AnalyticalMethods

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Simultaneous de

aCollege of Biotechnology and Pharmaceutic

Nanjing 211816, ChinabBioenergy Research Institute, Nanjing Te

E-mail: hellozheng@gmail.com

Cite this: Anal. Methods, 2014, 6, 8284

Received 29th May 2014Accepted 14th August 2014

DOI: 10.1039/c4ay01285b

www.rsc.org/methods

8284 | Anal. Methods, 2014, 6, 8284–8

termination of b-methyl-b-nitrostyrolene and its related substances by PS-DVBchromatographic column and structural analysis ofits photo-isomerization product

Yan Xu,a Xiao-ning Liu,a Jun Zhou,ab Ying Liub and Tao Zheng*b

A method of high performance liquid chromatography (HPLC) was established to determine b-methyl-b-

nitrostyrolene (MNS) and its related substances and to investigate the stability of MNS in acetonitrile

solution. In this chromatographic method, MNS and its related substances were separated using the

polystyrene-divinyl-benzene column (PS-DVB) (25 cm � 4.6 mm i.d. � 6 mm). Acetonitrile–water served

as the mobile phase with gradient elution of 0–8 min, ACN 74–92%, maintaining the flow rate at 1.0 mL

min�1 with the UV detection wavelength at 214 and 250 nm. The selected chromatographic conditions

were found to effectively separate MNS, benzaldehyde and nitroethane with retention times of 3.66, 5.51

and 8.94 min, and perfect resolutions of 6.76 and 8.69. The linearity range of MNS, benzaldehyde and

nitroethane were found in the range of 25–125, 0.16–15.71 and 0.16–15.67 mg mL�1, with the limits of

detection of 0.031, 0.006 and 0.011 mg mL�1, respectively. Recovery studies showed good results for all

examined compounds (from 96.5 to 99.9%) with RSD ranging from 0.31 to 0.94%. MNS was unstable in

acetonitrile when exposed to natural light, and a new product appeared. The new product was purified

and identified as a-methyl-styrolene nitrite by GC-MS and NMR, which was the photo-isomerization

product of MNS. The proposed method was found to be accurate, precise, reproducible and specific and

it could also be used for routine quality-control analysis of MNS.

1. Introduction

b-Methyl-b-nitrostyrene (MNS, Fig. 1) is usually synthesized viathe Henry reaction of aromatic aldehyde with nitroalkanes fol-lowed by dehydration of the resultant nitroalcohols.1 MNS is avital starting material for the synthesis of a variety of usefulbuilding blocks and valuable intermediates of numerousproducts. Since nitroethane is a strong electron-withdrawinggroup that can lead to polarization of conjugated double bondsand become readily transformed into a series of different typesof compounds, such as amine, hydroxylamine, nitrile, alcohol,aldehyde, ketone, etc.2,3 MNS also has effective antitumor andantibacterial functions.4–6 Normally, it is more inhibitive to theGram-positive than to the Gram-negative bacteria.7

The main related substances in the synthesis of MNS werebenzaldehyde and nitroethane. The determination of benzal-dehyde typically employs high-performance liquid chromatog-raphy (HPLC),8 UV absorption method with single wavelengthor gas chromatographic method.9 The general determination

al Engineering, Nanjing Tech University,

ch University, 211816 Nanjing, China.

290

method of nitroethane is gas chromatography.10 There has beenno other determination method about MNS except the highperformance liquid chromatography (HPLC) method which wasreported only in the literature.7 The chromatography conditionswere as follows: C18 chromatographic column was used, the UVdetection wavelength was 214 nm, acetonitrile–water served asthe mobile phase with the ow rate of 1.0 mL min�1, and agradient of 10–100% (v/v) ACN (0–35 min) was chosen. Theexistence of MNS detection is given in the literature but not theanalysis of the mixture of the three compounds. Furthermore,the detection process had no details. A notable issue was thatthe simultaneous determination of MNS and its relatedsubstances together using a single analytical tool had not beenreported.

High performance liquid chromatography (HPLC) acts as themost common and convenient technology in the chromatographic

Fig. 1 Chemical structures of MNS and related substances (A) nitro-ethane; (B) benzaldehyde; (C) MNS.

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world and is well known for providing high selectivity and sensi-tivity when it was used for determination recently. So the aim ofthis article was to develop a simple, sensitive, selective andreproducible RP-HPLC-UV method for the simultaneous determi-nation of MNS and its related substances for routine quality-control analysis using the polystyrene-divinyl-benzene (PS-DVB)column and to determine the stability of MNS in ACN solution inthe presence of natural light. In this study, double wavelengthdetection model and the optimization of the mobile phasegradient were investigated to obtain satisfactory consequence. TheGC-MS and NMR were used for further study of the unstableproduct.

2. Experimental section2.1 Chemicals and reagents

The MNS standard sample was purchased from Sigma-Aldrich(Germany); the MNS sample was synthesized by us; benzalde-hyde and nitroethane were purchased from Sinopharm Chem-ical Reagent Co., Ltd. (China); and ACN, of HPLC grade, waspurchased fromMerck (Germany). Throughout the whole study,ultrapure deionized (DI) water was used, which was made usingan Easypure RODI water system (Barnstead Int.).

2.2 RP-HPLC analysis

LC analyses were performed on a Dionex (USA) HPLC systemconsisting of a pump (P680 HPLC), an injection valve with 20 mLsample loop (7725i) and a UV detector (UVD-170U). Chromato-graphic separation was achieved on a polystyrene-divinyl-benzene column (PS-DVB) (25 cm � 4.6 mm i.d. � 6 mm).

The mobile phase containing acetonitrile and water wasprepared daily and ltered (Millipore, 0.45 mm) under vacuum.The ow-rate was 1 mLmin�1 and the detection wavelength wasset at 214 and 250 nm. All assays were performed at ambienttemperature.

2.3 GC-MS analysis

GC-MS analyses were carried out using a Thermo Trace DSQ/ISQequipped with a 30 m � 0.25 mm � 0.25 mm i.d. MS capillarycolumn (TR-5MS). The GC oven temperature ramp was: 80 �C,hold 1 min, increase to 300 �C at a rate of 20 �C min�1 and holdfor 20 min at the nal temperature. The interface temperaturewas 280 �C. Electron ionization (EI) spectra were obtained at70 eV at 280 �C. Helium carried gas at a constant ow-rate of1.0 mL min�1. The injection volume was 1 mL, with a splitlessmode. The MS was operating in FASST mode (SCAN and SIMmodes simultaneously) with the scan range of 50–600. All ofthese response data were analyzed by two soware standardmass spectral databases (Nist and Main Lab).

2.4 NMR analysis

NMR spectra were recorded at 37 �C on a 300 MHz Brukerspectrometer using CDCl3 as the solvent and tetramethylsilane(TMS) as the internal reference.

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2.5 Preparation of standard solutions

MNS standard stock solution was prepared by dissolving125 mg standard MNS in 10 mL acetonitrile. The stock solutionwas further diluted with acetonitrile to get standard MNSsolutions ranging between 25 and 125 mg mL�1.

Benzaldehyde and nitroethane standard stock solutionswere prepared in acetonitrile to give 10.4 and 10.5 mg mL�1,respectively. A mixed stock solution of benzaldehyde andnitroethane was prepared in acetonitrile in which the concen-trations of benzaldehyde and nitroethane were 104 and105 mg mL�1, respectively. The mixed stock solution was furtherdiluted with acetonitrile and MNS solution was added to getstandard mixture solutions. In those solutions, the concentra-tion of MNS maintains at 125 mg mL�1.

2.6 Preparation of MNS sample solutions

MNS sample stock solution was prepared by dissolving 125 mgMNS sample in 10 mL acetonitrile. The stock solution wasfurther diluted with acetonitrile to get MNS sample solutions.

3. Results and discussion3.1 Optimization of UV wavelength

The maximum absorption wavelengths of benzaldehyde andnitroethane were 214 and 250 nm, respectively.11–13 Consideringthe large wavelength gap between the two substances, themaximum detection wavelengths of benzaldehyde and nitro-ethane (214 and 250 nm) were both selected as the detectionwavelengths to improve the detection sensitivity. MNS couldachieve signicant absorption at this wavelength range becauseof the large yield. So there was no need to choose its maximumabsorption wavelength, as long as the good separating degreecan be achieved. The double-wavelength detection model ach-ieved the simultaneous estimation of the three substanceswithout worrying about the different amounts of every compo-nent and the low detection sensitivity.

3.2 Optimization of separation conditions

3.2.1 The preliminary selection of gradient elution. Basedon the detection method of MNS reported in the literature,7 thegradient of 10–100% (v/v) ACN (0–35 min) was chosen to sepa-rate the standard mixture solution, in which the concentrationsof nitroethane, benzaldehyde and MNS were 10.50, 10.40, and125.00 mg mL�1, separately. The wavelength of 214 nm waschosen. Although MNS could be detected (peak-4), but therelated substances (nitroethane and benzaldehyde, peak-1 andpeak-2) were overlapped partly as shown in Fig. 2. In addition,the separation analysis cost for a long time over 30 min.Therefore, the mobile phase gradient was optimized as followsin order to improve the separation efficiency and enhance thecolumn efficiency (number of theoretical plates, N).

3.2.2 Optimization of the gradient elution. Based on thendings of 3.2.1, the gradient elution system was optimized toimprove the resolution and to reduce the time needed foranalysis. In an attempt to achieve perfect peak symmetry,separation and column efficiency, different mobile phase

Anal. Methods, 2014, 6, 8284–8290 | 8285

Fig. 2 The HPLC chromatogram of ACN 10–100% (0–35 min). Fig. 3 The HPLC chromatograms of different gradients.1: ACN10–100% (v/v) (0–35 min); 2: ACN 30–92% (v/v) (0–20 min); 3: ACN50–92% (v/v) (0–15 min); 4: ACN 60–92% (v/v) (0–15 min); 5: ACN60–92% (v/v) (0–8 min); 6: ACN 74–92% (v/v) (0–8 min); 7: ACN 80–92% (v/v) (0–8 min); 8: ACN 92% (v/v) isocratic elution.

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gradients were tested. HPLC chromatograms of differentgradients are shown in Fig. 2. The perfect separation of nitro-ethane and benzaldehyde could not be achieved using thegradient of 10–100% (v/v) ACN (0–35 min). When preliminarilyincreasing the starting concentration of ACN and decreasingthe gradient time, every substance was separated well, and thecolumn efficiency was improved. Then further increasing thestarting concentration of ACN, such as the gradient of 74–92%(v/v) ACN (0–8 min), a better column efficiency was achieved.But when the gradient elution of 80–92% (v/v) ACN (0–8 min)was used, the number of theoretical plates reduced. From thediscussions above, it could be concluded that the best experi-mental condition was 74–92% (v/v) ACN (0–8 min), with whichthe three substances could be separated successfully and therun time was just 10 min. Thus the best condition was chosen totake simultaneous determination of MNS and its relatedsubstances.

Fig. 4 The HPLC chromatogram of MNS sample compared withstandard samples. A–C are shown in Fig. 1.

3.3 Specicity

To evaluate the specicity of the method, chromatograms of thepure standard solutions were compared with the MNS sampleunder the same determining conditions by injecting thefollowing solutions: (1) MNS sample solution aer light expo-sure for four days, (2) standard solution of MNS, (3) standardsolution of nitroethane, (4) standard solution of benzaldehydeand (5) the solvent (ACN).14 No peak was observed in the solventchromatogram, indicating an absence of interference from thesolvent. As could be seen from Fig. 3, the retention times of allsubstances were almost the same in both standard solutionsand MNS sample. The developed HPLC method had goodspecicity to MNS and its related substances and guaranteedobtaining well-shaped peaks both for the related substancesand coexisting photo-isomerization product (Fig. 4).

3.4 Linearity and limit of detection (LOD) of MNS and itsrelated substances

As mentioned above, it could not determinate the threesubstances sensitively unless 214 and 250 nm were chosen asthe detection wavelengths simultaneously, because of thedifference of the maximum absorption wavelength between

8286 | Anal. Methods, 2014, 6, 8284–8290

benzaldehyde and nitroethane. Considering the detectionsensitivity of both substances, the double wavelength detectionmodel was used to reduce the detection limitation.

To determine linearity, a series of standard mixture solu-tions containing all the analytes and MNS standard solutionwere injected in triplicate under optimum chromatographicconditions. A linear relationship existed between the peak area(Y) and the corresponding concentration (X) of MNS. In addi-tion, a linear relationship existed between the peak area (Y) andthe corresponding concentration (X) of the related substancesto obtain the calibration graphs. The limits of detection (LODs)were calculated from signal-to-noise ratios (S/N) of all thestandard solutions using the denition S/N ¼ 3.15 The LOD ofbenzaldehyde was 0.006 mg mL�1 and much lower than that inthe literature.16 Concentration ranges and calibration parame-ters are reported in Table 1. Good relationship was found asindicated by the coefficient of determination $0.9991.

3.5 Accuracy and precision

The accuracy of the method was assessed by detecting therecovery of known amounts of spiking analytes (A, B and C) in

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Table 1 The linearity, and limit of detection of MNS and its related substancesa (S/N ¼ 3)

Compound Regression equationCorrelationcoefficients

Linear range(mg mL�1)

Detection limit(mg mL�1)

A Y ¼ 0.814X + 0.034 0.9997 0.16–15.71 0.031B Y ¼ 2.2659X + 0.1364 0.9999 0.16–15.67 0.006C Y ¼ 0.738X + 0.5650 0.9991 25–125 0.011

a Y: peak area; X: mass concentration, mg L�1. A–C are shown in Fig. 1.

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the MNS sample. The concentration of MNS in the MNS samplewas 0.12 mg mL�1. Spiked samples were prepared in quintu-plicate at three levels and analyzed according to optimumchromatographic conditions. As it can be seen in Table 2,quantitative recovery was obtained in each instance (recovery ¼96.5–100.4%), and had low RSD values (RSD # 0.94%).17

3.6 Analysis of practical sample

The developed method was applied to a practical MNS sample.The sample contained nitroethane and benzaldehyde at rate of0.08% and 0.22%. The results showed that the method had thusbeen demonstrated to be a stability-indicating method and besuitable for assay for commercial products.

Fig. 5 The stability of benzaldehyde, nitroethane and MSN in standardmixture solution. A: nitroethane; B: benzaldehyde; I: peak-4 in darkcontrol; J: peak-4 upon natural light irradiation; K: peak-3 uponnatural light irradiation.

3.7 The light stability of MNS and its related substances

Stability is a key problem of every substance during their anal-ysis.18 The stabilities of standard MNS solution and standardmixture solution were evaluated with the effect of the naturallight, in order to nd the best storage method.

The standardmixture solution was prepared in triplicate andstored at ambient temperature, exposed to natural light andtested at intervals of 0, 4, 8, 12, 24 and 48 h. The concentrationsof nitroethane, benzaldehyde and MNS were 5, 2.5 and 125 mgmL�1, respectively. The results in Fig. 5A and B showed that nosignicant changes of the related substances of the stockstandard mixture solution were observed. A new product (peak-3) was observed at 7.45 min (Fig. 6), which had differentretention times compared with MNS. The conversion rates ofMNS to the new product were measured as 0, 2.24%, 5.35%,6.77%, 9.27% and 15.98% via this proposed HPLC method, andincreased with the increasing exposure time.

Table 2 Recovery, RSD of A, B and C

CompoundFortied level(mg mg�1)

Average of testin(mg mg�1)

A 6.72 6.4810.50 10.3420.10 19.79

B 6.66 6.5410.40 10.1220.08 20.06

C 756 757960 964

1165 1151

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The standard MNS solution was prepared and detectedunder the same condition of the above standard mixture solu-tion except for the dark control. No product was observed in the

gAverage of recovery (%) RSD (%, n ¼ 5)

96.5 0.7298.5 0.9498.5 0.3198.2 0.5198.0 0.3699.9 0.87

100.1 0.74100.4 0.7198.8 0.22

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Fig. 6 The HPLC chromatograms of fresh and storage (exposed tonatural light for 72 h) standard MNS solution. f: standard MNS solutionexposed to natural light for 72 h; g: fresh standard solution of MNS.

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dark control (Fig. 5I) suggesting that MNS was unstable underthe inuence of natural light and a new product appeared. TheRSD values of every detection point in Fig. 5 were between0.11% and 1.47%.

3.8 The structural analysis of peak-3

3.8.1 Preliminary study by GC-MS. A mixture of peak-3 andpeak-4 was obtained by placing the MNS standard solution innatural light for 7 days. The pure peak-3 was achieved aer theseparation and purication of the mixture with the HPLCmethod. MNS, the mixture and the pure peak-3 were detectedunder the conditions of section 2.3. A total ion current (TIC)

Fig. 7 Total ion current chromatograms (TIC) of MNS, the mixture and

Fig. 8 The full scan mass spectra of MNS and peak-3.

8288 | Anal. Methods, 2014, 6, 8284–8290

chromatogram for these three samples (Fig. 7) was obtainedand the selected-ion monitoring results are shown in Fig. 8.

It is apparent in Fig. 7 that MNS and the peak-4 of themixture held the same retention time of 6.84–6.86 min, and thepure peak-3 and the peak-3 of the mixture held the sameretention time of 6.21–6.25. In conclusion, the purication ofpeak-3 and peak-4 in the mixture achieved the expected result.Moreover, peak-3 and MNS were not the same substance andthey could be efficiently separated under the GC-MS detectingconditions. The most important, the retention time of peak-3was shorter than MNS, thus the boiling point of peak-3 waslower than MNS.

As shown in Fig. 8, the mass spectrogram of peak-3 behavedsimilar to those of MNS with the same m/z 163, 115, 91, 97 and146. According to the standard spectral library, the matchingresults of both peak-3 and peak-4 were MNS. The matching rateof peak-4 was 91%, but the matching rate of peak-3 was only79%. The result was incredible unless the matching rate wasabove 90%. Therefore, the molecular structure of peak-3 andMNS were very similar, but not the same. Peak-3 was suspectedto the isomerization of MNS.

The MNS structure contains double-bond and nitro, so itsisomers have two forms containing double-bond isomeride andnitro isomeride. The double-bond isomeride has the sameboiling point. But the boiling point of peak-3 was lower thanMNS, therefore peak-3 was not the double-bond isomeride ofMNS. The literature18,19 showed that photo-isomerization ofMNS was apt to generate nitrous acid ester, and the boilingpoint of nitrous acid ester was lower than nitro compounds. For

peak-3. a: MNS; b: the mixture; c: peak-3.

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Fig. 9 1H NMR spectrum of MNS and the mixture.

Table 3 The assignation of 1H NMR signals in the mixture

No. 5 4 3 2 1

Assignation H–C]C–NO2 Ar–H H–C]C–ON O2N–C–CH3 NO–C–CH3

MNS (ppm)/intensity ratio 8.1/(1) 7.3–7.45/(5) — 2.44–2.46/(3) —MNS (ref. 19) (ppm) 8.1/(1) 7.3–7.5/(5) — 2.46/(3) —D simulated (ppm) — 7.33–7.6/(5) 6.63/(1) — 1.99/(3)The mixture (ppm)/intensity ratio 8.08–8.1/(1) 7.3–7.47/(13) 6.46–6.47/(1.6) 2.44–2.46/(3) 2.34–2.35/(4.8)

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example, the boiling point of ethyl nitrite was 17.2 �C which wasmuch lower than nitroethane whose boiling point was 114 �C.By inference, the photo-isomeride of MNS was a-methyl-styr-olene nitrite whose proposed structure is shown in Fig. 9 (thephoto-isomeride).

3.8.2 Further validation by 1H NMR. According to thestructure inferred by GC-MS above, the mixture and MNS weretransferred into the NMR tube and dried with N2, dissolved with0.5 mL CDCL3 and detected by 1H NMR. 1H NMR chemicalshis of the peak-3 andMNS are shown in Fig. 9 and a summaryof the peaks observed in the spectra could be found in Table 3.

For the purposes of discussion, we started our analysis withMNS whose NMR signals could be assigned from the datareported in the literature.19

Thus, in 1H NMR of the mixture, the singlets at 2.44–2.46and 8.01–8.1 ppm were attributed to the methyl H anddouble-bond H on MNS, while the singlets at 2.34–2.35 and6.46–6.47 ppm were attributed to the methyl H and double-bond H on the photo-isomeride of MNS. The multiplet at 7.3–7.47 ppm was due to the aromatic group on MNS and itsphoto-isomeride.

Since the electron-withdrawing effect of the nitro group, N ofthe nitro group is electropositive, and O of the nitrite group iselectronegative, the nitrite group would promote the reso-nances absorbtion of H protons on its adjacent C to the upeld.So the methyl H and double-bond H of photo-isomeride dis-played at upeld compared with MNS. The displacement rule ofthe H proton was coincided with that simulated by ChemDrawsoware. Then it could be concluded that the photo-

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isomerization of MNS was a-methyl-styrolene nitrite (the photo-isomeride in Fig. 9) and the molar ratio of MNS and its photo-isomeride were detected as 1 : 1.6 with the area ratio of methylH (or double-bond H) between two compounds.

4. Conclusion

A fast, reliable, selective and effective RP-HPLC-UV methodwas developed using polystyrene-divinyl-benzene (PS-DVB)column and validated for the simultaneous determination ofMNS and its related substances. Suitable experimentalparameters for the separation and detection of the MNSsample were determined, and this method had beendemonstrated to have high sensitivity, specicity, linearityand precision. Moreover the method was applied to thepractical MNS sample and demonstrated to be a stability-indicating method. The method is simple and rapid, whichmakes it a candidate for use in routine analysis in monitoringprograms.

The MNS was unstable in acetonitrile solution whenexposed to natural light and underwent photo-isomerization.Photo-isomerization of MNS increased with increasingexposure time.

The studies performed by GC-MS and NMR allowed deter-mining the probable chemical structure of photo-isomerizationproduct of MNS, which could be determined using the devel-oped HPLC-UV method. The photo-isomerization product ofMNS was identied as a-methyl-styrolene nitrite.

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Acknowledgements

This study is supported by the National Basic Research Programof China (973 Program) and National High TechnologyResearch and Development Program of China (863 Program)(no. 2013CB733504 and 2012AA0212).

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