Forensic Science International, 59 (1993) 15 - 18 Elsevier Scientific Publishers Ireland Ltd.

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DETERMINATION OF COLCHICINE IN BIOLOGICAL FLUIDS BY REVERSE-PHASE HPLC. VARIATION OF COLCHICINE LEVELS IN RATS*

P. FERNANDEZ, A.M. BERMEJO, M.J. TABERNERO, M. LOPEZ-RIVADULLA and A. CRUZ

Department of Legal Medicine, Service of Forensic Toxicology, Faculty of Medicine, University of Santiago de Compostela (Spain)

(Received August 6th, 1992) (Revision received September 21st, 1992) (Accepted December 2nd, 1992)

Summary

A reverse-phase HPLC method for the determination of colchicine in biological fluids is proposed. Blood, liver and kidney colchicine concentrations were determined in rats at different times follow- ing intraperitoneal (i.p.) injection of 10 mg/kg of the drug. Colchicine was extracted from the samples studied using dichloromethane at pH 8. The dry extract was redissolved in the mobile phase and analyzed with simultaneous detection at 254 and 350 nm.

Key word-s: Colchicine; Reverse-Phase HPLC; Rats

Introduction

Colchicine is a natural alkaloid obtained from the bulb and seeds of Colchicum, which has been traditionally used in the treatment of gout.

The first serious studies that were carried out to determine colchicine levels involved the application of radioimmunoassay [l], spectrofluorimetry [2] and atomic absorption spectroscopy [3]. However, the need to separate colchicine from its metabolites requires the use of the other techniques such as thin layer chromatography (TLC) [4,5] and more recently high performance liquid chroma- tography (HPLC) and gas chromatography (GC) [6 - 81. This study describes an HPLC sensitive method to determine colchicine levels in biological fluids and to examine its variation as time elapses after an i.p. dose of 10 mgikg.

Correspondence to: P. Fernandez, Department of Legal Medicine, Service of Forensic Toxicology, Faculty of Medicine, University of Santiago de Compostela, C/San Francisco, S/N 15705 Santiago de Compostela, Spain. *Presented at the 26th International Meeting of TIAFT, Glasgow, August, 1989.

0379-0738/931$06.00 0 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Materials and Methods

Analytical procedure A Waters HPLC system with a manual injector, a 150 x 3.9 mm stainless

steel analysis column packed with 5 pm Lichrospher RP-18, a Waters 501 pump, a Waters 490 UV detector, a Professional 350 Digital Computer (for recording) and an Interface Module was used. The mobile phase used was metha- nol:acetonitrile:phosphate buffer, pH 7.6 (205:75:220, v/v/v).The flow rate of the eluent was 0.5 ml/min (1300 psi). Detection was carried out at 350 and 254 nm simultaneously.

Calibration curve The internal standard, quinidine, at a concentration of 10 @g/ml was added to

working solutions containing 1, 2, 3, 4 and 5 pglml of colchicine which were prepared by adding aliquots of a standard solution of the drug (1 mglml) in meth- anol to 1 ml of mobile phase. Twenty-five microliters of these mobile phase solu- tions were then injected onto the HPLC column and the calibration line was constructed by plotting the mean colchicine area/quinidine area ratios (Y) against the corresponding concentrations of colchicine (X).

Because of the light sensitivity of colchicine [5], the solutions were protected with aluminium foil. This approach was also necessary during the extraction pro- cedure in order to avoid the decomposition of colchicine and to obtain a good reproducibility of the results.

Extraction procedure Two-hundred microliters of the working plasma solutions containing colchicine

1,2, 3, 4 and 5 pg/ml, were adjusted to pH 8 with 1 N NaOH and then extracted with 3 ml of dichloromethane. The mixture was shaken gently for 15 min and then centrifuged for 5 min. An aliquot (2.5 ml) of the organic layer was separated and washed with 0.5 ml of phosphate buffer, pH 7.6. Two milliliters of the organ- ic layer were evaporated under a stream of nitrogen at 60°C and the dry residue was reconstituted in 100 ~1 of mobile phase containing quinidine (10 pg/ml). After mixing, samples (25 ~1) were then injected into the chromatograph and the cali- bration line was constructed similarly.

When the extraction was carried out on viscera (10 g), concentrated hydrochloric acid and ammonium sulphate were added until protein precipitation was completed. The solution was then filtered and an aliquot from the resulting solution was subjected to the extraction procedure described.

Animal experiments Sprague white rats ranging in weight from 250 to 300 g were used. The rats

were given an i.p. injection of colchicine (10 me/kg). After the experimental time had elapsed (1, 2 and 24 h), the animals were sacrificed and blood, liver and kid- ney samples were collected and processed according to the method described.

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Results and Discussion

Figure 1 shows the chromatogram obtained after injection of 25 ~1 of a solution containing 5 pglml of colchicine and 10 pglml of the internal standard. A good resolution was observed, with a retention times of 5.33 min for colchicine and 7.17 min for quinidine.

Consistent linear calibration curves were obtained; at 350 nm: water, Y= 0.418X -0.0190, r = 0.995; blood, Y = 0.281 X -0.0047, r = 0.990 and at 254 nm: water, Y = 0.0896X -0.0032, r = 0.905; blood, Y = 0.0718X -0.0015, r = 0.927. The higher correlation coefficients obtained at 350 nm allowed us to select this wavelength as the most suitable.

The mean absolute recovery over the colchicine concentration range of l- 5 pglml (n = 5) was 84.5 * 4.5%. The precision of the method was determined by repeated analyses of a plasma solution (n = 6). The results indicate a good reproducibility with a coefficient of variation of 3.92%.

Table 1 shows the average colchicine concentration obtained from three deter- minations carried out at the times studied (1, 2 and 24 h). The considerable decrease in plasma colchicine levels indicates the presence of a rapid biotransfor- mation process in the animals studied. These data agree with the results obtain- ed in humans, where plasma levels reached were significantly low 24 h after a fatal dose of colchicine was administered [9]. Waliace et al. [lo] demonstrated that the half-life (T1/2) of colchicine was 20 min in a patient who survived 40 h after ingesting the drug.

796.475

mV

393.693

0 4 5 6 7 8 9 Minutes

Fig. 1. Chromatogram from a solution containing colchicine (C) and quinidine (Q) (X = 350 nm).

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TABLE 1

COLCHICINE LEVELS IN RATS

Time (h)

1 2

24

Concentration of colchicine (pglml or @g/g)

Plo.sma Liver 1.720 1.056 0.230 0.940 0.040 0.266

Kidney 0.069 0.043 0.039

Colchicine levels in the liver remained relatively constant during the first 2 h. They later decreased, although at slower rate than the plasma levels. This shows that biotransformation processes are intense, mainly affecting the plasma and liver concentrations.

Colchicine levels in the kidney do not show great variations as time elapses. Levels seen 2 and 24 h after the dose is administered (10 mg/kg) are not too far apart.

These results justify the need to use a highly sensitive and fast analytical meth- od, such as the one described in this paper in order to detect low levels of col- chicine in the blood or viscera.

References

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