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Liquid-phase microextration combined with liquid chromatography–electrospray tandem mass spectrometry for detecting diuretics in urine
Available online at www.sciencedirect.com
Talanta 75 (2008) 658–665
Liquid-phase microextration combined with liquid chromatography– electrospray tandem mass spectrometry for detecting diuretics in urine Tzu-Feng Tsai a,b , Maw-Rong Lee a,∗ a
Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan b Department of Cosmetic Science, Providence University, Taichung 43301, Taiwan
Received 1 September 2007; received in revised form 25 November 2007; accepted 26 November 2007 Available online 8 January 2008
Abstract Trace amounts of diuretics were determined in human urine by hollow fiber liquid-phase microextraction (LPME) combined with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) in this study. Chromatography was performed on a C8 reversed-phase column. A 25 L n-octanol was used to extract analytes in urine. Extraction was optimized using a pH 2 solution spiked with 0.15 g/mL NaCl for 40 min at 40 ◦ C with 1010 rpm stirring. The limits of detection of diuretics in urine were 0.3–6.8 ng/mL, and linearity range was 1–1000 ng/mL. Recoveries of spiked 50 ng/mL diuretics were 97.7–102.5%. The intra-day precision and inter-day precision were 3–18% and 4–21%, respectively. The diuretics concentration profiles in patient urine were also determined. The results of this study reveal the adequacy of LPME–LC–MS/MS method for analyzing diuretics in urine and quantification limits exceed World Anti-Doping Agency requirements. © 2007 Elsevier B.V. All rights reserved. Keywords: Liquid-phase microextraction; Diuretics; Liquid chromatography–mass spectrometry; Urine
1. Introduction As therapeutic agents used to eliminate tissular liquids or enhance renal excretion of salt and water, diuretics are often used for treating congestive heart failure, hypertension, liver, kidney disease and lung disease [1,2]. Their effectiveness in water removal has led to misuse of diuretics by athletes attempting to reduce body weight. Diuretics have also been used to avoid detection of doping agents by diluting urine to reduce their concentration. Diuretics may also imbalance the thermoregulatory system, causing exhaustion, irregular heartbeat and ultimately heart failure or death [3]. Therefore, the Medical Commission of the International Olympic Committee (IOC) included diuretics as a banned substance since 1986 [4]. Diuretics drugs are available in several classes varying structures and physicochemical properties. The pKa value of diuretics is 2.8–9.5 and log P (octanol–water partition coefficient) value is −0.071 to 3.376 [5–7]. Effective methods of identification diuretics are vital for clinical research and doping control.
∗
Corresponding author. Tel.: +886 4 2285 1716; fax.: +886 4 2286 2547. E-mail address: [email protected] (M.-R. Lee).
0039-9140/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2007.11.058
Pharmacokinetic studies of urine samples after a single dose have shown that most diuretics are essential unchanged when excreted. Hence, procedures for screening diuretics in urine can be designed to detect the suspected parent compound. Many techniques have been developed for determining diuretics in human urine. High-performance liquid chromatography (HPLC), capillary electrophoretic (CE) or micellar electrokinetic capillary chromatography (MEKC) with UV or photodiode arrays detection (DAD) [8–14] were used widely. However, these methods suffer from significant interferences in urine samples matrix and offer limited qualitative information. Gas chromatography–mass spectrometry (GC–MS) can confirm the structure of diuretic compounds [15–18]. However, diuretics cannot be analyzed by GC without derivatization procedures for most substances containing polar functional groups. Generally, methylation or silylation derivatization procedures are time-consuming and unsuitable for some diuretics. For detecting polar substances and identifying analytes from complicate urine, liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) [19–21] has been proved to be sensitive, rapid and without doing any derivatization. Sample extraction clean-up from sample matrix and concentrate trace amounts of analyte are essential aspects of analysis.
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Liquid–liquid extraction (LLE) [9–15,20] and solid-phase extraction (SPE) [18,19,21] are the most common methods of preconcentration and clean-up. Solvents used for LLE and SPE normally require evaporation to concentrate and reconstitution of the sample for subsequent LC analysis. These steps are tedious and time-consuming and may cause loss of analytes. However, the major drawback in both techniques is the large amount of organic solvent consumed. Hollow fiber liquid-phase micrextraction (LPME), a fast, simple, inexpensive, less solvent sample preparation and disposal method has been devised to extract analytes from water and biologic fluids [22–38]. This method employs a membrane as an interface between the sample solution (donor) and the organic solvent (acceptor), which avoids mixing of the two phases and other problems encountered in classical liquid–liquid extraction. The advantages of LPME are single step enrichment/clean-up, very low organic solvent consumption and low cost. In this study, a hollow fiber LPME method was used to extract trace diuretics in urine samples. The analytes were analyzed by LC–MS with selected reaction monitoring (SRM) mode. The optimized parameters affecting LPME extraction efficiency including extraction temperature, extraction time, pH value of sample solution and ion strength were evaluated. Detection limits, linear ranges and precision of the technique as well as the feasibility of applying the proposed method in patient urine were studied.
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and glycerine were also obtained from Merck. Sodium hydroxide (99%), n-octanol, toluene, cyclohexane and sodium chloride (99.8%) were purchased from Riedel-deH¨aen Company (Seelze, Germany). Hydrochloric acid (36.5%) was from Fisher Scientific Company (Fair Lawn, NJ, USA). Diuretics analyzed in this study, bumetanide (BUM), chlorothiazide (CT), clopamide (CA), ethacrynic acide (EAA), hydrochlorothiazide (HCT), hydroflumethiazide (HFT), probenecid (PRO) and trichlormethiazide (TCT), were purchased from Sigma–Aldrich Company (St. Louis, MO, USA). The structures of diuretics are shown in Fig. 1. A stock solution of diuretics at 1.0 mg/mL was prepared by dissolving the solid bulk drug in methanol and stored at −30 ◦ C. This solution was further diluted with methanol to yield the appropriate working solutions. The eight point calibration curves were prepared by serial dilution of stock solution with blank urine at the concentration of 1–1000 ng/mL. Pooled drugfree human urine samples as blank urine were obtained from six healthy volunteers, stored at −30 ◦ C and thawed at room temperature before use. For optimum LPME study, urine samples were prepared by spiking with standard diuretics solution. The real urine samples collected from diuretics dosed patient were kept frozen at −30 ◦ C until further treatment. The laboratory purified water (>18 M) was obtained from a SG-Ultra Clear water purification system (SG Water Company, Germany). 2.2. Hollow fiber LPME device
2. Experimental 2.1. Chemicals and reagents An HPLC-grade of acetonitrile was obtained from TEDIA Company (Fairfield, OH, USA), and methanol was purchased from Merck Company (Darmstadt, Germany). Analytical grade formic acid, ammonium formate, acetic acid, ammonium acetate
The LPME device illustrated in Fig. 2 was used to preconcentrate the analyte from urine. The experimental apparatus employed an 8 cm Accurel Q 3/2 polypropylene tubular membrane (Membrana, Wuppertal, Germany) with 200 m wall thickness, 0.2 m pore size and 600 m internal diameter. The fiber was washed with acetone in ultrasonic bath for 30 min to remove contaminants then dried in an oven at 50 ◦ C before
Fig. 1. The structures of diuretics.
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Fig. 2. Schematic diagram hollow fiber-protected liquid-phase microextraction device.
use. For each extraction, a new piece of fiber with the U-shape configuration was employed to avoid possibility of carryover. Both a 7.5 mL urine solution and a 10 mm × 4 mm stir bar were placed in a 8 mL sample vial (diameter: 1.5 cm, height: 5.5 cm) with a screw top/silicone septum. The LPME device consisted of two 0.7 mm o.d. 24 Gauge intravenous catheter syringes (guiding needles) inserted through a silicon septum and connected with a section of polypropylene hollow fiber. Prior to extraction, the hollow fiber pores were dipped in organic solvent for 10 s to immobilize solvent in the pores. The excess solvent was then removed by ultrasonication in a water bath for 15 s. A 50 L syringe was used to fill the hollow fiber with 25 L of noctanol as extraction solvent (acceptor solution). The assembly was then placed into the sample vial and with a magnetic stirrer at approximately 630 rpm for 40 min under room temperature. After extraction, the fiber was removed from the guiding needles, and the acceptor solution was retracted into the 10 L Hamilton gas-tight syringe, (Hamilton, Bonaduz, Bonaduz, Switzerland) model 701 SN, with a beveled needle tip (length: 5.1 cm, o.d.: 0.071 cm, i.d.: 0.015 cm) from one end of the fiber. A 5 L portion of the extractant was injected into the LC–MS/MS system. 2.3. LC–MS/MS analysis A Varian 1200L triple-quadrupole LC–MS (Varian, Walnut Creek, CA, USA) coupled to the Dynamax ProStar 210 liquid chromatograph system was used. The analytes were separated on a Waters XTerra C8 (15 cm × 2.1 mm, 3.0 m) LC column (Waters Corp., Milford, MA, USA) preceded by an in-line precolumn filter of 3 mm frit (Supelco, Bellefonte, PA, USA). The mobile phase was filtered using a filter (0.22 m) and degassed by using vacuum followed by sonication. The gradient separation of LC was proceed. A pH 2.76 of mixture solution of 0.05 mM ammonium formate containing 0.1% formic acid (A) and acetonitrile (B) was used as mobile phase at a flow-rate of 0.2 mL/min. The initial composition was 35% B for 1.0 min, then increased to 65% for 6 min, then decreased to 35% for 5 min until equilibrium. The volume of injection loop was 5 L, and all the column effluent was directed into the mass spectrometer. The selected reaction monitoring (SRM) mode of MS/MS was used for quantization. The data acquisition software Varian MS workstation, Version 6.2, was used for instrument control,
data acquisition and data handling. Analysis was performed in both positive ionization and negative ionization modes. Voltages across the capillary, lens and the quadrupoles were tuned by an automated procedure to maximize the signal for analyzed ions. The parameters for the ionization efficiency were optimized by evaluating sensitivity using flow injection analysis (FIA) with the mobile phase at 0.2 mL/min. The instrumental parameters were optimized to maximize signal intensity of the deprotonated molecule ion [M−H]− or pseudomolecular ion [M+H]+ by injecting 5 L standard solution (1.0 g/mL) through a sample loop. The MS/MS was performed based on collision-induced dissociation (CID) of specific precursor ion and the generation of the characteristic fragment ions. The Q1 was scanned a range of m/z 50–500 for precursor ion optimization. In all deprotoned molecule ions of analytes observed were more stable, abundant and less fragmented during negative scan. Both Q1 and Q3 quadrupoles were set at unit resolution. During SRM analysis mode, mass peak width was 1.0 mass unit at half height for both Q1 and Q3, scan time was 1 s. The spray conditions of API interface were: housing temperature 65 ◦ C; nebulizing gas pressure 35 psi; drying gas temperature 250 ◦ C; drying gas pressure 20 psi. Argon was used as the collisional gas at pressure of 2.0 mTorr. 2.4. Method validation The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision (RSD), accuracy and recovery. Urine calibration standards were a set of spiked diuretics in drug-free urine at concentrations of 1, 5, 10, 50, 100, 250, 500 and 1000 ng/mL. Three replicate analyses were performed for each concentration to evaluate linearity. The peak areas were used for quantification through the calibration curves of diuretics. The LOD and LOQ were determined as the ratio of 3 times and 10 times standard deviation at the lowest concentration of linear range divided to the slope of linear equation, respectively. Precision of the assay was determined by generating intra- and inter-day variability data from a 50 ng/mL sample. The analytical accuracy of purposed method was determined by analyzing spiked diuretics in urine samples at a concentration of 50 ng/mL five times and expressed as the ratio of mean measured concentration to spiked concentration. The analytical recovery was determined by analyzing triplicate of spiked diuretics in urine sample at concentration of 50 ng/mL. To verify specificity, blank samples were prepared with drug-free urine samples from five different donors. 3. Results and discussion 3.1. Mass spectrometry of diuretics Both positive and negative ionization mode of ESI were used to evaluate the most sensitive ionization mode for the diuretics. The concentration of 1 g/mL diuretics was used in this study with FIA. The pseudomolecular ion, [M+H]+ , of all diuretics was obtained in positive ionization mode as the base ion. In the negative ionization mode, the peak of the deprotonated molecule
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Table 1 Precursor ion and product ion used in SRM of LPME–LC–MS/MS for detection diuretics Analytes Chlorothiazide C7 H6 ClN3 O4 S2 Hydrochlorothiazide C7 H8 ClN3 O4 S2 Hydroflumethiazide C8 H8 F3 N3 O4 S2 Clopamide C14 H20 ClN3 O3 S Trichlormethiazide C8 H8 Cl3 N3 O4 S2 Bumetanide C17 H20 N2 O5 S Probenecid C13 H19 NO4 S Ethacrynic acid C13 H12 Cl2 O4
Abb.
Precursor ion (m/z)
CT HCT HFT CA TCT BUM PRO EAA
[M−H]−
294 296 [M−H]− 330 [M−H]− 344 [M−H]− 378 [M−H]− 365 [M+H]+ 284 [M−H]− 301 [M−H]−
Product ion (m/z) 214 [M−H-SO2 NH2 ]− 205 [M−H-SO2 CNH]− 239 [M−H-SO2 CNH]− 189 [M-C8 N2 OH15 ]− 306 [M−H-2HCl]− 240 [M+H-CO2 SO2 NH2 ]+ 240 [M−H-CO2 ]− 243 [M−H-CO2 -CH2 ]−
ions, [M−H]− of all diuretics as the base ion and less fragment ions were observed. The structures of thiazide-base diuretics BUM, CA, CTA, HCT, HFT and TCT are a benzene ring and a sulfamonyl residue ortho-located to an electronegative substitute such as a chlorine atom (Fig. 1). In preliminary experiments for all thiazide-base diuretics except bumetanide indicated that the signal of [M−H]− was more sensitive than that of [M+H]+ . According to the paper described thiazides as acidic character, the use of negative ion electrospray ionization proved to be reasonable [39,40]. Therefore, the positive ionization mode was not considered further for using to analyze diuretics except for bumetanide. In MS/MS experiments, ESI interface was used in conjunction with the FIA for optimization of the MS/MS parameters. The product ion mass spectrum of deprotoned molecule ion was obtained in the scan range of m/z 50–500. For all thiazide diuretics, the characteristic fragment ion [M-SO2 NH2 ]− was observed in the product ion spectra. For CT, the ion [M-SO2 NH2 ]− , m/z 214 is the base peak. Therefore, in this assay, the mass transition pattern, m/z 294 → 214 was selected to monitor CT in negative ionization SRM analysis (Table 1). For other diuretics, the base peak ions were shown in Table 1. In negative ionization mode, the mobile phase of ACN exited the same mass transition m/z 363 → 319 as bumetanide. The pseudomolecular ion of bumetanide, [M+H]+ m/z 365 was chosen as precursor ion,
and the product ion, m/z 240, [M+H-CO2 SO2 NH2 ]+ was used for monitored in positive mode to avoid the interference from mobile phase. Therefore, in this assay both positive and negative ionization modes were used simultaneously to detect the diuretics in urine.
Fig. 3. Effect of salt concentration on the extraction efficiency of 50 ng/mL diuretics in urine produced by using LPME–LC–MS/MS.
Fig. 4. Effect of extraction temperature on the extraction efficiency of 50 ng/mL diuretics in urine produced by using LPME–LC–MS/MS.
3.2. The hollow-fiber LPME method In order to increase the sensitivity, the optimum LPME conditions for the determination of diuretics were required to evaluate the various parameters that affect the LPME extraction procedure. Extraction efficiency of the membrane may be affected by extraction solvent, extraction temperature, extraction time, solution pH, ionic strength of aqueous phase, hollow fiber length and stir rate. It is very important to optimize these parameters for enhance extraction efficiency and then to improve detection. For LPME, polypropylene membrane has proven effectively for increasing concentration and reducing matrix interference from urine [31,34,35]. The polypropylene fiber was selected in this study. The solvent used in LPME should have low water solubility and high solubility for analytes. Three organic solvents n-octanol, toluene and cyclohexane were used to evaluate the extraction efficiency of 50 ng/mL diuretics in urine at room temperature for 30 min. The experimental results indicated that the highest extraction efficiency was obtained with n-octanol. Therefore, n-octanol was chosen as extraction solvent in this study. The pH value of sample solution influences both the dissociation equilibrium and the solubility of organic acid or base
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analytes. For diuretics, BUM, EAA and PRO are highly acidic and the other five compounds are less acidic. A sample pH range from 1 to 10 was used to evaluate the extraction efficiency of 50 ng/mL diuretics in urine at 20 ◦ C for 30 min. The results indicated that lower pH value improved extraction efficiency of the target analytes. Hence, the pH of solution was adjusted to 2. Salt-out effect was often used to improve extraction efficiency of analytes in LPME as in SPME [41]. Ionic strength increases in the sample solution will decrease the solubility of target analytes
in water solution for salt-out effect. The NaCl is a conversional salt used to change the ionic strength in solution. The salt effect study was evaluated at salt concentrations from 0 to 0.35 g/mL. As Fig. 3 shows, extraction efficiency increased as NaCl concentration increased and reached a maximum at 0.15 g/mL and then decreased at higher NaCl concentrations. The decay in high concentration of NaCl was possible due to electrostatic interaction between polar analytes and NaCl ions in solution [41,42]. Hence, 0.15 g/mL NaCl was added in the further experiments.
Table 2 Estimated limits of detection and linear range for diuretics by LPME–LC–MS/MS Compound
Linear range (ng/mL)
Correction coefficient (r2 )
LODa (ng/mL)
LOQb (ng/mL)
Enrichment factorc
Extraction efficiency (%)
CT HCT HFT CA TCT BUM PRO EAA
5–1000 10–1000 2–1000 2–1000 2–1000 10–500 1–1000 5–1000
0.9994 0.9986 0.9938 0.9995 0.9989 0.9997 0.9969 0.9962
1.7 6.7 1.1 0.3 0.4 3.6 0.6 2.8
5.5 22.3 3.7 0.9 1.4 12 2.1 9.4
0.8 1.7 3.8 95.2 11.3 20.1 32.6 144.8
0.2 0.5 1.0 25.4 3.0 5.4 8.7 38.6
a b c
LOD, limit of detection, determined as 3S.D./slope, standard deviation of the lowest concentration of calibration curve divided by the slope (n = 3). LOQ, limit of quantification, determined as 10S.D./slope, standard deviation of the lowest concentration of calibration curve divided by the slope (n = 3). Urine was spiked at 50 ng/mL.
Fig. 5. Mass ion chromatograms of 50 ng/mL diuretics in urine produced by LPME–LC–MS/MS.
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Matrix interference is often an important problem in biological samples during trace analysis [28,31,42–44]. To evaluate the influence of matrix, the MS signal intensities of 50 ng/mL standard diuretic solutions diluted with artificial urine and ultrapure water were tested. Almost the same intensities of diuretics were obtained no matter what matrices used. It was shown that LPME can improve the sample clean-up to remove urine components and thereby decreasing the amount of matrix injected onto the column. Agitating stir bar can increase the mass transfer coefficient from the sample matrix to extraction phase. To trace the effect of stirring, eight stir rates (0–1200 rpm) were used to evaluate extraction efficiency of 50 ng/mL diuretics in urine at 40 ◦ C for 40 min. The high stirring speed increased extraction efficiency but also produced instability of the organic solvent in hollow fiber and the air bubbles would affected the precision and reproducibility of the extraction process. Therefore, a stirring speed of 1010 rpm was used for subsequent experiments. 3.2.1. Extraction temperature and time The LPME method employs a partition equilibrium process in target analytes between extraction phase (accept solution)
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and sample matrix (donor solution) [30]. Extraction temperature and time affect partition equilibrium. The higher extraction temperature normally has a positive effect on extraction yield by increases both solubility and diffusion of analytes into extraction phase. However, heating might also cause swelling in fibers and does not favor organic solvent immobilization in membrane pore. Extraction temperature was studied at a room temperature to 60 ◦ C. The experimental results in Fig. 4 show that extraction efficiency was maximum at 40 ◦ C. In optimization extraction time investigated, a spiked 50 ng/mL urine solution was used within 20–50 min at 40 ◦ C. The experimental results showed that the peak areas of diuretics were reached maximum at extraction for 40 min. When extraction time exceeded 40 min, the peak areas of analytes slowly decreased. Thus, 40 ◦ C extraction temperature and 40 min extraction time were selected for the following experiments. 3.2.2. Acceptor phase volume The ratio between the volume of the donor phase (V0 ) and the acceptor phase (Vorg ) governs the extraction efficiency (EE) and enrichment factor (EF) of analytes in the acceptor phase,
Fig. 6. Total ion chromatogram and mass ion chromatograms of dosed patient urine sample produced by LPME–LC–MS/MS.
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according to the expression: norg Vorg Corg EE = 100% = 100% n0 V0 C0
The concentrations of diuretics detected were HCT, 24.9 g/mL; BUM, 0.046 g/mL; PRO, 0.011 g/mL; EAA, 0.008 g/mL. 4. Conclusion
and EF =
Corg C0
where norg , Vorg and Corg are the mass, the volume and the concentration of analyte extracted in the organic solvent, respectively, and n0 , V0 and C0 are the mass, the volume and the concentration of analytes present in the original sample, respectively [18]. To optimize acceptor-to-donor phase volume ratio, the donor phase volume (V0 ) is kept constant, and the volume of organic solvent (Vorg ) is changed from 15–36 L by adjusting the length of the hollow fiber at 5, 6, 8, 10 and 12 cm. The experimental results showed the peak areas of the analytes were maximum at a 6.0 cm of hollow fiber with an acceptor phase volume of 20 L. The parameters of optimum condition of LPME were list in Table 2. 3.3. Assay validation Calibration curves for eight diuretics were established for the concentration range from 1 to 1000 ng/mL. The linear range, precision, limit of detection (LOD), and limit of quantification (LOQ) of this method were evaluated under the optimum condition as shown in Table 2. The linearity of the proposed method was with correlation coefficients (r2 ) being greater than 0.9938. The LOD and LOQ were 0.3–6.7 ng/mL and 0.9–22.3 ng/mL, respectively. The LOD of eight diuretics were found in low ng/mL level and lower than the minimum required performance limits (MRPL) of the World Anti-Doping Agency (WADA). The mass ion chromatograms of 50 ng/mL diuretics in urine analyzed by LPME-LC-MS/MS were shown in Fig. 5. The recovery and precision of this method expressed with % by analyzing 50 ng/mL diuretics spiked urine samples. The recovery of diuretics was 97.7–102.5%. The intra-day and interday relative standard deviations (R.S.D.s, n = 6) were 3–18 and 4–21%, respectively. LPME is a non-exhaustive extraction procedure as solid-phase microextraction, the extraction efficiency is defined as the ratio of the amount of analyte in acceptor phase to donor phase and dependant on the distribution coefficient of the compound. As shown in Table 2, the extraction efficiency of diuretics from spiked urine was 0.2–38.7%, the high enrichment factor could be obtained. For EAA, the high enrichment factor was reached 144.8-fold. 3.4. Application The diuretics dosed patient urine was analyzed by the LC–ESI–MS/MS method described. The LPME was operated at the optimum conditions studied. Representative SRM chromatograms of urine sample collected from the patient first urine after post-administration were shown in Fig. 6. The total ion chromatogram (TIC) and extracted ion chromatogram of urine sample obtained from a hypertension patient was shown in Fig. 6.
This study evaluated the coupling of LPME with LC–ESIMS/MS to determine the trace amount of diuretics in urine samples. A 6 cm Q 3/2 hollow fiber was used to extract trace diuretics from urine. The optimized procedure was revealed satisfactory intra-day and inter-day precision with R.S.D. of 3–18% and 4–21%, respectively. The limit of detection was 0.3–6.7 ng/mL and limit of quantification was 0.9–22.3 ng/mL. In addition, the feasibility of applying the methods to determine diuretics in real samples was also examined by analyzing urine samples from diuretics dose patient. The concentrations of diuretics detected were ranged from 0.008 g/mL (EAA) to 24.9 g/mL (HCT). The LPME method represents a valuable advance in sample preparation and offers numerous advantages, including procedural simplicity, low cost and organic solvent less used. Acknowledgment The authors would like to thank the National Science Council of the Republic of China for financially supporting this research under Contract No. NSC94-2113-M-005-001. References [1] A.S. Mehanna, M. Dowling, J. Pharm. Biom. Anal. 19 (1999) 967. [2] R. Faris, M. Flather, H. Purcell, M. Henein, P. Pool-Wilson, A. Coats, Int. J. Cardiol. 82 (2002) 149. [3] L.M. Antes, P.C. Fernandez, Disease-a-Month 44 (1998) 254. [4] Olympic Movement Anti-Doping Code, Prohibited Class of Substances and Prohibited Methods, IOC, Lausanne, 2001, Appendix A. [5] http://redpoll.pharmacy.ualberta.ca/drugbank. [6] The Merck Index, Merck & Co., Inc., tenth ed., 1983. [7] A. Berthod, Anal. Chem. 71 (1999) 879. [8] R. Ventura, J. Segura, J. Chromatogr. B 687 (1996) 127. [9] M.R. Taylor, P. Teale, J. Chromatogr. A 768 (1997) 89. [10] A. Rosado-Maria, A.I. Gasco-Lopez, A. Santos-Montes, R. IzquierdoHornillos, J. Chromatogr. B 748 (2000) 415. [11] D.L. Hertzog, J.F. McCafferty, X. Fang, R.J. Tyrrell, R.A. Reed, J. Pharm. Biomed. Anal. 30 (2002) 747. [12] M.L. Luis, S. Corujedo, D. Blanco, J.M.G. Graga, A.I. Jim´enez, J.J. Arias, Talanta 57 (2002) 223. [13] M.J. Ruiz-Angel, J.R. Torres-Lapasi´o, M.C. Garcia-Alvarez-Coque, J. Chromatogr. A 1022 (2004) 51. [14] J.A.M. Pulgarin, A.A. Molina, G.P.O. Nieto, Anal. Chim. Acta 518 (2004) 37. [15] D. Carreras, C. Imaz, R. Navajas, M.A. Garcia, C. Rodriguez, A.F. Rodriguez, R. Corres, J. Chromatogr. A 683 (1994) 195. [16] J. Segura, R. Ventura, C. Jurado, J. Chromatogr. B 713 (1998) 61. [17] R.K. M˝uller, J. Grosse, D. Thieme, R. Lang, J. Teske, H. Trauer, J. Chromatogr. A 843 (1999) 275. [18] L. Amendola, C. Colamonici, M. Mazzarino, F. Botr´e, Anal. Chim. Acta 475 (2003) 125. [19] D. Thieme, J. Grosse, R. Lang, R.K. Mueller, A. Wahl, J. Chromatogr. B 757 (2001) 49. [20] Y. Qin, X.B. Wang, C. Wang, M. Zhao, M.T. Wu, Y.X. Xu, S.Q. Peng, J. Chromatogr. B 794 (2003) 193. [21] C. Goebel, G.J. Trout, R. Kazlauskas, Anal. Chim. Acta 502 (2004) 65.
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Talanta 75 (2008) 658–665
Liquid-phase microextration combined with liquid chromatography– electrospray tandem mass spectrometry for detecting diuretics in urine Tzu-Feng Tsai a,b , Maw-Rong Lee a,∗ a
Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan b Department of Cosmetic Science, Providence University, Taichung 43301, Taiwan
Received 1 September 2007; received in revised form 25 November 2007; accepted 26 November 2007 Available online 8 January 2008
Abstract Trace amounts of diuretics were determined in human urine by hollow fiber liquid-phase microextraction (LPME) combined with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) in this study. Chromatography was performed on a C8 reversed-phase column. A 25 L n-octanol was used to extract analytes in urine. Extraction was optimized using a pH 2 solution spiked with 0.15 g/mL NaCl for 40 min at 40 ◦ C with 1010 rpm stirring. The limits of detection of diuretics in urine were 0.3–6.8 ng/mL, and linearity range was 1–1000 ng/mL. Recoveries of spiked 50 ng/mL diuretics were 97.7–102.5%. The intra-day precision and inter-day precision were 3–18% and 4–21%, respectively. The diuretics concentration profiles in patient urine were also determined. The results of this study reveal the adequacy of LPME–LC–MS/MS method for analyzing diuretics in urine and quantification limits exceed World Anti-Doping Agency requirements. © 2007 Elsevier B.V. All rights reserved. Keywords: Liquid-phase microextraction; Diuretics; Liquid chromatography–mass spectrometry; Urine
1. Introduction As therapeutic agents used to eliminate tissular liquids or enhance renal excretion of salt and water, diuretics are often used for treating congestive heart failure, hypertension, liver, kidney disease and lung disease [1,2]. Their effectiveness in water removal has led to misuse of diuretics by athletes attempting to reduce body weight. Diuretics have also been used to avoid detection of doping agents by diluting urine to reduce their concentration. Diuretics may also imbalance the thermoregulatory system, causing exhaustion, irregular heartbeat and ultimately heart failure or death [3]. Therefore, the Medical Commission of the International Olympic Committee (IOC) included diuretics as a banned substance since 1986 [4]. Diuretics drugs are available in several classes varying structures and physicochemical properties. The pKa value of diuretics is 2.8–9.5 and log P (octanol–water partition coefficient) value is −0.071 to 3.376 [5–7]. Effective methods of identification diuretics are vital for clinical research and doping control.
∗
Corresponding author. Tel.: +886 4 2285 1716; fax.: +886 4 2286 2547. E-mail address: [email protected] (M.-R. Lee).
0039-9140/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2007.11.058
Pharmacokinetic studies of urine samples after a single dose have shown that most diuretics are essential unchanged when excreted. Hence, procedures for screening diuretics in urine can be designed to detect the suspected parent compound. Many techniques have been developed for determining diuretics in human urine. High-performance liquid chromatography (HPLC), capillary electrophoretic (CE) or micellar electrokinetic capillary chromatography (MEKC) with UV or photodiode arrays detection (DAD) [8–14] were used widely. However, these methods suffer from significant interferences in urine samples matrix and offer limited qualitative information. Gas chromatography–mass spectrometry (GC–MS) can confirm the structure of diuretic compounds [15–18]. However, diuretics cannot be analyzed by GC without derivatization procedures for most substances containing polar functional groups. Generally, methylation or silylation derivatization procedures are time-consuming and unsuitable for some diuretics. For detecting polar substances and identifying analytes from complicate urine, liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) [19–21] has been proved to be sensitive, rapid and without doing any derivatization. Sample extraction clean-up from sample matrix and concentrate trace amounts of analyte are essential aspects of analysis.
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Liquid–liquid extraction (LLE) [9–15,20] and solid-phase extraction (SPE) [18,19,21] are the most common methods of preconcentration and clean-up. Solvents used for LLE and SPE normally require evaporation to concentrate and reconstitution of the sample for subsequent LC analysis. These steps are tedious and time-consuming and may cause loss of analytes. However, the major drawback in both techniques is the large amount of organic solvent consumed. Hollow fiber liquid-phase micrextraction (LPME), a fast, simple, inexpensive, less solvent sample preparation and disposal method has been devised to extract analytes from water and biologic fluids [22–38]. This method employs a membrane as an interface between the sample solution (donor) and the organic solvent (acceptor), which avoids mixing of the two phases and other problems encountered in classical liquid–liquid extraction. The advantages of LPME are single step enrichment/clean-up, very low organic solvent consumption and low cost. In this study, a hollow fiber LPME method was used to extract trace diuretics in urine samples. The analytes were analyzed by LC–MS with selected reaction monitoring (SRM) mode. The optimized parameters affecting LPME extraction efficiency including extraction temperature, extraction time, pH value of sample solution and ion strength were evaluated. Detection limits, linear ranges and precision of the technique as well as the feasibility of applying the proposed method in patient urine were studied.
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and glycerine were also obtained from Merck. Sodium hydroxide (99%), n-octanol, toluene, cyclohexane and sodium chloride (99.8%) were purchased from Riedel-deH¨aen Company (Seelze, Germany). Hydrochloric acid (36.5%) was from Fisher Scientific Company (Fair Lawn, NJ, USA). Diuretics analyzed in this study, bumetanide (BUM), chlorothiazide (CT), clopamide (CA), ethacrynic acide (EAA), hydrochlorothiazide (HCT), hydroflumethiazide (HFT), probenecid (PRO) and trichlormethiazide (TCT), were purchased from Sigma–Aldrich Company (St. Louis, MO, USA). The structures of diuretics are shown in Fig. 1. A stock solution of diuretics at 1.0 mg/mL was prepared by dissolving the solid bulk drug in methanol and stored at −30 ◦ C. This solution was further diluted with methanol to yield the appropriate working solutions. The eight point calibration curves were prepared by serial dilution of stock solution with blank urine at the concentration of 1–1000 ng/mL. Pooled drugfree human urine samples as blank urine were obtained from six healthy volunteers, stored at −30 ◦ C and thawed at room temperature before use. For optimum LPME study, urine samples were prepared by spiking with standard diuretics solution. The real urine samples collected from diuretics dosed patient were kept frozen at −30 ◦ C until further treatment. The laboratory purified water (>18 M) was obtained from a SG-Ultra Clear water purification system (SG Water Company, Germany). 2.2. Hollow fiber LPME device
2. Experimental 2.1. Chemicals and reagents An HPLC-grade of acetonitrile was obtained from TEDIA Company (Fairfield, OH, USA), and methanol was purchased from Merck Company (Darmstadt, Germany). Analytical grade formic acid, ammonium formate, acetic acid, ammonium acetate
The LPME device illustrated in Fig. 2 was used to preconcentrate the analyte from urine. The experimental apparatus employed an 8 cm Accurel Q 3/2 polypropylene tubular membrane (Membrana, Wuppertal, Germany) with 200 m wall thickness, 0.2 m pore size and 600 m internal diameter. The fiber was washed with acetone in ultrasonic bath for 30 min to remove contaminants then dried in an oven at 50 ◦ C before
Fig. 1. The structures of diuretics.
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Fig. 2. Schematic diagram hollow fiber-protected liquid-phase microextraction device.
use. For each extraction, a new piece of fiber with the U-shape configuration was employed to avoid possibility of carryover. Both a 7.5 mL urine solution and a 10 mm × 4 mm stir bar were placed in a 8 mL sample vial (diameter: 1.5 cm, height: 5.5 cm) with a screw top/silicone septum. The LPME device consisted of two 0.7 mm o.d. 24 Gauge intravenous catheter syringes (guiding needles) inserted through a silicon septum and connected with a section of polypropylene hollow fiber. Prior to extraction, the hollow fiber pores were dipped in organic solvent for 10 s to immobilize solvent in the pores. The excess solvent was then removed by ultrasonication in a water bath for 15 s. A 50 L syringe was used to fill the hollow fiber with 25 L of noctanol as extraction solvent (acceptor solution). The assembly was then placed into the sample vial and with a magnetic stirrer at approximately 630 rpm for 40 min under room temperature. After extraction, the fiber was removed from the guiding needles, and the acceptor solution was retracted into the 10 L Hamilton gas-tight syringe, (Hamilton, Bonaduz, Bonaduz, Switzerland) model 701 SN, with a beveled needle tip (length: 5.1 cm, o.d.: 0.071 cm, i.d.: 0.015 cm) from one end of the fiber. A 5 L portion of the extractant was injected into the LC–MS/MS system. 2.3. LC–MS/MS analysis A Varian 1200L triple-quadrupole LC–MS (Varian, Walnut Creek, CA, USA) coupled to the Dynamax ProStar 210 liquid chromatograph system was used. The analytes were separated on a Waters XTerra C8 (15 cm × 2.1 mm, 3.0 m) LC column (Waters Corp., Milford, MA, USA) preceded by an in-line precolumn filter of 3 mm frit (Supelco, Bellefonte, PA, USA). The mobile phase was filtered using a filter (0.22 m) and degassed by using vacuum followed by sonication. The gradient separation of LC was proceed. A pH 2.76 of mixture solution of 0.05 mM ammonium formate containing 0.1% formic acid (A) and acetonitrile (B) was used as mobile phase at a flow-rate of 0.2 mL/min. The initial composition was 35% B for 1.0 min, then increased to 65% for 6 min, then decreased to 35% for 5 min until equilibrium. The volume of injection loop was 5 L, and all the column effluent was directed into the mass spectrometer. The selected reaction monitoring (SRM) mode of MS/MS was used for quantization. The data acquisition software Varian MS workstation, Version 6.2, was used for instrument control,
data acquisition and data handling. Analysis was performed in both positive ionization and negative ionization modes. Voltages across the capillary, lens and the quadrupoles were tuned by an automated procedure to maximize the signal for analyzed ions. The parameters for the ionization efficiency were optimized by evaluating sensitivity using flow injection analysis (FIA) with the mobile phase at 0.2 mL/min. The instrumental parameters were optimized to maximize signal intensity of the deprotonated molecule ion [M−H]− or pseudomolecular ion [M+H]+ by injecting 5 L standard solution (1.0 g/mL) through a sample loop. The MS/MS was performed based on collision-induced dissociation (CID) of specific precursor ion and the generation of the characteristic fragment ions. The Q1 was scanned a range of m/z 50–500 for precursor ion optimization. In all deprotoned molecule ions of analytes observed were more stable, abundant and less fragmented during negative scan. Both Q1 and Q3 quadrupoles were set at unit resolution. During SRM analysis mode, mass peak width was 1.0 mass unit at half height for both Q1 and Q3, scan time was 1 s. The spray conditions of API interface were: housing temperature 65 ◦ C; nebulizing gas pressure 35 psi; drying gas temperature 250 ◦ C; drying gas pressure 20 psi. Argon was used as the collisional gas at pressure of 2.0 mTorr. 2.4. Method validation The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision (RSD), accuracy and recovery. Urine calibration standards were a set of spiked diuretics in drug-free urine at concentrations of 1, 5, 10, 50, 100, 250, 500 and 1000 ng/mL. Three replicate analyses were performed for each concentration to evaluate linearity. The peak areas were used for quantification through the calibration curves of diuretics. The LOD and LOQ were determined as the ratio of 3 times and 10 times standard deviation at the lowest concentration of linear range divided to the slope of linear equation, respectively. Precision of the assay was determined by generating intra- and inter-day variability data from a 50 ng/mL sample. The analytical accuracy of purposed method was determined by analyzing spiked diuretics in urine samples at a concentration of 50 ng/mL five times and expressed as the ratio of mean measured concentration to spiked concentration. The analytical recovery was determined by analyzing triplicate of spiked diuretics in urine sample at concentration of 50 ng/mL. To verify specificity, blank samples were prepared with drug-free urine samples from five different donors. 3. Results and discussion 3.1. Mass spectrometry of diuretics Both positive and negative ionization mode of ESI were used to evaluate the most sensitive ionization mode for the diuretics. The concentration of 1 g/mL diuretics was used in this study with FIA. The pseudomolecular ion, [M+H]+ , of all diuretics was obtained in positive ionization mode as the base ion. In the negative ionization mode, the peak of the deprotonated molecule
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Table 1 Precursor ion and product ion used in SRM of LPME–LC–MS/MS for detection diuretics Analytes Chlorothiazide C7 H6 ClN3 O4 S2 Hydrochlorothiazide C7 H8 ClN3 O4 S2 Hydroflumethiazide C8 H8 F3 N3 O4 S2 Clopamide C14 H20 ClN3 O3 S Trichlormethiazide C8 H8 Cl3 N3 O4 S2 Bumetanide C17 H20 N2 O5 S Probenecid C13 H19 NO4 S Ethacrynic acid C13 H12 Cl2 O4
Abb.
Precursor ion (m/z)
CT HCT HFT CA TCT BUM PRO EAA
[M−H]−
294 296 [M−H]− 330 [M−H]− 344 [M−H]− 378 [M−H]− 365 [M+H]+ 284 [M−H]− 301 [M−H]−
Product ion (m/z) 214 [M−H-SO2 NH2 ]− 205 [M−H-SO2 CNH]− 239 [M−H-SO2 CNH]− 189 [M-C8 N2 OH15 ]− 306 [M−H-2HCl]− 240 [M+H-CO2 SO2 NH2 ]+ 240 [M−H-CO2 ]− 243 [M−H-CO2 -CH2 ]−
ions, [M−H]− of all diuretics as the base ion and less fragment ions were observed. The structures of thiazide-base diuretics BUM, CA, CTA, HCT, HFT and TCT are a benzene ring and a sulfamonyl residue ortho-located to an electronegative substitute such as a chlorine atom (Fig. 1). In preliminary experiments for all thiazide-base diuretics except bumetanide indicated that the signal of [M−H]− was more sensitive than that of [M+H]+ . According to the paper described thiazides as acidic character, the use of negative ion electrospray ionization proved to be reasonable [39,40]. Therefore, the positive ionization mode was not considered further for using to analyze diuretics except for bumetanide. In MS/MS experiments, ESI interface was used in conjunction with the FIA for optimization of the MS/MS parameters. The product ion mass spectrum of deprotoned molecule ion was obtained in the scan range of m/z 50–500. For all thiazide diuretics, the characteristic fragment ion [M-SO2 NH2 ]− was observed in the product ion spectra. For CT, the ion [M-SO2 NH2 ]− , m/z 214 is the base peak. Therefore, in this assay, the mass transition pattern, m/z 294 → 214 was selected to monitor CT in negative ionization SRM analysis (Table 1). For other diuretics, the base peak ions were shown in Table 1. In negative ionization mode, the mobile phase of ACN exited the same mass transition m/z 363 → 319 as bumetanide. The pseudomolecular ion of bumetanide, [M+H]+ m/z 365 was chosen as precursor ion,
and the product ion, m/z 240, [M+H-CO2 SO2 NH2 ]+ was used for monitored in positive mode to avoid the interference from mobile phase. Therefore, in this assay both positive and negative ionization modes were used simultaneously to detect the diuretics in urine.
Fig. 3. Effect of salt concentration on the extraction efficiency of 50 ng/mL diuretics in urine produced by using LPME–LC–MS/MS.
Fig. 4. Effect of extraction temperature on the extraction efficiency of 50 ng/mL diuretics in urine produced by using LPME–LC–MS/MS.
3.2. The hollow-fiber LPME method In order to increase the sensitivity, the optimum LPME conditions for the determination of diuretics were required to evaluate the various parameters that affect the LPME extraction procedure. Extraction efficiency of the membrane may be affected by extraction solvent, extraction temperature, extraction time, solution pH, ionic strength of aqueous phase, hollow fiber length and stir rate. It is very important to optimize these parameters for enhance extraction efficiency and then to improve detection. For LPME, polypropylene membrane has proven effectively for increasing concentration and reducing matrix interference from urine [31,34,35]. The polypropylene fiber was selected in this study. The solvent used in LPME should have low water solubility and high solubility for analytes. Three organic solvents n-octanol, toluene and cyclohexane were used to evaluate the extraction efficiency of 50 ng/mL diuretics in urine at room temperature for 30 min. The experimental results indicated that the highest extraction efficiency was obtained with n-octanol. Therefore, n-octanol was chosen as extraction solvent in this study. The pH value of sample solution influences both the dissociation equilibrium and the solubility of organic acid or base
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analytes. For diuretics, BUM, EAA and PRO are highly acidic and the other five compounds are less acidic. A sample pH range from 1 to 10 was used to evaluate the extraction efficiency of 50 ng/mL diuretics in urine at 20 ◦ C for 30 min. The results indicated that lower pH value improved extraction efficiency of the target analytes. Hence, the pH of solution was adjusted to 2. Salt-out effect was often used to improve extraction efficiency of analytes in LPME as in SPME [41]. Ionic strength increases in the sample solution will decrease the solubility of target analytes
in water solution for salt-out effect. The NaCl is a conversional salt used to change the ionic strength in solution. The salt effect study was evaluated at salt concentrations from 0 to 0.35 g/mL. As Fig. 3 shows, extraction efficiency increased as NaCl concentration increased and reached a maximum at 0.15 g/mL and then decreased at higher NaCl concentrations. The decay in high concentration of NaCl was possible due to electrostatic interaction between polar analytes and NaCl ions in solution [41,42]. Hence, 0.15 g/mL NaCl was added in the further experiments.
Table 2 Estimated limits of detection and linear range for diuretics by LPME–LC–MS/MS Compound
Linear range (ng/mL)
Correction coefficient (r2 )
LODa (ng/mL)
LOQb (ng/mL)
Enrichment factorc
Extraction efficiency (%)
CT HCT HFT CA TCT BUM PRO EAA
5–1000 10–1000 2–1000 2–1000 2–1000 10–500 1–1000 5–1000
0.9994 0.9986 0.9938 0.9995 0.9989 0.9997 0.9969 0.9962
1.7 6.7 1.1 0.3 0.4 3.6 0.6 2.8
5.5 22.3 3.7 0.9 1.4 12 2.1 9.4
0.8 1.7 3.8 95.2 11.3 20.1 32.6 144.8
0.2 0.5 1.0 25.4 3.0 5.4 8.7 38.6
a b c
LOD, limit of detection, determined as 3S.D./slope, standard deviation of the lowest concentration of calibration curve divided by the slope (n = 3). LOQ, limit of quantification, determined as 10S.D./slope, standard deviation of the lowest concentration of calibration curve divided by the slope (n = 3). Urine was spiked at 50 ng/mL.
Fig. 5. Mass ion chromatograms of 50 ng/mL diuretics in urine produced by LPME–LC–MS/MS.
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Matrix interference is often an important problem in biological samples during trace analysis [28,31,42–44]. To evaluate the influence of matrix, the MS signal intensities of 50 ng/mL standard diuretic solutions diluted with artificial urine and ultrapure water were tested. Almost the same intensities of diuretics were obtained no matter what matrices used. It was shown that LPME can improve the sample clean-up to remove urine components and thereby decreasing the amount of matrix injected onto the column. Agitating stir bar can increase the mass transfer coefficient from the sample matrix to extraction phase. To trace the effect of stirring, eight stir rates (0–1200 rpm) were used to evaluate extraction efficiency of 50 ng/mL diuretics in urine at 40 ◦ C for 40 min. The high stirring speed increased extraction efficiency but also produced instability of the organic solvent in hollow fiber and the air bubbles would affected the precision and reproducibility of the extraction process. Therefore, a stirring speed of 1010 rpm was used for subsequent experiments. 3.2.1. Extraction temperature and time The LPME method employs a partition equilibrium process in target analytes between extraction phase (accept solution)
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and sample matrix (donor solution) [30]. Extraction temperature and time affect partition equilibrium. The higher extraction temperature normally has a positive effect on extraction yield by increases both solubility and diffusion of analytes into extraction phase. However, heating might also cause swelling in fibers and does not favor organic solvent immobilization in membrane pore. Extraction temperature was studied at a room temperature to 60 ◦ C. The experimental results in Fig. 4 show that extraction efficiency was maximum at 40 ◦ C. In optimization extraction time investigated, a spiked 50 ng/mL urine solution was used within 20–50 min at 40 ◦ C. The experimental results showed that the peak areas of diuretics were reached maximum at extraction for 40 min. When extraction time exceeded 40 min, the peak areas of analytes slowly decreased. Thus, 40 ◦ C extraction temperature and 40 min extraction time were selected for the following experiments. 3.2.2. Acceptor phase volume The ratio between the volume of the donor phase (V0 ) and the acceptor phase (Vorg ) governs the extraction efficiency (EE) and enrichment factor (EF) of analytes in the acceptor phase,
Fig. 6. Total ion chromatogram and mass ion chromatograms of dosed patient urine sample produced by LPME–LC–MS/MS.
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according to the expression: norg Vorg Corg EE = 100% = 100% n0 V0 C0
The concentrations of diuretics detected were HCT, 24.9 g/mL; BUM, 0.046 g/mL; PRO, 0.011 g/mL; EAA, 0.008 g/mL. 4. Conclusion
and EF =
Corg C0
where norg , Vorg and Corg are the mass, the volume and the concentration of analyte extracted in the organic solvent, respectively, and n0 , V0 and C0 are the mass, the volume and the concentration of analytes present in the original sample, respectively [18]. To optimize acceptor-to-donor phase volume ratio, the donor phase volume (V0 ) is kept constant, and the volume of organic solvent (Vorg ) is changed from 15–36 L by adjusting the length of the hollow fiber at 5, 6, 8, 10 and 12 cm. The experimental results showed the peak areas of the analytes were maximum at a 6.0 cm of hollow fiber with an acceptor phase volume of 20 L. The parameters of optimum condition of LPME were list in Table 2. 3.3. Assay validation Calibration curves for eight diuretics were established for the concentration range from 1 to 1000 ng/mL. The linear range, precision, limit of detection (LOD), and limit of quantification (LOQ) of this method were evaluated under the optimum condition as shown in Table 2. The linearity of the proposed method was with correlation coefficients (r2 ) being greater than 0.9938. The LOD and LOQ were 0.3–6.7 ng/mL and 0.9–22.3 ng/mL, respectively. The LOD of eight diuretics were found in low ng/mL level and lower than the minimum required performance limits (MRPL) of the World Anti-Doping Agency (WADA). The mass ion chromatograms of 50 ng/mL diuretics in urine analyzed by LPME-LC-MS/MS were shown in Fig. 5. The recovery and precision of this method expressed with % by analyzing 50 ng/mL diuretics spiked urine samples. The recovery of diuretics was 97.7–102.5%. The intra-day and interday relative standard deviations (R.S.D.s, n = 6) were 3–18 and 4–21%, respectively. LPME is a non-exhaustive extraction procedure as solid-phase microextraction, the extraction efficiency is defined as the ratio of the amount of analyte in acceptor phase to donor phase and dependant on the distribution coefficient of the compound. As shown in Table 2, the extraction efficiency of diuretics from spiked urine was 0.2–38.7%, the high enrichment factor could be obtained. For EAA, the high enrichment factor was reached 144.8-fold. 3.4. Application The diuretics dosed patient urine was analyzed by the LC–ESI–MS/MS method described. The LPME was operated at the optimum conditions studied. Representative SRM chromatograms of urine sample collected from the patient first urine after post-administration were shown in Fig. 6. The total ion chromatogram (TIC) and extracted ion chromatogram of urine sample obtained from a hypertension patient was shown in Fig. 6.
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