December, 1983 SHORT PAPERS

Simultaneous Determination of Fentiazac and 1535

p-Hydroxyfentiazac in Plasma by High-performance Liquid Chromatography with Ultraviolet Detection

Pauline S. Dowell* Wyeth Laboratories, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 OPH

Keywords : Fentiazac determination ; p-hydroxyfentiazac determination ; high- performance liquid chromatografihy ; ultraviolet detection

Fentiazac, [4-(4-chlorophenyl)-2-phenylthiazol-5-yl]acetic acid (I), has been shown to be an effective and well tolerated non-steroidal anti-inflammatory agent in man.l Gas-chromato- graphic methods have previously been reported for assay of fentiazac in plasma2s3; however, there have been no previous published reports of the quantitation in human plasma of p - hydroxyfentiazac (11), known to be a major metabolite of the drug in animal^.^ A method has been developed for the simultaneous determination of fentiazac and 9-hydroxyfentiazac in plasma.

The method described uses high-performance liquid chromatography (HPLC) with ultra- violet detection and has a lower detection limit of 20 ng ml-l for fentiazac and of 50 ng ml-l for p-hydroxyfen tiazac.

NYS

I OH

II

Experimental Equipment

The HPLC system consisted of a double reciprocating pump (Model 750/03, Applied Chromatography Systems, Luton, Bedfordshire), an automatic injection system (WISP, Model 710A, Waters Associates, Northwich, Cheshire) and an LC/UV detector (Pye Unicam, Cambridge). Quantitation was carried out on a computing integrator operating in the peak- height measurement mode (SP4100, Spectra Physics, St. Albans, Hertfordshire) . Glassware

All glassware was silanised by soaking in 2% dimethylchlorosilane in ethyl acetate for 30 min. The glassware was then dried, washed with distilled water, dried, soaked in methanol for 30 min and finally dried.

Reagents Methanol and dichloromethane, HPLC grade, were obtained from Rathburn Chemicals,

Walkerburn, Peeblesshire. Formic acid was supplied by BDH Chemicals Ltd., Poole, Dorset. The internal standard, [2,4-di-(@-methoxyphenyl) thiazol-5-yl]acetic acid, was synthesised in the Chemistry Department of Wyeth Laboratories.

* Present address : Glaxo Group Research Ltd., Greenford Road, Greenford, Middlesex.

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1536 SHORT PAPERS Analyst, Vol. 108

Solvent Extraction Procedure Internal standard

(250 ng in 25 p1 of methanol) was added. The plasma was adjusted to pH 2 with 25 pl of 5 N hydrochloric acid, and the mixture extracted with dichloromethane (3.5 ml) by vortexing for 1 min. Following centrifugation at 1500 rev min-l for 10 min, the upper aqueous phases were discarded and the lower organic phases were transferred into 5-ml flat-bottomed glass tubes. The extracts were evaporated to dryness a t 45 "C under a gentle stream of oxygen-free nitrogen. Residues were reconstituted in 150 pl of methanol - 1% V/V formic acid solution (77 + 23) before being chromatographed.

Aliquots of plasma (500 pl) were dispensed into silanised glass test-tubes.

Chromatography The extracts (120 pl) were injected on to a pBondapak C18 reversed-phase analytical

HPLC column (supplied by hplc Technology Ltd., Macclesfield, Cheshire), 0.5 cm i.d. and 25 cm long. This column was used in conjunction with a pre-column of the same material, 0.5 cm i.d. and 5 cm long (available from hplc Technology). The column was eluted with methanol - 1% V/V formic acid solution (77 + 23) at a flow-rate of 2.0 ml min-l. The pressure was approximately 1 700 p.s.i. Fentiazac, $-hydroxyfentiazac and the internal standard were detected by means of their absorbance at 310 nm.

Results and Discussion A typical chromatogram is shown in Fig. 1. The lower limit of detection is 20 ng ml-l for

Calibration graphs were linear over the ranges fentiazac and 50 ng ml-l for the metabolite.

i Time/min +

Fig. 1. Typical chro- matogram obtained from plasma containing 400 ng of fentiazac and p - hydroxyfentiazac, pro- cessed as described. Retention times: A ( p - hydroxyfentiazac), 5.1 : B (internal standard), 6.0; C (fentiazac), 7.7 min.

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December, 1983 SHORT PAPERS 1537

20-1 000 ng ml-l (fentiazac) and 50-1 000 ng ml-l (P-hydroxyfentiazac). The reproducibility of the method was investigated by analysing ten replicate samples of plasma at different concentrations. The resulting coefficients of variation of peak-height ratios for fentiazac were 10.7, 7.0 and 2.3% at 20, 50 and 1000 ng ml-l and for P-hydroxyfentiazac were 9.0 and 1.7% at 50 and 1000 ng ml-l, respectively. The method would appear to be specific for fentiazac and its +-hydroxylated metabolite. In a recent study by Mr. S. Rhenius of Life Science Research (personal communication), in which l4C-labelled fentiazac was administered orally t o volunteers, the only dry derived material detected in plasma by TLC was the unchanged drug and its 9-hydroxylated metabolite and their conjugates (glucuronide/sulphate) . As the conjugates would not be extracted from plasma, it is therefore unlikely that any other metabolite of fentiazac present in the plasma could interfere with the assay.

The method described here is a rapid, sensitive and specific method for the determination of fentiazac and P-hydroxyfentiazac and has been used successfully to monitor concentrations of the drug and its metabolite in plasma from subjects administered oral doses of 200 mg of the drug. A typical plasma concentration - time graph obtained using this method of analysis is shown in Fig. 2. Concentrations of P-hydroxyfentiazac after a single dose of fentiazac were of a comparable order of magnitude to those of the unchanged drug, maximum concentrations being about 40% of those of fentiazac.

I 1 I I I I

0 2 4 6 8 10 12

Time after dosing/h

Fig. 2. Typical plasma concentration versus time graph obtained after administration of a single 200-mg oral dose of fentiazac to a male volunteer. A, p - Hydroxyfentiazac ; B, fentiazac.

Fluorescence detection, using an excitation wavelength of 313 nm and an emission wave- length of 390 nm, has been briefly examined and has been found to be a good alternative to ultraviolet absorbance as a means of detection. It is as sensitive as ultraviolet detection, but the limit of detection has not been investigated. Therefore, it is not known whether this method offers any advantages in sensitivity.

The author thanks Dr. H. M. Norbury and Miss L. M. Whitfield for technical assistance and Dr. D. M. Pierce for helpful discussions.

References 1. 2.

3.

4.

Marmo, E., Curr. Med. Res. Opin., 1979, 6, 53. Quattrini, M., Zanolo, G., Mondino, A., Giachetti, C., and Silvestri, S., Arzneim. Forsch., 1981, 31,

Zanolo, G., Giachetti, C., Mondino, A., Silvestri, S., Bianchi, E., Segre, G., and Gomarasca, P.,

Fumero, S., Mondino, A., Silvestri, S., Zanolo, G., De Marchi, G., and Pedrazzini, S., Arzneim.

1046.

Avzneim. Forsch., 1981, 31, 1098.

Forsch., 1980, 30, 1253.

Received May 23rd, 1983 Accepted July 29th, 1983

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