Development and Validation of UV Spectrophotometric Method for the Estimation of Ceftriaxone Sodium in Nanoparticles

Der Pharma Chemica
Journal for Medicinal Chemistry, Pharmaceutical Chemistry, Pharmaceutical Sciences and Computational Chemistry

ISSN: 0975-413X
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Research Article - Der Pharma Chemica ( 2018) Volume 10, Issue 3

Development and Validation of UV Spectrophotometric Method for the Estimation of Ceftriaxone Sodium in Nanoparticles

Majani Ayushi V and Paradkar Mansi U*

Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology (CHARUSAT), Changa, Dist. Anand, Ta. Petlad, Gujarat-388421, India

*Corresponding Author:
Paradkar Mansi U
Ramanbhai Patel College of Pharmacy
Charotar University of Science and Technology (CHARUSAT)
Changa, Dist. Anand, Ta. Petlad, Gujarat-388421, India


The present work describes the simple, accurate and validated UV spectrophotometric method for determination of ceftriaxone sodium loaded in Nanoparticle formulation. The method was validated for different parameters like linearity, precision, specificity, accuracy, Limit of Detection (LOD), Limit of Quantification (LOQ) and Robustness as per ICH guidelines. A wavelength maximum of Ceftriaxone sodium in distilled water was found to be at 241 nm. The method was found to be linear in the range of 2 to 18 μg/ml with a correlation coefficient (r2) of 0.999. The accuracy of the method was studied by recovery study and % recovery was found in range of 99.27 to 100.06%. The method is simple, accurate and requires relatively inexpensive instrument. The proposed method can be successfully used for determination of Ceftriaxone sodium loaded in to Nanoparticle formulation.


UV spectrophotometry, Ceftriaxone sodium, %Assay, Chitosan nanoparticles


Ceftriaxone sodium is a third generation, semi synthetic cephalosporin antibiotic. Cephalosporins are derivatives of 7-aminocephalosporic acid and are closely related to penicillins in structure. Ceftriaxone sodium is a long acting, broad-spectrum cephalosporin antibiotic for parenteral use. It is effective in treatment of infections caused by accurate bacteria, blood poisoning, meningitis etc. The bactericidal activity of ceftriaxone sodium results from inhibition of cell wall synthesis. It exerts in vitro activity against a wide range of Gram-negative and Gram-positive microorganisms. It is highly stable to most beta-lactamases, both penicillinases and cephalosporinase of Gram-positive and Gram-negative bacteria [1].

Ceftriaxone sodium is chemically known as, (Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyiminoacetylamido]-3-[(2,5-dihydro-6-hydroxy-2-methyl- 5-oxo-1,2,4-triazin-3-yl)thiamethyl]-3-cephem-4-carboxylic acid, disodium salt. Ceftriaxone contains a highly acidic, heterocyclic system on the 3-thiamethyl group (Figure 1). This unusual dioxotriazine ring system is believed to confer the unique pharmacokinetic properties of this agent [2].

There are very much significant pharmacological activities exhibited by ceftriaxone sodium, several researchers have focused on the development of various analytical methods to determine ceftriaxone sodium in different matrices such as plasma, urine and saliva [3,4]. The commonly used methods are High Performance Liquid Chromatography (HPLC), spectrofluorimetric and chemilumescence has also been described [2,3,5]. As an alternative to HPLC assays, gas chromatography has also been proposed [6].

In this study, the UV spectrophotometric method was developed and validated for the determination of ceftriaxone sodium loaded into the nanoparticles by evaluating its assay.


Figure 1: Chemical structure of ceftriaxone sodium

Materials and Methods


UV- VIS double beam spectrophotometer (Shimadzu UV-1800, Shimadzu Corporation, Japan).


Ceftriaxone sodium was procured as a gift sample from JFL Life sciences Ltd, Ahmedabad, India. Chitosan used as the polymeric material was purchased from Sigma Aldrich (USA). Sodium Tripolyphosphate (TPP) and Glacial acetic acid were purchased from Loba Chemicals (Mumbai, India) respectively. All other reagents used were of analytical or equivalent grade.

Selection of wavelength maximum (λmax)

In order to ascertain the wavelength of maximum absorption (λmax) of the drug, a spectrum of 10 μg/ml was recorded using UV-Visible spectrophotometer by scanning in the range of 200 nm to 400 nm against distilled water. The λmax of the drug was noted. The absorption curve showed characteristic absorption maxima at 241 nm for ceftriaxone sodium.

Preparation of stock solution

Accurately weighed (10 mg) of ceftriaxone sodium was transferred to 100 ml amber colored volumetric flask. Small quantity of distilled water was added to ensure complete solubilization of drug and finally volume was made up to the mark with distilled water to produce 100 μg/ml solution.

Preparation of working standard solutions

With the help of pipette, 0.1 ml aliquot was withdrawn from stock solution and transferred to 10 mL amber colored volumetric flask. It was then diluted up to 10 ml with distilled water to produce 1 μg/mL solution. Similarly solutions of concentration 2, 6, 10, 14 and 18 μg/ml were prepared which were used for the construction of calibration curve.

Preparation of ceftriaxone sodium loaded in nanoparticles [7-9]

The formulation was prepared by Ionic crosslinking method. 2 mg/ml chitosan was dissolved in 2% glacial acetic acid solution. 1 mg/ml Sodium Tripolyphosphate (TPP) and drug was added in 10 ml of double distilled water. TPP solution was added to chitosan solution drop by drop at 0.5 ml/min under constant stirring at 800 rpm over magnetic stirrer. After complete addition of TPP solution, chitosan dispersion was allowed to cross linked for 100 min.

Preparation of nanoparticle test solution

Nanoparticle dispersion equivalent to 10 mg ceftriaxone sodium was weighed and transferred to 100 ml amber colored volumetric flask separately and the volume was made up to 100 ml with distilled water to produce 100 μg/ml. 1 ml of test solution was diluted up to 10 ml with distilled water and the absorbance of test solution (10 μg/ml) was recorded against distilled water as a blank at 241 nm.

Method validation [7,8]

Validation can be defined as establishing documented evidence which provides a high degree of assurance that a particular method will consistently produce a product meeting its predetermined specifications. The method was validated for different parameters like linearity, precision, accuracy, specificity, ruggedness, robustness, limit of detection, limit of quantification and robustness as per the ICH guidelines.


The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. For linearity study, six solutions of different concentration (2, 6, 10, 14 and 18 μg/ml) were prepared from the stock solution by withdrawing aliquots with the help of pipette and transferring to separate 10 ml amber coloured volumetric flasks and making volume up to the mark with distilled water. The absorbance of the solutions was measured at 241 nm. A graph of concentration versus absorbance was plotted and correlation coefficient (r2) was calculated.


The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision of the method was determined in terms of repeatability and intra-day and inter-day precision. Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability of the method was determined by analyzing six samples of same concentration of drug. For intra-day and inter-day precision studies six solutions of different concentration (2, 6, 10, 14 and 18 μg/ml) were prepared and analyzed three times a day and the same procedure was followed for next two days. The results were reported in terms of % standard deviation, % RSD.


The specificity of an analytical method represents its ability to assess unequivocally the analyte in presence of components which are expected to be present. It was checked by comparing the spectra of standard drug solution and nanoparticle test solution.


The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Recovery study was performed by standard addition method at three levels i.e. 80%, 100% and 120%. At each level, the determination was done in triplicate and the amount of drug recovered was calculated.

Limit of Detection (LOD)

It is the lowest concentration of analyte in sample that can be detected but not necessarily quantified. It was calculated based on standard deviation of response and slope of the curve using following equation:

LOD = 3.3 σ/s

Where, σ-standard deviation of the response, s-Slope of curve.

Limit of Quantification (LOQ)

It is the minimum concentration of analyte that can be quantified with suitable precision. It was calculated using following equation:

LOQ = 10 σ/s

Where, σ-is standard deviation of response, s-Slope of curve.


The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It was done by measuring absorbance of a 10 μg/mL solution at detection wavelength 241 ± 2 nm. Each measurement was done in triplicate.

Solution stability

For stability study solution was stored at room temperature for 24 h. The initial absorbance and absorbance at 24 h was measured and the difference was noted. The similarity factor was calculated as follows:


Results and Discussion

Selection of wavelength maximum (λmax)

The UV spectrum of ceftriaxone sodium in distilled water has maximum absorption (λmax) at 241 nm as per shown in Figure 2. The absorbance of excipients in Nanoparticle solution did not interfere with ceftriaxone sodium. As a result, 241 nm wavelengths was selected for quantitative analysis and validation of ceftriaxone sodium in nanoparticles.


Figure 2: UV spectrum of 10 μg/ml ceftriaxone sodium in distilled water


The drug obeyed Beer-Lambert’s law in the concentration range of 2-18 μg/ml with regression 0.999 at 241 nm with %RSD < 2% shown in Table 1. Overlay spectra of Ceftriaxone sodium are shown in Figure 3 and calibration curve is shown in Figure 4.

Concentration (mg/ml) Mean response ± SD (n=6) %RSD
2 0.1028 ± 0.00097 0.9838
6 0.3263 ± 0.00487 1.5099
10 0.517 ± 0.005 0.58164
14 0.7187 ± 0.00843 1.17901
18 0.9381 ± 0.00236 0.25284
  Linearity Equation y=0.051x + 0.004
  Correlation Coefficient 0.999
  Slope 0.051
  Intercept 0.004

Table 1: Linearity of ceftriaxone sodium (2-18 μg/ml) (λmax.: 241 nm)


Figure 3: Overlay of the spectra of ceftriaxone sodium (2-18 μg/ml)


Figure 4: Linearity curve of ceftriaxone sodium (2-18 μg/ml)


The developed method was found to be precise as the %RSD values for intraday (Table 2.1) and interday (Table 2.2) precision were found within limit (< 2%). Repeatability expresses the precision under the same operating conditions over a short interval of time. So, as per Table 3 this method is repeatable.

Absorbance Concentration
Mean absorbance ± SD (n=6) Mean concentration ± SD %RSD
2 0.1026 1.9333 0.1028 ± 0.0009 1.937 ± 0.0190 0.9838
0.1039 1.9588
0.102 1.9215
10 0.5169 10.0568 0.5170 ± 0.005 10.124 ± 0.0588 0.5816
0.5221 10.1588
0.5121 10.1588
18 0.810 18.3333 0.9381 ± 0.0023 18.317 ± 0.0463 0.2528
0.805 18.3529
0.798 18.2647

Table 2.1. Intraday precision

Absorbance Conc. (μg/ml) Mean absorbance ± SD Mean concentration ± SD %RSD
2 0.105 1.9803 0.1050 ± 0.0009 1.9801 ± 0.02 1.0100
0.106 2
0.1042 1.96
10 0.5123 9.9745 0.5070 ± 0.0048 9.8659 ± 0.0997 1.0107
0.5061 9.845
0.5027 9.7784
18 0.9402 18.3568 0.9256 ± 0.0039 18.2673 ± 0.0775 0.4247
0.9335 18.2254
0.9332 18.2196

Table 2.2. Interday precision

Table 2: Precision

S. No. Conc. (μg/ml) Absorbance Conc. (μg/ml)
1 10 0.5129 9.9784
2 0.5225 10.1666
3 0.5199 9.9588
4 0.5243 10.2019
5 0.5174 10.0666
6 0.5214 10.1450
Mean 0.5197 10.0862
S.D. 0.0040 0.1015
R.S.D. 0.7860 1.0066

Table 3: Repeatability


The excipients in nanoparticles did not interfere with absorbance of ceftriaxone sodium which indicates that the method is specific as shown in Figure 5.


Figure 5: Spectra of ceftriaxone sodium in nanoparticle formulation accuracy

Recovery study shows that the overall % recovery was found to be 99.83% as per in Table 4. So, this method is accurate.

Level of recovery Sample concentration (μg/ml) Std. Added (μg/ml) Total amount
Absorbance Amount
%Recovery Mean % recovery
80 5 4 9 0.4592 8.9254 99.17 99.27
5 4 9 0.4599 8.9392 99.32
5 4 9 0.46 8.9411 99.34
100 5 5 10 0.5122 9.0647 99.64 99.73
5 5 10 0.513 9.0647 99.8
5 5 10 0.5128 9.9803 99.76
120 5 6 11 0.569 11.078 100.7 100.6
5 6 11 0.5684 11.0666 100.6
5 6 11 0.5639 11.05686 100.51

Table 4: Accuracy


LOD calculated using the equation as mentioned earlier is 0.336805 μg/ml.


LOQ calculated using the equation as mentioned earlier is 1.020621 μg/ml.


The method was found to be robust when checked for the effect of change in detection wavelength (Table 5).

Concentration (μg/ml) Absorbance Concentration
10 239 nm 243 nm 239 nm 243 nm 239 nm 243 nm
0.5094 0.512 9.9098 9.9607 99.09 99.6
0.5102 0.5108 9.9254 9.9372 99.25 99.37
0.5099 0.5106 9.9196 9.9333 99.19 99.33
Average 0.5098 0.5111 9.9182 9.9437 9.9437 99.1766
S.D. 0.0004 0.0007 0.0078 0.0148 0.0148 0.0808
RSD 0.0792 0.0792 0.0794 0.1490 0.1490 0.0815

Table 5: Robustness

Solution stability

The stability of solution was evaluated by determining similarity factor (0.995) which was found within the acceptance criteria of 0.98-1.02. The developed method was found to be precise, specific and accurate (Table 6).

Solution stability
Concentration (μg/ml) Initial absorbance Initial concentration (μg/ml) Initial assay (%) Absorbance after 24 h Concentrate-ion after 24 h %Assay after 24 h Difference in % assay
10 0.5067 9.8568 98.56 0.5095 9.9117 99.11 0.55
0.5059 9.8411 98.41 0.5079 9.8803 98.8 0.39
0.5053 9.8294 98.29 0.5069 9.8607 98.6 0.31

Table 6: Solution stability

The overall summary of validation parameters is shown in Table 7.

Summary of validation parameters
Parameter Ceftriaxone sodium loaded nanoparticles
λmax 241 nm
Linearity 2 to 18 μg/ml
Equation y=0.051x + 0.004
R2 0.999
LOD 0.336805
LOQ 1.02062
Repeatability (%RSD, N=6) 0.7860
Intraday precision (%RSD, N=3) 0.6060
Interday precision (%RSD, N=3) 0.8151
% Recovery 99.83
Standard solution stability (Similarity factor) 0.995

Table 7: Summary of validation parameters


The proposed UV-Spectrophotometry method for estimation of ceftriaxone sodium in pharmaceutical dosage form that is nanoparticles was successfully developed and validated for its intended purpose. The method was shown to be linear, precise, repeatable, specific, accurate, robust and stable. Therefore it can be used for the determination of ceftriaxone sodium loaded into Nanoparticles.


We are thankful to JFL Life sciences Ltd, (Ahmedabad, India) for providing the gratis sample of Ceftriaxone sodium. We are also thankful toRamanbhai Patel College of Pharmacy, CHARUSAT for providing us the required facilities.


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