962 Xiaomei Mu Shuting Li Xin Lu Shulin Zhao Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Pharmacy, Guangxi Normal University, Guilin, China

Received October 7, 2013 Revised November 4, 2013 Accepted November 19, 2013

Electrophoresis 2014, 35, 962–966

Research Article

CE with chemiluminescence detection for the determination of thyroxine in human serum A sensitive and rapid approach to perform thyroxine (T4) assay by CE with chemiluminescence (CL) detection was developed. The sensitive detection was based on the enhancement effect of T4 on the CL reaction between luminol and potassium permanganate (KMnO4 ) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve maximum assay sensitivity. Optimal conditions were found to be 5.0 × 10−4 M luminol added to the CE running buffer and 9.2 × 10−5 M KMnO4 in 0.072 M NaOH solution introduced postcolumn. In the optimized experimental conditions, the linear range for T4 detection was 6.0 × 10−8 –6.0 × 10−6 M, with the detection limit of 2.0 × 10−8 M (S/N = 3). Six human serum samples from healthy subjects, hyperthyroid patients and hypothyroid patients were analyzed by the presented method. The serum level of T4 in healthy subjects was found be 9.0 × 10−8 M, whereas the T4 level was found to be 15.6 × 10−8 M in hyperthyroid patients and 1.3 × 10−8 M in hypothyroid patients. The results suggested a potential application of the proposed assay in rapid primary diagnosis of diseases such as hyperthyroid and hypothyroid. Keywords: CE / Chemiluminescence detection / Thyroxine / Human serum DOI 10.1002/elps.201300491

1 Introduction Thyroxine (3,5,3 ,5 -tetraiodo-L-thyronine, T4) is the primary active hormone synthesized within the follicular cells of the thyroid gland [1]. It is critically important for the normal development of the central nervous system in infants, the skeletal growth and the maturation in children as well as for the normal function of multiple organ systems in adult’s regulation of a number of biological processes [2]. It also affects metabolic activity in many tissues, leading to increased consumption of oxygen and stimulation of mitochondrial respiration. Experimental studies have demonstrated that the serum T4 level can be used for the diagnosis of thyroid gland diseases such as hypothyroid, hyperthyroid, and thyroiditis. Therefore, the analysis of T4 level in serum is of continuing interest to researchers. A series of analytical procedures based on HPLC [3, 4], immunoassay [5–7], GC–MS [8], chemiluminescence (CL) [9], and time-resolved fluoroimmunoassay [10] have been described to be effective for T4 determination during the past

Correspondence: Professor Shulin Zhao, College of Chemistry and Pharmacy, Guangxi Normal University, Guilin, 541004, China E-mail: [email protected] Fax: +86-773-5832294

Abbreviations: CL, chemiluminescence; DA, dopamine; E, epinephrine; T4, thyroxine  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

decade. Although each method has its advantages, many reported techniques involve still some tedious and timeconsuming procedure. Thus, it is highly desirable to develop simple, sensitive, and selective assay for T4 determination. CE is a microanalytical technique that provides advantages in term of simplicity, high separation efficiency, low cost and short analysis time. CE with ICP–MS detection has been applied for the determination of T4 [11]. CE with LIF detection has been also used in the T4 assay [12]. However, the ICP–MS detector is expensive, and LIF detection often requires precolumn derivatization with a fluorescence-tagging reagent. CL detection was one of the most sensitive detection techniques in CE [13]. CL can be defined as the emission of light from a molecule or atom in an electronically excited state produced by a chemical reaction. As advantages inherent to CL techniques, it is possible to remark the basic instrumentation required and the simplification of the optical system because no external light source is needed. The absence of strong background light level reduces the background signal and leads to improved detection limits [14]. Therefore, CE coupled with CL detection has become an attractive and alternative detection scheme for sensitive detection in CE [13,15–17]. In this work, we found that T4 could obviously enhance the CL reaction between luminol and potassium permanganate (KMnO4 ), and

Colour Online: See the article online to view Fig. 6 in colour.

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the enhanced CL intensity was proportional to the concentration of T4. Thus, a new CE–CL detection method was developed for T4 detection. The conditions for electrophoresis separation and CL detection were studied, and the quantification of the T4 in human serum was demonstrated.

2 Materials and methods 2.1 Reagents and apparatus T4 and luminol were purchased from Fluka (Buchs, Switzerland). KMnO4 was provided by Chengdou Kelong Reagents (Chengdou, China). H2 O2 was purchased from Taopu Chemicals (Shanghai, China). All other chemicals used in this work were of analytical grade. Water was purified by employing a Milli-Q plus 185 equipment from Millipore (Bedford, MA, USA), and used throughout the work. The postcolumn oxidizer solution was 72 mM NaOH solution containing 92 ␮M KMnO4 . The electrophoresis buffer was 14 mM sodium borate buffer solution (pH 7.8) containing 0.5 mM luminol. All solutions were filtered through 0.45-mm membrane filters before use. Experiments were carried out using a laboratory-built CE–CL system described previously [18]. Briefly, a highvoltage supply (0–30 kV, Beijing Cailu Science Instrument, Beijing, China) was used to drive the electrophoresis. Uncoated fused silica capillaries (55 cm × 75 ␮m id, Hebei Optical Fiber, China) were used for the separation. The polyimide on the 5 cm end section of the capillary was burned and removed. After etching with 10% hydrogen fluoride for 1 h, this end of capillary was inserted into the reaction capillary (530 ␮m id, Hebei Optical Fiber). A four-way Plexiglass joint held a separation capillary and a reaction capillary in place. The CL solution was siphoned into a tee. The grounding electrode was put in one joint of the tee. The CL solution flowed down to the detection window, which was made by burning 2 cm of the polyimide of the reaction capillary and was placed in front of the photomultiplier tube (R374 equipped with a C1556-50 DA-type socket assembly, Hamamatsu, Shizuoka, Japan). CL emission was collected by a photomultiplier tube and was recorded and processed with a computer using a CT-21 Chromatography Data System (Beijing Cailu Science Instrument Company).

2.2 Preparation of human serum samples Human serum samples were provided by the No. 5 People’s Hospital (Guilin, China). A 250 ␮L portion of serum sample was diluted with 500 ␮L of acetonitrile and shaken vigorously for 5 min to precipitate the proteins. After centrifuging at 14 000 rpm for 20 min, the supernatant was transferred into a 1.5 mL vial and dried with an N2 stream. The residue was redissolved in 50 ␮L of 14 mM borate buffer (pH 7.8). The solution was kept at 4°C until analysis.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Analysis of a standard T4 solution. Electrophoresis buffer solution was 14 mM borate buffer solution (pH 7.8) containing 0.5 mM luminol; oxidizer solution was 70 mM NaOH solution containing 92 ␮M KMnO4 ; separation capillary was 55 cm × 75 mm id; voltage applied was 16 kV; the concentration of T4 was 1.2 × 10−6 M.

2.3 CE–CL assay New capillary was preconditioned by flushing with 1M NaOH solution for 30 min before its first use. Between two consecutive injections, the capillary was rinsed sequentially with 0.1 M NaOH solution, water, and running buffer for 2 min each. The reaction capillary was rinsed with oxidation reagent solution for 2 min. Samples were injected into the capillary by hydrodynamic flow at a height differential of 20 cm for 10 s. Running voltage was 16 kV. Electrophoresis buffer was 14 mM borate buffer (pH 7.8) containing 0.5 mM luminol. The oxidizer solution was 72 mM NaOH solution containing 92 ␮M KMnO4 .

3 Results and discussion 3.1 CL reaction between luminol and KMnO4 in the presence of T4 It was found that T4 could obviously enhance the CL reaction between luminol and KMnO4 . CL enhancement resulted in a positive CE–CL peak in the electropherogram (Fig. 1), and the enhanced CL intensity (peak high) was proportional to the concentration of T4. To maximize the sensitivity of CL detection following CE separation, the conditions such as concentrations of NaOH, KMnO4 in postcolumn oxidizer solution and luminol in the electrophoresis buffer solution were optimized. In these experiments, a 1.2 × 10−6 M of T4 solution was injected into the CE–CL system and CL intensity was recorded. www.electrophoresis-journal.com

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Figure 2. The effects of NaOH concentration on CL intensity. The oxidizer solution was 92 ␮M KMnO4 solution containing NaOH at different concentrations. Other conditions were as in Fig. 1.

The luminol reacted with KMnO4 emitting CL in alkaline medium. NaOH solution was selected as the reaction medium. However, since the volume of the eluent from the separation capillary is very small compared with the volume of the postcolumn oxidizer solution, the acidity environment of CL reaction is mainly dependent on the postcolumn oxidizer solution. The effects of NaOH concentration on CL intensity for T4 were investigated by varying NaOH concentration from 0.03 to 0.12 M. Maximum peak height was recorded when the concentration of NaOH was at 0.07 M (Fig. 2). Thus, an oxidizer solution containing 0.07 M NaOH was selected for the postcolumn CL reaction. As oxidizer, the concentration of KMnO4 played an important role in the CL reaction. Therefore, the effect of KMnO4 concentration on CL intensity was investigated in the range of 2.0 × 10−6 –1.6 × 10−4 M. With the increase of KMnO4 concentration from 2.0 × 10−6 to 9.2 × 10−5 M, the CL intensity for T4 also increased, and further increasing KMnO4 concentration results in the decrease of CL (Fig. 3). So a 9.2 × 10−5 M KMnO4 solution was used in this experiment. The effect of luminol concentration was investigated in the range of 1.0 × 10−4 –8.0 × 10−4 M. With the increase of luminol concentration from 1.0 × 10−4 to 5.0 × 10−4 M, the CL intensity for T4 increased. Further increasing the luminol concentration, almost constant CL intensity was observed (Fig. 4). Thus, the concentration of 5.0 × 10−4 M luminol was elected for this CL reaction.

3.2 Optimization of the electrophoresis conditions The electrophoresis conditions including the pH of the running buffer, the concentration of buffer, and electrophoresis voltage affect the efficiency and the reproducibility of the CE separation and detection. Therefore, the effect of electrophoresis conditions on the CE detection was examined. The test results indicated that the increase in pH shortened  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 3. The effect of KMnO4 concentration on CL intensity. The oxidizer solution was 70 mM NaOH solution containing KMnO4 at different concentrations. Other conditions were as in Fig. 1.

Figure 4. The effect of luminol concentration on CL intensity. Electrophoresis buffer solution was 14 mM borate buffer solution (pH 7.8) containing luminol at different concentrations. Other conditions were as in Fig. 1.

the migration time of T4 in the range of 7.29.0 because of the increase in the electroosmotic flow. At pH 7.8, the electropherogram peak of T4 is sharp and symmetrical, and the CL intensity calculated by peak height is the highest. In addition, the concentration of borate in running solution also affects the detection. When the concentration of borate was 14 mM, the system had the maximal CL. Then, a 14 mM of borate solution at pH 7.8 was chosen as the running buffer. The effect of running voltage on the CL in the range of 12∼18 kV was tested. Results indicate that when the voltage was 16 kV, the system had the maximal CL, and stable basic line and symmetrical peak were obtained. Therefore, 16 kV electrophoresis voltage was chosen for the optimization. www.electrophoresis-journal.com

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pherogram obtained from the separation of a mixture containing the above-mentioned compounds. As can be seen, only DA, E, and tyrosine can enhance the CL reaction between luminol and KMnO4 , and T4 was well separated from DA, E, and tyrosine, which suggested that none of these endogenous amino acids, organic acids, and biogenic amines would interfere with the determination of T4 in biological samples. 3.4 Analytical figures of merit The method was evaluated in terms of response linearity, LOD, and reproducibility. Under the selected CE–CL conditions, seven standard T4 solutions at various concentrations were analyzed. Linear regression analysis of the results yielded the following equation: H = 0.3881 × 106 C + 0.5908, where H is the CL intensity (mV) from T4, and C is the concentration of T4 (M). The calibration curves showed excellent linearity from 6.0 × 10−8 to 6.0 × 10−6 M with a correlation coefficient of 0.9961. Based on an S/N of 3, the detection limit for T4 was estimated to be 2.0 × 10−8 M. Assay reproducibility was investigated by analyzing a standard solution of T4 at 1.2 × 10−6 M nine times. The results showed that the RSDsof the migration time and peak height were 0.8 and 2.1%.

Figure 5. Electropherogram obtained from the separation of a mixture containing uric acid, DA, E, ascorbic acid, and 20 protein amino acids. CE–CL conditions were as in Fig. 1.

3.5 Quantification of T4 in human serum Human serum samples taken from two healthy volunteers (euthyroid) and four patients suffering from different thyroid diseases were analyzed to demonstrate the feasibility of the proposed CE–CL method for the determination of T4 in complex biological samples. A typical electropherogram obtained from the analyses is shown in Fig. 6 (trace A). The peak corresponding to T4 was well identified. To verify the peak identification, the sample was spiked with T4 at 5.0 × 10−7 M and again analyzed. The electropherogram obtained is also shown in Fig. 6 (trace B). By comparing the two CE traces shown in Fig. 6, it can be seen that the peak corresponding to T4 increased in size without other major changes in the electropherogram. The analytical results are summarized in Table 1. The T4 level in the serum samples from healthy subjects was found to be 7.4 × 10−8 and 10.6 × 10−8 M. The highest level of T4 was detected for the hyperthyroid patient

Figure 6. Electropherograms obtained from the separation of a human serum sample (A) and a human serum sample spiked with T4 at 5.0 × 10−7 M (B). CE–CL conditions were as in Fig. 1.

3.3 Interference study Many endogenous amino acids, organic acids, and biogenic amines are present in biological samples and disturb the CL detection, which may interfere with the determination of T4. Therefore, the influence of co-existing compounds such as uric acid, DA, epinephrine (E), ascorbic acid, and 20 protein amino acids were investigated. Figure 5 shows the electro-

Table 1. Analytical results of T4 in human serum samples from healthy and patient subjects

Sample

Found (10−8 M)

Original concentration (10−8 M)

RSD (%, n = 5)

Added (10−8 M)

Total found (10−7 M)

Recovery (%)

Euthyroid Euthyroid Hyperthyroid Hyperthyroid Hypothyroid Hypothyroid

37.0 53.2 96.3 79.6 7.20 6.10

7.40 10.6 19.3 15.9 1.44 1.22

1.2 3.0 4.8 4.7 3.2 2.9

50.0 50.0 100 100 10.0 10.0

8.47 10.7 20.3 17.6 1.72 1.65

95.4 108 107 96.4 98.0 104

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at 19.3 × 10−8 M, and the lowest level was detected for the hypothyroid patient at 1.2 × 10−8 M. These results are in accordance with the data reported previously [7, 19]. The RSDs for the determination of T4 was in the range of 1.2–4.8%. Recovery of T4 from these samples was studied. T4 was spiked to each sample and analyzed. Recoveries were found to be in the range of 95.4–108%. The results suggested a potential application of the proposed assay in rapid primary diagnosis of diseases such as hyperthyroid and hypothyroid.

[2] Lum, S. M., Nicoloff, J. T., Spencer, C. A., Kaptein, E. M., Invest. J. Clin. 1984, 73, 570–575.

4 Concluding remarks

[7] Huang, Y., Zhao, S., Shi, M., Liu, Y. M., Anal. Biochem. 2010, 399, 72–77.

Based on the enhanced effect of T4 to the CL reaction between luminol and KMnO4 in alkalescence medium, a novel CE coupled with CL detection method was established for the determination of T4. The applicability of the present method was demonstrated by analyzing human serum samples. The present method shows high precision, sensitivity, and minimal sample handling, and can be easily applied to routine analysis in the clinical laboratory for the determination of T4 in serum.

[8] Hantson, A. L., Meyer, M. D., Guerit, N., J. Chromatogr. B 2004, 807, 185–192.

This work was supported by the National Natural Science Foundations of China (No. 21175030, 21311120056), and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Ministry of Education of China, as well as BAGUI Scholar Program. The authors have declared no conflict of interest.

5 References [1] Braverman, L. E., Utigen, R. D. (Eds.), Werner and Ingbar’s the Thyroid: A Fundamental and Clinical Text, 9th ed., Lippincott Williams & Wilkins, Philadelphia 2004.

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CE with chemiluminescence detection for the determination of thyroxine in human serum.

A sensitive and rapid approach to perform thyroxine (T4) assay by CE with chemiluminescence (CL) detection was developed. The sensitive detection was ...
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