Journal of Chromatography A, 1356 (2014) 23–31

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Ion pair hollow fiber liquid–liquid–liquid microextraction combined with capillary electrophoresis-ultraviolet detection for the determination of thyroid hormones in human serum Pingjing Li, Bin Hu ∗ , Man He, Beibei Chen Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China

a r t i c l e

i n f o

Article history: Received 5 March 2014 Received in revised form 7 June 2014 Accepted 16 June 2014 Available online 21 June 2014 Keywords: Ion pair hollow fiber liquid–liquid–liquid microextraction CE-UV Thyroid hormones and relevant compounds Human serum

a b s t r a c t In this study, a novel, inexpensive, sensitive and selective analytical method that combines ion pair hollow fiber liquid–liquid–liquid microextraction (IP-HF-LLLME) with capillary electrophoresisultraviolet detection (CE-UV) was developed for the simultaneous determination of six thyroid hormones (including diiodothyronine (T2 ), 3,3,5-triiodo-l-thyronine (T3 ), 3,5,3,5-tetraiodolthyronine (T4 ), 3,3,5triiodothyronine (rT3 ), monoiodotyrosine (MIT) and diiodotyrosine (DIT)) in human serum samples. By the addition of a low concentration of sodium dodecyl sulfate (SDS) into the donor phase as an ion pair reagent, octanol as the organic extraction solvent and 30 mmol/L Na2 CO3 as acceptor phase, six analytes with different polarity and water solubility were successfully extracted simultaneously using HF-LLLME. To the best of our knowledge, this is the first time that a liquid phase microextraction technique was proposed for the extraction of thyroid hormones in real samples. The CE separations were investigated in detail. When 20 kV of voltage was applied, the six compounds were separated within 13 min in 25 mmol/L phosphate buffer (pH 2.15) containing 10% (v/v) acetonitrile and 0.5% (m/v) polyethylene glycol (PEG). Under the optimized conditions, enrichment factors (EFs) ranging from 183- to 366-fold were obtained and the limits of detection (at a signal-to-noise ratio of 3) were at sub ␮g/L level. The established IP-HFLLLME-CE-UV method was successfully applied to simultaneous determination of thyroid hormones and relative compounds in human serum samples with good recoveries for the spiked samples. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The thyroid hormones, which have important roles in many physiological processes such as carbohydrate metabolism, oxygen consumption, protein synthesis and fetal neuro development, are important tyrosine-based hormones produced by the thyroid gland [1]. Among them, thyroxine (3,5,3,5-tetraiodolthyronine, T4 ) is the major thyroid hormone secreted from the thyroid gland. Liothyronine (3,3,5-triiodo-l-thyronine, T3 ), which is converted to T4 through deiodination at the phenolic ring in the kidneys and the liver, is the more active hormone but is present at approximately 60-fold lower concentrations than the total amount of T4 found in healthy human subjects [2]. The biotransformation of T4 also produces the inactive metabolite 3,3,5triiodothyronine (rT3 ), and further deiodination of T3 and rT3 yields

∗ Corresponding author. Tel.: +86 27 68752162; fax: +86 27 68754067. E-mail address: [email protected] (B. Hu). http://dx.doi.org/10.1016/j.chroma.2014.06.046 0021-9673/© 2014 Elsevier B.V. All rights reserved.

diiodothyronine (T2 ). Diiodotyrosine (DIT) and monoiodotyrosine (MIT) have no thyroxine-like activities but are still of great significance because they are involved in the formation process of T4 and are products of its metabolism [3]. Athyroid hormone deficiency can cause hypothyroidism, leading to myxoedema with struma and debility [4]. Development of a sensitive, specific and reliable method to determine the level of thyroid hormones in biological samples is thus of great significance in the diagnosis of hypothyroid disease. Several methods including immunoassays [5–7], high performance liquid chromatography (HPLC)/mass spectrometry (MS) [2,8–11] and capillary electrophoresis (CE) [1,12] have been developed for the determination of thyroid hormones in biological fluids or other samples. Immunoassay approaches have high sensitivity but sometimes lack specificity for thyroid hormones due to their endogenous factors (e.g., abnormal binding proteins, dialyzable protein binding competitors) or in vitro factors (free fatty acids, assay antibodies) [2]. HPLC and CE can be used for the analysis of different thyroid hormone species simultaneously, and they can

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provide relative high sensitivity and alleviate the issue of specificity encountered in immunoassay methods when MS is used as their detector [13]. Besides the development of highly efficient analytical instrumentation for end-point determination of analytes, sample pretreatment is promising to extract, isolate, and concentrate the analytes of interest from complex matrices. Specifically, liquid–liquid extraction (LLE) [14] and solid phase extraction (SPE) [3,10,15,16] were commonly applied to cleanup the sample matrix for the analysis of the target thyroid hormones in real-world samples with complex matrix. Besides conventional SPE and LLE, new miniaturized extractions schemes such as liquid phase microextraction (LPME) [17], solid phase microextraction (SPME) [18] and stir bar sorptive extraction (SBSE) [19] have been developed in recent years. These miniaturized extraction techniques have the merits of simplicity, selectivity, time-efficiency as well as a wide variety of configurations or modes available and have gained popularity in wide applications to various classes of analytes. Recently, our group developed a new method by coupling SBSE with HPLCinductively coupled plasma mass spectrometry (ICP-MS) for the determination of four thyroxine hormones in human urine samples [11]. The enrichment factors (EFs) in the range of 14.9–70.4 were obtained. Compared to other microextraction techniques, LPME has the advantages of good sample clean-up capability and high enrichment factors, requires only very simple and low cost devices, and does not suffer from any carry-over effect [17]. Among various LPME modes, hollow-fiber LPME (HF-LPME) is the most robust usually with good reproducibility, excellent clean-up ability, and high enrichment factor [20]. LPME can easily extract the non-polar and weak polar analytes with high partition coefficients by two phase or three phase mode. To those analytes with high polarity, ionpair (IP) LPME (also named as carrier-mediated LPME) has been developed to facilitate the extraction process [21–23]. In IP-LPME, the target analytes can form ion-pairs with the counter ions of opposite charge (ion-pair reagent). This enables the ion-pairs to possess higher partition coefficients than the native analytes. As a result, transfer of the target analytes into the extraction phase is enhanced. Ding et al. [24] developed a IP-HF-LPME method coupled with flow-injection electrospray ionization tandem mass spectrometry (ESI-MS/MS) method for the determination of perchlorate (ClO4 − ) in surface water samples using di-n-hexyl ammoniumacetate (DHAA) as ion pair reagent. Hultgren et al. [23] reported that surfactant extraction was facilitated by addition of carboxylic acid to the sample forming neutral ion pairs with target analyte (dicocodimethyl ammonium chloride). However, most applications of IP-LPME are still in two phase mode, limited reports are about ion-pair in three-phase LPME. Three-phase LPME is suitable to the subsequent liquid phase-based analytical methods such as HPLC or CE, as target analytes can be recovered in the aqueous acceptor phase. Xu et al. developed a very effective three phase IP-LPME system for the analysis of hydrophilic nerve agent degradation products including methylphosphonic acid (MPA), ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) by CE/contactless conductivity detection [25]. However, to the best of our knowledge, there is no report about the extraction of thyroid hormones by LPME. Thyroid hormones are zwitterionic amino acids containing iodine atoms and phenyl groups. According to their chemical structures as well as some of the chemical properties as listed in Table 1, it can be seen that their polarity and water solubility are quite different. Their log KOW (octanol/water partition coefficient) values range from −0.60 to 4.12. To extract thyroid hormones simultaneously by LPME, the use of ion pair to facilitate the extraction process would be a good choice. Therefore, the purpose of this work is to develop a sensitive and reliable method by combining ion pair

three phase LPME (hollow fiber-liquid–liquid–liquid microextraction, HF-LLLME) with CE-UV for the simultaneous determination of six thyroid hormones in human serum. The effect of the concentration of the IP reagent, the pH of the donor phase, the organic solvent, the acceptor solution and other relevant parameters on the IP-HF-LLLME were systematically studied. The developed IP-HFLLLME-CE-UV method was validated by the simultaneous analysis of six thyroid hormones in serum samples.

2. Experimental 2.1. Reagents, solutions and materials T3 (3,5,3-triiodo-l-thyronine, >99% purity) and DIT (3,5-diiodol-tyrosine, >99% purity) were purchased from Acros Organics (Geel, Belgium); rT3 (Reverse 3,3,5-triiodo-l-tyrosine, >95% purity) and T2 (3,5-Diiodo-l-thyronine dehydrate, >95% purity) were obtained from Toronto Research Chemical (North York, Canada) and Tokyo Chemical Industry (Tokyo, Japan), respectively; T4 (l-thyroxine, >98% purity) and MIT (3-iodo-l-tyrosine, >98% purity) were supplied by Shanghai Chemical Reagent Company, Shanghai, China. Each standard solution of six target analytes (T4 , T3 , rT3 , T2 , DIT and MIT) was prepared in a mixture of Na2 CO3 (50 mmol/L) and methanol (50/50, v/v) at a concentration of 250 mg/L, and a mixed standard solution containing 10 mg/L of each thyroxine was also prepared in the mixture of Na2 CO3 (50 mmol/L) and methanol (50/50, v/v). Working standard solutions were prepared by diluting the mixed standard solution with high purity water to the required concentrations. All standard stock solutions were kept in refrigerator at 4 ◦ C away from light. High purity water was obtained from a Milli-Q water purification system (18.2 M cm, Millipore, Bedford, MA, USA). 1-Heptanesulfonic acid sodium (HAS, >95% purity), 1octanesulfonic acid sodium (OAS, 95% purity), sodium dodecyl sulphate (SDS, >95% purity), methimazole and polyethylene glycol (PEG) with different molecular weights (MW) were purchased from the China Medicine Group, Sinopharmic Chemical Reagent Company (Shanghai, China). ␤-cyclodextrin (␤-CD) was purchased from Aldrich (Aldrich Chemical Company, St. Louis, MO, USA). All reagents were of analytical grade unless otherwise noted. Proteolytic enzyme was purchased from Merck (Darmstadt, Germany). Other reagents used were of analytical reagent grade. Q3/2 Accurel polypropylene hollow fiber (200 ␮m wall thickness, 600 ␮m inner diameter, 0.2 ␮m pore size) was obtained from Membrana (Wuppertal, Germany). The phosphate buffer (25 mmol/L, pH = 2.15) was prepared by first mixing the stock solutions of 200 mmol/L H3 PO4 and 200 mmol/L NaH2 PO4 in a ratio of 9:1 (v/v) and then diluting with high purity water to a final concentration of 25 mM H3 PO4 and 25 mM NaH2 PO4 .

2.2. Instruments All separation experiments were performed on a 3D CE system (Agilent, USA) equipped with a programmable, multiwavelength UV/Vis detector. The detection wavelength used was 214 nm. The separation capillary temperature was maintained at 25 ◦ C. Fusedsilica capillaries with the dimensions of 64.5 cm (56 cm to the detector) × 75 ␮m i.d. × 360 ␮m o.d. (Yongnian Optical Fiber, Hebei, China) were used for the CE separation. Before use, the capillaries was conditioned by successive flushing with 0.1 mol/L sodium hydroxide (NaOH) for 10 min, water for 10 min, and the running buffer for 10 min, respectively. Between runs, the capillary was rinsed with running buffer for 5 min. A modified CE automatic sample vial was used for sample injection into the CE for analysis [26].

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Table 1 Structure, pKa , calculated octanol/water partition coefficient (log KOW ) and water solubility of the studied thyroid gland hormones and relative compounds. Analyte

Molecular structure

pKa b

log KOW c

WSa (mg/L)c

T4 (CAS: 51-48-9) (l-thyroxine)

2.12, 8.94

4.12

0.0001047

T3 (CAS: 6893-02-3) (3, 5, 3-triiodo-l-thyronine)

2.13, 8.96

2.96

0.007185

rT3 (CAS: 5817-39-0) (reverse 3,3,5-triiodo-l-thyronine)

2.17, 9.08

T2 (CAS: 300-39-0) (3,5-diiodo-l-thyronine dihydrate)

2.15, 8.99

1.79

0.4747

DIT (CAS: 66-02-4) (3,5-diiodo-l-tyrosine)

2.17, 9.24

0.57

MIT (CAS: 70-78-0) (3-iodo-l-tyrosine)

2.21, 9.38

−0.60

a b c

20.05

1165

WS: water solubility. Data obtained from SciFinder Scholar. Data obtained from SRC PhysProp Database, http://epa.gov/oppt/exposure/pubs/episetup v400.exe.

2.3. IP-HF-LLLME procedure The set-up of the IP-HF-LLLME unit used in this work was similar to that described in our previous work [26]. In brief, the hollow fiber was cut into 4.5 cm segments. Each segment has an internal volume of 15 ␮L. Before use, the hollow fiber was cleaned with acetone by ultrasonication and air dried. First, the acceptor solution (10 ␮L of 30 mmol/L Na2 CO3 ) was slowly injected into the fiber lumen. Then, the hollow fiber, fixed on the end needles of two commercial 10 ␮L microsyringes (Gaoge, Shanghai, China), was dipped into octanol solvent for 25 s. After that, the microsyringes were placed and submerged into the donor phase (5 mL containing IP, pH 3.0) immediately. The whole extraction was performed in a 7-mL vial with 20 min of stirring at 1000 rpm on an 85-2A constant temperature magnetic stirrer (Ronghua, Jiangsu, China). After extraction, the hollow fiber was carefully removed from the sample solution, cut from the middle, then the acceptor solution was withdrawn back into the syringes. The collected acceptor solutions in both two syringes are then combined together in a modified CE automatic sample vial for further CE analysis. 2.4. Human serum sample pretreatment Human serum was collected from a local hospital. The preparation procedure of serum was adopted from Ref. [2] for the determination of total thyroid hormone species. In detail, an aliquot of 2 mL of thawed serum was placed into a 15-mL glass centrifuge tube. A protection solution containing the antioxidants ascorbic acid, citric acid, and dithiothreitol at concentrations of 25 g/L in water was prepared to prevent the degradation of the thyroid hormones during the sampling process, and 250 ␮L of this solution was added to prevent the potential conversions of the thyroid hormones. Usually, most of thyroid hormones bind with protein in biological samples. For the leaching of total thyroid hormones, 2 mL acetone was added to the centrifuge tube, and the mixture was thoroughly mixed and then allowed to stand for at least 30 min to deproteinate the serum. After vortex mixing, all the samples were centrifuged at 4000 rpm for 15 min. The supernatants were transferred to a 25-mL glass vial, and the precipitates in the test tube

were extracted twice more using 1.0 mL of a mixture of acetone and water (v/v: 1:1). After centrifugation for 5 min at 10,000 rpm, the supernatants were collected and all the supernatants were combined. After adding the appropriate amount of SDS and adjusting the pH to 3.0, the supernatant was diluted to 100 mL with high purity water. The spiked experiment was carried out by adding quantities of the target analytes to the supernatant after centrifugation for human serum samples.

3. Results and discussion 3.1. Optimization of the CE separation conditions All the analytes could be baseline separated using phosphate buffer (25 mmol/L, pH 2.15) as background electrolyte (BGE) buffer (Fig. 1A), but obvious tailing and broadening peaks were observed for rT3 , T3 and T4 . To improve the sensitivity and resolution of CE-UV for six target analytes, different organic solvents and additives were investigated as additives to the phosphate buffer BGE. As shown in electrophoretogram for six target analytes (Fig. 1C), the best sensitivity and resolution was obtained by using 25 mmol/L phosphate buffer (at pH 2.15) containing 10% (v/v) acetonitrile and 0.5% (m/v) of PEG with MW of 10,000 as BGE. The detail optimizations of separation conditions are provided as supporting information.

3.2. Optimization of the IP-HF-LLLME conditions The target thyroid hormones are zwitterionic amino acids containing iodine atoms and phenyl groups. They can bear a net positive charge, a net negative charge, or be electrically neutral in different pH environments. As a result, the adjustment of the acidity of the donor solution could enhance the extraction, as the dissociation equilibriums and the solubility of the target analytes can be affected by changing pH value of the donor solution. The difference in the pH value between the donor and acceptor phases can promote the extraction of the analytes from the donor phase to the acceptor phase.

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as octanol, and little difference in the solubility in acidic and basic aqueous solutions. Therefore, they are difficult to extract by only adjusting the pH values of the donor solution and the acceptor solution in HF-LLLPME. For T2 , T3 , rT3 and T4 , they could be extracted by passive diffusion because of their relatively large Kow values. Additionally, according to their characteristic pKa values, their isoelectric points (PIs), at which they bear no net charges and have the lowest solubility, should be between 4.0 and 6.0. Therefore, at pH values of 4.0–6.0, the maximum extraction efficiencies for T2 , T3 , rT3 and T4 were obtained for HF-LLLME without the addition of IP reagent in donor phase.

Fig. 1. Electrophoretograms of six thyroid hormones and relative compounds obtained by the CE conditions as follows: Injection time: 50 mbar × 5 s; applied voltage, 20 KV. (A) BGE, H3 PO4 :NaH2 PO4 (9:1, v/v), 25 mmol/L. Analytes concentration: T4 , 5 mg/L; T3 , 5 mg/L; rT3 , 5 mg/L; T2 , 10 mg/L; DIT, 10 mg/L; MIT, 15 mg/L. (B) BGE, H3 PO4 :NaH2 PO4 (9:1, v/v), 25 mmol/L with 10% acetonitrile. Analytes concentration: T4 , 5 mg/L; T3 , 5 mg/L; rT3 , 5 mg/L; T2 , 5 mg/L; DIT, 5 mg/L; MIT, 5 mg/L. (C) BGE, H3 PO4 :NaH2 PO4 (9:1, v/v), 25 mmol/L with 10% (v/v) acetonitrile and 0.5% (m/v) PEG. Analytes concentration: T4 , 5 mg/L; T3 , 5 mg/L; rT3 , 5 mg/L; T2 , 10 mg/L; DIT, 5 mg/L; MIT, 15 mg/L.

3.2.1. Effect of the sample pH in the absence of IP reagent Initially, the effect of the sample pH on the extraction efficiency of HF-LLLME (without adding IP in the donor solution) was studied in the pH range of 2.0–8.0, and the results are shown in Fig. 2A. In the absence of an IP reagent in the donor phase, T2 , T3 , rT3 and T4 could be extracted, and the maximum extraction efficiencies were obtained in the pH range of 4.0–6.0, while no CE signal of MIT and DIT was observed after the HF-LLLME process over the entire pH range of 2.0–8.0. These results can be easily explained by the structure, the pKa value, the calculated octanol/water partition coefficient (log KOW ) and the water solubility of the target thyroid hormones. As can be seen from Table 1, MIT and DIT have strong polarity (log KOW < 1) and high water solubility. As a result, they have low solubility in water-immiscible organic solvents, such

3.2.2. Effect of sample pH with the addition of IP reagent For the simultaneous extraction of six target thyroid hormones, an IP-HF-LLLME method was developed. As thyroid hormones belong to zwitterionic compounds, theoretically, it can be extracted both in cationic forms and anionic forms by selection of suitable ion pair reagent and pH gradient between sample solution and acceptor phase. However, according to their pKa values as well as our preliminary experiment results, if they are extracted in anionic forms, the pH value of acceptor solution should be less than 1. The injection of such acidic acceptor solution into the subsequent CE system will deteriorate the CE separation. Therefore, the IP-HF-LLLME was performed with the target analytes existing as the cationic forms in sample solutions in this work. For this purpose, different anionic IP reagents including SDS, HAS and OAS were selected to assist extraction of the target analytes by IP-HF-LLLME, as they can easily exist in anionic forms in acidic solutions benefiting from their sulfonic groups or sulfated group. Fig. 3 is the effect of different IP reagents including SDS, HAS and OAS on the extraction efficiency of six target analytes. As can be seen, with the use of SDS as IP, the highest enrichment factor was obtained for all the target analytes. By selecting SDS as the ion pair reagent, the effect of the sample pH on the extraction efficiencies of the six target analytes in IP-HF-LLLME was investigated with pH varying in the range of 2.0–8.0, and the experimental results are shown in Fig. 2B. With the addition of SDS as an ion pair reagent, MIT and DIT could be successfully extracted, and the best extraction efficiency was obtained at a pH of 2.5. However, the plot trends of the extraction efficiencies of the six target analytes obtained by IP-HF-LLLME are quite different. For T2 , the best CE signal response was observed at a pH of 3.5, while for T3 and

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55 50 45

50 40

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Area

35 Area

MIT DIT T2 rT3 T3 T4

30

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25 20

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15 10

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5 0

2

3

4

5 Sample pH

(B)

6

7

8

0

1

2

3

4 5 Sample pH

6

7

(A)

Fig. 2. Effect of sample pH on (A) HF-LLLME and (B) IP-HF-LLLME. Conditions: organic solvent, octanol; stirring rate, 1000 rpm; acceptor solution, 10 ␮L 30 mmol/L Na2 CO3 ; extraction time 20 min; without NaCl. (A) Analytes concentration, MIT 100 ␮g/L, DIT 100 ␮g/L, T2 100 ␮g/L, rT3 100 ␮g/L, T3 100 ␮g/L, T4 100 ␮g/L; (B) sample solution with 0.06 mmol/L SDS; analytes concentration, MIT 200 ␮g/L, DIT 200 ␮g/L, T2 100 ␮g/L, rT3 100 ␮g/L, T3 50 ␮g/L, T4 50 ␮g/L; n = 3.

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MIT DIT T2 rT3 T3 T4

50 40

Area

30 20 10 0

HAS

OAS

SDS

27

cyclohexane, carbon tetrachloride, or tributyl phosphate as the organic extraction solvent, and n-octanol was capable of simultaneously extracting all of the target analytes. This may be attributed to the formation of hydrogen bonds between octanol and the thyroid hormones and the relevantcompounds. For further investigation on the effect of aliphatic alcohols on the extraction, various chain aliphatic alcohols including n-hexanol, n-pentanol and ndocanol were examined and compared with 1-octanol for the simultaneous extraction of 300 ␮g/L of the target thyroid hormones. The results are shown in Fig. S1. As can be seen, n-octanol and n-decanol provided good extraction efficiency for target thyroid hormones, while no obvious signal was observed for target analytes by using n-pentanol and n-hexanol as extraction solvent, probably due to their relative high water solubility (21, 8.8 g/L) and polarity (log P 1.4, 1.9) over the other two solvents (1.2, 0.12 g/L, log P 2.9, 3.9 for n-octanol and n-decanol). Consequently, n-octanol was selected as the extraction solvent for further experiments.

Ion pair reagent Fig. 3. Effect of different kinds of ion pair reagent on IP-HF-LLLME. Conditions: analytes concentration, MIT 200 ␮g/L, DIT 200 ␮g/L, T2 100 ␮g/L, rT3 100 ␮g/L, T3 50 ␮g/L, T4 50 ␮g/L; sample solution, pH 3.0; the concentration of ion pair reagent, 0.06 mmol/L; organic solvent, octanol; stirring rate, 1000 rpm; acceptor solution, 10 ␮L 30 mmol/L Na2 CO3 ; extraction time 20 min; without NaCl; n = 3.

rT3 the best results were observed at a pH of 4.5. For T4 , the extraction efficiency was increased with the increase of the pH from 1.0 to 3.5 and remained constant at a pH from 3.5 to 7.4. The reason behind these results might be that the extraction of MIT and DIT can be mainly attributed to the formation of a relatively hydrophobic ion pair compound between SDS and MIT or DIT. At a lower pH value (pH 2.5), MIT and DIT are present as positive charges, which are helpful for ion pair extraction; in comparison, the extractions of T3 , rT3 and T4 are mainly facilitated by passive diffusion even in the presence of SDS, and thus the maximum extraction efficiencies were achieved at the pH value near their PIs. For T2 , both ion pair extraction and passive diffusion play important roles in the extraction process. As a compromise, a pH value of 3.0 was selected for subsequent experiments. 3.2.3. Effect of the concentration of SDS The effect of the SDS concentration in the range of 0–0.14 mmol/L on the extraction of six target analytes was studied. As the concentration range is less than its critical micelle concentration (CMC, 8.2 mmol/L), SDS was acted as an IP reagent rather than surfactant in this study. It was found that the extraction efficiencies of MIT and DIT were increased with increasing the SDS concentration up to 0.14 mmol/L, while the extraction efficiencies of T2 , T3 , rT3 and T4 , were slightly increased with the increase of the SDS concentration from 0 to 0.02 mmol/L, kept nearly constant with the further increase of the SDS concentration from 0.02 to 0.06 mmol/L, and decreased afterwards. These experimental results could be explained as follows: it is well known that SDS is an amphiphilic molecule, in which the head is polar or hydrophilic and the tail is hydrophobic. As a result, it may interact with T2 , T3 , rT3 and T4 electrostatically, hydrophobically or by a combination of both of these effects. According to the results, a 0.06 mmol/L SDS was selected as the IP reagent for the subsequent experiments. 3.2.4. Effect of organic solvent Several commonly used organic solvents in LPME with different polarity, including toluene, cyclohexane, carbon tetrachloride, 1-octanol, and tributyl phosphate, were employed for the simultaneous extraction of 100 ␮g/L of the target thyroid hormones from the aqueous solutions by IP-HF-LLLME. The experimental results show that no CE signals was observed by using toluene,

3.2.5. Effect of the concentration of the acceptor solution To ensure the extraction proceeds satisfactorily, pH gradient between sample solution and acceptor phase should be another major driving force in IP-HF-LPME. Therefore, alkaline solution should be chosen as acceptor solution. In our preliminary experiments, alkaline solutions of NaOH, NH3 ·H2 O and Na2 CO3 were tested as acceptor solution for extraction of the target analytes by IP-HF-LPME. It was found that the commonly used NaOH and ammonia solution as acceptor solution will lead to an increase of noise, unstable baseline and deterioration of electrophoresis peaks of the target analytes in subsequent CE-UV analysis even at low concentrations. Sodium carbonate (Na2 CO3 ) as acceptor solution can avoid such problems. Therefore, Na2 CO3 solution was selected as the acceptor solution for extraction of six target analytes by IP-HF-LPME, and the effect of the Na2 CO3 concentration on the extraction was then evaluated with its concentration changing over a range of 10–50 mmol/L. Based on the experimental results in Fig. S2, 30 mmol/L Na2 CO3 was chosen as the acceptor solution for extraction of six target analytes by IP-HF-LPME in this work. 3.2.6. Effect of the stirring rate and the ionic strength The effects of the stirring rate on the extraction efficiencies of the target thyroid hormones were investigated with stirring rates varying from 400 to 1400 rpm, and a stirring rate of 1000 rpm was selected for further studies according to the experimental results in Fig. S3. To evaluate the effect of the NaCl concentration on the extraction of the target analytes by IP-HF-LLLME, the extraction was performed using 5 mL of the sample solution containing various concentrations of NaCl (0–20% (m/v)). According to the experimental results in Fig. S4, all the extraction experiments were carried out without any salt additions in this work. 3.2.7. Effect of the extraction time Like other microextraction techniques, the established IPHF-LLLME is a process dependent on equilibrium rather than exhaustive extraction. The effects of the extraction times on the extraction performances of target analytes were examined with extraction times varying from 5 to 50 min, and the results are shown in Fig. 4A. The extractions for MIT and DIT did not attain equilibrium in 50 min. For T2 , T3 , rT3 and T4 , the extractions were obviously increased with the increase of extraction times from 0 to 25 min, while from 30 to 50 min a decrease in the extraction efficiencies were observed. These extraction time profiles were abnormal compared with that obtained by the usual HF-LLLME procedure. In comparison, the effect of the extraction times on the extraction efficiencies of HF-LLLME was also studied by the simultaneously extraction of T2 , T3 , rT3 and T4 in the absence of SDS at a pH of 5.0.

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45 MIT DIT T2 rT3 T3 T4

35

Area

30 25

35

25

20

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15

15

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10

5

5

0

0 0

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T2 rT3 T3 T4

30

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20

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40

50

0

10

20

30

40

50

Extraction time of HF-LLLME/min

Extraction time of IP-HF-LLLME/min

(A)

(B)

Fig. 4. Effect of extraction time on (A) IP-HF-LLLME and (B) HF-LLLME. Conditions: (A) analytes concentration, MIT 200 ␮g/L, DIT 200 ␮g/L, T2 100 ␮g/L, rT3 100 ␮g/L, T3 50 ␮g/L, T4 50 ␮g/L; sample solution, pH 3.0 with 0.06 mmol/L SDS; organic solvent, octanol; stirring rate, 1000 rpm; acceptor solution, 10 ␮L 30 mmol/L Na2 CO3 ; (B) analytes concentration, T2 100 ␮g/L, rT3 100 ␮g/L, T3 100 ␮g/L, T4 100 ␮g/L; sample solution, pH 5.0; organic solvent, n-octanol; stirring rate, 1000 rpm; acceptor solution, 10 ␮L 30 mmol/L Na2 CO3 ; n = 3.

As shown in Fig. 4B, no obvious signal decreases was observed. In detail, for T2 and T3 there was a corresponding increase of the CE signal from 5 to 20 min, followed by a period of stability. For rT3 and T4 , equilibrium was not attained even after 50 min. It can be implied that the specific tendencies of the extraction time plots can be correlated with the additions of the SDS in the donor phases. However, how the SDS affected the extraction time plots is not clear. Therefore, an extraction time of 25 min was selected for further studies. In summary, the optimized conditions for the simultaneous extraction of the target analytes by IP-HF-LLLME are as follows: 5 mL sample solution with 0.06 mmol/L of SDS adjusted to a pH of 3.0 as the donor phase; octanol as the organic solvent; 30 mmol/L of Na2 CO3 as the acceptor solution; stirring rate at 1000 rpm for 25 min; without a NaCl addition.

4. Performance of IP-HF-LLLME-CE/UV Under the optimized conditions, the analytical performance of the proposed method of IP-HF-LLLME-CE-UV for the determination of the six thyroid hormones was evaluated. All standards were prepared in high purity water and subjected to the overall method of IP-HF-LLLME-CE/UV for analytical performance study. Table 2 is the analytical performance data of the developed IP-HF-LLLME-CE/UV method for the target analytes. As can be seen, the relative standard deviations (RSDs) of corrected peak areas (peak area/migration

time) for the five replicate determinations of the targeted analytes at 10 ␮g/L were in the range of 3.2–9.0%. Good linearity with correlation coefficients ranging from 0.993 to 0.999 was obtained for the target analytes in the concentration range of 5–500 ␮g/L. The limits of detection (LODs), calculated on the basis of a ratio of a signal-to-noise of 3 (S/N = 3), were in the range of 0.54–1.43 ␮g/L. The sensitivity enhancement factors (EFs), which were obtained by simply calculating the ratio of the LODs obtained from the standard injection and that from the IP-HF-LLLME were between 183- and 366-fold. The extraction efficiencies of target analytes by IP-HFLLLME are calculated to be 36.6–73.2%.

5. Real sample analysis For the validation of developed IP-HF-LLLME-CE/UV method, pretreated samples according to Section 2.4 without IP-HF-LLLME were directly injected for the analysis by CE/UV firstly. Very mussy electrophoretic peaks, increased baseline and irreproducible results were obtained and no identified peaks were observed. We also studied the effect of serum matrix on the extraction and determination of the target analytes in real samples. The serum samples collected from healthy people were pretreated according to the process as specified in Section 2.4. After de-proteination, various amount of the combined supernatants were taken out, spiked with appropriate amount of SDS, appropriate amount of

Table 2 Analytical performance of IP-HF-LLLME-CE-UV for target thyroid hormones. Analyte

Linear range (␮g/L)

Linear equation

R2

RSDa (n = 5) (%)

MIT DIT T2 rT3 T3 T4

5–500 5–500 5–250 5–250 5–250 5–250

y = (2.105 ± 0.034)x + (5.09 ± 0.09) y = (2.392 ± 0.092)x + (10.11 ± 0.86) y = (2.709 ± 0.12)x − (5.35 ± 0.32) y = (2.982 ± 0.10)x − (8.89 ± 0.21) y = (2.767 ± 0.15)x − (9.82 ± 0.76) y = (2.763 ± 0.08)x + (4.90 ± 0.33)

0.993 0.996 0.998 0.995 0.999 0.998

8.21 4.77 6.25 3.19 8.98 7.67

a b c

Analytes concentration: MIT 10 ␮g/L, DIT 10 ␮g/L, T2 10 ␮g/L, rT3 10 ␮g/L, T3 10 ␮g/L and T4 10 ␮g/L. EF = LODs (CE-UV)/LODs (IP-HF-LLLME-CE-UV). EE% =

Cacceptor phase Cdonor phase

×

Vacceptor phase Vdonor phase

× 100% = EF ×

Vacceptor phase Vdonor phase

.

LOD (␮g/L) CE/UV

IP-HF-LLLME-CE-UV

262.2 225.7 231.9 209.4 203.6 197.6

1.43 0.82 0.75 0.68 0.65 0.54

EFb

EEc (%)

183 275 309 308 313 366

36.6 55.0 61.8 61.6 62.6 73.2

P. Li et al. / J. Chromatogr. A 1356 (2014) 23–31

29

Table 3 Analytical results of thyroid hormones in human serum samples (n = 3) obtained by IP-HF-LLLME-CE-UV. Analytes

MIT DIT T2 rT3 T3 T4

Human serum 1

Human serum 2

Detected (␮g/L)

Spiked (␮g/L)

Found (␮g/L)

N.D. N.D. N.D. N.D. N.D. 104.9 ± 12.8

200 200 200 200 200 200

186.3 184.2 166.8 202.5 162.1 291.1

± ± ± ± ± ±

8.2 13.9 4.9 9.1 10.4 12.0

Rec.a (%) 93 92 83 101 81 93

Detected (␮g/L)

Spiked (␮g/L)

Found (␮g/L)

N.D. N.D. N.D. N.D. N.D. 99.2 ± 10.9

200 200 200 200 200 200

176.3 218.9 174.1 184.4 160.4 301.3

± ± ± ± ± ±

12.8 18.7 8.3 4.8 10.3 23.1

Rec.a (%) 88 109 87 92 80 101

Peak area

Recovery = (the amount found in the spiked sample-the amount found in the sample) × 100%/the amount added. a Recovery: MIT, DIT, T2 , rT3 , T3 , T4 were spiked in the human serum sample.

Effect of serum matrix Fig. 5. Effect of serum matrix on IP-HF-LLLME. Conditions: analytes concentration, 300 ␮g/L; sample solution, pH 5.0; organic solvent, n-octanol; stirring rate, 1000 rpm; acceptor solution, 10 ␮L 30 mmol/L Na2 CO3 ; n = 3.

mixed target analytes, adjusted to pH 3.0, and diluted to a certain volume. The concentration of each of target analytes in the final solution for subsequent extraction was fixed as 300 ␮g/L. The dilution fold (final volume/original serum sample volume) was 2-, 10-, 40- and 100- folds, respectively. And the diluted serum samples were subjected to the proposed IP-HF-LLLME and the results were shown in Fig. 5. As can be seen, the signal intensities of target analytes in serum sample with 2-fold dilution are very low; when the dilution fold is increased from 2 to 40-fold, the signal of target analytes are increased obviously; no obvious difference are observed between 40-fold and 100-fold dilution. Considering the limited amount of available serum sample and possible effect of acetone on the extraction process, 50-fold dilution was employed for real sample analysis.

The applicability of the developed IP-HF-LLLME-CE-UV method was evaluated by the simultaneous analysis of thyroid hormones in human serum samples, and external standard method was utilized for quantification. Table 3 shows the analytical results of the target analytes and the recovery for the spiked samples. As can be seen, only T4 was detected in the two human serum samples with the concentration of 104.9 ± 12.8 ␮g/L in human serum sample 1 and 99.2 ± 10.9 ␮g/L in human serum sample 2, respectively. For the validation of the proposed method, the recovery experiments were carried out. As shown in Table 3, recoveries for the spiked human serum samples were in the range of 80–109%, indicating that the developed method of IP-HF-LPMECE-UV is reliable to measure thyroid hormones in human serum samples. The electrophoretograms of the human serum samples are shown in Fig. 6. As can be seen, the peaks of thyroid hormones are identified by the spiked experiments. The peaks detected at 5–6 min and 8–10 min may be the other compounds which are co-extracted with thyroid hormones. Besides, it should be noted that the total T4 were determined by the proposed method, while the determined values in clinic measurement by immunometric assays were for free T4 , which was not included herein. To further evaluate the developed IP-HF-LLLME-CE/UV method, LODs and the applicability of this method was compared with several other reported approaches used for thyroid hormones analysis. As can be seen from Table 4, the proposed method using inexpensive commercial CE-UV detection can provide sub-␮g/L levels of LODs and it is successfully applied for real serum samples. Also, it is worth noticing that miniaturized extraction method of IP-HF-LPME is developed for the thyroid hormones analysis, which has a great promising in clinic measurement of thyroid hormone species instead of only T4 and even for further analysis of much low abundance of free thyroid hormone species in biological samples if such miniaturized extraction method is coupled with high sensitivity detector such as MS/MS or ICPMS.

Table 4 Comparison of limits of detection for the analysis of thyroid hormones by different approaches. Analytes T4 , T3 , rT3 , T2 , T1 T3 , T4 T4 , T3 , T2 , rT3 , DIT, MIT T4 , T3 , T2 , T0 , DIT, MIT Iodide, iodide, T3 , T4 T4 , T3 , rT3 , T2 T4 , T3 , T2 , rT3 , DIT, MIT T2 , rT2 , T3 , rT3 , T4 T4 , T3 , rT3 , T2 T4 , T3 , T2 , rT3 , DIT, MIT

Preconcentration

Detection

LODs

Sample

Ref.

SPE SPE SPE SBSE IP-HF-LPME

LC–MS/MS CE-ADa HPLC-UV CE-UV CE-ICP-MS LC–MS/MS HPLC-UV LC–MS/MS HPLC-ICP/MS CE-UV

0.25–0.52 ng/mg (0.85–0.1) × 10−6 mol/L 40–380 ␮g/L 1300–1400 ␮g/L 7.5–61 ␮g/L 0.0003–0.0039 ␮g/L 20–100 ␮g/L 0.16–0.59 pg 0.0071–0.0355 ␮g/L 0.54–1.43 ␮g/L

Rat thyroid gland Pharmaceutical formulations Iodinated casein Iodinated casein Human serum, urine Tap water, waste water Pharmaceuticals, blood serum, urine Brain, liver, heart and thyroid homogenate Urine Human serum

[27] [28] [29] [30] [31] [1] [3] [16] [11] This work

Capillary electrophoresis enzyme immunoassay for alpha-fetoprotein and thyroxine in human serum with electrochemical detection. T1 : 3-iodo-l-thyronine. a AD: amperometric detection.

30

P. Li et al. / J. Chromatogr. A 1356 (2014) 23–31

Fig. 6. Electrophoretograms of thyroid hormones obtained by IP-HF-LLLME-CE-UV in (A) healthy human serum samples and (B) spiked human serum samples. 200 ␮g/L MIT, DIT, T2 , rT3 , T3 and T4 were spiked respectively in the sample solution after centrifugation in sample pretreatment procedures.

6. Conclusions In this work, a new IP-HF-LLLME technique was proposed for simultaneous extraction of six target analytes, four thyroid hormones (T2 , T3 , rT3 and T4 ) and two relevant compounds (MIT and DIT), with different polarity and water solubility. The extraction was facilitated by the introduction of a low concentration of SDS as an ion pair reagent in the donor phase. Accordingly, a new IP-HF-LLLME-CE-UV method was successfully established for the simultaneous analysis of the thyroid hormones in human serum samples. The merits of the developed approach include good clean-up ability, high sensitivity, and suitability for the determination of thyroid hormones in real-world biological samples. Acknowledgements Financial supports from the National Natural Science Foundation of China (Nos. 21375097, 21075095, 21175102), the Science Fund for Creative Research Groups of NSFC (Nos. 20621502, 20921062) and SRFDP (20110141110010) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.06.046. References [1] J. Svanfelt, J. Eriksson, L. Kronberg, Analysis of thyroid hormones in raw and treated waste water, J. Chromatogr. A 1217 (2010) 6469–6474. [2] D.L. Wang, H.M. Stapleton, Analysis of thyroid hormones in serum by liquid chromatography–tandem mass spectrometry, Anal. Bioanal. Chem. 397 (2010) 1831–1839. [3] H.G. Gika, V.F. Samanidou, I.N. Papadoyannis, Development of a validated HPLC method for the determination of iodotyrosines and iodothyronines in pharmaceuticals and biological samples using solid phase extraction, J. Chromatogr. B 814 (2005) 163–172. [4] D. Jin, A.P. Kumar, G.C. Song, Y.I. Lee, Determination of thyroxine enantiomers in pharmaceutical formulation by high-performance liquid chromatography–mass spectrometry with precolumn derivatization, Microchem. J. 88 (2008) 62–66.

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