ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 3, с. 316-320
ОРИГИНАЛЬНЫЕ СТАТЬИ =
DETERMINATION OF THREE PHENYLPHENOLS IN GRAPEFRUIT JUICE BY HPLC AFTER PRE-COLUMN DERIVATIZATION WITH 4-FLUORO-7-NITRO-2,1,3-BENZOXADIAZOLE © 2015 Yasuhiko Higashi1, Youichi Fujii
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan 1E-mail: email@example.com Received 10.04.2013; in final form 25.12.2013
o-Phenylphenol is generally utilized as a disinfectant for citrus fruits. The purpose of this study is to develop a high-performance liquid chromatography coupled with ultraviolet detection (380 nm) method for simultaneous determination of o-phenylphenol, and its analogues (m-phenylphenol and p-phenylphenol) in grapefruit juice after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). 2-Hydroxyfluorene was used as an internal standard (IS). Standard curves were obtained after derivatization with NBD-F in borate buffer (pH 8.0) at room temperature for 5 min. The three NBD-F derivatives were almost completely separated on a Cholester column (5 pm, 3.0 mm i.d. x 150 mm). Calibration plots were linear in the range of absolute amount of 1.04 ~ 2.08 to 41.6 ng/50 pL injection volume, with r2 values >0.9981, for the three compounds. The lower limits of detection were 0.3 to 0.7 ng/50 pL injection volume (signal-to-noise ratio of 3 : 1). The coefficients of variation were less than 11.1%. After extraction of grapefruit juice (2.0 mL) with n-pentane, the level of o-phe-nylphenol in the juice was estimated to be 20.2 ± 2.0 ng/mL (n = 6, mean ± SD), while m-phenylphenol and p-phenylphenol were below the lower limits of quantification. The recovery values of the three phenylphenols from samples spiked with a standard mixture of authentic compounds and IS were satisfactory (99.1 to 118.7%).
Keywords: phenylphenol, 4-fluoro-7-nitro-2,1,3-benzoxadiazole, high-performance liquid chromatography (HPLC), cholester column, grapefruit juice.
o-Phenylphenol (OPP) has bactericidal and virucidal activities, and is widely used in households, industry, and hospitals to disinfect surfaces, in addition to being utilized as a preservative in cosmetics, plastics, etc. [1, 2]. OPP exhibits low acute toxicity in animal experiments . The Japanese government approved its use as a food additive only for citrus fruits in 1977 with the permitted maximum residue level of 10 mg/kg in whole fruits [4, 5]. The WHO view on the toxicity of OPP is as follows  "A health-based value of 1 mg/L can be calculated for OPP on the basis ofanADI of 0.4 mg/kg of body weight, based on a NOAEL of39 mg/kg ofbody weight per day in a 2-year toxicity study for decreased body weight gain and hy-perplasia of the urinary bladder and carcinogenicity of the urinary bladder in male rats, using an uncertainty factor of 100. Because of its low toxicity, however, the health-based value derived for OPP is much higher than OPP concentrations likely to be found in drinking-water. Under usual conditions, therefore, the presence of OPP in drinking-water is unlikely to represent a hazard to human health."
To improve the selectivity and sensitivity of methods for determination of various analytes, derivatization with an ultraviolet (UV)-absorbing or fluorescent agent is one of the most useful techniques, and may make sample clean-up unnecessary. Yang et al.  developed a highly sensitive method of OPP determination by HPLC with electrochemical detection, using a microbore column; this afforded a limit of detection of 3.4 pg. GC—mass spectrometric methods for determination of OPP after derivatization with pentafluo-robenzoyl bromide and ferrocenecarboxylic acid chloride were applied to beer and citrus fruit samples, respectively [1, 2]. On the other hand, the WHO does not set guideline values for two positional isomers of OPP, m-phenylphenol (MPP) and ^-phenylphenol (PPP), and, to our knowledge, only one method has been reported for assay of MPP or PPP .
4-Fluoro-7-nitro-2,1,3-benzoxadiazole has been used as a fluorescent labeling agent for primary and secondary amino groups for HPLC—fluorescence detection [8—12]. It has also been used as a UV-labeling reagent reactive with the phenolic hydroxyl group of N-acetyltyrosine . In addition, we have previously
"N NO2 NBD-MPP
Fig. 1. Scheme illustrating the pre-column NBD-F derivatization of OPP, MPP and PPP in grapefruit juice, as well as the IS.
developed methods employing HPLC-UV after derivatization with NBD-F for the determination of phenolic compounds such as chlorophenols and eugenol [14, 15].
In this paper, we present a simple HPLC-UV method for simultaneous determination of three phe-nylphenols (PPs) (OPP, MPP and PPP) in grapefruit juice after pre-column derivatization with NBD-F. The derivatization scheme is shown in Fig. 1.
Chemicals and reagents. OPP and PPP were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). MPP and NBD-F was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo). 2-Hydroxyfluo-rene, used as IS, was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). «-Pentane and other general reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan). Grapefruit juice was bought at a market in Kanazawa City, Ishikawa Prefecture, Japan.
Chromatographic system. The HPLC system consisted of a model LC10-ATyp pump (Shimadzu, Kyoto, Japan), a Rheodyne injection valve (Cotati, CA, USA) with a 50-|L loop, and a model SPD-10Avp UV detector (Shimadzu) operating at 380 nm. The HPLC column was a Cholester column (Nacalai tesque, Kyoto), 150 x 3.0 mm i.d., containing 5 |m particles. Quantification of peaks was performed using a Chro-matopac Model C-R8A integrator (Shimadzu). The
mobile phase was prepared by the addition of acetoni-trile (600 mL) to 400 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 0.43 mL/min.
Preparation of standard solutions. Ultrapure water was from a Milli-Q water purification system (Simplicity® UV, Millipore Corporation, Bedford, MA, USA). Standard samples of the three PPs were dissolved in methanol to obtain concentrations of 1 mg/mL. Standard mixtures (each 0, 0.0833, 0.167, 0.333, 0.833, 1.67 and 3.33 |g/mL) were prepared by dilution as required with Milli-Q water.
Derivatization. Borate buffer (0.1 M) was adjusted to various pH values by the addition of NaOH. Borate buffer (100 |L) was added to mixtures of diluted standard samples (100 |L) and IS solution (100 |L, 1 |g/mL). Then, NBD-F solution in acetonitrile (2 mg/mL, 100 |L) was added. The mixture was vor-texed and allowed to react at room temperature, then an aliquot (50 |L, absolute amount of 0 to 41.6 ng each) was taken and injected into the HPLC system.
Application to grapefruit juice samples. A mixture of grapefruit juice (2.0 mL), a standard mixture of analytes (100 |L, 0, 0.0833, 0.167, 0.333, 0.833, 1.67 and 3.33 |g/mL each), HCl (100 |L, 0.1 M), and IS (100 |L, 1 |g/mL) was extracted with «-pentane (4 mL, twice). The organic layers were combined and evaporated. After the addition of water (200 |L) to the residue, derivatization was performed in the same
ce M <D
10 15 20 Time, min
7.5 8.0 8.5 9.0 9.5 pH of borate buffer
Fig. 2. Time courses of formation of NBD-F derivatives of OPP, MPP and PPP. Standard samples (each 3.33 ^g/mL) were reacted with NBD-F in borate buffer at pH 8.0 at room temperature. (O) - OPP, (A) - MPP, (□) - PPP.
Fig. 3. pH Dependency of the formation of NBD-F derivatives of OPP, MPP and PPP. Standard samples (each 3.33 ^g/mL, 41.6 ng/injection) were reacted with NBD-F for 5 min in various borate buffers at room temperature. (O) - OPP, (A) - MPP, (□) - PPP.
manner as described above and the products were analyzed.
Evaluation of relative recovery. Relative recovery was expressed as the ratio of the slope of the calibration curve prepared from a grapefruit juice sample spiked with the standard sample to that of the standard calibration curve prepared as described above. Relative recovery data were used to assess the accuracy of the method.
RESULTS AND DISCUSSION
Reaction time courses of the three PPs with NBD-F.
For the time course study, the reaction time was set at 1, 2, 5, 7, 10 and 20 min (Fig. 2). PP (100 |L, each 3.33 |g/mL), borate buffer (100 |L, pH 8.0), and NBD-F (100 |L, 2 mg/mL) were added to IS solution (100 |L), and each solution was left to stand for the appropriate time. Derivatives of the three PPs and IS reached a maximum at 5 min, while the peak area ratios of the three PPs almost reached a plateau at 1 min (Fig. 2). Thus, the derivatization time of 5 min was selected.
pH Dependency of derivatization of the three PPs with NBD-F. pH Dependency (pH 7.0 to 10.0) was examined at the derivatization time of 5 min (Fig. 3). The peak area ratio of derivatives showed little variation in the range of pH 7.0 to 9.0. However, the peak area ratios of the three derivatives at pH 9.5 and 10.0 were markedly reduced. Therefore, pH 8.0 was selected for the derivatization buffer.
Chromatograms. Figure 4 shows typical chromato-grams obtained from blank (A), a standard mixture (each 20.8 ng) (B), and a grapefruit juice sample (C) after derivatization with NBD-F. The retention times of NBD—OPP, NBD—MPP, NBD—PPP, and NBD— IS were 14.1, 17.4, 19.1 and 23.2 min, respectively. The resolution value between the NBD—MPP and NBD—PPP peaks was 1.32 using the Cholester col-
umn. Our preliminary tests showed that the value using the Cholester column was higher than those obtained with C18-MS-II (0.91) and C22-AR-II (0.99) columns (Nakalai t
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