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
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