Đề tài Quantitative Determination Of Phospholipid In Liposomal Amphotericin B For Lyophilized Injection By Uv-Vis Spectrophotometric Method - Nguyen Tuan Quang

Journal of military pharmaco-medicine n o 3-2019 104 QUANTITATIVE DETERMINATION OF PHOSPHOLIPID IN LIPOSOMAL AMPHOTERICIN B FOR LYOPHILIZED INJECTION BY UV-Vis SPECTROPHOTOMETRIC METHOD Nguyen Tuan Quang1; Nguyen Thi Kieu Anh3 Nguyen Thai Son2; Pham Thi Minh Hue3 SUMMARY Objectives: To validate the quantitative analysis of phospholipid in liposomal amphotericin B for lyophilized injection by UV-Vis spectrophotometer. Materials and methods: Liposomal amphotericin B for lyophilized injection produced by Department of Pharmaceutics, Hanoi University of Pharmacy is analyzed with Hitachi model U-1800 spectrophotometer by measuring the amount of phosphor in phospholipid. Results: The method was validated for specificity, compatibility, linearity, propriety, accuracy and the phosphor determined ranges from 45.36 to 68.05 µg/mL. As a result, the total amount of phospholipid measured in liposomal amphotericin B for lyophilized injection was 284.11 ± 4.41 mg/vial. Conclusion: The appraised procedure obtained all the requirements and can be used to measure the amount of phospholipid in liposomal amphotericin B for lyophilized injection. * Keywords: Phospholipid; Liposomal amphotericin B; Lyophilized injection; UV-Vis spectrophotometer. INTRODUCTION Liposome is a form of nano - structured microcyst. Nowadays, scientists have strong research interest in liposome for various functions, especially in the pharmaceutical industry [1]. Liposome’s main components include phospholipids (PL) and cholesterol. Many types of PL used in liposome preparation can be used seperately or in combination with others. Types and amount of PL in proportion to cholesterol plays a very important role in the sustainability and stability of composed liposome. Therefore, apart from criteria such as quality and quantity of active elements, measurement of particle size, the measurement of liposome products quality is required in PL quality control process. Liposomal amphotericin B (AmB) for lyophilized injection produced by Department of Pharmaceutics, Hanoi University of Pharmacy contains distearoyl phosphatidylglycerol (DSPG-C42H83O10P) and hydrogenated phosphatidylcholine (HSPC-C44H88NO8P). The combination of these PLs caused difficulties in measuring the total amount of PL in liposome injection AmB because DSPG and HSPC have similar molecular weights (DSPG was 779.076 g/moL, HSPC was 783.774 g/moL). 1. Vietnam Military Medical University 2. 103 Military Hospital 3. Hanoi University of Pharmacy Corresponding author: Nguyen Tuan Quang (dsquang2000@yahoo.com) Date received: 25/12/2018 Date accepted: 13/02/2019 Journal of military pharmaco-medicine n o 3-2019 105 Based on the composition of the two types of PL in liposome products and the principle of coloring reaction between phosphorus and molybdate, which was published in some studies [2, 3, 4], we have conducted experiments and presented in this article: The results of the process of measuring the total amount of phospholipid in AmB liposome lyophilized injection using UV-Vis spectrophotometric method. MATERIALS AND METHODS 1. Materials and instruments. - Liposomal AmB for lyophilized injection produced by Department of Pharmaceutics, Hanoi University of Pharmacy contains mainly AmB, DSPG, HSPC, and cholesterol. - Placebo sample contains liposomal AmB for lyophilized injection sample but not DSPG and HSPC. - Chemicals: Standardized kali dihydrophosphat (KH2PO4) (Merck, 99.9%), ammonium molybdate, ascorbic acid, percloric acid, sulfuric acid, and other standardized chemical reagents. - Hitachi U-1800 spectrum analyzer (Japan), Mettler Toledo analytical balance (Swiss, precision: 0.1 mg), 800B centrifuge (China) and other standardized analytical devices. 2. Methods. - Preparation for testing solutions: + Ammonium molybdate 1%: Dissolve 1 g of ammonium molybdate and fill up to 100 mL with distilled water. + Ascorbic acid 2%: Dissolve 2 g of ascorbic and fill up to 100 mL with distilled water. + Mixture B: Ammonium molybdate 1% and ascorbic acid solution 2% in ratio of 2:3 (v/v) (mix well before use). + HClO4 70% saturated with ammonium molybdate: Add ammonium molybdate to 20 mL of HClO4 70%, stir until being indissoluble, centrifuge and extract clear solution. - Sample preparation: + Standardized sample: Put 250.0 mg of KH2PO4 into a 100 mL volumetric flask, dissolve and fill up with distilled water. Suck 2 mL into a 20 mL volumetric flask, fill up with water. + Testing sample: Put 250.0 mg of the product into a beaker of 100 mL. Add 10 mL of solid HNO3, boil gently for evaporation until there’s about 5 mL left. Cool off and add 3 mL of HClO4, gently heat until HClO4 white smoke appears. Continue to boil until the solution become paler (in about 10 minutes). Cool off and move the solution into a 50 mL cooled vat, fill up the vat with water (cool off the vat and the solution while filling up). + Placebo sample: Carry out similarly with test sample but using 0.29 g of the placebo sample. + Blank sample: Distilled water. - Practical procedure: Suck 200 μL of standardized solutions, testing solutions, placebo solution and distilled water into a 100 mL heat - resistant triangular flask, add exactly 1.4 mL of HClO4 70% saturated with ammonium molybdate, lightly shake, heat gently for about 1 hour (the vase has milky white dregs), cool off. Add exactly 8 mL of mixture B, shake well and gently steam at 50 - 55 degrees Celsius for 1 hour, cool off. Move the Journal of military pharmaco-medicine n o 3-2019 106 solution into a 10 mL volumetric flask, fill up with distilled water and shake well. Filtrate using filtration paper; use the filtrated solution to conduct UV-Vis spectrometry. Scan the spectrum at the wavelength of 800 - 840 nm and measure the absorbance value at λmax = 820 nm. Calculate the results using the measured absorbance value. The content of phosphorus/vial was calculated by the following formula: At * Mc * HLc * 31 * 50 X = * Mtb (mg P/vial) Ac * 136.1 * 1000 * Mt In which: At, Ac: The absorbance of the sample and standardized sample; Mc and HLc: The amount (mg) and the content of standardized KH2PO4 sample; Mt, Mtb: The weight and average amount of sample (g); 31: The molecular weight of P in KH2PO4 (g); 136.1: The molecular weight of KH2PO4 (g); 50 and 1,000: The liquescency of sample and standardized samples. Measure the total content of PL (mg) in a testing sample vial using HSPC with the formula: X * 783.774 /31. - Measuring process appraisal: For the specificity, compatibility, linearity, propriety, accuracy and interval [5]. RESULTS AND DISSCUSION 1. Specificity. Measure the absorbance of the prepared standardized sample, placebo sample and blank sample. The results showed that the spectral chart of blank sample did not show maximum absorbance at 820 nm. The spectral chart of testing and standardized samples showed maximum absorbance at 820 nm. The spectral chart of placebo sample showed a response to absorbance at 820 nm, but the response was not greater than 1.0% compared to that of the standardized sample. Table 1: Effect of placebo sample on absorbance of active elements. Absorbance Number Standardized sample Placebo sample Effect of placebo sample (%) 1 0.833 0.006 0.716 2 0.844 0.006 0.716 3 0.837 0.007 0.835 Medium ± SD 0.838 0.006 0.756 ± 0.069 2. Linearity. Put 250.0 mg of KH2PO4 into a 100 mL of volumetric flask, dissolve and fill up with distilled water (original standardized solution). Suck certain amount of the standardized solution and dilute using distilled water (as described in table 2) to obtain a range of titrated solutions with concentration of about 50%, 80%, 100%, 120% and 150% Journal of military pharmaco-medicine n o 3-2019 107 compared to quantitative concentration. Suck 200 μL of standardized solutions, testing solution, placebo solution and distilled water into 100 mL of heat-resistant triangular flask, then conduct reaction (as above). The results of the correlation between the absorbance of the standardized sample and phosphorus concentration were presented in table 2 and figure 1. Table 2: Linearity of the measuring process. Number % compared to quantification Standardized weight (mg) Dilution (times) Phosphorus concentration (µg/ml) Absorbance 1 50 249.2 1
20 28.35 0.401 2 80 249.2 2
25 45.36 0.600 3 100 249.2 2
20 56.70 0.781 4 120 249.2 3
25 68.05 0.935 5 150 249.2 3
20 85.06 1.163 Regression equation: y = 0.0136 x + 0.0036. Correlation coefficient (r): 0.9982 Corner coefficient: 0.0136 Intercept coefficient: 0.0036 %Y: 0.4609 Figure 1: Linear correlation between phosphorus concentration and absorbance. Results showed that in the testing concentration ranging from 28.35 to 85.06 μg/mL, there was a strong linear correlation between phosphorus concentration and absorbance with correlation coefficient r ≈ 1. Intercept coeficient Y (at the concentration of 56.70 μg/mL) was 0.4609% (satisfaction < 2%). Concentration p (µg/ml) Absorbance Journal of military pharmaco-medicine n o 3-2019 108 3. Propriety. The appraisal of propriety was conducted by adding a standardized sample to the placebo sample to obtain the corresponding standardized phosphorus solutions of 80%, 100% and 120%, respectively to the quantitative concentration. Specifically: - Preparation of standardized solutions: Put about 0.5 g; 0.63 g and 0.75 g of KH2PO4 into three different 50 mL of volumetric flasks, dissolve and fill up with distilled water. - Put 0.29 g of the placebo sample into a 100 mL beaker. Add exactly 1.0 mL each of the original solution (repeat 3 times for each), mix well and fulfill as above (testing sample preparation). - Suck 200 μL of standard solutions, self - generated sample solution and distilled water into 100 mL heat-resistant flaks, then conduct the reaction (as above). Table 3: Results of the appraisal on the propriety of the measuring process. Number % compared to quantification Added standardized amount (mg) Absorbance Revovery amount (mg) % recovery 1 80 45.52 0.679 46.00 101.06 2 80 45.52 0.660 44.71 98.23 3 80 45.52 0.659 44.65 98.08 Medium: 99.12 RSD (%): 1.69 4 100 57.81 0.845 57.25 99.03 5 100 57.81 0.855 57.92 100.20 6 100 57.81 0.845 57.25 99.03 Medium: 99.42 RSD (%): 0.68 7 120 70.42 1.033 69.98 99.38 8 120 70.42 1.029 69.71 98.99 9 120 70.42 1.050 71.13 101.01 Medium: 99.79 RSD (%): 1.07 Medium (%) of 9 results (n = 9): 99.45 RSD (%) of 9 results (n = 9): 1.10 By using this method, the recovery level at each concentration level was within the allowance range of 98 - 102%, with the RSD of 9 results was 1.10% (within the limit of < 2%). Therefore, the method satisfied the requirements of propriety. Journal of military pharmaco-medicine n o 3-2019 109 4. Accuracy. * Repeatability: Repeatability of the measuring process was determined after 6 times of quantification made on one testing sample at the described conditions. Table 4: Results of the repeatability of measuring process (n = 6). Number Testing sample (g) Absorbance % quantification 1 0.2418 0.537 99.09 2 0.2526 0.559 98.74 3 0.2442 0.539 98.48 4 0.2567 0.584 101.50 5 0.2408 0.529 98.02 6 0.2712 0.615 101.18 Medium 99.50 RSD % 1.48 Standardized sample weight : 249.2 mg; standardized sample absorbance: 0.816 The method had high repeatability with RSD = 1.48% (satisfaction < 2%). * Intermediary accuracy: Intermediary accuracy of the measuring process was determined in the same way as that of repeatability but was conducted on a different date and by different testers. Table 5: Results of the intermediary accuracy of the measuring process. Tester 1 Tester 1 Standardized sample: 249.2 mg Standardized sample absorbance: 0.816 Standardized sample absorbance: 0.802 Number Testing sample weight (g) Absorbance % quantification Testing sample weight (g) Absorbance % quantification 1 0.2418 0.537 99.09 0.2425 0.528 98.84 2 0.2526 0.559 98.74 0.2498 0.546 99.22 3 0.2442 0.539 98.48 0.2423 0.525 98.36 4 0.2567 0.584 101.50 0.2556 0.572 101.59 5 0.2408 0.529 98.02 0.2394 0.519 98.41 6 0.2712 0.615 101.18 0.2714 0.608 101.70 Medium 99.50 Medium 99.69 RSD % 1.48 RSD % 1.55 Average quantification (12 testing samples): 99.59% RSD (%) (12 testing samples): 1.45 (%) Journal of military pharmaco-medicine n o 3-2019 110 5. Definite interval. Measuring the absorbance for 6 times of 80% and 120% standardized solutions compared to the quantitative concentration (in linearity). Table 6: Results of the accuracy of the measuring process. Number Concentration % compared to quantitative concentration Absorbance Number Concentration % compared to quantitative concentration Absorbance 1 80 0.641 1 120 0.979 2 80 0.666 2 120 0.976 3 80 0.649 3 120 0.986 4 80 0.674 4 120 0.986 5 80 0.661 5 120 0.962 6 80 0.672 6 120 0.959 Medium 0.661 Medium 0.975 RSD % 1.98 RSD % 1.20 The results showed that at both concentration levels of 80% and 120% compared to the quantitative concentrations gave an RSD (%) of < 2%. The phosphorus definite interval was from 45.36 to 68.05 μg/mL. 6. The total measured amount of PL in AmB liposome lyophilized injection. From the verified measuring process, we measured the total content of PL in AmB liposome lyophilized injection. The results showed that the total PL content in AmB liposome lyophilized injection was 284.11 ± 4.41 mg/vial (n = 6). CONCLUSIONS The process of measuring the PL proportion in liposomal AmB for lyophilized injection was appraised by UV-Vis spectrophotometric method using phosphorus amount measurement. The process was evaluated to ensure specificity, compatibility, linearity, propriety, accuracy as required and the defined phosphorous range was from 45.36 to 68.05 µg/mL. As a result, the determined proportion of PL in AmB liposome lyophilized injection was 284.11 ± 4.41 mg/vial. REFERENCES 1. Vo Xuan Minh, Pham Thi Minh Hue. Nanotechnology and liposome application in pharmaceuticals, cosmetics. Information Journal of military pharmaco-medicine n o 3-2019 111 Center - Library. Hanoi University of Pharmacy. 2013, pp.55-59, 84-100. 2. Samjhana Pradhan, Megh Raj Pokhrel. Spectrophotometric determination of phosphorus in sugarcane juice, fertilizer, detergent and water samples by molybdenum blue method. Scientific World. 2013, 11 (11), pp.58-62. 3. Sanjeevan J. Kharat, Sanjay D. Pagay. Determination of phosphate in water samples of Nashik District (Maharashtra State, India) river by UV-Visible spectroscopy. E-Journal of Chemistry. 2009, 6 (S1), pp.515-521. 4. Xiao-Lan Huang, Jia-Zhong Zhang. Kinetic spectrophotometric determination of submicron orthophosphate by molybdate reduction. Microchemical Journal. 2008, 89, pp.58-71. 5. Asean Guidelines for validation of analytical procedures. Adopted from ICH guidelines, ICH Q2A (1994), ICH Q2B (1996).