Validated HPLC Method for Determination of Contents of Residual Aminoguanidine in Drug Substances


Srivastava Bimal Kumar*, Kushwah Dharmendra Kumar, Kolhe Prakash Yashwant and Patel Nitin

CTX Life Sciences Pvt. Ltd., 251-252, Sachin Magdalla Road, Sachin, Surat, Gujarat-394230, India

*Corresponding Author E-mail:



This paper describes a validated method for the quantification of aminoguanidine in drug substances and their intermediates using High Performance Liquid Chromatography (HPLC), with a per column derivatization with 1-Naphthyl isothiocyanate. Derivative formed was analysed using a RP-18 HPLC column with pH 3 ortho-phosphoric acid and triethyleamine buffer and Methanol as mobile phase with a gradient elution. Wavelength 220 nm was used for detection. The method is validated for its Specificity, Precision, Accuracy, Linearity Ruggedness and Robustness. Aminoguanidine is linear from 0.015g/ml to 0.750 g/ml with correlation coefficient 0.99609. Limit of Quantitation of the method is 0.015 g/ml and Limit of Detection is 0.010 g/ml.


KEYWORDS: Thiourea derivative, HPLC, UV-Vis detection, RP-18 column, 1-Naphthyl isothiocyanate, Limit of Quantitation, and Limit of Detection.



Guanidine (aminomethanmidine) is found in beet juice, and it is one of the degradation products of purines1. Guinadine is made by addition of Me2NH to available cyanamide NH2.CN and keto ester in an aceto acetate product2. Aminoguanidine is used as an intermediate for the synthesis of pharmaceuticals, agrochemicals, dyestuffs and other organic derivatives; It is also used as an alternate to Gabriel synthesis, in which alkyl halides can be converted to primary amines in good yield, involves the treatment of the halides with the strong base guanidine followed by alkaline hydrolysis3. Aminoguanidine as Aminoguanidine Bicarbonate is used in the formation of Schiffs base, intermediate stage of lamotrigine.



Literature survey revealed that very few attempts are made for the determination of residual aminoguanidine in organic compounds especially in pharmaceuticals. Li, Y. L. Huang, et. al., developed a method for the determination of aminoguanidine bicarbonate by UV spectrophotometry4. M.A. Beaven, et. al. developed a specific and sensitive assay method for determination of aminoguanidine in animal tissues by derivatization of aminoguanidine with p-nitrobenzaldehyde and the yellow product thus formed is assayed on spectrophotometer5.


US patent No. US 5108930 describes a method for aminoguanidine assay and application thereof in plasma samples by derivatization of aminoguanidine with 6-methyl-2-pyridine-carboxaldehyde and further analysis on HPLC6.


Present study involves the development of a method for the estimation of aminoguanidine in different drug substances and their intermediates using high performance liquid chromatography (HPLC) and ultraviolet detector. Development of the method is based on the principle; N-Hydro-C-alkylamino-addition7, when ammonia, primary and secondary amines are added to isocynates or isothiocynates gives substituted ureas or thioureas, this is an excellent method of preparation of urea or thiourea e.g. R-N=C=O + R NH2 → R-NH-C=O NHR.

Aminoguanidine is non-chromophoric hence have no spectra or proper response in UV light. The thiourea derivative of aminoguanidine is UV active and λ max is about 220 nm.


Optimization of Derivatization conditions

For the optimization of derivatization parameters, samples with different reagents were exposed to different temperatures (25C to 90C) for different time (15 minutes to 3.0 hours). On exposure to higher temp and for longer time derivative formed shows degradation. Satisfactory derivatization takes place at when samples are exposed to 60 5C for about 60 minute, Derivative such formed is stabilized by addition of hydrochloric acid solution to neutralize the remaining sodium hydroxide.




1-Naphthyl isothiocyanate from Aldrich, Ortho phosphoric acid (HPLC grade) Merck, Triethylamine (HPLC grade) Merck, Methanol (HPLC grade) Merck, Hydrochloric acid (AR grade) Rankem, Sodium hydroxide (AR grade) Merck, Purified water (HPLC grade or equivalent) and 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine (Lamotrigine) procured in-house used as sample.



Waters 2695 Separation Module with PDA detector and Empower 2 software, Shimadzu LC-2010 with UV/PDA detector and LC solution software, Mettler Toledo XS 205 analytical balance. BDS Hypersil C-18 (250 x 4.6) mm, 5m HPLC column and all A grade glassware from Borosil and Rankem were used for separation and quantification.


Mobile phase:

Buffer solution for mobile phase was prepared by mixing 1.0 ml of ortho phosphoric acid in 1000 ml of water and pH adjusted to 3.0 with triethylamine, filtered and degassed methanol is used as solvent in gradient elution. Diluent is for sample preparation and blank solution is a mixture of buffer and methanol and mobile in ratio of 90:10, all the solutions prepared were filter through 0.45-m porosity membranes filters and degassed.


Analytical condition of HPLC:

Column oven temperature was kept at 30C, Injection volume: 20 l, Wavelength: 220 nm, Flow rate: 1.0 ml/min, Run time: 50 min., Gradient program used in analysis as given below, Table-1:




Time in minute



















Preparation of reagent and solutions:

10 mg/ml of 1-Naphthyl isothiocyanate is dissolved in methanol; sodium hydroxide solution was prepared by dissolving 4 g of sodium hydroxide to 100ml of volumetric flask with water. 2M hydrochloric acid solution was prepared by mixing 18 ml of hydrochloric with100 ml of water. A 0.005-mg/ml solution of aminoguanidine bicarbonate equivalent to aminoguanidine was prepared by diluting aminoguanidine bicarbonate stepwise with diluent.


Procedure to prepare derivatives of Blank, Standard and Samples:

2.0 ml of 1-Naphthyl isothiocyanate reagents added to a 10 ml of volumetric flasks each separately, containing 1.0 ml of Aminoguanidine standard stock solution, 10 mg of sample, and 1 ml of blank. To these flasks add 0.2 ml sodium hydroxide solution, 3.0 ml of methanol and sonicate for 1 to 2 minute. Keep this solution into an oven about 60 minute at 60 5C. Cool to attain room temperature, add 0.2 ml of hydrochloric acid solution to neutralize the remaining sodium hydroxide solution, and dilute up to mark with methanol. The solutions of blank, standard and samples are injected in HPLC for analysis.


System suitability check:

System suitable is checked with standard chromatograms standard solution is injected in six replicate. The Tailing factor of aminoguanidine peak is not more than 2.0 and column efficiency is not less than 2500 theoretical plates. Relative standard deviation for area of Aminoguanidine peak for six replicate injections of standard solution is not more than 5.0%.



For method validation studies 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine (Lamotrigine) is used as sample. Validation was performed with 0.05 % of aminoguanidine at specification level w.r.t. sample. Developed method is validated as per as per USP and ICH guidelines8,9.


Specificity and selectivity:

Blank, aminoguanidine individual standard, sample solution and spiked sample solution were prepared as per the method and injected separately into chromatograph. No interference from blank and peak due to any impurity was observed at the retention time of aminoguanidine peaks. The peak purity plot (Figure-1) also indicates that peak of aminoguanidine is pure and do not have any co-eluting peak. Peak Purity index; 3.515, Single Point Threshold; 4.137. Retention time of aminoguanidine is about 20 minutes.

Figure-1. Peak purity plot of aminoguanidine.


Linearity and calibration curve:

For linearity solutions of different concentrations ranging from LOQ level (0.015g/ml) to 100% level (0.500g/ml) and to 150% level (0.750 g/ml) of aminoguanidine were prepared in diluent and correlation coefficient, slop and intercept were determined, the data is provided in table below. (Table-2)


Table-2: Linearity Data





0.0150.750 g/ml

R2 = 0.99609









System precision (system repeatability), method precision (n=6) and intermediate precision (n=6) was studied by recovery, by spiking standard aminoguanidine in sample at the 0.50%w/w concentration, relative standard deviation of results was calculated. Ruggedness of the method was established by comparing the results obtained on different days by different analyst on different instrument and HPLC column. (Table-3)


Table-3: Precision data





(a) System Precision %RSD (n=6)


0.500 g/ml (standard)



(b) Method precision by recovery %RSD (n=6)

0.050 %w/w


(c) Intermediate precision by recovery %RSD (n=6)

0.050 %w/w


(d) Ruggedness %RSD (n=12)

0.050 %w/w




Accuracy of the method was studied by recovery, standard aminoguanidine was spiked with sample at different concentration levels (LOQ, 50%, 100%, and 150% levels of specification) and amount recovered was calculated (Table-4).


Table-4: Accuracy and recovery data





(Mean % Recovery %RSD)





(n =3) at LOQ level

0.05 %w/w

97.93 8.14 %

(n =3) at 50 % level

0.25 %w/w

100.68 2.21 %

(n =3) at 100 % level

0.50 %w/w

107.23 0.97 %

(n =3) at 150 % level

0.75 %w/w

100.09 0.56 %

(n = 12) Over all recovery


100.48 5.02 %


Limit of detection and quantification (LOQ and LOD):

LOD and LOQ of aminoguanidine were predicted from standard error and slop of linearity data and precision was established at the predicted concentrations. % Relative standard deviations are found within limits for LOQ at 0.015 g/ml (0.0015% w/w),% RSD=5.61 and LOD at 0.010 g/ml (0.0010% w/w), % RSD=16.23.


Stability of thiourea derivative formed:

Spiked sample at specification level was derivatized and the area of the peak of derivative formed was measured for about 24 hours at different time intervals. Cumulative RSD of the peak of derivative formed is found 0.59%. Hence the derivative formed is stable at ambient temperature for at least up to 24 hours.



Presented method is sensitive enough and is found to be, linearity in the concentration range of 0.0150.750 g/ml with R2 = 0.99609. Method is specific, as peak purity plot indicated no interference in the aminoguanidine thiourea derivative peak; Peak purity index (3.515) is less than single point threshold (4.137) is a significance of purity of aminoguanidine. Peak purity plot is presented as figure-1. Accuracy of method was established by recovery, the recovery values are within acceptable limits at different concentration levels, over all recovery is 100.48 5.02%, and this indicated that the method is accurate.


Figure-2. Representative chromatogram of Aminoguanidine by HPLC

Retention time of aminoguanidine is about 20 minutes.


Reparability, Reproducibility and Intra day precision are performed as System precision, Method Precision, Intermediate precision and Ruggedness, and the values obtained are well within limits. Representative chromatogram is presented in figure-2 and the different values of validation data; linearity, precision, ruggedness, accuracy, limit of quantification and limit of detection are given in table-2 to 4. Thiourea derivative formed with the reaction of aminoguanidine and 1-Naphthyl isothiocyanate is stables for at least up to 24 hours at ambient temperature, hence samples can be stored and analysed for at least 24 hours.



The presented work describes that developed reveres phase HPLC method for the determination of aminoguanidine is Specific, Rugged, Linear, Accurate, Precise, and can be applied for quantification of aminoguanidine in different drug products and their intermediates. This method can also be applied to other similar drug products with small modifications.



Authors are highly thankful to the CTX Life Sciences management for granting permission for the publication of this work.



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Received on 04.02.2010 Modified on 28.02.2010

Accepted on 20.03.2010 RJPT All right reserved

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1058-1060