simultaneous determination of hydrochlorothiazide and several inhibitors of angiotensin-converting...

924 (2001) 439–449Journal of Chromatography A,www.elsevier.com/ locate /chroma

Simultaneous determination of hydrochlorothiazide and severalinhibitors of angiotensin-converting enzyme by capillary

electrophoresisS. Hillaert*, K. De Grauwe, W. Van den Bossche

Laboratory of Pharmaceutical Chemistry and Drug Analysis, Department of Pharmaceutical Analysis,Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium

Abstract

A capillary electrophoresis method was developed for the simultaneous determination of hydrochlorothiazide and severalangiotensin-converting enzyme (ACE) inhibitors: enalapril, lisinopril, quinapril, fosinopril, ramipril, and cilazapril. The mostcritical parameter is the pH of the running buffer. Separation was performed on a fused-silica capillary (52 cm totallength375 mm I.D.) using a sodium phosphate buffer (pH 7.25; 100 mM). The method was successfully applied to thequantitative determination of these compounds in their corresponding pharmaceutical formulation. The method was validatedin terms of linearity of response, reproducibility and accuracy. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Pharmaceutical analysis; Hydrochlorothiazide; ACE inhibitors

1. Introduction used in the control of hypertension, either alone or incombination with other drugs such as ACE in-

Inhibitors of angiotensin-converting enzyme hibitors. Their hypotensive effect is believed to be(ACE) are widely used for the treatment of mild to due initially to the reduction of blood volume by

1moderate hypertension and heart failure either alone Na depletion, and later by direct relaxation ofor in conjunction with hydrochlorothiazide, a arteriolar smooth muscle [2,3].thiazide diuretic [1]. Hydrochlorothiazide increases Until now, high-performance liquid chromatog-the rate of urine excretion by the kidneys, primarily raphy has been the major technique used for thethrough decreased tubular reabsorption of sodium simultaneous determination of the concentration andand chloride and by increased osmotic transport of presence of hydrochlorothiazide (HCT) and enalaprilwater to the renal tubules. Thiazide diuretics are maleate [4–6], lisinopril [7,8] and benazepril [9].extremely useful in the treatment of oedema associ- Capillary electrophoresis (CE) offers an alternativeated with mild to moderate congestive heart failure. technique. Although analysis by means of CE hasMoreover, these diuretics are also primary agents been achieved for both hydrochlorothiazide [10–12]

and the different ACE inhibitors [13–18], thesestudies have usually been limited to the determi-*Corresponding author. Tel.: 132-9-264-8101; fax: 132-9-nation of a single component [13–16]. Only one264-8193.

E-mail address: [email protected] (S. Hillaert). study has reported the simultaneous determination of

0021-9673/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0021-9673( 01 )00714-2

924 (2001) 439–449440 S. Hillaert et al. / J. Chromatogr. A

hydrochlorothiazide and enalapril maleate by CE hydrochlorothiazide and a large number of structural-[19]. Therefore, the aim of the present study was to ly related ACE inhibitors by CE. The chemicaldevelop a selective method capable of separating structures of these compounds are shown in Fig. 1.

Fig. 1. Chemical structures of the ACE inhibitors and hydrochlorothiazide.

924 (2001) 439–449 441S. Hillaert et al. / J. Chromatogr. A

Table 1Reference solutions for the quantitative determination

Reference substance Reference solution Diluted reference(mg/100 ml) solution (mg/ml)

Enalapril maleate 660 60.24HCT 638 60.15

Lisinopril dihydrate 680 60.32HCT 650 60.20

Quinapril?HCl (10 mg/ tablet) 660 60.24HCT 670 60.28

Quinapril?HCl (20 mg/ tablet) 660 60.24HCT 635 60.14

Fosinopril sodium 656 60.22HCT 635 60.14

Ramipril 617.5 60.07HCT 687.5 60.35

Cilazapril?HCl 628 60.11HCT 670 60.28

2. Experimental disodium hydrogenphosphate dihydrate (both ana-lytical-reagent grade) were obtained from Merck

2.1. Instrumentation and electrophoresis procedure (Darmstadt, Germany). Enalapril maleate andlisinopril dihydrate were purchased from Sigma (St.

The method was developed and subsequent experi- Louis, MO, USA), and hydrochlorothiazide fromments were performed using a Waters Quanta 4000 Profarma (Belgium). Lisinopril dihydrate was ob-CE instrument (Millipore, Waters). The capillary tained from MSD and AstraZeneca, quinapril?HClused was a fused-silica capillary 52 cm in total from Parke-Davis, fosinopril sodium from Bristol-length (44.5 cm to the detector) with an internal Myers Squibb, ramipril from AstraZeneca, anddiameter of 75 mm. cilazapril from Roche.

Hydrostatic injections were performed by lifting The commercially available drugs Co-Renitecthe sample vial |10 cm above the height of the (MSD), Zestoretic (AstraZeneca), Accuretic (Parke-buffer vial for 10 s. For detection, absorbance was Davis), Foside (Solvay), Tritiazide (Hoechst Marionmeasured by means of an on-line fixed-wavelength Roussel) and Co-Inhibace (Roche) were used forUV detector with a zinc discharge lamp and a 214- quantitative determinations.nm filter. The experiments were performed at 20 kVat room temperature (20628C). Data were collected 2.3. Running bufferson a Hewlett-Packard Integrator (HP 3396, SeriesII), which was also used for calculating the areas During the development of the method, sodiumunder the peaks. phosphate buffers of different pH values were tested

as running buffers. A mixture of a sodium dihydro-2.2. Reagents genphosphate solution and a disodium hydro-

genphosphate solution was used with a pH range ofSodium dihydrogenphosphate monohydrate and 6.5–8.5.


A sodium phosphate buffer (pH 7.25; 100 mM) determination of lisinopril, another ACE inhibitorwas finally chosen as the running buffer. It was must be used. In this investigation, quinapril wasprepared by adjusting the pH of a 100 mM disodium chosen. An appropriate amount of the compoundhydrogenphosphate solution to pH 7.25 by the (Table 2) was dissolved in 10 ml of methanol andaddition of 100 mM sodium dihydrogenphosphate diluted to 100.0 ml with water.solution.

2.5. Choice of solvent2.4. Internal standard solutions

The running buffer cannot be used as a solvent forFor the quantitative determination of HCT and one the preparation of reference and sample solutions

of the six ACE inhibitors, another ACE inhibitor was because of the poor solubility of hydrochlo-always used as an internal standard. Selection had to rothiazide. Although an alkaline solution should bebe made based on the substance to be examined. suitable to dissolve both the diuretic and the ACELisinopril dihydrate was mostly chosen as the inter- inhibitors, this medium must be avoided, as it isnal standard, because of its baseline separation from unstable. Therefore, methanol was added to dissolveall the other ACE inhibitors and because of its ready the active substances, and the solutions were thenavailability as a bulk product on the market. For the diluted with water.

Table 2Sample preparation for the quantitative determination

Average Sample solution Internal standard Diluted samplemass (mg) (mg powder /50 ml) solution (mg/ml) solution (mg active

substance /ml)

Enalapril (E) 20 mg 200.4 6301 Lisinopril?2H O: E: 60.242

HCT 12.5 mg 1 HCT: 60.15(Co-Renitec, tablets)

Lisinopril (L) 20 mg 226.0 6452 Quinapril?HCl: L: 60.32HCT 12.5 mg 0.5 HCT: 60.20(Zestoretic, tablets)

Quinapril (Q) 10 mg 103.5 6285 Lisinopril?2H O: Q: 60.222

HCT 12.5 mg 1 HCT: 60.28(Accuretic 10/12.5, tablets)

Quinapril (Q) 20 mg 207.2 6290 Lisinopril?2H O: Q: 60.222

HCT 12.5 mg 1 HCT: 60.14(Accuretic 20/12.5, tablets)

Fosinopril (F) sodium 20 mg 201.9 6283 Lisinopril?2H O: F: 60.222

HCT 12.5 mg 0.8 HCT: 60.14(Foside, tablets)

Ramipril 5 mg 102.6 6180 Lisinopril?2H O: R: 60.072

HCT 25 mg 0.8 HCT: 60.35(Tritazide, tablets)

Cilazapril 5 mg 203.2 6570 Lisinopril?2H O: C: 60.112

HCT 12.5 mg 0.8 HCT: 60.28(Co-Inhibace, tablets)


2.6. Reference solutions them through a Millipore filter unit (0.45-mm poresize).

Reference solutions were prepared by weighingaccurately an appropriate amount of the corre-sponding reference substance, dissolving it in 10 ml 3. Results and discussionmethanol and diluting to 100.0 ml with water (Table1). A volume of 10.0 ml of each solution was mixed 3.1. Optimization of the methodwith 10.0 ml of the internal standard solution anddiluted to 25 ml with a methanol–water (1:9) The optimization of a selective capillary electro-mixture. phoretic separation of several ACE inhibitors was

described earlier [18]. Separation was performed by2.7. Sample preparations means of two phosphate buffers (each 100 mM) at

pH 7.0 and 6.25, respectively. This combination isA minimum of 20 tablets of each compound were necessary for the selective identification of the

weighed, ground, and mixed. An appropriate amount structurally related substances because of their simi-of the powder (Table 2) was mixed with 5 ml lar pK values. The addition of organic modifiers anda

methanol and diluted to 50.0 ml with water. Then 10 surfactants had a negative influence on peak symme-ml of the filtrate was mixed with 10.0 ml of the try, and selectivity was not improved [18]. Based onappropriate internal standard solution (Table 2) and these data, the pH was varied in the range of 6.5–diluted to 25 ml with a methanol–water (1:9) 8.5. Separation is greatly influenced by the pH of themixture. medium (Fig. 2). Below pH 7.0, hydrochlorothiazide

All samples and buffers were filtered by passing and the marker used (formamide) co-elute because

Fig. 2. Influence of pH. Experiments were carried out using a fused-silica capillary 52 cm (44.5 cm to the detector)375 mm I.D., andsodium phosphate buffer (100 mM) at varying pH as the running buffer, an applied voltage of 20 kV, and a detection wavelength of 214 nm.


they are both uncharged (i.e. there is no selectivity).From pH 8.0 and higher, the separation of hydro-chlorothiazide and the ACE inhibitors becomescritical because all species become negativelycharged and consequently some co-elute. The op-timum pH for the separation of HCT and the ACEinhibitors was in the range 7.0–8.0. The optimumconditions for the separation of HCT and the majori-ty of the ACE inhibitors were found using a sodiumphosphate buffer (pH 7.25).

As well as pH, the ionic strength of the runningbuffer is another parameter that controls the retentionof these compounds. We varied the molarity of thesodium phosphate buffers (see above) from 50 to 100mM. The selectivity of the separation was notinfluenced by the molarity, and only the migrationtimes increased. Because it had the highest bufferingcapacity and provided acceptable migration times, asodium phosphate buffer (100 mM) was chosen.

3.2. Quantitative determination in pharmaceuticalformulations

The proposed system (a sodium phosphate buffer,pH 7.25, 100 mM) may be applied for the quantita-tive determination of the combination of HCT/ACEinhibitors in tablets (Figs. 3–7).

3.3. Validation of the method

3.3.1. LinearityThe detector responses were found to be linear for

the different components in two concentrationranges, as described in Table 3. The amount of theinternal standard was adjusted according to theconcentration range used. Regression analysis datafor the calibration curves were calculated using thepeak areas.

3.3.2. PrecisionThe precision (repeatability) was determined by

the total analysis of six replicate samples under thesame operating conditions, by the same analyst, and

Fig. 3. Electropherogram of the quantitative determination ofon the same day. The mean value of the con-HCT and enalapril (Co-Renitec) on a fused-silica capillary,centration and the relative standard deviation areperformed on the Waters Quanta. Conditions: 52 cm (44.5 cm to

summarized in Table 4. the detector)375 mm I.D.; sodium phosphate buffer (pH 7.25; 100The error of the equipment, the accuracy of mM) as running buffer; applied voltage, 20 kV; detection at 214

electrophoretic separation, and the relative standard nm.


Fig. 4. Electropherogram of the quantitative determination of Fig. 5. Electropherogram of the quantitative determination ofHCT and quinapril (Accuretic) on a fused-silica capillary, per- HCT and fosinopril (Foside) on a fused-silica capillary, performedformed on the Waters Quanta. Conditions: 52 cm (44.5 cm to the on the Waters Quanta. Conditions: 52 cm (44.5 cm to thedetector)375 mm I.D.; sodium phosphate buffer (pH 7.25; 100 detector)375 mm I.D.; sodium phosphate buffer (pH 7.25; 100mM) as running buffer; applied voltage, 20 kV; detection at 214 mM) as running buffer; applied voltage, 20 kV; detection at 214nm. nm.


Fig. 6. Electropherogram of the quantitative determination of Fig. 7. Electropherogram of the quantitative determination ofHCT and ramipril (Tritiazide) on a fused-silica capillary, per- HCT and cilazapril (Co-Inhibace) on a fused-silica capillary,formed on the Waters Quanta. Conditions: 52 cm (44.5 cm to the performed on the Waters Quanta. Conditions: 52 cm (44.5 cm todetector)375 mm I.D.; sodium phosphate buffer (pH 7.25; 100 the detector)375 mm I.D.; sodium phosphate buffer (pH 7.25; 100mM) as running buffer; applied voltage, 20 kV; detection at 214 mM) as running buffer; applied voltage, 20 kV; detection at 214nm. nm.


Table 3 deviations of estimations were determined by per-Linearity forming ten consecutive injections of the same

Concentration Correlation sample (Table 5).2range (mg/ml) coefficient (r )

Enalapril maleate 0.026–0.320 0.99913.3.3. AccuracyHCT 0.016–0.200 0.9998

The accuracy of the method was determined byLisinopril dihydrate 0.026–0.520 0.9999 investigating the recovery of each component atHCT 0.020–0.400 0.9995 three levels, ranging from 80 to 120% of the

theoretical concentration, from placebo mixturesQuinapril?HCl 0.032–0.320 0.9994

spiked with the active substance (Table 6).HCT 0.020–0.400 0.9997

Fosinopril sodium 0.019–0.380 0.9991HCT 0.010–0.200 0.9992

4. ConclusionsRamipril 0.006–0.120 0.9997HCT 0.020–0.400 0.9997 The above results demonstrate that CE separation

of hydrochlorothiazide and one of the six ACECilazapril 0.008–0.160 0.9995

inhibitors can be achieved using a 100 mM sodiumHCT 0.020–0.400 0.9995phosphate buffer at pH 7.25. This system can be

Table 4Precision (repeatability) of the total analysis of six replicate samples

Substance Theoretical amount Amount found (mg) Relative standardto be examined (mg/ tablet) deviation (n56) (%)

Enalapril maleate 20 20.0560.14 or 100.2% 0.73HCT 12.5 12.5360.04 or 100.2% 0.30(Co-Renitec)

Lisinopril?2H O 20 20.4460.05 or 102.2% 0.222

HCT 12.5 12.6060.06 or 100.8% 0.48(Zestoretic)

Quinapril?HCl 10 10.1260.42 or 101.2% 0.42HCT 12.5 12.6060.77 or 100.8% 0.77(Accuretic 10/12.5)

Quinapril?HCl 20 20.1260.19 or 100.6% 0.92HCT 12.5 12.5360.06 or 100.2% 0.46(Accuretic 20/12.5)

Fosinopril sodium 20 19.4760.10 or 97.4% 0.53HCT 12.5 11.9760.07 or 95.8% 0.56(Foside)

Ramipril 5 4.8960.05 or 97.8% 0.92HCT 25 24.0160.05 or 96.0% 0.21(Tritiazide)

Cilazapril 5 5.2360.02 or 104.6% 0.38HCT 12.5 12.3560.05 or 98.8% 0.39(Co-Inhibace)


Table 5 AcknowledgementsRepeatability of ten consecutive injections of the same sample

Sample solution Relative standard The following firms are kindly acknowledged fordeviation (n510) (%) having supplied their products: MSD, AstraZeneca,

Enalapril maleate 0.76 Parke-Davis, Bristol-Myers Squibb and Roche.HCT 0.51

Lisinopril?2H O 0.562

HCT 0.52 References

Quinapril?HCl 0.50[1] G.H. Cocolas, in: J.N. Delgado, W.A. Remers (Eds.), Text-HCT 0.44

book of Organic Medicinal and Pharmaceutical Chemistry,10th ed, Lippincott-Raven, Philadelphia, 1998, p. 603.Fosinopril sodium 1.08

[2] W.O. Foye, Principles of Medicinal Chemistry, 2nd ed,HCT 0.47Henry Kimpton, London, 1981.

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Table 6Accuracy

Recovery Recovery Recoveryplacebo180% placebo1100% placebo1120%(n53) (%) (n53) (%) (n53) (%)

Enalapril 100.360.6 99.560.2 100.460.4HCT 99.460.1 101.260.1 99.160.1

Lisinopril 98.960.4 100.060.5 101.460.6HCT 102.160.9 103.060.6 97.760.6

Quinapril 10 mg 99.560.5 100.560.6 100.960.4HCT 101.360.2 100.160.5 99.860.5

Quinapril 20 mg 99.160.2 102.960.4 100.260.3HCT 99.360.3 100.060.4 100.360.4

Fosinopril 103.960.2 101.260.4 102.260.5HCT 98.760.4 101.660.9 99.660.7

Ramipril 101.060.3 100.460.6 98.660.4HCT 104.260.4 100.560.6 100.560.5

Cilazapril 102.860.1 102.460.8 101.860.7HCT 104.660.5 102.260.8 100.160.6


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simultaneous determination of hydrochlorothiazide and several inhibitors of angiotensin-converting...

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