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Microflow injection chemiluminescence system with spiral microchannel for the determination of cisplatin in human serum
Analytica Chimica Acta 678 (2010) 135–139
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Microflow injection chemiluminescence system with spiral microchannel for the determination of cisplatin in human serum Xiuzhong Wang, Xuefeng Yin ∗ , Heyong Cheng Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310027, China
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Article history: Received 1 July 2010 Received in revised form 6 August 2010 Accepted 8 August 2010 Available online 13 August 2010 Keywords: Microflow injection Chemiluminescence Cisplatin Serum Platinum
a b s t r a c t A new microflow injection chemiluminescence (FI-CL) system was described for the determination of cisplatin in human serum. By using the microchip with double spiral channel configuration, the sensitivity was greatly enhanced due to more efficient mixing of the analyte and reagent solutions. Experimental results revealed that common ions in human serum, such as Mn2+ , Co2+ , Fe3+ , Cu2+ , Zn2+ , Ni2+ , Na+ , K+ , Ca2+ , Cl− , NO3 − , Ac− , CO3 2− , PO4 3− , SO4 2− did not cause interference with the detection of Pt(II) by using 1,10-phenanthroline as the masking agent. Under the optimized conditions, a linear calibration curve (R2 = 0.998) over the range 2.0 × 10−8 to 2.0 × 10−6 mol L−1 was obtained with the detection limit of 1.24 × 10−9 mol L−1 . The relative standard deviation was found to be 3.46% (n = 12) for 2.0 × 10−7 mol L−1 . The sample consumption was only 2 L with the sample throughput of 72 h−1 . It had been used for trace platinum determination in cisplatin injection and human serum samples after the dosage of cisplatin. The recovery varied from 97.6 to 103.9%. The results proved that the proposed FI-CL system had the advantages of high sensitivity and precision, low sample and reagents consumption, and high analytical throughput. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cis-dichlorodiaminoplatinum(II), clinically known as cisplatin, is a platinum-based drug widely used in the clinical treatment of various cancers, including testicular, ovarian, lung, and head and neck cancer [1]. The success in cisplatin-based chemotherapy, however, strongly depends on how careful the drug’s dosages are monitored in order to reduce severe side-effects, such as nephrotoxicity, orthotoxicity, nausea, vomiting, neuropathy and allergy. For this reason, it is in urgent need to determine the platinum concentrations in biological fluids and tissues after the dosage of cisplatin [2]. A number of analytical methods have been proposed for the determination of platinum in drugs and biological samples, including graphite furnace atomic absorption spectrometry (GFAAS)
Abbreviations: FI-CL, microflow injection chemiluminescence; ICP-MS, inductively coupled plasma mass spectrometry; GFAAS, graphite furnace atomic absorption spectrometry; ICP-AES, inductively coupled plasma atomic emission spectrometry; HPLC, high performance liquid chromatographic; CE, capillary electrophoresis; GC, gas chromatography; FIA, microflow injection analysis; CL, chemiluminescence; SP, syringe pump; SV, six-port multiposition valve; PMT, photomultiplier tube; PTFE, polytetrafluoroethylene; HC, holding coil; FIA, flow injection analysis. ∗ Corresponding author. Tel.: +86 571 87991636; fax: +86 571 87952070. E-mail address: [email protected] (X. Yin). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.08.003
[2–4], inductively coupled plasma atomic emission spectrometry (ICP-AES) [2] and inductively coupled plasma mass spectrometry (ICP-MS) [1,5–8]. Although these methodologies have high sensitivity for the determination of platinum, they require complex and expensive instrumentation. Ultraviolet-visible spectroscopy [9], high performance liquid chromatographic (HPLC) [10,11], capillary electrophoresis (CE) [12], phosphorescence [13] and gas chromatography (GC) [14,15] are also used to detect the content of cisplatin. However, a selective derivatizing reaction is generally required for the detection of cisplatin in biological samples. The derivatizing processes are often tedious and time-consuming. Miniaturization is generally recognized as one of the most important trends in the development of analytical instrumentation. The microflow injection analysis (FIA) system has been developed for its low reagent consumption, reduced analysis time and suitability for miniaturized systems [16,17]. Chemiluminescence (CL) promises high sensitivity with simple instruments and does not need any light source. It has been used extensively in procedures based on FIA. However, to our best knowledge, the determination of cisplatin in human serum with FI-CL system has not been reported. In this paper, a novel, rapid and sensitive FI-CL system for the determination of cisplatin in pharmaceuticals and human serum was described based on platinum catalyzed luminol–hydrogen peroxide reaction in a basic aqueous solution. The effect of microchip
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depth of 50 m and a width of 300 m. Access holes were drilled into the etched plate with a 2.0 mm diameter diamond-tipped drill bit at the terminals of the channels. The first spiral channel was designed for CL reagents mixing with the inlets A, B for luminol and H2 O2 . The second spiral channel was reaction/detection channel, in which CL streams and the sample met together, mixed and chemiluminescent reaction occurred. A third inlet P at the end of the mixing channel prior to the reaction/detection channel was used for the introduction of the sample. At the end of the reaction/detection channel there was an outlet W for waste. Each spiral channel had a length of 10.0 cm from the entrance to the exit and a total volume of 1.5 L. The microchip was masked by black adhesive tape except the reaction/detection channel. Fig. 1. (a) Schematic diagram of the microchip (dimensions are given in mm); (b) schematic diagram of the experimental set-up for Pt(II) determination. SP, syringe pump; HC, holding coil; SV, six-port multiposition valve; R1 , water; R2 –R5 , samples 1–4; M1 , M2 , dual micropump; PMT, photo multiplier tube.
configuration on mixing efficiency was investigated. A spiral channel, in which analyte and CL reagents mixed and reacted, was suggested to enhance the mixing efficiency and the detective sensitivity. 1,10-Phenanthroline was proposed as the masking agent to remove the interference from other metal ions coexisted in the samples. The experimental results proved that the proposed FICL system had the advantages of high sensitivity and precision, low sample and reagents consumption, and high analytical throughput. 2. Materials and methods 2.1. Reagents and samples All the reagents were of analytical grade. Ultrapure water (Milli-Q Plus 185, Millipore Corporation) was used throughout. Luminol (3-aminophthalhydrazide) was purchased from Fluka (Milwaukee, USA) and hydrogen peroxide solution (30%) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 5.0 × 10−4 mol L−1 luminol and 0.02 mol L−1 hydrogen peroxide solution, both in 0.25 mol L−1 Na2 CO3 –NaHCO3 buffer (adjusted to pH 11.5 with 0.1 mol L−1 NaOH solution), were prepared daily. The solution of 0.02 mol L−1 hydrogen peroxide in 0.25 mol L−1 Na2 CO3 –NaHCO3 buffer is stable for at least 6 h. To all sample solutions, 1,10-phenanthroline (Shanghai SSS Reagent Co., Ltd., Shanghai, China) was added to eliminate the interference from other coexisted metal ions with the CL analysis and sodium chloride (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to enhance the CL intensity. Platinum standard solutions containing 3.0 mol L−1 NaCl and 1 × 10−4 mol L−1 1,10-phenanthroline were prepared daily from 1000 mg L−1 atomic absorption standard (Sigma–Aldrich, St. Louis, MO, USA). Cisplatin injection (freeze-dried) was purchased from Qilu pharmaceutical Co., Ltd. (Jinan, China). The blood samples of cancer patients on cisplatin chemotherapy were collected from Zhejiang Provincial People’s Hospital (Hangzhou, China) after infusion 1–2 h of cisplatin. The blood samples were collected with a 5 mL hypodermic syringe by vein puncture. Before use, all the utensils were soaked in 10% HNO3 for 24 h, rinsed thoroughly in Millipore purified water, and all solutions were filtered through 0.45 m membrane filters. 2.2. Microchip design and fabrication The microchip was fabricated on soda-lime glass using photolithographic and wet chemical etching procedures described elsewhere [18]. The channel design of the microchip device used for this work is shown in Fig. 1(a). The channels were etched to a
2.3. Instrumentation The design of the FI-CL system was depicted in Fig. 1(b). It consisted of a microchip, a dual micropump (Harvard 33 dual syringe pump, Harvard apparatus, USA), a syringe pump (SP) with a volume of 25 L injector and a 6-port multiposition valve (SV, Kloehn Co. Ltd., USA) and a photomultiplier tube (PMT, H10493003, Hamamatsu, Japan; control voltage: 1.2 V). The SP combined with the SV was controlled automatically by software written by Visual Basic and used for quantitative introduction of different samples in microliter amount into the microchip. The PMT was fixed beneath the reaction/detection channel as close as possible to the microchip for CL detection. The microchip and PMT were mounted in a light tight box to prevent external light from entering the detector. The dual micropump with polytetrafluoroethylene (PTFE) tubing (i.d. 0.5 mm, o.d. 1.5 mm) were used to drive luminol and H2 O2 streams through the microfluidic manifold via inlets A and B of the microchip. A length of 20 cm PTFE tubing of the same dimension bridged the SP and central port of the SV as the holding coil (HC). Ports 1–5 with PTFE tubing were used for sampling water and different samples (R1 –R4 ) via the SV into the holding coil. To reduce the dead volume, a 10 cm long flexible tube of 75 m i.d., and 1.5 mm o.d., which was made by inserting a quartz capillary (i.d. 75 m, o.d. 375 m) full way into a PTFE tubing (i.d. 0.5 mm, o.d. 1.5 mm) and sealing the gap between the capillary and PTFE tubing at both ends with glue, was used to connect port 6 and the inlet P of the microchip. ICP-MS instrument: The reference results were determined by ICP-MS (Thermo fishier XII, USA) method. 2.4. Experimental methods Holding coil and the flexible tube used to connect port 6 and the inlet P of the microchip were filled with the ultrapure water before the experiment. 5.0 × 10−4 mol L−1 luminol and 2.0 × 10−2 mol L−1 H2 O2 were pushed continuously to the microchip by the dual micropump with the individual flow rate of 50 L min−1 (100 L min−1 total). After the CL background signal of luminol and H2 O2 was stable, 18 L ultrapure water was aspirated within 25 s into the HC by SP as carrier with SV in position 1. Then, 2 L solution of sample 1 was aspirated within 5 s into the HC with SV in position 2. Finally, with SV in position 6, the aspirated liquids in the HC were pushed into the microchip via inlet P in 12 s. The sample mixed and reacted with the CL reagents in the reaction/detection channel, the emitted light was detected by the PMT and recorded by a computer. A sampling frequency of 72 h−1 was achieved with 2 L sample volume. By switching SV in position 3, 4 or 5 instead position 2 and repeating the procedure above, other three different samples (samples 2–4) could be determined, respectively.
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2.5. Sample digestion 2.5.1. Cisplatin injection Solid (1 mg) from cisplatin injection (freeze-dried) containing 0.05 mg cisplatin was added hydrochloric acid (2 mL, 37%) and contents were mixed well. The solution was heated to near dryness and the residue was dissolved in water [15]. The volume was adjusted to 50 mL with 3.0 mol L−1 NaCl containing a specified volume of 1,10phenanthroline for determination. The amount of platinum in the solution was evaluated from calibration curve. 2.5.2. Human serum Methanol (0.5 mL) was added into the serum sample (0.5 mL) and the mixture was centrifuged at 12,000 rpm for 15 min. Supernatant was transferred in a beaker and hydrochloric acid (2 mL, 37%) was added. The contents were heated to near dryness and the residue was dissolved in ultrapure water [15]. The volume was adjusted to 50 mL with 3.0 mol L−1 NaCl containing a specified volume of 1,10-phenanthroline for determination. The amount of platinum in serum was evaluated from calibration curve. 3. Results and discussion 3.1. Effect of the microchannel configuration on the CL intensity Chemiluminescence (CL) detection provides low background with excellent sensitivity. The optical systems for CL detection are simple and cheap. In addition, many CL reactions have been well characterized for a variety of species from the beginning of the last century. Therefore, CL detection has been used extensively as a detector in flow injection analysis (FIA) [19]. However, the intensity of chemiluminescence is proportional to the rate of the CL reaction rather than to the concentration of the product. In order to perform chemiluminescent reactions on a microfluidic chip, the analyte and CL reagents must be combined and mixed thoroughly. It has been reported that the flow in micro-scale conduits is laminar with Reynolds numbers well below the threshold for tur-
Fig. 2. An illustration of the microchannel configuration (dimensions are given in mm). (a) Double serpentine, (b) serpentine-spiral, (c) double spiral.
bulence. To achieve efficient mixing of the analyte and CL reagents in the detection conduit, different flow-cell designs have been explored and reviewed. These flow-through cells have achieved more sensitive chemiluminescence detection in flow analysis, but the manufacturing process was rather complex [20]. In our previous report [21], we have used a planar spiral channel to enhance the sensitivity of CL detection, which could be simply constructed using a single planar lithography step. In this paper, the planar spiral channel was modified and three microchannel configurations composed of serpentine channel and spiral channel as shown in Fig. 2 were designed and tested. To compare their mixing efficiency, NaOH and phenolphthalein solutions were introduced at the volumetric flow rate of 50 L min−1 each from A and B holes, respectively. The linear flow rate can be calculated to be 11 cm s−1 . Phenolphthalein is a pH indicator that changed its color from transparent to red when it mixed with NaOH solution. The color change profile indicates the mixing level of the two streams, which was calculated using the following equation. Mixing level = width of red color/width of chan-
Fig. 3. Comparison of mixing behaviour in different channels within the same distance of 10.0 cm with the same flow rate of 50 L min−1 . (a) Serpentine channel and (b) spiral channel. (c) Mixing intensity within serpentine channel (1) and spiral channel (2) as a function of channel length. Both channels had the same cross-sectional dimensions of 50 m × 300 m.
Fig. 4. Effect of width of the spiral channel on CL intensity. Experimental conditions: 1.0 × 10−7 mol L−1 Pt (II); 0.25 mol L−1 carbonate buffer, pH 11.5; 5.0 × 10−4 mol L−1 luminol; 0.02 mol L−1 H2 O2 .
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Fig. 5. (a) Effect of flow rate of CL reagents on CL intensity; (b) effect of flow rate of CL reagents on the peak shape: (1) 10 L min−1 , (2) 50 L min−1 , (3) 70 L min−1 . For other conditions, see Fig. 4.
nel × 100%. The images shown in Fig. 3 demonstrated that in the spiral channel, mixing level of up to 95% can be achieved, while less than 10% in the serpentine microchannel within the same distance of 10.0 cm. With the use of FI-CL methodology described in Section 2.4, the chemiluminescence intensities from the reaction of 1.0 × 10−7 mol L−1 Pt(II) with basic luminol–hydrogen peroxide using different microchannel configurations were further tested. The double spiral configuration (Fig. 2c) gave the greatest signal, about 20% higher than the serpentine-spiral configuration (Fig. 2b) and 110% higher than the double serpentine configuration (Fig. 2a). The results indicated that the spiral channel allowed greater quantities of light to be generated than a serpentine channel, due to more efficient mixing of the analyte and CL reagents within the detection channel. In spiral channels, transverse Dean flows arisen as a result of centrifugal effects experienced by fluids traveling along a curved trajectory. The centrifugal effects offered the attractive possibility of providing enhanced mixing [22]. The dependence of the CL intensity upon the width of the spiral channels was further investigated in the range of 150–400 m. The results are shown in Fig. 4, which demonstrated that the CL intensities exhibited maximum at the width of 300 m. In theory, a larger channel would allow more molecules to react and produce more photons within the measurable area of the PMT. However, with an increase in the width of channels, depth/width ratios of the channels decreased. The lower aspect ratio strongly suppressed vortex formation in the spiral channels [23] and reduced the mixing efficiency. Therefore, the double spiral configuration with the width of 300 m was used in the following experiments. 3.2. Effect of the flow rate of reagents on the CL intensity In order to ensure that the highest CL intensity appeared in the limited length of reaction/detection channel, the flow rates of CL reagents were optimized in the range of 10–70 L min−1 . The result is shown in Fig. 5(a). It can be seen that the CL intensity increased with increasing the flow rate, and reached a maximum at the flow rate of 50 L min−1 . Further increasing the flow rate to 70 L min−1 , the CL intensity decreased. It has been reported that the metal ion-catalyzed luminol–hydrogen peroxide reaction is rapid with the maximum peak height of the transient CL signal reached in approximately 500 ms [24]. With increasing the flow rate from 10 to 50 L min−1 , the mixing efficiency of the reactants improved, which was beneficial for increasing the CL intensity. However, the residence time of the reactants in the reaction/detection channel decreased with increasing the flow rate. When the flow rate increased from 50 to 70 L min−1 , the maximum emission did not occur in the limited resident time frame, resulting in the decrease of CL intensity.
The flow rate of the CL reagents had effects not only on the CL intensities, but also on the peak shape. As shown in Fig. 5(b) 1–3, peak tailing was serious at the flow rate of 10 L min−1 and peak width became narrow gradually when the flow rate increased. A symmetrical peak without tailing was obtained at a flow rate of 70 L min−1 , but the peak high reduced to some extent. Thus a flow rate of 50 L min−1 was used in the following experiments. 3.3. Investigation of interferences Ethylene diamine tetraacetic acid, tartaric acid, citric acid and 1,10-phenanthroline were tested as masking agents. The only potential masking agent was 1,10-phenanthroline, since 1.0 × 10−3 mol L−1 1,10-phenanthroline had almost no effect on the CL intensity of the platinum. Therefore, the interference of some common chemicals in human serum, such as Mn2+ , Co2+ , Fe3+ , Cu2+ , Zn2+ , Ni2+ , Na+ , K+ , Ca2+ , were investigated by adding relative chemicals to the solutions containing 1.0 × 10−7 mol L−1 Pt(II) and 1.0 × 10−4 mol L−1 1,10-phenanthroline. The tolerable concentration ratios for interference at the 5% level were over 10,000 for Na+ , K+ , Ca2+ ; 200 for Mn2+ , Pb2+ , 75 for Ni2+ , Fe2+ , Zn2+ ; 40 for Fe3+ , Cu2+ ; 20 for Co2+ , which were high enough to remove the interference. Anions have almost no any effect on Pt(II) determination. The tolerable concentration ratios were found to be over 10,000 for Cl− , NO3 − , Ac− , CO3 2− , PO4 3− and 800 for SO4 2− . The interference from the serum matrix was also been investigated. The amount of Pt(II) in the blank serum sample was undetectable. After digestion, Pt(II) standard solution was added in the blank serum sample to 3.00 mol L−1 , 2.95 mol L−1 could be recovered with the relative standard deviation of 2.46% (n = 3). The result indicated that after digestion the serum matrix had no significant interference on the determination of Pt(II). Thus, 1.0 × 10−4 mol L−1 1,10-phenanthroline was used as the masking agent. 3.4. Optimization of CL reaction conditions Since the optimal pH for metal ions to catalyze the oxidation reaction of luminol by H2 O2 is 11.0–11.8 [25] and carbonate buffer can enhance the CL signal in luminol–H2 O2 system [17], a 0.25 mol L−1 carbonate buffer with pH 11.5 was used to prepare the CL solutions. The bromide and chloride ion often acted as enhancers in the chemiluminescence of trace metal ion-catalyzed luminol oxidation by hydrogen peroxide [17,26]. The addition of chloride significantly enhanced the CL signal under our investigation. The effect of chloride concentration was examined between 0.10 and 3.50 mol L−1 . The results indicated that the CL signal reached a maximum and was stable within the concentration range of 2.50–3.50 mol L−1 . In this work, 3.0 mol L−1 NaCl was added in both standard solutions and samples to enhance the sensitivity.
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Table 1 Results of the determination of platinum in pharmaceutical and serum samples. Samples
Cisplatin injection S1 S2 S3 S4 S5 S6 S7 S8 S9 S 10
Proposed method
ICP-MS
Added (mol L−1 )
Found
Recovery (%)
Content (mol L−1 )
RSD (%) (n = 3)
Content (mol L−1 )
RSD (%) (n = 3)
Content (mol L−1 )
RSD (%) (n = 3)
3.08
2.22
3.15
2.04
3.00
6.02
2.20
98.1
2.81 2.75 5.48 4.25 3.23 2.51 3.41 2.63 1.87 3.26
2.52 2.05 2.01 2.29 2.26 2.31 2.28 2.20 2.14 2.20
3.64 2.77 5.49 4.31 3.28 2.82 3.95 2.92 2.26 3.54
2.18 2.01 1.90 1.87 1.86 2.13 1.89 1.97 2.03 1.98
3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00
5.80 5.70 8.60 7.30 6.25 5.45 6.50 5.73 4.85 6.80
2.28 2.25 1.95 2.00 2.06 2.30 2.04 2.07 2.34 2.10
99.5 98.4 102.1 101.2 100.7 97.6 102.6 103.9 98.9 98.1
The effects of luminol and H2 O2 concentration on the CL intensity were examined. The hydrogen peroxide concentration was kept at 0.01 mol L−1 while luminol concentration varied from 5.0 × 10−5 to 1.0 × 10−3 mol L−1 . The CL intensity was maximum when luminol concentration was 5.0 × 10−4 mol L−1 . When the luminol concentration was kept at 5.0 × 10−4 mol L−1 , the concentration of H2 O2 varied from 1.0 × 10−3 to 0.05 mol L−1 , a maximum CL intensity was achieved with the optimal concentration of H2 O2 to be 0.02 mol L−1 . 3.5. Performance of the FI-CL system Under the optimized conditions, a linear response was obtained over the range 2.0 × 10−8 to 2.0 × 10−6 mol L−1 with the regression equation of C = 1.67 × 10−9 I − 3.05 × 10−8 (r2 = 0.998, C: platinum (mol L−1 ), I: CL intensity (mV)). The relative standard deviation was found to be 3.46% (n = 12) for 0.2 mol L−1 with the detection limit of 1.24 × 10−9 mol L−1 (S/N = 3). The sample consumption was only 2 L, from which it can be calculated the absolute detection limit of 2.48 × 10−15 mol could be achieved. The sample throughput was 72 h−1 . 3.6. Application The sample solutions prepared in Section 2.5 were divided exactly in two equal parts. To one of the parts, 3.0 × 10−6 mol L−1 Pt(II) was added and both parts were analyzed by the proposed method. The amount of Pt(II) and recovery were calculated from the calibration curve and listed in Table 1. The recovery of the proposed method varied from 97.6 to 103.9%. The results indicated the proposed method could be used for the determination of cisplatinum concentration in the cisplatin injection and the serum samples of cancer patients. To further evaluate the sample pretreatment process of the proposed method, these samples were also analyzed by ICP-MS method and the corresponding results were also shown in Table 1. Since total platinum content was measured by ICP-MS, while only Pt(II) by the proposed CL method, the concentrations determined by the CL method are lower than that determined by ICP-MS. Nevertheless, most of the values determined were in accordance with ICP-MS method. This indicated that the biodegradation products of cisplatin in cancer patients may not be fully converted to detectable Pt(II) by the sample pretreatment process. 4. Conclusions The proposed FI-CL system was proven to be suitable for the determination of cisplatinum in the serum samples of cancer patients. The results indicated that the interference from the
common ions in human serum could be removed by using 1,10phenanthroline as the masking agent. Besides the high sensitivity and simple instrumentation, the system also offers other advantages, such as low sample and reagents consumption, and high sample throughput. These merits make the proposed system attractive for rapid point-of-care testing in the future. Acknowledgements This work was supported by the Zhejiang Provincial Natural Science Foundation of China under project No. Z407029, the National Natural Science Foundation of China under project No. 20890020, and the National Key Basic Research and Development (973) Program (No. 2007CB714503) of China. References [1] D.N. Bell, J.J. Liu, M.D. Tingle, M.J. McKeage, J. Chromatogr. B 837 (2006) 29–34. [2] M.E. Bosch, A.J.R. Sanchez, F.S. Rojas, C.B. Ojeda, J. Pharmaceut. Biomed. 47 (2008) 451–459. [3] M. Verschraagen, K. van der Born, T.H.U. Zwiers, W.J.F. van der Vijgh, J. Chromatogr. B 772 (2002) 273–281. [4] Y. Wang, S. Tinsen, Chin. Pharm. J. 30 (1995) 618–620. [5] D.G. Sar, M. Montes-Bayon, L.A. Ortiz, E. Blanco-Gonzalez, L.M. Sierra, A. SanzMedel, Anal. Bioanal. Chem. 390 (2008) 37–44. [6] D. Pozebon, V.L. Dressler, A. Matusch, J.S. Becker, Int. J. Mass Spectrom. 272 (2008) 57–62. [7] A. Standler, G. Koellensperger, W. Buchberger, G. Stingeder, S. Hann, Electrophoresis 28 (2007) 3492–3499. [8] V. Nischwitz, B. Michalke, A. Kettrup, J. Chromatogr. A 1016 (2003) 223–234. [9] B. Anilanmert, G. Yalcin, F. Arioz, E. Dolen, Anal. Lett. 34 (2001) 113–123. [10] S.N. Lanjwani, R.K. Zhu, M.Y. Khuhawar, Z.F. Ding, J. Pharmaceut. Biomed. 40 (2006) 833–839. [11] M.Y. Khuhawar, G.M. Arain, Talanta 66 (2005) 34–39. [12] Z. Huang, A.R. Timerbaev, B.K. Keppler, T. Hirokawa, J. Chromatogr. A 1106 (2006) 75–79. [13] R.A. Baumann, C. Gooijer, N.H. Velthorst, R.W. Frei, I. Klein, W.J.F. Vandervijgh, J. Pharmaceut. Biomed. 5 (1987) 165–170. [14] M.Y. Khuhawar, A.A. Memon, M.I. Bhanger, Chromatographia 49 (1999) 249–252. [15] A.J. Laghari, M.Y. Khuhawar, L.A. Zardari, A.G. Bhatti, Chromatographia 67 (2008) 749–754. [16] D.Y. He, Z.J. Zhang, Y. Huang, Y.F. Hu, Food Chem. 101 (2007) 667–672. [17] W. Som-Aum, J. Threeprom, H.F. Li, J.-M. Lin, Talanta 71 (2007) 2062–2068. [18] Y. Sun, X.F. Yin, J. Chromatogr. A 1117 (2006) 228–233. [19] J.M. Terry, J.L. Adcock, D.C. Olson, D.K. Wolcott, C. Schwanger, L.A. Hill, N.W. Barnett, P.S. Francis, Anal. Chem. 80 (2008) 9817–9821. [20] S. Mohr, J.M. Terry, J.L. Adcock, P.R. Fielden, et al., Analyst 134 (2009) 2233–2238. [21] X.Z. Wang, X.F. Yin, H.Y. Cheng, H. Shen, Analyst 135 (2010) 1663–1671. [22] A.P. Sudarsan, V.M. Ugaz, Lab Chip 6 (2006) 74–82. [23] J. Peter, B. Howell, D.R. Mott, J.P. Golden, F.S. Ligler, Lab Chip 4 (2004) 663–669. [24] A.A. Alwarthan, A. Townshend, Anal. Chim. Acta 196 (1987) 135–140. [25] B. Yan, P.J. Worsfold, Anal. Chim. Acta 236 (1990) 287–292. [26] C.A. Chang, H.H. Patterson, Anal. Chem. 52 (1980) 653–656.
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Microflow injection chemiluminescence system with spiral microchannel for the determination of cisplatin in human serum Xiuzhong Wang, Xuefeng Yin ∗ , Heyong Cheng Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310027, China
a r t i c l e
i n f o
Article history: Received 1 July 2010 Received in revised form 6 August 2010 Accepted 8 August 2010 Available online 13 August 2010 Keywords: Microflow injection Chemiluminescence Cisplatin Serum Platinum
a b s t r a c t A new microflow injection chemiluminescence (FI-CL) system was described for the determination of cisplatin in human serum. By using the microchip with double spiral channel configuration, the sensitivity was greatly enhanced due to more efficient mixing of the analyte and reagent solutions. Experimental results revealed that common ions in human serum, such as Mn2+ , Co2+ , Fe3+ , Cu2+ , Zn2+ , Ni2+ , Na+ , K+ , Ca2+ , Cl− , NO3 − , Ac− , CO3 2− , PO4 3− , SO4 2− did not cause interference with the detection of Pt(II) by using 1,10-phenanthroline as the masking agent. Under the optimized conditions, a linear calibration curve (R2 = 0.998) over the range 2.0 × 10−8 to 2.0 × 10−6 mol L−1 was obtained with the detection limit of 1.24 × 10−9 mol L−1 . The relative standard deviation was found to be 3.46% (n = 12) for 2.0 × 10−7 mol L−1 . The sample consumption was only 2 L with the sample throughput of 72 h−1 . It had been used for trace platinum determination in cisplatin injection and human serum samples after the dosage of cisplatin. The recovery varied from 97.6 to 103.9%. The results proved that the proposed FI-CL system had the advantages of high sensitivity and precision, low sample and reagents consumption, and high analytical throughput. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cis-dichlorodiaminoplatinum(II), clinically known as cisplatin, is a platinum-based drug widely used in the clinical treatment of various cancers, including testicular, ovarian, lung, and head and neck cancer [1]. The success in cisplatin-based chemotherapy, however, strongly depends on how careful the drug’s dosages are monitored in order to reduce severe side-effects, such as nephrotoxicity, orthotoxicity, nausea, vomiting, neuropathy and allergy. For this reason, it is in urgent need to determine the platinum concentrations in biological fluids and tissues after the dosage of cisplatin [2]. A number of analytical methods have been proposed for the determination of platinum in drugs and biological samples, including graphite furnace atomic absorption spectrometry (GFAAS)
Abbreviations: FI-CL, microflow injection chemiluminescence; ICP-MS, inductively coupled plasma mass spectrometry; GFAAS, graphite furnace atomic absorption spectrometry; ICP-AES, inductively coupled plasma atomic emission spectrometry; HPLC, high performance liquid chromatographic; CE, capillary electrophoresis; GC, gas chromatography; FIA, microflow injection analysis; CL, chemiluminescence; SP, syringe pump; SV, six-port multiposition valve; PMT, photomultiplier tube; PTFE, polytetrafluoroethylene; HC, holding coil; FIA, flow injection analysis. ∗ Corresponding author. Tel.: +86 571 87991636; fax: +86 571 87952070. E-mail address: [email protected] (X. Yin). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.08.003
[2–4], inductively coupled plasma atomic emission spectrometry (ICP-AES) [2] and inductively coupled plasma mass spectrometry (ICP-MS) [1,5–8]. Although these methodologies have high sensitivity for the determination of platinum, they require complex and expensive instrumentation. Ultraviolet-visible spectroscopy [9], high performance liquid chromatographic (HPLC) [10,11], capillary electrophoresis (CE) [12], phosphorescence [13] and gas chromatography (GC) [14,15] are also used to detect the content of cisplatin. However, a selective derivatizing reaction is generally required for the detection of cisplatin in biological samples. The derivatizing processes are often tedious and time-consuming. Miniaturization is generally recognized as one of the most important trends in the development of analytical instrumentation. The microflow injection analysis (FIA) system has been developed for its low reagent consumption, reduced analysis time and suitability for miniaturized systems [16,17]. Chemiluminescence (CL) promises high sensitivity with simple instruments and does not need any light source. It has been used extensively in procedures based on FIA. However, to our best knowledge, the determination of cisplatin in human serum with FI-CL system has not been reported. In this paper, a novel, rapid and sensitive FI-CL system for the determination of cisplatin in pharmaceuticals and human serum was described based on platinum catalyzed luminol–hydrogen peroxide reaction in a basic aqueous solution. The effect of microchip
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depth of 50 m and a width of 300 m. Access holes were drilled into the etched plate with a 2.0 mm diameter diamond-tipped drill bit at the terminals of the channels. The first spiral channel was designed for CL reagents mixing with the inlets A, B for luminol and H2 O2 . The second spiral channel was reaction/detection channel, in which CL streams and the sample met together, mixed and chemiluminescent reaction occurred. A third inlet P at the end of the mixing channel prior to the reaction/detection channel was used for the introduction of the sample. At the end of the reaction/detection channel there was an outlet W for waste. Each spiral channel had a length of 10.0 cm from the entrance to the exit and a total volume of 1.5 L. The microchip was masked by black adhesive tape except the reaction/detection channel. Fig. 1. (a) Schematic diagram of the microchip (dimensions are given in mm); (b) schematic diagram of the experimental set-up for Pt(II) determination. SP, syringe pump; HC, holding coil; SV, six-port multiposition valve; R1 , water; R2 –R5 , samples 1–4; M1 , M2 , dual micropump; PMT, photo multiplier tube.
configuration on mixing efficiency was investigated. A spiral channel, in which analyte and CL reagents mixed and reacted, was suggested to enhance the mixing efficiency and the detective sensitivity. 1,10-Phenanthroline was proposed as the masking agent to remove the interference from other metal ions coexisted in the samples. The experimental results proved that the proposed FICL system had the advantages of high sensitivity and precision, low sample and reagents consumption, and high analytical throughput. 2. Materials and methods 2.1. Reagents and samples All the reagents were of analytical grade. Ultrapure water (Milli-Q Plus 185, Millipore Corporation) was used throughout. Luminol (3-aminophthalhydrazide) was purchased from Fluka (Milwaukee, USA) and hydrogen peroxide solution (30%) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 5.0 × 10−4 mol L−1 luminol and 0.02 mol L−1 hydrogen peroxide solution, both in 0.25 mol L−1 Na2 CO3 –NaHCO3 buffer (adjusted to pH 11.5 with 0.1 mol L−1 NaOH solution), were prepared daily. The solution of 0.02 mol L−1 hydrogen peroxide in 0.25 mol L−1 Na2 CO3 –NaHCO3 buffer is stable for at least 6 h. To all sample solutions, 1,10-phenanthroline (Shanghai SSS Reagent Co., Ltd., Shanghai, China) was added to eliminate the interference from other coexisted metal ions with the CL analysis and sodium chloride (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to enhance the CL intensity. Platinum standard solutions containing 3.0 mol L−1 NaCl and 1 × 10−4 mol L−1 1,10-phenanthroline were prepared daily from 1000 mg L−1 atomic absorption standard (Sigma–Aldrich, St. Louis, MO, USA). Cisplatin injection (freeze-dried) was purchased from Qilu pharmaceutical Co., Ltd. (Jinan, China). The blood samples of cancer patients on cisplatin chemotherapy were collected from Zhejiang Provincial People’s Hospital (Hangzhou, China) after infusion 1–2 h of cisplatin. The blood samples were collected with a 5 mL hypodermic syringe by vein puncture. Before use, all the utensils were soaked in 10% HNO3 for 24 h, rinsed thoroughly in Millipore purified water, and all solutions were filtered through 0.45 m membrane filters. 2.2. Microchip design and fabrication The microchip was fabricated on soda-lime glass using photolithographic and wet chemical etching procedures described elsewhere [18]. The channel design of the microchip device used for this work is shown in Fig. 1(a). The channels were etched to a
2.3. Instrumentation The design of the FI-CL system was depicted in Fig. 1(b). It consisted of a microchip, a dual micropump (Harvard 33 dual syringe pump, Harvard apparatus, USA), a syringe pump (SP) with a volume of 25 L injector and a 6-port multiposition valve (SV, Kloehn Co. Ltd., USA) and a photomultiplier tube (PMT, H10493003, Hamamatsu, Japan; control voltage: 1.2 V). The SP combined with the SV was controlled automatically by software written by Visual Basic and used for quantitative introduction of different samples in microliter amount into the microchip. The PMT was fixed beneath the reaction/detection channel as close as possible to the microchip for CL detection. The microchip and PMT were mounted in a light tight box to prevent external light from entering the detector. The dual micropump with polytetrafluoroethylene (PTFE) tubing (i.d. 0.5 mm, o.d. 1.5 mm) were used to drive luminol and H2 O2 streams through the microfluidic manifold via inlets A and B of the microchip. A length of 20 cm PTFE tubing of the same dimension bridged the SP and central port of the SV as the holding coil (HC). Ports 1–5 with PTFE tubing were used for sampling water and different samples (R1 –R4 ) via the SV into the holding coil. To reduce the dead volume, a 10 cm long flexible tube of 75 m i.d., and 1.5 mm o.d., which was made by inserting a quartz capillary (i.d. 75 m, o.d. 375 m) full way into a PTFE tubing (i.d. 0.5 mm, o.d. 1.5 mm) and sealing the gap between the capillary and PTFE tubing at both ends with glue, was used to connect port 6 and the inlet P of the microchip. ICP-MS instrument: The reference results were determined by ICP-MS (Thermo fishier XII, USA) method. 2.4. Experimental methods Holding coil and the flexible tube used to connect port 6 and the inlet P of the microchip were filled with the ultrapure water before the experiment. 5.0 × 10−4 mol L−1 luminol and 2.0 × 10−2 mol L−1 H2 O2 were pushed continuously to the microchip by the dual micropump with the individual flow rate of 50 L min−1 (100 L min−1 total). After the CL background signal of luminol and H2 O2 was stable, 18 L ultrapure water was aspirated within 25 s into the HC by SP as carrier with SV in position 1. Then, 2 L solution of sample 1 was aspirated within 5 s into the HC with SV in position 2. Finally, with SV in position 6, the aspirated liquids in the HC were pushed into the microchip via inlet P in 12 s. The sample mixed and reacted with the CL reagents in the reaction/detection channel, the emitted light was detected by the PMT and recorded by a computer. A sampling frequency of 72 h−1 was achieved with 2 L sample volume. By switching SV in position 3, 4 or 5 instead position 2 and repeating the procedure above, other three different samples (samples 2–4) could be determined, respectively.
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2.5. Sample digestion 2.5.1. Cisplatin injection Solid (1 mg) from cisplatin injection (freeze-dried) containing 0.05 mg cisplatin was added hydrochloric acid (2 mL, 37%) and contents were mixed well. The solution was heated to near dryness and the residue was dissolved in water [15]. The volume was adjusted to 50 mL with 3.0 mol L−1 NaCl containing a specified volume of 1,10phenanthroline for determination. The amount of platinum in the solution was evaluated from calibration curve. 2.5.2. Human serum Methanol (0.5 mL) was added into the serum sample (0.5 mL) and the mixture was centrifuged at 12,000 rpm for 15 min. Supernatant was transferred in a beaker and hydrochloric acid (2 mL, 37%) was added. The contents were heated to near dryness and the residue was dissolved in ultrapure water [15]. The volume was adjusted to 50 mL with 3.0 mol L−1 NaCl containing a specified volume of 1,10-phenanthroline for determination. The amount of platinum in serum was evaluated from calibration curve. 3. Results and discussion 3.1. Effect of the microchannel configuration on the CL intensity Chemiluminescence (CL) detection provides low background with excellent sensitivity. The optical systems for CL detection are simple and cheap. In addition, many CL reactions have been well characterized for a variety of species from the beginning of the last century. Therefore, CL detection has been used extensively as a detector in flow injection analysis (FIA) [19]. However, the intensity of chemiluminescence is proportional to the rate of the CL reaction rather than to the concentration of the product. In order to perform chemiluminescent reactions on a microfluidic chip, the analyte and CL reagents must be combined and mixed thoroughly. It has been reported that the flow in micro-scale conduits is laminar with Reynolds numbers well below the threshold for tur-
Fig. 2. An illustration of the microchannel configuration (dimensions are given in mm). (a) Double serpentine, (b) serpentine-spiral, (c) double spiral.
bulence. To achieve efficient mixing of the analyte and CL reagents in the detection conduit, different flow-cell designs have been explored and reviewed. These flow-through cells have achieved more sensitive chemiluminescence detection in flow analysis, but the manufacturing process was rather complex [20]. In our previous report [21], we have used a planar spiral channel to enhance the sensitivity of CL detection, which could be simply constructed using a single planar lithography step. In this paper, the planar spiral channel was modified and three microchannel configurations composed of serpentine channel and spiral channel as shown in Fig. 2 were designed and tested. To compare their mixing efficiency, NaOH and phenolphthalein solutions were introduced at the volumetric flow rate of 50 L min−1 each from A and B holes, respectively. The linear flow rate can be calculated to be 11 cm s−1 . Phenolphthalein is a pH indicator that changed its color from transparent to red when it mixed with NaOH solution. The color change profile indicates the mixing level of the two streams, which was calculated using the following equation. Mixing level = width of red color/width of chan-
Fig. 3. Comparison of mixing behaviour in different channels within the same distance of 10.0 cm with the same flow rate of 50 L min−1 . (a) Serpentine channel and (b) spiral channel. (c) Mixing intensity within serpentine channel (1) and spiral channel (2) as a function of channel length. Both channels had the same cross-sectional dimensions of 50 m × 300 m.
Fig. 4. Effect of width of the spiral channel on CL intensity. Experimental conditions: 1.0 × 10−7 mol L−1 Pt (II); 0.25 mol L−1 carbonate buffer, pH 11.5; 5.0 × 10−4 mol L−1 luminol; 0.02 mol L−1 H2 O2 .
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Fig. 5. (a) Effect of flow rate of CL reagents on CL intensity; (b) effect of flow rate of CL reagents on the peak shape: (1) 10 L min−1 , (2) 50 L min−1 , (3) 70 L min−1 . For other conditions, see Fig. 4.
nel × 100%. The images shown in Fig. 3 demonstrated that in the spiral channel, mixing level of up to 95% can be achieved, while less than 10% in the serpentine microchannel within the same distance of 10.0 cm. With the use of FI-CL methodology described in Section 2.4, the chemiluminescence intensities from the reaction of 1.0 × 10−7 mol L−1 Pt(II) with basic luminol–hydrogen peroxide using different microchannel configurations were further tested. The double spiral configuration (Fig. 2c) gave the greatest signal, about 20% higher than the serpentine-spiral configuration (Fig. 2b) and 110% higher than the double serpentine configuration (Fig. 2a). The results indicated that the spiral channel allowed greater quantities of light to be generated than a serpentine channel, due to more efficient mixing of the analyte and CL reagents within the detection channel. In spiral channels, transverse Dean flows arisen as a result of centrifugal effects experienced by fluids traveling along a curved trajectory. The centrifugal effects offered the attractive possibility of providing enhanced mixing [22]. The dependence of the CL intensity upon the width of the spiral channels was further investigated in the range of 150–400 m. The results are shown in Fig. 4, which demonstrated that the CL intensities exhibited maximum at the width of 300 m. In theory, a larger channel would allow more molecules to react and produce more photons within the measurable area of the PMT. However, with an increase in the width of channels, depth/width ratios of the channels decreased. The lower aspect ratio strongly suppressed vortex formation in the spiral channels [23] and reduced the mixing efficiency. Therefore, the double spiral configuration with the width of 300 m was used in the following experiments. 3.2. Effect of the flow rate of reagents on the CL intensity In order to ensure that the highest CL intensity appeared in the limited length of reaction/detection channel, the flow rates of CL reagents were optimized in the range of 10–70 L min−1 . The result is shown in Fig. 5(a). It can be seen that the CL intensity increased with increasing the flow rate, and reached a maximum at the flow rate of 50 L min−1 . Further increasing the flow rate to 70 L min−1 , the CL intensity decreased. It has been reported that the metal ion-catalyzed luminol–hydrogen peroxide reaction is rapid with the maximum peak height of the transient CL signal reached in approximately 500 ms [24]. With increasing the flow rate from 10 to 50 L min−1 , the mixing efficiency of the reactants improved, which was beneficial for increasing the CL intensity. However, the residence time of the reactants in the reaction/detection channel decreased with increasing the flow rate. When the flow rate increased from 50 to 70 L min−1 , the maximum emission did not occur in the limited resident time frame, resulting in the decrease of CL intensity.
The flow rate of the CL reagents had effects not only on the CL intensities, but also on the peak shape. As shown in Fig. 5(b) 1–3, peak tailing was serious at the flow rate of 10 L min−1 and peak width became narrow gradually when the flow rate increased. A symmetrical peak without tailing was obtained at a flow rate of 70 L min−1 , but the peak high reduced to some extent. Thus a flow rate of 50 L min−1 was used in the following experiments. 3.3. Investigation of interferences Ethylene diamine tetraacetic acid, tartaric acid, citric acid and 1,10-phenanthroline were tested as masking agents. The only potential masking agent was 1,10-phenanthroline, since 1.0 × 10−3 mol L−1 1,10-phenanthroline had almost no effect on the CL intensity of the platinum. Therefore, the interference of some common chemicals in human serum, such as Mn2+ , Co2+ , Fe3+ , Cu2+ , Zn2+ , Ni2+ , Na+ , K+ , Ca2+ , were investigated by adding relative chemicals to the solutions containing 1.0 × 10−7 mol L−1 Pt(II) and 1.0 × 10−4 mol L−1 1,10-phenanthroline. The tolerable concentration ratios for interference at the 5% level were over 10,000 for Na+ , K+ , Ca2+ ; 200 for Mn2+ , Pb2+ , 75 for Ni2+ , Fe2+ , Zn2+ ; 40 for Fe3+ , Cu2+ ; 20 for Co2+ , which were high enough to remove the interference. Anions have almost no any effect on Pt(II) determination. The tolerable concentration ratios were found to be over 10,000 for Cl− , NO3 − , Ac− , CO3 2− , PO4 3− and 800 for SO4 2− . The interference from the serum matrix was also been investigated. The amount of Pt(II) in the blank serum sample was undetectable. After digestion, Pt(II) standard solution was added in the blank serum sample to 3.00 mol L−1 , 2.95 mol L−1 could be recovered with the relative standard deviation of 2.46% (n = 3). The result indicated that after digestion the serum matrix had no significant interference on the determination of Pt(II). Thus, 1.0 × 10−4 mol L−1 1,10-phenanthroline was used as the masking agent. 3.4. Optimization of CL reaction conditions Since the optimal pH for metal ions to catalyze the oxidation reaction of luminol by H2 O2 is 11.0–11.8 [25] and carbonate buffer can enhance the CL signal in luminol–H2 O2 system [17], a 0.25 mol L−1 carbonate buffer with pH 11.5 was used to prepare the CL solutions. The bromide and chloride ion often acted as enhancers in the chemiluminescence of trace metal ion-catalyzed luminol oxidation by hydrogen peroxide [17,26]. The addition of chloride significantly enhanced the CL signal under our investigation. The effect of chloride concentration was examined between 0.10 and 3.50 mol L−1 . The results indicated that the CL signal reached a maximum and was stable within the concentration range of 2.50–3.50 mol L−1 . In this work, 3.0 mol L−1 NaCl was added in both standard solutions and samples to enhance the sensitivity.
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Table 1 Results of the determination of platinum in pharmaceutical and serum samples. Samples
Cisplatin injection S1 S2 S3 S4 S5 S6 S7 S8 S9 S 10
Proposed method
ICP-MS
Added (mol L−1 )
Found
Recovery (%)
Content (mol L−1 )
RSD (%) (n = 3)
Content (mol L−1 )
RSD (%) (n = 3)
Content (mol L−1 )
RSD (%) (n = 3)
3.08
2.22
3.15
2.04
3.00
6.02
2.20
98.1
2.81 2.75 5.48 4.25 3.23 2.51 3.41 2.63 1.87 3.26
2.52 2.05 2.01 2.29 2.26 2.31 2.28 2.20 2.14 2.20
3.64 2.77 5.49 4.31 3.28 2.82 3.95 2.92 2.26 3.54
2.18 2.01 1.90 1.87 1.86 2.13 1.89 1.97 2.03 1.98
3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00
5.80 5.70 8.60 7.30 6.25 5.45 6.50 5.73 4.85 6.80
2.28 2.25 1.95 2.00 2.06 2.30 2.04 2.07 2.34 2.10
99.5 98.4 102.1 101.2 100.7 97.6 102.6 103.9 98.9 98.1
The effects of luminol and H2 O2 concentration on the CL intensity were examined. The hydrogen peroxide concentration was kept at 0.01 mol L−1 while luminol concentration varied from 5.0 × 10−5 to 1.0 × 10−3 mol L−1 . The CL intensity was maximum when luminol concentration was 5.0 × 10−4 mol L−1 . When the luminol concentration was kept at 5.0 × 10−4 mol L−1 , the concentration of H2 O2 varied from 1.0 × 10−3 to 0.05 mol L−1 , a maximum CL intensity was achieved with the optimal concentration of H2 O2 to be 0.02 mol L−1 . 3.5. Performance of the FI-CL system Under the optimized conditions, a linear response was obtained over the range 2.0 × 10−8 to 2.0 × 10−6 mol L−1 with the regression equation of C = 1.67 × 10−9 I − 3.05 × 10−8 (r2 = 0.998, C: platinum (mol L−1 ), I: CL intensity (mV)). The relative standard deviation was found to be 3.46% (n = 12) for 0.2 mol L−1 with the detection limit of 1.24 × 10−9 mol L−1 (S/N = 3). The sample consumption was only 2 L, from which it can be calculated the absolute detection limit of 2.48 × 10−15 mol could be achieved. The sample throughput was 72 h−1 . 3.6. Application The sample solutions prepared in Section 2.5 were divided exactly in two equal parts. To one of the parts, 3.0 × 10−6 mol L−1 Pt(II) was added and both parts were analyzed by the proposed method. The amount of Pt(II) and recovery were calculated from the calibration curve and listed in Table 1. The recovery of the proposed method varied from 97.6 to 103.9%. The results indicated the proposed method could be used for the determination of cisplatinum concentration in the cisplatin injection and the serum samples of cancer patients. To further evaluate the sample pretreatment process of the proposed method, these samples were also analyzed by ICP-MS method and the corresponding results were also shown in Table 1. Since total platinum content was measured by ICP-MS, while only Pt(II) by the proposed CL method, the concentrations determined by the CL method are lower than that determined by ICP-MS. Nevertheless, most of the values determined were in accordance with ICP-MS method. This indicated that the biodegradation products of cisplatin in cancer patients may not be fully converted to detectable Pt(II) by the sample pretreatment process. 4. Conclusions The proposed FI-CL system was proven to be suitable for the determination of cisplatinum in the serum samples of cancer patients. The results indicated that the interference from the
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