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Chemiluminescence determination of streptomycin in pharmaceutical preparation and its application to pharmacokinetic study by a flow injection analysis assembly
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 823–828
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Chemiluminescence determination of streptomycin in pharmaceutical preparation and its application to pharmacokinetic study by a flow injection analysis assembly Bin Du a, Hongyan Li a, Jianwen Jin a, Tiantian Wang a, Yang Li c, Guopeng Shen b,⇑, Xiaotian Li a,⇑ a b c
School of Pharmaceutical Science, Zhengzhou University, Zhengzhou 450001, China School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou 450001, China Henan Provincial Institute of Food and Drug Control, Zhengzhou 450003, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel NBS-eosin-streptomycin flow
600
CL Intensity
injection (FI) chemiluminescence (CL) system. A defined experimental design in order to find optimized conditions. The determination of streptomycin in vitro/vivo. High-throughput determination of the content of streptomycin. The possible mechanism for enhancement CL strength.
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a r t i c l e
i n f o
Article history: Received 2 May 2013 Received in revised form 23 June 2013 Accepted 1 July 2013 Available online 11 July 2013 Keywords: Chemiluminescence Streptomycin sulfate Eosin Pharmacokinetics
Blank sample Streptomycin sample
0
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6 t (s)
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a b s t r a c t A novel and rapid method for the determination of streptomycin has been established by chemiluminescence (CL) based on significant intensity enhancement of streptomycin on the weak CL of N-bromosuccinimide (NBS) and eosin in alkaline medium. The method is simple, rapid and effective to determine streptomycin in the range of 8.0 109–1.0 106 g mL1 with a determination limit of 2.25 109 g mL1. The relative standard deviation is 1.95% for the determination of 2.0 107 g mL1 streptomycin (n = 11). The pharmacokinetics of streptomycin in plasma of rat coincides with the two-compartment open model. The T1/2a, T1/2b, CL/F, AUC(0t), MRT, Tmax and Cmax were 18.83 ± 1.24 min, 82.14 ± 3.07 min, 0.0026 ± 0.0011 L kg1 min1, 36044.50 ± 105.02 mg min1 L1, 92.29 ± 8.21 min, 21.63 ± 1.26 min and 375.61 ± 8.50 lg mL1, respectively. There was no significant difference between the results obtained by CL and HPLC. The FI–CL method can be used to determine streptomycin in pharmaceutical preparation and biological samples. The established method is simple, rapid and sensitive without expensive instruments. The possible enhancement mechanism was also investigated. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Over the past few years, there has been increasing attention to the chemiluminescence (CL) as a sensitive, simple and fast assay ⇑ Corresponding authors. Tel.: +86 371 65293928; fax: +86 371 63886016 (G. Shen). E-mail address: [email protected] (G. Shen). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.07.007
for the research in many different fields, particularly in pharmaceutical and biomedical analysis [1–3]. Most of the CL methods involve inhibition or catalysis of the redox reaction of CL reagents such as luminol, rhodamine and fluorescein [4–6]. Therefore, the interests in the research of CL reagents and CL labeling reagents for pharmaceutical analysis have increased in recent years. Eosin as a type of xanthene dyes of luminescent material with unique optical properties, and has been successfully used in CL analysis. Mikuska et al. [7]
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and Yang et al. [8] reported the application of gallic acid and eosin Y for determination of ozone in air with a chemiluminescence aerosol detector and the analysis of dihydralazine sulfate based on hexacyanoferrate (III) oxidation sensitized by eosin Y, respectively. However, there is not any report for the CL determination of streptomycin with eosin as a luminophor up to now. Streptomycin belongs to the group of aminoglycoside antibiotics and mainly employed as a first effective remedy for tuberculosis [9], it is still a first line antibiotic in veterinary medicine for the treatment of Gram-negative bacteria in animals [10]. Several papers have been published proposing methods for determination of streptomycin, including UV spectrophotometry [11], LC–UV [12,13], LC–FLD [14], LC–MS [15–17], LC/MS/MS [18,19], capillary zone electrophoresis (CZE) [20,21], ion exchange high-performance thin-layer chromatography (IE-HPTLC) [22], electrochemical immunoassay [23], immunochromatographic [24]. Among these methods, HPLC is the most popular method for the analysis of real samples. However, the assay is sometimes time-consuming, because a pre-treatment of the biological sample (such as solidphase extraction or pre-column derivatization of analyte) is usually necessary prior to its injection into the instrument. In some cases complex gradient systems are also required, and long analytical times are needed due to prolonged elution times that add up to equilibration time between runs. Chemiluminescence is an attractive detection method for analytical determination because of the low detection limit and wide linear working range that can be achieved while using relatively simple instrumentation. In addition, compared with CL method, these methods require high commercial price and time-consuming. So CL method has been developed for the determination of streptomycin sulfate. The luminol – potassium periodate – Mn2+ and luminol – potassium ferricyanide – potassium ferrocyanide CL system were applied to the determination of streptomycin in milk and pharmaceutical preparations [25,26]. But to the best of our knowledge, no FI–CL analysis using N-bromosuccinimide (NBS) – eosin system in alkaline medium has been previously reported for the measurement of streptomycin in the open literature, and the present CL system has the lower limit of detection, compared with the above-mentioned CL methods for the determination of streptomycin. The new CL system of NBS-eosin-streptomycin was applied to complex biological samples for the first time. In this paper, a novel chemiluminescence reaction, NBS – eosin system, for the determination of streptomycin was described. The method was based on enhancement of CL emission of NBS – eosin system in the presence of streptomycin sulfate. The possible enhancement mechanism was also further investigated. In order to demonstrate the potential of the method, quantitative analysis of streptomycin sulfate in pharmaceutical preparations, and a preliminary pharmacokinetic study of streptomycin sulfate in rats was performed. The experimental results indicated that the method was reliable, simple, rapid and suitable for analysis of streptomycin sulfate in batch biological samples. This work gives a practical application of NBS – eosin CL reaction and will contribute to the determination of streptomycin sulfate in biomedical, pharmaceutical and clinical analysis.
Materials and methods Reagents Eosin was purchased from Beijing Chemical Plant (Beijing, China). NBS was provided by Sinopharm Chemical Reagent Limited Company (Shanghai, China). NaOH, Cl3CCOOH were purchased from Tianjin Chemical Experiment Plant (Tianjin, China). Powder-injection of streptomycin sulfate was purchased from
Shandong Lukang Pharmaceutical Limited Company (Shandong, China). Streptomycin reference substance was provided by the Control of Pharmaceutical and Biological Products (Beijing, China). The stock solution (0.1 mg mL1) was prepared by dissolving 1 mg of streptomycin in 10 mL water and stored in a brown bottle to avoid exposure to light and air. A series of working standard solutions of streptomycin in the range of 8.0 109 to 1.0 106 g mL1 were freshly prepared by dilution of the stock solution of streptomycin (0.1 mg mL1). Dissolving 415.2 mg of eosin in 100 mL water, a working solution of 6 103 mol L1 eosin was obtained. NBS solution (4.0 102 mol L1) was prepared by dissolving 356 mg of NBS in water, and diluting to 50 mL with water. All the reagents were of analytical reagent grade and all solutions were prepared with double-distilled water. Apparatus The CL analysis was conducted on a Model IFFM-E flow-injection chemiluminescence analyser (Xi’an Remex Analysis Instrument Company, Xi’an, China). The schematic diagram of the system was shown in Fig. 1. The steady injection system consists of two peristaltic pumps and a six-way valve. Two peristaltic pumps were used to deliver all the solutions. Polytetrafluoroethylene (PTFE) tubes (0.8 mm i.d.) were employed to connect all the components of the flow system. The CL emission produced in the flow cell (1 mm 25 cm spiral colorless glass tubing, three turns) was detected with a photomultiplier tube (Hamamatsu, Japan). The CL signal was recorded by using an IBM-compatible computer, equipped with a data acquisition interface. The UV–vis absorption spectra of the proposed system were investigated using a UV-2550 spectrophotometer (Shimadzu, Japan) with the wavelength range of 200–800 nm. FI–CL procedures As shown in Fig. 1 of the FI–CL system, the solutions containing oxidant NBS (R1), carrier NaOH (R2), eosin solution (R3) and blank or sample solution (R4) were pumped to flow cell via two peristaltic pumps at a flow rate of 2.84 mL min1 (P1) and 4.27 mL min1 (P2). The eosin solution and blank or sample solution were simultaneously pumped into a mixer and mixed. The mixed solution was carried into flow cell F in front of the PMT and reacted with the oxidant of NBS solution which was quantitatively injected into the carrier stream by a six-way valve in the flow cell F, and the CL emission produced was detected by sending the signal from the PMT to a detector and then to a computing integrator. The concentration of analyte was quantified by measuring the enhanced CL intensity, DI = IsI0, where I0 and Is are CL intensity in the absence and presence of streptomycin, respectively. Sample treatment Powder-injection sample treatment Sample solutions for analysis were prepared as follows. The average powder-injection weight was calculated from five powder-injections of streptomycin sulfate that were randomly selected. They were ground to a homogeneous fine powder. A portion of the powder corresponding to 1.0 mg was weighed and dissolved with 10 mL double-distilled water. The solution was suitably diluted with water before the determination so that the concentrations of the analyte were in the linear range of proposed method.
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Fig. 1. Schematic diagram of FIA-CL manifold used for the determination of streptomycin. R1, NBS solution; R2, NaOH solution; R3, eosin solution; R4, streptomycin solution; P1, P2, Peristaltic pump; V, injection valve; F, flow cell; PMT, Photo-multiplier tube; HV, High voltage; PC, Record computer.
Plasma sample treatment Plasma samples were obtained from blank rats. The protein of 100 lL plasma sample was removed by adding 100 lL 10% trichloroacetic acid (CCl3COOH) in a centrifuge tube, which was thoroughly vortex-mixed for 5 min. After centrifugation at 10,000g for 20 min, the supernatant was collected. Then the supernatant was evaporated to dryness under a stream of nitrogen at 25 °C. The residue was dissolved into 5 mL water for the CL analysis. A blank value was determined by treating streptomycin-free plasma in the same way. Pharmacokinetic study All experimental procedures comply with the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Zhengzhou University Health Science Center. A total of five Sprague–Dawley rats weighing 200 ± 20 g were supplied by the Henan Laboratory Animal Centre (Zhengzhou, China). The rats were maintained in an airconditioned animal room at a temperature of 22 ± 2 °C and relative humidity of 50 ± 10% and kept in the environmentally controlled breeding room for 2 days before starting the experiments. They were fed with standard laboratory food and water and then fasted for 12 h with free access to water prior to the experiments. Streptomycin sulfate prepared with sterile water for injection was administrated to the rats at a dose of 90.0 mg kg1 weight by intramuscular injection. An aliquot of plasma samples was collected in a 1.5 mL heparinized eppendorf tube at different time intervals from the rat eye socket vein. After separation by centrifugation (4000g) for 10 min at 4 °C, the plasma was separated immediately and frozen at 20 °C. Prior to analysis, 100 lL of the plasma was taken and pretreated as described above. Results and discussion Kinetic characteristics of CL system Before carrying out the flow-injection method, the kinetic characteristics of the proposed CL reaction were studied by using the 600
batch method. Upon adding eosin to NBS basic solution gave out a weak CL signal. On injection of the mixture of streptomycin and eosin, a strong CL emission was recorded. The CL kinetic curves for the CL reactions of NBS-eosin and NBS-eosin-streptomycin were shown in Fig. 2. The CL signal from the reaction of eosin with NBS will be the background of the flow-injection analysis. Experiments showed that the proposed CL reaction was a flash type luminescence and the time interval between the start of CL and its maximum is only about 2 s. It can be seen that the significant enhancement on CL peak of NBS-eosin was obtained after injection of streptomycin. This shows that the characteristic of the system is very suitable for FI-CL determination of streptomycin. Condition optimization of the CL system In order to determine the parameters that gave the optimum signal for the analysis of streptomycin, a series of univariate searches were performed on the basis of the net peak height (DI) and the ratio of the signal to the noise (S/N). To obtain a reliable assay of streptomycin, the effects of different chemical and instrumental variables were investigated. The influence of the concentration of NBS was examined in the range of 0.008–0.08 mol L1 (0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.08 mol L1). The result showed that the background noise level (I0) increased significantly with the increase of the concentration of oxidant, and the maximum value of DI and S/N ratio was obtained when 0.04 mol L1 NBS was used for the determination of streptomycin (Fig. 3). As a sensitizer of the proposed CL system, the influence of eosin concentration on the net CL intensity was investigated from 1.0 103 to 1.0 102 mol L1 (1.0 103, 2.0 103, 3 3 3 3 2 4.0 10 , 5.0 10 , 6.0 10 , 8.0 10 , 1.0 10 mol L1) and the result was given in Fig. 4. This figure showed that the maximum net CL intensity was obtained at the eosin concentration of 6.0 103 mol L1. When the eosin concentration was above this level, the net CL intensity decreased slightly. Thus, 6.0 103 mol L1 eosin was chosen for consequent research work. Preliminary studies showed that NBS could react with eosin to produce CL emission in alkaline solution and its CL intensity strongly depends on the concentration of alkaline medium. In the
Streptomycin sample 800
Net CL Intensity
CL Intensity
Blank sample
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0
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6
8
10
12
t (s)
600 400 200 0
0
0.02
0.04
0.06
0.08
0.1
NBS Concentration (mol/L) Fig. 2. Chemiluminescence kinetic curves of NBS-eosin-streptomycin system. Conditions: NBS, 0.04 M; eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
Fig. 3. Effect of NBS concentration on net CL intensity. Conditions: Eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
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800
Net CL Intensity
Net CL Intensity
800 600 400 200 0
0
2
4
6
8
10
P1 P2
600 400 200
12
0
-3
Eosin Concentration (10 mol/L)
0
2
4
6
8
Flow Rate (mL/min) Fig. 4. Effect of eosin concentration on net CL intensity. Conditions: NBS, 0.04 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
present study, NaOH was selected as the alkaline medium, and its concentration was tested in the range of 0.01–0.2 mol L1 (0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2 mol L1) with the presence of 2.0 107 g mL1 streptomycin. As shown in Fig. 5, the net CL intensity increased remarkably with the increase of the NaOH concentration until the latter reached 0.04 mol L1, and then decreased. Hence, 0.04 mol L1 was used as the optimum concentration of NaOH for further test. The total flow rate of the carrier stream and reagent streams was studied over the range from 1.60 to 3.70 mL min1 for P1 and 1.87 to 6.00 mL min1 for P2, respectively. The results were shown in Fig. 6. It can be seen from the figure that the net peak height increased sharply with the increasing flow rate until 2.84 mL min1 for P1 and 4.27 mL min1 for P2 was reached, where the net peak height had the highest value. When higher flow rates were used, DI decreased significantly. Thus, these values were chosen as the optimal flow rates. Analytical performance characteristics Under the optimized experimental conditions as mentioned above, the working curve was obtained. The net peak height (DI) was linearly proportional to the streptomycin concentration (C, g mL1) in the range of 8.0 109–1.0 106 g mL1. The regression equation was obtained as DI = 2.0 109 C–16.176 (r = 0.9997), the relative standard deviation (R.S.D.) was 1.95% for 11 replicate determinations of 2.0 107 g mL1 streptomycin. The detection limit was 2.25 109 g mL1. To the best of the authors’ knowledge, 2.25 ng mL1 is the lowest limit of detection among those already existing CL methods for the determination of streptomycin [25,26]. The proposed FIA method is fast and enables to determine 90 of samples per hour. Interference study In order to evaluate the selectivity of the proposed CL method for the determination of streptomycin, the interferences of various excipients and some possible common foreign species usually present in pharmaceutical preparations were investigated by using a 2.0 107 g mL1 streptomycin solution, which was spiked with
Fig. 6. Effect of flow rate on net CL intensity. Conditions: NBS, 0.04 M; eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
increasing amounts of the interfering constituents. A foreign substance was considered as non-interfering if its presence caused an analytical signal variation lower than ±5% regardless of the CL variation noted in its absence, and the results were listed in Table 1. The data showed that there was little interference. Method application Analysis of pharmaceutical preparation The proposed method was applied to the determination of streptomycin content in powder-injections according to the procedure described in Section 2.4.1. The results obtained (Table 2) were in good agreement with the nominal contents. In addition, the accuracy of the results obtained with the proposed method was evaluated through the recovery test. A known amount of standard solution of streptomycin was added to streptomycin sulfate powder-injections and the results were shown in Table 2. The mean recoveries were found to be 97.12–103.84% with RSD values of 0.61–2.07%. The results showed that the accuracy and precision of the proposed method were satisfactory. Analysis of streptomycin in plasma samples The method presented has a low detection limit. Therefore, the proposed method allows the determination of streptomycin in
Table 1 Tolerable concentration ratios with respect to 2.0 107 g/mL streptomycin for some interfering species (<5% error). Foreign interfering substances +
+
CO2 3 ; NO3 ,
Tolerable concentration ratio
K , Na , Ac , Cl , amylum Sucrose, fructose, lactose, glucose, HCO 3 H2 PO4 , b-cyclodextrin Ca2+, SO2 4 Mg2+, Zn2+ EDTA, Cu2+ Ascorbic acid, NHþ 4
1000 500 300 100 10 5 2
Table 2 Results of the analysis of streptomycin in pharmaceutical preparations (n = 5).
Net CL Intensity
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Sample No.
Labeled value (g)
Measured value (g)
Added (g)
Found (g)
Recovery (%)
RSD (%)
1
1.000
0.997
0.250 0.500 1.000
0.244 0.495 0.989
97.60 99.08 98.91
2.07 1.58 0.61
2
1.000
1.001
0.250 0.500 1.000
0.247 0.511 1.011
98.80 102.20 101.11
1.04 1.71 1.46
3
1.000
0.988
0.250 0.500 1.000
0.259 0.519 0.971
103.68 103.84 97.12
1.52 1.11 1.70
650
600
0
0.05
0.1
0.15
0.2
NaOH Concentration (mol/L) Fig. 5. Effect of NaOH concentration on net CL intensity. Conditions: NBS, 0.04 M; eosin, 6.0 103 M; streptomycin, 2.0 107 g/mL.
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B. Du et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 823–828 Table 3 Determination of streptomycin in plasma samples (n = 3). Sample
Content (107 g/mL)
Added (107 g/ mL)
Found (107 g/ mL)
Recovery (%)
RSD (%)
1
2.161
0.200 1.000 6.000
2.356 3.176 8.216
97.51 101.53 100.92
1.49 0.95 0.77
2
2.220
0.200 1.000 6.000
2.413 3.206 8.146
96.41 98.58 98.76
2.06 2.48 1.83
3
2.034
0.200 1.000 6.000
2.231 3.066 8.176
98.51 103.18 102.37
1.21 1.30 0.98
biological samples. The linearity for plasma analysis was in the range of 4.0 109 to 1.0 106 g mL1 with the regression equation DI = 4.0 109 C-195.69 (r = 0.9995; C, g mL1) with a limit of quantification of 4.48 109 g mL1 (LOQ, which is given as the concentration for which the analytical signal is 10 times higher than standard deviation of blank intensity). The recovery tests were carried out on samples to which three different known amounts of streptomycin were added. The recoveries for plasma at three different concentrations were 96.41–103.18% (Table 3). It is therefore suggested that the present method would be applicable to the pharmacokinetic study of streptomycin. Pharmacokinetic evaluation In this study, five rats were administered 90.0 mg kg1 of streptomycin sulfate for powder-injections by intramuscular injection. Blood samples were collected in a 1.5 mL heparinized eppendorf tube from the rat eye socket vein before administration of the drug and at intervals thereafter (5, 10, 20, 30, 40, 60, 90, 120, 240, 360 min) and the streptomycin content in the plasma was determined by the proposed method. Table 4 gives the corresponding pharmacokinetic parameters calculated using Kinetica 4.4.1 program (Thermo. Fisher Scientific Inc., MA, USA). The mean peak concentration (Cmax) was determined as 375.61 lg mL1 for streptomycin at 21.63 min after dosing. The values of AUC0-t representing the extent of absorption were calculated as 36044.50 mg min1 L1 and the T1/2b was calculated as 82.14 min. The distribution of streptomycin throughout the body conformed to a classical two-compartment open model with first-order absorption. The results were compared to the data obtained by using the method of HPLC for further ensuring the method’s application. From the pharmacokinetic parameters, it can be seen that there was no significant difference between the results obtained by the two methods (P > 0.05). Possible mechanism of CL reaction In order to obtain the possible mechanism of the CL reaction, the following experiments were performed. Various mixed Table 4 Mean pharmacokinetic parameters for streptomycin in healthy rats after administering 90.0 mg kg1 of streptomycin sulfate for powder-injections by intramuscular injection (n = 5). Parameters
CL
HPLC
T1/2a (min) T1/2b (min) Tmax (min) Cmax (lg mL1) CL/F (L kg1 min1) AUC(0t) (mg min1 L1) MRT (min)
18.83 ± 1.24 82.14 ± 3.07 21.63 ± 1.26 375.61 ± 8.54 0.0026 ± 0.0011 36044.50 ± 105.02 92.29 ± 8.21
20.86 ± 0.41 84.28 ± 0.92 22.42 ± 2.83 327.31 ± 29.45 0.0027 ± 0.0009 35907.90 ± 325.88 92.65 ± 2.63
Fig. 7. UV–vis spectra of streptomycin (orange curve), eosin (red curve), NBS-eosin (black curve), NBS-eosin-streptomycin (green curve). Conditions: NBS, 0.04 M; eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
methods were used to arrange the reagents in this CL system. The results indicated that almost no light emission was recorded when only NBS and streptomycin solution were mixed together and weak CL can be produced in NBS and eosin system. When NBS was mixed with streptomycin in the presence of eosin in alkaline condition, there was a luminescence peak at about 545 nm, which is similar to the fluorescence emission maximum of eosin (545 nm) reported in the literature [27]. Thus, it can be seen that the possible luminophor was eosin. The UV–vis spectra of streptomycin, eosin, NBS-eosin and NBS-eosin-streptomycin solutions were further observed in order to explore more details of the possible mechanism. From Fig. 7, it can be seen that streptomycin had absorption near 190 nm (Fig. 7, orange curve) and eosin had absorption peaks near 254, 301, 342 and 516 nm (Fig. 7, red curve). However, the absorption peaks of 254, 301 and 342 nm disappeared after NBS was added into eosin solution, and the maximum absorption at 516 nm decreased (Fig. 7, black curve), which meant NBS may have a redox reaction with eosin. Compared with the UV–vis spectroscopy of NBS-eosin (Fig. 7, black curve), UV–vis spectroscopy of NBS-eosin-streptomycin did not produce any new UV absorption peaks, but obviously enhanced the intensity of the absorption peak (Fig. 7, green curve), which indicated NBS may react with streptomycin. Hence, the UV–vis absorption spectra showed that the signal of NBS-eosin system was enhanced by streptomycin. The chemical structure of streptomycin is based on a streptose connected to streptidine and N-methyl-L-glucosamine in a glycoside linkage (Fig. 8), thus streptomycin is a polyhydroxyl compound. Therefore, streptomycin was extremely easy to be oxidized by some oxidants. In aqueous solutions, the oxidizing
H
OR'
O HO
CH2OH
H H OH
R
H
H
Fig. 8. The structure of streptomycin.
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property of NBS was attributed to hypobromous acid (HBrO) generated by its hydrolysis [28]. After NBS was hydrolyzed to HBrO, HBrO reacted with streptomycin and released energy to eosin. The energy then changed eosin to an activated one. When eosin returned from excited state to ground state, the energy was released as light. Conclusions In the work, a novel FI–CL method was established to determine streptomycin in pharmaceutical preparation and biological samples. The proposed method was used in a pharmacokinetic study, which further confirmed the potential of the method. Compared with previously published reports, the established procedure is simple, rapid and has the advantage of lower detection limit, it also uses low-cost instrumentation, which is easy to use and maintain, with a high sample throughput of 90 h1. These characteristics make the assay suitable not only for monitoring streptomycin plasma levels in animals and patients, but also for the rapid and precise measurement of low concentrations of streptomycin in a complex matrix. This is necessary both in the context of pharmacokinetic studies after a single dose and the ongoing discussion on adverse effects of aminoglycoside antibiotics, as well as impact on the environment. Acknowledgement Financial support from Project of Zhengzhou Science and Technology Innovation Team (Grant number: 121 PCXTD521) is gratefully acknowledged. References [1] L. Han, Y.M. Zhang, J. Kang, J.L. Tang, Y.H. Zhang, J. Pharm. Biomed. 58 (2012) 141–145.
[2] P.S. Francisa, J.L. Adcockb, J.W. Costinc, S.D. Purcelld, F.M. Pfeffera, N.W. Barnett, J. Pharm. Biomed. 48 (2008) 508–518. [3] M. Kaczmarek, J. Fluoresc. 21 (2011) 2201–2205. [4] D.H. Chen, Z.H. Song, H.R. Lv, Food Chem. 135 (2012) 2549–2553. [5] H.L. Wang, S.Q. Han, J. Chin. Chem. Soc-Taip. 59 (2012) 1130–1136. [6] Z.F. Fu, Z.J. Zhang, Z.P. Wang, W.F. Luo, L.Q. Fang, Chem. Anal-Warsaw. 49 (2004) 643–652. [7] P. Mikuska, Z. Vecera, Anal. Chim. Acta 374 (1998) 297–302. [8] X.F. Yang, H. Li, Talanta 64 (2004) 478–483. [9] A. Schatz, E. Bugie, S.A. Walkman, Proc. Soc. Exp. Biol. Med. 55 (1944) 66–69. [10] G. Goodman, Pharmakologische Grundlagen der Arzneimitteltherapie, second ed., McGraw-Hill International, London, 1998. [11] C.J. Wang, Y. He, China Pharm. 21 (2010) 452–453. [12] E. Adams, M. Rafiee, E. Roets, J. Hoogmartens, J. Pharm. Biomed. 24 (2000) 219–226. [13] M. Pendela, J. Hoogmartens, A. Van Schepdael, E. Adams, J. Sep. Sci. 32 (2009) 3418–3424. [14] B.J. Chen, H.Y. Zhang, B.X. Lin, J. Ge, L.H. Qiu, J. Aoac Int. 95 (2012) 523–527. [15] S.I. Kawano, Rapid Commun, J. Mass Spectrom. 23 (2009) 907–914. [16] U. Holzgrabea, C.J. Napb, N. Kunzb, S. Almeling, J. Pharm. Biomed. 56 (2011) 271–279. [17] A.M. Gremilogianni, N.C. Megoulas, M.A. Koupparis, J. Chromatogr. A 1217 (2010) 6646–6651. [18] D.A. Bohm, C.S. Stachel, P. Gowik, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess 29 (2012) 189–196. [19] M. Georg, S. Rosemarie, S.N. Irina, K.W. Thomas, Deut. Lebensm-Rundsch. 105 (2009) 101–105. [20] Y.M. Li, D. Debremaeker, A. Van, E. Schepdael, J. Roets, Hoogmartens, J. Liq. Chromatogr. R. T. 23 (2000) 2979–2990. [21] Y.M. Hsiao, J.L. Ko, C.C. Lo, J. Agr. Food Chem. 49 (2001) 1669–1674. [22] G. Vanik, S. Masood, W.H. Syed, JPC-J. Planar Chromat. 24 (2011) 520–523. [23] B.Q. Liu, B. Zhang, Y.L. Cui, H.F. Chen, Z.Q. Gao, D.P. Tang, ACS Appl. Mater. Interfaces 3 (2011) 4668–4676. [24] N.A. Byzovaa, E.A. Zverevaa, A.V. Zherdeva, S.A. Ereminb, P.G. Sveshnikovc, B.B. Dzantiev, Anal. Chim. Acta 701 (2011) 209–217. [25] G.H. Wan, H. Cui, H.S. Zheng, Y.Q. Pang, L.J. Liu, X.F. Yu, Luminescence 21 (2006) 36–42. [26] F.C. Cai, Y.M. Huang, Z.J. Zhang, J. Southwest China Normal Univ. (Natural Science Edition) 29 (2004) 79–83. [27] V. Balzani, P. Ceroni, S. Gestermann, M. Gorka, C. Kauffmann, F. Vogtle, Tetrahedron 58 (2002) 629–637. [28] Z.P. Wang, Z.J. Zhang, Z.F. Fu, W.F. Luo, X. Wang, Anal. Lett. 36 (2003) 2683– 2697.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Chemiluminescence determination of streptomycin in pharmaceutical preparation and its application to pharmacokinetic study by a flow injection analysis assembly Bin Du a, Hongyan Li a, Jianwen Jin a, Tiantian Wang a, Yang Li c, Guopeng Shen b,⇑, Xiaotian Li a,⇑ a b c
School of Pharmaceutical Science, Zhengzhou University, Zhengzhou 450001, China School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou 450001, China Henan Provincial Institute of Food and Drug Control, Zhengzhou 450003, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel NBS-eosin-streptomycin flow
600
CL Intensity
injection (FI) chemiluminescence (CL) system. A defined experimental design in order to find optimized conditions. The determination of streptomycin in vitro/vivo. High-throughput determination of the content of streptomycin. The possible mechanism for enhancement CL strength.
300
0
a r t i c l e
i n f o
Article history: Received 2 May 2013 Received in revised form 23 June 2013 Accepted 1 July 2013 Available online 11 July 2013 Keywords: Chemiluminescence Streptomycin sulfate Eosin Pharmacokinetics
Blank sample Streptomycin sample
0
2
4
6 t (s)
8
10
12
a b s t r a c t A novel and rapid method for the determination of streptomycin has been established by chemiluminescence (CL) based on significant intensity enhancement of streptomycin on the weak CL of N-bromosuccinimide (NBS) and eosin in alkaline medium. The method is simple, rapid and effective to determine streptomycin in the range of 8.0 109–1.0 106 g mL1 with a determination limit of 2.25 109 g mL1. The relative standard deviation is 1.95% for the determination of 2.0 107 g mL1 streptomycin (n = 11). The pharmacokinetics of streptomycin in plasma of rat coincides with the two-compartment open model. The T1/2a, T1/2b, CL/F, AUC(0t), MRT, Tmax and Cmax were 18.83 ± 1.24 min, 82.14 ± 3.07 min, 0.0026 ± 0.0011 L kg1 min1, 36044.50 ± 105.02 mg min1 L1, 92.29 ± 8.21 min, 21.63 ± 1.26 min and 375.61 ± 8.50 lg mL1, respectively. There was no significant difference between the results obtained by CL and HPLC. The FI–CL method can be used to determine streptomycin in pharmaceutical preparation and biological samples. The established method is simple, rapid and sensitive without expensive instruments. The possible enhancement mechanism was also investigated. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Over the past few years, there has been increasing attention to the chemiluminescence (CL) as a sensitive, simple and fast assay ⇑ Corresponding authors. Tel.: +86 371 65293928; fax: +86 371 63886016 (G. Shen). E-mail address: [email protected] (G. Shen). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.07.007
for the research in many different fields, particularly in pharmaceutical and biomedical analysis [1–3]. Most of the CL methods involve inhibition or catalysis of the redox reaction of CL reagents such as luminol, rhodamine and fluorescein [4–6]. Therefore, the interests in the research of CL reagents and CL labeling reagents for pharmaceutical analysis have increased in recent years. Eosin as a type of xanthene dyes of luminescent material with unique optical properties, and has been successfully used in CL analysis. Mikuska et al. [7]
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and Yang et al. [8] reported the application of gallic acid and eosin Y for determination of ozone in air with a chemiluminescence aerosol detector and the analysis of dihydralazine sulfate based on hexacyanoferrate (III) oxidation sensitized by eosin Y, respectively. However, there is not any report for the CL determination of streptomycin with eosin as a luminophor up to now. Streptomycin belongs to the group of aminoglycoside antibiotics and mainly employed as a first effective remedy for tuberculosis [9], it is still a first line antibiotic in veterinary medicine for the treatment of Gram-negative bacteria in animals [10]. Several papers have been published proposing methods for determination of streptomycin, including UV spectrophotometry [11], LC–UV [12,13], LC–FLD [14], LC–MS [15–17], LC/MS/MS [18,19], capillary zone electrophoresis (CZE) [20,21], ion exchange high-performance thin-layer chromatography (IE-HPTLC) [22], electrochemical immunoassay [23], immunochromatographic [24]. Among these methods, HPLC is the most popular method for the analysis of real samples. However, the assay is sometimes time-consuming, because a pre-treatment of the biological sample (such as solidphase extraction or pre-column derivatization of analyte) is usually necessary prior to its injection into the instrument. In some cases complex gradient systems are also required, and long analytical times are needed due to prolonged elution times that add up to equilibration time between runs. Chemiluminescence is an attractive detection method for analytical determination because of the low detection limit and wide linear working range that can be achieved while using relatively simple instrumentation. In addition, compared with CL method, these methods require high commercial price and time-consuming. So CL method has been developed for the determination of streptomycin sulfate. The luminol – potassium periodate – Mn2+ and luminol – potassium ferricyanide – potassium ferrocyanide CL system were applied to the determination of streptomycin in milk and pharmaceutical preparations [25,26]. But to the best of our knowledge, no FI–CL analysis using N-bromosuccinimide (NBS) – eosin system in alkaline medium has been previously reported for the measurement of streptomycin in the open literature, and the present CL system has the lower limit of detection, compared with the above-mentioned CL methods for the determination of streptomycin. The new CL system of NBS-eosin-streptomycin was applied to complex biological samples for the first time. In this paper, a novel chemiluminescence reaction, NBS – eosin system, for the determination of streptomycin was described. The method was based on enhancement of CL emission of NBS – eosin system in the presence of streptomycin sulfate. The possible enhancement mechanism was also further investigated. In order to demonstrate the potential of the method, quantitative analysis of streptomycin sulfate in pharmaceutical preparations, and a preliminary pharmacokinetic study of streptomycin sulfate in rats was performed. The experimental results indicated that the method was reliable, simple, rapid and suitable for analysis of streptomycin sulfate in batch biological samples. This work gives a practical application of NBS – eosin CL reaction and will contribute to the determination of streptomycin sulfate in biomedical, pharmaceutical and clinical analysis.
Materials and methods Reagents Eosin was purchased from Beijing Chemical Plant (Beijing, China). NBS was provided by Sinopharm Chemical Reagent Limited Company (Shanghai, China). NaOH, Cl3CCOOH were purchased from Tianjin Chemical Experiment Plant (Tianjin, China). Powder-injection of streptomycin sulfate was purchased from
Shandong Lukang Pharmaceutical Limited Company (Shandong, China). Streptomycin reference substance was provided by the Control of Pharmaceutical and Biological Products (Beijing, China). The stock solution (0.1 mg mL1) was prepared by dissolving 1 mg of streptomycin in 10 mL water and stored in a brown bottle to avoid exposure to light and air. A series of working standard solutions of streptomycin in the range of 8.0 109 to 1.0 106 g mL1 were freshly prepared by dilution of the stock solution of streptomycin (0.1 mg mL1). Dissolving 415.2 mg of eosin in 100 mL water, a working solution of 6 103 mol L1 eosin was obtained. NBS solution (4.0 102 mol L1) was prepared by dissolving 356 mg of NBS in water, and diluting to 50 mL with water. All the reagents were of analytical reagent grade and all solutions were prepared with double-distilled water. Apparatus The CL analysis was conducted on a Model IFFM-E flow-injection chemiluminescence analyser (Xi’an Remex Analysis Instrument Company, Xi’an, China). The schematic diagram of the system was shown in Fig. 1. The steady injection system consists of two peristaltic pumps and a six-way valve. Two peristaltic pumps were used to deliver all the solutions. Polytetrafluoroethylene (PTFE) tubes (0.8 mm i.d.) were employed to connect all the components of the flow system. The CL emission produced in the flow cell (1 mm 25 cm spiral colorless glass tubing, three turns) was detected with a photomultiplier tube (Hamamatsu, Japan). The CL signal was recorded by using an IBM-compatible computer, equipped with a data acquisition interface. The UV–vis absorption spectra of the proposed system were investigated using a UV-2550 spectrophotometer (Shimadzu, Japan) with the wavelength range of 200–800 nm. FI–CL procedures As shown in Fig. 1 of the FI–CL system, the solutions containing oxidant NBS (R1), carrier NaOH (R2), eosin solution (R3) and blank or sample solution (R4) were pumped to flow cell via two peristaltic pumps at a flow rate of 2.84 mL min1 (P1) and 4.27 mL min1 (P2). The eosin solution and blank or sample solution were simultaneously pumped into a mixer and mixed. The mixed solution was carried into flow cell F in front of the PMT and reacted with the oxidant of NBS solution which was quantitatively injected into the carrier stream by a six-way valve in the flow cell F, and the CL emission produced was detected by sending the signal from the PMT to a detector and then to a computing integrator. The concentration of analyte was quantified by measuring the enhanced CL intensity, DI = IsI0, where I0 and Is are CL intensity in the absence and presence of streptomycin, respectively. Sample treatment Powder-injection sample treatment Sample solutions for analysis were prepared as follows. The average powder-injection weight was calculated from five powder-injections of streptomycin sulfate that were randomly selected. They were ground to a homogeneous fine powder. A portion of the powder corresponding to 1.0 mg was weighed and dissolved with 10 mL double-distilled water. The solution was suitably diluted with water before the determination so that the concentrations of the analyte were in the linear range of proposed method.
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Fig. 1. Schematic diagram of FIA-CL manifold used for the determination of streptomycin. R1, NBS solution; R2, NaOH solution; R3, eosin solution; R4, streptomycin solution; P1, P2, Peristaltic pump; V, injection valve; F, flow cell; PMT, Photo-multiplier tube; HV, High voltage; PC, Record computer.
Plasma sample treatment Plasma samples were obtained from blank rats. The protein of 100 lL plasma sample was removed by adding 100 lL 10% trichloroacetic acid (CCl3COOH) in a centrifuge tube, which was thoroughly vortex-mixed for 5 min. After centrifugation at 10,000g for 20 min, the supernatant was collected. Then the supernatant was evaporated to dryness under a stream of nitrogen at 25 °C. The residue was dissolved into 5 mL water for the CL analysis. A blank value was determined by treating streptomycin-free plasma in the same way. Pharmacokinetic study All experimental procedures comply with the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Zhengzhou University Health Science Center. A total of five Sprague–Dawley rats weighing 200 ± 20 g were supplied by the Henan Laboratory Animal Centre (Zhengzhou, China). The rats were maintained in an airconditioned animal room at a temperature of 22 ± 2 °C and relative humidity of 50 ± 10% and kept in the environmentally controlled breeding room for 2 days before starting the experiments. They were fed with standard laboratory food and water and then fasted for 12 h with free access to water prior to the experiments. Streptomycin sulfate prepared with sterile water for injection was administrated to the rats at a dose of 90.0 mg kg1 weight by intramuscular injection. An aliquot of plasma samples was collected in a 1.5 mL heparinized eppendorf tube at different time intervals from the rat eye socket vein. After separation by centrifugation (4000g) for 10 min at 4 °C, the plasma was separated immediately and frozen at 20 °C. Prior to analysis, 100 lL of the plasma was taken and pretreated as described above. Results and discussion Kinetic characteristics of CL system Before carrying out the flow-injection method, the kinetic characteristics of the proposed CL reaction were studied by using the 600
batch method. Upon adding eosin to NBS basic solution gave out a weak CL signal. On injection of the mixture of streptomycin and eosin, a strong CL emission was recorded. The CL kinetic curves for the CL reactions of NBS-eosin and NBS-eosin-streptomycin were shown in Fig. 2. The CL signal from the reaction of eosin with NBS will be the background of the flow-injection analysis. Experiments showed that the proposed CL reaction was a flash type luminescence and the time interval between the start of CL and its maximum is only about 2 s. It can be seen that the significant enhancement on CL peak of NBS-eosin was obtained after injection of streptomycin. This shows that the characteristic of the system is very suitable for FI-CL determination of streptomycin. Condition optimization of the CL system In order to determine the parameters that gave the optimum signal for the analysis of streptomycin, a series of univariate searches were performed on the basis of the net peak height (DI) and the ratio of the signal to the noise (S/N). To obtain a reliable assay of streptomycin, the effects of different chemical and instrumental variables were investigated. The influence of the concentration of NBS was examined in the range of 0.008–0.08 mol L1 (0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.08 mol L1). The result showed that the background noise level (I0) increased significantly with the increase of the concentration of oxidant, and the maximum value of DI and S/N ratio was obtained when 0.04 mol L1 NBS was used for the determination of streptomycin (Fig. 3). As a sensitizer of the proposed CL system, the influence of eosin concentration on the net CL intensity was investigated from 1.0 103 to 1.0 102 mol L1 (1.0 103, 2.0 103, 3 3 3 3 2 4.0 10 , 5.0 10 , 6.0 10 , 8.0 10 , 1.0 10 mol L1) and the result was given in Fig. 4. This figure showed that the maximum net CL intensity was obtained at the eosin concentration of 6.0 103 mol L1. When the eosin concentration was above this level, the net CL intensity decreased slightly. Thus, 6.0 103 mol L1 eosin was chosen for consequent research work. Preliminary studies showed that NBS could react with eosin to produce CL emission in alkaline solution and its CL intensity strongly depends on the concentration of alkaline medium. In the
Streptomycin sample 800
Net CL Intensity
CL Intensity
Blank sample
300
0
0
2
4
6
8
10
12
t (s)
600 400 200 0
0
0.02
0.04
0.06
0.08
0.1
NBS Concentration (mol/L) Fig. 2. Chemiluminescence kinetic curves of NBS-eosin-streptomycin system. Conditions: NBS, 0.04 M; eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
Fig. 3. Effect of NBS concentration on net CL intensity. Conditions: Eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
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800
Net CL Intensity
Net CL Intensity
800 600 400 200 0
0
2
4
6
8
10
P1 P2
600 400 200
12
0
-3
Eosin Concentration (10 mol/L)
0
2
4
6
8
Flow Rate (mL/min) Fig. 4. Effect of eosin concentration on net CL intensity. Conditions: NBS, 0.04 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
present study, NaOH was selected as the alkaline medium, and its concentration was tested in the range of 0.01–0.2 mol L1 (0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2 mol L1) with the presence of 2.0 107 g mL1 streptomycin. As shown in Fig. 5, the net CL intensity increased remarkably with the increase of the NaOH concentration until the latter reached 0.04 mol L1, and then decreased. Hence, 0.04 mol L1 was used as the optimum concentration of NaOH for further test. The total flow rate of the carrier stream and reagent streams was studied over the range from 1.60 to 3.70 mL min1 for P1 and 1.87 to 6.00 mL min1 for P2, respectively. The results were shown in Fig. 6. It can be seen from the figure that the net peak height increased sharply with the increasing flow rate until 2.84 mL min1 for P1 and 4.27 mL min1 for P2 was reached, where the net peak height had the highest value. When higher flow rates were used, DI decreased significantly. Thus, these values were chosen as the optimal flow rates. Analytical performance characteristics Under the optimized experimental conditions as mentioned above, the working curve was obtained. The net peak height (DI) was linearly proportional to the streptomycin concentration (C, g mL1) in the range of 8.0 109–1.0 106 g mL1. The regression equation was obtained as DI = 2.0 109 C–16.176 (r = 0.9997), the relative standard deviation (R.S.D.) was 1.95% for 11 replicate determinations of 2.0 107 g mL1 streptomycin. The detection limit was 2.25 109 g mL1. To the best of the authors’ knowledge, 2.25 ng mL1 is the lowest limit of detection among those already existing CL methods for the determination of streptomycin [25,26]. The proposed FIA method is fast and enables to determine 90 of samples per hour. Interference study In order to evaluate the selectivity of the proposed CL method for the determination of streptomycin, the interferences of various excipients and some possible common foreign species usually present in pharmaceutical preparations were investigated by using a 2.0 107 g mL1 streptomycin solution, which was spiked with
Fig. 6. Effect of flow rate on net CL intensity. Conditions: NBS, 0.04 M; eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL.
increasing amounts of the interfering constituents. A foreign substance was considered as non-interfering if its presence caused an analytical signal variation lower than ±5% regardless of the CL variation noted in its absence, and the results were listed in Table 1. The data showed that there was little interference. Method application Analysis of pharmaceutical preparation The proposed method was applied to the determination of streptomycin content in powder-injections according to the procedure described in Section 2.4.1. The results obtained (Table 2) were in good agreement with the nominal contents. In addition, the accuracy of the results obtained with the proposed method was evaluated through the recovery test. A known amount of standard solution of streptomycin was added to streptomycin sulfate powder-injections and the results were shown in Table 2. The mean recoveries were found to be 97.12–103.84% with RSD values of 0.61–2.07%. The results showed that the accuracy and precision of the proposed method were satisfactory. Analysis of streptomycin in plasma samples The method presented has a low detection limit. Therefore, the proposed method allows the determination of streptomycin in
Table 1 Tolerable concentration ratios with respect to 2.0 107 g/mL streptomycin for some interfering species (<5% error). Foreign interfering substances +
+
CO2 3 ; NO3 ,
Tolerable concentration ratio
K , Na , Ac , Cl , amylum Sucrose, fructose, lactose, glucose, HCO 3 H2 PO4 , b-cyclodextrin Ca2+, SO2 4 Mg2+, Zn2+ EDTA, Cu2+ Ascorbic acid, NHþ 4
1000 500 300 100 10 5 2
Table 2 Results of the analysis of streptomycin in pharmaceutical preparations (n = 5).
Net CL Intensity
700
Sample No.
Labeled value (g)
Measured value (g)
Added (g)
Found (g)
Recovery (%)
RSD (%)
1
1.000
0.997
0.250 0.500 1.000
0.244 0.495 0.989
97.60 99.08 98.91
2.07 1.58 0.61
2
1.000
1.001
0.250 0.500 1.000
0.247 0.511 1.011
98.80 102.20 101.11
1.04 1.71 1.46
3
1.000
0.988
0.250 0.500 1.000
0.259 0.519 0.971
103.68 103.84 97.12
1.52 1.11 1.70
650
600
0
0.05
0.1
0.15
0.2
NaOH Concentration (mol/L) Fig. 5. Effect of NaOH concentration on net CL intensity. Conditions: NBS, 0.04 M; eosin, 6.0 103 M; streptomycin, 2.0 107 g/mL.
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B. Du et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 823–828 Table 3 Determination of streptomycin in plasma samples (n = 3). Sample
Content (107 g/mL)
Added (107 g/ mL)
Found (107 g/ mL)
Recovery (%)
RSD (%)
1
2.161
0.200 1.000 6.000
2.356 3.176 8.216
97.51 101.53 100.92
1.49 0.95 0.77
2
2.220
0.200 1.000 6.000
2.413 3.206 8.146
96.41 98.58 98.76
2.06 2.48 1.83
3
2.034
0.200 1.000 6.000
2.231 3.066 8.176
98.51 103.18 102.37
1.21 1.30 0.98
biological samples. The linearity for plasma analysis was in the range of 4.0 109 to 1.0 106 g mL1 with the regression equation DI = 4.0 109 C-195.69 (r = 0.9995; C, g mL1) with a limit of quantification of 4.48 109 g mL1 (LOQ, which is given as the concentration for which the analytical signal is 10 times higher than standard deviation of blank intensity). The recovery tests were carried out on samples to which three different known amounts of streptomycin were added. The recoveries for plasma at three different concentrations were 96.41–103.18% (Table 3). It is therefore suggested that the present method would be applicable to the pharmacokinetic study of streptomycin. Pharmacokinetic evaluation In this study, five rats were administered 90.0 mg kg1 of streptomycin sulfate for powder-injections by intramuscular injection. Blood samples were collected in a 1.5 mL heparinized eppendorf tube from the rat eye socket vein before administration of the drug and at intervals thereafter (5, 10, 20, 30, 40, 60, 90, 120, 240, 360 min) and the streptomycin content in the plasma was determined by the proposed method. Table 4 gives the corresponding pharmacokinetic parameters calculated using Kinetica 4.4.1 program (Thermo. Fisher Scientific Inc., MA, USA). The mean peak concentration (Cmax) was determined as 375.61 lg mL1 for streptomycin at 21.63 min after dosing. The values of AUC0-t representing the extent of absorption were calculated as 36044.50 mg min1 L1 and the T1/2b was calculated as 82.14 min. The distribution of streptomycin throughout the body conformed to a classical two-compartment open model with first-order absorption. The results were compared to the data obtained by using the method of HPLC for further ensuring the method’s application. From the pharmacokinetic parameters, it can be seen that there was no significant difference between the results obtained by the two methods (P > 0.05). Possible mechanism of CL reaction In order to obtain the possible mechanism of the CL reaction, the following experiments were performed. Various mixed Table 4 Mean pharmacokinetic parameters for streptomycin in healthy rats after administering 90.0 mg kg1 of streptomycin sulfate for powder-injections by intramuscular injection (n = 5). Parameters
CL
HPLC
T1/2a (min) T1/2b (min) Tmax (min) Cmax (lg mL1) CL/F (L kg1 min1) AUC(0t) (mg min1 L1) MRT (min)
18.83 ± 1.24 82.14 ± 3.07 21.63 ± 1.26 375.61 ± 8.54 0.0026 ± 0.0011 36044.50 ± 105.02 92.29 ± 8.21
20.86 ± 0.41 84.28 ± 0.92 22.42 ± 2.83 327.31 ± 29.45 0.0027 ± 0.0009 35907.90 ± 325.88 92.65 ± 2.63
Fig. 7. UV–vis spectra of streptomycin (orange curve), eosin (red curve), NBS-eosin (black curve), NBS-eosin-streptomycin (green curve). Conditions: NBS, 0.04 M; eosin, 6.0 103 M; NaOH, 0.04 M; streptomycin, 2.0 107 g/mL (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
methods were used to arrange the reagents in this CL system. The results indicated that almost no light emission was recorded when only NBS and streptomycin solution were mixed together and weak CL can be produced in NBS and eosin system. When NBS was mixed with streptomycin in the presence of eosin in alkaline condition, there was a luminescence peak at about 545 nm, which is similar to the fluorescence emission maximum of eosin (545 nm) reported in the literature [27]. Thus, it can be seen that the possible luminophor was eosin. The UV–vis spectra of streptomycin, eosin, NBS-eosin and NBS-eosin-streptomycin solutions were further observed in order to explore more details of the possible mechanism. From Fig. 7, it can be seen that streptomycin had absorption near 190 nm (Fig. 7, orange curve) and eosin had absorption peaks near 254, 301, 342 and 516 nm (Fig. 7, red curve). However, the absorption peaks of 254, 301 and 342 nm disappeared after NBS was added into eosin solution, and the maximum absorption at 516 nm decreased (Fig. 7, black curve), which meant NBS may have a redox reaction with eosin. Compared with the UV–vis spectroscopy of NBS-eosin (Fig. 7, black curve), UV–vis spectroscopy of NBS-eosin-streptomycin did not produce any new UV absorption peaks, but obviously enhanced the intensity of the absorption peak (Fig. 7, green curve), which indicated NBS may react with streptomycin. Hence, the UV–vis absorption spectra showed that the signal of NBS-eosin system was enhanced by streptomycin. The chemical structure of streptomycin is based on a streptose connected to streptidine and N-methyl-L-glucosamine in a glycoside linkage (Fig. 8), thus streptomycin is a polyhydroxyl compound. Therefore, streptomycin was extremely easy to be oxidized by some oxidants. In aqueous solutions, the oxidizing
H
OR'
O HO
CH2OH
H H OH
R
H
H
Fig. 8. The structure of streptomycin.
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property of NBS was attributed to hypobromous acid (HBrO) generated by its hydrolysis [28]. After NBS was hydrolyzed to HBrO, HBrO reacted with streptomycin and released energy to eosin. The energy then changed eosin to an activated one. When eosin returned from excited state to ground state, the energy was released as light. Conclusions In the work, a novel FI–CL method was established to determine streptomycin in pharmaceutical preparation and biological samples. The proposed method was used in a pharmacokinetic study, which further confirmed the potential of the method. Compared with previously published reports, the established procedure is simple, rapid and has the advantage of lower detection limit, it also uses low-cost instrumentation, which is easy to use and maintain, with a high sample throughput of 90 h1. These characteristics make the assay suitable not only for monitoring streptomycin plasma levels in animals and patients, but also for the rapid and precise measurement of low concentrations of streptomycin in a complex matrix. This is necessary both in the context of pharmacokinetic studies after a single dose and the ongoing discussion on adverse effects of aminoglycoside antibiotics, as well as impact on the environment. Acknowledgement Financial support from Project of Zhengzhou Science and Technology Innovation Team (Grant number: 121 PCXTD521) is gratefully acknowledged. References [1] L. Han, Y.M. Zhang, J. Kang, J.L. Tang, Y.H. Zhang, J. Pharm. Biomed. 58 (2012) 141–145.
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