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A highly sensitive fluorescence quenching method for perphenazine detection based on its catalysis of K2S2O8 oxidizing rhodamine 6G
Journal of Luminescence 156 (2014) 124–129
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A highly sensitive fluorescence quenching method for perphenazine detection based on its catalysis of K2S2O8 oxidizing rhodamine 6G Lihong Zhang a, Qitong Huang a, Changqing Lin a, Xiaofeng Lin b, Yiqun Huang c, Jiaming Liu b,n, Xudong Ma c,nn a
Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou, 363000, P.R. China College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000, P.R. China c Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 25 April 2014 Received in revised form 15 July 2014 Accepted 1 August 2014 Available online 11 August 2014
In this paper, the fluorescence spectra of Rhod 6G (rhodamine 6G)–K2S2O8–PPH (perphenazine) were studied. We found that Rhod 6G existed in the form of Rhod 6G þ under the conditions of 60 1C, 10 min and pH 5.42, and Rhod 6G þ can emit strong and stable fluorescence. Further study showed that when PPH and Rhod 6G þ coexisted, the ester exchange reaction carried out between –OH of PPH and –COOC2H5 of Rhod 6G þ to produced Rhod 6G þ –PPH compound. More interestingly, K2S2O8 could oxidize Rhod 6G þ and quench its RTP signal, while PPH was oxidized to red compound PPH0 by K2S2O8, and Rhod 6G þ –PPH0 and PPH were produced in the ester exchange reaction between the –OH of PPH0 and the –COOC2H5 of Rhod 6G þ –PPH. In the above process, PPH catalyzed K2S2O8 oxidizing Rhod 6G, which caused the fluorescence signal of the system to quench sharply. Hence, a catalytic fluorescence quenching method for the determination of residual PPH has been developed based on the its catalyzing K2S2O8 oxidize rhodamine 6G. This sensitive, accurate, simple and selective fluorescence quenching method was used to determine residual PPH in biological samples with the results consisting with those obtained by high performance liquid chromatography (HPLC), showing good accuracy. The structures of Rhod 6G þ , PPH and Rhod 6G þ –PPH were characterized by infrared spectra. The reaction mechanism of the determination of PPH was also discussed. & 2014 Elsevier B.V. All rights reserved.
Keywords: Perphenazine Ester exchange reaction Catalytic fluorescence quenching method
1. Introduction Up to now, more and more antipsychotic drugs were used in psychiatry [1–3]. As one of the antipsychotic drugs, the PPH has been widely used for many years due to its neuroleptic and antidepressive actions. It is effective in controlling the positive symptoms of schizophrenia (delusions, hallucinations and agitation) [4,5]. However, given that the use of too much PPH often leads to the onset of several side effects such as extrapyramidal effects, acute dystonias, neuroleptic malignant syndrome, angiocardiopathy and so on [6,7]. The therapeutic range and toxic threshold of PPH in serum were reported 4–30 and 50–100 ng mL 1 respectively [4]. Therefore, the determination of PPH becomes increasingly important in clinical medical practice. Till now, several analytical techniques have been applied for the determination of residual PPH, such as flow injection n
Corresponding author. Tel.: þ86 596 2591352. Corresponding author. Tel.: þ 86 596 2082021. E-mail addresses: [email protected], [email protected] (J. Liu), [email protected] (X. Ma). nn
http://dx.doi.org/10.1016/j.jlumin.2014.08.007 0022-2313/& 2014 Elsevier B.V. All rights reserved.
chemiluminescence method [4], coupled capillary electrophoresis [5], solid-phase extraction and liquid chromatography [7], electrochemiluminescent method [8], fluorophotometric method (the limit of detection (LOD): 0.020 mg mL 1) [9], molecularly imprinted polymer method [10], high performance liquid chromatography (HPLC) [11], HPLC–MS/MS (LOD: 0.010 ng mL 1) [12], enzyme-linked immunosorbent assay [13], LC/MS/MS [14] and so on. These methods have certain defects, such as enzyme-linked immunosorbent assay is easily affected by many factors such as low sensitivity and poor repeatability; electrochemical instrument is simple, but has a big error; the procedures used in HPLC, HPLC– MS/MS and LC/MS/MS are time-consuming, and their operation is more complicated. Besides, LOD of these methods only reach the ng level. Obviously, searching for a new method with high sensitivity and accuracy for the determination of PPH has important academic research value and practical prospect. In this paper, a highly sensitive catalytic fluorescence quenching method for the PPH detection has been developed based on the combination of catalytic reaction and fluorescence method due to the catalytic reaction could further improve the selectivity and sensitivity of fluorescence method. The sensitivity of this fluorescence quenching
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129
method (LOD: 3.3 10 14 g mL 1) was 6.1 105 and 3.0 102 times higher than that (LOD: 2.0 10 8 g mL 1, 1.0 10 11 g mL 1 ) of Refs. [9,12], and to our knowledge, a fluorescence quenching method for the PPH detection based on the catalyzing K2S2O8 oxidizing Rhod 6G has not been reported yet. This sensitive, accurate, selective and repeatable fluorescence quenching method has been applied successfully to determine trace PPH in human urine and human serum samples; showing better application prospects. The study not only developed a new method for the PPH detection, but also broadens the applications of catalytic reaction in some new fields, which promoted the progress in study of trace drugs analysis.
2.3. Preparation of analytical sample The patients with mental illness take orally 10 mg PPH tablet every day, and 5 mL urine and serum were taken after one week. They were centrifuged for 10 min, then the upper solution was diluted to 50 mL with NaH2PO4–H3 PO4 (pH 2.7). Then, 1.00 mL above test solution was diluted to 100 mL, and 1.00 mL solution was diluted to 109 mL for use. To a 10-mL conical centrifuge tube, 1.0 mL urine, 40 μL of 2 mgL 1 amitriptyline-methanol internal standard solution were added, and rapidly oscillated for a few seconds. The mixture was added 4.0 mL acetate-dichloromethane (80:20), rotating oscillated for 2 min and centrifuged at 4000 r min 1 for 4 min. The supernatant was absorbed into 5-mL conical centrifuge tube, and dried at 50 1C under vacuum condition. The mixture was rapidly oscillated for seconds to dissolve residue when adding 100 μL of methanol. And 20 μL of the test solution was introduced for HPLC analysis. 1.0 mL serum was treated for HPLC analysis in the same way mentioned above. The instrument's main parameters of HPLC are as follows: analysis column: ODS C18 (6 μm, 260 mm 4.8 mm); pre-column: ODS C18 (6 μm, 50 mm 4.8 mm); mobile phase: 0.03 mol L 1 ammonium acetate-methanol (23:77); flowing speed: 1 mL min 1; temperature: 40 1C; detect wavelength: 254 nm; AUFS: 0.01. The retention time of PPH and internal substance were 5.67 min and 5.12 min, respectively.
2. Experimental 2.1. Apparatus and reagents Fluorescence measurements were carried out on a PerkinElmer LS-55 luminescence spectrophotometer (PerkinElmer Corporation, U.S.A.). The instruments' main parameters are as follows: Ex. Slit: 10 nm; Em. Slit: 2.5 nm; scan speed: 1500 nm min 1. High performance liquid chromatograph (Agilent 1100) including G1315B diode array detector (DAD) and G1313A autosampler (Agilent Company), Nicolet-360X infrared spectrometer (Nicolet Company), a pHS-3B acidometer (Shanghai Medical Laser Instrument Plant) and an AE240 electronic analytical balance (Mettler-Toledo Instruments Company) were used in the experiment. PPH (Beijing Institute for the Control of Pharmaceutical and Biological Products, contents 499%) working solution: PPH of primary standard reagent was weighed and diluted to 0.10 mg mL 1 with 0.10 mol L 1 HCl. It was diluted to 100.00, 10.00 and 1.00 pg mL 1 with water as working solution before use. 1.0 10 4 mol L 1 reduced form of Rhod 6G (Sigma Company, contents498%) solution, 2 mg L 1 amitriptyline-methanol (Sigma Company, contents498%) as an internal standard solution, acetate-dichloromethane (80:20, Sigma Company), 0.10 mol L 1 K2S2O8 (Sigma Company, contents4 99%), KBr (Sigma Company, contents 499.99%) and 1.0 mol L 1 I were also used in the experiment. All the reagents were analytical grade except that PPH was primary standard and KBr was spectral pure. The water was prepared by thrice quartz sub-boiling distillation.
3. Results and discussion 3.1. Mechanism of PPH detection by catalytic fluorescence quenching method When PPH and Rhod 6G þ coexisted, the ester exchange reaction carried out between –OH of PPH and –COOC2H5 of Rhod 6G þ produced Rhod 6G þ –PPH compound (Scheme 1) [15]. In order to prove the probability of ester exchange reaction occurred between PPH and Rhod 6G þ , the infrared spectra of Rhod 6G þ , PPH and Rhod 6G þ –PPH were scanned by Nicolet-360 infrared spectrometer (KBr pellet) ranging from 200 cm 1 to 4000 cm 1. Results show that the characteristic absorption peak of ν–OH in Rhod 6G þ –PPH disappeared and the characteristic absorption peaks of νCQO and νC–O–C red-shifted while the characteristic absorption peak of other groups had little change, which proved the possibility of the occurrence for the ester exchange reaction between the –OH in PPH and the –COOC2H5 in Rhod 6G þ . In the Rhod 6G þ –K2S2O8 system, the fluorescence signal of Rhod 6G þ quenched (ΔF¼14.5, Fig. 1, curve 2.20 ). It might be explained that Rhod 6G þ was oxidized to Rhod 6G þ 0 (Scheme 2, [16]) and the π-electron density (δ) of carbon atom in the conjugated system decreased, which enhanced the non-radiation energy loss of excited Rhod 6G þ molecule of the triplet state [17], and caused the fluorescence signal of Rhod 6G þ to quench in the system.
2.2. Experimental method To a 25-mL colorimetric tube, proper amount of PPH working solution, 2.00 mL Rhod 6G and 2.00 mL K2S2O8 were added, diluted to 25 mL with water and mixed homogeneously. The mixture was kept at 60 1C for 10 min, cooled with flowing water for 5 min. At the same time, a blank test was also conducted. The fluorescence intensity of test solution (F) and reagent blank (F0) max were directly measured at 527/556 nm (λmax ex /λem ) in 1 cm fluorescence pool. Then, the change of the fluorescence intensity (ΔF ¼F0–F) was calculated.
S HNH5C2 H3C
N
+ NHC2H5 +
O
CH3 COOC2H5 Rhod.6G+
N Cl
N +
PPH
H+
125
OH HNH5C2 H3C
O
+ NHC2H5 CH3 COO
Cl N
Rhod.6G+-PPH
Scheme 1. Ester exchange reaction between PPH and Rhod 6G þ .
N
N
S
126
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129 800.0 700
1 2 3 5
1' 2' 3' 5'
4
4'
556.36,755.43
526.79,753.88
600 500
F
400
527.60,739.80
556.75,740.89
526.75,734.99
556.56,735.47
526.79,709.94
557.12,710.94
300 526.94,599.97
556.09,606.11
200 100 0.6 370.0
400
450
500
550
600
650.5
λ /nm Fig. 1. Fluorescence spectra of the Rhod 6G–K2S2O8–PPH system (curves 1–5 are the excitation spectra, and curves 10 –50 are the emission spectra. 1.10 , 2.20 , 3.30 , 4.40 and 5.50 are the number of the excitation spectra and the emission spectra.) 1.10 2.00 mL Rhod 6G þ 1.00 ml HCl 2.20 1.10 þ 2.00 mL K2S2O8 3.30 2.20 þ 2.50 pg PPH 4.40 2.20 þ 700.00 pg PPH 5.50 1.10 þ 700.00 pg PPH.
+ NHC2 H5
O
HNH5C2
CH3 + K S O + H+ 2 2 8 COOC2H5
H3C
+ NH2C2H5
O
HNH5C2
CH3 + K2SO4 + H O 2 COOC2H5
H3C
Rhod.6G+'
Rhod.6G+
Scheme 2. Oxidation-reduction reaction between K2S2O8 and Rhod 6G.
+ NHC2H5
O
HNH5C2
CH3 COO
H 3C
S
N
N
2
+
+ S 2O 8 + 2 H N
OH
O
S
Cl
H3C
+ OH
N
N
N
N
S
Rhod.6G+-PPH
Cl
PPH
HNH5C2
N
N
Cl
PPH'
O
+ NHC2H5 CH3 COO
Cl N
N
N
Rhod.6G+-PPH'
S
O
+
S
N
N
N
OH
Cl PPH
Scheme 3. Oxidation-reduction reaction between K2S2O8 and PPH, ester exchange reaction between PPH0 and Rhod 6G þ –PPH.
When PPH was added, PPH was oxidized to red compound PPH0 [18,19] in the presence of K2S2O8 while S2O28 was reduced to SO24 [20]. The property of PPH0 was similar to that of PPH, Rhod 6G þ –PPH0 and PPH were produced in the ester exchange reaction between the –OH of PPH0 [15] and the –COOC2H5 of Rhod 6G þ – PPH (Scheme 3). In the above process, PPH catalyzed K2S2O8 oxidizing Rhod 6G, which caused the fluorescence signal of the system to quench sharply. The content of PPH was liner to the ΔF of the system. Thus, trace PPH could be determined by the catalytic fluorescence quenching method.
3.2. Fluorescence spectra According to the experimental method, the fluorescence spectra of the Rhod 6G-K2S2O8–PPH system were scanned (Fig. 1). Under the conditions of 60 1C, 10 min and pH 5.42, Rhod 6G existed in the form of Rhod 6G þ [21]. In the research process, we found that Rhod max 6G þ can emit strong and stable fluorescence (λmax ex /λem ¼526.8/ 0 556.4 nm, F¼ 755.4, Fig. 1, curve 1.1 ), and K2S2O8 could oxidize max Rhod 6G and quench its fluorescence signal (λmax ex /λem ¼57.6/ 556.8 nm, F ¼ 740.9, Fig. 1, curve 2.20 ), while PPH catalyzed K2S2O8 oxidizing Rhod 6G, which caused the fluorescence signal
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129 max of the system to quench sharply (λmax ex /λem ¼526.9/556.1 nm, F¼606.1, Fig. 1, curve 4.40 ), and the △F (134.8) of the catalytic system was three times larger than that of the non-catalytic system (44.5, Fig. 1, curve 5.50 ), showing the signal amplification of catalytic reaction. Thus, the 527/556 nm was selected as the working wavelength to determine PPH.
127
2.64, 2.81, 3.04, 3.32, 3.82, 4.19, 5.42, 6.35, 7.44, 8.66, 9.42 and10.20), and standing time (5, 10, 15, 20, and 30 min) on the ΔF of the system were investigated in a univariate approach (Table 1 and Fig. 2). Though the ΔF of the system was high when FITC(A), Rhod 6G (B), eosin Y(C), calcein(D), orange yellow G (E) were chosen as luminescence substances they were still lower than that of Rhod 6G (Fig. 2a). Further investigations found that with the increasing of the concentration and dosage of Rhod 6G, the ΔF of the system enhanced gradually (Table 1). When the concentration and dosage of Rhod 6G were 2.00 mL of 1.0 10 4 mol L 1, the ΔF of the system reached the maximum, the main reason might be that the –COOC2H5 group of Rhod 6G þ and –OH group of PPH carried out the ester exchange reaction and produced Rhod 6G þ –PPH.
3.3. Optimum conditions for PPH detection For the system containing 4.00 pg mL 1 PPH, the effects of the dosage and concentrations of reagents, luminescence substances (FITC(A), Rhod 6G (B), eosin Y(C), calcein(D), orange yellow G (E), etc.), oxidants (H2O2(A), K2S2O8(B), KIO3(C), KClO3(D), NaIO4(E), etc.), temperature and time of reaction, reaction acidity (pH was
Table 1 Optimization of the concentration and dosage of reagents. Reagents Rhod 6G (mol L (mL) K2S2O8 (%) (mL)
Concentrations and dosage 1
)
–3
4
5
1.0 10 ,1.0 10 ,1.0 10 , 1.0 10 0.50, 1.00, 1.50, 2.00, 2.50, 3.00 0.01, 0.05, 0.1, 0.2, 0.3 0.50, 1.00, 1.50, 2.00, 2.50, 3.00
6
, 1.0 10
7
20
ΔF
0
A
B
C
D
1.0 10 4 2.00 0.1 2.00
20
0
E
A
B
20
ΔF
E
20
20
40
60
80
0
100
0
10
20
30
t/min
O
T/ C
30
30
20
ΔF
10 0
D
10
10
ΔF
C
30
30
0
9.4,31.5, 17.6, 10.7, 5.2 5.7, 11.3, 21.3, 28.9, 17.0, 10.3 9.4, 17.6, 30.5, 10.7, 5.2 5.8, 10.7, 21.2, 31.1, 14.0, 9.9
10
10
ΔF
Optimal
30
30 ΔF
The ΔF in Rhod 6G–K2S2O8– PPH system
20 10
2
3
4
5
6 7 pH
8
9 10
0
0
10
20 30 t/min
40
Fig. 2. (a) The effects of luminescence substances on the ΔF of the system: FITC (A), Rhod 6G (B), eosin Y (C), calcein (D), and orange yellow G (E); (b) the effects of oxidants on the ΔF of the system: H2O2 (A), K2S2O8 (B), KIO3 (C), KClO3 and (D), NaIO4 (E); (c) the effects of reaction temperature on the ΔF of the system; (d) the effects of reaction time on the ΔF of the system; (e) the effects of reaction acidity on the ΔF of the system; (f) the effects of standing time on the ΔF of the system.
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L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129
Compared with H2O2(A), K2S2O8(B), KIO3(C), KClO3(D) and NaIO4(E), K2S2O8 had the largest effect on the ΔF of the system (Fig. 2b), the main reason might be the strongest oxidity of K2S2O8 With the increase in the concentration and dosage of K2S2O8, the ΔF of the system enhanced gradually (Table 1). When the concentration and dosage of K2S2O8 were 2.00 mL of 1.0%, the ΔF of the system reached the maximum. As the reaction time and temperature increased, the ΔF of the system gradually enhanced (Fig. 2c and d), which might result from the gradual increase in the catalytic ability of PPH. When the reaction temperature and time were 60 1C and 10 min, respectively, the ΔF of the system reached the maximum, which might be that the catalytic ability of PPH reached the peak. Henceforth, with the continuous increase of the reaction temperature and time, the ΔF of the system decreased due to the decreasing of catalytic ability of PPH gradually. The ΔF of the system reached the maximum and remained stable when the pH value of the reaction solution was in the range of 3.82–6.35 (Fig. 2e) for the reason that the ester exchange reaction between the –COOC2H5 group of Rhod 6G þ and –OH group of PPH and the oxidation reaction between K2S2O8 and PPH completed. The ΔF of the system almost stayed invariable within 15–30 min after being cooled for 5 min with flowing water, while the ΔF of the system decreased sharp when the time was over 30 min (Fig. 2f). Obviously, the reproducibility of the system was the best within 15–30 min under the optimal conditions. Therefore, the ΔF of the system reached the maximum when the system containing 2.00 mL of 1.0 10 4 mol L 1 Rhod 6G, 2.00 mL of 0.10% (m/V) K2S2O8 carried out in the pH 5.42 at 60 1C for 10 min. Under the above optimal conditions, the ΔF of the system almost stayed invariable and had good repeatability within 15–30 min after being cooled for 5 min by flowing water in the system. 3.4. Linear range, working curve, sensitivity and precision Under the optimum conditions, the ΔF of the system was linear with the concentration of PPH in the range of 0.080–28.00 ( 10 12) g mL 1 (when the content of PPH were 0.080, 0.40, 2.0, 4.0, 12.0 and 28.0 pg mL 1, the F (ΔF) were 735.5(5.4), 729.0(11.9), 724.9(16.0), 710.9(30.0), 677.5(63.4), and 606.1(134.8) and the corresponding relative standard deviations (RSD, %, n ¼6) were 1.2, 1.9, 2.2, 2.8, 3.3, 3.8, and 4.1, respectively, Fig. 3), the regression equations was ΔF¼8.424þ4.535CPPH (pg mL 1), correlation coefficients (r) was 0.9988 (n ¼6). The systems containing 0.080 and 28.0( 10 12) g mL 1 PPH were measured for six times, their RSD (%) were 1.2 % and 4.1%, respectively, showing good precision. LOD was 3.3 10 14 g mL 1 (calculated by 3 Sb/k, here Sb represents the standard deviation of 11 blank measurements and the value was 0.050, k is the slope of standard curve), showing the higher sensitivity of the fluorescence quenching method. This was attributed to the catalyzing effect of PPH on the reaction of K2S2O8 oxidizing Rhod 6G greatly improves ΔF value. 3.5. Selectivity of the fluorescence quenching method To evaluate the selectivity of fluorescence quenching method towards the sample containing 4.00 pg mL 1 PPH, a series of coexistence ions (materials) in human urine were investigated under the optimum measurement conditions. When the relative error (Er) exceeded 75%, each ion is considered as interfering agent. Results show that the allowable concentrations (ng mL 1) of most ions are high: 10.50 for urea, 5.90 for soluble starch, 4.00 for uric acid and oxalate, 3.00 for sodium citrate and chlorpromazine hydrochloride, 2.50 for sodium carboxymethyl cellulose and
Fig. 3. Representative fluorescence emission spectra of Rhod 6G þ in the presence of increasing PPH concentrations (0.080–28.0 pg mL 1) in the HCl buffer at pH 5.42. Inset: fluorescence change (ΔF) titrated with PPH under the optimum conditions. (When the contents of PPH were 0.0, 0.080, 0.40, 2.0, 4.0, 12.0, 28.0, 32.0, 36.0 and 40.0 pg mL 1, the F (ΔF) were 740.9(0), 735.5(5.4), 729.0(11.9), 724.9 (16.0), 710.9(30.0), 677.5(63.4), 606.1(134.8), 590.5(150.4), 580.0(160.9) and 574.3 (166.6), and the corresponding RSD (%, n¼ 6) were 1.2, 1.9, 2.2, 2.8, 3.3, 3.8,4.1, 4.3, 4.6, and 4.8, respectively. The regression equations was ΔF¼ 10.35þ 4.183CPPH (pg mL 1), r was 0.9966, n ¼6).
fluoxetine, 2.00 for phenobarbital, 1.00 for sucrose, 0.90 for clonazepam, 0.80 for glucose, 0.70 for fructose, 0.60 for triazolam and amitriptyline, 3.00 for Na þ , 1.60 for NO3 , 1.50 for K þ , 1.40 for F , 1.00 for Ca2 þ ,0.90 for Sr2 þ and A l3 þ ,0.80 for CO23 and Co2 þ , 0.70 for Fe3 þ , 0.60 for Zn2 þ and 0.50 for Cu2 þ , showing that this catalytic fluorescence quenching method has excellent selectivity. 3.6. Analysis of samples The content of PPH in 1.00 mL test solution was examined by experimental method and HPLC [11], and additional recovery experiment was also conducted. The results are listed in Table 2. The significant difference analysis between the proposed method and HPLC for the determination of PPH is shown in Table 3. Seen from Table 2, this sensitive and accurate method could be used to determine the content of PPH in biological samples with the recovery of 97–102% and RSD of 1.8–4.3%. The results agreed with those obtained with the HPLC. As shown in the Table 3, the F was 1.6, 1.3 and 1.5 (human urine), 2.2, 2.1 and 1.8 (human serum), respectively, indicating that there was no significant differences between S1 and S2, and the corresponding t was 1.2, 1.6 and 1.5 (human urine),1.2, 1.6 and 1.7 (human serum), respectively, indicating that there was also no significant differences between X 1 and X 2 . Obviously, the proposed method was sensitive, accurate and reliable to the detection of PPH in the biological samples. According to the PPH content of A, B and C serum is in 50–100 (toxic threshold) ng mL 1 [4], we could forecast that A, B and C might be angiocardiopathy patients [6,7]. Therefore, this method could be applied to determine the PPH content in serum and to predict the human diseases.
4. Conclusion In this paper, the Rhod 6G þ –PPH compound formed in the ester exchange reaction between –OH of PPH and –COOC2H5 of Rhod 6G þ . PPH was oxidized to red compound (PPH0 ) in the presence of K2S2O8. Interestingly, fluorescence signal of Rhod 6G þ was quenched because the –OH of PPH0 reacted with –COOC2H5 of Rhod 6G þ –PPH to form Rhod 6G þ –PPH0 and PPH, which decreased the
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129
129
Table 2 Analysis results of PPH in samples. This method (n¼ 6) Sample Human Human Human Human Human Human
urine A urine B urine C serum A serum B serum C
HPLC (n¼ 5) Found (μg mL 1)
RSD (%)
Added (μg mL 1)
Add found(μg mL 1)
Obtaine (μg mL 1)
Recovery (%)
HPLC (μg mL 1)
Er (%)
4.52 4.90 4.60 4.78 4.57 4.81
3.2 3.4 2.6 4.3 1.8 2.3
0.050 0.050 0.050 0.050 0.050 0.050
4.57 4.95 4.65 4.84 4.62 4.86
0.0485 0.0510 0.0495 0.0505 0.0492 0.0496
97.0 102.0 99.0 101.0 98.5 99.2
4.69 4.81 4.72 4.71 4.78 4.70
–3.6 þ1.9 –2.5 þ1.5 –4.4 þ2.3
Table 3 Analysis of the significant differences for determination results (P ¼90%, f¼ n1 þ n2 2¼ 9, F0.90, 9 ¼6.3, t0.90, 9 ¼ 1.8). Sample
Human Human Human Human Human Human
This method (μg mL 1, n¼ 6)
urine A urine B urine C serum A serum B serum C
HPLC (μg mL 1, n¼ 5)
Statistical analysis
X1
S1
X2
S2
F
S
t
4.52 4.90 4.60 4.78 4.57 4.81
0.0322 0.0544 0.0603 0.0645 0.0597 0.0636
4.55 4.85 4.55 4.82 4.62 4.75
0.0510 0.0485 0.0495 0.0430 0.0412 0.0474
1.6 1.3 1.5 2.2 2.1 1.8
0.042 0.052 0.056 0.056 0.052 0.057
1.2 1.6 1.5 1.2 1.6 1.7
π-electron density (δ) of carbon atom in a Rhod 6G þ –PPH0 conjugated system and enhanced the non-radiation energy loss of the excited Rhod 6G þ of the triplet state. Thus, a new catalytic fluorescence quenching method for the determination of PPH was established based on the linear relativity between the ΔF of the system and the content of PPH. The simple and sensitive method was suitable for the analysis of residual PPH in biological samples, and has provided a new way for the clinical detection. Acknowledgments This work was supported by Fujian Province Natural Science Foundation (Grant No. 2012H6026), Fujian Province Education Committee (JA11311) and Fujian provincial bureau of quality and technical supervison (FJQI2011006). At the same time, we are very grateful to precious advices raised by the anonymous reviewers.
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
A highly sensitive fluorescence quenching method for perphenazine detection based on its catalysis of K2S2O8 oxidizing rhodamine 6G Lihong Zhang a, Qitong Huang a, Changqing Lin a, Xiaofeng Lin b, Yiqun Huang c, Jiaming Liu b,n, Xudong Ma c,nn a
Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou, 363000, P.R. China College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000, P.R. China c Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou 363000, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 25 April 2014 Received in revised form 15 July 2014 Accepted 1 August 2014 Available online 11 August 2014
In this paper, the fluorescence spectra of Rhod 6G (rhodamine 6G)–K2S2O8–PPH (perphenazine) were studied. We found that Rhod 6G existed in the form of Rhod 6G þ under the conditions of 60 1C, 10 min and pH 5.42, and Rhod 6G þ can emit strong and stable fluorescence. Further study showed that when PPH and Rhod 6G þ coexisted, the ester exchange reaction carried out between –OH of PPH and –COOC2H5 of Rhod 6G þ to produced Rhod 6G þ –PPH compound. More interestingly, K2S2O8 could oxidize Rhod 6G þ and quench its RTP signal, while PPH was oxidized to red compound PPH0 by K2S2O8, and Rhod 6G þ –PPH0 and PPH were produced in the ester exchange reaction between the –OH of PPH0 and the –COOC2H5 of Rhod 6G þ –PPH. In the above process, PPH catalyzed K2S2O8 oxidizing Rhod 6G, which caused the fluorescence signal of the system to quench sharply. Hence, a catalytic fluorescence quenching method for the determination of residual PPH has been developed based on the its catalyzing K2S2O8 oxidize rhodamine 6G. This sensitive, accurate, simple and selective fluorescence quenching method was used to determine residual PPH in biological samples with the results consisting with those obtained by high performance liquid chromatography (HPLC), showing good accuracy. The structures of Rhod 6G þ , PPH and Rhod 6G þ –PPH were characterized by infrared spectra. The reaction mechanism of the determination of PPH was also discussed. & 2014 Elsevier B.V. All rights reserved.
Keywords: Perphenazine Ester exchange reaction Catalytic fluorescence quenching method
1. Introduction Up to now, more and more antipsychotic drugs were used in psychiatry [1–3]. As one of the antipsychotic drugs, the PPH has been widely used for many years due to its neuroleptic and antidepressive actions. It is effective in controlling the positive symptoms of schizophrenia (delusions, hallucinations and agitation) [4,5]. However, given that the use of too much PPH often leads to the onset of several side effects such as extrapyramidal effects, acute dystonias, neuroleptic malignant syndrome, angiocardiopathy and so on [6,7]. The therapeutic range and toxic threshold of PPH in serum were reported 4–30 and 50–100 ng mL 1 respectively [4]. Therefore, the determination of PPH becomes increasingly important in clinical medical practice. Till now, several analytical techniques have been applied for the determination of residual PPH, such as flow injection n
Corresponding author. Tel.: þ86 596 2591352. Corresponding author. Tel.: þ 86 596 2082021. E-mail addresses: [email protected], [email protected] (J. Liu), [email protected] (X. Ma). nn
http://dx.doi.org/10.1016/j.jlumin.2014.08.007 0022-2313/& 2014 Elsevier B.V. All rights reserved.
chemiluminescence method [4], coupled capillary electrophoresis [5], solid-phase extraction and liquid chromatography [7], electrochemiluminescent method [8], fluorophotometric method (the limit of detection (LOD): 0.020 mg mL 1) [9], molecularly imprinted polymer method [10], high performance liquid chromatography (HPLC) [11], HPLC–MS/MS (LOD: 0.010 ng mL 1) [12], enzyme-linked immunosorbent assay [13], LC/MS/MS [14] and so on. These methods have certain defects, such as enzyme-linked immunosorbent assay is easily affected by many factors such as low sensitivity and poor repeatability; electrochemical instrument is simple, but has a big error; the procedures used in HPLC, HPLC– MS/MS and LC/MS/MS are time-consuming, and their operation is more complicated. Besides, LOD of these methods only reach the ng level. Obviously, searching for a new method with high sensitivity and accuracy for the determination of PPH has important academic research value and practical prospect. In this paper, a highly sensitive catalytic fluorescence quenching method for the PPH detection has been developed based on the combination of catalytic reaction and fluorescence method due to the catalytic reaction could further improve the selectivity and sensitivity of fluorescence method. The sensitivity of this fluorescence quenching
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129
method (LOD: 3.3 10 14 g mL 1) was 6.1 105 and 3.0 102 times higher than that (LOD: 2.0 10 8 g mL 1, 1.0 10 11 g mL 1 ) of Refs. [9,12], and to our knowledge, a fluorescence quenching method for the PPH detection based on the catalyzing K2S2O8 oxidizing Rhod 6G has not been reported yet. This sensitive, accurate, selective and repeatable fluorescence quenching method has been applied successfully to determine trace PPH in human urine and human serum samples; showing better application prospects. The study not only developed a new method for the PPH detection, but also broadens the applications of catalytic reaction in some new fields, which promoted the progress in study of trace drugs analysis.
2.3. Preparation of analytical sample The patients with mental illness take orally 10 mg PPH tablet every day, and 5 mL urine and serum were taken after one week. They were centrifuged for 10 min, then the upper solution was diluted to 50 mL with NaH2PO4–H3 PO4 (pH 2.7). Then, 1.00 mL above test solution was diluted to 100 mL, and 1.00 mL solution was diluted to 109 mL for use. To a 10-mL conical centrifuge tube, 1.0 mL urine, 40 μL of 2 mgL 1 amitriptyline-methanol internal standard solution were added, and rapidly oscillated for a few seconds. The mixture was added 4.0 mL acetate-dichloromethane (80:20), rotating oscillated for 2 min and centrifuged at 4000 r min 1 for 4 min. The supernatant was absorbed into 5-mL conical centrifuge tube, and dried at 50 1C under vacuum condition. The mixture was rapidly oscillated for seconds to dissolve residue when adding 100 μL of methanol. And 20 μL of the test solution was introduced for HPLC analysis. 1.0 mL serum was treated for HPLC analysis in the same way mentioned above. The instrument's main parameters of HPLC are as follows: analysis column: ODS C18 (6 μm, 260 mm 4.8 mm); pre-column: ODS C18 (6 μm, 50 mm 4.8 mm); mobile phase: 0.03 mol L 1 ammonium acetate-methanol (23:77); flowing speed: 1 mL min 1; temperature: 40 1C; detect wavelength: 254 nm; AUFS: 0.01. The retention time of PPH and internal substance were 5.67 min and 5.12 min, respectively.
2. Experimental 2.1. Apparatus and reagents Fluorescence measurements were carried out on a PerkinElmer LS-55 luminescence spectrophotometer (PerkinElmer Corporation, U.S.A.). The instruments' main parameters are as follows: Ex. Slit: 10 nm; Em. Slit: 2.5 nm; scan speed: 1500 nm min 1. High performance liquid chromatograph (Agilent 1100) including G1315B diode array detector (DAD) and G1313A autosampler (Agilent Company), Nicolet-360X infrared spectrometer (Nicolet Company), a pHS-3B acidometer (Shanghai Medical Laser Instrument Plant) and an AE240 electronic analytical balance (Mettler-Toledo Instruments Company) were used in the experiment. PPH (Beijing Institute for the Control of Pharmaceutical and Biological Products, contents 499%) working solution: PPH of primary standard reagent was weighed and diluted to 0.10 mg mL 1 with 0.10 mol L 1 HCl. It was diluted to 100.00, 10.00 and 1.00 pg mL 1 with water as working solution before use. 1.0 10 4 mol L 1 reduced form of Rhod 6G (Sigma Company, contents498%) solution, 2 mg L 1 amitriptyline-methanol (Sigma Company, contents498%) as an internal standard solution, acetate-dichloromethane (80:20, Sigma Company), 0.10 mol L 1 K2S2O8 (Sigma Company, contents4 99%), KBr (Sigma Company, contents 499.99%) and 1.0 mol L 1 I were also used in the experiment. All the reagents were analytical grade except that PPH was primary standard and KBr was spectral pure. The water was prepared by thrice quartz sub-boiling distillation.
3. Results and discussion 3.1. Mechanism of PPH detection by catalytic fluorescence quenching method When PPH and Rhod 6G þ coexisted, the ester exchange reaction carried out between –OH of PPH and –COOC2H5 of Rhod 6G þ produced Rhod 6G þ –PPH compound (Scheme 1) [15]. In order to prove the probability of ester exchange reaction occurred between PPH and Rhod 6G þ , the infrared spectra of Rhod 6G þ , PPH and Rhod 6G þ –PPH were scanned by Nicolet-360 infrared spectrometer (KBr pellet) ranging from 200 cm 1 to 4000 cm 1. Results show that the characteristic absorption peak of ν–OH in Rhod 6G þ –PPH disappeared and the characteristic absorption peaks of νCQO and νC–O–C red-shifted while the characteristic absorption peak of other groups had little change, which proved the possibility of the occurrence for the ester exchange reaction between the –OH in PPH and the –COOC2H5 in Rhod 6G þ . In the Rhod 6G þ –K2S2O8 system, the fluorescence signal of Rhod 6G þ quenched (ΔF¼14.5, Fig. 1, curve 2.20 ). It might be explained that Rhod 6G þ was oxidized to Rhod 6G þ 0 (Scheme 2, [16]) and the π-electron density (δ) of carbon atom in the conjugated system decreased, which enhanced the non-radiation energy loss of excited Rhod 6G þ molecule of the triplet state [17], and caused the fluorescence signal of Rhod 6G þ to quench in the system.
2.2. Experimental method To a 25-mL colorimetric tube, proper amount of PPH working solution, 2.00 mL Rhod 6G and 2.00 mL K2S2O8 were added, diluted to 25 mL with water and mixed homogeneously. The mixture was kept at 60 1C for 10 min, cooled with flowing water for 5 min. At the same time, a blank test was also conducted. The fluorescence intensity of test solution (F) and reagent blank (F0) max were directly measured at 527/556 nm (λmax ex /λem ) in 1 cm fluorescence pool. Then, the change of the fluorescence intensity (ΔF ¼F0–F) was calculated.
S HNH5C2 H3C
N
+ NHC2H5 +
O
CH3 COOC2H5 Rhod.6G+
N Cl
N +
PPH
H+
125
OH HNH5C2 H3C
O
+ NHC2H5 CH3 COO
Cl N
Rhod.6G+-PPH
Scheme 1. Ester exchange reaction between PPH and Rhod 6G þ .
N
N
S
126
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129 800.0 700
1 2 3 5
1' 2' 3' 5'
4
4'
556.36,755.43
526.79,753.88
600 500
F
400
527.60,739.80
556.75,740.89
526.75,734.99
556.56,735.47
526.79,709.94
557.12,710.94
300 526.94,599.97
556.09,606.11
200 100 0.6 370.0
400
450
500
550
600
650.5
λ /nm Fig. 1. Fluorescence spectra of the Rhod 6G–K2S2O8–PPH system (curves 1–5 are the excitation spectra, and curves 10 –50 are the emission spectra. 1.10 , 2.20 , 3.30 , 4.40 and 5.50 are the number of the excitation spectra and the emission spectra.) 1.10 2.00 mL Rhod 6G þ 1.00 ml HCl 2.20 1.10 þ 2.00 mL K2S2O8 3.30 2.20 þ 2.50 pg PPH 4.40 2.20 þ 700.00 pg PPH 5.50 1.10 þ 700.00 pg PPH.
+ NHC2 H5
O
HNH5C2
CH3 + K S O + H+ 2 2 8 COOC2H5
H3C
+ NH2C2H5
O
HNH5C2
CH3 + K2SO4 + H O 2 COOC2H5
H3C
Rhod.6G+'
Rhod.6G+
Scheme 2. Oxidation-reduction reaction between K2S2O8 and Rhod 6G.
+ NHC2H5
O
HNH5C2
CH3 COO
H 3C
S
N
N
2
+
+ S 2O 8 + 2 H N
OH
O
S
Cl
H3C
+ OH
N
N
N
N
S
Rhod.6G+-PPH
Cl
PPH
HNH5C2
N
N
Cl
PPH'
O
+ NHC2H5 CH3 COO
Cl N
N
N
Rhod.6G+-PPH'
S
O
+
S
N
N
N
OH
Cl PPH
Scheme 3. Oxidation-reduction reaction between K2S2O8 and PPH, ester exchange reaction between PPH0 and Rhod 6G þ –PPH.
When PPH was added, PPH was oxidized to red compound PPH0 [18,19] in the presence of K2S2O8 while S2O28 was reduced to SO24 [20]. The property of PPH0 was similar to that of PPH, Rhod 6G þ –PPH0 and PPH were produced in the ester exchange reaction between the –OH of PPH0 [15] and the –COOC2H5 of Rhod 6G þ – PPH (Scheme 3). In the above process, PPH catalyzed K2S2O8 oxidizing Rhod 6G, which caused the fluorescence signal of the system to quench sharply. The content of PPH was liner to the ΔF of the system. Thus, trace PPH could be determined by the catalytic fluorescence quenching method.
3.2. Fluorescence spectra According to the experimental method, the fluorescence spectra of the Rhod 6G-K2S2O8–PPH system were scanned (Fig. 1). Under the conditions of 60 1C, 10 min and pH 5.42, Rhod 6G existed in the form of Rhod 6G þ [21]. In the research process, we found that Rhod max 6G þ can emit strong and stable fluorescence (λmax ex /λem ¼526.8/ 0 556.4 nm, F¼ 755.4, Fig. 1, curve 1.1 ), and K2S2O8 could oxidize max Rhod 6G and quench its fluorescence signal (λmax ex /λem ¼57.6/ 556.8 nm, F ¼ 740.9, Fig. 1, curve 2.20 ), while PPH catalyzed K2S2O8 oxidizing Rhod 6G, which caused the fluorescence signal
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129 max of the system to quench sharply (λmax ex /λem ¼526.9/556.1 nm, F¼606.1, Fig. 1, curve 4.40 ), and the △F (134.8) of the catalytic system was three times larger than that of the non-catalytic system (44.5, Fig. 1, curve 5.50 ), showing the signal amplification of catalytic reaction. Thus, the 527/556 nm was selected as the working wavelength to determine PPH.
127
2.64, 2.81, 3.04, 3.32, 3.82, 4.19, 5.42, 6.35, 7.44, 8.66, 9.42 and10.20), and standing time (5, 10, 15, 20, and 30 min) on the ΔF of the system were investigated in a univariate approach (Table 1 and Fig. 2). Though the ΔF of the system was high when FITC(A), Rhod 6G (B), eosin Y(C), calcein(D), orange yellow G (E) were chosen as luminescence substances they were still lower than that of Rhod 6G (Fig. 2a). Further investigations found that with the increasing of the concentration and dosage of Rhod 6G, the ΔF of the system enhanced gradually (Table 1). When the concentration and dosage of Rhod 6G were 2.00 mL of 1.0 10 4 mol L 1, the ΔF of the system reached the maximum, the main reason might be that the –COOC2H5 group of Rhod 6G þ and –OH group of PPH carried out the ester exchange reaction and produced Rhod 6G þ –PPH.
3.3. Optimum conditions for PPH detection For the system containing 4.00 pg mL 1 PPH, the effects of the dosage and concentrations of reagents, luminescence substances (FITC(A), Rhod 6G (B), eosin Y(C), calcein(D), orange yellow G (E), etc.), oxidants (H2O2(A), K2S2O8(B), KIO3(C), KClO3(D), NaIO4(E), etc.), temperature and time of reaction, reaction acidity (pH was
Table 1 Optimization of the concentration and dosage of reagents. Reagents Rhod 6G (mol L (mL) K2S2O8 (%) (mL)
Concentrations and dosage 1
)
–3
4
5
1.0 10 ,1.0 10 ,1.0 10 , 1.0 10 0.50, 1.00, 1.50, 2.00, 2.50, 3.00 0.01, 0.05, 0.1, 0.2, 0.3 0.50, 1.00, 1.50, 2.00, 2.50, 3.00
6
, 1.0 10
7
20
ΔF
0
A
B
C
D
1.0 10 4 2.00 0.1 2.00
20
0
E
A
B
20
ΔF
E
20
20
40
60
80
0
100
0
10
20
30
t/min
O
T/ C
30
30
20
ΔF
10 0
D
10
10
ΔF
C
30
30
0
9.4,31.5, 17.6, 10.7, 5.2 5.7, 11.3, 21.3, 28.9, 17.0, 10.3 9.4, 17.6, 30.5, 10.7, 5.2 5.8, 10.7, 21.2, 31.1, 14.0, 9.9
10
10
ΔF
Optimal
30
30 ΔF
The ΔF in Rhod 6G–K2S2O8– PPH system
20 10
2
3
4
5
6 7 pH
8
9 10
0
0
10
20 30 t/min
40
Fig. 2. (a) The effects of luminescence substances on the ΔF of the system: FITC (A), Rhod 6G (B), eosin Y (C), calcein (D), and orange yellow G (E); (b) the effects of oxidants on the ΔF of the system: H2O2 (A), K2S2O8 (B), KIO3 (C), KClO3 and (D), NaIO4 (E); (c) the effects of reaction temperature on the ΔF of the system; (d) the effects of reaction time on the ΔF of the system; (e) the effects of reaction acidity on the ΔF of the system; (f) the effects of standing time on the ΔF of the system.
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L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129
Compared with H2O2(A), K2S2O8(B), KIO3(C), KClO3(D) and NaIO4(E), K2S2O8 had the largest effect on the ΔF of the system (Fig. 2b), the main reason might be the strongest oxidity of K2S2O8 With the increase in the concentration and dosage of K2S2O8, the ΔF of the system enhanced gradually (Table 1). When the concentration and dosage of K2S2O8 were 2.00 mL of 1.0%, the ΔF of the system reached the maximum. As the reaction time and temperature increased, the ΔF of the system gradually enhanced (Fig. 2c and d), which might result from the gradual increase in the catalytic ability of PPH. When the reaction temperature and time were 60 1C and 10 min, respectively, the ΔF of the system reached the maximum, which might be that the catalytic ability of PPH reached the peak. Henceforth, with the continuous increase of the reaction temperature and time, the ΔF of the system decreased due to the decreasing of catalytic ability of PPH gradually. The ΔF of the system reached the maximum and remained stable when the pH value of the reaction solution was in the range of 3.82–6.35 (Fig. 2e) for the reason that the ester exchange reaction between the –COOC2H5 group of Rhod 6G þ and –OH group of PPH and the oxidation reaction between K2S2O8 and PPH completed. The ΔF of the system almost stayed invariable within 15–30 min after being cooled for 5 min with flowing water, while the ΔF of the system decreased sharp when the time was over 30 min (Fig. 2f). Obviously, the reproducibility of the system was the best within 15–30 min under the optimal conditions. Therefore, the ΔF of the system reached the maximum when the system containing 2.00 mL of 1.0 10 4 mol L 1 Rhod 6G, 2.00 mL of 0.10% (m/V) K2S2O8 carried out in the pH 5.42 at 60 1C for 10 min. Under the above optimal conditions, the ΔF of the system almost stayed invariable and had good repeatability within 15–30 min after being cooled for 5 min by flowing water in the system. 3.4. Linear range, working curve, sensitivity and precision Under the optimum conditions, the ΔF of the system was linear with the concentration of PPH in the range of 0.080–28.00 ( 10 12) g mL 1 (when the content of PPH were 0.080, 0.40, 2.0, 4.0, 12.0 and 28.0 pg mL 1, the F (ΔF) were 735.5(5.4), 729.0(11.9), 724.9(16.0), 710.9(30.0), 677.5(63.4), and 606.1(134.8) and the corresponding relative standard deviations (RSD, %, n ¼6) were 1.2, 1.9, 2.2, 2.8, 3.3, 3.8, and 4.1, respectively, Fig. 3), the regression equations was ΔF¼8.424þ4.535CPPH (pg mL 1), correlation coefficients (r) was 0.9988 (n ¼6). The systems containing 0.080 and 28.0( 10 12) g mL 1 PPH were measured for six times, their RSD (%) were 1.2 % and 4.1%, respectively, showing good precision. LOD was 3.3 10 14 g mL 1 (calculated by 3 Sb/k, here Sb represents the standard deviation of 11 blank measurements and the value was 0.050, k is the slope of standard curve), showing the higher sensitivity of the fluorescence quenching method. This was attributed to the catalyzing effect of PPH on the reaction of K2S2O8 oxidizing Rhod 6G greatly improves ΔF value. 3.5. Selectivity of the fluorescence quenching method To evaluate the selectivity of fluorescence quenching method towards the sample containing 4.00 pg mL 1 PPH, a series of coexistence ions (materials) in human urine were investigated under the optimum measurement conditions. When the relative error (Er) exceeded 75%, each ion is considered as interfering agent. Results show that the allowable concentrations (ng mL 1) of most ions are high: 10.50 for urea, 5.90 for soluble starch, 4.00 for uric acid and oxalate, 3.00 for sodium citrate and chlorpromazine hydrochloride, 2.50 for sodium carboxymethyl cellulose and
Fig. 3. Representative fluorescence emission spectra of Rhod 6G þ in the presence of increasing PPH concentrations (0.080–28.0 pg mL 1) in the HCl buffer at pH 5.42. Inset: fluorescence change (ΔF) titrated with PPH under the optimum conditions. (When the contents of PPH were 0.0, 0.080, 0.40, 2.0, 4.0, 12.0, 28.0, 32.0, 36.0 and 40.0 pg mL 1, the F (ΔF) were 740.9(0), 735.5(5.4), 729.0(11.9), 724.9 (16.0), 710.9(30.0), 677.5(63.4), 606.1(134.8), 590.5(150.4), 580.0(160.9) and 574.3 (166.6), and the corresponding RSD (%, n¼ 6) were 1.2, 1.9, 2.2, 2.8, 3.3, 3.8,4.1, 4.3, 4.6, and 4.8, respectively. The regression equations was ΔF¼ 10.35þ 4.183CPPH (pg mL 1), r was 0.9966, n ¼6).
fluoxetine, 2.00 for phenobarbital, 1.00 for sucrose, 0.90 for clonazepam, 0.80 for glucose, 0.70 for fructose, 0.60 for triazolam and amitriptyline, 3.00 for Na þ , 1.60 for NO3 , 1.50 for K þ , 1.40 for F , 1.00 for Ca2 þ ,0.90 for Sr2 þ and A l3 þ ,0.80 for CO23 and Co2 þ , 0.70 for Fe3 þ , 0.60 for Zn2 þ and 0.50 for Cu2 þ , showing that this catalytic fluorescence quenching method has excellent selectivity. 3.6. Analysis of samples The content of PPH in 1.00 mL test solution was examined by experimental method and HPLC [11], and additional recovery experiment was also conducted. The results are listed in Table 2. The significant difference analysis between the proposed method and HPLC for the determination of PPH is shown in Table 3. Seen from Table 2, this sensitive and accurate method could be used to determine the content of PPH in biological samples with the recovery of 97–102% and RSD of 1.8–4.3%. The results agreed with those obtained with the HPLC. As shown in the Table 3, the F was 1.6, 1.3 and 1.5 (human urine), 2.2, 2.1 and 1.8 (human serum), respectively, indicating that there was no significant differences between S1 and S2, and the corresponding t was 1.2, 1.6 and 1.5 (human urine),1.2, 1.6 and 1.7 (human serum), respectively, indicating that there was also no significant differences between X 1 and X 2 . Obviously, the proposed method was sensitive, accurate and reliable to the detection of PPH in the biological samples. According to the PPH content of A, B and C serum is in 50–100 (toxic threshold) ng mL 1 [4], we could forecast that A, B and C might be angiocardiopathy patients [6,7]. Therefore, this method could be applied to determine the PPH content in serum and to predict the human diseases.
4. Conclusion In this paper, the Rhod 6G þ –PPH compound formed in the ester exchange reaction between –OH of PPH and –COOC2H5 of Rhod 6G þ . PPH was oxidized to red compound (PPH0 ) in the presence of K2S2O8. Interestingly, fluorescence signal of Rhod 6G þ was quenched because the –OH of PPH0 reacted with –COOC2H5 of Rhod 6G þ –PPH to form Rhod 6G þ –PPH0 and PPH, which decreased the
L. Zhang et al. / Journal of Luminescence 156 (2014) 124–129
129
Table 2 Analysis results of PPH in samples. This method (n¼ 6) Sample Human Human Human Human Human Human
urine A urine B urine C serum A serum B serum C
HPLC (n¼ 5) Found (μg mL 1)
RSD (%)
Added (μg mL 1)
Add found(μg mL 1)
Obtaine (μg mL 1)
Recovery (%)
HPLC (μg mL 1)
Er (%)
4.52 4.90 4.60 4.78 4.57 4.81
3.2 3.4 2.6 4.3 1.8 2.3
0.050 0.050 0.050 0.050 0.050 0.050
4.57 4.95 4.65 4.84 4.62 4.86
0.0485 0.0510 0.0495 0.0505 0.0492 0.0496
97.0 102.0 99.0 101.0 98.5 99.2
4.69 4.81 4.72 4.71 4.78 4.70
–3.6 þ1.9 –2.5 þ1.5 –4.4 þ2.3
Table 3 Analysis of the significant differences for determination results (P ¼90%, f¼ n1 þ n2 2¼ 9, F0.90, 9 ¼6.3, t0.90, 9 ¼ 1.8). Sample
Human Human Human Human Human Human
This method (μg mL 1, n¼ 6)
urine A urine B urine C serum A serum B serum C
HPLC (μg mL 1, n¼ 5)
Statistical analysis
X1
S1
X2
S2
F
S
t
4.52 4.90 4.60 4.78 4.57 4.81
0.0322 0.0544 0.0603 0.0645 0.0597 0.0636
4.55 4.85 4.55 4.82 4.62 4.75
0.0510 0.0485 0.0495 0.0430 0.0412 0.0474
1.6 1.3 1.5 2.2 2.1 1.8
0.042 0.052 0.056 0.056 0.052 0.057
1.2 1.6 1.5 1.2 1.6 1.7
π-electron density (δ) of carbon atom in a Rhod 6G þ –PPH0 conjugated system and enhanced the non-radiation energy loss of the excited Rhod 6G þ of the triplet state. Thus, a new catalytic fluorescence quenching method for the determination of PPH was established based on the linear relativity between the ΔF of the system and the content of PPH. The simple and sensitive method was suitable for the analysis of residual PPH in biological samples, and has provided a new way for the clinical detection. Acknowledgments This work was supported by Fujian Province Natural Science Foundation (Grant No. 2012H6026), Fujian Province Education Committee (JA11311) and Fujian provincial bureau of quality and technical supervison (FJQI2011006). At the same time, we are very grateful to precious advices raised by the anonymous reviewers.
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