RP-HPLC-UV Method for Simultaneous Estimation of Ceftriaxone

A Validated RP-HPLC-UV arrangement for Synchronous letter of Ceftriaxone and Sulbactum in Rat Plasma


A evolution bearing-liquescent chromatographic arrangement delay UV exposure is familiar for synchronous letter of ceftriaxone sodium and sulbactam sodium in rat plasma. Drugs were extracted from bare plasma by weak protein residuum technique. Chromatographic dissociation of these two garbages was done on Phenomenex C18 shaft (250mm X 4.6mm, i.d, 5μm) by using ductile bearing consisting of 10mM potassium dihydrogen orthophosphate buffer (pH- 5) and acetonitrile (90:10 % v/v). The familiar RP-HPLC arrangement had the delectable regular peaks amiable-natured-natured-tempered-natured-natured disjunction and garbages were eluted delay amiable-natured-natured-tempered-natured-natured contention age. The familiar bio-analytical arrangement was Linear, punctilious, and complimentary delay the ardor class of 20-150 μg mL-1 for ceftriaxone and 10-75 μg mL-1 for sulbactam. From the familiar arrangement we can moniter ceftriaxone and sulbactam sodium ardors in rat plasma.

Keywords: Ceftriaxone sodium, Sulbactam sodium, Liquescent chromatography, Rat plasma


Ceftriaxone[1] (CFX) is a third race 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 pungent. Sulbactam (SBM) chemically (2S,5R)-3,3-Dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0] heptane -2-carboxylic pungent 4,4-dioxide is used as a beta-lactamase inhibitor. Structural formulae of CFX and SBM are fond in Fig.1. These garbages are regularly associated in pharmaceutical formulations over meningitis, typhoid, gonorrhoea and urinary rely communicated [2].

Sulbactomax is a commercially adapted pharmaceutical fruit containing SBM and CFX. The fruit is adapted as a dry powder for introduction. The fruit is endowmented in contrariant strengths (250 mg+125 mg, 500mg+250 mg, 1gm+0.5gm, 2gm+1gm) of CFX and SBM respectively.

Fig.1.Chemical composition of CFX and SBM

Sulbactomax is a synergistic antimicrobial conformation delay plain in vitro antibacterial vital-force over a remote spectrum of organisms. SBM not singly increases the antibacterial vital-force of CFX but as-well shows a temperebuke antibacterial vital-force by forming a protein obscure delay beta-lactamas by irreversibly blockin their ruinous hydrolytic vital-force.

Thus, SBM increases the spectrum of vital-force of CFX. This SBM as-well binds delay some penicillin obligatory proteins, impressible strains are generally considered over capable to the Sulbactomax than CFX remaining. In bacterial strains that consent either low amounts of beta– lactamase, or none at all, a synergistic commodities is witnessed when SBM is associated delay CFX that has a complementary similarity for the target sites.

Sulbactomax has amiable-natured-natured-tempered-natured-natured erratic over all the microorganisms which are impressible/resistant to CFX. Further, it as-well reveals synergistic vital-force (retrench in stint inhibitory ardors for the confederacy versus those of each ingredient) in a multiplicity of organisms. So it has improved usefulness as compared to CFX remaining, lesser plane commoditiess, remoter spectrum coverage and amend fruits of bacterial MIC (stint inhibitory ardor) makes this fruit rare in the cosmos-people.

A learning review inspired a spectrophotometric [3], spectrofluorimetric in cosmical plasma [4], HPLC for the letter of trafficed formulations [5,6], in cosmical plasma [7] and for the satisfaction of pharmacokinetics in dogs [8], capillary electrophoresis [9] and GC-MS [10] arrangements for the letter of CFX and SBM partially and in entirely forms.

However, from the learning review there was no arrangement crop reputed for the synchronous letter of CFX and SBM by HPLC in rat plasma. The give message describes an isocratic liquescent chromatography (LC) arrangement for synchronous satisfaction of CFX sodium and SBM, which can be used for the kind guide of the formulation familiar and other biological applications.

  1. Experimental
    1. Chemicals and Reagents

    All chemicals and reagents used were of analytical compute singly. Milli-Q-insinuate was used throughout the mode and acetonitrile of HPLC compute were procured from Merck Chemical Laboratories, Bangalore, India. Commercial formulation, CetriaxS introduction containing ceftriaxone sodium 1gm and sulbactam sodium 0.5 gm were obtained from the national traffic. Bare rat plasma was obtained from JSS Medical College and Hospital, Mysore, India.

    1. Instrumentation and Analytical Conditions

    A HPLC delay the UV undeceiver was used for this learning labor. Here the dissociation was done using Phenomenex C-18 shaft. The ductile bearing was a conformation of phosphate Buffer (pH adjusted to 5 delay potassium hydroxide) and acetonitrile (90:10) v/v. The ductile bearing was depurateed through 0.45 μ membrane depurebuke antecedently its use, degassed delay a helium sparge for 15min at progress rebuke of 1.0 mL min-1. The shaft was maintained at capacity region 20±100C. The introduction tome of cases was 10 μL. The analyte was monitored at wavelength of 230 nm and optimized chromatographic provisions are shown in Table-1.

    2.3.Preparation of ductile bearing:

    Phosphate buffer of pH 5 was expert by dissolving 1.36 gm ofPotassiumdihydrogenorthophosphate in 1000 mL of insinuate and it was sonicated for 5 minutes, then the pH was adjusted using potassium hydroxide disconnection. It was than depurateed by vaccum depurateation. Finally the ductile bearing was expert by mixing phosphate buffer and acetonitrile in the harmony 90:10v/v.

    2.4.Preparation of flag and case disconnection

    SeparatelyweighedquantityofCFXsodium(10mg)andSBMsodium (10mg)was communicated into a 100mL tometricflaskandmadeupto100mLwithwatertoget100 µg mL-1 ofCFXsodiumand100 µg mL-1 ofSBM. From this, contrariant disconnections containing the conformation of CFXsodium(20-150 µg mL-1) and SBMsodium(10-75 µg mL-1) were expert.

    For the provision of case disconnection, Cetriax-Spowder for introduction(containing1gmof CFXand0.5gmof SBM)was communicated to a 100 mL tometric flask. Distilled insinuate was pretended, and then swirled to disperse it, feeble to 100 mL delay the selfselfsimilar solvent.

    2.5.Preparation of calibration deflexion:

    Five contrariant collected disconnections containing conformation of CFX (20-150 µg mL-1) and SBM (10-75 µg mL-1) were injected onto HPLC. A calibration deflexion was expert portico ardors on X-axis and Peak Area on Y-Axis.

    2.6.Preparation of plasma cases:

    Plasma cases of CFX and SBM was expert by the protein residuum arrangement. A bare was expert by portico 0.1mL of rat plasma and to this 1.9 mL of acetonitrile was pretended and case was expert by portico 0.1 mL of confederacy of CFX and SBM (which were adulterated in similar tomes) and 0.1 mL of rat plasma was pretended to the 2 mL Eppendorf tubes containing 1.8 mL of acetonitrile. These cases were centrifuged for 10 min at 10,000 rpm. The supernatant disconnection depurateed through 0.45µ syringe depurebuke and communicated to HPLC vials.


    3.1 Method Development

    Taking into compensation, the mutability of CFX and SBM in powerful alkaline and powerful pungentic requisite, the pH compute of the ductile bearing should be scant delayin the class of 3‐7, since moderate pungentic pH favours the contention and dissociation of two garbages on C‐18 shaft.

    After few trials, phosphate buffer delay pH 5 was finalized. The arrangement crop started delay the methanol and phosphate buffer as garbages did not elute in this ductile bearing, so the radical bearing was altered from methanol to acetonitrile. Twain CFX and SBM in the ductile bearing enjoy no symbolical UV ultimatum, the wavelength of 230 nm was assiduous for the exposure. Succeeding few trails Phenomenex C-18 shaft and binary conformation of phosphate buffer (pH 5) and acetonitrile (90:10 % v/v) was optimized as ductile bearing which consentd symmetric peak pattern, amiable-natured-natured-tempered-natured-natured disjunction and grave contention age for twain the garbages (Table 1). The contention ages of CFX and SBM for six repetitions were build to be 7.8 ± 0.02 min and 4.7 ± 0.006 respectively (Fig.2).



    Fig.2. LC chromatogram of rat bare plasma (a) plasma spiked delay flag CFX and SBM(b)

    Table 1. Optimized chromatographic provisions


    Optimized requisite


    HPLC delay UV- undeceiver


    C18 Column

    Mobile Phase

    Acetonitrile and pH-5 buffer in the harmony of 10:90(v/v)

    Flow rebuke

    1.00 mL min-1



    Injection tome

    10 μL

    Temperature shaft

    Room region

    3.2.Method validation

    Validation is a mode of establishing documented token, which offers a tall compute of selfreliance that a inequitable vital-force conciliate steadily consent anticipated fruit or fruit discussion its preimmovable inequitableations and kind features [11]. The arrangement was validated for contrariant parameters enjoy linearity, foresight, reanimation, foresight, selectivity and sensitivity [12].


    Selectivity is defined as, “the cleverness of an analytical arrangement to characterize and compute the analyte in the intercourse of other ingredients in the case [12]”. Selectivity is congenial by injecting extracted bare plasma and about delay the tally of extracted LLOQ cases. Twain the peaks of Ceftriaxone and Sulbactum did not interfere delay any endogenous ingredients.


    Sensitivity is computed using Lower Word of Quantification (LLOQ). LLOQ is the balanceest ardor of the flag deflexion that can be computed delay delectable foresight and foresight [12]”. The LLOQ was recurrent using five cases stubborn of flags and immovable the co-efficient of diversity and expend assurance period.

    3.2.3.Linearity of Response

    To demonstrebuke the linearity of tally, course of disconnections ranging from (20-150 µg mL-1) of CFX and SBM of (10-75 µg mL-1) were expert and injected onto the HPLC plan succeedingcited the forcible provisions. The graph was unnatural among ardor vs. peak area and it was build that interdependence co-efficient and return segregation were delayin the words and the fruits 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




    Retention age (min)



    Linear class (ppm) [n=6]

    (µg mL-1)



    Correlation coefficient (r2)









    Lowest word of quatification LLOQ (µg mL-1)




    “The reanimation of an analyte is the undeceiver tally achieved from an amount of the analyte pretended to and extracted from the biological matrix, corconnected to the undeceiver tally build for the gentleman ardor of the immaculate not spurious flag”[12]. “Recovery of the analyte is not expedient be 100%” [12]. This experiments were done by comparing the analytical fruits for extracted cases at three contrariant ardors (low, average, and tall) delay unextracted flags that regive 100% reanimation. Results are summarised in Table 3.

    Table 3. Reanimation studies of CFX sodium and SBM

    Concentration of CFX and sulbactam

    Amount recovered% for CFX

    Amount recovered% for SBM










    3.2.5.Accuracy and Precision

    For validation of this bioanalytical arrangement, foresight and foresight should be immovable using stint of five satisfactions per ardor smooth (beside bare cases). The balance compute should be delayin ± 15% of the hypothetical compute, bar at LLOQ, where it must not vary by over than ± 20%. The foresight and foresight about the balance compute should not be over 15% of the CV bar for LLOQ, where it should not yield by 20% of the CV.

    The foresight of the analytical arrangement defines the nearness of harmony among the banner compute and the relation compute. The foresight of the analytical arrangement describes the nearness of general idiosyncratic computes of analyte. Foresight is open in provisions of % obtained. Foresight is open in provisions of coefficient of diversity (CV). The statistical arrangement for satisfaction of the foresight and foresight should be predefined and congenial according flag exercise. Foresight and Foresight should be reveald for the low, average, tall and LLOQ QC cases, delayin a unique run and among contrariant runs fruits are summarised in Table 4 & 5.

    % CV (precision) =100 x Flag deviation/Mean

    Table 4. Accuracy and Precision of CFX

    Theoretical ardor


    Measured ardor (µg/mL)




    Accuracy (%)


    Accuracy (%)
















    Table 5. Accuracy and Precision of SLB

    Theoretical ardor

    µg mL-1

    Measured ardor (µg mL-1)




    Accuracy (%)


    Accuracy (%)
















    3.2.6.Stability studies

    Freeze and Dissolve Stability

    Stability of analyte was immovable delay three benumb and dissolve cycles. All the three aliquots at low, average and tall ardors were stored at the designed storage region for 24 hours and defrosted unassisted at capacity region. When altogether dissolveed, the cases were continually frozen for 12 to 24 hours inferior the selfselfsimilar provisions. The selfselfsimilar cycle was continual two over ages, and then analyzed succeeding the third cycle.

    Short-Term Region Stability

    Three aliquots of low, average and tall ardors were dissolveed at capacity region and at this region case was kept from 4 to 24 hours and analyzed.

    Long-Term Stability

    The storage age in a long-term stationariness assessment should surmount the age among the age of primary case assembly and the age of last case segregation. Long-term stationariness was immovable by storing three aliquots of the low, average and tall ardors inferior the selfselfsimilar provisions as that of the examine cases. The ardors of all the stationariness cases were connected to the balance of back-congenial computes for the flags at the decent ardors from the primary day of long-term stationariness bannering.

    Stock Disconnection Stability

    The stationariness of store disconnections of garbage was estimated at capacity region for 6 hours. Succeeding the desired storage age, the stationariness was open by comparing the tool tally delay that of newly expert disconnections Results are summarised in Table 6.

    Table 6. Stationariness studies of CFX and SBM


    µg mL-1 (fallacy %)


    µg mL-1 (fallacy %)















    Long term







    Short term







    Stock Solution







    1. SUMMARY

    In this labor, a weak, stationariness indicating, complimentary and validated stationariness indicating HPLC arrangement for the synchronous satisfaction of ceftriaxone and sulbactam in their pharmaceutical formulation was familiar. The arrangement was validated according to FDA guidelines. CFX and SBM were eluted at 7.3 min and 4.6 min respectively. The interdependence coefficient (r2) for CFX and SBM were build to be 0.996 and 0.9976 respectively. Lower Word of quantification (LLOQ) was build to be 0.87 µg mL-1 for ceftriaxone and 0.96 µg mL-1 for sulbactam. The %CV for the intraday and interday foresight were build to be <2.


    The arrangement includes weak and punctilious arrangement for synchronous satisfaction of CFX sodium and SBM. It consents regular peak pattern, amiable-natured-natured-tempered-natured-natured disjunction and grave contention age for twain garbages. So this arrangement can be expend for the  synchronous letter of CFX sodium and SBM in kind guide studies for uniformity segregation.


    The authors are indebted to The Principal, JSS College of Pharmacy, JSS University, Mysore for providing all expedient facilities to propel out the learning. The authors are as-well indebted to Strides Arco labs, Bangalore for providing the immaculate salbactum sodium and ceftriaxone sodium as endowment cases.


    1. Rang HP, Dale MM (1993). Pharmacology, (2nd ed.). Churchill Livingstone, ELBS.
    2. Physicians Desk Relation (1997). American Academy of Physician Assistants (51th ed).
    3. Patel FM, Dave JB , Patel CN, spectrophotometric arrangements for synchronous letter of cefuroxime sodium and sulbactam sodium in injecton, International narrative of pharmaceutical sciences and learning.2012; 3(9), 3513-3517.
    4. Jasmin Shah,Rasul Jan M,Sultan Shah,Naeem M, Spectrofluorimetric Protocol for Ceftriaxone in Commercial Formulation and Cosmical Plasma Succeeding Condensation delay Formaldehyde and Ethyl Acetoacetate, Journal of Fluorescence.2011; 21(6), 2155-2163.
    5. Shrivastava SM, Singh R, Ariq A, Siddiqui MR, Yadav J, Negi PS, Chaudhary M, A novel tall operation liquescent chromatographic arrangement for synchronous satisfaction of ceftriaxone and sulbactam in sulbactomax, Inter Narrative of Biomed Sci. 2009; 5(1), 37-43.
    6. Durairaj S, Annadurai T, Palani Kumar B, Arunkumar S, Synchronous Letter of Ceftriaxone Sodium and Sulbactam Sodium using Multi-Component Mode of Analysis, Inter Narrative of ChemTech Research. 2010; 2(4), 2177-2181.
    7. Huang Ying, Liang Maozhi, Yu Qin, Jiang Lei,Shi Yingkang, Satisfaction of ceftriaxone in cosmical plasma by HPLC, Chinease Narrative of Antibiotics.2000; 25,109.
    8. Zhao Xi, Zhang Dan, Chen Hong, Satisfaction of cefotaxime/sulbactam in plasma and pharmacokinetics in dogs by tall operation liquescent chromatography. Zhongguo Kangshengsu Zazhi. 2004; 29(10): 614-616.
    9. Jelinek I, Krejcirova H, Dohan J, Roubal Z, Satisfaction of sulbactam in cosmical serum using capillary electrophoresis,Cesk Farm.1990; 39,305–307.
    10. Foulds G, Gans DJ, Girard D, Whall TJ, Assays of sulbactam in the intercourse of ampicillin.Therm Garbage Monit.1986;2,223–237.
    11. Lloyd RS, Joseph JK, Joseph LG (1997). Practical HPLC Arrangement Crop (2ndedition) New York: Wiley-Blackwell.
    12. FDAUS. Guidance for Industry: Bioanalytical Arrangement Validation. Rockville, MD, USA: US Department of Health and cosmical services, US FDA/Center for Garbage Evaluation and Research; 2001.

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