Batch and Flow Injection Spectrophotometric Determination of Doxycycline Hyclate in Pharmaceutical Preparations

New, simple and sensitive batch and flow injection spectrophotometric methods for the determination of doxycycline hyclate (DCH) in pharmaceutical preparations were developed. These methods were based on diazotization-coupling reaction between diazotized o-nitroanline (DONA) and DCH in sodium carbonate medium to form yellow-orange colored azo dye product has a maximum absorbance at 448 nm. Calibration graphs of absorbance versus concentration show that Beerʼs law were obeyed over the concentration ranges 0.4-52, 5-200 μg mL of DCH, with limit of detection of 0.284, 1.117 μg mL of DCH for batch and flow injection analysis (FIA) procedure respectively, and sample throughput was 171 h for FIA method. All chemical and physical conditions affected on the developed colored product were carefully studied and the proposed methods were successfully applied for the determination of doxycycline in its pharmaceutical preparations. Keyword: Diazotized o-nitroanline, Flow injection analysis, Doxycycline hyclate. 1Introduction Doxycycline hyclate (DCH) chemically named as (4S,4aR,5S,5aR,6R,12aS)-4dimethylamino)-3,5,10,12,12a-pentahydroxy6-methyl-1,11-dioxo1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide hydrochloride hemiethanol hemihydrates and molecular weight 512.9 g.mol Fig.(1). DCH more soluble than doxycycline monohydrate, this is one of the main reasons for it’s more frequent use in pharmaceutical samples. DCH is one of the tetracycline derivatives has broad spectrum antibacterial agents effective against a host of Gram positive and Gram negative aerobic and anaerobic bacteria which obtained from oxytetracycline or metacycline. Doxycycline is preferred to other tetracyclines in the treatment of specific infections because of its fairly reliable absorption and its long half-time, it is used to treat chronic prostatic, Syphilis and pelvic inflammatory disease [1-2]. Several methods have been developed for determination DCH in pharmaceutical preparations such as spectrophotometric [3-6], high performance liquid chromatography HPLC [7,8], RP-HPLC [9], potentiometry[10], Chemliumincence [11] and thin layer Chromatography [12], however, some of these methods are time consuming and/or require expensive equipment. The present paper describe development of batch and FIA methods which based and diazotization-coupling reaction between DCH and diazotized o-nitroaniline (DONA) reagent in sodium carbonate medium, yellow-orange azo dye was produced and spectrophotometrically measured at 448 nm. Both batch and FIA procedures have applied for the determination DCH in its pharmaceutical preparations. Fig.(1) structure of doxycycline hyclate. 2Materials and methods 2-1: Apparatus All spectral and absorbance measurements were carried out by using a shimadzu UV– visible–260 digital double beam recording spectrophotometer (Tokyo–Japan), and using 1 cm quarts cells. A quartz flow cell with 50 μL internal volume and 1 cm bath length used for the absorbance measurements. A two channel manifold Fig.(2) was employed for the FIA spectrophotometer determinations of DCH. A peristaltic pump (Ismatec Lobortechnik – Mouayed Q. Al-Abachi 25 Analytic, CH–8512, Glatbragg–Zurich, Switzerland, Sixchannels) was used to transport the reagents solutions. Injection valve (Rheodyne, Altex 210, supeko use) was employed to provide appropriate injection volumes of standard solutions and samples, flexible vinyl tubing of 0.5 mm internal diameter was used for the peristaltic pump. Reaction coil (RC) was of Teflon with internal diameter of 0.5 mm. The diazotized onitroaniline (DONA) (A) stream was combined Fig.(2) with injected sample (DCH) and they merged with sodium carbonate (B) stream at T – link then mixed in reaction coil (RC) with length (50 cm), injection loop (200 μL), total flow rate 2.5 mLmin, the absorbance was measured at 448 nm at temperature 25 C. Fig.(2) A schematic diagram of FIA manifold where: (A) & (B), solutions of diazotized onitroaniline and sodium carbonate respectively; P = peristaltic pump; S= injection sample of DCH; IV = injection valve; RC=reaction coil; FC = flow cell; D=detector; W= waste. 2-2: Reagents and materials Analytical reagents grade chemicals and distilled water were used thoroughly. -Doxycycline hyclate stock solution (500 μg mL), (9.748 ×10 M): 0.05 g amount of pure DCH (SDIIraq) was dissolved in distilled water, and then completed to 100 mL in a volumetric flask with the same solvent. More dilute solutions were prepared by suitable dilution of the stock solution with distilled water. -Hydrochloric acid (BDH-England)(1M): Was prepared by diluting 21.5 mL of 11.64 M of concentrated hydrochloric acid with distilled water in 250 mL volumetric flask, and standardized against Na2CO3. -Diazotized o-nitroanaline (DONA) (10×10 M): Was prepared daily by dissolving 0.138 g of o-nitroaniline (BDH-England) in 5 mL ethanol and add 20 mL of distilled water, then 6mL of HCl (1M) was added. The mixture was transferred to 100 mL volumetric flask and placed in an ice-bath for 5 min, then 0.069 g of sodium nitrite was added to the mixture and shacked well, After 5 min, the volume was made up to the mark with distilled water. More dilute solutions were prepared by appropriate dilutions from stock solution with distilled water. -Sodium carbonate (BDH-England) (0.3M): 7.948 g of sodium carbonate Na2CO3 was dissolved in a 250 mL volumetric flask with distilled water. 0.1 M of sodium carbonate was prepared by dilution with distilled water. 2-3:Pharmaceutical preparations of doxycycline Pharmaceutical preparations were obtained from commercial sources. 1Doxycycline hyclate 8-capsules (ActavisU.K.), each capsule contain 100 mg of doxycycline. 2Medomycin 10-capsules (Medochemie Ltd.-cyprus), each capsule contain 100 mg of doxycycline. HCl. 3Tabocine 10-capsules (TabookK.S.A.), each capsule contain 100 mg of doxycycline hyclate. 2-4: General procedure 2-4-1: General batch procedure Into a series of 25 mL volumetric flasks, an increasing volumes of DCH working solution (100 μg mL) were transferred to cover the range 0.4-52 μg mL of calibration graph, 1mL of DONA (5×10M) reagent was added, and followed by adding 1 mL of Na2CO3 (0.1 M) for all flasks. The solutions were completed to the mark with distilled water, mixed well and left for 10 min at room temperature (25 C). The absorbance was measured at 448 nm versus the reagent blank was prepared in same way but without drug. A calibration graph was drawn and the statistical calculation was done for the analytical features obtained as shown in Table (1). For the optimization of conditions and in all subsequent experiments were carried out on 20 μg mL of DCH. Journal of Al-Nahrain University Vol.18 (3), September, 2015, pp.24-32 Science 26 2-4-2: General FIA procedure Working solution of DCH in the range 5200 μg mL was prepared from stock solution by appropriate dilution with distilled water. A 200 μL portion of DCH was injected into the stream of DONA (5×10M) reagent and was then combined with a stream of 0.1 M sodium carbonate with a total flow rate 2.5 mL min. A calibration graph was prepared and regression equation with other feature was calculated as shown in Table (1). Optimization of conditions was carried out on 40 μg mL of DCH.