ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
DEVELOPMENT AND VALIDATION OF RP-HPLC, HPTLC AND UV-VISIBLE SPECTROPHOTOMETRIC METHODS FOR SIMULTANEOUS ESTIMATION OF ALPRAZOLAM AND PROPRANOLOL HYDROCHLORIDE IN THEIR
COMBINED DOSAGE FORM © 2014 D. C. Patel, N. R. Patel, O. D. Sherikar, P. J. Mehta1
Department of Pharmaceutical Analysis, Institute of Pharmacy, Nirma University Sarkhej-Gandhinagar Highway, Ahmedabad, Gujarat, India — 382481 1E-mail: drpritimehta@nirmauni.ac.in Received 14.03.2012; in final form 03.09.2013
Three accurate, sensitive and reproducible methods are described for the quantitative determination of alprazolam (ALP) and propranolol hydrochloride (PNL) in their combined dosage form. The first method involves an RP—HPLC separation on the Ci8 column using acetonitrile — 25 mM ammonium acetate buffer and 0.2% triethylamine (pH of buffer adjusted to 4 with glacial acetic acid) in the ratio of 35 : 65 (v/v) as mobile phase. Symmetrical peaks with good separation, ALP at 9.3 min and PNL at 3.5 min, were achieved. Quantification was done with photo diode array detection at 255 nm over the concentration ranges of 0.5—50 and 10—250 p.g/mL for ALP and PNL, respectively. The second method is based on the separation of drugs by HPTLC using chlo-roform—methanol—ammonia 7 : 0.8 : 0.1 (v/v/v) as mobile phase. Quantification was achieved using UV detection at 248 nm over the concentration range of 100—600 ng/spot and 5—30 ^g/spot for ALP and PNL, respectively. The third method involves dual wavelength UV-visible spectrophotometric method. It is based on the determination of PNL at 319.4 nm using its absorptivity value and ALP at 258.2 nm after deduction of absor-bance due to PNL. Quantification was achieved over the concentration range of 1—40 and 80—200 p.g/mL for ALP and PNL, respectively. All methods were validated according to ICH guidelines and successively applied to marketed pharmaceutical formulation, and the results of all three methods were compared statistically as well. No interference from the tablet excipients was found.
Keywords: RP—HPLC, HPTLC, UV-visible spectrophotometry, alprazolam, propranolol hydrochloride. DOI: 10.7868/S0044450214070123
Alprazolam (8-chloro- 1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]-benzodiazepine) is an orally absorbed benzodiazepine. ALP is very effective in the short-term symptomatic relief of moderate to severe anxiety, essential tremor, and panic attacks [1]. Propranolol hydrochloride (-[(1-Methylethyl)ami-no]-3-(1-naphthalenyloxy)-2-propanol) is a non selective beta blocker, completely absorbed from gastrointestinal tract.
N
Cl
N
vi
Alprazolam
O
OH
Propranolol
PNL is indicated for the management of various conditions which include hypertension, angina pectoris, tachyarrhythmia, myocardial infarction, control of tachycardia/tremor associated with anxiety. Both the drugs are official in IP, EP and USP, but PNL is also official in BP. Literature survey revealed many chro-matographic and spectrophotometric methods for determination of ALP alone or in combinations with other drugs from pharmaceutical formulations and biological fluids [2—7]. Several chromatographic and spectrophotometric methods have also been reported for the determination of PNL from pharmaceutical formulations and biological fluids [8—17]. A single
RP—HPLC method is reported for the determination of ALP and PNL in a combined dosage form [18]. In the reported RP—HPLC method, more amount of organic phase was used and the optimized mobile phase is not LC—MS compatible. Hence it cannot be extended for characterization of degradation products and impurity profiling of drugs in combination. Therefore, it was endeavored to develop an accurate, precise and sensitive alternative RP—HPLC method along with UV-visible spectrophotometric and HPTLC to estimate both the drugs simultaneously in their combined dosage form.
EXPERIMENTAL
Chemicals and reagents. A standard drug sample of ALP was provided by Astron Research Centre (Ahmedabad, India) and of PNL by Torrent Research Centre (Ahmedabad, India). The pharmaceutical dosage form used in this study was LAM—PLUS tablets labeled to contain ALP 0.25 mg and PNL 20 mg (Tes Med India Pvt Ltd). Acetonitrile (HPLC grade), chloroform (AR grade), methanol (AR grade), triethy-lamine (AR grade), glacial acetic acid (AR grade) and ammonium acetate (AR Grade) were purchased from S.D. Fine Chemicals (Mumbai, India). Calibrated amber colored glasswares were used throughout the work.
Instrumentation. RP—HPLC method was developed on an HPLC system consisting of a pump (JASCO PU 2080, Japan) equipped with a PDA detector and a rheodyne injector of 20 ^L loop. Borwin PDA software was used for computational purpose; pH meter of model 111E/101E (Analabs Scientific Instruments Ltd, India) and range ofpH 0 to 14 with resolution ±0.01 pH, accuracy ±0.01 pH was used. The HPTLC instrumentation consisted of a Linomat V sample applicator with 100 ^L Hamilton syringe and TLC scanner III controlled by winCATS software (CAMAG, Muttenz, Switzerland). Merck TLC plates coated with silica gel 60F254 on aluminum sheets were used as the stationary phase. The plates were developed in a CAMAG twin trough chamber of 20 x 10 cm previously saturated for 30 min with the mobile phase. UV-visible spectrophotometric method was developed on a Shimadzu UV-visible double beam spectropho-tometer, model 2400 PC series with spectral width of 1 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cells (Shimadzu, Japan). All weighing was done on Citizen electronic balance Model CX 220 (Citizen India Ltd).
Chromatographic Conditions. RP—HPLC method. Phenomenex C18 column (150 x 4.6 mm, 5.0 ^m particle size) was used as stationary phase. Mobile phase composition was acetonitrile — 25 mM ammonium acetate buffer and 0.2% triethylamine, pH 4 adjusted with glacial acetic acid 35 : 65 (v/v). The flow rate was 1.0 mL/min. The mobile phase was filtered through a nylon 0.45 ^m, 47 mm membrane filter and degassed
before use. Detection was carried out at 255 nm with PDA detector. Mixture of acetonitrile and water in ratio of35 : 65 was used as diluent throughout the HPLC analysis.
HPTLC method. The solutions were spotted in the form of bands of 4 mm width with a Camag 100 ^L sample applicator syringe on precoated silica gel aluminium Plate 60 F254. The plates were activated at 110° C in oven for 20 min prior to sample application. A constant application rate of 0.1 ^L/sec was employed and space between two bands was 10 mm. The spotted plate was developed in twin trough chamber previously saturated for 30 min with mobile phase consisting of chloroform—methanol—ammonia 7 : 0.8 : 0.1 (v/v/v) to a distance of 8 cm. The developed plate was dried in a current of air with the help of an air dryer. The developed spots were scanned at 248 nm with slit dimension 5 mm x 0.45 mm and scanning speed of 10 mm/s.
Preparation of solution. Preparation of standard stock solution. ALP and PNL (25 mg each) were weighed accurately, transferred to individual 25 mL volumetric flasks and dissolved in methanol. The solutions were sonicated for 10 min. The flasks were shaken and volume was made up to the mark with methanol to get solutions containing 1000 |g/mL of ALP and PNL. Aliquot of 1 mL was pipetted further and diluted to 10 mL with methanol to obtain final concentration of 100 |g/mL of ALP and PNL, respectively, and labeled as standard stock solution.
Preparation of sample stock solution. A total of twenty tablets were weighed accurately and powdered. An amount of tablet powder equivalent to 100 mg of PNL (1.25 mg ofALP) was transferred to a 10 mL volumetric flask; 5 mL of methanol was added to flask and sonicated for 10 min. The solution was shaken, volume was made up to the mark with methanol and filtered through Whatmann filter paper No 41. An aliquot of1.0 mL was pipetted out from sample stock solution and diluted to 10 mL with diluent to obtain the solution containing 1000 |g/mL of PNL (12.5 |g/mL of ALP). The above solution was further diluted to obtain the solution containing 160 |g/mL of PNL (2 |g/mL of ALP) for RP-HPLC. An aliquot of 1.6 mL and 0.1 mL of sample stock solution was accurately transferred to two individual 10 mL volumetric flasks and diluted with methanol to obtain working solutions for HPTLC and UV-visible spectrophotometry.
Method validation. Preparation of calibration curves. For an RPHPLC calibration curve of ALP, solutions containing 0.5, 1, 5, 10, 20 and 50 |g/mL ALP with 20 |g/mL PNL, and for calibration curve of PNL, solutions containing 10, 50, 100, 150, 200 and 250 |g/mL PNL with 20 |g/mL ALP were prepared in diluent from standard stock solution. An aliquot (20 ^L) of each solution was injected under the operating chromatographic conditions.
For an HPTLC calibration curve of ALP, solutions containing 10, 20, 30, 40, 50 and 60 |g/mL ALP with
1600 |g/mL PNL and for calibration curve of PNL, solutions containing 500, 1000, 1500, 2000, 2500, 3000 |g/mL PNL with 20 |g/mL ALP were prepared in methanol. The plates were developed and scanned as described above.
Calibration curves for both methods were constructed by plotting peak areas versus concentrations of ALP and PNL, and the regression equations were calculated. Each response was the average of six determinations.
For UV-visible spectrophotometry the individual solutions containing 1, 5, 10, 20, 30 and 40 |g/mL of ALP as well as 80, 100, 120, 140, 160, 180 and 200 |g/mL of PNL were prepared in methanol and analyzed at 258.20 nm and 319.40 nm for ALP and PNL, respectively. Calibration curve was constructed by plotting absorbance versus concentration.
Accuracy (recovery). The accuracy of the method was determined by calculating recoveries of ALP and PNL by the standard addition method at three different levels (80, 100 and 120%) to preanalysed tablet sample. For RP—HPLC, known amounts of standard solutions of ALP (1.0, 1.25 and 1.5
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