Derivative spectrophotometric method for simultaneous determination of zofenopril and fluvastatin in mixtures and pharmaceutical dosage forms

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 66–71

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Derivative spectrophotometric method for simultaneous determination of zofenopril and fluvastatin in mixtures and pharmaceutical dosage forms Mariusz Stolarczyk ⇑, Anna Mas´lanka, Anna Apola, Wojciech Rybak, Jan Krzek Department of Inorganic and Analytical Chemistry, Jagiellonian University Medical College, Faculty of Pharmacy, 9 Medyczna Street, 30-688 Kraków, Poland

g r a p h i c a l a b s t r a c t

 A new method was developed for the

339.03

0,02

0,01

0

233

253

273

293

313

333

353

373

-0,01

D1

-0,02

-0,03

-0,04

-0,05

FLU

ZOF

D1

-0,06

-0,07

3

-0,08 Wavelengh (nm)

2,5

UV

2

0,0065

1,5

D2

Abs

1

0,0045

0,5

0,0025

0 -0,5

200

250

300 Wavelenght

350

400

D2

determination of zofenopril and fluvastatin.  The analytical method has been validated according to the ICH recommendations.  Our method was applied for the analysis of drugs in the mixtures and dosage forms.

252.57

h i g h l i g h t s

0,0005

215

235

255

275

295

315

335

355

375

-0,0015

-0,0035

-0,0055 Wavelength (nm)

D3 0,0015

258.50

0,001

D3

0,0005

0

218

238

258

278

298

318

338

358

-0,0005

-0,001

-0,0015 Wavelength (nm)

a r t i c l e

i n f o

Article history: Received 3 June 2014 Received in revised form 18 March 2015 Accepted 27 March 2015 Available online 2 April 2015 Keywords: Zofenopril Fluvastatin Derivative spectrophotometry

a b s t r a c t Fast, accurate and precise method for the determination of zofenopril and fluvastatin was developed using spectrophotometry of the first (D1), second (D2), and third (D3) order derivatives in two-component mixtures and in pharmaceutical preparations. It was shown, that the developed method allows for the determination of the tested components in a direct manner, despite the apparent interference of the absorption spectra in the UV range. For quantitative determinations, ‘‘zero-crossing’’ method was chosen, appropriate wavelengths for zofenopril were: D1 k = 270.85 nm, D2 k = 286.38 nm, D3 k = 253.90 nm. Fluvastatin was determined at wavelengths: D1 k = 339.03 nm, D2 k = 252.57 nm, D3 k = 258.50 nm, respectively. The method was characterized by high sensitivity and accuracy, for zofenopril LOD was in the range of 0.19–0.87 lg mL 1, for fluvastatin 0.51–1.18 lg mL 1, depending on the class of derivative, and for zofenopril and fluvastatin LOQ was 0.57–2.64 lg mL 1 and 1.56–3.57 lg mL 1, respectively. The recovery of individual components was within the range of 100 ± 5%. For zofenopril, the linearity range was estimated between 7.65 lg mL 1 and 22.94 lg mL 1, and for fluvastatin between 5.60 lg mL 1 and 28.00 lg mL 1. Ó 2015 Elsevier B.V. All rights reserved.

Introduction

Abbreviations: ZOF, zofenopril; FLU, fluvastatin; ICH, International Conference on Harmonisation. ⇑ Corresponding author. Tel.: +48 12 620 54 80; fax: +48 12 620 54 05. E-mail address: [email protected] (M. Stolarczyk). http://dx.doi.org/10.1016/j.saa.2015.03.100 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

Zofenopril (ZOF), (2S, 4R)-1-((S)-3-(benzoylthio)-2-methylpropanoyl)-4 (phenylthio) pyrrolidine-2-carboxylic acid (Fig. 1), is a cardioprotective drug from angiotensin-converting enzyme inhibitors group, used in the treatment of hypertension. In

67

M. Stolarczyk et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 66–71

a

b

comparison with older drugs of this group such as enalapril, captopril or perindopril, it is characterized by a higher hypotensive efficacy and fewer side effects. Fluvastatin (FLU), (3R, 5S, 6E)-7-[3-(4-fluorophenyl)-1-(propan2-yl)-1H-indol-2-yl]-3,5-dihydroxyhept-6-enoic acid (Fig. 1), belongs to a group of statins, selective inhibitors of 3-hydroxy-3methylglutaryl coenzyme-A reductase (HMG-CoA reductase). It is commonly used as one of the primary hypolipidemic drugs. Both, angiotensin converting enzyme inhibitors (ZOF) and statins (FLU) are recommended in the treatment of hypertension with concomitant dyslipidemia. The use of such pharmacological profile reduces the potential of the occurrence of changes in cardiovascular and respiratory system [1]. The development of simple and fast method for simultaneous determination of drugs from these pharmacological groups, seems to be useful. Review of the literature showed that UV spectrophotometry is mainly recommended for the determination of ZOF in pharmaceutical preparations, which is useless for the determination of ZOF in the presence of other components, e.g. FLU [2]. ZOF, similar to other substances from the group of angiotensinconverting inhibitors, is often used in the treatment of hypertension together with drugs characterized by diuretic action [3]. In such a form, it was determined in the presence of hydrochlorothiazide by reversed-phase liquid chromatography (RP-LC) [4]. For the determination of zofenopril and its active metabolite (zofenoprilate) in serum, LC–MS–MS was used [5]. TLC method was used in the analysis of zofenopril and fosinopril mixture [6]. For the determination of fluvastatin, a number of analytical methods were used. The most commonly used separation methods include: HPLC with spectrophotometric [7,8] or fluorimetric [9–11] detection, liquid chromatography (LC) [12] and capillary electrophoresis [13].

Fig. 1. Chemical structure (a) zofenopril (ZOF), and (b) fluvastatin (FLU).

2,75

2,25

mixture Fluvastatin Zofenopril

1,25

0,75

0,25

-0,25 200

220

240

260

280

300

320

340

360

380

400

Wavelength 1

Fig. 2. Zero-order absorption spectra of ZOF (22.94 lg mL and mixtures with the same concentrations.

0,01 0

233 -0,01

1

),

0,0065

339.03

0,02

), FLU (28.00 lg mL

0,0045 253

273

293

313

333

353

373 0,0025

252.57

Abs

1,75

D2

-0,03 11.20 16.80

16.80 22.40

235

255

275

295

315

335

355

-0,0015

22.40

-0,05

11.20

0,0005 215

5.60

-0,04

5.60

28.00 22.94

-0,06

-0,0035

-0,07 -0,0055

Wavelengh (nm)

Wavelength (nm)

0,0015

0,001 258.50

-0,08

5.60

0,0005

11.20 16.80

D3

D1

-0,02

0

22.40

218

238

258

278

298

318

338

358

28.00 22.94

-0,0005

-0,001

-0,0015

Wavelength (nm)

Fig. 3. The first D1, second D2 and third D3 derivative values for standard solutions of FLU (—) and ZOF (- - - -) (lg mL

1

).

375

28.00

22.94

68

M. Stolarczyk et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 66–71

Electroanalytical methods have also been widely used in the analysis of fluvastatin [13–17].

Procedures Standard and working solution

Experimental

ZOF – to a 10 mL volumetric flask, approximately 10 mg of zofenopril calcium was weighed using an analytical balance, and filled with methanol to a specified volume. FLU – to a 10 mL volumetric flask, approximately 10 mg of fluvastatin sodium was weighed using an analytical balance, and filled with methanol to a specified volume. For direct measurements, solutions were diluted with methanol in a 5 mL flask, to obtain concentrations between 7.65 lg mL 1 and 22.94 lg mL 1 for ZOF, and 5.6 lg mL 1 and 28.00 lg mL 1 for FLU, respectively.

Instruments Spectrophotometer UV–VIS Cary 100 (Varian), quartz cuvettes (l = 1 cm). Computer Dell Optiplex 755; Intel(R) Core(TM)2 Duo CPU; E4500 @ 2.20 GHz; 1.18 GHz, 1.95 GB Ram (Microsoft Office 2010, Statistica 10 edition 2012). Chemicals Zofenoprilum calcium – Chengdu Sino-Strong Pharmaceutical Co., Ltd. (Chengdu, PR China), Fluvastatin sodium – USP Rockville, MD LOT F0F015. Reagents of analytical grade quality: methanol.

Spectral characteristics of ZOF and FLU In the first stage of the study, absorption spectra for ZOF (22.94 lg mL 1), FLU (28.00 lg mL 1) and mixtures of these substances at given concentrations were recorded in quartz cuvettes (l = 1 cm), in the presence of methanol as a reference solution in the UV range. Recorded absorption spectra were characterized by little variation. Lack of well-defined absorption maxima and a clear interference of recorded spectra, especially in the range of 200– 270 nm, was observed. This fact hampers the determination of the substance directly using zero-order spectra (Fig. 2). Conversion of zero-order spectra into derivatives causes their significant variation. Applying ‘‘zero-crossing’’ method, zeros of function for one of the compound were determined on the curve

Pharmaceutical formulations Two pharmaceutical preparations were used for determinations: ZOFENIL 30 manufactured by Berlin-Chemie AG, Berlin, Germany. Each ZOFENIL 30 mg tablet contains 30 mg of zofenopril calcium as 28.7 mg of zofenopril. LESCOL 40 manufactured by Novartis Pharma GmbH, Nürnberg, Germany. One capsule contains 42.12 mg fluvastatin sodium equivalent to 40 mg fluvastatin free acid.

0,015 270.85

0,005

0,01

0,004

0,005 0,003 240

260

280

300

320

340

7.65

360

11.47 19.12 22.94

15.29 19.12

0

-0,015 -0,02

211

22.94 11.20

231

251

271

-0,001 -0,002

Wavelength (nm)

291

311

331

351

371

286.38

Wavelength (nm)

0,0005

0,0003

253.90

-0,025

11.47

0,001

11.20

-0,01

7.65

0,002

15.29

-0,005

D2

220

0,0001

7.65

11.47

D3

D1

0

220 -0,0001

240

260

280

300

320

340

360

15.29 19.12 22.94 11.20

-0,0003

-0,0005

-0,0007

Wavelength (nm)

Fig. 4. The first D1, second D2 and third D3 derivative values for standard solutions of ZOF (—) and FLU (- - - -) (lg mL

1

).

391

69

D3 (k 258.50 nm)

of derivatives. For quantitative analysis, the following wavelengths were selected: D1 k = 270.85 nm, D2 k = 286.38 nm, D3 k = 253.90 nm for ZOF, and D1 k = 339.03 nm, D2 k = 252.57 nm, D3 k = 258.50 nm for FLU, respectively. Recorded curves of derivatives for increasing concentrations of determined substance together with D1, D2, D3 plots against concentrations, are shown in Figs. 3 and 4.

5.60–28.00 y = 0.000056x 0.00002 0.9993 2.244 – 0.972 (0.903) 1.224 (0.349) 1.18 3.57 100.59 ± 1.193 101.89 1.19

M. Stolarczyk et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 66–71

Three concentrations of each analyte (7.50 lg mL 1, 15.00 lg mL 1, and 22.50 lg mL 1), repeated three times for each concentrate. Three concentrations of each analyte (12.00 lg mL 1, 15.00 lg mL 1, and 18.00 lg mL 1), repeated three times for each concentrate. a

b

5.60–28.00 y = 0.000572x 0.000243 0.9995 2.155 – 0.951 (0.750) 0.026 (0.883) 1.03 3.13 100.35 ± 1.064 99.85 1.06 7.65–22.94 y = 0.000016x 0.000002 0.9999 3.424 – 0.901 (0.382) 0.513 (0.526) 0.25 0.75 101.97 ± 2.005 101.52 1.97 7.65–22.94 y = 0.000041x + 0.000088 0.9993 3.019 1.645 (0.20) 0.949 (0.733) 0.231 (0.664) 0.87 2.64 98.77 ± 2.288 100.95 2.31 7.65–22.94 y = 0.000531x + 0.000325 0.9999 2.925 1.210 (0.27) 0.903 (0.39) 0.008 (0.93) 0.19 0.57 103.19 ± 1.573 102.70 1.52 Linearity range (lg mL 1) Regression equation Correlation coefficient (r) Durbin–Watson test Lagrange’s test (p) Shapiro–Wilk test (p) Mandel’s test (p) LOD (lg mL 1) LOQ (lg mL 1) Accuracya (%) Precisionb (%) RSD%

D1 (k 339.03 nm) D2 (k 286.38 nm)

D3 (k 253.90 nm)

Fluvastatin sodium

Laboratory prepared mixtures containing different ratios of ZOF and FLU (selectivity) To a series of 5 mL volumetric flasks, different amounts of working solutions were accurately transferred and filled with methanol to a specified volume, obtaining mixtures at concentrations of 13.60 lg mL 1, 17.00 lg mL 1, 20.40 lg mL 1 for ZOF, and 11.52 lg mL 1, 14.40 lg mL 1, 17.28 lg mL 1 for FLU. For such mixtures of the active compounds, absorption spectra in the range 200–400 nm were recorded. After converting spectra into derivatives of an appropriate order, the values of derivatives against experimentally determined wavelengths were read. The amount

Parameters

Limit of detection (LOD) and limit of quantitation (LOQ) LOD and LOQ were determined using standard error of the estimate (SY) and the slope of the calibration curve (a) and calculated according to the formulas: LOD = 3.3
SY/a and LOQ = 10.0
SY/a.

Table 1 Validation parameters of the proposed spectrophotometric methods.

Accuracy and precision The accuracy of the method was determined as a percentage of analyte recovery for the prepared solutions at three concentrations: 80%, 100% and 120%. The precision of the method was checked using the proposed procedure for the determination of solutions containing very specific analyte amounts at three concentrations: 50%, 100% and 150%. All measurements were repeated three times, accuracy and precision of the results were calculated using the appropriate regression equation.

Zofenopril calcium

Linearity Zero-order absorption spectra for methanol solution of ZOF were plotted within the range of 7.65 lg mL 1 and 22.94 lg mL 1 and for FLU between 5.6 lg mL 1 and 28.00 lg mL 1. After conversion of the absorption spectra into the D1, D2 and D3 curves of derivative, the value of derivative was read at an appointed wavelength. The graph of D1; 2; 3 = f(c) was plotted. In the tested concentration range, linearity was maintained. To evaluate the results, linear regression equation characterizing the intersection points, correlation coefficients, Mandel’s, Shapiro–Wilk, the Durbin–Watson and Lagrange tests were used. In the evaluation of the calibration method, linear and quadratic fit were tested. Both models were compared using Mandel’s test. The p-value <0.05 indicates a similarity of both fits. Normal distributions of the residuals were tested by the Shapiro–Wilk test. The value of W greater than the critical value allows to assume normal distribution. By applying Durbin–Watson test, the presence of residual autocorrelation was checked. If the tested values of DW are greater than the upper critical value no significant autocorrelation is observed, and if the DW values are lower than the lower critical value then autocorrelation appears. The test does not give an answer on the occurrence of autocorrelation, when DW is in the range between the lower and upper critical value and in such case, Lagrange test is recommended.

D1 (k 270.85 nm)

D2 (k 252.57 nm)

Validation procedure was done according to ICH recommendations for linearity, range, accuracy and precision, limit of detection (LOD), limit of quantitation (LOQ) and relative recovery [18].

5.60–28.00 y = 0.000164x 0.9999 2.029 – 0.922 (0.522) 0.545 (0.514) 0.51 1.56 99.63 ± 1.346 102.52 1.35

0.000091

Method validation

70

M. Stolarczyk et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 66–71

Table 2 Determination of the two analytes in their laboratory prepared mixtures by the proposed spectrophotometric methods. Conc. (lg/mL)

Zofenopril 17.00 20.40 13.60

Zofenopril (Recovery% ± Sx)

Fluvastatin 14.40 11.52 17.28

Fluvastatin (Recovery% ± Sx)

D1

D2

D3

D1

D2

D3

100.84 ± 0.304 96.16 ± 2.926 99.02 ± 1.852

104.02 ± 1.187 96.18 ± 0.766 98.63 ± 1.034

101.18 ± 1.175 97.0.4 ± 1.354 97.97 ± 1.609

100.69 ± 1.385 98.41 ± 1.272 100.95 ± 0.468

99.12 ± 2.187 95.38 ± 0.241 98.84 ± 0.606

100.14 ± 2.683 95.43 ± 0.447 100.17 ± 0.552

All calculations were done in triplicates, Sx – standard deviation.

Table 3 Determination of ZOF and FLU in pharmaceutical preparations by the proposed methods. Product

Determined content (Mean n = 5) (mg/tabl)

Statistical assessment

D1

D2

D3

D1

D2

D3

Zofenil 30 (ZOF)

30.80

30.41

30.90

Sx = 0.677 t0.95 = ±0.841 RSD = 2.19%

Sx = 1.122 t0.95 = ±1.393 RSD = 3.69%

Sx = 0.556 t0.95 = ±0.691 RSD = 1.80%

Lescol 40 (FLU)

39.77

40.34

39.74

Sx = 0.587 t0.95 = ±0.728 RSD = 1.47%

Sx = 1.220 t0.95 = ±1.515 RSD = 3.02%

Sx = 0.751 t0.95 = ±0.932 RSD = 1.89%

Sx – standard deviation; t0.95% – confidence interval; RSD – relative standard deviation.

of determined compound was calculated using an appropriate regression equation. Results along with statistical evaluation are shown in Tables 1 and 2. Application to pharmaceutical formulation To determine the amount of ZOF and FLU in commercial preparations (Zofenil 30 and Lescol 40), 10 tablets of an appropriate preparation were powdered and a portion of powder corresponding to one tablet was weighted on an analytical balance. The obtained sample was extracted with 10.0 mL of methanol. The extract was centrifuged for 15 min (1500 rpm) and filtered through a 0.5 lm Whatman filter paper. From such prepared solution, further dilutions were prepared, so that the concentration of the determined substance was in the tested linearity range. ZOF and FLU concentrations in the prepared samples were calculated from the appropriate regression equation; for each preparation five replicates were made and the results of measurements were shown in Table 3. Discussion of the results Due to high incidence of hypertension in combination with dyslipidemia, it became necessary to co-administrate antihypertensive drugs in combination with HMG-CoA reductase inhibitors. Such therapeutic system allows for better control of blood pressure with simultaneous lowering of lipid levels, particularly cholesterol. In this situation, it seems reasonable to develop accurate and fast method for the determination of substances from these pharmacological group in pharmaceutical preparations with the potential of possible application of the developed procedure for the determination of these substances in biological material. The fact that the absorption spectra of the tested substances interfere with each other, causes that direct analysis is impossible. It seems that the above-mentioned difficulties are the reason for replacing the spectrophotometric method by separation techniques, which is justified in many cases. Taking into account the advantages of the spectrophotometric method such as simple way of performing measurements, accuracy and availability as well as progress in the production of modern

apparatus, one succeeded in demonstrating, that this method has still a wide range of applications for fast and accurate quantitative analysis. This potential of wider application of spectrophotometry is possible thanks to new computational techniques, for which the use of derivative of the zero-order spectra, increase selectivity and sensitivity of analyses in comparison with the classical zero-order spectrophotometry. Conducted measurements indicate, that the application of derivative spectrophotometry and conversion of spectra into first, second or third order derivatives, for the determination of zofenopril and fluvastatin, enable the simultaneous analysis of active compounds in the studied preparations. Considering selected wavelengths, determined by the ‘‘zerocrossing’’ method, it was demonstrated, that the method is specific for the analyte, that is, tested compounds. No interference of matrix components was observed, which demonstrates a good selectivity of the method. The linearity was maintained over a wide range of concentrations between 7.65 lg mL 1 and 22.94 lg mL 1 for zofenopril, and between 5.60 lg mL 1 and 28.00 lg mL 1 for fluvastatin. The value of correlation coefficient (R) did not specify clearly the linearity of calibration method. Therefore, to evaluate the linearity, Mandel’s test was used. The obtained results of linear and quadratic fit, indicated a linear fit of the calibration curves for all cases. The normality of distribution of residuals was confirmed by the Shapiro–Wilk test. The points of line intersection of the appropriate curves did not significantly differ from zero. The results of the analysis of the Durbin–Watson test showed no significant autocorrelation of residuals of the fluvastatin and zofenopril linearity for D1 and D2. In terms of linearity for zofenopril, for the first and second derivative, no significant autocorrelation of residuals was demonstrated using the Lagrange test, because the Durbin–Watson test was not decisive. In the linear alignment of D3 zofenopril, the occurrence of autocorrelation was observed, however one decided to apply the least squares method, accepting less efficiency of estimators. The sensitivity of the developed method was high, LOD and LOQ values for particular substances were estimated at 0.19– 0.87 lg mL 1 for zofenopril, and 0.57–2.64 lg mL 1 for fluvastatin, respectively. Appropriate values for fluvastatin were in the range between 0.51 lg mL 1 and 1.18 lg mL 1, as well as 1.56 lg mL 1

M. Stolarczyk et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 148 (2015) 66–71

and 3.57 lg mL 1 depending on the class of derivative. Percent of the recovery of tested compounds presented as the average values for the three levels of concentration was high and was in the range of 100 ± 5%. All validation parameters were summarized in Tables 1 and 2. The results of the measurements of particular active compounds in pharmaceutical preparations such as Zofenil 30 and Lescol 40 did not differ from the values declared by the manufacturer, they were characterized by good precision and accuracy, as evidenced by Sx – standard deviation, t0.95% – confidence interval and RSD-relative standard deviation, presented in Table 3. Conclusion Proposed derivative spectrophotometric method is useful for the direct determination of zofenopril and fluvastatin. The application of ‘‘zero-crossing’’ method allowed for the determination of the appropriate wavelengths, at which no interference of determined analytes was observed. The proposed method is precise, accurate, specific at experimental conditions. It can be used for routine determinations of tested substances as an alternative to time-consuming and expensive separation techniques. References [1] J.B. Mancini et al., Reduction of morbidity and mortality by statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers in patients with chronic obstructive pulmonary disease, J. Am. Coll. Cardiol. 47 (2006) 2554–2560. [2] L. Sabarcea, L. Udrescu, et al., Validated UV spectrophotometric method for quantification of zofenopril in pharmaceutical formulations, Rev. Chim. 63 (2013) 562–564. [3] E. Agabiti-Rosei, A. Manolis, et al., Zofenopril plus hydrochlorothiazide and irbesartan plus hydrochlorothiazide in previously treated and uncontrolled diabetic and non-diabetic essential hypertensive patients, Adv. Ther. 31 (2014) 217–233. [4] A. Serap Saglik, Validated RP-LC method for simultaneous determination of zofenopril and hydrochlorothiazide in pharmaceutical preparations, J. Chromatogr. Sci. 49 (2011) 259–263.

71

[5] J. Yuan, Y. Fang, et al., Development and validation of a liquid chromatographytandem mass spectrometry method for the determination of zofenopril and its active metabolite zofenoprilat in human plasma, J. Pharm. Biomed. Anal. 55 (2011) 527–532. [6] L. Sabarcea, L. Udrescu, et al., Thin-layer chromatography analysis for cyclodextrins inclusion complexes of fosinopril and zofenopril, Farm 58 (2010) 478–484. [7] A. Nakashima, Ch. Saxer, M. Niina, N. Masuda, K. Iwasaki, K. Furukawa, Determination of fluvastatin and its five metabolites in human plasma using simple gradient reversed-phase high-performance liquid chromatography with ultra-violet detection, J. Chromatogr. B 760 (2001) 17–25. [8] F.P. Gomes, P.L. Garcia, J.M.P. Alves, A.K. Singh, et al., Development and validation of stability-indicating HPLC methods for quantitative determination of pravastatin, fluvastatin, atorvastatin and rosuvastatin in pharmaceuticals, Anal. Lett. 42 (2009) 1784–1804. [9] S. Alrawtthi, R.F. Hussein, A. Alzahranif, Sensitive assay for the determination of fluvastatin in plasma utilizing high-performance liquid chromatography with fluorescence detection, Ther. Drug Monit. 25 (2003) 88–92. [10] H. Toreson, B.M. Eriksson, Determination of fluvastatin enantiomers and the racemate in human blood plasma by liquid chromatography and fluorometric detection, J. Chromatogr. A 729 (1996) 13–18. [11] G. Kalafsky, H.T. Smith, M.G. Choc, High-performance liquid-chromatographic method for the determination of fluvastatin in human plasma, J. Chromatogr. B 125 (1993) 307–313. [12] R.V.S. Nirogi, V.N. Kandikere, W. Shirvastava, K. Mudigonda, et al., Liquid chromatography/negative ion electrospray tandem mass spectrometry method for the quantification of fluvastatin in human plasma: validation and its application to pharmacokinetic studies, Rapid Commun. Mass Spectrom. 20 (2006) 1225–1230. [13] A.K.D. Dogrukol, K. Kircali, M. Tuncel, H.Y. Aboul-Enein, et al., Validated analysis of fluvastatin in a pharmaceutical formulation and serum by capillary electrophoresis, Biomed. Chromatogr. 15 (2001) 389–392. [14] J.L. Yan, X.P. Lu, Determination of fluvastatin sodium by differential pulse voltammetry, J Chin. Clin. Med. 1 (2006) 33–36. [15] M.P.S. Neves, P.A. Nouws, C. Delerue-Matos, Direct electroanalytical determination of fluvastatin in a pharmaceutical dosage form: batch and flow analysis, Anal. Lett. 41 (2008) 2794–2804. [16] B. Dogan, S. Tuncel, B. Uslu, S.A. Ozkan, Selective electrochemical behavior of highly conductive boron-doped diamond electrodes for fluvastatin sodium oxidation, Diamond Relat. Mater. 16 (2007) 1695–1704. [17] S.A. Ozkan, B. Uslu, Electrochemical study of fluvastatin sodium analytical application to pharmaceutical dosage forms, human serum, and simulated gastric juice, Anal. Bioanal. Chem. 372 (2002) 582–586. [18] ICH, Validation of analytical procedures: methodology, in: International Conference on Harmonization, IFPMA, Geneva.