ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 11, с. 1219-1223
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
SIMPLE HPLC—FLUORESCENCE DETERMINATION OF EUGENOL IN CLOVE OIL AFTER PRE-COLUMN DERIVATIZATION WITH 4-(N-CHLOROFORMYLMETHYL-N-METHYLAMINO)-7-NITRO-2,1,3-BENZOXADIAZOLE © 2015 Yasuhiko Higashi
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan E-mail: y-higashi@hokuriku-u.ac.jp Received 31.03.2014; in final form 10.04.2015
Eugenol is a phenolic flavor constituent of various plants, including clove and cinnamon. Eugenol content in clove oil has been analyzed by HPLC—UV after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-ben-zoxadiazole (NBD-F). Here, we present a simple, more sensitive HPLC—fluorescence method for determination of eugenol in clove oil by pre-column derivatization with 4-(N-chloroformylmethyl-N-methylami-no)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl). The retention time of NBD-CO-eugenol was 14.9 min. A standard curve, obtained after derivatization with NBD-COCl in borate buffer (pH 9.0) at room temperature for 1 min, was linear in the range of 0.025 to 0.4 ^g/mL with r2 value of 0.9992. The limit of detection was 0.006 p.g/mL (signal-to-noise ratio of 3 : 1). This is 6.7-fold lower in terms of concentration and about 45-fold lower in terms of absolute amount, compared with the previous method using NBD-F. The coefficients of variation were less than 9.4%. The content of eugenol in clove oil (1.00 g) was 0.73 ± 0.05 g (range 0.65 to 0.79 g). Recovery tests were satisfactory (86 ± 5%; range 80 to 92%).
Keywords: eugenol, HPLC, 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole, derivatization, fluorescence.
DOI: 10.7868/S0044450215110183
Eugenol (4-allyl-2-methoxyphenol) is a phenolic constituent of clove (Syzygium aromaticum), cinnamon (Cinnnamomum cassia) and other plants [1, 2]. It is widely used as a flavoring agent for baked foods and beverages, as well as in dentistry for its analgesic properties [3]. In addition, it has a variety ofphysical and pharmacological effects, including antioxidant [4], anti-inflammatory [5], anti-histaminic [6], anti-anaphylactic [7], and DNA-protective properties [8].
Various methods for eugenol determination were reported based on HPLC and gas chromatigraphy (GC) with various detectors [9—16]. Beaudry et al. developed an liquid chromatography-electrospray qua-drupole ion trap mass spectrometry method after de-rivatization of eugenol with dansyl chloride, and reported that the lower limit of detection of eugenol was 0.5 pg injected on column (estimated to correspond to 0.44 ng/mL plasma concentration) [9]. However, their system is expensive and complicated. NBD-F was used as a fluorescent labeling agent for primary and
secondary amino groups for HPLC—fluorescence detection [17—21]. However, NBD-labeling at the phenolic hydroxyl group of N-acetyltyrosine, chlorophe-nols, or eugenol does not afford fluorescent derivatives, so NBD-F was utilized for labeling these compounds in combination with UV detection [22—25]. However, the lower limit of detection of eugenol was unsatisfactory (0.04 ^g/mL, absolute amount of 0.67 ng) [24]. Though the sensitivity was about 3-fold better than that of a previous HPLC method using a diode array detector [10], it was 12.5 to about 90-fold less than those of other methods [9, 13].
Therefore, we set out to develop a simple, more sensitive HPLC—fluorescence method for determination of eugenol in clove oil by pre-column derivatization with 4-(N-chloroformylmethyl-N-methylami-no)-7-nitro-2,1,3-benzoxadiazole, which is expected to be suitable as a fluorescent labeling agent for the phenolic hydroxyl group of eugenol. The derivatization is shown in reaction scheme:
CH
CH2
CH3O'
N
O
N
CH3
NBD-CO-eugenol
Cl O
NBD-COCl
Derivatization of eugenol with NBD-COCl.
EXPERIMENTAL
Chemicals and reagents. Eugenol (99%) was purchased from Sigma-Aldrich, Inc., (St. Louis, MO, USA.). Clove oil (Lot No. M5R8724) was obtained from Nacalai tesque (Kyoto, Japan). NBD-COCl and other general reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan).
Chromatographic system. The HPLC system consisted of a model L-6200 pump (Hitachi, Tokyo, Japan), a Rheodyne injection valve (Cotati, CA, USA.) with a 10-|L loop, and a model RF-10AXL fluorescence detector (Shimadzu, Kyoto, Japan) with excitation at 470 nm and emission at 540 nm. A 150 x 3.0 mm i.d. HPLC column (Inertsil® ODS-4, GL Science, Tokyo, Japan) containing 5 ^m particles of C18 packing material was used. Peak quantification was performed using a Chromatopac Model C-R3A integrator (Shimadzu). The mobile phase was prepared by the addition of ac-etonitrile (550 mL) to 450 mL of Milli-Q water containing trifluoroacetic acid (0.1%, v/v). Samples were eluted from the column at room temperature at a flow rate of 0.5 mL/min.
Preparation of standard solutions. Ultrapure water was from a Milli-Q water purification system (Simplicity® UV, Millipore Corporation, Bedford, MA, USA). Standard solution was prepared by dissolving eugenol (100 mg) in methanol (50 mL) and stored at 4°C. Working standard solutions (0, 0.025, 0.05, 0.1, 0.2, and 0.4 |g/mL) were prepared by dilution with 10% methanol. Borate buffer (0.1 M) was adjusted to pH 9.0 by addition of NaOH.
Derivatization. Borate buffer (100 |L) was added to diluted standard sample (100 |L), then NBD-COCl solution in acetonitrile (2 mg/mL, 100 |L) was added and the mixture was vortexed. After reaction for 1 min at room temperature, saturated L-aspartate solution (filtrate of 5 mg/mL of L-aspartate suspension) was
added to stop the reaction, and an aliquot (10 ^L) was injected into the HPLC system.
Application to clove oil samples. Oil samples were prepared according to the reported method with minor modifications [24]. Briefly, clove oil (each 100 mg) was dissolved in methanol (100 mL). The methanol solution (50 ^L) was 4000-fold diluted to 200 mL with 10% methanol to obtain a test sample. Derivatization was performed and the derivative was analyzed as described above.
Evaluation of recovery. Samples of clove oil (100 mg) spiked with 20 and 50 mg of eugenol standard were analyzed to determine recovery of the added standard in order to assess the accuracy of the method.
Recovery value, %
Total amount after spiking Original amount + spiked amount
x 100.
Detection of eugenol by HPLC—UV and validation data. HPLC—UV determination of eugenol in clove oil was performed as a control analysis. The HPLC—UV system consisted of a model LC-10ATyp pump (Shimadzu, Kyoto, Japan), a Rheodyne injection valve (Cotati, CA, USA) with a 20-|L loop, and a model SPD-10Avp UV-Vis detector (Shimadzu) operating at 280 nm. A 150 x 3.0 mm i.d. HPLC column (Inertsil® ODS-4, GL Science, Tokyo, Japan) containing 5 ^m particles of C18 packing material was used. Quantification of peaks was performed using a Chromatopac Model C-R8A integrator (Shimadzu). The mobile phase was prepared by the addition of acetonitrile (370 mL) to 630 mL of Milli-Q water containing trifluoroacetic acid (0.1%, v/v). The samples were eluted from the column at room temperature at a flow rate of 0.5 mL/min.
The retention time of eugenol was 10.1 min (data not shown). The standard curve of eugenol was constructed by plotting integrated peak area vs. concentration. The plot was linear in the range of 0.4 to 16 |g/mL with r2 value of0.9997 (y = 51.73*- 9.073). The values of the lower limits of quantification and detection were 0.23 |g/mL (signal-to-noise ratio of10 : 1) and 0.07 |g/mL (the ratio of 3 : 1), respectively. The level (%, w/w) of eugenol in clove oil was calculated by analysis of a clove oil sample (4 |g/mL in 10% methanol).
RESULTS AND DISCUSSION
Time courses of derivatization of eugenol with NBD-COCl at various pH values. For the time course study, the reaction time was set at 0.5, 1, 2, 3, 5, and 10 min at room temperature. Eugenol standard solution (100 |L, 0.2 |g/mL), borate buffer (100 |L, adjusted to pH 8.0 to 10.0), and NBD-COCl (100 |L, 2 mg/mL) were mixed appropriately and analysis was carried out as described in "Materials and methods".
SIMPLE HPLC-FLUORESCENCE DETERMINATION
1221
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ce <D
й
M
ce
P
800
700 -
600 -
500
0 12 3
4 5 6 Time, min
7 8 9 10
Fig. 1. Time courses of formation of eugenol derivative with NBD-COCl at various pH values. Standard sample (0.2 ^g/mL) was reacted with NBD-COCl in borate buffer at pH 8.0 (x), 8.5 (A), 9.0 (O), 9.5 (□), or 10 (O) at room temperature. Data are mean values of two experiments.
As shown in Fig. 1, the time courses at pH 8.5 to 9.5 were very similar, the peak area of NBD-CO-eugenol reached a maximum at 1 min and then tended to decrease. The decrease at pH 8.0 tended to be slower, while at pH 10.0 the peak area was lower throughout the time course. It is speculated that the decrease of
the peak area was due to degradation of the derivative, possibly involving OH ~. On the other hand, weak alkaline conditions may favor derivative formation in the initial stage up to 1 min, during which time the formation rate appears to be faster than the degradation rate. Further studies will be needed to identify the mechanisms involved.
Next, the temperature dependency was examined at 1 min and pH 9.0. Peak areas at 4°C (on ice), room temperature, 35, 45 and 55°C were 662, 811, 787, 703 and 622 mV s, respectively (mean values of two experiments). The peak area of NBD-CO-eugenol was maximum when the derivatization was performed at room temperature.
Thus, we selected 1 min and pH 9.0 as standard conditions. It is noteworthy that derivatization with NBD-COCl in the present method could be conducted at room temperature, while the previous method with NBD-F required a water-bath at 40°C [24].
Chromatogram. Figure 2 shows typical chromato-grams obtained from (A) blank, (B) standard sample (0.1 ^g/mL), and (C) test sample
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