Liquid chromatographic determination of some thiazidediuretics in pharmaceuticals with a sodium dodecylsulfate mobile phase

Samuel Carda Brocha, Josep S. Esteve-Romerob and M. Celia Garcıa Alvarez-Coque*a

a Departament de Quımica Analıtica, Facultat de Quımica, Universitat de Valencia, 46100Burjassot, Spainb Area de Quımica Analıtica, Universitat Jaume I, 12080 Castello, Spain

A rapid and simple liquid chromatographic procedurethat uses micellar mobile phases, containing only sodiumdodecyl sulfate (SDS) and phosphate buffer, is reportedfor the determination of some thiazide diuretics(althiazide, bendroflumethiazide, chlorothiazide,cyclothiazide, hydrochlorothiazide, hydroflumethiazideand trichloromethiazide) in pharmaceuticals. Aninterpretive optimization procedure, based on an equationthat relates the capacity factor with the micellarconcentration, indicated the SDS concentration at whichthe diuretics were best resolved. The determination ofhydrochlorothiazide and trichloromethiazide inpharmaceuticals could be achieved with a micellar mobilephase of 0.02 mol l21 SDS in 0.01 mol l21 phosphatebuffer (pH 7), with retention times below 5 min, but thedetermination of althiazide and bendroflumethiaziderequired a micellar mobile phase of 0.15 mol l21 SDS atthe same pH, with retention times below 7 min. Therecoveries from the pharmaceuticals were in the range97.2–104.0%. The results were compared with thoseobtained with methanol–water mobile phases.

Keywords: Thiazide diuretics; pharmaceuticals; micellarliquid chromatography; sodium dodecyl sulfate

The action of diuretics is based on the interference with themechanism of ionic transport along the complete length of thenephron. According to their action, diuretics are classified ashaving high, intermediate or low efficacy. A correlation hasbeen found between the retention of diuretics with a sodiumdodecyl sulfate (SDS) mobile phase in a C18 column and theirsite of action within the nephron.1

Micellar liquid chromatography has been reported as asuitable technique for the determination of diuretics inpharmaceuticals and physiological fluids. Micellar solutionscan replace conventional aqueous–organic mobile phases withgood results. A chromatographic procedure was first proposedfor the determination of eight diuretics (acetazolamide, bendro-flumethiazide, bumetanide, chlorthalidone, hydrochlorothia-zide, spironolactone, triamterene and xipamide) in severalpharmaceutical preparations, using a 0.05 mol l21 SDS–3%propanol micellar mobile phase.2 Other diuretics, such asamiloride, spironolactone and triamterene showed long reten-tion times, and therefore a new mobile phase with a highereluting strength, 0.07 mol l21 SDS–0.5% pentanol, was furtherrecommended for these diuretics.3 Bendroflumethiazide andchlorthalidone with intermediate retention times among thediuretics could also be determined under these conditions. A0.15 mol l21 SDS–7% propanol mobile phase at pH 3 was alsoused to separate a mixture of diuretics (amiloride, bendro-flumethiazide, chlorthalidone and hydrochlorothiazide) and b-blockers (atenolol, metoprolol, and oxprenolol).4

In other papers, direct injection procedures were proposed forthe control of several diuretics in urine samples. For bendro-flumethiazide and chlorthalidone, a 0.05 mol l21 SDS–5%methanol mobile phase was used.5 Other diuretics could not beassayed, as their peaks were overlapped by the high backgroundof the matrix at the beginning of the chromatogram, and theretention times of amiloride, spironolactone and triamterenewere too long. Later, the application of an interpretiveoptimization procedure6 indicated that the separation of amixture of amiloride, bumetanide, chlorthalidone, furosemide,spironolactone, triamterene and xipamide was possible with a0.042 mol l21–4% propanol mobile phase at pH 4.5.7

The retention times of several thiazide diuretics, such aschlorothiazide, hydrochlorothiazide, hydroflumethiazide andtrichloromethiazide, are short with both SDS–propanol andSDS–methanol mobile phases. The retention times of thesediuretics were increased through hydrolysis to the arylaminesand derivatization by coupling of the diazotized arylamines withN-(1-naphthyl)ethylenediamine to form the azo dyes. Thisavoided the overlapping of the chromatographic peaks of thesediuretics with the protein band in urine.8

In this work, it is demonstrated that mobile phases based onSDS without a modifier are suitable for the analysis ofpharmaceuticals containing several thiazide diuretics (althia-zide, bendroflumethiazide, chlorothiazide, cyclothiazide, hy-drochlorothiazide, hydroflumethiazide and trichloromethia-zide). A comparison of the performances of the micellar mobilephases and the aqueous–organic mobile phases recommendedin the USP,9 for the control of these diuretics, is also presented.The two main advantages of the micellar procedure are theelimination of the organic solvent and the simplification of thesample preparation step.

Experimental

Reagents

Sodium dodecyl sulfate (Merck, Darmstadt, Germany), sodiumdihydrogenphosphate (Panreac, Barcelona, Spain), HCl, NaOH,disodium hydrogenphosphate (Probus, Badalona, Spain), meth-anol (Scharlau, Barcelona, Spain) and ethanol (Prolabo, Paris,France) were used.

Stock standard solutions of 100 mg l21 of the diureticsalthiazide, chlorothiazide, cyclothiazide, hydroflumethiazide,trichloromethiazide (Sigma, St. Louis, MO, USA), bendro-flumethiazide (Davur, Madrid, Spain) and hydrochlorothiazide(Gayoso Welcome, Madrid, Spain) were prepared. The last twodiuretics were kindly donated by the Spanish pharmaceuticallaboratories indicated. The compounds were dissolved in a fewmilliliters of ethanol, with the aid of an ultrasonic bath (Model617, Selecta, Barcelona, Spain), and were conveniently dilutedwith 0.07 mol l21 SDS solution. Nanopure water (Barnstead,Sybron, Boston, MA, USA) was used throughout. The finalcontent of ethanol was very low, which guaranteed the

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N

NHSH2NO2S

CI

O O

CH2SCH2CHH

CH2

N

NHSH2NO2S

F3C

O O

CH2H

N

NHSH2NO2S

CI

O O

N

NHSH2NO2S

CI

O O

H

N

NHSH2NO2S

CI

O O

H

N

NHSH2NO2S

F3C

O O

H

N

NHSH2NO2S

CI

O O

CHCI2H

formation of micelles in the SDS solutions. Table 1 shows thestructures of the diuretics studied in this work.

Apparatus

The spectra of the diuretics were obtained with a UV/VIS/NIRspectrophotometer (Lambda 16, Perkin-Elmer, Norwalk, CT,USA) and pH measurements were made with a potentiometerprovided with a combined Ag/AgCl/glass electrode (MicropH2001, Crison, Barcelona, Spain).

An HP 1050 chromatograph (Hewlett-Packard, Palo Alto,CA, USA) provided with an isocratic pump and an autosamplerwas used. Injection of the solutions in the chromatograph wasmade through a Rheodyne (Cotati, CA, USA) valve with a 20 mlloop. A Spherisorb ODS-2 column (5 mm particle size, 120 mm3 4.6 mm id) was placed after a 30 mm long guard precolumnof similar characteristics (Scharlau). The SDS mobile phasesand the diuretic solutions were filtered through 0.45 mm nylonmembranes (Micron Separations, Westboro, MA, USA). Theflow rate was 1 ml min21. The dead time (t0 = 1.0 min) wastaken as the mean value of the first deviation of the baselineobtained in each chromatogram after injection of the micellarsolutions of the diuretics.

Monitoring was performed with a UV/VIS detector at 275nm. The signal was acquired by a PC connected to the

chromatograph through an HP 3396A integrator and using thePEAK-96 program (Hewlett-Packard, Avondale, PA, USA).The chromatographic data were treated with MICHROM, aprogram developed in our laboratory.11

Procedures

Micellar chromatography

Almost all the pharmaceuticals considered in this work weretablets, but Kalten capsules were also analysed. For theanalyses, 10 tablets were weighed, ground and homogenized,several portions were taken and weighed and each one wasdissolved with a small amount of ethanol with the aid of anultrasonic bath. A 0.07 mol l21 SDS solution was then added tofavor the extraction of the diuretics using the ultrasonic bath.Finally, a dilution was made with the same micellar solution togive a final concentration of 6–10 mg ml21 of the diuretics. Thecapsules were weighed before and after being carefullyemptied, to obtain the accurate mass of the capsule contents.Subsequently, the same procedure as above was followed.

The excipients were not soluble in the micellar medium,hence the sample solutions should be filtered before theirinjection into the chromatograph. The standard solutions of thedrugs were also filtered. However, the filtration was alwaysperformed directly into the autosampler vials through 0.45 mm

Table 1 Structures and protonation constants of the thiazide diuretics

Compound Manufacturer Structure Log K*

Althiazide Sigma, St. Louis, MO, USA –

Bendroflumethiazide Davur, Madrid, Spain 8.5

Chlorothiazide Sigma 9.5, 6.7

Cyclothiazide Sigma 10.5, 9.1

Hydrochlorothiazide Gayoso Welcome, Madrid, Spain 9.2, 7.0

Hydroflumethiazide Sigma 10.0, 8.5

Trichloromethiazide Sigma 10.6, 8.6, 7.3

* From ref. 10.

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nylon membranes of 13 mm diameter. For the analysis of thepharmaceuticals containing hydrochlorothiazide and trichloro-methiazide, a 0.02 mol l21 SDS–0.01 mol l21 Na2HPO4 mobilephase was used, whereas for those containing althiazide andbendroflumethiazide, 0.15 mol l21 SDS–0.01 mol l21 Na2HPO4was needed. The pH was adjusted to 7 with HCl.

Aqueous–organic reversed-phase chromatography

A methanol–0.01 mol l21 NaH2PO4 (22 + 78) mobile phase atpH 4.3 was also used for the determination of hydro-chlorothiazide and trichloromethiazide in the pharmaceuticals.For althiazide and bendroflumethiazide, methanol–0.05 mol l21

NaH2PO4 (40 + 60) at pH 3 was required. HCl was added to givea suitable pH. The procedure followed in the sample preparationwas similar to that described above for micellar chromatog-raphy, using the same aqueous–organic mobile phase to extractthe drugs from the samples.

Results and discussion

Optimization of the mobile phase composition

The possibility of using the same mobile phase for the analysisof pharmaceuticals containing thiazide diuretics was firstconsidered. Seven of these diuretics (althiazide, bendro-flumethiazide, chlorothiazide, cyclothiazide, hydrochlorothia-zide, hydroflumethiazide and trichloromethiazide) were in-cluded in this study. However, the retentions of chlorothiazide,hydrochlorothiazide, hydroflumethiazide and trichlorothiazidewere low with a 0.02 mol l21 SDS mobile phase (kA = 0.4, 2.1,3.1 and 3.4, respectively), whereas althiazide, bendroflumethia-zide and cyclothiazide, which have a voluminous substituent atthe C-3 position of the thiazide nucleus (see Table 1), showedhigh capacity factors under these conditions (kA = 22.6, 36.7and 37.2, respectively).

Propanol is a modifier usually added to micellar mobilephases to increase the efficiency of the chromatographic peaksand to decrease and control the retention. However, it is obviousthat the addition of this alcohol would not benefit the diureticsappearing at the head of the chromatogram, which could elutewith the dead volume. On the other hand, it was observed thatwhen the concentration of SDS was increased to accelerate theelution of the most retained diuretics, the capacity factors weresubstantially decreased. This means that these diuretics werestrongly associated with the micelle. Table 2 gives the values ofthe solute–micelle association constant, KMW , and the solutestationary phase–water partition constant, KSW , calculatedaccording to the Armstrong and Nome equation:12

1 1 1

′= + −

k K

K

Kφν

φSW

MW

SW

M( )

[ ] (1)

where [M] is the concentration of surfactant, f = VS/V0 (VS andV0 being the active surface of the stationary phase and columndead volumes, respectively) and n is the partial specific volumeof the monomers of surfactant in the micelle (0.246 l mol21 forSDS). To obtain the partition constants, the capacity factors ofthe diuretics eluted with four mobile phases, 0.02, 0.05, 0.1 and

0.15 mol l21 SDS, were taken. Fig. 1 shows the 1/kA versus[SDS] plots for four thiazide diuretics. The regression coeffi-cients of the fitted straight lines were usually r > 0.999. Theintercept of the plots for bendroflumethiazide and cyclothiazidewas close to zero: 23.5 3 1023 and 28.0 3 1024, respectively,which indicated the strong association of these diuretics withthe surfactant-modified stationary phases. This made thecalculation of KMW for these diuretics difficult.

The pH of the mobile phase was set to 7 with phosphatebuffer, since the retention of the thiazide diuretics did notexperience any change in the working pH range of a C18 column(2.5 < pH < 7.5).7 The protonation constants of thesecompounds in a water solution are given in Table 1. The anionicsurfactant in the micellar solution should increase the stabilityof the protonated species of the diuretics and, consequently,their protonation constants. Therefore, the same acid–basespecies will predominate over the whole 2.5–7.5 pH range.

It was decided to design a procedure for the determination ofthe thiazide diuretics using a pure micellar mobile phase, that is,without modifier. As commented upon above, according to theirretention, two groups of thiazide diuretics should be considered,which will require mobile phases with different elutionstrengths for adequate elution. In each case, however, theoptimum mobile phase should resolve the peaks of the differentdiuretics, since combinations of these diuretics could be foundin pharmaceutical products.

The optimum mobile phase for the separation of the diureticswas obtained through the application of an interpretiveprocedure, which needs the retention data of the solutes andconsiders the shape parameters of the chromatographic peaks inselected mobile phases of surfactant, adequately distributed inthe linear variable space. It should be taken into account that athigher concentrations of the surfactant the efficiency of thechromatographic peaks deteriorates. Previously, this inter-pretive procedure was applied to the optimization of thecomposition of hybrid micellar solutions of surfactant andmodifier.6

Eqn. (1) was rewritten as

′ =+

kc c

1

0 1[ ]M(2)

to predict the capacity factors for any SDS mobile phase ofvarying concentration. This equation, together with the effi-ciencies and asymmetry factors of the chromatographic peaks,was used to obtain a diagram for the resolution function,according to

rO

O

n

i

in

i

n

=

∑∏

=1

(3)

Table 2 Solute–micelle association and stationary phase partition con-stants

Compound KMW fKSW

Chlorothiazide 53.3 0.5Hydrochlorothiazide 44.9 2.5Hydroflumethiazide 56.8 3.8Trichloromethiazide 91.6 4.9Althiazide 867 115.5 Fig. 1 Armstrong and Nome plots for 1, hydrochlorothiazide; 2,

hydroflumethiazide; 3, althiazide; and 4, bendroflumethiazide.

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where Oi is the degree of overlap:

Ow

wii

i

= − ′1 (4)

wi being the total area of a given peak i and wAi the area of thepeak overlapped by other peaks.

For the four less retained diuretics, maximum and goodresolution was achieved for concentrations of SDS lower than0.02 mol l21. In contrast, the most retained diuretics, bendro-flumethiazide and cyclothiazide, could not be resolved underany conditions. The peak of althiazide, with an intermediateretention, was easily separated from the other compounds.

Fig. 2 shows the simulated chromatograms of a mixture ofchlorothiazide, hydrochlorothiazide, hydroflumethiazide andtrichloromethiazide for 0.02, 0.05 and 0.15 mol l21 SDS eluentsat pH 7. With variation in the SDS concentration, the peak oftrichloromethiazide changed its order of elution: for the 0.02mol l21 SDS mobile phase it eluted after chlorothiazide,hydrochlorothiazide and hydroflumethiazide, whereas at higherSDS concentrations it first overlapped with the peak ofhydroflumethiazide, and subsequently with hydrochloro-thiazide.

The results indicated that the determination of diureticsshowing low and high retentions required mobile phases ofdifferent elution strengths. For chlorothiazide, hydrochloro-thiazide, hydroflumethiazide and trichloromethiazide, 0.02mol l21 SDS was chosen, and for althiazide, bendroflumethia-zide and cyclothiazide, 0.15 mol l21 SDS gave an adequateretention.

Analysis of pharmaceuticals

Initially, the diuretics were extracted from the pharmaceuticalsusing ethanol–water (10 + 90). However, the recoveries werepoor and the repeatability was low. It was then decided toperform the extraction with ethanol–0.07 mol l21 SDS (10 +90). The selected mobile phases, indicated above, were used toanalyse several pharmaceutical preparations commerciallyavailable in Spain, which contained althiazide, bendroflume-thiazide, hydrochlorothiazide and trichloromethiazide.

Calibration curves in the 1–20 mg ml21 range wereconstructed for each drug from duplicate injections of fivesolutions with increasing concentration, with regression coeffi-cients r > 0.999. The areas of the chromatographic peaks weremeasured. Five replicates of the analyses of the pharmaceuticalswere made, each with duplicate injections of the solutions. Thevalues 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 wereusually in the range 97.2–104% for the micellar mobile phases.The RSDs were usually below 3.4%.

These results were validated by comparison with conven-tional reversed-phase liquid chromatography with the same C18column. A literature survey of the chromatographic proceduresused for the determination of the thiazide diuretics was made, in

order to select a suitable composition of the aqueous–organicmobile phase for the analysis of the pharmaceuticals. Therecommended procedures for the determination of bendro-flumethiazide, hydrochlorothiazide and trichloromethiazidemake use of aqueous mobile phases containing 10–20%acetonitrile or 20–40% methanol, usually buffered at pH2.8–4.5 with phosphate or acetate.9 A mobile phase ofmethanol–0.01 mol l21 Na2HPO4 (22 + 78) at pH 4.3 wasselected for hydrochlorothiazide and trichloromethiazide. Theelution of althiazide and bendroflumethiazide was effected witha mobile phase of higher elution strength, namely methanol–0.05 mol l21 Na2HPO4 (40 + 60) at pH 3. The results are givenin Table 3.

The retention times for hydrochlorothiazide andtrichloromethiazide, using a 0.02 mol l21 SDS mobile phasewere 3.1 and 4.4 min, respectively, and those for althiazide andbendroflumethiazide using 0.15 mol l21 SDS were 4.5 and 5.7min, respectively. The retention times for hydrochlorothiazideand trichloromethiazide, using methanol–water (22 + 78) at pH4.3 were 2.9 and 14.2 min, respectively, and those for althiazideand bendroflumethiazide using methanol–water (40 + 60) at pH3 were 6.7 and 15.4 min, respectively.

Fig. 3 shows the chromatograms of three pharmaceuticalsanalysed in this work. It can be seen that the pharmaceuticalcontaining a mixture of hydrochlorothiazide andtrichloromethiazide (Rulun) could be analysed in less than 5min with a 0.02 mol l21 SDS mobile phase, whereas 15 minwere required to elute the most retained of these diuretics withmethanol–water (22 + 78) at pH 4.3. An aqueous–organicmobile phase of higher elution strength, more suitable fortrichloromethiazide, would decrease excessively the retentionof hydrochlorothiazide, which would elute near the dead

Fig. 2 Chromatograms of mixtures of chlorothiazide (CHL), hydro-chlorothiazide (HYC), hydroflumethiazide (HYF) and trichloromethiazide(TRI), eluted with (a) 0.02, (b) 0.05 and (c) 0.15 mol l21 SDS.

Fig. 3 Chromatograms of the pharmaceuticals obtained using micellar(left) and aqueous–organic (right) mobile phases: (a) Rulun [hydro-chlorothiazide and trichloromethiazide, 0.02 mol l21 SDS at pH 7 andmethanol–water (22 + 78) at pH 4.3], (b) Aldactacine [althiazide, ALT, 0.15mol l21 SDS and methanol–water (40 + 60) at pH 3], and (c) NeatenolDiuvas [bendroflumethiazide, BEN, 0.15 mol l21 SDS and methanol–water(40 + 60) at pH 3].

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volume. In Rulun, hydrochlorothiazide was contained in thecoating of the tablets and trichloromethiazide was included inits nucleus.

Conclusions

The chromatographic procedure proposed here for thedetermination of some thiazide diuretics in pharmaceuticalswith a micellar mobile phase of SDS without a modifier is rapidand simple. The elution of the diuretics, except hydro-chlorothiazide, was more rapid with the micellar eluents thanwith the recommended aqueous–organic mobile phases, espe-cially for trichloromethiazide and bendroflumethiazide.

It is notable that the micellar mobile phases used in this workdid not require the addition of an organic solvent to achieveadequate retention of the diuretics. In most of the micellarchromatographic procedures reported in the literature, a smallamount of a short-chain alcohol must be added to accelerate andcontrol the elution of the solutes. A decrease in the organicsolvent content has been considered an interesting advantage ofmicellar mobile phases over aqueous–organic eluents. Theprocedure described here for the determination of the thiazide

diuretics has the advantage of complete elimination of thepolluting and flammable organic solvents.

In previous work, an optimization procedure was developed,based on the description of the retention of solutes in micellarmobile phases which contained a surfactant and a modifier.6 Wehave now demonstrated the application of this procedure usingsimpler mobile phases containing only the surfactant. In thiscase, an accurate description of the retention by the equationsuggested by Armstrong and Nome12 is employed. The elutionbehavior of the thiazide diuretics chlorothiazide, hydrochloro-thiazide, hydroflumethiazide and trichloromethiazide producedseveral changes in their elution order, which made theoptimization of their resolution with a procedure based on thesequential modification of the composition of the mobile phasedifficult. The interpretive procedure led to the optimum mobilephase using chromatographic data obtained with only two orthree mobile phases, which are necessary to obtain the two-variable equations that describe the elution behavior of eachdiuretic.

This work was supported by the DGICYT, Project PB94/967(Spain). Samuel Carda Broch thanks the Conselleria de Cultura,

Table 3 Results of analysis of pharmaceuticals with micellar and aqueous–organic reversed-phase liquid chromatography

Micellar Aqueous–organic

Found/ RSD (%) Found/ RSD (%)mg (n = 5) mg (n = 5)

Pharmaceutical* Laboratory Composition/mg per tablet or capsule

Hidrosaluretil Gayoso Welcome, Alcala deHenares, Madrid, Spain

Hydrochlorothiazide (50), lactose andother excipients

49.5 0.6 50.8† 0.6

Alopresin Diu Alonga, Alcala, Madrid, Spain Hydrochlorothiazide (25), captopril(25), lactose and other excipients

25.3 0.5 26.6† 1.9

Ameride DuPont Pharma, Madrid, Spain Hydrochlorothiazide (50), amiloridechlorhydrate (5), lactose (71),other excipients

50.5 0.4 53.0† 7.5

Selopresin Astra, Esplugues de Llobregat,Barcelona, Spain

Hydrochlorothiazide (12.5),metoprolol tartrate (100), lactoseand other excipients

12.7 1.6 12.8† 1.4

Kalten Zeneca Farma, Porrino,Pontevedra, Spain

Hydrochlorothiazide (25), atenolol(50), amiloride chlorhydrate (2.5),lactose and other excipients

24.8 1.2 24.6† 4.0

Picten Miquel, Barcelona, Spain Hydrochlorothiazide (40),methyldopa (70), triamterene (25),reserpine (0.2), lactose, sulfite andother excipients

40.1 1.0 39.6† 1.0

Neotensin Diu Cepa, Alcobendas, Madrid,Spain

Hydrochlorothiazide (12.5), enalaprilmaleate (20), lactose and otherexcipients

12.55 1.2 12.45† 1.2

Zestoretic Zeneca Farma, Porrino,Pontevedra, Spain

Hydrochlorothiazide (12.5), lisinopril(20), excipients

12.65 1.3 12.8† 1.0

Rulun Lacer, Sardenya, Barcelona,Spain

Tablet coating: hydrochlorothiazide(20), xanthinol nicotinate (40),saccharose (167.64), excipient

20.8 3.4 21.5† 5.6

Tablet nucleus: trichloromethiazide(3), alkaloid fraction of Rauwolfias. Dehra Dun (2.3), pentosansodium polysulfate (25), xanthinolnicotinate (60), lactose and otherexcipients

2.95 3.4 2.82† 2.8

Aldactacine Searle Industrie, Evreux, France Althiazide (15), spironolactone (25),lactose and other excipients

15.1 0.4 14.9‡ 0.9

Spirometon Belmac, Zaragoza, Spain Bendroflumethiazide (2.5),spironolactone (50), excipients

2.43 1.2 2.62‡ 1.5

Neatenol Diuvas Fides-Rottapharm, Almacera,Valencia, Spain

Bendroflumethiazide (5), hydralazinechlorhydrate (50), atenolol (100),excipients

5.01 1.0 4.92‡ 1.4

* All the pharmaceuticals were tablets, except Kalten, which was in the form of capsules. † Methanol–water (22 + 78) (pH 4.3) mobile phase.‡ Methanol–water (40 + 60) (pH 3) mobile phase.

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Educacio i Ciencia de la Generalitat Valenciana for an FPIGrant.

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Paper 7/05641IReceived August 4, 1997

Accepted October 1, 1997

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