Cardiovascular diseases are the leading cause of death globally [1]. It is estimated that 12.3 million deaths (25.8% of deaths of all ages (47.5 million)) were caused by cardiovascular disease in 1990 [2], and this has continued to increase significantly, especially in the developing countries [3]. For example, there were approximately 17.3 million deaths (31.5% of deaths of all ages (54.9 million)) from cardiovascular disease in 2013 [2]. Cyclovirobuxin D (CVB-D) is a steroidal alkaloid widely distributed in Buxus plants, including Buxus semperirens, that has been used for treating cardiovascular diseases since the 1990s [4]. Clinical data from 64 hospitals and more than 100 000 patients in 10 years showed that the tablet form of CVB-D was effective for treating angina pectoris, coronary heart disease, heat failure, and other cardiovascular disorders [4].

Although the tablet of CVB-D has been recorded in Chinese Pharmacopeia (CP) for many years [5], there is no HPLC method recommended for its quality control, possibly owing to the difficulty of CVB-D detection. Similar to other basic compounds without a natural UV chromophore, peak tailing and a very weak UV response are current bottlenecks for its quantitative analysis. Yu and co-workers [6] developed an ion-pair reversed-phase liquid chromatography (IP-RPLC) method with UV detection at 210 nm. This IP-RPLC method provided the improved peak symmetry for CVB-D, while the ion-pairing reagent produced an obvious baseline disturbance with low wavelength UV detection. A pre-column derivatization method was also developed to enhance the UV response for quantitative analysis of CVB-D [4]. However, derivatization is complex, and the derivatization reagent (phenyl isocyanate) used was toxic. A basic mobile phase combined with evaporative light scattering detection (ELSD) was also used to improve the peak shape and sensitivity for HPLC CVB-D detection [4]. However, peak tailing was still observed with such chromatographic conditions. Most importantly, the sensitivity and repeatability of ELSD was not sufficient for the detection of CVB-D at the low concentrations typically present in plasma and urine samples [7, 8]. The mass concentration of CVB-D in plasma is typically less than 60 μg/L [4]. However, none of the reported UV or ELSD methods provided a limit of quantification (LOQ) for CVB-D less than 3.41 mg/L because of peak tailing and limited detection sensitivity [4, 6]. Mass spectrometry (MS) is, by far, the most widely used technique for detection of low analyte concentrations owing to its high sensitivity. However, MS is costly, operationally complex, and can experience matrix effects such as from non-volatile salts present in biological samples. Therefore, a method that provides high sensitivity, ease of operation, and compatibility with complex matrices is necessary.

Electrochemical detection (ECD) is a simple and direct detection method. ECD has certain advantages over other detection methods [9, 10]. First, it does not require samples to be chemically derivatized. Second, it offers highly sensitive detection. For example, it is about 31 times more sensitive than UV [11]. However, the actual sensitivity enhancement varies from compound to compound. Only electroactive analytes can be detected via ECD. This property makes electroche-mical detection highly selective and cost effective. For example, it is less expensive than MS. It provides a good sensitivity comparable to, or even better than MS [12]. Furthermore, it is much less complicated than MS and can be used to detect analytes directly in complex samples [13]. Flanagan et al. [14] reported an HPLC-ECD method for detection of some basic drugs (e. g., amitriptyline), which included use of ion-pairing reagents to overcome the peak tailing of basic compounds. However, the addition of strong ion-pairing reagents to the eluent is often not desirable because it is difficult to recover the column after use since these additives tend to stick very strongly to the stationary phase. In addition, a glassy carbon (GC) working electrode was used. GC electrodes are difficult to use for detection of some basic compounds that have high over-potentials for oxidation. In recent years, boron-doped diamond (BDD) electrodes have received a great deal of attention owing to its attractive features including a wide electrochemical potential window in aqueous solutions, a very low voltammetric background current (1 order of magnitude lower than that of GC), and long-term response stability [15-17]. Because of their wide potential window, BDD electrodes can be used for detection of some analytes that cannot be detected by GC electrodes, such as polyamine [18, 19] and histamine [20], which exhibit high oxidative over-potentials.

In this study, a column with a positive charge-modified C18 stationary phase combined with a BDD electrode was used for the determination of CVB-D. The positive charge-modified column, C18HCE, was used to achieve symmetric and narrow peak shape for basic compounds without ion-pairing reagents [21-23]. Besides column type, other chromatographic conditions, including mobile phase composition, working electrode material, and applied potential, were also systematically investigated. The developed ECD method was compared with charged aerosol detection (CAD), UV methods, and other reported methods for the determination of CVB-D. Finally, we applied the present ECD method for analysis of CVB-D in tablets and human blood samples.

1 Materials and methods

1.1 Apparatus and reagents

The chromatographic system consisted of a DGP-3600RS pump, a WPS-3000RS autosampler, a TCC-3000RS column oven, a CAD Veo RS detector, a DAD 3000RS detector, and an ECD 3000RS detector. The design of the ECD cells is shown in Supporting Information 1 (http://www.chrom-China.com/UserFiles/File/1707020%20supporting%20information.pdf ). The ECD cell was a three-electrode cell that contained a working electrode, reference electrode, and counter electrode. In this cell, the counter electrode worked as a current divider, so only a very small portion of current was applied to the reference electrode. The iR drop (the characters of i and R are electric current and electric resistance, respectively) was minimized, and the potential applied to the working electrode was closer to the real potential from the power supply [9]. GC and BDD electrodes were used. Both electrodes can be reused, and the clean cell function can be used to clean the electrodes. Data were collected and analyzed by Chromeleon version 7.2. All of the abovementioned instruments and workstations were from Thermo Fisher Scientific Corporation (CA, USA). The C18HCE column (150 mm×2.1 mm, 3 μm) for basic compound separation was a gift from the Dalian Institute of Chemical Physics, Chinese Academy of Sciences [19]. The Zorbax Eclipse XDB-C18 column (150 mm×2.1 mm, 3.5 μm) was from Agilent Technologies (MA, USA). The CVB-D reference compounds (Supporting Information 2, http://www.chrom-China.com/UserFiles/File/1707020%20supporting%20information.pdf ) were purchased from the National Institutes for Food and Drug Control (Beijing, China). The two types of CVB-D tablets (Huangyangning tablet) used were from Longdu Pharmaceutical Company (Henan, China) and Baiyun Pharmaceutical Company (Henan, China). Human blood samples from 22-to 32-year-old healthy volunteers were collected into tubes containing 0.1 mol/L sodium citrate by peripheral venous puncture.

HPLC grade acetonitrile (ACN) and formic acid (FA) were obtained from Thermo Fisher Scientific Corporation (CA, USA). Sodium dihydrogen phosphate (NaH₂PO₄, purity 99%) and phosphoric acid (H₃PO₄, volume percentage 85%) were from Sigma Aldrich (MO, USA). Water was prepared by a Barnstead GenPure system (Sunnyvale, CA, USA).

1.2 Sample pretreatment

Huangyangning tablet: Huangyangning tablets were homogenized into a powder, and 0.5 g was mixed with 2 mL ACN-water (50 : 50, v/v). The mixture was sonicated for 30 min and then centrifuged at 10 000 r/min for 10 min. The supernatants were filtered through a 0.22 μm membrane and then analyzed by HPLC.

Human blood sample: a total of 200 μL of fresh human whole blood was mixed with 800 μL of ACN and 10 μL of 600 μg/L CVB-D standard solution, and the mixture was centrifuged for 10 min at 10 000 r/min to separate the precipitated proteins and other non-soluble matter. The supernatant (100 μL) was diluted to 200 μL with 100 mmol/L NaH₂PO₄ (adjusted to pH 2.8 by H₃PO₄). The sample was injected into the LC system without any further treatment.

1.3 Chromatographic conditions

The mobile phases for ECD and UV analysis were ACN (A), 100 mmol/L NaH₂PO₄ (adjusted to pH 2.8 by H₃PO₄) (B), and water (C). The column temperature was 30 ℃. The flow rate was 0.2 mL/min. The gradient was changed from 10% A to 30% A in 20 min, keeping the proportion of B constant at 20%. The optimum potential of the working electrode for amperometric measurement of CVB-D was 1 000 mV vs. a palladium hydrogen reference electrode. The chromatographic conditions for CVB-D analysis with CAD were as follows: mobile phase A was ACN-FA (1 000 : 1, v/v); mobile phase B was water-FA (1 000 : 1, v/v); the gradient was started from 10% A and changed to 30% A in 20 min. The detection conditions of CAD were: evaporation temperature 35 ℃, filter 5.0 s, data collection 10 Hz, power function value 1.0.

2 Results and discussion

2.1 Selection of column and working electrode

Peak tailing of basic compounds has been reported since the 1980s [24], and many strategies involving both mobile and stationary phase variables have been developed to improve peak symmetry. This includes use of mobile phases with higher ionic strength [25], ion-pairing reagents [26], and alkaline pH [27]. Improved peak shape for basic analytes has also been achieved with stationary phases bonded to highly pure silica (type B) [25] or to hybrid silica supports [28] and with polar group-modified [29] and positive charge-modified reversed-phase columns [21, 30]. Table 1 shows that with both low and high ionic strength mobile phases, a positive charge-modified column (C18HCE) produced a more symmetrical peak for CVB-D than a high purity C18 silica column. The C18HCE column was therefore selected for this study, and 20 mmol/L NaH₂PO₄ (pH 2.8) was chosen as a mobile phase additive, since ECD requires an electrolytically conductive solution, and UV detection of CVB-D requires use of a low wavelength. For CAD detection, water-FA (1 000 : 1, v/v) instead of phosphate was used because this technique requires a volatile mobile phase.

表 1 (Table 1)

Table 1 Asymmetry factor of CVB-D obtained with various methods Column Method Asymmetry factor Positive charge modified column (C18CHCE) 0-20 min, 10%-30%Aa) 1.04 0-20 min, 10%-30%A, 20%Bb) 1.07 High pure silica based column (XDB-C18) 0-20 min, 20%-50%Aa) 3.78 0-20 min, 20%-50%A, 20%Bb) 2.99 a) mobile phase A: ACN; mobile phase B: FA-water (1 : 1000, v/v). b) mobile phase A: ACN; mobile phase B: 100 mmol/L pH 2.8 NaH2PO4; mobile phase C: water. The mass concentration of CVB-D was 0.5 g/L. The injection volume was 10 μL.

Table 1 Asymmetry factor of CVB-D obtained with various methods

Although both BDD and GC electrodes can be used for detection with a potential from -1 000 to 1 000 mV, the background current and baseline noise obtained from these two electrodes were different. The background currents of GC and BDD obtained at 800 mV with 10% -30% (volume percentage) ACN are compared in Fig. 1, which illustrates three significant advantages of BDD compared to GC: 1) lower background current; 2) lower baseline noise; and 3) less baseline drift with increasing ACN eluent volume percentages (10% -30%). Besides the above advantages, BDD also provides a wider potential window (-3 to 3.5 V) than GC, thus extending its use to other electrochemical reactions in aqueous electrolyte solutions. For example, scopolamine and CVB-D had a weak or even no response when the potential was lower than 800 mV. Using 1 600 mV, all target compounds had a good response (Supporting Information 3, http://www.chrom-China.com/UserFiles/File/1707020%20supporting%20information.pdf ). Thus, a BDD electrode was chosen for further study.

Fig. 1

Fig. 1 Background current of GC and BDD electrodes obtained with different ACN volume percentages The concentration of phosphate buffer was constant (20 mmol/L pH 2.8 NaH2PO4). The potential was 800 mV.

2.2 Detection potential optimization

The detection potential was optimized by flow injection. As shown in Fig. 2a, there was no obvious CVB-D response when the potential was lower than 800 mV. When the detection potential was above 800 mV, both the peak height of CVB-D and baseline noise (data were not shown) increased with increasing potential. To achieve the highest sensitivity, a potential that provides the highest signal-to-noise (S/N) ratio should be applied for CVB-D detection. It can be seen in Fig. 2b that the highest S/N was acquired at 1 000 mV. Therefore, 1 000 mV was utilized for the detection of CVB-D in further studies. The electrochemical reaction of CVB-D on the BDD electrode was similar to that of lidocaine [31].

Fig. 2

Fig. 2 (a) Peak height and (b) signal-to-noise (S/N) ratio of CVB-D obtained with different potentials Mobile phase A: ACN; mobile phase B: 100 mmol/L NaH2PO4 (adjusted to pH 2.8 by H3PO4); mobile phase C: water. The column temperature was 30 ℃. The flow rate was 0.2 mL/min. The gradient was changed from 10% A to 30% A in 20 min, keeping the proportion of B constant at 20%. The potential range was from 200 to 1800 mV.

2.3 Mobile phase optimization

Phosphate buffer was selected as a mobile phase additive, since it produced a much lower baseline noise than other buffers, such as ammonium acetate [11]. Both steep and shallow ACN gradients were investigated for the analysis of CVB-D. The steep gradient was 0-20 min, 10% -50% ACN, and the shallow gradient was 0-20 min, 10% -30% ACN. In both cases, 20 mmol/L phosphate buffer (pH 2.8) was used as the mobile phase additive. The peak width (W_50%) obtained with the steep gradient (W_50%=0.075 min) was smaller than that obtained with the shallow gradient (W_50%=0.096 min). While the peak height acquired with the steep gradient was accordingly higher, a larger S/N was obtained with the shallow gradient (8 180 vs. 6 640) owing to less baseline drift. Thus, the shallow gradient was used for CVB-D analysis in the further studies.

2.4 Comparison of this HPLC-ECD method with other methods

While both low wavelength (210 nm) UV [4] and universal detection methods (e. g., ELSD) [4, 6] have been reported for analysis of CVB-D, the sensitivities of these methods were poor owing to peak tailing and interference of the mobile phase. In this study, a positive charge-modified column (C18HCE), which provided a symmetric peak shape for basic compounds, was used to develop new UV and universal detection methods. Furthermore, the performance of these methods was compared. CAD, a universal detection technique, which has been reported to provide higher sensitivity and better precision than ELSD [7, 21, 32], was used in this study.

The sensitivity of ECD, UV, and CAD methods are compared in Table 2. Compared to the LOQ value with reported UV methods [6], the sensitivity of the new UV method was 5.86 times better and benefited from the use of a positive charge-modified column. Compared to reported ELSD methods [4, 6], the sensitivity of the new deve-loped CAD method was 4.4-6.5 times better. The sensitivity of the new CAD method is 4.84 times better than the new UV method and, thus, could be more readily used for samples with higher CVB-D mass concentrations, such as in CVB-D tablets. Data in Table 2 also showed that the developed ECD method had much better sensitivity (2 630 and 12 727 times better) than developed CAD and UV methods, respectively. The present ECD method provides sensitivity comparable to [33] or even better than [12] MS methods, while it is less complicated than MS and can be used to detect analytes directly in complex samples. Thus, maybe the present ECD method can be used for the determination of CVB-D in low concentration samples including human blood samples.

表 2 (Table 2)

Table 2 LODs and LOQs of CVB-D obtained with different detectors by this method and other methods Detector LOQ/(μg/L) (S/N) LOD/(μg/L) (S/N) ECDa) 0.297(10.0) 0.198(6.4) CADa) 781.000(14.2) 195.000(3.0) UVa) 3780.000(17.2) 1890.000(5.4) ELSDb) 3410.000(10.0) 1020.000(3.0) UVb) 22154.000(10.0) 6736.000(3.0) a) methods developed in this study; b) data from Guo et al. [6].

Table 2 LODs and LOQs of CVB-D obtained with different detectors by this method and other methods

2.5 Linearity and precision of this HPLC-ECD method

The injection volume was 10 μL for CVB-D samples (approximately 0.297 to 1 891 μg/L). The peak areas of CVB-D obtained with 14 mass concentrations plotted versus the mass concentration were used to acquire calibration curve. Each concentration was injected for three times.Calibration curves for the resulting data were then fitted by linear regression. Compared to reported MS methods [12, 33], the developed HPLC-ECD method offered a much wider linear range. The dynamic range of the present method was demonstrated to span 5 orders of magnitude (A=0.932 4C+0.033 4) with a coefficient of determination (R²) equal to 0.999 4. A is the peak area and C is the mass concentration (μg/L). The wide linear range indicates that this HPLC-ECD method can be used to determine both low and high concentrations of CVB-D.

An experiment conducted over three days with six time points was performed to test intra-day and inter-day precision. The peak areas of two mass concentrations, 59.1 (mass concentration usually presented in blood sample) and 945.3 μg/L (mass concentration usually presented in tablet extract) [4], were analyzed for a total of 18 unique injections over a three-day study. The intra-day and inter-day precision results for peak areas are measured with two mass concentrations (945.3 and 59.1 μg/L). The intra-day precisions of 945.3 and 59.1 μg/L were 0.31% and 5.08% (n=6), respectively. The inter-day precisions of 945.1 and 59.1 μg/L were 4.83% and 5.57% (n=3), respectively. All RSD values for area variability are ≤5.57%, demonstrating that this HPLC-ECD method is robust for CVB-D detection.

2.6 Determination of CVB-D in CVB-D tablets and human blood samples

Once the analytical method was finalized, quantitative studies for CVB-D in CVB-D tablets (Huangyangning tablet) were conducted (Fig. 3). For these studies, CVB-D tablets from two diffe-rent manufacturers were processed under the same conditions, and each sample was analyzed by HPLC in triplicate. The CVB-D tablet samples were found to contain (0.46±0.025 6) and (0.57±0.031 7) mg (n=3) of CVB-D per tablet, respectively. The results agreed well with the amounts stated by the manufacturers. Metabolism studies are also important for new drug development [34, 35].In such study, the detection of the active compound in blood samples is necessary. The recovery of CVB-D from two human blood samples was investigated. As shown in Table 3, the recoveries from human blood samples were comparable to that of the CVB-D tablets.

Fig. 3

Fig. 3 Quantitative analysis of CVB-D from CVB-D tablet The chromatographic composition and ECD conditions were the same as those in Fig. 2.

表 3 (Table 3)

Table 3 Amounts and recoveries of CVB-D (n=3) Sample Amount/(mg/tablet) Recovery/% CVB-D tablet 1 0.46±0.0256 106.7 CVB-D tablet 2 0.57±0.0317 108.4 Human blood 1 - 99.6 Human blood 2 - 96.1 -: not detected.

Table 3 Amounts and recoveries of CVB-D (n=3)

The accuracy of the quantitative method was tested with five mass concentrations of CVB-D from 0.89 to 945.3 μg/L (Table 4). The calcula-ted concentrations were based on the average of two points for each sample run interlaced with standards. The linear fit technique for establishing mass concentration versus peak area was used for all measurements. The calculated sample amounts were then used to calculate an estimated percent recovery. The results indicated that the accuracy of the method was found to be acceptable; it could provide an accurate determination of CVB-D not only from high concentration samples, such as CVB-D tablets, but also from low concentration sample such as blood samples.

表 4 (Table 4)

Table 4 Accuracy results of CVB-D with various mass concentrations Mass concentration/(μg/L) Recovery/% 0.89 3.0 3.56 3.4 14.26 -4.8 118.15 -0.4 945.30 0.5

Table 4 Accuracy results of CVB-D with various mass concentrations

2.7 Other application

The current method, with a slight modification (acetonitrile concentration), could also be utilized to quantitatively determine other basic drugs, including scopolamine, amitriptyline, and strychnine (Supporting Information 2 and 3, http://www.chrom-China.com/UserFiles/File/1707020%20supporting%20information.pdf ). Furthermore, this HPLC-ECD method could also be applied to the quantitative determination of basic environmental pollutants such as 3, 3-dichlorobenzidine. The sensitivity of this HPLC-ECD method was 10 times better (Supporting Information 4, http://www.chrom-China.com/UserFiles/File/1707020%20supporting%20information.pdf ) than the quantitative method for 3, 3-dichlorobenzidine recommended by EPA 650 (United States Environmental Protection Agency Method 650) [36].

3 Conclusion

A HPLC-ECD method was optimized for quantitative analysis of CVB-D over a wide concentration range. The developed method provided comparable or even better sensitivity to reported MS methods, while being much less complex and not susceptible to matrix-related ionization suppression. The optimized method was successfully utilized for quantitation of CVB-D from tablets and blood samples. With a slight modification, the current HPLC-ECD method can also be applied to analysis of many other basic compounds including basic drugs and environmental pollutants. The results showed that the current HPLC-ECD method was an alternative to other techniques, such as MS, for basic compound determination.

Compliance with ethical standards

This manuscript complies with the ethical rules applicable for this journal. This article does not contain any studies with human participants performed by any of the authors. This research was not supported by any funding sources.

References

[1]	Mroczek T, Ndjoko L K, Glowniak K, et al. Anal Chim Acta, 2006, 5669(2): 157.
[2]	Naghavi M, Wang H, Lozano R, et al. Lancet, 2015, 385(9963): 117. doi: 10.1016/S0140-6736(14)61682-2
[3]	Fuster V, Kelly B B, Promoting Cardiovascular Health in the Developing World: a Critical Challenge to Achieve Global Health[M]. Washington D, C.: National Academies Press, 2010
[4]	Liang B, Zhang Z, Fang T, Cyclovirobuxine and Cardiovascular Disease[M]. Beijing: Chemical Industry Press, 2011
[5]	Chinese Pharmacopoeia Commission, Pharmacopoeia of the People 's Republic of China[M]. Beijing: China Medical Science Press, 2015: 406
[6]	Guo Q, Lu X, Wu N, et al. Chinese Journal of Pharmaceutical Analysis, 2009, 26: 929.
[7]	Ramos R G, Libong D, Rakotomanga M, et al. J Chromatogr A, 2008, 1209: 88. doi: 10.1016/j.chroma.2008.07.080
[8]	Takahashi K, Kinugasa S, Senda M, et al. J Chromatogr A, 2008, 1193: 151. doi: 10.1016/j.chroma.2008.04.012
[9]	Brad A J, Faulkner L R, Electrochemical Methods: Fundamentals and Applications[M]. New York: John Wiley & Sons Inc, 2000
[10]	Zhou Y, Yan H, Xie Q, et al. Analyst, 2013, 138: 7246. doi: 10.1039/c3an01437a
[11]	Long Z, Gamache P H, Pan Y Y. Chinese Journal of Chromatography, 2017, 35(8): 897.
[12]	Wei J B, Lai Q, Shumyak S P, et al. J Chromatogr B, 2015, 1002: 185. doi: 10.1016/j.jchromb.2015.08.022
[13]	Novak L, Seruga M, Komorsky L S. Food Chem, 2010, 122(4): 1283. doi: 10.1016/j.foodchem.2010.03.084
[14]	Jane L, McKinnon A, Flanagan R J. J Chromatogr A, 1985, 323: 191. doi: 10.1016/S0021-9673(01)90384-X
[15]	Gerling D, Schramm A. Anal Chem, 2007, 79(22): 8608. doi: 10.1021/ac071519h
[16]	Luong J H, Male K B, Glennon J D. Analyst, 2009, 134(10): 1965. doi: 10.1039/b910206j
[17]	Zhu X, Ni J, Peng L. Water Res, 2009, 43(17): 4347. doi: 10.1016/j.watres.2009.06.030
[18]	Miles D K, Malgorzata W, John B A, et al. Anal Chem, 1999, 71(6): 1188. doi: 10.1021/ac980697v
[19]	Witek M A, Swain G M. Anal Chim Acta, 2001, 440(2): 119. doi: 10.1016/S0003-2670(01)01055-8
[20]	Sarada B V, Rao T N, Tryk D A, et al. Anal Chem, 2000, 72(7): 1632. doi: 10.1021/ac9908748
[21]	Long Z, Guo Z M, Acworth L, et al. Talanta, 2016, 151: 239. doi: 10.1016/j.talanta.2016.01.027
[22]	Long Z, Guo Z M, Xue X Y, et al. J Sep Sci, 2013, 36: 3845. doi: 10.1002/jssc.v36.24
[23]	Long Z, Zhang Y, Guo Z M, et al. Planta Med, 2014, 80: 1124. doi: 10.1055/s-00000058
[24]	Snyder L R, Dolan J W, Gant J R. J Chromatogr, 1979, 165(1): 3. doi: 10.1016/S0021-9673(00)85726-X
[25]	McCalley D V. J Chromatogr A, 2010, 1217: 858. doi: 10.1016/j.chroma.2009.11.068
[26]	Nakanishi H, Imamori K, Iwasa A. J Pharm Soc Jap, 1992, 112(12): 944. doi: 10.1248/yakushi1947.112.12_944
[27]	Zhang J, Jin Y, Liu Y F, et al. J Sep Sci, 2009, 329(9): 1401.
[28]	Davies N H, Euerby M R, McCalley D V. J Chromatogr A, 2008, 1178: 71. doi: 10.1016/j.chroma.2007.11.040
[29]	O'Gara J E, Walsh D P, Phoebe C H, et al. LC GC North America, 2001, 19: 632.
[30]	Lucie N, Hana V, Solich P. Talanta, 2012, 93: 99. doi: 10.1016/j.talanta.2012.01.054
[31]	Jurva U, Wikstrom H V, Weidolf L, et al. Rapid Commun Mass SP, 2013, 17(8): 800.
[32]	Shaodong J, Lee W J, Ee J W, et al. J Pharm Biomed Anal, 2010, 51(4): 973. doi: 10.1016/j.jpba.2009.10.019
[33]	Mu L L, Yu P, He L H. Acta Pharm Sin B, 2011, 1(3): 184. doi: 10.1016/j.apsb.2011.06.012
[34]	Dong Y, Yu Z M, Li H Y. Chinese Journal of Chromatography, 2016, 34(8): 811. doi: 10.3724/SP.J.1123.2016.04009
[35]	Mai D T, Yang C, Wang Y. Chinese Journal of Chromatography, 2017, 35(6): 578. doi: 10.3724/SP.J.1123.2017.01026
[36]	Environmental Protection Agency. Appenix to Part 136, Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater, Method 605-Benzidines. [2017-07-16]. https://www.epa.gov/sites/production/files/2015-09/documents/method_605_1984.pdf