J S S
ISSN 1615-9306 · JSSCCJ 38 (9) 1441–1624 (2015) · Vol. 38 · No. 9 · May 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
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Zaifa Pan1 Xiaoya Huang1 Yuan Zhong1 Lili Wang1 Danhua Zhu2 Lanjuan Li2 ∗ 1 College
of Chemical Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang, P. R. China 2 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, Zhejiang, P. R. China Received January 4, 2015 Revised January 25, 2015 Accepted February 3, 2015
Research Article
Three-phase hollow-fiber microextraction combined with ion-pair high-performance liquid chromatography for the simultaneous determination of five components of compound ␣-ketoacid tablets in human urine The determination of ␣-ketoacid concentration is demanded to evaluate the absorption and metabolic behavior of compound ␣-ketoacid tablets taken by chronic kidney disease patients. To eliminate the interference of endogenous substance of urine and enrich the analytes, a three-phase hollow-fiber liquid-phase microextraction combined with ion-pair high-performance liquid chromatography method was established for the determination of D,L-␣-hydroxymethionine calcium, D,L-␣-ketoisoleucine calcium, ␣-ketovaline calcium, ␣-ketoleucine calcium, and ␣-ketophenylalanine calcium of compound ␣-ketoacid tablets in human urine samples. The extraction parameters, such as organic solvent, pH of donor phase and acceptor phase, stirring rate, and extraction time were optimized. Under the optimal conditions, the obtained enrichment factors were up to 11-, 110-, 198-, 202-, and 50fold, respectively. The calibration curves for these analytes were linear over the range of 0.1– 10 mg/L for ␣-ketovaline calcium, D,L-␣-ketoisoleucine calcium, and ␣-ketoleucine calcium, 0.5–10 mg/L for D,L-␣-hydroxymethionine calcium, and ␣-ketophenylalanine calcium with r > 0.99. The relative standard deviations (n = 5) were less than 6.27% and the LODs were 100.7, 10.0, 5.8, 7.8, and 8.6 g/L (based on S/N = 3), respectively. Good recoveries from spiked urine samples (92–118%) were obtained. The proposed method demonstrated excellent sample clean-up and analytes enrichment to determine the five components in human urine. Keywords: High-performance liquid chromatography / Hollow-fiber extraction / Human urine / ␣-Ketoacid DOI 10.1002/jssc.201401497
1 Introduction The number of patients of chronic kidney disease (CKD) is increasing, which becomes a global health challenge [1, 2]. It is essential to retard CKD evolution to end-stage renal disease [3, 4], and a low-protein diet supplemented with ␣-ketoacid is one of the efficient diet treatments of CKD [5–7]. ␣-Ketoacid could act as precursor of the amino acid, prevent degradation of the body’s own protein and reduce proteinuria, then relieve symp-
Correspondence: Dr. Zaifa Pan, College of Chemical Engineering, Zhejiang University of Technology, Chaowang Road 18#, Hangzhou 310014, China E-mail: [email protected] Fax: +86-571-88320797
Abbreviations: CKD, chronic kidney disease; EF, enrichment factor; HF-LPME, three-phase hollow-fiber LPME; HMACa, D,L-␣-hydroxymethionine calcium; KILCa, D,L-␣ketoisoleucine calcium; KLCa, ␣-ketoleucine calcium; KPACa, ␣-ketophenylalanine calcium; KVCa, ␣-ketovaline calcium; r, correlation coefficient; TBAH, tetrabutylammonium hydroxide C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
toms of uremia [5]. Clinically, the absorption and metabolic situation are different for individuals after patients taking compound ␣-ketoacid tablets. To evaluate the therapeutic effect and provide the basis data of doses of clinical treatment, it is demanded to develop an efficient determination method to monitor the ␣-ketoacid concentration in blood and urine. In addition, several aminoacidopathies also make the content of some ␣-ketoacid in human urine increasing abnormally, for instance, phenylketonuria results in the increasing of phenylalanine and its corresponding ␣-ketoacid concentration [8], maple syrup urine disease leads to the accumulation of the branched-chain ␣-ketoacids in urine [9]. Therefore, the determination of ␣-ketoacid concentration is also very important in diagnosis of these diseases. Compound ␣-ketoacid tablet contains four keto- and one hydroxy-amino acid in the form of calcium salts, including D,L-␣-hydroxymethionine calcium (HMACa), ␣-ketovaline calcium (KVCa), D,L-␣-ketoisoleucine calcium (KILCa), ␣-ketoleucine calcium (KLCa), and ∗ Additional
corresponding author: Professor Lanjuan Li.
E-mail: [email protected]
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␣-ketophenylalanine calcium (KPACa). Due to its complicated composition and high polarity, it is difficult to carry out quantitative analysis for ␣-ketoacid tablet by general HPLC. To date, besides the nonaqueous titration[10], IEC [11] and ion-pair RP-HPLC [12, 13] have been reported for the simultaneous determination of these five components in tablets. However, there is no report on the determination of the five components of compound ␣-ketoacid tablet in body fluid samples. Because of complex composition of biological fluids and low concentration of target analytes in biological samples, pretreatment and preconcentration step are generally required. A miniaturized extraction technology, hollow-fiber-based (HF) LPME was introduced by Pedersen-Bjergaard and Rasmussen in 1999 [14]. HF-LPME has high enrichment factor and sensitivity, and hollow fiber is disposable that can eliminate the possibility of carryover and cross-contamination [15]. Furthermore, HF-LPME is an eco-friendly and efficient alternative in sample preparation because of low consumption of organic solvent. Based on the advantages above, HF-LPME has been widely used to the medicine analysis in biological samples, including amino alcohols [16], tetracycline antibiotics [17], mirtazapine and its metabolites [18], strychnos alkaloids [19], antifungal drugs [20], and so on. Therefore, HFLPME is a powerful technique to eliminate the interference of endogenous substance of urine and to enrich the analytes. Because of the low partition coefficient between the organic phase and aqueous sample for most drugs, three-phase HFLPME is usually chosen for alkaline [21, 22] and acidic compounds [23, 24]. In this extraction type, various parameters may affect the extraction efficiency and accurate determination remains challenge. So it would be of great importance to develop a convenient and accurate pretreatment method for determination of the five ␣-ketoacid components in the urine. The present work was intended to establish an appropriate three-phase HF-LPME method combined with ion-pair RP-HPLC to determine HMACa, KVCa, KILCa, KLCa, and KPACa in human urine. Tetrabutylammonium hydroxide (TBAH) was selected and served as both acceptor solution and ion-pair reagent of mobile phase in this method. And the application of ion-pair reagent TBAH solution as the acceptor phase could avoid the procedure of neutralization with acetic acid when NaOH solution was used as the acceptor phase. Various parameters affecting the extraction efficiency such as organic solvent type, pH of donor phase and acceptor phase, ionic strength, stirring rate, and extraction time were investigated and optimized. Ultimately, the optimized and validated method was applied for simultaneous determination of these five analytes in human urine.
2 Materials and methods 2.1 Chemicals and reagents HMACa, KVCa, KILCa, KLCa, and KPACa were provided by Anglikang Pharmaceutical (Shaoxin, China). Tetrabuty C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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lammonium hydroxide (TBAH, 25%, w/v in water), hexyl acetate (99%), dodecanol, n-hexane, 1-octanol, and toluene were purchased from Aladdin (www.aladdin-e.com). Sulfuric acid (98%) was provided by Juhua Group (Quzhou, China) and sodium chloride was purchased from Shanghai reagent fourth factory (Shanghai, China). Sodium hydrogen phosphate and sodium dihydrogen phosphate were bought from Huzhou chemical reagent (Huzhou, China). Acetonitrile (HPLC grade) was provided by Tianjin Siyou Fine chemicals (Tianjin, China). Purified water was obtained from Wahaha Group (Hangzhou, China).
2.2 Apparatus and chromatographic conditions The HPLC system (Waters, USA) was equipped with a Waters 1525 binary HPLC pump, a Waters 2998 photodiode array detector, and a manual injector. Chromatographic data were recorded and analyzed by using a Waters BreezeTM 2 software. Q3/2 Accurel polypropylene hollow fiber (600 m id, 200 m wall thickness and 0.2 m pore size) was bought from Membrana (Wuppertal, Germany). Two 10 L GC microsyringes were obtained from Gaoge Industrial and Trading (Shanghai, China). The analytes were performed on a Symmetry C18 column (4.6 mm × 250 mm, particle size 5 m) at 35⬚C. The mobile phase consisted of acetonitrile (component A) and 20 mmol/L sodium dihydrogen phosphate buffer (component B, containing 15 mmol/L TBAH, pH 7.0) at a flow rate of 1.0 mL/min. A linear elution gradient was programmed from 8–25% A for 28 min and then the composition of 25–75% (A, B) was used in isocratic mode for 12 min. There was a 15 min interval between injections reaching reequilibration of the column to the initial conditions. The five compounds were baseline separated at the detection wavelength of 210 nm.
2.3 Three-phase LPME procedure Before the extraction, the hollow fibers were cut manually into segments of 5 cm, and then each piece of fiber was ultrasonically cleaned in acetone for 5 min to remove any contaminant in the fiber and then dried in air. For each experiment, a 10 mL aliquot of sample solution (pH = 1, adjusted with diluted H2 SO4 ) containing 30% m/v NaCl was poured into a 15 mL sample vial with a screw cap and a silicon septum, and the sample vial was fixed on the magnetic stirrer device. Then the fiber was immersed into the organic solvent (hexyl acetate) for 1 min to impregnate the pores and rinsed with purified water to remove the excess organic solvent from the surface of the fiber. Subsequently, 10 L of acceptor solution (0.5% TBAH) was injected into the lumen of the hollow fiber using a microsyringe, and another microsyringe was inserted into the other side of the fiber to close and support the fiber. The fiber was bent into a U-shape and immersed into the sample solution (donor phase) together with the microsyringes. Then the vial was capped and shaken www.jss-journal.com
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on a Magnetic stirrer (IKA C-MAG HS7, Germany) with an 8 mm × 3 mm magnetic stirrer bar for 30 min at 150 rpm. After extraction time out, the acceptor solution (6 L) was pushed out with a 1 mL disposable syringe and transferred into a 1.5 mL centrifuge tube. Then 18 L volume of purified water was added into the centrifuge tube by microsyringe and the acceptor phase was diluted to 24 L. Finally, 20 L of solution was injected into the HPLC system for analysis.
2.4 Preparation of sample solutions and biological samples The stock solution of HMACa, KVCa, KILCa, KLCa, and KPACa was prepared by dissolving accurately weighed standard substances in 10 mL purified water to get a concentration of 1.0 g/L. The working solution at 100 mg/L was prepared by diluting the stock solution with water. Human urine samples were collected from a healthy volunteer and stored at –18⬚C in the fridge and brought to room temperature before used. Before injecting, the urine samples without LPME extraction were centrifuged for 10 min (rotating speed of 4000 rpm), and then filtered through 0.45 m syringe filter (ProMax, Nylon). The urine calibration standards were made at concentrations of 0.1, 0.5, 2.0, 5.0, 7.0, and 10.0 mg/L by appropriate dilution of the stock solution with urine.
3 Results and discussion 3.1 Optimization of HPLC conditions A Symmetry C18 column (4.6 mm × 250 mm, particle size 5 m) was selected as working column at 35⬚C. TBAH was chose as ion-pair reagent and acetonitrile as organic component. The chromatographic conditions, such as the concentration of ion-pair reagent, the pH value of the mobile phase and the concentration of the phosphate buffer were investigated to obtain a satisfactory separation for the five compounds and endogenous substance of urine. The retention times of the five analytes became longer with the increasing of concentration of ion-pair reagents from 5, 10 to 15 mmol/L and good separation was obtained when the concentration of ion-pair reagent was 15 mmol/L. When the pH value was 3, the ion-pair could not be well formed and the five analytes could not be separated well. Good separation was not obtained for pH value 5, but achieved when the pH value equals 7. The concentration of phosphate buffer did not show obvious effect on separation and 20 mmol/L was selected. In consideration of the retention time, resolution, peak symmetry, and the protection of column, the optimized separation conditions were obtained as following: The mobile phase consisted of acetonitrile (component A) and 20 mmol/L sodium dihydrogen phosphate buffer (component B, containing 15 mmol/L TBAH, pH 7.0) at a flow rate of 1.0 mL/min. A linear elution gradient was programmed from 8 to 25% A for C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Basic principle of three-phase LPME.
28 min and then the composition of 25–75% (A, B) was used in isocratic mode for 12 min. All chromatographic separations were performed at the detection wavelength of 210 nm.
3.2 Extraction mechanism of three-phase LPME The extraction principle of the five components [(RCOO− )2 Ca2+ ] is illustrated in Fig. 1. The extraction efficiency of threephase LPME is based on the distribution of analytes in three phases (donor phase, organic phase, and acceptor phase), herein, the organic phase (hexyl acetate) is immobilized in the pores of the hollow fiber. In this case, the organic phase served as an effective barrier and intermediary layer between the two aqueous solutions. Before extraction, the donor phase was adjusted to acidic where [RCOO− ] was turned into [RCOOH], to increase the partition coefficient between the organic phase and donor phase. Subsequently, the analytes [RCOOH] were extracted from the aqueous sample (donor phase) through the organic phase into the alkaline aqueous solution (acceptor phase) inside the lumen of the hollow fiber. Because of the correspondingly high solubility of [RCOOH] in the alkaline acceptor solution and further formation of neutral ion-pairs [N(CH3 CH2 CH2 CH2 )4 OOCR] in the acceptor phase, the analytes were continuously transport to the acceptor phase until reached a partition equilibrium, resulting in a high enrichment of analytes in acceptor phase. Based on this principle, the factors of such three-phase LPME procedure were investigated and optimized in the following section.
3.3 Optimization of three-phase LPME procedure To obtain the optimal conditions in the proposed extraction procedure, the parameters including organic solvent, pH of donor phase and acceptor phase, ionic strength, stirring rate, and extraction time were investigated. The concentration of the analytes HMACa, KVCa, KILCa, and KLCa were 10 and 5 mg/L for KPACa. www.jss-journal.com
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3.3.1 Selection of organic solvent Choosing a suitable organic solvent plays an important role in achieving efficient enrichment of the analytes [14,25]. Firstly, organic solvent should have similar polarity with the hollow fiber, so it can be strongly immobilized within the pores of the fiber. Secondly, organic solvent should be able to provide high solubility for the target analytes, meanwhile the solubility should be higher in organic solvent than in donor solution but lower than in acceptor solution, so as to increase the extraction efficiency [26]. Thirdly, as a barrier between the donor and acceptor phase, organic solution should be immiscible with water and samples and stable enough during extraction to against dissolution. In addition, the solvent should be of low volatility, low viscosity, and no toxicity. Based on the above factors, five organic solvents (dodecanol, n-hexane, 1-octanol, toluene, and hexyl acetate) were evaluated for extraction of the five target analytes by three-phase LPME under the same conditions mentioned above. As can be seen in Fig. 2, although 1-octanol was mentioned as one of the most commonly used extraction solvents in this LPME extraction techniques [20,27,28], in this work, hexyl acetate was selected as the extraction solvent for subsequent experiments due to its highest extraction efficiency for present five components comparing to the other solvents. 3.3.2 Selection of acceptor phase The five target analytes are weak acidic compounds because of carboxyl groups, so the acceptor phase should be alkaline solution to keep the analytes ionized and promote their dissolution, which ensures the ionized molecules efficiently extracted from the organic phase into acceptor phase. In this work, TBAH solution was used as acceptor phase and NaOH solution was chose as control acceptor phase to compare the effect of extraction efficiency. Figure 3A showed that the extraction efficiency of the five analytes increased with the concentration of TBAH up to 0.5% in water, and then decreased
Figure 2. Effect of five organic solvents on the extraction efficiency. pH value of donor phase: 1, acceptor phase: 5% TBAH, extraction time: 30 min, stirring rate: 150 rpm, salt addition: null.
with further increase of TBAH concentration. Similar trends were observed for NaOH solution and the maxima extraction efficiency was obtained for 0.5 mol/L, as shown in Fig. 3B. The extraction recovery for HMACa, KVCa, KILCa, KLCa at 0.5% TBAH as acceptor phase were higher than 0.5 mol/L NaOH as acceptor phase, except for KPACa. To protect the C18 column, the NaOH acceptor phase solution should be neutralized with acetic acid before injected into HPLC system, but TBAH could be used as ion-pair reagent and did not need this further neutralization. Considering the above factors, 0.5% TBAH was selected to be the acceptor phase in the following studies and the acceptor phase was directly injected into HPLC system without a neutralization step. 3.3.3 Effect of pH value of donor phase The pH of donor phase also plays an important role in three phases LPME. The pH difference between donor phase and
Figure 3. Effect of the acceptor phase on the extraction efficiency: (A) TBAH as acceptor phase, (B) NaOH as acceptor phase. pH value of donor phase: 1, organic solvents: hexyl acetate, extraction time: 30 min, stirring rate: 150 rpm, salt addition: null.
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Figure 4. Other factors on the extraction: (A) Effect of pH value of donor phase, (B) effect of salt concentration, (C) effect of stirring speed, (D) effect of extraction time. Experimental conditions: pH value of donor phase: 1 for (B), (C), and (D); salt addition: 0.3 g/mL for (C), (D), and null for (A); extraction time: 30 min for (A), (B), and (C); stirring speed: 150 rpm for (A), (B), and (D); acceptor phase: 0.5% TBAH; organic solution: hexyl acetate.
organic phase can change the distribution coefficient of analytes, thereby improving the extraction efficiency. For acidic compounds, the sample solution should be acidic enough to keep all the analytes mainly in neutral form, to enhance the affinity of analytes to the organic solvent and reduce their solubility in the donor phase. Therefore, pH was tested for donor phase in the range of 0–5 (adjusted with sulfuric acid). Figure 4A showed that the highest extraction efficiency of all analytes was found at pH 1, and then with pH increasing the extraction efficiency decreased. At higher pH, protonation reaction was not complete and a great quantity of the analytes existed in ionic form with low affinity to the organic solvent [26]. Therefore, the optimum pH of the donor phase was selected as 1. 3.3.4 Effect of salt addition In the HF-LPME experiments, salt was often added into the sample solution to improve the extraction efficiency due to salting out effect [29]. A series of solutions with various concentrations of NaCl in the range of 0–0.35 g/mL were C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
added into donor phase to investigate the effect of the salt concentration on the extraction. As can be seen in Fig. 4B, the extraction efficiency increased with the NaCl concentration up to 20% for KLCa and 30% for others. This phenomenon can be explained with the two theories coexisted in the salt solution: (1) Salting out effect, it can increase the ionic strength and decrease the solubility of analytes in aqueous phase. This is because there are water molecules forming hydration spheres around the salt ions. These hydration spheres reduce the solubility of analyte molecules in the water [30]. (2) Electrostatic interactions, this force between polar molecules and salt ions in aqueous solution decreases mass transfer ability, thus decreasing the extraction efficiency for polar analytes. In the initial time, salting out effect played the predominant role. With the further increasing salt concentration, the electrostatic interactions between analytes and salt molecules overshadowed the salting out effect and became dominated, which led to the decline of response. Based on the results above, a 0.3 g/mL NaCl concentration was used as the final salt addition in the following studies.
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Table 1. Linear range, correlation equation, linear range, LOD, and enrichment factor of the method
Analytes
Liner range (mg/L)
Calibration curvea)
Correlation coefficient (r)
LOD (g/L)
RSD %, (n = 3)b)
Enrichment factor
HMACa KVCa KILCa KLCa KPACa
0.5–10 0.1–10 0.1–10 0.1–10 0.5–10
y = 27 704x + 28 788 y = 277 906x + 199 512 y = 482 013x + 317 373 y = 356 575x + 821 714 y = 323 251x + 150 420
0.9951 0.9903 0.9903 0.9923 0.9925
100.7 10.0 5.8 7.8 8.6
5.94 4.86 6.27 4.17 5.23
11 110 198 202 50
a) y: peak area, x: concentration (mg/L). b) All analytes at 5 mg/L.
3.3.5 Effect of stirring rate The transfer of target analytes between sample solution and organic phase was influenced by diffusion rate. Stirring process can decrease the thickness of diffusion layer around the interface between two phases, then accelerated the mass transfer and increased the extraction efficiency. Stirring also reduced the time required to attain a thermodynamic equilibrium [31, 32]. But at high stirring speed, extraction solvent could fall off from the lumen of hollow fiber. In addition, air bubbles could be produced and then adhered on the surface of the fiber, which can break the stability of acceptor phase and then decrease the extraction efficiency [33]. The effect of stirring rate varied in the range of 50–300 rpm was evaluated in Fig. 4C. At last, 150 rpm was applied in this study. 3.3.6 Effect of extraction time HF-LPME is an essentially dynamic mass transfer process, including the partitioning and diffusion of analytes between aqueous solution and organic solvent. To reach the maximum extraction efficiency of target analytes, extraction time was optimized from 10 to 50 min at the stirring speed of 150 rpm. Figure 4D showed that with the extraction time increasing in the range of 10–30 min, the amount of the extracted analytes was found to increase. After 30 min, the peak area of each compound started to decrease because of the loss and drop of organic solvent. Therefore, an extraction time of 30 min was chosen as the optimal condition.
3.4 Method evaluation Based on the research in the previous sections, the optimum extraction conditions were achieved: 5 cm hollow fiber immersed with hexyl acetate as the extraction solvent, 0.5% TBAH solution as the acceptor phase, 10 mL aqueous solution with pH 1 and 30% sodium chloride as the donor phase, 150 rpm stirring rate, 30 min extraction time at room temperature. Under these conditions, a series of experiments were carried out to determine the performance of this method, and the results were listed in Table 1. The calibration curves were obtained by plotting the measured peak areas against the concentrations of the analytes in the sample in the range of 0.1–10 mg/L (KVCa, KILCa, and KLCa) and 0.5–10 mg/L C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(HMACa and KPACa). Each level was extracted by the proposed method above and analyzed by ion-pair HPLC three times. All of these analytes exhibited good linearity with correlation coefficients (r) larger than 0.9903, and the RSDs for five replicate analyses of three target analytes at 5 mg/L were all below 6.27%. The LODs of these compounds in the urine sample were 5.8–100.7 g/L (S/N = 3). The enrichment factor (EF) was calculated according to the following equation: EF = CAP, final /CAP, initial (CAP, final : concentration of analytes in the acceptor phase after extraction, CAP, initial : initial concentration of analytes in the aqueous solution). The results indicated that the five analytes were extracted with satisfactory enrichment factors between 11 and 202. To evaluate the applicability of the proposed three phases LPME method to real samples, the established method was employed in the extraction of the five compounds in human urine. Each urine sample was diluted at 1:3 ratio with purified water to reduce matrix effect. After addition of 0.3 g/mL NaCl and adjustment of the pH at 1.0, the diluted urine and the sample spiked with definite value of standard was extracted under the optimum conditions. According to the obtained results (Table 2), the recoveries were in the range of 92–118% and RSDs were between 3.13 and 5.73% after five replicate analyses. Figure 5 shows the HPLC chromatograms of urine sample spiked with 7 mg/L (A) and blank urine sample (B) after HF-LPME, and those two without extraction (C) and (D). By comparison of Figure 5A and C, as well as B and D, we can see that the interference of endogenous substance of urine could be well eliminated by the HF-LPME, especially the polar components with the retention time less than 10 min. Furthermore, without the HF-LPME, it is hard to detect the five compounds in the spiked urine sample, as shown in Figure 5C, because of their low concentration. After extraction, Figure 5A shows that high enrichment were achieved for the five components, which is significant for detection of trace amount of analytes in urine. Therefore, the established method had high clean-up and enrichment power for determination of five components of compound ␣-ketoacid tablets in human urine.
4 Conclusions A new analytical method based on three-phase hollow-fiber microextraction coupled with ion-pair HPLC has been www.jss-journal.com
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Table 2. Recoveries and RSDs of the five components spiked to human urine samples
Analytes
Original amount (mg/L)
Spike amount (mg/L)
Found amount (mg/L)
Recovery (%)
RSD (%, n = 5)
HMACa
NDa)
KVCa
ND
KILCa
ND
KLCa
ND
KPACa
ND
2 7 2 7 2 7 2 7 2 7
2.19 6.42 2.23 7.17 2.34 7.26 2.36 7.1 1.87 8.08
110 92 112 102 117 104 118 101 94 115
3.72 3.44 4.36 4.40 5.46 5.73 4.56 4.43 3.35 3.13
a) ND, not detected.
Figure 5. The HPLC chromatograms of (A) diluted human urine spiked with 7 mg/L standards and (B) nonspiked diluted urine sample after the extraction under the optimized conditions, (C) diluted human urine spiked with 7 mg/L standards without extraction, (D) blank urine without extraction. Peak identifications: 1. HMACa; 2. KVCa; 3. KILCa; 4. KLCa; 5. KPACa.
developed to determine the five components of compound ␣-ketoacid tablet in human urine. Up to 202-fold enrichment factor was achieved, along with highly effective sample clean-up the interference of endogenous substance of urine, so were good linearity and recovery. And the ion-pair reagent TBAH was selected as the acceptor phase, which can avoid the neutralization step with acid for NaOH acceptor phase solution. Overall, this method is applicable to simultaneous determination of the five compounds of compound ␣-ketoacid tablet at low concentrations in human urine sample, and provides dose reference for the patients of CKD who are taking ␣-ketoacid tablets in clinical treatment. The established method could be further developed for the determination of ␣-ketoacid in the urine sample of the patients with aminoacidopathies diseases. The authors gratefully acknowledge the financial support of the open fund project of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (2010KF02). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The authors have declared no conflict of interest.
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[21] Saraji, M., Bidgoli, A. A. H., Farajmand, B., J. Sep. Sci. 2011, 34, 1708–1715. [22] Yang, Y., Chen, J., Shi, Y. P., J. Chromatogr. B 2010, 878, 2811–2816. [23] Mirzaei, M., Dinpanah, H., J. Chromatogr. B 2011, 879, 1870–1874. [24] Sanagi, M. M., Miskam, M., Ibrahim, W. A. W., Hermawan, D., Aboul-Enein, H. Y., J. Sep. Sci. 2010, 33, 2131–2139. [25] Farahani, H., Yamini, Y., Shariati, S., Khalili-Zanjani, M. R., Mansour-Baghahi, S., Anal. Chim. Acta 2008, 626, 166–173. [26] Shariati, S., Yamini, Y., Darabi, M., Amini, M., J. Chromatogr. B 2007, 855, 228–235. [27] Psillakis, E., Kalogerakis, N., TrAC, Trends Anal. Chem. 2003, 22, 565–574. [28] Xu, L., Basheer, C., Lee, H. K., J. Chromatogr. A 2007, 1152, 184–192. [29] Saraji, M., Mousavi, F., Food Chem. 2010, 123, 1310– 1317. [30] Boydboland, A. A., Pawliszyn, J. B., J. Chromatogr. A 1995, 704, 163–172. [31] Rasmussen, K. E., Pedersen-Bjergaard, S., TrAC, Trends Anal. Chem. 2004, 23, 1–10. [32] Jeannot, M. A., Cantwell, F. F., Anal. Chem. 1997, 69, 235– 239. [33] Yang, Y., Chen, J., Shi, Y. P., Biomed. Chromatogr. 2010, 24, 1350–1355.
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ISSN 1615-9306 · JSSCCJ 38 (9) 1441–1624 (2015) · Vol. 38 · No. 9 · May 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
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Zaifa Pan1 Xiaoya Huang1 Yuan Zhong1 Lili Wang1 Danhua Zhu2 Lanjuan Li2 ∗ 1 College
of Chemical Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang, P. R. China 2 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, Zhejiang, P. R. China Received January 4, 2015 Revised January 25, 2015 Accepted February 3, 2015
Research Article
Three-phase hollow-fiber microextraction combined with ion-pair high-performance liquid chromatography for the simultaneous determination of five components of compound ␣-ketoacid tablets in human urine The determination of ␣-ketoacid concentration is demanded to evaluate the absorption and metabolic behavior of compound ␣-ketoacid tablets taken by chronic kidney disease patients. To eliminate the interference of endogenous substance of urine and enrich the analytes, a three-phase hollow-fiber liquid-phase microextraction combined with ion-pair high-performance liquid chromatography method was established for the determination of D,L-␣-hydroxymethionine calcium, D,L-␣-ketoisoleucine calcium, ␣-ketovaline calcium, ␣-ketoleucine calcium, and ␣-ketophenylalanine calcium of compound ␣-ketoacid tablets in human urine samples. The extraction parameters, such as organic solvent, pH of donor phase and acceptor phase, stirring rate, and extraction time were optimized. Under the optimal conditions, the obtained enrichment factors were up to 11-, 110-, 198-, 202-, and 50fold, respectively. The calibration curves for these analytes were linear over the range of 0.1– 10 mg/L for ␣-ketovaline calcium, D,L-␣-ketoisoleucine calcium, and ␣-ketoleucine calcium, 0.5–10 mg/L for D,L-␣-hydroxymethionine calcium, and ␣-ketophenylalanine calcium with r > 0.99. The relative standard deviations (n = 5) were less than 6.27% and the LODs were 100.7, 10.0, 5.8, 7.8, and 8.6 g/L (based on S/N = 3), respectively. Good recoveries from spiked urine samples (92–118%) were obtained. The proposed method demonstrated excellent sample clean-up and analytes enrichment to determine the five components in human urine. Keywords: High-performance liquid chromatography / Hollow-fiber extraction / Human urine / ␣-Ketoacid DOI 10.1002/jssc.201401497
1 Introduction The number of patients of chronic kidney disease (CKD) is increasing, which becomes a global health challenge [1, 2]. It is essential to retard CKD evolution to end-stage renal disease [3, 4], and a low-protein diet supplemented with ␣-ketoacid is one of the efficient diet treatments of CKD [5–7]. ␣-Ketoacid could act as precursor of the amino acid, prevent degradation of the body’s own protein and reduce proteinuria, then relieve symp-
Correspondence: Dr. Zaifa Pan, College of Chemical Engineering, Zhejiang University of Technology, Chaowang Road 18#, Hangzhou 310014, China E-mail: [email protected] Fax: +86-571-88320797
Abbreviations: CKD, chronic kidney disease; EF, enrichment factor; HF-LPME, three-phase hollow-fiber LPME; HMACa, D,L-␣-hydroxymethionine calcium; KILCa, D,L-␣ketoisoleucine calcium; KLCa, ␣-ketoleucine calcium; KPACa, ␣-ketophenylalanine calcium; KVCa, ␣-ketovaline calcium; r, correlation coefficient; TBAH, tetrabutylammonium hydroxide C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
toms of uremia [5]. Clinically, the absorption and metabolic situation are different for individuals after patients taking compound ␣-ketoacid tablets. To evaluate the therapeutic effect and provide the basis data of doses of clinical treatment, it is demanded to develop an efficient determination method to monitor the ␣-ketoacid concentration in blood and urine. In addition, several aminoacidopathies also make the content of some ␣-ketoacid in human urine increasing abnormally, for instance, phenylketonuria results in the increasing of phenylalanine and its corresponding ␣-ketoacid concentration [8], maple syrup urine disease leads to the accumulation of the branched-chain ␣-ketoacids in urine [9]. Therefore, the determination of ␣-ketoacid concentration is also very important in diagnosis of these diseases. Compound ␣-ketoacid tablet contains four keto- and one hydroxy-amino acid in the form of calcium salts, including D,L-␣-hydroxymethionine calcium (HMACa), ␣-ketovaline calcium (KVCa), D,L-␣-ketoisoleucine calcium (KILCa), ␣-ketoleucine calcium (KLCa), and ∗ Additional
corresponding author: Professor Lanjuan Li.
E-mail: [email protected]
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␣-ketophenylalanine calcium (KPACa). Due to its complicated composition and high polarity, it is difficult to carry out quantitative analysis for ␣-ketoacid tablet by general HPLC. To date, besides the nonaqueous titration[10], IEC [11] and ion-pair RP-HPLC [12, 13] have been reported for the simultaneous determination of these five components in tablets. However, there is no report on the determination of the five components of compound ␣-ketoacid tablet in body fluid samples. Because of complex composition of biological fluids and low concentration of target analytes in biological samples, pretreatment and preconcentration step are generally required. A miniaturized extraction technology, hollow-fiber-based (HF) LPME was introduced by Pedersen-Bjergaard and Rasmussen in 1999 [14]. HF-LPME has high enrichment factor and sensitivity, and hollow fiber is disposable that can eliminate the possibility of carryover and cross-contamination [15]. Furthermore, HF-LPME is an eco-friendly and efficient alternative in sample preparation because of low consumption of organic solvent. Based on the advantages above, HF-LPME has been widely used to the medicine analysis in biological samples, including amino alcohols [16], tetracycline antibiotics [17], mirtazapine and its metabolites [18], strychnos alkaloids [19], antifungal drugs [20], and so on. Therefore, HFLPME is a powerful technique to eliminate the interference of endogenous substance of urine and to enrich the analytes. Because of the low partition coefficient between the organic phase and aqueous sample for most drugs, three-phase HFLPME is usually chosen for alkaline [21, 22] and acidic compounds [23, 24]. In this extraction type, various parameters may affect the extraction efficiency and accurate determination remains challenge. So it would be of great importance to develop a convenient and accurate pretreatment method for determination of the five ␣-ketoacid components in the urine. The present work was intended to establish an appropriate three-phase HF-LPME method combined with ion-pair RP-HPLC to determine HMACa, KVCa, KILCa, KLCa, and KPACa in human urine. Tetrabutylammonium hydroxide (TBAH) was selected and served as both acceptor solution and ion-pair reagent of mobile phase in this method. And the application of ion-pair reagent TBAH solution as the acceptor phase could avoid the procedure of neutralization with acetic acid when NaOH solution was used as the acceptor phase. Various parameters affecting the extraction efficiency such as organic solvent type, pH of donor phase and acceptor phase, ionic strength, stirring rate, and extraction time were investigated and optimized. Ultimately, the optimized and validated method was applied for simultaneous determination of these five analytes in human urine.
2 Materials and methods 2.1 Chemicals and reagents HMACa, KVCa, KILCa, KLCa, and KPACa were provided by Anglikang Pharmaceutical (Shaoxin, China). Tetrabuty C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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lammonium hydroxide (TBAH, 25%, w/v in water), hexyl acetate (99%), dodecanol, n-hexane, 1-octanol, and toluene were purchased from Aladdin (www.aladdin-e.com). Sulfuric acid (98%) was provided by Juhua Group (Quzhou, China) and sodium chloride was purchased from Shanghai reagent fourth factory (Shanghai, China). Sodium hydrogen phosphate and sodium dihydrogen phosphate were bought from Huzhou chemical reagent (Huzhou, China). Acetonitrile (HPLC grade) was provided by Tianjin Siyou Fine chemicals (Tianjin, China). Purified water was obtained from Wahaha Group (Hangzhou, China).
2.2 Apparatus and chromatographic conditions The HPLC system (Waters, USA) was equipped with a Waters 1525 binary HPLC pump, a Waters 2998 photodiode array detector, and a manual injector. Chromatographic data were recorded and analyzed by using a Waters BreezeTM 2 software. Q3/2 Accurel polypropylene hollow fiber (600 m id, 200 m wall thickness and 0.2 m pore size) was bought from Membrana (Wuppertal, Germany). Two 10 L GC microsyringes were obtained from Gaoge Industrial and Trading (Shanghai, China). The analytes were performed on a Symmetry C18 column (4.6 mm × 250 mm, particle size 5 m) at 35⬚C. The mobile phase consisted of acetonitrile (component A) and 20 mmol/L sodium dihydrogen phosphate buffer (component B, containing 15 mmol/L TBAH, pH 7.0) at a flow rate of 1.0 mL/min. A linear elution gradient was programmed from 8–25% A for 28 min and then the composition of 25–75% (A, B) was used in isocratic mode for 12 min. There was a 15 min interval between injections reaching reequilibration of the column to the initial conditions. The five compounds were baseline separated at the detection wavelength of 210 nm.
2.3 Three-phase LPME procedure Before the extraction, the hollow fibers were cut manually into segments of 5 cm, and then each piece of fiber was ultrasonically cleaned in acetone for 5 min to remove any contaminant in the fiber and then dried in air. For each experiment, a 10 mL aliquot of sample solution (pH = 1, adjusted with diluted H2 SO4 ) containing 30% m/v NaCl was poured into a 15 mL sample vial with a screw cap and a silicon septum, and the sample vial was fixed on the magnetic stirrer device. Then the fiber was immersed into the organic solvent (hexyl acetate) for 1 min to impregnate the pores and rinsed with purified water to remove the excess organic solvent from the surface of the fiber. Subsequently, 10 L of acceptor solution (0.5% TBAH) was injected into the lumen of the hollow fiber using a microsyringe, and another microsyringe was inserted into the other side of the fiber to close and support the fiber. The fiber was bent into a U-shape and immersed into the sample solution (donor phase) together with the microsyringes. Then the vial was capped and shaken www.jss-journal.com
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on a Magnetic stirrer (IKA C-MAG HS7, Germany) with an 8 mm × 3 mm magnetic stirrer bar for 30 min at 150 rpm. After extraction time out, the acceptor solution (6 L) was pushed out with a 1 mL disposable syringe and transferred into a 1.5 mL centrifuge tube. Then 18 L volume of purified water was added into the centrifuge tube by microsyringe and the acceptor phase was diluted to 24 L. Finally, 20 L of solution was injected into the HPLC system for analysis.
2.4 Preparation of sample solutions and biological samples The stock solution of HMACa, KVCa, KILCa, KLCa, and KPACa was prepared by dissolving accurately weighed standard substances in 10 mL purified water to get a concentration of 1.0 g/L. The working solution at 100 mg/L was prepared by diluting the stock solution with water. Human urine samples were collected from a healthy volunteer and stored at –18⬚C in the fridge and brought to room temperature before used. Before injecting, the urine samples without LPME extraction were centrifuged for 10 min (rotating speed of 4000 rpm), and then filtered through 0.45 m syringe filter (ProMax, Nylon). The urine calibration standards were made at concentrations of 0.1, 0.5, 2.0, 5.0, 7.0, and 10.0 mg/L by appropriate dilution of the stock solution with urine.
3 Results and discussion 3.1 Optimization of HPLC conditions A Symmetry C18 column (4.6 mm × 250 mm, particle size 5 m) was selected as working column at 35⬚C. TBAH was chose as ion-pair reagent and acetonitrile as organic component. The chromatographic conditions, such as the concentration of ion-pair reagent, the pH value of the mobile phase and the concentration of the phosphate buffer were investigated to obtain a satisfactory separation for the five compounds and endogenous substance of urine. The retention times of the five analytes became longer with the increasing of concentration of ion-pair reagents from 5, 10 to 15 mmol/L and good separation was obtained when the concentration of ion-pair reagent was 15 mmol/L. When the pH value was 3, the ion-pair could not be well formed and the five analytes could not be separated well. Good separation was not obtained for pH value 5, but achieved when the pH value equals 7. The concentration of phosphate buffer did not show obvious effect on separation and 20 mmol/L was selected. In consideration of the retention time, resolution, peak symmetry, and the protection of column, the optimized separation conditions were obtained as following: The mobile phase consisted of acetonitrile (component A) and 20 mmol/L sodium dihydrogen phosphate buffer (component B, containing 15 mmol/L TBAH, pH 7.0) at a flow rate of 1.0 mL/min. A linear elution gradient was programmed from 8 to 25% A for C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Basic principle of three-phase LPME.
28 min and then the composition of 25–75% (A, B) was used in isocratic mode for 12 min. All chromatographic separations were performed at the detection wavelength of 210 nm.
3.2 Extraction mechanism of three-phase LPME The extraction principle of the five components [(RCOO− )2 Ca2+ ] is illustrated in Fig. 1. The extraction efficiency of threephase LPME is based on the distribution of analytes in three phases (donor phase, organic phase, and acceptor phase), herein, the organic phase (hexyl acetate) is immobilized in the pores of the hollow fiber. In this case, the organic phase served as an effective barrier and intermediary layer between the two aqueous solutions. Before extraction, the donor phase was adjusted to acidic where [RCOO− ] was turned into [RCOOH], to increase the partition coefficient between the organic phase and donor phase. Subsequently, the analytes [RCOOH] were extracted from the aqueous sample (donor phase) through the organic phase into the alkaline aqueous solution (acceptor phase) inside the lumen of the hollow fiber. Because of the correspondingly high solubility of [RCOOH] in the alkaline acceptor solution and further formation of neutral ion-pairs [N(CH3 CH2 CH2 CH2 )4 OOCR] in the acceptor phase, the analytes were continuously transport to the acceptor phase until reached a partition equilibrium, resulting in a high enrichment of analytes in acceptor phase. Based on this principle, the factors of such three-phase LPME procedure were investigated and optimized in the following section.
3.3 Optimization of three-phase LPME procedure To obtain the optimal conditions in the proposed extraction procedure, the parameters including organic solvent, pH of donor phase and acceptor phase, ionic strength, stirring rate, and extraction time were investigated. The concentration of the analytes HMACa, KVCa, KILCa, and KLCa were 10 and 5 mg/L for KPACa. www.jss-journal.com
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3.3.1 Selection of organic solvent Choosing a suitable organic solvent plays an important role in achieving efficient enrichment of the analytes [14,25]. Firstly, organic solvent should have similar polarity with the hollow fiber, so it can be strongly immobilized within the pores of the fiber. Secondly, organic solvent should be able to provide high solubility for the target analytes, meanwhile the solubility should be higher in organic solvent than in donor solution but lower than in acceptor solution, so as to increase the extraction efficiency [26]. Thirdly, as a barrier between the donor and acceptor phase, organic solution should be immiscible with water and samples and stable enough during extraction to against dissolution. In addition, the solvent should be of low volatility, low viscosity, and no toxicity. Based on the above factors, five organic solvents (dodecanol, n-hexane, 1-octanol, toluene, and hexyl acetate) were evaluated for extraction of the five target analytes by three-phase LPME under the same conditions mentioned above. As can be seen in Fig. 2, although 1-octanol was mentioned as one of the most commonly used extraction solvents in this LPME extraction techniques [20,27,28], in this work, hexyl acetate was selected as the extraction solvent for subsequent experiments due to its highest extraction efficiency for present five components comparing to the other solvents. 3.3.2 Selection of acceptor phase The five target analytes are weak acidic compounds because of carboxyl groups, so the acceptor phase should be alkaline solution to keep the analytes ionized and promote their dissolution, which ensures the ionized molecules efficiently extracted from the organic phase into acceptor phase. In this work, TBAH solution was used as acceptor phase and NaOH solution was chose as control acceptor phase to compare the effect of extraction efficiency. Figure 3A showed that the extraction efficiency of the five analytes increased with the concentration of TBAH up to 0.5% in water, and then decreased
Figure 2. Effect of five organic solvents on the extraction efficiency. pH value of donor phase: 1, acceptor phase: 5% TBAH, extraction time: 30 min, stirring rate: 150 rpm, salt addition: null.
with further increase of TBAH concentration. Similar trends were observed for NaOH solution and the maxima extraction efficiency was obtained for 0.5 mol/L, as shown in Fig. 3B. The extraction recovery for HMACa, KVCa, KILCa, KLCa at 0.5% TBAH as acceptor phase were higher than 0.5 mol/L NaOH as acceptor phase, except for KPACa. To protect the C18 column, the NaOH acceptor phase solution should be neutralized with acetic acid before injected into HPLC system, but TBAH could be used as ion-pair reagent and did not need this further neutralization. Considering the above factors, 0.5% TBAH was selected to be the acceptor phase in the following studies and the acceptor phase was directly injected into HPLC system without a neutralization step. 3.3.3 Effect of pH value of donor phase The pH of donor phase also plays an important role in three phases LPME. The pH difference between donor phase and
Figure 3. Effect of the acceptor phase on the extraction efficiency: (A) TBAH as acceptor phase, (B) NaOH as acceptor phase. pH value of donor phase: 1, organic solvents: hexyl acetate, extraction time: 30 min, stirring rate: 150 rpm, salt addition: null.
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Figure 4. Other factors on the extraction: (A) Effect of pH value of donor phase, (B) effect of salt concentration, (C) effect of stirring speed, (D) effect of extraction time. Experimental conditions: pH value of donor phase: 1 for (B), (C), and (D); salt addition: 0.3 g/mL for (C), (D), and null for (A); extraction time: 30 min for (A), (B), and (C); stirring speed: 150 rpm for (A), (B), and (D); acceptor phase: 0.5% TBAH; organic solution: hexyl acetate.
organic phase can change the distribution coefficient of analytes, thereby improving the extraction efficiency. For acidic compounds, the sample solution should be acidic enough to keep all the analytes mainly in neutral form, to enhance the affinity of analytes to the organic solvent and reduce their solubility in the donor phase. Therefore, pH was tested for donor phase in the range of 0–5 (adjusted with sulfuric acid). Figure 4A showed that the highest extraction efficiency of all analytes was found at pH 1, and then with pH increasing the extraction efficiency decreased. At higher pH, protonation reaction was not complete and a great quantity of the analytes existed in ionic form with low affinity to the organic solvent [26]. Therefore, the optimum pH of the donor phase was selected as 1. 3.3.4 Effect of salt addition In the HF-LPME experiments, salt was often added into the sample solution to improve the extraction efficiency due to salting out effect [29]. A series of solutions with various concentrations of NaCl in the range of 0–0.35 g/mL were C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
added into donor phase to investigate the effect of the salt concentration on the extraction. As can be seen in Fig. 4B, the extraction efficiency increased with the NaCl concentration up to 20% for KLCa and 30% for others. This phenomenon can be explained with the two theories coexisted in the salt solution: (1) Salting out effect, it can increase the ionic strength and decrease the solubility of analytes in aqueous phase. This is because there are water molecules forming hydration spheres around the salt ions. These hydration spheres reduce the solubility of analyte molecules in the water [30]. (2) Electrostatic interactions, this force between polar molecules and salt ions in aqueous solution decreases mass transfer ability, thus decreasing the extraction efficiency for polar analytes. In the initial time, salting out effect played the predominant role. With the further increasing salt concentration, the electrostatic interactions between analytes and salt molecules overshadowed the salting out effect and became dominated, which led to the decline of response. Based on the results above, a 0.3 g/mL NaCl concentration was used as the final salt addition in the following studies.
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Table 1. Linear range, correlation equation, linear range, LOD, and enrichment factor of the method
Analytes
Liner range (mg/L)
Calibration curvea)
Correlation coefficient (r)
LOD (g/L)
RSD %, (n = 3)b)
Enrichment factor
HMACa KVCa KILCa KLCa KPACa
0.5–10 0.1–10 0.1–10 0.1–10 0.5–10
y = 27 704x + 28 788 y = 277 906x + 199 512 y = 482 013x + 317 373 y = 356 575x + 821 714 y = 323 251x + 150 420
0.9951 0.9903 0.9903 0.9923 0.9925
100.7 10.0 5.8 7.8 8.6
5.94 4.86 6.27 4.17 5.23
11 110 198 202 50
a) y: peak area, x: concentration (mg/L). b) All analytes at 5 mg/L.
3.3.5 Effect of stirring rate The transfer of target analytes between sample solution and organic phase was influenced by diffusion rate. Stirring process can decrease the thickness of diffusion layer around the interface between two phases, then accelerated the mass transfer and increased the extraction efficiency. Stirring also reduced the time required to attain a thermodynamic equilibrium [31, 32]. But at high stirring speed, extraction solvent could fall off from the lumen of hollow fiber. In addition, air bubbles could be produced and then adhered on the surface of the fiber, which can break the stability of acceptor phase and then decrease the extraction efficiency [33]. The effect of stirring rate varied in the range of 50–300 rpm was evaluated in Fig. 4C. At last, 150 rpm was applied in this study. 3.3.6 Effect of extraction time HF-LPME is an essentially dynamic mass transfer process, including the partitioning and diffusion of analytes between aqueous solution and organic solvent. To reach the maximum extraction efficiency of target analytes, extraction time was optimized from 10 to 50 min at the stirring speed of 150 rpm. Figure 4D showed that with the extraction time increasing in the range of 10–30 min, the amount of the extracted analytes was found to increase. After 30 min, the peak area of each compound started to decrease because of the loss and drop of organic solvent. Therefore, an extraction time of 30 min was chosen as the optimal condition.
3.4 Method evaluation Based on the research in the previous sections, the optimum extraction conditions were achieved: 5 cm hollow fiber immersed with hexyl acetate as the extraction solvent, 0.5% TBAH solution as the acceptor phase, 10 mL aqueous solution with pH 1 and 30% sodium chloride as the donor phase, 150 rpm stirring rate, 30 min extraction time at room temperature. Under these conditions, a series of experiments were carried out to determine the performance of this method, and the results were listed in Table 1. The calibration curves were obtained by plotting the measured peak areas against the concentrations of the analytes in the sample in the range of 0.1–10 mg/L (KVCa, KILCa, and KLCa) and 0.5–10 mg/L C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(HMACa and KPACa). Each level was extracted by the proposed method above and analyzed by ion-pair HPLC three times. All of these analytes exhibited good linearity with correlation coefficients (r) larger than 0.9903, and the RSDs for five replicate analyses of three target analytes at 5 mg/L were all below 6.27%. The LODs of these compounds in the urine sample were 5.8–100.7 g/L (S/N = 3). The enrichment factor (EF) was calculated according to the following equation: EF = CAP, final /CAP, initial (CAP, final : concentration of analytes in the acceptor phase after extraction, CAP, initial : initial concentration of analytes in the aqueous solution). The results indicated that the five analytes were extracted with satisfactory enrichment factors between 11 and 202. To evaluate the applicability of the proposed three phases LPME method to real samples, the established method was employed in the extraction of the five compounds in human urine. Each urine sample was diluted at 1:3 ratio with purified water to reduce matrix effect. After addition of 0.3 g/mL NaCl and adjustment of the pH at 1.0, the diluted urine and the sample spiked with definite value of standard was extracted under the optimum conditions. According to the obtained results (Table 2), the recoveries were in the range of 92–118% and RSDs were between 3.13 and 5.73% after five replicate analyses. Figure 5 shows the HPLC chromatograms of urine sample spiked with 7 mg/L (A) and blank urine sample (B) after HF-LPME, and those two without extraction (C) and (D). By comparison of Figure 5A and C, as well as B and D, we can see that the interference of endogenous substance of urine could be well eliminated by the HF-LPME, especially the polar components with the retention time less than 10 min. Furthermore, without the HF-LPME, it is hard to detect the five compounds in the spiked urine sample, as shown in Figure 5C, because of their low concentration. After extraction, Figure 5A shows that high enrichment were achieved for the five components, which is significant for detection of trace amount of analytes in urine. Therefore, the established method had high clean-up and enrichment power for determination of five components of compound ␣-ketoacid tablets in human urine.
4 Conclusions A new analytical method based on three-phase hollow-fiber microextraction coupled with ion-pair HPLC has been www.jss-journal.com
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Table 2. Recoveries and RSDs of the five components spiked to human urine samples
Analytes
Original amount (mg/L)
Spike amount (mg/L)
Found amount (mg/L)
Recovery (%)
RSD (%, n = 5)
HMACa
NDa)
KVCa
ND
KILCa
ND
KLCa
ND
KPACa
ND
2 7 2 7 2 7 2 7 2 7
2.19 6.42 2.23 7.17 2.34 7.26 2.36 7.1 1.87 8.08
110 92 112 102 117 104 118 101 94 115
3.72 3.44 4.36 4.40 5.46 5.73 4.56 4.43 3.35 3.13
a) ND, not detected.
Figure 5. The HPLC chromatograms of (A) diluted human urine spiked with 7 mg/L standards and (B) nonspiked diluted urine sample after the extraction under the optimized conditions, (C) diluted human urine spiked with 7 mg/L standards without extraction, (D) blank urine without extraction. Peak identifications: 1. HMACa; 2. KVCa; 3. KILCa; 4. KLCa; 5. KPACa.
developed to determine the five components of compound ␣-ketoacid tablet in human urine. Up to 202-fold enrichment factor was achieved, along with highly effective sample clean-up the interference of endogenous substance of urine, so were good linearity and recovery. And the ion-pair reagent TBAH was selected as the acceptor phase, which can avoid the neutralization step with acid for NaOH acceptor phase solution. Overall, this method is applicable to simultaneous determination of the five compounds of compound ␣-ketoacid tablet at low concentrations in human urine sample, and provides dose reference for the patients of CKD who are taking ␣-ketoacid tablets in clinical treatment. The established method could be further developed for the determination of ␣-ketoacid in the urine sample of the patients with aminoacidopathies diseases. The authors gratefully acknowledge the financial support of the open fund project of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (2010KF02). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The authors have declared no conflict of interest.
5 References [1] Meguid, E. l., Nahas, A., Bello, A. K., Lancet 2005, 365, 331–340. [2] Lysaght, M. J., J. Am. Soc. Nephrol. 2002, 13, S37–S40. [3] Piccoli, G. B., Ferraresi, M., Deagostini, M. C., Vigotti, F. N., Consiglio, V., Scognamiglio, S., Moro, I., Clari, R., Fassio, F., Biolcati, M., Porpiglia, F., Nephrol. Dial. Transplant. 2013, 28, 2295–2305. [4] Berger, A., Edelsberg, J., Inglese, G. W., Bhattacharyya, S. K., Oster, G., Am. J. Manag. Care 2009, 15, 509–528. ´ [5] Jaworska, M., Szulinska, Z., Wilk, M., J. Chromatogr. A 2003, 993, 165–172. [6] Walser, M., Am. J. Clin. Nutr. 1989, 49, 17–22. [7] Mou, S., Li, J., Yu, Z., Wang, Q., Ni, Z., J. Int. Med. Res. 2013, 41, 129–137.
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