Luminol System Yufei Hu,A,B Gongke Lia* and Zhujun Zhangb

Research article

Received: 17 April 2010, Revised: 14 June 2010, Accepted: 20 June 2010, Published online in Wiley Online Library: 23 October 2010

(wileyonlinelibrary.com) DOI 10.1002/bio.1230 A ﬂow injection chemiluminescence method for the determination of lincomycin in serum using a diperiodato-cuprate (III)–luminol system Yufei Hu,a,b Gongke Lia* and Zhujun Zhangb

ABSTRACT: In this paper, the novel trivalent copper–periodate complex {K5[Cu(HIO6)2], DPC} has been applied in a luminol- based chemiluminescence (CL) reaction. Coupled with ﬂow injection (FI) technology, the FI-CL method was proposed for the determination of lincomycin hydrochloride. The CL reaction between luminol and DPC occurred in an alkaline medium. The CL intensity could be greatly enhanced by lincomycin hydrochloride. The relative CL intensity was proportional to the concentration of lincomycin hydrochloride in the range of 1 ¥ 10-8 to 5 ¥ 10-6 gmL-1 and the detection limit was at the 3.5 ¥ 10-9 gmL-1 level. The relative standard deviation at 5 ¥ 10-8 gmL-1 was 1.7% (n = 9). The sensitive method was successfully applied to the direct determination of lincomycin hydrochloride (ng mL-1) in serum. A possible mechanism of the lumonol–DPC CL reaction was discussed by the study of the CL kinetic characteristics and the spectra of CL reaction. The oxidability of DPC was studied by means of its electrochemical response. Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: chemiluminescence; K5[Cu(HIO6)2]; ﬂow injection; lincomycin hydrochloride; serum

Introduction to the determination of lincomycin in pharmaceutical injection with a detection limit of 3.6 ¥ 10-8 gmL-1. There is no distinction Lincomycin [methyl 6,8-dideoxy-6-(1-methyl-4-propyl-2- of the detection limits between the proposed methods in this pyrrolidinecarboxamid-o)-1-thio-d-erythro-a-D-galactooc- study and that in Yang et al. (18). For the different CL systems, topyranoside] produced from Streptomycin Lincolnensis is a there are different mechanisms of reaction. The aim of the basic lincosamides antibiotic (1). It is a well-established broad- present study was to build a sensitive and selective chemilumi- spectrum antibiotic drug used in human and veterinary medi- nescence method for the direct determination of lincomycin in cine. It is particularly active against Gram-positive pathogens. real samples without any pretreatment and to deduce the pos- Lincomycin hydrochloride has been used in various illnesses, sible mechanism based on the electrochemical response data, including oral and upper respiratory tract and skin infections (2). the kinetic characteristics and the spectra of CL reaction. Drug residues from pharmaceutical products in patients may Chemiluminescence is an attractive analytical method that cause allergic reactions or bacterial resistance. Therefore, it is promises higher sensitivity, wider linear ranges and simpler important to develop an efficient analytical method for the instrumentation (19). Until now, CL has attracted increasing direct analysis of lincomycin in biological samples. Various attention in fields such as clinical and pharmaceutical science, determination methods of lincomycin have been proposed, environmental science and food analysis (20). The oxidation reac- including microbiological assay method (3), chemical assay (4) tion of luminol (3-aminophthalhydrazide) in alkaline medium is high-performance liquid chromatography (HPLC) with UV one of the most efficient CL reactions. It has been widely used in detection (5,6), electrochemical detection (7–9) or mass spec- quantitative analysis of many inorganic and organic compounds trometry detection (10–12), gas chromatography (13), capillary (21). In the luminol-based CL system, the commonly used electrophoresis (14,15) and chemiluminescence (16–18). The originally used microbiological assay method and chemical assay are non-specific and less accurate. The structure of lincomycin (Fig. 1) is characterized by poor UV absorbance in the low wavelength range (200–220 nm). Special derivatization pretreat- * Correspondence to: G.Li, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang Xi Road, Guangzhou 510275, PR China. ment is necessary for UV or fluorescence detection. These chro- E-mail: [email protected] matographic methods with spectrometric detection always suffer from limitations such as special instrumentation and com- a School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 plicated and time-consuming pretreatments. The chemilumi- Xingang Xi Road, Guangzhou 510275, People’s Republic of China nescence (CL) method (16) lays particular emphasis on the b Department of Chemistry, Institute of Analytical Science, Southwest Univer- set-up of the electrophoresis–electrochemiluminescence micro- sity, 2 Tiansheng Road, Beibei, Chongqing 400715, People’s Republic of chip, not on the CL system itself. This method (17) was applied China 313

Figure 1. Schematic diagram of the CL-FIA system.

chloride was purchased from Chongqing Institute for Drug Control (Chongqing, China). All chemicals were prepared from analytical-grade material using double distilled water through- out the work. Scheme 1. Structure of lincomycin. The 0.01 mol L-1 luminol stock solution was prepared by dis- solving 1.772 g luminol in 1 L 0.1 mol L-1 carbonate buﬀer and allowed to stand for approximately 24 h before use. The DPC oxidants include hydrogen peroxide, oxygen, potassium perman- stock solution (0.01 mol L-1) was prepared by oxidizing Cu (II) in ganate, ferricyanide, periodate, persulfate, hemoglobin and myo- the alkaline medium according the known method (35). In brief,

globin (22–28). In recent years, there has been renewed interest KIO4 (0.23 g), CuSO4·5H2O (0.125 g), Na2S2O8 (0.14 g) and KOH in the development of new oxidant reagents for the luminol- (0.8 g) were added to 30 mL water. The mixture was heated to based CL reaction, which can widen the application of CL analy- boiling for about 20 min on a hot plate with constant stirring. The sis. Transition metals in uncommon oxidation states such as boiling mixture turned intensely red and the boiling was contin- Ag(III) and Cu(III) have been reported. They can be stabilized by ued for another 20 min for the completion for the reaction. The chelation with suitable polydentate ligands (29,30). The studies mixture was then cooled and diluted to 50 mL with distilled mainly focus on the kinetics and mechanics (31,32) of the oxida- water. The stock solution obtained was stored under refrigeration tion reaction by calculating the rate constant and activation and was found to be fairly stable for several months. DPC solu- parameters. Diperiodatocuprate (III) {K5[Cu(HIO6)2], DPC} is a weak tions were freshly prepared before use. The complex was con- oxidizing agent in alkaline medium with the reduction potential firmed at 415 nm by UV–vis spectrum with a molar absorptivity 0.42 V (vs SCE) (33). For its unique catalysis effect, a sensitive and (e) of 6230 Ϯ 100 L3 mol-1 cm-1. selective CL method for H2O2 analysis has been built based on the reaction of luminol–H2O2–DPC (34). In the paper, it was found that the CL intensity of luminol–DPC Apparatus reaction can be greatly enhanced by lincomycin. DPC was obtained by complexation of trivalent oxidation of copper and The schematic diagram of the flow-injection CL employed in this periodate in strong alkaline medium. The CL reaction of luminol work is shown in Fig. 1. Two peristaltic pumps (HL-2, manufac- with a low concentration of 10-7 mol L-1 oxidized by the trivalent tured at Huxi Instrument and Meter Plant, Qingpu, Shanghai, copper–periodate complex, DPC, occurred in alkaline medium, China) were used to deliver all the chemicals at a flow rate of which made the CL system selective. The enhanced light emis- 2 mL min. The polytetrafluorothylene (PTFE) flow tubes (0.8 mm sion of luminol–DPC by lincomycin can be used for the quantita- i.d.) were used to connect all the components in the system. tive analysis of lincomycin concentration in serum sample. Injection was made using a 16-port injection valve (Hanzhou, Therefore, a novel more sensitive and selective chemilumines- China) equipped with a loop of 75 mL. The CL signal was moni- cence method has been developed for trace analysis of lincomy- tored by a BPCL ultra-weak luminescence analyzer (Institute of cin in serum by coupling flow injection analysis (FIA) techniques Biophysics, Chinese Academy of Science, Beijing, China) consist- without pretreatment of the sample. The electrochemical ing of a flat coil glass flow cell facing the window of the photo- response of DPC was used to describe its oxidation capacity. A multiplier tube (PMT). Data acquisition and treatment were possible mechanism of the proposed CL reaction is discussed by performed with BPCL software running under Windows 98. The the study of the CL kinetic characteristics and the chemilumines- UV-absorbance was detected with a UV–Vis-2001 spectropho- cence spectra. tometer (Hitachi Ltd, Japan). The CL spectrum was obtained with the LS45/55 fluorospectrophotometer (PerkinElmer Ltd, America). A CHI832 electrochemical workstation (ChenHua Ltd, Experimental Shanghai, China) was used to test the electrochemical characteristics of copper metal chelate complex. Reagents and chemical The reagents used were: cupric sulfate (CuSO .H O) and potas- 4 2 Procedure sium hydroxide (KOH) purchased from Chongqing Chemical Reagent Company (Chongqing, China); potassium periodate As shown in Fig. 2, the DPC solution, a standard lincomycin

(KIO4) purchased from Shanghai Chemical Reagent Company hydrochloride solution or a sample containing lincomycin hydro-

(Shanghai, China); and potassium persulfate (Na2S2O8) pur- chloride and luminol solution were respectively delivered chased from Shanghai Aijian Chemical Reagent Company through the system at the rate of 2.0 mL min-1 by the peristaltic 314 (Shanghai, China). The standard medicine of lincomycin hydro- pumps. The stream of DPC solution and luminol solution was

Figure 2. Kinetics curves of luminol–DPC-sample. Figure 4. Eﬀect of the DPC concentration.

Figure 3. Effect of the luminol concentration. Figure 5. Effect of medium pH of luminol solution. mixed and then the whole system was run until a stable baseline Optimization of the experiment procedure was recorded. The analyte was injected by means of the injection valve. The DPC solution carried 75 mL analyte solutions in the The effect of luminol solution. The oxidation of luminol in alka- sampling loop and ran directly through the flow cell, producing a line medium is one of the most important CL reactions. These CL CL emission. The CL signal was then recorded simultaneously. The reactions mostly result from the oxidation reaction of luminol by PMT operated at -800 V. The relative CL intensity DI (defined as typical oxidants, such as oxygen, hydrogen peroxide, potassium, the difference of CL intensity in the presence and in absence of permanganate, ferricyanide, tetravalent cerium ion, lead dioxide analyte) was proportional to the corresponding concentration of and oxygen free radicals. In our previous study (34), DPC was lincomycin hydrochloride solution. different from these other typical oxidants because the emitted CL could be clearly obversed when the luminol was lower than 1.0 ¥10-6 mol L-1. The CL reaction of luminol and those other routine oxidants occurred when the concentration of luminol Results and discussion wasupto1.0¥ 10-5 mol L-1. Therefore the effect of luminol concentration was examined over the range 1 ¥ 10-8 to 5 ¥ Kinetic curve of the CL reaction of luminol–DPC–lincomycin 10-7 mol L-1 when the DPC and lincomycin hydrochloride solu- hydrochloride tion were fixed at a certain concentration. As shown in Fig. 3, the In the primary experiment, the emitted CL was based on the CL intensity increased along with an increase in the luminol con- oxidation reaction of luminol by DPC in the presence of lincomy- centration in the presented range. The avoidance of PMT satura- cin hydrochloride. In batch mode, the typical response curve tion and minimal reagent consumption were considered, and the (intensity vs time) was used to describe the kinetic characteristics optimal concentration of luminol was 1 ¥ 10-7 mol L-1 for the of the CL system. Therefore the experimental parameters were subsequent experiment. kept constant, and the intensity–time curve of luminol–DPC in the presence of lincomycin hydrochloride was recorded as line 1 The effect of DPC solution concentration. Based on the kinet- in Fig. 2. In the same way, the typical response curve of luminol– ics curve of the proposed CL system, it is important to test the DPC in the absence of lincomycin hydrochloride was recorded as effect of DPC solution. A range concentrations of DPC from 1 ¥ line 2 in Fig. 2. From Fig. 3, the CL intensity peak appeared within 10-5 to 1 ¥ 10-3 mol·L-1 was investigated. The relative CL intensity 10 s from the injection of the solution of DPC and lincomycin as a function of the DPC concentration was plotted in Fig. 4. The hydrochloride into the luminol solution. The CL reaction was results showed that the CL intensity was increased along with an quick. The kinetic curve indicated that the rapid CL system was increase in the DPC concentration in a low-concentration range sensitive and the the FI-CL system was suitable for the determi- (5 ¥ 10-4 mol L-1), and reached a maximum. Above 5 ¥ nation of lincomycin hydrochloride. 10-4 mol L-1, the CL intensity decreased instead resulting from the 315

Table 1. Results of recovery tests of lincomycin hydrochloride in human serum

Sample Found Measured value (10-8 gmL-1) Recovery (%) RSD (%, n = 3) (10-8 gmL-1) Added Total found 1 3.25 1.00 4.32 107.0 0.55 3.00 6.18 97.67 0.84 5.00 8.42 103.40 1.71 2 5.43 4.00 9.58 103.75 2.75 6.00 11.36 98.83 3.24 8.00 13.52 101.13 2.16 3 12.06 6.00 18.13 101.17 1.44 10.00 22.15 100.90 2.21 14.00 25.98 99.43 3.71

self-absorption of the DPC with the higher concentration. There- fore the optimal concentration was 5 ¥ 10-4 mol L-1.

The effect of medium condition. It is well known that the CL oxidation of luminol often occurred in basic conditions. The lincomycin solution was acidic with a pH of 3.0–5.5. While DPC was obtained in strong alkaline solution (KOH solution), it is necessary to test the effect of pH medium. A solution of KOH (0.1 mol L-1) was added to the luminol solutions to offer different pH media for the CL system. The effects of pH (8–12) of the medium were investigated (Fig. 5). The result showed that the optimal pH of the reaction was 9.

Figure 6. CL spectrum of luminol–DPC–licomycin system. The analytical characteristic of the FI-CL method

Under the optimum experimental conditions, the calibration essential interferent in a luminol-based CL system because of its graph of change in CL intensity, DI, against lincomycin hydrochlo- catalytical effect on the oxidant of luminol. At a lower concentra- -8 ride concentration was linear in the range 1.0 ¥ 10 to 5.0 ¥ tion of luminol (10-7 mol L-1), the influence of HRP can be ignored, -6 -1 -9 -1 10 gmL with a detection limit of 3.5 ¥ 10 gmL (3s). The as shown in our previous study (34). The interference of protein in regression equation was DI = 48.05 + 214.67c (c being the linco- human serum can be ignored by ultra-filtration of the sample of -1 mycin hydrochloride concentration; mgmL ) over the given human serum. Therefore the present method could be applied range of lincomycin hydrochloride concentration. The relative for the directly determination of lincomycin hydrochloride in -8 -1 standard deviation was 1.7% for 5.0 ¥ 10 gmL lincomycin human serum. hydrochloride (n = 9). A different CL system for the determination of lincomycin (18) has been reported with a detection limit (DL) -9 -1 of 2.5 ¥ 10 gmL . The differences in CL systems result in differ- Analytical applications of the present FIA-CL method ent mechanisms of reaction. The proposed method was applied to the determination of lincomycin hydrochloride in human serum. A 2 mL blank serum Influence of coexisting foreign species sample was collected and transferred to a centrifugal filter unit, then centrifuged at 10,000 rpm for 10 min at 4°C. Then 1 mL fil- In order to apply the proposed method to the determination of trate was transferred to a 100 mL volumetric flask and diluted to lincomycin hydrochloride in a practical sample, the influence of the mark with double-distilled water. A known amount of linco- some possibly coexisting foreign inorganic ions and organic mycin hydrochloride standard solution was added to propose compounds was examined. The interferences studied were con- the recovery test. The obtained results of the recovery test are -8 -1 ducted by analyzing a standard solution of 5 ¥ 10 gmL linco- shown in Table 1. The proposed FI-CL method has been proved mycin hydrochloride solutions, to which varying amounts of sensitive and selective enough to be applied to the direct possible interference were added. The tolerated limit of each determination of lincomycin in serum sample without special foreign species was taken as a relative error not greater than pretreatment. Ϯ5%. The tolerable ratio for foreign species was 1000 for K+,Na+, 2+ 2+ 2+ + 3+ 2+ 4+ - - 2- - 2- Ca ,Mg ,Cu ,Cu,Al ,Cd ,NH ,Cl,Br,C2O4 ,NO3 ,SO4 , IO3-,SeO 2-,F- and Br-, 500 for Fe2+ and Fe3+, 200 for amylum, urea 3 Mechanistic studies of the CL reaction and lactate, 100 for citrate, 10 for lactose, ascorbic acid and lincomycin, 5 for glucose, Mn2+ and uric acid, and 2 for sucrose. For To determine the oxidant characteristic of DPC, we studied the 316 the serum sample, horseradish peroxide (HRP) is regarded as the electrochemical oxidant process of the copper (II) and metape-

Figure 7. The suggested reaction mechanism for the CL reaction between DPC and luminol. riodate anion in strong alkaline solution. A detailed description of Conclusion the electrochemical behavior has been reported in our previous study (36). The obvious oxidation peak at 0.42 V indicated that In the present study, a novel sensitive FI-CL method for the deter- copper(II) could be oxidanted to copper(III). The presence of poly- mination of lincomycin hydrochloride was proposed. It was - based on the enhancement of lincomycin hydrochloride in the dentate ligands (IO4 ) was helpful to stabilize the transitory oxidation state. DE (defined as the difference between oxidation oxidation reaction of DPC with luminol. As a novel oxidant potential and reduction potential) was 88 mV. This proved that reagent, the use of DPC made the oxidation reaction of luminol -7 -1 the oxidant reaction [Cu(II) → Cu(III)] was a single electron trans- occur with a lower concentration level (10 mol L ). The pro- fer process. In the oxidation reaction of DPC, the bivalent posed method proved to be an attractive analytical method complex was regarded as the final product. owing to its higher sensitivity, lower detection limits and wider The CL spectrum of the luminol–DPC system is recorded in linear ranges. The detection limit of lincomycin hydrochloride Fig. 6. Seen from Fig. 6, the fluorescence emission shows an was at the nanogram level. The luminol–DPC CL method has obvious peak at 430 nm when the luminol mixed with DPC. The been successfully applied to the direct determination of lincomy- obvious intensive peak appeared at the same wavelength when cin hydrochloride in serum sample without any pretreatment. the lincomycin joined the luminol–DPC system. This suggested This indicates that Cu(III) is a promoting oxidant in the CL system. that a reaction between DPC and luminol occurred, as well as the Moreover, the Cu(III) can widen the application of the CL method formation of intermediate states (excited aminophthalate) with in further studies. an emission maximum at 425 nm. The CL intensity can be enhanced in present of lincomycin. Based on prior studies (31,35), the formation of Acknowledgments 3- [Cu(H3IO6)2(OH)2] was suggested to be the reactive species of This study was supported by a Science Foundation of China post- water-soluble Cu(III) periodate complex in alkaline medium. From doctoral grant (no. 20080440789). the oxidation reaction of isoniazid (37) by DPC, the oxidation reaction proceeded via formation of a free radical derived from isoniazid. It was speculated that the mechanism of CL reaction for References the luminol has similar benzamide structural features to 1. Chen XQ, Jin YY, Tang G. New Edition of Pharmacology. Beijing: Peo- hydrazide. The mechanism could suffer from free radical inter- ple’s Heath Press, 2007; 94. vention. The CL reaction mechanism of DPC–luminol was pro- 2. National Pharmacopoeia Committee. Pharmacopoeia of the People’s posed as shown in Fig. 7. Republic of China. Part II. Beijing: Chemical Industry Press, 2005; 531. 317

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