ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
THREE-DIMENSIONAL VIEWED VALIDATED DIODE ARRAY UV-LC METHOD FOR ESTIMATING DOPAMINE IN LIPOSOMES
© 2015 V. Nagpal*, E. Joseph*, J. Abraham**, R. N. Saha*
*Industrial Research Laboratory, Department of Pharmacy, Faculty Division-III, Birla Institute
of Technology and Science (BITS) Pilani-333 031, Rajasthan, India 1E-mail: vibhunagpal2003@gmail.com **FnD (Discovery/NDDS), Torrent Pharmaceuticals Ltd. (TRL), Research Center Gandhinagar-382428, Gujarat, India Received 20.05.2013; in final form 05.03.2014
A new, sensitive, selective, rapid and validated high throughput liquid chromatographic method has been developed to estimate dopamine (DA) in bulk and formulations. Efficient chromatographic separation was achieved using a C18 reverse phase column with simple mobile phase combination, consisting of phosphate buffer (10 mM KH2PO4, pH 4.0) and methanol (96 : 4), delivered at 1 mL/min flow rate in an isocratic mode and quantitation was carried out using Photo Diode Array (PDA) detector. This method utilizes green chemistry approach in mobile phase optimization with less than 5% organic phase and only 10 min to run each sample. Thus our method is environmentally friendly. The method has demonstrated excellent linearity over the range of 25— 2000 ng/mL (regression equation: average peak area (mV s) = 72.091c (ng/mL) + 141.77, r2 = 0.9997). Moreover, the method was found to be sensitive with a low limits of detection (7 ng/mL) and quantitation (21 ng/mL). The method has shown good and consistent recoveries of 99.13—100.95% with low intra- and inter-day relative standard deviation (RSD) < 2.0%. Further, this method has demonstrated good stability of DA with antioxidants for longer period. Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The method was validated as per the International Conference on Harmonization (ICH) and United States Pharmacopoeia (USP) guidelines and successfully applied to the determination of dopamine in commercial and in-house prepared liposomal formulations.
Keywords: dopamine, experimental design, liposomes, stability, validation.
DOI: 10.7868/S0044450215030135
Dopamine, a typical neurotransmitter, is known to activate five types of dopamine receptors — Dx, D2, D3, D4, and D5 and their variants [1]. All are G protein coupled receptors and are grouped into two families: Drlike receptors (Dx and D5) are excitatory which act by increasing cAMP (3',5'-cyclic adenosine monophosphate) formation, whereas D2-like receptors (D2, D3 and D4) are inhibitory which act by inhibiting ade-nylyl cyclase [2]. It is well known that dopamine (3,4-dihydroxyphenylethylamine), due to presence of cate-chol group, is sensitive to air and light and is readily oxidisable to a quinoid form [3].
HO
HO
nh2
Chemical structure of dopamine.
DA plays an important role in brain functions. A low DA level causes Parkinson's disease, which is degeneration of neurons in substantia nigra pars com-
pacta and the nigrostriatal (dopaminergic) tract. This results due to deficiency of dopamine in the striatum which controls muscle tone and coordination of movements [2]. The dopamine is also used extensively in the treatment of shock, which may be caused by trauma, heart attack, open heart surgery, heart failure, kidney failure and severe bacterial infections of the blood [4, 5]. Despite its great clinical outcome, very few physicochemical investigations are reported. Thus not much work has been reported on sensitive analytical methods for estimation of DA. Due to more emphasis on DA formulations in recent times and its extensive use in several research investigations, there is a need for a simple, reliable and cost effective analytical method for routine analysis. Methods which are reported in literature have been mainly developed for the determination of dopamine in biological samples but not for bulk, formulations or related samples. Methods reported are spectrophotometry [6, 7], spectroflu-orimetry [5, 8—11], capillary electrophoresis [12], liquid chromatography [13, 14], chemiluminescence
[15] and electrochemical methods [4, 16, 17]. The spectrophotometry and spectrofluorimetry methods lack specificity and sensitivity as compared to liquid chromatography. Capillary electrophoresis methods need only a small amount of samples and their sensitivity is very low. The important drawback with electrochemical detector is interference from other elec-troactive species which have similar potentials. The reported liquid chromatography methods have some drawbacks, including the requirement for sample pre-treatment, poor chromatographic techniques, questionable uncharacterized peaks, long run time, complex mobile phase, long analysis time and high cost. These drawbacks have prevented these methods from being applied in routine analysis.
Extensive literature survey did not reveal any simple, sensitive and validated analytical liquid chromatography method for the determination of DA in bulk and pharmaceutical preparations and their evaluation. For dopamine determination, USP recommends the use of a HPLC with spectrophotometric detector which includes detection of DA at 280 nm. Moreover, use of multi component mobile phase with higher organic solvent content and high flow rate increased the cost of the method with less sensitivity [18].
The aim of this work was to develop an accurate, specific, precise and validated HPLC method for the determination of dopamine in bulk, commercial and in-house prepared formulations. Furthermore, due to the chemical instability of this compound, the addition of an antioxidant is required for automated analysis over a long period of time. The developed method was validated as per ICH as well as USP guidelines [19, 20]. Suitable statistical tests were performed to validate the developed method [21, 22].
EXPERIMENTAL
Reagents. Dopamine hydrochloride (assay 99.95%) was obtained from Sigma-Aldrich, India. HPLC grade methanol was purchased from Spectrochem, India. Fresh triple distilled water was prepared in quartz distillation assembly and filtered through a 0.22 ^m membrane using Millipore ultra-filtration system (Millipore, France) before use. Potassium dihydrogen orthophosphate, orthophosphoric acid, citrate, ammonium acetate, perchloric acid, sodium metabisulphite, acetic acid, oxalic acid, L-cysteine, ascorbic acid and chloroform were purchased from Merck, Mumbai, India. All other chemicals and reagents were of analytical grade. For preparation of liposomes soya phosphatidyl choline (PC) and cholesterol were purchased from Avanti Polar Lipids, Inc. Alabaster, Alabama and Sisco Research Lab, Mumbai, India, respectively. Ammonium sulphate and sucrose were purchased from Khimmed (Russia) and Sephadex G-50 from Sigma-Aldrich. A commercially available i.v. infusion — Dopamine Hydrochloride USP (Sterile Specialities India Pvt. Ltd. India) labeled 200 mg/5 mL dopamine HCl was procured from the lo-
cal Indian market. Liposomes loaded with DA and without it (blank) were prepared in-house using soya PC and cholesterol by thin film evaporation technique.
Instrumentation. An UFLC system (Shimadzu, Japan) consisting of two pumps (LC-20D), integrated system controller with Prominence auto-sampler (SIL-20AC) and Prominence diode array detector (SPD-20A) was used. Data acquisition and analysis were performed using 21 CFR part 11 compliant workstation LCsolutions1 (Shimadzu, Japan).
Analytical method development. In order to develop a selective and sensitive method, primary concern during development was to achieve high height to area ratio and peak symmetry. Different buffers of different pH, such as phosphate buffer (pH 3—7, 10 mM), citrate buffer (pH 3—5, 10 mM), ammonium acetate buffer (pH 3-5, 10 mM) and acetate buffer (pH 3-5, 10 mM) were studied in combinations with methanol (3, 4, 5 and 10%, v/v). The effects of various organic modifiers such as acetonitrile, methanol and their combinations, on peak properties (peak height and symmetry) and response function were investigated. Various antioxidants such as perchloric acid, acetic acid, oxalic acid, L-cysteine, ascorbic acid and sodium metabisulphite, were used to enhance drug stability. In addition, chromatographic parameters such as retention factor (k), number of theoretical plates (N), height equivalent to theoretical plate (HETP), tailing factor (Tf) etc. were also considered. Further, a robustness study was conducted during the final phase of method development by using design of experimentation (DOE) technique (Stat-Ease DOE).
Chromatographic conditions. Optimized mobile phase consisted of an aqueous 10 mM potassium dihy-drogen phosphate buffer and methanol (pH 4.0, 96 : 4, v/v), where the aqueous phase pH was adjusted using 0.1 M orthophosphoric acid. Mobile phase was delivered in isocratic elution mode at a flow rate of 1 mL/min. The chromatographic separation was achieved on BDS Hypersil C18 (250 mm x 4.6 mm, 5 ^m, 8 nm) double end-capped RP-HPLC column (Thermo Scientific, Mumbai, India). The quantitation was carried out at 227 nm with an injection volume of 50 ^L. Analysis was performed at ambient temperature (25°C) after baseline stabilization for at least 60 min.
Preparation of stock and standard solutions. A primary stock solution of DA was prepared in 0.1 M perchloric acid containing 0.1% (w/v) sodium metabisulphite having concentration of 100 ^g/mL. Working standards (1, 2, 4, 10, 20, 40, 60 and 80 ^g/mL) were prepared from primary stock solution using phosphate buffer of pH 4.0. Three separate series of eight calibration standards containing 25, 50, 100, 250, 500, 1000, 1500 and 2000 ng/mL of DA were prepared freshly by spiking 25 ^L of respective working standard in diluent on three different days of validation. Formulation standards were prepared by adding known amounts of drug to water for injection and to blank liposomes
preparatio
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