Liquid Chromatographic Determination of Some Thiazide Diuretics in Pharmaceuticals with a Sodium Dodecyl Sulfate Mobile Phase

Analyst, February 1998, Vol. 123 (301Ð306) 301

Liquid chromatographic determination of some thiazide diuretics in pharmaceuticals with a sodium dodecyl sulfate mobile phase

Samuel Carda Brocha, Josep S. Esteve-Romerob and M. Celia Garc«õa Alvarez-Coque*a a Departament de Qu«õmica Anal«õtica, Facultat de Qu«õmica, Universitat de Val`encia, 46100 Burjassot, Spain b Area de Qu«õmica Anal«õtica, Universitat Jaume I, 12080 Castell«o, Spain

A rapid and simple liquid chromatographic procedure In other papers, direct injection procedures were proposed for that uses micellar mobile phases, containing only sodium the control of several diuretics in urine samples. For bendro- dodecyl sulfate (SDS) and phosphate buffer, is reported flumethiazide and chlorthalidone, a 0.05 mol l21 SDSÐ5% for the determination of some thiazide diuretics methanol mobile phase was used.5 Other diuretics could not be (althiazide, bendroflumethiazide, chlorothiazide, assayed, as their peaks were overlapped by the high background cyclothiazide, hydrochlorothiazide, hydroflumethiazide of the matrix at the beginning of the chromatogram, and the and trichloromethiazide) in pharmaceuticals. An retention times of amiloride, spironolactone and triamterene interpretive optimization procedure, based on an equation were too long. Later, the application of an interpretive that relates the capacity factor with the micellar optimization procedure6 indicated that the separation of a concentration, indicated the SDS concentration at which mixture of amiloride, bumetanide, chlorthalidone, furosemide, the diuretics were best resolved. The determination of spironolactone, triamterene and xipamide was possible with a hydrochlorothiazide and trichloromethiazide in 0.042 mol l21Ð4% propanol mobile phase at pH 4.5.7 pharmaceuticals could be achieved with a micellar mobile The retention times of several thiazide diuretics, such as phase of 0.02 mol l21 SDS in 0.01 mol l21 phosphate chlorothiazide, hydrochlorothiazide, hydroflumethiazide and buffer (pH 7), with retention times below 5 min, but the trichloromethiazide, are short with both SDSÐpropanol and determination of althiazide and bendroflumethiazide SDSÐmethanol mobile phases. The retention times of these required a micellar mobile phase of 0.15 mol l21 SDS at diuretics were increased through hydrolysis to the arylamines the same pH, with retention times below 7 min. The and derivatization by coupling of the diazotized arylamines with recoveries from the pharmaceuticals were in the range N-(1-naphthyl)ethylenediamine to form the azo dyes. This 97.2Ð104.0%. The results were compared with those avoided the overlapping of the chromatographic peaks of these obtained with methanolÐwater mobile phases. diuretics with the protein band in urine.8 In this work, it is demonstrated that mobile phases based on Keywords: Thiazide diuretics; pharmaceuticals; micellar SDS without a modifier are suitable for the analysis of liquid chromatography; sodium dodecyl sulfate pharmaceuticals containing several thiazide diuretics (althiazide, bendroflumethiazide, chlorothiazide, cyclothiazide, hydrochlorothiazide, hydroflumethiazide and trichloromethiazide). A comparison of the performances of the micellar mobile The action of diuretics is based on the interference with the phases and the aqueousÐorganic mobile phases recommended mechanism of ionic transport along the complete length of the in the USP,9 for the control of these diuretics, is also presented. nephron. According to their action, diuretics are classified as The two main advantages of the micellar procedure are the having high, intermediate or low efficacy. A correlation has elimination of the organic solvent and the simplification of the been found between the retention of diuretics with a sodium sample preparation step. dodecyl sulfate (SDS) mobile phase in a C18 column and their site of action within the nephron.1 Micellar liquid chromatography has been reported as a Experimental suitable technique for the determination of diuretics in Reagents pharmaceuticals and physiological fluids. Micellar solutions can replace conventional aqueousÐorganic mobile phases with Sodium dodecyl sulfate (Merck, Darmstadt, Germany), sodium good results. A chromatographic procedure was first proposed dihydrogenphosphate (Panreac, Barcelona, Spain), HCl, NaOH, for the determination of eight diuretics (acetazolamide, bendro- disodium hydrogenphosphate (Probus, Badalona, Spain), meth- flumethiazide, bumetanide, chlorthalidone, hydrochlorothia- anol (Scharlau, Barcelona, Spain) and ethanol (Prolabo, Paris, zide, spironolactone, triamterene and xipamide) in several France) were used. pharmaceutical preparations, using a 0.05 mol l21 SDSÐ3% Stock standard solutions of 100 mg l21 of the diuretics propanol micellar mobile phase.2 Other diuretics, such as althiazide, chlorothiazide, cyclothiazide, hydroflumethiazide, amiloride, spironolactone and triamterene showed long reten- trichloromethiazide (Sigma, St. Louis, MO, USA), bendro- tion times, and therefore a new mobile phase with a higher flumethiazide (Davur, Madrid, Spain) and hydrochlorothiazide eluting strength, 0.07 mol l21 SDSÐ0.5% pentanol, was further (Gayoso Welcome, Madrid, Spain) were prepared. The last two recommended for these diuretics.3 Bendroflumethiazide and diuretics were kindly donated by the Spanish pharmaceutical chlorthalidone with intermediate retention times among the laboratories indicated. The compounds were dissolved in a few diuretics could also be determined under these conditions. A milliliters of ethanol, with the aid of an ultrasonic bath (Model 0.15 mol l21 SDSÐ7% propanol mobile phase at pH 3 was also 617, Selecta, Barcelona, Spain), and were conveniently diluted used to separate a mixture of diuretics (amiloride, bendro- with 0.07 mol l21 SDS solution. Nanopure water (Barnstead, flumethiazide, chlorthalidone and hydrochlorothiazide) and b- Sybron, Boston, MA, USA) was used throughout. The final blockers (atenolol, metoprolol, and oxprenolol).4 content of ethanol was very low, which guaranteed the 302 Analyst, February 1998, Vol. 123 formation of micelles in the SDS solutions. Table 1 shows the chromatograph through an HP 3396A integrator and using the structures of the diuretics studied in this work. PEAK-96 program (Hewlett-Packard, Avondale, PA, USA). The chromatographic data were treated with MICHROM, a program developed in our laboratory.11 Apparatus The spectra of the diuretics were obtained with a UV/VIS/NIR Procedures spectrophotometer (Lambda 16, Perkin-Elmer, Norwalk, CT, Micellar chromatography USA) and pH measurements were made with a potentiometer provided with a combined Ag/AgCl/glass electrode (MicropH Almost all the pharmaceuticals considered in this work were 2001, Crison, Barcelona, Spain). tablets, but Kalten capsules were also analysed. For the An HP 1050 chromatograph (Hewlett-Packard, Palo Alto, analyses, 10 tablets were weighed, ground and homogenized, CA, USA) provided with an isocratic pump and an autosampler several portions were taken and weighed and each one was was used. Injection of the solutions in the chromatograph was dissolved with a small amount of ethanol with the aid of an made through a Rheodyne (Cotati, CA, USA) valve with a 20 ml ultrasonic bath. A 0.07 mol l21 SDS solution was then added to loop. A Spherisorb ODS-2 column (5 mm particle size, 120 mm favor the extraction of the diuretics using the ultrasonic bath. 3 4.6 mm id) was placed after a 30 mm long guard precolumn Finally, a dilution was made with the same micellar solution to of similar characteristics (Scharlau). The SDS mobile phases give a final concentration of 6Ð10 mg ml21 of the diuretics. The and the diuretic solutions were filtered through 0.45 mm nylon capsules were weighed before and after being carefully membranes (Micron Separations, Westboro, MA, USA). The emptied, to obtain the accurate mass of the capsule contents. 21 flow rate was 1 ml min . The dead time (t0 = 1.0 min) was Subsequently, the same procedure as above was followed. taken as the mean value of the first deviation of the baseline The excipients were not soluble in the micellar medium, obtained in each chromatogram after injection of the micellar hence the sample solutions should be filtered before their solutions of the diuretics. injection into the chromatograph. The standard solutions of the Monitoring was performed with a UV/VIS detector at 275 drugs were also filtered. However, the filtration was always nm. The signal was acquired by a PC connected to the performed directly into the autosampler vials through 0.45 mm

Table 1 Structures and protonation constants of the thiazide diuretics

Compound Manufacturer Structure Log K*

O O

H2NO2S S Althiazide Sigma, St. Louis, MO, USA NH Ð

CI N CH2SCH2CH CH2 H O O

H2NO2S S NH Bendroflumethiazide Davur, Madrid, Spain 8.5

F3C N CH2 H

O O

H2NO2S S NH Chlorothiazide Sigma 9.5, 6.7 CI N

O O H2NO2S S NH Cyclothiazide Sigma 10.5, 9.1 CI N H

O O

H2NO2S S NH Hydrochlorothiazide Gayoso Welcome, Madrid, Spain 9.2, 7.0 CI N H O O

H2NO2S S NH Hydroflumethiazide Sigma 10.0, 8.5

F3C N H O O

H2NO2S S Trichloromethiazide Sigma NH 10.6, 8.6, 7.3

CI N CHCI2 * From ref. 10. H Analyst, February 1998, Vol. 123 303 nylon membranes of 13 mm diameter. For the analysis of the 0.15 mol l21 SDS, were taken. Fig. 1 shows the 1/kA versus pharmaceuticals containing hydrochlorothiazide and trichloro- [SDS] plots for four thiazide diuretics. The regression coeffi- 21 21 methiazide, a 0.02 mol l SDSÐ0.01 mol l Na2HPO4 mobile cients of the fitted straight lines were usually r > 0.999. The phase was used, whereas for those containing althiazide and intercept of the plots for bendroflumethiazide and cyclothiazide 21 21 23 24 bendroflumethiazide, 0.15 mol l SDSÐ0.01 mol l Na2HPO4 was close to zero: 23.5 3 10 and 28.0 3 10 , respectively, was needed. The pH was adjusted to 7 with HCl. which indicated the strong association of these diuretics with the surfactant-modified stationary phases. This made the calculation of K for these diuretics difficult. AqueousÐorganic reversed-phase chromatography MW The pH of the mobile phase was set to 7 with phosphate 21 A methanolÐ0.01 mol l NaH2PO4 (22 + 78) mobile phase at buffer, since the retention of the thiazide diuretics did not pH 4.3 was also used for the determination of hydro- experience any change in the working pH range of a C18 column chlorothiazide and trichloromethiazide in the pharmaceuticals. (2.5 < pH < 7.5).7 The protonation constants of these For althiazide and bendroflumethiazide, methanolÐ0.05 mol l21 compounds in a water solution are given in Table 1. The anionic NaH2PO4 (40 + 60) at pH 3 was required. HCl was added to give surfactant in the micellar solution should increase the stability a suitable pH. The procedure followed in the sample preparation of the protonated species of the diuretics and, consequently, was similar to that described above for micellar chromatog- their protonation constants. Therefore, the same acidÐbase raphy, using the same aqueousÐorganic mobile phase to extract species will predominate over the whole 2.5Ð7.5 pH range. the drugs from the samples. It was decided to design a procedure for the determination of the thiazide diuretics using a pure micellar mobile phase, that is, without modifier. As commented upon above, according to their Results and discussion retention, two groups of thiazide diuretics should be considered, Optimization of the mobile phase composition which will require mobile phases with different elution strengths for adequate elution. In each case, however, the The possibility of using the same mobile phase for the analysis optimum mobile phase should resolve the peaks of the different of pharmaceuticals containing thiazide diuretics was first diuretics, since combinations of these diuretics could be found considered. Seven of these diuretics (althiazide, bendro- in pharmaceutical products. flumethiazide, chlorothiazide, cyclothiazide, hydrochlorothia- The optimum mobile phase for the separation of the diuretics zide, hydroflumethiazide and trichloromethiazide) were in- was obtained through the application of an interpretive cluded in this study. However, the retentions of chlorothiazide, procedure, which needs the retention data of the solutes and hydrochlorothiazide, hydroflumethiazide and trichlorothiazide considers the shape parameters of the chromatographic peaks in were low with a 0.02 mol l21 SDS mobile phase (kA = 0.4, 2.1, selected mobile phases of surfactant, adequately distributed in 3.1 and 3.4, respectively), whereas althiazide, bendroflumethia- the linear variable space. It should be taken into account that at zide and cyclothiazide, which have a voluminous substituent at higher concentrations of the surfactant the efficiency of the the C-3 position of the thiazide nucleus (see Table 1), showed chromatographic peaks deteriorates. Previously, this inter- high capacity factors under these conditions (kA = 22.6, 36.7 pretive procedure was applied to the optimization of the and 37.2, respectively). composition of hybrid micellar solutions of surfactant and Propanol is a modifier usually added to micellar mobile modifier.6 phases to increase the efficiency of the chromatographic peaks Eqn. (1) was rewritten as and to decrease and control the retention. However, it is obvious 1 that the addition of this alcohol would not benefit the diuretics k′ = (2) + appearing at the head of the chromatogram, which could elute c0 c1[M] with the dead volume. On the other hand, it was observed that when the concentration of SDS was increased to accelerate the to predict the capacity factors for any SDS mobile phase of elution of the most retained diuretics, the capacity factors were varying concentration. This equation, together with the effi- substantially decreased. This means that these diuretics were ciencies and asymmetry factors of the chromatographic peaks, strongly associated with the micelle. Table 2 gives the values of was used to obtain a diagram for the resolution function, according to the soluteÐmicelle association constant, KMW , and the solute n stationary phaseÐwater partition constant, KSW , calculated according to the Armstrong and Nome equation:12 = Oi r ∏ n (3)  O  1 1 (K − 1)ν =  i  = + MW [M] (1) i 1 ∑  ′ φ φ n k KSW KSW where [M] is the concentration of surfactant, f = VS/V0 (VS and V0 being the active surface of the stationary phase and column dead volumes, respectively) and n is the partial specific volume of the monomers of surfactant in the micelle (0.246 l mol21 for SDS). To obtain the partition constants, the capacity factors of the diuretics eluted with four mobile phases, 0.02, 0.05, 0.1 and

Table 2 SoluteÐmicelle association and stationary phase partition constants

Compound KMW fKSW Chlorothiazide 53.3 0.5 Hydrochlorothiazide 44.9 2.5 Hydroflumethiazide 56.8 3.8 Trichloromethiazide 91.6 4.9 Althiazide 867 115.5 Fig. 1 Armstrong and Nome plots for 1, hydrochlorothiazide; 2, hydroflumethiazide; 3, althiazide; and 4, bendroflumethiazide. 304 Analyst, February 1998, Vol. 123

where Oi is the degree of overlap: order to select a suitable composition of the aqueousÐorganic ′ mobile phase for the analysis of the pharmaceuticals. The = − wi Oi 1 (4) recommended procedures for the determination of bendro- wi flumethiazide, hydrochlorothiazide and trichloromethiazide wi being the total area of a given peak i and wAi the area of the make use of aqueous mobile phases containing 10Ð20% peak overlapped by other peaks. acetonitrile or 20Ð40% methanol, usually buffered at pH For the four less retained diuretics, maximum and good 2.8Ð4.5 with phosphate or acetate.9 A mobile phase of 21 resolution was achieved for concentrations of SDS lower than methanolÐ0.01 mol l Na2HPO4 (22 + 78) at pH 4.3 was 0.02 mol l21. In contrast, the most retained diuretics, bendro- selected for hydrochlorothiazide and trichloromethiazide. The flumethiazide and cyclothiazide, could not be resolved under elution of althiazide and bendroflumethiazide was effected with any conditions. The peak of althiazide, with an intermediate a mobile phase of higher elution strength, namely methanolÐ 21 retention, was easily separated from the other compounds. 0.05 mol l Na2HPO4 (40 + 60) at pH 3. The results are given Fig. 2 shows the simulated chromatograms of a mixture of in Table 3. chlorothiazide, hydrochlorothiazide, hydroflumethiazide and The retention times for hydrochlorothiazide and trichloromethiazide for 0.02, 0.05 and 0.15 mol l21 SDS eluents trichloromethiazide, using a 0.02 mol l21 SDS mobile phase at pH 7. With variation in the SDS concentration, the peak of were 3.1 and 4.4 min, respectively, and those for althiazide and trichloromethiazide changed its order of elution: for the 0.02 bendroflumethiazide using 0.15 mol l21 SDS were 4.5 and 5.7 mol l21 SDS mobile phase it eluted after chlorothiazide, min, respectively. The retention times for hydrochlorothiazide hydrochlorothiazide and hydroflumethiazide, whereas at higher and trichloromethiazide, using methanolÐwater (22 + 78) at pH SDS concentrations it first overlapped with the peak of 4.3 were 2.9 and 14.2 min, respectively, and those for althiazide hydroflumethiazide, and subsequently with hydrochloro- and bendroflumethiazide using methanolÐwater (40 + 60) at pH thiazide. 3 were 6.7 and 15.4 min, respectively. The results indicated that the determination of diuretics Fig. 3 shows the chromatograms of three pharmaceuticals showing low and high retentions required mobile phases of analysed in this work. It can be seen that the pharmaceutical different elution strengths. For chlorothiazide, hydrochloro- containing a mixture of hydrochlorothiazide and thiazide, hydroflumethiazide and trichloromethiazide, 0.02 trichloromethiazide (Rulun)« could be analysed in less than 5 mol l21 SDS was chosen, and for althiazide, bendroflumethia- min with a 0.02 mol l21 SDS mobile phase, whereas 15 min zide and cyclothiazide, 0.15 mol l21 SDS gave an adequate were required to elute the most retained of these diuretics with retention. methanolÐwater (22 + 78) at pH 4.3. An aqueousÐorganic mobile phase of higher elution strength, more suitable for trichloromethiazide, would decrease excessively the retention Analysis of pharmaceuticals of hydrochlorothiazide, which would elute near the dead Initially, the diuretics were extracted from the pharmaceuticals using ethanolÐwater (10 + 90). However, the recoveries were poor and the repeatability was low. It was then decided to perform the extraction with ethanolÐ0.07 mol l21 SDS (10 + 90). The selected mobile phases, indicated above, were used to analyse several pharmaceutical preparations commercially available in Spain, which contained althiazide, bendroflumethiazide, hydrochlorothiazide and trichloromethiazide. Calibration curves in the 1Ð20 mg ml21 range were constructed for each drug from duplicate injections of five solutions with increasing concentration, with regression coeffi- cients r > 0.999. The areas of the chromatographic peaks were measured. Five replicates of the analyses of the pharmaceuticals were made, each with duplicate injections of the solutions. The values found agreed well with those declared by the manu- facturers, as shown in Table 3. No interferences from accom- panying compounds were observed. The recoveries were usually in the range 97.2Ð104% for the micellar mobile phases. The RSDs were usually below 3.4%. These results were validated by comparison with conventional reversed-phase liquid chromatography with the same C18 column. A literature survey of the chromatographic procedures used for the determination of the thiazide diuretics was made, in

Fig. 3 Chromatograms of the pharmaceuticals obtained using micellar (left) and aqueousÐorganic (right) mobile phases: (a) Rulun« [hydrochlorothiazide and trichloromethiazide, 0.02 mol l21 SDS at pH 7 and methanolÐwater (22 + 78) at pH 4.3], (b) Aldactacine [althiazide, ALT, 0.15 Fig. 2 Chromatograms of mixtures of chlorothiazide (CHL), hydro- mol l21 SDS and methanolÐwater (40 + 60) at pH 3], and (c) Neatenol chlorothiazide (HYC), hydroflumethiazide (HYF) and trichloromethiazide Diuvas [bendroflumethiazide, BEN, 0.15 mol l21 SDS and methanolÐwater (TRI), eluted with (a) 0.02, (b) 0.05 and (c) 0.15 mol l21 SDS. (40 + 60) at pH 3]. Analyst, February 1998, Vol. 123 305 volume. In Rulun,« hydrochlorothiazide was contained in the diuretics has the advantage of complete elimination of the coating of the tablets and trichloromethiazide was included in polluting and flammable organic solvents. its nucleus. In previous work, an optimization procedure was developed, based on the description of the retention of solutes in micellar mobile phases which contained a surfactant and a modifier.6 We Conclusions have now demonstrated the application of this procedure using simpler mobile phases containing only the surfactant. In this The chromatographic procedure proposed here for the case, an accurate description of the retention by the equation determination of some thiazide diuretics in pharmaceuticals suggested by Armstrong and Nome12 is employed. The elution with a micellar mobile phase of SDS without a modifier is rapid behavior of the thiazide diuretics chlorothiazide, hydrochloro- and simple. The elution of the diuretics, except hydro- thiazide, hydroflumethiazide and trichloromethiazide produced chlorothiazide, was more rapid with the micellar eluents than several changes in their elution order, which made the with the recommended aqueousÐorganic mobile phases, espe- optimization of their resolution with a procedure based on the cially for trichloromethiazide and bendroflumethiazide. sequential modification of the composition of the mobile phase It is notable that the micellar mobile phases used in this work difficult. The interpretive procedure led to the optimum mobile did not require the addition of an organic solvent to achieve phase using chromatographic data obtained with only two or adequate retention of the diuretics. In most of the micellar three mobile phases, which are necessary to obtain the two- chromatographic procedures reported in the literature, a small variable equations that describe the elution behavior of each amount of a short-chain alcohol must be added to accelerate and diuretic. control the elution of the solutes. A decrease in the organic solvent content has been considered an interesting advantage of micellar mobile phases over aqueousÐorganic eluents. The This work was supported by the DGICYT, Project PB94/967 procedure described here for the determination of the thiazide (Spain). Samuel Carda Broch thanks the Conselleria de Cultura,

Table 3 Results of analysis of pharmaceuticals with micellar and aqueousÐorganic reversed-phase liquid chromatography

Micellar AqueousÐorganic

Found/ RSD (%) Found/ RSD (%) mg (n = 5) mg (n = 5) Pharmaceutical* Laboratory Composition/mg per tablet or capsule Hidrosaluretil Gayoso Welcome, Alcala« de Hydrochlorothiazide (50), lactose and 49.5 0.6 50.8† 0.6 Henares, Madrid, Spain other excipients Alopresin Diu Alonga, Alcala,« Madrid, Spain Hydrochlorothiazide (25), captopril 25.3 0.5 26.6† 1.9 (25), lactose and other excipients Ameride DuPont Pharma, Madrid, Spain Hydrochlorothiazide (50), amiloride 50.5 0.4 53.0† 7.5 chlorhydrate (5), lactose (71), other excipients Selopresin Astra, Esplugues de Llobregat, Hydrochlorothiazide (12.5), 12.7 1.6 12.8† 1.4 Barcelona, Spain metoprolol tartrate (100), lactose and other excipients Kalten Zeneca Farma, Porrino,˜ Hydrochlorothiazide (25), atenolol 24.8 1.2 24.6† 4.0 Pontevedra, Spain (50), amiloride chlorhydrate (2.5), lactose and other excipients Picten Miquel, Barcelona, Spain Hydrochlorothiazide (40), 40.1 1.0 39.6† 1.0 methyldopa (70), triamterene (25), reserpine (0.2), lactose, sulfite and other excipients Neotensin Diu Cepa, Alcobendas, Madrid, Hydrochlorothiazide (12.5), enalapril 12.55 1.2 12.45† 1.2 Spain maleate (20), lactose and other excipients Zestoretic Zeneca« Farma, Porrino,˜ Hydrochlorothiazide (12.5), lisinopril 12.65 1.3 12.8† 1.0 Pontevedra, Spain (20), excipients Rulun« Lacer,« Sardenya, Barcelona, Tablet coating: hydrochlorothiazide 20.8 3.4 21.5† 5.6 Spain (20), xanthinol nicotinate (40), saccharose (167.64), excipient Tablet nucleus: trichloromethiazide 2.95 3.4 2.82† 2.8 (3), alkaloid fraction of Rauwolfia s. Dehra Dun (2.3), pentosan sodium polysulfate (25), xanthinol nicotinate (60), lactose and other excipients Aldactacine Searle Industrie, Evreux, France Althiazide (15), spironolactone (25), 15.1 0.4 14.9‡ 0.9 lactose and other excipients Spirometon« Belmac, Zaragoza, Spain Bendroflumethiazide (2.5), 2.43 1.2 2.62‡ 1.5 spironolactone (50), excipients Neatenol Diuvas Fides-Rottapharm, Almacera,« Bendroflumethiazide (5), hydralazine 5.01 1.0 4.92‡ 1.4 Valencia, Spain chlorhydrate (50), atenolol (100), excipients * All the pharmaceuticals were tablets, except Kalten, which was in the form of capsules. † MethanolÐwater (22 + 78) (pH 4.3) mobile phase. ‡ Methanol–water (40 + 60) (pH 3) mobile phase. 306 Analyst, February 1998, Vol. 123

Educacio« i Ciencia` de la Generalitat Valenciana for an FPI 7 Bonet Domingo, E., Torres Lapasio,« J. R., Medina Hernandez,« M. J., Grant. and Garc«õa Alvarez-Coque, M. C., Anal. Chim. Acta, 1994, 287, 201. References 8 Carda Broch, S., Garc«õa Alvarez-Coque, M. C., Simo« Alfonso, E. F., and Esteve Romero, J. S., Anal. Chim. Acta, in the press. 1 Medina Hernandez,« M. J., Bonet Domingo, E., Ramis Ramos, G., and 9 US Pharmacopeia, XXII Revision, US Pharmacopeial Convention, Garc«õa Alvarez-Coque, M. C., Anal. Lett., 1993, 26, 1881. Rockville, MD, 1990. 2 Bonet Domingo, E., Medina Hernandez,« M. J., Ramis Ramos, G., and 10 Hansch, C. C., in Comprehensive Medicinal Chemistry, ed. Garc«õa Alvarez-Coque, M. C., Analyst, 1992, 117, 843. Sammes, R. G., and Taylor, J. B., Pergamon Press, Oxford, 1990, vol. 3 Bonet Domingo, E., Medina Hernandez,« M. J., and Garc«õa Alvarez- 6. Coque, M. C., J. Pharm. Biomed. Anal., 1993, 11, 711. 11 Torres Lapasio,« J. R., Garc«õa Alvarez-Coque, M. C., and Baeza 4 Rapado Mart«õnez, I., Garc«õa Alvarez-Coque, M. C., and Villanueva Baeza, J. J., Anal. Chim. Acta, 1997, 348, 187. Camanas,˜ R. M., Analyst, 1996, 121, 1677. 12 Armstrong, D. W., and Nome, F., Anal. Chem., 1981, 53, 1662. 5 Bonet Domingo, E., Medina Hernandez,« M. J., Ramis Ramos, G., and Garc«õa Alvarez-Coque, M. C., J. Chromatogr., 1992, 582, 189. 6 Torres Lapasio,« J. R., Villanueva Camanas,˜ R. M., Sanchis Mallols, Paper 7/05641I J. M., Medina Hernandez,« M. J., and Garc«õa Alvarez-Coque, M. C., Received August 4, 1997 J. Chromatogr., 1994, 677, 239. Accepted October 1, 1997