Novel spectrophotometric method for determination of cinacalcet hydrochloride in its tablets via derivatization with 1,2-naphthoquinone-4-sulphonate
© Darwish et al 2012
Received: 12 November 2011
Accepted: 3 February 2012
Published: 3 February 2012
This study represents the first report on the development of a novel spectrophotometric method for determination of cinacalcet hydrochloride (CIN) in its tablet dosage forms. Studies were carried out to investigate the reaction between CIN and 1,2-naphthoquinone-4-sulphonate (NQS) reagent. In alkaline medium (pH 8.5), an orange red-colored product exhibiting maximum absorption peak (λmax) at 490 nm was produced. The stoichiometry and kinetic of the reaction were investigated and the reaction mechanism was postulated. This color-developing reaction was employed in the development of a simple and rapid visible-spectrophotometric method for determination of CIN in its tablets. Under the optimized reaction conditions, Beer's law correlating the absorbance with CIN concentration was obeyed in the range of 3 - 100 μg/ml with good correlation coefficient (0.9993). The molar absorptivity (ε) was 4.2 × 105 l/mol/cm. The limits of detection and quantification were 1.9 and 5.7 μg/ml, respectively. The precision of the method was satisfactory; the values of relative standard deviations (RSD) did not exceed 2%. No interference was observed from the excipients that are present in the tablets. The proposed method was applied successfully for the determination of CIN in its pharmaceutical tablets with good accuracy and precisions; the label claim percentage was 100.80 - 102.23 ± 1.27 - 1.62%. The results were compared favorably with those of a reference pre-validated method. The method is practical and valuable in terms of its routine application in quality control laboratories.
KeywordsCinacalcet HCl 1,2-Naphthoquinone-4-sulphonate Kinetic Spectrophotometry Pharmaceutical analysis
Cinacalcet hydrochloride (CIN); N-[1-(R)-(-)-(1-naphthyl) ethyl]-3-[3-(trifluoromethyl) phenyl]-1-aminopropane HCl is a selective calcimimetic agent, which acts on a calcium-sensing receptor of the parathyroid gland. This principal negative regulator of parathyroid hormone release increases its selectivity to activation by extracellular calcium, thus decreasing the parathyroid hormone levels [1, 2]. CIN is effective in clinical setting and it has been approved for the treatment of secondary hyperthyroidism in patients with chronic kidney disease placed on dialysis , and for the treatment of elevated calcium levels in patients with parathyroid carcinoma .
The effective and safe therapy with CIN is basically depending on the quality of its pharmaceutical preparations (tablets), and assessing its concentrations in tablets for the purposes of quality control. Literature review revealed that there were only two methods were employed for thin-layer  and liquid chromatographic [5, 6] enantiomeric separation of CIN enantiomers in laboratory-made racemic mixtures. However, no reports have been found for describing the development of analytical methods for the quantitative determination of CIN in its tablets. Therefore, this study was devoted to the development of a new method for determination of CIN in its tablets for the purpose of its pharmaceutical quality control.
Spectrophotometry is considered the most convenient technique because of its inherent simplicity, low cost and wide availability . As well, 1,2-Naphthoquinone-4-sulphonic sulphonate (NQS) has been used as a color-developing reagent in the spectrophotometric determination of many pharmaceutical amines [8–11]. The reaction between NQS and CIN has not been investigated yet. Therefore, the present study was devoted to investigate this and its employment in the development of a new simple and rapid spectrophotometric method for determination of CIN in its tablets.
Double beam V-530 (JASCO Ltd., Kyoto, Japan) ultraviolet-visible spectrophotometer with matched 1-cm quartz cells was used for all the spectrophotometric measurements. pH meter, model 350 (Bibby Scientific Ltd., T/As Jenway, Essex, England). MLW type thermostatically controlled water bath (Memmert GmbH Co., Schwabach, Germany).
Reagents and Materials
Cinacalcet hydrochloride (Amgen Inc., Thousand Oaks, CA, USA) was obtained and used as received; its purity was > 99%. A solution of 0.5% (w/v) of NQS (Aldrich Chemical Co., St. Louis, USA) was freshly prepared and protected from light during use. Clark and Lubs buffer solution was prepared by mixing 50 ml of 0.2 M aqueous solution of boric acid and potassium chloride (1 liter containing 12.368 g of boric acid and 14.90 g of potassium chloride) with 21.3 ml of 0.2 M sodium hydroxide in 200 ml standard flask , and adjusted by pH meter. Tris buffer was prepared by mixing 100 ml 0.1 M tris(hydroxymethyl)aminomethane with 29.4 ml of 0.1 M HCl . Britton-Robinson buffer composed of 0.04 M boric acid, 0.04 M phosphoric acid and 0.04 M acetic acid adjusted at pH 8.5 by using 0.2 M sodium hydroxide . Phosphate buffer composed of 0.1 M disodium hydrogen phosphate (14.2 g/l) and 0.1 M sodium hydroxide . Sensipar® and Mimpara® tablets (Amgen Inc, Thousand Oaks, CA, USA) were labeled to contain 60 mg CIN per tablet. Double distilled water was obtained through WSC-85 water purification system (Hamilton Laboratory Glass Ltd., Kent, USA) and used throughout the work. All solvents and materials used throughout this study were of analytical grade.
Preparation of Solutions
Standard CIN Solution
An accurately weighed amount (50 mg) of CIN was quantitatively transferred into a 25-ml calibrated flask, dissolved in 20 ml distilled water, completed to volume with the same solvent to obtain a stock solution of 2 mg/ml. The stock solution was found to be stable for at least two weeks when kept in a refrigerator. The stock solution was further diluted with water to obtain working solutions in the range of 3 - 100 μg/ml.
Tablet Sample Solution
Twenty tablets were weighed and finely powdered. An accurately weighed quantity of the powder equivalent to 100 mg of the active ingredient was transferred into a 100-ml calibrated flask, and dissolved in about 40 ml of distilled water. The contents of the flask were swirled, sonicated for 5 minutes, and then completed to volume with water. The contents were mixed well and filtered; the first portion of the filtrate was rejected. The filtered solution was diluted quantitatively with distilled water to obtain suitable concentrations for the analysis by the proposed spectrophotometric method.
General Recommended Procedures
One milliliter of CIN solution containing 3 - 100 μg/ml was transferred into separate 10-ml calibrated flask. A 0.5 ml of tris buffer solution of pH 8.5 and 1 ml of NQS solution (0.5%, w/v) were added. The reaction solution was allowed to proceed for 10 min at room temperature (25 ± 2°C) and completed to volume with water. The resulting solution was measured at 490 nm against reagent blank treated similarly.
Determination of Stoichiometric Ratio
The Job's method of continuous variation  was employed. Master equimolar (1 × 10-4 M) aqueous solutions of CIN and NQS were prepared. Series of 10-ml portions of the master solutions of CIN and NQS were made up comprising different complementary proportions (0:10, 1:9,..., 9:1, 10:0, inclusive) in 10-ml calibrated flasks containing 0.5 ml of tris buffer solution (pH 8.5). The solutions were further manipulated as described under the general recommended procedures.
Limiting Logarithmic Method
The limiting logarithmic method  was employed. Two sets of experiments were carried out employing the general recommended procedures described above. The first set of experiments was carried using varying concentrations of the analytical reagent (1.9 × 10-3 - 9.6 × 10-3 M) at a fixed CIN concentration (1 × 10-4 M). The second set of experiments was carried using varying concentrations of CIN (7.6 × 10-6 - 2.5 × 10-4 M) at a fixed concentration of NQS (1.9 × 10-2 M). The logarithms of the obtained absorbances for the reaction of CIN with NQS were plotted as a function of the logarithms of the concentrations of the reagent and CIN in the first and second sets of experiments. The slopes of the fitting lines in both sets of experiments were calculated.
Results and Discussion
Optimization of Reaction Conditions
Effect of NQS Concentration
Effect of pH and Buffer Components
The influence of pH on the reaction of CIN with NQS was investigated by carrying out the reaction in buffer solution of varying pH values. The results revealed that CIN has difficulty to react with NQS in acidic media (Figure 2). This was possibly due to the existence of the amino group of CIN in the form of hydrochloride salt, thus it loses its nucleophilic substitution affinity. As the pH increased, the readings increased rapidly, as the amino group of CIN (in the hydrochloride salt) turns into the free amino group, thus facilitating the nucleophilic substitution. The maximum readings were attained at pH values of 8.5. At higher pH, sharp decrease in the readings occurred. This was attributed probably to the increase in the amount of hydroxide ion that holds back the reaction of CIN with NQS, and the instability of NQS reagent .
In order to investigate the effect of buffer components on the reaction, different buffer solutions of pH 8.5 were tested: Clark, Robinson, phosphate, borate and tris buffers. The highest absorbances were obtained when tris buffer was used, thus it was used in all the subsequent experiments.
Effect of Temperature and Time
In order to determine the optimum time that is required for completion the reaction, it was allowed to proceed at room temperature for varying periods of time. It was found that the reaction goes to almost completion within 5 min (Figure 3), however for higher precision readings, the reaction was allowed to proceed for quite longer time; reactions in all the subsequent experiments were carried out for 10 min.
Effect of Diluting Solvent
Stability of the Chromogen
The effect of time on the stability of the CIN-NQS chromogen was studied by following the absorption intensity of the reaction solution (after dilution) at different time intervals. It was found that the absorbance of the chromogen remains stable for at least 1 h. This allowed the processing of large batches of samples, and their comfortable measurements with convenience. This gives the high throughput property to the proposed method when applied for analysis of large number of samples in quality control laboratories.
Stoichiometry, Kinetics and Mechanism of the Reactions
Summary for the optimization of variables affecting the reaction of CIN with NQS reagent employed in the development of the proposed spectrophotometric method
NQS concentration (%, w/v)
0.1 - 0.9
6 - 10
25 - 60
5 - 35
Measuring wavelength (nm)
400 - 600
where K is reaction rate, K' is the rate constant, C is the molar concentration of CIN and n (slope of regression line) is the order of the reaction. The values of the slope (≈1) confirmed that the reaction was first order. However under the optimized reaction conditions, the concentrations of NQS were in much more excess than that of CIN in the reaction solution. Therefore, the reaction was regarded as pseudo-first order reaction.
Validation of the Proposed Method
Calibration and Sensitivity
Parameters for the performance of the proposed spectrophotometric method for determination of CIN
Measurement wavelength (nm)
Linear range (μg/ml)
3 - 100
Standard deviation of the intercept
Standard deviation of the slope
Correlation coefficient (r)
Limit of detection, LOD (μg/ml)
Limit of quantification, LOQ (μg/ml)
Molar absorptivity, ε (l/mol/cm)
4.2 × 105
Precision of the proposed assay at different CIN concentrations
Relative standard deviation
Intra-assay, n = 5
Inter-assays, n = 6
Accuracy and Interference Liabilities
Recovery studies for determination of CIN by the proposed method
Recovery (% ± SD)a
98.60 ± 0.12
99.25 ± 0.21
99.88 ± 0.12
99.91 ± 0.01
99.96 ± 0.04
Analysis of CIN in presence of the excipients that are present in its tablets by the proposed method
Recovery (% ± SD)a
Pregelatinized starch (50)b
102.09 ± 0.26
Microcrystalline cellulose (50)
102.0 ± 0.57
99.28 ± 0.27
Cross povidone (10)
99.25 ± 0.25
Colloidal silicon dioxide (10)
98.34 ± 0.26
Magnesium stearate (10)
101.16 ± 0.95
Average ± SD
100.57 ± 1.51
Robustness and Ruggedness
Influence of small variations in the assay conditions on the analytical performance of the proposed spectrophotometric method for determination of CIN using NQS reagent
Recovery (% ± SD)a
100.57 ± 1.51
NQS concentration (%, w/v)
100.06 ± 0.53
100.20 ± 0.34
Buffer solution (pH)
98.95 ± 0.67
100.55 ± 0.68
99.24 ± 1.04
101.15 ± 0.48
Reaction time (min)
99.87 ± 0.26
101.41 ± 0.66
Ruggedness was also tested by applying the proposed methods to the assay of CIN using the same operational conditions but using two different instruments at two different laboratories and different elapsed time. Results obtained from lab-to-lab and day-to-day variations were reproducible, as the relative standard deviations (RSD) did not exceed 2%.
Application of the Proposed Method to Analysis of CIN in Tablets
Determination of CIN in tablets by the proposed and reference methods
Recovery (% ± SD)b
102.23 ± 1.27
101.43 ± 1.35
100.80 ± 1.60
100.10 ± 1.30
The present study described, for the first time, the successful evaluation of NQS as an analytical reagent in the development of simple and rapid spectrophotometric method for the accurate determination of CIN in its dosage forms. The method described herein has many advantages: it does not need expensive sophisticated apparatus, it is simple and rapid, and it has high sensitivity. The proposed method used inexpensive reagents with excellent shelf life, and is available in any analytical laboratory. Therefore, the method is practical and valuable for its routine application in quality control laboratories for analysis of CIN.
1,2-naphthoquinone-4-sulphonate; wavelength of maximum absorption
molar absorptivity: ε
The international Conference on Harmonization
limit of detection
limit of quantification
relative standard deviation
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group No. RGP-VPP-065.
- Franceschini N, Joy MS, Kshirsagar A: Cinacalcet HCl: a calcimimetic agent for the management of primary and secondary hyperparathrodism. Exp Opin Invest Drugs. 2003, 2: 1413-1421.View ArticleGoogle Scholar
- Torres PU: Cinacalcet HCl: a novel treatment for secondary hyperparathyroidism caused by chronic kidney disease. J Ren Nut. 2006, 16: 253-258. 10.1053/j.jrn.2006.04.010.View ArticleGoogle Scholar
- Amgen: Sensipar for Parathyroid Carcinoma. Amgen. 2009, [http://www.sensipar.com/Sensipar_for_HPC.html]Google Scholar
- Block GA, Martin KJ, de Francisco AL: Cinacalcet for secondary hyperparathyroidism in patients receiving hemodialysis. N Engl J Med. 2004, 350: 1516-1525. 10.1056/NEJMoa031633.View ArticleGoogle Scholar
- Bhushan R, Dubey R: Indirect reversed-phase high-performance liquid chromatographic and direct thin-layer chromatographic enantioresolution of (R, S)-cinacalcet. Biomed Chromatogr. 2011, 25: 674-679. 10.1002/bmc.1502.View ArticleGoogle Scholar
- Ravinder V, Ashok S, Varma MS, Babu CVR, Shanker K, Balaswam G: A validated chiral LC method for the enantiomeric separation of cinacalcet hydrochloride. Chromatographia. 2009, 70: 229-232. 10.1365/s10337-009-1129-5.View ArticleGoogle Scholar
- Görög S: Ultraviolet-Visible Spectrophotometry in Pharmaceutical Analysis. 1994, CRC Press, New YorkGoogle Scholar
- Kumar CHA, Kumar TA, Gurupadayya BM, Sloka SN, Reddy BMR: Novel spectrophotometric determination of valacyclovir and cefotaxime using 1, 2-napthaquinone-4-sulfonic acid sodium in bulk and pharmaceutical dosage form. Arch Appl Sci Res. 2010, 2: 278-287.Google Scholar
- Mahmoud AM, Khalil NY, Darwish IA, Aboul-Fadl Y: Selective spectrophotometric and spectrofluorometric methods for the determination of amantadine hydrochloride in capsules and plasma via derivatization with 1,2-naphthoquinone-4-sulphonate. Int J Anal Chem. 2009, 2009: 8-Article ID 810104Google Scholar
- Padmarajaiah N, Kumar HRA, Vasantha RA, Yathirajan HS: Novel reagents for the sensitive spectrophotometric determination of flutamide, an anticancer drug in pharmaceutical preparations. Int J Pharm. 2002, 235: 113-120. 10.1016/S0378-5173(01)00975-9.View ArticleGoogle Scholar
- Al-Momani IF: Spectrophotometric determination of selected cephalosporins in drug formulations using flow injection analysis. J Pharm Biomed Anal. 2001, 25: 751-757. 10.1016/S0731-7085(01)00368-5.View ArticleGoogle Scholar
- Pesez M, Bartos J: Colorimetric and fluorimetric analysis of organic compounds and drugs. 1974, Marcel Dekker Inc., New York, 628-630.Google Scholar
- Robinson RA, Stokes RH: Electrolyte solutions, the measurement and interpretation of conductance, chemical potential, and diffusion in solutions of simple electrolytes. 1968, London, Butterworths, 2Google Scholar
- Job P: Advanced Physicochemical Experiments. 1964, Oliner and Boyd, Edinburgh, 54-2Google Scholar
- Rose J: Advanced Physicochemical Experiments. 1964, Pitman, LondonGoogle Scholar
- Foster R: Organic charge-transfer complexes. 1969, London, New York, Academic Press, 470-Google Scholar
- Starczewska B, Jasińska A, Białous B: Study and analytical application of ion-pair formation in the system fluoxetine-pyrocatechol violet and fluvoxamine-pyrocatechol violet. Pharmazie. 2003, 58: 245-248.Google Scholar
- Starczewska B, Mielech K: Application of chrome azurol S for the extractive spectrophotometric determination of fluoxetine and fluvoxamine. J Pharm Biomed Anal. 2000, 23: 243-247. 10.1016/S0731-7085(00)00296-X.View ArticleGoogle Scholar
- Starczewska B, Puzanowska-Tarasiewicz H, Baranowska K: Investigation and analytical application of the reactions of eriochrome cyanine R with fluvoxamine and fluoxetine. J Pharm Biomed Anal. 2000, 23: 477-481. 10.1016/S0731-7085(00)00323-X.View ArticleGoogle Scholar
- Onal A, Kepekçi SE, Oztunç AA: Spectrophotometric methods for the determination of the antidepressant drug paroxetine hydrochloride in tablets. J AOAC Int. 2005, 88: 490-495.Google Scholar
- Darwish IA: Kinetic spectrophotometric methods for determination of trimetazidine dihydrochloride. Anal Chim Acta. 2005, 551: 222-231. 10.1016/j.aca.2005.07.027.View ArticleGoogle Scholar
- Darwish IA, Abdine HH, Amer SM, Al-Rayes LI: Simple spectrophotometric method for the determination of paroxetine in tablets using 1,2-naphthoquinone-4-sulphonate as a chromogenic reagent. Int J Anal Chem. 2009, 2009: 8-Article ID 237601Google Scholar
- Darwish IA, Abdine HH, Amer SM, Al-Rayes LI: New spectrophotometric and fluorimetric methods for determination of fluoxetine in pharmaceutical formulations. Int J Anal Chem. 2009, 2009: 9-Article ID 257309Google Scholar
- Darwish IA, Abdine HH, Amer SM, Al-Rayes LI: Spectrophotometric Study for the Reaction of Fluvoxamine 1,2-naphthoquinone-4-sulphonate: Kinetic, Mechanism, and Use for Determination of Fluvoxamine in its Dosage Forms. Spectrochim Acta A. 2009, 72: 897-902. 10.1016/j.saa.2008.12.009.View ArticleGoogle Scholar
- Saurina J, Hernandez-Cassou S: Continuous-flow spectrophotometric determination of amino acids with 1,2-naphthoquinone-4-sulphonate reagent. Anal Chim Acta. 1993, 283: 414-420. 10.1016/0003-2670(93)85252-F.View ArticleGoogle Scholar
- Fidler AT, Baker EL, Letz RE: Neurobehavioural effects of occupational exposure to organic solvents among construction painters. Br J Indust Med. 1987, 44: 292-308.Google Scholar
- Kristensen P, Hilt B, Svendsen K, Grimsrud TK: Incidence of lymphohaematopoietic cancer at university laboratory: a cluster investigation. Eur J Epidemiol. 2008, 23: 11-15. 10.1007/s10654-007-9203-5.View ArticleGoogle Scholar
- Lindbohm ML, Taskinen HT, Sallman M, Hemminki K: Spontaneous abortions among women exposed to organic solvents. Am J Indust Med. 2007, 17: 449-463.View ArticleGoogle Scholar
- Wennborg H, Bonde JP, Stenbeck M, Olsen J: Adverse reproduction outcomes among employee in biomed-ical research laboratories. Scand J Work Environ Health. 2002, 28: 5-11. 10.5271/sjweh.640.View ArticleGoogle Scholar
- Wennborg H, Lennart B, Harri V, Gösta A: Pregnancy outcome of personnel in swedish biomedical re-search laboratories. J Occup Environ Med. 2000, 42: 438-446. 10.1097/00043764-200004000-00022.View ArticleGoogle Scholar
- International Conference on Harmonisation Q2(R1): Validation of analytical procedures: text and methodology. London. 2005, [http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf]Google Scholar
- Amgen: Sensipar® (cinacalcet) Tablets. 2010, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, V6: 91320-1799.Google Scholar