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Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors
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Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors Tao Chen a , Jun Fan a,∗ , Ruiqi Gao a , Tai Wang b , Ying Yu a , Weiguang Zhang a,b,∗ a b
School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China Guangzhou Research & Creativity Biotechnology Co., Ltd, Guangzhou 510663, China
a r t i c l e
i n f o
Article history: Received 17 February 2016 Received in revised form 2 July 2016 Accepted 5 July 2016 Available online xxx Keywords: Metalaxyl Enantiomeric ratio HPLC chiral analysis Chromatographic detectors Quantitative study
a b s t r a c t Chiral stationary phase-high performance liquid chromatography coupled with various detectors has been one of most commonly used methods for analysis and separation of chiral compounds over the past decades. Various detectors exhibit different characteristics in qualitative and quantitative studies under different chromatographic conditions. Herein, a comparative evaluation of HPLC coupled with ultraviolet, optical rotation, refractive index, and evaporative light scattering detectors has been conducted for qualitative and quantitative analyses of metalaxyl racemate. Effects of separation conditions on the peak area ratio between two enantiomers, including sample concentration, column temperature, mobile phase composition, as well as flow rate, have been investigated in detail. In addition, the limits of detection, the limits of quantitation, quantitative range and precision for these two enantiomers by using four detectors have been also studied. As indicated, the chromatographic separation conditions have been slight effects on ultraviolet and refractive index detections and the peak area ratio between two enantiomers remains almost unchanged, but the evaporative light scattering detection has been significantly affected by the above-mentioned chromatographic conditions and the corresponding peak area ratios varied greatly. Moreover, the limits of detection, the limits of quantitation, and the quantitative ranges of two enantiomers with UV detection were remarkably lower by 1–2 magnitudes than the others. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chirality is ubiquitous in life sciences, pharmaceuticals, fine chemicals, and materials, etc. [1–5]. Enantiomers may exhibit different physiological, pharmacological, and toxic activities in the chiral environment, although having same physical and chemical properties. Only one or several enantiomers in many chiral pharmaceuticals exhibit pharmacologically active, and the presence of enantiomeric impurities could lead to potentially serious consequences. Thus, separation, quantitative determination, in-process quality control, and pharmacological studies of chiral compounds have been one of hotspots in research of novel pharmaceuticals. In recent years, chiral stationary phase-high performance liquid chromatography (HPLC) coupled with various detectors, e.g. ultraviolet (UV) detector, optical rotation (OR) detector, refractive index (RI) detector and evaporative light scattering (ELS) detector, has been one of the most commonly used methods in chiral
∗ Corresponding authors at: School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China. E-mail addresses: [email protected] (J. Fan), [email protected] (W. Zhang).
separation studies [1,2,6–15]. Due to high sensitivity, high stablity, and low cost, UV detector has been widely applied in detection of analytes with typical chromophoric groups, and showed some potential drawbacks, such as unsuitablity for analytes without typical chromophores and limit of solvent [16–19]. As being a universal detector, evaporative light scattering detector is suitable for analytes with less volatility than mobile phase [17,20–25]. Most of analytes with/without typical chromophores are able to be detected through ELS detector. For refractive index detector, sample is detected on the basis of difference between its refraction and mobile phase’s refraction [26–29], and it has some potential shortages, such as low sensitivity, susceptibility to disturbance from eluents, baseline instability, and gradient incompatibility. Moreover, optical rotation detector as a kind of selective detector is only suitable to detection of optically active substances and nonoptically active compunds couldn’t interfere with determintion of enantiomers [30–33]. Chromatographic conditions have some potential impacts on sensitivity and precision of these detectors and peak area normalization method can’t be directly used for quantitation of enantiomers under some conditions. Toussaint group reported a comparative study on determination of chiral non-aromatic alco-
http://dx.doi.org/10.1016/j.chroma.2016.07.007 0021-9673/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: T. Chen, et al., Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.07.007
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hols by HPLC coupled with four different detectors [33]. The peak area ratio between enantiomers by using the ELS detector was remarkably different with the others. Wipf and his co-worker reported HPLC determinations of enantiomeric ratios through three detectors, namely, UV, ELS, and electrical aerosol detector [29]. ELS detector didn’t reflect real enantiomeric purity under different elution conditions in comparison with the others, and might be complemented through adopting a suitable calibration protocol. HPLC coupled with ELS detector was applied in detection and determination of a mixture of enantiomers, and the effects of some factors on integration of chromatographic peaks, such as molecular weight and volatility of analytes and peak shape, have been discussed in detail [34,35], which indicated that non-linear response for ELS detector has been the main factor leading to deviation. Metalaxyl is an acetanilide-type fungicide and contains an asymmetrically substituted C atom, thereby having two enantiomers (Fig. 1). It shows high activity against fungi of the order Peronosporales and good effectiveness against diseases caused by oomycetes, and has been widely employed in the agriculture field from 1977, for seed treatment, banded or broadcast soil application, and foliar spray [36,37]. Usually, plant treatment is performed with the racemic mixture. However, some results indicated that the R-metalaxyl has been the primary active ingredient relative to the S-enantiomer and the anti-fungicidal activity mostly originated from the R-enantiomer [37]. In addition, metalaxyl is photolytically and hydrolytically stable with about a half-time of 400 days under natural sunlight, and 7–118 days at 20 ◦ C from pH 5 to pH 9 in hydrolysis [37–39]. Thus, its long-term accumulation in soils and groundwater could cause a potentially serious consequence to the environment and public health [40,41]. Herein, a comparative evaluation of HPLC coupled with UV, RI, OR and ELS detectors has been conducted for qualitative and quantitative determinations of metalaxyl enantiomers. Effects of chromatographic conditions on the peak area ratio, such as concentration, column temperature, mobile phase composition, and flow rate, have been investigated in detail. 2. Experimental 2.1. Chemicals and materials Racemic metalaxyl (purity > 98%) was bought from Wuhan Fengzhulin Chemexpress Co., Ltd (Wuhan, China). Optically pure S-(+)-metalaxyl and R-(−)-metalaxyl were obtained through preparative separation of metalaxyl racemate by simulated moving bed chromatography [42]. Ethanol and n-hexane were of HPLC-
grade and purchased from Lab Science, Inc (Houston, Texas, US). Nitrogen with purity higher than 99.999% was used as the evaporation gas. EnantioPak OD column (150 mm × 4.6 mm, 5 m) was friendly provided by Guangzhou Research & Creativity Biotechnology Co., Ltd (Guangzhou, China).
2.2. Instruments Chromatographic analysis experiments have been performed with a 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, a quaternary pump, an auto injector with a 100 L sample loop, a column oven, a multiple wavelength UV detector, a RI detector, and the Agilent Chemstation software. Polarimetry of chiral compounds has been carried out by using IBZ Chiralyser-MP optical rotation detector (IBZ Messtechnik GMBH, Hannover, Germany). In addition, an Alltech Model 3300 evaporative light scattering detector (Grace Davison Discovery Sciences, Deerfield, IL, USA) was used in this study. These four detectors were individually connected to the same HPLC system. Minor difference between retention times of two enantiomers was present in the obtained chromatograms because of different flow cells and pipelines of these four detectors.
2.3. Chromatography Solutions of racemic metalaxyl, S-(+)-metalaxyl, and R-(−)metalaxyl were prepared through dissolving the corresponding samples and diluting with the mobile phase, respectively. Mixture of n-hexane/ethanol was freshly prepared and degassed in an ultrasonic bath before use. Enantioseparation of metalaxyl was achieved on the EnantioPak OD column by normal-phase HPLC at 25 ◦ C, without otherwise specified. Sample solutions (20 L) were injected twice and the flow rate was 1.0 mL min−1 . The UV detection wavelength of metalaxyl was set to 220 nm. The optical unit’s temperature for refractive index detector was 35 ◦ C. For evaporative light scattering detector, the flow rate of nitrogen was set at 1.5 L min−1 , the temperature of the drift tube was 35 ◦ C, and the gain was set at 1. Optical rotation detector was used for identification of the enantiomers. The average and the range of optical rotation detector were set at off and 4.0 m deg, respectively.
Fig. 1. Molecular structures of two metalaxyl enantiomers.
Please cite this article in press as: T. Chen, et al., Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.07.007
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Fig. 2. Effect of sample concentration on peak area ratio between two enantiomers.
3. Results and discussion 3.1. Effects of chromatographic conditions on determination of peak area ratio The amount of two enantiomers is equal in a racemic mixture, and their peak area ratio is very close to 1 when resolved by chiral HPLC. However, the experimental results might seriously deviate from the theoretical value by using different detectors (UV, RI, and especially ELS) in the HPLC studies [29,33,34]. Thus, a series of experiments have been run to explore the potential effects of chromatographic conditions on enantioseparation of metalaxyl. 3.1.1. Effect of sample concentration A series of metalaxyl racemate solutions, which their concentrations varied from 0.2 mg mL−1 to 1.8 mg mL−1 , have been separated with the mixture of n-hexane and ethanol (70:30, v/v) at 25 ◦ C, and the UV, RI, ELS, and OR detectors were used, respectively. The obtained results are shown in Table 1 and Fig. 2, and the selected chromatograms were depicted in Fig. 3. Under these above-mentioned conditions, S-(+)-metalaxyl was earlier eluted than R-(−)-enantiomer. The peak area ratios between S- and R-enantiomers under different sample concentrations are in the range of 0.960–1.013 for the UV detector, 0.978–1.063 for the RI detector, and 0.920–1.066 for the OR detector, respectively, which are very close to the theoretic value, and the corresponding RSD values are 1.95%, 2.77%, and 4.49%. However, the results obtained from ELS detector are relatively poor. The peak area ratios between two enantiomers ranging from 1.357 to 2.077 are deviated significantly away from 1, and the RSD value is 16.15%. Obviously, the UV detection is less influenced than the others with the
Fig. 3. Representative chromatograms of metalaxyl by HPLC with four detectors. Chromatographic conditions: sample concentration, 0.8 mg mL−1 ; column temperature, 25 ◦ C; mobile phase, Hex-EtOH (70/30, v/v); flow rate, 1.0 mL min−1 . * denoted as chiral separation at 45 ◦ C.
change of sample concentration, and the ELS detection has been a much greater impact than the others under the similar condition. Good linear relationships between sample concentration and signal responses of UV detector, RI detector, and/or OR detection were observed in certain range of concentrations, but the response from ELS detection is incompletely linear [34,40]. At relatively low concentration level (0.20–0.80 mg mL−1 , herein), obvious nonlinear response by ELS detection was shown and the peak areas related to two enantiomers were clearly different. With increasing sample’s concentration (1.00–1.80 mg mL−1 ), the correlation between the concentration and the signal response turned good and the peak area ratios remained more than 1. Furthermore, separations of metalaxyl with different concentrations by HPLC coupled with ELS detector have been rerun at 45 ◦ C, as presented in Table 1. Clearly, the retention of two enantiomers became weak in the column and the chromatographic peaks turned narrow. With increasing the concentration, the peak area ratio between two enantiomers ranged from 1.468 to 0.821 and was trending downward. The ratio is 1.004 at the concentration of 1 mg mL−1 . More importantly, the results obtained at 45 ◦ C are much closer to the theoretic value than those at 25 ◦ C, which indicated that ELS detector showed better response at the higher column temperature. One thing to note here, the peak shape for two enantiomers has become sharp at both sides and flat in the middle when the sample concentration exceeded 1.60 mg mL−1 , which might be attributed that the signal strength under relative high concentration was over the signal range of this detector.
Table 1 Effect of sample concentration on peak area ratio between two enantiomers. Peak area ratio
a
S/R (UV) S/R (RI)a S/R (OR)a S/R (ELS)a S/R (ELS)b
Concentration (mg mL−1 ) 0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
RSD (%)
1.013 1.063 1.024 2.077 1.468
0.998 1.057 0.920 1.749 1.279
0.986 1.029 0.975 1.612 1.223
0.978 0.978 0.975 1.438 1.138
0.960 1.003 0.964 1.389 1.004
0.964 1.014 1.032 1.378 0.975
0.964 1.034 1.066 1.357 0.926
0.962 1.011 0.975 1.370 0.866
0.962 0.993 1.026 1.369 0.821
1.95 2.77 4.49 16.15 19.88
*Mobile phase: n-hexane: ethanol = 70:30 (v/v); flow rate, 1.0 mL min−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm; column temperature, a, 25 ◦ C, b, 45 ◦ C.
Please cite this article in press as: T. Chen, et al., Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.07.007
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Fig. 4. Effect of column temperature on peak area ratio between two enantiomers.
Fig. 6. Effect of mobile phase composition on peak area ratio between two enantiomers.
Fig. 5. Selected chromatograms of metalaxyl by HPLC with four detectors. Chromatographic conditions: sample concentration, 1.0 mg mL−1 ; column temperature, 45 ◦ C; mobile phase, Hex-EtOH (70/30, v/v); flow rate, 1.0 mL min−1 .
3.1.2. Effect of column temperature The effect of column temperature on enantioseparation of metalaxyl has been investigated in this section (Table 2 and Fig. 4), and the representative chromatograms were shown in Fig. 5. Both the retention factors and resolution gradually decreased with raising the column temperatures from 20 to 60 ◦ C. Under these experimental conditions, the peak area ratios between S- and R-enantiomers are in the small range of 0.924–0.960 for the UV detector, 0.962–1.028 for the RI detector, 0.926–1.165 for the OR detector, and 0.970–1.402 for the ELS detector, respectively, and the corresponding RSD values are 1.51%, 2.17%, 8.18% and 15.20%. Clearly, changing temperature has been less impact on the results obtained from both UV and RI detectors, while has much more influenced on the others. When the column temperature was set to be 50 ◦ C and more, resolution of two enantiomers was poor by HPLC coupled with the OR detector and the corresponding peaks weren’t accurately integrated, thus giving unfavorable results. In addition, the results from the ELS detector showed a downward trend in the same temperature range, similar to the effect of sample con-
Fig. 7. Selected HPLC chromatograms of metalaxyl from four detectors. Chromatographic conditions: sample concentration, 1.0 mg mL−1 ; column temperature, 25 ◦ C; mobile phase, Hex-EtOH (0/100, v/v); flow rate, 1.0 mL min−1 . * denoted as chiral separation at 45 ◦ C.
centration. Lower column temperatures led to worse results with bigger deviation, which might be explained that the signals related to the samples clearly strengthened along with raising the column temperature and the non-linear responses became fewer [24]. 3.1.3. Effect of the eluent composition Composition of mobile phases has significant effect on retention behaviors and separations of chiral analytes. In the following-up work, the effect of eluent composition on resolution of metalaxyl has been studied (Table 3 and Fig. 6), and the selected chromatograms were displayed in Fig. 7. The retention of metalaxyl enantiomers turned weak with increasing ethanol content in the mobile phase. The peak area ratios between S- and R-enantiomers under the mobile phases with dif-
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Table 2 Effect of column temperature on peak area ratio between two enantiomers. Peak area ratio
S/R (UV) S/R (RI) S/R (OR) S/R (ELS)
Column temperature (◦ C) 20
25
30
35
40
45
50
55
60
RSD(%)
0.942 1.013 0.989 1.402
0.960 1.003 0.964 1.389
0.936 1.028 0.974 1.198
0.960 0.969 0.926 1.120
0.925 0.978 1.101 1.046
0.924 0.962 1.058 1.004
0.925 0.993 1.084 1.006
0.929 0.981 1.147 0.970
0.939 0.981 1.165 0.976
1.51 2.17 8.18 15.2
*Mobile phase: n-hexane: ethanol = 70:30 (v/v); flow rate, 1.0 mL min−1 ; concentration of metalaxyl, 1.0 mg mL−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm. Table 3 Effect of mobile phase composition on peak area ratio between two enantiomers. Eluent composition(volume ratio)
90:10 80:20 70:30 60:40 50:50 40:60 30:70 20:80 10:90 0:100 RSD (%)
Peak area ratio S/R (UV)a
S/R (RI)a
S/R (OR)a
S/R (ELS)a
S/R (ELS)b
1.000 0.994 0.960 0.989 1.000 1.013 1.035 0.985 0.972 0.942 2.68
1.024 0.959 1.003 0.962 1.034 1.035 0.980 0.991 1.013 1.027 2.86
1.039 0.985 0.964 1.012 1.074 1.080 1.046 0.990 0.978 0.909 5.28
1.604 1.379 1.389 1.242 1.218 1.165 1.168 1.120 1.107 1.052 13.5
0.703 0.891 1.004 1.039 1.013 1.015 0.996 1.028 1.025 1.003 10.6
*Mobile phase: n-hexane-ethanol; flow rate, 1.0 mL min−1 ; concentration of metalaxyl, 1.0 mg mL−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm; column temperature, a, 25 ◦ C; b, 45 ◦ C.
Fig. 8. Effects of flow rate on peak area ratio between two enantiomers.
ferent composition were in the range of 0.942–1.035 for the UV detector, 0.959–1.034 for the RI detector, 0.909–1.074 for the OR detector, and 1.052–1.604 for the ELS detector, and the corresponding RSD values were 2.68%, 2.86%, 5.28% and 13.5%, respectively. The results from the UV and RI detectors were close to 1 under the experimental conditions, which meant that change of eluent composition had slight effect on determination of two enantiomers with UV or RI detector. In addition, the peak area ratio between two enantiomers from ELS detection showed a downward tendency with increasing ethanol, that is, declining rapidly in the 10–40% range and slightly changed in the 50%–100% range, and the deviation is bigger than the others. The ratio was close to 1 with 100% ethanol as the eluent. Retention of two enantiomers became
Fig. 9. Representative HPLC chromatograms of metalaxyl from four detectors. Chromatographic conditions: sample concentration, 1.0 mg mL−1 ; column temperature, 25 ◦ C; mobile phase, Hex-EtOH (70/30, v/v); flow rate, 0.5 mL min−1 . * denoted as chiral separation at 45 ◦ C.
weaker and weaker in the eluent with higher content of ethanol, possibly leading to good linear response [29,34]. Moreover, separations of metalaxyl have been conducted by HPLC coupled with ELS detector at 45 ◦ C to further investigate the effect of eluent composition (Table 3 and Fig. 6). Obviously, the retention of two enantiomers turned weak and the chromatographic peaks became narrow. The peak area ratio between two enantiomers ranged from 0.703 to 1.039, and kept almost unchanged when the content of ethanol in the mobile phase was over 30%. More importantly, these results obtained at 45 ◦ C were
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Table 4 Effects of flow rate on peak area ratio between two enantiomers. Flow rate (mL min−1 )
Peak area ratio
S/R (UV)a S/R (RI)a S/R (OR)a S/R (ELS)a S/R (ELS)b
0.50
0.80
1.00
1.20
1.50
1.80
2.00
RSD (%)
0.937 0.995 1.025 0.909 0.790
0.943 0.974 1.033 1.298 0.995
0.960 1.003 0.964 1.389 1.004
0.965 0.989 0.928 1.341 1.024
0.953 0.970 0.897 1.363 1.072
0.957 1.021 1.083 1.403 1.082
0.947 0.998 0.978 1.439 1.135
1.04 1.74 6.54 13.8 10.9
*Mobile phase: n-hexane: ethanol = 70:30 (v/v); concentration of metalaxyl, 1.0 mg mL−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm; column temperature, a, 25 ◦ C; b, 45 ◦ C.
Table 5 LOD and LOQ results of metalaxyl enantiomers through four detectors. Detectors
UV
OR
LOD *
S-(+)-metalaxyl R-(−)-metalaxyl* *
LOQ −4
1.28 × 10 1.31 × 10−4
RI
LOD −4
4.22 × 10 4.32 × 10−4
LOQ −2
2.06 × 10 2.51 × 10−2
ELS
LOD −2
6.79 × 10 8.28 × 10−2
LOQ −2
1.71 × 10 1.79 × 10−2
LOD −2
5.64 × 10 5.91 × 10−2
LOQ −3
2.80 × 10 1.32 × 10−2
9.24 × 10−3 4.36 × 10−2
Unit, mg mL−1 .
much closer to the theoretic value than those at 25 ◦ C, which indicated that ELS detector showed better response at the higher column temperature.
3.1.4. Effect of flow rate In order to further investigate the potential influence of flow rate on separation of metalaxyl, injections has been run at increasing flow rates from 0.50 mL min−1 to 2.0 mL min−1 in this work (Table 4 and Fig. 8), and the representative chromatograms were presented in Fig. 9. The elution of metalaxyl enantiomers turned fast along with increasing the flow rate. Under these conditions, the peak area ratios between two enantiomers are in the range of 0.937–0.965 for the UV detector, 0.970–1.021 for the RI detector, 0.897–1.083 for the OR detector, and 0.909–1.439 for the ELS detector, respectively, and the corresponding RSD values are 1.04%, 1.74%, 6.54%, and 13.8%. The results from the UV, RI or OR detector are close to the theoretic value and remained almost unchanged, indicating that they are less affected by the change of flow rate. In addition, change of flow rate exhibited also slightly effect on separations of metalaxyl with ELS detector at 45 ◦ C (Table 4 and Fig. 8). However, similar to the effect from eluent composition, peak area ratios obtained through ELS detection at 25 ◦ C exhibited much serious deviation from 1 than at 45 ◦ C and might be explained that nonlinear response for ELS detector enhanced along with increasing flow rate of the mobile phase. Under the lower flow rate conditions, peak shape for the first-eluted component turned a bit changed, that is, sharp at both sides and flat in the middle, and the responses related with the enantiomers exceeded the detector scale, thereby leading to inaccurate integral [24].
3.2. Quantitative analysis studies of metalaxyl enantiomers 3.2.1. Limits of detection and quantitation Limit of detection (LOD) is defined as the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions, and the limit of quantitation (LOQ) is the lowest concentration of substance in a sample can be quantitatively detected in accordance with acceptable accuracy and precision, which are closely related to signal to noise ratios (S/N). Herein, LOD is considered as the concentration of metalaxyl enantiomer when S/N is 3, and LOQ corresponds to the concentration when S/N is 10.
As shown in Table 5, the LOD and LOQ of two metalaxyl enantiomers through the UV detector are 1–2 orders of magnitude lower than the others, followed by the ELS and RI detectors. The OR detector seems not to be well suitable for quantitative analysis study, due to relatively higher LOD and LOQ than the others. In addition, the LOD and LOQ for the S-enantiomer are much lower than the R-enantiomer, especially for ELS detection.
3.2.2. Linear range study In order to further compare difference in quantitative study by using four kinds of detectors, a series of metalaxyl enantiomers with different concentrations were prepared by diluting a mother solution and injected into the chromatographic system in sequence of concentration. Then, the relationship between the enantiomer’s concentration and the corresponding chromatographic peak area has been obtained by fitting with power function model, linear model, and quadratic polynomial model, respectively (Table 6). As discussed above, the UV detection for metalaxyl enantiomers has been slightly influenced by chromatographic conditions, namely, concentration, column temperature, mobile phase composition as well as flow rate, and show much more advantages in quantitative study than the others, namely a much lower LODs and LOQs, as well as a quite wider linear range. In Table 6, the concentration range of S-(+)-metalaxyl is from 1.030 × 10−3 to 0.8824 mg mL−1 with the UV detector, and the corresponding range for R-(−)-metalaxyl is from 1.260 × 10−3 to 1.005 mg mL−1 , which is a bit wider than the S-enantiomer. In addition, these correlation coefficients fitted by three models exceed 0.998. For the RI detection, the quantitative ranges for two enantiomers have been also much wider than the OR and ELS detections (Table 6), and their correlation coefficients fitted by three models are over 0.998, due to less affects from the chromatographic conditions. In addition, it seems that for these data, both the power function and quadratic polynomial models are more suitable than the linear model. Due to relative high LOD and LOQ for OR detection, the obtained quantitative ranges for two metalaxyl enantiomers are narrower significantly than the UV and RI detections (Table 6). In addition, the quantitative ranges and correlation coefficients fitted by three different models are almost the same. For that ELS detection shows some nonlinear response characteristic and has been much easily affected by the chromatographic conditions as described above, a piecewise fitting method is adopted herein. For S-(+)-metalaxyl, both the power function
Please cite this article in press as: T. Chen, et al., Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.07.007
S-(+)-metalaxyl
R-(−)metalaxyl
S-(+)-metalaxyl
R-(−)metalaxyl
S-(+)-metalaxyl
R-(−)metalaxyl
S-(+)-metalaxyl
R-(−)-metalaxyl
1.030 × 10−3 ∼8.224 × 10−1 6
1.260 × 10−3 ∼1.005 6
5.300 × 10−2 ∼2.120 6
5.825 × 10−2 ∼2.336 6
0.1074 ∼1.074 6
0.1260 ∼1.512 6
1.056 × 10−2 ∼4.224 × 10−1 6
5.120 × 10−2 ∼ 8.192 × 10−1 7
y = 2.924 × 104 × x1.007 0.9984
y = 3.058 × 10−3 × x0.9824 0.9999
y = 1.954 × 105 × x1.081 0.9994
y = 1.906 × 105 × x0.9349 0.9993
y = 1.404 × 106 × x1.0895 0.9996
y = 1.304 × 106 × x1.0155 0.9993
y = 5.356 × 104 × x1.655 0.9984
y = 3.511 × 104 × x1.777 0.9988
1.030 × 10−3 ∼8.224 × 10−1 6
1.260 × 10−3 ∼1.005 6
5.300 × 10−2 ∼2.120 6
5.825 × 10−2 ∼2.336 6
0.1074 ∼1.074 6
0.1260 ∼1.512 6
0.2112 ∼0.8448 5
0.4096 ∼1.024 4
y = 2.903 × 104 × x + 88 0.9984
y = 3.061 × 104 × x + 51 0.9999
y = 2.086 × 105 × x −7226 0.9983
y = 1.799 × 105 × x-5434 0.9983
y = 1.452 × 106 × x-52208 0.9994
y = 1.312 × 106 × x-5515 0.9992
y = 3.965 × 104 × x-4284 0.9983
y = 4.389 × 104 × x-11122 0.9924
1.030 × 10−3 ∼8.224 × 10−1 6
1.260 × 10−3 ∼1.005 6
5.300 × 10−2 ∼2.120 6
5.825 × 10−2 ∼2.336 6
0.1074 ∼ 1.074 6
0.1260 ∼1.512 6
1.056 × 10−2 ∼ 4.224 × 10−1 6
5.120 × 10−2 ∼ 8.192 × 10−1 7
y = 2871 × x + 26729× x + 213
y = −987 × x + 31572 × x-14
0.9985
0.9999
y = 9.232 × 10 × x + 1.894 × 105 × x-2755 0.9988
y = −8.281 × 102 × x2 + 1.894 × 105 × x-2755 0.9983
y = 4.091 × 104 × x2 + 1.244 × 106 × x + 12776 0.9992
y = 5.173 × 104 × x2 + 8.992 × 104 × x-137 0.9979
y = 2.815 × 104 × x2 + 7.889 × 104 × x-723 0.9987
Power function model Calibration range (mg·mL−1 ) Calibration points Equations Correlation coefficient Linear model Calibration range (mg mL−1 ) Calibration points Equations Correlation coefficient Quadratic polynomial model Calibration range (mg mL−1 ) Calibration points Equations
Correlation coefficient
2
RI detector
2
OR detector
3
2
y = −9.847 × 10 × x2 + 2.024 × 105 × x-307 0.9996 3
ELS detector
*x, the concentration of the enantiomer, mg mL−1 ; y, peak area of the enantiomer in the chromatogram.
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Table 6 Fit forms for S- and R-enantiomers by HPLC coupled with different detectors.
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Table 7 Recovery and precision results of metalaxyl enantiomers by using four detectors. detector UV
S-form added (g) 0.2056 1.028 8.224
R-form added (g) 0.2512 2.512 10.05
S-form found (g) 0.1655 1.058 7.838
R-form found (g)
RSD for S-form (%)
RSD for R-form (%)
Recovery for S-form (%)
Recovery for R-form (%)
0.1993 2.642 10.12
1.3 0.04 0.08
0.33 0.36 0.05
80.50 102.9 95.31
79.34 105.2 100.7
RI
1.696 10.60 25.44
1.864 11.65 27.96
1.983 9.134 24.21
2.41 12.82 29.34
1.6 1.3 2.6
3.1 1.7 0.99
116.9 86.17 95.17
121.5 110.0 104.9
OR
4.496 6.444 16.11
5.040 12.60 20.16
4.321 6.501 16.13
4.505 12.44 20.40
0.14 1.3 3.2
0.40 0.44 0.73
96.11 100.9 100.1
89.38 98.73 101.2
ELS
2.112 5.280 12.67
8.192 12.29 16.38
2.362 5.200 12.87
8.478 11.82 16.26
2.3 1.7 1.4
0.50 0.57 0.64
111.8 98.48 101.6
103.5 96.18 99.27
model and quadratic polynomial model are more suitable in the low concentration range from 1.056 × 10−2 to 4.224 × 10−1 mg mL−1 than the linear model, while under the relative high concentration range from 0.2112 to 0.8448 mg mL−1 , the linear model seems to be better for fitting. For R-(−)-metalaxyl, similar phenomena are observed (Table 6). Moreover, the quantitative ranges for R-(−)metalaxyl is a bit wider than S-(+)-metalaxyl.
Acknowledgements Financial supports from Natural Science Foundation of China (Nos. 21171059, 21275056, and 21571070), Guangdong Provincial Science and Technology Project (Nos. 2014A010101145, 2012B010900043, and 2016B090921005), Guangzhou Science and Technology Project (Nos. 2013J4400027 and 201508020093) are gratefully acknowledged. References
3.2.3. Recovery and precision In order to investigate recovery and precision of quantitative method for metalaxyl enantiomers through four detectors, determination assay has been conducted. Each sample solution with different concentration was injected repeatedly for six times under the optimal experimental conditions. As listed in Table 7, the found concentration data of two enantiomers display a bit big deviation with the actual at the low concentration. Increasing the sample concentration remarkably reduces the error of recovery data and most of the recovery data are close to 100%. In addition, the results from the RI detection seem poorer than the others.
4. Conclusion Recently, chiral stationary phases-HPLC coupled with different detectors (UV, RI, OR, and ELS detectors) have been widely applied to separation and determination of chiral substances with/without chromophoric groups. Herein, effect of chromatographic conditions on the peak area ratio between two metalaxyl enantiomers, namely, concentration, column temperature, mobile phase composition and flow rate, have been investigated in detail. Among of them, the UV and RI detections have been slightly affected and most of the peak area ratios are very close to the theoretic value, but the results from the ELS detection show a much bigger deviation than the others. Moreover, the LOD, LOQ, linear range, recovery and precision for two metalaxyl enantiomers have been further studied by using four kinds of detectors under optimal conditions. Among of them, the UV detection shows some advantages, namely the lowest LOD and LOQ, as well as the wider quantitative range, better precision and recovery. Due to relative high LOD, the developed method described herein may be not suitable for studies of long-term accumulation in soils and groundwater, as well as for consequences to the environment and public health. Thus, a much more sensitive determination method for metalaxyl enantiomers in soils and groundwater by chiral HPLC needs to be developed in the future.
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Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors Tao Chen a , Jun Fan a,∗ , Ruiqi Gao a , Tai Wang b , Ying Yu a , Weiguang Zhang a,b,∗ a b
School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China Guangzhou Research & Creativity Biotechnology Co., Ltd, Guangzhou 510663, China
a r t i c l e
i n f o
Article history: Received 17 February 2016 Received in revised form 2 July 2016 Accepted 5 July 2016 Available online xxx Keywords: Metalaxyl Enantiomeric ratio HPLC chiral analysis Chromatographic detectors Quantitative study
a b s t r a c t Chiral stationary phase-high performance liquid chromatography coupled with various detectors has been one of most commonly used methods for analysis and separation of chiral compounds over the past decades. Various detectors exhibit different characteristics in qualitative and quantitative studies under different chromatographic conditions. Herein, a comparative evaluation of HPLC coupled with ultraviolet, optical rotation, refractive index, and evaporative light scattering detectors has been conducted for qualitative and quantitative analyses of metalaxyl racemate. Effects of separation conditions on the peak area ratio between two enantiomers, including sample concentration, column temperature, mobile phase composition, as well as flow rate, have been investigated in detail. In addition, the limits of detection, the limits of quantitation, quantitative range and precision for these two enantiomers by using four detectors have been also studied. As indicated, the chromatographic separation conditions have been slight effects on ultraviolet and refractive index detections and the peak area ratio between two enantiomers remains almost unchanged, but the evaporative light scattering detection has been significantly affected by the above-mentioned chromatographic conditions and the corresponding peak area ratios varied greatly. Moreover, the limits of detection, the limits of quantitation, and the quantitative ranges of two enantiomers with UV detection were remarkably lower by 1–2 magnitudes than the others. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Chirality is ubiquitous in life sciences, pharmaceuticals, fine chemicals, and materials, etc. [1–5]. Enantiomers may exhibit different physiological, pharmacological, and toxic activities in the chiral environment, although having same physical and chemical properties. Only one or several enantiomers in many chiral pharmaceuticals exhibit pharmacologically active, and the presence of enantiomeric impurities could lead to potentially serious consequences. Thus, separation, quantitative determination, in-process quality control, and pharmacological studies of chiral compounds have been one of hotspots in research of novel pharmaceuticals. In recent years, chiral stationary phase-high performance liquid chromatography (HPLC) coupled with various detectors, e.g. ultraviolet (UV) detector, optical rotation (OR) detector, refractive index (RI) detector and evaporative light scattering (ELS) detector, has been one of the most commonly used methods in chiral
∗ Corresponding authors at: School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China. E-mail addresses: [email protected] (J. Fan), [email protected] (W. Zhang).
separation studies [1,2,6–15]. Due to high sensitivity, high stablity, and low cost, UV detector has been widely applied in detection of analytes with typical chromophoric groups, and showed some potential drawbacks, such as unsuitablity for analytes without typical chromophores and limit of solvent [16–19]. As being a universal detector, evaporative light scattering detector is suitable for analytes with less volatility than mobile phase [17,20–25]. Most of analytes with/without typical chromophores are able to be detected through ELS detector. For refractive index detector, sample is detected on the basis of difference between its refraction and mobile phase’s refraction [26–29], and it has some potential shortages, such as low sensitivity, susceptibility to disturbance from eluents, baseline instability, and gradient incompatibility. Moreover, optical rotation detector as a kind of selective detector is only suitable to detection of optically active substances and nonoptically active compunds couldn’t interfere with determintion of enantiomers [30–33]. Chromatographic conditions have some potential impacts on sensitivity and precision of these detectors and peak area normalization method can’t be directly used for quantitation of enantiomers under some conditions. Toussaint group reported a comparative study on determination of chiral non-aromatic alco-
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hols by HPLC coupled with four different detectors [33]. The peak area ratio between enantiomers by using the ELS detector was remarkably different with the others. Wipf and his co-worker reported HPLC determinations of enantiomeric ratios through three detectors, namely, UV, ELS, and electrical aerosol detector [29]. ELS detector didn’t reflect real enantiomeric purity under different elution conditions in comparison with the others, and might be complemented through adopting a suitable calibration protocol. HPLC coupled with ELS detector was applied in detection and determination of a mixture of enantiomers, and the effects of some factors on integration of chromatographic peaks, such as molecular weight and volatility of analytes and peak shape, have been discussed in detail [34,35], which indicated that non-linear response for ELS detector has been the main factor leading to deviation. Metalaxyl is an acetanilide-type fungicide and contains an asymmetrically substituted C atom, thereby having two enantiomers (Fig. 1). It shows high activity against fungi of the order Peronosporales and good effectiveness against diseases caused by oomycetes, and has been widely employed in the agriculture field from 1977, for seed treatment, banded or broadcast soil application, and foliar spray [36,37]. Usually, plant treatment is performed with the racemic mixture. However, some results indicated that the R-metalaxyl has been the primary active ingredient relative to the S-enantiomer and the anti-fungicidal activity mostly originated from the R-enantiomer [37]. In addition, metalaxyl is photolytically and hydrolytically stable with about a half-time of 400 days under natural sunlight, and 7–118 days at 20 ◦ C from pH 5 to pH 9 in hydrolysis [37–39]. Thus, its long-term accumulation in soils and groundwater could cause a potentially serious consequence to the environment and public health [40,41]. Herein, a comparative evaluation of HPLC coupled with UV, RI, OR and ELS detectors has been conducted for qualitative and quantitative determinations of metalaxyl enantiomers. Effects of chromatographic conditions on the peak area ratio, such as concentration, column temperature, mobile phase composition, and flow rate, have been investigated in detail. 2. Experimental 2.1. Chemicals and materials Racemic metalaxyl (purity > 98%) was bought from Wuhan Fengzhulin Chemexpress Co., Ltd (Wuhan, China). Optically pure S-(+)-metalaxyl and R-(−)-metalaxyl were obtained through preparative separation of metalaxyl racemate by simulated moving bed chromatography [42]. Ethanol and n-hexane were of HPLC-
grade and purchased from Lab Science, Inc (Houston, Texas, US). Nitrogen with purity higher than 99.999% was used as the evaporation gas. EnantioPak OD column (150 mm × 4.6 mm, 5 m) was friendly provided by Guangzhou Research & Creativity Biotechnology Co., Ltd (Guangzhou, China).
2.2. Instruments Chromatographic analysis experiments have been performed with a 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, a quaternary pump, an auto injector with a 100 L sample loop, a column oven, a multiple wavelength UV detector, a RI detector, and the Agilent Chemstation software. Polarimetry of chiral compounds has been carried out by using IBZ Chiralyser-MP optical rotation detector (IBZ Messtechnik GMBH, Hannover, Germany). In addition, an Alltech Model 3300 evaporative light scattering detector (Grace Davison Discovery Sciences, Deerfield, IL, USA) was used in this study. These four detectors were individually connected to the same HPLC system. Minor difference between retention times of two enantiomers was present in the obtained chromatograms because of different flow cells and pipelines of these four detectors.
2.3. Chromatography Solutions of racemic metalaxyl, S-(+)-metalaxyl, and R-(−)metalaxyl were prepared through dissolving the corresponding samples and diluting with the mobile phase, respectively. Mixture of n-hexane/ethanol was freshly prepared and degassed in an ultrasonic bath before use. Enantioseparation of metalaxyl was achieved on the EnantioPak OD column by normal-phase HPLC at 25 ◦ C, without otherwise specified. Sample solutions (20 L) were injected twice and the flow rate was 1.0 mL min−1 . The UV detection wavelength of metalaxyl was set to 220 nm. The optical unit’s temperature for refractive index detector was 35 ◦ C. For evaporative light scattering detector, the flow rate of nitrogen was set at 1.5 L min−1 , the temperature of the drift tube was 35 ◦ C, and the gain was set at 1. Optical rotation detector was used for identification of the enantiomers. The average and the range of optical rotation detector were set at off and 4.0 m deg, respectively.
Fig. 1. Molecular structures of two metalaxyl enantiomers.
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Fig. 2. Effect of sample concentration on peak area ratio between two enantiomers.
3. Results and discussion 3.1. Effects of chromatographic conditions on determination of peak area ratio The amount of two enantiomers is equal in a racemic mixture, and their peak area ratio is very close to 1 when resolved by chiral HPLC. However, the experimental results might seriously deviate from the theoretical value by using different detectors (UV, RI, and especially ELS) in the HPLC studies [29,33,34]. Thus, a series of experiments have been run to explore the potential effects of chromatographic conditions on enantioseparation of metalaxyl. 3.1.1. Effect of sample concentration A series of metalaxyl racemate solutions, which their concentrations varied from 0.2 mg mL−1 to 1.8 mg mL−1 , have been separated with the mixture of n-hexane and ethanol (70:30, v/v) at 25 ◦ C, and the UV, RI, ELS, and OR detectors were used, respectively. The obtained results are shown in Table 1 and Fig. 2, and the selected chromatograms were depicted in Fig. 3. Under these above-mentioned conditions, S-(+)-metalaxyl was earlier eluted than R-(−)-enantiomer. The peak area ratios between S- and R-enantiomers under different sample concentrations are in the range of 0.960–1.013 for the UV detector, 0.978–1.063 for the RI detector, and 0.920–1.066 for the OR detector, respectively, which are very close to the theoretic value, and the corresponding RSD values are 1.95%, 2.77%, and 4.49%. However, the results obtained from ELS detector are relatively poor. The peak area ratios between two enantiomers ranging from 1.357 to 2.077 are deviated significantly away from 1, and the RSD value is 16.15%. Obviously, the UV detection is less influenced than the others with the
Fig. 3. Representative chromatograms of metalaxyl by HPLC with four detectors. Chromatographic conditions: sample concentration, 0.8 mg mL−1 ; column temperature, 25 ◦ C; mobile phase, Hex-EtOH (70/30, v/v); flow rate, 1.0 mL min−1 . * denoted as chiral separation at 45 ◦ C.
change of sample concentration, and the ELS detection has been a much greater impact than the others under the similar condition. Good linear relationships between sample concentration and signal responses of UV detector, RI detector, and/or OR detection were observed in certain range of concentrations, but the response from ELS detection is incompletely linear [34,40]. At relatively low concentration level (0.20–0.80 mg mL−1 , herein), obvious nonlinear response by ELS detection was shown and the peak areas related to two enantiomers were clearly different. With increasing sample’s concentration (1.00–1.80 mg mL−1 ), the correlation between the concentration and the signal response turned good and the peak area ratios remained more than 1. Furthermore, separations of metalaxyl with different concentrations by HPLC coupled with ELS detector have been rerun at 45 ◦ C, as presented in Table 1. Clearly, the retention of two enantiomers became weak in the column and the chromatographic peaks turned narrow. With increasing the concentration, the peak area ratio between two enantiomers ranged from 1.468 to 0.821 and was trending downward. The ratio is 1.004 at the concentration of 1 mg mL−1 . More importantly, the results obtained at 45 ◦ C are much closer to the theoretic value than those at 25 ◦ C, which indicated that ELS detector showed better response at the higher column temperature. One thing to note here, the peak shape for two enantiomers has become sharp at both sides and flat in the middle when the sample concentration exceeded 1.60 mg mL−1 , which might be attributed that the signal strength under relative high concentration was over the signal range of this detector.
Table 1 Effect of sample concentration on peak area ratio between two enantiomers. Peak area ratio
a
S/R (UV) S/R (RI)a S/R (OR)a S/R (ELS)a S/R (ELS)b
Concentration (mg mL−1 ) 0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
RSD (%)
1.013 1.063 1.024 2.077 1.468
0.998 1.057 0.920 1.749 1.279
0.986 1.029 0.975 1.612 1.223
0.978 0.978 0.975 1.438 1.138
0.960 1.003 0.964 1.389 1.004
0.964 1.014 1.032 1.378 0.975
0.964 1.034 1.066 1.357 0.926
0.962 1.011 0.975 1.370 0.866
0.962 0.993 1.026 1.369 0.821
1.95 2.77 4.49 16.15 19.88
*Mobile phase: n-hexane: ethanol = 70:30 (v/v); flow rate, 1.0 mL min−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm; column temperature, a, 25 ◦ C, b, 45 ◦ C.
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Fig. 4. Effect of column temperature on peak area ratio between two enantiomers.
Fig. 6. Effect of mobile phase composition on peak area ratio between two enantiomers.
Fig. 5. Selected chromatograms of metalaxyl by HPLC with four detectors. Chromatographic conditions: sample concentration, 1.0 mg mL−1 ; column temperature, 45 ◦ C; mobile phase, Hex-EtOH (70/30, v/v); flow rate, 1.0 mL min−1 .
3.1.2. Effect of column temperature The effect of column temperature on enantioseparation of metalaxyl has been investigated in this section (Table 2 and Fig. 4), and the representative chromatograms were shown in Fig. 5. Both the retention factors and resolution gradually decreased with raising the column temperatures from 20 to 60 ◦ C. Under these experimental conditions, the peak area ratios between S- and R-enantiomers are in the small range of 0.924–0.960 for the UV detector, 0.962–1.028 for the RI detector, 0.926–1.165 for the OR detector, and 0.970–1.402 for the ELS detector, respectively, and the corresponding RSD values are 1.51%, 2.17%, 8.18% and 15.20%. Clearly, changing temperature has been less impact on the results obtained from both UV and RI detectors, while has much more influenced on the others. When the column temperature was set to be 50 ◦ C and more, resolution of two enantiomers was poor by HPLC coupled with the OR detector and the corresponding peaks weren’t accurately integrated, thus giving unfavorable results. In addition, the results from the ELS detector showed a downward trend in the same temperature range, similar to the effect of sample con-
Fig. 7. Selected HPLC chromatograms of metalaxyl from four detectors. Chromatographic conditions: sample concentration, 1.0 mg mL−1 ; column temperature, 25 ◦ C; mobile phase, Hex-EtOH (0/100, v/v); flow rate, 1.0 mL min−1 . * denoted as chiral separation at 45 ◦ C.
centration. Lower column temperatures led to worse results with bigger deviation, which might be explained that the signals related to the samples clearly strengthened along with raising the column temperature and the non-linear responses became fewer [24]. 3.1.3. Effect of the eluent composition Composition of mobile phases has significant effect on retention behaviors and separations of chiral analytes. In the following-up work, the effect of eluent composition on resolution of metalaxyl has been studied (Table 3 and Fig. 6), and the selected chromatograms were displayed in Fig. 7. The retention of metalaxyl enantiomers turned weak with increasing ethanol content in the mobile phase. The peak area ratios between S- and R-enantiomers under the mobile phases with dif-
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Table 2 Effect of column temperature on peak area ratio between two enantiomers. Peak area ratio
S/R (UV) S/R (RI) S/R (OR) S/R (ELS)
Column temperature (◦ C) 20
25
30
35
40
45
50
55
60
RSD(%)
0.942 1.013 0.989 1.402
0.960 1.003 0.964 1.389
0.936 1.028 0.974 1.198
0.960 0.969 0.926 1.120
0.925 0.978 1.101 1.046
0.924 0.962 1.058 1.004
0.925 0.993 1.084 1.006
0.929 0.981 1.147 0.970
0.939 0.981 1.165 0.976
1.51 2.17 8.18 15.2
*Mobile phase: n-hexane: ethanol = 70:30 (v/v); flow rate, 1.0 mL min−1 ; concentration of metalaxyl, 1.0 mg mL−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm. Table 3 Effect of mobile phase composition on peak area ratio between two enantiomers. Eluent composition(volume ratio)
90:10 80:20 70:30 60:40 50:50 40:60 30:70 20:80 10:90 0:100 RSD (%)
Peak area ratio S/R (UV)a
S/R (RI)a
S/R (OR)a
S/R (ELS)a
S/R (ELS)b
1.000 0.994 0.960 0.989 1.000 1.013 1.035 0.985 0.972 0.942 2.68
1.024 0.959 1.003 0.962 1.034 1.035 0.980 0.991 1.013 1.027 2.86
1.039 0.985 0.964 1.012 1.074 1.080 1.046 0.990 0.978 0.909 5.28
1.604 1.379 1.389 1.242 1.218 1.165 1.168 1.120 1.107 1.052 13.5
0.703 0.891 1.004 1.039 1.013 1.015 0.996 1.028 1.025 1.003 10.6
*Mobile phase: n-hexane-ethanol; flow rate, 1.0 mL min−1 ; concentration of metalaxyl, 1.0 mg mL−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm; column temperature, a, 25 ◦ C; b, 45 ◦ C.
Fig. 8. Effects of flow rate on peak area ratio between two enantiomers.
ferent composition were in the range of 0.942–1.035 for the UV detector, 0.959–1.034 for the RI detector, 0.909–1.074 for the OR detector, and 1.052–1.604 for the ELS detector, and the corresponding RSD values were 2.68%, 2.86%, 5.28% and 13.5%, respectively. The results from the UV and RI detectors were close to 1 under the experimental conditions, which meant that change of eluent composition had slight effect on determination of two enantiomers with UV or RI detector. In addition, the peak area ratio between two enantiomers from ELS detection showed a downward tendency with increasing ethanol, that is, declining rapidly in the 10–40% range and slightly changed in the 50%–100% range, and the deviation is bigger than the others. The ratio was close to 1 with 100% ethanol as the eluent. Retention of two enantiomers became
Fig. 9. Representative HPLC chromatograms of metalaxyl from four detectors. Chromatographic conditions: sample concentration, 1.0 mg mL−1 ; column temperature, 25 ◦ C; mobile phase, Hex-EtOH (70/30, v/v); flow rate, 0.5 mL min−1 . * denoted as chiral separation at 45 ◦ C.
weaker and weaker in the eluent with higher content of ethanol, possibly leading to good linear response [29,34]. Moreover, separations of metalaxyl have been conducted by HPLC coupled with ELS detector at 45 ◦ C to further investigate the effect of eluent composition (Table 3 and Fig. 6). Obviously, the retention of two enantiomers turned weak and the chromatographic peaks became narrow. The peak area ratio between two enantiomers ranged from 0.703 to 1.039, and kept almost unchanged when the content of ethanol in the mobile phase was over 30%. More importantly, these results obtained at 45 ◦ C were
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Table 4 Effects of flow rate on peak area ratio between two enantiomers. Flow rate (mL min−1 )
Peak area ratio
S/R (UV)a S/R (RI)a S/R (OR)a S/R (ELS)a S/R (ELS)b
0.50
0.80
1.00
1.20
1.50
1.80
2.00
RSD (%)
0.937 0.995 1.025 0.909 0.790
0.943 0.974 1.033 1.298 0.995
0.960 1.003 0.964 1.389 1.004
0.965 0.989 0.928 1.341 1.024
0.953 0.970 0.897 1.363 1.072
0.957 1.021 1.083 1.403 1.082
0.947 0.998 0.978 1.439 1.135
1.04 1.74 6.54 13.8 10.9
*Mobile phase: n-hexane: ethanol = 70:30 (v/v); concentration of metalaxyl, 1.0 mg mL−1 ; the injection volume, 20 L; the UV detection wavelength, 220 nm; column temperature, a, 25 ◦ C; b, 45 ◦ C.
Table 5 LOD and LOQ results of metalaxyl enantiomers through four detectors. Detectors
UV
OR
LOD *
S-(+)-metalaxyl R-(−)-metalaxyl* *
LOQ −4
1.28 × 10 1.31 × 10−4
RI
LOD −4
4.22 × 10 4.32 × 10−4
LOQ −2
2.06 × 10 2.51 × 10−2
ELS
LOD −2
6.79 × 10 8.28 × 10−2
LOQ −2
1.71 × 10 1.79 × 10−2
LOD −2
5.64 × 10 5.91 × 10−2
LOQ −3
2.80 × 10 1.32 × 10−2
9.24 × 10−3 4.36 × 10−2
Unit, mg mL−1 .
much closer to the theoretic value than those at 25 ◦ C, which indicated that ELS detector showed better response at the higher column temperature.
3.1.4. Effect of flow rate In order to further investigate the potential influence of flow rate on separation of metalaxyl, injections has been run at increasing flow rates from 0.50 mL min−1 to 2.0 mL min−1 in this work (Table 4 and Fig. 8), and the representative chromatograms were presented in Fig. 9. The elution of metalaxyl enantiomers turned fast along with increasing the flow rate. Under these conditions, the peak area ratios between two enantiomers are in the range of 0.937–0.965 for the UV detector, 0.970–1.021 for the RI detector, 0.897–1.083 for the OR detector, and 0.909–1.439 for the ELS detector, respectively, and the corresponding RSD values are 1.04%, 1.74%, 6.54%, and 13.8%. The results from the UV, RI or OR detector are close to the theoretic value and remained almost unchanged, indicating that they are less affected by the change of flow rate. In addition, change of flow rate exhibited also slightly effect on separations of metalaxyl with ELS detector at 45 ◦ C (Table 4 and Fig. 8). However, similar to the effect from eluent composition, peak area ratios obtained through ELS detection at 25 ◦ C exhibited much serious deviation from 1 than at 45 ◦ C and might be explained that nonlinear response for ELS detector enhanced along with increasing flow rate of the mobile phase. Under the lower flow rate conditions, peak shape for the first-eluted component turned a bit changed, that is, sharp at both sides and flat in the middle, and the responses related with the enantiomers exceeded the detector scale, thereby leading to inaccurate integral [24].
3.2. Quantitative analysis studies of metalaxyl enantiomers 3.2.1. Limits of detection and quantitation Limit of detection (LOD) is defined as the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions, and the limit of quantitation (LOQ) is the lowest concentration of substance in a sample can be quantitatively detected in accordance with acceptable accuracy and precision, which are closely related to signal to noise ratios (S/N). Herein, LOD is considered as the concentration of metalaxyl enantiomer when S/N is 3, and LOQ corresponds to the concentration when S/N is 10.
As shown in Table 5, the LOD and LOQ of two metalaxyl enantiomers through the UV detector are 1–2 orders of magnitude lower than the others, followed by the ELS and RI detectors. The OR detector seems not to be well suitable for quantitative analysis study, due to relatively higher LOD and LOQ than the others. In addition, the LOD and LOQ for the S-enantiomer are much lower than the R-enantiomer, especially for ELS detection.
3.2.2. Linear range study In order to further compare difference in quantitative study by using four kinds of detectors, a series of metalaxyl enantiomers with different concentrations were prepared by diluting a mother solution and injected into the chromatographic system in sequence of concentration. Then, the relationship between the enantiomer’s concentration and the corresponding chromatographic peak area has been obtained by fitting with power function model, linear model, and quadratic polynomial model, respectively (Table 6). As discussed above, the UV detection for metalaxyl enantiomers has been slightly influenced by chromatographic conditions, namely, concentration, column temperature, mobile phase composition as well as flow rate, and show much more advantages in quantitative study than the others, namely a much lower LODs and LOQs, as well as a quite wider linear range. In Table 6, the concentration range of S-(+)-metalaxyl is from 1.030 × 10−3 to 0.8824 mg mL−1 with the UV detector, and the corresponding range for R-(−)-metalaxyl is from 1.260 × 10−3 to 1.005 mg mL−1 , which is a bit wider than the S-enantiomer. In addition, these correlation coefficients fitted by three models exceed 0.998. For the RI detection, the quantitative ranges for two enantiomers have been also much wider than the OR and ELS detections (Table 6), and their correlation coefficients fitted by three models are over 0.998, due to less affects from the chromatographic conditions. In addition, it seems that for these data, both the power function and quadratic polynomial models are more suitable than the linear model. Due to relative high LOD and LOQ for OR detection, the obtained quantitative ranges for two metalaxyl enantiomers are narrower significantly than the UV and RI detections (Table 6). In addition, the quantitative ranges and correlation coefficients fitted by three different models are almost the same. For that ELS detection shows some nonlinear response characteristic and has been much easily affected by the chromatographic conditions as described above, a piecewise fitting method is adopted herein. For S-(+)-metalaxyl, both the power function
Please cite this article in press as: T. Chen, et al., Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.07.007
S-(+)-metalaxyl
R-(−)metalaxyl
S-(+)-metalaxyl
R-(−)metalaxyl
S-(+)-metalaxyl
R-(−)metalaxyl
S-(+)-metalaxyl
R-(−)-metalaxyl
1.030 × 10−3 ∼8.224 × 10−1 6
1.260 × 10−3 ∼1.005 6
5.300 × 10−2 ∼2.120 6
5.825 × 10−2 ∼2.336 6
0.1074 ∼1.074 6
0.1260 ∼1.512 6
1.056 × 10−2 ∼4.224 × 10−1 6
5.120 × 10−2 ∼ 8.192 × 10−1 7
y = 2.924 × 104 × x1.007 0.9984
y = 3.058 × 10−3 × x0.9824 0.9999
y = 1.954 × 105 × x1.081 0.9994
y = 1.906 × 105 × x0.9349 0.9993
y = 1.404 × 106 × x1.0895 0.9996
y = 1.304 × 106 × x1.0155 0.9993
y = 5.356 × 104 × x1.655 0.9984
y = 3.511 × 104 × x1.777 0.9988
1.030 × 10−3 ∼8.224 × 10−1 6
1.260 × 10−3 ∼1.005 6
5.300 × 10−2 ∼2.120 6
5.825 × 10−2 ∼2.336 6
0.1074 ∼1.074 6
0.1260 ∼1.512 6
0.2112 ∼0.8448 5
0.4096 ∼1.024 4
y = 2.903 × 104 × x + 88 0.9984
y = 3.061 × 104 × x + 51 0.9999
y = 2.086 × 105 × x −7226 0.9983
y = 1.799 × 105 × x-5434 0.9983
y = 1.452 × 106 × x-52208 0.9994
y = 1.312 × 106 × x-5515 0.9992
y = 3.965 × 104 × x-4284 0.9983
y = 4.389 × 104 × x-11122 0.9924
1.030 × 10−3 ∼8.224 × 10−1 6
1.260 × 10−3 ∼1.005 6
5.300 × 10−2 ∼2.120 6
5.825 × 10−2 ∼2.336 6
0.1074 ∼ 1.074 6
0.1260 ∼1.512 6
1.056 × 10−2 ∼ 4.224 × 10−1 6
5.120 × 10−2 ∼ 8.192 × 10−1 7
y = 2871 × x + 26729× x + 213
y = −987 × x + 31572 × x-14
0.9985
0.9999
y = 9.232 × 10 × x + 1.894 × 105 × x-2755 0.9988
y = −8.281 × 102 × x2 + 1.894 × 105 × x-2755 0.9983
y = 4.091 × 104 × x2 + 1.244 × 106 × x + 12776 0.9992
y = 5.173 × 104 × x2 + 8.992 × 104 × x-137 0.9979
y = 2.815 × 104 × x2 + 7.889 × 104 × x-723 0.9987
Power function model Calibration range (mg·mL−1 ) Calibration points Equations Correlation coefficient Linear model Calibration range (mg mL−1 ) Calibration points Equations Correlation coefficient Quadratic polynomial model Calibration range (mg mL−1 ) Calibration points Equations
Correlation coefficient
2
RI detector
2
OR detector
3
2
y = −9.847 × 10 × x2 + 2.024 × 105 × x-307 0.9996 3
ELS detector
*x, the concentration of the enantiomer, mg mL−1 ; y, peak area of the enantiomer in the chromatogram.
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Table 6 Fit forms for S- and R-enantiomers by HPLC coupled with different detectors.
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Table 7 Recovery and precision results of metalaxyl enantiomers by using four detectors. detector UV
S-form added (g) 0.2056 1.028 8.224
R-form added (g) 0.2512 2.512 10.05
S-form found (g) 0.1655 1.058 7.838
R-form found (g)
RSD for S-form (%)
RSD for R-form (%)
Recovery for S-form (%)
Recovery for R-form (%)
0.1993 2.642 10.12
1.3 0.04 0.08
0.33 0.36 0.05
80.50 102.9 95.31
79.34 105.2 100.7
RI
1.696 10.60 25.44
1.864 11.65 27.96
1.983 9.134 24.21
2.41 12.82 29.34
1.6 1.3 2.6
3.1 1.7 0.99
116.9 86.17 95.17
121.5 110.0 104.9
OR
4.496 6.444 16.11
5.040 12.60 20.16
4.321 6.501 16.13
4.505 12.44 20.40
0.14 1.3 3.2
0.40 0.44 0.73
96.11 100.9 100.1
89.38 98.73 101.2
ELS
2.112 5.280 12.67
8.192 12.29 16.38
2.362 5.200 12.87
8.478 11.82 16.26
2.3 1.7 1.4
0.50 0.57 0.64
111.8 98.48 101.6
103.5 96.18 99.27
model and quadratic polynomial model are more suitable in the low concentration range from 1.056 × 10−2 to 4.224 × 10−1 mg mL−1 than the linear model, while under the relative high concentration range from 0.2112 to 0.8448 mg mL−1 , the linear model seems to be better for fitting. For R-(−)-metalaxyl, similar phenomena are observed (Table 6). Moreover, the quantitative ranges for R-(−)metalaxyl is a bit wider than S-(+)-metalaxyl.
Acknowledgements Financial supports from Natural Science Foundation of China (Nos. 21171059, 21275056, and 21571070), Guangdong Provincial Science and Technology Project (Nos. 2014A010101145, 2012B010900043, and 2016B090921005), Guangzhou Science and Technology Project (Nos. 2013J4400027 and 201508020093) are gratefully acknowledged. References
3.2.3. Recovery and precision In order to investigate recovery and precision of quantitative method for metalaxyl enantiomers through four detectors, determination assay has been conducted. Each sample solution with different concentration was injected repeatedly for six times under the optimal experimental conditions. As listed in Table 7, the found concentration data of two enantiomers display a bit big deviation with the actual at the low concentration. Increasing the sample concentration remarkably reduces the error of recovery data and most of the recovery data are close to 100%. In addition, the results from the RI detection seem poorer than the others.
4. Conclusion Recently, chiral stationary phases-HPLC coupled with different detectors (UV, RI, OR, and ELS detectors) have been widely applied to separation and determination of chiral substances with/without chromophoric groups. Herein, effect of chromatographic conditions on the peak area ratio between two metalaxyl enantiomers, namely, concentration, column temperature, mobile phase composition and flow rate, have been investigated in detail. Among of them, the UV and RI detections have been slightly affected and most of the peak area ratios are very close to the theoretic value, but the results from the ELS detection show a much bigger deviation than the others. Moreover, the LOD, LOQ, linear range, recovery and precision for two metalaxyl enantiomers have been further studied by using four kinds of detectors under optimal conditions. Among of them, the UV detection shows some advantages, namely the lowest LOD and LOQ, as well as the wider quantitative range, better precision and recovery. Due to relative high LOD, the developed method described herein may be not suitable for studies of long-term accumulation in soils and groundwater, as well as for consequences to the environment and public health. Thus, a much more sensitive determination method for metalaxyl enantiomers in soils and groundwater by chiral HPLC needs to be developed in the future.
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Please cite this article in press as: T. Chen, et al., Analysis of metalaxyl racemate using high performance liquid chromatography coupled with four kinds of detectors, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.07.007