignesh Chavda2, Vidya Sabale3

1Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University,

Mahatma Jyotiba Fuley Shaikshanik Parisar, Amravati Road, Nagpur-440 033 India

2Department of Pharmaceutical Quality Assurance, Parul Institute of Pharmacy, Limda-391 760, Vadodara, Gujarat, India

3Department of Pharmaceutics, Parul Institute of Pharmacy and Research, Limda-391 760, Vadodara, Gujarat, India

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

 

ABSTRACT:

The present research work describes the development of a new simple, precise, sensitive and validated RP-HPLC method for Zn-sparfloxacin 1,10-phenanthroline metal complex. The chromatographic conditions used for the separation was Phenomenex Luna C18 (250mm × 4.6mm, 5μm particle size) and mobile phase comprised of methanol: phosphate buffer (30:70 v/v) and pH was adjusted to 3 by o-phosphoric acid. The flow rate was 1.0 ml/min with detection at 293 nm. The retention time was found to be 4.357 min. The linearity was found to be in the range of 2-10 μg/ml for Zn-spar-phe metal complex with correlation coefficient of 0.9996. The proposed method is accurate with 98-102 % recovery and precise (%RSD of repeatability, intra-day and inter-day variations were 0.240, 0.7989-1.1386 and 1.09-1.53 respectively). The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.2658 and 0.8056 μg/ml respectively. Proposed method was validated as per ICH guidelines for precision, accuracy and linearity for estimation of Zn-spar-phe Metal complex. Results of the validation were found satisfactory.

 

KEYWORDS: RP-HPLC, Method development, Validation, Accuracy, Metal complex, Sparfloxacin, Zn-sparfloxacin-1,10-phenanthroline metal complex.

 

 


INTRODUCTION:

Sparfloxacin (C19H22F2N4O3) is a synthetic fluoroquinolone broad spectrum antibiotics used in the treatment of bacterial infections. It exerts its antibacterial activity by inhibiting DNA gyrase, a bacterial topoisomerase.  DNA gyrase is an essential enzyme which controls DNA topology and assists in DNA replication, repair, deactivation, and transcription. It is 5-amino-1-cyclopropyl-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Sparfloxacin has in vitro activity against a wide range of gram-negative and gram-positive microorganisms [1, 2]. There are numbers of antibiotics called metallo-antibiotics that require metal ions to function properly. Thus complexation with metals enhances the biological activity of quinolone ligand antibiotics which induces lipophilicity and facilitate in greater permeability of the drug into the site of action [3].

 

Metal complexes are known as coordination compounds, consisting of a central atom or ion (metal) bonded with anions (ligands). The environment around the metal center coordination geometry, number of coordinated ligands and their donor group is the key factor for metalloprotein to carry out a specific physiological function [4]. Interaction of various metal ions with antibiotics may enhance their antimicrobial activity as compared to that of free ligands [5]. Due to the presence of vicinal oxo and carboxyl groups, Fluoroquinolones like sparfloxacin, ofloxacin, levofloxacin, pefloxacin, norfloxacin and gatifloxacin are capable of forming a well-defined metal-fluoroquinolone complex. The presence of metal ion results in a higher uptake of quinolones by bacterial cells compared to that of the drug alone [6-8].

 

Literature survey reveals that there are few methods for estimation based on spectrophotometric and RP-HPLC techniques developed for sparfloxacin alone and also with other combinations [9-13]. Till now there were no RP-HPLC method reported for metal complex of sparfloxacin.

 

Our research group from Pharmaceutical chemistry department working on metalloantibiotics, have synthesized different complexes with N-N donars such as 2,2’-Bipyridyl and 1,10-Phenanthroline and characterized on the basis of spectral interpretations. Metal complexes evaluated for antimicrobial activity have also exhibited moderate activity against Escherichia coli and Staphylococcus aureus, as compared to parent fluroquinolones.  The preliminary studies has been carried out at the Dept. considering the fact based on its biological and synthetic studies and it gave better biological activity than its Parent Drugs [14-16]. Various analytical methods were also developed and validated for metal complexes [17-20]. In this article we used Zinc metal to complex with sparfloxacin and N-N donor 1,10-pheanthroline to form Zn-spar-phe metal complex as shown in (Fig. 1).

 

Figure 1: Zn-Sparfloxacin-1, 10-Phenanthroline

 

MATERIALS AND METHODS:

Apparatus and Instrumentation

The HPLC system was of LC-20 AT (Shimadzu) with single wavelength UV detector. The chromatographic analysis was performed using EZ-Chrom software on a Phenomenex Luna C18 (250 mm x 4.6 mm), 5μ column. UV Spectrophotometer Pharmspec-1700 (Shimadzu). In addition, an electronic analytical balance (Mettler Toledo, model XP205, Switzerland), and a Sonicator (Ultrasonic cleaner, model OU-72SPL), a pH meter model: L1610 (Elico) and a vacuum filter assembly were used in this study.

 

Reagents and Materials

Sparfloxacin was a received as gift sample from Alembic Research Center, Vadodara. Zn-spar-phe metal complex was synthesized and its structure was studied with spectral studies at Dept of Pharmaceutical Chemistry, Parul Institute of Pharmacy, Vadodara. Methanol (HPLC Grade) from Fischer scientific was used. All other chemicals were purchased from s.d. Fine Chemicals Ltd.

 

Selection of mobile phase

Based on sample solubility, stability and suitability various mobile phase compositions were tried to get a good resolution and sharp peaks. The standard solution was run in different mobile phases. From the various mobile phases methanol: phosphate buffer [30:70 v/v] pH 3 adjusted with o-phosphoric acid was chosen with detection wavelength 293 nm, since it gave sharp peak with good symmetry within limits. The mobile phase and samples was filtered using 0.2 µm membrane filter before injecting in HPLC system. Mobile phase was degassed by ultrasonicator. All determinations were performed at ambient temperature. The chromatogram obtained with the above mentioned mobile phase is shown in (Fig. 2).

 

Figure 2: Chromatogram of Zn-spar-phe metal complex ( MeOH : Phosphate Buffer 30:70,  pH 3.0 adjusted with o Phopharic acid )

 

Chromatographic conditions

The optimized parameters which were used as a final method for the estimation of Zn-spar-phe metal complex were represented in Table 1.

 

Table 1: Optimised chromatographic conditions

 Mobile phase

Methanol: Phosphate Buffer (30:70 v/v)

 Stationary phase

Phenomenex Luna C18 (4.6 x 250 mm, 5μ) column

 Wavelength

293 nm

 Run time

10 min

 pH of the mobile phase

3

 Flow rate

1 ml/min

 Injection volume

20 µl

 Temperature

Ambient

 Mode of operation

Isocratic elution

 

 

PREPARATION OF SOLUTIONS:

Preparation of standard stock solution

Standard drug solution of Zn-spar-phe metal complex was prepared by dissolving 10 mg Zn-spar-phe metal complex in 10 ml distilled methanol. This solution was then sonicated for 10 min to prepare the 1000 μg/ml stock solution. Then transfer 1 ml from stock solution and dilute upto the mark with distilled methanol to prepare working standard stock solution.

 

Preparation of calibration curve

Aliquots of 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard stock solution (100 μg/ml) was transferred to 10 ml of volumetric flasks and made up to the mark with distilled methanol to get concentration of 2, 4, 6, 8 and 10 μg/ml of Zn-spar-phe metal complex. Solutions were scanned in the range of 400-200 nm against blank. The absorption maxima were found to be at 293 nm against blank. Calibration curve was constructed by plotting the peak areas versus the concentration and the regression equation was calculated as shown (Fig. 3).

 

Figure 3: Linearity curve of Zn-Spar-Phe complex

 

System suitability test

20 μL of the standard solution was injected under optimized chromatographic conditions to evaluate the suitability of system. The values of system suitability parameters were shown in Table 2.

 

Table 2: System Suitability Test Parameters

Parameter

Zn-spar-phe metal complex

Retention Time (min.) ± SD

4.357 ± 0.07

Theoretical Plates ± SD

8732 ± 159

Tailing factor± SD

1.27 ± 0.09

 

 

METHOD DEVELOPMENT AND VALIDATION [21, 22]:

Selection of wavelength

From the working standard stock solution of Zn-spar-phe metal complex, a 10 µg/ml of solution was prepared and then scanned over the wavelength range of 200-400 nm. The wavelength maximum was selected by observing the obtained spectra for highest absorbance. The UV spectrum showing wavelength selected for detection at 293 nm in Distilled Methanol was shown (Fig. 4).

 

Linearity

The linearity of the response of Zn-spar-phe metal complex was verified at 2-10 μg/ml concentration as shown in   Table 3. The calibration curve was obtained by plotting the Concentration versus Area and linear regression analysis also obtained as shown (Fig. 5).

 

Figure 4: UV spectra of Zn-Spar-Phe complex

 

Table 3: Linearity data for Zn-spar-phe complex

Concentration (μg/ml)

Area

2

156.23

4

1545.36

6

3212.57

8

4716.41

10

6423.12

 

Figure 5: Calibration curve of Zn-Spar-Phe complex

 

Precision

The precision of the method was established by carrying out the analysis of the analytes (n=6) using the proposed developed methods. It was verified by repeatability, Inter-day and intra-day precision. Precision was carried out on 3 different concentrations, 80%, 100% and 120% of nominal concentration of both drugs. The experiment was repeated 3 times at same time for repeatability, 3 times in a day for intra-day and on 3 different days for inter-day precision. Precision was expressed in terms of %RSD. The low value of standard deviation showed that the methods were precise. Details of precision in terms of repeatability, interday and intraday precision were recorded in Table 4-6.

 


 

Table 4: Repeatability for Zn-Spar-Phe complex

Sr. no.

Zn-Spar-Phe complex (μg/ml)

1

2

3

4

5

6

Mean

SD

%RSD

1

6

3189.16

3182.72

3179.34

3165.78

3179.26

3180.48

3179.45

7.6544

0.2407

 

Table 5: Intraday Precision for Zn-Spar-Phe complex

Zn-Spar-Phe complex

Area

1 hr

2 hr

3 hr

Mean

SD

%RSD

4

1535.52

1519.16

1554.12

1536.267

17.49196

1.138602

6

3175.16

3127.36

3165.18

3155.9

25.21505

0.798981

8

4698.39

4622.17

4667.3

4662.62

38.32491

0.821961

Range of %RSD                                                                                                                                                                         0.798-1.138

 

Table 6: Interday Precision for Zn-Spar-Phe complex

Zn-Spar-Phe complex

Area

1 day

2 day

 3 day

Mean

SD

%RSD

4

1545.52

1504.16

1544.12

1531.27

23.4855

1.53373

6

3189.16

3113.36

3165.18

3155.9

38.7427

1.22763

8

4678.39

4592.17

4682.3

4650.95

50.9454

1.09538

Range of %RSD                                                                                                                                                                         1.09-1.533

 


Accuracy

For the accuracy of proposed method, recovery studies were performed by standard addition method at three different levels (80%, 100% and 120% of final concentration). A known amount of Zn-spar-phe metal complex was added to pre-analyzed drug  sample and  then analyzed by proposed method. Results of recovery studies were found to be satisfactory and reported in Table 7. % Recovery is calculated by Following Equation.

 

Table 7: Accuracy of Zn-Spar-Phe complex

Sr. no.

Concentration

Area

Amount Found

Mean

%

Recovery

1

4

1587.39

3.92

3.95

99.15

2

4

1596.82

3.94

3

4

1625.47

3.97

4

6

3146.78

5.91

5.99

99.87

5

6

3294.28

6.09

6

6

3192

5.97

7

8

4682.12

7.86

7.88

98.58

8

8

4698.37

7.88

9

8

7.92

7.92

 

 

Robustness

To evaluate robustness of the developed method, few parameters were deliberately varied. These parameters included variation in flow rate, organic phase ratio, detection wavelength and pH of mobile phase. The robustness of the method was determined by calculating area at different flow rate, area at different mobile phase ratio of same solvent system and also robustness is determined by calculated area at three different wavelengths. The average value of robustness expressed in % RSD for all parameters were shown in Table 8-10.

 

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Limit of Detection (LOD) and Limit of Quantification (LOQ) were estimated from the signal-to-noise ratio. The LOD defined as the lowest concentration that gave a peak area with signal-to-noise ratio. It may be expressed as the concentration that gives a signal to noise ratio of 2:1 or 3:1. LOD was calculated by using the formula:

 

LOD = 3.3SD/𝑆

Where,    SD = standard deviation of the response

𝑆 = slope of calibration curve of the analyte.

 

LOQ defined as is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy.  It may be expressed as a signal to noise ratio 10:1. LOQ was calculated by using the formula:

 

LOQ = 10SD/𝑆

Where,    SD = standard deviation of the response

𝑆 = slope of calibration curve of the analyte.

 

The 10 blank samples were injected into the HPLC column and then the mean of the areas obtained is calculated. Then find the standard deviation (SD). Plot the calibration curve (n=3) and the slope (S) of regression equation is obtained. The results of the same were shown in Table 11.

 


 

 

Table 8: Robustness [Area at Different Flow Rate for Zn-Spar-Phe complex]

Zn-Spar-Phe complex

Area

0.8ml/min

1ml/min

1.2 ml/min

  Mean

SD

%RSD

4

1536.36

1535.52

1538.64

1536.84

1.614

0.105

6

3187.29

3148.71

3199.17

3195.057

6.730

0.2106

8

4639.53

4698.39

4667.12

4668.347

29.44

0.6308

Range of % RSD                                                                                                                                                                        0.105-0.630

 

Table 9: Robustness [Area at Different Mobile Phase Ratio for Zn-Spar-Phe complex]

Zn-Spar-Phe complex

Area

28:72

30:70

32:68

Mean

SD

%RSD

4

1544.76

1545.36

1559.72

1549.94

8.469

0.5464

6

3155.23

3176.22

3192.12

3174.523

18.503

0.5828

8

4651.37

4671.81

4676.48

4673.48

22.588

0.4833

Range of % RSD                                                         0.4833-0.5828

 

Table 10: Robustness [Area at Different Wavelength for Zn-Spar-Phe complex]

Zn-Spar-Phe complex

Area

 

291

293

295

Mean

SD

%RSD

4

1577.38

1578.32

1557.12

1570.94

11.97

0.7621

6

3178.47

3189.16

3165.56

3177.73

11.81

0.3718

8

4681.52

4698.34

4676.25

4683.72

13.70

0.2925

Range of % RSD                                                                                                                                                                                            0.292-0.762

 

 


Table 11: LOD and LOQ

Parameter

Zn-Spar-Phe complex

SD of intercepts

62.425

Mean slope

774.86

LOD (μg/ml)

0.2658

LOQ (μg/ml)

0.8056

 

 

RESULT AND DISCUSSION:

The objective of the study was to develop and validate a simple, selective, rapid, accurate, precise RP-HPLC method for the estimation of Zn-spar-phe metal complex using Phenomenex Luna C18 (4.6 x 250 mm, 5μ) column with U.V detection. Initially various mobile phase compositions were tried to elute complex but mobile phase comprising of methanol: phosphate buffer (30:70 v/v) pH-3 adjusted with o-phosphoric acid was found to be giving better resolution with run time 10 min and flow rate 1ml/min. A working standard solution of particular concentration was injected and the chromatogram was recorded as shown (Fig. 2). In this method, first the conditions were optimized to obtain the elution of complex as mentioned in Table 1. The system suitability parameters like tailing factor (1.27 ± 0.09), theoretical plates (8732 ± 159) and retention time (4.357 ± 0.07) were found within limits as shown in Table 2. The linearity of the method was studied by injecting 20 µl of working standard solution of concentration ranging from 2-10 µg/ml into the column, the linearity results were obtained as described in Table 3. A calibration curve was constructed by plotting concentration against peak area and a good linear relationship (r2 = 0.9996) was obtained as shown (Fig. 3, 5). For precision, the % RSD of repeatability study was found to be 0.2407, which was within limit as mentioned in Table 4. The intraday precision studied in terms of range of % RSD, was found in the range of 0.798-1.138 which is acceptable as shown in Table 5. The interday precision studied was found to be in range of 1.09-1.533 which was within acceptable criteria as shown in Table 6. Accuracy of the method was determined from recovery experiments. The recovery studies were carried out at 80%, 100% and 120% of final concentration and % recovery obtained was within the range of 98-102 which was acceptable as described in Table 7. In robustness, a deliberate change in the flow rate, mobile phase ratio and wavelength was made to evaluate the impact on the method. The range of % RSD obtained for different flow rate was 0.105-0.630, for different mobile phase ratio was 0.4833-0.5828, for different wavelength was 0.292-0.762. The results of % RSD less than 2% revealed the robustness of the method as described in Table 8-10. The LOD and LOQ values found were 0.2658 and 0.8056 respectively as shown in Table 11. The developed method is found to be accurate and precise as indicated by recovery studies and % RSD not more than 2 and were found to be within limits as per ICH guidelines. The summary of validation parameter for RP-HPLC method for Zn-spar-phe complex was shown in Table 12.

 

 

Table 12: Summary of Validation Parameter for RP-HPLC method

Sr. No.

Validation Parmeter

Zn-Spar-Phe complex

1

Linearity

Regression Equation

y = 781.3x - 1498

Regression Coefficient

0.9996

2

Range

2-10(μg /ml)

3

Precision (%RSD)

Repeatability

0.240

Intraday

0.798-1.13

Interday

1.09-1.53

4

Accuracy

(% Recovery)

98-102%

5

Robustness (%RSD)

Different Flow Rate

0.105-0.630

Different Mobile Phase Ratio

0.483-0.582

Different Wavelengths

0.292-0.762

6

LOD (μg /ml)

0.2658

7

LOQ (μg /ml)

0.8056

 

 

 

CONCLUSION:

The developed RP-HPLC method was proved to be simple, rapid, and reproducible. The validation data indicate good precision, accuracy, and reliability of the method. This makes the method more specific & reliable for estimation of Zn-spar-phe Metal Complex. There were no reported methods developed for such metal complexes; hence, this method has an advantage of being unique and novel. Statistical analysis proves that the method is suitable for routine analysis of Zn-spar-phe metal complex.

ACKNOWLEDGMENTS:

We would like to thanks Dr. Devanshu J Patel, Managing Trustee Parul Trust for providing necessary infrastructure and Dr. Rajesh K. S. Principal, Parul Institute of Pharmacy, Limda, Vadodara for offering precious suggestions.

 

REFERENCES:

1.       Drug bank: open data drug and drug database. Available at http://www.drugbank.ca/drugs/DB01208 Assessed on 05.08.2013

2.       Patel MN, Joshi HN, Patel CR. Cytotoxic, antibacterial, DNA interaction and superoxide dismutase like activities of sparfloxacin drug based copper(II) complexes with nitrogen donor ligands. Spectrochim Acta A Mol Biomol Spectrosc, 104, 2013 ; 48-55.

3.       Klement R, Stock F, Elias H, Paulus H, Pelikan P, Valko M,Mazur M. “Copper(II) complexes    with derivatives of salen and tetrahydrosalen: a spectroscopic, electrochemical and structural study”. Polyhedron, 18, 1999 ; 3617- 3628.

4.       Abd El-Wahab Z H, El-Sarrag M R. “Derivatives of phosphate Schiff base transition metal complexes: synthesis, studies and biological activity”. Spectrochimica Acta, 60, 2004 ; 271-277.

5.       Ming L J. Structure and function of metalloantibiotics. Med Res Rev, 23,  2003 ; 697- 762,.

6.       Wallis S C, Gahan L R, Charles B G, Hambley T W and Duckworth P A. Copper(I) Complexes of the Fluoroquinolone Antimicrobial Ciprofloxacin.Synthesis, X-Ray Structural Characterization, and Potentiometric Study. J.Inorg. Biochem; 62, 1996 ; 1-16,.

7.       Sabale PM, Kaur P, Patel Y, Patel J and Patel R. Metalloantibiotics in Therapy: An overview. J Chem Pharm Res, 4(11), 2012 ; 4921-4936.

8.       Sabale PM, Patel Y, Patel J and Patel R. Metal Complexes: Current Trends and Future Potential. Int J Pharma, Chem Biol Sci., 2(3), 2012 ; 251-265.

9.       Shah J, Jan MR, Khan I, Khan MN. Quantification of sparfloxacin in pharmaceutical dosages and biological samples. Pak J Pharm Sci. 25, 2012 ; 823-29.

10.     Borner K, Borner E, Lode H. Determination of sparfloxacin in serum and urine by high-performance liquid chromatography. J Chromatogr.579, 1992 ; 285-89,.

11.     Gupta H, Aqil M, Khar RK, Ali A, Sharma A, Chander P. Development and validation of a stability-indicating RP-UPLC method for the

12.     quantitative analysis of sparfloxacin. J Chromatogr Sci.48, 2010 ; 1-6,.

13.     Cao SX, Zhang JY, Ji XM, Liu HM. Quantitative analysis of sparfloxacin injection by high performance liquid chromatography. Se Pu.; 19, 2001 ; 454-56,.

14.     Salem MY, El-Guindi NM, Mikael HK, Abd-El-Fattah Lel-S. Stability indicating methods for the determination of some fluoroquinolones in the presence of their decarboxylated degrades. Chem Pharm Bull (Tokyo).54, 2006 ; 1625-32,.

15.     Patel Roshani, M. Pharm Thesis. Synthesis and biological evaluation of some Metal complexes; Gujarat Technological University, 2011.

16.     Patel Jahanvi, M. Pharm Thesis. Synthesis and Bioactivity of metal complexes with fluroquinolones and donors. Gujarat Technological University, 2012.

17.     Patel Yogini, M. Pharm Thesis. Synthesis, characterization and antibacterial activity of some fluroquinolone,-N-V donor Ferric (III) Complex. Gujarat Technological University; 2012.

18.     Sabale PM, Chavda J. Development and validation of spectrophotometric method of Bismuth-Ciprofloxacin- 2,2-Bipyridyl Metal Complex, Inventi Rapid: Pharm Analysis and Quality Assurance, 2013: Article ID- " Inventi:ppaqa/805/13 , 2013

19.     Sabale PM, Chavda J, Patel R. Development and validation of RP-HPLC Method for Bismuth Ciprofloxacin 2,2-Bipyridyl Metal Complex, Pharmagene,; 01,  2013 ; 41-47,.

20.     Sabale PM, Chavda Jignesh, Vidya Sabale. Simple Spectrophotometric Methods for the Determination of Zn-Sparfloxacin 1, 10-Phenanthroline Metal complex. Res J Pharm Tech; 6, 2013 ; 614-20.

21.     Urbaniak B, Kokot ZJ. Spectroscopic investigations of fluoroquinolones metal ion complexes. Acta Pol Pharm.;70, 2013 ; 621-29.

22.     ICH Q2A; Guidelines on validation of analytical procedure; Definitions and terminology, Federal Register, 60, 11260,1995,ICH Q2B; Guidelines on validation of analytical procedure; Methodology, Federal Register, 60, 27464, 1996.

 

 


 

 

Received on 29.09.2013       Modified on 25.10.2013

Accepted on 02.11.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 7(2): Feb. 2014; Page   155-160