ASSAYS OF DOXYCYCLINE ANTIBIOTIC USED IN TASIKMALAYA CITY HEALTH CENTER FOR EVALUATION CAUSE ANTIBIOTIC RESISTANCE

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Ramdhani et al. SJIF Impact Factor Volume 7, Issue 8, Research Article ISSN ASSAYS OF DOXYCYCLINE ANTIBIOTIC USED IN TASIKMALAYA CITY HEALTH CENTER FOR EVALUATION CAUSE ANTIBIOTIC RESISTANCE Danni Ramdhani* and Resmi Mustarichie Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Padjadjaran University, Indonesia. Article Received on 21 June 2018, Revised on 10 July 2018, Accepted on 31 July 2018 DOI: /wjpps *Corresponding Author Danni Ramdhani Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Padjadjaran University, Indonesia. ABSTRACT An acute respiratory tract infection (ARIs) is a disease attacking one or more parts of the respiratory tract. ARI is mostly caused by bacteria than viruses. For treatment of ARI, people tend to give some antibiotics. But there are many cases of bacterial resistance to some antibiotics have been reported. Testing levels of antibiotics is one of the evaluation to determine the quality of antibiotics used. This quality evaluation to determine the role of antibiotics in causing antibiotic resistance. Method: Samples were obtained from Tasikmalaya City Health Department. Samples derived from only one pharmaceutical factory in Indonesia, with one production batch number. Tests carried out using UV-Visible spectrophotometer instrument, with validation parameters: linearity, precision, accuracy, limit of detection (LOD) and limit of Quantification (LOQ). Result: The test results showed that the levels of doxycycline antibiotics that are used to meet the requirements that exist in the Indonesian Pharmacopoeia grading 115,16%, and the validation parameters that meet the requirements, namely koefesisen correlation of , reproducibility 0,01013%, percent recovery 99,827%, LOD 5,810, and LOQ 19,368. KEYWORDS: Doxycycline, UV-Visible Spectrophotometer, Validation Parameters, Indonesia Pharmacopoeia. Vol 7, Issue 8,

2 INTRODUCTION Acute Respiratory Infection (ARIs) was known as one of the leading causes of mortality for all ages, especially in children month age group is the age group most vulnerable to experiencing respiratory infection. Based on data from the WHO and the Ministry of Health of the Republic of Indonesia in 2008, one of ISPA is the main cause of death in children under five are pneumonia (Nasution et al., 2009). This relates to the data ARI in Tasikmalaya, who reported that the main causes of infant and child mortality are pneumonia (DinKes Prov, West Java, 2003). Based on the results of basic medical research in West Java province, the prevalence of ARI in Tasikmalaya city occupies third position after Karawang and Cirebon. This respiratory disease is an infectious disease that is deadly if not treated. Therefore, prevention and control of ARIs is one of the main priorities in health development in Tasikmalaya (Riskesdas, 2007). Respiratory diseases can be caused by viruses or bacteria that enter the human respiratory system (Mennenghetti, 2007). Reported that the incidence of acute respiratory infection caused by a lot more bacteria than viruses. During this time of ARI treatment is done with antibiotics. However, cases of bacterial resistance to some antibiotics that are important have been reported during the period (WHO, 2011). This can happen due to the irrational use of antibiotics and the potential reduction of antibiotics used and the precise levels of antibiotics in the preparation (Kakkilaya, 2010). Based on data from previous studies, it is known that some bacteria of clinical isolates from patients with ARIs in Tasikmalaya City Health Center Purbaratu have responded resistant to some antibiotics used to treat respiratory infections, ie 68.03% resistant to amoxicillin; Resistant to cefadroxil 70.25% and 43.03% were resistant to ciprofloxacin (Alfaeira, et al., 2016). MATERIALS AND METHODS. Tools The tools used in this study is an autoclave (Hirayama), micropipette ml volume (Eppendrof), tip micropipette, uv-visble spektrotometer ( SPECORD ), glass vials, ultrasonic bath (NEY-1510), a glass vial of 10 ml, 100 ml brown bottle and glass tools commonly used in the Laboratory Analysis. Vol 7, Issue 8,

3 Test Materials Materials tested were Doxycycline from PT. Yarindo Farmatama used in Community Health Center in Tasikmalaya, West Java, Indonesia. HCL p.a (Merck), aqua bidestilation (Ikapharmindo Putramas). METHOD A. Preparation of Doxycycline Standard Solution Into a series of 20 ml volumetric flask, aliquots of standard solution of doxycycline (100 mg / ml in 0.1 M HCl) is equivalent to 5-15 mg / ml doxycycline measured and put in a flask and then added 0.1 M HCl to mark boundaries. After mixing the measured absorbance at the maximum wavelength and compared with 0.1 M HCl. After it was made the standard curve so obtained line equation in the form y = ax + b (Ramesh et al., 2011). B. Determination of Wavelength Maximum Determination of the maximum wavelength done by measuring the standard solution of doxycycline 20 mg / ml were prepared from stock solutions of doxycycline 100 mg / ml. Solution of maximum absorbance is observed using a UV-Vis spectrophotometer. C. Method Validation Validation of the analytical methods used consisted of a test of linearity, limit of detection (LOD), Limit of Quantification (LOQ), precision, and accuracy. 1. Linearity Test Linearity test is done by calculating the correlation coefficient (r2) with a linearity parameter whose value > 0.99 at least four different concentrations in the range of % of the content of the analyte in the sample (Riyanto, 2014). 2. Precision Test Made standard solution with 5 variations of concentration is 2 mg / ml, 5 mg / ml, 10 mg / ml, 12.5 mg / ml, and 15 mg / ml. Furthermore, each of these concentrations measured absorbance at 271 nm wavelength. 0.1N HCl is used as a blank. Precision expressed as relative standard deviation or coefficient of variation. Parameters acceptable precision of <2% (Harmita, 2004). Vol 7, Issue 8,

4 3. Accuracy Test Made standard solution with 5 variations of concentration is 2 mg / ml, 5 mg / ml, 10 mg / ml, 12.5 mg / ml, and 15 mg / ml. Furthermore, each of these concentrations measured absorbance at 271 nm wavelength. Accuracy is obtained through the% recovery (% recovery) by the equation. % Recovery = (levels of analysis results) / (actual content) x 100% Parameters required accuracy is % (Harmita, 2004). 4. Determining LOD and LOQ From the standard curve which has been obtained, calculated the amount of analyte smallest detectable (LOD) by the equation. LOD = Besides LOD, also calculated the smallest quantity of analyte can still be detected (LOQ) by the equation. LOQ = E. Assays Doxycycline Samples are prepared and weighed as much as 5 mg and dissolved in 50 ml volumetric flask with 0.1 N HCl (concentration of 100 ug / ml). After the sample is diluted to obtain a concentration of 10 mg / ml. The absorbance was observed at the maximum wave UV- Visible Spectrophotometer. RESULT AND DISCUSSION A. Determination of Wavelength Maximum Determination of the maximum wavelength needs to be done before determination of antibiotics. It is intended to look at the value of absortivitas which provides the highest measurement sensitivity. Determination the maximum wavelength is done by making the stock standard solution 100 ppm. Making the stock is done by weighing the raw doxycycline as much as 10 mg and then diluted with 0.1N HCl in 100 ml volumetric flask. Stock standard solution is then diluted to obtain a concentration 20 ppm. Standard solution with a concentration of 20 ppm and then measured the absorbance maximum in the wavelength Vol 7, Issue 8,

5 Absorbance Ramdhani et al. range nm. spectrum results the maximum wavelength measurements with the raw doxycycline. Figure 1: Spectrum Wavelength Doxycycline 20 ppm Maximum Baku. The measurement results show that the maximum wavelength of doxycycline is 270 nm. Wavelength obtained in accordance with literature (USP, 2014). B. Determination of a Standard Curve Preparation of standard curve equation is to obtain a standard solution in the assay sample. This curve is a relationship between the absorbance with concentration, ie by plotting the concentration of standard solution as the x-axis and absorbance as the y-axis. The concentration used is 5 ppm; 7.5 ppm; 10 ppm, 12.5 ppm and 15 ppm and aborban sinya measured at a wavelength of 270 nm. Doxycycline standard curve is shown in Figure 2. Standard Curve Doxycycline Concentration Figure 2: Standard Curve Doxycycline. Vol 7, Issue 8,

6 Obtained line equation y = x with R2 = Standard curve above in accordance with the Lambert-Beer law which states that the intensity transmitted by the sample solution is proportional to the thickness and concentration and inversely proportional to transmittance (Day, 2002). R value close to 1 proved that the regression equation are linear and small standard deviation indicates a fairly high accuracy (Andayani, et al, 2008). C. Method Validation Validation of analytical methods performed to prove that certain parameters still meet the requirements for its use. The parameters tested were linearity, accuracy, precision and LOD and LOQ. 1. linearity Test Linearity test is done with a series of standard solutions which consist of at least four different concentrations in the range of % of the content of the analyte in the sample (Riyanto, 2014). The concentration used in the assay was 5 ppm; 7.5 ppm; 10 ppm; 12.5 ppm; and 15 ppm. The linearity of the method can describe the precision of the analysis of a method shown by the coefficient of determination of> 0.99 (Chan, 2004). The equation of a straight line obtained standard curve is y = x with R2 = Correlation coefficient obtained meets the requirements (Chan, 2004). R value close to 1 proved that the regression equation is linear and small standard deviation indicates a fairly high accuracy (Andayani, et al, 2008). 2. Accuracy Test Accuracy indicates the degree of closeness of the results of a series of measurements obtained from a homogeneous sample under specified conditions (ICH, 1995). Accuracy expressed as a percent recovery (recovery) the analyte is added. Testing is done by five different of concentration is 5 ppm; 7.5 ppm; 10 ppm; 12.5 ppm and 15 ppm. Calculation% recovery can be seen in Table 1. Vol 7, Issue 8,

7 Table 1: Precision Test Calculation Results. The average value of% recovery (recovery) obtained is %. These results can be accepted because they are within the required range, ie % (Harmita, 2004). 3. Precision Test Precision is a measurement repeatability of analytical methods derived from multiple measurements on the same sample (Gandjar and rohman, 2007). Precision is measured as the standard deviation or relative standard deviation (coefficient of variation) (Harmita, 2004). According to Sherri (2008) precision test criteria can be distinguished as follows. Table 2: Criteria of Precision Test. The concentration of the test used is 5 ppm; 7.5 ppm; 10 ppm; 12.5 ppm and 15 ppm. The test results are shown in Table 3. Vol 7, Issue 8,

8 Table 3: Results Calculation Accuracy. Results% RSD obtained amounted to %. The results are included in the criteria very carefully which shows that this method has a high degree of accuracy ffor testing of samples. 4. LOD and LOQ Test The limit of detection is the smallest amount of analyte in a sample that can be detected which still provides significant response compared to the blank and the test parameters limits. Values obtained detection limit is mg / ml. Quantification limit is a parameter on the analysis of trace and is defined as the smallest quantity of analyte in the sample were still able to meet the criteria of a careful and thorough (Riyanto, 2014). Values obtained quantification limit was mg / ml. LOD and LOQ calculation results can be seen in Table 4. Table 4: Calculation Result LOD and LOQ. Vol 7, Issue 8,

9 D. Assays Doxycycline Doxycycline assay samples was done by UV spectophotometric instrument. Absorbance is read at the spectrophotometer is between 0.2 to 0.8 or 15% to 70% when read as transmittance. This is due to the range of photometric absorbance value of the error that occurred was the most minimal (Rohman, 2007). Measurements done by making a sample of 100 ppm stock solution in 50 ml then diluted to a concentration of 10 ppm. Measurements were taken at maximum wavelength that has been obtained before that at 270 nm. The average yield obtained absorbance is The average is then inserted into a standard curve which has been obtained is y = 0,0325x and the results obtained concentration is equal to mg / ml. Levels of sample obtained is equal to %. Results are still within the range required by the USP 97% -120% (2014). CONCLUTION Levels of antibiotic Doxycycline samples used in Tasikmalaya City Health Center is 115, 16%. Results are still within the range required by the USP 97% -120% (2014). ACKNOWLEDGMENTS The authors are deeply grateful to the subjects participating in this study. The author would like to thank Tasikmalaya City Health Office. The author also thanked Amalia Octa Permatasari for its cooperation in this study. REFERENCES 1. Alfaeira, C.H, Kusuma, S.A. dan Ramdhani, D Pengujian Resistensi Isolat Klinis dari Apusan Rongga Mulut Pasien Infeksi Saluran Pernapasan Akut (ISPA) di Puskesmas Purbaratu Kota Tasikmalaya terhadap Antibiotik Amoksisilin, Sefadroksil, dan Siprofloksasin. [Skripsi]. Fakultas Farmasi Universitas Padjadjaran. 2. Amin, L.Z Pemilihan Antibiotik yang Rasional. Medical review PPDS Ilmu Penyakit Dalam, Fakultas Kedokteran Universitas Indonesia/RSUPN Cipto Mangunkusumo. 3. Day R.A. dan Underwood, A.L Analisis Kimia Kuantitatif. Jakarta: Penerbit Erlangga. 4. Departemen Kesehatan RI Farmakope Indonesia. Edisi IV. Jakarta: Depkes RI. 5. Departemen Kesehatan RI Informasi pada Balita tentang ISPA. Jakarta: Pusat Penyuluhan Kesehatan Masyarakat. Vol 7, Issue 8,

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11 22. Wijayanti, A.D., Rahardjo, S., dan Satria, G.D Perbandingan profil farmakokinetik doksisiklin aplikasi intravena dan intramuskular pada ular sanca (Phyton reticulatus). J. Sain Vet, 27(1). 23. World Health Organization Promoting Rational Use of Medicine. Geneva: Core Components. 24. World Health Organization Bibliography of Scientific Publication on Antimicrobial Resistence from South-East Asia Region Available online at [8 September 2016]. 25. Yulianti, N.T., Werdiningsih, S., Isriyanthi, N.M.R., Patriana, U., dkk Profil Farmakokinetik Beberapa Sediaan Antibiotik Doksisiklin pada Ayam Boiler. Tersedia online di [24 Agustus 2016]. Vol 7, Issue 8,