RP-HPLC-UV Method for Simultaneous Estimation of Ceftriaxone

RP-HPLC-UV Method for Simultaneous Estimation of Ceftriaxone A Validated RP-HPLC-UV method for Simultaneous estimation of Ceftriaxone and Sulbactum in Rat Plasma ABSTRACT: A reverse phase-liquid chromatographic method with UV detection is developed for simultaneous estimation of ceftriaxone sodium and sulbactam sodium in rat plasma. Drugs were extracted from blank plasma by simple protein precipitation technique. Chromatographic separation of these two drugs was done on Phenomenex C18 column (250mm X 4.6mm, i.d, 5ÃŽ ¼m) by using mobile phase consisting of 10mM potassium dihydrogen orthophosphate buffer (pH- 5) and acetonitrile (90:10 % v/v). The developed RP-HPLC method had the acceptable symmetrical peaks good resolution and drugs were eluted with good retention time. The developed bio-analytical method was Linear, precise, and accurate with the concentration range of 20-150 ÃŽ ¼g mL-1 for ceftriaxone and 10-75 ÃŽ ¼g mL-1 for sulbactam. From the developed method we can moniter ceftriaxone and sulbactam sodium concentrations in rat plasma. Keywords: Ceftriaxone sodium, Sulbactam sodium, Liquid chromatography, Rat plasma INTRODUCTION Ceftriaxone[1] (CFX) is a third generation cephalosporin. Chemically it is (6R,7R)-7-{2-(2-amino-4-thiazolyl)-(Z)-2- [methoxyiminuteo-acetamido]-3{[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)thio]methyl}-8-oxo-5-thia-l-azobicyclo [4,2,0] oct-2-ene-2-carboxylic acid. Sulbactam (SBM) chemically (2S,5R)-3,3-Dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0] heptane -2-carboxylic acid 4,4-dioxide is used as a beta-lactamase inhibitor. Structural formulae of CFX and SBM are given in Fig.1. These drugs are frequently associated in pharmaceutical formulations against meningitis, typhoid, gonorrhoea and urinary tract infections [2]. Sulbactomax is a commercially available pharmaceutical product containing SBM and CFX. The product is available as a dry powder for injection. The product is supplied in different strengths (250 mg+125 mg, 500mg+250 mg, 1gm+0.5gm, 2gm+1gm) of CFX and SBM respectively. Fig.1.Chemical structure of CFX and SBM Sulbactomax is a synergistic antimicrobial mixture with clear in vitro antibacterial activity against a wide spectrum of organisms. SBM not only increases the antibacterial activity of CFX but also shows a moderate antibacterial activity by forming a protein complex with beta-lactamas by irreversibly blockin their destructive hydrolytic activity. Thus, SBM increases the spectrum of activity of CFX. This SBM also binds with some penicillin binding proteins, sensitive strains are often considered more susceptible to the Sulbactomax than CFX alone. In bacterial strains that produce either low amounts of beta lactamase, or none at all, a synergistic effect is witnessed when SBM is associated with CFX that has a complementary affinity for the target sites. Sulbactomax has good active against all the microorganisms which are sensitive/resistant to CFX. Further, it also demonstrates synergistic activity (decrease in minimum inhibitory concentrations for the combination versus those of each component) in a variety of organisms. So it has improved efficacy as compared to CFX alone, lesser side effects, wider spectrum coverage and better results of bacterial MIC (minimum inhibitory concentration) makes this product unique in the world. A literature survey revealed a spectrophotometric [3], spectrofluorimetric in human plasma [4], HPLC for the estimation of marketed formulations [5,6], in human plasma [7] and for the determination of pharmacokinetics in dogs [8], capillary electrophoresis [9] and GC-MS [10] methods for the estimation of CFX and SBM individually and in combined forms. However, from the literature survey there was no method development reported for the simultaneous estimation of CFX and SBM by HPLC in rat plasma. The present communication describes an isocratic liquid chromatography (LC) method for simultaneous determination of CFX sodium and SBM, which can be used for the quality control of the formulation developed and other biological applications. Experimental Chemicals and Reagents All chemicals and reagents used were of analytical grade only. Milli-Q-water was used throughout the process and acetonitrile of HPLC grade were procured from Merck Chemical Laboratories, Bangalore, India. Commercial formulation, CetriaxS injection containing ceftriaxone sodium 1gm and sulbactam sodium 0.5 gm were obtained from the local market. Blank rat plasma was obtained from JSS Medical College and Hospital, Mysore, India. Instrumentation and Analytical Conditions A HPLC with the UV detector was used for this research work. Here the separation was done using Phenomenex C-18 column. The mobile phase was a mixture of phosphate Buffer (pH adjusted to 5 with potassium hydroxide) and acetonitrile (90:10) v/v. The mobile phase was filtered through 0.45 ÃŽ ¼ membrane filter before its use, degassed with a helium sparge for 15min at flow rate of 1.0 mL min-1. The column was maintained at room temperature 20 ±100C. The injection volume of samples was 10 ÃŽ ¼L. The analyte was monitored at wavelength of 230 nm and optimized chromatographic conditions are shown in Table-1. 2.3.Preparation of mobile phase: Phosphate buffer of pH 5 was prepared by dissolving 1.36 gm ofPotassiumdihydrogenorthophosphate in 1000 mL of water and it was sonicated for 5 minutes, then the pH was adjusted using potassium hydroxide solution. It was than filtered by vaccum filteration. Finally the mobile phase was prepared by mixing phosphate buffer and acetonitrile in the ratio 90:10v/v. 2.4.Preparation of standard and sample solution SeparatelyweighedquantityofCFXsodium(10mg)andSBMsodium (10mg)was transferred into a 100mL volumetricflaskandmadeupto100mLwithwatertoget100  µg mL-1 ofCFXsodiumand100  µg mL-1 ofSBM. From this, different solutions containing the mixture of CFXsodium(20-150  µg mL-1) and SBMsodium(10-75  µg mL-1) were prepared. For the preparation of sample solution, Cetriax-Spowder for injection(containing1gmof CFXand0.5gmof SBM)was transferred to a 100 mL volumetric flask. Distilled water was added, and then swirled to dissolve it, diluted to 100 mL with the same solvent. 2.5.Preparation of calibration curve: Five different concentrated solutions containing mixture of CFX (20-150  µg mL-1) and SBM (10-75  µg mL-1) were injected onto HPLC. A calibration curve was prepared taking concentrations on X-axis and Peak Area on Y-Axis. 2.6.Preparation of plasma samples: Plasma samples of CFX and SBM was prepared by the protein precipitation method. A blank was prepared by taking 0.1mL of rat plasma and to this 1.9 mL of acetonitrile was added and sample was prepared by taking 0.1 mL of combination of CFX and SBM (which were mixed in equal volumes) and 0.1 mL of rat plasma was added to the 2 mL Eppendorf tubes containing 1.8 mL of acetonitrile. These samples were centrifuged for 10 min at 10,000 rpm. The supernatant solution filtered through 0.45 µ syringe filter and transferred to HPLC vials. RESULTS AND DISCUSSION 3.1 Method Development Taking into consideration, the instability of CFX and SBM in strong alkaline and strong acidic condition, the pH value of the mobile phase should be limited within the range of 3à ¢Ã¢â€š ¬Ã‚ 7, since mild acidic pH favours the retention and separation of two drugs on Cà ¢Ã¢â€š ¬Ã‚ 18 column. After few trials, phosphate buffer with pH 5 was finalized. The method development started with the methanol and phosphate buffer as drugs did not elute in this mobile phase, so the organic phase was altered from methanol to acetonitrile. Both CFX and SBM in the mobile phase have no significant UV maximum, the wavelength of 230 nm was employed for the detection. After few trails Phenomenex C-18 column and binary mixture of phosphate buffer (pH 5) and acetonitrile (90:10 % v/v) was optimized as mobile phase which produced symmetric peak shape, good resolution and reasonable retention time for both the drugs (Table 1). The retention times of CFX and SBM for six repetitions were found to be 7.8  ± 0.02 min and 4.7  ± 0.006 respectively (Fig.2). (a) (b) Fig.2. LC chromatogram of rat blank plasma (a) plasma spiked with standard CFX and SBM(b) Table 1. Optimized chromatographic conditions Parameter Optimized condition Chromatograph HPLC with UV- detector Column C18 Column Mobile Phase Acetonitrile and pH-5 buffer in the ratio of 10:90(v/v) Flow rate 1.00 mL min-1 Detection 230nm Injection volume 10 ÃŽ ¼L Temperature column Room temperature 3.2.Method validation Validation is a process of establishing documented evidence, which offers a high degree of assurance that a specific activity will steadily yield anticipated result or product meeting its predetermined specifications and quality features [11]. The method was validated for different parameters like linearity, precision, recovery, accuracy, selectivity and sensitivity [12]. 3.2.1Selectivity Selectivity is defined as, the capability of an analytical method to distinguish and measure the analyte in the presence of other components in the sample [12]†. Selectivity is calculated by injecting extracted blank plasma and relating with the response of extracted LLOQ samples. Both the peaks of Ceftriaxone and Sulbactum did not interfere with any endogenous components. 3.2.2Sensitivity Sensitivity is measured using Lower Limit of Quantification (LLOQ). LLOQ is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision [12]†. The LLOQ was established using five samples independent of standards and determined the co-efficient of variation and appropriate confidence interval. 3.2.3.Linearity of Response To demonstrate the linearity of response, series of solutions ranging from (20-150  µg mL-1) of CFX and SBM of (10-75  µg mL-1) were prepared and injected onto the HPLC system following the described conditions. The graph was constructed between concentration vs. peak area and it was found that correlation co-efficient and regression analysis were within the limits and the results are summarized in the Table 2, and the calibration graphs are shown in Fig. 3 and Fig. 4 for CFX and SBM respectively. Fig.3. Calibration graph of CFX Fig.4. Calibration graph of SBM Table 2. Linearity of CFX and SBM Parameters CFX SBM Retention time (min) 7.3 4.6 Linear range (ppm) [n=6] ( µg mL-1) 20-150 10-75 Correlation coefficient (r2) 0.996 0.997 Slope 1513.1 155.58 Intercept 272333 61596 Lowest limit of quatification LLOQ ( µg mL-1) 0.87 0.96 3.2.4.Recovery â€Å"The recovery of an analyte is the detector response achieved from an quantity of the analyte added to and extracted from the biological matrix, correlated to the detector response found for the true concentration of the pure authentic standard†[12]. â€Å"Recovery of the analyte is not necessary be 100%† [12]. This experiments were performed by comparing the analytical results for extracted samples at three different concentrations (low, medium, and high) with unextracted standards that represent 100% recovery. Results are summarised in Table 3. Table 3. Recovery studies of CFX sodium and SBM Concentration of CFX and sulbactam Amount recovered% for CFX Amount recovered% for SBM Low 98.7% 99.9% Medium 96.8% 98.9% High 99.3% 98.6% 3.2.5.Accuracy and Precision For validation of this bioanalytical method, precision and accuracy should be determined using minimum of five determinations per concentration level (excluding blank samples). The mean value should be within  ± 15% of the theoretical value, except at LLOQ, where it must not differ by more than  ± 20%. The accuracy and precision around the mean value should not be beyond 15% of the CV except for LLOQ, where it should not exceed by 20% of the CV. The accuracy of the analytical method defines the closeness of agreement between the test value and the reference value. The precision of the analytical method describes the closeness of frequent individual measures of analyte. Accuracy is expressed in terms of % obtained. Precision is expressed in terms of coefficient of variation (CV). The statistical method for determination of the accuracy and precision should be predefined and calculated according standard practise. Accuracy and Precision should be demonstrated for the low, medium, high and LLOQ QC samples, within a single run and between different runs results are summarised in Table 4 5. % CV (precision) =100 x Standard deviation/Mean Table 4. Accuracy and Precision of CFX Theoretical concentration ( µg/mL) Measured concentration ( µg/mL) Intra-day Inter-day %CV Accuracy (%) %CV Accuracy (%) 20 0.98 98.4 1.42 96.1 100 0.76 103.7 1.32 102.3 150 1.34 99.5 1.7 98.7 Table 5. Accuracy and Precision of SLB Theoretical concentration  µg mL-1 Measured concentration ( µg mL-1) Intra-day Inter-day %CV Accuracy (%) %CV Accuracy (%) 10 0.96 101.7 0.76 95.6 50 1.00 99.8 1.2 103.4 75 1.02 97.3 1.04 97.4 3.2.6.Stability studies Freeze and Thaw Stability Stability of analyte was determined with three freeze and thaw cycles. All the three aliquots at low, medium and high concentrations were stored at the proposed storage temperature for 24 hours and defrosted unassisted at room temperature. When completely thawed, the samples were again frozen for 12 to 24 hours under the same conditions. The same cycle was repeated two more times, and then analyzed after the third cycle. Short-Term Temperature Stability Three aliquots of low, medium and high concentrations were thawed at room temperature and at this temperature sample was kept from 4 to 24 hours and analyzed. Long-Term Stability The storage time in a long-term stability assessment should surpass the time between the date of first sample collection and the date of last sample analysis. Long-term stability was determined by storing three aliquots of the low, medium and high concentrations under the same conditions as that of the study samples. The concentrations of all the stability samples were related to the mean of back-calculated values for the standards at the suitable concentrations from the first day of long-term stability testing. Stock Solution Stability The stability of stock solutions of drug was estimated at room temperature for 6 hours. After the desired storage time, the stability was confirmed by comparing the instrument response with that of newly prepared solutions Results are summarised in Table 6. Table 6. Stability studies of CFX and SBM Stability  µg mL-1 (error %) CFX  µg mL-1 (error %) SBM 20 100 150 10 50 75 Freeze-thaw 84.5 93.3 94.9 88.5 96.3 97.9 Long term 100.5 100.6 100.8 100.5 101.6 100.8 Short term 93.9 97.6 101.4 93.9 93.6 103.4 Stock Solution 95.6 97.6 93.2 95.3 96.8 98.5 SUMMARY In this work, a simple, stability indicating, accurate and validated stability indicating HPLC method for the simultaneous determination of ceftriaxone and sulbactam in their pharmaceutical formulation was developed. The method was validated according to FDA guidelines. CFX and SBM were eluted at 7.3 min and 4.6 min respectively. The correlation coefficient (r2) for CFX and SBM were found to be 0.996 and 0.9976 respectively. Lower Limit of quantification (LLOQ) was found to be 0.87  µg mL-1 for ceftriaxone and 0.96  µg mL-1 for sulbactam. The %CV for the intraday and interday precision were found to be CONCLUSION The method includes simple and precise method for simultaneous determination of CFX sodium and SBM. It produces symmetrical peak shape, good resolution and reasonable  retention time for both drugs. So this method can be appropriate for the   simultaneous estimation of CFX sodium and SBM in quality control studies for routine analysis. AKNOWLEDGMENT The authors are thankful to The Principal, JSS College of Pharmacy, JSS University, Mysore for providing all necessary facilities to carry out the research. The authors are also thankful to Strides Arco labs, Bangalore for providing the pure salbactum sodium and ceftriaxone sodium as gift samples. References Rang HP, Dale MM (1993). Pharmacology, (2nd ed.). Churchill Livingstone, ELBS. Physicians Desk Reference (1997). American Academy of Physician Assistants (51th ed). 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