RP-HPLC-UV Method for Simultaneous Estimation of Ceftriaxone

RP-HPLC-UV Method for Simultaneous Estimation of ceftriaxoneA Validated RP-HPLC-UV regularity for Simultaneous theme of Ceftriaxone and Sulbactum in Rat PlasmaABSTRACTA reverse phase-liquid chromatographical recordical 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 tower (250mm X 4.6mm, i.d, 5m) by using mobile phase consisting of 10mM potassium dihydrogen orthophosphate dampen (pH- 5) and acetonitrile (9010 % v/v). The developed RP-HPLC method had the acceptable symmetrical peaks nice resolution and drugs were eluted with good retention magazine. The developed bio-analytical method was Linear, precise, and complete with the concentration range of 20- one hundred fifty g mL-1 for ceftriaxone and 10-75 g mL-1 for sulbactam. From the developed method we spate moniter ceftriaxone and sulbactam sodium concentrations in rat plasma.Keywords Ceftriaxone sodium, Sulbactam sodium, Liquid chromatography, Rat plasmaINTRODUCTIONCeftriaxone1 (CFX) is a third generation cephalosporin. Chemic whollyy it is (6R,7R)-7-2-(2-a secondo-4-thiazolyl)-(Z)-2- methoxyi momentuteo-acetamido-3(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazin-3-yl)thiomethyl-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- azabicyclo3.2.0 heptane -2-carboxylic acid 4,4-dioxide is apply 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+ one hundred twenty-five mg, 500mg+250 mg, 1gm+0.5gm, 2gm+1gm) of CFX and SBM respectively.Fig.1.Chemical structure of CFX and SBMSulbactomax is a synergistic antimicrobial form with clear in vitro antibacterial natural action 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 two(prenominal) penicillin binding proteins, sensitive strains are often con placementred 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 sensit ive/resistant to CFX. Further, it also plants 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 determin ation of CFX sodium and SBM, which can be utilize for the quality control of the formulation developed and other biological applications.ExperimentalChemicals and ReagentsAll 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. technical 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 exam College and Hospital, Mysore, India.Instrumentation and Analytical ConditionsA HPLC with the UV sensing element 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 (9010) v/v. The mobile phase was filtered through 0.45 membrane filter before its use, degassed with a hel ium spit for 15min at flow rate of 1.0 mL min-1. The column was maintained at room temperature 20 ascorbic acidC. The injection volume of samples was 10 L. The analyte was monitored at wavelength of 230 nm and optimized chromatographic conditions are shown in display board-1.2.3.Preparation of mobile phasePhosphate buffer of pH 5 was prompt 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 mix phosphate buffer and acetonitrile in the ratio 9010v/v.2.4.Preparation of standard and sample solutionSeparatelyweighedquantityofCFXsodium(10mg)andSBMsodium (10mg)was transferred into a 100mL volumetricflaskandmadeupto100mLwithwatertoget100 g mL-1 ofCFXsodiumand100 g mL-1 ofSBM. From this, different solutions containing the mixture of CFXsodium(20-150 g mL-1) and SBMsodium(10-75 g 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 wrickFive different concentrated solutions containing mixture of CFX (20-150 g mL-1) and SBM (10-75 g 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 samplesPlasma 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 syringe filter and transferred to HPLC vials.RESULTS AND DISCUSSION3.1 Method DevelopmentTaking into consideration, the in perceptual constancy of CFX and SBM in watertight alkaline and strong acidic condition, the pH value of the mobile phase should be limited within the range of 37, since mild acidic pH favours the retention and separation of two drugs on C18 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 eat 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 (9010 % v/v) was optimized as mobile phase which produced symmetric peak shape, good resolution and reasonable ret ention 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 conditionChromatographHPLC with UV- detectortowerC18 ColumnMobile PhaseAcetonitrile and pH-5 buffer in the ratio of 1090(v/v)Flow rate1.00 mL min-1Detection230nmInjection volume10 LTemperature columnRoom temperature3.2.Method validationValidation is a process of establishing documented evidence, which offers a blueschool degree of assurance that a specific activity will steadily yield anticipated result or product meeting its pre determine specifications and quality features 11. The method was validated for different parameters like linearity, clearcutness, recovery, accuracy, selectivity and sensitivity 12.3.2.1SelectivitySelectivity is defined as, the apti tude 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 ResponseTo demonstrate the linearity of response, series of solutions ranging from (20-150 g mL-1) of CFX and SBM of (10-75 g 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 correlativity 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 CFXFig.4. Calibration graph of SBMTable 2. Linearity of CFX and SBMParametersCFXSBMRetention time (min)7.34.6Linear range (ppm) n=6(g mL-1)20-15010-75Correlation coefficient (r2)0.9960.997Slope1513.1155.58Intercept27233361596Lowest limit of quatification LLOQ (g mL-1)0.870.963.2.4. convalescenceThe 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 standard12. Recovery of the analyte is not needed be 100% 12. This experiments were performed by comparing the analytical results for extracted samples at three different concentrations (low, medium, and high) with un extracted standards that represent 100% recovery. Results are summarised in Table 3.Table 3. Recovery studies of CFX sodium and SBMConcentration of CFX and sulbactamAmount recovered% for CFXAmount recovered% for SBMLow98.7%99.9%Medium96.8%98.9%High99.3%98.6%3.2.5.Accuracy and PrecisionFor 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(prenominal) 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/MeanTable 4. Accuracy and Precision of CFXTheoretical concentration(g/mL)Measured concentration (g/mL)Intra-dayInter-day%CVAccuracy (%)%CVAccuracy (%)200.9898.41.4296.11000.76103.71.32102.31501.3499.51.798.7Table 5. Accuracy and Precision of SLBTheoretical concentrationg mL-1Measured concentration (g mL-1)Intra-dayInter-day%CVAccuracy (%)%CVAccuracy (%)100.96101.70.7695.6501.0099.81.2103.4751.0297.31.0497.43.2.6.Stability studiesFreeze and Thaw StabilityStability 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 restate two more times, and then analyzed after the third cycle. Short-Term Temperature StabilityThree 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 StabilityThe 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 concent rations from the first day of long-term stability testing.Stock Solution StabilityThe 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 SBMStabilityg mL-1 (error %)CFXg mL-1 (error %)SBM20100150105075Freeze-thaw84.593.394.988.596.397.9Long term100.5100.6100.8100.5101.6100.8Short term93.997.6101.493.993.6103.4Stock Solution95.697.693.295.396.898.5SUMMARYIn 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 0.9 96 and 0.9976 respectively. Lower Limit of quantification (LLOQ) was found to be 0.87 g mL-1 for ceftriaxone and 0.96 g mL-1 for sulbactam. The %CV for the intraday and interday precision were found to be findingThe method includes simple and precise method for simultaneous determination of CFX sodium and SBM. It produces symmetrical peak shape, good resolution and reasonableretention 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.AKNOWLEDGMENTThe authors are thankful to The Principal, JSS College of Pharmacy, JSS University, Mysore for providing all necessary facilities to carry out the research. 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