New developingand Validation method to Determine Lidocaine Hydrochloride in Raw Material and Injection Forms by Using RP-HPLC

ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 149 ةداملا يف ديرولكورديه نيئاكوديللا ديدحتل ةديدجلا ةقيرطلا ةيحلاصو ريوطت ةءافكلا ةيلاع ةلئاسلا ايفارغوتاموركلا مادختساب ةيقرزلا لاكشلأا يفو ماخلا سوكعملا روطلاب يلكطنا دعس .د.أ 1 صخلملا : ةيفارغوتاموركلا ةقيرطب ةقيقدو ةساسح ،ةعيرس ،ةطيسب ةقيرط ريوطت ىلإ ثحبلا اذه فدهي مت .ةيقرزلا اهتارضحتسم يفو ماخ ةدامك ديرولكورديه نيئاكوديللا ريدقتل ،ةءافكلا ةيلاع ةلئاسلا (روفسوفلا ضمح:ليرتنوتيسأ نم جيزم مادختساب لئاسلا يفارغوتاموركلا لصفلا قيقحتpH = 3( ةيمجح جزم ةبسنب )70:30 دومع دوجوب ،كرحتملا روطلا نم )Shim-pack clcC8 column (25 cm x 4.6 mm i.d., 5 μm) يجسفنبلا قوف لاجملا يف فشكلا مت . ةجوملا لوط دنعλ = 212 nm قفدتبو ،1 mL/min.ةنوقحملا ةنيعلا مجح ،20 µL مت لا ،ةيطخلا ديدحتب ةقيرطلا ةيحلاص نم ققحتلا ي رايعلا ينحنملا رهظأ .ةيعونلاو ةحصلا ،ةقد ( نيب ام ةيطخ2.5 – 250 )µg/mL دحو فشكلا دح ناك .يلخاد ي رايعك نيئفاكلا دوجوب يمكلا ديدحتلا0.31 𝜇g/mL و0.94 𝜇g/mL .لسلستلا ىلع رادقمب ديرولكورديه نيئاكوديلل ظافتحا نمز عم ةعيرس ةقيرطلا تناك4.5 min قيبطت نكمي . طلا .يقرزلا ينلاديصلا لكشلا يف نيئاكوديللا ةبقارمل ةينيتورلا ليلاحتلا ىلع ةروطملا ةقير :ةيحاتفملا تاملكلا ليلحتلا ،ديرولكورديه نيئاكوديل ،ةءافكلا ةيلاع ةيفارغوتاموركلا .يلخادلا ي رايعلا ،ةيحلاصلا ،يفارغوتاموركلا ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 150 ________________________ 1 ايروس ،بلح ةعماج ،مولعلا ةيلك ،ءايميكلا مسق ،ةيليلحتلا ءايميكلا ،ذاتسأ New developing and Validation method to Determine Lidocaine Hydrochloride in Raw Material and Injection Forms by Using RP-HPLC. 1 . SAAD ANTAKLIProf. Dr ABSTRACT: This work was aimed to develop a rapid, simple, sensitive and precise high performance liquid chromatography (HPLC) method for the estimation of Lidocaine hydrochloride (Lido) in both injection dosage forms and raw materials . The chromatographic separation was achieved with acetonitrile:phosphoric acid (pH = 3) in ratio of 30:70 (v/v) as mobile phase on a Shim-pack clc-C8 column (25 cm x 4.6 mm i.d., 5 μm) with UV detection at 212 nm, pump flow rate 1.0 mL/min and sample injection volume 20 μL . The method was validated with respect to linearity, precision, accuracy and specificity. The calibration curve showed good linearity over the concentration range of (2.5 - 250) µg/mL in presence of Caffeine (Caff) as internal standard with limit of detection and limit of quantification were to be 0.31 𝜇g/mL and 0.94 𝜇g/mL, respectively . ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 151 The developed method was very rapid with a run time of 4.5 min and found to be successively applied for the quality control of (Lido) in pharmaceutical formulations. KEYWORDS: HPLC, Lidocaine hydrochloride, Chromatographic analysis, Validation, Internal standard. ________________________ 1 Professor, Analytical Chemistry, Department of Chemistry, Faculty of Science, University of Aleppo, Syria 1. Introduction Lidocaine hydrochloride: Acetamide, 2-(Diethylamino)-N-(2,6-dimethylphenyl)-, hydrochloride, monohydrate or Lidocaine hydrochloride (Lido) is a white or almost white, crystalline powder. Very soluble in water, freely soluble in ethanol (96 percent) [1 .] Lidocaine-HCl is a local anesthetic material with strong and fast acting. It has a high permeability of the tissue and is suitable for external use to relieve the pain, itching and inflammation [2 .] Several methods have been applied in the literature for the determination of (Lido) in dosage forms and in biological fluids. Techniques such as spectrophotometry [3,4,5,6], high performance liquid chromatography (HPLC) [1,7,8], liquid chromatography [9,10], electrochemical analysis [11] and electrophoresis analysis [12] were used to determine (Lido) in different pharmaceutical forms. 2. Materials and Method Apparatus: HPLC analysis was performed on a YL 9100 HPLC system equipped with a binary pump YL9111, vacuum degasser series YL9101, YL9130 column ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 152 compartment and YL9120, UV/Vis. Detector (Korea). Chromatographic separations were obtained by using Shim-pack clc-C8 column (25 cm × 4.6 mm i.d., 5 µm) (Shimadzu, Japan). Ultrasonic bath (Daihan, USA), analytical balance TE64 Sartorius (Germany) sensitivity 0.1 mg. Germany digital pipettes (Isolab). Chemicals: Ssolvents and materials were used as analytical grade: water, acetonitrile (Isolab, Germany), all were HPLC grade and Phosphoric acid (Isolab, Germany). Caffeine (Caff) pure drug substance, its purity was 99.84 % (Abbott health care, India) and (Lido) purity 99.21%, (Abbott health care, India). Stock standard preparations: 2.5 mg/mL of (Lido) was prepared as stock standard solution by dissolving an appropriate weight of this material in bi-distillation water, by taking the purity of the material into consideration. 2.5 mg/mL of (Caff) was prepared as stock standard solution by dissolving an appropriate amount of this material in bidistillation water, by taking the purity of the material into consideration. Calibration Curve: To construct the calibration curve, seven standard solutions (2.5, 25, 50, 100, 150, 200, 250) 𝜇g/mL for (Lido) were prepared and the area of peaks was measured of each solution five times . Samples preparation: Two Syrian products were studied: • Containing five "Obarcaine" vials (each vial contains 1000 mg Lido/50 mL) was transferred to a beaker then a 2.5 mL of it was transferred to a 10 mL volumetric flask. The volume was completed to 10 mL using HPLC-grade water. Then 0.1 mL was taken to 10 mL volumetric flask (which equivalent to 50 μg/mL theoretically) which contains 25 μg/mL Caff and adjusted to volume with mobile phase and filtered through a 0.45 µm nylon syringe filter ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 153 and degassed by ultrasonication, then 20 μL of the sample was injected into the chromatograph system. • Containing five "Lidosol" ampule (each ampule contains 50 mg Lido/5 mL) was transferred to a beaker then a 5 mL of it was transferred to a 10 mL volumetric flask, the volume was completed to 10 mL using HPLC-grade water. Then 0.1 mL was taken to 10 mL volumetric flask (which equivalent to 50 μg/mL theoretically) which is contains 25 μg/mL Caff and adjusted to volume with mobile phase and filtered through a 0.45 µm nylon syringe filter and degassed by ultrasonication, then 20 μL of the sample was injected into the chromatograph system. 3. Results and Discussion One of the chromatograms of seven different standard concentrations mixtures (Lido) and (Caff) as internal standard is presented in figure (1). Each concentration was injected five times under optimized method conditions. The chromatogram showed that (Lido) was well separated from (Caff) with a good resolution and the time of analysis was achieved in less than 5 min. Figure (1): Chromatogram of standard solution: 1- (Lido) 50 µg/mL, 2- (Caff) 25 µg/mL. Chromatographic conditions: C8 column; mobile phase: acetonitrile:phosphoric acid (pH = 3) 30:70 (v/v) flow rate 1.0 mL/min, temperature 30 °C and detection at 212 nm. ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 154 Optimization of the HPLC conditions: Selection of λmax wavelength: The wavelength at which the maximum absorption (212 nm) occurs is selected for further analysis. A definite concentration of Lido solution was scanned in UV range of 200 – 300 nm. Water was used as a blank. The absorbance of solutions in HPLC method was measured at 212 nm and calibration curve of Lido was built up accordingly. Mobile phase effect: The effect of composition of the mobile phase (using C8 column 25 cm × 4.6 mm i.d., 5 µm) on the retention time of (Lido) and the internal standard (Caff) was investigated in figure (2). The percentage of Acetonitrile (30 – 60) % in the mobile phase had a significant effect on the retention behavior of the studied compounds. An increase in the percentage of acetonitrile has decreased the retention of compounds; Lido and Caff. A satisfactory separation of Lido and Caff with satisfactory resolution was obtained with a mobile phase containing 30% acetonitrile. Figure (2): Mobile phase effect on the retention time of: (Lido) 50 µg/mL and (Caff) 25 µg/mL. Chromatographic conditions: C8 column; mobile phase: variable ratio of acetonitrile:phosphoric acid (pH = 3) , flow rate 1.0 mL/min, temperature 30 °C and detection at 212 nm. ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 155 pH effect of mobile phase: Effect of pH on the chromatographic elution of both compounds (Lido) and (Caff) was investigated by changing the pH values of the aqueous component of the mobile phase from (2.5 to 5) by H3PO4, figure (3). It was observed that the pH of mobile phase values ranged from (2.5 to 4) permitted the elution of drugs in the following order (Lido) and (Caff), but when the pH values range became from (4 to 5) the order was (Caff) and (Lido). pH = 3 was chosen for the optimum separation of these compounds. Figure (3): pH effect of mobile phase on the retention time (tR) of (Lido) 50 µg/mL and (Caff) 25 µg/mL. Chromatographic conditions: C 8 column; mobile phase: acetonitrile:phosphoric acid (variable value of pH) 30:70 (v/v); flow rate 1.0 mL/min, temperature 30 °C and detection at 212 nm. \ Flow rate effect: The optimum flow rate was identified by changing flow rate from 0.8 to 1.3 mL/min. It was concluded that each two peaks were completely separated, with a fine and symmetrical aspect at flow rate (1 mL/min) which also corresponds to the deviation point as seen in figure (4). ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 156 Figure (4): Flow rate effect of mobile phase on the retention time of (Lido) 50 µg/mL and (Caff) 25 µg/mL. Chromatographic conditions: C8 column; mobile phase: acetonitrile:phosphoric acid (pH = 3) 30:70 (v/v); flow rate (variable value), temperature 30 °C and detection at 212 nm. Optimum chromatographic conditions: Table (1) presents the chromatographic method conditions, which were applied for simultaneous determination of (Lido) in presence an internal standard (Caff). Table (1): (Lido) determination chromatographic conditions, in presence (Caff) as internal standard. HPLC method Specification Shim-pack clc-C8 (25 cm × 4.6 mm, 5 μm) Column Phosphoric acid (pH = 3):acetonitrile 70:30 (v/v), Mobile phase Caff Internal standard 1 mL/min Flow rate 30 °C Temperature UV 212 nm Detector 20 µL Injection volume METHOD VALIDATION: The method was validated according to ICH guidelines [13]. The following validation characteristics were addressed: ةصاخلا ةينطولا ةعماجلا ةلجم– دلجملا1 – ددعلا1 -2023 .أ د . يلكطنا دعس 1 157 Linearity: Standard solutions containing (Lido) were prepared in a mixture of acetonitrile:phosphoric acid (pH = 3) in ratio of 30:70 (v/v) from a fresh stock solution (2.5 mg/mL) to construct the calibration curve. The least square regression analysis was carried out for the obtained data. Calibration curve consisted of seven different concentrations in the range (2.5 – 250) 𝜇g/mL for (Lido) with correlation coefficient of the regression equation greater than 0.999. Each concentration level was performed five times. The equation of the calibration curve attained was y = 0.0176 x + 0.0017. It was obtained by plotting the peak area ratio of (Lido) to the internal standard (Caff) (y) as a function of analyte concentration (x) in 𝜇g/mL as seen in figure (5). Figure (5): Linear relationship for (Lido): C1: 2.5 𝜇g/mL, C2: 25 𝜇g/mL, C3: 50 𝜇g/mL, C4: 100 𝜇g/mL, C5: 150 𝜇g/mL, C6: 200 𝜇g/mL, C7: 250 𝜇g/mL. n = 5 for each concentration Limit of detection (LOD) and Limit of quantification (LOQ): The (LOD) and (LOQ) were obtained from the calibration curves. 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