Difference in Lymphatic Function in Health and Disease State Essay

ABSTRACTHigh Performance Liquid Chromatography has been employ to evolve an analytical procedure for the evaluation of the content of paracetamol in the bulk, window pane forms and in water system, a body fluid. Sepa proportionateityn and cloture have been achieved with a combination of wood alcohol and 2.5% acetic acerbic (1585) on a reversed- phase tug at ambient temperature. Elution was isocratic with UV signal detection at 257nm. Internal precedent normalization rule acting was utilise for quantitation with caffeine as the internal exemplification. represent retention cartridge clips for paracetamol and caffeine were respectively 2.61 0.13 min and 11.98 0.72 min . The calibration curve was linear over the range 0.1-5.0g/ml. The method acting was withal suitable for the assay of paracetamol-codeine combination dose as well as estimation of the amount of constituents in piss when the wavelength of UV detection was 245 nm with acetanilide as the internal hackne yed. Keywords Chromatography, isocratic, internal standard, in vivo and in vitro doorParacetamol (N-(4-hydroxyphenyl) acetamide) tablets are listed among the essential doses selected for the health care delivery brass in Ghana.OHNHCOCH3Figure 1 Chemical Structure of ParacetamolParacetamol is very much used for antipyresis and analgesia without prescription. The medicate is useful in mild to moderate pain such(prenominal) as headache, myalgia and postpartum pain. It is a very good preference for mild to moderate pain in patients who can non take aspirin because of allergy,haemophilia, recital of peptic ulcer and asthma. (Katzung, 1989).As a result of the Ghana political relations policy of generic prescribing, the liberalization of trade and import laws, and the ever-increasing spot of pharmaceutical industries, a wide range of paracetamol overlaps appear on the Ghanian market. According to the Ghana National Drugs Policy, only drugs conforming to nationally trus 2rthy and/ or internationally recognized quality standards shall be permitted to be procured and distributed in the country (Ghana National Drugs Policy, 1999). Any study therefore designed to superintend and improve the quality evaluation of pharmaceutical point of intersections both at the term of registration and post-market is very essential in the policy and technical guidelines of drug regulatory authorities such as the Food and Drugs Board. Such a study overly benefits the Ghana Health Service in the smell that procurement staff, prescribers, dispensers and patients have access to high-quality and efficacious drug products. pharmaceutic industries may also have simple analytical procedures for both in-process and finished product evaluations.The HPLC has been used to determine paracetamol in tablets. Franeta et al (2002) used the HPLC for the19coinciding determination of acetylsalicylic acrid, paracetamol, caffeine and purple heart in tablets on a reversed-phase tugboat explo itation a mixture of acetonitrile and water (2575 v/v) familiarized to pH 2.5 with phosphoric acid.The Bio Rad 1801 UV-Vis detector was used (207 nm). Ramos-Martos et al (2001) also described a rapid reversed-phase HPLC method with UV detection for the coincident determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine and thiamine in pharmaceutical preparations using cardinal successive eluants of water for 5 minutes and acetonitrile-water (75 25 v/v) for 9 minutes, both eluants adjusted to pH 2.1 with phosphoric acid. Codeine was determined at 240 nm whilst the rest were detect at 285 nm. Okine et al (2003) used a mixture of methanol and 0.05M NaH2PO4 (1783), pH2.0 with UV detection (273 nm) for eluting ascorbic acid, paracetamol and caffeine combined in a tablet. Apart from the high cost of acetonitrile for routine analysis, the systems were not selective for unvaried paracetamol in water supply. It therefore becomes imperative to evolve a system th at is cost effective and selective for paracetamol in the bulk mill, window pane forms and biologic fluids such as blood and urine for easier routine in vitro and in vivo monitoring of drug samples.EXPERIMENTALComponents of the Liquid ChromatographPump Spectra System P100 (Spectra Physics) sensor Spectra 100 Variable Wavelength Detector (Spectra Physics) Integrator CR501 Chromatopac (Schimadzu)Sample Injector Syringe fill sample injector fitted with an external 20l loop (Model no. 8125-095) unmoving phase Spherisorb HPLC editorial, S10 ODS2 (10cm, 4.6mm) MaterialsPure paracetamol powder (Chemcon GmbH, Germany), Paracetamol tablets (Phyto-Riker Ltd., Ghana), Paracetamol tablets ( PZ Co Ltd., Ghana), Paracetamol tablets (Tylenol Forte, Cilag Ltd., Switzerland), Paracetamol-codeine combination product (Paracod, Phyto-Riker Ltd., Ghana), Paracetamol-codeine combination product (Co-codamol, Alpharma, UK), pinhead urine sample, deionised water, urine samples with unchanged drug and drug metabolites, methanol (BDH), acetic acid (BDH), potassium dihydrogen phosphate (BDH), caffeine (BDH), salbutamol sulfate (Shubhmets, Mumbai, India), citric acid (Acid India) and phenyl ethanolamine (Blue Bird, Mumbai, India)Method Design ConsiderationsInformation on the physico- chemic properties of paracetamol and the otherwise chemical substances above were searched. Details considered include solubility properties, chemical structures, acid dissociation constants (pka), level of purity, stability in net result and ultraviolet light submergence pattern in acidic, basic and neutral media with their respective wavelengths of maximum compactness (M shootat, 1986 British Pharmacopoeia, 2000). The substances were make up moderately icy. Based on their frozenity, reversed-phase HPLC wasconsidered more relevant because in this mode, a nonpolar stationary phase and a polar bustling phase were utilised so that more polar substances were eluted in front the comparatively n onpolar. The differences in the physico-chemical properties of paracetamol and the other chemicals aided in selecting an internal standard for the study since they all interacted variantly with a chosen combination of industrious phase to give a chromatogram of various withdrawals, resolutions and retention quantify.Various combinations of methanol/phosphate buffer and methanol/water (pH and bonce strength marked with acetic acid) were tried in order to optimise tower contentedness factor for separation and resolution. individually submergence of phosphate buffer or water (various pH) was combined with methanol in various proportions, starting with a 5050 combination and gradually increasing and decreasing the aqueous content while monitoring their respective effects on separation and resolution. tout ensemble the mobile phase combinations tried could elute both paracetamol in the bulk powder and tablet matrix with reasonable retention, but not all the other chemicals b eing considered for an internal standard (caffeine, salbutamol, citric acid and phenyl ethanolamine). Some had poor resolution and tailing visors while others had poor resolution and unduly long retention times.Some of the mobile phase combinations that could handily separate and resolve paracetamol in vitro could not separate and resolve distance urine (urine from a healthy person ahead drug was administered) bar with a standard solution of paracetamol. Further altering the combination ratio, ionic strength and pH of the mobile phase produced the optimum system that could satisfactorily resolve paracetamol in the bulk powder, tablet matrix, spiked blank urine and unchanged paracetamol and other paracetamol metabolitesexcreted in urine. Among the list of chemicals for an internal standard, caffeine was found the best beneath the optimum chromatographic conditions of the study. The best mobile phase combination was methanol/2.5 % acetic acid (1585). Elution was isocratic becaus e a single mobile phase combination was used. aft(prenominal) other investigations, the best wavelength of maximum preoccupation for UV detection was 257 nm, absorption unit fraction scale (aufs) for vicenary detection of the analyte at very abject tightfistednesss , 0.5, flow rate of mobile phase, 1.5 ml/min and chart recorder speed, 5 mm/min. cooking of mobile phaseThe volume of mixtures do not usually concern the sum of the separate volumes do up the mixture as a result of differences in density and other physical factors such as volume expansion and contraction. The mobile phase was therefore alert by measuring separately the volume of from each one component and mixing them unneurotic. All mobile phases prepared were filtered through a membrane filter before use.Validation of Analytical MethodVarious parameters can be evaluated for validating any refreshedly developed analytical system. These include linearity, precision, accuracy, sensitivity and resemblance to ot her standard methods. Comparison of upstart method with standard spectrophotometric method, (BP, 2000) The method was applied to paracetamol products from three pharmaceutical companies. Twenty tablets of each of the experimental paracetamol products were weighed together and finely powdered. A quantity of the powder containing 0.15g of paracetamol (0.1692g of Phyto-Riker Paracetamol, 0.1578g of PZ Paracetamol and 0.2001g of Tylenol Forte) was weighed and quantitatively transferred into a 200ml volumetric flask with 50ml of 0.1M NaOH and and then diluted with 100ml of distilled water and shaken railroad carmatically for 15 minutes. Sufficient distilled water was then added to produce 200ml. After filtration, go on dilutions were made with distilled water such that the final concentration of paracetamol in solution was 0.00075 %w/v and the NaOH, 0.01 M. The absorbance of the resulting solution was then taken in reproduce with the Cecil 7020 double beam UV spectrophotometer at a wavelength of 257nm with quartz cuvette of agency length 1 cm using 0.01 M NaOH as the blank solvent.New MethodFor each of the experimental brands, sample preparation was make by crushing 20 tablets. A quantity of the powder alike to 0.1g of paracetamol (0.1128g of Phyto-Riker Paracetamol, 0.1052g of PZ Paracetamol and 0.1333g of Tylenol Forte) was weighed and quantitatively transferred into a clean 100ml volumetric flask with 20ml of methanol. It was then mechanicallyshaken for 10 minutes. It was diluted to the 100ml mark with deionised water. Insoluble excipients were filtered off through a medium porosity sintered glass filter. A 0.1%w/v aqueous solution of caffeine was also prepared as a stock internal standard solution. A final solution containing 0.00025% paracetamol and 0.001 %w/v caffeine was prepared for the HPLC analysis.Triplicate injections onto the column were successively done for each of the experimental brands. Average bakshis discipline ratios (test sample/int ernal standard) for the various samples were calculated from their chromatograms. The actual concentration of paracetamol in each of the samples analysed was interpolated from a calibration curve using the average peak nation ratios. The students t-test was used to compare the means of the deuce methods while the variances were compared with the f-test.Calibration GraphThe range of concentrations used was 0.000005%-0.001%w/v. The new HPLC method was used for the determinations. Injections were done in triplicate for each of the concentrations in the above range. A graph of average peak area ratio was plotted against concentration. All the concentrations used gave signals but not all the signals were relative to concentration. Concentrations that were not detected proportionally defined the limits of detector linearity under the given set of experimental conditions. Before preparing the solutions for the calibration curve, the paracetamol reference powder was identified and charact erised according to BP 2000. ratiocination of inter-day interlingual rendition ofanalytical methodThe inter-day variation was investigated to assess the likely variations in results from day to day when the new method is used. This involved the HPLC assay of paracetamol of some the analogous concentrations on two antithetic days under same conditions. The results for the two different days were paired and the f-test applied to determine likely significant difference in their variances.Determination of intra-day variation of the analytical method Several assays of paracetamol were done within the same day to determine the repeatability of the new method. Seven sets of solutions of approximately the same concentration (0.0001 %w/v) from the same homogenous stock were prepared and successively analysed with the HPLC. Each set was run three times and the average peak area ratio taken to represent that set. Actual concentrations of solutions were interpolated from the calibration graph. The relative standard deviation of results was calculated to determine the level of repeatability. The concentration of paracetamol solution was so chosen to ensure that readings were taken within the linear region of the calibration curve. Application of analytical procedure to urine, a body fluidAfter following all pharmacokinetic protocols (Shargel and Andrew, 1993) six healthy male volunteers between the ages of 26 and 32 years were each given 1.0g of paracetamol tablets and had their urine samples collected at specific time intervals for 24 hours. All subjects had the same time points of urine collection after administration of the tablets and these were 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 9.0, 12.0, 18.0 and 24.0 hours. The entire volume of urine voided during each sampling interval was pooled together and recorded and the analysis done immediately. Where urine samples had to be kept overnight, quantities were kept tight in sample tubes and frozen. otherwise applica tionsThe new HPLC method was also applied to a combination product containing paracetamol and codeine both for assay and determination of unchanged forms of the two constituents in urine. RESULTS AND handlingSince paracetamol is a compound of moderate polarity as shown by its chemical structure in Figure 1, a reversed-phase column with a polar mobile phase was used. The mobile phase was methanol/2.5% acetic acid (1585). In reversed-phase separation, compounds were separated based on their hydrophobicity. Retention increased as the solutes decreased in polarity thus, polar species were eluted first. Hence, eluting time increased by increasing the polarity (water content) of the eluent. The pH of the eluentas well as the pka of the drug being separated affected the elution profile. Figures 2 (a-c) therefore show different elution profiles and retention times because of the differences in the physicochemical properties of the analytes under review. The retention time of caffeine (11.9 8 0.72 min) was greater than paracetamol (2.61 0.13 min) because it was greatly retained on the column as evidenced by the tailing nature of the caffeine peak because of its relatively greater hydrophobicity. The components of the blank urine though poorly resolved, had shorter retention times ( 2.5 min) than paracetamol because they were relatively more polar and interacted interrupt with the polar mobile phase, resulting in decreased retention.The presence of aromatic rings together with auxochromes in the chemical structures of paracetamol and caffeine made UV absorption possible for monitoring the column effluent. As regards the intra-day precision of the new method, the relative standard deviation (RSD) of perennial assay of separate identical samples of concentration 1g/ml was 2.17% ( duck 1 and 2). According to Dong (2000), only HPLC analysis with modern auto samplers yields RSD of less than 2.0%. Manual sample injections with RSD of 2.17 can therefore not be said to have a poor potential to give ordered data under the same experimental conditions. Random errors from analysts might as well have contributed to the RSD value being greater than 2.0%. The degree of concordance among the individual utterances was indicated by the value of the imperative precision (0.02). This appears small and suggests a good level of agreement between test results. The inter-day precision from Table 3 and 4 was high as there was no statistical difference between the variances of the set of analytical data generated for two different days at a confidence level of 95%. The method therefore was reproducible and could produce data for peer analysis.There was a positive correlation between peak area ratios and the concentrations of analyte (Figure 3). Correlation coefficient (r2) of 0.9998 implies that the spatter presented in Figure 3 was faithful enough for predictable purposes within limits of detector linearity. From Table 5, the range of detector linearity was est ablished as 0.1-5.0 g/ml. Under the set of experimental conditions, the lowest concentration of paracetamol that was detected but did not necessarily produce a signal that was proportional to concentration was 0.05 g/ml. However, signal for 0.2 g/ml paracetamol solution was approximately twice that of 0.1 g/ml. Since the minimum concentration of paracetamol that started varying proportionally with peak area ratio was 0.1 g/ml, it was chosen as the limit of quantitation of the method. This observation was in good agreement with what has been reported that in many cases, the limit of quantitation is approximately twice the limit of detection (Sethi, 1993 Olaniyi, 2000). The upper limit of quantitation was also set at 5.0g/ml because there was no proportional increase in peak area on increasing paracetamol concentration from 5 to 10 g/ml.Evidence of correlation between the new method and that of British Pharmacopoeia (2000) for the assay of paracetamol tablets was positive. The F-test at 95% confidence level, showed no significant difference between the variances of both the HPLC and UV methods (Table 6). This means that within certain limits, both methods have comparable precisions. However, the absolute precision of the two methods at the same confidence level indicates that the HPLC method has a better precision. The absolute precision values were respectively 1.90 and 2.12 (Table 7). As regards accuracy, even though the results of both methods complied with BP (2000) limits for content of paracetamol in tablets (Table 8), a significant difference was find between the means of the two methods when the students t-test was applied (Table 6). Assessing the absolute error of the mean for the two methods, the HPLC results (2.3%) was found to be more accurate than the UV (4.3%) (Table 7).Moffat (1986) reported that when a dose of paracetamol tablets is administered orally, close to 5% is excreted unchanged in urine. As found in this study, only a small fraction of the absorbed dose was excreted unchanged for all the paracetamol products. These were 5.30.9%, 5.31.2% and 5.00.7% respectively for Paracetamol CoA, Paracetamol great black-backed gull and Paracetamol CoC (Table 9). These values are closely in agreement with what has already been reported, making the new method suitable for the detection and quantitation of paracetamol in urine.CONCLUSIONSParacetamol in the bulk, dosage form and urine has been analysed accurately and precisely by HPLC with Methanol / 2.5% acetic acid (15 85) in the reversed-phase mode at a wavelength of 257 nm using caffeine as the internal standard. The method has also been used for the detection and quantitation of codeine and paracetamol in urine as well as codeine-paracetamol combination tablet. The wavelength of detection in this case was 245 nm with acetanilide as the internal standard.REFERENCESBritish Pharmacopoeia (BP) (2000). Volumes I &II, CD-ROM, The British Pharmacopoeial Commission. Dong, W. M. (2000) . Precision in HPLC. In Todays Chemist at Work (2000), 9 (8) 28-32. Franeta, J. T., Agbaba, D., Eric, S., Pavkov, S., Aleksi, M. and Vladimirov, S. (2002). HPLC assay of acetylsalicylic acid, paracetamol, caffeine and phenobarbital in tablets, Farmaco Sep 57 (9) 709-13 Ghana National Drugs Policy (1999). Ministry of Health, Ghana. pp 4, 7, 12 and 19 Katzung, G. B. (1989). Basic and Clinical Pharmacology, 4th edition, Appleton and Lange, Norwalk, CT. p 444Moffat, A. C. (1986). Clarkes closing off and Identification of Drugs, second edition, the Pharmaceutical Press, London. pp 420-421, 849-850Okine, N.N.A., Asiedu, K.S. and Acheampong, J. (2003). RP-LC determination of ascorbic acid, paracetamol and caffeine in multicomponent anti-cold preparation, journal of Science and Technology, 23 (1) 55Olaniyi, A. A. (2000). Principles of Drug Quality Assurance and Pharmaceutical Analysis, Monsuro Publishers, Ibadan, Nigeria.Ramos-Martos, N., Aguirre-Gomez, F., Molina-Diaz, A., Capitan-Valley, L. F. (2001). Application of liquid chromatography to the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine and thiamine in pharmaceutical preparations. J.A.O.C. Int. May-Jun 84 (3) 676-83Sethi, P. D. (1993). Quantitative Analysis of Drugs in Pharmaceutical Formulations, 2nd edition, C.B.S Publishers and Distributors, New Delhi. pp 33-37Shargel, L., Andrew, B. C. Y. (1993). Applied Biopharmaceutics and Pharmacokinetics, 3rd edition, Appleton and Lange, Norwalk, CT. pp 205-209