Subhash chaudhary 2.pmd

Vol. 26(1), 247-250 (2010)
Field method for the micro-quantitative determination
of tetracycline in human urine and blood serum
SUBHASH CHAUDHARY, SYED KASHIF ALI and Y.P. SINGH
Department of Chemistry, D.S. College, Aligarh - 202 001 (India).
(Received: July 12, 2009; Accepted: August 07, 2009) ABSTRACT
This method described the determination of tetracycline in blood serum and it is based on the formation of an Eu(III)-tetracycline complex. Another spectrophotometric method2 utilizing the reactionof tetracycline with p-N, N-dimethylphenylenediamine and chloramines T was described. A sensitiveliquid chromatographic method (10) has been adopted for the simultaneous determination oftetracycline, oxytetracycline and minocycline in serum. Tetracyclines were separated from otherserum components by RPLC with buffered MeOH mobile phase. UV absorbance of the columneffluent was monitered at 267 nm.
Key words: Determination, tetracycline, urine and blood.
INTRODUCTION
spectrophotometr y at 353 and 365 nm fordetermining tetracycline and oxytetracycline, respectively. The ion-pair extraction of tetracyclines quantitative estimation has been of a great interest from body fluids using dyes as counter ions was also described4. Tetracyclines form ion-pair complexwith dyes that can be extracted in CHCl and re- extracted in HCl and finally quantified by available for the analysis of tetracycline in biological spectrophotometry. 98% of tetracycline from the body fluids containing 10µg/ml of tetracycline can spectrophotometry1. This method described the determination of tetracycline in blood serum and it chromatography has also been used for the is based on the formation of an Eu(III)-tetracycline determination of tetracycline in biological fluids.
complex. Another spectrophotometric method2 Reeuwijk and Tjaden5 have explored the possibilities utilizing the reaction of tetracycline with p-N, N- of using two non-ionogeni resins (α AD-2 and dimethylphenylenediamine and chloramines T was PRP-1) in the chromatograph of tetracyclines and described. The tetracycline was then extracted by their degradation products in biological samples.
BUOH, and the absorbance measurement carried Another HPLC procedure which allowed the rapid out at 640 nm. The method was applied for the determination of tetracycline in blood was also estimation of tetracycline in blood and urine with a described6. The detection limit was 10-500 ng/ml.
relative standard deviation ranging between 1.63- Another HPLC method was also reported7. A rapid 2.11 and recovery 98.0 – 99.3%. Essien et al. 3 and accurate determination of tetracycline in human serum by reversed-phase HPLC with fluorescence polarographic and UV spectrophotometric methods detection was developed8, based on protein for the analysis of tetracyclines in urine. The precipitation in serum. The detection limits of this polarographic one combining d. c. and differential method were 10-35 ng/ml for three different pulse polarography was more suitable than tetracyclines. Tetracycline was also determined in Chaudhary et al., Orient. J. Chem., Vol. 26(1), 247-250 (2010)
human urine by HPLC with PLRP-S column and determination of tetracycline hydrochloride using guard column, a mobile phase of 7.5 mM H PO - Dowex 1 x 8 resin beads as detection. The methods MeCN-MeOH (20:33), and detection at 355 nm 9.
has been successfully applied to human urine andserum samples.
method10 has been adopted for the simultaneous EXPERIMENTAL
determination of tetracycline, oxytetracycline andminocycline in serum. Tetracyclines were separated Reagents
buffered MeOH mobile phase. UV absorbance of India) 1, 3, 5-Trinitrobenzene (Fluka, guaranteed the column effluent was monitered at 267 nm.
reagent), Sodium Hydroxide (Qualigens, India), Concentrations as low as 0.2 µg/m. of tetracyclines Dowex 1 x 8 resin beads (BDH, England) and in serum were quantitable. Wenzel and co-workers dimethyl sulfoxide (Emerk India Ltd.) were used.
11 have discussed a liquid chromatographic and flowinjection analysis for the deter mination of Solutions
tetracycline in uring and serum using sensitized europium (III) luminescence detection. The method hydrochloride was prepared by dissolving 2.0mg was highly selective for tetracycline since few in1.00 ml urine. Another stock solution of tetracycline compounds are capable for transferring energy to hydrochloride was prepared by dissolving 2.0mg in Eu(III). Novaka12-13 has presented a thin layer 1.00 ml serum 0.01 mol 1-1 of sodium hydroxide chromatographic method for the identification of was prepared in distilled water. 1.0% 1, 3, 5- tetracycline in urine. Samples were extracted with Trinitrobenzene was prepared in distilled ethanol.
Et acetate (with or without acidic hydrolysis) and Double distilled water was used throughout.
the extractions were separated on Silufol platesusing a mobile phase of Chloroform-methanol-0.1 Procedure
Determination of tetracycline hydrochloride visualized by UV light at 254 nm or by spraying in urine 0.50 ml tetracycline hydrochloride from the with aqueous solution of Fast Blue B. A differential stock solution was pippitted out made upto the mark pulse polarographic method for the determination with distilled water in 10ml calibrated flask. From of oxytetracycline in human urine and serum in acid this flask, 20-100 µg were taken in a 50ml breakers.
media was also proposed14. The detection limit was To the aliquots, 1 ml of 1.0% 1, 3, 5-trinitrobenzene, 5.5 × 10-6 mol 1-1. Adsorptive stripping voltammetric 2ml dimethyl sulfoxide and little amount of Dowex 1 method15 was also developed for the Quantative x 8 resin beads were added. The solutions are then determination of tetracyclines in urine based on titrated using a micro-burettle, against 0.01 mol 1-1 controlled adsorptive preconcentration of the sodium hydroxide. Transition in colour of the resin antibiotic on the hanging mercury drop electrode beads from light yellow to deep brown signifies the (HMDE), followed by tracing the volatammogram end point. It was prepared with a blank titrated under in a cathodic potential scan. The modes used were same set of conditions of observe the sharp d. c. stripping voltammetry (DCSV) and differential distinction in colour change of the resin beads.
pulse striping voltammetry (DPSV). Fluorimetricmethods have been extensively used for the Determination of tetracycline hydrochloride in
determination of tetracycline in biological fluids and recently appeared in the literature16-23. Moreover, titrimetric methods of analysis and the use of ion hydrochloride was pipetted out from the stock exchange resin beads in color reactions are still solution and diluted up to the volumes with distilled very widely used owing to their simplicity and wide water in a 10ml volumetric flask. From the flask 20- 100 µg were taken in a 50ml beakers. To the aliquots,1 ml of 1.0% 1, 3, 5-trinitrobenzene, 2ml dimethyl In this chapter, we describe a sensitive and sulfoxide and little amount Dowex 1 x 8 resin beads accurate titrimetric method for the micro-quantitative were added. The remaining procedure is the same Chaudhary et al., Orient. J. Chem., Vol. 26(1), 247-250 (2010)
as in the case of tetracycline hydrochloride in urine unknown amounts of tetracycline hydrochloride can be completed either from Fig. 3.1 and 3.2 or directlyfrom the equation summarized in Table 3.2. The RESULTS AND DISCUSSION
method was successfully applied to thedetermination of tetracycline hydrochloride in 10 human urine and 10 serum samples of healthy by taking solutions of varied amounts of tetracycline hydrochloride in urine and serum. Each solution wastitrated with sodium hydroxide, as described inthe After evaluating the developed procedure, experimental part, and the end point recorded. A the % recovery was found to be 99.90 and 99.47% straight line calibration curves were obtained when with a standard deviation of 0.64 and 0.44 µg/ml the varying amount of tetracycline hydroxide in urine and % relative standard deviation of 1.06 and 0.74% and serum samples were plotted against the volume at 95% confidence level for the determination of tetracycline hydrochloride in urine and serumrespectively. The correlation coefficient in both the determination was equal to 1.0000. Therefore the tetracycline hydroxhloride in urine. Fig. 3.2 shows linearity of the calibration graphs and conformity of the calibration curves for tetracycline hydrochloride the systems to Beer’s law are proved by the high in serum. The data used in plotting both the value of the correlation coefficients of the regression calibration graphs are listed in Table 3.1. The that tetracycline forms charge-transfer complex with1, 3, 5-Trinitrobenzene. On addition of sodiumhydroxide, the charge-transfer complex between 1,3, 5-Trinitrobenzene is first formed. When a slightexcess of sodium hydroxide added 12, 3, 5-Trinitrobenzene like other polynitro aromatics, isexpected to form anionic sigma complexes withbases. This seems all the more likely becausedimethyl sulfoxide stabilizes the colour. The deepbrown coloured anionic sigma complex is absorbedon the resin beads indicating the end point.
Fig. 3.1: Calibration graph for the
determination of tetracycline
importance of tetracycline hydrochloride, it was hydrochloride (TCHC) in urine
Table 3.1: Data used in plotting the calibration
graphs for the determination of tetracycline
hydrochloride (TCHC) in urine and serum
Concentration of
Volume of NaOH (ml)
TCHC (µg)
Fig. 3.2: Calibration graph for the
determination of tetracycline
hydrochloride (TCHC) in serum
Chaudhary et al., Orient. J. Chem., Vol. 26(1), 247-250 (2010)
thought in the first instance to develop a titrimetric consuming method than the existing ones.
method for its determination in human urine and Therefore, a complimentary method was devised blood serum, which can be used in the routine analysis and provides more simple and less time REFERENCES
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