Cu-Creatinine- Metol system

Transcription

1 Quantification of Creatinine in Human Serum using Metol as a Chromogenic Probe Materials and methods 6.1. Reagents N-methyl-p-aminophenol sulfate N-methyl-p-aminophenol sulfate also denoted as Metol (Molecular formula = (C 7 H 10 NO) 2 SO 4, Molecular mass = 344.5) is a purple coloured solid with a melting point of 260 o C [1]. Metol was first synthesized by Alfred Bogisch [2]. Metol was initially used as a developing agent in black and white photography. Metol is prepared by the reaction of hydroquinone with methylamine, followed by neutralization with sulfuric acid [3]. Metol can be selectively oxidized to yield a chromogenic product [4]. The author has made an attempt for the first time in developing a chromogenic probe in the quantification of creatinine by spectrophotometric assay method. The main objective in developing this method was to obtain higher sensitivity, more convenience and simultaneously overcoming the need for costly equipment and 150

2 reducing the time gap. During this experimental study linearity, precision, accuracy, standard deviation and interference tests, applicability of the method using serum sample and other related data were compared with the routine pathological kit method. 151

3 6.2. Instrumentation A JASCO model UVIDEC 610 PC spectrophotometer with 1 cm matched quartz cells was used for all measurements. Solution ph was monitored using chemlabs (Nairobi, Kenya) ph meter Reagents and solutions All chemicals used in the assay were of analytical grade and double distilled water was used throughout the assay N-methyl-p-aminophenol sulfate (Metol) Metol was purchased from Himedia. Ltd., India. Solution of 58 mm was prepared by dissolving 200 mg of Metol in 10 ml of double distilled water. The prepared solution was protected from the sunlight by wrapping in a carbon paper. The sample was refrigerated until use. Fresh solution of metol was prepared for every 24 h Copper (Cu) Copper (II) sulphate pentahydrate was purchased from Himedia (India). Stock solution of copper (2.06 mm) was prepared by dissolving 5 mg of copper (II) sulphate pentahydrate in 10 ml of distilled water Acetate/Acetic acid buffer solution (0.5 M, ph 5.4) This solution was prepared by mixing 0.5 M solutions of acetic acid and 152

4 sodium acetate (trihydrate) in 1:3 v/v ratio Creatinine (Cr) Creatinine was purchased from Himedia Laboratories, Mumbai, India. The stock solution was prepared by dissolving 75 mg of Creatinine in 10 ml of distilled water. It was used as standard stock solution and stored in refrigerator at 4 o C. Further dilution with distilled water was made freshly as and when required Analytical method development Different media were tried to develop an appropriate UV spectrophotometric method for the analysis of creatinine concentration. The different criteria employed for the selection of media were sensitivity of the method at particular ph, ease of sample preparation, interference of excipients, cost of reagents and applicability of the method for different samples. Absorbance of reaction product in the selected medium at respective wavelength was determined; apparent molar absorptivity, Sandell s sensitivity, robustness and ruggedness were calculated according to the standard formulae General procedure for the quantification of creatinine Calibration graph for the quantification of creatinine was performed in a final 3 ml reagent mixture containing 1.93 mm Metol, 68.6 µm copper and 1 mm aceticacid / sodium acetate buffer of ph 5.4. The reaction was initiated at 25 o C by adding 100 μl of varying concentrations of creatinine. The reaction mixture was allowed to stand for 30 min at room temperature. Absorbance of the coloured solution 153

5 was recorded at 530 nm with respect to blank containing all reagents of optimized concentration except creatinine Imprecision studies Imprecision studies were carried out to evaluate the magnitude of total imprecision of the proposed method. A final 3 ml solution containing optimized reagent concentration along with varying amounts of creatinine within linearity range was employed for the precision studies. Creatinine with concentrations of 88, 265, 442 μm were selected to analyse the imprecision of the proposed method. The study included 10 runs in a day with a time interval of 1 h for within day precision and 20 days run for day-to-day precision with a time interval of h. All solutions were prepared freshly every day Sandell s sensitivity (S) Sandell s sensitivity is a suitable parameter for expressing the sensitivity of developed UV-spectrophotometric method. It represents the number of micrograms or nanogram of the determinant per milliliter of a solution having an absorbance (A) of for a path length (d) of 1 cm. Thus, S=10-3 /ε s =µg cm -2 where ε s is the specific absorptivity and its value (in ml g -1 cm -1 ) corresponds to the determinant in a cuvette with an optical length of 1cm. Also, ε s = (ε /molecular weight of creatinine) 1000, where ε = molar absorptivity = A/Cd, where, C is the molar concentration of the determinant and d = 1cm path length. 154

6 6.7. Results and Discussion Absorption spectra The absorption spectra of the reaction were investigated using three different concentrations of creatinine in the wavelength range of nm. The reagents were prepared as described in the general procedure. The results obtained showed maximum absorbance at 530 nm as shown in Figure 6.1. Spectral interference of the reagent was eliminated by measuring the coloured solution against a reagent blank containing all reagents except creatinine. Figure.6.1. Absorption spectra of the product with 3 different concentrations of creatinine 155

7 Temperature sensitivity The effect of temperature on color development was investigated. There were almost constant values of absorbance at 530 nm in the temperature range C. Any further increase in the temperature beyond 35 C resulted in a decrease in the absorbance due to the colour development of blank, and decrease in temperature below 20 C resulted in decrease in absorbance due to the slow development of colour. Hence, in the present work, all absorbance measurements were done at 25 C ph for maximum absorbance The effect of ph on the absorption maxima at 530 nm was studied for solutions containing fixed concentration of creatinine and prepared as described in the general procedure in the ph range The different buffers used at 0.1 to 100 mm included, citric acid / potassium citrate at ph , acetate/acetic acid at ph , potassium dihydrogen phosphate/sodium hydroxide at ph , and potassium dihydrogen orthophosphate / dipotassium hydrogen orthophosphate at ph Increase in absorbance of the reagent mixture was observed in acetate/acetic acid buffer. The results obtained showed gradual increase in absorbance of the coloured solution with increase in the ph up to 5.4, above this ph showed decrease in the absorbance up to ph 6 and beyond that no colour was formed. Therefore, total concentration of 1 mm acetate/acetic acid buffer of ph 5.4 was selected as optimum ph for further studies. The change in absorbance with respect to change in ph of buffer is shown in Figure

8 Figure.6.2. Effect of ph on absorbance Effect of time The optimum reaction time was determined by following the colour development spectrophotometrically at temperature of 25 o C. It was found that, complete colour development was attained after 30 min and the colour so developed remained stable for 6 h (Figure.6.3). Figure.6.3. Effect of time 157

9 Effect of reagent concentrations The effect of varying concentrations of metol and copper was studied under optimum experimental conditions with 3 ml solution by varying the concentration of one at each experiment. The results showed increase in the absorbance with increase in each reagent concentration, but it remained constant beyond 1.93 mm and μm for metol and copper respectively. Hence the final optimal concentration was fixed at this level for all further experiments Robustness and Ruggedness Robustness is described as the reproducibility of the analytical method under different circumstances without the occurrence of unexpected differences in the obtained result(s), whereas ruggedness is defined as the degree of reproducibility of the test results obtained under a variety of normal test conditions. For the evaluation of the method robustness, an important experimental variable, reaction time was slightly varied deliberately. The analysis was performed at ±2 min of the optimum reaction time by taking three different concentrations of creatinine. The results were found to remain unaffected as shown by the RSD values, which were in the range of 0.78 to 2.12 %. Ruggedness was expressed as the RSD of the same procedure applied by four different analysts as well as by using three different instruments. The interanalysts RSD were within 0.92 % whereas the inter-instruments RSD for the same creatinine concentration ranged from 1.32 to 3.46 % suggesting that the developed method was rugged. 158

10 6.8. Analytical parameters The calibration graph for creatinine was carried out by fixed time assay method in 3 ml of solution containing 1.9 mm metol, and 68.6 μm copper in 1 mm acetic acid / sodium acetate buffer of ph 5.4. Varying concentrations of creatinine were added and allowed to stand for 30 min at 25 o C temperature. The absorbance of the coloured product was recorded and plotted against the concentration of creatinine as shown in Figure.6.4. Figure.6.4. Calibration graph for the quantification of creatinine Absorbance of coloured solution increased with increase in the concentration of creatinine. The calibration graph of creatinine showed excellent linearity over the concentration range of μm. The linearity graph yielded a regression equation as absorbance = C cr , r 2 = The molar absorption coefficient and Sandell s sensitivity were L mol -1 cm -1, and μg cm -2, respectively and the RSD was % (n = 10). Limit of detection and limit of quantification were μm and μm, respectively. Precision studies were carried out by analyzing solutions containing known amounts of creatinine within the Beer s law 159

11 range; results are shown in Table.6.1. Within day imprecision range was % (n = 10) and day-to-day imprecision range was % (n = 20). The results obtained are summarized in Table.6.1. Imprecision studies Within day precision* Day - to - day precision* X μm SD CV n X μm SD CV n Note: n = number of runs, SD = Standard deviation, CV = Coefficient of variation, X = Creatinine concentration * duplicate measurements 6.9 Proposed reaction mechanism In the proposed reaction metol is selectively oxidized by copper in presence of creatinine leading to the formation of imine group which provides intense purple coloured product. The proposed reaction mechanism is as shown in figure. 160

12 6.9. Interference studies Interference study was performed to assess selectivity of the method for creatinine. Spectral and chemical interferences may occur because of reagents and matrix effects during analytical procedure. Selected potential interference compounds were chosen according to matrix composition and added reagents. Known quantities of many chemical species were added to the solution containing 176 μm of creatinine. A deviation of 3% from the original value of absorbance reading was considered tolerable. The resultant tolerance ratio are summarised in Table.6.2. It can be clearly observed that some compounds like creatinine, glucose, ammonia, nitrate, urea and common inorganic ions showed reliable tolerance under the given conditions, whereas hemoglobin, ascorbic acid, Fe (II), (III), uric acid, nitrite and some drugs showed low tolerance. 161

13 Table.6.2. Interference study Interferants Tolerance ratio a Nickel, Calcium, Iron +2, +3, Nitrite, Phosphate 15 Magnesium, Alluminium, Sodium, Potassium 30 Hemoglobin, Ascorbic acid 35 Ammonia 70 Bicarbonate, Uric acid 190 EDTA 200 Glucose 310 Nitrate 380 Nitrite 510 Sulphamic acid 950 Carbonate 980 Chloride, Creatine 1650 Urea 39,176 Note a Tolerance ratio corresponds to the ratio of limit of inhibiting species concentration to that of 176 μm creatinine used. 162

14 6.10. Recovery and applicability Recovery experiments were performed in order to prove the specificity of our method and the accuracy in pathological creatinine concentration ranges. Serum samples previously analysed by modified Jaffe s kit method in the range of 54.9 to µm were analysed by the proposed method. The results obtained indicated a linear correlation with the diagnostic kit method. Analytical recovery was evaluated by spiking stock creatinine solution to serum samples and analyzing concentration recovered. The mean analytical recovery was 106 %. There was minimal interference and good reproducibility of the assay procedure with creatinine recovery of 101.4% % in serum sample. The results obtained are summarized in Table.6.3. Table.6.3. Recovery of creatinine from human serum sample by the proposed method. Sample Creatinine μm Added Found Recovered Recovery* number Proposed Standard μm μm μm % method a method Note a mean of duplicate measurements * [(final concentration initial concentration)/added concentration] 163

15 6.11. Conclusion Quantification of creatinine by direct spectrophotometric procedure has been attemted. In the optimization of different parameters the proposed method, the reaction ph and the concentration of all components were taken into account; optimal conditions were considered to be those affording maximum absorbance, greatest possible color stability, and maximum linearity. The problems encountered in setting up the procedure involved several aspects of the reaction, namely, the assay of creatinine, test sensitivity, and competition by some interfering substances with the chromogenic system, reflecting the relative non specificity of the proposed system. This study is the first to report on the quantification of creatinine by oxidation of metol in the presence of copper and creatinine. The reagents used for the assay are versatile and economical, the coupled product has maximum absorbance wavelength in the visible region. The reaction requires only room temperature. Buffer studies helped to attain the best absorbance with selective assay thereby avoiding the addition of strong base or acids which results in multiple interferences caused by precipitation. The creatinine catalyzed oxidation of metol in the presence of copper allowed spectrophotometric determination of creatinine within the range of 4.4 to 620 µm. Fixed time assay provides the ease of manual analysis with negligible errors. Interference by foreign substances is negligible compared to other methods. The LOD and LOQ data indicated that microgram quantity of creatinine can be accurately determined. The applicability of the proposed method was investigated through the recovery test in serum samples in which known amount of analyte was added, recovery values showed good reproducibility and accuracy and independent of matrix effects, and is not affected by the sample matrix. These values show that, proposed 164

16 method is suitable for the routine quantification of creatinine in serum sample. Within day and day to day precision showed very less imprecision indicating that the method is highly reproducible. The ease in reagent s preparation and relatively low instrumentation cost make the proposed method potentially feasible and more attractive. 165

17 Literature cited [1] accessed on 15/03/2012 [2] N. H. Furman, R. N. Adams, Anal. Chem., 25 (1953) [3] N. R. Harger, J. Am., Chem. Soc. 41 (1919) 270. [4] M. J. C. Allinson, J. Biol. Chem., 147 (1943)