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E-mail: smennick udec. The linearity, precision, detection and quantification limits, accuracy and selectivity of the method were validated.
The method was linear between 0. The intra-assay variation repeatability was between 5. The detection limit was 0. The method proved accurate, with a recovery percentage of In conclusion, is a good method for the quantitative determination of carbamazepine in saliva.
Key Words: carbamazepine, HPTLC high performance thin layer chromatography or instrumental thin layer chromatography or instrumental planar chromatography , quantitative analysis, saliva, analytical parameters, drugs, epilepsy.
Periodically, neurons depolarize abnormally causing paroxysmal alteration in motor, sensory, or behavioral activity 1 - 3. Carbamazepine is one of the most widely used anticonvulsive drugs in the treatment of epilepsy 2 - 4 , especially for the control of partial and tonicoclonic seizures 2 , 4 , 5.
This drug has a low solubility and its metabolism is self-induced or induced by other drugs as well. This produces a poor relationship between dosage and effect. Therefore, it is fundamental to monitor plasma concentrations of carbamazepine in order to obtain safe, non-toxic and effective therapeutic regimens 2 , 6 , 7 , 8.
This study deals with the use of saliva as a biologic fluid in the analysis of carbamazepine, using instrumental planar chromatography HPTLC as the analytical technique. Saliva is a fluid that can be easily obtained with the aid of non-invasive methods, an aspect of crucial importance in the studies of children, elderly patients and pregnant women, as well as in all situations where samples need to be obtained without exposing a patient to discomfort, skin irritation and risk of infection.
The use of saliva instead of blood is a help for the adherence of the patients to their controls. Furthermore, the rationale for measuring drug concentration in saliva is that the pharmacological action depends on the unbound fraction of the drug in the plasma; this fraction is usually excreted by the salivary gland into the saliva 8 - Carbamazepine is non-ionized at the pH range of saliva, an important factor in the analysis of this fluid 8.
Also, this drug is widely distributed throughout the body in areas such as the brain, duodenal fluids, bile and saliva 7. The reported saliva concentrations are between 1. HPTLC is a technique carried out within a short period of time, requires few mobile phases and allows for the analysis of a large number of samples simultaneously. The ability of HPTLC to analyse many samples in parallel has the advantage over other techniques in that the separation of 10 or 20 samples only takes the same amount of time as the separation of 1 sample.
Amounts on the order of nanograms UV and smaller than picograms fluorescence can be detected All of the reagents were of pro-analysis quality Merck. Carbamazepine solutions were prepared in ethanol Saliva of volunteer donors was used for the recovery study. Sample application was carried out on 3 mm bands using a semiautomatic Linomat IV device.
The length of development was 5cm ocurring in a period of 10 minutes. Horizontal development chambers were used. Determinations were performed at a wavelength of nm.
Validation was performed in compliance with international standards 13 , 15 , 16 , using adequate statistic estimates relative standard deviation, Student t, variance analysis 15 - Each solution was applied in triplicate. Concentrations solutions of 0. The amount of analyte by spot versus average response peak area was graphed and the equation for this curve was determined, thereby obtaining an estimate of the target response: ybl The ybl value corresponds to the intersection of the curve.
Subsequently, a second curve was graphed showing the amount of analyte by spot versus standard deviation of the responses. From the equation of this curve, we obtained an estimate of the standard deviation for target: sbl 15 , which corresponds to the intersection of this curve.
These cover the linear range determined applying 1 uL. To assess the repeatability of the analytical method, five solutions of every concentration were prepared, each of them being applied one time 13 , 15 Repeatability of the system, however, was done using one solution of every concentration and each was spotted in triplicate All applications were made on the same plate, by the same analyst, on the same instrument and on the same day. To assess reproducibility, one solution of every concentration was applied five times during five days, using a different plate each day.
Accuracy was evaluated by means of recovery assays. The solutions were centrifuged at rpm and the supernatant was used. The recovery of the active principle was determined by comparison of a prepared concentration and an obtained concentration. Each solution was applied three times in order to obtain the mean recovery. The obtained concentration was calculated from the equation of concentration versus peak areas graph.
The latter two are the main metabolites of carbamazepine and can therefore be found in saliva samples. The solution was prepared at a concentration of 0. This range include to the expected carbamazepine concentrations in saliva 1. The calibration curve for carbamazepine had a regression coefficient of 0. The sensitivity of this method is adequate for the detection and quantification of carbamazepine in saliva, since the detection limit was 0.
Figure 2 shows the curve for the amount of analyte by spot versus average response peak area in this study. The regression coefficient of this curve was 0. Quantity of carbamazepine by spot versus means of peak areas for each concentration. Curve for determination of detection and quantification limits. These values are adequate for the analysis of drugs in biological fluids The method allows separation of carbamazepine from its main metabolites with a resolution of 2.
Figure 3. The fact that no extraction is required gives advantages that need not be emphasized. The very small amounts of sample required for the assay make the method ideally suitable for the study of carbamazepine levels in body fluids that usually can be obtained only in small amounts e. Although we made an exhaustive search at libraries and the internet, we were unable to find other studies on carbamazepine similar to ours, however we found some investigations done on drug determination in saliva involving other drugs or other analytical methods 17 - The method showed high sensitivity, within the ng order, and was selective for the active principle studied, allowing for good separation of its metabolites.
Therefore, we propose this method for the quantitative determination of carbamazepine in saliva. Garnett, Pharmacotherapy. Hardman, L. Gorrel, U. Ryan et al, J. Child Adolesc. Page et al. Pichini et al, Clin. Malamud, BMJ. Lifhitz, Z. Ben-Zui, R. Gorodischer, Ther.
Drug Monit. Rylance and T. Moreland, Archives of Disease in Chilhood. Wilson, Ther. Quattrochi, S. Abelaira, R. Tomlin, I. McKinlay and I. Pichini, I. Altieri, P. Zuccaro and R. Pacifici, Clin. Klitgaard and O. Kristensen, Eur. Ohkubo, M. Suno, M. Kudo, T. Uno and K. Sugawara, Ther. Thijssen, C. Gispen-De Wied, G. Veeman, Clinical Chemistry. Rojas, I. Riberos, M. Lifshitz, Z.
Introducción A La Hplc Aplicación Y Práctica (o. A. Quattrocchi, S. A. De Andrizzi & R. F. Laba)
The procedure consists on the direct injection of filtered and diluted samples in a reverse phase system with ultraviolet detection nm. The separation of peaks of parabens is achieved without any interference of the matrix. The relative standard deviation was 0. The method is quick and simple being recommended its use as quality control of the previous formulation, previous validation of it self. Preservatives are substances added to formulation in order to avoid the multiplication or development of microorganisms in the preparation. They should be used in a concentration high enough to protect the preparation and at the same time not to irritate the skin or other tissues in which they are applied.
2012, Number 4
Bioelectrochemistry Laboratory. In this work an HPLC stability-indicating method was developed and applied to study the hydrolytic behaviour of lovastatin in different simulated fluids. The developed method exhibited an adequate repeatability and reproducibility CV 0. Furthermore, the detection and quantitation limits were 9. Lovastastin exhibited a pH-dependent degradation with an instantaneous hydrolysis in alkaline media at room temperature. One or two degradation products could be observed when lovastatin is hydrolyzed in alkaline or acid medium, respectively. The degradation products from lovastatin retain the UV-spectra of the parent drug, evidencing that the chromophore structure remains unaltered.
Introducción a la HPLC Aplicación y Práctica (O. A. Quattrocchi, S. A. de Andrizzi & R. F. Laba)
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Introduccion a La HPLC Quattrocchi
E-mail: smennick udec. The linearity, precision, detection and quantification limits, accuracy and selectivity of the method were validated. The method was linear between 0. The intra-assay variation repeatability was between 5. The detection limit was 0. The method proved accurate, with a recovery percentage of