Corresponding Author:
R. R. Raju
Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur−522 510, India
E-mail: [email protected]
Date of Submission 02 November 2013
Date of Revision 08 December 2014
Date of Acceptance 18 April 2015
Indian J Pharm Sci 2015;77(3):343-347  

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A simple and rapid reversed phase-high performance liquid chromatographic method was developed for simultaneous determination of imipramine hydrochloride and diazepam in pharmaceutical formulations. The elution was done in isocratic mode utilizing a mobile phase consisting of methanol:water:0.1M sodium acetate (30:50:20 v/v/v) on Chromosil C18 column with a flow rate of 1.0 ml/min and with detection at 243 nm. The measured retention time was 3.33±0.02 min for imipramine hydrochloride and 4.64±0.02 min for diazepam. Linearity was measured in the range 25-150 μg/ml for imipramine hydrochloride (r 2 =0.999) and in the range 5-30 μg/ml for diazepam (r 2 =0.9994), respectively. The limits of detection and quantitation were 0.03 and 0.1 μg/ml for imipramine hydrochloride and 0.02 and 0.07 μg/ml for diazepam. Satisfactory validation was also obtained from recovery (100.95-101.52% for imipramine hydrochloride and 99.47-100.33% for diazepam) studies, intraday and interday precision (<2%) and robustness results. The reported method was the first study of these drugs in combination and could be employed for routine quantitative determination of imipramine hydrochloride and diazepam in tablets.


Imipramine HCL, Diazepam, RP-HPLC, UV detection, Tablet dosage form

Imipramine hydrochloride (IPM) is a tricyclic antidepressant [1]. Its chemical name is 10,l1-dihydro- 5Hdibenz[b,f]azepine-5-(dimethylaminopropy1) hydrochloride. The molecular formula is C19H24N2. HCl and the molecular mass is 316.9 g/mol. It is officially recognized in Indian Pharmacopoeia (IP) [1], British Pharmacopoeia (BP) [2] and the United States Pharmacopoeia (USP) [3]. Diazepam (DZM) an antianxiety, antiepileptic drug with the IUPAC name, 7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H- 1,4-benzodiazepin-2-one. The molecular formula of diazepam is C16H13ClN2O and the molecular weight is 284.7 g/mol. It is also officially recognized in IP [1], BP [2] and USP [3].

IPM was studied as a single drug and in combination with other drugs [4-6]. Different methods like secondorder derivative spectrophotometry [5], chemometric method [6], spectrophotometry [7,8], high performance liquid chromatography (HPLC) [4,9,10], fluorescence polarization immunoassay [10], HPLC-diode array detection (HPLC-DAD) [11], surface ionization organic mass spectrometry [12] and HPLC tandem mass spectrometry (HPLC-MS/MS) [13] have been employed to measure IPM. It was also determined in human serum [4,9,13] and urine [4,9].

Diazepam has been studied separately, in combination with other benzodiazepines and other drugs [14-20] and its metabolites [21]. It was studied in whole blood [16], plasma [17,18], urine, hair and oral fluids [19,22] and in pharmaceutical form [23-26]. These studies were carried out using analytical methods such as secondderivative spectrophotometry [5], chromatography [15], HPLC [16-18,26], LC-MS/MS [18,19], HPLC electrospray tandem mass spectrometry [20], capillary electrophoresis and HPLC-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) [21], gas chromatography-negative ion chemical ionization mass spectrometry [22], spectrophotometry [23] fluorimetry [24], differential pulse polarographic determination [25] and capillary electrophoresis [26].

IPM and DZM combination is available as Imipam (La Pharma), Imidep (Sunrise Remedies), Imilor (Intra Doxis) and Imizonic plus (Acme) and many others. The motivation for the present work is that benzodiazepines and tricyclic antidepressants are often administered to patients in combination for the treatment of anxiety and depression related disorders. This clinical use necessitates quantitative estimation of these drugs in combination using a simple and rapid method.

In this paper, the development of a method for quantitative and simultaneous estimation of imipramine hydrochloride and diazepam in combination is reported. The developed method is validated by measuring linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, robustness, recovery, ruggedness and specificity based on ICH guidelines [27]. To the best of our knowledge, this is the first work reporting simultaneous determination of IPM and DZM drug combination by RP-HPLC method using ultraviolet (UV) detection.

The chemicals methanol and water (pH between 5 and 8) were of HPLC grade and procured from Merck Limited, Mumbai. Analytical grade sodium acetate and acetic acid were used (Merck Limited, Mumbai). Formulations of the drug were purchased from a local pharmacy. For filtering the prepared solutions 0.45 µm nylon membrane filter paper (Ultipore, Mumbai) was used.

A Peak HPLC system operated in isocratic mode was utilized for the presented investigations. A Chromosil C18 column (250×4.6 mm, 5 µm particles) was used as a stationary phase. The HPLC system was equipped with a LC 20AT pump and a variable wavelength programmable UV/Vis detector (Shimadzu, SPD-10Avp). The samples were injected with a 20 µl Hamilton syringe. The degassing of the mobile phase was done with an ultrasonic bath sonicator and a digital electronic balance was used for weighing the materials. Chromatograms were recorded and integrated by Peak LC 7000 software (MSP Lab instruments, India). Analysis of the data was done with Microsoft Excel software. The detection wavelength of IPM and DZM was determined with an ultraviolet UV/Vis spectrophotometer (Techcomp, UV 2301) provided with Hitachi software.

To prepare the stock solution, 10 mg of IPM and DZM was weighed and dissolved in 10 ml of methanol separately in a 10 ml volumetric flask. Then the drug was sonicated for 2 min to dissolve completely. After cooling, the drug was filtered through 0.45 µm nylon membrane filter paper. A 1000 µg/ml solution was prepared, from this 2 ml was further diluted to 20 ml to get a stock concentration of 100 µg/ml solution. Required concentrations were prepared by selective dilution from the standard solution.

The RP-HPLC method with UV detection was started with the development of the mobile phase composition, flow rate, wavelength and pH. These were optimized for sharper peaks with less asymmetry and for good resolution of IPM and DZM drugs. Initially, mobile phase volume ratio was developed. Methanol and water were tested separately and in combination as a mobile phase. Further, sodium acetate was added to methanol and water, which increased the pH of the mobile phase. pH was decreased to 3.8 by using dilute acetic acid. After several chromatographic runs, methanol:water:0.1 M sodium acetate (30:50:20 v/v/v) showed better peak symmetry, good signal to noise (S/N) ratio and well separated chromatographic peaks. Similarly, different pump pressures were tried and the pressure was set to 12.5 MPa. Optimum flow rate was determined by running the solutions at different flow rates and was set to 1.0 ml/min. The elution runtime was limited to ten minutes after checking for the interference and influence of the excipients.

The optimum chromatographic conditions obtained during the method development were: mobile phasemethanol: water:0.1M sodium acetate (30:50:20 v/v/v); detection wavelength-243 nm; stationary phase-Chromosil C18 column (250×4.6 mm, 5µm); pH of the mobile phase-3.8 adjusted with diluted acetic acid; active pharmaceutical ingredient concentration-75 µg/ml (IPM) and 15 µg/ml (DZM); flow rate-1.0 ml/min; pump pressure-12.5±0.5 MPa and runtime-10 min.

The spectra of diluted solutions of IPM and DZM in methanol were recorded on an UV spectrophotometer by scanning the wavelength from 200 to 400 nm. The peak of maximum absorbance for both IPM and DZM showed a wavelength of 243 nm (fig. 1). For simultaneous analysis of the two drugs; first the single drugs were scanned individually and then maximum absorption wavelength was chosen from the overlay spectra. By complete separation of IPM and DZM the specificity of the developed RP-HPLC method was validated with retention time, tailing factor and resolution. The measured peaks for both the drugs were sharper and were well separated. The chromatogram of IPM and DZM from standard solution was shown in fig. 2. The retention time was 3.33±0.02 for IPM and 4.64±0.02 min for DZM for 10 min runtime, respectively. The tailing factors of IPM and DZM were 0.79 and 1.03 and these values fall in the range of recommended values. Furthermore, the theoretical plates of IPM (12184) and DZM (2862) were well above the recommended values.


Fig. 1:Absorption spectrum of IPM and DZM. Absorption spectrum of imipramine hydrochloride (IPM) and diazepam (DZM). Absorbance as a function of wavelength. The maximum absorption was observed at 243 nm.


Fig. 2:Chromatogram of IPM and DZM. Chromatogram of imipramine hydrochloride (IPM) and diazepam (DZM), whose retention times were 3.33 and 4.64 min, respectively.

The linearity measurements were carried out for six concentrations in the range 25-150 µg/ml in 25 µg/ml steps for IPM and in the range 5-30 µg/ml in 5 µg/ml steps for DZM, respectively. These measurements were fitted with a linear regression of the form y=ax+b and the values of regression parameters for the curves were r2=0.999 for IPM and r2=0.9994 for DZM. All the linear regression parameters were statistically significant.

The precision studies of the two drugs IPM (75 µg/ml) and DZM (15 µg/ml) were done by intraday and interday precision (for three successive days) measurements. The data obtained from intraday precision and interday precision was given in Table 1. The peak areas were expressed in term of mean normalized area of six samples. Table 1 gives standard deviation (SD) and relative standard deviation (RSD) along with the recommended values of these parameters. The RSD of intraday analysis of all the samples was 0.31 and 0.61, and interday analysis was 0.85 and 0.99 for IPM and DZM, respectively. This was less than 1% indicating the method was precise. Ruggedness of IPM (75 µg/ml) and DZM (15 µg/ml) was checked by two different analysts. In total six samples were analyzed and the results were given in Table 1. The RSD was less than 2% indicating the ruggedness of the method.

Parameter IPM     DZM     Recommended
  Normalized are mean±SD (n=6)a % RSD Normalized aremean±SD (n=6)a % RSD RSD
Intraday precision 1.01±0.02 1.79   0.97±0.02 1.62   <2 %
Intraday precision 1.01±0.01 1.1   1.014±0.01 1.23   <2 %
Ruggedness 0.98±0.01 1.22   0.99±0.01 1.24   <2 %

Table 1: Validation parameters

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated from the calibration curves of IPM and DZM drugs. These were determined from the sensitivity during linearity measurements. For IPM, LOD was 0.03 µg/ml and LOQ was 0.1 µg/ml. For DZM, LOD was 0.02 µg/ml and LOQ was 0.07 µg/ml.

The developed method was validated for robustness by changing the mobile phase volume ratio, flow rate and wavelength from the optimal chromatographic parameters. The percentage change of peak areas was calculated for the above three parameters. The observed change was less than 2% from the standard value (Table 2). The results indicate that the developed method was robust.

Parameter Value IPMa   DZMb
    Area (mAU) % change   Area (mAU) % change
Standard   535134 - 248816 -
Mobile phase 35:45:20 535841 0.69 249418 0.24
Methanol:water:0.1M sodium acetate 25:55:20 532425 0.13 249993 0.47
pH (adjusted with dil. acetic acid) 3.7 532173 -0.50 250394 0.63
  3.9 525043 -0.55 249562 0.29
Wavelength (nm) 241 542416 -1.8 249724 0.36
  245 538835 1.3 253108 1.72

Table 2: Robustness studies of IPM and DZM

The accuracy of the proposed method was verified using standard addition technique by adding known amount of standard to samples (spiked samples). Three different percentage determinations (50, 100 and 150%) were used for the recovery studies. For each percentage level the analysis was repeated three times (n=3) for IPM and DZM. The recovery percentage was compared with the actual amount. The results were presented in Table 3 as the mean concentrations and their standard deviation (SD) for each percentage level. The relative standard deviation (RSD) or the coefficient of variation (CV) and calculated percentage error was also given (Table 3). The good recovery of the two drugs IPM and DZM in the range of 100.59-101.52% and 99.47-100.33% satisfy the ICH guidelines [27].

Analyte Recovery (%) Target Conc. (µg/ml) Spiked Conc. (µg/ml) Final Conc. (µg/ml) Conc. obtained (µg/ml)Mean±SD RSD % Recovery Mean±SD % Errora
IPM 50 50 25 75 76.14±0.51 0.67 101.52±0.8 0.38
  100 50 50 100 100.59±0.9 0.9 100.59±0.91 0.52
  150 50 75 125 126.19±1.2 0.98 100.95±0.99 0.57
DZM 50 10 5 15 14.94±0.25 1.7 99.63±1.67 0.98
  100 10 10 20 19.89±0.20 1.0 99.47±1.03 0.58
  150 10 15 25 25.17±0.31 1.2 100.33±1.23 0.69

Table 3: Recovery studies of spiked samples of IPM and DZM

Formulation assay was performed on commercial Imipam (La Pharma) tablets. The procedure was repeated two times, separately. Twenty tablets were powdered and weighed. The powdered drug equal to 1 mg was taken and dissolved in 10 ml of methanol. From a concentration of 100 µg/ml solution, 75 and 15 µg/ml were prepared. From formulation assay studies 74.6 and 14.9 µg/ml were found, respectively. The assay results of the two drugs were expressed as percentage of label claims. The recovery percentages were 99.4% for IPM and 99.9% for DZM that were in good agreement within the 90 to 100% of the label claim. The chromatogram peaks of the IPM and DZM drugs were predominant in the drug sample with negligible interference from excipients, normally present in the tablets.

In summary, the described RP-HPLC method in this paper was simple and sensitive, fast and specific to carry out. The reported method facilitates for simultaneous determination of imipramine hydrochloride and diazepam with good resolution and sharper chromatographic peaks within a run time of 10 min. The presented results of validation parameters intraday and interday were precise and recovery results were accurate. They were established by statistical parameters and satisfy the ICH guidelines. Hence, this method can be used for routine quantitative analysis of these drugs in combination and in pharmaceutical preparations in industry and laboratories.

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Conflict of interest

There are no conflicts of interest.