Development and Validation of Difference Spectrophotometric Method for Quantitative Estimation of Gemcitabine Hydrochloride Injection

 

P Ravi Kumar*, P Spandana, Amani Shareef, K Sahithi, N Raghavendra Babu

 

ABSTRACT:

Gemcitabine hydrochloride is used as an anti-cancer drug. The aim of the present study is to develop difference spectrophotometric method for the estimation of gemcitabine in bulk and lyophilized powder for injection. Difference spectrophotometric method has been developed for quantification of gemcitabine in injection formulation. The method was developed by measuring the difference spectral characteristics of the drug in 0.1N HCL and 0.1N NaOH at absorption maximum of 236nm. Beers law was obeyed in the concentration range of 5-25μg/ml. The LOD and LOQ were found to be 0.43 and 1.30μg/ml respectively with coefficient of correlation 0.999. The proposed method has been validated for linearity, accuracy and precision as per ICH guidelines and applied for quantitative analysis of gemcitabine hydrochloride injection.

 

 

 

INTRODUCTION:

Gemcitabine is used in treatment of non-small cell lung cancer, pancreatic cancer, bladder cancer and breast cancer, esophageal cancer and is used experimentally in lymphomas and various other tumor types. UV Visible spectrophotometry is widely used for qualitative and quantitative analysis which involves measuring the amount of radiation absorbed by a substance.

 

 

HCl

Fig.1:  Chemical Structure Gemcitabine Hydrochloride

 

Difference spectroscopy provides a sensitive method for detecting small changes in the environment of a chromophore or it can be used to demonstrate ionization of a chromophore leading to identification and quantitation of various components in a mixture. The essential feature of a difference spectrophotometric assay is that the measured value is the difference absorbance (Δ A) between two equimolar solutions of the analyte indifferent forms which exhibit different spectral characteristics. In this method, the simplest and most commonly employed technique for altering the spectral properties of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. The ultraviolet-visible absorption spectra of many substances containing ionisable functional groups, e.g. phenols, aromatic carboxylic acids and amines depends  on the pH of the solution .A substance whose spectrum is unaffected by changes of pH may be determined by a difference spectrophotometric procedure if it can be quantitatively converted by means of a suitable reagent to a chemical species that has different spectral properties to its unreacted parent substance. The ∆A between equimolar solutions of the unreacted substance and its derivative is free of interference if the irrelevant absorption is unaffected by the reagent.

 

MATERIALS AND METHODS:

Instruments:

ShimadzuUV- Visible spectrophotometer, Contech Digital Balance, Sonica ultrasonicator.

 

Chemicals and Reagents:

sodium hydroxide, hydrochloric acid, sodium nitrite, ammonium sulphamate, brattonmarshall reagent

 

Standards and Sample:

API as gift sample from Drug Control Administration and Gempower, powder for injection (Khandelwal Laboratories) purchased from local market.

 

METHOD DEVELOPMENT:

Preparation of standard stock-I & II solutions (1000μg/ml &100μg/ml)

Standard gemcitabine hydrochloride of 1000μg/ml solution was prepared by dissolving 10mg of gemcitabine hydrochloride working standard in 10ml of 0.1NHCl and 0.1N NaOH separately.1.0ml of standard stock-I solution transferred to 10ml volumetric flask and volume adjusted to the mark with respective solvents.

 

Determination of absorption maximum (λmax):

From stock-II solution, 10µg/ml solutions prepared, NaOH solution placed in sample compartment and Hcl solution placed in reference cell and scanned in the range of 200-400nm to determine the absorption maximum for the drug. The absorption spectrum was noted down.

 

Selection of analytical concentration ranges (5-25μg/ml) and Construction of Calibration Curve to check Linearity:

Various concentrations were prepared from the working standard stock solution of gemcitabine hydrochloride (100µg/ml), appropriate aliquots like 0.5, 1.0, 1.5, 2.0, 2.5ml solutions were pipetted in 10ml graduated tubes. The solution in each tube wasmade up with 0.1N NaOH of one set and with 0.1N HCl of another set to obtainworking standard concentration ranging from 5-25µg/ml. The absorbance of these solutions was measured at 236nm by taking 0.1N NaOH prepared concentrations in sample cell and 0.1 N HCl prepared concentrations in reference cell. Difference absorbance measured and calibration curve was plotted.

 

METHOD VALIDATION:

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The sensitivity of proposed method was estimated in terms of LOD & LOQ, calculated using standard deviation method.

 

Precision:

The precision of an analytical method is the degree of agreement among individual test results.

(A) System Precision (Repeatability):

The repeatability was checked by measuring the absorbance of six replicates of same concentration  (15μg/ml) of standard solution  and % RSD was calculated.

(B) Method precision:

The method precision was checked as intra and interday precision.

 

Intra-day precision & Inter-day precision (Intermediate):

The intra-day precision was determined by analyzing the corresponding concentrations (12 μg/ml, 15μg/ml and  18μg/ml) 3 times on the same day and inter-day precision determined by measuring absorbance on 3 consecutive days. The results were reported in terms of relative standard deviation.

 

Accuracy:

The accuracy of an analytical method describes the extent to which the test results deviate from the expected results and it is a measure of the exactness of an analytical method. Accuracy may be reported as the percentage recovery of a known added amount of analyte to a sample or as the difference between the mean obtained and the accepted true of a sample, together with an associated confidence interval. Accuracy for drug substance was determined on samples of drug solutions at varying concentration levels in the range of 80%-120% (16μg/ml, 20μg/ml and 24μg/ml) by analyzing three levels of each sample as a batch in a single assay. The % relative standard deviation was calculated at each level.

 

ANALYSIS OF GEMCITABINE HYDROCHLORIDE INJECTION:

Lyophilized powder was weighed, drug powder equivalent to 20.45mg of gemcitabine hydrochloride was weighed and transferred into two 10ml volumetric flasks separately. To, one flask, .01N HCl and to the second flask 0.1 N NaOH were added, the drug dissolved and volume was made up to the mark. These solutions were filtered through Whatmann filter paper (no.41) and collected in another volumetric flask. Appropriate dilutions were carried out on filtrate as in case of standard drug solutions and difference absorbance was measured at 236nm. Gemcitabine hydrochloride quantity in the injection (Gempower) was calculated.

 

RESULTS AND DISCUSSION:

Difference spectrum of Gemcitabine hydrochloride:

Detection of absorbance maximum (λ_max) of 236nm with differential absorption was determined which was suitable for the analysis of gemcitabine hydrochloride for difference spectrophotometric method.

 

Fig.2 Absorption spectrum of gemcitabine

 

Fig.3: Overlay spectrum of gemcitabine

 

Fig.4 : Calibration Curve for Gemcitabine

 

 

Table 1: Calibration Curve Data

 

Table 2:  Optimized UV conditions

λmax	236nm
Beer’s law range	5-25µg/ml
Molar absorptivity (liter/moles/cm)	7.2 x 105 lit/mol/cm
Limit of detection (μg/ml)	0.10μg/ml
Limit of quantification (μg/ml)	0.32μg/ml
Regression equation	Y=0.031x
Slope	0.031
Correlation coefficient(r2)	0.999

 

Method validation:

The developed method was validated as per ICH Q2(R1) guidelines.

 

Linearity:

A linear and proportional relationship was observed between the concentration range of 5-25μg/ml at 236nm. The coefficient of correlation was found to be 0.996. The results were shown in Ttable 1.

 

Limit of Detection (LOD) and Limit of Quantization (LOD):

The LOD and LOQ reported as a means to analyze the sensitivity. The LOD and LOQ for gemcitabine hydrochloride were found to be 0.10 μg/ml and 0.32 μg/ml respectively

 

Precision:

The precision of the developed analytical method was assessed by checking repeatability, intra-day precision and inter-day precision. The results are expressed in Table 3,4 and 5.

 

Table 3: Repatability data for Gemcitabine

 

The results revealed that % RSD values were within the limits i.e., not more than 2% hence the method is repeatable. This indicates that the instrument performance was good.

 

The results revealed that the %RSD values were within limits hence the method is precise. This indicates that the method is reproducible at any time during the day.

Table 4: intra-day precision data for Gemcitabine

 

Table 5: Inter-day precision data for Gemcitabine

 

 

The results shows that all the calculated % RSD values are below 2%, therefore the method is precise. This indicates that the method is reproducible on different days.

 

Accuracy:

The analytical accuracy is the nearness of the results obtained against the real values at each level of gemcitabine concentration. The results obtained for accuracy studies for the drug substance and drug product were reported in terms of % RSD and % recovery respectively. Accuracy data for gemcitabine drug substance was shown in Table 6.

 

Table 6: Accuracy data for Gemcitabine drug substance

Concentration level (μg/ml)	Absorbance	Mean	Standard Deviation	% RSD
16	0.420	0.418	0.002	0.47
	0.416
	0.418
20	0.531	0.527	0.0035	0.66
	0.524
	0.527
24	0.631	0.635	0.004	0.62
	0.639
	0.635

 

From the above data it was found that the % RSD (acceptance criteria < 2%) values are within the acceptance limits. This indicates that the method is accurate.

 

Table 7: Recovery study for drug product

S. no	Amount of sample μg/ml	Amount of standard μg/ml	Total concentra-tion μg/ml	Total concentration found (μg/ml)	% Recovered
1	20	16	36	35.5	98.6
2	20	20	40	39.2	98
3	20	24	44	43.6	99.0

 

The recovery was assessed by determining the agreement between the measured standard concentration and added known concentration to the sample. The test was done by spiking the pre-analyzed lyophilized powder with pure gemcitabine hydrochloride at three different levels (80%, 100% and 120%).

 

Assay of Gemcitabine lyophilized powder:

The average absorbance of the three dilutions of concentration (20μg/ml) of GEMPOWER estimated and % purity was calculated. The results are shown in Table 8.

 

 

Table 8: Assay of Gemcitabine injection

Formulation	Labeled amount (mg)	Amount obtained (mg)	% Recovery
GEMPOWER (lyophilized powder)	200	197	98%

Limit (IP): 95-105%

 

CONCLUSISON:

A difference spectrophotometric method was developed for the quantitative estimation of gemcitabine hydrochloride injection. The developed method was optimised and validated as per ICH Q2A (R1) guidelines. All the results were found to be within the limits. From the results, it can be concluded that the proposed method was valid and can be applied for the routine quantitative estimation of gemcitabine hydrochloride in bulk and pharmaceutical dosage forms.

 

REFERENCES:

1.      K.D. Tripathi. Essentials of Medical Pharmacology. 7th edition Jaypee Brothers MedicalPublishers Pvt Ltd; 2008

2.      H. P, Rang, M. M. Dale, J. M. Ritter, R. J. Flower. Principles of Pharmacology. Published by Churchill Livingstone; 7th edition; 2012

3.      G.R. Chatwal, S.K. Anand. Instrumental Methods of Chemical Analysis. 5th Edition. Himalaya Publishing House; 2002

4.      Y.R. Sharma. Organic Spectroscopy. Instrumental methods of chemical analysis. Published by S. Chand & Company ltd; 2011

5.      A.H. Beckett, J.B. Stenlake. Practical Pharmaceutical Chemistry: Ultraviolet-visible absorption spectrophotometry; 2002

6.      Pavia, Lampman, Kriz, Vyvyan. Introduction to spectroscopy. Brookescole publishers; 2008

7.      Indian Pharmacopoeia, Indian Pharmacopoeia Commission, Ghaziabad, 2014, volume II,1261-1263

8.      P Ravi Kumar, Y Padmavathi, P Niveditha, N. Raghavendra Babu; Development and Validation of Difference Spectrophotometric Method for the Quantitative Estimation of Mesalamine in Bulk Drug and Dosage forms; Asian J. Pharm. Ana. 2017; 7(4): 225-228.

9.      Malladi Srinivas Reddy and Sadiya Firadous; Spectrophotometric Estimation of Gemcitabine HCl in Pharmaceutical Dosage form via Oxidative Coupling Reaction, Chemical Science Transactions; 2015

10.   A Santosh Kumar Sreevatsav, K. Shyam Sunder and JayapalMaleraju; RP-HPLC method for estimation of Gemcitabine HCl 1gm injection in parenteral dosage form, International Journal of Pharmaceutical Chemistry; 2013

11.   Subhashini Edla and B. Syama Sundhar; RP-HPLC method for the quantification of gemcitabine in formulations, International Journal of Pharma and Bio Sciences; 2013.

12.   V.Rajesh, B. Anupama, V. Jagathi And P. Sai Praveen Simultaneous Estimation of Gemcitabine Hydrochloride and CapecitabineHydrochloride in Combined Tablet Dosage Form by RP-HPLC Method; E-Journal of Chemistry; 2011

 

 

 

 

Received on 12.06.2019                 Accepted on 11.07.2019

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Asian J. Pharm. Ana. 2019; 9(4):219-223.