Original Article

Spectrophotometric Determination of Dopamine in Bulk and Dosage Forms Using 2,4-Dinitrophenylhydrazine


  • Mai Abed Alrahman RAMADAN
  • Ihab ALMASRI
  • Ghada KHAYAL

Received Date: 23.09.2019 Accepted Date: 21.11.2019 Turk J Pharm Sci 2020;17(6):679-685 PMID: 33389979


Dopamine (DA) hydrochloride is a sympathomimetic agent used therapeutically for the correction of hemodynamic disorders associated with shock episodes. Although several analytical methods have been described, a spectroscopic assay of DA after chemical derivatization with 2,4-dinitrophenylhydrazine (DNP) is still unexamined. Therefore, the optimization of the reaction parameters and validation of developed method were required.

Materials and Methods:

The method is based on coupling of DA as a phenolic compound with a diazonium salt to produce an intensely colored azo derivative. DNP was oxidized with potassium periodate to produce a diazonium salt that coupled with DA in basic media. The experimental parameters were then optimized. The developed method was validated according to International Conference on Harmonisation Guidelines and was applied to dosage forms. The results were compared with the data of a reference method.


The method was linear in a concentration range between 5 and 50 μg/mL. The regression line equation was Y=0.042±0.0003X+0.0672±0.0015 with a regression coefficient of 0.9944 (n=5). The limit of detection and limit of quantification were 0.32 and 0.97 μg/mL, respectively. The precision was satisfactory; the percentage relative standard deviation did not exceed 2%. The average values of the recovery study were in the range 98.90- 100.40±0.31-1.21%. The developed method was applied successfully for the determination of DA in injection and infusion fluid.


The method is accurate, sensitive, and practical for DA analysis in quality control laboratories.

Keywords: Dopamine hydrochloride, 2,4-dinitrophenylhydrazine, spectrophotometric, validation


The chemical name for dopamine (DA) hydrochloride is 1,2-benzenediol,4-(2-amonoethyl) hydrochloride. DA is an endogenous catecholamine that is a sympathomimetic agent with prominent dopaminergic and β1-adrenergic effects at low to moderate doses and α-adrenergic effects at high doses. It is used for the correction of hemodynamic disorders associated with shock episodes.1,2

A literature survey of DA revealed several methods for its determination in injection. Spectrophotometric methods using bromanil, 2,6-dichloroquinone-4-chloroimide, 3-amiopyridine, chloramine T, and various oxidative coupling based methods were published.3,4,5,6,7,8,9,10 In addition, different high performance liquid chromatography (HPLC),11,12,13,14,15 flow injection,16,17 fluorimetric,18 capillary electrophoresis,19 chemiluminescence,20 and electrochemical21,22,23,24,25,26 methods were reported.

Spectrophotometry is considered the most practical analytical procedure in quality control laboratories, since it does not need costly instrumentation or toxic solvents like chromatography does. 2,4-dinitrophenylhydrazine (DNP) is a derivatizing agent used in the analysis of many drugs.27,28,29,30,31 Chemical derivatization prior to spectroscopic analysis enhances both sensitivity and selectivity.32

The current study was performed, in continuation of our interest in the development and validation of simple, sensitive, and rapid spectrophotometric methods for the analysis of drugs,33,34 to determine DA depending on a derivatization reaction with DNP in pharmaceuticals.



The spectrophotometers used were a Shimadzu ultraviolet (UV)-1601 with UV-Pro software (Shimadzu, Japan) and a Lambda 25 with V5 ES software (PerkinElmer, USA) and 1-cm quartz cells (Innovative Lab Supply, USA).


All chemicals used were of analytical grade. DA hydrochloride standard was purchased from Merck (Germany). DA dosage forms were ampoules for infusion (200 mg/5 mL) and DA hydrochloride with 5% dextrose infusion fluid (800 µg/mL DA) obtained from a local hospital pharmacy (Gaza, Palestine).

Preparation of reagents

DNP, 0.005 M reagent: 0.10 g of DNP was accurately weighed and transferred into a 100 mL volumetric flask, dissolved in 2.5 mL of concentrated sulfuric acid, and completed up to the volume with distilled water. The solution was freshly prepared and protected from light during use because it is light sensitive.

Potassium periodate [(PPI), 0.0065 M] reagent: 0.15 g of PPI was accurately weighed and transferred into a 100 mL volumetric flask, dissolved, and completed up to the volume with distilled water.

Sodium hydroxide [(NaOH), 10 M]: 40.00 g of NaOH was accurately weighed and transferred into a volumetric flask, dissolved, and completed up to the volume of 100 mL with distilled water.

Standard stock solution

It was prepared by dissolving 0.02 g of DA hydrochloride standard in 100 mL of distilled water (200 µg/mL). Working solutions were prepared by diluting the stock solution. The stock solution was freshly prepared during use.

General procedure

An aliquot of standard stock solution was transferred into a 10 mL volumetric flask followed by 1.0 mL of DNP, 1.0 mL of PPI, and 0.5 mL of NaOH reagents. The mixture was mixed well and diluted to 10 mL with distilled water at room temperature. The absorbance was measured at absorption maximum (λmax) 560 nm against a blank.

Determination of stoichiometric ratio (Job’s method)

Job’s method of continuous variation was employed.35 Equimolar (3x10-3 M) aqueous solutions of DA and DNP were prepared. Series of 1.0 mL portions of DA and DNP were made up comprising different complementary volumes (0.0:1.0, 0.1:0.9, 0.2:0.8, 0.3:0.7, 0.4:0.6, 0.5:0.5, 0.6:0.4, 0.7:0.3, 0.8:0.2, 0.9:0.1, 1.0:0.0) in 10 mL volumetric flasks. The process followed the general procedure. Absorbance was plotted against DNP molar fraction.

Optimization of reaction conditions

Different reaction parameters were studied. They included concentration and volume of DNP, PPI, and NaOH; temperature; reaction time; order of addition; and stability of the developed chromogen. The study was carried out by altering one factor and keeping the others constant.

Method validation

Validation parameters were determined according to International Conference on Harmonisation (ICH) guidelines.36

Assay of pharmaceutical formulations

The content of three ampoules for DA was mixed and an accurately measured volume equivalent to 0.020 g of DA was transferred to a 100 mL volumetric flask. Distilled water was added to bring the volume up to 100 mL.

For DA and 5% dextrose infusion fluid, the content of three bottles was mixed and an accurately volume equivalent to 0.02 g was transferred into a 100 mL volumetric flask and diluted with water. It was further diluted to get a concentration of working solutions. Analysis was performed as described in the general procedure.

Statistical analysis

Data analysis was performed using SPSS version 17 to calculate the regression equation, coefficient factor, standard deviation, relative standard deviation (RSD), t-test, and p value.


In order to enhance sensitivity a derivatization reaction of DA using DNP was performed. A red shifted DA derivative was produced, which showed an λmax at 560 nm (Figure 1). The reaction parameters were optimized (Table 1). The reaction was completed immediately at room temperature. Heating was not advantageous due to intermediate (diazonium) instability. To DA solution was added DNP followed by PPI and NaOH (Table 2). DNP was oxidized by PPI to form a diazonium ion and the pH was still acidic. Once diazonium formed it attacked the electron-rich phenolic DA. The last step required basic media. Addition of PPI to DA (Table 2) was unsuitable since it can lead to oxidation of the catechol moiety of DA. When NaOH was added to DA the absorption was decreased. This can be explained by phenoxide formation and inappropriate media for diazonium salt formation.37,38,39 The best result was achieved when 1 mL of both DNP and PPI reagents and 0.5 mL of NaOH were added to DA in the mentioned order. After dilution the DA derivative was stable for at least 15 min (Figure 2), which allowed processing of samples and their comfortable measurement. The stoichiometric ratio of the DA-derivatization reaction was studied by Job’s method. The molar ratio was 1:1 for DA and DNP (Figure 3). Accordingly, a proposed mechanism of the reaction is illustrated in Figure 4 depending on the result for the molar ratio and the mechanism of azo formation.

Method validation

Linearity and sensitivity

For evaluation of linearity DA was determined at optimized conditions for five concentrations. The calibration curve was Y=0.042±0.0003X+0.0672±0.0015 (r=0.9944, n=5), where Y is the absorbance at 560 nm and X is the concentration of DA (µg/mL). The linear range was 5-50 µg/mL. The molar absorptivity (ε) was 7.9x104 L/mol.cm. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as 3.3σ/S and 10σ/S, respectively,36 where s is the residual standard deviation of the regression line and S is the slope of the regression line. The LOD and LOQ were 0.32 and 0.97 (µg/mL), respectively.

Accuracy, precision, and specificity

The accuracy was evaluated by recovery studies for added standard concentrations to a pre-analyzed product at low, intermediate, and high concentrations. The recovery values were 99.5-101.9±0.21-1.12% (Table 3), indicating the accuracy of the method. Intraday precision was assessed at three different concentrations by analyzing five replicates per concentration on the same day. Interday precision was determined by analyzing samples for 6 consecutive days within a week (Table 4). The percentage of RSD did not exceed 2%, proving the high precision of the method. Before proceeding with the analysis of DA in its dosage forms, interference liabilities were determined to examine the effect of excipients that might be added during formulation. Samples were prepared by mixing 10 and 40 mg of DA with excipients like sodium bisulfate (0.05 g) and dextrose (5.0 g). These laboratory-prepared samples were analyzed by the developed method. The recovery values were 98.9-100.4±0.31-1.21% (Table 5). These data confirmed the absence of interference from excipients with DA determination by the developed method.

Robustness and ruggedness

Robustness was evaluated by studying the influence of small variations in the method variables on its analytical performance. One parameter was changed while the others were kept unchanged and the recovery values were calculated each time. The recovery values were 98.6-101.1±0.31-1.12 (Table 6). This indicated the reliability of the method. Regarding ruggedness, lab-to-lab variations were examined by performing DA analysis using the same operational conditions but using two different instrumentations. The results obtained were reproducible, as RSD did not exceed 1.43% (Table 7).

Application of the method

DA pharmaceutical dosage forms (ampoule, infusion fluid) were analyzed successfully by the developed method. The results comply with the USP 29 specifications of DA content in injection (95-105%).40 Comparison of the result with the reference data5 by statistical analysis with respect to accuracy by t-test showed that there was no significant difference at the 95% confidence level. This confirms similar accuracy in the determination of DA by the two methods (Table 8).


DA contains a catechol group, which can be coupled with a diazonium cation in basic solution to produce a red shifted azo derivative. A simple one-step procedure was achieved for spectrophotometric analysis of DA after derivatization with DNP. The reaction was completed immediately at room temperature, which is advantageous in comparison to other spectroscopic DA assays. Heating at 30, 40, and 75°C and adjustment of pH using a buffer were required for 4-aminoantipyrine-,10 bromanil-,6 and 2-hydroxynaphthaldehyde-based11 spectroscopic analysis of DA, respectively. A derivatization reaction was described for spectroscopic analysis of DA based on the formation of intensely colored Prussian blue that required 35 min for completion.9 In addition, the developed method exhibited enhanced sensitivity (ε 7.9x104 L/mol.cm). The recorded molar absorptivities (ε) for spectrophotometric DA determination were 3.475x103, 6.47x103, and 7.4x103.4,6 A HPLC assay for DA analysis showed a range (12-40 µg/mL) comparable with that of the developed method.12 Chromatographic methods require highly sophisticated instruments and expensive solvents.

The results of the validation studies were in good agreement with ICH guidelines. The method was accurate and precise. The results for interference liabilities proved the specificity of the developed method; thus it can be applied for DA analysis in its dosage forms. The statistical analysis showed that the developed method is comparable with a reference method5 for analysis of DA.


The developed method has the advantages of being simple, rapid, sensitive, accurate, and low cost, and does not require any pretreatment of the drug. The method was applied successfully for analysis of DA in dosage forms without interference from excipients. Therefore, it can be suitable for routine analysis of DA in quality control laboratories.


I would like to thank Al-Shifa hospital pharmacy (Gaza,Palestine) for supplying the DA dosage forms.

Conflicts of interest: No conflict of interest was declared by theauthors. The authors alone are responsible for the content andwriting of the paper.
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