148 The determination of phenazone in blood plasma for obtained sistem suitable test of monitoring drug level Mochamad lazuardi Veterinary Pharmacy Subdivision, Veterinary Basic Science Veterinary Faculty, Airlangga University Surabaya - Indonesia abstract The determining of Phenazone to human blood plasma from healthy man after separated by solid phase extraction (SPE) and spectroscopic measurements has been investigated. The objective of that research was to obtain system suitable test for determine the Phenazone level in biological fluids (human blood plasma), for new performed dosage regimented in clinical dentistry. The method can be divided into the following four steps. 1. Centrifugation the blood sample, 2. Extraction from blood plasma and, 3. Separation by SPE with manual pressured, 4. Elution to SPE followed by the measurement on a spectrophotometer in the ultra violet region. The critical value of t at the 5% confidence level indicates that there is no systematic error in the linearity proposed method. Recoveries for this research were obtained at ranging 93.460 to 95.598%. The coefficient variation precision of this procedure was clearly good at smallest than 2%. The analytical procedure can be carried out in one working operation as a monitored therapeutic activity. Key words: phenazone, solid phase extraction, spectrophotometer UV-Vis Correspondence: Mochamad Lazuardi, c/o: Veterinary Faculty, Airlangga University. Jln. Mulyorejo (Kampus C) Universitas Airlangga Surabaya, 60115. e-mail: ardiunair@hotmail.com introduction Phenazone (as an analgesic and antipyretic properties) has been given by mouth.1 Topically, solution containing 5 % of Phenazone have been used locally as ulcers drops in disorders such as acute gingivitis.2 The dentist was usually approved with that dosage form as an analgesic and anti- inflammatory agent. In clinical cases, the re-arrangement of regimentation dosage of Phenazone for treated pediatrics patient was usually problems. Those problems were appeared after showed that Phenazone at long period’s usage can be induced Reye syndrome of red blood cell. That problem would be reduced if the dentistry were using re-designing dosage form concepts by monitored drug level during the therapeutic periods of pediatric patients for obtained satisfaction dosage form. For obtained a suitable drug levels, the method of drug level determination was usually researching. During recent years several papers have been published on the Phenazone.3-7 However, the results obtained are often in poor agreement. The accuracy and the precision of the analytical method used may give the contribution on the variation of the result. The new method of Nuraini et al.,8 was reported that recoveries was obtained at ranging 86.010 to 88.069% and their coefficient variation of precision at ranging 0.433 to 3.871%. The recovery of new method at above was not satisfaction yet, although their precision was nearly exactly. Therefore, the purpose of this work was to develop an accurate method for the determination of Phenazone in human blood plasma using fourth-step sample preparation method. material and methods The analytical method used in the present work incorporates a partition-adsorption of drug-biological matrix separation concepts.9 The separation apparatus of that equipment was used Solid Phase extraction ODS (- CH17-CH3) reverse phase at 3 ml capacity (Sigma Chem. Corp.,). Plastic pestil 0f 3 ml SPe ODS-C18 was used as a manual mobile phase pressure. A pure Phenazone from Sigma Corp. Chem., was used as reference drug material. The Spectronic Hitachi 1100 UV-Vis was used as a measurement drug concentration. The healthy human blood plasma (Indonesian man) was used as an artificial human blood plasma. The human blood plasma was obtained from Indonesia Red Cross Unit. The research protocol was used four step procedures as follows; the 1st step was analyzed linearity of Phenazone in distillated water from stock solution. The standard stock solutions were made up as 1000 µg.ml-1 from 100 mg of Phenazone pure. The standard solutions of 0.5 to 100 µg.ml-1 were prepared by sequential dilution of the standard stock solution. Both standard and stock solutions were placed in test tube. The linearity was analysis during two hours from 0.5 to 100 µg.ml-1 of the standard solution vs. absorbance (Å) on 230 nm at five times replications. The result data of the step one on graphic illustration of the concentration solution series vs. absorbance unit full scale (AUF) will be used as an additional standard solution data. 10 The 2nd step was produced artificial samples in human blood plasma. The pure of Phenazone were weighing of 100 mg and dissolved 149Lazuardi: The determination of phenazone with human blood plasma to produce samples stock at 1000 ppm. The stock samples were prepared at ranging series additional standard concentration of 0.5-100 ppm.10-12 The 3rd step was prepared artificial sample. The samples were extracted by SPe as described Akira et al.,13 and Nuraini et al.,8 as follows; one ml plasma samples were added 1 ml chloroform and shake up well at 10 minutes. Their solutions were added 2 ml with distillated water and centrifuge 3000 rpm (15 minutes). The supernatant were removed sample vials and keep on dark room about 5 minutes. The SPe were activated with inserting 1 ml methanol and 1 ml distillated water consists of 5% glacial acetic acid. The supernatant were inserted to SPe and vacuum drying the SPe at about 30 minutes. The SPe were added with 3 ml elution solution at rate 0.05 ml.second-1 and pressured gentle with plastic pestil. Filtrate from elution were drying by N2 gas and added 3 ml distillated water and analyzing to obtained absorbance values at maximum wave length (230 nm). The graphic of the additional standard solution series vs. absorbance was using for calculation drug concentration on human blood plasma after plotted their absorbance values to the graph. The Vx0 equation was used for measurement of linearity at series 0,5 to 100 ppm. The Vx0 (Vervahrenvariationk oeffizient) equation was described at equation 1 with Sx0 (vervahrenstandardabweichung) and Sy (mean residual deviation of the function) refereed to equation 2.14 equation 1. 100%• × =× X S V 0 0 equation 2, where 2 2 − ∑ − − =       N iYiY yS for Yi = a + bXi b Sy S =× 0 In order to investigate accuracy and precision of the method the samples were spiked with standard solutions and then analyzed by the mentioned method at below. The accuracy expressed as percent recovery was obtained by comparing the results between the Phenazone found and the Phenazone standard.15 The precision expressed as percent coefficient variation was obtained by divided the values of standard deviation with the mean of concentration at five times replicates.16 result The result procedures at the first step was appeared good correlation at ranging of the mean concentrations 0.5 to 100 mg.ml–1 (p < 0.05, Table 1, Figure 1). 0 0,5 1 1,5 2 2,5 3 0 10 20 30 40 50 60 Concentration (ug/ml) A bs or ba n ce (A U F) Series1 Series2 figure 1. The linearity analysis of Phenazone dissolved in distillated water (¨) at mean of 1.002 to 50.002 mg.ml–1 of 0,502 to 100 mg.ml–1. The linearity analysis of Phenazone series at 1.002 to 50.002 mg.ml–1 in human blood plasma (n). But the best purposed for additional standard was obtained at 1 to 50 mg.ml–1 (CV of absorbency < 5%, Table 2 and Table 3). The V × 0 analysis of serial concentration (0.5–100 ppm) was not necessary linear (various) as referred to table 2 at below. The best serial concentration was obtained at 1.002 mg.ml–1 to 50.001 mg.ml–1. The result research of accuracy and precision of that procedures were table 1. The result analysis of linearity Phenazone in distillated water Concentration in mg.ml–1 Absorbance (Å) in AUF Mean ± (%CV) n–1 n–2 n–3 n–4 n–5 Mean± (%CV) n–1 n–2 n–3 n–4 n–5 0.502 0.501 0.501 0.504 0.505 0.503 ± 0.36 0.002 0.003 0.002 0.003 0.002 0.002 ± 22.83 1.002 1.002 1.003 1.002 1.002 1.002 ± 0.045 0.048 0.049 0.051 0.052 0.051 0.050 ± 3.273 2.504 2.503 2.503 2.504 2.503 2.503 ± 0.023 0.136 0.137 0.136 0.137 0.135 0.136 ± 0.615 5.103 5.102 5.103 5.102 5.102 5.102 ± 0.022 0.271 0.270 0.269 0.270 0.271 0.270 ± 0.310 10.003 10.002 10.002 10.003 10.002 10.002 ± 0.005 0.545 0.546 0.544 0.545 0.543 0.545 ± 0.209 20.002 20.002 20.001 20.002 20.002 20.002 ± 0.002 1.091 1.091 1.090 1.091 1.091 1.091 ± 0.041 30.002 30.001 30.001 30.002 30.001 30.001 ± 0.002 1.638 1.637 1.638 1.637 1.638 1.638 ± 0.033 40.001 40.001 40.001 40.002 40.001 40.001 ± 0.001 2.180 2.181 2.180 2.182 2.181 2.181 ± 0.038 50.001 50.002 50.001 50.002 50.001 50.001 ± 0.001 2.728 2.729 2.726 2.727 2.726 2.727 ± 0.048 60.001 60.001 60.001 59.999 60.002 60.001 ± 0.002 3.268 3.272 3.269 3.888 2.865 3.112 ± 6.293 70.001 70.001 70.001 70.001 70.000 70.001 ± 0.001 3.789 3.798 3.811 2.989 3.790 3.635 ± 9.945 80.000 80.001 80.000 80.000 80.002 80.001 ± 0.001 4.365 4.333 3.994 3.345 3.911 3.990 ± 10.336 90.000 90.002 90.000 90.001 90.002 90.001 ± 0.001 4.911 4.912 4.231 3.881 4.001 4.387 ± 11.280 100.000 100.001 100.002 100.001 100.000 100.000 ± 0.001 5.214 5.111 4.895 4.021 4.233 4.695 ± 11.421 150 Dent. J. (Maj. Ked. Gigi), Vol. 40. No. 3 July-September 2007: 149-151 table 2. The Linear analysis of Phenazone in distillated water The mean of serial Conct. (mg.ml–1) Sy (mg.ml–1) Coefficient Correlation* (r) Vx0** (%) Xp*** (mg.ml–1) 0.503-100.00 0.446 0.971, p < 0.05 (Good) 22.431 (Not good) 33.534 (Not good) 0.503-90.001 0.099 0.998, p < 0.05 (Good) 5.585 (Not good) 7.622 (Not good) 0.503-80.001 0.076 0.999, p < 0.05 (Good) 4.841 (Moderate) 5.834 (Not good) 0.5 to 70.001 0.050 0.999, p < 0.05 (Good) 3.627 (Moderate) 3.802 (Not good) 0.5 to 60.001 0.045 0.999, p < 0.05 (Good) 3.830 (Moderate) 3.412 (Not good) 0.5 to 50.001 0.008 0.999, p < 0.05 (Good) 0.809 (Good) 0.599 (Not good) 0.5 to 40.001 0.008 0.999, p < 0.05 (Good) 1.102 (Good) 0.650 (Not good) 0.5 to 30.001 0.008 0.999, p < 0.05 (Good) 1.579 (Good) 0.706 (Not good) 1.002-100.00 0.132 0.997, p < 0.05 (Good) 6.372 (Not good) 10.656 (Not good) 1.002-90.001 0.100 0.998, p < 0.05 (Good) 5.241 (Not good) 7.917 (Not good) 1.002-80.001 0.077 0.999, p < 0.05 (Good) 4.528 (Moderate) 6.107 (Not good) 1.002-70.001 0.051 0.999, p < 0.05 (Good) 3.355 (Moderate) 3.989 (Not good) 1.002-60.001 0.046 0.999, p < 0.05 (Good) 3.557 (Moderate) 3.651 (Not good) 1.002-50.001 0.003 0.999, p < 0.05 (Good) 0.272 (Good) 0.238 (Good) * Good: r observation >r table at significance 5% ** Not good: at > 5%, moderate: at ranging 2–5%, good: at < 5%. *** Not good: at more than the lowest conc. of their series, good: at least than the lowest conc. of their series. table 3. Analysis of accuracy and precision of procedure determination of Phenazone in human blood plasma Phenazone in distillated water Absorbance (Å) Phenazone in blood plasma (AUF) Replication-1 Replication-2 Replication-3 Replication-4 Replication-5 Intraday precision (% CV) Drug conc. (mg.ml–1) Absorbance (AUF) (Å) AUF Recovery % (Å) AUF Recovery % (Å) AUF Recovery % (Å) AUF Recovery % (Å) AUF Recovery % 1.002 0.050 0.043 86.000 0.047 94.000 0.041 82.000 0.042 84.000 0.046 92.000 5.913 2.503 0.136 0.127 92.029 0.125 91.912 0.123 90.441 0.128 94.118 0.126 92.647 1.508 5.102 0.270 0.259 95.926 0.257 95.185 0.255 94.444 0.258 95.555 0.256 94.815 0.615 10.002 0.545 0.528 96.887 0.527 96.697 0.525 96.330 0.524 96.147 0.521 95.596 0.522 20.002 1.091 1.067 97.800 1.066 97.708 1.063 97.433 1.061 97.250 1.062 97.342 0.243 30.012 1.638 1.601 97.741 1.600 97.680 1.597 97.497 1.598 97.558 1.596 97.436 0.116 50.002 2.727 2.616 95.929 2.618 96.003 2.620 96.076 2.622 96.150 2.630 96.443 0.206 Recovery (mean ± % CV) 94.616 ± 4.514 95.598 ± 2.197 93.460 ± 5.998 94.397 ± 5.002 95.183 ± 2.278 0.862 151Lazuardi: The determination of phenazone apparently good at mean 94.651 percent of recoveries and 0.862 percent of coefficient variation as illustrated at Table 3. discussion The method described here is based partly on the method development by Nuraini et al.,8 but by pressuring of SPe during the elution process, It was make a good recovery in determination of Phenazone in human blood plasma.17 Our recovery was apparently 1.5 to 3% highest than Nuraini et al.,8 method at ranging 93.460 to 95.598%. The bias of precision described here was shown lowest than their protocol, although we saw both of them still satisfaction (The CV < 2%). each step in the assay method has been examined to give optimal results, provided the following precautions are observed. Separation by SPe should be carried out at room temperature by methanol: glacial acetic acid 0.5% (v/v) dissolve in distillated water 2 : 80 for at least 0.05 ml.second–1 rate pressure to ensure complete separation of Phenazone. After drying by N2, all samples must be added by distillated water not more than 15 minutes. That process was doing to kept the chance of pH solution from acid to basic condition. The wavelength of 230 nm will be maximum detecting to aqueous sample in acid condition (5 to 6 pH solution) at not more than 2 hours.4,8 Accurate timing and adequate mixing between the addition of each reagent is essential. If these precautions are taken, then reproducibility between experiments is 2%, and it is possible to determine accurately of 1 mg.ml–1 to 50 mg.ml–1 samples (Figure 1). The sensitivity of the method is limited at least 1 to 50 mg.ml–1 sample concentration, because of a decrease in the yield of Phenazone during the sample preparation especially in pH factor. Our method could be of use in monitoring drug concentrations in human undergoing treatment of Phenazone. Phenazone as an analgesic and anti-inflammatory purposed or for dentistry cases was available effective in human blood bodies at about 250 mg.ml–1 to 500 mg.ml–1. That ranging concentration at above can be monitored by our procedures after diluted 10 times for reduced their concentration up to 50 mg.ml–1 as mentioned at additional standard procedure (Table 1). These procedures could be of use for monitored therapeutics of Phenazone for obtained suitable drug level at ranging 1 to 50 mg.ml–1. 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